PIGT-CDG, a Disorder of Glycosylphosphatidylinositol Anchors: Description of Fourteen Novel Patients and Expansion of the Clinical Characteristics

University of Southern Denmark

PIGT-CDG, a disorder of the glycosylphosphatidylinositol anchor description of 13 novel patients and expansion of the clinical characteristics Bayat, Allan; Knaus, Alexej; Juul, Annika Wollenberg; Dukic, Dejan; Gardella, Elena; Charzewska, Agnieszka; Clement, Emma; Hjalgrim, Helle; Hoffman-Zacharska, Dorota; Horn, Denise; Horton, Rachel; Hurst, Jane A.; Josifova, Dragana; Larsen, Line H.G.; Lascelles, Karine; Obersztyn, Ewa; Pagnamenta, Alistair; Pal, Deb K.; Pendziwiat, Manuela; Ryten, Mina; Taylor, Jenny; Vogt, Julie; Weber, Yvonne; Krawitz, Peter M.; Helbig, Ingo; Kini, Usha; Møller, Rikke S.; the DDD Study Group Published in: Genetics in Medicine

DOI: 10.1038/s41436-019-0512-3

Publication date: 2019

Document version: Accepted manuscript

Citation for pulished version (APA): Bayat, A., Knaus, A., Juul, A. W., Dukic, D., Gardella, E., Charzewska, A., Clement, E., Hjalgrim, H., Hoffman- Zacharska, D., Horn, D., Horton, R., Hurst, J. A., Josifova, D., Larsen, L. H. G., Lascelles, K., Obersztyn, E., Pagnamenta, A., Pal, D. K., Pendziwiat, M., ... the DDD Study Group (2019). PIGT-CDG, a disorder of the glycosylphosphatidylinositol anchor: description of 13 novel patients and expansion of the clinical characteristics. Genetics in Medicine, 21(10), 2216-2223. https://doi.org/10.1038/s41436-019-0512-3

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PIGT-CDG, a disorder of glycosylphosphatidylinositol anchors: description of fourteen novel patients and expansion of the clinical characteristics.

Allan Bayat 1 γ*, Alexej Knaus 2,3,4 *, Annika Wollenberg Juul 1 *, Usha Kini 5, Elena Gardella 6,10, Agnieszka Charzewska 7, Emma Clement 8, Helle Hjalgrim 6,9,10, Dorota Hoffman-Zacharska 7, Denise Horn2, Rachel Horton 11, Jane A Hurst 8, Dragana Josifova 12, Peter Krawitz 2,3,4, Line HG Larsen9, Karine Lascelles 13, Ewa Obersztyn7, Deb K Pal 14, Manuela Pendziwiat 15, Mina Ryten12,16, Julie Vogt 17, Yvonne Weber 18, Ingo Helbig 16,19, Rikke S. Møller9,10

1 Department of Pediatrics, University Hospital of Hvidovre, Hvidovre, Denmark.

2 Institute for Medical Genetics and Human Genetics, Charité Universitätsmedizin Berlin, Berlin, Germany.

3 Berlin-Brandenburg School for Regenerative Therapies (BSRT), Charité, Germany.

4 Institute for Genomic Statistics and Bioinformatics, Rheinische Friedrich-Wilhelms-University of Bonn, Germany

5 Department of Clinical Genetics, Oxford University Hospitals NHS Trust, Oxford, United Kingdom.

6 Danish Epilepsy Centre, Dianalund, Denmark.

7Institute of Mother and Child, Department of Medical Genetics, Kasprzaka 17A, 01-211 Warsaw, Poland

8 North East Thames Regional Genetics Service, Great Ormond Street Hospital for Children, London WC1N 3JH, United

Kingdom.

9 Amplexa Genetics, Odense, Denmark.

10 Institute for Regional Health Services Research, University of Southern Denmark, Odense, Denmark.

11 To be investigated

12 The Guy's and St. Thomas NHS Trust, Clinical Genetics Department, Great Maze Pond, London SE1 9RT, United

Kingdom.

13 Department of Neuroscience, Evelina London Children's Hospital, St Thomas' Hospital, Westminster Bridge Road, London SE1 7EH, United Kingdom.

14 Department of Basic and Clinical Neuroscience, Institute of Psychiatry, Psychology and Neuroscience, King’s College London, London , United Kingdom.

15 Department of Neuropediatrics University Medical Center Schleswig-Holstein Christian Albrechts University Kiel, Germany.

16 Reta Lila Weston Research Laboratories, Department of Molecular Neuroscience, University College London, Institute of Neurology, London, United Kingdom.

17 Clinical Genetics Unit, Birmingham Women's Hospital, Birmingham, United Kingdom.

18 Department of Neurology and Epileptology, Hertie Institute of Clinical Brain Research, University of Tübingen, Tübingen, Germany.

19 Division of Neurology, The Children's Hospital of Philadelphia, Philadelphia, PA, United States.

γ Corresponding author

* Shared first authors

Abstract

PIGT-CDG, an autosomal recessive syndromic form of a glycosylphosphatidylinositol biosynthesis defect (GPIBD) with intellectual disability, has so far only been described in seven independent families and all but one patient presented with an epileptic encephalopathy. PIGT encodes phosphatidylinositol-glycan biosynthesis class T, a subunit of the heteropentameric transamidase complex that facilitates the transfer of proteins to the GPI anchor. The GPI anchor links proteins to the cell membrane in all tissues. We describe fourteen novel patients from nine unrelated families with homozygous (NM_015937.5: c.709G>C, p.Glu237Gln and c.550G>A; p.Glu184Lys) or compound heterozygous (c.1472T>A, p.L491H; c.1484+2T>A and c.494-2A; c.1582G>A; p.Val528Met) mutations in PIGT. All patients had hypotonia, severe global developmental delay and epilepsy. Epilepsy onset ranged from first day of life to 2 years of age, and the severity of the seizure disorder varied from treatable seizures to severe neonatal onset epileptic encephalopathies. Furthermore, nine patients presented with ophthalmologic anomalies and four patients had skeletal anomalies such as long slender bones, scoliosis, pectus excavatum and dysplastic distal phalanxes. Congenital fractures were only found in one patient which is a feature not described previously. In addition, abnormal body-hair distribution was observed in eight out of the fourteen patients. In this study we provide a detailed description of the phenotype of PIGT-CDG. Furthermore we investigate the onset and severity of epilepsy determined by the different genetic subtypes and our results emphasize that GPI anchor-related congenital disorders of glycosylation (CDGs) should be considered in both subjects with a developmental delay plus epilepsy and in patients with an epileptic encephalopathy even in the presence of a normal carbohydrate-deficient transferrin pattern and N-glycan profiling. Currently available screening for CDGs will not reliably detect this family of disorders, and our cases reaffirm that the use of genetic testing is essential for diagnosis in this group of disorders.

Key Words PIGT-CDG, Congenital disorder of glycosylation, Transamidase, GPI, Exome.

Key Point Box (…) 1. Introduction Glycosylphosphatidylinositol (GPI) is a glycolipid that is synthetized and transferred to proteins in the membrane of the endoplasmic reticulum1. Biogenesis of GPI anchored proteins (GPI-APs) is a conserved post-translational mechanism in eukaryotes and is important for attaching these proteins to the cell membrane, for protein sorting, trafficking, and dynamics2. It also plays an essential role in embryogenesis, immune responses and neurogenesis3-6. GPI synthesis and GPI- anchored protein (GPI-AP) modification are mediated by at least 29 genes and loss-of-function mutations in 16 of these genes may lead to neurological impairments including seizures, intellectual disability (ID), developmental delay (DD) and multiple congenital anomalies7-22. PIGT [MIM, 610272] encodes phosphatidylinositol-glycan biosynthesis class T, which is a subunit of the heteropentameric GPI transamidase complex that facilitates the attachment of GPI anchors to proteins23;24. In 2013 Kvarnung et al. reported a homozygous PIGT mutation in four patients from a consanguineous Turkish family with Multiple Congenital Anomalies-Hypotonia-Seizures Syndrome 3 (MCAHS3) [MIM, 615398]10. Subsequently compound heterozygous mutations in PIGT were identified in six unrelated families with a similar clinical presentation25-28. Recently Skauli et al. presented two somalic brothers with typical features of MCAHS3, but in addition, pyramidal tract neurological signs27. The predominant presentation for the 12 patients described so far is that of an epileptic encephalopathy including profound ID, hypotonia, intractable seizures, cortical visual impairment, and nystagmus and/or strabismus. Here we describe fourteen additional patients from nine unrelated families with PIGT mutations presenting with seizures, ID, and congenital anomalies.

2. Methods 2.1. Variant filtering and analysis.

We screened a cohort of 793 patients with various childhood-onset epilepsies for mutations in the PIGT gene using a previously described next generation sequencing panel29. The panel included targeted capture of all exons and at least 10 base pairs of flanking intronic sequence of PIGT. Genomic DNA from blood was extracted with standard methods, and a next-generation sequencing panel screening method was applied that was based on the Ion Torrent PGM platform. Ion AmpliSeq (kit version 2.0) or Sureselect library building methods were used with subsequent clonal amplification and enrichment on an Ion OneTouch 2 system with the Ion PGM Template OT2 200 Kit, followed by sequencing on the Ion Torrent system with the Ion PGM 200 Sequencing Kit. Variants resulting from the gene panel sequencing were reduced by allele frequency ≥2% and SNPs observed in more than 2 samples for each analyzed sample batch were filtered out. Genetic non-synonymous/splice site variants were evaluated through database searches such as dbSNP, Exome Variant Server, the Exome Aggregation Consortium database (ExAC), and HGMD Professional. One homozygous missense mutation (NM_015937.5: c.709G>C, p.Glu237Gln) that leads to an exchange of a highly conserved amino acid was predicted by SIFT, MutationTaster, and PolyPhen-2 to be pathogenic. 16 heterozygos carriers were identified in the gnomAD database of this variant and Sanger sequencing confirmed biparental inheritance.

In parallel, we ascertained additional, previously unreported PIGT patients through epilepsy and genetic centers in Europe and the United States. The probands and their families underwent detailed clinical examinations, review of the medical files, MRIs, and EEG investigations.

2.2. Standard protocol approvals, registrations, and patient consents. The study was approved by the local ethics committees. All probands or, in case of minors, their parents or legal guardians gave informed consent. The clinical information has been collected from hospital journals of the patients and their family members.

3. Results

In our screening cohort of 793 patients we identified one patient with a likely pathogenic homozygous PIGT mutation. Furthermore, we recruited twelve additional patients with PIGT mutations from other research and diagnostic programs. The pedigrees of the twelve newly identified individuals with homozygous or compound heterozygous PIGT mutations are shown in figure 1. An overview of the clinical, MRI and genetic features of the fourteen novel patients and of the twelve previously published PIGT-CDG cases is provided in Table 1.

