Exome Sequencing Identifies a Founder Frameshift Mutation in an Alternative Exon of USH1C as the Cause of Autosomal Recessive Retinitis Pigmentosa with Late-Onset Hearing Loss

We used a combined approach of homozygosity mapping and whole exome sequencing (WES) to search for the genetic cause of autosomal recessive retinitis pigmentosa (arRP) in families of Yemenite Jewish origin. Homozygosity mapping of two arRP Yemenite Jewish families revealed a few homozygous regions. A subsequent WES analysis of the two index cases revealed a shared homozygous novel nucleotide deletion (c.1220delG) leading to a frameshift (p.Gly407Glufs*56) in an alternative exon (#15) of USH1C. Screening of additional Yemenite Jewish patients revealed a total of 16 homozygous RP patients (with a carrier frequency of 0.008 in controls). Funduscopic and electroretinography findings were within the spectrum of typical RP. While other USH1C mutations usually cause Usher type I (including RP, vestibular dysfunction and congenital deafness), audiometric screening of 10 patients who are homozygous for c.1220delG revealed that patients under 40 years of age had normal hearing while older patients showed mild to severe high tone sensorineural hearing loss. This is the first report of a mutation in a known USH1 gene that causes late onset rather than congenital sensorineural hearing loss. The c.1220delG mutation of USH1C accounts for 23% of RP among Yemenite Jewish patients in our cohort.     

Exome Sequencing Reveals Novel and Recurrent Mutations with Clinical Significance in Inherited Retinal Dystrophies

This study aimed to identify the underlying molecular genetic cause in four Spanish families clinically diagnosed of Retinitis Pigmentosa (RP), comprising one autosomal dominant RP (adRP), two autosomal recessive RP (arRP) and one with two possible modes of inheritance: arRP or X-Linked RP (XLRP). We performed whole exome sequencing (WES) using NimbleGen SeqCap EZ Exome V3 sample preparation kit and SOLID 5500xl platform. All variants passing filter criteria were validated by Sanger sequencing to confirm familial segregation and the absence in local control population. This strategy allowed the detection of: (i) one novel heterozygous splice-site deletion in RHO, c.937-2_944del, (ii) one rare homozygous mutation in C2orf71, c.1795T>C; p.Cys599Arg, not previously associated with the disease, (iii) two heterozygous null mutations in ABCA4, c.2041C>T; p.R681* and c.6088C>T; p.R2030*, and (iv) one mutation, c.2405-2406delAG; p.Glu802Glyfs*31 in the ORF15 of RPGR. The molecular findings for RHO and C2orf71 confirmed the initial diagnosis of adRP and arRP, respectively, while patients with the two ABCA4 mutations, both previously associated with Stargardt disease, presented symptoms of RP with early macular involvement. Finally, the X-Linked inheritance was confirmed for the family with the RPGR mutation. This latter finding allowed the inclusion of carrier sisters in our preimplantational genetic diagnosis program.     

Next generation sequencing-based molecular diagnosis of retinitis pigmentosa: identification of a novel genotype-phenotype correlation and clinical refinements

Retinitis pigmentosa (RP) is a devastating form of retinal degeneration, with significant social and professional consequences. Molecular genetic information is invaluable for an accurate clinical diagnosis of RP due to its high genetic and clinical heterogeneity. Using a gene capture panel that covers 163 of the currently known retinal disease genes, including 48 RP genes, we performed a comprehensive molecular screening in a collection of 123 RP unsettled probands from a wide variety of ethnic backgrounds, including 113 unrelated simplex and 10 autosomal recessive RP (arRP) cases. As a result, 61 mutations were identified in 45 probands, including 38 novel pathogenic alleles. Interestingly, we observed that phenotype and genotype were not in full agreement in 21 probands. Among them, eight probands were clinically reassessed, resulting in refinement of clinical diagnoses for six of these patients. Finally, recessive mutations in CLN3 were identified in five retinal degeneration patients, including four RP probands and one cone-rod dystrophy patient, suggesting that CLN3 is a novel non-syndromic retinal disease gene. Collectively, our results underscore that, due to the high molecular and clinical heterogeneity of RP, comprehensive screening of all retinal disease genes is effective in identifying novel pathogenic mutations and provides an opportunity to discover new genotype-phenotype correlations. Information gained from this genetic screening will directly aid in patient diagnosis, prognosis, and treatment, as well as allowing appropriate family planning and counseling.     

