Hereditary Spastic Paraplegias: Clinical Spectrum in Sudan, Further Deciphering of the Molecular Bases of Autosomal Recessive Forms and New Genes Emerging

Hereditary spastic paraplegias : clinical spectrum in Sudan, further deciphering of the molecular bases of autosomal recessive forms and new genes emerging Liena Elbaghir Omer Elsayed

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Liena Elbaghir Omer Elsayed. Hereditary spastic paraplegias : clinical spectrum in Sudan, further deciphering of the molecular bases of autosomal recessive forms and new genes emerging. Neurons and Cognition [q-bio.NC]. Université Pierre et Marie Curie - Paris VI; University of Khartoum, 2016. English. NNT : 2016PA066056. tel-01438739

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Université Pierre et Marie Curie University of Khartoum

Cerveau-Cognition-Comportement (ED3C) Institut du Cerveau et de la Moelle Epinière / Equipe Bases Moléculaires, Physiopathologie Et Traitement Des Maladies Neurodégénératives

Hereditary spastic paraplegias: clinical spectrum in Sudan, further deciphering of the molecular bases of autosomal recessive forms and new genes emerging

Par Liena ELBAGHIR OMER ELSAYED

Thèse de doctorat de Neuroscience

Dirigée par Dr. Giovanni STEVANIN, Professor. Ammar ELTAHIR AHMED

Présentée et soutenue publiquement le 27 avril 2016, devant un jury composé de:

Pr.LEGUERN, Eric, PU-PH Représentant de l’UPMC Pr.KLEBE, Stephan, PU-PH Rapporteur Pr.KOENIG, Michel, PU-PH Rapporteur Pr.BOESPFLUG-TANGUY, Odile, PU-PH Examinateur Pr.ELTAYEB IBRAHIM, Mutaser, PU Examinateur Pr.ELTAHIR AHMED, Ammar, PU-PH Co-directeur de thèse Dr.STEVANIN, Giovanni, DR Directeur

Dedication To my parents, husband, kids.

Acknowledgement

I am grateful to the members of my thesis defense jury who have kindly accepted to read and assess my PhD manuscript.

I would also like to express my sincere gratitude to my thesis directors Dr.Giovanni Stevanin and professor Ammar Eltahir Ahmed for kindly accepting to guide me throughout my journey in this challenging PhD project. They have been quite supportive at each step and have given me a great deal of independence which has allowed me to gain the required skills in project management and trouble shooting and be prepared for the next step. Dr.Giovanni Stevanin has struggled to get me the budget necessary for my experiments and has gently helped me at each step despite his rather busy schedule. I enjoyed much his humble friendly attitude despite his vast knowledge in our weekly discussions that we had in the last two months of my stay in France. Pr.Ammar Eltahir has kindly taken the burden of the neurophysiological investigations and opened his heart and clinic for all patients included in the cohort. He has been quite supportive and enthusiastic to help me by all means and efficiently contributed to the recruitment of new families.

I would like to thanks Pr.Alexis Brice for accepting to collaborate and acquaint me in his team and even taking all the effort to come to Sudan to finalize the collaboration. My gratitude is great for Prof. Victor Patterson who has practically started the collaboration by introducing me to Pr.Brice. He has been extremely supportive and encouraging.

All my appreciation is conduced to my mentors Pr.Muntaser Eltayeb Ibrahim and Pr.Mustafa Idris Elbashir who embraced and supported me from the beginning when the project was just an ambitious day dreaming.

Our collaborating Sudanese clinicians are the corner stone in this research. The three lively neuropaediatricians (Dr.Inaam Gashey, Dr.Maha Elseed and Dr.Ahlam Hamed ), the humane Dr.Sarah Misbah, Pr.Mustafa Salih, Pr.Mohamed Nagib and his team, Dr. Hassab Elrasoul Siddig, Pr. Farooq Yasin and all the referring physicians, the radiologists, the ophthalmologists and the neurophysiologists all have contributed significantly to establish this project. My sincere gratitude is conducted to the Sudanese patients and their families who were as kind as a Sudanese can be expected.

Prof. Alexandra Durr and Prof. Odile Boespflug-Tanguy (and her team especially Imen Dorboz) have not saved any effort to help me build my expertise in clinical neurogenetic and neuropaediatrics. Pr.Eric Leguern valuable tips with his sense of humour were priceless.

Prof. Anjon Audhya team (USA) has kindly performed all the biochemical functional studies for the TFG protein and with the Parkinson team (Dr.Suzanne Lesage and her team) has yielded our successful reports so thanks to their efforts.

The assistants of Alexis Brice team (Lydia Guennec, Marie-Luce Poupinel-Descambres and Antoine Guisier) have been professional and if not for the efforts of Lydia Guennec in my registration for the PhD I would have been unable to continue.

The French embassy in Sudan and Campus France and the University of Khartoum provided me with the scholarship that has enabled me to come and do the PhD study in France.

III

My acknowledgement to the funding agents of the study: the France-Parkinson Association (to AB), the Agence Nationale de la Recherche (SPATAX-QUEST project, to GS), the Verum Foundation (to GS and AB), the Roger de Spoelberg Foundation (R12123DD, to AB), the French Academy of Sciences (to AB), the European Union (Neuromics projects, OMICS call, to AB) and the European Joint Programme - Neurodegenerative Disease Research (JPND-COURAGE-PD) project and Programme d’Investissement d’Avenir IHU-A-ICM (ANR-10-IAIHU-06).

I would like to transmit my gratitude to team of the DNA and Cell bank (with special regards to the friendly Sylvie Forlani and Christelle Dussert), the laboratory and bioinformatics staff of the sequencing platform (ICM) in which we performed all the next generation techniques and the bioinformatic analysis, the platform P3S where we had the homozygosity mapping experiments performed with the help of Dr.Wassila Carpentier (who has done big efforts not only to explain and train but also has given me valuable tips for my thesis writing).

Working with the team of Dr.Giovanni Stevanin has been a pleasure. They have become my second family. Professionally, Marie Coutelier and Christelle Tesson helped me in the first steps of my PhD and have been quite cooperative whenever I needed. The moments that I had with Marie were great and extremely pleasant. With Marie I discovered that Paris is truly described the city of light and because of that I stated to fall in love with the quiet lighted streets at night. The help that I have received from Mathilde Mairey with a beautiful smile and a comforting attitude of a friend cannot be described by words. Her efforts to help me in the last months of my PhD make me run out of expressions. Sawssan Ben Romdhan has been the support (with a friendly spirit) that I needed at a time when I was so desperate because of the accumulating work. Dr. Frédéric Darios and his professional group have helped and trained me on the basics of manipulations of cells and functional experiments. Special thanks are conducted to the nice Typhaine Esteves. Livia Parodi, Federica Barreca, Graziella Mongone and Paola have made me believe that Italians are all kind. The special memories, help, great laughter and the discussions I had with Khalid Hamid El-Hachimi, Livia Parodi, Celine Lustremant, Valérie Drouet, Claire Pujol, Claire Sopie Davoine, , Khadija Tahiri, Sara Morais, Oriane Trouillard, Annie Sittler, Remi Valter, Julien Branchu make me grateful to them and I feel sad and tearful when I imagine that we can’t be together anymore as we used to be. Special thanks to Claire Pujol for helping me with the final editing of the manuscript. She helped me at the critical moments of extreme fatigue and exhaustion with unforgettable friendship spirit.

My team members in Sudan have been the scaffolding unit of all the work. Ashraf Kambalawy, Mahmoud Koko, Rayan Abubakar established the team with me and their professional attitude, hard working, highly committed and loyalty made me surmount all the obstacles. All the younger members of my team have worked extremely hard just to make our work successful without waiting for any reward and they have represented our team in conferences and workshops in a distinct way (Arwa Babai, Zulfa Mohamed, Hiba Malik, Israa Zuhair, Amal Salah, Ahmed Khalid, Elhami Abdalmuti, Lina Salah, Duaa Mustafa, Mayada Osama, Nabila Mohamed Hasan, Abdelhamid Mohamed and the two under-graduates Malka Osman and Eman Osama).

Finally but most importantly, I would like to say honestly and proudly say that if not for the support of my family I would have done literally nothing. They have set an example of a great family who would sacrifice for the sake of others. My husband I would describe as a great man. Not only has he sacrificed all his rights of a stable situation with a settled wife but also

IV he supported me psychologically and financially. My parents ElBaghir Omer Elsayed and Fatma Elsaraj have taken care of my kids and I would say my mother deserves a medal of honour for taking in charge the responsibilities of my kids’ studies and have made them brilliant youngsters better than what I could have done. My kids (Tull, Abdelaziz and Yusr) have encouraged me to go on when I was in despair and they insisted that I continue to help the patients. My parents in law (Abdelaziz Tangasawi and Asia Abdallah) took in charge a significant deal of my responsibly towards my husband and kids in parenting manner. The precious professional advices of my father in law have been extremely useful.

My brother Omer Elbaghir has shown me what it means to be a brother. He supported me psychologically and supported my research financially with real love. His wife Dina Saklah used to call me to encourage me. I would like to thank all my dear sisters (Lubaba, Lubna and Shayamaa) and their families for the continuous supporting love. All my appreciation Shaymaa who helped my mother in her care for my kids in silent way.

Finally, my thanks are to other friends and family that I could not list all. The combined efforts of everyone have helped me overcome the sorrow that I used to experience upon separation from my family over long months I spent alone in France.

Liena Elbaghir Omer Elsayed

University of Khartoum/University of Paris 6

February 2016

Table of Contents Introduction

1. State of the art 1

1.1. Clinical spectrum of HSP 3 1.1.1. Associated signs and clinical phenotypes 3

1.1.2. Age at onset 4

1.1.3. Disease progression 5

1.2. History of Spastic Neurogenetic Disorders 5

1.3. A continuum of spastic neurogenetic disorders 9

1.4. Pathophysiology of HSP 13

1.4.1. Basic pathophysiologic mechanisms 13

1.4.2. HSPs are mainly due to neuronal degeneration 13

1.4.3. Pathways involved in HSP pathogenesis 14

1.4.4. Functional heterogeneity of HSP proteins 15

1.5. Assessment and diagnostic approach of HSP patients 15

1.6. Treatment options for HSP 18

1.7. Genetic counseling 19

2. Materials and methods 20

2.1. Ethics 20

2.2. Subjects recruitment 20

2.2.1. Inclusion and exclusion Criteria 20

2.2.2. Recruitment Rate 21

2.3. Clinical phenotyping 21

2.3.1. Questionnaire and auditing 21

2.3.2. Clinical Assessment 21

2.4. Sampling 23

2.5. DNA Extraction, and quality/quantity check 23

2.5.1. DNA Extraction: 23

2.5.2. DNA quality/quantity check 24

2.6. Genetic linkage analysis using traditional microsatellite markers 27

2.7. Primer design, PCR amplification and control 27

2.7.1. Primer Design 27

2.7.2. PCR 28

2.8. Genome-wide genotyping, homozygosity mapping and whole 28 genome linkage analysis

2.8.1. General description 28

2.8.2. Laboratory protocol 29

2.8.3. Bioinformatic analysis (downstream) 30

2.9. Screening HSP genes using targeted Next Generation Sequencing 30

2.9.1. General description 30

2.9.2. Laboratory protocol 31

2.9.3. Bioinformatic analysis 35

2.10. Whole Exome Sequencing (WES) 39

2.10.1. General description 39

2.10.2. Laboratory protocol 40

2.10.3. Bioinformatic analysis 43

2.11. Basic Functional Studies (provisional post-genetic workup) 46

2.11.1. Biochemistry 46

2.11.2. Culture of fibroblasts 46

3. Results 49

3.1. General features of the cohort 49

III

3.2. Genetic results 54

3.2.1. Identification of mutations in known genes 54

3.3. Identification of new causative HSP Genes 108

3.3.1. ABHD16A mutation in family F37 108

3.3.2. Candidate genes in family in F41 115

3.3.3. Exploration of family F50 123

3.3.4. ST7L mutation in family FM3 130

3.3.5. PANK4 mutation in branch 1 of family FM7 135

3.4. Causes unidentified 143

3.5. Comments and summary 159

4. Discussion 161

4.1. Overview of the cohort 161

4.2. Diagnostic NGS-based multigene panels for HSP 170

4.3. Comparison of the yield of genetic results 172

4.4. Towards the identification of new genes 173

4.4.1. ABHD16A and complex TCC HSP in family F37 (The flip coin of 173 ABHD12/PHARC)

4.4.2. CAMSAP3, MINK1, ZNF334 candidate genes in family F41 177

4.4.3. BIRC5 and C21ORF91 variants in family F50 181

4.4.4. ST7L unique mutation responsible for the pathology in family 185 FM3

4.4.5. PANK4 unique variant in branch1 of family FM7 187

4.5. Cellular pathogenic mechanisms proposed for the novel genes: a 191 resumé

4.5.1. Microtubules/cytoskeletal related cellular mechanisms 191

4.5.2. Neurite outgrowth 191

4.5.3. Glucose metabolism 191

4.5.4. Apoptosis and regulation of cell growth 192

4.5.5. Cytokinesis 192

4.6. A comprehensive overview of pathogenetic Mechanisms of HSP 192

4.6.1. General Observations 193

4.6.2. Correlations 193

4.7. A new nosology and objective case definition of HSP: a question 211 raising itself ?

4.8. Challenges, milestones and particularities of the Study 212

4.8.1. Cohort building 212

4.8.2. Diagnostic Capacities 214

4.8.3. Huge work / One pair of hands/ Time 214

4.8.4. Budget management 214

4.8.5. WES analysis 215

4.8.6. Administrative processes 215

5. Conclusion and prospective work 216

References 218

Annexes 237

List of Figures 265

List of Tables 269

Résumé 271

Abstract 272

List of Abbreviations

HA Hereditary ataxia HSP Hereditary spastic paraplegia NBIA Neurodegeneration with brain iron accumulation INAD Infantile neuroaxonal dystrophy PLAN PLA2G6 associted neurodegeneration P Pure C Complex UL Upper limb LL Lower limb AD Autosomal dominant AR Autosomal recessive X-L X- linked Mito Mitochondrial SPGn Spastic paraplegia gene “n” ALS Amyotrophic lateral sclerosis CP Cerebral palsy SLS Sjogren-Larsson Syndrome MSS Marinesco Sjogren Syndrome ARSACS AR spastic ataxia of Charlevoix-Saguenay NGS Next generation sequencing LHON Leber hereditary optic neuropathy PLS Primary lateral sclerosis PD Parkinson disease CPSQ Cerebral palsy spastic quadriplegia IEM Inborn errors of metabolism E-Pyr Extrapyramidal syndrome PN Peripheral neuropathy PNS Peripheral nervous system CNS Central nervous system AIS Axon initial segment MRI Magnetic resonance imaging TCC Thin corpus callosum VEP Visual evoked potential AEP Auditory evoked potential NCS Nerve conduction studies SEP Somatosensory evoked potential EMG Electromyogram EEG Electroencephalogram HZM Homozygosity mapping WES Whole exome sequencing DRD Dopa responsive dystonia ITB Intrathecal baclofen

PCR Polymerase chain reaction Amyo Amyotrophy Cog Cognitive CI Cognitive Impairment OA Optic atrophy RD Retinal degeneration MD Macular degeneration

VII

Introduction

Hereditary spastic paraplegias (HSP) are a heterogeneous group of neurodegenerative disorders with the core defining clinical features of insidiously progressive weakness and spasticity of the lower extremities. They impose diagnostic challenges due to the multiple overlaps with other neurological disorders in which spastic paraplegia/quadriplegia is present. The rapidly expanding surge of new causative genes (named spastic paraplegia gene “n”or SPGn) that accompanied the emergence of multiple next generation sequencing (NGS) techniques further contributed to this diagnostic dilemma. In addition, the concept of neurodegeneration being a continuum of disorders is illustrated by 2 facts in HSP:

- Many clinico-genetic HSP entities are associated with other neurological signs that can be the major clinical features of overlapping diseases. For example, hereditary ataxias (HA) represent the other extreme of the spectrum of spinocerebellar neurodegenerative disorders. HAs may sometimes be difficult to differentiate clinically from HSPs as cerebellar manifestations are present in more than 30 clinico-genetic SPG entities. Many HSP subtypes present with peripheral neuropathy [SPG3A, SPG5A, SPG11, SPG17, SPG30, SPG31, SPG57, SPG70, FAM134B]. A third example is leukodystrophy (non-neurodegenerative disorders). Although it is classified as a differential diagnosis of HSP that should be excluded in early steps of clinico-genetic investigations, white matter abnormalities are described as a complicating feature for at least 28 HSP subtypes, making differential diagnosis a real challenge.

- Mutations in multiple SPG genes can account for HSP but also for allelic phenotypes. This is the case for motor neuron disease [MND] [KIAA1840 (SPG11), ERLIN2 (SPG18)] and the reverse situation is also true (ALS2 is a major cause of amyotrophic lateral sclerosis but few HSP families have also been reported mutated in this gene). Moreover, mutations in seven SPG genes are associated with leukodystrophy as an allelic disorder [PLP1(SPG2), HSPD1 (SPG13), FA2H (SPG35), GJC2 (SPG44), RNASEH2B, ADAR1, IFIH1]. The same applies for juvenile Parkinson disease [SPG11], HA [SPG7, SPG39], spastic ataxia [SPG39, SPG46, SPG58] and epilepsy [MT-ATP6]. These observations following the explosion of results from NGS techniques suggest that clinico-genetic classifications are imperfect. Along the same line, recently characterized novel clinico-genetic forms implicating lipidic or amino acid metabolism now raises a question of whether or not metabolic spastic disorders (previously

VIII classified as non HSP conditions [eg. ARG1] should now be reclassified as HSP or if a larger rebuilt of the nosology of neurological conditions has to be performed.

In this study, I explored the clinical varieties and genetic and molecular pathways behind spastic neurodegeneration in a familial Sudanese cohort. The Sudanese population is highly consanguineous with inter- and intra-generational consanguinity loops. Although a lot is known about it from clinical neurologic point of view, it remains almost completely unexplored from a neurogenetic point of view. Statistics estimates about incidence, prevalence and relative frequencies of HSP genes are not available to the present time as in most sub-Saharan countries.

With the help of clinicians from Sudan, we recruited 41 nuclear and extended Sudanese families representing 337 total number of individuals of whom 106 were affected patients. First, full clinical examination was performed by myself and/or my colleagues in Sudan. Then I have established a DNA bank. To do so, saliva samples were collected for extraction of genomic DNA. When necessary for validation experiments, fibroblasts were also obtained from skin biopsies. The second aspect of my thesis consisted of deciphering the genetic bases explaining the disease in these families. Screening of several known SPG genes was done in five families using a phenotype-based candidate gene approach. I applied an NGS-based screening strategy for 74 SPG-related genes as the main screening method in all remaining 36 families and the two families with negative candidate gene approach result. Whole exome sequencing (WES) was then done in search for novel genes in families with negative screening results in which DNA of two or more patients were available. Homozygosity mapping using genotyping array was done to further consolidate gene search process. In certain conditions, functional studies were necessary, depending on the feasibility and novelty of expected outcome.

I identified the genetic cause in 17/41 families. In 12 families the genes where I found a segregating mutation, were known HSP genes. In three families, novel genes were identified mutated whereas five candidate genes were found segregating with disease in two other unrelated families requiring additional experiments to conclude. In these highly inbred families, multiple genes /genetic disorders were found running in parallel in different branches [five families] or in the same branch [three families] of the family. Analysis of the NGS screening panel and of WES data, thus imposed certain challenges specific to these families with elevated consanguinity rates. Another peculiar phenomenon was the appearance

IX of clinical differences between Sudanese patients and patients of other origins even when caused by mutations in the same genes and in rare conditions even when caused by the same variant which contributed to the phenotypic heterogeneity already reported in these disorders.

1. State of the art

Hereditary spastic paraplegia (HSP) is considered as the second most frequent motor neuron disease (MND) with a prevalence of 3-10/100000 in most populations (Noreau et al., 2014). HSP is transmitted via all modes of inheritance which include: autosomal dominant (AD), autosomal recessive (AR), X-linked (X-L), mitochondrial/maternal (Mito) and mixed inheritance (AD/AR; AR/? AD). The Prevalence of (AD) HSP ranges between 0.5- 5.5/100000 and (AR) HSP from 0.3-5.3/100000 (Ruano et al., 2014). HSP shows remarkable genetic and clinical heterogeneity [Figure (1-1), (1-2), (1-3)]. To date there are more than 88 loci/72 genes implicated in the pathogenesis of HSP. 75 clinico-genetic forms are assigned the designation spastic paraplegia gene “n”or SPGn but other forms remain unclassified yet (Tesson et al., 2015) (Klebe et al., 2015). The category of mixed inheritance has emerged recently with increasing number of genes showing this pattern (five genes with frank AD/AR inheritance: SPG3A, SPG9, SPG72, BICD2, SPG30 and two showing AR inheritance with milder/ partial phenotype in individuals with heterozygous genotype (possible dose dependent phenotype): SPG7,SPG58) [Table (1-1)].

1.1. Clinical spectrum of HSP

1.1.1. Associated signs and clinical phenotypes There is huge phenotypic heterogeneity associated with HSP. The core clinical presentation of patients is with pyramidal syndrome which includes increased tone (spasticity with scissoring and clonus in its most severe form), hyperreflexia and extensor plantar response (Babinski sign). It can be classified as pure (uncomplicated) and complex (complicated) according to the absence or presence of additional neurological and extra-neurological manifestations [Figure (1-4)]. Pure forms can be associated with involvement of the dorsal column and diminuted or even abolished vibration sense. Sphincteric involvement (mainly urinary urgency) can occur in pure HSP too due to increased bladder muscle tone (Fink, 2000) (Tesson et al., 2015) (Klebe et al., 2015). Complicating neurological clinical features include cognitive/mental function deterioration, cerebellar syndrome, peripheral neuropathy, features of lower motor neuron involvement (amyotrophy), signs of bulbar/ pseudobulbar palsy, optic neuropathy, psychiatric symptoms, extrapyramidal signs, auditory neuropathy as well as brain imaging abnormalities. MRI of the brain may show atrophy (focal: thin corpus callosum, atrophy of the cortex and cerebellum or generalized), features of dysmyelination (hypo- and de-myelination) and leukodystrophic changes (white matter

3 hypersignal intensity lesions). Extraneurologic systems involved are the eye, heart, skeletal system, skin, hair and gastrointestinal system.

Cognitive and mental impairment are the most frequent clinical features complicating various HSP forms (≈38 forms) followed by cerebellar signs, peripheral neuropathy and/or amyotrophy and eye signs taken collectively (cataract, retinal/macular degeneration and optic atrophy) [Annexes: Tables 1 to 11]

The rapidly expanding genetic heterogeneity has resulted in an even more increase of phenotypic heterogeneity. The spectrum of HSP has broadened largely to include additional atypical phenotypes that were not reported previously in the earlier cohorts of HSP (Harding, 1983) [Table (1-1)]. Non-exhaustive illustrative examples include tetraplegic cerebral palsy (SPG50) (Verkerk et al., 2009), HSP with hyperbilirubinemia and persistent vomiting due to hiatus hernia (SPG29) (Orlacchio et al., 2005) and atypical HSP presenting with areflexia, ventriculomegaly, apnoea, hypoventilation and gastrooesophageal reflux disease associated with SPG49/ TECPR2 (Oz-Levi et al., 2012).

The list of reported complicating neurological and non-neurological clinical signs has also extensively expanded to reach the present range. In addition to the signs associated with the above mentioned phenotypes; the mitochondrial genes (MT-ATP6, MT-TI) cause with HSP associated with cardiomyopathy (Verny et al., 2011). Another mitochondrial gene MT-CO3 has been linked to HSP but with a Leigh syndrome- like lesion in the brain (Tiranti V. et al., 2000).

The greatest part of clinical heterogeneity can be attributed to the AR HSP forms which mainly present as complicated HSP in contrast to the tendency of AD HSP for pure presentation [Figure (1-4)] [Table (1-1)].

1.1.2. Age at onset Age at onset of HSP varies widely. It ranges from birth in some HSP forms to more than 40 years (in some rare cases the disease started at the age of 80 years). The variability of age at onset not only occurs between various forms but also occurs within single HSP forms and even within families in patients carrying the same mutation [Figure (1-5)].

4 It is often difficult to assess the age at onset precisely in HSP. This is sometimes attributed to the subtlety of the presenting motor symptoms particularly in complex forms. In pure forms, some patients do not complain of the disease but have clinical signs at examination. This results in an underestimated and overestimated incidence, prevalence of HSP.

The age at onset of AR HSP tends to cluster in childhood in 80% (45/57) of the genetic forms. More than 34 AR HSP forms as well as (3/5) X-linked forms occur in toddlers (0 - <5 years). AD forms do not show similar clustering but tend to have variable age at onset. Some forms show substantial variability. Of these forms with widely variable age at onset are four AD HSP subtypes [including three of the commonest AD HSP forms (SPG4, SPG3A, SPG10)], two AR HSP (SPG11 and SPG48) and an X-linked HSP (SPG2) [Figure 1-5].

1.1.3. Disease progression Most HSP subtypes are slowly progressive. However, significant variability exists regarding disease severity and progress. Some forms deteriorate rapidly and multiple subtypes are very slowly progressive that can be fairly considered as non-progressive. Surprisingly, late onset forms are more often associated with a more rapidly progressive evolution. In many cases the disease is progressive at the start of the disease then reach a static plateau. Rarely, the disease improves before reaching this plateau.

1.2. History of Spastic Neurogenetic Disorders It is important to go back to the history of HSP and the evolution of its definition and classification over time in order to better understand the dimensions of the HSP. This will help identify the relations among all overlapping neurogenetic disorders which present with pyramidal syndrome as whole or part of their phenotypic spectrum.

HSPs were described first in 1883 by the German neurologist Adolph Strümpell and later described more extensively in 1888 by the French physician Maurice Lorrain.

5 Complex Pure AD HSP AD HSP SPG17, SPG36 AD HSP SPG12, SPG13 SPG29 SPG4, SPG6, SPG8 SPG19, SPG33 , SPG10, SPG31, SPG38 SPG37, SPG40, SPG9 SPG41, SPG42, AR HSP SPG73

SPG3A, BICD SPG14, SPG18, SPG20, SPG30 SPG58 SPG 72 SPG21 SPG23, SPG30, SPG35, SPG7 SPG44, SPG45, SPG46, AR HSP

SPG53, SPG54, SPG55, AR HSP Mito SPG56, SPG57, SPG59, SPG5, SPG11 MT-ATP6 SPG62, SPG71 SPG60, SPG61, SPG64, MT-TI SPG24, No SPG: SPG65, SPG66, SPG68, SPG15, SPG27 SPG28, SPG48 ADAR1, IFIH1, SPG69, SPG70, SPG74, RNASEH2B SPG75, LYST, EXOSC3 SPG65

X-L SPG2, SPG16 X-L Mito SPG34 MT-CO3 X -L SPG1, SPG22

Figure 1-4: Correlation between the modes of inheritance and the clinical phenotype (pure or complex). Font colour codes correspond to various patterns of inheritance: AD HSP, AR HSP, Mito, X-L, Frank mixed inheritance and allelic dose mixed inheritance.

6 Table 1-1: Clinical classification of various HSP forms. HSP: hereditary spastic paraplegia, HA: hereditary ataxia, CP: cerebral palsy, ALS: amyotrophic lateral sclerosis, NBIA: neurodegeneration with brain iron accumulation.Font colour codes represent the modes of inheritance: AD Forms, AR Forms, X-L and Mito, mixed Inheritance: ( Frank AD/AR; Allelic dose dependent presentation ?AD/AR).

Clinical Summary No. of HSP HSP FORMS FORMS

CLASSICAL HSP with predominant pyramidal syndrome

Pure HSP 15 SPG4, SPG6, SPG31, SPG33, SPG38, SPG12, SPG19, SPG40, SPG41, SPG42, SPG73,

SPG62, SPG71, SPG24, SPG15, SPG11, No SPG: ADAR1, IFIH1, RNASEH2B

SPG30 SPG72 SPG34

Complicated HSP (minimal 17 SPG4 SPG8, SPG10,SPG13, SPG17, SPG31 complexity: one additional SPG36, No SPG: VCP SPG3A, No SPG: feature only) BICD2 SPG25 SPG39, SPG48, SPG63, SPG67, SPG32, No SPG: CCT5, FAM134B

Complicated HSP (more than 16 SPG4, SPG6, SPG33, SPG38 one complicating features) SPG5, SPG14, SPG15, SPG11, SPG18, SPG20, SPG21, SPG23, SPG27, SPG28, SPG35, SPG44, SPG45, SPG53, SPG54, SPG55, SPG56, SPG57, SPG59, SPG60, SPG61, SPG64, SPG65, SPG66, SPG68, SPG69, SPG70, SPG74, SPG75, LYST, EXOSC3

MT-TI, MT-CO3, MT-ATP6

SPG30, SPG7, SPG1, SPG16

Atypical forms

Spastic Ataxia (equal burden)/ 1 SPG39, SPG46, ARSACS, SPG58 HA

ALS like 1 SPG11

HSP (secondary to Metabolic 1 SPG5A, SPG9 disorder)

CP like 4 SPG47, SPG50, SPG51, SPG52

Complex presentation with 3 SPG29 (probably metabolic) pyramidal signs SPG49(TECPR2), SPG22

NBIA / Leukodystrophy 3 SPG43, SPG44 SPG2

7 Birth (0)-1 yr 1-4 yrs 4-18 yrs 18-40 yrs >=40 yrs

X-L: SPG1 SPG16 SPG22 Mixed AD: SPG38 AR: SPG14 AD: SPG33, SPG40, Mito: MT-CO3 Inheritance: AR: SPG15 No SPG: VCP Mixed Inheritance: No SPG58 SPG72 SPG32 AR: SPG21, SPG: BICD2 AR: SPG43 No SPG (LYST) AD Forms: SPG29 SPG53 SPG64 , SPG28 Mito: No SPG(MT-TI) AR: SPG68 SPG20 SPG23 SPG24 No SPG: ADAR1, SPG45 SPG47 SPG49/ FAM134B, IFIH1, TECPR2 SPG50 SPG51 RNASEH2B. SPG52 SPG57 SPG59 SPG60 SPG61 SPG62 SPG63 SPG65 SPG66 SPG69 SPG70 SPG71 SPG75/ MAG No SPG: CCT5, EXOSC3, , ARSACS.

1-19 yrs: AD: SPG38 18 ->40 yrs AR: SPG27(C), SPG46, SPG55, SPG74 AD: SPG13, SPG19 AR: SPG25 , SPG27(P) /IBA57, SPG26, SPG35. Mito: No SPG(MT-ATP6)

0-4 yrs: AR:SPG67, SPG56/CYP2U1 4-40yrs: X-L: SPG34 AD/AR: SPG30 AD: SPG12, SPG6, SPG36 , SPG41 AR: SPG44

1-40yrs: Mixed Inheritance: SPG9 :SPG9 SP 4->40 yrs: AD/AR: SPG7 AD: SPG8,SPG37 AR: SPG5, SPG42 0-18 yrs: AR: SPG39

1->40 yrs: AD: SPG4, SPG10, SPG17 AR: SPG48

0->40yrs: X-L: SPG2 Mixed Inheritance: SPG3A AR: SPG11

X-L

Mito

Figure 1-5: Regrouping of the age at onset of various HSP subtypes. Font colour codes represent the modes of inheritance: AD Forms, AR Forms, X-L and Mito, Mixed Inheritance: ( Frank AD/AR; Allelic dose dependent presentation ?AD/AR).

8 In 1983, A. E. Harding suggested classification of hereditary ataxias (HAs) and HSPs. In this classification, HSP was classified into pure and complicated HSP and each was further grouped into subtypes based on the mode of inheritance in the pure HSP and on clinical syndromic presentation in the complicated forms. It is noteworthy that many of the syndromes included in the complicated forms are reclassified at present as subtypes of other overlapping syndromes. Examples are Sjogren-Larsson Syndrome (SLS; now considered as leukodystrophy) and AR HA of Charlevoix-Saguenay syndrome (ARSACS; now considered as AR ataxia). Other subclasses were broadly described as could be predicted at that era when the genetic studies were just starting for more precise classification (Harding, 1983).

Only five loci and one gene were described until 1996 (Fink JK et al., 1996). By 1999, 12 loci, of which, 6 AD loci and two X-linked genes, were linked to HSP. (Fink JK et al., 1999)

By 2003, 20 loci and 9 genes were identified (Fink JK, 2003). The completion of the human genome sequence project (publication of the draft genome in 2001 and the completion of the sequence in 2004) heralded a new era of medical and genetic research and neurogenetic research was not an exception (Venter et al., 2001) (International Human Genome Sequencing Consortium, 2004) (Stein et al., 2004). The recent advent of the next generation sequencing (NGS) technologies in the late 2000s has resulted in identification of a large number of genes, instead of loci in the previous linkage-dependent era, since 2010 (Mardis, 2008) (Metzker, 2010).

1.3. A continuum of spastic neurogenetic disorders Many hereditary spastic paraplegia genes (including mitochondrial genes) have been found to result in more than one allelic phenotype. These phenotypes have occasionally closely related clinical scenarios. Two obvious examples of genes that present with phenotypic alleles include two mitochondrial genes: MT-ATP6 and MT-CO3. MT-ATP6 as stated earlier has recently been related to HSP (Verny et al., 2011). Previously, MT- ATP6 has been linked to Leigh syndrome, Leber hereditary optic neuropathy (LHON) and NARP syndrome [Neuropathy, Ataxia, And Retinitis Pigmentosa (Rahman et al., 1996) (Lamminen, 1995) (Holt et al., 1990). Another example is the mitochondrial gene MT- CO3 has been linked to HSP but with a Leigh syndrome- like lesion in the brain (Tiranti

9 V. et al., 2000). MT-CO3 has also been associated with Leber optic atrophy (Johns and Neufeld, 1993). Further examples are illustrated in [Figure (1-6)].

There are also numerous genes that are located in the overlap zone with related clinical scenarios. These genes are classified to be related to HSP, HA, leukodystrophy, neurodegeneration with brain iron accumulation (NBIA), Parkinson disease/parkinsonism (PD), primary lateral sclerosis and amytrophic lateral sclerosis (PLS and ALS), cerebral palsy spastic quadriplegia (CPSQ), hereditary dystonia, mental retardation, peripheral neuropathy (PN) and even some inborn errors of metabolism (IEM) as illustrated earlier. Clearly not all the above mentioned categories are neurodegenerative as exemplified by the overlapping metabolic disorders (eg. leukodystrophy and IEM). This phenomenon is sometimes inseparable from the above observation about one gene presenting with multiple phenotypic alleles. A number of HSP focused examples are illustrated in [Figure (1-7)]

The recent genetic advances identified some genes that correspond to loci previously linked to HSP. SPG9 is a clear illustration for this fact. The identification of the gene and the study of the proposed protein function has demonstrated an amino acid metabolic pathway quite related to what has long been considered as a differential diagnosis of HSP (as the case of other genes/proteins eg ARG1 related to the urea cycle or other genes related to lipid metabolism) (Coutelier et al, 2015a) (Coutelier et al, 2015b).

Consequent to all the above phenomena and if we consider all the above mentioned examples, we observe that multiple atypical forms of HSP have emerged either as a result of an atypical underlying cause or an atypical clinical phenotype [Table (1-1)].

There appears a lack of an objective case definition and too much complexity of the closely related spastic neurogenetic disorders. This has led to the rise of many reviews that highlight these overlaps (Klebe et al., 2015) and suggest reclassification (Garcia-Cazorla et al., 2014) (Vallat et al., 2016) or even expert case definition and classification of some of these entities (leukodystrophies and leukoencephalopathies) (Vanderver et al., 2015).

Figure 1-6: Schematic illustration of the phenomenon of genes presenting with closely related allelic phenotypes throughout the spectrum of the neurodegenerative disorders.

HSP: hereditary spastic paraplegia, HA: hereditary ataxia, PN: peripheral neuropathy and E-Pyr.: extrapyramidal syndrome. Opaque curves stand for clinical presentation as pure category (HSP,HA,PN,E-Pyr.). Interrupted lines imply that the gene manifests the clinical category only in complex form

11 PLS/ALS/ PN/CPSQ Hereditary Ataxias

with pyramidal HA complicated

ARSAL, FRDA, SCA1, SCA3 PN: SPG3A, SPG5A, SPG11, SCA7, SCA12, SCA19/22

Mixed phenotypeMixed SPG17, SPG30, SPG31, SPG57, (KCND3), SCA20, SCA30, SPG70, FAM134B SCA35, SCA36, SCA40, ALS: ALS2, ALS4, SCA28, MSS (SIL1), PHARC SPG11/ALS5, VCP ( ABHD12 ), SCAR7 (TPP1) PLS: SPG18

phenotype Mixed CPSQ: SPG47/CPSQ5,

SPG50/CPSQ3, SPG51/ CPSQ4, ARSACS (SACS), SPG39 SPG52/CPSQ6 (PNPLA6) SPG58

PN HSP complicated with ataxia HSP

HSP+PN: SPG36, SPG9, SPG4 SPG58, SPG5, SPG7, SPG11

complicated with

SPG6, SPG8, SPG10, SPG14, SPG15, SPG20, SPG21, SPG26 SPG15, SPG23, SPG25, SPG26, SPG27, SPG28, SPG30, SPG35 SPG28, SPG39, SPG43, SPG49 SPG44, SPG46, SPG49 SPG55, SPG60, SPG61, SPG66 (TECPR2), SPG54, SPG59 SPG68, SPG74, CCT5, LYST, SPG60, SPG64, SPG68, SPG75 FAM134B, ARSACS (SACS), MT- (MAG) EXOSC3, LYST ATP6

WMH HSP complicated with SPG4, SPG5, SPG8, SPG11 SPG12, SPG15, SPG16 HSP SPG20, SPG21, SPG26

with Extra complicated HSP SPG22, SPG47, SPG48 SPG49, SPG50, SPG53 SPG1, SPG13, SPG21 SPG54, SPG55, SPG56 SPG22, SPG35, SPG56 SPG58, SPG63, SPG64 (CYP2U1), SPG58 - SPG65, SPG67, SPG74

phenotype Mixed

phenotype Mixed SPG11, SPG26 SPG13 (HSPD1), (B4GALNT1), SPG43, RNASEH2B, ADAR1, IFIH, PLA2G6 SPG2 (PLP1), SPG35

(FA2H), SPG44 (GJC2)

Extrapyramidal syndromes (NBIA, PD, Leukodystrophy Dystonias, Choreas)

Figure 1-7: Figure showing the overlap zone between spastic neurogenetic disorders: evidence for the need of new nosology and objective case definition. HSP: hereditary spastic paraplegia, PLS: primary lateral sclerosis, ALS: amyotrophic lateral sclerosis, PN: peripheral neuropathy, CPSQ: Cerebral palsy spastic quadriplegia, NBIA: neurodegeneration with brain iron accumulation, PD: Parkinson disease.

1.4. Pathophysiology of HSP

1.4.1. Basic pathophysiologic mechanisms

 The core phenotypic features of HSP can be explained by alterations of the pyramidal motor system responsible for the voluntary movements in Human beings. The neurons of the pyramidal tracts extend from the cerebral motor cortex to innervate the skeletal muscles at the neuromuscular junctions. These neurons can be injured in a length dependent manner as travel very long way to their destination. The resulting axonal degeneration occurs mainly via a dying back mechanism of the longest neurons and therefore leads to clinical involvement of the lower limbs (LLs) and to a lesser extent of the upper limbs (ULs) (DeLuca et al., 2004)( Blackstone, 2012).  The pyramidal motor tracts are arranged in two stages: 1. The corticospinal spinal tract which originates from layer V of the motor cortex (neuronal cell bodies). The axons of these large pyramidal neurons pass down through the medullary pyramids where the majority of fibers decussate in the caudal part of medulla to descend as the lateral corticospinal tract within the spinal cord. Some of these upper motor neurons synapse directly with the lower motor neurons at the anterior horn of the spinal cord whereas the vast majority synapse with the spinal interneurons which then relay with the lower motor neurons. 2. The lower motor neurons terminate on the skeletal muscle at the neuromuscular junctions distributed all over the body with the exception of the areas innervated by cranial nerves which have other pathways ( Blackstone, 2012).  In complex clinical phenotypes other regions of the central and peripheral nervous systems (CNS and PNS) as well as other extra-neurological organs and systems can also be affected.

1.4.2. HSPs are mainly due to neuronal degeneration The contribution of oligodendrocytes and other glial cells to the pathology cannot be ignored. However, with the exception of certain genetic entities including those in

13 which myelin proteins are affected, the main category of cells that degenerate in HSP are neurons. Neurons have two principal morphological, molecular and functional compartments: the axons and the somatodendritic compartment. Dendrites are multiple short and highly branched neurites that function in receiving and integrating electrical synaptic inputs from thousands of neurons. There is only a single axon responsible for transmitting the integrated information in the form of an action potential that travels along the axonal membrane. The axon has a unique cytoskeletal organization. It contains several specialized structures which include the axon initial segment (AIS) and presynaptic boutons (Kevenaar et al., 2015).

Specific proteins accumulate at the proximal part of the axon to assemble the AIS which separates the axon from the rest of the neuron. The AIS serves to maintain neuronal polarity. The assembly of presynaptic protein complexes leads to presynaptic differentiation (Kevenaar et al., 2015).

1.4.3. Pathways involved in HSP pathogenesis

Not only the genetic technologies but also the closely linked functional studies have advanced substantially in the late 2000s. The latter includes all genetic-manipulation- based techniques (cell lines, animal models), protein modeling, biochemical techniques, electrophysiological cellular studies, cellular and organ imaging and counting. This is illustrated by the immense knowledge gained about HSP and other spastic neurogenetic disorders in recent years. Several cellular pathways have been associated with HSP. Multiple attempts have been done to regroup these pathways according to their shared functional role (Blackstone, 2012) (Noreau et al. in 2014). Blackstone has identified seven principal pathogenic themes including axon-path finding, myelination, endoplasmic reticulum network morphology, lipid synthesis and metabolism, endosomal dynamics, motor based transport and mitochondrial functions defects, in addition to three minor themes [anterograde transport, Golgi apparatus and microtubules related functions] (Blackstone, 2012) [Figure (1-8)]. Noreau et al. later have adopted slightly different functional localization themes including principally morphogenesis of endoplasmic reticulum (ER), disturbances of lipid metabolism (fatty acid, cholesterol and phospholipid), endosomal trafficking and mitochondrial regulation abnormalities in addition to five more categories of DNA repair, gap junctions, autophagy and

14 lysosomal metabolism and myelination (Noreau et al. in 2014) [Figure (1-9)]. However; given the fact that a lot remains to be explained about the underlying pathogenesis of HSP, the possible odds for reclassification are unlimited.

1.4.4. Functional heterogeneity of HSP proteins Many proteins produced from SPG genes are now found to be associated with multiple cellular functions and known to interact with various proteins involved in the same pathway (or inter-digitations of pathways) or sharing the same gene ontology function. This demonstrates how far we stand back from understanding the mechanisms of these disorders and why one gene/protein results in certain phenotype in an individual and with another phenotype when the background genetics changes.

1.5. Assessment and diagnostic approach of HSP patients Positive family history is the main point that strongly suggests HSP. However, in sporadic cases, HSP remains a diagnosis of exclusion. The patient assessment requires proper history and clinical examination. Spastic Paraplegia Rating Scale (SPRS) is a scale used to assess the severity of the motor symptoms (Schüle et al., 2006). Variable disability scores are also used for assessment of the patient’s disability stage. Additional radiological, electrophysiological and laboratory testing will give further evidence on the nature of the disease and help to exclude important differential diagnoses. The principal radiological tests used are magnetic resonance imaging (MRI) of the brain and spinal cord. While MRI of the spine does not show significant abnormalities, the MRI of the brain (plus or minus CT scan of the brain) can give essential information that can aid in exclusion of many of the differential diagnoses (eg. leukodystrophies and leukoencephalopathies, NBIA, MND/ALS, infectious and immune causes) and give further clue to the category of HSP (eg. TCC-HSP) and probable genes beyond it (eg. SPG11 is the most probable gene in TCC HSP).

Electrophysiological testing includes electromyogram/ nerve conduction studies (EMG/NCS), evoked potentials: somatosensory evoked potential (SEP) and other relevant evoked potentials as determined by clinical presentation of individual cases [visual evoked potential (VEP) and auditory evoked potential (AEP)].

Figure (1-8): Common pathogenic themes in the HSPs (Blackstone, 2012).

