A 46,XX testicular disorder of sex development caused by a Wilms' tumour Factor-1 (WT1) pathogenic variant

Molecular diagnosis is rarely established in 46,XX testicular (T) disorder of sex development (DSD) individuals with atypical genitalia. The Wilms' tumour factor-1 (WT1) gene is involved in early gonadal development in both sexes. Classically, WT1 deleterious variants are associated with 46,XY disorders of sex development (DSD) because of gonadal dysgenesis. We report a novel frameshift WT1 variant identified in an SRY-negative 46,XX testicular DSD girl born with atypical genitalia. Target massively parallel sequencing involving DSD-related genes identified a novel heterozygous WT1 c.1453_1456del; p.Arg485Glyfs*14 variant located in the fourth zinc finger of the protein which is absent in the population databases. Segregation analysis and microsatellite analysis confirmed the de novo status of the variant that is predicted to be deleterious by in silico tools and to increase WT1 target activation in crystallographic model. This novel and predicted activating frameshift WT1 variant leading to the 46,XX testicular DSD phenotype includes the fourth zinc-finger DNA-binding domain defects in the genetic aetiology of 46,XX DSD.     

Whole exome sequencing combined with linkage analysis identifies a novel 3?bp deletion in NR5A1

Disorders of sex development (DSDs) encompass a broad spectrum of conditions affecting the development of the gonads and genitalia. The underlying causes for DSDs include gain or loss of function variants in genes responsible for gonad development or steroidogenesis. Most patients with DSD have an unknown genetic etiology and cannot be given an accurate diagnosis. We used whole exome capture and massively parallel sequencing to analyse a large family with 46,XY DSD and 46,XX premature ovarian insufficiency. In addition, we used a recently developed method for linkage analysis using genotypes extracted from the MPS data. This approach identified a unique linkage peak on chromosome 9 and a novel, 3 bp, in-frame deletion in exon six of NR5A1 (steroidogenic factor-1 or SF1) in all affected individuals. We confirmed that the variant disrupts the SF1 protein and its ability to bind and regulate downstream genes. NR5A1 has key roles at multiple points in gonad development and steroidogenic pathways. The variant described here affects the function of SF1 in early testis development and later ovarian function, ultimately leading to the 46,XY DSD and 46,XX premature ovarian insufficiency phenotypes, respectively. This study shows that even at low coverage, whole exome sequencing, when combined with linkage analysis, can be a powerful tool to identify rapidly the disease-causing variant in large pedigrees.     

Massively parallel sequencing for early molecular diagnosis in Leber congenital amaurosis

PurposeLeber congenital amaurosis (LCA) is a rare congenital retinal dystrophy associated with 16 genes. Recent breakthroughs in LCA gene therapy offer the first prospect of treating inherited blindness, which requires an unequivocal and early molecular diagnosis. While present genetic tests do not address this due to a tremendous genetic heterogeneity, massively parallel sequencing (MPS) strategies might bring a solution. Here, we developed a comprehensive molecular test for LCA based on targeted MPS of all exons of 16 known LCA genes.MethodsWe designed a unique and flexible workflow for targeted resequencing of all 236 exons from 16 LCA genes based on quantitative PCR (qPCR) amplicon ligation, shearing, and parallel sequencing of multiple patients on a single lane of a short-read sequencer. Twenty-two prescreened LCA patients were included, five of whom had a known molecular cause.ResultsValidation of 107 variations was performed as proof of concept. In addition, the causal genetic defect and a single heterozygous mutation were identified in 3 and 5, respectively, of 17 patients without previously identified mutations.ConclusionWe propose a novel targeted MPS-based approach that is suitable for accurate, fast, and cost-effective early molecular testing in LCA, and easily applicable in other genetic disorders.Genet Med 2012:14(6):576–585     

Clinical applications of high-throughput genetic diagnosis in inherited retinal dystrophies: Present challenges and future directions

