ABSTRACT We aimed to identify the genetic cause of the devastating neurodegenerative disease amyotrophic lateral sclerosis (ALS) in a German family with two affected individuals, and to

assess the prevalence of variants in the identified risk gene, _FIG4_, in a central European ALS cohort. Whole-exome sequencing (WES) and an overlapping data analysis strategy were performed

in an ALS family with autosomal dominant inheritance and incomplete penetrance. Additionally, 200 central European ALS patients were analyzed using whole-exome or targeted sequencing. All

patients were subjected to clinical, electrophysiological, and neuroradiological characterization to explore genotype–phenotype relationships. WES analysis of the ALS family identified the

overall _FIG4_ variant frequency of 3% in our cohort. Four of six variants identified were previously associated with ALS or the motor and sensory neuropathy Charcot-Marie-Tooth disease type

4J (CMT4J), whereas two variants were novel. In _FIG4_ variant carriers, disease duration was longer and upper motor neuron predominance was significantly more frequent compared with ALS

patients without _FIG4_ variants. Our study provides evidence for _FIG4_ as an ALS risk gene in a central European cohort, adds new variants to the mutational spectrum, links ALS to CMT4J on

a genetic level, and describes a distinctive ALS phenotype for _FIG4_ variant carriers. SIMILAR CONTENT BEING VIEWED BY OTHERS LOSS-OF-FUNCTION VARIANTS IN _NEK1_ ARE ASSOCIATED WITH AN

INCREASED RISK OF SPORADIC ALS IN THE JAPANESE POPULATION Article 12 September 2020 GENETIC EPIDEMIOLOGY OF AMYOTROPHIC LATERAL SCLEROSIS IN CYPRUS: A POPULATION-BASED STUDY Article Open

access 28 December 2024 GENETIC ANALYSIS OF ALS CASES IN THE ISOLATED ISLAND POPULATION OF MALTA Article Open access 07 January 2021 INTRODUCTION Amyotrophic lateral sclerosis (ALS) is the

most common adult-onset motor neuron disease characterized by progressive degeneration of upper motor neurons (UMNs) and lower motor neurons (LMNs), leading to paralysis of voluntary muscles

and ultimately death due to respiratory failure within 3–5 years from disease onset.1, 2 While 5–10% of ALS patients have a positive family history (fALS), the majority of patients are

sporadic cases (sALS).3 The distinction between fALS and sALS, however, is currently being questioned by the awareness that apparently sALS can be inherited.4 Since 1993, when the superoxide

dismutase 1 (_SOD1_) gene was found to be mutated in fALS patients, an increasing number of disease genes, designated ALS 1–22, have been reported.5 In 2009, ALS11 was identified as a rare

autosomal dominant form of ALS associated with heterozygous deleterious variants of _FIG4_ in North American patients.6 _FIG4_ encodes a phosphoinositide 5-phosphatase regulating

phosphatidylinositol-3,5-bisphosphate, an intracellular signaling lipid with a key role in endosomal vesicle trafficking.7, 8 A loss of function _Fig4_ variant was found to cause neuronal

degeneration in the central nervous system including spinal motor neurons, and peripheral neuropathy in the ‘pale tremor’ mouse.9 Biallelically mutated _FIG4_ was detected in patients with

(i) Charcot-Marie-Tooth disease type 4J (CMT4J),9 a recessively inherited form of hereditary motor and sensory neuropathy, (ii) Yunis-Varón syndrome,10 an autosomal recessive disorder with

skeletal anomalies and severe neurological involvement, and (iii) familial epilepsy with polymicrogyria.11 Here, we present known and novel heterozygous deleterious _FIG4_ variants in a

cohort of 201 central European fALS and sALS patients, and provide clinical, electrophysiological, and neuroradiological information on _FIG4_ variant carriers. SUBJECTS AND METHODS PATIENTS

The study was approved by the Ethics Board of Hannover Medical School. Each patient and family member provided informed consent for participation in the study. All patients were examined at

least once at the motor neuron disease outpatient clinic of Hannover Medical School by a neurologist specialized in ALS. Extensive clinical workup including magnetic resonance imaging