3.1. Phenotypic analysis. All the fourteen patients suffered from ID/DD, either profound (patients 1-3, 7-10, and 12-14) or severe (patients 4-6 and 11), and from epilepsy with neonatal / infancy onset. The median age at seizure onset was six months (range 1 day – 18 months). All the patients had myoclonic and/or tonic seizures, often with apnea, sometimes evolving to bilateral tonic-clonic seizures. Subtle focal seizures were also described (patients 1-3, 9-10 and 12). Fever-associated seizures were reported in all patients. The symptoms varied from profound intellectual disability and severe drug resistant, neonatal or infantile onset epilepsy (patients 1-3, 7-10 and 12-14) with recurrent episodes of convulsive status epilepticus (patients 1-2), to severe intellectual disability and treatable epilepsy with later age at onset (8-18 months) (patients 4-6 and 11). The interictal EEG was available in 13 out of 14 patients (no data available for patient 8) and was severely abnormal in 12 of them (see table 1). One single patient had a normal EEG at epilepsy onset (patient 9) and no further EEG controls were available. In patients (1-3, 13, 14) with neonatal epilepsy onset, the EEG showed initially a burst suppression pattern and at follow up (patients 1-2) was characterized by background slowing/destructuration, with frequent multifocal spike and slow waves. Patients (7, 9, 10, 12) with early infantile epilepsy onset and severe drug resistant epilepsy might have a normal EEG at epilepsy onset (patients 9-10) and developed background slowing, focal theta-delta activity in the fronto-temporal regions or in the posterior quadrants, and frequent multifocal spike and slow waves at follow up. The EEG of patients (4-6 and 11) with treatable epilepsy with later age at onset showed only background slowing (patient 11) or background slowing with sporadic (patients 4-6) epileptiform abnormalities in the fronto- temporal regions.

A cortical visual impairment was diagnosed in six patients (patients 1, 2, 5, 6, 8 and 12). None of the patients were diagnosed with hearing loss, and they all exhibited normal alkaline phosphatase, plasma calcium, plasma phosphate and parathyroid hormone values. Congenital heart defects were identified in two individuals and both resolved spontaneously: a persistens foramin ovale (PFO) in one patient (patient 9) and the combination of an PFO and an atrial septal aneurysm in another (patient 7). Patients 2 and 8 both died due to a pneumonia at respectively 11 and 15 months of age. Patient 3 died at six months of age following a cardiac arrest and patient 4 was operated on at the age of two years for a suprasellar adamantinomatous craniopharyngioma. Our novel cases shared similar craniofacial features that included a high forehead with bitemporal narrowing, a depressed nasal bridge, a short anteverted nose, distinct philtrum, full cheeks and an open mouth consistent with general hypotonia. Scalp hair, eyebrows, and eyelashes were sparse in eight of the fourteen patients (patient 1, 2, 4-8 and 12) (figure 2 and supplementary figure S1). Since clinical pictures were not available it has not been possible to evaluated whether or not the remaining patients shared this feature. Cerebral MRI was available in all fourteen patients and was abnormal in all but two patients (patients 6 and 13). MRI abnormalities included prominent cortical and subcortical volume loss with brainstem atrophy (patients 1, 2, 4, 9, and 11-12), white matter immaturity (patients 1, 2, 3), hypoplastic cerebellum (patients 4-6, 9 and 12) and an abnormal corpus callosum (patients 1 and 2) (table 1). A detailed clinical description of all fourteen patients is available in the supplementary file S-1.

3.2. Mutational analysis We identified twelve different mutations including three novel mutations. One of these three mutations was a missense mutation and two of the novel mutations affected a splice site. Concerning the inheritance mode, in all families the mutation was inherited in a recessive fashion from unaffected parents. All mutations were predicted to be damaging by 1 or 2 prediction tools (Polyphen2 or SIFT; supplementary file S-2). The protein positions of the different PIGT mutations are shown in figure 3. The mutation c.709G>C was found in a girl from Asian heritage (patient 3) and in two Bangladeshi siblings (patient 13 and 14) while the mutation was found c.1582G>A in

four Polish patients (patient 4-6 and 11). While c.709G>C has been previously published in an Afghanistani male30 the variant c.1582G>A has not previously been published. This leads us to conclude that c.1582G>A seems to be a European variant while c.709G>A is a Middle-Eastern or Asian variant. The variant c.1582G>A was also found in the four patients classified as having a severe developmental delay with a treatable epilepsy (patients 4-6 and 11). Since this variant has also previously been described in an affected female with a developmental delay and epilepsy27 (Table 1). We contacted Dr. Kini Usha and Dr. Rachel Horton who informed us that this patient had a global developmental delay with seizures, and that she became seizure-free with a combination of antiepileptic drugs. This suggests that the missense variant c.1582G>A could be associated with a milder phenotype.

3.3. Overall mutational landscape Mining the available literature and databases, we were able to identify 12 previously reported PIGT cases (Table 1)10;25-27;30. From the complete dataset of 24 patients with PIGT mutations, 14 different mutation sites emerged: c.250G>T (n=2); c.494-2A, (n=1); c.547A>C (n=2); c.550G>A (n=2); c.709G>C (n=4); c.918dupC (n=1); c.1079G>T (n=1); c.1096G>T (n=1); c.1342C>T (n=2); c.1472T>A (n=2); 1484+2T>A (n=2); c.1582G>A (n=6); c.1724_1725insC (n=1); c.1730dupC (n=1)

4. Discussion So far only 12 patients from seven different families have been described with a GPI anchor deficiency due to recessive PIGT mutations10;25-27;30 and all but one patient27 presented with an epileptic encephalopathy. We describe an additional fourteen patients from nine unrelated families. The patients show a broad clinical spectrum and share several common features (Table 1). The neurological findings include a severe/profound ID, epilepsy with variable onset and severity and a severe congenital hypotonia. The symptoms varied from profound intellectual disability and severe drug resistant epilepsy with neonatal-infantile onset epilepsy (9/13 patients), to severe intellectual disability and treatable epilepsy with later age at onset (8-24 months) (4/13 patients). Based on previously published and our novel findings we conclude that the missense variant c.1582G>A seems to be associated with a milder phenotype. Ophthalmological features

including nystagmus and/or strabismus and a cortical visual impairment were also observed in nine out of fourteen patients (Table 1).

The bone and endocrine features in our patients differed from the initial five patients reported by Nakashima et al.28 and Kvarnung et al.10, however their features overlapped with the four patients presented by Lam et al.25 and Skauli et al.27. Our patients exhibited normal alkaline phosphatase, plasma calcium, plasma phosphate and parathyroid hormone values. Bone age was only available in patient 1 and was normal. Except of the brothers presented by Skauli et al.27 all previously described patients had reduced bone mineralization and scoliosis, features that could arise secondary to their neurologic complications. X-rays were only available in three of our patients (patient 1 and 2) and only two patients (patient 1 and 13) underwent a systematic skeletal survey. Scoliosis was found in one of our patients (patient 5), long bones with reduced mineralization were found in three patients (patient 1, 2 and 13) and in patient 13 the reduced mineralization was present already at birth (pictures not shown). Dysplastic distal phalanges were found in one patient (patient 1) (pictures not shown). Congenital fractures located at the right humeral, the right femoral diaphyses and along the midshaft of the left humerus and also subtle contour abnormality at the anterior ends of the left seventh and eighth ribs were only found in one patient (patient 13) which is a feature not described previously (pictures not shown). So far craniosynostosis has only been described in two patients by Kvarnung et al.10 however a metopic and a saggital ridge was found in one of our patients (patient 9) and did not require surgical intervention.

One of the affected sibpairs had abnormal dentition (patient 1 and 2), similar to the patients described by Kvarnung et al.10 which has previously been reported in one additional family. This finding could be unrelated to PIGT, could reflect a genotype phenotype effect, or could arise from differences in genetic background. Five patients (patient 1-2,5-6 and 12) also exhibited significant joint hypermobility which was also reported by Lam et al.25.

Ten of the published PIGT deficient patients had an MRI done and in all patients cortical and cerebellar atrophy was evident. Kvarnung et al.10 presented two patients in whom MRI at the age of 2.7 years demonstrated global atrophy with predominant vermis and cerebellar atrophy.

The patient described by Nakashima et al.28 demonstrated progressive atrophy of the cerebral hemisphere, cerebellum and brainstem at the age of three years. The two brothers published by Skauli et al.27 were examined respectively at 14 months and 2.8 years of age. Both demonstrated cortical atrophy and cerebellar atrophy, primarily affecting the vermis. Finally Lam et al.25 presented MRI observations supporting that atrophy in the cerebellum starts earlier and proceeds more rapidly than atrophy elsewhere in the brain. So far published data suggests that the disease preferentially affects the cerebellum. In contrast to previously reported patients four of our patients (patients 1-3, 6-8, 10 and 14) showed no signs of cerebellar atrophy (Figure 4, pictures not available for patient 3 and 6). Patients 2, 3, 6 and 14 had the MRI done at a very early age, which could perhaps explain why the cerebellar atrophy was not yet evident, however patient 1 had an MRI done at three days (Figure 4A-B), three months (Figure 4C-D), nine month (Figure 4E-F) and four years (Figure 4G-H) of age. Based on the previously published MRI findings in PIGT patients we would have expected a cerebellar hypoplasia to be detectable at four years of age. Although cortical atrophy was evident we detected no signs of cerebellar atrophy in patient 1. This further expands the clinical spectrum of PIGT-CDG.

Congenital heart defects are were indentified in only two individuals and both resolved spontaneously: a persistent foramin ovale in one and a combined persistent foramin ovale and an atrial septum aneurysm in another.

The majority of the published patients had onset of febrile-induced seizures between four and six months of age followed by unprovoked and poorly controlled seizures. In the article by Kvarnung et al.10 the age at onset of seizures was not specified. We therefore contacted Dr. Kvarnung who informed us that onset of febrile-induced seizures in their patients was around 12-18 months of age. In our cohort the onset of epileptic seizures did also include the neonatal period leading us to conclude that PIGT-CDG should be considered in patients with hypotonia, severe global developmental delay, and neonatal seizures. EEG recordings were severly abnormal in patients with neonatal onset epilepsy, and might also be normal or almost normal in the other patients early in life. At follow up, the most common EEG features were the slowing of the background activity, often associated with focal slowing and multifocal epileptiform abnormalities, predominant in the fronto-

temporal regions. The severity of the EEG abnormalities seemed to correlate with the severity of the phenotypes.

