The FGF14 GAA repeat expansion in Greek patients with late-onset cerebellar ataxia and an overview of the SCA27B phenotype across populations.

A pathogenic GAA repeat expansion in the first intron of the fibroblast growth factor 14 gene (FGF14) has been recently identified as the cause of spinocerebellar ataxia 27B (SCA27B). We herein screened 160 Greek index cases with late-onset cerebellar ataxia (LOCA) for FGF14 repeat expansions using a combination of long-range PCR and bidirectional repeat-primed PCRs. We identified 19 index cases (12%) carrying a pathogenic FGF14 GAA expansion, a diagnostic yield higher than that of previously screened repeat-expansion ataxias in Greek LOCA patients. The age at onset of SCA27B patients was 60.5 ± 12.3 years (range, 34-80). Episodic onset (37%), downbeat nystagmus (32%) and vertigo (26%) were significantly more frequent in FGF14 expansion-positive cases compared to expansion-negative cases. Beyond typical cerebellar signs, SCA27B patients often displayed hyperreflexia (47%) and reduced vibration sense in the lower extremities (42%). The frequency and phenotypic profile of SCA27B in Greek patients was similar to most other previously studied populations. We conclude that FGF14 GAA repeat expansions are the commonest known genetic cause of LOCA in the Greek population and recommend prioritizing testing for FGF14 expansions in the diagnostic algorithm of patients with LOCA.

Mutational screening of Greek patients with axonal Charcot-Marie-Tooth disease using targeted next-generation sequencing: Clinical and molecular spectrum delineation.

BACKGROUND AND AIMS: Axonal forms of Charcot-Marie-Tooth disease (CMT) are classified as CMT2, distal hereditary motor neuropathy (dHMN) or hereditary sensory neuropathy (HSN) and can be caused by mutations in over 100 genes. We presently aimed to investigate for the first time the genetic landscape of axonal CMT in the Greek population. METHODS: Sixty index patients with CMT2, dHMN or HSN were screened by a combination of Sanger sequencing (GJB1) and next-generation sequencing custom-made gene panel covering 24 commonly mutated genes in axonal CMT. RESULTS: Overall, 20 variants classified as pathogenic or likely pathogenic were identified in heterozygous state in 20 index cases, representing 33.3% of the cohort. Of these, 14 were known pathogenic/likely pathogenic and six were designated as such according to ACMG classification, after in silico evaluation, testing for familial segregation and further literature review. The most frequently involved genes were GJB1 (11.7%), MPZ (5%) and MFN2 (5%), followed by DNM2 (3.3%) and LRSAM1 (3.3%). Single cases were identified with mutations in BSCL2, HSPB1 and GDAP1. INTERPRETATION: A wide phenotypic variability in terms of severity and age of onset was noted. Given the limited number of genes tested, the diagnostic yield of the present panel compares favourably with studies in other European populations. Our study delineates the genetic and phenotypic variability of inherited axonal neuropathies in the Greek population and contributes to the pathogenicity characterization of further variants linked to axonal neuropathies.

Screening for the FMR1 premutation in Greek patients with late-onset movement disorders.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset, X-linked, neurodegenerative disorder that affects premutation carriers of the FMR1 gene. FXTAS is often misdiagnosed as spinocerebellar ataxia (SCA) or Parkinson's disease (PD). Herein, we sought to investigate the frequency, genotypic and phenotypic profile of FXTAS in two cohorts of Greek patients with late-onset movement disorders, one with cerebellar ataxia and the other with PD. In total, 90 index patients with late-onset cerebellar ataxia and 171 with PD were selected. None of the cases had male-to-male transmission. Genetic screening for the FMR1 premutation was performed using standard methodology. The FMR1 premutation was detected in two ataxia patients (2.2%) and two PD patients (1.2%). Additional clinical features in FXTAS patients from the ataxia cohort included neuropathy, mild parkinsonism, cognitive impairment and pyramidal signs. The FXTAS patients from the PD cohort had typical PD. We conclude that, in the Greek population, the FMR1 premutation is an important, albeit rare, cause of late-onset movement disorders. Routine premutation screening should be considered in SCA panel-negative late-onset ataxia cases. Directed premutation screening should be considered in all ataxia and PD cases with additional features suggestive of FXTAS. Our study highlights the importance of FMR1 genetic testing in the diagnosis of late-onset movement disorders.

A Greek National Cross-Sectional Study on Myotonic Dystrophies.