Whole Exome Analysis Identifies Frequent CNGA1 Mutations in Japanese Population with Autosomal Recessive Retinitis Pigmentosa

Objective

The purpose of this study was to investigate frequent disease-causing gene mutations in autosomal recessive retinitis pigmentosa (arRP) in the Japanese population.

Methods

In total, 99 Japanese patients with non-syndromic and unrelated arRP or sporadic RP (spRP) were recruited in this study and ophthalmic examinations were conducted for the diagnosis of RP. Among these patients, whole exome sequencing analysis of 30 RP patients and direct sequencing screening of all CNGA1 exons of the other 69 RP patients were performed.

Results

Whole exome sequencing of 30 arRP/spRP patients identified disease-causing gene mutations of CNGA1 (four patients), EYS (three patients) and SAG (one patient) in eight patients and potential disease-causing gene variants of USH2A (two patients), EYS (one patient), TULP1 (one patient) and C2orf71 (one patient) in five patients. Screening of an additional 69 arRP/spRP patients for the CNGA1 gene mutation revealed one patient with a homozygous mutation.

Conclusions

This is the first identification of CNGA1 mutations in arRP Japanese patients. The frequency of CNGA1 gene mutation was 5.1% (5/99 patients). CNGA1 mutations are one of the most frequent arRP-causing mutations in Japanese patients.     

Mutations of 60 known causative genes in 157 families with retinitis pigmentosa based on exome sequencing

Retinitis pigmentosa (RP) is the most common and highly heterogeneous form of hereditary retinal degeneration. This study was to identify mutations in the 60 genes that were known to be associated with RP in 157 unrelated Chinese families with RP. Genomic DNA from probands was initially analyzed by whole exome sequencing. Sanger sequencing was used to confirm potential candidate variants affecting the encoded residues in the 60 genes, including heterozygous variants from genes that are related to autosomal dominant RP, homozygous or compound heterozygous variants from genes that are related to autosomal recessive RP, and hemizygous variants from genes that are related to X-linked RP. Synonymous and intronic variants were also examined to confirm whether they could affect splicing. A total of 244 candidate variants were detected by exome sequencing. Sanger sequencing confirmed 240 variants out of the 244 candidates. Informatics and segregation analyses suggested 110 potential pathogenic mutations in 28 out of the 60 genes involving 79 of the 157 (50 %) families, including 31 (39 %, 31/79) families with heterozygous mutations in autosomal dominant genes, 37 (47 %, 37/79) families with homozygous (9) or compound heterozygous (28) mutations in autosomal recessive genes, and 11 (14 %, 11/79) families with hemizygous mutations in X-linked genes. Of the 110 identified variants, 74 (67 %) were novel. The genetic defects in approximately half of the 157 studies families were detected by exome sequencing. A comprehensive analysis of the 60 known genes not only expanded the mutation spectrum and frequency of the 60 genes in Chinese patients with RP, but also provided an overview of the molecular etiology of RP in Chinese patients. The analysis of the known genes also supplied the foundation and clues for discovering novel causative RP genes.     

Exome sequencing identifies RDH12 compound heterozygous mutations in a family with severe retinitis pigmentosa

Objective

Retinitis pigmentosa (RP) is the most prevalent type of inherited retinal degeneration and one of the commonest causes of genetically determined visual dysfunction worldwide. To date, approximately 35 genes have been associated with nonsyndromic autosomal recessive RP (arRP), however the small contribution of each gene to the total prevalence of arRP and the lack of a clear genotype–phenotype correlation complicate the genetic analysis in affected patients. Next generation sequencing technologies are powerful and cost-effective methods for detecting causative mutations in both sporadic and familial RP cases.