(MRI), cerebral spinal fluid analysis, electromyography (EMG), and nerve conduction studies (NCS) was performed to exclude ALS-mimicking conditions. Longitudinal information over a number of

years was available for most individuals. A total of 124 male and 77 female patients of almost exclusively central European origin were enrolled in this study, including 6 fALS and 195 sALS

cases. The mean age of onset was 58.6±12.5 years (range 19–84 years). Initial symptoms occurred in the bulbar region in 44 patients (21.9%) and the spinal cord in 157 patients (78.1%). UMN

and LMN involvement was assessed in all patients as described previously.12 Increased muscle tone, jaw jerk, increased deep tendon reflexes, spread of reflexes, clonus, and Babinski’s sign

were classified as signs of UMN affection. Muscle atrophy, fasciculations, and weakness were classified as LMN signs. Regarding bulbar involvement, brisk masseter reflex and tongue

spasticity were interpreted as UMN signs, whereas tongue atrophy and tongue fasciculations were considered to be LMN signs. The extent and distribution of clinically inapparent LMN affection

was also assessed by EMG in all patients. Muscle strength, muscle tone, reflexes, and the degree of atrophy and fasciculations were graded as detailed in Körner _et al._12 Using these

criteria, 13 patients (6.5%) had a predominant UMN phenotype, eight of which were diagnosed with primary lateral sclerosis (PLS). In the index patient, FamALS006-01, the following genetic

tests had been performed before this study: (1) sequence analysis of the genes _SOD1_, _FUS_, and _TARDBP_ using conventional chain termination protocols, (2) analysis of the hexanucleotide

repeat in the _C9orf72_ gene by repeat prime PCR and Southern blot analysis. GENETIC ANALYSIS Whole-exome sequencing (WES) was performed on peripheral blood from the index patient, father

and mother of ALS family FamALS006, 22 sALS patients, and 48 controls not affected by ALS using Agilent SureSelect Human All Exon v4 Target Enrichment System on an Illumina HiSeq 2000

(Oxford Gene Technology, Begbroke, UK) as described previously.13 All samples were sequenced to a mean target coverage of >50 ×. WES data from ALS family FamALS006 were analyzed using

INGENUITY Variant Analysis (INGENUITY Systems; QIAGEN, Redwood City, CA, USA) and our in-house NGS data analysis workflow as described in Results, Supplementary Table S1, and Supplementary

Table S2. In 22 sALS patients, _FIG4_ variants were retrieved from WES data. To verify selected _FIG4_ variants identified by WES and to screen the coding exons (1–23) and adjacent intronic

regions of _FIG4_ (NG_007977.1 and NM_014845.5) for variants in 178 further ALS patients, conventional chain termination protocols were used (for primer sequences see Supplementary Table

S3). Minor allele frequency (MAF) was retrieved from Variant Analysis (INGENUITY Systems) and Exome Aggregation Consortium (ExAC) Browser Beta (http://exac.broadinstitute.org/;

http://biorxiv.org/content/early/2015/10/30/030338). For prediction of variant deleteriousness, the tools SIFT and PROVEAN (http://sift.jcvi.org/), PolyPhen-2

(http://genetics.bwh.harvard.edu/pph2/), and MutationTaster (http://www.mutationtaster.org) were used. The six rare non-silent _FIG4_ variants predicted to be deleterious identified in ALS

patients were submitted to ClinVar (SCV accession nos SCV000299294–SCV000299299; http://www.ncbi.nlm.nih.gov/clinvar/). RESULTS ANALYSIS OF WES DATA REVEALED A RARE HETEROZYGOUS _FIG4_

