Congenital hearing loss (CHL) is diagnosed in 1 – 2 newborns in 1000, genetic factors contribute to two thirds of CHL cases in industrialised countries. Mutations of the GJB2 gene located in the DFNB1 locus (13q11-12) are a major cause of CHL worldwide. The aim of this cross-sectional study was to assess the contribution of the DFNB1 locus containing the GJB2 and GJB6 genes in the development of early onset hearing loss in the affected group of participants, to determine the population-specific mutational profile and DFNB1-related HL burden in Lithuanian population. Clinical data were obtained from a collection of 158 affected participants (146 unrelated probands) with early onset non-syndromic HL. GJB2 and GJB6 gene sequencing and GJB6 gene deletion testing were performed. The data of GJB2 and GJB6 gene sequencing in 98 participants in group of self-reported healthy Lithuanian inhabitants were analysed. Statistic summary, homogeneity tests, and logistic regression analysis were used for the assessment of genotype-phenotype correlation. Our findings show 57.5 % of affected participants with two pathogenic GJB2 gene mutations identified. The most prevalent GJB2 mutations were c.35delG, p. (Gly12Valfs*2) (rs80338939) and c.313_326del14, p. (Lys105Glyfs*5) (rs111033253) with allele frequencies 64.7 % and 28.3 % respectively. GJB6 gene mutations were not identified in the affected group of participants. The statistical analysis revealed significant differences between GJB2(−) and GJB2(+) groups in disease severity (p = 0.001), and family history (p = 0.01). The probability of identification of GJB2 mutations in patients with various HL characteristics was estimated. The carrier rate of GJB2 gene mutations – 7.1 % (~1 in 14) was identified in the group of healthy participants and a high frequency of GJB2-related hearing loss was estimated in our population. The results show a very high proportion of GJB2-positive individuals in the research group affected with sensorineural HL. The allele frequency of c.35delG mutation (64.7 %) is consistent with many previously published studies in groups of affected individuals of Caucasian populations. The high frequency of the c.313_326del14 (28.3 % of pathogenic alleles) mutation in affected group of participants was an unexpected finding in our study suggesting not only a high frequency of carriers of this mutation in our population but also its possible origin in Lithuanian ancestors. The high frequency of carriers of the c.313_326del14 mutation in the entire Lithuanian population is supported by it being identified twice in the ethnic Lithuanian group of healthy participants (a frequency 2.0 % of carriers in the study group). Analysis of the allele frequency of GJB2 gene mutations revealed a high proportion of c. 313_326del14 (rs111033253) mutations in the GJB2-positive group suggesting its possible origin in Lithuanian forebears. The high frequency of carriers of GJB2 gene mutations in the group of healthy participants corresponds to the substantial frequency of GJB2-associated HL in Lithuania. The observations of the study indicate the significant contribution of GJB2 gene mutations to the pathogenesis of the disorder in the Lithuanian population and will contribute to introducing principles to predict the characteristics of the disease in patients.

CHARGE syndrome (MIM# 214800)—which is characterised by a number of congenital anomalies including coloboma, ear anomalies, deafness, facial anomalies, heart defects, atresia choanae, genital hypoplasia, growth retardation, and developmental delay—is caused by a heterozygous variant in the CHD7 (MIM# 608892) gene located on chromosome 8q12. We report the identification of a novel c.5535-1G > A variant in CHD7 and provide the evaluation of its effect on pre-mRNA splicing. In this study, we report on a female presenting features of CHARGE syndrome. A novel heterozygous CHD7 variant c.5535-1G > A located in the acceptor splice site of intron 26 was identified in the proband’s DNA sample after analysis of whole exome sequencing data. In silico predictions indicating that the variant is probably pathogenic by affecting pre-mRNA splicing were verified by genetic analysis based on reverse transcription of the patient’s RNA followed by PCR amplifications performed on synthesised cDNA and Sanger sequencing. Sanger sequencing of cDNA revealed that the c.5535-1G > A variant disrupts the original acceptor splice site and activates a cryptic splice site only one nucleotide downstream of the pathogenic variant site. This change causes the omission of the first nucleotide of exon 27, leading to a frameshift in the mRNA of the CHD7 gene. Our results suggest that the alteration induces the premature truncation of the CHD7 protein (UniProtKB: Q9P2D1), thus resulting in CHARGE syndrome. Genetic analysis of novel splice site variant underlines its importance for studying the pathogenic splicing mechanism as well as for confirming a diagnosis.

Summary Next-generation sequencing (NGS) became an effective approach for finding novel causative genomic variants of genetic disorders and is increasingly used for diagnostic purposes. Public variant databases that gather data of pathogenic variants are being relied upon as a source for clinical diagnosis. However, research of pathogenic variants using public databases data could be carried out not only in patients, but also in healthy people. This could provide insights into the most common recessive disorders in populations. The study aim was to use NGS and data from the ClinVar database for the identification of pathogenic variants in the exomes of healthy individuals from the Lithuanian population. To achieve this, 96 exomes were sequenced. An average of 42 139 single-nucleotide variants (SNVs) and 2306 short INDELs were found in each individual exome. Pooled data of study exomes provided a total of 243 192 unique SNVs and 31 623 unique short INDELs. Three hundred and twenty-one unique SNVs were classified as pathogenic. Comparison of the European data from the 1000 Genomes Project with our data revealed five pathogenic genomic variants that are inherited in an autosomal recessive pattern and that statistically significantly differ from the European population data.

