A case of Usher syndrome type IIA caused by a rare USH2A homozygous frameshift variant with maternal uniparental disomy (UPD) in a Chinese family

Usher syndrome encompasses a group of genetically and clinically heterogeneous autosomal recessive disorders with hearing deficiencies and retinitis pigmentosa. The mechanisms underlying the Usher syndrome are highly variable. In the present study, a Chinese family with Usher syndrome was recruited. Whole exome sequencing (WES), Sanger sequencing, homozygosity mapping, short tandem repeat (STR) analysis and segregation analysis were performed. Functional domains of the pathogenic variant for USH2A were analysed. We identified a homozygous frameshift variant c.99_100insT (p.Arg34Serfs*41) in the USH2A gene in the proband that showed discordant segregation in the father. Further homozygosity mapping and STR analysis identified an unusual homozygous variant of proband that originated from maternal uniparental disomy (UPD). The p.Arg34Serfs*41 variant produced a predicted truncated protein that removes all functional domains of USH2A. The variant was not included in the 1000 Human Genomes Project database, ExAC database, HGMD or gnomAD database, but was included in the ClinVar databases as pathogenic. Although USH2A is an autosomal recessive disease, the effects of UPD should be informed in genetic counselling since the recurrence risk of an affected child is greatly reduced when the disease is due to the UPD mechanism. To test potential patients, WES, combined with STR analysis and homozygosity mapping, provides an accurate and useful strategy for genetic diagnosis. In summary, our discoveries can help further the understanding of the molecular pathogenesis of Usher syndrome type IIA to advance the prevention, diagnosis and therapy for this disorder.     

Whole Exome Sequencing Identifies a Troponin T Mutation Hot Spot in Familial Dilated Cardiomyopathy

Dilated cardiomyopathy (DCM) commonly causes heart failure and shows extensive genetic heterogeneity that may be amenable to newly developed next-generation DNA sequencing of the exome. In this study we report the successful use of exome sequencing to identify a pathogenic variant in the TNNT2 gene using segregation analysis in a large DCM family. Exome sequencing was performed on three distant relatives from a large family with a clear DCM phenotype. Missense, nonsense, and splice variants were analyzed for segregation among the three affected family members and confirmed in other relatives by direct sequencing. A c.517T C>T, Arg173Trp TNNT2 variant segregated with all affected family members and was also detected in one additional DCM family in our registry. The inclusion of segregation analysis using distant family members markedly improved the bioinformatics filtering process by removing from consideration variants that were not shared by all affected subjects. Haplotype analysis confirmed that the variant found in both DCM families was located on two distinct haplotypes, supporting the notion of independent mutational events in each family. In conclusion, an exome sequencing strategy that includes segregation analysis using distant affected relatives within a family represents a viable diagnostic strategy in a genetically heterogeneous disease like DCM.     

Pseudotrisomy 13 syndrome: use of homozygosity mapping to target candidate genes

Pseudotrisomy 13 syndrome is characterised by holoprosencephaly with or without polydactyly, but with a normal karyotype. The genetic cause of this syndrome remains unclear, but it is thought to be autosomal recessive. In order to identify possible candidate genes, we identified regions of homozygosity in the DNA of an affected foetus, which was the seventh pregnancy of a healthy non-consanguineous Cook Island Maori couple; this ethnic group derives from a small founder population. Several large regions of homozygosity were identified using a high density array. We excluded two candidate genes that lay within these regions, and suggest that Pseudotrisomy 13 syndrome might not be monogenic and that a larger cohort of patients should be analysed using high density dosage/SNP arrays as well as whole exome sequencing in order to clarify the genetic underpinning of this rare syndrome.     

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.     

