Investigating the Molecular Basis of Retinal Degeneration in a Familial Cohort of Pakistani Decent by Exome Sequencing

Purpose

To define the molecular basis of retinal degeneration in consanguineous Pakistani pedigrees with early onset retinal degeneration.

Methods

A cohort of 277 individuals representing 26 pedigrees from the Punjab province of Pakistan was analyzed. Exomes were captured with commercial kits and sequenced on an Illumina HiSeq 2500. Candidate variants were identified using standard tools and analyzed using exomeSuite to detect all potentially pathogenic changes in genes implicated in retinal degeneration. Segregation analysis was performed by dideoxy sequencing and novel variants were additionally investigated for their presence in ethnicity-matched controls.

Results

We identified a total of nine causal mutations, including six novel variants in RPE65, LCA5, USH2A, CNGB1, FAM161A, CERKL and GUCY2D as the underlying cause of inherited retinal degenerations in 13 of 26 pedigrees. In addition to the causal variants, a total of 200 variants each observed in five or more unrelated pedigrees investigated in this study that were absent from the dbSNP, HapMap, 1000 Genomes, NHLBI ESP6500, and ExAC databases were identified, suggesting that they are common in, and unique to the Pakistani population.

Conclusions

We identified causal mutations associated with retinal degeneration in nearly half of the pedigrees investigated in this study through next generation whole exome sequencing. All novel variants detected in this study through exome sequencing have been cataloged providing a reference database of variants common in, and unique to the Pakistani population.     

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.     

Homozygosity Mapping and Targeted Sanger Sequencing Reveal Genetic Defects Underlying Inherited Retinal Disease in Families from Pakistan

Background

Homozygosity mapping has facilitated the identification of the genetic causes underlying inherited diseases, particularly in consanguineous families with multiple affected individuals. This knowledge has also resulted in a mutation dataset that can be used in a cost and time effective manner to screen frequent population-specific genetic variations associated with diseases such as inherited retinal disease (IRD).

Methods

We genetically screened 13 families from a cohort of 81 Pakistani IRD families diagnosed with Leber congenital amaurosis (LCA), retinitis pigmentosa (RP), congenital stationary night blindness (CSNB), or cone dystrophy (CD). We employed genome-wide single nucleotide polymorphism (SNP) array analysis to identify homozygous regions shared by affected individuals and performed Sanger sequencing of IRD-associated genes located in the sizeable homozygous regions. In addition, based on population specific mutation data we performed targeted Sanger sequencing (TSS) of frequent variants in AIPL1, CEP290, CRB1, GUCY2D, LCA5, RPGRIP1 and TULP1, in probands from 28 LCA families.

Results

Homozygosity mapping and Sanger sequencing of IRD-associated genes revealed the underlying mutations in 10 families. TSS revealed causative variants in three families. In these 13 families four novel mutations were identified in CNGA1, CNGB1, GUCY2D, and RPGRIP1.

Conclusions

Homozygosity mapping and TSS revealed the underlying genetic cause in 13 IRD families, which is useful for genetic counseling as well as therapeutic interventions that are likely to become available in the near future.     

Exome Sequencing of Index Patients with Retinal Dystrophies as a Tool for Molecular Diagnosis

Background

Retinal dystrophies (RD) are a group of hereditary diseases that lead to debilitating visual impairment and are usually transmitted as a Mendelian trait. Pathogenic mutations can occur in any of the 100 or more disease genes identified so far, making molecular diagnosis a rather laborious process. In this work we explored the use of whole exome sequencing (WES) as a tool for identification of RD mutations, with the aim of assessing its applicability in a diagnostic context.

Methodology/Principal Findings

We ascertained 12 Spanish families with seemingly recessive RD. All of the index patients underwent mutational pre-screening by chip-based sequence hybridization and resulted to be negative for known RD mutations. With the exception of one pedigree, to simulate a standard diagnostic scenario we processed by WES only the DNA from the index patient of each family, followed by in silico data analysis. We successfully identified causative mutations in patients from 10 different families, which were later verified by Sanger sequencing and co-segregation analyses. Specifically, we detected pathogenic DNA variants (∼50% novel mutations) in the genes RP1, USH2A, CNGB3, NMNAT1, CHM, and ABCA4, responsible for retinitis pigmentosa, Usher syndrome, achromatopsia, Leber congenital amaurosis, choroideremia, or recessive Stargardt/cone-rod dystrophy cases.