Our novel cases shared similar facial features with previous patients leading us to conclude that there is a common facial gestalt that includes a high forehead with bitemporal narrowing, a depressed nasal bridge, a short anteverted nose, distinct philtrum, full cheeks and an open mouth consistent with general hypotonia. Scalp hair, eyebrows, and eyelashes were sparse in eight of fourteen patients (patient 1, 2, 4-8 and 12). After reviewing the pictures of the previously described patients we believe that the patients V-1, V-2, V-4 described by Kvarnung et al.10 and the siblings reported by Lam et al.25 all show signs of sparse scalp hair including a high and thin anterior hairline. Abnormal distribution of hair on the body has not been described as a feature of any of the GPI anchor disorders25;31 and the hypotrichosis as a clinical feature has not been previously described in patients with PIGT-CDGs. We furthermore identified hypertrichosis in patient 1 which is a possible novel feature in this disease. Therefore abnormal body-hair distribution including hypotrichosis might be a recurrent but overlooked feature of PIGT-CDGs.

There are no current FDA approved therapies for GPI anchor disorders, but the progressive nature of PIGT-CDG is attractive for therapies that slow or halt the neurologic deterioration37. Identification of additional PIGT-CDG patients should further define the clinical spectrum and assist in developing diagnostic criteria.

Conflict of interest The authors declare no conflict of interest.

Acknowledgments We would like to thank the families for participating in this study. We thank Dr. Malin Kvarnung for kindly sharing previously unpublished data regarding the onset of epileptic seizures in their patients. We thank Dr. Kini Usha and Dr. Rachel Horton for kindly sharing previously unpublished data regarding the outcome of epileptic seizures in their patient.

Ingo Helbig was supported by intramural funds of the University of Kiel, and by a grant from the German Research Foundation (DFG, HE5415/6-1). Yvonne Weber was supported by a grant from the DFG (WE4896/3-1). Agnieszka Charzewska was sponsored by Polish National Science Centre grant No 2014/15/D/NZ5/03426.

Legends Figure 1: Two-generation pedigree of the nine affected families. Clinically affected family members are shown as shaded squares and circles. P - patient.

Figure 2: Clinical pictures of patient 1 at 3½ years (A), patient 2 at 11 months (B), patient 4 at 2½ years (C), patient 5 at 7 years (D), and patient 6 at 19 months (E). Sparse scalp hair, bitemporal narrowing, a high forehead, a distinct philtrum, a tented mouth, full cheeks, a depressed nasal bridge, and a short nose in all patients is notable. Eyebrows were either straight (A, B, C) or arched (D, E).

Figure 3:

A: Locations of mutations in the PIGT gene. Novel mutations described in this study are represented in red with the patient ID in superscript. Known pathogenic mutations are shown with corresponding reference numbers. B: Conservationanalysis. Amino acid comparison of PIGT of different species. Mutations affect highly conserved amino acids (black boxes).

Figure 4. MRI of patient 1 and patient 2 showing sagital T1W and axial T1W. MRI exams of patient 1 were performed at three days (4A-B), three months (4C-D), nine month (4E-F) and four years of age (4G- H). MRI of patient 2 were performed only once at thirteen days of age (4I-J). MRI at three days (4A-B) shows delayed myelination, including insufficient myelinated PLIC (posterior limb internal capsule), normal sulcations and no atrophy, at three month (4C-D) atrophy

of the splenium of the corpus callosum without growth, supratentorial atrophy of white matter, delayed myelination, including unmyelinated ALIC (anterior limb of the internal capsule) and only PLIC (posterior limb internal capsule) fully myelinated, at nine months (4E-F) somewhat progressed myelination, still delayed, resembling a three month old on T1W with a little, but still insufficient growth of corpus callosum and supratentorial atrophy of white matter, at four years (4G-H) clearly progressed myelination, but still not completed, supratentorial atrophy of white matter and corpus callosum. No atrophy of the cerebellum. Magnetic Resonance Spectroscopy (TE:135 ms) at 3T performed in Patient 1 , nine months old , from right parietal white matter and bioccipital grey matter showed near normal N-acetylaspartate/choline, choline/creatine-ratios, no lactate or other unusal metabolites. T1W sagittal (4I) and axial (4J) brain MRI exam of patient 2 taken at thirteen days of age revealed global delayed myelination, including insufficient myelinated PLIC (shown), normal sulcation and no atrophy.

Table 1: Clinical features of fourteen novel PIGT-CDG cases and the clinical features in the twelve previously published PIGT-CDG cases

Supplementary figure S1: Sparse scalp hair (A - C), hypertrichosis of the legs (D) and back (E), and delayed tooth eruption with small and widely spaced teeth (F) of patient 1.

Supplementary figure S2: Patient 2 presented with a pectus excavatum (A), short and friable nails and slightly clinidactyly (B and C). Reference List

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Table 1 Clinical features of twelve novel PIGT-CDG cases and the clinical features in the fourteen previously published PIGT-CDG cases.

Nakas Patient 5 Patient 6 Patient 7 Patient 8 Patient Patient Patient Patient Patient Patient

Physiologic Kvarnung hima Kohashi Patient 4 9 10 11 12 13 14 Lam et al. Pagnamenta Patient 1 System Clinical et al. et Skauli et 2017 Patient 2 Patient 3 2015 [25] 2017 [30] 2013 [10] al.201 al. [26]

4 [28] 2016 [27] Caucasian Patient 1: Patient 1: Japanese Asian Polish Polish Polish Somalian Somalian Femal Male, Polish Pakistani Banglade Banglade mother African Caucasion male, female, female, female, female, male, male, e deceas female female, 2- shi shi and an male, female, age 11 deceased 5-years- 9-years-old 4-years- 11-years 22- (family ed at / 4- years-old female, 6- female, 4- African 9-years- unknown months- at 6 old (family 4) old old months- 6) 26 years- (family 8) months- months- American old old months (family 3) (family 4) (family 5) old month old old old Danish Turkish Japane father Patient 2: of age (family 5) s of (family (family (family 9) Danish male, family. se Patient 2: Afghanistani (family 2) age 7) 9)- female, deceased Sex / age at 4 affected female Patient 1: African male, age (family 7-years- at 11 investigation females. . 12- A 7-years- male, unknown 6) old months Ages 1-3 years- old 7-years- (family 1) of age years old female old (family 1)

Patient 2: A 6-years- old male

Whole Whole Whole Whole Targeted Whole Sanger Exome Sanger Exome Target Exome Targeted Targeted Whole exome exome exome exome NGS exome sequenci sequenci sequenci seque ed seque NGS NGS exome seque sequenci sequenci sequenci panel sequencing, ng ng via the ng ncing Sanger ncing panel panel sequenci Whole ncing ng ng ng Whole Whole Whole DDD via the seque via the after ng Whole Genetic exome exome exome exome study DDD ncing DDD result in exome investigation sequenci sequenci sequenci sequenci study after study patient sequencing ng ng ng ng result 10 in patien t 9 Patient 1: c.1097 c.1079G> c.1582G>A; c.250G>T; c709G>C; c.494-2A, c.1582G>A; c.1582G> G>T; c.550G>A c.250G c.918dup T; c.1730dupC c.1096G> c.1472T> c.1472T> c709G>C, c.1582G> c.1582G>A A; c.1079 c.1079 c.1079 c.1097 c.1724 ; c.709 c.709 Genetic c.547A>C; >T; C; c.1079G> T A; A; A; c.1582G> G>T; G>T; G>T; G>T _1725i c.550G>A G>C; G>C;

result c.547A>C c.1342 c.1342C> T Patient 2: 1484+2T> 1484+2T> A c.1079G> c.1079G> C.1079 nsC; c. 709G>C c. 709G>C C>T T c.709G>C; A A T T G>T c.1582 c.709G>C G>A

Gestation 37–40 40 40 weeks Unknown Unknown 39 weeks 39 weeks 38 weeks 37 weeks 42 weeks 29 weeks 40 weeks 42 36w 40 weeks 39 weeks Neonatal 31 weeks 40 weeks 39 weeks al age weeks weeks weeks weeks

50– 10th and Birth 69–99th 50th Unknown Unknown 90th 50th 0.4th >90th >95th >95th 95th Unknown 50th 99th 91th 72th <0.4th 0.4th 90th 90th weight centile centile centile centile centile centile centile centile centile centile centile centile centile centile centile centile centile

Birth 93–99th 50th 10-90th 50th Unknown Unknown 95th 90th Not >97th >95th >75th Not Unknown Unkno Un Unkno Unknown Unknown Unknown length centile centile centile centile centile centile recorded centile centile centile known wn known wn

10th and Unknown Unknown Not >95th >95th 69th Unknown <0.4th >95th Not Not <0.4th <0.4th Birth 50– >97.5th recorded 50th centile centile centile centile centile record recorded centile centile head 84–99th <10-75th >95th 50th 90th centile centile ed circumfer centile centile centile centile centile ence

Other No Polyhy 1/2 No No Electiv dramni Elevated Cesarean Forceps No No No Unknown Cesare e Vagina Born by Forceps Multiple os AFP on No Unknown section assisted No an cesare l assited assisted bone 2nd due to delivery sectio an bleedi vaginal delivery, fractures trimester breech n due sectio ng at delivery Bilateral screen presentat to 6-8 elbow ion breech weeks contractu presen and res & tation 12wee distal ks of finger pregna contractu ncy res Unknown Unknown Not at Unknown Unknown Unknown Brachycepha No Saggital No Metop birth Norma Brachyce Normal Normal

Dysmorphic Brachyce Unkno Brachyce (2/2) Brachyce Brachyce ly ridge ic and Later. l phaly Skull features phaly wn phaly phaly phaly saggita Scaph

(4/4) (2/2) l ridge oceph aly Unknown High Unkno Yes Yes Unknown Yes Yes Yes Yes Yes Yes Yes Yes Yes Unknown Yes 4/4 2/2 Yes Yes forehead wn (patient 1)

Bitempor Unknown NO Yes No No

al Unkno Yes Yes Unknown Yes Yes No Yes No No No 4/4 2/2 Yes Yes narrowin wn (patient 1)

g, Unknown No Mild Unknown Unknown Yes Yes Yes Yes No Mild teleca No Yes

Telecanth Unknown (patient 1) (and also Yes teleca nthus Unkno Yes Yes us Unknown Unknown epicanthu nthus wn s)

Unknown Norma

Straight Straight Unknown Straight, Straight Upslanting Upslantin Mildly No Straigh l Straigh Normal Normal Upslantin Palpebral Upslan Upslantin Straight, Straight (2/2) (patient 1) normal g downslati t, t, g fissures ting g normal (4/4) length ng normal normal (2/2) length lenghtl length Unknown Norma

Arched Unkno Arched Arched Straight Arched Straight Arched Arched Straight Unknown Straigh l Norma Normal Normal Normal Eyebrows Straight Straight (3/4) wn (2/2) (1/2) (patient 1) t l

Unknown Norma

Short, Short, Short with Short, Small Short, Short, Short, Unknown Unkno l Broad Short Normal Normal Short, Short, Short, Short, antevere antevere broad nasal antevere anteverted anteverte antevere wn nasal anteverte Nose anteve antevere antevere antevere d d tip (patient d d d tip d red d d d (4/4) (2/2) 1) (2/2)