Challenges in whole exome sequencing: an example from hereditary deafness

Whole exome sequencing provides unprecedented opportunities to identify causative DNA variants in rare Mendelian disorders. Finding the responsible mutation via traditional methods in families with hearing loss is difficult due to a high degree of genetic heterogeneity. In this study we combined autozygosity mapping and whole exome sequencing in a family with 3 affected children having nonsyndromic hearing loss born to consanguineous parents. Two novel missense homozygous variants, c.508C>A (p.H170N) in GIPC3 and c.1328C>T (p.T443M) in ZNF57, were identified in the same ～6 Mb autozygous region on chromosome 19 in affected members of the family. Both variants co-segregated with the phenotype and were absent in 335 ethnicity-matched controls. Biallelic GIPC3 mutations have recently been reported to cause autosomal recessive nonsyndromic sensorineural hearing loss. Thus we conclude that the hearing loss in the family described in this report is caused by a novel missense mutation in GIPC3. Identified variant in GIPC3 had a low read depth, which was initially filtered out during the analysis leaving ZNF57 as the only potential causative gene. This study highlights some of the challenges in the analyses of whole exome data in the bid to establish the true causative variant in Mendelian disease.     

Decreased Levels of BAG3 in a Family With a Rare Variant and in Idiopathic Dilated Cardiomyopathy

The most common cause of dilated cardiomyopathy and heart failure (HF) is ischemic heart disease; however, in a third of all patients the cause remains undefined and patients are diagnosed as having idiopathic dilated cardiomyopathy (IDC). Recent studies suggest that many patients with IDC have a family history of HF and rare genetic variants in over 35 genes have been shown to be causative of disease. We employed whole‐exome sequencing to identify the causative variant in a large family with autosomal dominant transmission of dilated cardiomyopathy. Sequencing and subsequent informatics revealed a novel 10‐nucleotide deletion in the BCL2‐associated athanogene 3 (BAG3) gene (Ch10:del 121436332_12143641: del. 1266_1275 [NM 004281]) that segregated with all affected individuals. The deletion predicted a shift in the reading frame with the resultant deletion of 135 amino acids from the C‐terminal end of the protein. Consistent with genetic variants in genes encoding other sarcomeric proteins there was a considerable amount of genetic heterogeneity in the affected family members. Interestingly, we also found that the levels of BAG3 protein were significantly reduced in the hearts from unrelated patients with end‐stage HF undergoing cardiac transplantation when compared with non‐failing controls. Diminished levels of BAG3 protein may be associated with both familial and non‐familial forms of dilated cardiomyopathy. J. Cell. Physiol. 229: 1697–1702, 2014. © 2014 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.     

Exome sequencing and functional analysis identifies BANF1 mutation as the cause of a hereditary progeroid syndrome

Accelerated aging syndromes represent a valuable source of information about the molecular mechanisms involved in normal aging. Here, we describe a progeroid syndrome that partially phenocopies Hutchinson-Gilford progeria syndrome (HGPS) but also exhibits distinctive features, including the absence of cardiovascular deficiencies characteristic of HGPS, the lack of mutations in LMNA and ZMPSTE24, and a relatively long lifespan of affected individuals. Exome sequencing and molecular analysis in two unrelated families allowed us to identify a homozygous mutation in BANF1 (c.34G>A [p.Ala12Thr]), encoding barrier-to-autointegration factor 1 (BAF), as the molecular abnormality responsible for this Mendelian disorder. Functional analysis showed that fibroblasts from both patients have a dramatic reduction in BAF protein levels, indicating that the p.Ala12Thr mutation impairs protein stability. Furthermore, progeroid fibroblasts display profound abnormalities in the nuclear lamina, including blebs and abnormal distribution of emerin, an interaction partner of BAF. These nuclear abnormalities are rescued by ectopic expression of wild-type BANF1, providing evidence for the causal role of this mutation. These data demonstrate the utility of exome sequencing for identifying the cause of rare Mendelian disorders and underscore the importance of nuclear envelope alterations in human aging.     

