Molecularly confirmed pontocerebellar hypoplasia in a large family from Slovakia with four severely affected children.

BACKGROUND: Pontocerebellar hypoplasia type 1 (PCH1) is characterized by a central and peripheral motor dysfunction associated with anterior horn cell degeneration, similar to spinal muscular atrophy (SMA). OBJECTIVES: We analysed three probands (later discovered to be siblings) suspected to have severe SMA, however, not confirmed by genetic test. METHODS: Clinical-exome analysis (Illumina) was performed to identify causative variants, followed by Sanger sequencing confirmation in probands and other 10 family members. RESULTS: Homozygous pathogenic variant c.92G>C (p.(Gly31Ala)) in the Exosome Component 3 (EXOSC3) gene was found in all 3 probands, thus confirming the diagnosis of a severe form of PCH1B. The parents and six siblings were carriers, while one sibling was homozygous for a reference allele. This variant is frequent in the Czech Roma population, where it is considered a founder mutation. Haplotype analysis in this largest reported PCH1B family showed that our patients inherited from their father (of Roma origin) a haplotype identical to that found in the Czech Roma population, thus indicating these alleles have a common origin. CONCLUSION: This EXOSC3 variant is rare among the general population but most likely frequent also among Roma people in Slovakia. PCH1B should be considered for a differential diagnosis in infants manifesting SMA-like phenotype, especially if of Roma origin (Tab. 1, Fig. 1, Ref. 22). Text in PDF www.elis.sk Keywords: pontocerebellar hypoplasia, PCH1B, EXOSC3, SMA plus syndromes, pathogenic sequence variant.

A common founder effect of the splice site variant c.-23 + 1G > A in GJB2 gene causing autosomal recessive deafness 1A (DFNB1A) in Eurasia.

Mutations in the GJB2 gene are known to be a major cause of autosomal recessive deafness 1A (OMIM 220290). The most common pathogenic variants of the GJB2 gene have a high ethno-geographic specificity in their distribution, being attributed to a founder effect related to the Neolithic migration routes of Homo sapiens. The c.-23 + 1G > A splice site variant is frequently found among deaf patients of both Caucasian and Asian origins. It is currently unknown whether the spread of this mutation across Eurasia is a result of the founder effect or if it could have multiple local centers of origin. To determine the origin of c.-23 + 1G > A, we reconstructed haplotypes by genotyping SNPs on an Illumina OmniExpress 730 K platform of 23 deaf individuals homozygous for this variant from different populations of Eurasia. The analyses revealed the presence of common regions of homozygosity in different individual genomes in the sample. These data support the hypothesis of the common founder effect in the distribution of the c.-23 + 1G > A variant of the GJB2 gene. Based on the published data on the c.-23 + 1G > A prevalence among 16,177 deaf people and the calculation of the TMRCA of the modified f2-haplotypes carrying this variant, we reconstructed the potential migration routes of the carriers of this mutation around the world. This analysis indicates that the c.-23 + 1G > A variant in the GJB2 gene may have originated approximately 6000 years ago in the territory of the Caucasus or the Middle East then spread throughout Europe, South and Central Asia and other regions of the world.

Characterisation of Non-Pathogenic Premutation-Range Myotonic Dystrophy Type 2 Alleles.

Myotonic dystrophy type 2 (DM2) is caused by expansion of a (CCTG)n repeat in the cellular retroviral nucleic acid-binding protein (CNBP) gene. The sequence of the repeat is most commonly interrupted and is stably inherited in the general population. Although expanded alleles, premutation range and, in rare cases, also non-disease associated alleles containing uninterrupted CCTG tracts have been described, the threshold between these categories is poorly characterised. Here, we describe four families with members reporting neuromuscular complaints, in whom we identified altogether nine ambiguous CNBP alleles containing uninterrupted CCTG repeats in the range between 32 and 42 repeats. While these grey-zone alleles are most likely not pathogenic themselves, since other pathogenic mutations were identified and particular family structures did not support their pathogenic role, they were found to be unstable during intergenerational transmission. On the other hand, there was no observable general microsatellite instability in the genome of the carriers of these alleles. Our results further refine the division of CNBP CCTG repeat alleles into two major groups, i.e., interrupted and uninterrupted alleles. Both interrupted and uninterrupted alleles with up to approximately 30 CCTG repeats were shown to be generally stable during intergenerational transmission, while intergenerational as well as somatic instability seems to gradually increase in uninterrupted alleles with tract length growing above this threshold.

