Two types of recessive hereditary spastic paraplegia in Roma patients in compound heterozygous state; no ethnically prevalent variant found

Journal Pre-proof Two types of recessive hereditary spastic paraplegia in Roma patients in compound heterozygous state; no ethnically prevalent variant found Anna Uhrova Meszarosova, Pavel Seeman, Jan Jencik, Jana Drabova, Renata Cibochova, Julia Stellmachova, Dana Safka Brozkova

PII:

S0304-3940(20)30070-7

DOI:

https://doi.org/10.1016/j.neulet.2020.134800

Reference:

NSL 134800

To appear in:

Neuroscience Letters

Received Date:

27 March 2019

Revised Date:

9 January 2020

Accepted Date:

29 January 2020

Please cite this article as: Meszarosova AU, Seeman P, Jencik J, Drabova J, Cibochova R, Stellmachova J, Brozkova DS, Two types of recessive hereditary spastic paraplegia in Roma patients in compound heterozygous state; no ethnically prevalent variant found, Neuroscience Letters (2020), doi: https://doi.org/10.1016/j.neulet.2020.134800

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Two types of recessive hereditary spastic paraplegia in Roma patients in compound heterozygous state; no ethnically prevalent variant found

Anna Uhrova Meszarosova(1), Pavel Seeman(1), Jan Jencik(1), Jana Drabova(2), Renata Cibochova(3), Julia Stellmachova(4), Dana Safka Brozkova(1)

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(1) DNA laboratory, Department of Paediatric Neurology, 2nd Faculty of Medicine Charles University and University Hospital Motol, Prague (2) Department of Biology and Medical Genetics, 2nd Faculty of Medicine Charles University and University Hospital Motol, Prague (3) Department of Paediatric Neurology, 2nd Faculty of Medicine Charles University and University Hospital Motol, Prague (4) Department of Medical Genetics, Palacky University Hospital, Olomouc

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Two first Czech Roma patients with hereditary spastic paraplegia genetically diagnosed. Third patient/family with pure phenotype in SPG77 described worldwide. None of detected pathogenic variants is prevalent in the Czech Roma population. Both patients are compound heterozygotes, therefore higher heterogeneity of HSP pathogenic variants could be among the Czech Roma minority. Gene panel sequencing with robust CNV analysis is the most effective approach for HSP Roma patients.

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Highlights:

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Abstract:

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Hereditary spastic paraplegia (HSP or SPG) is a group of rare upper motor neuron diseases. As some ethnically-specific, disease-causing homozygous variants were described in the Czech Roma population, we hypotesised that some prevalent HSP-causing variant could exist in this population. Eight Czech Roma patients were found in a large group of Czech patients with suspected HSP and were tested using gene panel massively parallel sequencing (MPS). Two of the eight were diagnosed with SPG11 and SPG77, respectively. The SPG77 patient manifests a pure HSP phenotype, which is unusual for this SPG type. Both patients are compound heterozygotes for two different variants in the SPG11 (c.1603-1G>A and del ex. 16-18) and FARS2 (c.1082C>T and del ex.1-2) genes respectively; the three variants are novel. In order to find a potential ethnically-specific, disease-causing variant for HSP, we tested the heterozygote frequency of these variants among 130 anonymised DNA samples of

Czech Roma individuals without clinical signs of HSP (HPS-negative). A novel deletion of ex.16-18 in the SPG11 gene was found in a heterozygous state in one individual in the HSPnegative group. Haplotype analysis showed that this individual and the patient with SPG11 shared the same haplotype. This supports the assumption that the identified SPG11 deletion could be a founder mutation in the Czech Roma population. In some Roma patients the disease may also be caused by two different biallelic pathogenic mutations. Keywords: hereditary spastic paraplegia, Czech Roma population, prevalent variant, SPG11, SPG77 Introduction:

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The Roma population represents an important ethnic minority in the Czech Republic with a high rate of endogamy 1. In endogamous populations, a higher frequency of autosomal recessive disorders is observed as well as a higher frequency of healthy carriers of pathogenic variants in a heterozygous state. The affected patients are usually homozygous for pathogenic variants, that are extremely rare in the general population, but frequent in the Roma population. Previous studies, published by our group and others, reported multiple ethnically-specific, disease-causing variants that are responsible for a spectrum of neurological disorders, such as neuropathies (HK1, NDRG1, CTDP1 genes), congenital myasthenic syndromes (CHRNE), deafness (GJB2) and others 2-7 in the Czech Roma population. Therefore, a patient’s ethnicity can provide important guidance in the process of genetic testing. Hereditary spastic paraplegia (HSP/SPG) is a group of rare upper motor neuron diseases that manifests with progressive spasticity and weakness of the lower limbs leading to progressive gait impairment. HSP is caused by variants in more than 90 genes or gene loci 8,9. According to Czech government statistics, in 2017 the population of Roma in the Czech Republic is 240,300. The prevalence of HSP in the Czech Republic is not known, but based on the prevalence from various populations (1-10 : 100,000) 10 the estimated number of Czech Roma HSP patients is between 2-24. We hypothesised that certain gene variants associated with HSP might be more prevalent in the Czech Roma population, similar to the variants associated with hereditary motor and sensory neuropathy type Lom and Russe or CCFDN or others 4,11. We tested Czech Roma patients with suspected HSP using gene panel massively parallel sequencing (MPS). Further, in order to describe variants for first-line genetic testing in Roma patients with HSP, we aimed to evaluate the prevalence of potential ethnicallyspecific pathogenic variants causing hereditary spastic paraplegia (HSP) in the Czech Roma population. For HSP, there were no ethnically-specific types of disease-causing variants described previously. Patients and methods: Study design:

In order to detect pathogenic variants associated with HSP, we first performed a MPS comprising a gene panel of 38 genes associated with uncomplicated HSP in all Czech Roma patients (n=8) with HSP. Next, we analysed and anonymised ethnically-matched 130 DNA samples from HSP-negative Czech Roma patients for the presence of the variants detected in the patient’s group.

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Ethnically-matched HSP-negative DNA samples

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Patient selection: As a part of a larger diagnostic genetic study of HSP, eight Czech patients of Roma origin (Roma ethnicity was mentioned in anamnestic data) with suspected uncomplicated HSP were examined using a targeted MPS of a gene panel of selected HSP genes (Supp.1). The patients were selected from a group, drawn from the entire Czech Republic, who had been referred originally by clinical geneticists and neurologists for SPG4 testing between the years 2005 – 2019. Only patients initially manifesting the uncomplicated form of HSP (at the time of referring for the first genetic testing) were available. Patients with atypical presentation of HSP (e.g. those with asymmetrical presentation, prominent ataxia, predominant affection of the upper limbs) and those with point mutations or gross deletions in SPAST gene were not included for MPS (all 17 coding exons Sanger sequenced and MLPA P-0165 previously performed).

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The HSP-negative group consisted of DNA samples from individuals and their relatives of Roma origin collected over 10 years from the entire Czech Republic who had been referred originally for genetic testing for deafness or congenital myasthenic syndromes. Individuals with clinical signs or suspected HSP were excluded from the group as well as those below 30 years of age due to early presentation of HSP. For multiple samples from one family, we only selected one single sample in order to minimize the risk of bias in variant frequencies, and when possible, we preferred the healthy parent’s sample. Altogether, 130 unique DNA samples were included in the HSP-negative group of individuals and subsequently anonymised.

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All patients and/or their legal representatives provided informed consent with the genetic testing. Methods:

SureSelect Target Enrichment Kit (Agilent Technologies, US) and Hiseq (Illumina, Inc., US) was used for DNA library preparation and MPS. Raw FASTQ data were analysed by two independent alignment software SureCall (Agilent Technologies, US) and NextGene (Softgenetics, US). Called variants were annotated by AlamutBatch software (Interactive Biosoftware, France).

Two approaches for CNV analysis were used (both NextGene). The first type of analysis (NextGene CNV Tool) is based on the Hidden Markov model comparison and three control individuals were used for comparison. This analysis was repeated three times and always with three different control individuals. The second type of CNV analysis (NextGene BetaBatch) compares and normalizes the read count of all the samples from the same sequencing run together.