Identification of a Novel p.Q1772X ANK1 Mutation in a Korean Family with Hereditary Spherocytosis

Hereditary spherocytosis (HS), a common form of inherited hemolytic anemia, is a heterogeneous group of disorders with regard to clinical severity, protein defects, and mode of inheritance. Causal mutations in at least five genes have been reported so far. Because multiple genes have been associated with HS, clinical genetic testing that relies on direct sequencing will be a challenge. In this study, we used whole exome sequencing to identify a novel nonsense mutation in ANK1 (p.Q1772X, NM_020476) that resulted in a truncated protein in a Korean patient with HS. Sanger sequencing confirmed the two affected individuals in the patient’s family were heterozygous for the mutation. This is the first report of a Korean family that carries an ANK1 mutation responsible for HS. Our results demonstrate that next generation sequencing is a powerful approach for rapidly determining the genetic etiology of HS.     

Whole Exome Sequencing of Distant Relatives in Multiplex Families Implicates Rare Variants in Candidate Genes for Oral Clefts

A dozen genes/regions have been confirmed as genetic risk factors for oral clefts in human association and linkage studies, and animal models argue even more genes may be involved. Genomic sequencing studies should identify specific causal variants and may reveal additional genes as influencing risk to oral clefts, which have a complex and heterogeneous etiology. We conducted a whole exome sequencing (WES) study to search for potentially causal variants using affected relatives drawn from multiplex cleft families. Two or three affected second, third, and higher degree relatives from 55 multiplex families were sequenced. We examined rare single nucleotide variants (SNVs) shared by affected relatives in 348 recognized candidate genes. Exact probabilities that affected relatives would share these rare variants were calculated, given pedigree structures, and corrected for the number of variants tested. Five novel and potentially damaging SNVs shared by affected distant relatives were found and confirmed by Sanger sequencing. One damaging SNV in CDH1, shared by three affected second cousins from a single family, attained statistical significance (P = 0.02 after correcting for multiple tests). Family-based designs such as the one used in this WES study offer important advantages for identifying genes likely to be causing complex and heterogeneous disorders.     

Whole‐exome sequencing identifies a novel mutation of GPD1L (R189X) associated with familial conduction disease and sudden death

Cardiac conduction disease (CCD) is a serious disorder and the leading cause of mortality worldwide. It is characterized by arrhythmia, syncope or even sudden cardiac death caused by the dysfunction of cardiac voltage‐gated channel. Previous study has demonstrated that mutations in genes encoding voltage‐gated channel and related proteins were the crucial genetic lesion of CCD. In this study, we employed whole‐exome sequencing to explore the potential causative genes in a Chinese family with ventricular tachycardia and syncope. A novel nonsense mutation (c.565C>T/p.R189X) of glycerol‐3‐phosphate dehydrogenase‐like (GPD1L) was identified and co‐segregated with the affected family members. GPD1L is a crucial interacting protein of SCN5A, a gene encoded sodium channel α‐subunit Na_v1.5 and mainly associated with Brugada syndrome (BrS). The novel mutation (c.565C>T/p.R189X) may result in a premature stop codon at position 189 in exon 4 of the GPD1L gene and lead to functional haploinsufficiency of GPD1L due to mRNA carrying this mutation will be degraded by nonsense‐mediated mRNA decay, which has been confirmed by Western blot in HEK293 cells transfected HIS‐GPD1L plasmid. The levels of GPD1L decreasing may disturb the function of Na_v1.5 and induce arrhythmia and syncope in the end. In conclusion, our study not only further supported the important role of GPD1L in CCD, but also expanded the spectrum of GPD1L mutations and will contribute to the genetic diagnosis and counselling of families with CCD.     