Myotonic Dystrophies (DM, Dystrophia Myotonia) are autosomal dominant inherited myopathies with a high prevalence across different ethnic regions. Despite some differences, mainly due to the pattern of muscle involvement and the age of onset, both forms, DM1 and DM2, share many clinical and genetic similarities. In this study, we retrospectively analyzed the medical record files of 561 Greek patients, 434 with DM1 and 127 with DM2 diagnosed in two large academic centers between 1994-2020. The mean age at onset of symptoms was 26.2 ± 15.3 years in DM1 versus 44.4 ± 17.0 years in DM2 patients, while the delay of diagnosis was 10 and 7 years for DM1 and DM2 patients, respectively. Muscle weakness was the first symptom in both types, while myotonia was more frequent in DM1 patients. Multisystemic involvement was detected in the great majority of patients, with cataracts being one of the most common extramuscular manifestations, even in the early stages of disease expression. In conclusion, the present work, despite some limitations arising from the retrospective collection of data, is the first record of a large number of Greek patients with myotonic dystrophy and emphasizes the need for specialized neuromuscular centers that can provide genetic counseling and a multidisciplinary approach.

Expanding the spectrum of C9ORF72-related neurodegenerative disorders in the Greek population.

The C9ORF72 hexanucleotide repeat expansion is an established cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) and has also been associated with Huntington disease (HD)-like syndromes and rarely with Parkinson's disease (PD) and Alzheimer's disease (AD). In the present study we aimed to investigate the genotypic and phenotypic profile of C9ORF72-related disorders in Greece. For this reason, 957 patients (467 with ALS, 53 with HD-like syndromes, 247 with dementia, 175 with PD and 15 with hereditary spastic paraplegia, HSP) and 321 controls were tested for the C9ORF72 repeat expansion. Forty-nine patients with ALS (10.5%), 2 with HD-like syndromes (3.8%), 13 with FTD (11.5%), 1 with AD (1.6%), and 2 with PD (1.1%) were expansion-positive. The expansion was not detected in the HSP or control groups. The results of this study provide an update on the spectrum of C9ORF72-related neurodegenerative diseases, emphasizing the importance of C9ORF72 genetic testing in Greek patients with familial and sporadic ALS and/or FTD and HD-like syndromes.

Variant transthyretin amyloidosis (ATTRv) polyneuropathy in Greece: a broad overview with a focus on non-endemic unexplored regions of the country.

Comprehensive data on variant transthyretin amyloidosis polyneuropathy (ATTRv-PN) in Greece are lacking. We presently provide an overview of ATTRv-PN in Greece, focusing on unexplored non-endemic regions of the country. In total, we identified 57 cases of ATTRv-PN diagnosed over the past 25 years, including 30 from the island of Crete, an apparent endemic region. Patients carried 10 different TTR mutations (C10R; P24S; V30M; R34G; R34T; I68L; A81T; E89Q; E89K and V94A). Carriers of the common V30M mutation constituted 54.3 % of the cohort. A known founder effect for the V30M mutation was present on the island of Crete. Non-endemic cases identified outside the island of Crete are presently reported in more detail. The age of onset ranged from 25 to 77 years, with a mean of 51.1 years. A mean diagnostic delay of 3.2 years was observed. V30M patients had earlier onset and less cardiac involvement than patients carrying other mutations. Genotype-phenotype correlations were largely consistent with published data. We conclude that, with the exception of the Cretan cluster, ATTRv-PN is not endemic in the Greek population. This makes timely diagnosis more challenging, yet absolutely essential given the availability of therapies that can alter the long-term course of the disease.

HINT1-related neuropathy in Greek patients with Charcot-Marie-Tooth disease.

Autosomal recessive axonal neuropathy with neuromyotonia (ARAN-NM) is a rare hereditary neuropathy within the Charcot-Marie-Tooth disease (CMT) spectrum, linked to mutations in the histidine triad nucleotide-binding protein 1 (HINT1) gene. HINT1-related neuropathy is particularly common in selected populations from Central and Eastern Europe but rare in Western European cohorts. It has not been investigated to date in the Greek population. We presently investigated the frequency of HINT1-neuropathy in a selected cohort of 42 Greek index patients with autosomal recessive or sporadic axonal hereditary neuropathy according to standard molecular genetics procedures. We identified 4 patients with biallelic mutations in HINT1, comprising 9.5% of all cases and 44.4% of cases also displaying neuromyotonia. The c.110G> C (p.Arg37Pro) HINT1 mutation was present in all cases (2 homozygous) and the c.250T> C (p.Cys84Arg) in 2 cases (compound heterozygous). HINT1-related neuropathy patients were characterized by early onset and neuromyotonia. Two patients had noteworthy clinical features, one case developing myoclonic epilepsy and the other displaying "adducted thumbs." We conclude that HINT1-related neuropathy is common in selected Greek patients with hereditary neuropathy within the CMT spectrum, in accordance with some, but not all, European populations.