Unknown Mildy Depressed Depresse Broad Unknown Depres depres Norma Normal

Nasal Depresse Depres Depresse Unknown Depresse Depresse Depresse Depresse d and sed sed l Broad Normal Depresse bridge d sed d (2/2) d d d d depresse d (2/2) (4/4) d

Unknown No Malar Yes Yes Yes Unknown Unknown Yes Yes Yes No No Yes No Yes No No Yes Yes Yes flattening (4/4) (2/2) (2/2)

Unknown Normal Norma

Short Short Long Normal Long and Long and Normal Short l Distinc Distinct Normal Normal Long and Unkno Long Long and Long and Philtrum (2/2) (patient 1) distinct distinct t distinct wn (2/2) distinct distinct

(4/4) Unknown No Yes Yes Unkno Yes Cupid Unknown Unknown Unknown Yes Yes Yes Yes Yes No Yes Yes Yes Normal Unknown (4/4) wn (2/2) bow lip (tented lip)

Unknown Tented Downt Tented Wide mouth Unknown Prominent Prominen Tented Tented Tented upper Unkno Tented Unknown Unknown urned 2/2 upper lip (patient 1) Tented Tented Tented and everted t and upper lip upper lip upper lip wn upper lip corner Downtur (2/2) upper lip, upper lip, upper lip lower lip everted Downtur lip Mouth Unknown s ned microsto microsto lower lip ned of corners mia mia corners mouth of mouth Unknown Unkno High Yes Unknown Yes Not Yes Yes Yes Yes Unkno wn No Yes Yes Yes Unknown arched Unknown Yes (2/2) Yes No recorded wn (2/2) palate

Unknown Gum Teeth Unknown Unknown Unknown No No Yes Unknown No hypert No Yes Unknown Unknown Yes abnormal No Unknown Yes Yes Unknown rophy (4/4) ities

Unknown No No

Microgna No No Yes No No No No No No No No No Yes Yes No Yes Yes thia (4/4)

Low set Unknown Norma

Low Large Low set Low set Unknown and Normal Normal Normal Low set No Norma l Norma Normal Normal Low set Ears Unknown Normal set ears and (patient 1) upliftet l l

(2/2) (1/2) earlobes Unknown Unkno Unknw Unkno Hypotrich Unknown Unknown Hypotrich Hypotrich Hypotrichosi Hypotrich Hypothric Hypothric wn on wn Hypothric Unknown Unknown Hypotrich Hypotrich osis osis Hypotrich osis, high s, osis, osis. Thin osis. Thin osis. Thin osis Ingen osis Thin, high Thin, high osis frontal Thin, high Thin, high frontal frontal frontal

Hair Thin, high billede Thin, high frontal frontal Thin, high hairline frontal frontal hairline. hairline. hairline. frontal r frontal hairline. hairline. frontal hairline. hairline. hairline. hairline. (2/2) Hypertric hairline. hosis Norma Body Short Unkno Normal Normal Unknown Unknown Unknown Normal Relative Relative Normal Normal Norma l Norma Normal Normal Normal proportio arms Normal Normal wn (2/2) macrocephal macrocep l l ns (4/4) y haly

Intellectu Profound Profou Profound Profound Profound Profound Severe Severe Severe Profound Profound Profou Profou Severe Profound Profound Profound Neurological al Profound Profound Profound (4/4) nd (2/2) (2/2) nd nd disability (2/2) 12 Patient 1: 2 months 2 weeks 18 11 months 8 months 11 14 10 12 12 5 1st day of 1th day months 12 months of life of life months of age of age months months month month month months life of life 4 of age of age of age of age of age s of s of s of 12-18 Seizure month 5 months 1th day 1th day age age age months onset s of of life Patient 2 of life of life of age life Neonatal onset

Myocloni Tonic-clonic Myocloni Myocloni Myocloni Generalized Myocloni c and Tonic- and Tonic and Generaliz Myocloni Myocl Myocl Gener Generalis Myocloni Generalis Myocl Tonic and Tonic and c (2/4). c, tonic, c, tonic, tonic clonic c, tonic subtle clonic generalized myocloni ed tonic c and onic onic alised ed tonic c jerks. ed tonic onic, myocloni myocloni and tonic and tonic (2/2) with focal and c clonic focal and and tonic and seizures. tonic c with c with Generaliz clonic clonic apnea (febrile / generaliz occasiona and seizures subtle focal clonic myocloni with apnea. apnea. Epileptic ed tonic that that that can upper ed lly atonic focal seizure seizure c. Focal apnea Subtle Subtle seizures clonic occasiona occasiona generaliz respirator generaliz seizures seizure s, s myocloni that focal focal (1/4). lly lly e y ed with s. Some c can seizures. seizures. generaliz generaliz infection) apnea Febrile genera seizures. genera Recurrent Recurrent Absence+ e e seizure lised. Febrile lize CSE CSE head (2/2) (2/2) s. Febrile seizures. jerks & seizure blinking s. (1/4) Multifocal Multifoca (2 wks) (4 yrs) (3 and 7 yrs) (18 mo) (22 mo) NA (10 (12 (12 (6 mo) (neonatal (age epileptiform l burst slowing BG slowing BG bilateral mo) mo) mo) BG ) burst unknown) (2 wks) Multifoca abnormalitie epileptifo (2 wks) suppressi and slowing, bursts of normal bilater BG slowing, suppressi BG bilateral Multif Multifoca l s rm bilateral on, (poly)SW sporadic SW al fast slowin posterior on slowing Multifoca burst of ocal l epileptifo (2/2) abnormal burst of myocloni in the left SW in the activit g, epileptifo pattern and burst l SW epilept epileptifo rm ities SW c jerks, FT region left FT y (f.u) rm suppressi EEG epileptifo (6 mo) BG iform rm abnormal (4 yrs) BG focal sz region or Multif abnormal on features rm slowing, abnor abnormal ities slowing, (right and F bilat ocal ities pattern abnormal focal malitie ities (2/2) focal FT left T) epilept ities posterior s (2/2) slowing iform (3/4) slowing and SW abnor and SW malitie s

Patient 1: Intractabl Favorable Favora Intractabl e Intractabl Intractabl (seizure-free Intractabl Intractabl Favorable Favorable Favorable Intractabl Intractabl Intract Intract ble e e

e with AED). e e e on AEDs e able able Seizure Intractabl Intract Intractabl Intractabl Intractabl but outcome e able e e e Patient 2 almost

Intractable seizure- free on ketogenic diet Epilept Epilept Patient 1: Epileptic Develop Developmen Develop Epileptic Epileptic ic ic Develo Epileptic

Developmen encephal mental tal delay mental encephal encephal encep encep pment Epileptic Epileptic encephal Epileptic Epileptic tal delay Epileptic opathy delay with delay opathy opathy halopa halopa al encephal encephal opathy encephal Epilept encephal with encephal with epilepsy with thy thy delay opathy opathy opathy or Epileptic ic Epileptic Epileptic Epileptic opathy epilepsy opathy epilepsy epilepsy with developm encephal encep encephal encephal encephal epileps ental opathy halopa opathy opathy opathy Patient 2 y delay thy Epileptic with encephalopa epilepsy thy

Yes Yes Yes but Yes Yes Yes Yes Yes (2/2) Yes Yes Yes Yes Yes Yes but with Yes Hypotoni Yes Yes Yes (2/2) Yes Yes with brisk a (4/4) (2/2) brisk reflexe reflexe s s Yes Yes Was an Yes Yes Yes MRI Yes Yes Yes Yes Yes (2/2) Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes available (4/4) (2/2) (2/2)

Brain imaging Progressive Progressi No Progre Global Unknown No — Global isolated ve No (only Yes ssive cerebral cerebral cerebellar isolated No (only mild isolate and and Yes atrophy cerebellar No Yes Yes No slight ventricula d cerebellar cerebellar (2/2) affecting atrophy asymmetr r cerebe atrophy. atrophy vermis and affecting y of the dilatation llar with Yes Yes Yes cerebellar vermis No No hippocam ) atroph predomin (3/4) (2/2) hemispheres and pi) y ant (patient 1) cerebellar affecti vermis hemisphe ng and res vermis cerebellar and hemisphe hemis re pheres atrophy (at 4 years of age). Brain Unknown Unknown Unknown No Unknown Unknown imaging No No No No Normal — No No No No Unkno Possible 2/4 0/2 No No wn neuronal migration defect Unknown Unkno Unknown Unknown Unkno CSF wn Normal Unknown Norma wn Norma Normal Normal Not

(glucose, l l performe High protein, Yes Normal Unknown Unknown Unknown d Normal protein cell (2/2) Unknown (2/2) count count,

oligoclon al bands) CSF Unknown Unkno Unknown Unknown Unknown Norma Norma Unknown

albumin wn Unknown Unknown l l Unkno Unknown Normal Low & Unknown Normal Unknown Unknown Unknown wn (2/2) High albumin

quotient Unknown Unknown Unknown Unknown Not EMG Normal perfor and Not Normal Not Not Unknown Not Not med nerve perfor performe performe performed performe condu med Not d d d ction performe did not d show any eviden

Periphera ce of

l nerve Not Not neuro Unknown Unkno Normal conductio performe performe muscul wn (2/2) n studies d d ar

proble m but neurog enic change in bulbar muscul ature Unknown Unknown No No No but Unkno ophthalm Unknown wn Yes No Yes ological Yes Yes No Yes No Cortical (2/2) examinati Ophthalmol visual Yes Yes Yes Yes Yes on was ogic impairme (4/4) (2/2) done nt once at three weeks of age.