Rapid gene identification in a Chinese osteopetrosis family by whole exome sequencing

Osteopetrosis is a rare genetically heterogeneous disorder of bone metabolism characterized by increased skeleton density. In the past, standard methods for genetic diagnosis of osteopetrosis have primarily been performed by candidate gene screening and positional cloning. However, these methods are time and labor consumptive; and the genetic basis of approximately 30% of the cases is yet to be elucidated. Here, we employed whole exome sequencing of two affected individuals from an osteopetrosis family to identify a candidate mutation in CLCN7 (Y99C). It was identified from a total of 1757 and 1728 genetic variations found in either patient, which were then distilled using filtering strategies and confirmed using Sanger sequencing. We identified this mutation in six family members, while not in population matched controls. This mutation was previously found in osteopetrosis patients by other researchers. Our evolutionary analysis also indicated that it is under extremely high selective pressure, and is likely to be critical for the correct function of ClC-7, and thus is likely to be the responsible cause of disease. Collectively, our data further indicated that mutation (Y99C) may be a cause of osteopetrosis, and highlights the use of whole exome sequencing as a valuable approach to identifying disease mutations in a cost and time efficient manner.     

A missense mutation in myelin oligodendrocyte glycoprotein as a cause of familial narcolepsy with cataplexy

Narcolepsy is a rare sleep disorder characterized by excessive daytime sleepiness and cataplexy. Familial narcolepsy accounts for less than 10% of all narcolepsy cases. However, documented multiplex families are very rare and causative mutations have not been identified to date. To identify a causative mutation in familial narcolepsy, we performed linkage analysis in the largest ever reported family, which has 12 affected members, and sequenced coding regions of the genome (exome sequencing) of three affected members with narcolepsy and cataplexy. We successfully mapped a candidate locus on chromosomal region 6p22.1 (LOD score = 3.85) by linkage analysis. Exome sequencing identified a missense mutation in the second exon of MOG within the linkage region. A c.398C>G mutation was present in all affected family members but absent in unaffected members and 775 unrelated control subjects. Transient expression of mutant myelin oligodendrocyte glycoprotein (MOG) in mouse oligodendrocytes showed abnormal subcellular localization, suggesting an altered function of the mutant MOG. MOG has recently been linked to various neuropsychiatric disorders and is considered as a key autoantigen in multiple sclerosis and in its animal model, experimental autoimmune encephalitis. Our finding of a pathogenic MOG mutation highlights a major role for myelin and oligodendrocytes in narcolepsy and further emphasizes glial involvement in neurodegeneration and neurobehavioral disorders.     

Role of the newer p53 family proteins in malignancy

The most recently identified members of the p53 family, p63 and p73, share certain structural and functional similarities with p53. Both p63 and p73 can bind to canonical p53-DNA-binding sites, transactivate the promoters of known p53 target genes and induce apoptosis. Despite these similarities there are many important differences. In contrast to p53, p63 and p73 give rise to multiple distinct protein isoforms that have different functional properties. Upstream signaling pathways involved in the activation of p63 and p73 differ from those involved in p53 activation. Only a subset of the DNA damaging agents that induce p53 can induce p73. Cellular and viral oncoproteins can discriminate between p53 and the newer family members. In addition, the levels of p63 and p73 are affected by certain states of cellular differentiation. Finally, it is becoming clear that the newest members of the p53 family are not classical tumor suppressor genes. In contrast to the high prevalence of p53 mutations in human cancers, p63 and p73 mutations are rare. Indeed, levels of p73 increase during malignant progression. In addition, unlike p53-/- mice, mice lacking p63 and p73 do not develop tumors, but instead have significant developmental abnormalities. Mutations in p63 have also been detected in humans with the ectodermal dysplastic syndrome EEC. Further studies are required to determine whether qualitative or quantitative differences in the expression of p63 and p73 isoforms are important in the development of human cancers.     