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An evaluation of variant pathogenicity was performed in accordance with the ACMG criteria 12 . For in silico prediction of variant effects, we used the following tools for missense variants: PolyPhen2 13 and SIFT 14; and for splicing variants MaxEntScan 15, HSF 16 and NNSplice 17. We further filtered the variants based on their presence in the HGMD Professional database and their frequency (<1%) in population databases (GnomAD,dbSNP). Segregation analysis in families was performed where possible.

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All detected sequence variants as well as segregation analysis in the families was confirmed by Sanger sequencing. Reference sequences NM_025137.3 (SPG11) and NM_006567.4 (FARS2) were used. MLPA kit P306-SPG11 with Coffalyzer software (both MRC Holland, Netherlands) or SurePrint CGH 4x180K G3 ISCA Array Chip and CytoGenomics 4.0.3.12 software (Agilent Technologies, US) were used for SPG11 and FARS2 gene deletion confirmation.

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The frequency of detected single nucleotide variants (SNVs) (SPG11 c.1603-1G>A; and FARS2 c.1082C>T) in ethnically-matched HSP-negative DNA samples was tested by real-time PCR allelic discrimination TaqMan®Assay (ThermoFischer Scientific, US) with custom-designed probes. ABI PRISM 7000 Sequence Detecting System and ABI PRISM 7000 SDS Software (both Applied Biosystems, US) were used for real-time PCR and data evaluation. MLPA kit P306-SPG11 and Coffalyzer software (both MRC Holland, Netherlands) were used for testing ex.16-18 del in SPG11 gene.

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Results:

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Haplotype analysis of six STS loci surrounding the SPG11 gene was performed to assess the kinship. Fragment analysis with FAM/HEX labeled primers of D15S1044, D15S994, D15S641 and D15S161, D15S208, D15S990 STSs markers were used.

All eight Czech Roma patients with suspected HSP tested by gene panel MPS were index patients from independent families and, of the eight, four were classed as sporadic patients (neurological negative history in two successive generations), three are probably with autosomal recessive (AR) and one is probably with autosomal dominant (AD) mode of inheritance in family. The age of the tested patients ranged from 5 to 63 years; the age at onset was in the first two decades of life except for the patient with AD inheritance (details in Tab.1).

Using the gene panel MPS, the cause of their HSP was elucidated in two patients and they were genetically diagnosed with SPG11 and SPG77, respectively. Surprisingly both patients are not homozygotes, but compound heterozygotes for 2 different pathogenic variants. The patient with SPG11 carries a missense variant c.1603-1G>A and a deletion of exons 16-18 (ex.16-18 del) in SPG11. The patient with SPG77 carries a missense variant c.1082C>T (p.Pro361Leu) and a deletion of exons 1-2 (ex.1-2 del) in FARS2. Patient with two heterozygous variants in the SPG11 gene:

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The splice site SNV c.1603-1G>A at the acceptor splice site of intron 7 and the gross deletion of exons 16, 17 and 18 in SPG11 gene in a heterozygous state were found in a 26-year-old male patient. Variants are in the trans position because the variant c.1603-1G>A was confirmed in the mother, and the ex. 16-18 del in the father (both heterozygous). Both variants are novel, have not been described previously and both have zero frequency in population databases (GnomAD,dbSNP). A single nucleotide change c.1603-1G>A effects the invariant -1 acceptor splice site. Deletion of exons 16-18 is out-of-frame and, except for the missing three coding exons, predicts a premature termination codon. According to ACMG criteria, both variants are classified as pathogenic (PVS1, PM2, PM3). We assume the combination of these two variants is the cause of patient´s spastic paraparesis.

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Data on the patient’s clinical history and previous neurological findings are limited. He reported that his gait problems started at the age of 15. He complained of muscle weakness and pain in the lower limbs. Neurological examination showed progressive gait impairment, spasticity, bilateral hyperreflexia and present Babinski sign. A brain MRI performed at the age of 26 years showed thin corpus callosum. Signs of mild cognitive impairment were described in testing, but no specific scale was used to quantify this. No other clinical information is currently available. The SPG11 phenotype manifested in our patient (disease onset in the second decade of life, rapid progression and typical thin corpus callosum) corresponds to the originally described clinical picture of SPG11 18-20.

