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Original Article SERAC1 deficiency causes complicated HSP: evidence from a novel splice mutation in a large family Benjamin Roeben,1,2 Rebecca Schüle,1,2 Susanne Ruf,3 Benjamin Bender,4 Bader Alhaddad,5 Tanja Benkert,6 Thomas Meitinger,5,7 Selina Reich,1 Judith Böhringer,3 Claus-Dieter Langhans,8 Frédéric M Vaz,9 Saskia B Wortmann,5,7,10 Thorsten Marquardt,11 Tobias B Haack,5,7 Ingeborg Krägeloh-Mann,3 Ludger Schöls,1,2 Matthis Synofzik1,2

►► Additional material is Abstract DDHD1 (SPG28), FA2H (SPG35), PNPLA6 published online only. To view Objective to demonstrate that mutations in the (SPG39),4 DDHD2 (SPG54)5 or CYP2U1 (SPG56)) please visit the journal online phosphatidylglycerol remodelling enzyme SERAC1 can and corresponding pathways have been identified (http://dx.​ ​doi.org/​ ​10.1136/​ ​ 2 6–9 jmedgenet-2017-​ ​104622). cause juvenile-onset complicated hereditary spastic in the past few years. paraplegia (cHSP) clusters, thus adding SERAC1 to the Here we provide evidence from five affected For numbered affiliations see increasing number of complex lipid cHSP . subjects from two different branches of a large end of article. Methods combined genomic and functional validation family that mutations in the phosphatidylglyc- studies (whole-exome sequencing, mRNA, cDNA and erol (PG) remodelling enzyme SERAC1 cause Correspondence to cHSP, thus adding SERAC1 to the increasing list Dr Matthis Synofzik, Department ), biomarker investigations (3-methyl-glutaconic of Neurodegenerative Diseases, acid, filipin staining and phosphatidylglycerols PG34:1/ of genes involved in lipid metabolism associated Hertie-Institute for Clinical PG36:1), and clinical and imaging phenotyping were with cHSP phenotypes. The juvenile-onset cHSP Brain, Research, University of performed in six affected subjects from two different phenotype observed in the five subjects, and in Tübingen, Hoppe-Seyler-Str. branches of a large consanguineous family. particular the oligosymptomatic, relatively benign 3, 72076 Tübingen, Germany; ​ matthis.synofzik@​ ​uni-​ Results 5 of 6 affected subjects shared cHSP as a cHSP cluster observed in the three family members tuebingen.de​ common disease phenotype. Three subjects presented from the first family branch, strikingly diverges with juvenile-onset oligosystemic cHSP, still able to from all 17 SERAC1 cases reported in the litera- Received 22 February 2017 walk several miles at age >10–20 years. This benign ture so far and yields a redefinition of the pheno- Revised 24 August 2017 typic spectrum associated with SERAC1 deficiency. Accepted 31 August 2017 phenotypic cluster and disease progression is strikingly Published Online First divergent to the severe infantile phenotype of all The classic severe, infantile 3-methylglutaconic 15 September 2017 SERAC1 cases reported so far. Two family members aciduria, deafness, , Leigh-like showed a more multisystemic juvenile-onset cHSP, syndrome (MEGDEL syndrome), described for indicating an intermediate phenotype between the SERAC1 so far, is only one (infantile) presentation benign oligosystemic cHSP and the classic infantile of the now-unfolding SERAC1 deficiency spectrum; SERAC1 cluster. The homozygous splice mutation led we here show that it includes also juvenile-onset to loss of the full-length SERAC1 protein and impaired cHSP on the other end of the spectrum. phosphatidylglycerol PG34:1/PG36:1 remodelling. These phosphatidylglycerol changes, however, were milder than Methods in classic infantile-onset SERAC1 cases, which might Subjects and clinical and imaging phenotyping partially explain the milder SERAC1 phenotype. Six affected subjects from two different branches Conclusions Our findings addSERAC1 to the of a large consanguineous Iraqi index family were increasing list of complex lipid cHSP genes. At the same assessed (pedigree figure 1A). Nerve conduction time they redefine the phenotypic spectrum of SERAC1 studies, electromyography and 1.5 Tesla cerebral deficiency. It is associated not only with the severe MRI investigations were performed in all patients infantile-onset ’Methylglutaconic aciduria, Deafness, available for these investigations. One patient Encephalopathy, Leigh-like’ syndrome (MEGDEL who had already died at the time of the study was syndrome), but also with oligosystemic juvenile-onset assessed based on written medical records. cHSP as part of the now unfolding SERAC1 deficiency An independent subject with a classic infan- spectrum. tile-onset MEGDEL phenotype (subject SERAC1 MEGDEL #1; mutations: c.1102C>T; p.Arg368*; c.1822_1828+11 delinsACCAACAGGT; p.?; Introduction NM_032861.3) (table 1) was recruited as a disease The genetic and molecular spectrum of complicated control for comparative analysis of the functional hereditary spastic paraplegias (cHSP) is rapidly and biomarker findings from the index family. expanding, with more than 70 genes implicated up to now.1 2 Biosynthesis and remodelling of complex Genomic studies, functional validation (DNA, To cite: Roeben B, Schüle R, lipids has thereby emerged as a central pathophys- mRNA, cDNA and protein) and biomarker studies Ruf S, et al. J Med Genet iological hub. A large variety of new cHSP lipid Whole-exome sequencing (WES) was performed on 2018;55:39–47. genes (eg, CYP7B1 (SPG5), B4GALNT1 (SPG26),3 four affected individuals (II.6 and III.2–5) using the