FRAMESHIFT VARIANT SHARED BY PATIENT AND FATHER OF FAMALS006 WES was performed in ALS family FamALS006 comprising the index patient and his affected paternal great-aunt, thus displaying

autosomal dominant inheritance with incomplete penetrance (Figure 1a). Before WES, analysis of the most common fALS genes (_SOD1_, _FUS_, _TARDBP_, and _C9orf72_) in the index patient had

revealed no pathogenic aberrations. For WES data analysis (Supplementary Table S1), we used an overlapping strategy to detect variants shared by the patient and his father because the

affected great-aunt was related on the father’s side, and DNA from her or the clinically asymptomatic grandfather was not available. Of 1861 coding variants shared by the index patient and

his father and not present in the healthy mother, 1425 variants were non-silent (ie, canonical±1 or 2 splice site, frameshift, in-frame indels, stop gained/lost, and missense variants), and

of these 96 variants were rare (MAF of <1%) and not present in 48 in-house controls (Supplementary Table S2). To focus on known ALS-associated genes, we then extracted only variants in

genes (_n_=126) listed in the ALS Online Genetics Database (http://alsod.iop.kcl.ac.uk/),14 resulting in one variant. This variant, a deletion of a single nucleotide in exon 7 of the _FIG4_

gene, _FIG4_:c.759delG, p.(F254Sfs*8), causes a frameshift predicted to result in a truncated protein lacking the catalytic domain including the active center. This variant was not found in

WES data from 48 neurologically unaffected German controls and is very rare (MAF of 0.00009001) in Europeans (non-Finnish) according to the ExAC database. By Sanger sequencing, the variant

was confirmed in the index patient and his father, but not detected in the 62-year-old healthy daughter of the affected great-aunt or the mother of the index patient (Figure 1b). The

affected paternal great-aunt and the paternal grandfather have passed away, and the sister of the index patient was also not available for genetic testing, thereby limiting segregation

analysis. Therefore, it cannot be excluded that the _FIG4_ frameshift variant was inherited from the paternal grandmother of the index patient. SEQUENCE ANALYSIS OF _FIG4_ IN 200 ADDITIONAL

ALS CASES REVEALED FIVE RARE HETEROZYGOUS _FIG4_ MISSENSE VARIANTS PREDICTED TO BE DELETERIOUS Next, we assessed the frequency of _FIG4_ variants in an almost exclusively central European

cohort of 200 unrelated ALS patients using WES and targeted sequencing. Thereby, we identified novel or rare heterozygous missense variants in the coding region of _FIG4_ in five additional

ALS patients (Table 1 and Figure 2a). None of these patients had a family history of neurodegenerative disorders, and no parental DNA was available for segregation analysis. All five

affected amino acids were highly or very highly conserved across species (Figure 2b), and the variants were predicted to have a deleterious effect on protein structure or function by at

least two of four prediction tools, that is MutationTaster, PROVEAN, SIFT, or PolyPhen-2 (Table 1). The affected amino acids were located in the N-terminal SAC domain (_n_=1), the catalytic

domain (_n_=2), or in regions of FIG4 not covered by structural data (_n_=2; Figure 2c). Three of the missense variants had previously been described in ALS or CMT patients, whereas two

variants have not been associated with these disorders (Table 1). The latter is true for the novel variant _FIG4_:c.1619C>T, p.(T540I) that is neither listed in the single-nucleotide

polymorphism database nor in the ExAC browser, and for the very rare variant _FIG4_:c.919G>A, p.(D307N) with a MAF of 0 in non-Finnish Europeans according to ExAC. The six rare

deleterious _FIG4_ variants we identified in total in 201 ALS patients were distributed throughout the gene without any specific clustering region (Figure 2d). All _FIG4_ variants detected

in our ALS cohort are summarized in Supplementary Table S4, including common, noncoding or silent variants that were not considered deleterious. When comparing the _FIG4_ variant frequency

in our 201 central European ALS patients with ethnicity-matched controls (non-Finnish Europeans from the ExAC database, _n_=33 370 individuals), 6 of 201 ALS cases (3%) carried rare

(MAF<1%) non-silent deleterious (all loss of function variants, that is, frameshift, stop gained/lost, canonical ±1 or 2 splice site, and initiation codon variants, as well as missense

variants predicted to be deleterious by either SIFT or PolyPhen-2 prediction tools) _FIG4_ variants _versus_ 441 of 33 370 controls (1.32%). This difference is statistically significant