Methods

A Mexican family with 5 members affected from arRP was studied. All patients underwent a complete ophthalmologic examination. Molecular methods included genome-wide SNP homozygosity mapping, exome sequencing analysis, and Sanger-sequencing confirmation of causal mutations.

Results

No regions of shared homozygosity among affected subjects were identified. Exome sequencing in a single patient allowed the detection of two missense mutations in the RDH12 gene: a c.446T>C transition predicting a novel p.L149P substitution, and a c.295C>A transversion predicting a previously reported p.L99I replacement. Sanger sequencing confirmed that all affected subjects carried both RDH12 mutations.

Conclusions

This study adds to the molecular spectrum of RDH12-related retinopathy and offers an additional example of the power of exome sequencing in the diagnosis of recessively inherited retinal degenerations.     

The diagnostic application of targeted re-sequencing in Korean patients with retinitis pigmentosa

Background

Identification of the causative genes of retinitis pigmentosa (RP) is important for the clinical care of patients with RP. However, a comprehensive genetic study has not been performed in Korean RP patients. Moreover, the genetic heterogeneity found in sensorineural genetic disorders makes identification of pathogenic mutations challenging. Therefore, high throughput genetic testing using massively parallel sequencing is needed.

Results

Sixty-two Korean patients with nonsyndromic RP (46 patients from 18 families and 16 simplex cases) who consented to molecular genetic testing were recruited in this study and targeted exome sequencing was applied on 53 RP-related genes. Causal variants were characterised by selecting exonic and splicing variants, selecting variants with low allele frequency (below 1 %), and discarding the remaining variants with quality below 20. The variants were additionally confirmed by an inheritance pattern and cosegregation test of the families, and the rest of the variants were prioritised using in-silico prediction tools. Finally, causal variants were detected from 10 of 18 familial cases (55.5 %) and 7 of 16 simplex cases (43.7 %) in total. Novel variants were detected in 13 of 20 (65 %) candidate variants. Compound heterozygous variants were found in four of 7 simplex cases.

Conclusion

Panel-based targeted re-sequencing can be used as an effective molecular diagnostic tool for RP.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1723-x) contains supplementary material, which is available to authorized users.     

A major locus for autosomal recessive retinitis pigmentosa on 6q, determined by homozygosity mapping of chromosomal regions that contain gamma-aminobutyric acid-receptor clusters.

Retinitis pigmentosa (RP) is the most common inherited retinal dystrophy, with extensive allelic and nonallelic genetic heterogeneity. Autosomal recessive RP (arRP) is the most common form of RP worldwide, with at least nine loci known and accountable for approximately 10%-15% of all cases. Gamma-aminobutyric acid (GABA) is the major inhibitory transmitter in the CNS. Different GABA receptors are expressed in all retinal layers, and inhibition mediated by GABA receptors in the human retina could be related to RP. We have selected chromosomal regions containing genes that encode the different subunits of the GABA receptors, for homozygosity mapping in inbred families affected by arRP. We identify a new locus for arRP, on chromosome 6, between markers D6S257 and D6S1644. Our data suggest that 10%-20% of Spanish families affected by typical arRP could have linkage to this new locus. This region contains subunits GABRR1 and GABRR2 of the GABA-C receptor, which is the effector of lateral inhibition at the retina.     