Roeben B, et al. J Med Genet 2018;55:39–47. doi:10.1136/jmedgenet-2017-104622 39 Downloaded from http://jmg.bmj.com/ on February 1, 2018 - Published by group.bmj.com Neurogenetics

Figure 1 Pedigree and cerebral MRIs of the SERAC1 family, and overview on the unfolding phenotypic spectrum of SERAC1 deficiency. (A) Pedigree. Arrows in the pedigree indicating subjects who have undergone genetic sequencing (wt=wildtype; mut=c.91+6T>C variant). (B) MRI. Fluid Attenuated Recovery (FLAIR) images from two exemplary subjects of the SERAC1 family (subject II.6 left; subject III.5 right) reveal progressive atrophy of the head of the caudate nucleus (arrows) and progressive T2-hyperintense lesions of the putamen (arrow heads) with increasing disease duration (dd; in years) across subjects. (C) MEGDEL and slowly progressive oligosymptomatic cHSP represent two extreme clusters along a gradual phenotypic spectrum of SERAC1 deficiency.C lassically, SERAC1 mutations have been associated with the severe, infantile onset ‘MEGDEL’ phenotypic cluster, comprising of hypoglycaemia, liver failure/sepsis, sensorineural deafness, and cognitive deficits, which rapidly evolve during the first decade of life, leading to early death (left side of the figure; see also patient SERAC1 MEGDEL #1, table 1). Here we show that on the other end of the disease spectrum, SERAC1 deficiency can also present with slowly progressive, oligosymptomatic cHSP starting in adolescence (right side of the figure), as observed in three affected members of family branch I. The two family members of family branch II presenting with a more multisystemic juvenile-onset cHSP indicate an intermediate phenotype between the oligosystemic cHSP and the classic infantile cluster along this gradual spectrum of SERAC1 deficiency. Other diseases with a similar gradual phenotypic spectrum ranging from frequent, multisystemic infantile-onset phenotypes to more rare, oligosystemic juvenile-onset phenotypes are lysosomal storage diseases and in particular Niemann-Pick type C, which can be conceptualised similarly to SERAC1.20 cHSP, complicated hereditary spastic paraplegia; MEGDEL, 3-methylglutaconic aciduria, deafness, encephalopathy, Leigh-like syndrome.

SureSelect Human All Exon 50 Mb V5 kit (Agilent, Santa Clara, subjects from the index family (III.4 and III.5)12 13 (for detailed California, USA) for in-solution enrichment and the Hiseq2500 description, see online supplementary file 4). To evaluate the instrument (Illumina, San Diego, California, USA) as described sensitivity of filipin staining for underlying SERAC1 disease, before10 (for filter settings, see online supplementary file 1). To filipin staining was performed in fibroblasts of subject III.3 and confirm the effect of the non-canonical near-splice variant, anal- compared with filipin stainings of SERAC1 MEGDEL #1 and a yses on both cDNA/mRNA and protein level were performed patient with another cholesterol trafficking disorder, Niemann- in subject III.3 and—for comparative purposes—in SERAC1 Pick type C (NP-C) (for detailed methods, see online supplemen- MEGDEL #1 (for detailed methods, see online supplementary tary file 5). file 2). To investigate whether the near-splice variant impairs the PG remodelling function of the SERAC1 enzyme,11 levels of PG34:1 and PG36:1 were analysed in fibroblasts from subject Results III.3 and compared with n=3 controls as well as n=8 SERAC1 Genomic studies patients with the classic MEGDEL phenotype (including subject Filtering of WES variants (for filtering strategy, see online supple- SERAC1 MEGDEL #1; for detailed description of the methods, mentary file 1) revealed a homozygous intronic variant see online supplementary file 3). Functional relevance of the c.91+6T>C, p.(?) in SERAC1 (NM_032861.3). This variant novel near-splice variant was further confirmed by laboratory was absent from 8000 in-house exomes as well as public data- testing of the organic acids 3-methyl-glutaconic acid (3-MGA) bases (1000 Genomes, dbSNP 142, ExAC Server (Cambridge, and 3-methyl-glutaric acid (3-MGC) in urine of two affected MA (02/2016)) and was observed in all four affected subjects

40 Roeben B, et al. J Med Genet 2018;55:39–47. doi:10.1136/jmedgenet-2017-104622 Downloaded from http://jmg.bmj.com/ on February 1, 2018 - Published by group.bmj.com Neurogenetics istory of H sepsis or liver failure Choreo Choreo athetosis ensori S neural deafness – + – – + ––––– Optic atrophy . limb