Clinical manifestation of CDKL5 deficiency disorder and identified mutations in a cohort of Slovak patients.

CDKL5 deficiency disorder (CDD) is an independent clinical entity associated with early-onset encephalopathy, which is often considered the type of epileptic encephalopathy with CDKL5 mutation also found in children diagnosed with early-onset seizure (Hanefeld) type of Rett syndrome, epileptic spasms, West syndrome, Lennox-Gastaut syndrome, or autism. Since early seizure onset is a prominent feature, in this study, a cohort of 54 unrelated patients consisting of 26 males and 28 females was selected for CDKL5 screening, with seizures presented before 12 months of age being the only clinical criterion. Five patients were found to have pathogenic or likely pathogenic variants in CDKL5 while 1 was found to have a variant of uncertain significance (p.L522V). Although CDKL5 variants are more frequently identified in female patients, we identified three male and three female patients (11.1 %, 6/54) in this study. Missense variant with unknown inheritance (p.L522V), de novo missense variant (p.E60 K), two de novo splicing (IVS15 + 1G > A, IVS16 + 2 T > A), and one de novo nonsense variant p.W125* were identified using Sanger sequencing. Whole exome analysis approach revealed de novo frameshift variant c.1247_1248delAG in a mosaic form in one of the males. Patient clinical features are reviewed and compared to those previously described in related literature. Variable clinical features were presented in CDKL5 positive patients characterised in this study. In addition to more common features, such as early epileptic seizures, severe intellectual disability, and gross motor impairment, inappropriate laughing/screaming spells and hypotonia appeared at the age of 1 year in all patients, regardless of the type of CDKL5 mutation or sex. All three CDKL5 positive males from our cohort were initially diagnosed with West syndrome, which suggests that the CDKL5 gene mutations are a significant cause of West syndrome phenotype, and also indicate the overlapping characteristics of these two clinical entities.

Technical and Methodological Aspects of Cell-Free Nucleic Acids Analyzes.

Analyzes of cell-free nucleic acids (cfNAs) have shown huge potential in many biomedical applications, gradually entering several fields of research and everyday clinical care. Many biological properties of cfNAs can be informative to gain deeper insights into the function of the organism, such as their different types (DNA, RNAs) and subtypes (gDNA, mtDNA, bacterial DNA, miRNAs, etc.), forms (naked or vesicle bound NAs), fragmentation profiles, sequence composition, epigenetic modifications, and many others. On the other hand, the workflows of their analyzes comprise many important steps, from sample collection, storage and transportation, through extraction and laboratory analysis, up to bioinformatic analyzes and statistical evaluations, where each of these steps has the potential to affect the outcome and informational value of the performed analyzes. There are, however, no universal or standard protocols on how to exactly proceed when analyzing different cfNAs for different applications, at least according to our best knowledge. We decided therefore to prepare an overview of the available literature and products commercialized for cfNAs processing, in an attempt to summarize the benefits and limitations of the currently available approaches, devices, consumables, and protocols, together with various factors influencing the workflow, its processes, and outcomes.

A Study among the Genotype, Functional Alternations, and Phenotype of 9 SCN1A Mutations in Epilepsy Patients.