(two-tailed _P_-value<0.05, _χ_2 test), whereby it should be considered that the two cohorts do not match in size and may not match in age and gender distribution. PHENOTYPIC SPECTRUM OF

SIX ALS PATIENTS WITH DELETERIOUS VARIANTS OF _FIG4_: DISEASE DURATION WAS LONGER AND UMN PREDOMINANCE WAS SIGNIFICANTLY MORE FREQUENT IN _FIG4_ VARIANT _VERSUS_ NON-VARIANT CARRIERS

Clinical, electrophysiological, and neuroradiological characteristics of ALS patients carrying deleterious _FIG4_ variants are summarized in Table 2. The index patient, FamALS006-01,

carrying the frameshift variant _FIG4_:c.759delG, p.(F254Sfs*8) developed ALS at 40 years of age, with atrophic paresis of the left upper limb and increased deep tendon reflexes as initial

symptoms. Within 3 years, atrophies and weakness progressed to the right upper and both lower extremities. No marked respiratory deficiency was observed. Cranial MRI at the age of 41 years

showed pronounced frontoparietal atrophy (Figure 1c), whereas the Edinburgh Cognitive and Behavioral ALS Screen15 revealed no abnormalities. FamALS006-03, the index patient’s father, also

carrying _FIG4_:c.759delG, p.(F254Sfs*8) did not develop neurological symptoms until he died of cardiovascular disease at 75 years of age several months after the genetic test. The

great-aunt of the index patient, who was diagnosed with ALS, had slowly progressive atrophic weakness of upper and lower limbs with dysphagia and died ∼30 years after symptom onset. Patients

MD072 carrying _FIG4_:c.122T>C, p.(I41T) and VALS007 carrying _FIG4_:c.1619C>T, p.(T540I) had initially been diagnosed with UMN predominant ALS. In both cases, the diagnosis later

changed to PLS because of slow disease progression over 3 years, no clinical signs of LMN involvement, and only mild LMN signs detectable by needle EMG in one body region in patient VALS007.

Similarly, patient VALS042 carrying _FIG4_:c.919G>A, p.(D307N) had an UMN predominant clinical phenotype, but in contrast to MD072 and VALS007, progressive LMN signs developed after 3

years of stable disease. Comparable to MD072, VALS007, and VALS042, patient VALS012 carrying _FIG4_:c.1940A>G, p.(Y647C) had a relatively long disease duration (5 years) before he died of

a traumatic subdural hemorrhage. He had been diagnosed with flail arm syndrome with no UMN signs. In contrast, patient VALS015 carrying _FIG4_:c.2558C>T, p.(S853L) suffered from

classical Charcot-type ALS with rapid disease progression and death from respiratory failure within 12 months. Remarkable neurological non-motor neuron symptoms in the _FIG4_ variant

carriers were mild cognitive impairment in VALS007 and sensory deficits in three of six patients. By initial neurophysiological studies, no major slowing of nerve conduction velocity

indicating peripheral demyelination, as seen in CMT4J, was detected in the six _FIG4_ variant carriers. However, amplitude reduction or lack of detectable response of the sural nerve was

observed in three patients indicating sensory axonal neuropathy in addition to the ALS-related motor axonal impairment. Varying degrees of brain atrophy, particularly in the frontoparietal

region, were found in all _FIG4_ variant carriers by cranial MRI. When comparing the phenotype in ALS patients from our cohort with (_n_=6) _versus_ without (_n_=195) deleterious _FIG4_

variants, predominance of UMN signs was significantly more frequent in _FIG4_ variant carriers (3/6: 50%) compared with non-_FIG4_ variant carriers (10/195: 5% _P_=0.004, Fisher’s exact

test, two-sided). Bulbar onset was not significantly more frequent in patients with (2/6: 33%) _versus_ without (42/195: 21.5%) _FIG4_ variants (_P_=0.614, Fisher’s exact test, two-sided).