– Peripheral Peripheral neuropathy UL, upper UL, Febrile Febrile ystonia + N/A N/A N/A N/A N/A N/A N/A N/A + – +c – – – – – ––/ – + +b – – – – – – + +b – – – – – – + – c – – – – – D to

active site containing protein 1;

educed +progression mutism verbal fluency R Cognitive delay mutation and the classic MEGDEL subject studied here

pasticity UL and LL + N/A UL and LL + – + + LL + + LL + + LL + + S 1.5 years LL + + died at age of 25 27 21 Age at last examination (years) hyperammonaemia, hyperammonaemia, lactic acidosis Cognitive delay/spasticity Cognitive delay/spasticity Age of onset (years) clinical symptom First ex C1 family with the homozygous intronic c.91+6T>C M neonatal Hyperbilirubinaemia, M 7 F 5 M 7 Cognitive delay 10 F 6 Cognitive delay 17 M 6 Cognitive delay 20 F 2 Tremor S b=trophic changes of distal extremities and/or pallhypaesthesia and c=electrophysiologically by nerve conduction studies. delins

areflexia,

c.1102C>T, p.Arg368*; p.Arg368*; c.1102C>T, c.1822_1828+10 ACCAACAGG, p.? ACCAACAGG, N/A c.(91+6T>C); p.? (91+6T>C), (91+6T>C), p.? (91+6T>C), (91+6T>C), p.? (91+6T>C), (91+6T>C), p.?) (91+6T>C), (91+6T>C), p.? (91+6T>C), Genotype Clinical findings of the SERA

D III.1 SERAC1 MEGDEL #1 wildtype Wildtype; F Healthy II.5 II.6 III.5 c.(91+6T>C); III.4 c.[91+6T>C]; III.3 c.(91+6T>C); III.2 c.(91+6T>C); F, female; LL, lower limb; M, male; MEGDEL, 3-methylglutaconic aciduria, deafness, encephalopathy, Leigh-like syndrome; SERAC1, serine syndrome; Leigh-like encephalopathy, deafness, 3-methylglutaconic aciduria, MEGDEL, M, male; lower limb; LL, female; F, I patients (‘MEGDEL syndrome’) but that are not present in the subjects from index family reported here. Listed are those items that have been usually associated with classical SERAC1 neuropathy: a=clinical Peripheral Table 1 Table