Mutations in the voltage-gated sodium channel Nav1.1 (SCN1A) are linked to various epileptic phenotypes with different severities, however, the consequences of newly identified SCN1A variants on patient phenotype is uncertain so far. The functional impact of nine SCN1A variants, including five novel variants identified in this study, was studied using whole-cell patch-clamp recordings measurement of mutant Nav1.1 channels expressed in HEK293T mammalian cells. E78X, W384X, E1587K, and R1596C channels failed to produce measurable sodium currents, indicating complete loss of channel function. E788K and M909K variants resulted in partial loss of function by exhibiting reduced current density, depolarizing shifts of the activation and hyperpolarizing shifts of the inactivation curves, and slower recovery from inactivation. Hyperpolarizing shifts of the activation and inactivation curves were observed in D249E channels along with slower recovery from inactivation. Slower recovery from inactivation was observed in E78D and T1934I with reduced current density in T1934I channels. Various functional effects were observed with the lack of sodium current being mainly associated with severe phenotypes and milder symptoms with less damaging channel alteration. In vitro functional analysis is thus fundamental for elucidation of the molecular mechanisms of epilepsy, to guide patients' treatment, and finally indicate misdiagnosis of SCN1A related epilepsies.

Alkaptonuria: Current Perspectives.

The last 15 years have been the most fruitful in the history of research on the metabolic disorder alkaptonuria (AKU). AKU is caused by a deficiency of homogentisate dioxygenase (HGD), the enzyme involved in metabolism of tyrosine, and is characterized by the presence of dark ochronotic pigment in the connective tissue that is formed, due to high levels of circulating homogentisic acid. Almost 120 years ago, Sir Archibald Garrod used AKU to illustrate the concept of Mendelian inheritance in man. In January 2019, the phase III clinical study SONIA 2 was completed, which tested the effectiveness and safety of nitisinone in the treatment of AKU. Results were positive, and they will serve as the basis for the application for registration of nitisinone for treatment of AKU at the European Medicines Agency. Therefore, AKU might become a rare disease for which a cure will be found by 2020. We understand the natural history of the disease and the process of ochronosis much more, but at the same time there are still unanswered questions. One of them is the issue of the factors influencing the varying severity of the disease, since our recent genotype-phenotype study did not show that differences in residual homogentisic acid activity caused by the different mutations was responsible. Although nitisinone has proved to arrest the process of ochronosis, it has some unwanted effects and does not cure the disease completely. As such, enzyme replacement or gene therapy might become a new focus of AKU research, for which a novel suitable mouse model of AKU is available already. We believe that the story of AKU is also a story of effective collaboration between scientists and patients that might serve as an example for other rare diseases.

Structural and Functional Impact of Seven Missense Variants of Phenylalanine Hydroxylase.

The molecular genetics of well-characterized inherited diseases, such as phenylketonuria (PKU) and hyperphenylalaninemia (HPA) predominantly caused by mutations in the phenylalanine hydroxylase (PAH) gene, is often complicated by the identification of many novel variants, often with no obvious impact on the associated disorder. To date, more than 1100 PAH variants have been identified of which a substantial portion have unknown clinical significance. In this work, we study the functionality of seven yet uncharacterized PAH missense variants p.Asn167Tyr, p.Thr200Asn, p.Asp229Gly, p.Gly239Ala, p.Phe263Ser, p.Ala342Pro, and p.Ile406Met first identified in the Czech PKU/HPA patients. From all tested variants, three of them, namely p.Asn167Tyr, p.Thr200Asn, and p.Ile406Met, exerted residual enzymatic activity in vitro similar to wild type (WT) PAH, however, when expressed in HepG2 cells, their protein level reached a maximum of 72.1% ± 4.9%, 11.2% ± 4.2%, and 36.6% ± 7.3% compared to WT PAH, respectively. Remaining variants were null with no enzyme activity and decreased protein levels in HepG2 cells. The chaperone-like effect of applied BH4 precursor increased protein level significantly for p.Asn167Tyr, p.Asp229Gly, p.Ala342Pro, and p.Ile406Met. Taken together, our results of functional characterization in combination with in silico prediction suggest that while p.Asn167Tyr, p.Thr200Asn, and p.Ile406Met PAH variants have a mild impact on the protein, p.Asp229Gly, p.Gly239Ala, p.Phe263Ser, and p.Ala342Pro severely affect protein structure and function.