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Regarding his family history, his parents are healthy and the patient has no siblings. Two paternal cousins are similarly affected (Fig.1; III/3 and III/4), but they were not available for genetic analysis. Based on their neurological report, they both suffer from a similar pattern of gait impairment. One cousin is wheelchair-bound and her brain MRI showed thin corpus callosum. We could not obtain more detailed clinical information or the brain MRI of the second cousin. No other affected individuals have been reported from either paternal or maternal families and there is no known consanguinity in the family. Patient with two heterozygous variants in the FARS2 gene: Biallelic pathogenic variants in the FARS2 gene cause a very rare type of HSP, the SPG77. We detected two heterozygous variants, c.1082C>T (p.Pro361Leu) and ex.1-2 del in the FARS2 gene in a 22-year-old male patient. The variant c.1082C>T (P361L) in exon 6 affects a highly conserved nucleotide as well as an amino acid. In silico tools predict this variant to be

deleterious (PolyPhen Hum Div Score 1.000 Probably Damaging, SIFT deleterious) and, based on ACMG criteria, is classified as likely pathogenic (class 4). The variant has a low frequency of 0.014 % according to population databases (GnomAD, dbSNP). Recently, the variant c.1082C>T (P361L) has been associated with a complicated spastic paraplegia phenotype 21.

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The gross deletion of ex.1-2 del in the FARS2 gene was detected by CNV analysis. ArrayCGH chip testing confirmed the extent of the deletion: a 6p25.1 heterozygous deletion in the range of 100.4 kb-151.6 kb encompassing ex.1-2 of FARS2 gene (exon 1 is noncoding/untranslated) and ex.1 of the neighboring LYRM4 gene. Deletion of this extent has not been described previously and, according to the ACMG criteria the deletion is classified as pathogenic (PVS1, PM2, PM3 od PM6). The patient´s healthy mother carries only the heterozygous c.1082C>T (P361L) variant (Fig.2). We were unable to obtain either the father´s DNA sample, or DNA samples from other relatives. We presume that the gross deletion was inherited from the healthy father or occurred de novo. We therefore assume that both variants are in trans position in the patient and this fact supports their causality.

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The patient’s gait problems were initially noticed at 5 years of age, otherwise he had normal psychomotor development. The symptoms progressed slowly for, as a child, he was even able to play football. At the age of 12 and 14 years, he underwent Achilles tendon prolongation surgery. In 2008, a muscle biopsy (at the age of 12 years) showed there were no myopathic changes. The MRI, EEG, EMG and SEP (somatosensory evoked potentials) examinations performed over several years of disease have consistently shown normal results and his last brain and spine MRIs were done in 2014 as, since then, the patient has refused further examinations. The most recent neurological examination performed in 2018 showed a bilateral hyperreflexia, present Babinski sign, ankle clonus, feet deformities (pes cavus) and mild muscle atrophy on both lower limbs, with mildly increased muscle tonus in the legs (Fig.3). Vibration sense was not affected and the upper limbs are without neurological impairment. Other features (e.g. nystagmus, dysarthria, sphincter deficits) were not reported. A cognitive deficit was not observed, the patient completed secondary education. Today, after having the condition for 16 years, the patient complains only of pain and weakness of the lower limbs and is able to walk independently without support, although his gait has spastic features.

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Based on the above-mentioned features, we concluded that the patient manifests a pure HSP phenotype without any complicating signs. The patient is the only affected family member since his older brother and both parents are healthy and show no signs of spastic paraplegia nor are there any other known neurological problems in the larger family. There is no consanguinity in the pedigree. TaqMan and MLPA testing in ethnically-matched HSP-negative group

From the 130 ethically matched HSP-negative individuals, the gross deletion del ex. 16-18 in the SPG11 gene was present in only one sample in the heterozygous state. Neither c.16031G>A (SPG11) nor c.1082C>T (FARS2) were observed in the HSP-negative cohort.