Roeben B, et al. J Med Genet 2018;55:39–47. doi:10.1136/jmedgenet-2017-104622 41 Downloaded from http://jmg.bmj.com/ on February 1, 2018 - Published by group.bmj.com Neurogenetics that had received WES. Presence of this homozygous SERAC1 III.5) for these organic acids. Compared with the reference rage variant was subsequently confirmed by Sanger sequencing also of healthy controls, a nearly 10-fold increase of 3-MGA and in the fifth affected subject were DNA was available, while it was up to threefold increase of 3-MGC was observed (patient III.5: absent in a healthy sibling, thus demonstrating full segregation of 3-MGA 132 mmol/mol creatinine, 3-MGC: 34 mmol/mol creat- the variant with disease in five subjects from different branches of inine; patient III.4: 3-MGA: 39 mmol/mol creatinine, 3-MGC: the family and absence of the variant in healthy family members 10 mmol/mol creatinine; reference range healthy controls (for pedigree and segregation, see figure 1A). Biallelic location of 3-MGA: 0–15 mmol/mol creatinine; reference range healthy the variants was confirmed by testing the parents of the affected controls 3-MGC: 0–10 mmol/mol creatinine). family members from family branch I. Changes in complex lipids in SERAC1 have been suggested to The intronic variant was located close to the canonical splice lead to free, unesterified cholesterol accumulation in late endo- donor of exon 2, the first coding exon in the NM_032861.3 somes, as visualised by filipin staining, similar to NP-C, where transcript. A splice effect of this variant was predicted by two unesterified cholesterols are known to accumulate.11 However, independent in silico splicing predictions tools (MaxEntScan we here observed that—compared with a positive NP-C disease [16] and SpliceSiteFinder-like). Indeed, RT-PCR from fibroblast control—filipin staining was negative in SERAC1 subject III.3, as mRNA and subsequent sequencing of amplicons revealed pres- it was in SERAC1 MEGDEL #1 (figure 4). ence of two aberrantly spliced transcripts in patient III.3 lacking either both exon 2 and 3 (exon2_3del) or just exon 2 (exon2_ Clinical phenotype del) (figure 2A). The expected full-length amplicon of 253 bp, A total of five out of six affected subjects from the two family containing the first four exons of SERAC1, as well as a weaker branches shared cHSP as a common disease phenotype (family product of 216 bp lacking exon 3, were present in controls but branch I: three out of four subjects; family branch: two out of absent in patient III.3 (figure 2A,B). To test whether the mutant two subjects). In family branch I, the three siblings affected by transcript in patient III.3 is subject to nonsense-mediated decay, cHSP presented with slowly progressive lower limb spasticity we performed a qPCR (figure 2C). SERAC1 mRNA levels in starting in adolescence. Only the youngest sibling, examined at patient III.3 did not differ from levels in controls, indicating that age 10 years, did not yet show spasticity. At a mean age of 20 the novel splice mutation does not impair mRNA expression or years all of the three siblings with cHSP were still able to walk stability. In contrast, SERAC1 mRNA levels was severely reduced unaided over a distance of several miles. Also in family branch II, in the disease control subject with a classic infantile-onset both affected siblings presented with juvenile-onset progressive MEGDEL phenotype (SERAC1 MEGDEL #1) who carried two spasticity, which progressed more quickly to tetraspasticity. other truncating SERAC1 mutations (table 1). The cHSP phenotype in family branch I was complicated by Western blot analysis demonstrated a strong wild-type infantile-onset mild cognitive developmental delay (on the level SERAC1 band at 74 kDa in controls (figure 2E), which corre- of a learning disability), febrile seizures and mild unspecific sponds to the full-length SERAC1 protein (NP_116250.3). This motor abnormalities, in particular slight impairment of fine 74 kDa band is lost in the SERAC1 patient III.3, confirming that motor skills (table 1). Cognitive status of all four siblings from the splice mutation c.91+6T>C leads to the absence of a full- family branch I was static without progression of deficits in the length SERAC1 protein. It hereby paralleled the finding from the prospective longitudinal observation period of up to 6 years. In disease control SERAC1 MEGDEL #1 patient with two other family branch II, the cHSP phenotype was complicated by more truncating SERAC1 mutations, where the full-length SERAC1 severe features, in particular generalised dystonia, progressive protein was likewise absent (figure 2E). reductions in speech, dysphagia and sensorimotor axonal-demy- elinating neuropathy in the 33-year-old female (subject II.6). As all patients spent the first years of life in rural Iraq, no detailled The novel splice mutation impairs PG remodelling medical investigations are available from the neonatal period; The SERAC1 enzyme remodels PG34:1 to PG36:111 also according to the parents’ report, there were no signs or (figure 3D–E). We assessed whether the novel c.91+6T>C splice medical results indicating possible metabolic acidosis, hyperam- variant impairs this central PG remodelling function of the monaemia or lactic academia in this period. enzyme. Compared with healthy controls, SERA1C III.3 fibro- MRI in all affected subjects (available for five of six affected blasts showed a median 170% increase in PG34:1 concentra- subjects) showed the characteristic striking SERAC1 basal tion, whereas the median PG36:1 concentration was 42% of ganglia imaging pattern, consisting of bilateral T2-hyperintensity that in control fibroblasts, resulting in a mean PG34:1/PG36:1 of the putamen and caudate nucleus as well as bilateral atrophy ratio of 2.2 in SERAC1 III.3 fibroblasts as compared with 0.5 of caudate nucleus head. It also included the so-called ‘puta- in control fibroblasts (figure 3A-C), Interestingly, PG34:1 and minal eye’ (=sparing of T2-hyperintense lesions in the poste- PG36:1 changes in patient SERAC1 III.3 were milder than in rior putamen) in all 5/5 subjects, a pathognomonic feature of classic SERAC1 MEGDEL cases. Here PG34:1 concentrations SERAC1 disease15 (for two exemplary subjects, see figure 1B; for were increased by 332% and PG36:1 concentrations decreased MRIs of all subjects, see online supplementary file 6). to 15% of controls, resulting in a mean PG34:1/PG36:1 ratio of 14.2 (figure 3A, B, C). Taken together, this indicates that Discussion SERAC1 PG remodelling activity is impaired in SERAC1 patients SERAC1 as a novel cHSP with the c.91+6T>C splice variant, yet less severe than in classic Here we show that cHSP phenotypes can be caused by reces- SERAC1 MEGDEL patients. sive mutations in SERAC1, acting via an impairment of the PG remodelling function (PG34:1 to PG36:1) of the encoded Fluid and fibroblast biomarker analysis SERAC1 enzyme. Juvenile-onset paraspasticity, complicated SERAC1 deficiency has been repeatedly demonstrated to lead by non-progressive mild cognitive deficits, was observed in to accumulation of 3-MGA and 3-MGC.11 14 To further corrob- five of six affected subjects, including a relatively benign cHSP orate the loss of function effect of the novel c.91+6T>C muta- disease course in three of them. This phenotypic combination tion, we tested urine of two affected family members (III.4 and is fully compatible with the findings seen in many cHSPs where

42 Roeben B, et al. J Med Genet 2018;55:39–47. doi:10.1136/jmedgenet-2017-104622 Downloaded from http://jmg.bmj.com/ on February 1, 2018 - Published by group.bmj.com Neurogenetics