On the critical evaluation and confirmation of germline sequence variants identified using massively parallel sequencing.

Although massively parallel sequencing (MPS) is becoming common practice in both research and routine clinical care, confirmation requirements of identified DNA variants using alternative methods are still topics of debate. When evaluating variants directly from MPS data, different read depth statistics, together with specialized genotype quality scores are, therefore, of high relevance. Here we report results of our validation study performed in two different ways: 1) confirmation of MPS identified variants using Sanger sequencing; and 2) simultaneous Sanger and MPS analysis of exons of selected genes. Detailed examination of false-positive and false-negative findings revealed typical error sources connected to low read depth/coverage, incomplete reference genome, indel realignment problems, as well as microsatellite associated amplification errors leading to base miss-calling. However, all these error types were identifiable with thorough manual revision of aligned reads according to specific patterns of distributions of variants and their corresponding reads. Moreover, our results point to dependence of both basic quantitative metrics (such as total read counts, alternative allele read counts and allelic balance) together with specific genotype quality scores on the used bioinformatics pipeline, stressing thus the need for establishing of specific thresholds for these metrics in each laboratory and for each involved pipeline independently.

Detection of mitochondrial haplogroups in a small avar-slavic population from the eigth-ninth century AD.

OBJECTIVES: In the sixth century AD, Avars came to Central Europe from middle Eurasian steppes and founded a strong Empire called the Avar Khagante (568-799/803 AD) in the Pannonian basin. During the existence of this empire, they undertook many military and pugnacious campaigns. In the seventh century, they conquered the northern territory inhabited by Slavs, who were further recruited in Avar military and were commissioned with obtaining food supplies. During almost 200 years of Avar domination, a significant influence by the Avar culture (especially on the burial rite) and assimilation with indigenous population (occurrence of "East Asian"cranial features) could be noticed in this mixed area, which is supported by achaeological and anthropologcal research. Therefore we expected higher incidence of east Eurasian haplogroups (introduced by Avars) than the frequencies detected in present-day central European populations. MATERIALS AND METHODS: Mitochondrial DNA from 62 human skeletal remains excavated from the Avar-Slavic burial site Cífer-Pác (Slovakia) dated to the eighth and ninth century was analyzed by the sequencing of hypervariable region I and selected parts of coding region. Obtained haplotypes were compared with other present-day and historical populations and genetic distances were calculated using standard statistical method. RESULTS AND DISCUSSION: In total, the detection of mitochondrial haplogroups was possible in 46 individuals. Our results prooved a higher frequency of east Eurasian haplogroups in our analyzed population (6.52%) than in present-day central European populations. However, it is almost three times lower than the frequency of east Eurasian haplogroups detected in other medieval Avar populations. The statistical analysis showed a greater similarity and the lowest genetic distances between the Avar-Slavic burial site Cifer-Pac and medieval European populations than the South Siberian, East and Central Asian populations. CONCLUSION: Our results indicate that the transfer of Avar genetic variation through their mtDNA was rather weak in the analyzed mixed population.

Comprehensive genetic study of cystic fibrosis in Slovak patients in 25 years of genetic diagnostics.