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To assess the potential kinship between the SPG11 patient, and the HSP-negative individual with detected heterozygous SPG11 ex.16-18 del, we performed a haplotype analysis of six STS markers neighbouring the SPG11 gene; the selected markers covered the region of 8.1 Mb (Fig.4). In the SPG11 family, we found the haplotype, inherited from the father, segregating with the deletion (Fig.5A). The same unique combination of alleles/same haplotype is present in the HSP-negative sample. We didn’t know the frequencies of the particular alleles in tested STS markers, so we were unable to assess how frequently this combination of alleles/haplotype occurs in the studied population. Therefore, we performed a haplotype analysis in 10 randomly selected samples from the HSP-negative individuals. The same combination of alleles/haplotype, as in the individuals with SPG11 ex16-18 del (patient, healthy father, HSP-negative sample with deletion), was not present in any of the 10 randomly tested samples; it is, therefore, unique (Fig.5B).

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Discussion:

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Given that only eight index Roma patients were included in our study, it is possible that this number may underestimate slightly the actual frequency of HSP in the Czech Roma population. Moreover, patients were selected from the large group of individuals originally referred for SPG4 testing and some patients or families with autosomal recessive inheritance could be missed. However, as our centre is the only facility in the Czech Republic providing genetic testing for HSP, we assume that our collected sample of cohort individuals from all ethnicities in the Czech Republic is representative. We were unable to obtain data on ethnicity of all individuals, nevertheless we selected all patients where there Roma ethnicity was clear yet this might have introduced a selection bias.

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Two patients among the eight tested (25 %) were genetically diagnosed with SPG11 and SPG77, respectively. Other studies using the same diagnostic approach (gene panel MPS of non-SPG4 HSP patients) clarified 19 % and 25 % of tested patients, respectively 22,23. Both genetically diagnosed patients reported in this study are compound heterozygotes for two different pathogenic variants in the causal genes. This is a rather unusual finding since in endogamous populations we would expect a higher frequency of autosomal recessive disorders with pathogenic variants in a homozygous state 4-6. Homozygosity mapping, frequently used in Roma families for the detection of causal variants, would fail to detect the cause of the disease in these two families. Of the four detected variants, three are reported here for the first time and have not been described previously: c.1603-1G>A, ex.16-18 del (SPG11 gene) and ex.1-2 del (FARS2 gene). The variant c.1082C>T (P361L) in the FARS2 gene was reported previously 21, in two index patients from Belgium who had a complicated phenotype (including developmental delay,

wheelchair dependency, seizures, very early loss of walking, severe dysarthria, problems with chewing and swallowing and changes on MRI) (Tab.2). None of the two patients described were of Roma origin and no consanguinity was reported. The SPG77 patient, who manifested with a pure HSP phenotype, developed a gait disturbance but only the lower limbs were affected. He showed no other clinical signs during the 16 years of his condition, even though the last brain and spine MRI was performed in 2014 (with normal results). Thus, we report the third family with a pure HSP phenotype caused by biallelic pathogenic variants in the FARS2 gene. Our results confirm the previous findings of a pure HSP caused by the FARS2 gene variants.

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Overall, the pathogenic variants in the FARS2 gene have been described in only a few families worldwide, and they were associated mostly with a very severe phenotype, including encephalopathy, cortical atrophy, delayed psychomotor development with dysmorphic features, seizures, etc. 24,25 and reviewed in 21. Only eight pathogenic variants in the FARS2 gene have been described as causative for the SPG77 type of HSP in five families worldwide (Tab.2; references listed in the table) and of these, two SPG77 patients presented with severe complicated phenotype (above) 21. In addition, Yang at al. described four SPG77 siblings from China all with pure phenotype, progressive lower limb spasticity, muscle weakness with hyperreflexia, extensor plantar responses (Babinski) and scissors gait 26. Sahai at al. described SPG77 in 9-year-old boy who, with the pure phenotype, had progressive lower limb spasticity and normal cognition associated with bilateral signal abnormalities in the dentate nuclei 27. Two SPG77 siblings described by Forman et al. showed a complex HSP phenotype with dysphonia 28.