Two novel mutations in the EYS gene are possible major causes of autosomal recessive retinitis pigmentosa in the Japanese population

Retinitis pigmentosa (RP) is a highly heterogeneous genetic disease including autosomal recessive (ar), autosomal dominant (ad), and X-linked inheritance. Recently, arRP has been associated with mutations in EYS (Eyes shut homolog), which is a major causative gene for this disease. This study was conducted to determine the spectrum and frequency of EYS mutations in 100 Japanese arRP patients. To determine the prevalence of EYS mutations, all EYS exons were screened for mutations by polymerase chain reaction amplification, and sequence analysis was performed. We detected 67 sequence alterations in EYS, of which 21 were novel. Of these, 7 were very likely pathogenic mutations, 6 were possible pathogenic mutations, and 54 were predicted non-pathogenic sequence alterations. The minimum observed prevalence of distinct EYS mutations in our study was 18% (18/100, comprising 9 patients with 2 very likely pathogenic mutations and the remaining 9 with only one such mutation). Among these mutations, 2 novel truncating mutations, c.4957_4958insA (p.S1653KfsX2) and c.8868C>A (p.Y2956X), were identified in 16 patients and accounted for 57.1% (20/35 alleles) of the mutated alleles. Although these 2 truncating mutations were not detected in Japanese patients with adRP or Leber's congenital amaurosis, we detected them in Korean arRP patients. Similar to Japanese arRP results, the c.4957_4958insA mutation was more frequently detected than the c.8868C>A mutation. The 18% estimated prevalence of very likely pathogenic mutations in our study suggests a major involvement of EYS in the pathogenesis of arRP in the Japanese population. Mutation spectrum of EYS in 100 Japanese patients, including 13 distinct very likely and possible pathogenic mutations, was largely different from the previously reported spectrum in patients from non-Asian populations. Screening for c.4957_4958insA and c.8868C>A mutations in the EYS gene may therefore be very effective for the genetic testing and counseling of RP patients in Japan.     

Mutation of CERKL, a novel human ceramide kinase gene, causes autosomal recessive retinitis pigmentosa (RP26)

Retinitis pigmentosa (RP), the main cause of adult blindness, is a genetically heterogeneous disorder characterized by progressive loss of photoreceptors through apoptosis. Up to now, 39 genes and loci have been implicated in nonsyndromic RP, yet the genetic bases of >50% of the cases, particularly of the recessive forms, remain unknown. Previous linkage analysis in a Spanish consanguineous family allowed us to define a novel autosomal recessive RP (arRP) locus, RP26, within an 11-cM interval (17.4 Mb) on 2q31.2-q32.3. In the present study, we further refine the RP26 locus down to 2.5 Mb, by microsatellite and single-nucleotide polymorphism (SNP) homozygosity mapping. After unsuccessful mutational analysis of the nine genes initially reported in this region, a detailed gene search based on expressed-sequence-tag data was undertaken. We finally identified a novel gene encoding a ceramide kinase (CERKL), which encompassed 13 exons. All of the patients from the RP26 family bear a homozygous mutation in exon 5, which generates a premature termination codon. The same mutation was also characterized in another, unrelated, Spanish pedigree with arRP. Human CERKL is expressed in the retina, among other adult and fetal tissues. A more detailed analysis by in situ hybridization on adult murine retina sections shows expression of Cerkl in the ganglion cell layer. Ceramide kinases convert the sphingolipid metabolite ceramide into ceramide-1-phosphate, both key mediators of cellular apoptosis and survival. Ceramide metabolism plays an essential role in the viability of neuronal cells, the membranes of which are particularly rich in sphingolipids. Therefore, CERKL deficiency could shift the relative levels of the signaling sphingolipid metabolites and increase sensitivity of photoreceptor and other retinal cells to apoptotic stimuli. This is the first genetic report suggesting a direct link between retinal neurodegeneration in RP and sphingolipid-mediated apoptosis.     