Unknown Unknown Anteri

Strabismu Yes No No Yes Yes No Yes Yes or No Yes Unknown Unknown Yes s, Yes (2/2) conver (4/4) Yes Yes Yes nystagmu (2/2) gent

s Down

beat nystag mus Unknown Unknown No No Yes Unknown No Yes Yes Yes No No Unknown Unknown Unknown Hyperopi Unkno Yes 1/2 No Unknown a wn (4/4)

Unknown Unknown No No No No Unkno Myopia 0/4 1/2 Unknown Yes Unknown No No No No No Unknown Unknown Unknown wn

Unknown No No Deep Vertica Unknown Unknown Astigmati Oculomotor No optic l No sm apraxia No No Astigmatism Astigmati discs oculo (2/2) (patient 1). sm motor Astigmati Nystagmus apraxi sm Down and optic a, (1/2) gaze Unkno atrophy previo Other Posterior palsy wn (patient 2) us staphylo (1/4) episod ma es of (1/2) tonic upgaze deviati on Unknown Unknown No Normal Unkno Unkno No Normal Normal Unknown Audiologic Hearing Unkno 0/4 2/2 Unknown No No No Unknown No No wn wn loss wn

Unknown PFO with No Small Unkno No No No Patent No No No No No No an atrial PDA wn No Cardiologic ductus 1/4 Yes 0/2 No No septal arteriosus aneurysm

Unknown No No No Unkno No Restrictiv No No No No No wn No No No e No 1/4 No 0/2 No No cardiomy opathy

Unknown No No No Recurr No No No No Unknown Unknown No ent No No No bradyc ardia and atrial ectopi Increased cs 1/4 No 1/2 No No atrial load noted on ECG- no cardiac echo perfor med Atrial Unknown No No Unkno Unkno No

Septal 0/4 No 1/2 No No No No No No No No wn wn No No No

defect Unknown No Unkno No No Borderlin Unkno Unknown 1/2 Unknown No Unknown Unknown Unknown No No No No wn Unkno No Unknown e long QT wn wn Atypical Unknown Mild Obstru Central Central lung Unkno Mixed No No None obstructiv ctive apnea None Central No No Respiratory sleep sleep lobulatio wn central Unknown Central None Unknown e sleep sleep sleep apnea apnea n and apnea apnea apnea apnea (1/4) obstructiv e sleep apnea (1/2)

Unknown No No No No No No No No Unknown No Gastro- Unkno Obesity No Yes Unknown No No No No No intestinal wn (2/2)

Unknown Unknown Yes No Unkno Yes Gastroes Unknown Unkno Yes Unknown Unknown Unknown No No Yes No wn Yes Unknown ophageal Unknown Unknown wn (2/2) reflux

Unknown Impaired Yes Yes Aspiratio Unknown Unkno Unknown Yes Yes Unknown Unknown No swallow Unkno No Unknown Yes

n risk + wn Unknown wn Yes secondar (2/2) Yes Yes y G-tube

depende nce Unknown Unknown Unknown Unkno Unkno Unknown Hypertrigl Unknown Unkno Unknown Unknown Unknown Yes Unknown Unknown wn wn Unkno No Unknown yceridemi 1/2 Unknown Unknown wn wn a

Unknown Unknown Unkno No No No Nephrolithia No Unknown No No No wn Left Unknown No Urologic/Re Nephroca Yes (but + No sis (patient No No No ureteri nal lcinosis (4/4) urolith 1) c iasis) stone Unknown Unknown Unknown Unkno Unknown Urine Norma High Normal Unknown Unknown Unknown Unknown Unknown Unknown Unknown No No wn Norma No Unknown calcium l l Unknown Unknown No No Unknown Not Yes Ureteral Yes No No No known No No Unknown Yes 0/2 No No No dilation (3/4) No

Unknown Unknown Unknown Unkno No Renal No No No No No No wn No Unknown No Cysts and ¼ No 0/2 No No No dysplasia

Prematur Unknown Unknown Unknown No Musculo- e loss of 2/4 No 0/2 Unknown No Unknown Unknown No No No No No No No No Unknown skeletal teeth Unknown Unknown Unknown Unkno Not No Slender No (but Unknown No No Unknown wn known No Unknown Yes Unkno long 4/4 2/2 pictures Yes Yes Unknown wn bones not shown) Unknown Unknown No No Unkno No No No No

Scoliosis 2/4 Yes 2/2 No No No No No Yes No wn No

Unknown Unknown No No Unknown No No No Unknown No Metop No Unknown No Craniosyn 2/4 No Unknown Unknown Unknown ic and ostosis saggita l ridge Unknown Unknown No No Unkno No No No No No Short Unkno No No No Unknown Unknown wn 4/4 0/2 No No arms wn

Pectus Unknown Unknown No No Unkno No No Yes Unknown No 1/4 ½ No No excavatu Unkno No No No No No wn m wn

Joint Unknown Unknown No No Unkno Unkno Unkno Yes No Unknown

hypermo Unknown Unkno 2/2 Unknown Yes Yes No No Yes Yes wn wn wn

bility wn Unknown Unknown Unknown Unknwon Unkno Neuro Unkno No Unknown No Unknown Unknown Unknown Unknown Unknown wn genic wn Unknown Unkno change wn in the Reduced bulbar muscle muscul Electromy bulk, but Unknown Unknown ature ography normal other motor wise units normal EMG and NC Advanced Unknown Unknown Unknown Unknwon Unkno Not Unkno Unknown Unknown Unknown Delayed Unkno (1/2), Normal Unknown Unknown Unknown Normal Unknown wn known wn Endocrine Bone age Normal (4/4 wn Normal (2/2) (1/2) Unknown Unknown Unknown No Unkno Not Unkno Unknown Yes Unknown Unknown wn known wn

Osteopen No Unknown Unknown Normal 4/4 Yes 2/2 Yes Yes ia (2/2)

Normal Norma Unknown Plasma Normal Normal Normal l alkaline Normal (patient 1) Low Normal Normal Normal Norma Norma Normal Normal Low Low Normal Normal Normal phosphat Low l l ase (patient 2)

Unknown Normal Norma Unknown Not Normal Normal Norma l specified l Norma Normal Normal Normal but Normal Unknown Normal l Plasma High/high Norma laborator Normal Normal Normal calcium normal l y tests were descibed as normal

Unknown Normal No Unkno Norma Norma Unknown Normal Normal Not Normal Normal Normal wn l l Unknown specified Normal but Plasma laborator Unkno phosphat Normal Normal y Normal Normal wn e tests were descibed as normal

Unkno Unknown Unknown Unknown Unkno Unkno Unknown wn Not Unknown Unknown Unknown wn wn Unknown specified Unknown Norma Parathyro but Normal l Normal id laborator Low Normal Normal Normal hormone y tests values were descibed as normal

Unkno Unknown Normal Unkno Not Unknown wn Not Normal Normal wn done specified Normal at Normal but Normal Normal GOSH Norma Unknown Normal Thyroid laborator l function Normal Normal Normal Normal y tests tests were descibed as normal

Unknown Unkno Unkno No Not wn wn specified Unknown Unknown No No Unknown Unkno Unknown but Unknown Unknown Not No Unknown Precociou wn laborator applicabl 0/4 ½ Unknown s puberty y tests e were descibed as normal

Unknown Unkno Unknown Unknown Normal Unkno No wn Not wn specified Unknown Unknown Unknown Unkno IgA Unknown Unknown IgA and but IgA and Unknown Unknown wn low, Immunologi IgM laborator IgM IgM ½ Unknown c deficienc y tests deficienc lower y were y limit of descibed normal as normal

Unknown Unkno Unknown Unknown Not Unknown Unknown No No Unknown Pneumoc Unkno Bronch Unkno Unknown Unknown wn specified occal wn iolitis wn but meningiti and Unknown Other laborator s and chest immune 0/2 y tests No No sepicaemi infecti deficienci were a ons es descibed as normal

Unknown Unkno Unknown Unknown No Unkno Unknown wn Not wn specified Unknown Unknown Normal Normal Unknown Unknown Unkno Unknown Unknown Hyper/hy but wn po laborator Hematologic 0/2 Normal Normal coagulabi y tests lity were descibed as normal

Unknown Unkno Unknown Unknown Unkno Unkno Unknown Unknown wn Slightly Not wn wn decrease specified Unknown d Factors but Unknown Unknown Unknown Unknown Unknown Unknown Unkno V & XI laborator wn Factor (1/2) y tests excess or were deficienc Mildly descibed y Unknown No increased as normal Factor VIII (1/2)

Abbreviations: AFP: Alpha-fetoprotein BG: background CDG: Congenital disorders of glycosylation CSE: Convulsive status epilepticus DDD: Deciphering Developmental Disorders EMG: Electromyography F: frontal NGS: Next generation sequencing PFO: Persistent foramen ovale PIGT: Phosphatidylinositol-glycan class T SW: spike and waves Sz: seizures T: temporal

Clinical report Patient 1: A 7-year-old girl, the first child of non-consanguineous healthy Caucasian parents, was born at 40 weeks of gestation after a normal pregnancy. Birth weight 4010 gram (90th centile), birth length 54 cm (95th centile) and head circumference 38 cm (95th centile). APGAR scores were 8 at both 1 and 5 min of life. Within a few hours after birth, she was admitted to the neonatal intensive care unit due to poor sucking, severe hypotonia and epileptic seizures. She presented with tonic and myoclonic seizures with apnea, both of which repeatedly turned to convulsive status epilepticus (CSE). Seizures were refractory to multiple antiepileptic drugs including phenobarbital (PB), levetiracetam (LEV), valproic acid (VPA) and midazolam (MDZ). After two months, she was discharged from the neonatal ward but she was still experiencing recurrent seizures despite treatment with a combination of PB, LEV, VPA and oxcarbazepine (OXC). In the following years she had intractable myoclonic and tonic seizures with secondarily generalization despite combinations of PB, vigabatrin, LEV, VPA, topiramate (TPM), OXC, MDZ and diazepam. Currently she is treated with TPM, OXC and clobazam and has approximately twenty monthly events with either brief tonic seizures and deviating eye movements or tonic-clonic seizures with apnea that last around one minute. Her initial electroencephalogram (EEG) performed at two weeks of age showed multiple bilateral bursts of spike-and-waves. She was frequently admitted to the hospital because of CSE induced by fever, recurrent episodes of respiratory infections or urinary tract infections. The interictal EEG at the age of four years of age showed diffuse background slowing and focal slowing as well as frequent spike and slow waves predominant in the fronto- temporal regions, bilaterally asynchronously. On video EEG polygraphic recordings have been documented frequent episodes of central apnea (op to 10 sec), mainly during NREM sleep but sometimes also during wakefulness, without EEG correlate. By the age of nine months, significant developmental delay was evident: she had poor head control, was not fixing or following, had head lag when pulled to sit and made no attempt to elevate her head when prone. At the age of 7 years she is profoundly delayed. She is unable to sit or stand on her own but she is able to make eye contact and smile. She has no meaningful words and her sleep cycle is disorganized. Since the age of twenty months she has been dependent on a permanent feeding tube. Ophthalmological examination revealed cortical visual impairment and abnormal motility of the eyes including strabismus and nystagmus. Auditory examination showed normal hearing.