Conclusions/Significance

Despite the absence of genetic information from other family members that could help excluding nonpathogenic DNA variants, we could detect causative mutations in a variety of genes known to represent a wide spectrum of clinical phenotypes in 83% of the patients analyzed. Considering the constant drop in costs for human exome sequencing and the relative simplicity of the analyses made, this technique could represent a valuable tool for molecular diagnostics or genetic research, even in cases for which no genotypes from family members are available.     

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.     

Analysis of archived residual newborn screening blood spots after whole genome amplification

Background

Deidentified newborn screening bloodspot samples (NBS) represent a valuable potential resource for genomic research if impediments to whole exome sequencing of NBS deoxyribonucleic acid (DNA), including the small amount of genomic DNA in NBS material, can be overcome. For instance, genomic analysis of NBS could be used to define allele frequencies of disease-associated variants in local populations, or to conduct prospective or retrospective studies relating genomic variation to disease emergence in pediatric populations over time. In this study, we compared the recovery of variant calls from exome sequences of amplified NBS genomic DNA to variant calls from exome sequencing of non-amplified NBS DNA from the same individuals.

Results

Using a standard alignment-based Genome Analysis Toolkit (GATK), we find 62,000–76,000 additional variants in amplified samples. After application of a unique kmer enumeration and variant detection method (RUFUS), only 38,000–47,000 additional variants are observed in amplified gDNA. This result suggests that roughly half of the amplification-introduced variants identified using GATK may be the result of mapping errors and read misalignment.

Conclusions

Our results show that it is possible to obtain informative, high-quality data from exome analysis of whole genome amplified NBS with the important caveat that different data generation and analysis methods can affect variant detection accuracy, and the concordance of variant calls in whole-genome amplified and non-amplified exomes.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1747-2) contains supplementary material, which is available to authorized users.     

Identifying Likely Transmission Pathways within a 10-Year Community Outbreak of Tuberculosis by High-Depth Whole Genome Sequencing

Background

Improved tuberculosis control and the need to contain the spread of drug-resistant strains provide a strong rationale for exploring tuberculosis transmission dynamics at the population level. Whole-genome sequencing provides optimal strain resolution, facilitating detailed mapping of potential transmission pathways.

Methods

We sequenced 22 isolates from a Mycobacterium tuberculosis cluster in New South Wales, Australia, identified during routine 24-locus mycobacterial interspersed repetitive unit typing. Following high-depth paired-end sequencing using the Illumina HiSeq 2000 platform, two independent pipelines were employed for analysis, both employing read mapping onto reference genomes as well as de novo assembly, to control biases in variant detection. In addition to single-nucleotide polymorphisms, the analyses also sought to identify insertions, deletions and structural variants.

Results

Isolates were highly similar, with a distance of 13 variants between the most distant members of the cluster. The most sensitive analysis classified the 22 isolates into 18 groups. Four of the isolates did not appear to share a recent common ancestor with the largest clade; another four isolates had an uncertain ancestral relationship with the largest clade.

Conclusion

Whole genome sequencing, with analysis of single-nucleotide polymorphisms, insertions, deletions, structural variants and subpopulations, enabled the highest possible level of discrimination between cluster members, clarifying likely transmission pathways and exposing the complexity of strain origin. The analysis provides a basis for targeted public health intervention and enhanced classification of future isolates linked to the cluster.     

Evaluation of the Genetic Basis of Familial Aggregation of Pacemaker Implantation by a Large Next Generation Sequencing Panel

Background

The etiology of conduction disturbances necessitating permanent pacemaker (PPM) implantation is often unknown, although familial aggregation of PPM (faPPM) suggests a possible genetic basis. We developed a pan-cardiovascular next generation sequencing (NGS) panel to genetically characterize a selected cohort of faPPM.

Materials and Methods

We designed and validated a custom NGS panel targeting the coding and splicing regions of 246 genes with involvement in cardiac pathogenicity. We enrolled 112 PPM patients and selected nine (8%) with faPPM to be analyzed by NGS.