Exome-Based Mapping and Variant Prioritization for Inherited Mendelian Disorders

Exome sequencing in families affected by rare genetic disorders has the potential to rapidly identify new disease genes (genes in which mutations cause disease), but the identification of a single causal mutation among thousands of variants remains a significant challenge. We developed a scoring algorithm to prioritize potential causal variants within a family according to segregation with the phenotype, population frequency, predicted effect, and gene expression in the tissue(s) of interest. To narrow the search space in families with multiple affected individuals, we also developed two complementary approaches to exome-based mapping of autosomal-dominant disorders. One approach identifies segments of maximum identity by descent among affected individuals; the other nominates regions on the basis of shared rare variants and the absence of homozygous differences between affected individuals. We showcase our methods by using exome sequence data from families affected by autosomal-dominant retinitis pigmentosa (adRP), a rare disorder characterized by night blindness and progressive vision loss. We performed exome capture and sequencing on 91 samples representing 24 families affected by probable adRP but lacking common disease-causing mutations. Eight of 24 families (33%) were revealed to harbor high-scoring, most likely pathogenic (by clinical assessment) mutations affecting known RP genes. Analysis of the remaining 17 families identified candidate variants in a number of interesting genes, some of which have withstood further segregation testing in extended pedigrees. To empower the search for Mendelian-disease genes in family-based sequencing studies, we implemented them in a cross-platform-compatible software package, MendelScan, which is freely available to the research community.     

A novel mutation in <Emphasis Type="Italic">RDH5</Emphasis> gene causes retinitis pigmentosa in consanguineous Pakistani family

Retinitis pigmentosa (RP) is the most frequent genetically and clinically heterogeneous inherited retinal degeneration. To date, more than 80 genes have been identified that cause autosomal dominant, autosomal recessive and X linked RP. However, locus and allelic heterogeneity of RP has not been fully captured yet. This heterogeneity and lack of an accurate genotype phenotype correlation makes molecular dissection of the disease more difficult. The present study was designed to characterize the underlying pathogenic variants of RP in Pakistan. For this purpose, a large consanguineous family with RP phenotype showing autosomal recessive mode of inheritance was selected after a complete ophthalmological examination. Next generation sequencing was used for the identification of molecular determinant followed by Sanger-sequencing for confirmation. After sequence analysis a novel homozygous missense mutation, (c.602 C?>?T) in exon 4 of the RDH5 gene (MIM: 601617) was identified. This mutation resulted in substitution of phenyl alanine for serine at amino acid 201 (p.Ser201Phe) of the RDH5 gene. The same mutation was not detected in the 200 ethnically-matched control samples by Sanger sequencing. The identified mutant allele segregated in homozygous fashion in all the affected individuals of pedigree. Identification of this mutation reveals the allelic heterogeneity of RDH5 in patients with RP phenotype. The findings of this study demonstrate the clinical significance of next generation sequencing to understand the molecular basis of diseases and would help to reveal new proteins and their function in visual cycle will pave the way for early diagnosis, genetic counseling and better therapeutic inventions.     

Severe autosomal recessive retinitis pigmentosa maps to chromosome 1p13.3–p21.2 between D1S2896 and D1S457 but outside ABCA4

A severe form of autosomal recessive retinitis pigmentosa (arRP) was identified in a large Pakistani family ascertained in the Punjab province of Pakistan. All affected individuals in the family had night blindness in early childhood, early complete loss of useful vision, and typical RP fundus changes plus macular degeneration. After exclusion of known arRP loci, a genome-wide scan was performed using microsatellite markers at about 10 cM intervals and calculating two-point lod scores. PCR cycle dideoxynucleotide sequencing was used to sequence candidate genes inside the linked region for mutations. RP in this family shows linkage to markers in a 10.5 cM (8.9 Mbp) region of chromosome 1p13.3-p21.2 between D1S2896 and D1S457. D1S485 yields the highest lod score of 6.54 at theta=0. Sequencing the exons and intron-exon boundaries of five candidate genes and six ESTs in this region, OLFM3, GNAI3, LOC126987, FLJ25070, DKFZp586G0123, AV729694, BU662869, BU656110, BU171991, BQ953690, and CA397743, did not identify any causative mutations. This novel locus lies approximately 4.9 cM (7.1 Mbp) from ABCA4, which is excluded from the linked region. Identification and study of this gene may help to elucidate the phenotypic diversity of arRP mapping to this region.     