Cytokinin dehydrogenase: a genetic target for yield improvement in wheat

The plant hormone group, the cytokinins, is implicated in both qualitative and quantitative components of yield. Cytokinins have opposing actions in shoot and root growth—actions shown to involve cytokinin dehydrogenase (CKX), the enzyme that inactivates cytokinin. We revise and provide unambiguous names for the CKX gene family members in wheat, based on the most recently released wheat genome database, IWGSC RefSeq v1.0 & v2.0. We review expression data of CKX gene family members in wheat, revealing tissue‐specific gene family member expression as well as sub‐genome‐specific expression. Manipulation of CKX in cereals shows clear impacts on yield, root growth and orientation, and Zn nutrition, but this also emphasizes the necessity to unlink promotive effects on grain yield from negative effects of cytokinin on root growth and uptake of mineral nutrients, particularly Zn and Fe. Wheat is the most widely grown cereal crop globally, yet is under‐research compared with rice and maize. We highlight gaps in our knowledge of the involvement of CKX for wheat. We also highlight the necessity for accurate analysis of endogenous cytokinins, acknowledging why this is challenging, and provide examples where inadequate analyses of endogenous cytokinins have led to unjustified conclusions. We acknowledge that the allohexaploid nature of bread wheat poses challenges in terms of uncovering useful mutations. However, we predict TILLING followed by whole‐exome sequencing will uncover informative mutations and we indicate the potential for stacking mutations within the three genomes to modify yield components. We model a wheat ideotype based on CKX manipulation.     

Whole-exome sequencing reveals a recurrent mutation in the cathepsin C gene that causes Papillon–Lefevre syndrome in a Saudi family

Papillon–Lefevre syndrome (PALS) is a rare, autosomal recessive disorder characterized by periodontitis and hyperkeratosis over the palms and soles. Mutations in the cathepsin C gene (CTSC) have been recognized as the cause of PALS since the late 1990s. More than 75 mutations in CTSC have been identified, and phenotypic variability between different mutations has been described. Next generation sequencing is widely used for efficient molecular diagnostics in various clinical practices. Here we investigated a large consanguineous Saudi family with four affected and four unaffected individuals. All of the affected individuals suffered from hyperkeratosis over the palms and soles and had anomalies of both primary and secondary dentition. For molecular diagnostics, we combined whole-exome sequencing and genome-wide homozygosity mapping procedures, and identified a recurrent homozygous missense mutation (c.899G>A; p.Gly300Asp) in exon 7 of CTSC. Validation of all eight family members by Sanger sequencing confirmed co-segregation of the pathogenic variant (c.899G>A) with the disease phenotype. This is the first report of whole-exome sequencing performed for molecular diagnosis of PALS in Saudi Arabia. Our findings provide further insights into the genotype–phenotype correlation of CTSC pathogenicity in PALS.     

Barley whole exome capture: a tool for genomic research in the genus Hordeum and beyond

Advanced resources for genome‐assisted research in barley (Hordeum vulgare) including a whole‐genome shotgun assembly and an integrated physical map have recently become available. These have made possible studies that aim to assess genetic diversity or to isolate single genes by whole‐genome resequencing and in silico variant detection. However such an approach remains expensive given the 5 Gb size of the barley genome. Targeted sequencing of the mRNA‐coding exome reduces barley genomic complexity more than 50‐fold, thus dramatically reducing this heavy sequencing and analysis load. We have developed and employed an in‐solution hybridization‐based sequence capture platform to selectively enrich for a 61.6 megabase coding sequence target that includes predicted genes from the genome assembly of the cultivar Morex as well as publicly available full‐length cDNAs and de novo assembled RNA‐Seq consensus sequence contigs. The platform provides a highly specific capture with substantial and reproducible enrichment of targeted exons, both for cultivated barley and related species. We show that this exome capture platform provides a clear path towards a broader and deeper understanding of the natural variation residing in the mRNA‐coding part of the barley genome and will thus constitute a valuable resource for applications such as mapping‐by‐sequencing and genetic diversity analyzes.     