Elevated Serum α-Synuclein Levels in Huntington's Disease Patients.

Recent evidence suggests a potential role for mixed proteinopathies in the development of clinical manifestations in patients with Huntington's disease (HD). A possible cross-talk between mutant huntingtin and α-synuclein aggregates has been postulated. Serum α-synuclein has been evaluated as a potential biomarker in patients with Parkinson's disease (PD). We presently sought to investigate serum α-synuclein levels in 38 HD patients (34 symptomatic and 4 premanifest) and compare them to 36 controls. We found that α-synuclein was elevated in HD patients vs. controls (2.49 ±â€¯1.47 vs. 1.40 ±â€¯1.16, p = 0.001). There was no difference in α-synuclein levels between symptomatic vs. premanifest HD, nor between HD patients receiving medication vs. treatment-naïve. Furthermore, α-synuclein levels showed no correlation with CAG2, Unified HD Rating Scale (UHDRS) motor score, age, disease duration or disease burden score. Our results provide evidence for elevated serum α-synuclein in HD and lend support to further investigating the role of α-synuclein in this disorder.

Evidence for Cognitive Deficits in X-Linked Charcot-Marie-Tooth Disease.

OBJECTIVE: X-linked Charcot-Marie-Tooth disease (CMTX) is an hereditary neuropathy caused by mutations in GJB1 coding for connexin-32, found in Schwann cells, but also expressed in oligodendrocytes. Reports have identified CNS involvement in CMTX, but no systematic study of cognitive function has been published. METHODS: We assessed 24 CMTX patients (13 males; 9GJB1 mutations) with a comprehensive neuropsychological battery, including tests of memory, language, and executive functions. RESULTS: No differences in cognitive performance were observed between males and females. A case-by-case investigation revealed selective deficits in individual patients. One subgroup (29%) demonstrated executive abnormalities; and a non-overlapping subgroup (29%), prominent reading (decoding) abnormalities. CONCLUSIONS: The present data provide evidence for cognitive deficits in CMTX. Emerging neuropsychological patterns are also discussed.

Mutational screening of the SH3TC2 gene in Greek patients with suspected demyelinating recessive Charcot-Marie-Tooth disease reveals a varied and unusual phenotypic spectrum.

Charcot-Marie-Tooth disease type 4 C (CMT4C) is an autosomal recessive form of demyelinating peripheral neuropathy caused by mutations in SH3TC2, characterized by early onset, spine deformities, and cranial nerve involvement. We screened SH3TC2 in 50 unrelated Greek patients with suspected demyelinating Charcot-Marie-Tooth disease and pedigree compatible with recessive inheritance. All patients had been previously screened for PMP22, GJB1, and MPZ mutations. We found five previously identified pathogenic mutations in SH3TC2 distributed among 13 patients in homozygosity or compound heterozygosity (p. Arg954Stop, Arg1109Stop, Gln892Stop, Ala878Asp, and Arg648Trp). Although most cases had early onset and spine deformities were almost omnipresent, a wide phenotypic spectrum was observed. Particularly notable were two siblings with Roussy-Lévy syndrome and one patient with young-onset trigeminal neuralgia. In conclusion, mutations in SH3TC2 are responsible for 26% of Greek patients with suspected CMT4, identifying CMT4C as the most common recessive demyelinating neuropathy in the Greek population, in accordance with other Mediterranean cohorts.

X linked Charcot-Marie-Tooth disease and multiple sclerosis: emerging evidence for an association.