Whole-Exome Sequencing to Decipher the Genetic Heterogeneity of Hearing Loss in a Chinese Family with Deaf by Deaf Mating

Inherited deafness has been shown to have high genetic heterogeneity. For many decades, linkage analysis and candidate gene approaches have been the main tools to elucidate the genetics of hearing loss. However, this associated study design is costly, time-consuming, and unsuitable for small families. This is mainly due to the inadequate numbers of available affected individuals, locus heterogeneity, and assortative mating. Exome sequencing has now become technically feasible and a cost-effective method for detection of disease variants underlying Mendelian disorders due to the recent advances in next-generation sequencing (NGS) technologies. In the present study, we have combined both the Deafness Gene Mutation Detection Array and exome sequencing to identify deafness causative variants in a large Chinese composite family with deaf by deaf mating. The simultaneous screening of the 9 common deafness mutations using the allele-specific PCR based universal array, resulted in the identification of the 1555A>G in the mitochondrial DNA (mtDNA) 12S rRNA in affected individuals in one branch of the family. We then subjected the mutation-negative cases to exome sequencing and identified novel causative variants in the MYH14 and WFS1 genes. This report confirms the effective use of a NGS technique to detect pathogenic mutations in affected individuals who were not candidates for classical genetic studies.     

Genome arrays for the detection of copy number variations in idiopathic mental retardation, idiopathic generalized epilepsy and neuropsychiatric disorders: lessons for diagnostic workflow and research

We review the contributions and limitations of genome-wide array-based identification of copy number variants (CNVs) in the clinical diagnostic evaluation of patients with mental retardation (MR) and other brain-related disorders. In unselected MR referrals a causative genomic gain or loss is detected in 14-18% of cases. Usually, such CNVs arise de novo, are not found in healthy subjects, and have a major impact on the phenotype by altering the dosage of multiple genes. This high diagnostic yield justifies array-based segmental aneuploidy screening as the initial genetic test in these patients. This also pertains to patients with autism (expected yield about 5-10% in nonsyndromic and 10-20% in syndromic patients) and schizophrenia (at least 5% yield). CNV studies in idiopathic generalized epilepsy, attention-deficit hyperactivity disorder, major depressive disorder and Tourette syndrome indicate that patients have, on average, a larger CNV burden as compared to controls. Collectively, the CNV studies suggest that a wide spectrum of disease-susceptibility variants exists, most of which are rare (<0.1%) and of variable and usually small effect. Notwithstanding, a rare CNV can have a major impact on the phenotype. Exome sequencing in MR and autism patients revealed de novo mutations in protein coding genes in 60 and 20% of cases, respectively. Therefore, it is likely that arrays will be supplanted by next-generation sequencing methods as the initial and perhaps ultimate diagnostic tool in patients with brain-related disorders, revealing both CNVs and mutations in a single test.     

Homozygosity Mapping of Autosomal Recessive Retinitis Pigmentosa Locus (RP22) on Chromosome 16p12.1–p12.3

Autosomal recessive retinitis pigmentosa (arRP) is a genetically and clinically heterogeneous and progressive degenerative disorder of the retina, leading usually to severe visual handicap in adulthood. To date, disease loci/genes have been mapped/identified only in a minority of cases. DNA samples were collected from 20 large consanguineous Indian families, in which arRP segregated and that were suitable for homozygosity mapping of the disease locus. After excluding linkage to all known arRP loci, a genome-wide scan was initiated. In two families, homozygosity mapping, haplotype analysis, and linkage data mapped the disease locus (RP22) in an approximately 16-cM region between D16S287 and D16S420 on the proximal short arm of chromosome 16. No mutation has been found by direct sequencing in the gene (CRYM) encoding μ crystallin, which maps in the critical region.     