Results

Our NGS panel covers 95% of the intended target with an average of 229x read depth at a minimum of 15-fold depth, reaching a SNP true positive rate of 98%. The faPPM patients presented with isolated cardiac conduction disease (ICCD) or sick sinus syndrome (SSS) without overt structural heart disease or identifiable secondary etiology. Three patients (33.3%) had heterozygous deleterious variants previously reported in autosomal dominant cardiac diseases including CCD: LDB3 (p.D117N) and TRPM4 (p.G844D) variants in patient 4; TRPM4 (p.G844D) and ABCC9 (p.V734I) variants in patient 6; and SCN5A (p.T220I) and APOB (p.R3527Q) variants in patient 7.

Conclusion

FaPPM occurred in 8% of our PPM clinic population. The employment of massive parallel sequencing for a large selected panel of cardiovascular genes identified a high percentage (33.3%) of the faPPM patients with deleterious variants previously reported in autosomal dominant cardiac diseases, suggesting that genetic variants may play a role in faPPM.     

Targeted Next Generation Sequencing as a Reliable Diagnostic Assay for the Detection of Somatic Mutations in Tumours Using Minimal DNA Amounts from Formalin Fixed Paraffin Embedded Material

Background

Targeted Next Generation Sequencing (NGS) offers a way to implement testing of multiple genetic aberrations in diagnostic pathology practice, which is necessary for personalized cancer treatment. However, no standards regarding input material have been defined. This study therefore aimed to determine the effect of the type of input material (e.g. formalin fixed paraffin embedded (FFPE) versus fresh frozen (FF) tissue) on NGS derived results. Moreover, this study aimed to explore a standardized analysis pipeline to support consistent clinical decision-making.

Method

We used the Ion Torrent PGM sequencing platform in combination with the Ion AmpliSeq Cancer Hotspot Panel v2 to sequence frequently mutated regions in 50 cancer related genes, and validated the NGS detected variants in 250 FFPE samples using standard diagnostic assays. Next, 386 tumour samples were sequenced to explore the effect of input material on variant detection variables. For variant calling, Ion Torrent analysis software was supplemented with additional variant annotation and filtering.

Results

Both FFPE and FF tissue could be sequenced reliably with a sensitivity of 99.1%. Validation showed a 98.5% concordance between NGS and conventional sequencing techniques, where NGS provided both the advantage of low input DNA concentration and the detection of low-frequency variants. The reliability of mutation analysis could be further improved with manual inspection of sequence data.

Conclusion

Targeted NGS can be reliably implemented in cancer diagnostics using both FFPE and FF tissue when using appropriate analysis settings, even with low input DNA.     

Mutations in the gene DNAJC5 cause autosomal dominant Kufs disease in a proportion of cases: study of the Parry family and 8 other families

Background

The Neuronal Ceroid Lipofuscinoses (NCL) comprise at least nine progressive neurodegenerative genetic disorders. Kufs disease, an adult-onset form of NCL may be recessively or dominantly inherited. Our study aimed to identify genetic mutations associated with autosomal dominant Kufs disease (ADKD).

Methodology and Principal Findings

We have studied the family first reported with this phenotype in the 1970s, the Parry family. The proband had progressive psychiatric manifestations, seizures and cognitive decline starting in her mid 20 s. Similarly affected relatives were observed in seven generations. Several of the affected individuals had post-mortem neuropathological brain study confirmatory for NCL disease. We conducted whole exome sequencing of three affected family members and identified a pLeu116del mutation in the gene DNAJC5, which segregated with the disease phenotype. An additional eight unrelated affected individuals with documented autosomal dominant or sporadic inheritance were studied. All had diagnostic confirmation with neuropathological studies of brain tissue. Among them we identified an additional individual with a p.Leu115Arg mutation in DNAJC5. In addition, a pAsn477Ser change in the neighboring gene PRPF6, a gene previously found to be associated with retinitis pigmentosa, segregated with the ADKD phenotype. Interestingly, two individuals of the Parry family did report visual impairment.