Disease duration was compared in the subset of 189 patients with disease onset between 2004 and 2014. Mean disease duration was longer in the six _FIG4_ variant carriers (4.93±3.94 years)

compared with that in the 183 non-_FIG4_ variant carriers (3.18±2.21 years), although this difference did not reach statistical significance (_P_=0.212, Mann–Whitney _U_-test, two-tailed).

DISCUSSION Heterozygous deleterious variants in the _FIG4_ gene have first been implicated in sALS and fALS in 2009.6 To date, at least 14 rare nonsynonymous _FIG4_ variants have been

detected in ALS cases (Figure 2d). However, the contribution of _FIG4_ variants to ALS pathogenesis has been debated because in smaller cohorts no deleterious _FIG4_ variants were found, and

some non-penetrant _FIG4_ variant carriers have been described.16, 17, 18 Our sequence analysis of the _FIG4_ gene in 201 almost exclusively central European ALS patients confirms known and

identifies novel or rare heterozygous variants. Several lines of evidence suggest that the six _FIG4_ variants detected in our cohort are pathogenic and associated with an ALS phenotype.

The frameshift variant, _FIG4_:c.759delG, p.(F254Sfs*8), was identified by WES in the fALS patient and his father, which is consistent with the fact that the other ALS patient in the family,

the great-aunt of the index patient who was not available for genetic testing, was in the paternal line. This frameshift variant is very rare and predicted to truncate the protein at the

C-terminal end of the β10-strand. Therefore, the frameshift variant could result in a substantially shortened protein lacking the catalytic domain including the P-loop, which corresponds to

the active center, or in the loss of expression at the mRNA level because of nonsense-mediated decay. The variant _FIG4_:c.122T>C, p.(I41T) that we detected in sALS patient MD072 was

reported previously in one of 349 sALS patients.19 According to Manford _et al._,20 it may affect local folding or protein stability. Isoleucine at position 41 is located at the C-terminal

end of the β3-strand, is surrounded by hydrophobic residues of the adjacent β-strands, and might stabilize folding by hydrophobic interactions. Using a yeast two-hybrid system, the p.I41T

variant impaired interaction of mutant FIG4 with the scaffold protein VAC14, causing an unstable protein and low protein levels in an animal model and patient fibroblasts.21 The very rare

variant _FIG4_:c.919G>A, p.(D307N) was identified in sALS patient VALS042. Aspartic acid at position 307 is located at the C-terminal end of the β12-strand, directly upstream of the

protruding loop 3. The residue is surface exposed and, therefore, might affect protein folding and stability. In a previous study, a missense variant in close proximity, p.E302K, was shown

to act as a functional null allele of _FIG4._16 The substitution by lysine did not rescue the phenotype of Fig4p-null yeast, possibly due to reduced protein stability.16 The novel variant

_FIG4_:c.1619C>T, p.(T540I) was detected in sALS patient VALS007. Threonine at position 540 is part of the N-terminal turn of the α9-helix. The residue stabilizes the α-helix by a

hydrogen bond of the hydroxyl side chain with the backbone carbonyl oxygen of H537 (Figure 2c). The change of threonine at position 540 to a nonpolar residue prevents this important

interaction and might destabilize the initial α-turn of the helix, which is in close vicinity of the catalytic P-loop, thereby directly affecting the active site. The very rare variant

_FIG4_:c.1940A>G, p.(Y647C) identified in sALS patient VALS012 was previously reported in one of 473 ALS patients.6 Taken together, the frequency of rare non-silent deleterious _FIG4_

variants identified here in central European ALS patients (3%) is significantly higher compared with that in ethnicity-matched controls from the ExAC database (1.32%), whereas there is no

lower-than-expected number of _FIG4_ LoF, that is, stop-gained and essential splice site, variants in the ExAC cohort. Our findings are in line with previous results reporting _FIG4_ gene

variants in 2% of North American ALS patients of European ancestry.6 Comparably, variants in _FIG4_ were identified as ALS risk factors in two recent large-scale sequencing studies.19, 22