Background and Objectives: Heterozygous pathogenic variants in the MED13L gene cause impaired intellectual development and distinctive facial features with or without cardiac defects (MIM #616789). This complex neurodevelopmental disorder is characterised by various phenotypic features, including plagiocephaly, strabismus, clubfoot, poor speech, and developmental delay. The aim of this study was to evaluate the clinical significance and consequences of a novel heterozygous intragenic MED13L deletion in a proband with clinical features of a MED13L-related disorder through extensive clinical, molecular, and functional characterisation. Materials and Methods: Combined comparative genomic hybridisation and single-nucleotide polymorphism array (SNP-CGH) was used to identify the changes in the proband’s gDNA sequence (DECIPHER #430183). Intragenic MED13L deletion was specified via quantitative polymerase chain reaction (qPCR) and Sanger sequencing of the proband’s cDNA sample. Western blot and bioinformatics analyses were used to investigate the consequences of this copy number variant (CNV) at the protein level. CRISPR-Cas9 technology was used for a MED13L-gene-silencing experiment in a culture of the control individual’s skin fibroblasts. After the MED13L-gene-editing experiment, subsequent functional fibroblast culture analyses were performed. Results: The analysis of the proband’s cDNA sample allowed for specifying the regions of the breakpoints and identifying the heterozygous deletion that spanned exons 3 to 10 of MED13L, which has not been reported previously. In silico, the deletion was predicted to result in a truncated protein NP_056150.1:p.(Val104Glyfs*5), partly altering the Med13_N domain and losing the MedPIWI and Med13_C domains. After MED13L gene editing was performed, reduced cell viability; an accelerated aging process; and inhibition of the RB1, E2F1, and CCNC gene expression were found to exist. Conclusions: Based on these findings, heterozygous intragenic 12q24.21 deletion in the affected individual resulted in MED13L haploinsufficiency due to the premature termination of protein translation, therefore leading to MED13L haploinsufficiency syndrome.

Every next generation sequencing (NGS) platform relies on proprietary and open source computational tools to analyze sequencing data. NGS tools for Illumina platforms are well documented which is not the case with AB SOLiD systems. We applied several computational and variant calling pipelines to analyse targeted exome sequencing data obtained using AB SOLiD 5500 system. Our investigated tools comprised proprietary LifeScope’s pipeline in combination with open source color-space competent mapping programs and a variant caller. We present instrumental details of the pipelines that were used and quantitative comparative analysis of variant lists generated by LifeScope’s pipeline versus open source tools. Sufficient coverage of targeted regions was achieved by all investigated pipelines. High variability was observed in identities of variants across the mapping programs. We observed less than 50 % concordance of variant lists produced by approaches based on different mapping algorithms. We summarized different approaches with regards to coverage (DP) and quality (QUAL) properties of the variants provided by GATK and found that LifeScope’s computational pipeline is superior. Fusion of information on mapping profiles (pileup) at genomic positions of variants in several different alignments proved to be a useful strategy to assess questionable singleton variants. We quantitatively supported a conclusion that Lifescope’s pipeline is superior for processing sequencing data obtained by AB SOLiD 5500 system. Nevertheless the use of alternative pipelines is encouraged because aggregation of information from other mapping and variant calling approaches helps to resolve questionable calls and increases the confidence of the call. It was noted that a coverage threshold for variant to be considered for further analysis has to be chosen in data-driven way to prevent a loss of important information.

Background and Objectives: Pathogenic variants of PIGN are a known cause of multiple congenital anomalies-hypotonia-seizures syndrome 1 (MCAHS1). Many affected individuals have clinical features overlapping with Fryns syndrome and are mainly characterised by developmental delay, congenital anomalies, hypotonia, seizures, and specific minor facial anomalies. This study investigates the clinical and molecular data of three individuals from two unrelated families, the clinical features of which were consistent with a diagnosis of MCAHS1. Materials and Methods: Next-generation sequencing (NGS) technology was used to identify the changes in the DNA sequence. Sanger sequencing of gDNA of probands and their parents was used for validation and segregation analysis. Bioinformatics tools were used to investigate the consequences of pathogenic or likely pathogenic PIGN variants at the protein sequence and structure level. Results: The analysis of NGS data and segregation analysis revealed a compound heterozygous NM_176787.5:c.[1942G>T];[1247_1251del] PIGN genotype in family 1 and NG_033144.1(NM_176787.5):c.[932T>G];[1674+1G>C] PIGN genotype in family 2. In silico, c.1942G>T (p.(Glu648Ter)), c.1247_1251del (p.(Glu416GlyfsTer22)), and c.1674+1G>C (p.(Glu525AspfsTer68)) variants are predicted to result in a premature termination codon that leads to truncated and functionally disrupted protein causing the phenotype of MCAHS1 in the affected individuals. Conclusions: PIGN-related disease represents a wide spectrum of phenotypic features, making clinical diagnosis inaccurate and complicated. The genetic testing of every individual with this phenotype provides new insights into the origin and development of the disease.