Conclusions

Our study confirmed the recently reported association of DNAJC5 mutations with ADKD in two out of nine well-defined families. Sequence changes in PRPF6 have not been identified in other unrelated cases. The association of vision impairment with the expected PRPF6 dysfunction remains possible but would need further clinical studies in order to confirm the co-segregation of the visual impairment with this sequence change.     

Genetic Identification Is Critical for the Diagnosis of Parkinsonism: A Chinese Pedigree with Early Onset of Parkinsonism

Background

A number of hereditary neurological diseases display indistinguishable features at the early disease stage. Parkinsonian symptoms can be found in numerous diseases, making it difficult to get a definitive early diagnosis of primary causes for patients with onset of parkinsonism. The accurate and early diagnosis of the causes of parkinsonian patients is important for effective treatments of these patients.

Methods

We have identified a Chinese family (82 family members over four generations with 21 affected individuals) that manifested the characterized symptoms of parkinsonism and was initially diagnosed as Parkinson’s disease. We followed up with the family for two years, during which we carried out clinical observations, Positron Emission Tomography-Computed Tomography neuroimaging analysis, and exome sequencing to correctly diagnose the case.

Results

During the two-year follow-up period, we performed comprehensive medical history collection, physical examination, and structural and functional neuroimaging studies of this Chinese family. We found that the patient exhibited progressive deteriorated parkinsonism with Parkinson disease-like neuropathology and also had a good response to the initial levodopa treatment. However, exome sequencing identified a missense mutation, N279K, in exon 10 of MAPT gene, verifying that the early parkinsonian symptoms in this family are caused by the genetic mutation for hereditary frontotemporal lobar dementia.

Conclusions

For the inherited parkinsonian patients who even show the neuropathology similar to that in Parkinson’s disease and have initial response to levodopa treatment, genetic identification of the molecular basis for the disease is still required for defining the early diagnosis and correct treatment.     

Efficient In Silico Identification of a Common Insertion in the MAK Gene which Causes Retinitis Pigmentosa

Background

Next generation sequencing (NGS) offers a rapid and comprehensive method of screening for mutations associated with retinitis pigmentosa and related disorders. However, certain sequence alterations such as large insertions or deletions may remain undetected using standard NGS pipelines. One such mutation is a recently-identified Alu insertion into the Male Germ Cell-Associated Kinase (MAK) gene, which is missed by standard NGS-based variant callers. Here, we developed an in silico method of searching NGS raw sequence reads to detect this mutation, without the need to recalculate sequence alignments or to screen every sample by PCR.

Methods

The Linux program grep was used to search for a 23 bp “probe” sequence containing the known junction sequence of the insert. A corresponding search was performed with the wildtype sequence. The matching reads were counted and further compared to the known sequences of the full wildtype and mutant genomic loci. (See https://github.com/MEEIBioinformaticsCenter/grepsearch.)

Results

In a test sample set consisting of eleven previously published homozygous mutants, detection of the MAK-Alu insertion was validated with 100% sensitivity and specificity. As a discovery cohort, raw NGS reads from 1,847 samples (including custom and whole exome selective capture) were searched in ~1 hour on a local computer cluster, yielding an additional five samples with MAK-Alu insertions and solving two previously unsolved pedigrees. Of these, one patient was homozygous for the insertion, one compound heterozygous with a missense change on the other allele (c. 46G>A; p.Gly16Arg), and three were heterozygous carriers.

Conclusions

Using the MAK-Alu grep program proved to be a rapid and effective method of finding a known, disease-causing Alu insertion in a large cohort of patients with NGS data. This simple approach avoids wet-lab assays or computationally expensive algorithms, and could also be used for other known disease-causing insertions and deletions.     

Design of DNA Pooling to Allow Incorporation of Covariates in Rare Variants Analysis

Background

Rapid advances in next-generation sequencing technologies facilitate genetic association studies of an increasingly wide array of rare variants. To capture the rare or less common variants, a large number of individuals will be needed. However, the cost of a large scale study using whole genome or exome sequencing is still high. DNA pooling can serve as a cost-effective approach, but with a potential limitation that the identity of individual genomes would be lost and therefore individual characteristics and environmental factors could not be adjusted in association analysis, which may result in power loss and a biased estimate of genetic effect.