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To date, five gross deletions in the FARS2 gene, though all to a different extent, have been described. The deletions are associated mainly with encephalopathy, epilepsy or mitochondrial dysfunction, and also, very recently, with a complicated spastic paraplegia 25,29-31. (Tab.3, Fig.6). Intronic presence of Alu repeats have been described repeatedly as the cause of rearrangements/gross deletions in various genes 32-34. We don’t know the actual breakpoints of the deletion in our patient, therefore we inspected in detail the part of chromosome 6 bordered by two undeleted points (g.5,244,167 and g.5,395,818) identified by the ArrayCGH test. AluSq2 (chr6:5,256,441-5,256,753), in intron 1 of the LYRM4 gene, and two repeats of AluSc (chr6:5,385,644-5,385,955) and AluSz (chr6:5,390,092-5,390,396), in intron 2 of the FARS2 gene with 81% and 86% of homology (compared with AluSq2), were found (Fig.6). These highly homologous Alu repeats could be the cause of the rearrangement that lead to the gross deletion detected in our patient. In the group of 130 ethnically-matched HSP-negative individuals, the presence of all three identified pathogenic variants (c.1603-1G>A plus del ex. 16-18 in SPG11 and c.1082C>T (P361L) in FARS2) was tested. We did not test for the presence of del ex.1-2 in the FARS2 gene because the exact range of the deletion is not known so using long-range PCR is therefore not possible. An ArrayCGH could be used for testing but the costs would be

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prohibitive. The gross deletion ex.16-18 del in SPG11 was detected in a heterozygous state in one sample among the tested group. Although the whole SPG11 gene was not sequenced in this sample, we did not expect this individual to have spastic paraplegia because of the selection criteria. The haplotype analysis showed that the SPG11 patient and the HSPnegative sample carrying the heterozygous SPG11 deletion had a common ancestor since their shared haplotype was unique and could not be found among ten randomly selected samples from ethnically-matched HSP-negative individuals. Based on these findings, we hypothesise that the SPG11 ex. 16-18 del could be a founder mutation in the Czech Roma population and it is similar to the p.G31A variant in the EXOSC3 gene associated with pontocerebellar hypoplasia type 1 35. Since the SPG11 deletion was found only in a single HSP-negative Roma individual, we cannot comment on the actual frequency of the variant in the Czech Roma population.

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We have not found a possible ethnically-specific variant associated with HSP in the Czech Roma population that would justify the preferential genetic testing of such a variant. In addition, the SPG11 gene itself is a long gene consisting of 40 coding exons; under these circumstances, Sanger sequencing of each coding exons combined with MLPA testing would be prohibitively expensive. In addition, the prevalence of HSP in the Czech Republic is relatively low. Therefore, we argue for targeted gene panel MPS, including the CNV analysis. Both gross deletions were detected by the CNV analysis tool for MPS data, which highlights its importance in the process of data evaluation. Conclusion:

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The number of Roma individuals with hereditary spastic paraplegia in the Czech Republic is relatively low and they can manifest very rare types of this heterogeneous disease. We found eight Roma individuals with suspected HSP among a large group of Czech patients and two were diagnosed with SPG11 and SPG77, which is very rare. The SPG77 patient manifested the pure type of the disease. Testing for the presence of detected variants in an ethnically-matched HSP-negative group did not lead to the discovery of an ethnically-specific variant responsible for HSP in the Czech Roma patients. Both diagnosed patients are compound heterozygotes for two different pathogenic variants; this finding provides evidence for a higher heterogeneity of HSP pathogenic variants among the Czech Roma minority. Therefore, we argue that gene panel MPS with robust CNV analysis represents the most effective genetic testing approach for these patients.

Acknowledgments: All authors declare that there is no conflict of interest. We would like to thank all participants and physicians for sending the DNA samples. The patient agreed to the use of the photo for publication. This study was supported by the Ministry of Health of the Czech

Republic MH CZ nr. 15-31899A and the University Hospital Motol, Prague, Czech Republic MH CZ-DRO 00064203.

