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Biallelic loss‐of‐function variants in the thrombospondin‐type laminin G domain and epilepsy‐associated repeats (TSPEAR) gene have recently been associated with ectodermal dysplasia and hearing loss. The first reports describing a TSPEAR disease association identified this gene is a cause of nonsyndromic hearing loss, but subsequent reports involving additional affected families have questioned this evidence and suggested a stronger association with ectodermal dysplasia. To clarify genotype–phenotype associations for TSPEAR variants, we characterized 13 individuals with biallelic TSPEAR variants. Individuals underwent either exome sequencing or panel‐based genetic testing. Nearly all of these newly reported individuals (11/13) have phenotypes that include tooth agenesis or ectodermal dysplasia, while three newly reported individuals have hearing loss. Of the individuals displaying hearing loss, all have additional variants in other hearing‐loss‐associated genes, specifically TMPRSS3, GJB2, and GJB6, that present competing candidates for their hearing loss phenotype. When presented alongside previous reports, the overall evidence supports the association of TSPEAR variants with ectodermal dysplasia and tooth agenesis features but creates significant doubt as to whether TSPEAR variants are a monogenic cause of hearing loss. Further functional evidence is needed to evaluate this phenotypic association.

Purpose The ability of a single technology, next-generation sequencing, to provide both sequence and copy number variant (CNV) results has driven the merger of clinical cytogenetics and molecular genetics. Consequently, the distinction between the definition of a sequence variant and a CNV is blurry. As the 2015 American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) standards and guidelines for interpretation of sequence variants address CNV classification only sparingly, this study focused on adapting ACMG/AMP criteria for single-gene CNV interpretation. Methods CNV-specific modifications of the 2015 ACMG/AMP criteria were developed and their utility was independently tested by three diagnostic laboratories. Each laboratory team interpreted the same 12 single-gene CNVs using three systems: (1) without ACMG/AMP guidance, (2) with ACMG/AMP criteria, and (3) with new modifications. A replication study of 12 different CNVs validated the modified criteria. Results The adapted criteria system presented here showed improved concordance and usability for single-gene CNVs compared with using the ACMG/AMP interpretation guidelines focused on sequence variants. Conclusion These single-gene CNV criteria modifications could be used as a supplement to the ACMG/AMP guidelines for sequence variants, allowing for a streamlined workflow and a step toward a uniform classification system for both sequence and copy number alterations.

Facioscapulohumeral muscular dystrophy (FSHD) is the third most common form of muscular dystrophy. The disease is named by the initial pattern of weakness, with patients developing facial weakness, scapular winging, and foot drop. As the disorder progresses, other muscles, particularly truncal muscles, are affected and may lead to loss of ambulation. The genetic mechanism has been more clearly elucidated in the past decade. It is clear that the central pathogenic mechanism is epigenetic derepression. A downstream consequence of depression of the D4Z4 units on the long arm of chromosome 4 is expression of DUX4 , which is known to only be naturally expressed early in embryogenesis. In FSHD, expression of DUX4 is the key and leads to muscle damage and fibrosis. The most common mechanism resulting in DUX4 derepression is a deletion of multiple integral copies of the D4Z4 repetitive element on chromosome 4.1 What has been evolving is the discovery of other less common causes of the epigenetic derepression, including those in other genes that have a role in chromatin repression ( SMCHD1 and DNMT3B ).2,3

Next-generation sequencing (NGS) is now routinely used to interrogate large sets of genes in a diagnostic setting. Regions of high sequence homology continue to be a major challenge for short-read technologies and can lead to false-positive and false-negative diagnostic errors. At the scale of whole-exome sequencing (WES), laboratories may be limited in their knowledge of genes and regions that pose technical hurdles due to high homology. We have created an exome-wide resource that catalogs highly homologous regions that is tailored toward diagnostic applications. This resource was developed using a mappability-based approach tailored to current Sanger and NGS protocols. Gene-level and exon-level lists delineate regions that are difficult or impossible to analyze via standard NGS. These regions are ranked by degree of affectedness, annotated for medical relevance, and classified by the type of homology (within-gene, different functional gene, known pseudogene, uncharacterized noncoding region). Additionally, we provide a list of exons that cannot be analyzed by short-amplicon Sanger sequencing. This resource can help guide clinical test design, supplemental assay implementation, and results interpretation in the context of high homology. Genet Med 18 12, 1282–1289.