Figure (1-9): Illustration of the pathogenic mechanisms in hereditary spastic paraplegia (HSP) regrouped in nine functional and cellular localization categories (Noreau et al., 2014)

17 Biochemical analysis of very long chain fatty acids (VLCFA), plasma amino/organic acids (IEM of amino acid metabolism), lipoprotein (abeta lipoproteinemia), vitamin B (B12) and E levels (vitamin deficiencies), white cell enzymes, homocysteine, copper and ceruloplasmin (Wilson disease) levels as well as metabolic screening for diseases which present with pyramidal signs as part of their clinical phenotype like Krabbe disease, metachromatic leukodystrophy, gangliosidosis (GM1, GM2), and Tay-Sachs, Sandhoff or Gaucher disease are all considered for exclusion of diferential diagnoses.. In addition, exclusion must be performed for acquired spastic syndromes due to infectious disease [treponema pallidum, human T-cell leukemia virus 1 (HTLV1) (tropical spastic paraplegia) and human immunodeficiency virus (HIV) (HIV related neurological syndromes)]. Dopa responsive dystonia (DRD) should also be excluded by treatment with low doses of L-dopa as it can present with spastic paraplegia early in the disease course (Wijemanne and Jankovic, 2015) (Klebe et al., 2015).

As a result of the immense genetic/phenotypic heterogeneity it is becoming extremely difficult to suggest the gene beyond the HSP using candidate gene approach based on the clinical phenotype. With the advent of NGS techniques, multi-gene panels have become the favorite screening tool as they allow massive screening for genes responsible for spastic neurogenetic disorders. These panels are time/effort effective but pose the important question about cost effectiveness as with its use in other diseases (Robson, 2014).

1.6. Treatment options for HSP Unfortunately there is no curative treatment for HSP and all the available options are symptomatic treatment. Simple walking aids and wheel chairs, hearing aids and air matrices can be of great use to improve the quality of life. Physiotherapy, speech therapy, occupational therapy are used based upon the clinical presentation. Two studies have been done recently on a new trend to support the use of robot gait training. Provisional evidence from one study suggests long term improvement of walking and balance in HSP patients (Bertolucci et al., 2015). However, despite the improvement in gait in the second study there was no change in kinetics (Seo et al., 2015). Oral medications of antispasticity drugs (baclofen, tizanidine, tolperisone or dantrolene) as well as intramuscular botulinum toxin injections and continuous intrathecal baclofen application (ITB) can decrease the

functional disability especially when the spasticity is predominant over the weakness (Klebe et al., 2015) (Fink et al., 1993).

ITB is a widely used in children as well as in adults for treatment of spastic disorders at variable doses. Recently provisional evidence has shown that spastic HSP may require low doses compared to other spastic disorders (Dressler et al., 2015).

Encouraging results with significant improvement of SPRS in HSP patients have been observed in a recent study conducted on 12 patients to support the use of dalfampridine. Dalfampridine is a potassium channel blocker that has recently been approved to improve walking in patient with multiple sclerosis (Béreau et al., 2015).

Anti-cholinergic medications can help with the urinary urgency. Treatment for complicating clinical signs follows their own guidelines (Klebe et al., 2015).

Surgerical interference can be implemented through orthopedic surgical procedures (for contractures) and ophthalmological surgeries (cataract extraction) to protect against augmentation of disability by blindness.

The emergence of multiple metabolic pathways associated with HSP may provide targets for more specific options in certain HSP subtypes.

1.7. Genetic counseling Genetic counseling depends on the pattern of inheritance. High consanguinity in certain communities (Africa, Middle East..) can be an important pointer towards the tendency to have AR HSP over AD HSP although the latter can’t be entirely excluded. Explanation of the expected risk of having an affected child associated with each pattern of inheritance depends mainly on good interpretation of family pedigree although it can still be ambiguous in certain cases until the causative gene/variant is found (Fink, 1993).

Materials and methods

2.1. Ethics This study was approved by the Paris Necker Ethics Committee (France) and the Ethical Committee of the University of Khartoum, Medical Campus (Sudan). A written informed consent was obtained from all participants [Annex 13].

2.2. Subjects recruitment Our cohort was composed of 106 patients and 337 healthy related individuals from 41 extended Sudanese families, almost all consanguineous with multiple consanguinity loops.

Patients were referred from several principal tertiary neurology and neuro-pediatric clinics in Khartoum (list of the departements…). They belong to various ethnic groups distributed in different regions of Sudan. For each kindred we recruited the proband, all other accessible diseased family members and, when possible, available unaffected first, second and third degree relatives.

1. Inclusion and exclusion Criteria 2.2.1.1. Inclusion Criteria: 1. Clinical presentation suggestive of spastic neurodegeneration. 2. Favorable criteria were familial consanguinity and positive family history of neurodegenerative disorders. 2.2.1.2. Exclusion criteria: 3. Alternative acquired causes of pyramidal syndrome should all be excluded. 4. Other overlapping genetic disorders that clearly belong to a differential diagnosis as overt leukodystrophy/leukoencephalopathy are to be excluded when necessary data are available. N.B

In born errors of metabolism (IEM) were excluded on clinical ground and metabolic screening when the test was feasible. However, it could not be included as an exclusion criteria as the tests were not always available.

2. Recruitment Rate: The rate of recruitment was slower at the start. Later, as the neurogenetic team was created in Sudan, the recruitment became more efficient. We also managed to enhance the referral system by the help of the young doctors of the team who were distributed in various hospitals and tertiary neurology clinics in Khartoum to consolidate the principle of referral to our neurogenetic clinic/team and provided the physicians with printed referral forms that we prepared.

2.3. Clinical phenotyping

1. Questionnaire and auditing o Patients were clinically examined and diagnosed by the referring consultant neurologist/neuro-pediatrician. We performed a second scrutinized phenotyping and filled a standard questionnaire [the standard questionnaire of the SPATAX network (https://spatax.wordpress.com/)]. Later a version of the questionnaires with minor modifications was created and adopted. This was in particular, because our cohort contained mainly pediatric cases and consequently we had to adapt the clinical history and examination to this category [Annex 12]. o Professional video-taping was performed to enable auditing and documentation of the physical examination and clinical signs. We examined all healthy related controls to exclude subtle disorders.

2. Clinical Assessment:

2.3.2.1. History: Points covered in history include:

1. Personal data including origin and contact details 2. Family history of medical disorders including similar conditions or other neurological or non-neurological disorders 3. Consanguinity (pedigree obtained)

4. Detailed natural history of the disease since the age at onset till the date of examination (Patients motor disability using a Seven-Stage Disability Score also obtained) (https://spatax.wordpress.com/) (Table 2-1) 5. In pediatric cases or adult patients with early onset, disease pregnancy, delivery, early development (gross motor and intellectual disability)

2.3.2.2. Examination: General and neurological assessment included the following: 1. Centiles especially head perimetry in pediatric patients or when microcephaly was suspected 2. UMN features (pyramidal syndrome) 3. LMN feature (sensory and motor peripheral neuropathy features) 4. Cerebellar syndrome (UL, LL, eye movements, speech, gait ataxia, trunk and head tremor and neck ataxia) 5. Extrapyramidal signs (bradykinesia, rigidity, postural instability, dystonia, chorea, athetosis and related features depending on the preliminary findings) 6. Eye examination (including fundoscopy) and visual acuity assessment. 7. Skeletal deformities, dysmorphic features and any skin or hair changes. 8. Any additional clinical sign that was necessary to elaborate based on the provisional history and examination even if not directly related to the above points

2.3.2.3. Investigations: Magnetic resonance imaging (MRI) of the brain and spine was performed in at least one patient per family in most families. Electrophysiological studies including nerve conduction studies (NCS), electromyography (EMG), electroencephalography (EEG) were performed when indicated and feasible. Other laboratory tests were done based on individualized differential diagnoses.

 Disability Stage  Disability Description

 0  No Functional Handicap

 1  No Functional Handicap but Signs at Examination

 2  Mild, Able to Run, Walking Unlimited

 3  Moderate, Unable to Run, Limited walking without Aid

 4  Severe Walking with one Stick

 5  Walking with two Sticks

 6  Unable to Walk requiring Wheelchair

 7  Confined to Bed

Table (2-1): Disability Scoring system used in the SPATAX standard questionnaire.

2.4. Sampling Two milliliters of saliva were collected using Oragene.Discover® DNA self-collection kits (OGR- 500) from each patients and healthy related and unrelated controls. For infants and markedly disabled patients Oragene.Discover® DNA assisted-collection kits (OGR- 575) was used to collect around one milliliter of saliva (Genotek Inc., Canada).

Skin biopsies were collected in certain cases where an interesting genetic result was obtained and needed further functional studies. They were taken from the inner part of the upper arm after 20-30 minutes of local anesthesia batch using sterilized disposable kits.

2.5. DNA Extraction, and quality/quantity check

1. DNA Extraction: DNA purification out of 0.5 ml of saliva was done according to the prepIT®.L2P manual protocol provided by the producer.

2. DNA quality/quantity check: DNA quality control were performed using three laboratory methods: the standard agarose gel electrophoresis, Caliper® bioanalyzer to check for degradation of high molecular weight DNA and the NanoDrop® 8000Uv-Vis spectrophotometer (Thermo Scientific, Wilmington, USA) to assess the purity of the DNA.

DNA quantity was measured with the use of Qubit® fluorometer (Promega®, Wisconsin, USA) as well as the NanoDrop® 8000Uv-Vis spectrophotometer (Thermo Scientific, Wilmington, USA).

2.5.2.1. Standard Agarose gel electrophoresis Agarose gel electrophoresis allows the separation and identification of 0.5- to 25-kb DNA fragments. It can thus be used for quality checking of genomic DNA to assess its integrity and to exclude its degradation. It was also used to separate PCR products (see below) according to their precise molecular weight. 1. The protocol summary can be divided into three stages: - preparation of an agarose gel concentration appropriate for the size of DNA fragments to be separated - the DNA samples are loaded together with a graded band size ladder into the sample wells and the gel is run at a voltage and for a time period that will achieve optimal separation - Visualization of gel under UV illuminator and capture of necessary images 2. In practice: 1% gel was prepared using standard method: heating one gram of agarose powder dissolved in 100 mL of (0.5-1x) TBE (Tris/Boris/EDTA) buffer solution in a microwave oven for 3-5 minutes. Addition of 5 µL (concentration=10 mg/mL in H2O) of an intercalating stain as Ethidium bromide or cyber safe stain under the safety hood after cooling the solution to less than 60° C using all good laboratory practice guidelines for safety use of biohazardous substances. The buffer improves the conductivity of the DNA by adding 24

negative charges to it and thus improves the DNA migration and protects the DNA through its EDTA component from degradation.

We used 0.8% gel for genomic DNA control and 1% agarose gel was used for PCR products analysis. A higher agarose percentage enhances resolution of smaller bands; conversely, a lower agarose percentage gives better resolution and separation of higher molecular-weight bands. If the wrong percentage is used, it can be difficult to visualize the DNA bands reliably. Low-percentage agarose gels are typically weak and more prone to breakage so they need careful handling. DNA/PCR product were loaded mixed with a loading dye (bromophenol blue+ xylene cyanol+ glycerol) [1-2 µL of DNA + 4-5µL of dye]. The gel was then run for 30 minutes at 120 mV voltage for genomic DNA and for 15-20 minutes at little higher voltage for PCR products.

2.5.2.2. Caliper® is a PerkinElmer® apparatus (Hopkinton, Massachusetts, USA): Performs a reproducible high resolution electrophoretic separation using a microfluidic chip instead of an agarose gel. It utilizes a data analysis software to allow visualization of results through a virtual gel.  In practice:

20 µL of PCR product /dil PCR product (18 µL H2O+ 2 µL PCR product) was vortexed and centrifuged then loaded into the Caliper and the software launched. Resulting gel image was finally exported in a common image format for its interpretation (JPEG).

2.5.2.3. NanoDrop® 8000Uv-Vis spectrophotometer NanoDrop® was used to measure between 1 and 8 microvolume DNA samples simultaneously. It uses a sample retention system that allows you to assess the concentration and purity of DNA by deposition of 1-2 µL samples. It utilizes a spectral range from 220 to 750 nm. It measures a dsDNA concentration range between 2.5 and 3,700 ng/µL. DNA and RNA are measured at wavelength 260 nm so the DNA measured is usually 25

overestimated using this method unless purified samples are used. Small changes in the pH of the solution will cause the 260/280 to vary: acidic solutions will under-estimate the 260/280 ratio by 0.2-0.3, while basic solutions will over-estimate the ratio by 0.2-0.3. Sample purity ratios (260/280) and (260/230) are used to indicate the presence of protein or contaminants that absorb at 280nm or excess of EDTA, carbohydrates and phenol that absorb at 230 nm. Ratio of 260/280 around 1.8 and of 260/230 in the range of 2.0-2.2 are usually accepted for DNA purity.

2.5.2.4. Qubit® fluorometer The Qubit® fluorometer is a ThermoFisher SCIENTIFIC Life Technologies Invitrogen® product (Carlsbad, California, USA) that was used to measure dsDNA precise concentration. It offered two types of assays; the Qubit dsDNA HS (for High Sensitivity) Assay and the Qubit ®dsDNA BR (for Broad Range) Assay. The quantification method uses a fluorescent dye (Qubit reagent) which binds specifically to dsDNA and not to ssDNA. The intensity of the resulting fluorescence is directly proportionate to the quantity of dsDNA. The assessment of sample concentration is based on plotting the relationship between the two dsDNA Standards used in calibration (standard #1 at 0 µg/mL and standard #2 at 100 µg/mL). The Qubit® fluorometer produced final concentration values in either µg/mL (ng/µL). This concentration corresponded to the concentration calculated from the Qubit® fluorometer value (QF) and the DNA dilution factor we used (practically 1/200). It used the equation: Concentration of your sample = QF value × (200)/X where X is the number of microliters of sample we added to the assay tube.

 Assay protocol was provided by Invitrogen® as summarized below: i. A working solution is prepared from 199µL Qubit® buffer per sample/standard plus 1 µL Qubit® reagent per sample/standard. ii. The working solution is then distributed into special Qubit® tubes provided from the manufacturer (190 µL/standard tube and 199 µL/sample ( 180-199 µL based on the dilution factor chosen) )

iii. 10µL of Standard #1 and Standard #2 are added and 1µL (1-20µL optional) of each to their corresponding tubes vortexed slightly incubated for two minutes. iv. Measurement is then done by choosing the specific assay (HS vs BR), calibration using Standard #1 then #2. Samples measurement is done by following a wizard indicated by the device. v. Sample concentration is given as QF value (an option to provide calculation of final concentration is available if the value of the starting volume of the sample is entered.

2.6. Genetic linkage analysis using traditional microsatellite markers We performed linkage analysis to screen two families (10 patients and 31 healthy related controls) for candidate loci. For each gene we used four microsatellite markers flanking: CYP7B1 (SPG5), KIAA1840 (SPG11), ZFYVE26 (SPG15), GBA2 (SPG46), CYP2U1 (SPG49 or SPG56 according to HUGO and NCBI nomenclatures respectively) and SACS genes. Classical procedures were used for the PCR amplification. Allelic resolution was run in an ABI3730 sequencer (Applied Biosystems, US). Peak Scanner® software (Applied Biosystems®, USA) was then utilized for analysis of product length which varied according to the number of nucleotide repeats. Haplotypes were then manually reconstructed for each locus and pedigree in order to obtain the minimal number of recombination events.

2.7. Primer design, PCR amplification and control 1. Primer Design Specific primers were designed using primer3plus software (ref). The sequence used as an input was obtained from the reference sequence downloaded from any of the genome browsers (ensembl genome browser was preferentially used). Primer specificity was tested using the BLAT and BLAST tools of the genome browsers (UCSC and NCBI). In difficult cases where specificity was doubted, the in silico PCR tool (UCSC genome browser) was used.

2. PCR amplification of microsatellite markers or of specific exons/regions of specific genes was performed using a classical procedure that included preparation of a master mix composed of a buffer, dNTPs, Taq polymerase, specific primers

(forward/F and reverse/R), H2O and the DNA template (DNA sample).  Touchdown protocol was used to amplify the whole exon where the variant of interest was located or, if that was too big to get the optimal amplification we amplify around 100-150 bp up- and down-stream of the variant. The touchdown protocol is, as any standard PCR protocol, composed of three steps: 1. Denaturation (94-95°C) 2. Hybridization (annealing) (Range including the melting temperature (Tm) of the primer), typically 65°C to 54°C 3. Extension (synthesis) (72°C) The advantage of the touchdown is that the first 10-30 cycles are performed at high annealing temperature to maintain the specificity of the polymerization and then reduced it gradually to cover a wider range of annealing temperatures making it easier to obtain a PCR product in adequate quantity.

PCR products were then checked for specific amplification at the expected size using a caliper® Lapchip® GX bioanalyzer (Hopkinton, Massachusetts, USA).

2.8. Genome-wide genotyping, homozygosity mapping and whole genome linkage analysis

1. General description HumanCore-24 BeadChip kit v1-0® (WG-330-2001) (Illumina®, San Diego, USA) was used for genome-wide genotyping. It is a silicon-based array device. The Beadchip provided contains 306,670 genome-wide tag SNPs of which over 20,000 high-value markers. The exome-focused SNPs are 41,518. The mutations tested include 9,800 nonsense, 6,707 missense, 12,286 indels and 160 mitochondrial plus other types of variations spaced at a mean distance of 9.4 Kb and a median distance of 5.7 Kb. 28

An automated Infinium high throughput screening (HTS) based assay protocol provided by the manufacturer was used. The assay was performed in a specialized Illumina platform in a local facility. A special MSA3 plate based protocol was used with a starting DNA quantity of 200 ng (4µL at 50ng/µL). The maneuver was conducted in two separate laboratory sections (Pre-Amp and Post-Amp) as indicated in the Illumina guidelines. The compatible iScan® technology was used to obtain the necessary SNP images. The SNP image data sets obtained allowed us to perform a variety of downstream applications, including the search for loss of heterozygosity (mapping of homozygous regions) and CNV detection studies. In addition the data analysis would allow identification of common variants (including in mtDNA), ancestry determination, loss-of-variant and indel identification, as well as sex confirmation.

2. Laboratory protocol : 2.8.2.1. Practical summary of the principal steps of the process: 1. DNA Amplification (Pre-Amp): After the DNA samples are run on gel electrophoresis to be sure that they are not degreaded and a test PCR is done to exclude the presence of PCR inhibitors. The DNA samples are chemically denatured and neutralized to prepare them for amplification. The denatured DNA is isothermally amplified in an overnight step. As the process is isothermal (non-PCR amplification) the whole-genome amplification uniformly increases the amount of the DNA sample by several thousand-fold without significant amplification bias. 2. DNA Fragmentation (Post-Amp): The fragmentation is a controlled enzymatic process which fragments the amplified product. The process uses an endpoint fragmentation to avoid over- fragmenting the sample. 3. DNA Precipitation (Post-Amp): This is achieved via isopropanol precipitation followed by centrifugation at 4°C to collect the fragmented DNA. 4. DNA Resuspension (Post-Amp): The precipitated DNA is resuspended in hybridization buffer. 5. Hybridization to BeadChip (Post-Amp): 29

At this step the samples are applied to a BeadChip and separated by an IntelliHyb seal (or gasket). The amplified and fragmented DNA samples anneal (hybridized) to locus-specific 50-mers [in contrast to the traditional oligomers (~20-mers) used in PCR]. The loaded BeadChip is incubated in the Illumina Hybridization Oven for an overnight. 6. BeadChip Wash (Post-Amp): The aim of this step is to prepare the BeadChip for staining and extension. All unhybridized and non-specifically hybridized DNA are washed away to leave only the specifically hybridized DNA fragments. 7. BeadChip Extend and Stain(XStain) (Post-Amp): This includes single-base extension of the oligos on the BeadChip, using the captured DNA as a template. It incorporates the detectable labels on the BeadChip and determines the genotype call for the sample. X-Stain occurrs in a capillary flow-through chamber. 8. BeadChip Imaging (Post-Amp): The Illumina iScan System is used to scan the BeadChip. It uses a laser to excite the fluorophore of the single-base extension product on the beads. The scanner records high resolution images of the light emitted from the fluorophores.

3. Bioinformatic analysis (downstream): GenomeStudio V2011.1 (1) software was used for data analysis. After genotyping quality check, the genotypes for all markers were determined. Two additional plug- in and algorithm (CNV region and cnvpartition, respectively) were used for the analysis of CNVs and loss of heterozygosity regions.

2.9. Screening HSP or Parkinson genes using targeted Next Generation Sequencing 1. General description: I used 2 multi-gene targeted next generation sequencing panels to screen index patients from the 41 families (at least 1-2 patients/family). The HSP kit targets all exonic regions of 67 genes known to be related to a variety of spastic disorders and 7 unpublished spastic candidate genes (Annex 14). The total number of target regions is 30

1042 with a total length of 220095 bps. The PARK kit targets all exons of six genes known to be involved in Parkinson disease (PARK2, PINK1, DJ-1/PARK7, ATP13A2, PLA2G6, SYNJ1, DNAJC6, FBXO7, VPS13C). Each region corresponds to exons plus 20 base pairs of the up- and down-stream flanking non-coding or intronic regions. A capture enrichment strategy is used (Roche NimbleGen®/SeqCap Ez®, USA). Sequencing is performed using MiSeq platform (Illumina®, San Diego, USA) on 250 bp paired-end reads. In this maneuver, 24 DNA samples are processed (multiplexed) at a time after proper sample indexing is done for each sample. Base calling and quality control are performed using Genomics Workbench (CLC Bio®, Denmark). Variants are detected using probability- and quality- based algorithms.

2. Laboratory protocol: 2.9.2.1. Library preparation An illustration of the basic steps of library preparation protocol used for the NGS is provided in figures [Figure (2-1)] and [Figure (2-2)].

1. Genomic (DNA) preparation and fragmentation: A starting quantity of DNA of 1µg (50 µL at a concentration of 20ng/µL) is used as recommended by Roche NimbleGen. DNA quality is tested using nanodrop® and gel electrophoresis. As the process includes many PCR dependent steps, samples are also passed in a test PCR before the start to exclude the presence of any PCR inhibitors. The genomic DNA fragmentation is performed using a Covaris shearing instrument. It is a focused ultrasonicator that utilizes ultrasonic acoustic energy to mechanically fragment the DNA. It employs an AFA technology [Adaptive Focused Acoustics™]. The fragmentation is achieved by directing bursts of ultrasonic waves towards discrete zones within the sample vessel immersed in a water bath. The two conditions required for successful fragmentation are the use of special Covaris tubes (52.5 µL) and conducting the maneuver at isothermal condition.

2. End Repair:

In this step both and 3 end of the fragmented dsDNA are turned into blunt ends with the use of Kapa® end repair master mix. The mix which contains an end repair enzyme and buffer is incubated in a thermocycler with the DNA fragments. The fragments are then cleaned using Agencourt AMPure XP reagent®. This is a kit that employs the highly efficient PCR cleaning system that utilizes the Solid Phase Reversible Immobilization (SPRI) magnetic-based technology with the help of paramagnetic beads. The cleaning system allows easy removal of contaminants as primers, primer dimers, salts and dNTPs to leave only the proper amplified dsDNA fragments.

3. A-tailing: It is an intermediate step necessary for the adapter ligation (the step that follows). Addition of a stretch of dAMP to the 3 end of the amplified and cleaned dsDNA fragments is performed using a Kapa® A-tailing buffer and enzyme in a thermocycler based step. An SPRI cleaning step followed.

4. Adapter ligation: SeqCap® adapter Kits A and B (with 12 adapters each) allows indexing of the 24 samples with 24 different adapters. The index adapters are linkers specific to sequencing platforms that allow library fragments to attach to the flow cell surface called P5 and P7. Each adapter has an index that contains a unique sequence that identifies samples at sequencing and allows their multiplexing. A mix of KAPA® T4 DNA Ligase and buffer is then incubated with dsDNA sample and the specific index adapter in a thermocycler at 20˚. This is followed by an SPRI cleaning step.

5. Dual-SPRI® size selection: Fragments with size between 250-450 bp are selected by dual binding to the magnetic beads. This is important because this size range corresponds to the target sequences that can be processed in the MiSeq sequencing system.

6. Double capture steps: The concept of the capture enrichment is shown in [Figure (2-1)] whereas illustration of the steps of the double capture technique is shown in [Figure (2-2)]. i. Pre-Captured LM-PCR followed by SPRI cleaning step. ii. Quality and quantity measurements of the product. Using NanoDrop® spectrophotometer, we check that the sample library yield is > 1 μg, with an A260/ A280 between 1.7 and 2.0. The quantity is then rechecked by Qubit® fluorometer for more accurate measurement. It is then run in Caliper® Bioanalyzer DNA 1000 chip to assure that the fragments size ranges between 150-500 bp (average 300 bp). iii. Multiplexing where each 12 samples are multiplexed in one tube. They are mixed together at equal amounts (by mass) of each of the amplified DNA sample libraries to obtain a single pool with a combined mass of at least 1.2 μg (“Multiplex DNA Sample Library Pool”). 1 μg of the multiplex pool is used in the sequence capture hybridization step. The remaining 250 ng of the Multiplex Pool is stored at -15 to -25°C. iv. Hybridization of the biotinylated specific target probes (oligos that are complementary to the sequencing primer site inside the paired end adapter sequence of Kit A and B) [Figure (2-1)]. After addition of the Multiplex Hybridization Enhancing Oligo pool (dedicated to blocking of non-specific hybridization sites), the multiplex DNA samples are dried using DNA vacuum concentrater then resuspended using a mix of the hybridization component A and hybridization buffer. The sample is incubated for 10 min at 95° in a heat block to allow denaturation of the double stranded DNA multiplex. The denatured sample is then transferred to an aliquot of COT Human enriched in repeated DNA sequences which block repeated sequences (contained in the SeqCap EZ® Accessory Kit v2). An overnight incubation in a thermocycler at 47°C (with heated lid turned on at 57°C) allows the hybridization to take place overnight.

v. First capture: capture of the sample hybridized to biotinylated probes is performed using Streptavidin Dynabeads® incubated with the sample at 47°C for 45 min followed by a wash process. vi. Middle-captured LM-PCR followed by SPRI cleaning step. vii. Second hybridization reaction with biotinylated probes at 47°C for overnight. viii. Second capture with streptavidin capture bead followed by a wash process. ix. Post-captured LM-PCR followed by SPRI cleaning step. 7. The product is measured using NanoDrop® spectrophotometer to determine the concentration. The proposed sample library yield is expected to be around 0. μg ( 00 ng), with an A260/ A280 between 1.7 and 2.0. The quantity is then confirmed by Qubit® fluorometer and run in Caliper® Bioanalyzer DNA 1000 chip to assure that the fragments size ranges between 150-500 bp (average 300bp).

8. Library preparation for Miseq sequencing: This is based on the concentrations obtained with Qubit® fluorometer. The different libraries are combined in one tube with the more concentrated multiplexed amplified captured sample being diluted to the concentration of the less concentrated one (C1*V1=C2*V2). Then with the use of the elution buffer tween (EBT), the library is diluted to 4nM to obtain the appropriate concentration of the Miseq sampling using a formula provided by the manufacturer. The sample is chemically denatured using NaOH followed by addition of a special hybridization buffer (HT1) supplied with the Miseq sequencing kit to dilute the library to a final concentration of 9 pM. The library is then combined with Phix library control (internal quality control for cluster generation, sequencing and alignment) and loaded into the designated reservoir in the prefilled cartridge reagent provided by Illumina.

2.9.2.2. Miseq sequencing The Illumina reagent cartridge (Illumina reagent kit v2), the thoroughly washed and dried flow cell and the PR2 bottle are loaded into the Miseq

machine and the sequencing process is launched after assuring that the waste bottle is empty. Illumina sequencing is based on the concept of bridge amplification of the captured DNA fragments from the denatured library [Figure (2-3)]. The DNA fragments bind to the surface of the flow cell through annealing of the P5 and P7 sequences of the adapters to complementary sequences on the flow cell. The bridge amplification technique is considered a solid phase sequencing method. DNA polymerase then produces clusters of 1 million copies of clonal DNA fragments originating from each single DNA molecule. As sequencing is carried out from both end it is called a paired end sequencing and the reads obtained from each end are called R1 and R2. The index sequence of each adapter allows the machine to affiliate each sequence to the correct sample. In principle, Illumina sequencing technology is sequencing by synthesis (SBS). In this technology, a fluorescently labeled reversible terminator is imaged as each dNTP is added, and then cleaved to allow incorporation of the next base. Since all 4 reversible terminator-bound dNTPs are present during each sequencing cycle, natural competition minimizes incorporation bias.

3. Bioinformatic analysis

2.9.3.1. Read mapping and variant detection The Miseq output data is in the form of two FastQ files per sample corresponding to Read 1 and Read2 data (≈forward and reverse reads). The files are exported to be analyzed by CLC Genomics Workbench 7.0 software (CLC Bio®, Denmark). This software has a re-sequencing tool that allows alignment of the genetic variation data to a reference sequence. The software first maps the reads to the reference sequence (based on GRCh37 assembly) and thus the coverage of each region is determined as well as the on-target specificity. Consequently, two coverage files are produced per sample: - Coverage report which provides a general summary about each sample coverage including the mean coverage of all regions/sample. - Coverage analysis (OA) which provides detailed coverage data about each region.

2.9.3.2. Variant annotation (detection): Is achieved through two algorithms: quality-based variant detection (QBVD) and probability-based variant detection (PBVD): this results in production of two excel sheets named as corresponding variant analysis files. 2.9.3.3. Variant prioritization Analysis was done through excel sheet filter tools. For Gene/Variant Prioritization a minimal threshold depth of 30x was chosen. To ensure inclusion of all valid variants, we first set a relaxed minor allele frequency (MAF) filter of <1.5% at the offset of our analysis. Indeed, allele frequency can vary greatly from one population to another and we preferred filtering on this criteria on later steps, including the frequency in our local database. The filter was tightened at a downstream step to <0.1% for more stringency. Functional consequences of variants considered were stop codons, frameshift, splice site variants and missense changes. Regarding missense alterations, five pathogenicity prediction algorithms were used (SIFT, Polyphen-2, Mutation Taster, Align GVGD, KD4v). The conservation score was calculated by PhastCons and PhyloP. Moreover, for the prediction of splicing sites, we used five tools (Human Splicing Finder, SpliceSiteFinder–Like, Gene Splicer, NNSPLICE, MaxEntScan). All tools were collectively provided by AlamutVisual® software (Interactive Biosoftware, France).

Figure (2-1): Sequence Capture Protocol: Basic illustration of the protocol for library preparation for next generation sequencing (NGS) using capture enrichment technique

(https://lifescience.roche.com/shop/CategoryDisplay?catalogId=10001&tab=&identifier=Sequence+C apture+Overview&langId=-1&storeId=15006 )

50 μL Fragmented DNA SPRI® bead cleanup

End Repair SPRI® bead cleanup Both

Miseq A-Tailing SPRI® bead cleanup /Next

Adapter Ligation SPRI® bead cleanup seq

Dual-SPRI® size selection 250-450 bp 500 500

Pre-Capture LM-PCR SPRI®bead cleanup

Multiplexing 12 samples/tube

panelonly Miseq

Hybridization to biotinylated probes SPRI®bead cleanup /Gene Capture: of biotinylated sample using Streptavidin Dynabeads®

Middle-captured LM-PCR SPRI®bead cleanup Both Both Overnight/Miseq- 64-72

Miseq Hybridization to biotinylated probes hrs/Nextseq

® /Next Capture: of biotinylated sample using Streptavidin Dynabeads

seq Post-captured LM-PCR SPRI®bead cleanup 500 500 Library preparation for Miseq/Nextseq 500 sequencing Bridge Miseq (Gene panel / Nextseq (WES) Sequencing Amplificatoion

Bioinformatic analysis Read mapping/ variant detection Variant prioritization

Sanger Sequencing Variant Validation Cosegregation of variant with disease distribution

Figure (2-2): Summary of the principal steps of next generation sequencing (NGS)-based genetic investigations including: library preparation, NGS, bioinformatics and the genetic validation of selected variants. The figure also illustrates the basic differences between the library preparation protocols of the Miseq sequencing used in the Gene panel and Nextseq 500 sequencing used for the whole exome sequencing (WES) methods.

Figure (2-3): Illustration of the bridge amplification sequencing technique (Illumina Sequencig technology) (http://nextgen.mgh.harvard.edu/IlluminaChemistry.html)

2.10. Whole Exome Sequencing (WES)

1. General description In this maneuver, 10 DNA samples are processed (multiplexed) at a time after proper sample indexing is done for each sample. Sequencing is performed using

the Hiseq property of the NextSeq-500 sequencer (Illumina®, San Diego, USA) available in our local facility, on 150 bp paired-end reads. 2. Laboratory protocol:

2.10.2.1. Library preparation: Library preparation is basically the same as for targeted NGS [Figure (2-1)] and [Figure (2-2)].

The exception is that there is only one capture instead of two implying that there is a single hybridization, one capture and no middle-captured PCR. The hybridization is carried over longer period of 64-72 hours instead of overnight [Figure (2-2)].

2. End Repair: In this step both and 3 end of the fragmented dsDNA are turned into blunt ends with the use of Kapa® end repair master mix.

3. A-tailing: Addition of a stretch of dAMP to the 3 end of the amplified and cleaned dsDNA fragments is performed using a Kapa® A-tailing buffer and enzyme in a thermocycler based step. An SPRI cleaning step follows.

4. Adapter ligation: SeqCap® adapter Kit A and B (with 12 adapters each) allows indexing of the 10 samples with 10 different adapters.

5. Dual-SPRI size selection: 40

Fragments with size between 250-450 bp are selected.

6. Capture steps: Illustration is provided in [Figure (2-1)]. i. Pre-Captured LM-PCR is followed by SPRI cleaning step. ii. Measurment of the product using NanoDrop® spectrophotometer to determine the concentration. The proposed sample library yield is > 1 μg, with a A260/ A280 between 1.7 and 2.0. The quantity is rechecked by Qubit® fluorometer for more accurate measurement. It is run in Caliper® Bioanalyzer DNA 1000 chip to assure that the fragments size ranged between 150-500 bp (average 300 bp). iii. Multiplexing in one tube: They are mixed together at equal amounts (by mass) of each of the amplified DNA sample libraries to obtain a single pool with a combined mass of at least 1.2 μg (“Multiplex DNA Sample Library Pool”). 1 μg of the multiplex pool will be used in the sequence capture hybridization step. The remaining 250 ng of the Multiplex Pool was stored at -15 to - 25°C. iv. Hybridization of the biotinylated specific target probes (oligos that are complementary to the sequencing primer site inside the paired end adapter sequence of Kit A and B). After addition of the Multiplex Hybridization Enhancing Oligo pool the multiplex DNA samples are dried using DNA vacuum concentrater then resuspended using a mix of the hybridization component A and hybridization buffer. The sample for 10 min at 95° is incubated in a heat block to allow denaturation of the double stranded DNA multiplex. Then transferred the denatured sample to an aliquot of COT Human (contained in the SeqCap EZ® Accessory Kit v2). Incubation in a thermocycler at 47°C (with heated lid turned on at 57°C) allows the hybridization to take place over 64-72 hours. v. Capture: capture of the sample hybridized with biotinylated probes by Streptavidin Dynabeads® incubated with the sample at 47°C for 45 min followed by a wash process.

vi. Post-captured LM-PCR followed by SPRI cleaning step. 7. The product is measured using NanoDrop® spectrophotometer and run in Caliper® Bioanalyzer DNA 1000 chip to assure that the fragments size ranged between 150-500 bp (average 300bp). 8. Library preparation for NextSeq-500 sequencing: 9. Based on the concentrations obtained with Qubit® fluorometer the more concentrated multiplexed amplified captured sample is diluted to the concentration of the less concentrated one (C1*V1=C2*V2). Then with the use of the elution buffer tween (EBT), the library is diluted to 4nM to obtain the appropriate concentration of the NextSeq sampling using a formula provided by the manufacturer. The sample is chemically denatured using NaOH followed by addition of a special hybridization buffer (HT1) supplied with the NextSeq sequencing kit to dilute the library to a final concentration of 1.8 pM. The library is then loaded into the designated reservoir in the prefilled cartridge reagent provided by Illumina.

10. NextSeq-500 sequencing: The Illumina reagent cartridge (NextSeq 500 HighOutput kit 150 cycles), the thoroughly washed and dried flow cell and the buffer cartridge are loaded into the NextSeq500 machine and the sequencing process is launched after assuring that the waste cartridge is empty. The DNA fragments bind to the surface of the flow cell through annealing of the P5 and P7 sequences of the adapters to complementary sequences on the flow cell. The bridge amplification technique is considered a solid phase sequencing method. DNA polymerase then produces clusters of 1 million copies of clonal DNA fragments originating from each single DNA molecule. As sequencing is carried out from both end it is called a paired end sequencing and the reads obtained from each end are called R1 and R2. The index sequence of each adapter allows the machine to affiliate each sequence to the correct sample. In principle, Illumina sequencing technology in the NextSeq500 is also sequencing by synthesis (SBS), but uses a 2- 42

channels sequencing (based on only 2 colors instead of 4 in the MiSeq chemistry) which allows more efficient acquisition of data: base A emits 50% red and 50% green intensity, base C emits 100% red intensity, base G is dark and doesn’t emit any intensity, and base T emits 100% green intensity [Figure (2-4)]

Figure (2-4): Nextseq 500 emission.

(http://support.illumina.com/content/illumina-support/us/en/training/online- courses/sequencing.html)

3. Bioinformatic Analysis 2.10.3.1. Read mapping and variant detection The NextSeq-500 output data is in form of eight FastQ files per sample (four for Read 1 and four for Read 2: Nextseq 500 flow cell contains four physical lanes). Base calling read mapping and quality control are performed using a locally developed bioinformatic pipeline. The reference sequence for read mapping and alignment is based on GRCh37 assembly. The output is a variant calling format (VCF) and a BAM (for alignment, reads and coverage

data) file. The files are uploaded into the interface based software Polyweb (an unpublished software developed by Paris Descartes University, France).

2.10.3.2. Variant selection and prioritization

1. Polyweb software Analysis was carried out with the use of the interface based polyweb tool. For Gene/Variant Prioritization a minimal threshold depth of 20x was selected. To ensure inclusion of all valid variants, we set a relaxed minor allele frequency (MAF) filter of <1% at the offset of our analysis, in public and also in our local database. The filter was tightened at a downstream step to <0.1% for more stringency. Functional consequences of variants selected were stop codons, frameshift, splice site variants and missense changes.

2. Alamut Regarding missense alterations, five pathogenicity prediction algorithms were used (SIFT, Polyphen-2, Mutation Taster, Align GVGD, KD4v). The conservation score was calculated by PhastCons and PhyloP. Moreover, for the prediction of splicing sites, we used five tools (Human Splicing Finder, SpliceSiteFinder–Like, Gene Splicer, NNSPLICE, MaxEntScan). All tools were collectively provided by AlamutVisual® software (Interactive Biosoftware, France).

3. Further Tools to enhance the search  Gene prioritization software  Genome Browsers (ensemble, UCSC, NCBI, ExAC)  Databases (EVS, dbSNP, ExAC, ClinVar, 1000 genome)  Genic intolerance assessment (RVIS, ExAC)  Expression level (Allen Brain, Ensembl link)  Mouse model (MGI)

 Supporting literature about gene and protein (OMIM, Genecards, Uniprot, PDB, Pubmed..) 

2.10.3.3. Validation of Variants 1. Primer design (See section 2.7.1) 2. Polymerase chain reaction (PCR) (See section 2.7.2) 3. ExoSAP purification: ExoSap purification is an enzymatic purification method to clean the PCR product from primers and dNTPs. After mix of specific buffer and two enzymes: the mix was added to the PCR product to obtain a final concentration of the Exonuclease I (EI) (20 U/µl) and the Alkaline phosphatase (AP) (1 U/µl), the product was incubated for 15 min at 37°C (activate enzyme) then at 80°C for 5 min (inactivate the enzyme). This allowed the EI to remove single stranded primers, DNA and PCR product. It also allowed the AP to remove the phosphates of the remaining dNTP to prevent their incorporation at the sequencing reaction leading to background noise.

4. Sanger Sequencing Sanger sequencing is still the gold standard in sequencing. This is the reason why ssubsequent to all approaches, we performed Sanger sequencing for validation of selected variants and to confirm cosegregation of the appropriate zygosity of the variant with the disease distribution in the family. Sanger sequencing is also used to sequence regions of low coverage in certain genes suggested by the clinical phenotype. Sanger sequencing is done at the GATC® platform. One sense primer solution (forward or reverse) at 5 pM concentration combined with a diluted PCR product/ sample are prepared in 98-well plate and sent to the external facility. The BIGDYE chemistry on an ABI3730 sequencer (Applied Biosystems) is used for sequencing. Bioinformatic sequence analysis is performed using Seqscape® (Applied Biosystems®, USA) and Chromas lite® software (Technelysium, Australia).

2.11. Basic Functional Studies (provisional post-genetic workup) 1. Biochemistry: Through our collaborator in USA biochemical tests were achieved to illustrate the effect of a mutation on TFG protein (Elsayed et al., under revision):

Recombinant proteins were expressed as fusions to GST using BL21 (DE3) Escherichia coli, and purifications were conducted using glutathione agarose beads. Proteins were cleaved from resin with GST- HRV3C protease overnight at 4°C and purified further using ion exchange chromatography. Light-scattering data were collected. Purified proteins were applied onto a high-resolution size-exclusion chromatography column (Wyatt WTC-030S5), which was coupled to a Wyatt miniDAWN TREOS three-angle light-scattering detector. Data were collected at a flow rate of 0.5 mL/min and analyzed (ASTRA software) to determine the molecular mass of proteins (Elsayed et al., under revision).

2. Culture of fibroblasts 2.11.2.1. Skin biopsies: They are collected in culture medium for growth of fibroblasts. Components of the medium are indicated in [Table (2-2)]: Table (2-2): Components of culture medium used for collection of skin biopsy and harvest of fibroblasts.

Principal Reagent Original Final Concentartion Concentration

Dulbecco’s Modified 1X 1X Eagle Medium (DMEM) (ThermoFisher)

Fetal Bovine Serum (FBS) 100% 15%

Penicillin/Streptomycin 10 000 U/mL (10 50 U/mL (50 mg/mL) μg/mL)

L-glutamine 200 mM 2 mM

2.11.2.2. Fibroblasts culture protocol summary: Fibroblast culture is the base for most of the downstream procedures in functional studies such as immune-fluorescence, protein extraction and protein based processes as western blotting, immunoprecipitation.

The processing is all conducted in a sterile atmosphere under sterile hood:

1. The skin biopsy is cut into small pieces in a Petri dish with the use of two scalpels. 2. 3 mL of the culture medium is added and the fragments are homogenized by pipetting up and down. 3. 2 mL of the suspension is transferred into a culture flask T25. 4. The flasks are incubated in 37 degrees incubator. 5. Day 2 : 0.5 mL of medium is added 6. Day 8/9: the fibroblast are observed under microscope and culture medium is changed 7. Day 10: the flask are observed daily and trypsanization done when the fibroblast are dense and confluent (passages are started)

2.11.2.3. Passages and Trypsanization: Trypsanization and new passages is done when fibroblasts are condensed to avoid cellular death due to accumulation of waste products and change in pH.

The protocol is the same for all passage and the only unique criterion of the first trypsanization is that the fibroblasts are transterred from T25 to the larger T75 flask and all passages that follow are into T75 flasks.

Summary of trypsanization protocol:

1. Culture medium is eliminated and the cellular layer is washed by 3 mL of Dulbecco’s phosphate buffered saline (DPBS) ) (ThermoFisher SCIENTIFIC Life Technologies Invitrogen® product (Carlsbad, California, USA)) 2. 2 mL of Trypsin EDTA are added and the flask is incubated in 37 degrees for 3-5 min (to detach the cells from the walls of the flask. 47

3. The trypsin is inactivated by addition of 8 mL of culture medium and the suspension is homogenized and 2 mL is transferred into a new T75 (labeled a new passage number: n+1) flask and 8mL of medium are added. 4. The flask is observed under microscope for check of cells viability. 5. The flask is incubated again in 37 degrees and observed daily. 6. The process is repeated whenever cells are confluent.

3. Results

3.1. General features of the cohort Our cohort consisted of 41 nuclear and extended Sudanese families. The cohort included 23 families with predominant pyramidal signs (more like a classical HSP) (23/41; ≈ 7%). There were families where other neurological features (mainly ataxia including spastic ataxia cases) were as severe as the upper motor neuron lesion (UMNL) signs (12/41; ≈ 29%). There were as well families with the pyramidal tract likely affected as part of other diseases (juvenile onset Parkinson disease - JOPD, inborn errors of metabolism -IEM or NBIA) (6/41; ≈14%).

There was a rather high consanguinity rate (37/41: 90%) [Figure (3-1)]. The families originated from almost all regions of Sudan but correlated with the regions/tribes where the traditional culture of consanguineous marriages was the rule for many generations. The highly inbred communities and tribes from Al Jazira (central Sudan) predominated our cohort as could well be predicted (eight families), followed by the White Nile region (An Nil Alabyad) and Northern region (Ash Shamalyah) [Figure (3-2)]. In the four non- consanguineous families (2AD, 1 sporadic and one AR), the parents were not related but consanguinity existed in both maternal/paternal branches, separately.