However, there is incomplete penetrance in the ALS family described here, which includes an unaffected father carrying a _FIG4_ frameshift variant, suggesting that _FIG4_ variants are not

causative alone, but that ALS patients may need to carry rare variants in multiple genes to show disease. This notion is corroborated by the finding that at least 30 genes are implicated in

ALS pathogenesis with a low percentage of ALS cases explained by variants in each of these.22 In addition, there is direct evidence for an oligogenic basis of ALS, for example, 7 of 14 ALS

patients with _TARDBP_ variants carried variants in 4 other ALS genes sequenced,23 and 15 of 391 (3.8%) ALS cases had variants in more than one of 17 analyzed ALS genes.19 Our data provide

evidence for a possible link between the motor neuron disease ALS and the hereditary motor and sensory neuropathy CMT4J at the genetic and phenotypic level, comparable to the detection of

deleterious variants in the _DYNC1H1_ (dynein, cytoplasmic 1, heavy chain 1) gene previously associated with classical CMT, in patients with UMN syndromes.24 Deleterious biallelic variants

of _FIG4_ were initially identified in CMT4J,9 which is an autosomal recessive form of CMT with severe combined axonal and demyelinating peripheral neuropathy and potentially severe muscle

weakness and wasting.16 Four of the _FIG4_ variants identified here in ALS patients were either previously found in a compound heterozygous state with other _FIG4_ variants in CMT4J patients

(c.122T>C and c.759delG),9 or as heterozygous variants in patients from CMT disease cohorts (c.1940A>G and c.2558C>T).16, 25 In contrast to ALS, the typical CMT phenotype involves

a sensory deficit. We, therefore, examined _FIG4_ variant carriers with respect to sensory impairment. Clinical and electrophysiological examination of the six _FIG4_ variant carriers

revealed four cases with sensory impairment or sensory–motor axonal neuropathy, whereby two patients were older than 70 years. Although lower extremity sensory nerve response can be

difficult to elicit in the elderly, our findings suggest that ALS patients carrying _FIG4_ variants may show sensory deficits as a sign of a clinical overlap with CMT, however, without

neurophysiological evidence of demyelination as in CMT4J.16 Interestingly, Fig4 loss in the ‘pale tremor’ mouse results in both neuronal degeneration in the central nervous system including

spinal motor neurons and in sensory and autonomic ganglia, as well as in electrophysiological and pathological abnormalities in peripheral nerves.9 While a peripheral neuronopathy is

detectable in the ‘pale tremor’ mouse, an ALS phenotype in later life cannot be excluded because Fig4 loss causes juvenile lethality.9 Similar to neuronal loss in the cerebral cortex and

ventricular enlargement in the ‘pale tremor’ mouse, all ALS patients with _FIG4_ variants studied here showed varying degrees of brain atrophy of the frontal or parietal cortex, some

combined with enlarged ventricles upon neuroradiological evaluation. Frontoparietal atrophy was particularly severe in the fALS patient carrying the _FIG4_ frameshift variant, while the

temporal lobe was unaffected. Comparably, frontal lobe atrophy was described in two siblings with a homozygous _FIG4_ frameshift variant and Yunis-Varón syndrome,10 whereby extramotor, in

particular frontotemporal, affection occurs in the majority of fALS as well as sALS cases.2, 26, 27 By clinical and electrophysiological assessment, we found that UMN predominance was

significantly more frequent in patients with _versus_ without _FIG4_ variants of our cohort. Similarly, very prominent corticospinal tract findings were previously reported in four of nine

ALS patients carrying heterozygous _FIG4_ variants.6 Among these, two of nine _FIG4_ variant carriers were diagnosed with PLS.6 Consistently, in our study, two of six patients carrying

heterozygous _FIG4_ variants exhibited a PLS phenotype. PLS involves UMNs only, represents 1–4% of cases with motor neuron disease,28 and is now considered a restricted phenotype of ALS in

the most recent revision of the El Escorial diagnostic criteria.29 As PLS is associated with a better prognosis than other ALS forms,2, 30 we compared disease duration in _FIG4_ variant