INTRODUCTION: Cystic fibrosis (CF) has one of the longest histories in hereditary disease molecular diagnostics. However, identification of causative mutations in the CFTR gene is complicated by over 2000 currently identified mutations; with more still being discovered. Knowledge of mutation spectrum may improve effective routine diagnostics and is obligatory in mutation-specific treatment. OBJECTIVES: This study presents comprehensive mutation screening of the CFTR gene; with 275 unrelated, clinically confirmed and treated cystic fibrosis (CF) patients diagnosed in 25 years genetic testing in Slovakia. METHODS: Detection of the most common CFTR mutations was performed by ELUCIGENE 29 and ELUCIGENE CF EU2 kits. HRM and dHPLC mutation screening methods with subsequent Sanger sequencing were applied for minor mutation screening, and MLPA analysis for deletion/duplication detection. RESULTS: A total of 70 different mutations were identified, from which the most common mutation F508del accounted for 60.36% of all disease alleles and 8 mutations have currently been observed only in Slovak patients. Two large deletions identified on chromosomes 2 and 22 were further characterized to identify breakpoints. Based on mutation screening results and neonatal screening we estimated incidence in Slovakian newborns at approximately 1:6000-7000. CONCLUSION: In our study, we identified mutations in 98.54% of all disease chromosomes, while 86.54% were identified using ELUCIGENE kits, 0.54% by MLPA analysis and 11.46% by sequencing analysis. Knowledge of the mutation spectrum in genetically diagnosed patients improves possibilities of genetic counseling and cascade screening in the affected families and Slovak population.

Functional and structural characterisation of 5 missense mutations of the phenylalanine hydroxylase.

Phenylketonuria (PKU) and hyperphenylalaninemia (HPA) are a group of genetic disorders predominantly caused by mutations in the phenylalanine hydroxylase (PAH) gene. To date, more than 950 variants have been identified, however the pathogenic mechanism of many variants remains unknown. In this study, in silico prediction and in vitro prokaryotic and eukaryotic expression systems were used to functionally characterize five PAH missense variants (p.F233I, p.R270I, p.F331S, p.S350Y, and p.L358F) previously identified in Slovak and Czech patients. p.F233I, p.R270I, and p.S350Y were classified as deleterious mutations since they showed no specific activity in functional assay and no response to chaperone co-expression. Protein levels of these PAH variants were very low when expressed in HepG2 cells, and only p.S350Y responded to BH4 precursor overload by significant increase in PAH monomer, probably due to reduced rate of protein degradation as the result of proper protein folding. Variants p.F331S and p.L358F exerted residual enzymatic activity in vitro. While the first can be classified as probably pathogenic due to its very low protein levels in HepG2 cells, the latter is considered to be mild mutation with protein levels of approximately 17.85% compared to wt PAH. Our findings contribute to better understanding of structure and function of PAH mutated enzymes and optimal treatment of PKU patients carrying these mutations using BH4 supplementation.

Correction: A European Spectrum of Pharmacogenomic Biomarkers: Implications for Clinical Pharmacogenomics.

[This corrects the article DOI: 10.1371/journal.pone.0162866.].

The first Slovak Legius syndrome patient carrying the SPRED1 gene mutation.

Autosomal dominant disorder Legius syndrome (NF1- like syndrome) shows phenotype features that overlap with neurofibromatosis type 1 (NF1), such as CALMs, freckling, macrocephaly and learning disability. Mutation analysis provides an important tool in order to distinguish two entities that have different clinical implications. We analyzed SPRED1 gene by cDNA and/or gDNA sequencing in a cohort of 46 Slovak patients in whom previously NF1 mutation was excluded. In one case we identified a nonsense mutation c.46C>T (p.Arg16*) in exon 2 of SPRED1 gene, confirming diagnosis of Legius syndrome. This mutation was reported previously.

A European Spectrum of Pharmacogenomic Biomarkers: Implications for Clinical Pharmacogenomics.