;

Figure (3-2): The political map of the Sudan showing the origin of 41 families included in the cohort.

In ten families, there were sporadic cases (24%) whereas the pattern of inheritance was autosomal recessive in 27 families (66%) and autosomal dominant in four families (10%). Branch 2 of family FM7 showed possible AD inheritance but was not included in the four AD families [Figure (3-3)]. However, pseudo-autosomal dominant could not be excluded in the majority of AD families. Pure HSP constituted only 10% (4/41) of the cohort, while complicated HSP with minimal complexity (one additional complicating feature only) was only found in two families (5%). The remaining 35 families (85%) were complicated with at least two additional clinical signs (on top of the pyramidal features) which made the high

level of complexity the rule in the cohort. 17 out of the 35 families with high complexity clinical picture (17/41; 41.5%) showed classical HSP whereas 18 families were atypical (43.5%) [Figure (3-4)] [Table (3-1)]. Age at onset was in childhood (<18 years) in 80% of families, adulthood (>18 years) in 16% and variable among related patients (childhood to adulthood) in 4 %. The two families within the latter category of variable age at onset were both dominant with anticipation which could explain the large variability [Figure (3-5)]. [Table (3-2)].

Figure (3-3): Patterns of inheritance in 41 families.

Table (3-1): Clinical categories in the 41 families. HSP: hereditary spastic paraplegia, HA: hereditary ataxia, PD:Parkinson disease, INAD: infantile neuroaxonal dystrophy.

Clinical Summary No. Families /41 % Families

CLASSICAL HSP with predominant pyramidal syndrome

Pure HSP 4 10% F2, F7, F17, F48

Complicated HSP (minimal 2 5% F34, F35 complexity: one additional feature only)

Complicated HSP (more than one 17 41.5% F1, F6, F14, F16, F19, F23, complicating features) F28, F36, F37, F42, F44, F49, F50, FM1, FM3, FM5, FM6

ATAXIA AND HSP (equal weight)

Spastic Ataxia (equal burden) 2 5% F21, F41

ATAXIC syndromes with pyramidal features

Complicated HA 10 24% F12, F24, F26, F30, F45, F46, F47, F31, F38, FM7

Spasticity as a complicating feature of ANOTHER syndrome

HSP (secondary to Metabolic 1 2% F15 disorder)

PD with pyramidal signs 1 2% FM4

Complex presentation with pyramidal 2 5% F11, F22 signs

INAD with pyramidal signs 1 2% F25

Peculiar Syndromes (probably new) 1 2% F27

Figure (3-5): Distribution of age at onset in 41 extended families (44 nuclear families: 3 branches were treated as separate families).

Table (3-2): Distribution of age at onset in 41 extended families (44 nuclear famiies:3 branches were treated as separate families)

Category Age at onset No of % Family codes families/44 (additional three branches)

1 Birth-1 yr 9 F1 branchD, F12, F37, F41, 20% F44, F45, FM3, FM6, FM7 2 1 yr- 5 yrs 11 F1 branchC, F7, F11, F15, F19, 25% F22, F23, F25, F27, F30, F42

3 5 yrs- 18 yrs 11 F1 branchB, F2, F6, F14, F16, 25% F24, F28, F36, F38, F48, FM4

4 18 yrs -40 yrs 6 F1 branch A, F17, F35, F47, 14% F49, FM1

5 >= 40 yrs 1 F21 2%

Variable: category 1-2 Birth-5yrs 2 F50, FM5 5%

Variable: category 2-3 1 – 18 yrs 2 F34, F46 5%

Variable: category 3-4 5-40 yrs 1 F26 2%

Variable: category 2-4 1-40 yrs 1 2% F31

3.2. Genetic results 3.2.1. Identification of mutations in known genes According to inheritance and clinical presentation, 41 families were screened for mutations in known genes as a preliminary step. In 3 families, because of their complex phenotype, WES was performed. In one additional case, the phenotype was compatible with a spinocerebellar ataxia due to nucleotide expansion and a genotyping approach was then used.

3.2.1.1. Identification of a case (sporadic family) with Parkinson disease associated with pyramidal signs due to DNAJC6 mutation

a) Summary: Because of the specific association of pyramidal signs (deep tendon reflexes, spasticity) in lower limbs and Parkinson’s disease (PD) in a sporadic patient in family FM4, we screened nine autosomal recessive (AR) PD genes (PARK2, PINK1, DJ-1/PARK7, ATP13A2, PLA2G6, SYNJ1, DNAJC6, FBXO7, VPS13C) using a targeted next generation sequencing gene panel. The technique utilized a specific capture enrichment strategy (Roche NimbleGen®/SeqCap Ez®, USA) followed by massive parallel sequencing using the MiSeq platform (Illumina®, San Diego, USA). A nonsense mutation in DNAJC6 (exon 16: c.2365C>T, p.(Gln789*)) was found in a homozygous state in the patient, representing the sixth family so far reported.

b) Manuscript o Article 1: Elsayed LE, Drouet V, Usenko T, Mohammed IN, Hamed AA, Elseed MA, Salih MA, Koko ME, Mohamed AY Siddig RA, Elbashir MI, Ibrahim ME, Durr A, Stevanin G, Lesage S, Ahmed AE, Brice A. A novel nonsense mutation in DNAJC6 expands the phenotype of autosomal recessive juvenile-onset Parkinson disease. Ann Neurol 2016 (advance online 2015 dec 24).

3.2.1.2. Identification of families mutated in known HSP genes a) Summary: We tested 65 patients from 25 consanguineous HSP families originating from Sudan for HSP candidate genes using next generation sequencing on patients from 23 families and candidate gene sequencing in two families. We could establish a genetic diagnosis in seven families (28%) with autosomal recessive complex HSP. Truncating homozygous mutations were identified in SPG11 and in SACS genes in three and one families, respectively. Three missense mutations were also found in one family each in TFG/SPG57, ATL1 and ALS2. All mutations were novel except one in SPG11 previously reported with earlier onset compared to our patients. Interestingly, the TFG missense mutation (exon 2: c.64C>T p.(Arg22Trp) / PB1 domain) is the third identified worldwide, and we demonstrated its effect on TFG oligomerization in vitro. Patients did not present visual impairment, as observed in the previously reported SPG57 family (p.(Arg106Cys) / coiled coil domain), suggesting unique contributions of the PB1 and coiled coil domains in TFG complex formation function. Further genetic heterogeneity is expected in HSP Sudanese families. b) Manuscript Article 2: Liena Elbaghir Omer Elsayed, Inaam N. Mohammed, Ahlam Abd Alrahman Ahmed Hamed, Maha Abdelmoneim Elseed, Adam Johnson, Mathilde Mairey, Hassab Elrasoul Siddig Ali Mohamed, Mohamed Nagib Idris, Mustafa A.M. Salih, Sarah Misbah El-sadig, Mahmoud E. Koko, Ashraf Yahia Osman Mohamed, Laure Raymond, Marie Coutelier, Frédéric Darios, Rayan Abubaker Siddig, Ahmed Khalid Mohamed Albashir Ahmed, Arwa Mohammed Abbker Babai, Hiba Mohamed Osman Malik, Zulfa Mohammed Babikir Mohammed Omer, Eman Osama Eldirdiri Mohamed, Hanan Babikir Eltahir, Nasr Aldin Ali Magboul, Elfatih Elfadl Bushara, Abdelrahman Elnour, Salah Mohamed Abdel Rahim, Abdelmoneim Alattaya, Mustafa Idris Elbashir, Muntaser Eltayeb Ibrahim, Alexandra Durr, Anjon Audhya, Alexis Brice, Ammar Eltahir Ahmed, and Giovanni Stevanin. Hereditary spastic paraplegias: Identification of novel SPG57 mutation affecting TFG oligomerization and description of HSP mutations in Sudan. EJHG 2016 (under revision). 58

1 Hereditary spastic paraplegias: Identification of novel SPG57 mutation affecting 2 TFG oligomerization and description of HSP mutations in Sudan 3 Running title: Novel spastic paraplegia mutations in Sudan 4 Liena Elbaghir Omer Elsayed,1,2,3 Inaam N. Mohammed,3 Ahlam Abd Alrahman 5 Ahmed Hamed,3 Maha Abdelmoneim Elseed, Adam Johnson,4 Mathilde Mairey,1,2 6 Hassab Elrasoul Siddig Ali Mohamed,5,6 Mohamed Nagib Idris,3,6 Mustafa A.M. Salih,7 7 Sarah Misbah El-sadig,3,8 Mahmoud E. Koko,9 Ashraf Yahia Osman Mohamed,10 Laure 8 Raymond,1,2,11 Marie Coutelier,1,2 Frédéric Darios,1 Rayan Abubaker Siddig,12 Ahmed 9 Khalid Mohamed Albashir Ahmed,3 Arwa Mohammed Abbker Babai,3 Hiba Mohamed 10 Osman Malik,3 Zulfa Mohammed Babikir Mohammed Omer,3 Eman Osama Eldirdiri 11 Mohamed,3 Hanan Babikir Eltahir,13 Nasr Aldin Ali Magboul,14 Elfatih Elfadl Bushara,3 12 Abdelrahman Elnour,15 Salah Mohamed Abdel Rahim,14 Abdelmoneim Alattaya,16 13 Mustafa Idris Elbashir,3 Muntaser Eltayeb Ibrahim,9 Alexandra Durr,1,11 Anjon 14 Audhya,4 Alexis Brice,*1,11 Ammar Eltahir Ahmed,3,6 and Giovanni Stevanin.1,2,11 15 16 17 *Correspondance: Professor Alexis Brice, Institut du Cerveau et de la Moelle épinière, 18 CHU Pitié-Salpêtrière, 75013 Paris, France. Email: [email protected] 19 20 1Institut du Cerveau et de la Moelle, INSERM U1127, CNRS UMR7225, Sorbonne 21 Universités – UPMC Université Paris VI UMR_S1127, Paris, France. 2 Ecole Pratique 22 des Hautes Etudes, Pitié-Salpêtrière Hospital, ICM, Paris, France. 3Faculty of Medicine, 23 University of Khartoum, Khartoum, Sudan. 4Department of Biomolecular Chemistry, 24 University of Wisconsin-Madison School of Medicine and Public Health, 440 Henry 25 Mall, Madison, WI, 53706, USA. 5Alnelain Medical Center, Khartoum, Sudan. 6Sudan 26 Medical Council, Neurology, Sudan. 7Division of Pediatric Neurology, Department of 27 Pediatrics, College of Medicine, King Saud University, Riyadh, Saudi Arabia. 28 8Department of Neurology, Soba University Hospital, Khartoum, Sudan. 9Department 29 of Molecular Biology, Institute of Endemic Diseases, University of Khartoum, 30 Khartoum, Sudan. 10Department of Biochemistry, Faculty of Medicine, National 31 University, Khartoum, Sudan. 11APHP Pitié-Salpêtrière Hospital, Department of 32 genetics, 75013 Paris, France. 12Faculty of Science, Neelain University, Khartoum, 33 Sudan. 13Department of Biochemistry, Faculty of Medicine, El Imam EL Mahdi 34 University, Kosti, Sudan. 14Department of Radiology, Alamal National Hospital, 35 Khartoum, Sudan. 15Department of Radiology, Ribat University Hospital. 16Antalya 36 Medical Center, Khartoum, Sudan. 37 Conflicts of interest: Authors declare no conflicts of interest. 38 39 Funding and support: This study was supported financially by the Agence Nationale de 40 la Recherche (SPATAX-QUEST project, to Giovanni Stevanin), the Verum Foundation 41 and Roger de Spoelberg Foundation (to Alexis Brice), the European Union (Neuromics 42 projects, OMICS call, to Alexis Brice, Alexandra Durr and Giovanni Stevanin) and the 43 National Institutes of Health (GM110567 to Anjon Audhya) and benefited from the 44 Programme d’Investissement d’Avenir IHU-A-ICM. Liena Elsayed was the recipient of 45 a Campus France and University of Khartoum fellowships.

46 Abstract 47 Hereditary spastic paraplegias (HSP) are the second most frequent motor neuron 48 diseases. We used next generation sequencing to screen 74 HSP genes on 51 patients 49 from 23 consanguineous families from Sudan. Two other families were studied by 50 candidate gene sequencing. We established a genetic diagnosis in seven families (28%) 51 with autosomal recessive complex HSP. Truncating homozygous mutations were 52 identified in SPG11 and SACS genes in three and one families, respectively. Three 53 missense mutations were also found, each in one family, in TFG/SPG57, ATL1 and 54 ALS2. All were novel except one in SPG11 previously reported with earlier onset 55 compared to our patients. The TFG missense mutation (exon 2: c.64C>T p.Arg22Trp / 56 PB1 domain) is the second identified worldwide, and we demonstrated its effect on 57 TFG oligomerization in vitro. Patients did not present with visual impairment, as 58 observed in the previously reported SPG57 family (p.Arg106Cys / coiled coil domain), 59 suggesting unique contributions of the PB1 and coiled coil domains in TFG complex 60 formation/function. Our seven families manifested inter- and intra-familial variations 61 implying the possibility of modifier factors, complicated by high inbreeding that 62 precipitated the phenomenon of multiple genes segregating in different subfamilies. 63 Further genetic heterogeneity is expected in HSP Sudanese families. 64 Key Words: Hereditary Spastic Paraplegia, SPG57/TFG, SPG11, SACS, ATL1, ALS2. 65 Introduction: 66 Pure Hereditary Spastic Paraplegias (HSP) and Hereditary ataxias (HA) represent the 67 extremes of the spectrum of spinocerebellar neurodegenerative disorders. They are often 68 correlated and impose diagnostic difficulty specially with other neurodegenerative 69 disorders with close clinical presentation as motor neurone disease (MND).1 They can 70 be transmitted by 70 all modes of inheritance.1–5 HSP is a group of disorders with the core 71 defining clinical features of insidiously progressive weakness and spasticity of the lower 72 extremities. It is the second most important motor neuron disease with a prevalence of 73 3-10 per 100000 in most populations.6 Prevalence of autosomal dominant (AD) HSP 74 ranges between 0.5-5.5 per 100000 individuals and that of autosomal recessive (AR) 75 HSP between 0.3-5.3 per 100000 individuals.7 HSP can be pure (uncomplicated) or 76 complex according to the absence or presence of additional neurological and non 77 neurological manifestations. To date there are more than 67 known HSP genes.1 AD 78 HSP forms are the most frequent in western populations. SPG4, SPG3A, SPG10 and 79 SPG31 account for up to 50% of AD HSP. SPG3A (ATL1) alone accounts for 10% of 80 AD HSP, with a frequency depending on the age at onset.8 On the contrary, AR HSP 81 predominates in highly consanguineous communities.7,9,10 Thin corpus callosum (TCC)- 82 associated HSP represents a distinct subgroup of HSP accounting for approximately a 83 third of AR HSP.11 At least nine genes have been identified to be responsible for this 84 category. They include SPG1, SPG11, SPG15, SPG18, SPG21, SPG32, SPG46 and 85 SPG49/56 (SPG49 according to HUGO and SPG56 according to OMIM nomenclature) 86 and in rare cases, SPG7. Mutations in SPG11 (also known as KIAA1840 and FLJ21439) 87 (OMIM gene *: 610844) constitute the most frequent cause of TCC-associated HSP 88 (41-77%).12 SPG11 is responsible for 10-20% of all AR HSP [Spastic Paraplegia 11; 89 OMIM phenotype #: 604360], particularly in the Mediterranean basin.6,10,13–16 Next 90 generation sequencing aided the identification of rare genes causing HSP.1,17 91 TFG/SPG57 stands as an example of this genetic surge. Mutations in TFG/SPG57 were

92 implicated in SPOAN-like HSP (spastic paraplegia, optic atrophy, neuropathy)18 and 93 hereditary sensory motor neuropathy with proximal predominant involvement (HSMN4P).19 The 94 TFG protein is a highly conserved regulator of protein secretion, which 95 functions at the interface between the endoplasmic reticulum (ER) and ER-Golgi 96 intermediate compartments. It acts as an oligomer in vivo.18 97 Spastic ataxias now stand as a distinct category with few genes recognized to date. AR 98 spastic ataxia of Charlevoix Saguenay (ARSACS) (OMIM phenotype #: 270550) is a 99 challenging phenotypic overlap between HSP and HA, with a remarkable clinical 100 diversity.20–22 ALS2 (Alsin) (OMIM gene*: 606352), first described by Yang et al., is 101 another prominent example of a gene implicated in pyramidal syndrome, with 102 overlapping roles in other neurodegenerative conditions, including amyotrophic lateral 103 sclerosis.23 104 Relatively little is known about the genetics of spinocerebellar degenerations (SCD) in 105 the Sudanese population.24 In this article, we describe 25 unrelated families with 106 progressive spastic neurodegenerative disorders and their genetic profiling to determine 107 the genes involved. We report the first seven Sudanese families carrying novel and rare 108 mutations in KIAA1840 (SPG11), TFG/SPG57, ATL1 (SPG3A), ALS2 and SACS genes. 109 Furthermore, we demonstrate the pathological effect of the novel TFG mutation 110 reported here using in vitro experiments. Further genetic heterogeneity of HSP is also 111 highlighted by our study. 112 Materials and Methods: 113 Ethics approval: This study was prospectively reviewed and approved by the Paris 114 Necker Ethics Committee (France) and the Ethical Committee of the University of 115 Khartoum, Medical Campus (Sudan). It was conducted in accordance with the 116 recommendation of the Helsinki declaration. A written informed consent was obtained 117 from all participants. 118 Subjects Recruitment: Our cohort was composed of 65 patients from 25 extended 119 Sudanese families, all consanguineous with multiple consanguinity loops belonging to 120 various ethnic groups distributed in different regions of Sudan. For each kindred we 121 recruited the proband, all other accessible diseased family members and, when possible, 122 available unaffected first, second and third degree relatives. Inclusion criteria for 123 probands were clinical presentation suggestive of spastic neurodegeneration, preferably 124 with familial consanguinity and positive family history of neurodegenerative disorders. 125 Clinical Phenotyping: Patients were clinically examined and diagnosed by the 126 referring consultant neurologist/neuro-pediatrician, followed by a second scrutinized 127 phenotyping by the research team. We examined all available healthy related controls to 128 exclude subtle disorders. Patients were assessed for motor disability using a Seven- 129 Stage Disability Score (supplementary I and II). Magnetic resonance imaging (MRI) of 130 the brain was performed for at least one patient per family in most families. 131 Sampling and DNA Purification: Two milliliters of saliva were collected using 132 Oragene.Discover DNA collection kits (Genotek Inc., Canada). DNA purification was 133 done according to the prepIT.L2P manual protocol provided by the manufacturer. DNA 134 quality and quantity control were performed using standard Agarose gel electrophoresis 135 as well as the NanoDrop spectrophotometer (Thermo Scientific, Wilmington, USA) and 136 the Qubit fluorometer (Promega, Wisconsin, USA). 137 Genetic linkage analysis: We performed linkage analysis to screen two families (10

138 patients and 31 healthy related controls) for candidate loci based on patients’ clinical 139 and brain imaging presentation or based on their known relative frequencies. We used 140 microsatellite markers (list available upon request) flanking the CYP7B1 (SPG5), 141 KIAA1840 (SPG11), ZFYVE26 (SPG15), GBA2 (SPG46), CYP2U1 (SPG49 or SPG56 142 according to HUGO and NCBI nomenclatures respectively) and SACS genes. Classical 143 procedures were used for their PCR amplification and allelic resolution in an ABI3730 144 sequencer (Applied Biosystems, CA, USA). Peak Scanner software (Applied 145 Biosystems, CA, USA) was utilized for analysis of product length. Haplotypes were 146 then manually reconstructed for each locus and pedigree in order to obtain the minimal 147 number of recombination events. 148 Targeted Next Generation Sequencing Screening Panel (NGS Panel): We used a 149 multi-gene targeted next generation sequencing panel to screen 23 index patients for 67 150 genes known to be related to a variety of spastic disorders1 and 7 unpublished candidate 151 genes (Supplementary III). NimbleGen/SeqCap Ez enrichment system was used (Roche, 152 CA, USA). Sequencing was performed using MiSeq platform (Illumina, San Diego, 153 USA) on 250 bp paired-end reads. Base calling and quality control were performed 154 using Genomics Workbench (CLC Bio, Denmark). Variants were detected using 155 probability-based and quality-based algorithms. For gene/variant prioritization, stop 156 codons, frameshift, splice site and missense variants with a minimum depth of 30x were 157 selected. Minor allele frequency (MAF) cut-off of 1.5% was used first then decreased 158 to 0.1% for more stringency. Five pathogenicity prediction algorithms were used for 159 missense variants (SIFT, Polyphen-2, Mutation Taster, Align GVGD, KD4v). 160 PhastCons and PhyloP conservation scores were used. For splicing sites prioritization, 161 five tools (Human Splicing Finder, SpliceSiteFinder–Like, Gene Splicer, NNSPLICE, 162 MaxEntScan) were used. All tools were collectively provided by AlamutVisual 163 software (Interactive Biosoftware, France). 164 Sanger Sequencing: We performed Sanger sequencing using the BIGDYE chemistry 165 on an ABI3730 sequencer (Applied Biosystems, CA, USA) to confirm the segregation 166 of culprit variants with the disease. Sequences were analyzed using Seqscape (Applied 167 Biosystems, CA, USA) and Chromas lite software (Technelysium, Australia). 168 Biochemistry: Recombinant proteins were expressed as fusions to GST using BL21 169 (DE3) Escherichia coli, and purifications were conducted using glutathione agarose 170 beads. Proteins were cleaved from resin with GST-HRV3C protease overnight at 4°C 171 and purified further using ion exchange chromatography. Light-scattering data were 172 collected as described previously.18 Briefly, purified proteins were applied onto a high 173 resolution size-exclusion chromatography column (Wyatt WTC-030S5), which was 174 coupled to a Wyatt miniDAWN TREOS three-angle light-scattering detector. Data were 175 collected at a flow rate of 0.5 mL/min and analyzed (ASTRA software) to determine the 176 molecular mass of proteins. 177 Results: 178 Genetic Results: In 25 studies families, autosomal recessive inheritance was observed 179 in 60% (15/25 families). Seven cases (28%) were sporadic and three were from 180 autosomal dominant pedigrees (12%). In two families, we first performed linkage 181 analysis using microsatellites for seven AR HSP loci and found homozygous haplotypes 182 segregating with the disease surrounding the SPG11 locus. Sanger sequencing 183 confirmed the segregation of a homozygous mutation in SPG11 with the disease 184 distribution in these two families (F1 subfamily I and F6). The other 23 families were 185 screened for mutations in 74 HSP genes using targeted capture followed by massive 186 parallel sequencing. In total, genetic diagnosis was established in seven of the 25 187 families (28%). Causative mutations were identified in five genes: SPG11 (three 188 families), TFG/SPG57, ATL1, ALS2 and SACS (one family each). The SPG11 mutations 62

189 were two homozygous nonsense mutations and a homozygous frameshift single base 190 deletion (Figure (1)). In three genes (TFG/SPG57, ATL1, ALS2), missense mutations 191 were identified as the cause of the disease (Figure (2) and Figure (3)). A nonsense 192 mutation was detected in exon 10 of the SACS gene (Figure (3)). Details of these 193 mutations are found in table (1). All the variants showed segregation with the disease 194 and were absent from large sets of exomes (n=68,251) in local data base, 1000 195 Genomes, EVS and Broad institute ExAC cohorts. The missense variants were 196 predicted to be deleterious by at least three pathogenicity prediction algorithms and 197 affected conserved amino-acids in highly conserved protein regions (Table 1). All 198 mutations were novel except a frameshift deletion in SPG11 (c.6709del, 199 p.Ala2237Glnfs*7) that was previously reported in Somalia.25 200 Biochemical effect of TFG novel missense mutation (p.Arg22Trp): This is the 201 second TFG/SPG57 mutation in HSP families. This p.Arg22Trp mutation lies within the 202 Phox and Bem1p (PB1) domain of the TFG protein. The PB1 domain has been shown 203 previously to self-associate in yeast 2-hybrid studies.26 To determine whether the 204 mutation impacts the homo-oligomeric properties of the TFG PB1 domain, we 205 conducted a series of size exclusion chromatography studies, coupled to multi-angle 206 light scattering, which enables an accurate determination of absolute protein molecular 207 mass. Consistent with a previous structural characterization of TFG,27 we found that the 208 PB1 domain, at concentrations ranging from 18-45 μM, formed octamers in solution 209 (~92 kD). In contrast, the p.Arg22Trp mutant of the PB1 domain exhibited a 210 significantly reduced molecular mass of ~24 kD throughout the same range of protein 211 concentrations, indicating it was only capable of forming dimers. Additionally, the 212 elution time of the p.Arg22Trp mutant PB1 domain was extended relative to the wild 213 type protein, consistent with the idea that the mutant failed to self-assemble properly 214 (Figure (2)). Together, these studies demonstrate that the p.Arg22Trp mutation disrupts 215 the ability of the PB1 domain to oligomerize highlightING a mechanistic basis for 216 HSP. 217 Phenotype-genotype correlations: A complex phenotype was predominant in this 218 cohort (21/25 families, 84%). Clinical data from 23 patients (7 families) with 219 established genetic diagnosis is presented in table (2) and table (3). A summary of the 220 MRI and electro-physiological investigations is provided in table (4). 221 In three nuclear families (F34, FM5 and F19), with mutations in ATL1, ALS2 and TFG 222 respectively, we observed homogeneous clinical patterns in all patients. Family F34 223 represented a rare example of both pure HSP phenotype and AD inheritance in our 224 cohort, despite the high consanguinity in this family. The presentation (Table 3)matched 225 the classical clinical pattern and the early age at onset associated with SPG3A.28 In 226 family FM5, four patients from two branches presented with infantile onset progressive 227 pyramidal syndrome with pseudobulbar palsy. This is in conformity with what was 228 reported in Infantile onset ascending spastic paralysis (IAHSP) associated with Alsin 229 mutations (OMIM #607225) (Table 3).29–31 Interestingly, phenotypes in family F19 230 extended the signs associated with mutations in TFG/SPG57. The mean age at onset in 231 the three affected sibs was around one year and mild cognitive impairment was also 232 observed. They presented with signs of progressive pyramidal tract involvement. In 233 contrast to the SPOAN-like HSP reported by Beetz et al.18 in the first SPG57 family 234 (c.316C>T, p.R106C), patients in this family had intact optic nerve and normal fundi. 235 The electrophysiological studies confirmed variable degrees of motor neuronal 236 demyelination in addition to motor axonal degeneration matching the previous report.18 237 Although brain MRI revealed no significant abnormalities in the first family, we report 238 thinning of the corpus callosum, variable degrees of cerebellar atrophy and mild white 239 matter hyperintensities, as part of the SPG57 clinical spectrum. 63

240 In other families, the phenotypic presentation varied between patients from different 241 branches. Patients in family F14 carried a SACS truncating mutation. Age at onset was 242 late childhood, in agreement with Baets et al.32 and higher than what was reported in 243 Tunisian patients.33 Although this was not genetically analyzed, it is strongly suggested 244 that there were at least two different segregating disease genes within the same nuclear 245 sibship as our index patient had a family history of three older brothers who died all 246 from renal failure and were clinically diagnosed as Laurence Moon Biedl (LMB) 247 syndrome years ago. Furthermore, the two diseased direct cousins who had the same 248 damaging SACS mutations showed interesting clinical differences. They both had 249 spastic ataxia. However, the proband had predominant ataxia and peripheral neuropathy 250 whereas the cousin had clinical features of a predominant spastic paraparesis. Both 251 patients had absent fundal striations in accordance with previous reports of Italian 252 patients 34,35 and Japanese sibship.36 253 Overall, patients from the three families with mutations in SPG11 presented with a 254 complex HSP frequently associated with cognitive deficit. MRI of the brain showed 255 atrophy of the corpus callosum in all six tested cases often associated with 256 periventricular white matter abnormalities (Figure (1)) and signs of motor 257 demyelination frequently mixed with axonal degeneration at electrophysiological 258 examination. This agrees with previous reports.37,38 It is worth noting that of the 18 259 families (72%) in which the genetic diagnosis is not yet established, 2 families only 260 presented as pure HSP. Six families showed HSP complicated with stereotypic 261 movement (2/18), bulbar signs and sensory motor polyneuropathy (1/18), cognitive 262 decline (1/18), dystonia (1/18) or severe cortical atrophy (1/18). Ten families presented 263 with spastic ataxia, complicated in six families with dysmorphic features (2/18), chorea 264 (1/18) and visual impairment (3/18). 265 Discussion: 266 In line with the studies done on other inbred communities,9,10 we found that autosomal 267 recessive HSP inheritance is predominant in Sudan. However, in our series, the complex 268 phenotype is predominant (84%), in disagreement with Coutinho et al. who elicited a 269 predominant pure phenotype,9 but in agreement with a study in Tunisia10 that reported 270 69% of complex cases. Such complex forms are often misdiagnosed due to overlapping 271 symptoms with other diseases. The use of a targeted NGS panel provided an efficient 272 and reliable screening method to test HSP and HSP-related genes. We screened 53 273 patients from 21 families and identified culprit variants in five families. Mutations in 274 known genes were identified in 28% of index cases (7/25) in line with other reports 275 using candidate gene screening or exome sequencing in heterogeneous diseases.38 All 276 mutations, except one, are novel with convincing evidence of pathogenecity. The cohort 277 of Sudanese families with autosomal recessive HSP showed high frequency of SPG11 278 mutations (3/22; 13%), in conformity with other populations.15 Of note, SPG7 was 279 absent in the present cohort where SPG57, ARSACS, and ALS2 accounted for 1 family 280 each (4%). Regarding dominant forms, SPG3 was seen in one family while SPG4 was 281 absent from our series. While the phenotype was homogeneous between patients in 282 several families (F34, F19, FM5 showing mutations in ATL1, TFG/SPG57 and ALS2 283 respectively), it was more often heterogeneous in other extended families (F1, F6, F14). 284 These differences ranged between subtle and overt, including motor and cognitive 285 domains, onset and progression, and were evident within nuclear and extended families. 286 Of major interest, we report the second SPG57 family with three siblings carrying the 287 novel c.64C>T, p.Arg22Trp mutation. We observed significant clinical differences that 288 may be attributed to the different domains affected in the two families and the 289 functional sequel. The previously identified p.Arg106Cys mutation is located in the 290 coiled coil (CC) domain of TFG. The novel p.Arg22Trp mutation lies in the PB1 64

291 domain. Although both mutations resulted in perturbed oligomerization of the protein, 292 the mechanism and consequent functional effect are distinct based on our biochemical 293 evidence. Structural studies indicate that full length TFG self-assembles into octameric 294 ring-like complexes in solution.18 Our analysis of the PB1 domain indicates that it 295 exhibits a similar propensity to form octamers. Thus, the coiled coil domain may play a 296 distinct role in promoting TFG complex assembly, potentially facilitating the assembly 297 of the ring-like organization, which is likely under considerable strain. The relation 298 between phenotypic spectrum and mutation localization is well known in HSP genes 2 including PNPLA6 reported by Schabh ttl et al.39 and TFG/SPG57 reported by Beetz et 300 al.18 Mutations in the proline and glutamine-rich domain (carboxyl terminus) of 301 TFG/SPG57 result in AD HSMN-P.19 On the contrary, mutations in the amino terminus 302 result in AR HSP as shown by Beetz et al.18 and in this study. 303 Conclusions: We were able to identify the first Sudanese families carrying novel 304 mutations in SPG11, ATL1, ALS2, SACS and TFG/SPG57 genes and establish their 305 relative frequencies. However, the difficulty to reach a genetic diagnosis in the majority 306 of studied families suggests the possibility of new genes or noncoding variations 307 underlying spinocerebellar degeneration. The phenotypes of the undiagnosed families 308 and the high frequency of spastic ataxic phenotypes imply a high possibility to expand 309 the phenotypes and molecular basis of HSP. Genetic screening using exome or genome 310 sequencing is therefore highly recommended to explore these families. TFG novel 311 mutation resulting in p.Arg22Trp change was found to disrupt the ability of the PB1 312 domain to oligomerize highlighting a mechanistic basis for HSP. 313 Acknowledgment: We thank the members of the DNA and cell bank of the ICM, 314 Federica Barreca, Emeline Mundwiller, Khalid El Hachimi and Delphine Bouteiller for 315 their valuable contribution. This study was supported by the Agence Nationale de la 316 Recherche SPATAX-QUEST project (to GS), Verum Foundation, Roger de Spoelberg 317 Foundation (to AB), European Union Neuromics projects - OMICS call (to AB, AD, 318 GS), NIH (grant GM110 67 to AA) and the Programme d’Investissement d’Avenir 319 IHU-A-ICM. LE received Campus France and University of Khartoum fellowships. 320 Conflicts of Interests: The authors declare no conflicts of interests. 321 Supplementary information is available at European Journal of Human Genetics’s 322 website. 323 References 324 1. Tesson C, Koht J, Stevanin G. Delving into the Complexity of Hereditary Spastic 325 Paraplegias: How Unexpected Phenotypes and Inheritance Modes Are 326 Revolutionizing Their Nosology. Hum Genet 2015; 134 (6): 511–38. 327 doi:10.1007/s00439-015-1536-7. 328 2. Fink J. Hereditary Spastic Paraplegia Overview. In: Pagon R, Adam M, Ardinger H, 329 et al. (eds). GeneReviews(®). University of Washington: Seattle (WA), USA, 1993. 330 http://www.ncbi.nlm.nih.gov/books/NBK1509/. 331 3. Schmit -H bsch T, Klockgether T. An Update on Inherited Ataxias. Current 332 Neurology and Neuroscience Reports 2008; 8 (4): 310–19. doi: 10.1007/s11910- 333 008-0048-4. 334 4. Finsterer J. Ataxias with Autosomal, X-Chromosomal or Maternal Inheritance. Can 335 J Neurol Sci 2009; 36 (4): 409–28. doi: 10.1017/S0317167100007733. 336 5. Coutelier M, Stevanin G, Brice A. Genetic Landscape Remodelling in 337 Spinocerebellar Ataxias: The Influence of next-Generation Sequencing. J Neurol 338 2015; 262 (10): 2382-95. doi:10.1007/s00415-015-7725-4. 339 6. Noreau A, Dion P, Rouleau G. Molecular Aspects of Hereditary Spastic Paraplegia. 340 Exp Cell Res 2014; 325 (1): 18–26. doi:10.1016/j.yexcr.2014.02.021. 341 7. Ruano L, Melo C, Silva M, Coutinho P. The Global Epidemiology of Hereditary 65

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444 doi:10.1016/j.ejmg.2010.10.006. 44 3 . Schabh ttl M, Wieland T, Senderek J, et al. Whole-Exome Sequencing in Patients 446 with Inherited Neuropathies: Outcome and Challenges. J Neurol 2014; 261 (5): 447 970–82. doi:10.1007/s00415-014-7289-8. 448 Titles and legends to figures: 449 Figure (1): Three families (F1, F6, and F16) with autosomal recessive hereditary 450 spastic paraplegia (HSP) with thin corpus callosum (TCC) caused by SPG11. (A I) 451 Pedigree of Family F1. Subfamily II (branch C and D) presents with two different HSP 452 phenotypes. (A II) MRI of the brain (Sagittal section T1) of index individual (arrow) 453 showing severe TCC. (B I) Pedigree of Family F6. (B II) MRI of the brain (Sagittal 454 section T1) of index individual showing severe TCC and cortical atrophy. (C I) 455 Pedigree of Family F16. (C II) MRI of the brain (Axial section T2 FLAIR) of index 456 individual showing TCC and periventricular hypersignal intensity lesions. In all 457 families, example of Sanger sequencing results of the mutation site in a patient and a 458 heterozygous carrier is shown. HTZG: heterozygous, * sampled individual, black 459 shading indicates affected individuals, ++ homozygous reference, M+ heterozygous 460 genotype, MM homozygous mutant. 461 Figure (2): One family (F19) with autosomal recessive hereditary spastic 462 paraplegia (AR HSP) caused by TFG/SPG57. (A) Pedigree of Family F19. Example 463 Sanger sequencing of the mutation site in a patient, a heterozygous carrier and a control 464 homozygous reference allele with conserved amino acid sequence, is shown along with 465 aminoacid conservation. HTZG: heterozygous, * sampled individual, black shading: 466 individuals with HSP, ++ homozygous reference genotype, M+ heterozygous, MM 467 homozygous mutant genotype. (B) Purified, untagged forms of TFG (amino acids 1- 468 96; wild-type or mutated to include the p.R22W substitution), were separated over a gel 469 filtration column (Wyatt TWC-030S5) that was coupled to a multi-angle light scattering 470 device. Representative light scattering profiles (wild type TFG , blue; TFG p.R22W, 471 red) are plotted (B I) and eluted fractions were separated by SDS-PAGE and stained 472 using Coomassie to hig hlight the elution profiles of the both forms of TFG (B II). 473 Based on 3 independent experiments for each protein, wild type TFG (amino acids 1- 474 96) exhibits a molecular mass of 92.4 kD +/- 3.7 kD, and p.R22W TFG (amino acids 1- 475 96) exhibits a molecular mass of 24.4 kD +/- 1.0 kD. (C) MRI of the brain (Sagittal 476 section T1) of individual 19172 showing thinning of the body of the corpus callosum 477 (C I) and MRI of the brain (Axial section T2 FLAIR) of individual 19171 showing thin 478 corpus callosum and very mild periventricular hypersignal intensity lesions at the 479 occipital pole (C II). 480 Figure (3): Three families (F14, F34, and FM5) with spastic ataxia and hereditary 481 spastic paraplegia (HSP) caused by SACS, SPG3A and ALS2, respectively. (A I) 482 Pedigree of Family F14 with AR spastic ataxia of Charlevoix Saguenay (ARSACS) 483 caused by nonsense mutation of SACS gene. Example Sanger sequencing of the 484 mutation in a patient and in a heterozygous carrier is shown. (A II) MRI of the brain 485 (Sagittal section T1) of individual 14133 showing thinning of the posterior half of the 486 body of corpus callosum and moderate thinning of cerebellum. (B) Pedigree of Family 487 F34 with Autosomal Dominant HSP caused by SPG3A/ATL1 gene. Example Sanger 488 sequencing of the mutation site in a patient and in a control (homozygous reference) is 489 shown. (C) Pedigree of Family FM5 with Autosomal Recessive HSP caused by 490 missense mutation of ALS2 gene. Example Sanger sequencing of the mutation site in a 491 patient and in a heterozygous carrier is shown. In all pedigrees: HTZG: heterozygous, * 492 sampled individual, black shading: affected individuals, ++ Homozygous reference 493 genotype, M+ Heterozygous, MM Homozygous mutant.

3.2.1.3. Whole exome sequencing approach In three complex HSP families, WES was used as a first approach.

 Family F15 Family F15 was an extended family composed of three branches. The age at onset ranged between one to three years. The patients had history of regression of milestones following a normal early development till the age at onset which ranged from one to three years. The clinical picture was dominated by spastic quadriplegia complicated by cognitive impairment in the majority of patients and epilepsy in two patients. Despite the homogeneity of the clinical presentation in all branches, stereotypic clapping distinguished the patient from branch 1 [Figure (3- 6)] [Table (3-3)] [Table (3-4)].

Using WES in the five patients and one healthy parent, only one homozygous variant was found shared by patients from all three branches, remained after filtering out the common and non-coding variants. This was a novel missense variant in the ARG1 gene. The mutation [Exon 4: g. 131902487T>A, c.434T>A, p.(Val145Glu)] co-segregated with the disease distribution [Figure (3-6)]. It was a transversion variation of a highly conserved nucleotide. The mutation was in a homozygous state in five patients from three branches of this extended family. All other seven sampled healthy related individuals were homozygous reference or heterozygous carriers.

This mutation was predicted pathogenic by four algorithms [SIFT, Polyphen2, mutation taster and align GVGD] [Table (3-8)]. The variation resulted in a moderate physiochemical difference [Grantham distance of 121 (0-215)] from the highly conserved (12 species) hydrophobic amino acid valine to the acidic amino acid glutamate. The variant was located in the protein domains: Ureohydrolase, Arginase subgroup.

Arginases (EC 3.5.3.1) are proteins involved in last reaction of the urea cycle in which they catalyze the hydrolysis reaction of L-Arginine to form the urea and ornithine. There are two forms of the arginase enzyme. Arginase 1 which predominates in the liver whereas arginase 2 is the main arginase in the kidneys.

The first case in whom the genetic basis of argininemia was characterized was a Japanese patient with compound heterozygous mutations (Haraguchi et al., 1990)

The combination of progressive spastic tertraplegia, regression of milestones, epilepsy in our patients is in agreement with what was reported about the presentation of a loss of function mutation on the Arginase 1 (Wu et al., 2015) (Schlune et al., 2015)(Sin et al., 2015) . In contrast to the Japanese patient described by Haraguchi in 1990, the five Sudanese patients in family F15 did not have microcephaly which was reported in many patients of hyperargininemia (Haraguchi et al., 1990) (Wong et al., 2015). In contrast to what was reported in five Chinese patients with poor physical growth associated with argininemia the Sudanese patients had normal physical growth despite the mental impairment with variable severity witnessed in all of them (Wu et al., 2013)

Biochemistry assessment of amino acid levels in the serum of the patients showed an increased level of Arginine in all patients in a first assessment but it was normal in another occasion. A laboratory error was considered in the second occasion as arginine was not reported as normal in patients with ARG1 mutation to suggest oscillating level of arginine with occasionally normal level and peaks. In a set of Brazilian patients the level of arginine was approximately near borderline in some patients (Carvalho et al., 2012). Retesting of erythrocyte Arginase activity and plasma Arginine level is planned in the near future to further confirm the diagnosis due to the availability of treatment and protection options. In the absence of other evidence of the involvement of any other variants, I considered the ARG1 mutation as causative in this family.

Figure (3-6): Pedigree of family F15 caused by missense mutation in ARG1 segregating with the disease distribution in whole family presenting with spastic tetraplegia and mental retardation. Whole exome sequencing was performed for five affected individuals (138, 142, 143, 148, and 149) and the mother (137) of patient 138. Example sanger sequencing of the mutation in patient (homozygous mutant), a heterozygous carrier and a control homozygous reference allele with conserved amino acid sequence, is shown. Pedigree symbols: * sampled individual; yellow square indicates the two index patients; Phenotype symbols: half-fill black color: affected individuals; Genotype symbols: ++ Homozygous reference genotype; M+ Heterozygous genotype; MM Homozygous mutant genotype. Others are standard medical pedigree symbols.

Table (3-3): Clinical data of five patients from family F15 with hyperargininemia due to a mutation in ARG1 gene. UL: upper limbs. LL: lower limbs. PUL/PLL: proximal upper/lower limbs. DUL/DLL: distal upper/lower limbs. Cog: cognitive. Psych: psychological. Clinical signs severity: – Absent, + Mild, ++ Moderate, +++ Severe. Tendon Reflexes: ↑Increased Tendon Reflex. Extensor Planter Response: ↑ Unilateral, ↑↑ Bilateral, ↔ Mute, ↓ Flexor. N: Normal. NA: Not Applicable due to Patients Condition.

Family Code F 15 F 15 F 15 F15 F15

Mutated Gene ARG1

Individual Code 138 142 143 148 149

Gender F M M M F

Clinical Diagnosis Spastic quadriplegia

Origin North of Sudan, Gaalia tribe

Age at Onset of Motor 1 year 3 years 2 years/ 9 3 years 2 years 8 Symptoms months months

Age at Initial 8 year 10 year 6 year 10 year 8 year Examination

Spasticity UL/LL ++/+++ ++/++ +++/+++ ++/+++ +++/+++

Motor Deficit +/+ +/+ +/+ +/- -/- PUL/DUL

Motor Deficit ++/++ +/+ ++/++ +++/+++ -/- PLL/DLL

Tendon Reflexes ↑/↑ ↑/↑ ↑/↑ ↑/↑ ↑/↑ UL/LL Patellar

Ankle Reflex/ Planter ↑/↑↑ ↑/↑↑ ↑/↔ ↑/↑↑ ↑/↑↑ Response

Ataxia Eye/UL/LL/ - /-/-/- -/-/-/- -/-/-/- -/-/-/- -/-/-/- Gait

Dysarthria +++/- +/- +/ - +++/- +++/- Spastic/Cerebellar

Muscle Atrophy +/+ +/+ -/+ -/+ -/- DUL/DLL

Facial Atrophy - - - - -

Cog/ Psych signs +/+ +/+ +/+ -/- +/+

Individual Code 138 142 143 148 149

Sensory Loss - - - - -

Optic Atrophy - - - - -

Extrapyramidal - - - - - Signs

Other Signs Severe Severe Anal Pes Pes urinary/ anal urinary/ anal incontinence cavus/ cavus/ incontinence incontinence Scoliosis Scoliosis

Pes cavus/ Epilepsy

Scoliosis

Epilepsy

Rt. Hearing impairment

Disability Score 5 6 5 6 4

MRI of the brain NAD NAD NAD NAD NAD

Electrophysiological ND ND ND ND ND Studies (NCS)

Plasma amino acids ↑Arginine in ↑Arginine in ↑Arginine in ↑Arginine ↑Arginine level one one one in one in one occasion, occasion, occasion, occasion, occasion, normal in a normal in a normal in a normal in normal in second test second test second test a second a second test test

Table (3-4): Clinical summary of family F15. HSP:hereditary spastic paraplega, UL: upper limbs, LL: lower limbs, ND: not done, CT: computerized tomography scan, MRI: magnetic resonance imaging, EEG: electroencephalogram, EMG: electromyogram, NCS: nerve conduction studies.