Analysis of pathogenic variants in 605 Chinese children with non-syndromic cardiac conotruncal defects based on targeted sequencing

ObjectiveDeleterious genetic variants comprise one cause of cardiac conotruncal defects (CTDs). Genes associated with CTDs are gradually being identified. In the present study, we aimed to explore the profile of genetic variants of CTD-associated genes in Chinese patients with non-syndromic CTDs.MethodsThirty-nine CTD-related genes were selected after reviewing published articles in NCBI, HGMD, OMIM, and HPO. In total, 605 patients with non-syndromic CTDs and 300 healthy controls, all of Han ethnicity, were recruited. High-throughput targeted sequencing was used to detect genetic variants in the protein-coding regions of genes. We performed rigorous variant-level filtrations to identify potentially damaging variants (Dvars) using prediction programs including CADD, SIFT, PolyPhen-2, and MutationTaster.ResultDvars were detected in 66.7% (26/39) of the targeted CTD-associated genes. In total, 11.07% (67/605) of patients with non-syndromic CTDs were found to carry one or more Dvars in targeted CTD-associated genes. Dvars in FOXH1, TBX2, NFATC1, FOXC2, and FOXC1 were common in the CTD cohort (1.5% [9/605], 1.2% [7/605], 1.2% [7/605], 1% [6/605], and 0.5% [3/605], respectively).ConclusionTargeted exon sequencing is a cost-effective approach for the genetic diagnosis of CTDs. Our findings contribute to an understanding of the genetic architecture of non-syndromic CTDs.     

Targeted massive parallel sequencing: the effective detection of novel causative mutations associated with hearing loss in small families

ABSTRACT: BACKGROUND: Hereditary hearing loss is one of the most common heterogeneous disorders, and genetic variants that can cause hearing loss have been identified in over fifty genes. Most of these hearing loss genes have been detected using classical genetic methods, typically starting with linkage analysis in large families with hereditary hearing loss. However, these classical strategies are not well suited for mutation analysis in smaller families who have insufficient genetic information. METHODS: Eighty known hearing loss genes were selected and simultaneously sequenced by targeted next-generation sequencing (NGS) in 8 Korean families with autosomal dominant non-syndromic sensorineural hearing loss. RESULTS: Five mutations in known hearing loss genes, including 1 nonsense and 4 missense mutations, were identified in 5 different genes (ACTG1, MYO1F, DIAPH1, POU4F3 and EYA4), and the genotypes for these mutations were consistent with the autosomal dominant inheritance pattern of hearing loss in each family. No mutational hot-spots were revealed in these Korean families. CONCLUSION: Targeted NGS allowed for the detection of pathogenic mutations in affected individuals who were not candidates for classical genetic studies. This report is the first documenting the effective use of an NGS technique to detect pathogenic mutations that underlie hearing loss in an East Asian population. Using this NGS technique to establish a database of common mutations in Korean patients with hearing loss and further data accumulation will contribute to the early diagnosis and fundamental therapies for hereditary hearing loss.     

Dependable and Efficient Clinical Molecular Diagnosis of Chinese RP Patient with Targeted Exon Sequencing

Retinitis pigmentosa (RP) is the most common inherited retinal disease. It is a clinically and genetically heterogeneous disorder, which is why it is particularly challenging to diagnose. The aim of this study was to establish a targeted next-generation sequencing (NGS) approach for the comprehensive, rapid, and cost-effective clinical molecular diagnosis of RP. A specific hereditary eye disease enrichment panel (HEDEP) based on exome capture technology was used to collect the protein coding regions of 371 targeted hereditary eye disease genes, followed by high-throughput sequencing on the Illumina HiSeq2000 platform. From a cohort of 34 Chinese RP families, 13 families were successfully diagnosed; thus, the method achieves a diagnostic rate of approximately 40%. Of 16 pathogenic mutations identified, 11 were novel. Our study demonstrates that targeted capture sequencing offers a rapid and effective method for the molecular diagnosis of RP, which helps to provide a more accurate clinical diagnosis and paves the way for genetic counseling, family planning, and future gene-targeted treatment.