Use of Targeted Exome Sequencing in Genetic Diagnosis of Chinese Familial Hypercholesterolemia

Familial hypercholesterolemia is an autosomal dominant inherited disease characterized by elevated plasma low-density lipoprotein cholesterol (LDL-C). It is mainly caused by mutations of the low-density lipoprotein receptor (LDLR) gene. Currently, the methods of whole genome sequencing or whole exome sequencing for screening mutations in familial hypercholesterolemia are not applicable in China due to high cost. We performed targeted exome sequencing of 167 genes implicated in the homozygous phenotype of a proband pedigree to identify candidate mutations, validated them in the family of the proband, studied the functions of the mutant protein, and followed up serum lipid levels after treatment. We discovered that exon 9 c.1268 T>C and exon 8 c.1129 T>G compound heterozygous mutations in the LDLR gene in the proband derived from the mother and father, respectively, in which the mutation of c.1129 T>G has not been reported previously. The mutant LDL-R protein had 57% and 52% binding and internalization functions, respectively, compared with that of the wild type. After 6 months of therapy, the LDL-C level of the proband decreased by more than 50% and the LDL-C of the other family members with heterozygous mutation also reduced to normal. Targeted exome sequencing is an effective method for screening mutation genes in familial hypercholesterolemia. The exon 8 and 9 mutations of the LDLR gene were pedigree mutations. The functions of the mutant LDL-R protein were decreased significantly compared with that of the wild type. Simvastatin plus ezetimibe was proven safe and effective in this preschool-age child.     

Whole exome sequencing identifies rare biallelic ALMS1 missense and stop gain mutations in familial Alström syndrome patients

Alström syndrome (AS, OMIM ID 203800) is a rare childhood multiorgan disorder, which is widely studied in non-Arab ethnic patients. The clinical and molecular basis of AS and the mode of disease inheritance in consanguineous Arab populations is not well investigated. Therefore, to identify the molecular basis of AS in familial forms, the present study performed whole exome sequencing of 5 AS patients belonging to 2 different Bedouin families from Saudi Arabia. The present study identified the AS causative rare biallelic mutations in ALMS gene:T376S in exon 5 and S909* in exon 8 for family A and an R2721* in exon 10 (R2721*) for family B. ALMS1 targeted genetic sequencing of healthy population controls and family members has confirmed its extremely rare frequency and autosomal recessive mode of inheritance. The truncating mutations S909* and R2721* could cause the loss of CC domains and ALMS motif on C-terminal end of the protein and creates unstable protein, which eventually undergoes intracellular degradation. The premature protein truncating mutations described in our study may eventually provide further insight into the functional domains of the ALMS1 protein and contribute to the understanding of the phenotypic spectrum of AS. Whole exome sequencing based molecular diagnosis is expected to rule out ambiguity surrounding clinical diagnosis of suspected AS cases.     

A novel locus for split-hand/foot malformation associated with tibial hemimelia (SHFLD syndrome) maps to chromosome region 17p13.1-17p13.3

Split-hand/foot malformation (SHFM) associated with aplasia of long bones, SHFLD syndrome or Tibial hemimelia-ectrodactyly syndrome is a rare condition with autosomal dominant inheritance, reduced penetrance and an incidence estimated to be about 1 in 1,000,000 liveborns. To date, three chromosomal regions have been reported as strong candidates for harboring SHFLD syndrome genes: 1q42.2–q43, 6q14.1 and 2q14.2. We characterized the phenotype of nine affected individuals from a large family with the aim of mapping the causative gene. Among the nine affected patients, four had only SHFM of the hands and no tibial defects, three had both defects and two had only unilateral tibial hemimelia. In keeping with previous publications of this and other families, there was clear evidence of both variable expression and incomplete penetrance, the latter bearing hallmarks of anticipation. Segregation analysis and multipoint Lod scores calculations (maximum Lod score of 5.03 using the LINKMAP software) using all potentially informative family members, both affected and unaffected, identified the chromosomal region 17p13.1–17p13.3 as the best and only candidate for harboring a novel mutated gene responsible for the syndrome in this family. The candidate gene CRK located within this region was sequenced but no pathogenic mutation was detected.