OBJECTIVE: X linked Charcot-Marie-Tooth disease (CMTX) is a hereditary neuropathy caused by mutations in GJB1 coding for connexin-32, a gap junction protein expressed in Schwann cells, but also found in oligodendrocytes. Four patients with CMTX developing central nervous system (CNS) demyelination compatible with multiple sclerosis (MS) have been individually published. We presently sought to systematically investigate the relationship between CMTX and MS. METHODS: Over 20 years, 70 consecutive patients (36 men) with GJB1 mutations were identified at our Neurogenetics Unit, Athens, Greece, and assessed for clinical features suggestive of MS. Additionally, 18 patients with CMTX without CNS symptoms and 18 matched controls underwent brain MRI to investigate incidental findings. Serum from patients with CMTX and MS was tested for CNS immunoreactivity. RESULTS: We identified three patients with CMTX who developed clinical features suggestive of inflammatory CNS demyelination fulfilling MS diagnostic criteria. The resulting 20-year MS incidence (4.3%) differed significantly from the highest background 20-year MS incidence ever reported from Greece (p=0.00039). The search for incidental brain MRI findings identified two CMTX cases (11%) with lesions suggestive of focal demyelination compared with 0 control. Moreover, 10 cases in the CMTX cohort had hyperintensity in the splenium of the corpus callosum compared with 0 control (p=0.0002). No specific CNS-reactive humoral factors were identified in patients with CMTX and MS. CONCLUSIONS: We have demonstrated a higher than expected frequency of MS in patients with CMTX and identified incidental focal demyelinating lesions on brain MRI in patients with CMTX without CNS symptoms. This provides circumstantial evidence for GJB1 mutations acting as a possible MS risk factor.

Replication study of GWAS risk loci in Greek multiple sclerosis patients.

OBJECTIVES: To validate in an ethnically homogeneous Greek multiple sclerosis (MS) cohort, genetic risk factors for the disease, identified through a number of previous multi-ethnic genome-wide association studies (GWAS). METHODS: A total of 1228 MS cases and 1014 controls were recruited in the study, from 3 MS centers in Greece. We genotyped 35 susceptibility SNPs that emerged from previous GWAS or meta-analyses of GWAS. Allele and genotype single locus regression analysis, adjusted for gender and site, was performed. Permutation testing was applied to all analyses. RESULTS: Six polymorphisms reached statistical significance (permutation p value < 0.05). In particular, rs2760524 of LOC105371664, near RGS1 (permutation p value 0.001), rs3129889 of HLA-DRA, near HLA-DRB1 (permutation p value < 1.00e-04), rs1738074 of TAGAP (permutation p value 0.007), rs703842 of METTL1/CYP27B1 (permutation p value 0.008), rs9596270 of DLEU1 (permutation p value < 1.00e-04), and rs17445836 of LincRNA, near IRF8 (permutation p value 0.001) were identified as susceptibility risk factors in our group. CONCLUSION: The current study replicated a number of GWAS susceptibility SNPs, which implies that some similarities between the examined Greek population and the MS genetic architecture of the GWAS populations do exist.

The different faces of the p. A53T alpha-synuclein mutation: A screening of Greek patients with parkinsonism and/or dementia.

BACKGROUND: The p. A53T mutation in the alpha-synuclein (SNCA) gene is a rare cause of autosomal dominant Parkinson's disease (PD). Although generally rare, it is particularly common in the Greek population due to a founder effect. A53T-positive PD patients often develop dementia during disease course and may very rarely present with dementia. METHODS: We screened for the p. A53T SNCA mutation a total of 347 cases of Greek origin with parkinsonism and/or dementia, collected over 15 years at the Neurogenetics Unit, Eginition Hospital, University of Athens. Cases were classified into: "pure parkinsonism", "pure dementia" and "parkinsonism plus dementia". RESULTS: In total, 4 p. A53T SNCA mutation carriers were identified. All had autosomal dominant family history and early onset. Screening of the "pure parkinsonism" category revealed 2 cases with typical PD. The other two mutation carriers were identified in the "parkinsonism plus dementia" category. One had a diagnosis of PD dementia and the other of behavioral variant frontotemporal dementia. Screening of patients with "pure dementia" failed to identify any further A53T-positive cases. CONCLUSIONS: Our results confirm that the p. A53T SNCA mutation is relatively common in Greek patients with PD or PD plus dementia, particularly in cases with early onset and/or autosomal dominant family history.

Screening for the C9ORF72 repeat expansion in a greek frontotemporal dementia cohort.

The C9orf72 repeat expansion is a common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) in European populations. A previous study has reported a high frequency of the expansion in Greek ALS. However, no data have been reported on the frequency of the expansion in Greek FTD. Currently, we investigated the frequency of the C9orfF72 expansion in a well-characterized cohort of 64 Greek FTD patients. We detected the C9orf72 repeat expansion in 9.3% of cases. Overall, 27.7% of familial and 2.2% of sporadic cases were expansion-positive. Five out of 6 cases had a diagnosis of behavioral variant FTD. All expansion-positive cases had fairly typical FTD presentations. Clinical features included motor neuron disease, Parkinsonism and hallucinations. We conclude that the overall frequency of C9orf72-positive cases in Greek FTD is high, comparable to Greek ALS, similar to some Western European, but significantly higher than some Mediterranean FTD populations.