Minor dysmorphic features was noted shortly after birth including a depressed nasal bridge, an anteverted nose and a long philtrum. However her dysmorphic features became more evident over time and at latest evaluation (Figure 2A) they included brachycephaly with bitemporal narrowing, telecanthus, a long and distinct philtrum, a small tented mouth with a high and arched palate, full cheeks, a depressed nasal bridge and a short anteverted nose. Scalp hair and eyebrows were sparse, the eyebrows were straight (Figure 2A and figure S1). She also presented with hypertrichosis (Figure S1). Tooth eruption was delayed and the teeth were small and widely spaced (Figure 2I). She also exhibited joint hypermobility, overlapping toes and skeletal abnormalities. A systematic skeletal survey performed at 76 months of age illustrated skeletal abnormalities including an S-shaped scoliosis, slender osteopenic long bones, bilateral dislocated hips with a shallow acetabulum and dysplastic distal phalanxes in both feet (pictures not shown). The bone age was normal. An echocardiography and an ultrasound of the kidneys performed at thirteen months of age were normal. Brain MRI was performed at three days (Figure 4A-B), three months (Figure 4C-D), nine month (Figure 4E-F) and four years of age (Figure 4G-H) showed delayed myelination, atrophy of the white matter supratentorially and a severely hypoplastic corpus callosum. There were no signs of cerebellar atrophy. Metabolic screening included plasma amino acids, lactic acid, pyruvic acid, organic acids, lysosomal enzymes and these were all unremarkable. Analysis of the urine for mucopolysaccharides and oligosaccharides also showed normal results. Standard karyotyping and a SNP 6.0 array were both normal. She had normal levels of alkaline phosphatase, plasma calcium, plasma phosphate and parathyroid hormone. Due to the hypertrichosis blood levels of 17- hydroxyprogesterone, cortisol and adrenal steroids were measured and were found to be normal.

Patient 2: Patient 2 was the younger brother of patient 1. He was born at 39 weeks of gestation following an uneventful pregnancy. Birth weight 3685 gram (50th centile), birth length 54 cm (90th centile) and head circumference 36 cm (50th centile). APGAR scores were 8 at both 1 and 5 min of life.

Following birth, he was admitted to the neonatal intensive care unit due to severe hypotonia and within a few hours he developed epileptic seizures. He showed tonic and myoclonic seizures with apnea and frequent episodes of CSE. His seizures were also refractory to multiple antiepileptic drugs including PB, LEV, VPA and midazolam while the seizures decreased with the combination of PB, LEV, VPA, TPM and OXC. After 1½ months he was discharged from the neonatal ward. In the following months he experienced treatment resistant brief or longer lasting myoclonic and tonic seizures with secondary generalization. Prior to his dead, he was treated with LEV, carbamazepine and PB. A ketogenic diet was never tried. EEG performed at two weeks of age showed multiple bilateral bursts of spike-and-waves. The EEG at follow up showed diffuse background slowing/destructuration and continuous delta activity and spike-and- slow waves bilaterally with clear predominance in the posterior quadrants. He had (non-epileptic) central apneas during NREM sleep.

Following the neonatal period there were no significant psychomotor gains. He had a poor head control, was not fixing or following, had head lag when pulled to sit and made no attempt to elevate his head when prone. Ophthalmological examination revealed cortical visual impairment and abnormal motility of the eyes including strabismus and nystagmus. Auditory examination showed normal hearing. He had dysmorphic features including brachycephaly with bitemporal narrowing, telecanthus, a long and distinct philtrum, a small tented mouth with a high and arched palate, full cheeks, a depressed nasal bridge and a short anteverted nose (Figure 2B). Scalp hair, eyebrows, and eyelashes were sparse, the eyebrows were straight (Figure 2B), and the nails were short and friable (Figure S2). Tooth eruption was delayed and the teeth were small and widely spaced (pictures not available). Furthermore, he exhibited pectus excavatum (Figure S2) and joint hypermobility. At eleven months of age he died of pneumonia.

Brain MRI performed at thirteen days of age (Figure 4I-J) showed delayed myelination and a hypoplastic corpus callosum. Ultrasound of the kidneys and an echocardiography was never performed.

Metabolic screening included plasma amino acids, lactic acid, pyruvic acid, organic acids, lysosomal enzymes and these were all unremarkable. He had normal levels of alkaline phosphatase, plasma calcium, plasma phosphate and parathyroid hormone. A systematic skeletal survey was never performed, however X-ray of the chest revealed a slender long right humerus bone (Figure 4I).

Patient 3: First female child of healthy consanguineous Asian parents born at 39+5 weeks gestation by forceps assisted delivery. Intrauterine growth retardation was noted during the pregnancy and her birth weight was 2380g (0.4th percentile). Head circumference at birth was not recorded but at two weeks of age it was 33cm (9th percentile) and her weight was 2690g (below 0.4th percentile). Apgar scores were 3 at 1 min and 10 at 5 min and she required stimulation and oxygen but no ventilatory support. At birth she was noted to be centrally and peripherally hypotonic, weak and areflexic with multiple joint contractures. She required nasogastric tube feeding. She was transferred to the department of pediatrics at 2 weeks of age for further evaluation of a suspected neuromuscular disorder. Brief myoclonic jerks were noted and an EEG showed a burst suppression pattern with brief myoclonic jerks as well as subtle focal seizures of right and left temporal onset. She did not respond to trials of biotin, pyridoxal phosphate, TPM and LEV. She remained severely hypotonic, encephalopathic and did establish neither a social smile nor meaningful visual behavior. At six months of age she died following central sleep apnea and cardiac arrest at home.

Her MRI scan showed absence of myelination in the posterior limbs of the internal capsule and signal change in the basal ganglia bilaterally indicating a possible neuro-metabolic disorder (pictures not shown). Her electromyography and nerve conduction patterns were normal. An echocardiography and an ultrasound of the kidneys were normal. A skeletal survey was never performed. An ophthalmological examination was done at three weeks of age and was not followed up again.

An extensive neurometabolic screen including bloods for creatinine kinase, lactate, ammonia, aminoacids, transferrin isoforms, very long chain fatty acids, urate, acylcarnitines, vacuolated lymphocytes, electron microscopy of the buffy coat, urine for purine screen, and organic acids, CSF lactate, neurotransmitters, pyridoxal phosphate and aminoacids were all negative. She had normal levels of alkaline phosphatase. Clinical pictures of this patient were not available and the description of her dysmorphic features was based on the patient files.

Patient 4: This patient was a 2-year-old girl, the first child of non-consanguineous parents of Polish origin. The family history was unremarkable for birth defects, intellectual disability or other genetic diseases. The girl was born spontaneously at 39 weeks of gestation with a length of 56 cm (97th centile), weight of 4150 g (90-97th centile) and head circumference at 36 cm (50th centile). Weight, length and head circumference progressed in the normal range and her current measures are length 91 cm (75th centile), weight 12.7 kg (50th centile) and headcircumference OFC 50 cm (75-90th centile). At 18 months of age she experienced her first tonic-clonic seizure. She was initially treated with VPA and responded well but did not become seizure free. Therefore LEV was added to the treatment which has so far kept her free from seizures. Her initial EEG performed at the age of 18 months was normal. At the age of 4 years the EEG showed focal slowing and spike/polyspikes-and slow waves in the left fronto-centro-temporal region. During the first year of life a muscular hypotonia and a marked psychomotor developmental delay was noticed. At 28 months her development was profoundly delayed. She had just learned to sit but was unable to stand on her own and had no meaningful words. An ophthalmologic evaluation had revealed a severe bilateral hyperopia. Her MRI scan showed a mild cerebellar hypoplasia and in addition a suprasellar cystic tumor. The tumor was surgically removed and subsequent histological investigation showed that it was an adamantinomatous craniopharyngioma (WHO grade 1). An extensive neurometabolic screen including bloods for creatinine kinase, lactate, ammonia, aminoacids, transferrin isoforms, very long chain fatty acids, urate, acylcarnitines, urine for purine screen, and organic acids were all negative. She had normal levels of alkaline phosphatase. Her dysmorphic features included a high forehead with sparse hair, a high frontal hairline, and bitemporal narrowing. A small nose with a depressed nasal bridge, tented upper lip, cupid bow lips were prominent (Figure 2C).

Patient 5: The proband, a currently 7-years-old girl, was born after an uneventful second pregnancy at 38 weeks of gestation by cesarean section because of mother’s orthopedic indications. Body weight was 4810 g (>95th centile), length was 57 cm (>95th centile), and occipito-frontal head circumference was 38 cm (>95th centile). The family history was unremarkable for birth defects, intellectual disability or other genetic diseases. There is healthy daughter from the first pregnancy. The parents were young, healthy and non-consanguineous. Both Apgar’s scores were normal and the neonatal period was uneventful. Horizontal nystagmus and astigmatism (+8.5D) with visual impairment were diagnosed at 8 months of age. Clinical evaluation at 10 months showed psychomotor retardation with muscular hypotonia and a severe delay in motor development. At the age of 11 months she started to experience febrile generalized febrile tonic-clonic seizures coincident with upper respiratory infection. The seizures typically lasted 2-5 minutes. Due to a high frequency of epileptic seizures a treatment with VPA was initiated at the age of 18 months. Currently she is still on VPA and has been seizure free since the treatment was initiated although at the age of 6 years following a head injury she experienced a short generalized tonic-clonic. Her EEG in awake performed at 3 and 7 years of age showed background slowing 4-7 Hz (the basal function of the parietal-occipital region consists of amplitude up to 70uV with low-voltage fast action) without epileptiform abnormalities. At the age of 7 years, an MRI showed a mild hypoplasia of the lower part of cerebellar vermis, mild hypoplasia of cerebral hemispheres with a cystic dilatation of the fourth ventricle and an enlarged posterior fossa corresponding to a Dandy - Walker malformation. Ultrasonography of the abdomen as well as an echocardiographic evaluation was normal. EEG investigation indicated abnormal excess of slow waves in the temporo-occipital region with multifocal epileptiform abnormalities. An extensive neurometabolic screen including bloods for creatinine kinase, lactate, ammonia, aminoacids, transferrin isoforms, very long chain fatty acids, urate, acylcarnitines, vacuolated lymphocytes, electron microscopy of the buffy coat, urine for purine screen, and organic acids, CSF lactate, neurotransmitters, pyridoxal phosphate and aminoacids were all negative. She had normal levels of alkaline phosphatase. Slight hyperglyceridemia was noted. Chromosome analysis showed a normal karyotype of 46, XX at 500-band, and no CNVs were found in microarray-based comparative genomic hybridization (array CGH – CytoSure Constitutional v3 resolution 120kb, Oxford Gene Technology). Neurologic examination showed mild hypotonia with joint hypermobility with tremor of the hands. Deep tendon reflexes were normal and pathologic reflexes were absent. At 7 years of age she was still unable to walk. She had a mild degree of intellectual disability and difficulties with speech and articulation. Her dysmorphic features included a brachycephalic headshape with a high forehead. Scalp hair and eyebrows were sparse with a high frontal hairline. She had upslanting palpebral features, telecanthus, arched eyebrows, slightly depressed nasal bridge, prominent and long philtrum, cupid bow lips with prominent lower lip and a high palate (Figure 2D).