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Reese MG, Eeckman FH, Kulp D, Haussler D. Improved splice site detection in Genie. J Comput Biol. 1997;4(3):311-323. Stevanin G, Santorelli FM, Azzedine H, et al. Mutations in SPG11, encoding spatacsin, are a major cause of spastic paraplegia with thin corpus callosum. Nat Genet. 2007;39(3):366-372. Hehr U, Bauer P, Winner B, et al. Long-term course and mutational spectrum of spatacsinlinked spastic paraplegia. Ann Neurol. 2007;62(6):656-665. Denora PS, Schlesinger D, Casali C, et al. Screening of ARHSP-TCC patients expands the spectrum of SPG11 mutations and includes a large scale gene deletion. Hum Mutat. 2009;30(3):E500-519. Vantroys E, Larson A, Friederich M, et al. New insights into the phenotype of FARS2 deficiency. Mol Genet Metab. 2017;122(4):172-181. Iqbal Z, Rydning SL, Wedding IM, et al. Targeted high throughput sequencing in hereditary ataxia and spastic paraplegia. PLoS One. 2017;12(3):e0174667. Kumar KR, Blair NF, Vandebona H, et al. Targeted next generation sequencing in SPASTnegative hereditary spastic paraplegia. J Neurol. 2013;260(10):2516-2522. Elo JM, Yadavalli SS, Euro L, et al. Mitochondrial phenylalanyl-tRNA synthetase mutations underlie fatal infantile Alpers encephalopathy. Hum Mol Genet. 2012;21(20):4521-4529. Raviglione F, Conte G, Ghezzi D, et al. Clinical findings in a patient with FARS2 mutations and early-infantile-encephalopathy with epilepsy. Am J Med Genet A. 2016;170(11):3004-3007. Yang Y, Liu W, Fang Z, et al. A Newly Identified Missense Mutation in FARS2 Causes Autosomal-Recessive Spastic Paraplegia. Hum Mutat. 2016;37(2):165-169. Sahai SK, Steiner RE, Au MG, et al. FARS2 mutations presenting with pure spastic paraplegia and lesions of the dentate nuclei. Ann Clin Transl Neurol. 2018;5(9):1128-1133. Forman EB, Gorman KM, Ennis S, King MD. FARS2 Causing Complex Hereditary Spastic Paraplegia With Dysphonia: Expanding the Disease Spectrum. J Child Neurol. 2019;34(10):621. Almalki A, Alston CL, Parker A, et al. Mutation of the human mitochondrial phenylalaninetRNA synthetase causes infantile-onset epilepsy and cytochrome c oxidase deficiency. Biochim Biophys Acta. 2014;1842(1):56-64. Vernon HJ, McClellan R, Batista DA, Naidu S. Mutations in FARS2 and non-fatal mitochondrial dysfunction in two siblings. Am J Med Genet A. 2015;167A(5):1147-1151. Jou C, Ortigoza-Escobar JD, O'Callaghan MM, et al. Muscle Involvement in a Large Cohort of Pediatric Patients with Genetic Diagnosis of Mitochondrial Disease. J Clin Med. 2019;8(1). Conceicao Pereira M, Loureiro JL, Pinto-Basto J, et al. Alu elements mediate large SPG11 gene rearrangements: further spatacsin mutations. Genet Med. 2012;14(1):143-151. Gunther S, Elert-Dobkowska E, Soehn AS, et al. High Frequency of Pathogenic Rearrangements in SPG11 and Extensive Contribution of Mutational Hotspots and Founder Alleles. Hum Mutat. 2016;37(7):703-709. Ticha I, Kleibl Z, Stribrna J, et al. Screening for genomic rearrangements in BRCA1 and BRCA2 genes in Czech high-risk breast/ovarian cancer patients: high proportion of population specific alterations in BRCA1 gene. Breast Cancer Res Treat. 2010;124(2):337-347. Schwabova J, Brozkova DS, Petrak B, et al. Homozygous EXOSC3 mutation c.92G-->C, p.G31A is a founder mutation causing severe pontocerebellar hypoplasia type 1 among the Czech Roma. J Neurogenet. 2013;27(4):163-169.

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17.

35.

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Fig.1. The pedigree of Patient with two heterozygous variant in the SPG11 gene (rhomb shape-unknown gender, blacked shape-affected, WT-wild type, N.T. – genetically not tested; the number inside the rhombus shape represents the number of siblings).

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Fig.2: The pedigree of the Patient 2 with two heterozygous variants in the FARS2 gene (blacked shape-affected, WT-wild type, N.T. – genetically not tested).

Fig. 3. The patient with SPG77 at the age of 21. Feet deformities and mild muscle atrophy is present on the lower limbs.

ro of -p re lP

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Fig.4: STS markers used in the haplotype analysis and their localization in chromosome 15.