Figure 2 Effects of the homozygous non-canonical splice site SERAC1 mutation on SERAC1 mRNA and protein levels. (A) PCR of SERAC1 cDNA: A PCR spanning SERAC1 exon1 to exon4 reveals that the intronic c.91+6T>C mutation results in two shorter splice variants of SERAC1 mRNA (subject III.3) compared with two control cDNAs (cntr-1 and cntr-2). (B) Sequencing of PCR products: sequencing of these two distinct products revealed the lack of exon2 (161 bp product) and of exon2 and exon3 (124 bp product), respectively. (C) qRT-PCR of SERAC1 mRNA: qPCRs with two primer pairs (blue=primer 1; red=primer 2) from fibroblast mRNA isolated at two independent time points show similar SERAC1 mRNA levels in subject III.3 compared with three control cell lines (cntr-1 to cntr-3), whereas mRNA levels are severely reduced in SERAC1 MEGDEL #1. mRNA expression is normalised to the housekeeping genes RNF10, RNF111 and GAPDH. Data presented as dot plot with mean±SEM. (D) SERAC1 protein structure: schematic representation of the SERAC1 protein domains (bottom row) in relation to SERAC1 cDNA exons (middle row) and SERAC1 amino acids (top row, number bar; in total 654 amino acids). The intronic c.91+6T>C SERAC1 mutation leads to loss of the N-terminal end of the protein, including the TMD domain. Sources of the protein domains: Uniprot (TMD), EMBL- EBI InterPro (armadillo-like helical domain) and Wortmann et al 201216 (serine- domain). (E) Immunodetection of SERAC1 protein in patient and control fibroblasts. Three control fibroblast lines (cntr-1 to cntr-3) demonstrate a wild-type SERAC1 band at 74 kDa, which is lost in both SERAC1 patient III.3 and SERAC1 patient MEGDEL #1. A weak 66 kDa band is still seen in SERAC1 patient III.3 and healthy controls but also faintly in patient SERAC1 MEGDEL #1 (for discussion, see discussion section of main text). As loading control, the reference gene GAPDH was used. Bp, ; GAPDH, glyceraldehyde-3-phosphat-dehydrogenase; kDa, kilo Dalton; MEGDEL, 3-methylglutaconic aciduria, deafness, encephalopathy, Leigh-like syndrome; qRT- PCR, quantitative real-time PCR; SERAC1, serine active site containing protein 1.

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Figure 3 Impaired remodelling of phosphatidylglycerol (PG) PG34:1 to PG36:1 in SERAC1 patients. (A–C) PG concentrations in a mildly affected SERAC1 patient compared with controls and classic SERAC1 MEGDEL patients. Compared with healthy controls, PG34:1 concentrations were increased (A) and PG 36:1 concentrations decreased (B) in both the mildly affected SERAC1 patient III.3 and the SERAC1 MEGDEL patients, resulting in increased mean PG34:1/PG36:1 ratios (C). Compared with the SERAC1 MEGDEL patients, PG34:1 and PG36:1 changes were milder in the SERAC1 patient III.3., falling in an intermediate range between SERAC1 MEGDEL patients and healthy controls. (D and E) Remodelling of PG34:1 to PG36:1 depends on intact SERAC1 activity. The increased PG34:1 levels and decreased PG36:1 levels observed in the SERAC1 patients can be explained as follows. In presence of intact SERAC1 (D), PG34:1 is remodelled to PG36:1 with subsequent processing to (CL) in the mitochondria and processing to bis-monoacylglycerol- phosphate (BMP) in the endosomes/lysosomes. In case of impaired SERAC1 activity (E), unremodelled PG34:1 accumulates, leading to deficiency of PG36:1 and derived metabolites. a.u., arbitrary units; CLS, cardiolipin synthase; ER, endoplasmatic reticulum; IMM, inner mitochondrial membrane; IMS, intermembrane space; MAM, mitochondrial-associated membranes; MEGDEL, 3-methylglutaconic aciduria, deafness, encephalopathy, Leigh-like syndrome; OMM, outer mitochondrial membrane; SERAC1, serine active site containing protein 1. Figure drawn according to ref 7. early-onset cognitive impairment often accompanies and even failure, spasticity, dystonia, hearing loss and truncal , precedes juvenile-onset spasticity, for example, in SPG1116 or with death usually occurring before age 10–20 years. Individ- SPG15. It is also seen in several complex lipid cHSPs like SPG26 uals suffering from MEGDEL syndrome typically miss motor (B4GALNT1)3 or SPG54 (DDHD2),5 thus adding SERAC1 to development milestones, for example, learning to sit and walk the increasing list of complex lipid HSP genes.1–7 16 independently in infancy,6 and show subsequent early-child- hood motor and cognitive regression. Such a disease trajectory The unfolding spectrum of SERAC1 deficiency: from infantile could also be observed in our disease control subject SERAC1 MEGDEL to adolescent cHSP MEGDEL #1. In contrast, the findings from our index family So far, SERAC1 mutations have been largely associated with the 11 demonstrate that SERAC1 mutations can also present with a severe, complex MEGDEL syndrome, comprising an infan- more oligosystemic and later onset phenotype. None of the six tile-onset phenotype characterised by feeding problems, liver

44 Roeben B, et al. J Med Genet 2018;55:39–47. doi:10.1136/jmedgenet-2017-104622 Downloaded from http://jmg.bmj.com/ on February 1, 2018 - Published by group.bmj.com Neurogenetics