Fam. Origin/ Inherita Phenotype Age at Electrophys Brain Imaging Clinical features summary code Consan nce/ summary onset/Exam. iological guinity Gene studies EMG/NCS, EEG)

F15- consanguineous Gaalia/ North AR/ Complicated Two sibs (one ND MRI: Normal 1- Pyramidal signs(LL &/> UL: spasticity, hyperreflexia BRANC ARG1 HSP (metabolic) died) : and extensor planter response) 2-Sterotypical behaviour 3- H-1 Mental retardation 4-Epilepsy (atonic initiated by laughter) 1yr/8 yrs 5-Skeletal deformities (pes cavus, scoliosis) 6-Rt conductive deafness 7-Psychiatric symptoms

(hyperactivity, distractability) 7-Sphincter disturbance 8- Regression of milestone (aggravation at 2.5 yrs age) 9- Abnormal way of looking at objects

F15- consanguineous Gaalia/ North AR/ Complicated Two sibs : ND CT scan: 1- Pyramidal signs(LL &/> UL: spasticity, hyperreflexia BRANC HSP (metabolic) Normal and extensor planter response) 2- Mental retardation 3- H-2 ARG1 2yrs Epilepsy (generalized tonic clonic epilepsy) 4- Sphincter 9months,3yrs/6,10y MRI: Normal disorder 5-Abnormal way of looking at objects rs

F15- consanguineous Gaalia/ North AR/ Complicated Two sibs : ND CT scan: 1- Pyramidal signs(LL &/> UL: spasticity, hyperreflexia BRANC ARG1 HSP (metabolic) Normal and extensor planter response) 2- Mental retardation 3- H-3 2yrs 8months, 3 Psychiatric symptoms 4-Sphincter disturbance 5- yrs/ 8,10yrs Regression of milestones 6-Skeletal deformities (pes cavus, scoliosis) 7-Abnormal way of looking at objects

 Family F25 The two patients presented with homogenous disease presentation with the mean age at onset at 22 months (18, 24 months) and history of regression of milestones. Examination revealed marked hypotonia, pyramidal signs (hyperreflexia in both individuals and extensor planter response in one patient). Additional features included dysphagia and aphasia, mental impairment and psychiatric manifestation (blunt mood). The older sib (proband) had optic atrophy. There was no extrapyramidal signs or evidence of PN in both sibs. MRI of the brain was also homogenous but with more overt changes in the older sib. T2 Weighted FLAIR images showed hyperintense signals in periventricular areas, basal ganglia including globus pallidus and to lesser degree head of caudate nucleus and putamen as well as the cerebellum. Mild generalized cerebellar atrophy was found in both patients but there was no evidence of brain iron accumulation (hypointense signals in T2 and hyperintense signals in T1 weighted images) [Figure (3-7)] [Table (3-5)].

WES in patient 210 combined to homozygosity mapping analysis identified three variants novel or rare with convincing evidence of pathogenicity. Among these, the novel variant (c.1427+2T>C) in the PLA2G6 gene acting as a splice site donor was the most convincing and the clinical scenario of the patients matched with what was reported in the literature about the presentation of PLA2G6. The mutation very likely led to the skipping of exon10 [Table (3-8)]. The predicted change at the donor site, which is 2 bps upstream, is -100.0% (MaxEnt: -100.0%, NNSPLICE: -100.0% and HSF: -100.0%). The mutation was found to co- segregate with the disease distribution. It was found in a homozygous state in two patients and homozygous reference or heterozygous in five healthy related individuals [Figure (3-7)].

The clinical presentation of the two patients was compatible with infantile neuroaxonal dystrophy (INAD) due to neurodegeneration associated with mutations in PLA2G6 gene (PLAN) [Table (3-5)].

In INAD the age at onset is between six months and three years. Its less frequent variant has later onset: atypical neuroaxonal dystrophy (atypical NAD). The hallmark in both conditions is the presence of cerebellar atrophy and optic atrophy

(Gregory and Hayflick, 2016). In literature, MRI feature suggestive of NBIA were found absent in half of the cases of INAD. The MRI finding of the Sudanese patient was quite in accordance with this. (Kurian et al., 2008) (Tonelli et l., 2010) (Gregory and Hayflick, 2016)

Figure (3-7): Pedigree and MRI of the index patient of family F25 caused by splice donor mutation in PLA2G6 segregating with the disease distribution in whole family presenting with pyramidal signs and features associated with infantile neuroaxonal dystrophy (INAD). MRI shows no evidence of brain iron accumulation but neurodegeneration with white matter hyperintensities (WMH) in periventricular areas, basal ganglia including globus pallidus and to lesser degree head of caudate nucleus and Putamen. Example sanger sequencing of the mutation in patient (homozygous mutant), a heterozygous carrier and a control homozygous reference allele with conserved amino acid sequence, is shown. Pedigree symbols: * sampled individual; Phenotype symbols: half-fill black color: affected individuals; Genotype symbols: ++ Homozygous reference genotype; M+ Heterozygous genotype; MM Homozygous mutant genotype. Others are standard medical pedigree symbols. 95

Table (3-5): Clinical data of two patients from family F25 with infantile neuroaxonal dystrophy (INAD) due to a mutation in PLA2G6 gene. UL: upper limbs. LL: lower limbs. PUL/PLL: proximal upper/lower limbs. DUL/DLL: distal upper/lower limbs. Cog: cognitive. Psych: psychological. Clinical signs severity: – Absent, + Mild, ++ Moderate, +++ Severe. Tendon Reflexes: ↑Increased Tendon Reflex. Extensor Planter Response: ↑ Unilateral, ↑↑ Bilateral, ↔ Mute, ↓ Flexor. N: Normal. NA: Not Applicable due to Patients Condition. WMH: white matter hyperintensities.

Family Code F25 F25

Mutated Gene PLA2G6

Individual Code 209 210

Gender M F

Clinical Diagnosis INAD

Origin White Nile/ Kwahla tribe

Age at Onset of Motor Symptoms 2 years 1 year 8 months

Age at Initial Examination 2.5 years 3 years 8months

Spasticity UL/LL -/- -/-

Motor Deficit PUL/DUL ++/++ ++/++

Motor Deficit PLL/DLL NA/NA ++/++

Tendon Reflexes UL/LL Patellar ↑/↑ ↑/↑

Ankle Reflex/ Planter Response N/↑↑ N /↓

Ataxia Eye/UL/LL/ Gait -/++/NA/NA -/NA/NA/NA

Dysarthria Spastic/Cerebellar Aphasic Aphasic

Muscle Atrophy DUL/DLL -/- +/+

Facial Atrophy - -

Cog/ Psych signs +/+ +/+

Optic Atrophy - +

Extrapyramidal Signs - -

Sensory Loss NA NA

Other Signs Dysphagia Dysphagia

Disability Score 5 7

MRI of the brain WMHS in periventricular WMHS in periventricular areas, basal ganglia areas, basal ganglia including including globus pallidus globus pallidus and to lesser and to lesser degree head of degree head of caudate caudate nucleus and nucleus and Putamen plus the Putamen plus the cerebellum. Mild Cerebellar cerebellum. Mild Cerebellar atrophy atrophy.

Electrophysiological Studies Normal Normal (NCS)

CT scan of the brain ND ND

 Family F30 The four patients (sisters) showed homogeneous clinical scenarios with age at onset of motor symptoms at around 2 years and the age at development of cataract at 4-6 years. They had delayed motor and normal intellectual development. The main clinical features were ataxia complicated by short stature, cataract, pyramidal signs, mild cognitive impairment and hypogonadism. The pyramidal syndrome was more prominent in LL than in UL. There was mild cognitive impairment and clinical evidence of hypogonadism in three sibs (lack of menarche in one and early menopause in the 2nd decade of life in two of them) whereas the fourth was too young to assess [Figure (3-8)] [Table (3-6)].

An extremely rare missense variant in the SIL1 gene was found among seven others that were identified upon analysis of WES of two patients (258,259). It was the only one that was found to co-segregate with the disease distribution (homozygous in four patients whereas five healthy related individuals were homozygous reference or heterozygous) [Figure (3-8)].

The variant was extremely rare and was found only once in a heterozygous state and never in the homozygous state in the 60,706 WES in the broad institute ExAC browser. It was not found in EVS, dbSNP or 1000 genome databases. The mutation [Exon 8: g.138356887 G>A, c.740C>T / p.(Ala247Val)] was a transition mutation of a highly conserved nucleotide. The variation was predicted pathogenic

97 by the two algorithms Polyphen2 and mutation taster [Table (3-8)]. It resulted in a small physiochemical change [Grantham distance of 64 (0-215)] of the highly conserved hydrophobic amino acid alanine (12 species) (located in the protein domains: Armadillo-type fold) to another hydrophobic amino acid valine.

The clinical presentation of the patients was in accordance with what had been reported in the majority of Marinesco-Sjogren syndrome (MSS) cases due to mutations in the SIL1 gene (*608005). Marinesco-Sjogren syndrome (OMIM # 248800) is an AR disorder characterized by cerebellar ataxia, progressive myopathy, and cataract (Anttonen et al., 2005). Hypogonadism had been described in MSS in male and females in many studies (Senderek et al., 2005) (Karim et al., 2006). The spasticity is a rare manifestation of MSS as it was only reported in three Japanese sibs (Takahata et al., 2010). We could not detect any evidence of myopathy and no supporting investigations could be performed (serum creatine kinese, EMG/NCS and muscle biopsy). Serum immunoglobulin levels could not be measured although low serum IgG and IgA had been reported in MSS (Hasegawa et al., 2014).

Figure (3-8): Pedigree and MRI of the index patient of family F30 caused by missense mutation in SIL1 segregating with the disease distribution in whole family presenting with pyramidal signs associated with Marinesco Sjogren Syndrome (MSS). MRI shows marked cerebellar atrophy. Example sanger sequencing of the mutation in patient (homozygous mutant), a heterozygous carrier and a control homozygous reference allele with conserved amino acid sequence, is shown. Pedigree symbols: sampled individual are numbered; Phenotype symbols: black color: affected individuals; Genotype symbols: ++ Homozygous reference genotype; M+ Heterozygous genotype; MM Homozygous mutant genotype. Others are standard medical pedigree symbols.

Table (3-6): Clinical data of four patients from family F30 with Marinesco Sjogren Syndrome due to a mutation in SIL1 gene. UL: upper limbs. LL: lower limbs. PUL/PLL: proximal upper/lower limbs. DUL/DLL: distal upper/lower limbs. Cog: cognitive. Psych: psychological. Clinical signs severity: – Absent, + Mild, ++ Moderate, +++ Severe. Tendon Reflexes: ↑Increased Tendon Reflex. Extensor Planter Response: ↑ Unilateral, ↑↑ Bilateral, ↔ Mute, ↓ Flexor. N: Normal. NA: Not applicable due to patients condition.

Family Code F30 F30 F30 F30

Mutated Gene SIL1

Individual Code 257 258 259 260

Gender F F F F

Clinical Diagnosis Complex HA

Origin Gazira, Almanagil

Age at Onset of Motor 2 Yrs 2 Yrs 2 Yrs 2 Yrs Symptoms

Age at Initial 37 Yrs 25 Yrs 15Yrs 13 Yrs Examination

Motor Development Delayed Delayed Delayed Delayed

Intellectual N N N N development

Spasticity +/++ -/++ -/+ -/+

UL/LL

Motor Deficit -/- -/- -/- -/- PUL/DUL

Motor Deficit -/- -/- -/- -/- PLL/DLL

Tendon Reflexes ↑/↑ N/ N N/ N N/ ↑ UL/LLPatellar

Ankle Reflex/ Planter ↑/↓ N/ ↓ N/ ↓ ↑/↓ Response

Ataxia Eye/UL/LL/ +/-/-/+++ +/++/++/++ +/++/++/+++ +/++/++/+++ Gait

100

Family Code F30 F30 F30 F30

Individual Code 257 258 259 260

Dysarthria ++/++ ++/++ -/- ++/++ Spastic/Cerebellar

Muscle Atrophy -/- -/- -/- -/- DUL/DLL

Facial Atrophy - - - -

Cog/ Psych signs +/- -/- +/- +/-

Cataract Bilateral Cataract Bilateral Cataract Bilateral Cataract Bilateral Cataract at 4 yrs (operated on the at 6 yrs ( at 4 yrs right side) operated on the left side)

Extrapyramidal - - - -

Signs

Sensory Loss - - - -

Other Signs of Eye ↓Visual acuity, ↓Visual acuity, ↓Visual acuity, ↓Visual acuity vertical vertical vertical vertical ophthalmoplegia ophthalmoplegia ophthalmoplegia ophthalmoplegia (Restricted (Restricted (Restricted (Restricted upgaze), saccadic upgaze), saccadic upgaze), saccadic upgaze), saccadic pursuit pursuit pursuit ,Slow pursuit saccades

Signs of Menarche at Never had Menarche at NA hypogonadism 15yrs menopause menstrual cycles 12yrs menopause at 16 yrs at 15 yrs

Other signs Urinary Short stature Short stature Epilepsy at age Incontinence of 7 months

Dysphagia to Short stature liquids (mild)

Short stature

Disability Score 5 5 5 6

MRI of the brain Global cerebellar Global cerebellar Global cerebellar Global cerebellar atrophy atrophy atrophy atrophy

Electrophysiological ND ND ND ND Studies (NCS)

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3.2.1.4. Genotyping approach  Family F26 Because of the specific association of pyramidal signs with AD cerebellar ataxia and macular degeneration [Table (3-7)], the DNA of the proband of this family was sent to the diagnostic unit in the hospital (Dr. Cécile Cazeneuve, UF de neurogénétique, APHP) for expansions in known AD cerebellar ataxias (SCA1, SCA2, SCA3, SCA6, SCA7, SCA17 and DRPLA) using a PCR-based approach followed by genotyping to determine the number of CAG repeats carried by each genes. A deleterious allele with 60 +/- 3 CAG trinucleotide repeats in a heterozygous state in the ATXN7 gene was found [Figure (3-9)]. Clear anticipation was observed with the age at onset 18.5 years younger in the second generation (son and daughter) than the first generation (mother) and the disease showing more severe and rapidly progressive course in the kids [Figure (3-9)]. The anticipation is well known in SCA7 although it was more linked to paternal transmission (Benomar et al., 1994). The clinical presentation, summarized as a combination of AD ataxia, pyramidal signs, extrapyramidal features and macular degeneration [Table (3-7)], matched what was reported in the literature about SCA7. SCA7 was referred as progressive cerebellar ataxia with pigmentary macular degeneration (type II autosomal dominant cerebellar ataxia (ADCA) in the classification of Harding in 1983. It was found to be due to unstable CAG repeat in the gene ATXN7 (David et al., 1997).

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Figure (3-9): Pedigree and colour image of the retina of the index patient of family F26 caused by pathogenic CAG repeat in ATXN7 family presenting with pyramidal signs associated with spinocerebellar ataxia type 7 (SCA7). Colour image of the retina shows macular degeneration and pale optic disc. Pedigree symbols: * sampled individual; Phenotype symbols: half-fill black color: affected individuals; Others are standard medical pedigree symbols.

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Table (3-7): Clinical data of three patients from family F26 with spinocerebellar ataxia (SCA7) due to a pathologic CAG repeat in ATXN7. UL: upper limbs. LL: lower limbs. PUL/PLL: proximal upper/lower limbs. DUL/DLL: distal upper/lower limbs. Cog: cognitive. Psych: psychological. Clinical signs severity: – Absent, + Mild, ++ Moderate, +++ Severe. Tendon Reflexes: ↑Increased Tendon Reflex. Extensor Planter Response: ↑ Unilateral, ↑↑ Bilateral, ↔ Mute, ↓ Flexor. N: Normal. NA: Not Applicable due to Patients Condition.

Family Code F26 F26 F26

Mutated Gene SCA7

Individual Code 216 218 220

Gender F F M

Clinical Diagnosis Complicated HA

Origin Khartoum - Alilfoon

Age at Onset of Motor 34 yrs 17Yrs 14 Yrs Symptoms

Age at Initial 49 Yrs 26 Yrs 17 Yrs Examination

Spasticity ++/++ -/- -/+

UL/LL

Motor Deficit +/+ -/- -/- PUL/DUL

Motor Deficit +/+ -/- -/- PLL/DLL

Tendon Reflexes ↑/↑ ↑/↑ ↑/↑ UL/LL Patellar

Ankle Reflex/ Planter ↑/↔ N /↔ ↑/↓ Response

Ataxia Eye/UL/LL/ NA +/ /-/+ +/+++/+++/+++ Gait (blind)/+++/+++/NA

Dysarthria ++/++ +/+ -/++ Spastic/Cerebellar

Muscle Atrophy ++/++ -/- ++/++ DUL/DLL

Facial Atrophy - - -

104

Family Code F26 F26 F26

Individual Code 216 218 220

Cog/ Psych signs +/- -/- +/-

Optic Atrophy + + -

Extrapyramidal Signs Dystonia, - Marked bradykinesia Hypokinesia

Sensory Loss - - -

Other Signs and Eye: optic atrophy. Eye: optic Eye summary: optic specifications Ankle clonus, atrophy and atrophy, macular urinary incontinence macular degeneration, degeneration. /urgency diminished visual Mild pes cavus, mild hearing acuity, slow

impairment, saccades, mild Urinary horizontal & vertical incontinence ophthalmoplegia

(limitation) Myoclonus

Disability Score 7 3 3

Brain MRI ND Mild diffuse ND brain atrophy (cortical, cerebellar)

Eye colour Image ND Pale optic disc Pale optic disc with

with vessel vessel attenuation, attenuation. salt and pepper Maccular appearance of the degeneration. retina wity thinning (Retinitis pigmentosa sine pigmentosa), macular degeneration.

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3.2.1.5. Comment In 12 families the genetic cause was found to be a gene previously reported to cause spastic syndrome. Three of them were found through the classical candidate gene approach. These included two families (F1 and F6) with SPG11 (Elsayed et al, in revision) and one family (F26/SCA7) through the analysis of pathological expansions in spinocerebellar ataxia genes (part 3.2.1.4). The remaining nine were identified using an NGS-based method (gene panel or WES) (Elsayed et al, 2016; Elsayed et al, under revision; unpublished data). The success rate for families with classical HSP (Elsayed et al., in revision) when using NGS-based diagnostic kits was 22% (5 out of 23 families) [Figure (3-10)]. The use of combined approaches for screening of known genes in families with classical HSP presentation (candidate gene approach and linkage analysis) gave us a higher diagnostic power of around 30% (7/23) [Figure (3-11)].

Screening panel success rate; 22%

Panel diagnostic Failure ; 78%

Figure (3-10): Screening Panel success rate in classical HSP (23 families): numbers and percentages are based on families .

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Table (3-8): Summary of mutations in known genes found in three families with pyramidal features. N.S.: Nonsense, M.S: Missense, Hom: homozygous, Het: heterozygous, Deleter: Deleterious, Prob Damag: Probably Damaging, NA: Not Applicable. NF not found. PhastCons score ranges from zero (low conservation) to 1 (high conservation), Phylop score from -14.1 (low conservation) to 6.4 (high conservation).

Predicted Change Predicted Conservation Predicted Pathogenicity MAF Novelty/Reference

Protein Change Protein Browser EXAC

Consequence

Family Code Family

Exon/Intron Exon/Intron

Involved

Zygosity

Align GVGD Align

Gene

PhastCons

Polyphen2

Mutation Mutation

Genomes

PhyloP

dbSNP

Taster

SIFT

1000

EVS

F15 ARG1 c.434T>A p.(Val145Glu ) Hom 1.0 4.64 Causing C35 NF NF NF NF

Deleter.

Damag.

Disease Disease

Exon 4 Exon

Novel

Prob. Prob.

M.S.

site Intron 10 siteIntron F25 PLA2G6 Splice Donor c.1427+2T> Skip of exon Hom 1.0 2.38 NA NA NA NA NF NF NF NF

C 10 is very Novel likely S.D

NMD possible

F30 SIL1 c.740C>T p.(Ala247Val) Hom 0.99 4.64 C0 NF NF NF Heterozygous

Tolerated

0.00082%

Databases

Causing

Damag.

Disease Disease

Exon 8 Exon

Prob. Prob. M.S.

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3.3. Identification of new causative HSP Genes

Ten families previously excluded through gene panel explorations were subjected to WES in search for new potential genes. These families were selected on the basis of their informativity (available full clinical data, multiple sampled patients to allow segregation analysis). In families where the pedigree suggested AR inheritance WES was performed in one patient only per family or family branch in case of extended families. In families where AD inheritance was suggested WES was done in two patients at least. There were rare conditions in which we chose to perform WES to more than one individual per branch.

3.3.1. ABHD16A mutation in family F37 3.3.1.1. Case report The affected individuals were two boys (314, 315) who were born to first degree consanguineous parents after an uneventful pregnancy and delivery. The two sibs showed homogeneous disease progression. A little after birth, the family observed some delay in their psychomotor development followed by significant regression of the poorly acquired milestones at around 3-4 years. They were not floppy at birth or at their early months. They had delayed acquisition of minimal speech skill. They started to walk late unaided but limited. When they were at 3-4 years of age they started to show regression of the limited walking and poor speaking skills they acquired where they reverted to crawling (314) and aided walking (315). At around 7-10 years of age they became both wheel chair bound. They acquired sphincter control at 2 and 3 years of age (younger and older sib, respectively). They did not develop sphincter disturbance with the disease progression.

Table (3-9): Summary of mutations in putative candidate new genes found in five families with spastic neurogenetic disorder. N.S.: Nonsense, M.S: Missense, Hom: homozygous, Het: heterozygous, Deleter: Deleterious, Prob Damag: Probably Damaging, NA: Not Applicable. NF not found. PhastCons score ranges from zero (low conservation) to 1 (high conservation), Phylop score from -14.1 (low conservation) to 6.4 (high conservation).

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Conservation Predicted Pathogenicity MAF

Protein Change Protein EXAC

c DNA Change DNA c

Exon Involved Exon

Consequence

Family Code Family

Zygosity

Novelty

Align GVGD Align

Freq./Count

Gene

PhastCons

Polyphen2

Mutation Mutation

Genomes

(AA/EA)

Browser

PhyloP

dbSNP

Taster

SIFT

1000

EVS

5 c.340C>T p.(Arg114*) Hom 1.0 2.71

ABHD16A

Novel

N.S.

F37

NA NA NA NA

NF NF NF NF

12 c.1264C>T p.(Arg422Trp) Hom 0.94 1.42 Causing Disease

Prob. Damag. Prob.

Heterozygous

0.03/0.08%

CAMSAP3

Databases

<0.001/0

Deleter.

0.098%

Benign

M.S.

F41

C 0 0 C

F41 12 c.1207G>C p.(Glu403Gln) Hom 3.35

Tolerated

Causing

Causing Causing

MINK1

Disease Disease Disease

Benign Benign

Novel

M.S.

C 0 0 C

NF NF NF NF

F41 ZNF433 4 c.707G>A p.(Ser236Asn) Hom 1.01

Poss.. Damag. Poss..

Tolerated

Benign

Novel

M.S.

C 0 0 C

NF NF NF NF

109

Gene

Freq./Count

Consequenc

PhastCons

Polyphen2

Mutation Mutation

Genomes

(AA/EA)

Involved

Zygosity

Browser

Change Change Novelty

Protein Protein

PhyloP

Family Family GVGD dbSNP

c DNA c

EXAC

Taster

Align Align

Code Exon SIFT

1000

EVS

4 c.327C>G p.(Phe109Leu) Hom 1.0 2.06

Causing

Deleter.

Damag.

Disease Disease

Benign

BIRC5

Novel

Prob. Prob.

M.S.

F50

C 0 0 C NF NF NF NF

3 c.430G>C p.(Ala144Pro) Hom. 2.63 % 0.06295

C21ORF91

Databases

Causing

Disease Disease

Deleter

0.002/0

Benign Benign

M.S.

F50

C 0 0 C

0.095 % 0.095

Heterozygou

1 c.202G>T p.(Ala68Ser) Hom 1.0 2.95 Tolerated

Databases

Causing

Disease Disease

0.002/0

Benign

ST7L

FM3

M.S.

C 0 0 C

NF NF

Heterozygou

0.00/0.01%

15 c.1798G>A p.(Val600Met) Hom 0.99 4.73 % 0.00083

Databases

Causing

Damag.

PANK4

Disease Disease

Deleter

Benign

Prob. Prob.

FM7 FM7

M.S.

C 0 0 C

NF NF

110

3.3.1.2. Last clinical examination: LL examination could not be performed for the older sib (314) because of post- corrective surgery for ankle deformity. The younger sib (315) had talipes equinovarus more pronounced on the left leg. Both brothers had spastic paraplegia presenting with severe bilateral spasticity, hyperreflexia, extensor plantar response and motor deficit in the LL (distal more than proximal). Extrapyramidal signs in form of significant choreoathetosis were detected. They were both mentally retarded. They had psychiatric signs in form of irritability, distractability, bouts of crying and the ease of getting upset and frightened. Sterotypic behavior were marked and included clapping and head slapping. LL wasting both peripherally and distally was a sign of lower motor neuron pathology [Table (3-10)].

3.3.1.3. Clinical Summary: 1- Complicated HSP (TCC HSP) 2- Age at onset of two sibs: within the 1st year of life. 3- Delayed psychomotor development followed by further regression of milestones 4- Pyramidal signs (LL: spasticity, hyperreflexia and bilateral extensor plantar response) 5- Mental retardation 6- Sterotypic behavior (clapping and head slapping) 7- Extrapyramidal signs (choreoathetotic movement) 8- Psychiatric symptoms (distractability, irritability, bouts of crying, ease of upset and frightened attitude) 9- Brain MRI: TCC, subcortical and periventricular WMH signals.

3.3.1.4. Gene identification Genetic exploration using WES in patient (314), focusing on rare/novel gene with direct effect on the protein/mRNA, identified 13 rare variants at the homozygous state . Only one segregated with the disease in the family, a nonsense mutation in the ABHD16A gene: [Exon 5: g.31668722G>A, c.340C>T, p.Arg114*].

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The mutation was found to completely co-segregate with the disease distribution where it was found in a homozygous state in the two affected siblings. The parents were confirmed to be obligate heterozygous carriers [Figure (3-12)].

This mutation was predicted to result in nonsense mediated decay (NMD) of the mRNA and absence of the protein with complete loss of function in the homozygous state [Table (3-9)].

 Functional interest of the gene ABHD16A (also known as Human lymphocyte antigen B-associated transcript 5 (BAT5))is located on the short arm of chromosome 6 (6p21.33) [Ensembl: Chromosome 6: 31,686,949-31,703,444 (GRCh38); Chromosome 6: 31,654,726- 31,671,221 (GRCh37)].

ABHD16A (Abhydrolase domain containing 16A) has recently been shown to be the main Phosphatidyl serine lipase in the brain and constitutes the second protein in the ABHD12/ABHD16 axis (Kamat et al, 2015).

ABHD16A is correlated in STRING network with three of the proteins that are included in the ataxia interactome (SAFB, PTGS2, and RNF5) which can be considered as an added pointer high probability of its involvement in a neurodegenerative disorder (Lim J et al., 2006).

112

Figure (3-12): Pedigree and MRI of the index patient of family F37 with a nonsense mutation in ABHD16A segregating with the disease distribution in the whole family. MRI shows thin corpus callosum (TCC). Example sanger sequencing of the mutation in patient (homozygous mutant) and in a healthy heterozygous carrier. Pedigree symbols: * sampled individual; Phenotype symbols: half-fill black color: affected individuals; Genotype symbols: M+ Heterozygous genotype; MM Homozygous mutant genotype. Others are standard medical pedigree symbols.

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Table (3-10): Clinical data of two patients from family F37 with a nonsense mutation in ABHD16A. UL: upper limbs. LL: lower limbs. PUL/PLL: proximal upper/lower limbs. DUL/DLL: distal upper/lower limbs. Cog: cognitive. Psych: psychological. Clinical signs severity: – Absent, + Mild, ++ Moderate, +++ Severe. Tendon Reflexes: ↑Increased Tendon Reflex. Extensor Planter Response: ↑ Unilateral, ↑↑ Bilateral, ↔ Mute, ↓ Flexor. N: Normal. NA: Not Applicable due to Patients Condition. PVWMH: periventricular white matter hyperintensities.

Family Code F37 F37

Mutated Gene ABHD16A

Individual Code 314 315

Gender M M

Clinical Diagnosis HSP with TCC

Origin Kosti/ Sudan

Age at Onset of Motor Symptoms 6 months 6 months

Age at Initial Examination 10 years 7 years

Spasticity UL/LL -/+++ -/+++

Motor Deficit PUL/DUL -/- -/-

Motor Deficit PLL/DLL -/NA -/+++

Tendon Reflexes UL/LLPatellar N/↑ N/↑

Ankle Reflex/ Planter Response NA/NA N/↑↑

Ataxia Eye/UL/LL/ Gait -/-/-/- -/-/-/-

Dysarthria Spastic/Cerebellar -/- -/-

Muscle Atrophy DUL/DLL - /- - / ++

Facial Atrophy - -

MR/ Psych signs +/+ +/ +

Optic Atrophy NA NA

Family Code F37 F37

Individual Code 314 315

Extrapyramidal Signs + Choreoathetosis + Choreoathetosis

Sensory Loss NA NA

Other Signs Sterotypic Sterotypic behavior :

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behavior :head slapping clapping

Disability Score 6 6

MRI of the brain TCC , Subcortical and TCC , Subcortical PVWMHS and PVWMHS

Electrophysiological Studies (NCS) ND ND

3.3.2. Candidate genes in family in F41 3.3.2.1.Case report Three Sudanese sibs (one boy and two girls) were born to first degree consanguineous parents. They were born after uneventful pregnancy and delivery. Since birth they were noted to be abnormally developing. They were hypotonic at first and the parents noted delayed development. The proband (349) sat without support and sustained head control at 8 months. He could stand up at 2 years and two months. He had delayed speech; he said two words at 1.5 years then few more words at 3 years but till now no full sentence. He was not dry by day and sustained anal control till recently and he has still nocturia. Anal sphincter was well controlled. His visual acuity diminished since the age of 7 months and at 2 years he developed bilateral cataract and was operated. He had been diagnosed with absence epilepsy (age not specified). Just after two years the parents noted that he started to develop imbalance and then progressively more motor symptoms started to occur. They noted that his neck fell back as if the patient lost neck control. At 10 years they noted that he developed some tremors when reached for an object. The eldest and youngest sisters had basically the same natural history of the disease but cataract developed earlier at 7 months and 1 year 8 months, respectively. They had both excessive bouts of crying at <2 months and 4 months, respectively. While the youngest sib did not have any type of seizures the oldest had history of febrile convulsions once at the age of 4 years.

3.3.2.2. Last clinical examination: The index case had dysmorphic features including elongated face and malocclusion of mouth. Some pigmented spots were noted in the trunk (“café au lait”) as well as some pustules which were described as repetitive by the parents. Skeletal deformities were found including scoliosis and enhanced lumber lordosis.

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Lower motor neuron features were found (trunkal hypotonia and marked UL and LL wasting both proximally and distally). He had pyramidal signs (LL predominantly with spasticity and hyperreflexia but flexor plantar response) and features of cerebellar dysfunction in UL, wide based gait and eye (saccadic pursuit). Extrapyramidal signs in form of significant choreoathetosis and dystonia (retrocolis) as well as titubation of the whole upper half of the body and pill rolling (like) tremor were detected. There was cognitive impairment and psychiatric signs as irritability and the ease of getting upset.

Only the youngest sister was available for examination. She was irritable and had bouts of continuous crying. She had the same trunkal “café au lait” but no pustules. She had left eye divergent squint. Lumber lordosis was also found. She had some dysmorphic features including minor prominent upper and lower jaws and some milder malocclusion of the mouth. She had trunkal hypotonia but the tone of the limbs was normal. There was UL and LL hyperreflexia with flexor plantar responses. Mild cerebellar ataxia of the UL was observed on reaching for objects [Table (3-11)].

3.3.2.3. Clinical Summary:

1- Complicated spastic ataxia 2- Age at onset: birth 3- Trunkal hypotonia and UL / LL wasting. 4- Delayed psychomotor development 5- Pyramidal signs (vary between patients): spasticity, hyperreflexia 6- Cerebellar signs: UL, Gait, trunk and eyes 7- Congenital cataract 8- Dysmorphic features 9- « Café au lait » pigmented spots and pustules 10- Extrapyramidal signs: choreoathetosis, dystonia (retrocolis), titubation and resting tremors 11- Skeletal deformities: scoliosis, lordosis 12- Cognitive impairment 13- Psychiatric symptoms: irritability and excessive crying 14- Sphincter disturbance

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15- Brain MRI : cerebellar atrophy

3.3.2.4. Genetic exploration: WES analysis identified 3 rare variants at the homozygous state that segregated with the disease in genes MINK1, ZNF433 and CAMSAP3.

1. CAMSAP3 The most convincing variant was a known missense variant rs180741228 [exon 12: g.7676462C>T, c.1264C>T, p.Arg422Trp] (based on the longest transcript (NM_001080429.2)) in CAMSAP3 (OMIM *612685). CAMSAP3 is located on the short arm of chromosome 19 (19p13.2) [Ensembl: Chromosome 19:7,595,902-7,618,304 (GRCh38); Chromosome 19:7,660,788- 7,683,190(GRCh37)] and stands for calmodulin regulated spectrin-associated protein family, member 3.

The variant rs180741228 passed the GATK filter with high confidence and was covered at a depth of 41x. The variant had an ExAC frequency of 0.09773% and was present only at the heterozygous state. The variant fully co-segregated with the disease distribution in family F41 and was homozygous in the two sampled affected siblings whereas other five healthy related individuals were heterozygous carriers or homozygous reference as in controls [Figure (3-13)].

The mutation resulted in a change from the basic amino acid arginine to the hydrophobic amino acid tryptophan (moderate physicochemical difference [Grantham distance of 101 (0-215)]). Arginine is moderately conserved considering 18 species, but conserved in mammals.

The change was predicted as pathogenic by three different algorithms (SIFT, Polyphen2 and Mutation taster) [Table (3-9)].

The gene extreme intolerance to missense and loss of function (LOF) mutations is suggested by the ExAC z score for missense mutations (4.29) and ExAC pLI for LOF mutations (1.0) (0-1; 1 = the maximum intolerant) and by RVIS score (another way of calculating genic intolerance) of 0.1% [ratio -1.13]: the gene is ranked among the 6.57% most intolerant genes.

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Moreover, homozygous variants are rather rare in this gene based on the ExAC browser database with only 16 homozygous variants in 60,706 WES (with the exception of the quite frequent polymorphism (rs3745358)

Total number of observed missense mutations is low (306) compared to the expected (502.8). The number of the observed stops/loss of function is only 1 (expected is 31.9).

A good supporting evidence for the pathogenicity of CAMSAP3 is its extreme genic intolerane as expected in rare neurodevelopmental disorders and its interactions with many genes in the two neurodegenerative networks (HSP network: TUBB4A, GSK3B KIF3C (Novarino et al., 2014) and ataxia interactome: GSK3B, PPP1CA, MAGOH, SSSCA1 and NCF2 (Lim J et al., 2006)

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Figure (3-13): Pedigree of family F41 showing segregation of missense mutation in CAMSAP3 with the disease distribution in the whole family. Example sanger sequencing of the mutation in patient (homozygous mutant), a heterozygous carrier and a control homozygous reference allele with conserved amino acid sequence, is shown. Pedigree symbols: * sampled individual; Phenotype symbols: half-fill black color: affected individuals; Genotype symbols: ++ Homozygous reference genotype; M+ Heterozygous genotype; MM Homozygous mutant genotype. Others are standard medical pedigree symbols.

2. MINK1 A novel mutation in exon 12 of the gene MINK1 [g.4791062G>C, c.1207G>C, p.Glu403Gln] (based on the longest transcript NM_153827.4) cosegregated with the disease distribution in family F41. MINK1 (misshapen- like kinase 1) is also known as MAP4K6,GN MINK, YSK2, hMINK, ZC3, MINK, MAP4K6, B55 and hMINKbeta.

MINK1 is located on the short arm of chromosome 17 (17p13.2) [Ensembl: 17: 4,833,388-4,898,061 (GRCh38); 17: 4,736,683-4,801,356 (GRCh37)].

The variant completely co-segregated with the disease distribution where it was homozygous in the affected sibs and heterozygous carriers or homozygous reference in three healthy related individuals [Figure (3-14)].

The variant passed the GATK filter with high confidence and coverage at depth of 24X. The mutation of this moderately conserved nucleotide [PhastCons : 1.0, (phyloP: 3.35 (-14.1;6.4)] was predicted pathogenic by MutationTaster which designated it as disease causing (p-value: 1) [Table (3- 9)]. There is a moderate physicochemical difference [Grantham distance of 29 (0-215)] between both hydrophilic amino acid glutamate and glutamine. The amino acid is a highly conserved amino acid in all mammals and vertebrates up to the Tetraodon nigroviridis.

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Figure (3-14): Pedigree of family F41 showing segregation of missense mutation in MINK1 with the disease distribution in the whole family. Example sanger sequencing of the mutation in patient (homozygous mutant), a heterozygous carrier and a control homozygous reference allele with conserved amino acid sequence, is shown. Pedigree symbols: * sampled individual; Phenotype symbols: half-fill black color: affected individuals; Genotype symbols: ++ Homozygous reference genotype; M+ Heterozygous genotype; MM Homozygous mutant genotype. Others are standard medical pedigree symbols.

3. ZNF433 A mutation in exon 4 of the gene ZNF433 [g.12126975, c.707G>A, p.Ser236Asn] (based on the longest transcript NM_001080411.2) cosegregated with the disease distribution in family F41.

ZNF433 (zinc finger protein 433) is also known as FLJ40981. It is located on the short arm of chromosome 19 (19p13.2) [Ensembl: 19: 12,014,732- 12,035,741 (GRCh38); 19: 12,125,547-12,146,556 (GRCh37)].

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The variant passed the GATK filter with high confidence and was covered at a depth of 52x. The variant has an ExAC frequency of 0.00083% only at the heterozygous state. The variant completely co-segregated with the disease distribution where it was homozygous in the two sampled affected sibs and heterozygous carriers or homozygous reference in two healthy related individuals [Figure (3-15)].

The mutation of this weakly conserved nucleotide [PhastCons : 1.0, (phyloP: 1.01 (-14.1;6.4)] was predicted possibly damaging by PolyPhen2 (p-value: 1) [Table (3-9)]. There is a small physicochemical difference [Grantham distance of 46 (0-215)] between both hydrophilic amino acid serine and asparagine. The amino acid is a moderately conserved amino acid in mammals and vertebrates (considering 11 species : not conserved in pigs).

Figure (3-15): Pedigree of family F41 showing segregation of missense mutation in ZNF433 with the disease distribution in the whole family. Example sanger sequencing of the mutation in patient (homozygous mutant), a heterozygous carrier and a control homozygous reference allele with conserved amino

121 acid sequence, is shown. Pedigree symbols: * sampled individual; Phenotype symbols: half-fill black color: affected individuals; Genotype symbols: ++ Homozygous reference genotype; M+ Heterozygous genotype; MM Homozygous mutant genotype. Others are standard medical pedigree symbols Table (3-11): Clinical data of two patients from family F41. UL: upper limbs. LL: lower limbs. PUL/PLL: proximal upper/lower limbs. DUL/DLL: distal upper/lower limbs. Cog: cognitive. Psych: psychological. Clinical signs severity: – Absent, + Mild, ++ Moderate, +++ Severe. Tendon Reflexes: ↑Increased Tendon Reflex. Extensor Planter Response: ↑ Unilateral, ↑↑ Bilateral, ↔ Mute, ↓ Flexor. N: Normal. NA: Not Applicable due to Patients Condition.

Family Code F41 F41

Mutated Gene CAMSAP3 or MINK1 or ZNF433

Individual Code 349 351

Gender M F

Clinical Diagnosis Complex spastic ataxia

Origin White Nile State

Age at Onset of Motor Symptoms Birth Birth

Age at development of cataract 2 Yrs 20 months

Age at Initial Examination 11 yrs 1 month 20 months

Spasticity +/++ -/-

UL/LL

Motor Deficit PUL/DUL ++/+ -/-

Motor Deficit PLL/DLL ++/+ -/-

Tendon Reflexes UL/LL Patellar N/↑ ↑/↑

Ankle Reflex/ Planter Response ↑/↓ ↑/↓

Ataxia Eye/UL/LL/ Gait + sacc.pur/-/-/+ N/-/-/NA

Dysarthria Spastic/Cerebellar -/- NA

Muscle Atrophy DUL/DLL +++/+++ -/-

Facial Atrophy + -

Cog/ Psych signs +/+ NA/+

Optic Atrophy - -

Extrapyramidal Tetubation & Resting Tremor -

Signs Choreoathetosis Neck dystonia 122 (retrocolis) Family Code F41 F41

LMN signs Trunkal hypotonia Moderate Trunkal hypotonia UL/LL muscle wasting (distal and proximal)

Sensory Loss - -

Dysmorphic features Elongated face Malocclusion of Mild Protrusion of upper and mouth lower jaws

Malocclusion of the mouth

Skin Café au lait spots Pustules Café au lait spots

Skeletal deformities Exaggerated lumber lordosis , Exaggerated lumber lordosis Scoliosis

Other Signs Scoliosis Lt divergent squint

Nocturia

Disability Score 5 3

MRI of the brain Cerebellar atrophy Cerebellar atrophy

Electrophysiological Studies (NCS) ND ND

3.3.3. Exploration of family F50 3.3.3.1.Case report: The affected individuals were two girls (402, 403) who were born to first degree consanguineous parents after an uneventful pregnancy and delivery. They looked healthy for few months after birth. The disease started with epilepsy. The index (older, 402) had earlier onset at 7 months of age whereas the younger had it at 2 years. They had myoclonic epilepsy with myoclonus occurring mainly when awake. Delayed psychomotor development was observed by the family. This was evident in the delayed social smile, speech, apparent decreased intellect and gross motor development. The younger sib (403) showed regression of speech and she became aphasic after she could say a few words. They couldn’t walk until the age of 4-5 years. They acquired sphincter control just around the age of examination (4-5 years). The parents noticed that the index developed cerebellar symptoms (eg.

123 imbalance) when she was 3 years whereas the younger had predominant cognitive impairment and psychological symptoms at the same age.

3.3.3.2.Last clinical examination: On assessment of the patients they both had convergent squint. They were both wheel-chair bound. The older had talipes equina varus. They were tetraspastic with hyperreflexia. The plantar response was extensor in the younger sib. The motor deficit in both patients was mild and did not match the spasticity in severity. Unfortunately the cerebellar function could not be assessed properly in the younger sib but signs of cerebellar ataxia were found in the older girl who had ataxia in UL, LL, at gait and in the eyes (saccadic pursuit). The cerebellar ataxia was less prominent than the pyramidal features. Horizontal ophthalmoplegia was found in the two sibs but the younger (403) had oculomotor apraxia in addition. The younger sib had marked mental retardation, drooling of saliva and significant psychiatric signs which include irritability, emotional lability and persistent crying. On the contrary, the older showed only minor mental subnormality and blunt mood. None of them had signs of extrapyramidal or lower motor neuron lesions [Table (3-12)].

3.3.3.3.Clinical Summary: 1- Complicated HSP 2- Age at onset of two sibs: 7 months and two years 3- Pyramidal signs: Tetraspastic, hyperreflexia 4- Cerebellar ataxia: one patient showed UL, LL, speech, gait and eye (saccadic pursuit) 5- Delayed psychomotor development 6- Mental retardation 7- Psychiatric symptoms as irritability, emotional lability, blunt mood and persistent crying 8- Myoclonic epilepsy 9- Occulomotor apraxia 10- Horizontal ophthalmoplegia (convergent squint) 11- Drooling of saliva 12- MRI of the brain: Cerebellar atrophy, hypomyelination, TCC and mild prominence of internal and external CSF space.

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3.3.3.4.Genetic exploration: WES filtering in patient (402) identified ten variants at the homozygous state and predicted deleterious. Only two variants from two genes (BIRC5 and C21ORF91) co-segregated with the disease distribution.

1. BIRC5 The most convincing one was a missense transversion mutation in BIRC5 [exon 4: g. 76212781C>G, c.327C>G, p.Phe109Leu] (based on the longest transcript: NM_001012271.1); [exon 4: c.258C>G, p.Phe86Leu] in the transcript (NM_001168.2) which codes for the canonical isoform 1 of the survivin protein).