Fig.5A: Haplotype analysis of the SPG11 family and control sample with SPG11 ex.16-18 del; Alleles inherited from the father segregating with deletion are marked in blue. The control sample has the same unique combination of alleles/the same unique haplotype. The marker D15S208 is not informative.

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Fig.5B: Haplotype analysis in 10 randomly selected HSP-negative individuals. None has the same combination of alleles as individuals with SPG11 ex.16-18 del. Number means the absolute length of PCR product.

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Fig.6: Schematic picture of the FARS2 gene with all gross deletions described to date. The deletion found in the Czech patient is in red. Alu repeats with high percentage of homology in intron 1 of the LYRM4 gene and intron 2 of the FARS2 gene are highlighted in the detailed picture.

Age at Age at examination disease (years) onset (years)

Supposed inheritance

Affected family members

in family

Patient 1

5

1*

sporadic case

Patient 2

11

1*

AR ?

Patient 3

13

2

sporadic case

ro of

Tab.1. Characteristics of eight Czech Roma patients tested by targeted gene panel MPS

Patient 4

15

4

AR

brother affected similarly

Patient 5

22

6

sporadic case

-

Patient 6

26

15

AR

two cousins affected similarly

Patient 7

35

13

Sporadic case

-

Patient 8

63

?

AD

? **

-

mild gait problems observed in a cousin

lP

re

-p

-

ur

na

AR-autosomal recessive, AD-autosomal dominant, * abnormal gait since onset (tip-toe walking), ** not mentioned in detail

Tab.2: All SPG77 patients described to date Variants

Jo

Patient

HSP type

Phenotype Gait impairment, progressive limb spasticity, hyperreflexia, pyramidal weakness Spastic paraplegia, wheelchair dependency, developmental delay, MRI changes, seizures, neurogenic bladder, cryptorchism Delayed motor development, spastic paraplegia, severe lower limb spasticity, wheelchair dependency, MRI changes, severe dysarthria, tremor,

1.-4. (siblings)

D142Y homozygous

Pure

5.

A154V P361L

Complicated

6.

V174del P361L

Complicated

Sex Age F (3x); M (1x) 23 – 41 years

Onset 1–5 years

Yang et al.

M 20 years

6 month

Vantroys et al.

F 16 years

10 month

Vantroys et al.

7.

P136H Q216X

Pure

8.-9. (siblings)

G141E ex 6 del

10. (this report)

P361L Ex1-2 deletion

Complex with dysphonia Pure

dystonia Spastic gait, lower limb spasticity, brisk deep tendon reflexes; lesions of dentate nuclei in MRI Spasticity, weakness, delayed walking, tremor on upper limbs, dysphonia in the boy Gait impairment, progressive limb spasticity, hyperreflexia, slow progression

M

2.5 years

Sahai et al.

M,F

24 and 18 month 5 years

Forman et al.

M 21 years

Meszarosova et al.

Tab. 3: All gross deletions in the FARS2 gene described to date

~ 134 kb incl. ex 1 (coding)

2. - 3. (siblings)

~ 116 kb incl. ex 6

R419C

4.

88 kb incl. prom ex1 (noncoding) and 3´exons of LYRM4 ex 3

D325Y

6.-7. (siblings)

ex 6

G141E

8.

~ 151 kb incl ex1 (coding), prom ex1 (noncoding) and 3´exons of LYRM4

P361L

Sex Age

Onset

Encephalopathy, early infantile with seizures; axial hypotonia, distal hypertonia, psychomotor delay Non-fatal mitochondrial dysfunction, developmental delay, facial dysmorphism, dysarthria Epilepsy, infantile-onset; Developmental delay, facial dysmorphism

? 3 years in 2016 (when reported)

3 month

Raviglione et al.

F, M 5 and 13 years in 2015 (when reported) M

-

Vernon et al.

6 month

Almalki et al.

Encephalopathy and spastic paraparesis Spastic paraplegia with dysphonia

F ? M, F 13 and 7 years M 21 years

7

Jou et al.

24 and 18 month 5 years

Forman et al.

lP

Pure spastic paraplegia; no complicating signs, normal psychomotor development, normal MRI

na

Jo

ur

(this report)

R490H

Phenotype

ro of

1.

5.

Second variant in FARS2 R386G

-p

Deletion type

re

Patient

Meszarosova et al.