_versus_ non-variant carriers. In our ALS cohort, mean disease duration was longer in patients carrying deleterious _FIG4_ variants compared with non-_FIG4_ variant carriers (4.9 _versus_

3.2 years). Although not statistically significant, this difference in mean disease duration is particularly striking because one of six variant carriers died prematurely because of an

accident and two others did not show a PLS/UMN predominant phenotype. Mean disease duration of our ALS patients with deleterious _FIG4_ variants was also longer than median survival from

onset in a recent epidemiologic study of 1217 Dutch ALS patients (4.9 _versus_ 2.9 years).31 This finding is corroborated by the even longer mean disease duration, that is, 9.1 years,

reported by Chow _et al._6 for eight ALS patients with heterozygous deleterious variants of _FIG4_. From this and previous studies, the notion emerges that ALS patients carrying _FIG4_

variants may often have a distinctive phenotype, although our six patients do include cases with rapid progression and mainly LMN involvement. In summary, our data demonstrate heterozygous

deleterious _FIG4_ variants in 3% of ALS patients from a central European cohort providing further evidence that variants of _FIG4_ are associated with ALS and suggesting that it may be

worthwhile to perform _FIG4_ genetic testing in ALS patients, particularly in patients with a predominant UMN phenotype. If identified in ALS patients, _FIG4_ variants may serve as markers

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sporadic amyotrophic lateral sclerosis. _PLoS Genet_ 2014; 10: e1004704. Article  Google Scholar  Download references ACKNOWLEDGEMENTS We thank the patients and their families for

participating in this study, and are indebted to Christopher Baum, Reinhard Dengler, and Brigitte Schlegelberger for generous support and helpful discussions. Isolde Rangnau received a

scholarship from the Klin-StrucMed program of Hannover Medical School funded by the Else Kröner-Fresenius-Stiftung. Anne Kosfeld and Ruthild G Weber received research support from the Else

Kröner-Fresenius-Stiftung (Grant No. 2014_A234). Alma Osmanovic, Susanne Petri, and Ruthild G Weber received research support from the Petermax-Müller-Stiftung. AUTHOR INFORMATION Author

notes * Alma Osmanovic and Isolde Rangnau: These authors contributed equally as first authors to this work. * Susanne Petri and Ruthild G Weber: These authors contributed equally as senior

authors to this work. AUTHORS AND AFFILIATIONS * Department of Human Genetics, Hannover Medical School, Hannover, Germany Alma Osmanovic, Isolde Rangnau, Anne Kosfeld, Bernd Auber & 

Ruthild G Weber * Department of Neurology, Hannover Medical School, Hannover, Germany Alma Osmanovic, Isolde Rangnau, Susanne Abdulla, Claas Janssen & Susanne Petri * Department of

Neurology, University Hospital Magdeburg, Magdeburg, Germany Susanne Abdulla * Department of Neuroradiology, Hannover Medical School, Hannover, Germany Peter Raab * Institute for Biophysical

Chemistry, Hannover Medical School, Hannover, Germany Matthias Preller * Center for Structural Systems Biology, German Electron Synchrotron (DESY), Hamburg, Germany Matthias Preller Authors

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also search for this author inPubMed Google Scholar * Susanne Petri View author publications You can also search for this author inPubMed Google Scholar * Ruthild G Weber View author

publications You can also search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to Ruthild G Weber. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare

no conflict of interest. ADDITIONAL INFORMATION Supplementary Information accompanies this paper on European Journal of Human Genetics website SUPPLEMENTARY INFORMATION SUPPLEMENTARY

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_et al._ _FIG4_ variants in central European patients with amyotrophic lateral sclerosis: a whole-exome and targeted sequencing study. _Eur J Hum Genet_ 25, 324–331 (2017).

https://doi.org/10.1038/ejhg.2016.186 Download citation * Received: 13 July 2016 * Revised: 18 November 2016 * Accepted: 24 November 2016 * Published: 04 January 2017 * Issue Date: March

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