Figure 4 Filipin staining of SERAC1 patients compared with a subject with Niemann-Pick Type C (NP-C) disease and a healthy control fluorescence microscopy images of free, unesterified cellular cholesterol accumulation in late endosomes after filipin staining. Wheras filipin staining was positive in a positive control patient with NP-C disease (A), it was negative in subjects SERAC1 III.3 (B) and SERAC1 MEGDEL #1 (C), similar to a healthy control (D), except for a slightly higher, unspecific background fluorescence. subjects presented here had a history of infantile feeding prob- located in exon 4, which is spared by the c.91+6T>C variant lems, liver failure, hearing loss or truncal hypotonia, and only (see figure 2B) and predicted to be active by two independent one out of six subjects (the most severely affected one) showed translation start site predictors (Net Start 1.017; DNA TIS Miner signs of dystonia. output18). The size of the (weak) 66 kDa band observed in the Our findings also show that the disease course associated with western blot of SERAC1 patient III.3 as well as healthy controls SERAC1 deficiency can be much more benign than reported so (figure 2E) would indeed be compatible with a naturally occur- far. The three subjects of family branch I showing cHSP were ring, N-terminally truncated SERAC1 isoform translated from still able to walk several miles without assistance at age >10–20 the (apparently less efficient) second ATG start codon (predicted years, and the youngest sibling did not even show any motor protein XP_011534500.1; p.Ser2_Met71del). Expression of problems at age 10 years. This demonstrates that the classic this isoform might vary with tissue and be, for example, more severe MEGDEL syndrome is only one (infantile, severe) presen- relevant in brain tissue than in fibroblasts. However, a very faint tation at one end of the now unfolding SERAC1 deficiency spec- 66 kDa band was also observed in the SERAC1 MEGDEL #1 trum, with relatively benign juvenile-onset cHSP phenotypes patient (figure 2E), where mRNA levels were severely reduced on the other end. The two affected family members of family (figure 2C), allowing for the possibility that this band might also branch II presenting with a more multisystemic juvenile-onset be unspecific. cHSP indicate an intermediate phenotype between the oligosys- temic cHSP and the classic infantile cluster along a gradual spec- trum of SERAC1 deficiency disease (see figure 1C). Biomarker and imaging markers for detecting ‘atypical’ phenotypes along the SERAC1 disease spectrum The milder phenotype is associated with milder PG changes Our finding of cHSP as one end of the SERAC1 disease spectrum The milder phenotype observed in the index family corresponds has direct clinical diagnostic implications. 3-MGA screening to milder PG34:1/PG36:1 changes than found in classic, severe seems to be helpful to identify even ‘atypical’ SERAC1 families SERAC1 MEGDEL patients. The milder PG changes in the as shown here. The 3-MGA levels observed in our index family c.91+6T>C family might result from more efficient compen- fall within (the relatively wide) increased range of 3-MGA levels satory mechanisms in PG remodelling and/or more residual observed in MEGDEL patients (16–196 mmol/mol).19 However, SERAC1 remodelling function. Residual SERAC1 function would in contrast to the neuropaediatric work-up of complex neuro- be conceivable by translation of residual SERAC1 protein, which logical syndromes, 3-MGA screening is not done routinely in might not necessarily be detected by the western blot given its cHSP patients, in particular if not starting before teenage years. limitations in sensitivity. Translation of residual SERAC1 protein This implicates the possibility that SERAC1 patients might be would still be possible—at least in principle—in c.91+6T>C overlooked if presenting with oligosystemic presentations (eg, patients, given that this splice mutation does not impair mRNA cHSP presentations) to adult clinics and that additional diag- expression or stability (in contrast to the SERAC1 MEGDEL #1 nostic methods are needed to identify these patients. patient studied here) (figure 2C). c.91+6T>C patients might Filipin staining might not be helpful here. Whereas it has still use, for example, the alternative second ATG start codon been reported to show the classical staining in SERAC1 patients