Patient 6: She is the five years younger sister of patient 5. She was born after an uneventful third pregnancy at 37 weeks of gestation by cesarean section because of mother’s orthopedic indications. Body weight was 3380 g (>75th centile), length was 53 cm (>95th centile), and occipito-frontal head circumference was 36 cm (>95th centile). Apgar scores were normal. Shortly after birth nystagmus with strabismus and poor visual contact was found. At the age of 7 months, the first myoclonic-tonic generalized seizures with breathing disturbances and ocular movements occurred. Treatment with VPA was immediately implemented. At the age of 18 months, her EEG showed discrete background slowing and sporadic high amplitude spike-and-slow wave complexes in the left fronto-temporal region or bilaterally in the frontal regions. During the neonatal period, an abdominal ultrasound, an echocardiography and a brain MRI did not reveal any abnormalities. Results of biochemical, hormonal and metabolic investigations, among others plasma amino acids, lactic acid, pyruvic acid, organic acids, lysosomal enzymes were all unremarkable. She had normal levels of alkaline phosphatase, plasma calcium, plasma phosphate and parathyroid hormone. Neurologic examination at 19 months of age showed muscle hypotonia and an inability to sit without support. She is still seizure free but is kept on antiepileptic treatment. Her dysmorphic features included a brachycephalic headshape with a high forehead. Scalp hair and eyebrows were sparse with a high frontal hairline. She had upslanting palpebral features, telecanthus, arched eyebrows, slightly depressed nasal bridge, prominent and long philtrum, cupid bow lips with prominent lower lip and a high palate (Figure 2E).

Patient 7: The proband is an 11 years old male born to a consanguineous Somali parents. He was born following a normal pregnancy by emergency Caesarean section at 42 weeks of gestation because of fetal distress. His birth weight was 4.32 kg (95th centile) and his occipito-frontal head circumference was 35.5 cm (69th centile). There were no perinatal problems. He had vomiting and gastro-oesophageal relux from 2 months of age. He developed generalized seizures initially associated with fever from 6 months of age however from one year of age the seizures were atypical and associated with a prolonged recovery phase. By 22 months he had developed atonic seizures. An EEG at that time showed suspicious paroxysmal discharges and associated sharp waves. A repeat EEG showed multiple bilateral bursts of spike-and-waves discharges. An MRI brain scan was normal. His seizures were refractory to LEV, VPA and LMT and they continued on combined treatment with zonisamide, clobazam (CBZ) and TPM. His seizures have almost completely ceased on commencing a ketogenic diet and he has discontinued his anti-convulsant medication. He has had recurrent chest infections. He had a patent ductus arteriosus and an aneurysmal fossa ovalis however a follow up echocardiogram at the age of 2 years was normal. The results of metabolic and biochemical investigations including calcium and alkaline phosphatase levels were normal. He has a normal recent renal ultrasound scan.

He was referred for assessment at 6½ months of age because of developmental delay and hypotonia. At 18 months he had head lag when pulled to sit. He could however sit with support, push up on his arms when prone and roll. He could reach out with a palmar grasp and transfer object and put them into his mouth. He had no hand dominance. He had five words and understood simple instructions. He could finger feed and eat lumpy foods. At 22 months he could sit independently and weight bear. He has been able to crawl from 4 or 5 years of age. Currently he can stand holding onto furniture for short periods of time. He communicates using simple signs and by pointing and he understands simple instructions. He makes high pitched sounds and he has repetitive mannerisms and will clap his hands together if excited. His hearing and ophthalmological assessments have been normal. He attends special school.

He had a weight of 40 kg (73rd centile) and a head circumference of 51.5 cm (2nd centile). He had marked truncal hypotonia and increased peripheral tone and reflexes. He had ridging of the sagittal suture. He had a wide forehead and bitemporal narrowing. He had slightly wiry hair, straight eyebrows, mildly downslanting palpebral fissures and infraorbital creases. He had a broad nasal bridge and short anteverted nose. He had a tented mouth with down turned corners and a high arched palate. His ears were round, low set and posteriorly rotated. He had slightly puffy hands and feet. His chest, abdomen and spine were normal.

Patient 8: Patient 8 was the younger brother of patient 7. He was born at 29 weeks gestation. He had developmental delay hypotonia and myoclonic seizures. He had visual impairment, a left hemiplegia, and increased hypotonia following pneumococcal meningitis and septicaemia. He appeared facially similar to patient 7. He had the familial homozygous PIGT mutation. He also had an older sister with a similar presentation including developmental delay, hypotonia and seizures and who died at 15 months of age from a chest infection. His parents had a son born at 30 weeks gestation that died at 3 weeks of age with multiple problems related to prematurity and four first trimester miscarriages. They have one healthy developmentally normal daughter. Further clinical data was not available.

Patient 9: This girl, now 6 years and 9 months, is the elder sister of patient 10. Her parents are from Somalia and recently became aware that they are consanguineous. She was born by elective Cesarean section for breech presentation with a birth weight of 3450 g (>99th centile). and head circumference 37.4 cm (>99th centile). When reviewed in the Genetics Clinic at 1 year 9 months she is able to sit for a few minutes and weight bear with some help from her parents. On examination we noted head circumference was on the 90th centile with a length between 71st to 91st and weight on the 91st centile. She was able to interact visually, though convergent squint was noted. She was still centrally hypotonic. The prominence of the forehead was less than previously noted. Facially, we noted the relatively flat nasal bridge, mild epicanthus and tented upper lip. Her first seizure was around 10 months affecting the left side and then progressed to more frequent seizure with myoclonic jerks. Video fluoroscopy had shown aspiration, therefore she was on thickened feeds. She had tonsils and adenoids removed to help her mild obstructive sleep apnoea. She did not have any history of early tooth loss, she had cardiology review which showed small patent ductus but no abnormality of cardiac muscle, check renal ultrasound treatment for epilepsy sodium valproate and Levetiracetam.

Patient 10 This male baby was born at 41 weeks by elective Caesarean section with birth weight of 3580 g (>99th centile) and head circumference of 37 cm (>96th centile). There was a history of polyhydramnios in pregnancy. He developed persistent vomiting at 8 hours of age with abdominal distention and he was observed by the surgical team but did not require any intervention. Neurological features at that time were poor fixing and following, tremulous, jerky movement and hyperreflexia at both the upper and lower limbs. He had an EEG which did not reveal any epileptic activity and an MRI reporting widely patent cavum septum pellucidum, some dilatation of the fourth ventricle and rotation of the cerebellar vermis away from the brainstem though the vermis was anatomically normal. On review at three months of age, he was unable to hold his head steady. He had increased tone in both upper and lower limbs bilaterally. Bilateral brisk reflexes more pronounced in the lower limbs. Examination of the spine was normal and he was not thought to have any dysmorphic features. When he was reviewed in genetics at seven months of age, he was getting less stiff and trying to reach for objects but not yet sitting or rolling. As his neurological and physical features (relative macrocephaly with scaphocephaly and tented upper lip) were in keeping with his older sister an exome analysis was arranged. His growth and clinical features are outlined in the table. He developed seizures from just under a year of age which became more difficult to control. He had a combination of myoclonic jerks and generalised tonic-colonic seizures.

He did become increasingly unwell with recurrent chest problems and bronchiolitis and difficult to control epilepsy, and died at the age of two years and two months during one of these emergency admissions to hospital.

Patient 11: This patient is the second child of unrelated parents. She was born by vaginal delivery at 42 weeks after a pregnancy complicated by vaginal bleeding at around 7 and 12 weeks. She weighed 3990 g (91th centile) and there were no immediate concerns at birth. She had some tremulous movements as a young baby and was noted to have unusual eye movements from the first week of life, with upward deviation of her eyes and poor fixing and following.

She had three seizures in the context of a febrile illness when she was around a year old. Initially this was attributed to encephalitis but CSF studies were normal. At this time her EEG was noted to be slow, and a convalescent EEG also showed bilateral slow activity but no clear epileptiform abnormalities. She was later diagnosed with epilepsy after she went on to experience further seizures, with her predominant seizure type being generalised tonic clonic seizures (often associated with febrile illnesses). Over time she achieved good seizure control with VPA.

She developed a left renal stone when she was around a year old. X-rays of her wrist and knee showed no evidence of metabolic bone disease, and a 24-hour urine collection for citrate, calcium, oxalate and urate showed normal excretion of all of these. Her plasma calcium phosphate and urate were normal as was her calcium and magnesium. The renal stone was initially conservatively managed but this became impacted aged 3 years, at which point she had left ureteroscopy and fragmentation of the stone.

She was diagnosed with global developmental delay as she was late to start rolling and unable to sit until she was 15 months old. She went on to develop marked ataxia and serial MRI scans demonstrated progressive isolated cerebellar atrophy affecting the vermis and hemispheres. When reviewed by neurology aged 5 years she had an alternating convergent squint, oculomotor apraxia of vertical gaze, and marked gait ataxia with bilateral limb ataxia. She has significant dysarthria and delayed expressive language. Aged 9 years, she could speak around twenty clear words. However she was very good at communicating her needs using sign language and communication aids.

On examination in the genetics clinic aged 9 years, her head circumference was 53 cm (25th-50th centile), her height was 135.5 cm (75th centile) and her weight was 32.4 kg (75th centile). She had slightly deep set eyes, a broad nasal tip, a 'cupid's bow' shape to her top lip and a pointed chin. Her hands were structurally normal. Her spine was straight. There was no unusual shape to her chest. Her palate, skin and teeth were normal.

Extensive neurometabolic investigations including full blood count, liver function tests, thyroid function, vitamin E, creatinine kinase, very long chain fatty acids, phytanic acid, urate, urine amino acids, caeruloplasmin, transferrin glycoforms, white cell enzymes were normal. ERGs and VEPs were normal. Array CGH and Angelman testing was normal.

Patient 12: First female child of healthy consanguineous Pakistani consanguineous parents born at 36 weeks gestation by forceps assisted delivery. Her birth weight was 3000g (75th percentile). Height and head circumference at birth was not recorded. Apgar scores were normal. She presented with focal seizures at 6 months of age, associated with fever and necessitating admission to the intensive care unit. These initially responded to buccal midazolam. She progressed to having generalized tonic clonic seizures. CT on admission showed general reduction of brain volume with open opercula and thin commissures. Prior to the onset of seizures there was a history of global developmental delay and bilateral hydronephrosis with multi resistant recurrent urinary tract infections. She has a variable lymphopenia with no underlying immunological defect identified. MRI prior to onset of seizures showed delayed myelination and EEG was abnormal with generalised slowing throughout with posterior epileptiform discharges. Subsequent seizures have usually been associated with underlying infection or fever, may start as erratic myoclonus with no consistent focality. There was an improvement in seizures after introduction of VPA in addition to LVT and CBZ. Seizure frequency now approximately 25 episodes a day, up to 17 seconds and self resolving. Ophthalmological examination revealed cortical visual impairment and abnormal motility of the eyes including strabismus and nystagmus. Auditory examination showed normal hearing. She had dysmorphic features including brachycephaly and a high forhead, telecanthus, a distinct philtrum, a tented mouth with a high and arched palate, cubid bow shaped lip and a short anteverted nose. Scalp hair were sparse. An echocardiography was normal. The results of metabolic and biochemical investigations including calcium and alkaline phosphatase levels were normal. At almost 2 years of age she remains globally delayed, hypotonic and unable to fix and follow. She has an unsafe swallow and is dependent of a permanent feeding tube.