The gene BIRC5 or baculoviral (IAP) repeat containing 5 (OMIM *603352) encodes for the apoptosis inhibitor 4 (API4), survivin and EPR-1.

BIRC5 is located on the long arm of chromosome 17 (17q25.3) [Ensembl: 17: 78,214,186-78,225,636 (GRCh38) 76,210,267-76,221,717(GRCh37)].

The variant had passed the GATK filter with high confidence and good coverage with 79x depth). Co-segregation with the disease distribution in family F50 was confirmed where it was homozygous in the two sampled affected siblings whereas both parents were heterozygous carriers [Figure (3- 16)].

The mutation resulted in a change from phenylalanine to leucine. The two are branched hydrophobic amino acids. There is a small physicochemical difference [Grantham distance is 22 (0-215)] but leucine lacks the aromatic ring. The amino acid is located in the core of the zinc finger of the conserved baculoviral inhibition of apoptosis protein (IAP) repeat domain. This change was predicted pathogenic by SIFT, Polyphen2 and Mutation taster [Table (3- 9)].

The concerned nucleotide has a PhastCons conservation score of 1.00 (0-1) and a PhyloP score of 2.06 (-14.1-6.4)]. The amino acid is highly conserved in 11 species up to C.elegans.

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BIRC5 is intolerant to missense (ExAC z score = 0.62) and LOF muatations (ExAC pLI = 0.22) (0-1; 1 = the maximum intolerant).

Homozygous variants are rare (12 homozygous) in 60,706 WES in ExAC browser (With the exception of the frequent polymorphism rs2071214). The total number of missense mutations is only 55 (expected 65.2) and Stops (loss of function/LOF) is 2 (expected 6.8).

BIRC5 has connections constructed in STRING software with TUBG1 from the HSP network and with NCOR1, BCL10, ITGB1, CASP9, RANGAP1, CASP7 and DIABLO. Both networks would increase the likelihood of the gene to have a role in related neurodegenerative disorders.

2. C21ORF91 A known but rare variant rs36095555 in exon 3 of the gene C21ORF91 [g. 19169133C>G, c.430G>C, p.Ala144Pro] (based on the longest transcript NM_001100420.1) cosegregated with the disease distribution in the family [Figure (3-16)].

C21ORF91 (chromosome 21 open reading frame 91) is also known as EURL, C21orf14, CSSG1, YG81, C21orf38. It is located on the long arm of chromosome 21 (21q21.1) [Ensembl: 21: 17,788,967-17,819,386 (GRCh38); 21: 19,161,284-19,191,703 (GRCh37)].

The rs36095555 had an ExAC frequency of 0.0629 but existed only at the heterozygous state. The variant completely co-segregated with the disease distribution where it was homozygous in the two sampled affected siblings whereas both parents were heterozygous carriers [Figure (3-16)].

The variant passed the GATK filter with high confidence and coverage at depth of 61X. The mutation of this moderately conserved nucleotide [PhastCons : 1.0, (phyloP: 2.63 (-14.1;6.4)] was predicted pathogenic by both SIFT (deleterious; Score :0) and MutationTaster which designated it as disease causing (p-value: 1) [Table (3-9)]. There is a small physicochemical difference [Grantham distance of 27 (0-215)] between both hydrophilic amino acid alanine and proline. The amino acid is a highly conserved amino acid considering 12 species up to the frog.

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C21ORF91 is relatively tolerant to missense (ExAC z score = 0.19) and LOF mutations (ExAC pLI = 0.002) (0-1; 1 = the maximum intolerant).

In 60,706 WES in ExAC browser, the total number of observed missense mutations is more than the expected (82 observed variants compared to 78.6 expected).

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Figure (3-16): Pedigree and MRI of two patients of family F50 showing missense mutations in two putative candidate genes BIRC5 and C21ORF91 segregating with the disease distribution in whole family. MRI of patient 402 shows cerebellar atrophy and hypomylination. MRI of patient 403 (lower two images) shows hypomyelination, thin corpus callosum (TCC) and mild prominence of internal and external CSF space. Example sanger sequencing of the BIRC5 mutation in patient (homozygous mutant), a heterozygous carrier and a control homozygous reference allele with conserved amino acid sequence, is shown. Pedigree symbols: * sampled individual; Phenotype symbols: half-fill black color: affected individuals; Genotype symbols: ++ Homozygous reference genotype; M+ Heterozygous genotype; MM Homozygous mutant genotype. Others are standard medical pedigree symbols.

Table (3-12): Clinical data of two patients from family F50. UL: upper limbs. LL: lower limbs. PUL/PLL: proximal upper/lower limbs. DUL/DLL: distal upper/lower limbs. Cog: cognitive. Psych: psychological. Clinical signs severity: – Absent, + Mild, ++ Moderate, +++ Severe. Tendon Reflexes: ↑Increased Tendon Reflex. Extensor Planter Response: ↑ Unilateral, ↑↑ Bilateral, ↔ Mute, ↓ Flexor. N: Normal. NA: Not Applicable due to Patients Condition. TCC: thin corpus allosum. CSF: cerebrospinal fluid.

Family Code F50 F50

Mutated Gene BIRC5 or C21orf91

Individual Code 402 403

Gender F F

Clinical Diagnosis Complicated HSP

Origin Neyala, Western sudan

Age at Onset 7 months 2 Yrs

Age at Initial Examination 5 Yrs 4 Yrs

Spasticity ++/ +++/+++

UL/LL ++ (Rt)+++ (Lt)

Motor Deficit PUL/DUL +/+ -/-

Motor Deficit PLL/DLL +/+ -/-

Tendon Reflexes UL/LLPatellar ↑/↑ ↑/↑

Ankle Reflex/ Planter Response ↑/↓ NA/ ↑↑

Ataxia Eye/UL/LL/ Gait +(saccadic pursuit)/++/+/++ -/NA/NA/NA

Dysarthria Spastic/Cerebellar NA/NA NA/NA

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Muscle Atrophy DUL/DLL -/- -/-

Facial Atrophy - -

Cog/ Psych signs +/+ +/+

Optic Atrophy NA NA

Extrapyramidal - -

Signs

Sensory Loss NA NA

Other Signs Myoclonic epilepsy Myoclonic epilepsy

Horizontal ophthalmoplgia Bilateral Talipus Equina Varus (convergent squint) Occulomotor apraxia Horizontal ophthalmoplegia (convergent squint)

Disability Score 6 6

MRI of the brain Cerebellar atrophy and Hypomyelination, TCC and hypomyelination mild prominence of internal and external CSF space for age

Electrophysiological Studies (NCS) ND ND

3.3.4. ST7L mutation in family FM3 3.3.4.1.Case report: The affected individuals were three sibs (2020, 2021, 2022) presented to the clinic at the ages of 5 years, 4 years 4 months and 2 yrs 3 months. They were born to first degree consanguineous parents. The two girls (2021, 2022) were born after an uneventful pregnancy and delivery but for the boy (2020) the mother noted decreased fetal movement at the end of the third trimester, which implied the possibility of prenatal onset. The two affected girls (2021 and 2022) looked healthy for few months after birth. Over all the three sibs showed homogenous disease course. The parents then noted delayed psychomotor development of their milestones as all kids could not sit till the age of presentation to the clinic and could only turn in bed. They also observed abnormal movement in all three kids. Speech skill was not acquired and there was also obvious poverty of movement. Sphincter control was acquired normally. There was evidence of mental

130 retardation and psychological symptoms in form of blunt mood. There was no evidence of dysmorphism. 3.3.4.2.Last clinical examination: On assessment of the patients they were aphasic. They had predominant pyramidal signs (UL and LL spasticity, hyperreflexia and bilateral extensor plantar response). All three had skeletal deformity in form of pes cavus and mild scoliosis. They had generalized sever dystonia and bradykinesia. There was no sign of cerebellar syndrome. Provisional ophthalmoscopy showed bilateral optic atrophy in all three patients but this was not reassessed by the ophthalmologist. They had marked mental retardation, drooling of saliva and they had blunt mood and lack of interaction with others [Table (3-13)].

3.3.4.3.Clinical Summary:

1- Complicated HSP with Dystonia. 2- Age at onset of three sibs: prenatal (end of last trimester: boy), 4 month (two girls) 3- Delayed psychomotor development 4- Pyramidal signs: UL and LL spasticity, hyperreflexia and extensor plantar response 5- Extrapyramidal signs: generalized severe dystonia, bradykinesia and poverty of movement 6- Mental retardation 7- Skeletal deformities: pes cavus and kyphoscoliosis 8- Drooling of saliva 9- MRI of the brain: mild increase of internal CSF spaces (mild subcortical atrophy) and PVWMH

3.3.4.4.Genetic exploration: There was 15 homozygous rare variant identified upon analysis of the WES data of patients (2020, 2022). Only one known missense variant rs116335992 (ExAC frequency of 0.09498) in exon 1 of the gene ST7L [g.113161534C>A, c.202G>T, p.Ala68Ser] (based on the longest transcript NM_017744.4) cosegregated with the

131 disease distribution in the family. ST7L (Suppression Of Tumorigenicity 7 Like) is also known as ST7R, FAM4B, STLR, FLJ20284. ST7L is located on the short arm of chromosome 1 (1p13.2) [Ensembl: 1: 112,523,518-112,620,825 (GRCh38); 1: 113,066,140-113,163,447 (GRCh37)]. The variant completely co-segregated with the disease distribution where it was homozygous in the affected sibs and heterozygous carriers or homozygous reference in four healthy related individuals [Figure (3-17)]. The variant passed the GATK filter with high confidence and coverage (depth of 26X). The mutation of this moderately conserved nucleotide [PhastCons : 1.0, (phyloP: 2.95 (-14.1;6.4)] was predicted pathogenic by MutationTaster which designated it as disease causing (p-value: 1) [Table (3-9)]. There is moderate physicochemical difference [Grantham distance of 99 (0-215)] between the hydrophobic amino acid Alanine that changed to the polar uncharged amino acid Serine. The affected amino acid (located in the protein domain: ST7) is a moderately conserved amino acid (considering 12 species) but highly conserved in mammals [Figure (3-17)].

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Figure (3-17): Pedigree and MRI of the index patient of family FM3 with missense mutation in ST7L segregating with the disease distribution in the whole family. MRI of the brain shows cortical and cerebellar vermial atrophy, periventricular subcortical white matter hyperintensities (WMH). Example sanger sequencing of the mutation in patient (homozygous mutant), a heterozygous carrier and a control homozygous reference allele with conserved amino acid sequence, is shown. Pedigree symbols: * sampled individual; Phenotype symbols: half-fill black color: affected individuals; Genotype symbols: ++ Homozygous reference genotype; M+ Heterozygous genotype; MM Homozygous mutant genotype. Others are standard medical pedigree symbols.

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Table (3-13): Clinical data of three patients from family FM3. UL: upper limbs. LL: lower limbs. PUL/PLL: proximal upper/lower limbs. DUL/DLL: distal upper/lower limbs. Cog: cognitive. Psych: psychological. Clinical signs severity: – Absent, + Mild, ++ Moderate, +++ Severe. Tendon Reflexes: ↑Increased Tendon Reflex. Extensor Planter Response: ↑ Unilateral, ↑↑ Bilateral, ↔ Mute, ↓ Flexor. N: Normal. NA: Not Applicable due to Patients Condition. PVWMH: periventricular white matter hyperintensities. WMH: white matter hyperintensities.

Family Code FM3 FM3 FM3

Mutated Gene ST7L

Individual Code 2022 2020 2021

Gender F M F

Clinical Diagnosis Complicated HSP (Dystonia)

Origin Gezira/ Awamra tribe

Age at Onset of Motor 4 months Before birth 4month Symptoms (decreased movement in late pregnancy)

Age at Initial 2 Yrs 7 Yrs 4 Years Examination

Spasticity +++/+++ +++/+++ +++/+++

UL/LL

Motor Deficit NA NA NA PUL/DUL

Motor Deficit PLL/DLL NA NA NA

Tendon Reflexes UL/LL ↑/↑ ↑/↑ ↑/↑ Patellar

Ankle Reflex/ Planter N/↑↑ ↑/↑↑ ↑/↑↑ Response

Ataxia Eye/UL/LL/ Gait -/-/-/- -/-/-/- +(Slow saccades)/-/- /-

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Dysarthria Aphasic Aphasic Aphasic Spastic/Cerebellar

Muscle Atrophy -/- ++/++ /+ DUL/DLL

Facial Atrophy - - -

MR/ Psych signs +/- +/- +/-

Optic Atrophy - - -

Extrapyramidal Severe generalized Severe generalized Severe generalized dystonia dystonia dystonia Signs

Skeletal deformities Pes cavus Pes cavus Kyphoscoliosis

Sensory Loss NA NA NA

Other Scarce movement Scarce movement Scarce movement

Disability Score 7 7 7

MRI of the brain MRI of the brain: ND MRI of the brain: mild increase of cortical and internal CSF spaces cerebellar vermial (mild subcortical atrophy, PVWMH atrophy?) and and subcortical PVWMH WMH.

Electrophysiological ND Normal ND Studies (NCS)

3.3.5. PANK4 mutation in branch 1 of family FM7 In this family (FM7), there were two branches. Patients presented with a complex neurological disorder that we found different despite the multiple shared points. In branch 1 there were five affected sibs whereas in branch 2 there were two patients (a father and his daughter). The two branches were far related few generations upstream [Figure (3-18)].

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Family FM7/ Branch 1

3.3.5.1.Case report The affected individuals were five sibs (2047, 2048, 2049, 2050, 2051) presented to the clinic at the ages ranging between 13 to 29 years. They were born to consanguineous parents. The parents noted the disease onset since birth : they didn’t cry and were then suspected to be mute. The two had difficult prolonged delivery (2050, 2051) and there was antepartum hemorrhage in late pregnancy of the individual (2051). All other sibs were born after an uneventful pregnancy. With the exception of patient (2048) all individuals were floppy at birth and had delayed psychomotor development. The five sibs had homogenous clinical picture but with marked variability of the disease severity. Two affected boys (2050, 2051) and one girl (2049) had the most aggressive disease manifestations whereas their sister (2048) had the mildest form. The patients developed clumsiness indicating cerebellar involvement at 15 to 18 months. Psychological symptoms were present in a severe form in three of them (2049, 2050, 2051) (nightmares, aggressive attacks, emotional lability) and less prominent in patient (2047, 2048). The two boys (2050, 2051) were diagnosed with epilepsy at the age of 12 years.

3.3.5.2.Last clinical examination:

On assessment of the patients they were all deaf or almost deaf, mute and with apparent mental retardation. In three of them there was microcephaly. Psychiatric signs included remarkable euphoria in four patients (2047, 2049, 2050, 2051) and blunt mood in their sister (2048). All patients had cerebellar ataxia and the patients with microcephaly had additional pyramidal signs (UL and LL spasticity, hyperreflexia) [Table (3-14)].

3.3.5.3.Clinical Summary: 1- Complicated HA/ HSP 2- Age at onset of five sibs: birth 3- Pyramidal signs: spasticity, hyperreflexia but plantar response is mostly mute (flexor in two occasions) 4- Cerebellar ataxia: All sibs (started at age 15-18 months)

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5- Delayed psychomotor development (all sibs except in patient 2048) 6- Deafness/deep hearing impairment and mutism 7- Severe mental retardation in three (2049, 2050, 2051), milder impairment in two (2047, 2048). 8- Psychological symptoms and signs: severe in three (2049, 2050, 2051) (nightmares, aggressive attacks, emotional lability ), less prominent in the other two (blunt mood (2048) and emotional lability, nightmares and mild euphoria in individual (2047)) 9- Microcephaly in the three patients with more severe manifestations (2049, 2050, 2051) 10- Epilepsy at 12 years of age (2050, 2051) 11- MRI of the brain: Severe cortical atrophy especially the frontal region, cerebellar atrophy, TCC, hypomyelination and WMH

Family FM7 branch 2

In this branch the affected case were a father and his daughter who presented at the ages of 29 and 9 years, respectively. They did not complain of any neurological symptoms and were found upon interrogation of the members of first branch about family history of deafness and mutism. They were mute and had deep hearing impairment and mild cerebellar ataxia. They had normal development and were intellectually sound. No additional symptoms or signs were detected.

3.3.5.4. Genetic exploration: A single known missense transition mutation (rs151240697) in exon15: g.2442812C>T, c.1822G>A, p.(Val608Met) of PANK4 (based on the longest transcript: NM_018216.2) was found in patient 2049 and 2051. Co-segregation with the disease distribution in branch 1 of family FM7 was confirmed where it was homozygous in all the five affected siblings and heterozygous or homozygous reference in seven healthy individuals [Figure (3- 18)]. The variant was not the cause in branch 2 of family FM7 with a different clinical phenotype despite some shared signs. In this branch, the two patients where a homozygous reference and heterozygous carrier [Figure (3-18)].

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Further analysis using homozygosity mapping data was done. HZM did not show any shared region of homozygosity shared between patients of both branches although a low threshold for detection of homozygous region of 100000 bp was utilized. No genomic rearrangement was found too. PANK4 (pantothenate kinase 4; also known as FLJ10782] (OMIM *606162) is located on the short arm of chromosome 1 (1p36.32) [Ensembl: 1: 2,508,533- 2,526,628 (GRCh38) 2,439,972-2,458,067 (GRCh37)] The variant had passed the GATK filter with high confidence and good coverage depth at 56x, 76x, 84xThis variant was found only once in the heterozygous state in 60,607 WES in ExAC browser and only once in 6501 samples in EVS also in heterozygous state. It has a frequency of 0.001 in a sample size of 5871 also in heterozygous only in dbSNP. The mutation resulted in a change from valine to methionine. The two are hydrophobic amino acids. There is thus a small physicochemical difference [Grantham distance is 21 (0-215)]. The amino acid is located in the domain of unknown function DUF89 (Pantothenate kinase, acetyl-CoA regulated, two-domain type). This change was predicted pathogenic by SIFT, Polyphen2 and Mutation taster. The nucleotide where the variant is located is conserved and has a PhastCons conservation score of 0.99 (0-1) and a PhyloP score of 4.73 (-14.1-6.4)]. The amino acid is moderately conserved in 12 species but highly conserved in mammals.

138

Figure (3-18): Pedigree and MRI of the two index patients from the two branches of family FM7. Branch 1 of the family was associated with a missense mutation with full segregation with the disease distribution in branch 1 (blue rectangles) but not segregating in branch 2 (red rectangles). MRI of the index of branch 1 (T1 sagittal and T2 FLAIR axial) shows cortical atrophy more prominent in the frontal lobe in addition to cerebellar atrophy. MRI of the index of branch 2 (T1 sagittal and T2 FLAIR axial) shows mild cortical and significant cerebellar atrophy Example sanger sequencing of the mutation in patient (homozygous mutant), a heterozygous carrier and a control homozygous reference allele with conserved amino acid sequence, is shown. Pedigree symbols: * sampled individual; Phenotype symbols: half-fill black color: affected individuals; Genotype symbols: ++ Homozygous reference genotype; M+ Heterozygous genotype; MM Homozygous mutant genotype. Others are standard medical pedigree symbols.

139

Table (3-14): Clinical data of five patients from family FM7 branch 1. UL: upper limbs. LL: lower limbs. PUL/PLL: proximal upper/lower limbs. DUL/DLL: distal upper/lower limbs. Cog: cognitive. Psych: psychological. Clinical signs severity: – Absent, + Mild, ++ Moderate, +++ Severe. Tendon Reflexes: ↑Increased Tendon Reflex. Extensor Planter Response: ↑ Unilateral, ↑↑ Bilateral, ↔ Mute, ↓ Flexor. N: Normal. NA: Not Applicable due to Patients Condition.

Family Code FM7 branch 1

Mutated Gene PANK4

Individual 2047 2048 2049 2050 2051 Code

Gender M F F M M

Clinical Complicated HA Diagnosis

Khartoum Gamooia tribe

Age at Onset Birth

Age at Initial 29yrs 26yrs 20yrs 13yrs 18yrs Examination

Spasticity -/- -/- -/+ Rt. +/+/+++ ++/++

UL/LL/ Gait

Motor Deficit -/- -/- 3+ (difficult to 3+ (difficult to 3+ (difficult to PUL/DUL assess) assess) assess)

Motor Deficit -/- -/- 3 (difficult to 3 (difficult to (difficult to PLL/DLL assess) assess) assess)

Tendon ↑/↑ ↑/↓ ↑/↑ ↑/↑ ↑/↑ Reflexes UL/LL Patellar

Ankle Reflex/ ↑Rt. /↔ ↓/↔ N /↔ ↑/Rt.↔ Lt. ↓ ↑/↓Lt. ↔Rt. Planter Response

Ataxia -/++/-/++ -/++/-/++ Interrupted +/+/+/+ Nystagmus/+/+/ Eye/UL/LL/ saccadic + Gait pursuit /++/- /++

Dysarthria NA/NA NA/NA NA/NA NA/NA NA/NA Spastic/Cerebel lar

Muscle -/- -/- -/- -/- +/+ 140 Atrophy DUL/DLL Facial Atrophy - - - - -

Cog/ Psych +/+ +/+ +++/+ +++/ + +++/+ signs Nightmares, Nightmares. emotional Blunt mood Nightmares Nightmares, emotional lability and emotional temper, lability and euphoria lability and aggression euphoria euphoria episodes and euphoria aggressive episodes

Optic Atrophy - - - - -

Extrapyramidal - - - - Hypokinesia

Signs

Sensory Loss NA - NA NA NA

Other Signs Hearing Hearing Microcephaly Microcephaly Microcephaly impairment impairment and mutism and mutism Hearing Urinary Urinary impairment incontinence, incontinence, and mutism Dysphagia, Progressive Hearing Hearing impairment and impairment and mutism mutism

Disability Score 2 1 2 7 6

MRI of Brain ND ND Severe cortical Severe cortical Severe cortical atrophy atrophy atrophy especially the especially the especially the frontal region frontal region frontal region Cerebellar Cerebellar Cerebellar atrophy TCC atrophy TCC atrophy TCC

Hypomyelinati Hypomyelinatio Hypomyelinatio on WMH n WMH n WMH

Electrophysiolo N N N N N gical Studies (NCS)

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Table (3-15): Clinical data of two patients from family FM7 branch2. UL: upper limbs. LL: lower limbs. PUL/PLL: proximal upper/lower limbs. DUL/DLL: distal upper/lower limbs. Cog: cognitive. Psych: psychological. Clinical signs severity: – Absent, + Mild, ++ Moderate, +++ Severe. Tendon Reflexes: ↑Increased Tendon Reflex. Extensor Planter Response: ↑ Unilateral, ↑↑ Bilateral, ↔ Mute, ↓ Flexor. N: Normal. NA: Not Applicable due to Patients Condition.

Family code FM7 branch 2

Gene ?

Individual code 2056 2059

Gender M F

Clinical Diagnosis Complicated HA

Khartoum Gamooia tribe

Age at onset Birth

Age at Initial 29yrs 9yrs Examination

Spasticity -/- -/-

UL/LL

Motor Deficit -/- -/- PUL/DUL

Motor Deficit -/- -/- PLL/DLL

Tendon Reflexes N/N N/↓ UL/LL Patellar

Ankle Reflex/ ↑/↓Rt. ↔Lt. N /↔ Planter Response

Ataxia Saccadic Slow saccades /+/- Eye/UL/LL/ Gait pursuit /+/-/+ /+

Dysarthria NA/NA NA/NA Spastic/Cerebella r

Muscle Atrophy -/- -/- DUL/DLL

Facial Atrophy - - 142

Cog/ Psych signs - - Optic Atrophy - -

Extrapyramidal - -

Signs

Sensory Loss - -

Other Signs Hearing Hearing

impairment impairment and and mutism mutism

Disability Score 2 1

MRI of Brain Mild cortical ND

and significant cerebellar atrophy

Electrophysiologi N N cal Studies (NCS)

3.4. Causes unidentified

A summary of the families in which the genetic cause was not identified is in [Table (3- 16)] and statistics represented in [Figure (3-19)].

The cause was not identified in 24 families in the cohort. Negative results upon analysis of WES data were found in five families. In the remaining 19 families the cause was the absence of WES data due to the families prioritization policy that we followed where only high yielding families, in which two or more affected individuals were sampled, were selected for WES. Five families were exception as they were not included in the cohort for WES because of logistic reasons such as the cost and the time factor.

143

Table (3-16): Clinical summary of 23 families with unidentified genetic cause (family 27 is presented in a separate table). HSP:hereditary spastic paraplega, UL: upper limbs, LL: lower limbs, ND: not done, CT: computerized tomography scan, MRI: magnetic resonance imaging, EEG: electroencephalogram, EMG: electromyogram, NCS: nerve conduction studies.

144

Fam. Origin/ Inheritan Phenotype Age at Electrophysiol Brain Imaging Clinical features summary code Consan ce/ Gene summary onset/E ogical studies guinity xam. EMG/NCS ,EEG)

s consanguineou Jazira/ Al

F1- Sporadic Complicated 1.5 ND ND 1- Pyramidal signs(LL &/> UL: spasticity, hyperreflexia and extensor BRANC HSP yrs/ 15 planter response) 2- Cerebellar signs (UL, dysarthria and eyes) 3- H-C yrs Congenital hip dislocation 4- Cognitive Impairment

5-Optic atrophy 6-Scoliosis 7-Distal muscle wasting (UL &LL)

F1- consanguine / Jazira Al Sporadic Complicated Birth/1 ND ND 1- Pyramidal signs (LL:spasticity, hyperreflexia ) 2- Cerebellar signs BRANC HSP 0.5 yrs (LL & dysarthria) 3- Recurrent congenital cataract and postoperative H-D retinal detachment (almost blind) 4- Cognitive Impairment 5-floppy after general anaesthesia 6-Generalized muscle wasting

ous

F2 consanguineous non (Dwem)/ State Nile White Sporadic Pure HSP 14yrs/ ND Normal CT scan 1- Pyramidal signs (LL: spasticity, hyperreflexia and extensor planter 23 yrs of the brain response)2- Cerebellar signs (Mild:UL & nystagmus)

3-Vibration sense diminished

145

Fam. Origin/ Inheritanc Phenotype Age at Electrophysio Brain Imaging Clinical features summary code Consan e/ Gene summary onset/E logical guinity xam. studies EMG/NCS ,EEG)

F7 Kurdufan/ AR/Pure Pure HSP Three ND ND 1- Pyramidal signs(LL: spasticity, hyperreflexia and extensor planter HSP sibs(tw response) o of

consanguineous them identica l twins):

4yrs/

34-40 yrs

F11- Jazira Al Sporadic Complex 1.4 yrs / EEG: MRI brain:Mild 1- Pyramidal signs (Moderate LL and UL: spasticity, hyperreflexia but BRANC disorder with 5 yrs 4 evidence of global cerebellar flexor planter response) 2- Matching level of motor deficit (UL and LL) H-1 HSP months generalized atrophy and mild

/ epilepsy TCC 3- Cerebellar signs (Moderate Gait, trunk: limbs and eye movement could consanguineous not be assessed properly) 4- Regression of milestones 5- Cognitive deterioration 6-Mild urinary urgency 7-Epilepsy at 4 yrs 3 months 8- Dysphagia 9-Mild psychiatric symptoms 10-Optic atrophy,milde lid retraction and decreased visual acuity 11- Auditory test: mild to moderate sensory neural hearing loss

F11- consanguineous Jazira Al Sporadic Complex 1.5 yrs/ ND MRI brain:Mild 1- Pyramidal signs (mild:UL > LL: spasticity, hyperreflexia but flexor BRANC disorder with 5 yrs global cerebellar planter response) 2- Matching level of motor deficit (UL and LL) H-2 HSP atrophy and mild

/ TCC 3- Cerebellar signs (Moderate Gait, trunk: limbs and eye movement could not be assessed properly) 4- Regression of milestones (speech and motor)

5- Cognitive deterioration 6-Deterioration following a febrile illness then improved gradually 7- Auditory test: mild to moderate conductive hearing loss associated with Eustachian tube obstruction (? Temporary)

146

F12 consanguineous Darfur/ , Elfashir AR Complicated Two ND MRI of the 1- Pyramidal signs (In one individual: LL and />UL:, hyperreflexia but HA sibs : brain: Cerebellar normal tone and flexor planter response) 2-Delayed psychomotor atrophy and development 3- Cerebellar signs (severe: trunk, LL, UL, gait and eye 4- Birth / PVWMH Mental retardation 5-Drooling of saliva 17

month, 3 yrs

F17 ous consanguin Falata/ Sennar Sporadic/ Pure HSP 28yrs/3 ND Normal 1- Pyramidal signs(LL: spasticity, hyperreflexia)

Pure HSP 8yrs

F21 consanguineous Danagla/ North Sporadic Spastic 44yrs/4 ND Mild cerebellar 1- Pyramidal signs(LL &/> UL: spasticity, hyperreflexia & indifferent Ataxia 7yrs lobes and planter response) vermial atrophy with prominent 2- Cerebellar signs (UL, LL, Gait, trunk and eyes(nystagmus & saccadic folia. Few pursuit)) 3-Vitiligo secondary to botulinum toxin injection as treatment for lacunar high spasticity) 4-Urinary urgency signal intensities

147

Fam. Origin/ Inheritanc Phenotype Age at Electrophysio Brain Imaging Clinical features summary code Consan e/ Gene summary onset/E logical guinity xam. studies EMG/NCS ,EEG)

F22 consanguineous State(Atbara,Gaalia)/ Nile River AR Complex Two EEG: active Brain MRI: 1- Pyramidal signs (LL and UL: spasticity, hyperreflexia and extensor disorder with sibs generalized generalized brain planter response) 2-Cerebellar sigs (trunkal)(limbs could not be assessed) HSP (one epilepsy atrophy (cortical, 3- Stereotypic behavior: head movements and teeth clenching 4- died) : cerebellar and Extrapyramidal signs (dystonia) 5-UL and LL wasting (distal and TCC) PVWMH proximal) 6- Mental retardation 7-Psychiatric symptoms: bouts of 1.5 yrs / and cerebellar excessive crying 8-Epilepsy: generalized tonic clonic since 1.5 yrs 9- 8 yrs hyperintensity Pale optic disc, thin retinal background and hypermetropic fundi. signals 10- Skeletal deformities: talipes equina varus 11-Regression of milestones

F23 consanguineous (DwemNile State Kawahla)/ White AR Comlicated Two ND MRI Brain : 1- Pyramidal signs (LL and UL: spasticity, hyperreflexia, speech and HSP (with sibs mild cortical extensor planter response) (spasticity predominant and intellectual Dementia) (only atrophy. deterioration) one Cerebellar PLP1 like sampled atrophy. 2-Intellectual deterioration (dementia like with loss of memory) 3-

Cerebellar signs (gait, speech and eye:nystagmus, saccadic pursuit) 4- 4.5 yrs PVWMH signal Regression of milestones 5-Mild horizontal eye movement /7 yrs lesions limitation(ophthalmoplegia) 6-Generalized tonic clonic epilepsy 7- Sphincter disturbance 8-Generalised mild muscle wasting

148

Fam. Origin/ Inheritanc Phenotype Age at Electrophy Brain Imaging Clinical features summary code Consan e/ Gene summary onset/Exam. siological guinity studies EMG/NCS ,EEG)

F24 consanguineous (Danagla)/ North Identical Complicated Identical EMG/NC Normal MRI 1- Pyramidal signs(Mild to moderate: LL &/> UL spasticity, twins : HA twins : S: low hyperreflexia) 2- Cerebellar signs (Moderate: UL, LL, Gait, trunk and sporadic motor eyes(saccadic pursuit)) 3-Epilepsy (seizures controlled by treatment) 4- 11 yrs/18 amplitude Decreased visual acuity but normal fundus (myopia) 5-Minimal cognitive yrs in lower impairment limb (axonal motor PN)

EEG :phot osensitive

generalize d epilepsy (1 Pt)

F28 consanguineous Kurdufan/ Sporadic Complicated 7 yrs/12.5yr ND Cerebellar 1- Pyramidal signs (moderate to severe: LL &>UL: spasticity and HSP atrophy extensor planter response but absent other reflexes) 2- Cerebellar signs (prominent (moderate: UL, LL, Gait, trunk and eyes (saccadic pursuit) 3- foli, vermial Extrapyramidal signs (choreoathetotic movement) 4-Cognitive Impairment atrophy and 5-Psychiatric symptoms (irritable mood) 6-Skeletal deformities and mega cistern contractures (scoliosis, Lt talipes equinovarus, fixed flexion deformity of magna) the hip joints Lt>Rt) 7-Dysphagia

149

Fam. Origin/Co Inheritanc Phenotype Age at Electrophysio Brain Imaging Clinical features summary code nsanguini e/ Gene summary onset/E logical ty xam. studies EMG/NCS ,EEG)

F31 State/ Nile River AD Complicate Father ND ND 1- Cerebellar signs (UL, LL, Gait, trunk) 2- Pyramidal signs (LL &/>UL: d HSP/HA and two spasticity, hyperreflexia and extensor planter response) spectrum daughte rs 3-Convergent squint (horizontal ophthalmoplegia) 4-Psychiatric symptoms (irritability: in one) 5-The father (previously not known to have 38, 4, 2 a disease) only has horizontal opthalmoplegia/ mild UL ataxia / mild pes

consanguineous yrs/ 38 cavus yrs, 7m/7m

F35 consanguineous Darfur Nyala/ Sporadic Complex 19 yrs/ ND ND 1- Pyramidal signs ( LL &/> UL: spasticity, hyperreflexia) 2-Hearing HSP 27 yrs Impairment (minimal complexity

)

F36 consanguineous Falata/ DarfurNyala AR Complex One ND ND 1- Pyramidal signs(LL & UL: spasticity, hyperreflexia and extensor BRANC HSP patient : planter response) 2-Cerebellar signs (could not be examined appropriately) H-1 3-Blindness 4-Severe proximal & disal muscle wasting (UL & LL) 5- 13 yrs/ Contracture(fixed flexion of knee) 6-Sphincter disturbance 7- Impaired 26 yrs sexual function 8-Hearing impairment 8-Dysphagia 8-Normal cognition

150

Fam. Origin/ Inheritanc Phenotype Age at Electrophysio Brain Imaging Clinical features summary code Consan e/ Gene summary onset/E logical guinity xam. studies EMG/NCS ,EEG)

F38- consanguineous Nile State/ White AR Complex Three ND MRI brain : 1- Cerebellar signs (UL, LL, Gait, trunk) 2- Pes cavus (one) 3- Seasonal BRANC HA sibs : skin rash (winter: one patient) H-1 (minimal Mild comlexity) 7, 7, 12 hypomyelination yrs/ 15, features

18, 21 yrs Mild cerebellar atrophy

F38- consanguineous Nile State/ White AR Complicate Two ND ND BRANC d HSP/HA sibs : H-2 spectrum  One sib(the younger): Cerebellar signs (UL, LL, Gait, trunk) 2- Pes 11, 13 cavus (one) 3- Seasonal skin rash (winter: one patient) yrs / 20,  The other (older): 1-Pyramidal signs (LL(Lt>Rt): spasticity,

17 yrs hyperreflexia and unilateral Lt extensor planter response (indifferent in the other) 2-Intellectual impairment 3-ataxia, 4- Generalized tonic clonic epilepsy at 6 yrs 5-Scoliosis Lt sided pes cavus and wrist deformity 6-LL wasting (proximal and distal)

F42 consanguineous Jazira Al AR Complicate One EMG/NCS: MRI: ND 1-Pyramidal signs predominant(spasticity, hyperreflexia and flexor planter d HSP patient Normal response) 2-Cerebellar signs (UL, LL, Gait, trunk and eyes:slow (one saccades)) 3-Regression of milestones 4-Sphincter disturbances 5-

/ died) : Epilepsy: myoclonic epilepsy (awake or asleep) 6-Skeletal deformity: Lumbar kyphosis 7-Dysphagia (drooling of saliva) 8-MR 9-Psychiatric 2 yrs / 8 symptoms: irritability 10- Skin: After he received omega3 fatty acids: yrs 4 hypopigmented areas non scaly maccules on the face,neck and UL months

151

Fam. Origin/ Inheritanc Phenotype Age at Electrophysio Brain Imaging Clinical features summary code Consan e/ Gene summary onset/E logical guinity xam. studies EMG/NCS ,EEG)

F44 consanguineous Jazira Al AR Complicate Two EMG/NCS: ND 1-Pyramidal signs (LL & UL: spasticity, hyperreflexia but flexor planter d HSP male prolonged response) sibs : distal motor

/ latency in LL 2- Cerebellar signs (UL, LL, Gait, neck, trunk and eyes) (spastisity Birth/ predominant) 3-Delayed psychomotor development 4-Drooling of saliva

4,9 5-Eye:slow saccades, saccadic pursuit, nystagmus, horizontal years ophthalmoplegis and occulomotor apraxia (one) 6-Sphincters intact 7- Normal cognition 8- Febrile convulsions once (one)

F45 consanguineous Jazira Al Sporadic Complicate Birth/ 4 ND ND 1-Floppy at birth 2-Cerebellar signs (UL, LL, Gait, speech, trunk and d HA yrs eyes:nystagmus) 3-Pyramidal signs (spasticity: UL and LL (proximal more than distal), hyperreflexia and flexor planter response) (ataxia

/ predominant) 4-Delayed psychomotor development 5-Recurrent febrile convulsions since 3 months (mother noted eye movement increased with fever) 6-Sphincters: not yet dry by day

F46 consanguineous Gaalia/ Khartoum AR Complicate Two ND ND 1-Cerebellar signs (Both: UL, LL, speech, gait, trunk and eyes: d HA sibs : nystagmus) 2-Pyramidal signs (In the older the individual: spasticity, hyperreflexia and extensor planter response) 3- Scoliosis 4-Sphincter 3, 13 disturbance (the younger individual) 5-Normal cognition 6-Epilepsy yrs (partial with secondary generalization: the younger individual)

/12,25 yrs

152

Fam. Origin/Co Inheritanc Phenotype Age at Electrophysio Brain Imaging Clinical features summary code nsanguini e/ Gene summary onset/E logical ty xam. studies EMG/NCS ,EEG)

F47 consanguineous / Kurdufan AR Complicat Boy EMG/NCS: MRI of the 1-Cerebellar ataxia (one patient: UL, LL, speech, gait and eye (saccadic ed HA and his brain: Cerebellar pursuit)) 2-Mild Pyramidal signs (In the younger individual: spasticity, materna Sensorimotor atrophy (the hyperreflexia and flexor planter response) (ataxia predominant) 3- l uncle : axonal younger patient) Extrapyramidal signs in the older individual (mild dystonia and chorea) neuropathy

35, 38 yrs / 38, 52 yrs

F48 consanguineous (Mahas)/ North AR/Pure Pure HSP Two ND Spinal MRI : no Pure Pyramidal signs (LL only in the older and mild UL and LL in the HSP sibs : spinal anomaly younger: spasticity, hyperreflexia and Rt unilateral extensor planter or compression response in the older) 2-Mild scoliosis (older sib) 15, 16yrs MRI brain: ND

7month s / 15 /16 yrs 10 months

F49 non Darfur (Elfashir)/ AD Complicate Two EMG/NCS MRI Brain 1-Pyramidal signs (UL and LL: severe spasticity, hyperreflexia and

consanguineous d HSP male (older (older patient): extensor planter response) sibs : patient): Mild sensorimotor Cerebellar 2-Cerebellar signs (UL, LL, Gait, trunk and eyes: slow saccades) 20 , 24 axonal atrophy and (spastisity predominant) 3- LL muscle wasting (distal and proximal) 4- yrs /24, neuropathy PVWMH signal Hypopigmented spots 1-2cm in diameter behind RT knee- and in LT axilla 33 yrs lesions (one patient) 5-Dysphagia to fluids mainly (same individual)

153

Fam. Origin/C Inheritan Phenotype Age at Electrophysi Brain Imaging Clinical features summary code onsangui ce/ Gene summary onset/E ological nity xam. studies EMG/NCS ,EEG)

FM1 consanguineous Nile ,Gezira states/ White AR/MND Complicate One ND Hypomyelination, 1-Pyramidal signs (LL &/> UL: spasticity, hyperreflexia and extensor like d HSP patient TCC and mild planter response) 2- Mild cerebellar signs (UL, LL, Gait) 3-Pseudobulbar (ALS like) sampled prominence of palsy (dysphagia to both liquid and solids, spastic anarthria, spastic : internal and tongue) 4-Lower motor neuron signs (generalized LL and UL muscle external CSF space wasting: distal > proximal, fasciculations) 5- Subjective feeling or for age 39 yrs/ respiratory muscle weakness. 6-Facial muscle atrophy (temporalis) 7-Pes 40 yrs cavus

FM5 consanguineous (Elfashir)/non Darfur Shaaygia/ North AR/ALS2 Complicated Four ND ND 1-Pyramidal signs (LL &/> UL: spasticity, hyperreflexia and extensor HSP patients planter response) (Juvenile (three sibs ALS) and a 2-Dysphagia 3-Involuntary laughter 4-Distal muscle wasting (variable in cousin): two individuals)

3-18 months/ 11- 13 yrs

154

Fam. Origin/ Inheritanc Phenotype Age at Electroph Brain Imaging Clinical features summary code Consan e/ Gene summary onset/Exam. ysiologic guinity al studies EMG/NC S ,EEG)

FM6 GadarifGaalia / AR Complicated Two sibs : ND MRI brain : 1- Pyramidal signs (LL: spasticity, hyperreflexia and extensor HSP Moderate TCC planter response) 2- Nystagmus (2 BEATS), saccadic pursuit 4, 8 months/ (posteriorly more) and slow saccades 3-Epilepsy (one: febrile convulsions at 8 15,18 yrs Features of months and diagnosed and started treatment at 10 month) 4- hypomyelination Extrapyramidal signs (in one:hypokinesia) 5-Delayed motor Gradual consanguineous development and speech. 6-Normal intelligence. amelioration and disease progression plateau reached

by 4, 5 yrs

FM7- consanguineous Gamooia/ Khartoum AD Complicated Father and EMG/N CT scan : Normal 1-Deafness 2-Mutism 3-Mild ataxia BRANC HA daughter : CS: H-2 Normal MRI : Birth / 29,9 years

155

 Family 27, syndromic presentation Among the 24 families without genetic results, one family emerged, with a peculiar phenotype, suggesting a common genetic origin:

Clinical summary

In this family, three sibs presented at the ages of 15, 6.5 and 5 years of age with homogenous clinical picture of globally retarded growth with apparent developmental entrapment/arrest since around 1-3 years of age. They had facial dysmorphism more prominent in the older sib. Hair colour was lighter than their parents and healthy sibs sibs and hair texture smoother. They had scaly seasonal hypopigmented skin rash. Mental retardation and psychiatric signs of irritability and emotional lability were observed. Remarkably, the three sibs were extremely friendly, interactive and welcoming. The neurological syndrome was of complex spastic ataxia with bradykinesia (extrapyramidal signs). Ophthalmological assessment revealed optic atrophy with retinitis punctata albescence (in all three sibs) (mottled retina and a variant from retinitis pigmentosa) and hypermetropia in the older sib with most advanced disease. The older girl was diabetic. In one occasion the boy showed altered liver function test but there was no further investigations to exclude other causes nor a follow up tests [Figure (3-20)] [Table (3-17)].

This phenotype presentation has never been reported previously, to our knowledge, and might represent a new syndrome.

156

Table (3-17): Clinical summary of family F27 with unidentified genetic cause. HSP:hereditary spastic paraplega, UL: upper limbs, LL: lower limbs, ND: not done, CT: computerized tomography scan, MRI: magnetic resonance imaging, EEG: electroencephalogram, EMG: electromyogram, NCS: nerve conduction studies.

Fam. sanguinity Origin/Con Gene/ Inheritance summary Phenotype /Exam. onset at Age ,EEG) EMG/NCS studies siological Electrophy Imaging Brain Clinical features summary code

F27 non Kurdufan/ AR Syndromic Three sibs: EEG: Normal Severe 1- Pyramidal signs (LL &/> UL: spasticity, hyperreflexia and flexor planter features (one) generalized response) 2- Cerebellar signs (UL = LL, Gait, trunk and eyes) 3-Extrapyramidal ( ?new 1.5-3 yrs / brain atrophy signs (hypokinesia) 4-Facial dysmorphism 5-Mental retardation 6-Short stature syndrome) 5-15 yrs EMG/NCS : ND with PVWMH and severely delayed psychomotor development (general growth entrapment/arrest signals and since around 1-3 yrs of age) 7-Psychiatric symptoms (irritability, emotional

- consanguineous features of lability) 8-Fundus: optic atrophy with retinitis punctata albescence (in all three hypomyelinatio sibs) (mottled retina and is a variant from retinitis pigmentosa); hypermetropia in n. the older sib with most advanced disease 9-Skin rash (scaly seasonal) 10-Hair lighter colour and smoother texture than sibs. 11-DM in the older sib with more advanced. 12-Impaired liver function in one (mild increased liver enzymes:

ALT,AST,ALPand increased bilirubin)

157

Figure (3-20): Pedigree of family F27. Pedigree symbols: * sampled individual; Phenotype symbols: half-fill black color: affected individuals, others are standard medical pedigree symbols.