Roeben B, et al. J Med Genet 2018;55:39–47. doi:10.1136/jmedgenet-2017-104622 45 Downloaded from http://jmg.bmj.com/ on February 1, 2018 - Published by group.bmj.com Neurogenetics with classic infantile phenotypes, it was negative for our subject Ethics approval Ethics committee of the Medical Faculty of the University of with a mild juvenile-onset cHSP phenotype as it was in subject Tübingen. SERAC1 MEGDEL #1 with a classic infantile-onset phenotype. Provenance and peer review Not commissioned; externally peer reviewed. This suggests a limited sensitivity of filipin staining as a diag- © Article author(s) (or their employer(s) unless otherwise stated in the text of the nostic biomarker for underlying SERAC1 disease, both with article) 2018. All rights reserved. No commercial use is permitted unless otherwise classic and with benign disease trajectories. These findings are expressly granted. in line with the concept of SERAC1 deficiency as a primary disorder of PG remodelling, where disturbances of cholesterol References trafficking (as visualised by abnormal filipin staining) are only 1 tesson C, Koht J, Stevanin G. Delving into the complexity of hereditary spastic secondary downstream effects, which might be non-obligatory paraplegias: how unexpected phenotypes and inheritance modes are revolutionizing and more variable. It hereby mirrors findings well-known from their nosology. Hum Genet 2015;134:511–38. another disease with secondary cholesterol trafficking deficits— 2 lamari F, Mochel F, Saudubray JM. An overview of inborn errors of complex lipid NP-C—where filipin staining is also often variable, in particular biosynthesis and remodelling. J Inherit Metab Dis 2015;38:3–18. 20 3 Boukhris A, Schule R, Loureiro JL, Lourenço CM, Mundwiller E, Gonzalez MA, in late-onset, mild phenotypes. Charles P, Gauthier J, Rekik I, Acosta Lebrigio RF, Gaussen M, Speziani F, Ferbert While comprehensive HSP gene panels or WES might ulti- A, Feki I, Caballero-Oteyza A, Dionne-Laporte A, Amri M, Noreau A, Forlani S, Cruz mately help to identify causal SERAC1 variants (as shown VT, Mochel F, Coutinho P, Dion P, Mhiri C, Schols L, Pouget J, Darios F, Rouleau here), we also demonstrate that MRI might facilitate to guide GA, Marques W, Brice A, Durr A, Zuchner S, Stevanin G. Alteration of ganglioside the genetic sequencing of SERAC1 in patients with so far unex- biosynthesis responsible for complex hereditary spastic paraplegia. Am J Hum Genet 2013;93:118–23. plained cHSP. All five subjects where MRI was available showed 4 Synofzik M, Gonzalez MA, Lourenco CM, Coutelier M, Haack TB, Rebelo A, Hannequin the characteristic striking SERAC1 imaging pattern D, Strom TM, Prokisch H, Kernstock C, Durr A, Schöls L, Lima-Martínez MM, Farooq (figure 1B and online supplementary file 5). This also indicates A, Schüle R, Stevanin G, Marques W, Züchner S. PNPLA6 mutations cause Boucher- that the underlying MRI changes in SERAC1 disease might Neuhauser and Gordon Holmes syndromes as part of a broad neurodegenerative spectrum. Brain 2014;137(Pt 1):69–77. evolve in a rather uniform pattern, even if the presenting clin- 5 gonzalez M, Nampoothiri S, Kornblum C, Oteyza AC, Walter J, Konidari I, Hulme ical features evolve and progress very heterogeneously along the W, Speziani F, Schöls L, Züchner S, Schüle R. Mutations in phospholipase DDHD2 unfolding spectrum of SERAC1 disease. cause autosomal recessive hereditary spastic paraplegia (SPG54). Eur J Hum Genet Author affiliations 2013;21:1214–8. 1Department of , Hertie Institute for Clinical Brain Research (HIH), 6 Wortmann SB, Espeel M, Almeida L, Reimer A, Bosboom D, Roels F, de Brouwer University of Tübingen, Tübingen, Baden-Württemberg, Germany AP, Wevers RA. Inborn errors of metabolism in the biosynthesis and remodelling of 2German Center for Neurodegenerative Diseases (DZNE), Tübingen, Germany . J Inherit Metab Dis 2015;38:99–110. 3Department of Pediatric Neurology and Developmental Medicine, University 7 lu YW, Claypool SM. Disorders of metabolism: an emerging class of Children’s Hospital Tübingen, Tübingen, Germany mitochondrial disease due to defects in nuclear genes. Front Genet 2015;6:3. 4Department of Neuroradiology, University of Tübingen, Tübingen, Baden- 8 edvardson S, Hama H, Shaag A, Gomori JM, Berger I, Soffer D, Korman SH, Taustein Württemberg, Germany I, Saada A, Elpeleg O. Mutations in the fatty acid 2-hydroxylase gene are associated 5Institute of Human Genetics, Technische Universität München, Munich, Germany with leukodystrophy with spastic paraparesis and dystonia. Am J Hum Genet 6Institute of Medical Genetics and Applied Genomics, University of Tübingen, 2008;83:643–8. Tübingen, Germany 9 tsaousidou MK, Ouahchi K, Warner TT, Yang Y, Simpson MA, Laing NG, Wilkinson PA, 7Institute of Human Genetics, Helmholtz Zentrum München, Neuherberg, Germany Madrid RE, Patel H, Hentati F, Patton MA, Hentati A, Lamont PJ, Siddique T, Crosby AH. 8Division of Neuropediatrics and Pediatric Metabolic Medicine, University Children’s Sequence alterations within CYP7B1 implicate defective cholesterol homeostasis in Hospital Heidelberg, Heidelberg, Germany motor-neuron degeneration. Am J Hum Genet 2008;82:510–5. 