Methods

For case-control studies, we propose a design strategy for pool creation and an analysis strategy that allows covariate adjustment, using multiple imputation technique.

Results

Simulations show that our approach can obtain reasonable estimate for genotypic effect with only slight loss of power compared to the much more expensive approach of sequencing individual genomes.

Conclusion

Our design and analysis strategies enable more powerful and cost-effective sequencing studies of complex diseases, while allowing incorporation of covariate adjustment.     

Re-Examining the Association between Vitamin D and Childhood Caries

Background

Previous studies have reported an inverse association between vitamin D and childhood dental caries, but whether this is causal is unclear.

Objective

To determine the causal effect of circulating 25-hydroxyvitamin D concentration on dental caries experience, early caries onset and the requirement for a dental general anesthetic.

Design

A Mendelian randomization study was undertaken, using genetic variants known to be associated with circulating 25-hydroxyvitamin D concentrations in 5,545 European origin children from the South West of England. Data on caries and related characteristics were obtained from parental and child completed questionnaires between 38 and 91 months and clinical assessments in a random 10% sample at 31, 44 and 61 months.

Results

In multivariable confounder adjusted analyses no strong evidence for an association of 25-hydroxyvitamin D with caries experience or severity was found but there was evidence for an association with early caries onset, or having a general anesthetic for dental problems. In Mendelian randomization analysis the odds ratio for caries experience per 10 nmol/L increase in 25-hydroxyvitamin D was 0.93 (95% confidence interval: 0.83, 1.05; P = 0.26) and the odds ratio for dental general anaesthetic per 10 nmol/L increase in 25-hydroxyvitamin D was 0.96 (95% confidence interval: 0.75, 1.22; P = 0.72).

Conclusions

This Mendelian randomization study provides little evidence to support an inverse causal effect of 25-hydroxyvitamin D on dental caries. However, the estimates are imprecise and a larger study is required to refine these analyses.     

Increased Missense Mutation Burden of Fatty Acid Metabolism Related Genes in Nunavik Inuit Population

Background

Nunavik Inuit (northern Quebec, Canada) reside along the arctic coastline where for generations their daily energy intake has mainly been derived from animal fat. Given this particular diet it has been hypothesized that natural selection would lead to population specific allele frequency differences and unique variants in genes related to fatty acid metabolism. A group of genes, namely CPT1A, CPT1B, CPT1C, CPT2, CRAT and CROT, encode for three carnitine acyltransferases that are important for the oxidation of fatty acids, a critical step in their metabolism.

Methods

Exome sequencing and SNP array genotyping were used to examine the genetic variations in the six genes encoding for the carnitine acyltransferases in 113 Nunavik Inuit individuals.

Results

Altogether ten missense variants were found in genes CPT1A, CPT1B, CPT1C, CPT2 and CRAT, including three novel variants and one Inuit specific variant CPT1A p.P479L (rs80356779). The latter has the highest frequency (0.955) compared to other Inuit populations. We found that by comparison to Asians or Europeans, the Nunavik Inuit have an increased mutation burden in CPT1A, CPT2 and CRAT; there is also a high level of population differentiation based on carnitine acyltransferase gene variations between Nunavik Inuit and Asians.

Conclusion

The increased number and frequency of deleterious variants in these fatty acid metabolism genes in Nunavik Inuit may be the result of genetic adaptation to their diet and/or the extremely cold climate. In addition, the identification of these variants may help to understand some of the specific health risks of Nunavik Inuit.     

Low-Frequency IL23R Coding Variant Associated with Crohn’s Disease Susceptibility in Japanese Subjects Identified by Personal Genomics Analysis

Background

The common disease-common variant hypothesis is insufficient to explain the complexities of Crohn’s disease (CD) genetics; therefore, rare variants are expected to be important in the disease. We explored rare variants associated with susceptibility to CD in Japanese individuals by personal genomic analysis.