158

3.5. Comments and summary

The overall genetic diagnostic power was 41% since a genetic diagnosis was established in 17 families (known, potential new genes) out of the 41 Sudanese families included in the cohort (in two families the causative gene/variant was identified in a branch of the family: F1 and FM7) [Figure (3-21)] [A summary of the thesis method and genetic results is in [Figure (3-23)].

WES was collectively performed in 14 families. In three of them, the genetic diagnoses were mutations in genes known to cause spastic neurogenetic disorders. In five families we had potential new genes for further confirmation of pathogenic effect through functional studies [Figure (3-22)].

Figure (3-22): WES success rate for known and new genes in the Sudanese cohort

159

Classical HSP families Spastic ataxia HA complicated with Pyramidal signs as 23 families 2 families pyramidal signs part of other 10 families syndromes 6 families

Candidate gene approach for 3 families (F1. F6. F7)

F1 branch A/B, F6: SPG11

Screening of 41 families for HSP genes using NGS panel (39 families +F1 branch C/D)

Screening FM4: F14: SACS, F16: SPG11 Candidate ADCA PD genes NGS F19: SPG57/TFG gene approach for panel F34: SPG3A F26 FM5: ALS2 F26: SCA7 FM4:DNAJC6

WES for F7, F27, F37, F48, WES for F41 WES for F30, F46 WES for F15, F25 F50,FM3, FM6, FM7

F37: ABHD16A F41: F30: SIL1 F15: ARG1, FM3: ST7L CAMSAP3, MINK1 F25:PLA2G6 FM7 branch1: PANK4 ZNF433 F50: BIRC5, C21ORF91

Figure (3-23): Schematic summary of the thesis method pipeline and genetic results.

Colour code: in blue are the families and their clinical stratification, in violet are the methods used, in green are the genetic results obtained.

160

Discussion

4.1. Overview of the cohort The cohort is heterogeneous but within limits that could be expected from the broad inclusion criteria as well as the huge phenotypic and genetic heterogeneity of the HSP and its overlap with other closely related differential diagnoses. The core clinical characteristic of the Sudanese familial cohort is the increased level of complexity which is noted in the majority of families.

The most frequent complicating clinical features are signs of cerebellar syndrome, cognitive impairment and various eye involvement (neuronal and non neuronal). This matches what we found in our analysis of the clinical presentation of various forms of HSP. Peripheral neuropathy is underestimated given the fact that EMG/NCS has not been performed in many patients and the clinical examination alone is not satisfactory especially on assessment of pediatric and mentally impaired patients [Figure (4-1)] [Table (4-1)].

Extrapyramidal signs and more specifically chorea/athetosis are more prevalent in our cohort (4/41 families: F28, F37, F41 and F47) than what we found on analyzing the known HSP subtypes. To date, SPG58 is the only type of HSP in which chorea was reported. MRI of the brain was not performed in all families but in the cases where it was done it showed complex abnormalities [Figure (4-1)] [Figure (4-2)] [Table (4-1)]. In the methodology chapter we explained in details, the limitations that we encountered with the brain imaging.

Examples have been illustrated where we have broadened the phenotypic spectrum associated with certain known genes [F19/TFG (SPG57), FM4/DNAJC6 (JOPD), F30/SIL1 (MSS), F15/ARG1 (hyperargininemia)]. In rare conditions even the same mutation is associated with minor differences in their clinical presentation (family F6/SPG11).

The scene is further complicated by the fact that in at least seven families multiple phenotypes coexisted. In at least three of these families (F1, F6, FM7) we could provide provisional proof that these phenotypes are distinct genetically. This is not always easy especially when the candidate genes in these families are new (FM7). Yet when the family (or branch) under question is sporadic the decision will be even harder (F1 branch C and 161

D) (Elsayed et al., under revision). In three families (F14, F16, F21) [Figure (4-4)] [Figure (4-5)] [Figure (4-3)], respectively) (Elsayed et al., under revision) the other coexisting phenotype is non-neurological and was based on clinical diagnosis and we have not explored the genetic background of these disorders as they are out of the scope of the study.

Figure (4-1): Main clinical signs complicating the 41 families included in the cohort.

Table (4-1): Detailed clinical signs complicating the 41 families included in the cohort.

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Figure (4-2): Brain MRI abnormal signs encountered in the cohort.

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 Family F27, a peculiar syndrome An intriguing condition was found in family F27. In this family a peculiar syndrome was observed as indicated in the results. Preliminary clinical assessment implied a new clinical syndrome described and to be reported after full work up. The gene behind the family was not identified. WES has been performed; analysis excluded homozygous variants in all protein coding regions of genes covered in the WES. As the family showed recessive inheritance with three sibs affected and the parents are non- consanguineous, compound heterozygous variants were a possibility. Yet although none was identified, this possibility cannot be excluded given the fact that the second incriminated variant may be located in non-coding region or in region not well covered. Proper complete phenotyping is required including reexamination of the eyes, orthopedic consultation, standard IQ testing, endocrinological assessment, cardiac assessment, full metabolic screening, test and repeat liver functions for all patients, electrophysiological studies (visual and auditory evoked potentials, EMG/NCS, EEG) and follow up by brain imaging. Reanalysis of the WES and functional assays for the fibroblasts just to assess the organellar morphology even in absence of genetic diagnosis will be considered.

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Figure (4-3): Pedigree of family F21. Pedigree symbols: * sampled individual; Phenotype symbols: half-fill black color: spastic ataxia individual, other filling patterns correspond to various clinical phenotypes in the family. Others are standard medical pedigree symbols.

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Figure (4-4): Pedigree of family F14. Pedigree symbols: * sampled individual; Phenotype symbols: half-fill black color: spastic ataxia/SACS individuals, other filling patterns correspond to various clinical phenotypes in the family. Others are standard medical pedigree symbols.

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Figure (4-5): Pedigree of family F16. Pedigree symbols: * sampled individual; Phenotype symbols: half-fill black color: HSP/SPG11 individuals, other filling patterns correspond to various clinical phenotypes in the family. Others are standard medical pedigree symbols.

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4.2. Diagnostic NGS-based multigene panels for HSP Phenotype directed choice of culprit genes can be used in this era of NGS to facilitate the selection of probable genes when more than one variant is obtained on bioinformatic analysis of output data of the diagnostic panels.

Below is a proposed flow chart based on phenotype/ genotype correlation data to aid in the selection of variants/genes [Figure (4-6)].

Evidently, this approach can mask unexpected phenotypes or inheritance models associated to mutations in a given gene. Therefore, the use of flow charts has to be done with caution in typical cases and is of interest mainly when the geneticist has to deal with various putative mutations in various known genes.

Patient presenting with pyramidal signs with suggested genetic aetiology

Inheritance Pattern suggested by pedigree

Autosomal X-Linked Maternal

Sporadic Dominant Recessive Figure2

Consanguinity P C

C: +PN/Amyo SPG4, SPG3 A No Yes SPG31, SPG10 s SPG6, SPG8 SPG12, SPG13 Only + Other Signs More likely SPG19, SPG33 features SPG37, SPG38 SPG41, SPG42 SPG58, SPG72 SPG31, SPG10 SPG31, SPG10 AD AR SPG73, No SPG17, SPG36 SPG6, SPG17 SPG: (BICD2) SPG8, SPG58, SPG9, SPG58 NO SPG4, SPG3A

SPG(BICD2) SPG8, SPG36, SPG3A NO SPG (BICD2)

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Autosomal Recessive

C P

C: +TCC C: +CI C: +PN/Amyo C: +Ataxia EYE HSP

SPG11, SPG15 SPG7, SPG5 SPG11, SPG15 SPG58, SPG5 PN + Amyo SPG7, OA SPG24, SPG28 SPG18, SPG21 SPG14, SPG18 SPG3A, SPG9 ARSACS SPG7 SPG30, SPG48 SPG32, SPG35 SPG20, SPG21 SPG11, SPG39 /SACS, SPG58, SPG35 SPG51, SPG27 SPG46, SPG47 SPG23, SPG26 SPG43, SPG55 SPG5, SPG11, SPG57 SPG52, SPG11 SPG3A, SPG48 SPG27, SPG32 SPG66, SPG68 SPG15, SPG20 SPG45 SPG62, SPG65 SPG56/CYP2U1 SPG35, SPG44 SPG74 SPG21, SPG26, SPG54 SPG15, SPG71 SPG55, SPG54 SPG45, SPG46 PN - Amyo SPG27, SPG28, SPG55 SPG3, SPG72 SPG63, SPG65 SPG47, SPG48 SPG14, SPG15 SPG30, SPG35, SPG68 SPG73 SPG66, SPG67 SPG49/SPG56 SPG23, SPG25, SPG39, SPG44, SPG74 No SPG: ADAR1 SPG71, SPG74 SPG51, SPG50 SPG26, SPG28 SPG46, SPG54, IFIH1 SPG49/ TECPR2 SPG52, SPG53 SPG30 SPG59, SPG60, RNASEH2B SPG54, SPG59 SPG56/CYP2U1 SPG64, SPG68, SPG55, SPG64 SPG57, SPG60 SPG75/MAG SPG65, SPG69 SPG61, NoSPG: No SPG: No SPG: GAD1 CCT5 LYST EXOSC3, RD/MD EXOSC3 ARSACS/SACS LYST, SPG11 SPG49/TECPR FAM134B SPG49/TECPR SPG15

2 Amyo – PN 2 SPG5, SPG20, SPG35 SPG47, SPG52 SPG51, Cataract SPG64 SPG65, SPG9 SPG67 SPG70, No SPG46 SPG: EXOSC3 SPG69 BICD2

MAG

Ataxia + PN/Amyo + CI SPG11, SPG15, SPG20, SPG22, SPG26 SPG35, SPG64 No SPG : EXOSC3 Ataxia + PN/Amyo – CI SPG28, SPG30, SPG60, SPG68 SPG75/ MAG No SPG: LYST ARSACS

Figure (4-6): Suggested flow chart designed to assist in the variant/gene prioritization process of gene panel data (based on inheritance and the most frequent clinical presentations). P: pure, C: complex, AR: autosomal recessive, AD: autosomal dominant, PN: peripheral neuropathy, Amyo: amyotrophy, TCC:thin corpus callosum, CI:cognitive impairment, OA: optic atrophy, RD: retinal degeneration, MD: macular degeneration.

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4.3. Comparison of the yield of genetic results As described earlier, the overall success rate was 41% whereas in 24/41 families (59%) the cause could not be identified. In 5/41 families (12%), the causing genes were not associated with the disease before (potential new genes).The genes identified were known in 12/41 families (29%).

This power went significantly higher with the use of WES as it reached 57% (3.5x the diagnostic kit approach and 2.7x the combined screening methods approach). This could be explained by the percentage of families with potentially new genes causing the disease which was 36% of the diagnosed families (5/14 families). The failure rate of the WES approach was 43% and this could be attributed to the fact that in these families the variant causing the disease was located in a noncoding/intronic region or a compound heterozygous with one of the heterozygous mutations present in a badly covered region or a noncoding region. In all cases genomic rearrangements could not be completely excluded though preliminary results on coverage evaluation in WES data showed that they were unlikely. The rates are comparable to what has been reported about success rates in other studies [Figure (4-7)] (Novarino et al., 2014).

Figure (4-7): Comparison of the success rates of approaches that were utilized in genetic investigation. WES: whole exome sequencing, HSP:hereditary spastic paraplegia.

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4.4. Towards the identification of new genes We identified variants in eight new genes corresponding to five novel genetic isoforms of HSP. Although additional genetic and functional data are required to definitely validate their causality on the spastic paraplegia segregating in patients, the function of some of them can be linked to physiopathology of HSP.

4.4.1. ABHD16A and complex TCC HSP in family F37 (The flip coin of ABHD12/PHARC) The single mutation segregating in family F37 is a loss of function mutation in ABHD16A.

 Gene/protein isoforms and splice variants of ABHD16A

This gene has 16 transcripts (splice variants) but only two are protein coding (two isoforms)

NM_021160 is the transcript that codes for the canonical isoform1 (identifier: O95870-1) which is composed of 20 exons and is 558 amino acid long and 63,243 in Mass (Da).

 Function Abhydrolase domain containing 16A (ABHD16A) is a phosphatidylserine (PS) lipase that generates lyso-PS in mammalian systems. It has been suggested that it is the main PS lipase in the CNS (brain and spinal cord). Lysophosphatidylserines (lyso-PSs) are a category of lipids that are involved in regulation of immunological processes (Kamat et al., 2015). ABHD16A is localized on the cytoplasmic face of cellular membranes. This is consistent with its role as a PS lipase, PS are mainly confined to the inner part of the lipid bilayer in healthy cells (Kamat et al., 2015). ABHD16A represent the one of the two pillars of the ABHD16A/ABHD12 axis [Figure (4-8)]. This axis dynamically regulates lyso-PS metabolism in vivo ( Kim, 2015) (Kamat et al., 2015). Lyso-PS is generated by ABHD16A and becomes accessible to hydrolytic degradation by ABHD12 (OMIM *613599). ABHD12 is an integral membrane protein whose active site is predicted to face the lumen/extracellular space

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(Blankman et al., 2007). Disruption of ABHD12 and ABHD16A in mouse macrophages causes an increase and a decrease in both lipopolysaccharide-induced cytokine production and lyso-PSs, respectively. C18:0 lyso-PS is a lyso-PS that is considered to be the most abundant species. It is secreted by macrophages and potentiates the above mentioned release of cytokines (Kamat et al., 2015).

Figure (4-8): Schematic representation of the ABHD12/ABHD16A axis (Kim, 2015). Nonsense mutation causing TCC HSP in family F37 mimics a knock out of ABHD16A (added to the illustration).

As expected, ABHD16A−/− Knockout mice have decreased brain lyso-PSs whereas ABHD12−/− Knockout mice have increased brain lyso-PSs. ABHD16A−/− mice were born at a much lower frequency than expected for Mendelian distribution. ≈30% of the knock out mice are smaller than the ABHD16A+/+ and +/− mice. Nevertheless, they appeared normal in their cage behavior with no observed evidence of increased postnatal lethality in these animals. ABHD12 has been associated with PHARC (OMIM #612674) (PHARC stands for Polyneuropathy, Hearing loss, Ataxia, Retinitis pigmentosa, and cataract) a rare syndrome of AR complex HA (Fiskerstrand et al. (2010). PHARC is a neurodegenerative syndrome presumably attributed to a neuroinflammatory process

174 accompanying the elevated level of lyso-PS and the production of the lipopolysaccharide-induced cytokines. The phenotypes associated with ABHD12 were later expanded to nonsyndromic presentation (Nishiguchi et al. (2014)). On the contrary to ABHD12 related clinical phenotypes (Kamat et al., 2015), loss of function mutation in the Sudanese family [ABHD16A (-/-)] lead to complex HSP with TCC with no evidence of cerebellar involvement. If the PHARC is due to an exaggerated neuroinflammatory response due to accumulation of lyso-PS, the question asked is why the decrease in lyso-PS results in TCC HSP. Lyso-PS is necessary for [Figure (4-9)]:

- mast cell degranulation-stimulating activity. This is not induced by other lysophospholipids LysoGPs including lysophosphatidic acid (LPA), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylglycerol (LPG), and lysophosphatidylinositol (LPI). It strictly requires the serine residue of LysoPS. It also strictly requires the structure of the serine residue of LysoPS. Modification of the serine residue entirely abolishes the degranulation-stimulating activity of the mast cells (Makide et al, 2014). Whether this is part of the phenotype in patient remain to be explored either by the study of macrophages from patients or by knock down experiments in macrophage strains in vitro. - Nerve Growth Factor (NGF)-induced neurite outgrowth in PC12 cells. Neurite outgrowth is one of the important pathways involved in HSP. It is the major function involved in the X-linked SPG1 and involved in axonal guidance. The presentation of SPG1 resembles to some extent the presentation of our family as both are associated with TCC (hypoplasia of CC) and mental retardation. Neurite outgrowth is also indirectly associated with the functions of many of the spastic paraplegia proteins. Lyso-PS is required for nerve growth factor (NGF)-induced secretion of histamine from rat mast cells. Lyso-PS alone is ineffective in that process but strongly promoted the NGF-induced differentiation. Lyso-PS colocalizes with NGF in sites of tissue damage and therefore may be a naturally-occurring modifier of neuronal structure and/or function (Lourenssen and Blennerhassett, 1998).

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Figure (4-9): Biological functions of lysophosphatidyl serine (LysoPS) (Makide et al, 2014).

- Lyso-PS also regulates cytochrome P450 activity. Lyso-PS interaction with P450 1A2 (CYP1A2) and 2E1 (CYP2E1) increases their phospholipase D (PLD) activity in a concentration-dependent manner and decreases the monooxygenase (MMO) activity of both CYPs (Cho EY et al., 2008). Of note, cytochrome P450 activity was recently linked to SPG49/56 (CYP2U1) which is also associated with TCC and mental impairment (Tesson et al., 2012). - Lyso-PS (C18:1/OH lyso-PLs bearing the phosphoserine) also enhances apoptotic cell-engulfment by macrophages by activation of human neutrophil through binding and mobilization of G2A latent in the plasma membrane/secretory vesicle fraction ( Frasch et al., 2007) It is quite evident that lyso-PS induces various cellular responses in a Lyso-PS- specific manner, these actions may be mediated by different Lyso-PS receptors (Makide et al., 2014). - stimulates migration of fibroblasts (Park et al., 2006).

 Potential Treatment ? As was speculated that PHARC can be treated by inhibition of ABHD16A to decrease the Lyso-PS level. ABHD16A associated TCC HSP could be potentially treated by blocking the degradation of lyso-PS by ABHD12 or possibly by blocking

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the ABHD16A-counteracting acyltransferase enzyme. Unfortunately to present no selective inhibitor of ABHD12 has been characterized (Hoover et al., 2008) (Navia- Paldanius et al., 2012). Tetrahydrolipstatin (THL/orlistat) is a drug of clinical utility as an anti-obesity agent as it functions as a pancreatic lipases blocker in the intestine. It has an inhibitory effect on ABHD12 but it is non-selective. It exerts its inhibition on other enzymes including ABHD16A (Hoover et al., 2008). Another inhibitor, KC02, has significant inhibitory effect on ABHD12 but has two partial off-targets which are ABHD11 (94%) and LYPLA1 (63%) (Kamat et al., 2015). In conclusion, the genetic and function data strongly support the implication of the loss of function mutations in ABHD16A in family F37. Further exploration of fibroblasts and macrophages of patients would be necessary to provide cellular functional argument in support of what we have hypothesized as underlying mechanisms.

4.4.2. CAMSAP3, MINK1, ZNF334 candidate genes in family F41 Three missense variants with convincing genetic arguments are segregating with the disease in family F41. Additional biological data, from fibroblasts of patients, will be required to determine which mutation is responsible for the phenotype. The function of these 3 genes is also informative and will define which mechanisms to carefully look for in the fibroblasts.

4.4.2.1. CAMPSAP3

This gene has 4 transcripts (splice variants) but only two are protein coding

NM_020902.1 canonical transcript. It codes for the canonical isoform1 (identifier: Q9P1Y5-1) which is composed of 17 exons and is 1,249 amino acid long and 134,750 in Mass (Da).

NM_001080429.2 is the longest transcript. It codes for isoform 2 (identifier: Q9P1Y5-2) which is composed of 19 exons and is 1,276 amino acid long and 137,878 in Mass (Da).

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 Mouse model

Interestingly a knockout (KO)mouse model was found (C57BL/6NTac/Camsap3tm1a(EUCOMM)Wtsi). The preliminary data in the knock out mouse model showed eye morphology signs as fused cornea and lens, abnormal retinal pigmentation and persistence of hyaloid vascular system. As well as dysmorphlogy signs as abnormal coat/hair pigmentation abnormal snout morphology, asymmetric snout and malocclusion (https://www.mousephenotype.org/data/genes/MGI:1916947#order2). As these were provisional data, verification for more phenotypic details may be needed.

In our patients there were skin pigmentations that resembled the “café au lait” spots, cataract, some dysmorphic features including protrusion of upper and lower jaws and malocclusion of mouth. These features are then reminiscent of what is described in the KO mouse model.

 Expression

CAMSAP3 is a cytoplasmic protein mainly localized along the zonula adherens (Meng et al., 2008). Very high expression was detected in adult and fetal brain and liver in addition to adult ovary, kidney, testis and pancreas. Very high expression was also detected in all regions examined of the adult brain. There is low expression in adult heart, lung, skeletal muscle, spleen, and spinal cord (Nagase et al., 2000).

 Identification and structure of CAMSAP3

No crystal structure is available at present. Interestingly, CAMSAP3 protein was identified de novo and renamed several times:

First it was identified as KIAA1543, which was named Nezha after a deity in Chinese mythology, as a PLEKHA7-binding protein. The human Nezha protein contained an N-terminal calponin homology domain (CH domain), two central coiled-coil regions, and a DUF1781 domain at the C-terminal end of the protein. In human Caco-2 cell line, major and minor Nezha proteins were detected using western blot analysis. They had molecular masses around 150 and 160 kD, respectively (Meng et al., 2008).

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Later, KIAA1543 was re-identified and called CAMSAP3. The deduced protein contains a calponin homology domain, 2 coiled-coil regions, followed by a proline-rich region, a coiled-coil region, and a C-terminal DUF1781 domain. The DUF1781 domain was renamed as the CKK domain, since it is found in KIAA1078 (CAMSAP1L1), CAMSAP1 and KIAA1543 (CAMSAP3) (Baines et al., 2009).

Zheng et al. identified it as marshalin. The protein was shown to have multiple protein interacting domains including a C-terminal CKK domain (DUF1781) and an N-terminal calponin homology (CH) domain, and three coiled-coil (CC) and two proline-rich (PR) domains in the middle (Zheng et al., 2013)

The protein was also independently discovered and named patronin by another group (Goodwin and Vale, 2010).

 Function

CAMSAP3 acts as a microtubule minus-end binding protein. It acts as a regulator of non-centrosomal microtubule dynamics and organization. It is required for the biogenesis and the maintenance of zonula adherens. It anchors the minus-end of microtubules to zonula adherens. It does so by recruiting the kinesin KIFC3 to the junctional sites. CAMSAP3 may regulate the nucleation and the polymerization of microtubules (Meng et al., 2008). CAMSAP3 was later found to bind to the minus-end of MTs through its CKK domain (Baines et al., 2009). Indirectly, through the microtubule cytoskeleton, the protein may regulate the organization of cellular organelles including the Golgi and the early endosomes (genecards). Based upon differences in patterns of expression of marshalin seen in the organ of Corti (sense organ for hearing), eight isoforms were identified. Their size ranged from 863 to 1280 amino acids. Marshalin-L and marshalin-S isoforms induced different MT-bundle structures and/or composition. The two isoforms differ by 416 aa, which include protein- protein interaction domains CC and PR (Zheng et al, 2013). The Golgi network, normally located near the nucleus, was found to be disrupted and separated into fragments in cells expressing marshalin-Ld. These Golgi-

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membrane fragments colocalized with marshalin protein. This was justified by the fact that the distribution and the localization of the Golgi network is connected closely to the cytoskeleton (Sandoval et al., 1984), Golgi disruption could be due to the changes in MTs which were caused by marshalin-Ld expression. Marshalin had multiple isoforms. It acted as a scaffolding protein and was capable of modifying the cytoskeletal networks, and consequently organelle positioning like Golgi apparatus, through interactions with various protein partners present in different cells (Zheng et al., 2013). This is not the first gene in which microtubules and Golgi apparatus are associated with complex spastic paraplegia. Microtubules are involved in the pathogenesis of some HSP subtypes, particularly SPG4. Subcellular organelles and more specifically the Golgi apparatus (as part of the ER-Golgi axis) are behind the occurrence of SPG21, SPG57, SPG58 and SPG associated with FAM134B. Further functional studies will illustrate the real mechanisms of CAMSAP3 that we can consider to represent a good candidate in family 41.

4.4.2.2. MINK1 MINK1 (Misshapen-like kinase1) is a germinal center kinase. It has been found that MINK1 is composed of a kinase domain at the N-terminus, a proline-rich region, and a GCK homology region at the C-terminus. It is highly expressed in the developing mouse brain (Dan et al., 2000). MINK1 is involved in several crucial biological roles in the cells. It has been found to be necessary for completion of cytokinesis (Hyodo et al., 2012). It plays a role in the activation of p38 pathways and cJun N-terminal kinase (JNK) (Dan et al., 2000). MINK1 is involved in cell motility and reorganization of cytoskeleton. It colocalized with golgi apparatus (Hu et al., 2004). Cytokinesis has been associated with HSP (Hazan et al., 1999) as well as with the main role of some HSP genes in organization of cytoskeleton.

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4.4.2.3. ZNF433 Little is known about ZNF433. It is shown to be expressed in the cerebral cortex (http://www.proteinatlas.org/ENSG00000197647-ZNF433/tissue). In one study it has been shown that ZNF433 together with another new gene VAV2 have suggestive evidence of association with multiple sclerosis but did not show genome-wide significance level (Nischwitz et al., 2010).

4.4.2.4. Conclusion The CAMSAP3 mouse phenotype similarity to some features we have had in our patients supports its role in pathogenesis, rather than MINK1. Knockdown and rescue experiments in zebrafish could help to determine which variant is causal.

4.4.3. BIRC5 and C21ORF91 variants in family F50

4.4.3.1. BIRC5 Two missense variants with convincing genetic arguments are segregating with the disease in family F50. Additional biological data, based on the known function of the genes, is required to determine which mutation is responsible for the phenotype. Fibroblasts of patients will be used for such exploration.

 Gene/protein isoforms and splice variants of BIRC5 BIRC5 has 11 transcripts of which only 6 are protein coding. The canonical isoform 1 of the protein is 142 amino acids in length and is the product of the transcript NM_001168.2 (4 exons). The longest transcript (NM_001012271.1 ) is composed of 5 exons and encodes for variant 3/ isoform 3 of the protein with 165 aa also known as Survivin 28 but is not well characterized.

 Biochemical structure of survivin Survivin is considered the smallest member of the proteins family of inhibitor of apoptosis (IAP). Unlike other members of IAP family it contains only one N-terminal Baculovirus IAP repeat (BIR) domain and lacks the C-terminal ring finger domain. This domain is necessary for ubiquitination dependant proteosomal degradation of caspases (Srinivasula, S. M. & Ashwell, 2008). In survivin, a c-terminal coiled-coil α-helix domain replaces the ring finger

181 domain, which in turn is responsible for its regulation of cell division (Wheatley, S. P, 2015).

The overall shape of a survivin has been studied using x-ray crystallography (Chantalat et al., 2000) (Muchmore et al., 2000). The BIR and Helix domains has been found packed tightly together. The BIR domain is characterized by a zinc-coordinating Cys/His motif shown to be stabilized by hydrophobic interactions involving the amino acid residues: F22, W25, F27, M38, F43, F58, L64, W67, and F86. The BIR domain in survivin is used for homodimerization and interaction with other chromosome passenger proteins (Verdecia et al., 2000). The BIR motif as well plays an important antiapoptotic role (Sun et al., 1999).

The mutation we report here changes the above mentioned phenylalanine (F86) into leucine (L86). Although both amino acids are branched hydrophobic with small physiochemical difference, leucine lacks the aromatic ring of phenylalanine and this could well modify the crystal structure of the survivin protein. Further modeling workup of the effect of the p.Phe86Leu is required to demonstrate if that change will disrupt the above mentioned hydrophobic interactions and affects the stability of the core of the zinc finger domain with alteration of its functions.

Function

BIRC5/Survivin is a multitasking protein with a myriad of protein-protein interactions and then with potentially multiple functions. It has dual roles in preventing apoptosis and promoting cell mitotic proliferation. It has been demonstrated that survivin cannot inhibit apoptosis by directly binding to caspases under physiological conditions (Eckelman, 2006). However, it inhibits apoptosis indirectly through interaction with both pro- and antiapoptotic proteins that promotes cell survival (Dohi, et al, 2004) (Ceballos-Cancino et al., 2007).

Survivin is possibly implicated in the process of DNA repair (Chakravarti, et al., 2004). The multiplicy of survivin functions have been attributed in part to differences in cells types and the variable subcellular localization of the

182 protein (Altieri, D., 2008). It has been postulated that mitochondrial Survivin is associated to antiapoptotic function and nuclear survivin has been connected to the role in mitosis and protection against ionizing radiation (Colnaghi, et al., 2006).

Survivin has a short half-life of about 30 minutes (Zhao et al., 2000). Its expression is dynamic and can be modified in response to many intracellular and extracellular stimuli. Examples for factors that can alter survivin expression include cell cycle stage (Altieri, 2006) cytokines ( Mahboubi et al. , 2001) hormones (Nabilsi et al., 2010), certain bacteria (Valenzuela et al., 2013) noise (Knauer et al., 2010),hypoxia (Conway et al., 2003), medications (Cheng et al., 2014), radiation (Dallaglio et al., 2012), and DNA damage (Capalbo et al., 2010) among others.

Targeted deletion of survivin gene in neural progenitor cells in mouse developing brain has shown that survivin is critically important for early CNS development (Jiang et al., 2005). This supports the suggested effect of the p.Phe86Leu incriminated in the pathogenesis of F50 with early onset at 1-2 years of age. Further accumulating evidence supports the possible involvement of survivin in causing neurological disorder with complex and severe manifestations. Deletion of survivin gene is associated with apoptosis which is severe and multifocal involving the retina and many regions of the central nervous system including the spinal cord, cerebrum, cerebellum and brain steam (Jiang et al., 2005). In a transgenic mouse model, postnatal expression of survivin was detected in two key sites of adult neurogenesis (the subventricular zone (SVZ) and the subgranular zone (SGZ)) (Coremans et al., 2010). Lack of expression of survivin in the neural precursor cells during embryonic development, was associated with profound postnatal defects in neurogenesis and loss of interneurons that manifest by major deficits in learning and memory, and augmented sensitivity to seizures (Coremans et al., 2010). In human adults survivin expression has been detected in the cochlea and its expression was found to be dysregulated in a transcriptomic study in HSP patients with SPAST mutations (Abrahamsen et al., 2015). The study found that in the patient derived cells, there was a major dysregulation of gene expression with 57% of all mRNA transcripts affected including BIRC5 among

183 other genes with functions related to microtubules dynamics (Abrahamsen et al., 2015). All this goes in concert with the presentation of our two sibs with complicated HSP. From another point of view, defective DNA repair may be one of the mechanisms by which survivin is implicated in causing HSP complicated with cerebellar syndrome. Compared to other tissues, the developing brain is particularly sensitive to DNA damage (O'Driscoll and Jeggo, 2008). Neurons are metabolically active and are susceptible to DNA oxidative damage (Jackson and Bartek, 2009). Accumulation of DNA damage in the terminally differentiated, irreplaceable neurons increases the vulnerability of the nervous system to DNA damaging insults (Jackson and Bartek, 2009) . Furthermore, being in G0, neurons do not repair DNA double strand breaks by homologous recombination and use the more error prone non-homologous end joining (NHEJ) mechanism (Jackson and Bartek, 2009).

Defects in DNA repair mechanisms can usually present with problems in the nervous, reproductive and immune systems in addition to premature aging and increased susceptibility to cancer (Ciccia and Elledge, 2010). Neurons of the cerebellum account for roughly 50% of brain neurons (Katyal & McKinnon , 2008). Many defective DNA repair syndromes present with degeneration of cerebellar neurons (Ciccia and Elledge, 2010).

Survivin is thought to have a role in DNA double strand repair mechanism, particularly non-homologous end joining (NHEJ) repair machinery through accumulation of survivin into the nucleus and its physical interaction with the mediator of DNA damage checkpoint protein 1 (MDC1), the histone protein γ- H2AX in addition to the DNA dependant protein kinase catalytic subunit (DNAPKcs) (Capalbo et al., 2010) .

Survivin, now known to be a component of the chromosome passage protein complex (CPC) which is crucial for chromosome alignment and segregation during cytokinesis and mitosis (Lens et al., 2006). Although its role as promitotic was particularly proved in cancer cells, we can never ignore the fact that this may apply to the highly proliferating fetal cell and this may have contributed to the importance of survivin for the survival of developing central nervous system (Jiang et al., 2005). Further cellular and functional studies will

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allow us to shed light on the role of survivin that lead to HSP in the two sibs opening a new passage between cancer and neurodegeneration.

4.4.3.2. C21orf91  Gene/protein isoforms and splice variants of C21orf91 It codes for the protein EURL homolog. The gene has 6 transcripts (splice variants) but only four are protein coding.

NM_001100420 is the transcript that codes for the canonical isoform1 (identifier: Q9NYK6-1). The transcript is composed of 5 coding exons and is 297 amino acid long and 33,948 in Mass (Da).

 Function Not much is known about C21ORF91 and the protein EURL homolog function. The protein is a cytosolic protein with unknown function. The gene was found to be strongly correlated with the infection with Herpes simplex labialis (cold sore) (Kriesel et al., 2011). In a study about hepatocellular carcinoma, C21ORF91 was found as a target for miR-194 among a few other genes (Bao et al., 2015). In a genome-wide association study about conducted in African Americans to identify candidate genes associated with systolic and diastolic blood pressure and (SBP/DBP). rs2258119 in the gene C21orf91transmitted the strongest signal for SBP (Fox et al., 2011)

4.4.3.3. Conclusion Given what is known about the functions of the 2 candidates genes, the genetic and functional elements are in support of the involvement of BIRC5 variant in HSP pathology in family F50.

4.4.4. ST7L unique mutation responsible for the pathology in family FM3

In family FM3, a single rare and predicted pathogenic variant was segregating, in ST7L.

This gene has 28 transcripts (splice variants). Of them 8 are protein coding (for 8 isoforms produced by alternative splicing) (Uniprot: Q8TDW4).

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NM_017744.4 longest transcript: which is composed of 15 exons and codes for the canonical isoform 1 (Uniprot identifier: Q8TDW4-1). The isoform is 575 amino acids and is 64,779 Da in Mass.

Unfortunately little is known about the structure and the functions of ST7L. It is considered a multi-pass membrane protein as it is predicted to be composed of two helical transmembrane domains. (Uniprot: Q8TDW4).

With the use of northern blotting ST7R mRNA was found in various normal tissues, and especially large amounts in testis.

In the Fantom 5 project (fetal) ST7L was found relatively highly expressed in the spinal cord, various regions of the brain (occipital, parietal and temporal lobes), skeletal muscles and the eye. The Fantom 5 project (adult) showed that the gene expression expanded to involve the cerebellum, basal ganglia, diencephalon and thalamus. It was not detected in the eye, skeletal muscle and temporal lobe (http://www.ensembl.org/Homo_sapiens/Gene/ExpressionAtlas?db=core;g=ENS G00000076826;r=19:7595902-7618304).

Broadly speaking, ST7L seems involved in negative regulation of cell growth (GO:0030308).

In a single study, luciferase reporter assay provided evidence that the expression of ST7L is suppressed by overexpression of miR-24 and that led to enhanced cell proliferation and invasion. The study demonstrated through sequence analysis that miR-24 could form complementary base pairing with the 3 prime UTR region of ST7L. Apoptosis assay performed showed that the apoptotic cell fractions were significantly increased when ST7L was restored compared to control cells, as observed after miR-24 suppression. The hypothesis suggested that ST7L decreases cell proliferation and increased apoptosis. The tumor suppressor effects of ST7L were in part mediated by negative regulation of b- catenin/Tcf4 signaling (Chen et al., 2013).

Murine erythroleukemia (MEL) cells were used to study erythroid differentiation due to their ability of in vitro terminal differentiation in response to chemical induction. ST7L was identified among several genes that could be 186

used as markers for erythropoiesis with the use of semi-quantitative qPCR. They suggested that these genetic markers could act as potential regulators in cellular functional studies of erythroid differentiation, or studied as straightforward cell type marker. (Heo et al., 2005)

The onset of the disease in family FM3 was just after birth and in one individual the onset was suggested to be prenatal implying a defect in development occurring in the proliferating neuronal cell lines. Many neurological disorders especially leukodystrophies have been associated with haemopoeitic cancer. In some (adrenoleukodystrophy) stem cell therapy may help to improve even the neurological symptoms and signs.

In the absence of other variants or rearrangement, we can reasonably consider the ST7L variant as causative in the family but further biological elements are required to firmly validate its involvement in the pathology and decipher the underlying mechanism.

4.4.5. PANK4 unique variant in branch1 of family FM7 In branch 1 of family FM7, the single rare and pathogenic variant remaining after bioinformatics filtering was found in PANK4. The variant did not segregate in branch 2 of family FM7 and thus we hypothesize that it was not involved in causation of the disease in this branch. Although both branches had ataxia and hearing defect and mutism, we could distinguish them as two different phenotypes. As mentioned earlier this is not the only example of extended family in which patients from different branches present with closely related clinic-genetic entities. In family F1 spasticity and ataxia were shared clinical features in all four branches (A, B, C and D). SPG11 was found to cause the disease in patients of branch A/B with TCC HSP but not in the relatives branch C and D for whom the natural history was different (Elsayed et al., under revision).

 Gene/protein isoforms and splice variants

This gene has 12 transcripts (splice variants) but only two are protein coding. NM_018216 is the transcript that generate the canonical isoform1 (identifier:

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Q9NVE7-1) which is composed of 19 exons and is 773 amino acid long and 85,991 in Mass (Da).

PanK4 belongs to the type II pantothenate kinase family. Unfortunately no crystal structure is available.

 Pantothenate kinases expression and function

There are four isoforms of pantothenate kinase: PanK1, PanK2, PanK3 and PanK4 (Li et al., 2005).

PanK2 is expressed ubiquitously, with high levels in retinal and infant basal ganglia PanK1 is found predominantly in heart, liver and kidney; PanK3 is limited to the liver, but expressed at a high level. The expression of PanK4 is ubiquitous, with a high level in muscles [Figure (4-1)] (Zhou B et al., 2001) PanK1, PanK3 and PanK4 isoforms are cytosolic whereas PanK2 is mitochondrial. Human PanK2 is also detected in the nucleus (Alfonso-Pecchio et al., 2012).

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Figure (4-10): Expression of pantothenate kinases (PanK 1, PanK2, PanK3 and PanK4) (Zhou et al, 2001)

The pantothenate kinases (PanK) catalyze a rate limiting step (the first committed step) in coenzyme A (CoA) biosynthesis. Coenzyme A (CoA) is a cofactor that plays a critical role in intermediary metabolism in all organisms. It functions as a carrier for carboxylic acid substrates. It supports multiple essential biochemical pathways which include the tricarboxylic acid cycle (TCA), biosynthesis of sterol, metabolism of amino acid, and both anabolic and catabolic pathways of fatty acids and complex lipids (Leonardi et al., 2005).

Although putative PanK4 was claimed to be catalytically inactive (Zhang et al., 2007), evidence is progressively accumulating for its in vivo activity. It was shown in Drosophila that human cytosolic PanK3 and PanK4 could mostly, but not fully, rescue Pank2/Fbl lack. PanK2 is more potent than the two cytosolic PanK4 and PanK3 in vivo but the ability of either PanK3 or PanK4 to significantly complement Pank2/fbl mutation indicated they were both functional pantothenate kinases. Furthermore, it was shown that the human PANK4 gene can complement mutant of E.coli deficient in pantothenate kinase. This reflects the high degree of conservation of the pantothenate kinase and thus provides evidence for a probable compensatory mechanism in PKAN patients (Hӧrtnagel et al., 2003).

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PanK4 has been shown to be upregulated in glucose-challenged rat muscles suggesting that PanK4 expression is stimulated to allow flux at a higher rate through the TCA (Li et al., 2005).

The role that carbohydate metabolism plays in normal brain development and proper function can’t be ignored. This has been extensively studied over decades. Glucose is an essential energy source for the adult human brain. In the adult brain, neurons have the highest energy requirements (Dienel, 2012). Neurons as well as cancer cells are among the cell types that depend on glucose metabolism almost completely for energy generation.

Tight regulation of glucose metabolism is essential for brain physiology. Disruption of normal glucose metabolism and its correlation with cell death pathways forms the basis for many brain disorders. Not only glucose is required for provision of the ATP as energy source for their actions and for production of the precursors for neurotransmitter synthesis but also to satisfy the brain’s energy demands. Synaptic activity accounts for most of the energy consumption of the brain (Mergenthaler et al., 2013). The human cortex alone requires approximately 3×1023 ATP /s/m3 [Dienel, 2012].

Aerobic glycolysis (AG) was shown to support developmental processes, and especially important for synapse growth and formation. Meta-analysis of brain glucose and oxygen metabolism studies demonstrated that AG increases during childhood, corresponding to the highest synaptic growth. High AG, was found to correlate with increased expression of genes related to formation and growth of synapses. In contrast, regions with elevated oxidative glucose metabolism showed high expression of genes more related to mitochondria and synaptic transmission (Goyal et al., 2014)

Interestingly, paralog PANK2 is incriminated in Hallervorden-Spatz syndrome (HSS)/ pantothenate kinase associated neurodegeneration (PKAN). HSS is an AR neurodegenerative disorder associated with brain iron accumulation. Histologic studies for HSS reveals iron accumulation in basal ganglia via secondary metabolite accumulation. The differential expression of PanK genes discussed earlier may explain why PANK2 causes HSS.

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In family FM7 branch 1, the affected sibs presented with developmental defect and age at onset since birth. We therefore suggest a different mechanism behind the pathogenesis of the disorder. PanK4 has been associated with glucose metabolism in muscles in addition to its role partially compensating PanK2 deficiency. In all cases Pank4 is highly unexplored and a lot of functional work may be required to prove this hypothetical role in development and to explain the variable clinical severity witnessed in our five affected siblings. Zebrafish modeling may represent a good opportunity to explore the normal and pathological functions at the same time.

4.5. Cellular pathogenic mechanisms proposed for the novel genes: a resumé In the following paragraph we will highlight the pathways proposed earlier for the above mentioned novel genes.

4.5.1. Microtubules/cytoskeletal related cellular mechanisms Microtubules is a major component of the cytoskeleton that support the subcellular organization of organelles especially Golgi apparatus. Disruption of the organization and dynamics of the microtubules is the proposed pathogenetic mechanism for CAMSAP3. BIRC5 is proposed to cause the disease through antiapoptotic, promitotic and DNA repair associated with microtubules formation and dynamics. MINK1 has been associated with organization of cell cytoskeleton and motility.

4.5.2. Neurite outgrowth This mechanism is the major process for neuronal cell formation and development of CNS. It is involved in a family while a mutation in ABHD16A together with possible effects on neuroinflammatory response.

4.5.3. Glucose metabolism Glucose metabolism and defect in synthesis of CoA is suggested as the mechanism behind PanK4 despite the lack of sufficient studies. Its paralog, Pank2 is responsible for another neurodegenerative condition.

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4.5.4. Apoptosis and regulation of cell growth Minimal data and studies are available about ST7L. The only evidence designates ST7L as proapoptotic and negative regulator of cell growth (decreases cell proliferation).

4.5.5. Cytokinesis MINK1 has been shown to plays a crucial role in many cellular processes including cytokinesis, a process overlapping microtubules/cytoskeletal dynamics.

4.6. A comprehensive overview of pathogenetic Mechanisms of HSP The rapidly expanding surge of discovery of new genes revealed that we can just see the top of the iceberg and that we are lagging far beyond the appreciation of the full picture that links the disorders along the spectrum of the neurogenetic disorders and their mechanisms. As mentioned earlier multiple trials have been done to regroup and create categories of the functions attributed to HSP genes and their protein products. This is always not an easy task as HSP genes/proteins have been implicated in a wide variety of functions and what is unknown seems to exceed what we managed to disclose. In this section an attempt is done to create a modified version of functional categorization [Figure (4-11)] [Table (4-2)]. Around 11 major functional themes can be distinguished based on the known ultimate role HSP genes play/modify in the cell. Most of the HSP genes/proteins participate in 1 to 3 functional themes [Figure (4-11)]. An overall primary and a secondary (when relevant) functional category has been assigned to each HSP protein to summarize the principal functional theme of the SPG gene/protein [Table (4-2)]. As the phenotypic features has been almost exhaustively listed (Annexe Tables 1-11), a step further has been taken to observe the possible links between the pathways and the clinical presentation (pure versus complex), signs, age at onset and patterns of inheritance. This has been done using descriptive observations with basic statistical regression analysis to create provisional correlations. In this regression, the functional themes have been considered as the independent variables (X) and the phenotypes and signs have been treated as the dependent variables (Y). The statistical significance is set as P-value < 0.05. It is quite clear that this analysis will just provide basic crude correlations but it can be regarded as

192 the first step to deduce preliminary clues to guide a second more specialized analysis with more functional data (cell types where the gene is expressed, putative function based on partners of these proteins…) which if accompanied with better fine tuning of the data analysed and may lead to more conclusive results. A problem encountered is the small number of forms included in certain functional categories. This point of weakness may suggest that some biased conclusions may occur from a statistical point of view.