Patient 13: A 6 month-old girl now deceased, who was the first child of consanguineous healthy Bangladeshi parents. She was born at 40 weeks gestation, after a pregnancy complicated by intrauterine growth restriction. Birth weight 2380g (<0.4th centile) and head circumference 32 cm (<0.4th centile). She required inflation and ventilation breaths at birth and APGAR scores were 3 at 1 minute and 10 at 5 minutes of life. At birth she was noted to have bilateral elbow contractures and contracture of the distal finger joints. She was admitted to the neonatal intensive care unit due to poor sucking, areflexic severe hypotonia and a presumed neuromuscular disorder. She was noted to have facial twitching and myoclinic jerks, which prompted an EEG demonstrating a burst suppression pattern. Seizures were refractory to multiple antiepileptic drugs with trialing of pyridoxal phosphate, LEV and TPM. She was discharged from the neonatal ward with ongoing seizures. By the age of 3 months she had developed intermittent dystonic movements of the lower limbs and had suffered recurrent chest infections. She died at the age of 6 months. She was thought to be non-dysmorphic on examination at 3 weeks of age. An echocardiography and an ultrasound of the kidneys performed at three weeks of age were normal. Brain MRI was performed at 2 weeks of age and showed abnormal T1 signals in the globi pallidi bilaterally and absent myelination in the posterior limbs of the internal capsule Metabolic screening included plasma amino acids, lactic acid, pyruvic acid, organic acids, lysosomal enzymes and were all unremarkable. Analysis of the urine for mucopolysaccharides and oligosaccharides also showed normal results. CSF neurotransmitters and biotinidase were normal. She had normal levels of alkaline phosphatase, plasma calcium and plasma phosphate.

Patient 14: Patient 13 was the older sister of patient 12. She was born at 39 weeks gestation by normal vaginal delivery. Her birth weight was 2400g (0.4th percentile) and birth length was 32cm (<0.4th percentile). The head circumference at birth was not recorded. Apgar scores were normal. Within a few hours after birth, she was admitted to the neonatal intensive care unit due to poor sucking, severe hypotonia and epileptic seizures. She presented with generalized tonic seizures with apnea and was treated with LEV. Do to pain and swelling of the extremities a series of x-ray were carried out and showed that she had three major fractures of her humerus, femur and wrist. A supplementary skeletal survey found signs of healing fractures of the right humeral and right femoral diaphyses. Subtle periosteal reaction along the midshaft of the left humerus and subtle contour abnormality at the anterior ends of the left seventh and eighth ribs were also found and could represent fractures. Bone density appeared diffusely reduced. During her hospital stay, her condition remained the same and she was finally discharged home with palliative care plan. She continued to have seizures although they are short lived and mainly involve eye flickering and facial grimacing. She is currently 4 months old and has made only little developmental progress since. She has not sustained further fractures. The EEG showed diffusely attenuated background activity with burst-suppression pattern and interburst intervals lasting up to 8 seconds. Bursts contained multifocal sharp elements and clinically correlated with eye blinking, oral movement and twitching of the extremities. Her MRI scan performed during the first month of life was normal. An echocardiography and an ultrasound of the kidneys were normal. Due to feeding difficulties she is dependent on a permanent feeding tube. Ophthalmological examination was normal but an auditory examination has not been carried out. She had normal levels of alkaline phosphatase. Clinical pictures of this patient were not available and the description of her dysmorphic features was based on the patient files. Next generation sequencing (NGS) Genomic DNA was extracted from full blood using standard methods. For whole exome sequencing (WES) of patient 1, 2, 12, 13 and their respective parents, exome targets were enriched using Nextera Rapid Capture Exome Kit and products were sequenced on Illumina HiSeq 2000 instrument using version 4 chemistry with a paired end 2 x125 base-paired protocol.

In patient 3 the homozygous PIGT mutation was detected by using an epilepsy gene panel that tested 85 genes. Genomic DNA was extracted from full blood using standard methods. A targeted NGS library was prepared from 15 ng of template DNA using the Ion AmpliSeq library 2.0 kit and custom primers following the manufacturer’s instructions (Life Technologies). The library DNA was clonally amplified onto the Ion Spheres Particles (ISPs) by emulsion PCR using an Ion OneTouch 2 system and the Ion PGM Template OT2 200 kit (Life Technologies). ISPs were sequenced on an Ion PGM sequencer as per the manufacturer’s instructions (Life Technologies). Sequences were mapped to hg19 in the Torrent suite software (Life Technologies) and variant calling was achieved in the Strand NGS software (Avadis) with a minimum of 20-fold read depth.

WES sequencing identified the homozygous mutation in patient 4. Coding regions were enriched with the Human All Exons Kit SureSelect XT (Agilent) and the library was sequenced with 150bp paired-end reads on a HiSeq 2000 (Illuimina). Reads were mapped to the reference genome GRCh37/hg19 using BWA (0.7.12-r1039). FreeBayes-Software (Version 0.9.20) was used for variant calling.

WES was performed on DNA from patient 5 using Agilent Sure SelectQXT Human All Exon v6 enrichment kit (Agilent). Enriched library was sequenced on Illumina HiSeq 1500 platform (Illumina). Variant calling and alignment was performed using SureCall software (Agilent) and annotations were performed using Variant Studio software (Illumina). For the analysis of quality and read coverage IGV tool was applied29.

WES was performed on DNA from patient 7, 9 and 11 as part of the Deciphering Developmental Commented [B1]: Firth HV, Wright CF (2011). The Deciphering Disorders project (reference). Developmental Disorders (DDD) study. Dev Med Child Neurol 53:702-703.

In patient 12 a trio WGS was performed as part of NIHR GOSH/UCL BRC funded RaPS project ormbrc-2012-1. DNA was extracted using Chemagic-STAR (Hamilton USA). Whole genome gDNA libraries were prepared using TruSeq DNA PCR-Free Library Prep (Illumina, USA) following manufacturer’s advice starting with 1ug of sheared gDNA. Parental samples were pooled at equimolar concentrations and sequenced on Illumina NextSeq 550 High-Output Mode (29 hours). Patient samples were sequenced on Illumina HighSeq 2500 Dual Flow Cell, Rapid Run Mode (27 hours) Mapping and variant calling were performed using a GENALICE appliance running GENALICE Map 2.5.5 including Mapping, Variant Calling and the Population Calling module for trio analysis (GENALICE BV, Netherlands). GENALICE default configuration files were used for WGS mapping, and trio variant detection.

Variant identification, prediction and verification Exome data from patient 1, 2, 13 and 14 was analyzed and variants prioritized using the Cologne Center for Genomics' VARBANK database analysis tool (https://varbank.ccg.uni-koeln.de). wAnnovar30 annotated variants were filtered for recessive mode of inheritance according to the family history. In patients 1 and patient 2 we identified two compound heterozygous variants in PIGT (NM_015937.5: c.1472T>A, p.Leu491His, and c.1484+2T>A) that segregated in biparental mode. The paternally inherited missense mutation of a highly conserved amino acid was predicted to be probably damaging by PolyPhen-231 and MutationTaster32 and had a CADD score33 of 24.2. The maternally inherited (c.1484+2T>A) was a splice variant with a CAAD score of 22.9. Both variants were yet not found in healthy individuals (gnomAD). In patients 13 and patient 14 one homozygous missense mutation (NM_015937.5: c.709G>C, p.Glu237Gln) that leads to an exchange of a highly conserved amino acid was predicted by SIFT, MutationTaster, and PolyPhen-2 to be pathogenic. 16 heterozygos carriers were identified in the gnomAD database of this variant and Sanger sequencing confirmed biparental inheritance.

Variants from patient 3 resulting from epilepsy gene panel sequencing were reduced by allele frequency ≥2% and SNPs observed in more than 2 samples for each analyzed sample batch were filtered out. Genetic non-synonymous/splice site variants were evaluated through database searches such as dbSNP, Exome Variant Server, the Exome Aggregation Consortium database (ExAC), and HGMD Professional. One homozygous missense mutation (NM_015937.5: c.709G>C, p.Glu237Gln) that leads to an exchange of a highly conserved amino acid was predicted by SIFT, MutationTaster, and PolyPhen-2 to be pathogenic. 16 heterozygos carriers were identified in the gnomAD database of this variant and Sanger sequencing confirmed biparental inheritance.

Rare variants (allele frequency < 1%) in patient 4 were filtered out based on healthy individuals from ExAC and 1KGP. Prioritization of the remaining variants was done with PhenIX (http://compbio.charite.de/ PhenIX/)34. MutationTaster was used to predict the pathogenic effect of the compound heterozygous mutatations (NM_015937.5: c.494-2A and c.1582G>A; p.Val528Met). The maternally inherited c494-2A>G variant affects a consecutive splice site and was predicted to be pathogenic by MutationTaster. This variant was observed in 17 healthy in heterozygous carriers (gnomAD). The paternally transmitted c.1582G>A transition was shown to be pathogenic by Pagnamenta, et al.35

Variants with a MAF below 0.01 % in genes associated with ID36 remained after filtering and were assessed with bioinformatic tools (Polyphen2, Mutation Taster and Sift). The biparental transmission of the homozygous pathogenic variant (NM_015937.5: c.1582G>A; p.Val528Met) was confirmed in the affected sister by Sanger sequencing.

In case 7 a homozygous sequence variant c.1079G>T was identified using trio WES performed by the Deciphering Developmental Disorders project. The c.1079G>T p.(Gly360Val) variant is predicted to result in the substitution of a highly conserved glycine at position 360 for valine. It was absent or at extremely low frequency in controls in Exome Sequencing Project, 1000 Genomes or ExAC and there are multiple lines of computational evidence support a deleterious effect. This has been supported by functional data with reduced levels of GPI-anchored proteins reported in leukocytes in two clinically affected brothers homozygous for this variant. Sanger sequence analysis of exon 9 of the PIGT gene confirmed homozygosity for sequence variant c.1079G>T p.(Gly360Val). Segregation analysis showed that the variant was biparentally inherited from parents who are both heterozygous for this variant. The same sequence variant was also detected in the homozygous state in a clinically affected sibling. In accordance with ACMG guidelines the homozygous sequence variant c.1079G>T was classified as likely to be pathogenic, and explained the clinical features in the proband and his affected siblings.

In case 12 the Ingenuity Variant Analysis™ software (QIAGEN, USA) was used to identify rare variants predicted to result in loss of function, or to have a functional effect on the protein. Variants with a frequency of ≤ 0·5% in 1000 Genomes, ExAC , and Exome Variant Server were investigated. A homozygous PIGT variant c.550G>A was identified. This is a highly conserved residue present in low freqency in Exac with pathogenicity supported by the majority of in silico tools.