The advent of next generation sequencing (NGS) techniques has greatly simplified the molecular diagnosis and gene identification in very rare and highly heterogeneous Mendelian disorders. Over the last two years, these approaches, especially whole exome sequencing (WES), alone or combined with homozygosity mapping and linkage analysis, have proved to be successful in the identification of more than 25 new causative retinal dystrophy genes. NGS-approaches have also identified a wealth of new mutations in previously reported genes and have provided more comprehensive information concerning the landscape of genotype-phenotype correlations and the genetic complexity/diversity of human control populations. Although whole genome sequencing is far more informative than WES, the functional meaning of the genetic variants identified by the latter can be more easily interpreted, and final diagnosis of inherited retinal dystrophies is extremely successful, reaching 80%, particularly for recessive cases. Even considering the present limitations of WES, the reductions in costs and time, the continual technical improvements, the implementation of refined bioinformatic tools and the unbiased comprehensive genetic information it provides, make WES a very promising diagnostic tool for routine clinical and genetic diagnosis in the future.     

Solving the molecular diagnostic testing conundrum for Mendelian disorders in the era of next-generation sequencing: single-gene,gene panel,or exome/genome sequencing

Next-generation sequencing is changing the paradigm of clinical genetic testing. Today there are numerous molecular tests available, including single-gene tests, gene panels, and exome sequencing or genome sequencing. As a result, ordering physicians face the conundrum of selecting the best diagnostic tool for their patients with genetic conditions. Single-gene testing is often most appropriate for conditions with distinctive clinical features and minimal locus heterogeneity. Next-generation sequencing–based gene panel testing, which can be complemented with array comparative genomic hybridization and other ancillary methods, provides a comprehensive and feasible approach for heterogeneous disorders. Exome sequencing and genome sequencing have the advantage of being unbiased regarding what set of genes is analyzed, enabling parallel interrogation of most of the genes in the human genome. However, current limitations of next-generation sequencing technology and our variant interpretation capabilities caution us against offering exome sequencing or genome sequencing as either stand-alone or first-choice diagnostic approaches. A growing interest in personalized medicine calls for the application of genome sequencing in clinical diagnostics, but major challenges must be addressed before its full potential can be realized. Here, we propose a testing algorithm to help clinicians opt for the most appropriate molecular diagnostic tool for each scenario.Genet Med17 6, 444–451.     

Comprehensive next-generation sequence analyses of the entire mitochondrial genome reveal new insights into the molecular diagnosis of mitochondrial DNA disorders

PurposeThe application of massively parallel sequencing technology to the analysis of the mitochondrial genome has demonstrated great improvement in the molecular diagnosis of mitochondrial DNA–related disorders. The objective of this study was to investigate the performance characteristics and to gain new insights into the analysis of the mitochondrial genome.MethodsThe entire mitochondrial genome was analyzed as a single amplicon using a long-range PCR–based enrichment approach coupled with massively parallel sequencing. The interference of the nuclear mitochondrial DNA homologs was distinguished from the actual mitochondrial DNA sequences by comparison with the results obtained from conventional PCR–based Sanger sequencing using multiple pairs of primers.ResultsOur results demonstrated the uniform coverage of the entire mitochondrial genome. Massively parallel sequencing of the single amplicon revealed the presence of single-nucleotide polymorphisms and nuclear homologs of mtDNA sequences that cause the erroneous and inaccurate variant calls when PCR/Sanger sequencing approach was used. This single amplicon massively parallel sequencing strategy provides an accurate quantification of mutation heteroplasmy as well as the detection and mapping of mitochondrial DNA deletions.ConclusionThe ability to quantitatively and qualitatively evaluate every single base of the entire mitochondrial genome is indispensible to the accurate molecular diagnosis and genetic counseling of mitochondrial DNA–related disorders. This new approach may be considered as first-line testing for comprehensive analysis of the mitochondrial genome.     