4.6.1. General Observations:  Age at onset: With the exception of the tendency for early onset associated with lipid and nucleotide metabolism, age at onset is not obviously linked to any functional category. This might well be due to the role of all HSP genes during the development of the nervous system.

 Mode of inheritance: - AR forms are observed to predominate the metabolic pathways (lipids and nucleotide metabolism) as well as degradation pathways and endomembrane trafficking. - AD HSP are mainly clustering in the organelle shaping, development, microtubule dynamics and active cellular transport (often with mixed transmission) functional categories.

4.6.2. Correlations:

4.6.2.1. Microtubules dynamics, Development, Amino acid metabolism, Transport and Organelle shaping: Significant correlation exists [Figure (4-12)] between microtubules dynamics and three other functions: the development, organellar shaping and transport. This can be well explained by the role that the microtubules play in the neurons with regard to these functions:

1. MTs keep the organellar organization and thus maintain the shape of the subcellular organelles (Zheng et al., 2013) 2. MTs play an essential role in neuronal development through various models that have been suggested including the role of MTs in polarized 193

axonal transport (Faivre et al., 1985) (Dent et al., 1999) (Brown, 2000) (Nakata and Hirokawa , 2003)( Witte et al., 2008) (Hoogenraad and Bradke, 2009). In addition, MTs contribute to the neuronal and synaptic plasticity in the process of memory and learning (Penazzi et al., 2016)

On a clinical point of view, microtubule dynamics shows significant correlation with PN and deafness. The role of microtubules in AD HSP (SPG4, SPG10 and SPG31), which are mainly complicated with PN, can explain this association. The correlation of MT dynamics and hearing loss has been described in many studies about deafness and its genetic causes (Hasson, 1997)

Deafness, on the other hand, is significantly correlated with cataract, although linked to amino acid metabolism and organelle shaping but not to microtubule dynamics. As cataract is a common clinical feature in IEM, this makes the link between amino acid and metabolism logical.

4.6.2.2. Transport and microtubule dynamics: Cellular transport is linked to the pure phenotype. This may well be because of the predominance of the AD forms where the pure presentation is the commonest.

4.6.2.3. Endomembrane trafficking: Significant links can be observed between CI and other phenotypes including TCC, PN, cerebellar ataxia as well as with the complex presentation per se. This observation is expected given the fact that these are the commonest complicating signs in HSP as mentioned earlier in the introduction (Annex 1-11). Interestingly, CI has shown significant correlation with the endomembrane trafficking.

Endomembrane trafficking is associated with 14 HSP forms. all with a complex presentation with five forms rarely presenting as pure HSP (SPG11, SPG15, SPG48, SPG6 and SPG8). Moreover, although both AD and AR inheritance can be observed in this category, the AR HSPs are predominant (11/14 forms).

There is obvious overlap between the degradation pathway and the endomembrane trafficking. This overlap occurs in five HSP forms [SPG11, SPG15, SPG48/AP5Z1, SPG59/USP8 and VCP spastic paraplegia].

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4.6.2.4. Degradation pathways: Of the 10 forms associated with degradation pathway, one form presents as pure HSP and nine are complex (three of them has an alternative pure phenotype). The majority had AR inheritance (9/10) and present cerebellar ataxia (6/10).

The association between cerebellar syndrome and the degradation pathways is well known in the pathogenesis of AD cerebellar ataxias. Degradation of cellular inclusion is the basis of many types of HAs especially the polyglutamine AD cerebellar ataxias (PolyQ: SCA). In these disorders, the proteasomal and lysosomal degradation pathways are the main cellular defense mechanisms against the aggregates (the hallmark of the polyQ disorders) (Seidel et al., 2015) (Mori et al., 2016)

4.6.2.5. Mitochondrial functions: Out of 12 forms associated with mitochondrial functions, 11 have complex, with four having additional pure phenotypes.

There is no apparent tendency for association with certain age at onset or inheritance pattern. But there is statistically significant association with optic atrophy (p value : 0.007) and PN with amytrophy. This apparently matches what is known about the association in many mitochondrial inherited disorders between mitochondrial dysfunction and optic atrophy.

4.6.2.6. Lipid metabolism influence on myelination: An obvious but interesting statistically significant observation with well known theoretical background is the effect of lipid metabolism on myelin. Such observations that emerged upon analysis would give a positive control to give the remaining links some sort of confidence.

4.6.2.7. “Take home” message: Not all functions of the HSP proteins are known. Based on the state of the art in 2016, it is clear that:

 Age at onset is not discriminant of altered functions except for lipid and nucleotide metabolism, probably because of need during development.  There is not a clear clustering of functions according to inherence with the exception of enzymatic functions in AR HSP.

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 There are very few connections between signs, or groups of signs and certain functions: cerebellar ataxia with degradation; myelination/development and lipid metabolism; ER shaping and nucleotide metabolism with pure forms or endomembrane traffic and CI. Understanding these links and coupling these data with expression levels and profiles could be useful for better deciphering of the mechanisms involved in theses diseases.

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Myelination Degradation pathways SPG42 (AD) SLC33A1 No SPG (AD) VCP Metabolism SPG5 (AR) CYP7B1 SPG11 (AR) KIAA1840 SPG35 (AR) FA2H Mitochondrial functions SPG15 (AR) ZFYVE26 SPG39 (AR) PNPLA6 SPG9 (AD/AR) ALDH18A1 SPG18 (AR) ERLIN2 Lipid metabolism SPG44 (AR) GJC2 SPG7 (AR/ ?AD) SPG7 SPG48 (AR) AP5Z1 SPG42 (AD) SLC33A1 SPG75 (AR) MAG SPG13 (AD) HSPD1 SPG49 (AR) TECPR2 SPG5 (AR) CYP7B1 SPG2 (X-L ) PLP1 SPG31 (AD) REEP1 SPG59 (AR) USP8 SPG73 (AD) CPT1C SPG20 (AR) KIAA0610 SPG60 (AR) WDR48 SPG18 (AR) ERLIN2 SPG43 (AR) C19orf12 SPG62 (AR) ERLIN1 SPG26 (AR) B4GALNT1 SPG55 (AR) C12orf65 No SPG (AR) LYST SPG28 (AR) DDHD1 SPG74 (AR) IBA57 SPG35 (AR) FA2H Developement No SPG (AR) SACS Transport SPG39 (AR) PNPLA6 SPG3A (AD/AR) ATL1 No SPG (Mito) MT-ATP6 SPG30 (AR/AD) KIF1A SPG44 (AR) GJC2 SPG4 (AD) SPAST No SPG (Mito) MT-CO3 SPG (AR/AD) BICD2 SPG46 (AR) GBA2 SPG6 (AD) NIPA1 No SPG (Mito) MT-TI SPG58 (AR/ ?AD) K IF1C SPG54 (AR) DDHD2 SPG8 (AD) KIAA0196 SPG4 (AD) SPAST SPG56 (AR) CYP2U1 SPG10 (AD) KIF5A SPG10 (AD) KIF5A No SPG2 (X-L) PLP1 SPG33 (AD) ZFYVE27 SPG20 (AR) KIAA0610 Organelle shaping Endo-membrane Amino acid metabolism SPG59 (AR) USP8 SPG3A (AD/AR) ATL1 trafficking SPG9 (AD/AR) SPG68 (AR) FLRT1 SPG72 (AR/AD) REEP2 SPG6 (AD) NIPA1 ALDH18A1 SPG1 (X-L) L1CAM SPG58 (AR/ ?AD) KIF1C SPG8 (AD) KIAA0196 SPG22 (X-L) SLC16A2 SPG4 (AD) SPAST No SPG (AD) VCP Nucleotide metabolism SPG12 (AD) RTN2 SPG59 (AR) USP8 SPG63 (AR) AMPD2 SPG17 (AD) BSCL2 SPG11 (AR) KIAA1840 Microtubule dynamics SPG64 (AR) ENTPD1 SPG31 (AD) REEP1 SPG15 (AR) ZFYVE26 SPG4 (AD) SPAST SPG65 (AR) NT5C2 SPG33 (AD) ZFYVE27 SPG48 (AR) AP5Z1 SPG10 (AD) KIF5A SPG70 (AR) MARS SPG20 (AR) KIAA0610 SPG57 (AR) TFG SPG31 (AD) REEP1 SPG71 (AR) ZFR SPG46 (AR) GBA2 PG21 (AR) ACP33 No SPG (AR) ADAR1 Signaling SPG57 (AR) TFG SPG47 (AR) AP4B1 No SPG (AR) EXOSC3 SPG6 (AD) NIPA1 SPG61 (AR) ARL6IP1 SPG50 (AR) AP4M1 No SPG (AR) IFIH1 SPG18 (AR) ERLIN2 SPG67 (AR) PGAP1 SPG51 (AR) AP4E1 No SPG (AR) RNASEH2B SPG68 (AR) FLRT1 SPG69 (AR) RAB3GAP2 SPG52 (AR) AP4S1 SPG53 (AR) VPS37A No SPG (Mito) MT-TI SPG22 (X-L) SLC16A2 No SPG (AR) FAM134B

Figure (4-11): Summary of pathogenic mechanisms of hereditary spastic paraplegia (HSP).

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Lipid Metabolism TCC Myelination

Nucleotide Mito PN Optic Atrophy metabolism

Organellar Shaping Microtubules Amyotrophy Dynamics

Amino Acid Metabolism Complex CI Deafness Cataract

Development Signaling Endomembrane Transport trafficking Pure

Degradation Cerebellar Pathway ataxia

Figure (4-12): Statistical correlations identified using regression test. Filled boxes represent pathogenic themes while white boxes are clinical phenotypes/signs.

Table (4-2): Table summarizing the 88 HSP clinico-genetic entities with special focus on the functions of their proteins and our suggested primary and secondary functional categories. P: pure, C: complex, AD: autosomal dominant, AR: autosomal recessive.

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SPG code OMIM # Gene Age at onset P/C Protein Function Functional Second Reference (Inheritance) / % category functional category

SPG3A #606439 ATL1 <1 to 51 P/C Atlasin GTPase1 Dynamin GTPase: ER ER Dynamics Zhao et al., (AD/AR) (mainly <10) shaping, ER and lipid 2001 droplet fusion, Inhibit BMP signaling

SPG9 (AD/AR) #601162 ALDH18A1 1–30 C Pyrroline-5-carboxylate Enzyme: Pyrroline-5- Metabolism Mitochondria Seri et al., synthase (P5CS) protein carboxylate synthase with 1999 glutamate kinase (GK) and γ-glutamyl phosphate Coutelier et reductase activities al., 2015 (amino-acid metabolism)

SPG30 #610357 KIF1A 10–39 P/C Kinesin-like protein KIF1A Motor protein, axonal Intracellular Klebe et al., (AR/AD) anterograde transport Trasnport 2006

Erlich et al., 2011

SPG72 #615625 REEP2 3–4 P Receptor expression-enhancing ER membranous protein: ER dynamics Esteves et (AR/AD) protein 2 ER shaping al., 2014

No SPG #615290 BICD2 Infancy P/C Bicaudal D Homolog 2 Adaptor protein of the Intracellular Oates et al., (AR/AD) dynein–dynactin motor Trasnport 2013 complex Novarino et al., 2014

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SPG7 (AR/ #607259 SPG7 4–42 P/C Paraplegin Component of the Mitochondria Casari et al., ?AD) mitochondrial AAA 1998 protease

SPG58 (AR/ No KIF1C 2–4 P/C Kinesin family member 1C Motor protein (retrograde Intracellular Novarino et ?AD) OMIM # Golgi to ER transport) Trasnport al., 2014 for HSP Dor et al., 2014

SPG4 (AD) #182601 SPAST 1–80 P/C Spastin AAA protein: Microtubule ER Dynamics Endolysosome Hazan et al., dynamics (Microtubule Trafficking 1999 severing), inhibits BMP signaling ER morphogenesis, Endosomal trafficing (MIT domain), Cytokinesis(abscission)

SPG6 (AD) #600363 NIPA1 8–37 P/C NIPA1/ Non-imprinted in Mg2+ transporter: Endolysosome Rainier et al., Prader Willi/ Angelman Inhibitor of BMP pathway, Trafficking 2003 syndrome 1 Endosomal trafficking

SPG8 (AD) #603563 KIAA0196 10–60 P/C Strumpellin Cytoskeleton/Actin Not clear Valdmanis et remodeling, Endosomal al., 2007 traffic

SPG10 (AD) #604187 KIF5A 2–51 P/C Kinesin heavy chain isoform 5 Motor protein: Intracellular Reid et al., A microtubule dependent Trasnport 2002 ATPASE, axonal transport

SPG12 (AD) #604805 RTN2 7–24 P Reticulon 2 ER shaping protein ER dynamics Montenegro at al., 2012

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SPG13 (AD) #605280 HSPD1 17–68 P Heat shoch protein 60 Kda Mitochondrial chaperonin / Mitochondria Hansen et al., protein 1/chaperonin Mitochondrial regulation 2002

SPG17 (AD) #270685 BSCL2 2–60 C Seipin ER scaffolding protein for ER stress in HSP Windpassinger lipid metabolism, lipid cases et al., 2004 droplet biogenesis at ER

SPG31 (AD) #610250 REEP1 Variable P/C Receptor expression-enhancing ER-shaping protein, ER dynamics Züchner et al., protein 1 mitochondrial-ER interface 2006 functions, ER-microtubule interaction

SPG33 (AD) #610248 ZFYVE27/ 42–50 P Protrudin ER morphology protein, ER dynamics Mannan et al., Protrudin regulates neurite 2006 outgrowth

SPG42 (AD) #612539 SLC33A1 4–42 P Acetyl-coenzyme A transporter Acetyl-CoA transporter Metabolism Mitochondria Lin et al., 1 (role in glycolipid 2008 metabolism)

SPG73 (AD) #616282 CPT1C 19-48 P Carnitine palmitoyl-transferase Enzyme : neuronal Oxidative Rinaldi et al., isoform of Carnitine metabolism 2015

Palmitoyltransferase-1c

No SPG (AD) NO VCP ? C Valosin-Containing Protein AAA protein member; endolysosome Degradation De Bot et al., OMIM # Role in the ubiquitin- trafficking Pathway 2012 for HSP proteasome system

SPG19 (AD) 607152% 9q33-q34 36–55 P Protein not identified Protein not identified Protein not Valente et al., identified 2002

SPG29 (AD) 609727% 1p31.1-21.1 Infancy C Protein not identified Protein not identified Protein not Orlacchio et identified al., 2005

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SPG36 (AD) 613096% 12q23-24 14–33 C Protein not identified Protein not identified Protein not Schüle et al., identified 2009

SPG37 (AD) 611945% 8p21.1- 8–60 P Protein not identified Protein not identified Protein not Hanein et al., q13.3 identified 2007

SPG38 (AD) 612335% 4p16-p15 16–19 P Protein not identified Protein not identified Protein not Orlacchio et identified al., 2008

SPG40 (AD) No ? adulthood P/C Protein not identified Protein not identified Protein not Subramony et OMIM # identified al., 2009 / %

SPG41 (AD) 613364% 11p14.1- Mean 17 ± 3 p Protein not identified Protein not identified Protein not Zhao et al., 11p.2 identified 2008

SPG5/SPG5A #270800 CYP7B1 4–47 P/ C 25-hydroxycholesterol 7-alpha- Enzyme: Hydroxylase, Lipid Metabolism Tsaousidou et (AR) hydroxylase cholesterol and al., 2008 neurosteroïd

SPG11 (AR) #604360 KIAA1840 <1 to 33 P/ C Spatacsin Lysosome recycling Endolysosome Degradation Stevanin et al., protein, involved in trafficking Pathway 2007 endosomal traffic

SPG15 (AR) #270700 ZFYVE26 4–19 P/ C Zinc finger FYVE domain- Lysosome recycling Endolysosome Degradation Hughes et al., containing protein 26 protein, involved in Trafficking Pathway 2001 cytokinesis, autophagy, endosomal traffic Hanein et al., 2008

SPG18 (AR) #611225 ERLIN2 <2 C Erlin-2 ER-associated degradation ER Stress Alazami et al., pathway (ERAD) 2011

Yıldırım et al.,

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2011

SPG20 (AR) #275900 KIAA0610 Infancy C Spartin Protein with multiple Mitochondria Patel et al., Spartin functions such as 2002 Cytokinesis, BMP signaling, Lipid droplet maintenance, Mitochondrial Ca2+ homeostasis

SPG21 (AR) #248900 ACP33 Adulthood C Maspardin Protein associated Endolysosome Simpson et predominantly with Trafficking al., 2003 markers for the trans-Golgi and endocytic compartments (Endosomal traffic )

SPG26 (AR) #609195 B4GALNT1 2–19 C Beta-1,4 N- Enzyme: GM2 synthase, Lipid Metabolism Wilkinson et acetylgalactosaminyltransferase Ganglioside metabolism al., 2005 1 Boukhris et al., 2013

SPG28 (AR) #609340 DDHD1 7–15 P/C Phospholipase DDHD1 Enzyme: Phospholipase Lipid Metabolism Bouslam et al., A1, lipid metabolism 2005

Tesson et al., 2012

SPG35 (AR) #612319 FA2H 2–17 (1 C Fatty acid 2-hydroxylase Enzyme: involved in Lipid Metabolism Dick et al., family late Myelin stability, cell 2008 onset) differentiation Dick et al., 2010

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SPG39 (AR) #612020 PNPLA6 Infancy, C Neuropathy target esterase Enzyme: Neuropathy Lipid Metabolism Rainier et al., adolescence target esterase (NTE) of 2008 lipid metabolism, involved in membrane curvature, Synofzik et axonal maintenance, al., 2014 phospholipid homeostasis

SPG43 (AR) #615043 C19orf12 7–12 C Protein C19orf12 Mitochondrial protein with Mitochondrial Meilleur et unknown functions Protein With al., 2010 Unknown Functions Landouré et al., 2013

SPG44 (AR) #613206 GJC2 1st or 2nd C Gap junction gamma-2 protein Oligodendrocyte connexin Lipid Metabolism Orthmann- decade (intercellular gap junction Murphy et al., channel 2009

SPG46 (AR) #614409 GBA2 1–16 C Non-lysosomal Enzyme: Non-lysosomal Lipid Metabolism Boukhris et glucosylceramidase b-glucosidase of the al., 2010 ganglioside metabolism Martin et al., 2013

SPG47 (AR) #614066 AP4B1 Birth C AP-4 complex subunit beta-1 Member of the trafficking Endolysosome Abou Jamra et adaptor protein complex 4 Trafficking al., 2011

Bauer et al., 2012

SPG48 (AR) #613647 AP5Z1 2–50 P/C AP-5 complex subunit zeta-1 Member of the trafficking Endolysosome Degradation Słabicki et al., Adaptor protein complex Trafficking Pathway 2010 5, involved in DNA repair response

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SPG49 (AR) #615031 TECPR2 Infancy C Tectonin beta-propeller repeat- Protein involved in Lipid Endolysosome Degradation Oz-Levi et al., containing protein 2 metabolism and Trafficking Pathway 2012 Autophagy

SPG50 (AR) #612936 AP4M1 Infancy C AP-4 complex subunit mu-1 Member of the trafficking Endolysosome Verkerk et al., adaptor protein complex 4 Trafficking 2009

SPG51 (AR) #613744 AP4E1 Infancy C AP-4 complex subunit epsilon- Member of the trafficking Endolysosome Abou Jamra et 1 adaptor protein complex 4 Trafficking al., 2011

Moreno-De- Luca et al., 2011

SPG52 (AR) #614067 AP4S1 Infancy C AP-4 complex subunit sigma-1 Member of the trafficking Endolysosome Abou Jamra et adaptor protein complex 4 Trafficking al., 2011

SPG53 (AR) #614898 VPS37A 1–2 C Vacuolar protein sorting- Member of the ESCRT-I Endolysosome Zivony- associated protein 37A complex involved in Trafficking Elboum et al., Endosomal trafficking 2012

SPG54 (AR) #615033 DDHD2 <2 C Phospholipase DDHD2 Enzyme: Phospholipase Lipid Metabolism Al-Yahyaee et (lipid metabolism) al., 2006

Schuurs- Hoeijmakers et al., 2012

SPG55 (AR) #615035 C12orf65 2–7 C Probable peptide chain release Member of the mediated Mitochondria Shimazaki et factor C12orf65, mitochondrial ribosome rescue system in al., 2012 mitochondria

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Mitochondrial protein syntheis, uncertain Function

SPG56 (AR) #615030 CYP2U1 <1–8 P/C Cytochrome P450 2U1 Enzyme of lipid Lipid Metabolism Tesson et al., metabolism 2012

SPG57 (AR) #615658 TFG Infancy C Protein TFG Protein of the ER ER-Golgi Beetz et al., morphology, necessary for Trafficking 2013 vesicle transport between (Synthesis ER and Golgi Pathway)

SPG59 (AR) No USP8 Infancy C Ubiquitin carboxyl-terminal Deubiquitination enzyme Degradation Novarino et OMIM # hydrolase 8 Pathway al., 2014 / %

SPG60 (AR) No WDR48 Infancy C WD repeat-containing protein Deubiquitination Degradation Novarino et OMIM # 48 regulation protein Pathway al., 2014 / %

SPG61 (AR) #615685 ARL6IP1 Infancy C ADP-ribosylation factor-like ER morphology protein ER Dynamics Novarino et protein 6-interacting protein 1 al., 2014

SPG62 (AR) No ERLIN1 Infancy P Erlin-1 ER-associated degradation ER Stress Degradation Novarino et OMIM # Pathway al., 2014 / %

SPG63 (AR) #615686 AMPD2 Infancy C AMP deaminase 2 Enzyme: Deaminates AMP Nucleotide Novarino et to IMP in purine Metabolism al., 2014 nucleotide metabolism

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SPG64 (AR) #615683 ENTPD1 1–4 C Ectonucleoside triphosphate Enzyme: Hydrolyzes ATP Nucleotide Novarino et diphosphohydrolase 1 and other nucleotides to Metabolism al., 2014 regulate purinergic transmission

SPG45 #613162 NT5C2 Infancy P/C Cytosolic purine 5'- Enzyme: Hydrolyses IMP Nucleotide Dursun et al., (SPG65) (AR) nucleotidase in both purine/pyrimidine Metabolism 2009 nucleotide metabolism Novarino et al., 2014

SPG66 (AR) NO ARSI Infancy C Arylsulfatase I Enzyme: Arylsulfatase 1, Novarino et OMIM # Hydrolyses sulfate esters, al., 2014 for HSP hormone biosynthesis

SPG67 (AR) No PGAP1 <1–4 C GPI inositol-deacylase GPI-Anchor synthesis Lipid Metabolism ER Novarino et OMIM # pathway / GPI-AP sorting (Biosynthesis al., 2014 / % by ERES Pathways)

SPG68 (AR) No FLRT1 2–3 C Leucine-rich repeat Transmembrane protein of Signalling Novarino et OMIM # transmembrane protein FLRT1 the FGF pathway Pathways al., 2014 / %

SPG69 (AR) No RAB3GAP2 <1 C Rab3 GTPase-activating ER morphology protein ER Dynamics Novarino et OMIM # protein non-catalytic subunit al., 2014 / %

SPG70 (AR) No MARS <1 C Methionine--tRNA synthetase, Enzyme: Cytosolic Nucleotide Novarino et OMIM # cytoplasmic methionyl-tRNAsynthetase Metabolism al., 2014 / %

SPG71 (AR) No ZFR Infancy P Zinc finger RNA-binding RNA binding protein Nucleotide Novarino et OMIM #

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/ % protein Metabolism al., 2014

SPG74 (AR) #616451 IBA57 3–12 C Putative transferase CAF17, Member of the iron–sulfur Mitochondria Lossos et al., mitochondrial cluster (ISC) assembly 2015 machinery in mitochondria

SPG75 (AR) MAG Infancy C Myelin-associated glycoprotein Myelin component Myelin Development Novarino et #616680 al., 2014

SPG14 (AR) #605229 3q27-q28 ~30 C Protein not identified Protein not identified Protein not Vazza et al., identified 2000

SPG23 (AR) 270750% 1q24-q32 Infancy C Protein not identified Protein not identified Protein not Blumen et al., identified 2003

SPG24 (AR) 13q14 Infancy P Protein not identified Protein not identified Protein not Hodgkinson et %607584 identified al., 2002

SPG25 (AR) #608220 6q23-24.1 30–46 C Protein not identified Protein not identified Protein not Zortea et al., identified 2002

SPG27 (AR) 609041% 10q22.1- P: 25–45 C: P/C Protein not identified Protein not identified Protein not Meijer et al., q24.1 2–7 identified 2004

SPG32 (AR) 611252% 14q12-q21 6–7 C Protein not identified Protein not identified Protein not Stevanin et al., identified 2007b

No SPG (AR) No ADAR1 2 P Double-stranded RNA-specific RNA binding protein Nucleotide Crow et al., OMIM # adenosine deaminase Metabolism 2014 for HSP

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No SPG (AR) #256840 CCT5 Infancy C T-complex protein 1 subunit Cytosolic chaperonin of Protein Folding? Bouhouche et epsilon the ER, mitochondria, Degradation? al., 2006 cytoskeleton, proteasome and apoptosome

No SPG (AR) #614678 EXOSC3 Infancy C Exosome complex component Core component of the Nucleotide Zanni et al., RRP40 RNA exosome complex Metabolism 2013

Halevy et al., 2014

No SPG (AR) No FAM134B 2–3 C Reticulophagy receptor Golgi protein Unclear Ilgaz Aydinlar OMIM # FAM134B et al., 2014 for HSP

No SPG (AR) No IFIH1 2 P Interferon-induced helicase C RNA binding protein Nucleotide Crow et al., OMIM # domain-containing protein 1 Metabolism 2014 for HSP

No SPG (AR) #214500 LYST Late (48–58) C Lysosomal-trafficking Protein involved in Endolysosome Degradation Shimazaki et regulator Lysosome fusion/fission Trafficking Pathway al., 2014 regulation

No SPG (AR) No RNASEH2B 18– 21 P Ribonuclease H2 subunit B Enzyme of the metabolism Nucleotide Crow et al., OMIM # months of ribonucleotides Metabolism 2014 for HSP

No SPG (AR) No SACS Infancy C Sacsin Chaperone Mitochondria Engert et al., OMIM # 2000 for HSP

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No SPG (Mito) No MT-ATP6 30–50 P/C ATP synthase subunit a Respiratory chain complex Mitochondria Verny et al., OMIM # V subunit 2011 for HSP

No SPG (Mito) No MT-CO3 Infancy C Mitochondrial Cytochrome c Respiratory chain complex Mitochondria Tiranti et al., OMIM # oxidase subunit III IV subunit 2000 for HSP

No SPG (Mito) No MT-TI Adulthood P/C Mitochondrial tRNA for Mitochondria tRNA Mitochondria Nucleotide Corona et al., OMIM # isoleucine isoleucine Metabolism 2002 for HSP

SPG1 (X- #303350 L1CAM Congenital C Neural cell adhesion molecule Cell adhesion and Axonal Guidance Rosenthal et linked) L1 signaling protein involved al., 1992 in axonal guidance, neurite outgrowth ,neuronal cell Jouet et al., migration and survival 1995

SPG2 (X- #312920 PLP1 Variable P/C Proteolipid protein 1 Myelin component Myelin Development Saugier-Veber linked) Oligodendrocyte et al., 1994 progenitor cell migration

SPG22 (X- #300523 SLC16A2 Early infancy C Monocarboxylate transporter 8 Thyroid hormone Development Signalling Schwartz et linked) transporter MCT8 Pathways al., 2005

SPG16 (X- 300266% Xq11.2 Early infancy P/C Protein not identified Protein not identified Protein Not Steinmüller et linked) Identified al., 1997

SPG34 (X- 300750% Xq24-q25 16–25 P Protein not identified Protein not identified Protein Not Macedo-Souza linked) Identified at al., 2008

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4.7. A new nosology and objective case definition of HSP: a question raising itself ? As highlighted in the introduction, HSPs share multiple overlap zones with other spastic neurogenetic disorders. Neurodegeneration is now forcefully imposing the concept of being a continuum of disorders through the huge amount of knowledge gained in the last ten years and the rapidly expanding numbers of HSP and overlapping disorders subtypes.

The question about the urging need for a new nosology with a wider umbrella that includes all closely related neurological disorders is raised by the factors we stated earlier: the large number of HSP subtypes associated with other neurological signs that can be the major clinical feature of overlapping disoders, the phenomenon of multiplicity of allelic phenotypes of SPG genes together with the recently characterized novel HSP forms implicating lipid or amino acid metabolic pathways.

The second point raised is the need for improved and more objective case definition to minimize the subjectivity in diagnosis especially with regards to the overlap zones and to decrease the technical diagnostic difficulties due to the lack of sufficient expertise in the rapidly growing field of neurogenetics. Difficulties are mainly encountered by less experienced clinical practioners or even by high clinicians and neurologists with less expertise in the field of neurogenetics. The problem is further augmented where the setting does not provide the necessary multisystemic medical neurogenetic centers with equipped facilities and required expertise for neurogenetic workup. A clear example for the latter situation is the practice in developing countries where minimal is known about neurogenetic disorders. If we consider that many of these communities are highly inbred with a predominant AR patterns of inheritance and with high chances of new genes discovery, then the importance of guideline and classification to facilitate proper phenotypic stratification with minimal cost would unveil. This point has been highlighted by the big number of genes discovered through international collaborations between the highly privileged teams with vast neurogenetic facilities and teams/specialists from developing countries. An easy working frame for neurogenetic disorders will increase the chance of even faster discovery rates for genes, better understanding of the physiopathological background and emergence of new phenotypes and will ensure at the same time that proper science will be provided the thing that will have great repercussions on our understanding of these disorders.

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4.8. Challenges, milestones and particularities of the Study

4.8.1. Cohort building The first step that was required to start the study was the cohort building. This comprises the building of a network of specialists, a referral system (as illustrated earlier), a trained neurogenetic team and optimized clinical examination/sampling method. All these steps required hard work and keen determination to be achieved and to build the first neurogenetic team in Sudan and to continue despite all the challenges in a field that was previously unexplored.

Continuous Optimization of the Phenotyping Process:

1. Questionnaire: As mentioned earlier we had to adapt the comprehensive standard questionnaire of the SPATAX network (https://spatax.wordpress.com/) to our local needs indicated by the nature of our cohort.

2. Video Documentation: With the help of the small team that I created in Sudan, we managed to optimize the video documentation system to create video archiving system for the major and minor clinical signs that we encountered since the start of our recruitment. This was especially important for diagnosis establishment by the second viewer in order to eliminate alternate disorders. This had many advantages including increasing our expertise and training of the new members that joined our team to warrantee that they would be more efficient and accurate when they perform the clinical assessment.

3. National collaborations with experts in related fields: To further ameliorate the process of phenotyping, we created further collaborations with experts in the fields of neuroradiology (Departments of Radiology in: Alamal Hospital, Police hospital, Antalia Medical center, Dar Alelaj Hospital), neurophysiology (Department of physiology: University of Khartoum, Soba University hospital) , ophthalmology (Department of Ophthalmology: University of Khartoum, Mekka Eye Charity Hospital) and psychiatry (Department of Psychiatry: University of Khartoum, Taha Baashar Hospital for Psychiatic disorders).

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i. Neuroradiology: With the help of three neuroradiology teams and departments, we managed to set a new magnetic resonance imaging allowing storing them in databases. We also obtained a set of free MRIs to be dedicated to poor patients as a part of collaboration between our team representing the university of Khartoum/ faculty of medicine and the radiology departments of two governmental hospitals: Alamal Hospital, Police hospital (check the list of collaborations above). We also managed to provide means of transportation for several patients far from medical centers. These two steps allowed us to partially solve the two main problems that we encountered in MRI: unavailability due to financial inability and the badly conserved or lost films.

o Unsolved part of the problem of MRI: 1. We had to accept films in patients who already did the MRI and were reluctant to repeat them because of many reasons including: - Logistic difficulties as lack of time for families living in the peripheral regions in Sudan and came to the capital for short time only - Fear of anesthesia in kids and mentally subnormal patients who could not stay still during the imaging process. 2. Anesthesia staff unavailable in certain imaging centers. 3. Patients far from equipped hospitals and unable to travel

ii. Other Fields: The collaboration with the ophthalmology and neurophysiology departments allowed us to establish a steady referral system of our patients to three of the most advanced centers in our settings for free specialized assessment.

4.8.2. Diagnostic Capacities Most importantly was building our clinical judgment capacity and the improvement of our diagnostic approach. As I was in charge of the final analysis of the clinical

213 assessment this was a burden especially that most of our cohort was pediatric and needed special training considering my background training of adult neurology. Even in adult cases the neurogenetic disorders needed vast knowledge and expertise in that branch of neurology. The pediatric team in Sudan as well as the clinical neurogeneticist/ leukodystrophy (neuropediatrics) experts in France provided me with invaluable expertise.

4.8.3. Huge work / One pair of hands/ Time The time factor was rather stressing as a lot of tasks had to be done in short time. Almost half of the study time was dedicated to the cohort recruitment and organization of the work flow to make it smoother. In the remaining half, all laboratory work from the DNA extraction to the bioinformatic analysis and confirmation of results as well as some functional workup had to be achieved. As we needed to do functional studies, skin biopsy system had to be started and this needed training of our team, optimization and coordination with the DNA bank in France in order to be launched successfully. The formation of the team in Sudan and the teaming with cooperative colleagues in France especially towards the end of the study had the best effect on the yield. This reinforces the notion that the best results are achieved via team work.

4.8.4. Budget management As the case with every research project finance was an obstacle.

4.8.4.1. Sudan It was a challenge to provide budget for the research cost in Sudan. This included the expenses for sampling sessions (gloves, sterilizers, needles .. etc) as well as providing travel/transportation cost to the poor families to the sampling center [and to the team members in case the reverse (home visits) occurs] in the peripheral regions of the country. It also included the sample shipment and custom cost. It included as well the cost for investigations for the poor noninsured patients (Imaging, electrophysiological studies and specialist consultation (ophthalmologists, audiologists). While the latter could be partially covered by building collaborations, the former had to be provided from the researcher personal wages and familial support.

4.8.4.2. France

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There was no specific finance for the French/Sudanese collaborative project so it was an extra-burden for both the researcher and staff. To overcome the restricted budget for WES, a serious decision had to be taken for AR families. That was to do one WES per family or per family branch in case of extended families. This strategy worked quite well since we looked for homozygous variants manly. In AD families we had to do WES for two affected individuals. Families had to be prioritized for WES based on predicted yield. WES was thus not performed in sporadic families.

4.8.5. WES analysis In addition to the difficulties that are usually encountered in analyzing WES data for prioritization of variants and genes, WES data analysis had to be adapted to the particular nature of the Sudanese families. One of the most important criterion that had to be considered is the probability of occurrence of two clinical entities in the same families. This probability increases when analyzing patients belonging to two branches of the same family or even when analyzing data of affected siblings. In fact, rather rarely that we find a causative mutation segregating in more than one branch of our extended families with more than one branch despite the closely related phenotypic presentation.

4.8.6. Administrative processes This thesis was the first joint project between University of Khartoum (UofK) and the University of Paris 6 (UPMC). Moreover, it was the first joint PhD approved by the senate of the UofK. This conferred certain difficulty to the PhD administrative process. However, this thesis demonstrates the feasibility of such European-African collaborative works and opens the way for increase collaborations and exchange of researchers in the near future.

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5. Conclusion and prospective work

HSP in Sudan is a rare condition. We are far from the full characterization of all cases but considering that we collected 106 patients in an area of 30 million inhabitants, the minimal prevalence can be established as ≈ 0.4/100000 (≈ 0.3/100000 if families with pyramidal syndrome complicating PD, SCA7 are excluded). These roughly calculated figures although won’t reflect the true epidemiological prevalence but they are in accordance with what has been reported about the prevalence of HSP in other countries (even more compatible with the reports of AR HSP being almost all AR HSP) (Ruano et al., 2014) 41% of Sudanese families were solved through gene screening, a frequency also similar to other studies of the host laboratory on European cases. In the case of F15, the identification of the causative gene will be of interest for better follow-up and therapy (Arginase 1 deficiency) Interestingly, following WES, I have potentially identified the causative gene in two families for which the effects of multiple variants need to be distinguished. The screening of additional cases of the SPATAX cohort to add new mutations and therefore genetic arguments, and the modeling in Zebrafish by knock down, including rescue experiments, will be crucial steps before definite conclusions. The study of fibroblasts of patients may also reveal cytological abnormalities that could help to define the causative genes among the candidates. In three other families, a single variant with convincing evidence of pathogenicity in at least two (one is a paralog of another gene involved in neurodegeneration, one is a stop mutation), were identified. Similar experiments are in progress to determine the frequency of these new genetic entities and the functional consequences in vitro. Finally, WES is to be performed to the remaining families in which known genes have been excluded. There are also two families still under WES analysis, potentially with new causative genes to come. In case of negative results in WES, a genome analysis will be considered first to analyze the exome data better covered in that case than in WES, and intronic/structural regions. The need for new nosology: time has come. As illustrated in the introduction and discussion, the spasticity world is huge and overlaps with many disorders. Many genes with HSP gene name and number are more frequently accounting for other diseases when mutated and the reverse is true. My results also highlight this fact with HSP patients found mutated in genes mutated in classical HSP families and not fulfilling perfectly the typical HSP presentation and the other way around. A new nosology may emerge in the near future thanks to WES data. The development of WES or large gene

216 panels covering multiple pathologies will allow better assessment of the full clinical spectrum of neurological conditions.

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Annexes

Annex 1: Table 1 a: HSP forms complicated with clinical signs of cerebellar syndrome.

1- SPG 2- Inheritance 3- Gene 4- Comment

5- SPG30 6- AD/AR 7- KIF1A 9- Cerebellar atrophy 8-

10- SPG58 11- AR, AD? 13- KIF1C 15- Cerebellar atrophy 12- 14-

16- SPG7 17- AR, AD? 19- SPG7 21- Cerebellar atrophy 18- 20-

22- SPG4 24- AD 25- SPAST 26- No reported cerebellar 23- atrophy

27- SPG5/SPG5A 29- AR 30- CYP7B1 32- No reported cerebellar 28- 31- atrophy

33- SPG11 34- AR 35- KIAA1840 36- Cerebellar atrophy

37- SPG15 38- AR 39- ZFYVE26 40- Cerebellar atrophy

41- SPG20 42- AR 43- 45- No reported cerebellar 44- KIAA0610 atrophy

46- SPG21 47- AR 48- ACP33 49- Cerebellar atrophy

50- SPG26 51- AR 52- B4GALNT1 53- No reported cerebellar atrophy

54- SPG27 55- AR 56- 10q22.1-q24.1 57- Cerebellar atrophy

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58- SPG 59- Inheritance 60- Gene 61- Comment

62- SPG28 63- AR 64- DDHD1 66- - 65-

67- SPG35 68- AR 69- FA2H 71- Cerebellar atrophy 70-

72- SPG44 73- AR 74- GJC2 76- - 75-

77- SPG46 78- AR 79- GBA2 80- Cerebellar atrophy

81- SPG49 83- AR 84- TECPR2 85- Cerebellar atrophy 82-

86- SPG54 87- AR 88- DDHD2 89- -

90- SPG59 91- AR 92- USP8 94- - 93-

95- SPG60 96- AR 97- WDR48 99- - 98-

100- SP 101- AR 102- ENT 103- - G64 PD1

104- SP 105- AR 106- FLR 108- - G68 T1 107-

109- SP 110- AR 111- MAG 113- - G75 112-

114- No 115- AR 116- EXO117- Cerebella SPG SC3 r atrophy

118- No 119- AR 120- LYST121 - Cerebella SPG r atrophy

122- No 123- AR 124- SAC125- Cerebella SPG S r atrophy

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126- SP127- Inherit128- Gene129 - Commen G ance t

130- SP 131- X- 132- PLP1 133- - G2 linked

134- SP 135- X- 136- SLC1 137- - G22 linked 6A2

138- No 139- MITO 140- MT- 141- - SPG ATP6

142- No 143- MITO 144- MT- 146- - SPG TI 145-

Table 1 b: Cerebellar atrophy with no clinical signs of cerebellar syndrome

SPG32 AR 14q12-q21 No cerebellar signs reported

SPG50 AR AP4M1 No cerebellar signs reported

SPG66 AR ARSI No cerebellar signs reported

SPG67 AR PGAP1 No cerebellar signs reported

SPG74 AR IBA57 No cerebellar signs reported

No SPG Mito MT-CO3 No cerebellar signs reported

Annex 2:

Table 2: HSP forms complicated with cognitive/mental function impairment.

147- SPG148 - Inherit149- Gene150 - Comment ance 151-

152- SPG5153- AR, 154- KIF1C155 - developmental delay 8 AD? or MR

156- SPG4157 - AD 158- SPAST159 - cognitive impairment

160- SPG6161 - AD 162- NIPA1163 - memory 164- impairment

165- SPG3166- AD 167- 4p16- 168- cognitive impairment 8 p15

169- SPG5170 - AR 171- CYP7B172- cognitive

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1 173- impairment

174- SPG1175- AR 176- KIAA18177- cognitive decline 1 40

178- SPG1179- AR 180- 3q27- 181- mild MR and memory 4 q28 deficiency

182- SPG183 - Inherita184- Gene185 - Comment nce 186-

187- SPG1188- AR 189- ZFYVE190- cognitive decline 5 26

191- SPG1192- AR 193- ERLIN194- ID 8 2

195- SPG2196- AR 197- KIAA06198- mild ID 0 10

199- SPG2200- AR 201- ACP33202 - Mast syndrome: 1 203- cognitive decline

204- SPG2205- AR 206- 1q24- 207- Lisonsyndrome:MR 3 q32

208- SPG2209- AR 210- B4GAL211- ID 6 NT1

212- SPG2213- AR 214- 10q22.215- MR 7 1-q24.1

216- SPG3217- AR 218- 14q12-219- Mild MR 2 q21

220- SPG3221- AR 222- FA2H 223- cognitive decline 5

224- SPG4225- AR 226- GJC2 227- mental impairment 4

228- SPG4229- AR 230- 10q24.231- MR 5 3-q25.1

232- SPG4233- AR 234- GBA2 235- MR 6

236- SPG4237- AR 238- AP4B1239 - Severe ID 7

240- SPG4241- AR 242- AP5Z1243 - cognitive impairment 8 or MR

244- SPG4245- AR 246- TECPR247- Severe ID 9 2

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248- SPG5249- AR 250- AP4M1251 - MR 0

252- SPG5253- AR 254- AP4E1255 - Severe ID 1

256- SPG5257- AR 258- AP4S1259 - Severe ID 2

260- SPG5261- AR 262- VPS37263- delays in cognition 3 A

264- SPG5265- AR 266- DDHD267- ID or developmental 4 2 delay

268- SPG5269- AR 270- C12orf271- ID 5 65

272- SPG5273- AR 274- CYP2U275- Mental impairment 6 1 276- SPG56 by OMIM

277- SPG5278- AR 279- USP8 280- mild MR 9

281- SPG6282- AR 283- ENTPD284- moderate ID 4 1

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285- SPG286 - Inherit 287- Gene288 - Comment ance 289-

290- SPG6291- AR 292- NT5C2293 - learning disability 5

294- SPG6295- AR 296- RAB3G297- ID 9 AP2

298- No 299- AR 300- EXOSC301- Mild cognitive SPG 3 impairment

302- SPG1303 - X- 304- L1CAM305 - MASA syndrome linked

306- SPG2307 - X- 308- PLP1 309- MR and sometimes linked dementia

310- SPG1311- X- 312- Xq11.2313 - mild MR 6 linked

314- SPG2315- X- 316- SLC16317- severe MR 2 linked A2

318- No 319- mito 320- MT- 321- Dementia SPG ATP6 322-

Annex 3

Table 3a: HSP forms complicated with peripheral neuropathy without evidence of amyotrophy.