Pharmacogenomics aims to correlate inter-individual differences of drug efficacy and/or toxicity with the underlying genetic composition, particularly in genes encoding for protein factors and enzymes involved in drug metabolism and transport. In several European populations, particularly in countries with lower income, information related to the prevalence of pharmacogenomic biomarkers is incomplete or lacking. Here, we have implemented the microattribution approach to assess the pharmacogenomic biomarkers allelic spectrum in 18 European populations, mostly from developing European countries, by analyzing 1,931 pharmacogenomics biomarkers in 231 genes. Our data show significant inter-population pharmacogenomic biomarker allele frequency differences, particularly in 7 clinically actionable pharmacogenomic biomarkers in 7 European populations, affecting drug efficacy and/or toxicity of 51 medication treatment modalities. These data also reflect on the differences observed in the prevalence of high-risk genotypes in these populations, as far as common markers in the CYP2C9, CYP2C19, CYP3A5, VKORC1, SLCO1B1 and TPMT pharmacogenes are concerned. Also, our data demonstrate notable differences in predicted genotype-based warfarin dosing among these populations. Our findings can be exploited not only to develop guidelines for medical prioritization, but most importantly to facilitate integration of pharmacogenomics and to support pre-emptive pharmacogenomic testing. This may subsequently contribute towards significant cost-savings in the overall healthcare expenditure in the participating countries, where pharmacogenomics implementation proves to be cost-effective.

Novel SCN1A variants in Dravet syndrome and evaluating a wide approach of patient selection.

Voltage-gated sodium channels are essential for generation and propagation of the action potential mainly in nerve and muscle cells. Causative variants in SCN1A gene which codes the main, pore-forming subunit of the channel expressed in central nervous system are associated predominantly with Dravet syndrome (DS), as well as with generalized epilepsy with febrile seizures plus (GEFS+) making it one of the most significant epilepsy gene. Our goal was to determine whether SCN1A screening is relevant in patients with a broad range of epileptic syndromes. 52 patients diagnosed with DS, generalized epilepsy with GEFS+ or similar types of epileptic syndromes were included. Sequencing of the protein coding parts of the gene complemented with MLPA analysis was carried out. One already described nonsense variant, four novel protein truncating variants and a deletion encompassing the whole SCN1A gene were revealed, all in heterozygous state. All identified variants were found in DS patients with 85.7% sensitivity, thus supporting the role of profound SCN1A gene variants in etiology of DS phenotype. No causative variants were identified in any of non-DS epileptic patients in our cohort, suggesting a minor, but not irrelevant role for SCN1A in patients with other types of childhood epilepsy.

The prevalence of melanocortin-4 receptor gene mutations in Slovak obese children and adolescents.

Melanocortin-4 receptor (MC4R) deficiency is the most frequent monogenic form of obesity. The contribution of MC4R mutations to the Slovak population has not been investigated as yet. We screened the coding sequence of the MC4R gene in a cohort of 210 Slovak obese children and adolescents. We identified four different mutations in four patients, giving a mutation detection rate of 0.95%. Of these, three were missense mutations previously identified and characterized by other research groups (p.R7C, p.S127L and p. R305W, respectively). One was a novel nonsense mutation p.W174* detected in a severely obese 7-year-old boy. This mutation was further analyzed in family segregation analysis and exhibited variable penetrance. Two known amino acid polymorphisms (p.V103I and p.I251L) were also identified in seven subjects of our cohort group. We also performed multifactorial statistical analysis to determine the influence of genotypes on standard biochemical blood markers. No significant influence was observed in carriers of DNA variants on tested parameters. We conclude that rare heterozygous MC4R mutations contribute to the onset of obesity only in a few cases in the Slovak population.

Twelve novel HGD gene variants identified in 99 alkaptonuria patients: focus on 'black bone disease' in Italy.