Novel myosin mutations for hereditary hearing loss revealed by targeted genomic capture and massively parallel sequencing

Hereditary hearing loss is genetically heterogeneous, with a large number of genes and mutations contributing to this sensory, often monogenic, disease. This number, as well as large size, precludes comprehensive genetic diagnosis of all known deafness genes. A combination of targeted genomic capture and massively parallel sequencing (MPS), also referred to as next-generation sequencing, was applied to determine the deafness-causing genes in hearing-impaired individuals from Israeli Jewish and Palestinian Arab families. Among the mutations detected, we identified nine novel mutations in the genes encoding myosin VI, myosin VIIA and myosin XVA, doubling the number of myosin mutations in the Middle East. Myosin VI mutations were identified in this population for the first time. Modeling of the mutations provided predicted mechanisms for the damage they inflict in the molecular motors, leading to impaired function and thus deafness. The myosin mutations span all regions of these molecular motors, leading to a wide range of hearing phenotypes, reinforcing the key role of this family of proteins in auditory function. This study demonstrates that multiple mutations responsible for hearing loss can be identified in a relatively straightforward manner by targeted-gene MPS technology and concludes that this is the optimal genetic diagnostic approach for identification of mutations responsible for hearing loss.     

Mammalian sex determination—insights from humans and mice

Disorders of sex development (DSD) are congenital conditions in which the development of chromosomal, gonadal, or anatomical sex is atypical. Many of the genes required for gonad development have been identified by analysis of DSD patients. However, the use of knockout and transgenic mouse strains have contributed enormously to the study of gonad gene function and interactions within the development network. Although the genetic basis of mammalian sex determination and differentiation has advanced considerably in recent years, a majority of 46,XY gonadal dysgenesis patients still cannot be provided with an accurate diagnosis. Some of these unexplained DSD cases may be due to mutations in novel DSD genes or genomic rearrangements affecting regulatory regions that lead to atypical gene expression. Here, we review our current knowledge of mammalian sex determination drawing on insights from human DSD patients and mouse models.     

Ambiguous genitalia: what prenatal genetic testing is practical?

Concern for ambiguous genitalia or chromosome-phenotype discordance detected in a prenatal setting has increased over the last two decades. Practitioners faced with this prenatal finding have a variety of genetic tests available to them; however, it is unclear to what extent prenatal testing for disorders of sex development (DSD) is useful or practical. We undertook a retrospective review of the medical records of 140 individuals evaluated through the DSD clinic at Seattle Children's Hospital with birthdates from 01/01/1994 through 08/16/2011 to determine the rate of prenatal detection of ambiguous genitalia in individuals with DSD, what prenatal diagnostic workup was undertaken, and the postnatal outcome, including whether a postnatal genetic diagnosis was confirmed. Of all 140 subjects, 34 (24%) were identified prenatally. The most common postnatal diagnoses were penoscrotal hypospadias with transposition of the scrotum with no known genetic cause (24/140; 17%) and 21-hydroxylase deficiency (20/140; 14%). Apart from these, no single diagnosis comprised more than a few cases. Prenatal diagnostic testing varied widely, from no tests to multiple molecular tests with amniotic fluid hormone concentrations. In the absence of other fetal anomalies or growth retardation on ultrasound, prenatal karyotype with fluorescence in situ hybridization for the SRY gene is the most useful test when ambiguous genitalia is suspected. Further prenatal testing for Smith-Lemli-Opitz syndrome in 46,XY individuals and congenital adrenal hyperplasia in 46,XX individuals may be considered. However, targeted molecular testing for rare DSD conditions in the absence of a family history of DSD has a low yield.     

Review of genetic testing in kidney disease patients: Diagnostic yield of single nucleotide variants and copy number variations evaluated across and within kidney phenotype groups

Genetic kidney disease comprises a diverse group of disorders. These can roughly be divided in the phenotype groups congenital anomalies of the kidney and urinary tract, ciliopathies, glomerulopathies, stone disorders, tubulointerstitial kidney disease, and tubulopathies. Many etiologies can lead to chronic kidney disease that can progress to end-stage kidney disease. Despite each individual disease being rare, together these genetic disorders account for a large proportion of kidney disease cases. With the introduction of massively parallel sequencing, genetic testing has become more accessible, but a comprehensive analysis of the diagnostic yield is lacking. This review gives an overview of the diagnostic yield of genetic testing across and within the full range of kidney disease phenotypes through a systematic literature search that resulted in 115 included articles. Patient, test, and cohort characteristics that can influence the diagnostic yield are highlighted. Detection of copy number variations and their contribution to the diagnostic yield is described for all phenotype groups. Also, the impact of a genetic diagnosis for a patient and family members, which can be diagnostic, therapeutic, and prognostic, is shown through the included articles. This review will allow clinicians to estimate an a priori probability of finding a genetic cause for the kidney disease in their patients.     