SPG code inheritance Gene SPG30 AR/AD KIF1A SPG4 AD SPAST SPG6 AD NIPA1 SPG31 AD REEP1 SPG36 AD 12q23-24 SPG14 AR 3q27-q28 SPG23 AR 1q24-q32 SPG25 AR 6q23-24.1 SPG26 AR B4GALNT1 SPG28 AR DDHD1 SPG56 AR CYP2U1 SPG57 AR TFG

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SPG61 AR ARL6IP1 SPG60 AR WDR48 No SPG AR SACS No SPG AR FAM134B No SPG AR LYST No SPG AR CCT5 No SPG MITO MT-ATP6

Table 3b: HSP forms complicated with amyotrophy with/without peripheral neuropathy

SPG code Inheritance Gene Comment No SPG AR/AD BICD2 No PN SPG3A AD/AR ATL1 + PN SPG10 AD KIF5A + PN SPG17 AD BSCL2 + PN SPG9 AD ALDH18A1 + PN SPG68 AR FLRT1 + PN SPG39 AR PNPLA6 + PN SPG66 AR ARSI + PN SPG11 AR KIAA1840 + PN SPG15 AR ZFYVE26 + PN SPG43 AR C19orf12 + PN SPG55 AR C12orf65 + PN SPG74 AR IBA57 + PN SPG20 AR KIAA0610 / Spartin No PN

SPG5/SPG5A AR CYP7B1 No PN

SPG35 AR FA2H No PN

SPG75 AR MAG No PN

SPG64 AR ENTPD1 No PN

SPG65 AR NT5C2 No PN

SPG70 AR MARS No PN

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SPG67 AR PGAP1 No PN

SPG47 AR AP4B1 No PN

SPG51 AR AP4E1 No PN

SPG52 AR AP4S1 No PN

No SPG AR EXOSC3 No PN

SPG22 X-linked SLC16A2 No PN

Annex 4:

Table 4: Eye

Table 4a: Cataract

SPG Inheritance Gene

SPG9 AD/AR 10q23.3-q24.2

SPG46 AR GBA2

SPG69 AR RAB3GAP2

Table 4b: Retinal/macular degeneration

SPG Inheritance Gene Comment

SPG11 AR KIAA1840 Macular degeneration

SPG15 AR ZFYVE26 Macular degeneration

No SPG AR SACS Retinal striation

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Table 4c: Optic atrophy

SPG Inheritance Gene

SPG7 AR SPG7

SPG35 AR FA2H

SPG55 AR C12orf65

SPG57 AR TFG

SPG68 AR FLRT1

SPG74 AR IBA57

SPG45 AR 10q24.3-q25.1

SPG54 AR DDHD2

Annex 5:

Table 5: Characteristic syndromes of some HSP subtypes.

SYNDROME SIGNS and SYMPTOMS GENES ASSOCIATED WITH IT

MASA syndrome Mental retardation Aphasia Shuffling gait and SPG1 Adducted thumb

CRASH syndrome Corpus callosum hypoplasia Retardation Adducted SPG1 thumb Spastic paraplegia and Hydrocephalus

Silver syndrome neuropathy, amyotrophy SPG10, SPG17

`Kjellin syndrome Spastic paraplegia ,cognitive decline, neuropathy, SPG11, SPG15 retinopathy

Troyer Syndrome dysarthria, distal amyotrophy in SPG20 hands and feet, cerebellar signs, mild ID and skeletal abnormalities (short stature)

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Mast syndrome speech decline leading to akinetic SPG21 mutism, personality disturbances, psychotic episodes, cognitive decline and cerebellar dysfunction (incoordination and dysdiadochokinesia

Allan–Herndon–Dudley spastic quadriplegia, severe MR, central hypotonia, SPG22 syndrome muscle hypoplasia, dystonia, ataxia

Lison syndrome abnormal skin and hair pigmentation, ± SPG23 dysmorphisms, skeletal deformities, MR or sensorimotor neuropathy

Tetraplegic cerebral SPG50 palsy with MR

(SPOAN-like Spastic paraplegic optic atrophy neuropathy SPG57, SPG74 phenotype)

Hereditary spastic - No SPG/ VCP paraplegia with Paget’s disease of bone

Annex 6:

Table 6a: Skeletal deformities reported in HSP.

SPG Inheritance Gene Comment

SPG18 AR ERLIN2 Contractures, Troyer Syndrome SPG20 AR SPG20/ short stature KIAA0610 SPG23 AR 1q24-q32 skeletal deformities, Lison syndrome SPG27 AR 10q22.1-q24.1 skeletal abnormalities SPG47 AR AP4B1 foot deformity SPG49 AR TECPR2 dysmorphic features SPG51 AR AP4E1 foot deformity SPG52 AR AP4S1 foot deformity SPG53 AR VPS37A marked kyphosis

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SPG59 AR USP8 pes equinovarus

SPG65 AR NT5C2 pes equinovarus

SPG66 AR ARSI pes equinovarus

Table 6b: Microcephaly in HSP.

SPG Inheritance Gene Comment

SPG47 AR AP4B1

SPG49 AR TECPR2 brachycephalic microcephaly

SPG51 AR AP4E1

SPG52 AR AP4S1

SPG64 AR ENTPD1

Table 6c: Short stature in HSP.

SPG Inheritance Gene

SPG58 AR, AD? KIF1C

SPG20 AR KIAA0610

SPG27 AR 10q22.1-q24.1

SPG52 AR AP4S1

SPG54 AR DDHD2

SPG56 AR CYP2U1

SPG63 AR AMPD2

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Annex 7:

Table 7: Psychiatric symptoms associated with HSP.

SPG Inheritance Gene Comment

SPG 21 AR ACP33 Mast syndrome: personality disturbances, psychotic episodes SPG26 AR B4GALNT1 One family presents behavioral problems SPG47 AR AP4B1 shy character, stereotypic laughter

SPG51 AR AP4E1 shy character, stereotypic laughter SPG52 AR AP4S1 shy character, stereotypic laughter SPG64 AR ENTPD1 aggressiveness

Annex 8:

Table 8: Extrapyramidal signs associated with HSP.

SPG Inheritance Gene Comment

SPG58 AR, AD? KIF1C Chorea

SPG21 AR ACP33 NCBI By isra SPG35 AR FA2H Dystonia

SPG56 AR CYP2U1 Dystonia

SPG1 X-linked L1CAM Shuffling gait

SPG22 X-linked SLC16A2 Allan–Herndon–Dudley syndrome: dystonia

Annex 9:

Table 9: Deafness in HSP.

248

SPG Inheritance Gene

SPG29 AD 1p31.1-21.1

SPG10 AD KIF5A

SPG69 AR RAB3GAP2

No SPG MITO MT-TI

Annex 10:

Table 10: Developmental delay in HSP.

SPG Inheritance Gene

SPG58 AR, AD? KIF1C

SPG47 AR AP4B1

SPG51 AR AP4E1

SPG52 AR AP4S1

SPG53 AR VPS37A

SPG54 AR DDHD2

Annex 11:

Table 11: Hypogonadism and infertility in HSP

SPG Inheritance Gene Comment

SPG46 AR GBA2 infertility in males

SPG64 AR ENTPD1 delayed puberty

SPG5 AR CYP7B1 27-hydroxy-cholesterol accumulation in blood and CSF.(decreased libido)

No SPG Mito MT-ATP6 hypogonadism

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Annex 12: DIAGNOSTIC FORM FOR GNIDARNNNGNDORUEN RUDADRNDD (modified SPATAX form)

Date: ___ / ___ / ___ Center: ______Neurologist: ______Stick the identification tag Code ID patient: ______Birth date: ____ / ____ / ____

Proband: Yes No Sex: female male

Initial exam: Yes No Follow up n°: ______

A. FAMILIAL HISTORY (add pedigree) B. AGE No Yes Spastic paraparesis and/or ataxia in the

family? Age at ONSET: ______Other familial disease Specify:______

Consanguinity Specify: ______Age at examination: ______Parental inheritance of the disease Paternal Maternal Geographical origin of the transmitting ______parent:

C. esoisiHrPcnengerP E. lonteoNPcnenger

lgeroN laigeroN yfsHnpr Signs at onset lg sse los yfsHnpr

ycgeegerP • rgcgegeeec

RgevigcP • yPcemvree

• roeevnvoe

• yaPrgoeoevree

• Dcggrg . snsNgfrsinP • Pain and/or cramps

Normal Delayed dsoeseesR At age Specify: • NcvegcaP Motor

Development • NancecPcemvree Intellectual •RPamoccgve rervee Development Mile Stones • Others :

F. PREDOMINANT SIGNS at examination SPASTICITY CEREBELLAR ATAXIA

None Mild egRseons Severe Not Applicable None Mild egRseons Severe Not Applicable • Upper limbs • Lower limbs* • Gait • Dysarthria * at rest for spasticity and knee-heel for ataxia

G- DISABILITY STAGE At age At age 0: no functional handicap 4: severe, walking with one stick 1: no functional handicap but signs at examination 5: walking with two sticks 2: mild, able to run, walking unlimited 6: unable to walk, requiring wheelchair 3: moderate, unable to run, limited walking without aid 7: confined to bed

250

- OTHER CLINICAL SIGNS

1. Muscle wasting 2. Facial dysmorphia None Mild Moderate Severe No Yes Describe: • Proximal UL

• Distal UL • Proximal LL ______

• Distal LL

3. Fasciculations or Myokymias 4. Skeletal abnormalities PPP(Facial contraction fasciculations) please circle None Mild Moderate Severe No Yes Localisation: • Scoliosis

• Pes cavus

______• Other: ______

5. Centiles 6P. gisP

esoetesrsin CsinnNs los yfsHnpr Hypotonic lgeroN yfoennH dnonR Head eglnroNPrP Circumferance nenoNPrP

esnornPpgePlos eglnroNPPP

losPcnornPpge nenoNPPP llnoN

7. Motor deficit 8. Reflexes Mild Moderat Severe None 4/5 2-3/5 <2/5 Normal Increased Diffused Decreased Absent Clonus • Facial palsy/ atrophy • Jaw jerk • Proximal UL • Biceps • Distal UL • Finger

• Proximal LL flexor • Distal LL • Patellar

9. Extra-pyramidal symptoms • Adductor

None Mild Moderate Severe Specify: • Ankle • Resting tremor • Postural tremor Absent Present • Chorea •Hoffmann’s

sign • Dystonia Unilat. Flexor Indifferent Bilat.  • Myoclonus  • Plantar • Hypokinesia reflex • Rigidity

11. Sensory deficit None Mild Moderate Severe Abolished

• Vibration sense

 (ankles) (8/8) (> 5/8) (2-5/8) (<2/8) (0/8)

• Superficial No Touch Prick Cold sensory loss

251

11. Ophtalmological signs

No Yes * Oculomotor No Yes

• Diplopia • Nystagmus Describe: ______

• Ptosis • Saccadic pursuit Describe: ______• Eye lid retraction • Slow saccades (bulging eyes) • Diminished visual acuity At age: ______• Ocular motor apraxia

* Fundus No Yes • Vertical ophthalmoplegia Describe: ______Optic atrophy • Abnormal Retinis Pigmentosa • Hori ontal ophthalmoplegia Describe: ______Other:

12. Mental status 13. Sphincter and sexual disturbances No Yes None Mild Moderate Severe Comment • Intellectual At age: ______Type: ______deterioration • Urinary urgency • Mental At age: ______Type: ______retardation • Psychiatric Describe: • Urinary incontinence symptoms ______• Urinary retention

• Anal incontinence

• Impaired sexual function

• Early menopause No Yes Not Applicable:_____

11. Other signsPPPP No Yes Describe • Dysphagia ______Severity: ______• Skin problems ______• Hearing impairment ______• Epilepsy At age: ______Type: ______• Others

11: Other medical complaints: ______

G- FUNCTIONAL CLINICAL EVALUATION - Please perform ALL tests listed in annexes and indicate scores below

- SPRS (annex 1) : _____/ 52 - 25 feet ambulatory test (annex 4): _____sec

- SARA (annex 2) : _____/ 40 - UHDRS – functional part IV (annex 5): _____/ 25

- CCFS (annex 3) : _____

252

H- CLINICAL DIAGNOSTIC CONCLUSION

Cerebellar ataxia Spastic paraparesis

Autosomal Pure form Definitely affected Autosomal Pure form Definitely affected dominant dominant Autosomal Complicated Probably affected Autosomal Complicated Probably affected (enhanced or very brisk recessive form (only dysarthria) recessive form LL reflexes +/- Babinski)

Isolated case Possibly affected (only Isolated case Possibly affected (enhanced LL

mild gait ataxia) reflexes) X-linked X-linked

I- MOLECULAR DIAGNOSIS Genes/Loci to test: Diagnosis:

J- COMPLEMENTARY INVESTIGATIONS

NOT NOR- AB- NOT NOR- AB- EXAMINATION EXAMINATION SPECIFY DONE MAL NORMAL SPECIFY DONE MAL NORMAL

1. Cerebral MRI 2. Medullar MRI

None Mild Moder. Severe ATROPHY

- Cerebrum None Mild Moder. Severe - Upper spinal

- Cerebellum cord

- Brainstem

- Corpus callosum

NOT NOR- AB- NOT NOR- AB- EXAMINATION SPECIFY EXAMINATION SPECIFY DONE MAL NORMAL DONE MAL NORMAL

3. EMG + NCV UL 9. VLCFA

4. EMG + NCV LL 10. -foetoprotein

5. VEP 11. Cholesterol 12. Serum protein 6. AEP electrophoresis

7. MEP 13. Vitamin E 14. Apolipo- 8. SEP protein A, B

NOT NOR- AB- EXAMINATION SPECIFY DONE MAL NORMAL K- STORED MATERIAL 15. Muscle biopsy Yes No 16. Skin biopsy • DNA 17. ERG 18. Fundus • Immortalized cell lines examination • Muscle tissue 19. Neuropsycho- logical exam • Skin biopsy - IQ • Nerve biopsy 20. Urodynamics • Other: ______21. Urine density

253

ANNEX 1: Spastic Paraplegia Rating Scale (SPRS)

(1) Walking distance without pause Due to history, walking aids allowed (8) Spasticity -knee flexion (Modified Ashworth scale) 0: Normal, unlimited Score more severely affected side 1: Abnormal exhaustion due to spasticity after more than 500m 0: No increase in muscle tone 2: Walking distance less than 500m 1: Slight increase in muscle tone, manifested by a catch and release 3: Walking distance less than 10 m 2: More marked increase in muscle tone through most of the range 4: Unable to walk of motion 3: Considerable increase in muscle tone - passive movement is (2) Gait quality difficult Patient is asked to walk as fast as possible a 10 meter distance 4: Limb stiff in flexion or extension including one turn 0: Normal (9) Wealkness -hip abduction (Medical Research Council 1976) 1: Mild stiffness, running still possible 0: No weakness 2: Clearly spastic gait, interfering with running 1: Mild weakness (4/5) 3: Spastic gait requiring use of canes/walker 2: Moderate weakness (3/5) 4: Unable to walk for a 10 meter distance even with maximal 3: Severe weakness (1-2/5) support 4: Plegia (0/5)

(3) Maximum gait speed (10) Weakness -foot dorsiflexion (Medical Research Council 1976) Time for a 10 meter distance including one turn, taken by stop 0: No weakness watch Timing: 1: Mild weakness (4/5) 0: Normal 2: Moderate weakness (3/5) 1: Slightly reduced (10m: ≥ s) sec 3: Severe weakness (1-2/5) 2: Moderately reduced (10m: ≥ 10s) 4: Plegia (0/5) 3: Severely reduced (10m: ≥ 20s) 4: Unable to walk for a 10m distance or time ≥ 40s (11) Contractures of lower limbs Score in supine position (4) Climbing stairs  Hip extension: lumbar spine and thighs touch the underlay. Hip 5 steps upstairs - turn - 5 steps downstairs abduction: abduction up to an angle of >60° between the legs 0: Normal: needs no support of the banister possible 1: Mild impairment: needs intermittent support of the banister  Knee extension: thigh and calf touch the underlay 2: Moderate impairment: needs permanent support of the banister  Ankle dorsal extension: > 10° possible. Ankle pronation: > 10° 3: Severe impairment: needs support of another person or possible additional walking aid to perform task 0: No contracture 4: Unable to climb stairs 1: Mild, not fixed abnormal position of one joint (unilaterally or bilaterally) (5) Speed of stair climbing 2: Fixed contracture of one joint (unilaterally or bilaterally) Time for 5 steps upstairs - turn - 5 steps downstairs, taken by stop- 3: Fixed contracture of two joints (unilaterally or bilaterally) watch Timing: 4: Fixed contracture of more than two joints (unilaterally or 0: Normal bilaterally) 1: Slightly reduced (≥ s to perform task) sec 2: Moderately reduced (≥ 10s to perform task) (12) Pain due to SP related symptoms 3: Severely reduced (≥ 20s to perform task) 0: None 4: Unable to climb stairs 1: ≤ 0% of waking day present AND intensity 0 - 3 points on visual analogue scale (6) Arising from chair 2: ≤ 0% of waking day present AND intensity 4 - 10 points on Patient attempts to arise from a straight-back wood or metal chair visual analogue scale with arms folded across chest 3: > 50% of waking day present AND intensity 0 - 3 on visual 0: Normal analogue scale 1: Slow, or may need more than one attempt. 4: > 50% of waking day present AND intensity 4 - 10 points on 2: Pushes self up from arms of seat. visual analogue scale 3: Tends to fall back and may have to try more than one time but can get up without help. (13) Bladder and bowel function 4: Unable to arise without help. 0: Normal bladder and bowel function 1: Urinary or fecal urgency (difficulties to reach toilet in time) (7) Spasticity -hip adductor muscles (Modified Ashworth scale) 2: Rare and mild urge incontinence (no nappy required) Score more severely affected side 3: Moderate urge incontinence (requires nappy or catheter when 0: No increase in muscle tone out of the house) 1: Slight increase in muscle tone, manifested by a catch and release 4: Permanent catheterization or permanent nappy 2: More marked increase in muscle tone through most of the range of motion 3: Considerable increase in muscle tone - passive movement is difficult 4: Limb stiff in adduction Total SPRS Score: /52

254

ANNEX 2: Scale for the Assessment and Rating of Ataxia (SARA)

1) Gait 2) Stance Proband is asked (1) to walk at a safe distance parallel to a wall Proband is asked to stand (1) in natural position, (2) with feet including a half-turn (turn around to face the opposite direction of together in parallel (big toes touching each other), and (3) in gait) and (2) to walk in tandem (heels to toes) without support. tandem (both feet on one line, no space between heel and toe). Proband does not wear shoes, eyes are open. For each condition, three trials are allowed. Best trial is rated. 0 Normal, no difficulties in walking, turning and walking tandem (up 0 Normal, able to stand in tandem for > 10 s to one misstep allowed) 1 Slight difficulties, only visible when walking 10 consecutive steps in 1 Able to stand with feet together without sway, but not in tandem tandem for > 10s 2 Clearly abnormal, tandem walking >10 steps not possible 2 Able to stand with feet together for > 10 s, but only with sway 3 Considerable staggering, difficulties in half-turn, but without support 3 Able to stand for > 10 s without support in natural position, but not with feet together 4 Marked staggering, intermittent support of the wall required 4 Able to stand for >10 s in natural position only with intermittent support 5 Severe staggering, permanent support of one stick or light support by 5 Able to stand >10 s in natural position only with constant one arm required support of one arm 6 Walking > 10 m only with strong support (two special sticks or 6 Unable to stand for >10 s even with constant support of one stroller or accompanying person) arm 7 Walking < 10 m only with strong support (two special sticks or stroller or accompanying person) 8 Unable to walk, even with supported Score : ______Score : ______

3) Sitting 4) Speach disturbance Proband is asked to sit on an examination bed without support of feet, Speech is assessed during normal conversation. eyes open and arms out stretched to the front. 0 Normal, no difficulties sitting > 10 sec 0 Normal 1 Slight difficulties, intermittent sway 1 Suggestion of speech disturbance 2 Constant sway, but able to sit for > 10 s without support 2 Impaired speech, but easy to understand 3 Able to sit for > 10 s only with intermittent support 3 Occasional words difficult to understand 4 Unable to sit for >10 s without continuous support 4 Many words difficult to understand 5 Only single words understandable 6 Speech unintelligible / anarthria Score : ______Score : ______

255

5) Finger chase (Rated separately for each side) 6) Nose-finger test (Rated separately for each side) Proband sits comfortably. If necessary, support of feet and trunk is Proband sits comfortably. If necessary, support of feet and allowed. Examiner sits in front of proband and performs 5 trunk is allowed. Proband is asked to point repeatedly with his consecutive sudden and fast pointing movements in unpredictable index finger from his nose to examiner’s finger which is in directions in a frontal plane, at about 50 % of proband’s reach. front of the proband at about 90% of proband’s reach. Movements have an amplitude of 30 cm and a frequency of 1 Movements are performed at moderate speed. Average movement every 2 s. Proband is asked to follow the movements with performance of movements is rated according to the his index finger, as fast and precisely as possible. Average amplitude of the kinetic tremor. performance of last 3 movements is rated. 0 No dysmetria 0 No tremor 1 Dysmetria, under/ overshooting target <5 cm 1 Tremor with an amplitude < 2 cm 2 Dysmetria, under/ overshooting target < 15 cm 2 Tremor with an amplitude < 5 cm 3 Dysmetria, under/ overshooting target > 15 cm 3 Tremor with an amplitude > 5 cm 4 Unable to perform 5 pointing movements 4 Unable to perform 5 pointing movements Score Right: Left: Score Right: Left:

Mean of both sides (R+L)/2 Mean of both sides (R+L)/2 7) Fast alternating hand movements (Rated separately for each side) 8) Heel-shin slide (Rated separately for each side) Proband sits comfortably. If necessary, support of feet and trunk is Proband lies on examination bed, without vision of his legs. allowed. Proband is asked to perform 10 cycles of repetitive Proband is asked to lift one leg, point with the heel to the alternation of pro- and supinations of the hand on his/her thigh as fast opposite knee, slide down along the shin to the ankle, and to and as precise as possible. Movement is demonstrated by examiner at lay the leg back on the examination bed. The task is a speed of approx. 10 cycles within 7 s. Exact times for movement performed 3 times. Slide-down movements should be execution have to be taken. performed within 1 s. 0 Normal, no irregularities (performs <10s) 0 Normal 1 Slightly irregular (performs <10s) 1 Slightly abnormal, contact to shin maintained 2 Clearly irregular, single movements difficult to distinguish or 2 Clearly abnormal, goes off shin up to 3 times during 3 cycles relevant interruptions, but performs <10s 3 Very irregular, single movements difficult to distinguish or relevant 3 Severely abnormal, goes off shin 4 or more times during 3 interruptions, performs >10s cycles 4 Unable to complete 10 cycles 4 Unable to perform the task Score Right: Left: Score Right: Left:

Mean of both sides (R+L)/2 Mean of both sides (R+L)/2

ANNEX 3: Composite Cerebellar Functional Severity Score (CCFS)

Dominant hand Right Left

Nine-hole Pegboard test – dominant hand

The patient is seated and holds nine dowels (9mm in diameter and 32-mm long) in one hand and places them randomly, one by one, with the other hand in a board with nine holes. Timing begins when the first peg is placed in a hole and ends when the last peg is placed. The examiner holds the board steady on the table during the test. The trial is performed once only with the dominant hand. If the patient drops a peg the examiner stops the timer and the patient starts the test again once from the beginning.

Timing dominant hand: sec

256

Click test – dominant hand

The patient is seated facing the examiner across a table on which is placed a device composed of two mechanical counters fixed on a wooden board 39 cm apart. The patient uses his index finger to press the buttons on the counters alternately 10 times. Timing begins when the first button is pressed and stops when the second counter reaches 10. The trial is performed once only with the dominant hand.

Timing dominant hand: sec

Z pegboard dominant hand = Pegboard DH – (13.4-0.16*age +0.002*age2) =

Z click dominant hand = click DH – (8+0.05*age) =

CCFS = log10 (7 + Z pegboard dominant hand /10 + 4*Z click dominant hand /10) =

(Mean normal values 0.85 ± 0.05 (0.64 – 0.94))

ANNEX 4: 25 feet ambulatory test (from the Friedreich Ataxia Rating Scale)

To test gait, place markers 25 feet apart in hallway with no furniture within reach of 1 m/3 ft. and no loose carpet. Patient walks 7.62 m/25 ft at normal pace, turns around using single step pivot and return to start. The activity is timed. Note if the gait was achieved with or without helping device and serial examinations should be done with the same device as in the first examination.

Helping device No Yes Describe:

Time: sec

ANNEX 5: Unified Huntington's Disease Rating Scale (UHDRS) part IV: Functional Assessment

IV. UHDRS - FUNCTIONAL ASSESSMENT NO YES

43. Could subject engage in gainful employment in his/her accustomed work? 0 1

44. Could subject engage in any kind of gainful employment? 0 1

257

45. Could subject engage in any kind of volunteer or non gainful work? 0 1

46. Could subject manage his/her finances (monthly) without any help? 0 1

47. Could subject shop for groceries without help? 0 1

48. Could subject handle money as a purchaser in a simple cash (store) transaction? 0 1

49. Could subject supervise children without help? 0 1

50. Could subject operate an automobile safely and independently? 0 1

51. Could subject do his/her own housework without help? 0 1

52. Could subject do his/her own laundry (wash/dry) without help? 0 1

53. Could subject prepare his/her own meals without help? 0 1

54. Could subject use the telephone without help? 0 1

55. Could subject take his/her own medications without help? 0 1

56. Could subject feed himself/herself without help? 0 1

57. Could subject dress himself/herself without help? 0 1

58. Could subject bathe himself/herself without help? 0 1

59. Could subject use public transportation to get places without help? 0 1

60. Could subject walk to places in his/her neighbourhood without help? 0 1

61. Could subject walk without falling? 0 1

62. Could subject walk without help? 0 1

63. Could subject comb hair without help? 0 1

64. Could subject transfer between chairs without help? 0 1

65. Could subject get in and out of bed without help? 0 1

66. Could subject use toilet/commode without help? 0 1

67. Could subject’s care still be provided at home? 0 1

Functional Assessment Score: _____/25

68. Information Sources 1 Subject only

Was the Functional Assessment information obtained from: 2 Subject and family/companion

258

Annex 13:

Informed Consent

Diagnosis

Name

Family code /Serial No.

Phone No.

إقـــرار بالموافقة

نطلب من سيادتكم المشاركة فى الدراسة الخاصة باالمراض العصبية الوراثية و التى تهدف إلى دراسة األسباب التى تزيد من قابلية بعض األشخاص لإلصابة بالمرض وعدم إصابة البعض اآلخر من ناحية جينية، علماً بأن الدراسة ستضم أشخاصاً سليمين لمقارنتهم بالمصابين. تحصلنا على اسمك من الطبيب المعالج لك وقد أرشدنا إلى طلب المشاركة منك. مشاركتك فى الدراسة طوعية ويمكنك رفض المشاركة أو تطلب إلغاءك من الدراسة فى أى وقت متى رغبت ولن يؤثر على الخدمات العالجية التى تتلقاها. نرجو أن تقرأ اإلقرار أو أن يقرأ لك بحرص قبل التوقيع عليه.

بناءاً على موافقتك سوف نقوم بأخذ بعض المعلومات عنك عن تاريخ المرض و االسرة وسوف نطلب أيضاً منك أن تطلع على معلومات سجلك الطبي الخاص وسوف نأخذ عينة من اللعاب او مضمضة من الفم و قد وكذلك قد نأخذ عينة من الدم ) 01 سيسى(

سيتم ملء اإلستبيان و فحصك سريريا و سيتم تصوير الفحص بالفيديو لعرضه على مختصين لمزيد من المزايا التشخيصية وستأخذ العينة بينما أنت فى المستشفى ، العيادة أو المنزل وسوف تقوم بأخذ الدم ممرضة متمرسة أو طبيب من الذراع وسوف نفحص هذه العينة من ناحية جينية وخلوية لمعرفة األسباب المحتملة لتكون المرض.

تستخدم العينات الحيوية حاليا لألغراض البحثية و لكن سيتم ابالغك بنتائج الفحوص عند الوصول اليها و قد تتم اضافة بعض الفحوصات االخرى ذات االستخدام التشخيصي قريبا ولكن بعض نتائج هذا البحث ليس لها إستخدام حالى كفحص معملى روتيني ولذلك قد ال تكون ذات قيمة بالنسبه لك في المستقبل القريب لكن له فوائد على المدى البعيد في حال وجود بعض العالجات حاليا او في المستقبل و قد تساعد ذريتك في تفادي المرض .

لن تكون مشاركتك بمقابل عدا الزمن الذى سيأخذه اإلستبيان وأخذ العينة. قد تشعر بألم بسيط عند وخزك باإلبرة ألخذ عينة الدم منك كما هو الحال بالنسبه ألخذ الدم بالطريقة الروتينية وقد يحدث نادراُ تورم بسيط أو خروج للدم فى مكان الطعنة.

هذا البحث ربما لن تكون له فائدة مباشرة لك ولكنه ذو قيمة كبيرة لألجيال القادمة. 259

المعلومات الخاصة بك ستكون سرية وسوف تستخدم للألغراض البحثية فقط ولن يطلع عليها خالف الباحثون فى هذا المجال كما سيتعامل الباحثون مع العينة الخاصة بك فى المعمل كرقم ولن يكتب اسمك على العينة.

شهادةPالتوضيحPالخاص:PP

لقد قرأت التوضيح الخاص بهذه الدراسة وأعطيت فرصة للسؤال أو اإلستيضاح وأنا

أوافق ال أوافق

0- أخذ عينة من الدم

2- أخذ عينة من الفم.

3- اإلطالع على السجل الطبي.

4- ملء اإلستبيان:

توقيع المشارك: التاريخ:

توقيع الشاهد: التاريخ:

نشكر لك تعاونك فى هذا البحث الهام، إذا كانت لديك أى إستفسارات أو أسئلة يمكنك اإلتصال بـ:

.- معهد األمراض المتوطنة - جامعة الخرطوم - كلية الطب

تـلفون المشارك: ......

……………………………………………………………….. E. mail:

260

Annex 14:

Genes included of the Targeted NGS Panel (excluding the confidential genes)

Gene SPG code

ACP33 SPG21

ALS2 No SPG

AMPD2 SPG63

AP4B1 SPG47

AP4E1 SPG51

AP4M1 SPG50

AP4S1 SPG52

AP5Z1 SPG48

ARL6IP1 SPG61

ARSI SPG66

ATL1 SPG3A

B4GALNT1 SPG26

BICD2 No SPG

BSCL2 SPG17

C12orf65 SPG55

C19orf12 SPG43

CCT5 No SPG

CPT1C SPG73

CYP2U1 SPG56

CYP7B1 SPG5A

DDHD1 SPG28

DDHD2 SPG54

ENTPD1 SPG64

ERLIN1 SPG62

ERLIN2 SPG18

FA2H SPG35

FLRT1 SPG68

GAD1 No SPG

GBA2 SPG46

GJA1 No SPG

GJC2 SPG44

GRID1 No SPG

HSPD1 SPG13

KIAA0196 SPG8 261

KIAA0610 SPG20 KIAA1840 SPG11

KIF1A SPG30

KIF1C SPG58

KIF5A SPG10

L1CAM SPG1

MAG SPG75 Gene SPG code

MARS SPG70

MCM7 No SPG

MT-ATP6 No SPG

NIPA1 SPG6

NT5C2 SPG65

PGAP1 SPG67

PLP1 SPG2

PNPLA6 SPG39

RAB3GAP2 SPG69

REEP1 SPG31

REEP2 SPG72

RTN2 SPG12

SACS No SPG

SETX No SPG

SLC16A2 SPG22

SLC33A1 SPG42

SPAST SPG4

SPG7 SPG7

TECPR2 SPG49

TFG SPG57

USP8 SPG59

VCP No SPG

VPS37A SPG53

WDR48 SPG60

ZFR SPG71

ZFYVE26 SPG15

262

Annex 15: List of webtools and software:  1000 Genomes: http://www.1000genomes.org  Alamut Visual: http://www.interactive-biosoftware.com  Chromas Lite: http://sur.ly/o/technelysium.com.au/chromas_lite.html/AA001290  Ensembl Genome Browser: http://www.ensembl.org/index.html  EVS : http://evs.gs.washington.edu/EVS  Exome Aggregation Consortium (ExAC), Cambridge, MA: http://exac.broadinstitute.org  GeneCards®: www.genecards.org  NCBI, ClinVar: http://www.ncbi.nlm.nih.gov/clinvar/  NCBI Genome Browser: http://www.ncbi.nlm.nih.gov/genome/  NCBI Home: http://www.ncbi.nlm.nih.gov  NCBI, dbSNP: http://www.ncbi.nlm.nih.gov/SNP  NCBI, Pubmed: http://www.ncbi.nlm.nih.gov/pubmed  NCBI,Genereviews®: http://www.ncbi.nlm.nih.gov/books/NBK1116/?term=genereviews  Neuromuscular Disease Center: http://neuromuscular.wustl.edu  OMIM®: http://omim.org  Primer3Plus: http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi  Protein Data Bank: http://www.rcsb.org/pdb/home/home.do  UCSC Genome Browser: https://genome.ucsc.edu

 UniProtKB/Swiss-Prot: http://www.uniprot.org  Exomiser: https://www.sanger.ac.uk/resources/software/exomiser/  Ingenuity variant analysis: http://www.ingenuity.com/products/variant-analysis  Sift: http://siftdna.org/www/Extended_SIFT_chr_coords_submit.html  Polyphen2: http://genetics.bwh.harvard.edu/pph2/  Mutation Taster : http://www.mutationtaster.org/  Mutation Assessor : http://mutationassessor.org/  AlighnGVGD : http://agvgd.iarc.fr/index.php

263

 Human splicing finder: http://www.umd.be/HSF3/  NNSPLICE: http://www.fruitfly.org/seq_tools/splice.html  MaxEntScan: http://genes.mit.edu/burgelab/maxent/Xmaxentscan_scoreseq.html  GeneSplicer: http://www.cbcb.umd.edu/software/GeneSplicer/gene_spl.shtml  Splice Predictor: http://bioservices.usd.edu/splicepredictor/  Endeavour:http://homes.esat.kuleuven.be/~bioiuser/endeavour/tool/endeavourweb. php

 Suspect: http://www.cgem.ed.ac.uk/resources/suspects/

 Genedistiller: www.genedistiller.org

 Genic Intolerance: http://genic-intolerance.org/

 Collective tools: http://omictools.com/toppgene-s7336.html

 Human Protein Atlas: http://www.proteinatlas.org/

 Allen Brain: http://www.brain-map.org/

 Mouse Genome Informatics: http://www.informatics.jax.org/

 Phenomin: http://www.phenomin.fr/

 Orphanet: http://www.orpha.net/consor/cgi-bin/index.php

 Human Phenotype Ontology: http://www.ontobee.org/index.php

 Neuromuscular disease : http://neuromuscular.wustl.edu/

 Zotero: https://www.zotero.org/

264

List of figures Figure Title Page

Figure (1-1) Relative Frequencies of AD HSPs in European Populations: 1 based on SPATAX network statistics Figure (1-2) Relative Frequencies of AD HSPs in European Populations: 2 based on SPATAX network statistics

Figure (1-3) Proportion of HSP genes/loci according to inheritance mode 3

Figure (1-4) Correlation between the modes of inheritance and the clinical 6 phenotype (pure or complex).

Figure (1-5) Regrouping of the age at onset of various HSP subtypes. 8

Figure (1-6) Schematic illustration of the phenomenon of genes presenting 11 with closely related allelic phenotypes throughout the spectrum of the neurodegenerative disorders. Figure (1-7) Figure showing the overlap zone between spastic neurogenetic 12 disorders: evidence for the need of new nosology and objective case definition

Figure (1-8) Common pathogenic themes in the HSPs 16

Figure (1-9) Illustration of the pathogenic mechanisms in hereditary spastic 17 paraplegia (HSP) regrouped in nine functional and cellular localization categories

Figure (2-1) Sequence Capture Protocol: Basic illustration of the protocol for 37 library preparation for next generation sequencing (NGS) using capture enrichment technique Figure (2-2) Summary of the principal steps of next generation sequencing 38 (NGS)-based genetic investigations including: library preparation, NGS, bioinformatics and the genetic validation of selected variants

Figure (2-3) Illustration of the bridge amplification sequencing technique 39

Figure (2-4) Nextseq 500 emission. 43 Figure (3-1) Consanguinity rate in 41 families 49

Figure (3-2) The political map of the Sudan showing the origin of 41 families 50 included in the cohort

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Figure (3-3) Patterns of inheritance in 41 families 51

Figure (3-4) Clinical categories of 41 families 51

Figure (3-5) Distribution of age at onset in 41 extended families 53

Figure (3-6) Pedigree of family F15 caused by missense mutation in ARG1 90 segregating with the disease distribution in whole family presenting with spastic tetraplegia and mental retardation

Figure (3-7) Pedigree and MRI of the index patient of family F25 caused by 95 splice donor mutation in PLA2G6 segregating with the disease distribution in whole family presenting with pyramidal signs and features associated with infantile neuroaxonal dystrophy (INAD).

Figure (3-8) Pedigree and MRI of the index patient of family F30 caused by 99 missense mutation in SIL1 segregating with the disease distribution in whole family presenting with pyramidal signs associated with Marinesco Sjogren Syndrome (MSS).

Figure (3-9) Pedigree and colour image of the retina of the index patient of 103 family F26 caused by pathogenic CAG repeat in ATXN7 family presenting with pyramidal signs associated with spinocerebellar ataxia type 7 (SCA7).

Figure (3-10) Screening Panel success rate in classical HSP (23 families) 106

Figure (3-11) Combined Panel /Candidate Gene Approach: classical HSP 106 cohort (23 families)

Figure (3-12) Pedigree and MRI of the index patient of family F37 with a 113 nonsense mutation in ABHD16A segregating with the disease distribution in the whole family

Figure (3-13) Pedigree of family F41 showing segregation of missense 118 mutation in CAMSAP3 with the disease distribution in the whole family.

Figure (3-14) Pedigree of family F41 showing segregation of missense 120 mutation in MINK1 with the disease distribution in the whole

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family

Figure (3-15) Pedigree of family F41 showing segregation of missense 121 mutation in ZNF433 with the disease distribution in the whole family

Figure (3-16) Pedigree and MRI of two patients of family F50 showing 128 missense mutations in two putative candidate genes BIRC5 and C21ORF91 segregating with the disease distribution in whole family.

Figure (3-17): Pedigree and MRI of the index patient of family FM3 with 133 missense mutation in ST7L segregating with the disease distribution in the whole family

Figure (3-18) Pedigree and MRI of the two index patients from the two 139 branches of family FM7. Branch 1 of the family was associated with a missense mutation with full segregation with the disease distribution in branch 1 (blue rectangles) but not segregating in branch 2 (red rectangles).

Figure (3-19) Clinical Summary of the 24 families with unidentified genetic 144 cause

Figure (3-20) Pedigree of family F27. 158

Figure (3-21): Overall genetic diagnostic power of the study 159

Figure (3-22) WES success rate for known and new genes in the Sudanese 159 cohort

Figure (3-23) Schematic summary of the thesis method pipeline and genetic 160 results. Figure (4-1) Main clinical signs complicating the 41 families included in the 162 cohort

Figure (4-2) Brain MRI abnormal signs encountered in the cohort. 165 Figure (4-3) Pedigree of family F21. 167 Figure (4-4) Pedigree of family F14. 168 Figure (4-5) Pedigree of family F16. 169

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Figure (4-6) Suggested flow chart designed to assist in the variant/gene 170-171 prioritization process of gene panel data (based on inheritance and the most frequent clinical presentations).

Figure (4-7) Comparison of the success rates of all approaches that were 172 utilized in genetic investigation. Figure (4-8) Schemtic representation of the ABHD12/ABHD16A axis 174 Figure (4-9) Biological functions of lysophosphatidyl serine 176

Figure (4-10) Expression of pantothenate kinases (PanK 1, PanK2, PanK3 and 189 PanK4) Figure (4-11) Summary of pathogenic mechanisms of hereditary spastic 197 paraplegia (HSP).

Figure (4-12) Statistical correlations identified using regression test 198

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List of Tables Table Title Page

Table (1-1) Clinical classification of various HSP forms 7 Table (2-1) Disability Scoring system used in the SPATAX standard questionnaire Table (2-2) Components of culture medium used for collection of skin biopsy and 46 harvest of fibroblasts. Table (3-1) Clinical categories in the 41 families 52

Table (3-2) Distribution of age at onset in 41 extended families 53

Table (3-3) Clinical data of five patients from family F15 with 91 hyperargininemia due to a mutation in ARG1 gene

Table (3-4) Clinical summary of family F15 93

Table (3-5) Clinical data of two patients from family F25 with infantile 96 neuroaxonal dystrophy (INAD) due to a mutation in PLA2G6 gene.

Table (3-6) Clinical data of four patients from family F30 with Marinesco 100 Sjogren Syndrome due to a mutation in SIL1 gene

Table (3-7) Clinical data of three patients from family F26 with 104 spinocerebellar ataxia (SCA7) due to a pathologic CAG repeat in ATXN7.

Table (3-8) Summary of mutations in known genes found in three families 107 with pyramidal features.

Table (3-9) Summary of mutations in putative candidate new genes found in 109 five families with spastic neurogenetic disorder.

Table (3-10) Clinical data of two patients from family F37 with a nonsense 114 mutation in ABHD16A.

Table (3-11) Clinical data of two patients from family F41 122

Table (3-12) Clinical data of two patients from family F50. 129

Table (3-13) Clinical data of three patients from family FM3 134

Table (3-14) Clinical data of five patients from family FM7 branch 1 140

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Table (3-15) Clinical data of two patients from family FM7 branch2 142

Table (3-16) Clinical summary of 23 families with unidentified genetic cause 145 (family 27 is presented in a separate table).

Table (3-17) Clinical summary of family F27 with unidentified genetic cause 157

Table (4-1) Detailed clinical signs complicating the 41 families included in 163 the cohort. Table (4-2) Table summarizing the 88 HSP clinico-genetic entities with 199 special focus on the functions of their proteins and our suggested primary and secondary functional categories.

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Paraplégies spastiques héréditaires : exploration clinique au Soudan, études des origines moléculaires des formes autosomiques récessives et identification de nouveaux gènes en cause.

Résumé :

Les paraplégies spastiques héréditaires (PSH) font partie d’un groupe plus large de pathologies neurodégénératives associant une spasticité. J’ai exploré la variabilité clinique et moléculaire de ces pathologies à l’aide d’une cohorte de familles soudanaises. Nous avons recruté 41 familles soudanaises [337 individus/106 atteints de PSH]. J’ai extrait l’ADN génomique et constituer une banque. Le criblage de gènes candidats a été réalisé dans 4 familles en fonction du phénotype des patients. La technologie de séquençage de nouvelle génération (SNG) appliquée à 74 gènes de PSH a ensuite été appliquée aux 37 cas restants. Enfin, le séquençage de l’exome a permis de rechercher les gènes en cause dans les cas négatifs. Dans certains cas, des études fonctionnelles ont été utilisées afin de valider l’effet biologique des mutations. J’ai pu identifier la cause génétique dans 17 familles. Pour 12 familles, la mutation concernait un gène de PSH connu. Dans 3 familles, un nouveau gène a pu être incriminé tandis que dans 2 autres, 5 gènes candidats restent à départager. Il est à noter que parfois, de multiple mutations ou maladies génétiques ségrégaient dans nos familles soit dans la même ou dans des branches séparées. La complexité de ces familles fortement consanguines a rendu l’analyse des données du SNG difficile. Une autre particularité a été l’hétérogénéité clinique associée à des mutations du même gène entre patients de la même famille ou en comparaison avec la littérature. Ce travail est la première étude à grande échelle de patients soudanais avec PSH et rapporte de nouveaux gènes en cause, prérequis pour mieux comprendre dans le futur les mécanismes sous-jacents. Mots clés : [paraplégies spastiques héréditaires; famille Soudanaise ; consanguinité ; séquençage de nouvelle génération ; le séquençage de l’exome ; nouveaux gènes]

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Hereditary spastic paraplegias: clinical spectrum in Sudan, further deciphering of the molecular bases of autosomal recessive forms and new genes emerging

Abstract:

Hereditary spastic paraplegias (HSP), a heterogeneous group of spastic neurodegenerative disorders which impose diagnostic challenges. I explored the clinical varieties and genetic pathways of spastic neurodegeneration in a familial Sudanese cohort. We recruited 41 Sudanese families [337 individuals/106 HSP patients]. I have established a genomic DNA bank and when necessary, skin biopsies and fibroblasts were also obtained. A phenotype-based candidate gene approach was followed in 4 families. A targeted next generation sequencing (NGS) for 74 HSP-related genes was the main screening strategy in all remaining 37 families. Whole exome sequencing (WES) was done in search for novel mutations in new genes in families with negative screening results. Occasionlly, functional studies were conducted when feasible and relevant. I identified the genetic cause in 17/41 families. In 12 families, the mutated genes were known HSP genes. In 3 families, novel genes were identified mutated. 5 candidate genes segregated with disease in 2 other families with more experiments needed to conclude. Analysis of the NGS screening panel and of WES data imposed certain challenges as multiple genetic disorders were sometimes found running in parallel in the same/ different branches of highly inbred families. We could expand the phenotypic heterogeneity of these disorders due to clinical differences observed between Sudanese patients and patients of other origins even when caused by mutations by the same gene/variant. This is the first genetic screening in a large set of HSP families in Sudan. It describes new causative genes, paving the way for further deciphering of the underlying mechanisms. Key Words: [Hereditary spastic paraplegias; Sudanese families; consanguinity; next generation sequencing (NGS); Whole exome sequencing; novel genes]

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