Alkaptonuria (AKU) is an autosomal recessive disorder caused by mutations in homogentisate-1,2-dioxygenase (HGD) gene leading to the deficiency of HGD enzyme activity. The DevelopAKUre project is underway to test nitisinone as a specific treatment to counteract this derangement of the phenylalanine-tyrosine catabolic pathway. We analysed DNA of 40 AKU patients enrolled for SONIA1, the first study in DevelopAKUre, and of 59 other AKU patients sent to our laboratory for molecular diagnostics. We identified 12 novel DNA variants: one was identified in patients from Brazil (c.557T>A), Slovakia (c.500C>T) and France (c.440T>C), three in patients from India (c.469+6T>C, c.650-85A>G, c.158G>A), and six in patients from Italy (c.742A>G, c.614G>A, c.1057A>C, c.752G>A, c.119A>C, c.926G>T). Thus, the total number of potential AKU-causing variants found in 380 patients reported in the HGD mutation database is now 129. Using mCSM and DUET, computational approaches based on the protein 3D structure, the novel missense variants are predicted to affect the activity of the enzyme by three mechanisms: decrease of stability of individual protomers, disruption of protomer-protomer interactions or modification of residues in the region of the active site. We also present an overview of AKU in Italy, where so far about 60 AKU cases are known and DNA analysis has been reported for 34 of them. In this rather small group, 26 different HGD variants affecting function were described, indicating rather high heterogeneity. Twelve of these variants seem to be specific for Italy.

Deregulation of energetic metabolism in the clear cell renal cell carcinoma: A multiple pathway analysis based on microarray profiling.

Clear cell renal cell carcinoma (ccRCC) is the most frequent type of kidney cancer. In order to better understand the biology of ccRCC, we accomplished the gene profiling of fresh tissue specimens from 11 patients with the renal tumors (9 ccRCCs, 1 oncocytoma and 1 renal B-lymphoma), in which the tumor-related data were compared to the paired healthy kidney tissues from the same patients. All ccRCCs exhibited a considerably elevated transcription of the gene coding for carbonic anhydrase IX (CAIX). Moreover, the ccRCC tumors consistently displayed increased expression of genes encoding the glycolytic pathway enzymes, e.g. hexokinase II (HK2) and lactate dehydrogenase A (LDHA) and a decreased expression of genes for the mitochondrial electron transport chain components, indicating an overall reprogramming of the energetic metabolism in this tumor type. This appears to be accompanied by altered expression of the genes of the pH regulating machinery, including ion and lactate transporters. Immunohistochemical staining of tumor tissue sections confirmed the increased expression of CAIX, HK2 and LDHA in ccRCC, validating the microarray data and supporting their potential as the energetic metabolism-related biomarkers of the ccRCC.

MARVELD2 (DFNB49) mutations in the hearing impaired Central European Roma population--prevalence, clinical impact and the common origin.

BACKGROUND: In the present study we aimed: 1) To establish the prevalence and clinical impact of DFNB49 mutations in deaf Roma from 2 Central European countries (Slovakia and Hungary), and 2) to analyze a possible common origin of the c.1331+2T>C mutation among Roma and Pakistani mutation carriers identified in the present and previous studies. METHODS: We sequenced 6 exons of the MARVELD2 gene in a group of 143 unrelated hearing impaired Slovak Roma patients. Simultaneously, we used RFLP to detect the c.1331+2T>C mutation in 85 Hungarian deaf Roma patients, control groups of 702 normal hearing Romanies from both countries and 375 hearing impaired Slovak Caucasians. We analyzed the haplotype using 21 SNPs spanning a 5.34Mb around the mutation c.1331+2T>C. RESULTS: One pathogenic mutation (c.1331+2T>C) was identified in 12 homozygous hearing impaired Roma patients. Allele frequency of this mutation was higher in Hungarian (10%) than in Slovak (3.85%) Roma patients. The identified common haplotype in Roma patients was defined by 18 SNP markers (3.89 Mb). Fourteen common SNPs were also shared among Pakistani and Roma homozygotes. Biallelic mutation carriers suffered from prelingual bilateral moderate to profound sensorineural hearing loss. CONCLUSIONS: We demonstrate different frequencies of the c.1331+2T>C mutation in hearing impaired Romanies from 3 Central European countries. In addition, our results provide support for the hypothesis of a possible common ancestor of the Slovak, Hungarian and Czech Roma as well as Pakistani deaf patients. Testing for the c.1331+2T>C mutation may be recommended in GJB2 negative Roma cases with early-onset sensorineural hearing loss.