DNA repair pathways underlie a common genetic mechanism modulating onset in polyglutamine diseases.

OBJECTIVE: The polyglutamine diseases, including Huntington's disease (HD) and multiple spinocerebellar ataxias (SCAs), are among the commonest hereditary neurodegenerative diseases. They are caused by expanded CAG tracts, encoding glutamine, in different genes. Longer CAG repeat tracts are associated with earlier ages at onset, but this does not account for all of the difference, and the existence of additional genetic modifying factors has been suggested in these diseases. A recent genome-wide association study (GWAS) in HD found association between age at onset and genetic variants in DNA repair pathways, and we therefore tested whether the modifying effects of variants in DNA repair genes have wider effects in the polyglutamine diseases. METHODS: We assembled an independent cohort of 1,462 subjects with HD and polyglutamine SCAs, and genotyped single-nucleotide polymorphisms (SNPs) selected from the most significant hits in the HD study. RESULTS: In the analysis of DNA repair genes as a group, we found the most significant association with age at onset when grouping all polyglutamine diseases (HD+SCAs; p = 1.43 × 10(-5) ). In individual SNP analysis, we found significant associations for rs3512 in FAN1 with HD+SCAs (p = 1.52 × 10(-5) ) and all SCAs (p = 2.22 × 10(-4) ) and rs1805323 in PMS2 with HD+SCAs (p = 3.14 × 10(-5) ), all in the same direction as in the HD GWAS. INTERPRETATION: We show that DNA repair genes significantly modify age at onset in HD and SCAs, suggesting a common pathogenic mechanism, which could operate through the observed somatic expansion of repeats that can be modulated by genetic manipulation of DNA repair in disease models. This offers novel therapeutic opportunities in multiple diseases. Ann Neurol 2016;79:983-990.

Hereditary spastic paraplegia in Greece: characterisation of a previously unexplored population using next-generation sequencing.

Hereditary Spastic Paraplegia (HSP) is a syndrome characterised by lower limb spasticity, occurring alone or in association with other neurological manifestations, such as cognitive impairment, seizures, ataxia or neuropathy. HSP occurs worldwide, with different populations having different frequencies of causative genes. The Greek population has not yet been characterised. The purpose of this study was to describe the clinical presentation and molecular epidemiology of the largest cohort of HSP in Greece, comprising 54 patients from 40 families. We used a targeted next-generation sequencing (NGS) approach to genetically assess a proband from each family. We made a genetic diagnosis in >50% of cases and identified 11 novel variants. Variants in SPAST and KIF5A were the most common causes of autosomal dominant HSP, whereas SPG11 and CYP7B1 were the most common cause of autosomal recessive HSP. We identified a novel variant in SPG11, which led to disease with later onset and may be unique to the Greek population and report the first nonsense mutation in KIF5A. Interestingly, the frequency of HSP mutations in the Greek population, which is relatively isolated, was very similar to other European populations. We confirm that NGS approaches are an efficient diagnostic tool and should be employed early in the assessment of HSP patients.

A novel ABCD1 mutation detected by next generation sequencing in presumed hereditary spastic paraplegia: A 30-year diagnostic delay caused by misleading biochemical findings.

OBJECTIVES: To present a Greek family in which 5 male and 2 female members developed progressive spastic paraplegia. Plasma very long chain fatty acids (VLCFA) were reportedly normal at first testing in an affected male and for over 30 years the presumed diagnosis was hereditary spastic paraplegia (HSP). Targeted next generation sequencing (NGS) was used as a further diagnostic tool. METHODS: Targeted exome sequencing in the proband, followed by Sanger sequencing confirmation; mutation segregation testing in multiple family members and plasma VLCFA measurement in the proband. RESULTS: NGS of the proband revealed a novel frameshift mutation in ABCD1 (c.1174_1178del, p.Leu392Serfs*7), bringing an end to diagnostic uncertainty by establishing the diagnosis of adrenomyeloneuropathy (AMN), the myelopathic phenotype of X-linked adrenoleukodystrophy (ALD). The mutation segregated in all family members and the diagnosis of AMN/ALD was confirmed by plasma VLCFA measurement. Confounding factors that delayed the diagnosis are presented. CONCLUSIONS: This report highlights the diagnostic utility of NGS in patients with undiagnosed spastic paraplegia, establishing a molecular diagnosis of AMN, allowing proper genetic counseling and management, and overcoming the diagnostic delay that can be rarely caused by false negative VLCFA analysis.