9Laboratory Genetic Metabolic Diseases, Academic Medical Center, Amsterdam, 10 Haack TB, Hogarth P, Kruer MC, Gregory A, Wieland T, Schwarzmayr T, Graf E, Sanford Noord Holland, The Netherlands L, Meyer E, Kara E, Cuno SM, Harik SI, Dandu VH, Nardocci N, Zorzi G, Dunaway T, 10Department of Pediatrics, Salzburger Landeskliniken (SALK) and Paracelsus Medical Tarnopolsky M, Skinner S, Frucht S, Hanspal E, Schrander-Stumpel C, Héron D, Mignot University (PMU), Salzburg, Austria C, Garavaglia B, Bhatia K, Hardy J, Strom TM, Boddaert N, Houlden HH, Kurian MA, 11Department of Pediatrics, University Hospital of Muenster, Muenster, Germany Meitinger T, Prokisch H, Hayflick SJ. Exome sequencing reveals de novo WDR45 mutations causing a phenotypically distinct, X-linked dominant form of NBIA. Am J Hum Genet 2012;91:1144–9. Correction notice This article has been corrected since it was published Online 11 Hoffmann G, Aramaki S, Blum-Hoffmann E, Nyhan WL, Sweetman L. Quantitative First. The name of Saskia B Wortmann has been updated. analysis for organic acids in biological samples: batch isolation followed by gas Contributors Collection and analysis of data: BR, SuR, BB, TBH, SeR, RS and MS. chromatographic-mass spectrometric analysis. Clin Chem 1989;35:587–95. Drafting of the manuscript: BR and MS. Critical review of the manuscript: BR, SuR, 12 . Sweetman L. Analysis Organic Acids. Wiley-Liss New York:, 1991. BB, BA, TB, TM, C-DL, TBH, SeR, RS, IK-M, LS and MS. 13 Pedersen AG, Nielsen H. Neural network prediction of translation initiation sites in eukaryotes: perspectives for EST and genome analysis. Proc Int Conf Intell Syst Mol Funding This research was supported by the European Union within the 7th Biol 1997;5:226–33. European Community Framework Programme through funding for the E-RARE-3 14 liu H, Wong L. Data mining tools for biological sequences. J Bioinform Comput Biol Joint Transnational Call grant ’Preparing therapies for autosomal recessive ’ 2003;1:139–67. (PREPARE) (BMBF, 01GM1607 to MS), the e-Rare Network NEUROLIPID (BMBF, 15 tort F, García-Silva MT, Ferrer-Cortès X, Navarro-Sastre A, Garcia-Villoria J, Coll 01GM1408B to RS) and a Marie Curie International Outgoing Fellowship (grant MJ, Vidal E, Jiménez-Almazán J, Dopazo J, Briones P, Elpeleg O, Ribes A. Exome PIOF-GA-2012-326681 to RS), and the Else Kröner-Fresenius-Stiftung (to MS). It sequencing identifies a new mutation in SERAC1 in a patient with 3-methylglutaconic was further supported by the National Institute of Neurological Disorders and Stroke aciduria. Mol Genet Metab 2013;110:73–7. of the National Institutes of Health under Award Number R01NS072248 (RS). We 16 Wortmann SB, Vaz FM, Gardeitchik T, Vissers LE, Renkema GH, Schuurs-Hoeijmakers thank the participants and their family for participating in this study. This work JH, Kulik W, Lammens M, Christin C, Kluijtmans LA, Rodenburg RJ, Nijtmans was supported by the German Federal Ministry of Education and Research (BMBF) LG, Grünewald A, Klein C, Gerhold JM, Kozicz T, van Hasselt PM, Harakalova M, within the framework of the e:Med research and funding concept (to TBH; grant Kloosterman W, Barić I, Pronicka E, Ucar SK, Naess K, Singhal KK, Krumina Z, Gilissen #01ZX1405C). C, van Bokhoven H, Veltman JA, Smeitink JA, Lefeber DJ, Spelbrink JN, Wevers RA, Disclaimer Dr BR has received travel expenses from Actelion Pharmaceuticals Morava E, de Brouwer AP. Mutations in the phospholipid remodeling gene SERAC1 Ltd., unrelated to this manuscript and research. Dr BB has received travel expenses impair mitochondrial function and intracellular cholesterol trafficking and cause from Bayer Vital. Drs TMa and MS have received travel expenses and presentation dystonia and deafness. Nat Genet 2012;44:797–802. honoraria from Actelion Pharmaceuticals Ltd, unrelated to this manuscript and 17 Wortmann SB, van Hasselt PM, Barić I, Burlina A, Darin N, Hörster F, Coker M, Ucar SK, research. All other authors report no disclosures. Krumina Z, Naess K, Ngu LH, Pronicka E, Riordan G, Santer R, Wassmer E, Zschocke J, Competing interests None declared. Schiff M, de Meirleir L, Alowain MA, Smeitink JA, Morava E, Kozicz T, Wevers RA, Wolf NI, Willemsen MA. Eyes on MEGDEL: distinctive basal ganglia involvement in dystonia Patient consent Obtained. deafness syndrome. Neuropediatrics 2015;46:98–103.

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Roeben B, et al. J Med Genet 2018;55:39–47. doi:10.1136/jmedgenet-2017-104622 47 Downloaded from http://jmg.bmj.com/ on February 1, 2018 - Published by group.bmj.com

SERAC1 deficiency causes complicated HSP: evidence from a novel splice mutation in a large family Benjamin Roeben, Rebecca Schüle, Susanne Ruf, Benjamin Bender, Bader Alhaddad, Tanja Benkert, Thomas Meitinger, Selina Reich, Judith Böhringer, Claus-Dieter Langhans, Frédéric M Vaz, Saskia B Wortmann, Thorsten Marquardt, Tobias B Haack, Ingeborg Krägeloh-Mann, Ludger Schöls and Matthis Synofzik

J Med Genet2018 55: 39-47 originally published online September 15, 2017 doi: 10.1136/jmedgenet-2017-104622

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