Molecular genetics of hypospadias and cryptorchidism recent developments

During the last decade, a tremendous amount of work has been devoted to the study of the molecular genetics of isolated hypospadias and cryptorchidism, two minor forms of disorders of sex development (DSD). Beyond the genes involved in gonadal determination and sex differentiation, including those underlying androgen biosynthesis and signaling, new genes have been identified through genome-wide association study and familial clustering. Even if no single genetic defect can explain the whole spectrum of DSD, these recent studies reinforce the strong role of the genetic background in the occurrence of these defects. The timing of signaling disruption may explain the different phenotypes.     

Clinical utility of a targeted next generation sequencing panel in severe and pediatric onset Mendelian diseases

Next generation sequencing has provided great advancements in genetic diagnosis of Mendalian disorders. Simultaneous sequencing of many genes has become increasingly cheaper and faster. Recently, a number of gene panels have been established for the diagnosis of specific disease groups. The aim of this study is to evaluate the utility of an inherited disease panel in pediatric onset Mendelian diseases. Two hundred and seventeen probands and 10 carriers molecularly analyzed using a TruSight Inherited Disease^® Panel which included 552 genes responsible for pediatric onset Mendelian disorders, were enrolled in the study. The clinical phenotype, sequencing data, pretest and posttest diagnoses were evaluated. The patients in the study were classified into two groups. Group 1 (n:209) included the patients having a clinical diagnosis prior to molecular analysis. Group 2 (n:18) included the patients undiagnosed clinically prior to molecular analysis. Targeted panel provided a molecular diagnosis in 37% (84 of 227 cases) of all cases. The molecular diagnostic rate was 40.2% in patients with a specific prior clinical diagnosis. However, in patients having no primary clinical diagnosis no pathogenic variants were found. In 14 patients, a molecular diagnosis differing from the established clinical diagnosis was made. In conclusion, a targeted panel covering a high number of genes responsible for broad phenotypic spectrum can provide improved levels of diagnosis in patients with pediatric onset Mendelian diseases. A careful clinical evaluation of patients prior to the application of a next generation sequencing method leads to the best alternative approach for a conclusive molecular diagnosis.     

Novel candidate genes for 46,XY gonadal dysgenesis identified by a customized 1 M array-CGH platform

Half of all patients with a disorder of sex development (DSD) do not receive a specific molecular diagnosis. Comparative genomic hybridization (CGH) can detect copy number changes causing gene haploinsufficiency or over-expression that can lead to impaired gonadal development and gonadal DSD. The purpose of this study was to identify novel candidate genes for 46,XY gonadal dysgenesis (GD) using a customized 1 M array-CGH platform with whole-genome coverage and probe enrichment targeting 78 genes involved in sex development. Fourteen patients with 46,XY gonadal DSD were enrolled in the study. Nine individuals were analyzed by array CGH. All patients were included in a follow up sequencing study of candidate genes. Three novel candidate regions for 46,XY GD were identified in two patients. An interstitial duplication of the SUPT3H gene and a deletion of C2ORF80 were detected in a pair of affected siblings. Sequence analysis of these genes in all patients revealed no additional mutations. A large duplication highlighting PIP5K1B, PRKACG and FAM189A2 as candidates for 46,XY GD, were also detected. All five genes are expressed in testicular tissues, and one is shown to cause gonadal DSD in mice. However detailed functional information is lacking for these genes.     