Methods

Two-step analyses were performed. The first step was a trio analysis with whole-exome sequence (WES) analysis and the second was a follow-up case-control association study. The WES analysis pipeline comprised Burrows-Wheeler Aligner, Picard, Genome Analysis Toolkit, and SAMTOOLS. Single nucleotide variants (SNVs)/indels were annotated and filtered by using programs implemented in ANNOVAR in combination with identity-by-descent (IBD), subsequently were subjected to the linkage based, and de novo based strategies. Finally, we conducted an association study that included 176 unrelated subjects with CD and 358 healthy control subjects.

Results

In family members, 234,067–297,523 SNVs/indels were detected and they were educed to 106–146 by annotation based filtering. Fifty-four CD variants common to both individuals of the affected sib pair were identified. The linkage based strategy detected five candidate variants whereas the de novo based strategy identified no variants. Consequently, five candidates were analyzed in the case-control association study. CD showed a significant association with one variant in exon 4 of IL23R, G149R [rs76418789, P = 3.9E-5, odds ratio (OR) 0.21, 95% confidence interval (CI) 0.09–0.47 for the dominant model (AA + AG versus GG), and P = 7.3E-5, OR 0.21, 95% CI 0.10–0.48 for AG versus GG, and P = 7.2E-5, OR 0.23, 95% CI 0.10–0.50 for the allele model].

Conclusions

The present study, using personal genomics analysis of a small CD pedigree, is the first to show that the low-frequency non-synonymous variant of IL23R, rs76418789, protects against CD development in Japanese subjects.     

Molecular Genetics of the Usher Syndrome in Lebanon: Identification of 11 Novel Protein Truncating Mutations by Whole Exome Sequencing

Background

Usher syndrome (USH) is a genetically heterogeneous condition with ten disease-causing genes. The spectrum of genes and mutations causing USH in the Lebanese and Middle Eastern populations has not been described. Consequently, diagnostic approaches designed to screen for previously reported mutations were unlikely to identify the mutations in 11 unrelated families, eight of Lebanese and three of Middle Eastern origins. In addition, six of the ten USH genes consist of more than 20 exons, each, which made mutational analysis by Sanger sequencing of PCR-amplified exons from genomic DNA tedious and costly. The study was aimed at the identification of USH causing genes and mutations in 11 unrelated families with USH type I or II.

Methods

Whole exome sequencing followed by expanded familial validation by Sanger sequencing.

Results

We identified disease-causing mutations in all the analyzed patients in four USH genes, MYO7A, USH2A, GPR98 and CDH23. Eleven of the mutations were novel and protein truncating, including a complex rearrangement in GPR98.

Conclusion

Our data highlight the genetic diversity of Usher syndrome in the Lebanese population and the time and cost-effectiveness of whole exome sequencing approach for mutation analysis of genetically heterogeneous conditions caused by large genes.     

Applicability and Efficiency of NGS in Routine Diagnosis: In-Depth Performance Analysis of a Complete Workflow for CFTR Mutation Analysis

Background

Actually, about 2000 sequence variations have been documented in the CFTR gene requiring extensive and multi-step genetic testing in the diagnosis of cystic fibrosis and CFTR-related disorders. We present a two phases study, with validation and performance monitoring, of a single experiment methodology based on multiplex PCR and high throughput sequencing that allows detection of all variants, including large rearrangements, affecting the coding regions plus three deep intronic loci.

Methods

A total of 340 samples, including 257 patients and 83 previously characterized control samples, were sequenced in 17 MiSeq runs and analyzed with two bioinformatic pipelines in routine diagnostic conditions. We obtained 100% coverage for all the target regions in every tested sample.

Results

We correctly identified all the 87 known variants in the control samples and successfully confirmed the 62 variants identified among the patients without observing false positive results. Large rearrangements were identified in 18/18 control samples. Only 17 patient samples showed false positive signals (6.6%), 12 of which showed a borderline result for a single amplicon. We also demonstrated the ability of the assay to detect allele specific dropout of amplicons when a sequence variation occurs at a primer binding site thus limiting the risk for false negative results.

Conclusions

We described here the first NGS workflow for CFTR routine analysis that demonstrated equivalent diagnostic performances compared to Sanger sequencing and multiplex ligation-dependent probe amplification. This study illustrates the advantages of NGS in term of scalability, workload reduction and cost-effectiveness in combination with an improvement of the overall data quality due to the simultaneous detection of SNVs and large rearrangements.     

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.