Identification of the first homozygous 1‐bp deletion in GDF9 gene leading to primary ovarian insufficiency by using targeted massively parallel sequencing

Targeted massively parallel sequencing (TMPS) has been used in genetic diagnosis for Mendelian disorders. In the past few years, the TMPS has identified new and already described genes associated with primary ovarian insufficiency (POI) phenotype. Here, we performed a targeted gene sequencing to find a genetic diagnosis in idiopathic cases of Brazilian POI cohort. A custom SureSelect^XT DNA target enrichment panel was designed and the sequencing was performed on Illumina NextSeq sequencer. We identified 1 homozygous 1‐bp deletion variant (c.783delC) in the GDF9 gene in 1 patient with POI. The variant was confirmed and segregated using Sanger sequencing. The c.783delC GDF9 variant changed an amino acid creating a premature termination codon (p.Ser262Hisfs*2). This variant was not present in all public databases (ExAC/gnomAD, NHLBI/EVS and 1000Genomes). Moreover, it was absent in 400 alleles from fertile Brazilian women screened by Sanger sequencing. The patient's mother and her unaffected sister carried the c.783delC variant in a heterozygous state, as expected for an autosomal recessive inheritance. Here, the TMPS identified the first homozygous 1‐bp deletion variant in GDF9. This finding reveals a novel inheritance pattern of pathogenic variant in GDF9 associated with POI, thus improving the genetic diagnosis of this disorder.     

Massively parallel sequencing and identification of genes for primary lymphoedema: a perfect fit

Primary lymphoedema is a clinically and genetically heterogeneous group of disorders characterized by disruption of the lymphatic system. To date, the majority of the causative genes in primary lymphoedema have been identified through linkage analysis in large families with multiple affected subjects. Studies aimed at isolating additional genes responsible for primary lymphoedema have been hampered by cohorts comprised primarily of sporadic cases and small affected kindreds. In the absence of genetic heterogeneity, recent development of massively parallel DNA sequencing technology, specifically exome sequencing, has provided novel paradigms for disease gene identification in such cohorts. In this review, we summarize the novel approaches to disease gene discovery with massively parallel sequencing also known as Next Generation Sequencing (NGS), and show how the selection of unrelated affected cases from clinically homogenous phenotypic subclassifications is proving to be a successful approach for disease gene discovery in primary lymphoedema.     

Comprehensive massive parallel DNA sequencing strategy for the genetic diagnosis of the neuro-cardio-facio-cutaneous syndromes

Variants in 11 genes of the RAS/MAPK signaling pathway have been causally linked to the neuro-cardio-facio-cutaneous syndromes group (NCFCS). Recently, A2ML1 and RIT1 were also associated with these syndromes. Because of the genetic and clinical heterogeneity of NCFCS, it is challenging to define strategies for their molecular diagnosis. The aim of this study was to develop and validate a massive parallel sequencing (MPS)-based strategy for the molecular diagnosis of NCFCS. A multiplex PCR-based strategy for the enrichment of the 13 genes and a variant prioritization pipeline was established. Two sets of genomic DNA samples were studied using the Ion PGM System: (1) training set (n=15) to optimize the strategy and (2) validation set (n=20) to validate and evaluate the power of the new methodology. Sanger sequencing was performed to confirm all variants and low covered regions. All variants identified by Sanger sequencing were detected with our MPS approach. The methodology resulted in an experimental approach with a specificity of 99.0% and a maximum analytical sensitivity of ≥98.2% with a confidence of 99%. Importantly, two patients (out of 20) harbored described disease-causing variants in genes that are not routinely tested (RIT1 and SHOC2). The addition of less frequently altered genes increased in ≈10% the diagnostic yield of the strategy currently used. The presented workflow provides a comprehensive genetic screening strategy for patients with NCFCS in a fast and cost-efficient manner. This approach demonstrates the potential of a combined MPS–Sanger sequencing-based strategy as an effective diagnostic tool for heterogeneous diseases.     

Diagnostic Exome Sequencing to Elucidate the Genetic Basis of Likely Recessive Disorders in Consanguineous Families

Rare, atypical, and undiagnosed autosomal‐recessive disorders frequently occur in the offspring of consanguineous couples. Current routine diagnostic genetic tests fail to establish a diagnosis in many cases. We employed exome sequencing to identify the underlying molecular defects in patients with unresolved but putatively autosomal‐recessive disorders in consanguineous families and postulated that the pathogenic variants would reside within homozygous regions. Fifty consanguineous families participated in the study, with a wide spectrum of clinical phenotypes suggestive of autosomal‐recessive inheritance, but with no definitive molecular diagnosis. DNA samples from the patient(s), unaffected sibling(s), and the parents were genotyped with a 720K SNP array. Exome sequencing and array CGH (comparative genomic hybridization) were then performed on one affected individual per family. High‐confidence pathogenic variants were found in homozygosity in known disease‐causing genes in 18 families (36%) (one by array CGH and 17 by exome sequencing), accounting for the clinical phenotype in whole or in part. In the remainder of the families, no causative variant in a known pathogenic gene was identified. Our study shows that exome sequencing, in addition to being a powerful diagnostic tool, promises to rapidly expand our knowledge of rare genetic Mendelian disorders and can be used to establish more detailed causative links between mutant genotypes and clinical phenotypes.     

Phenotypic predictors and final diagnoses in patients referred for RASopathy testing by targeted next-generation sequencing

IntroductionRASopathies include disorders generally characterized by developmental delay, specific heart defects, short stature, cardiac hypertrophy, and facial dysmorphisms. Next-generation sequencing (NGS)-based panels have widespread acceptance as a diagnostic tool for RASopathies.Materials and methodsThe first 126 patients evaluated by clinical examination and the NGS RASopathy panel at the Children’s Hospital of Philadelphia were enrolled. We calculated diagnosis rate, correlated reported clinical findings with positive or negative test results, and identified final molecular diagnoses in 28/96 patients who tested negative for RASopathies.ResultsTwenty-four patients had pathogenic variants on the RASopathy panel, for a diagnostic yield of 19%. Reported features of pulmonic stenosis and ptosis were significantly correlated with a positive test result; no reported features were significantly correlated with a negative test result. We identified 27 different alternative diagnoses for patients originally suspected of having RASopathies.DiscussionThis study provides information that can assist in guiding differential diagnosis and genetic testing for patients suspected of having a RASopathy disorder.Genet Med advance online publication 20 October 2016     

A Post‐Hoc Comparison of the Utility of Sanger Sequencing and Exome Sequencing for the Diagnosis of Heterogeneous Diseases

The advent of massive parallel sequencing is rapidly changing the strategies employed for the genetic diagnosis and research of rare diseases that involve a large number of genes. So far it is not clear whether these approaches perform significantly better than conventional single gene testing as requested by clinicians. The current yield of this traditional diagnostic approach depends on a complex of factors that include gene‐specific phenotype traits, and the relative frequency of the involvement of specific genes. To gauge the impact of the paradigm shift that is occurring in molecular diagnostics, we assessed traditional Sanger‐based sequencing (in 2011) and exome sequencing followed by targeted bioinformatics analysis (in 2012) for five different conditions that are highly heterogeneous, and for which our center provides molecular diagnosis. We find that exome sequencing has a much higher diagnostic yield than Sanger sequencing for deafness, blindness, mitochondrial disease, and movement disorders. For microsatellite‐stable colorectal cancer, this was low under both strategies. Even if all genes that could have been ordered by physicians had been tested, the larger number of genes captured by the exome would still have led to a clearly superior diagnostic yield at a fraction of the cost.     

Mutational spectrum of Korean patients with corneal dystrophy

Corneal dystrophy typically refers to a group of rare hereditary disorders with a heterogeneous genetic background. A comprehensive molecular genetic analysis was performed to characterize the genetic spectrum of corneal dystrophies in Korean patients. Patients with various corneal dystrophies underwent thorough ophthalmic examination, histopathologic examination, and Sanger sequencing. A total of 120 probands were included, with a mean age of 50 years (SD = 18 years) and 70% were female. A total of 26 mutations in five genes (14 clearly pathogenic and 12 likely pathogenic) were identified in 49 probands (41%). Epithelial–stromal TGFBI dystrophies, macular corneal dystrophy and Schnyder corneal dystrophy (SCD) showed 100% mutation detection rates, while endothelial corneal dystrophies showed lower detection rates of 3%. Twenty six non‐duplicate mutations including eight novel mutations were identified and mutations associated with SCD were identified genetically for the first time in this population. This study provides a comprehensive characterization of the genetic aberrations in Korean patients and also highlights the diagnostic value of molecular genetic analysis in corneal dystrophies.