The field of clinical genetics has advanced at an unprecedented pace. Today, with the aid of several high-resolution and high-precision technologies, physicians are able to make molecular genetic diagnoses for many infants affected with genetic disease. It is imperative, however, that perinatologists and neonatologists understand the strengths and limitations of genetic testing. This article discusses the different genetic testing options available for perinatal and neonatal diagnostics, along with their clinical utilities and indications. From variant-specific testing to whole-exome and genome sequencing, the article covers the whole gamut of genetic testing, with some thoughts on the changing paradigm of medical genetics.

Wagner syndrome and erosive vitreoretinopathy together constitute the phenotypic continuum of an autosomal dominant vitreoretinopathy, with clinical findings typically isolated to the eye. The disease is caused by pathogenic variants in the VCAN gene and all such variants reported to date are those that plausibly result in haploinsufficiency of exon 8 containing vcan transcripts. Here, we report the molecular findings and long-term follow-up of a 16-year-old female with a history of retinal detachments and pigmentary retinal changes. Next-generation sequencing and microarray analysis of 141 genes established a diagnosis of Wagner syndrome in this individual, by detection of an 11.7 kilobase (kb) deletion encompassing exon 8 of VCAN. In light of the emerging functions and roles of versican protein in human disease, we discuss how variants within exon 8 of the VCAN gene can be compared to those in exon 2 of the COL2A1 gene that cause atypical Stickler syndrome and propose that variants in other regions of the gene can be expected to present with a more systemic disease. The distinctive facial features and atypical gastrointestinal symptoms observed in this long-term follow-up study support the possibility that individuals with VCAN-related vitreoretinopathy may have extra-ocular clinical features.

South Asian Indians represent a sixth of the world's population and are a racially, geographically, and genetically diverse people. Their unique anthropological structure, prevailing caste system, and ancient religious practices have all impacted the genetic composition of most of the current-day Indian population. With the evolving socio-religious and economic activities of the subsects and castes, endogamous and consanguineous marriages became a commonplace. Consequently, the frequency of founder mutations and the burden of heritable genetic disorders rose significantly. Specifically, the incidence of certain autosomal-recessive disorders is relatively high in select Indian subpopulations and communities that share common recent ancestry. Although today clinical genetics and molecular diagnostic services are making inroads in India, the high costs associated with the technology and the tests often keep patients from an exact molecular diagnosis, making more customized and tailored tests, such as those interrogating the most common and founder mutations or those that cater to select sects within the population, highly attractive. These tests offer a quick first-hand affordable diagnostic and carrier screening tool. Here, we provide a comprehensive catalog of known common mutations and founder mutations in the Indian population and discuss them from a molecular, clinical, and historical perspective.

Dihydropyrimidine dehydrogenase (DPD) encoded by DPYD gene is the major enzyme involved in metabolism of 5-flurouracil (5-FU), a pyrimidine analogue used in cancer chemotherapy. Although very effective as a cancer therapeutic drug, if not rapidly metabolized, 5-FU may prove lethal. Single nucleotide variants (SNVs) within DPYD that modulate DPD enzyme activity contribute to 5-FU toxicity.This study looked for DPYD SNVs common in the Indian population that might be associated with variable DPD activity and drug toxicity. To achieve this, sequencing analysis was performed of all 23 exons and flanking intronic regions of the DPYD gene in a cohort of 50 healthy adult Indians. This study detected 22 SNVs including intronic, synonymous and non-synonymous changes in the DPYD gene, of which six have not been documented before. Allelic frequency was calculated for the observed variants and linkage disequilibrium (LD) analysis was performed on variants with frequency ≥0.1 to identify haplotypes.This study provides a brief overview of the genetic polymorphism in DPYD in Indians and emphasizes the need for a large scale extensive study to establish markers associated with the frequently observed variable drug metabolism.

Dysferlinopathies are autosomal recessive inherited muscular dystrophies caused by mutations in the gene DYSF. Dysferlin is primarily expressed in skeletal muscle, cardiac muscle, and peripheral blood monocytes. Expression in skeletal muscle and monocytes strongly correlates in healthy and disease states. We evaluated the efficiency of the monocyte assay to detect carriers and to determine the carrier frequency of dysferlinopathies in the general population. We enrolled 149 healthy volunteers and collected peripheral blood samples for protein analysis. While 18 of these individuals with protein levels in the range of 40%-64% were predicted to be carriers by the monocyte assay, subsequent DYSF sequencing analysis in 14 of 18 detected missense variants in only four. Analysis of DNA methylation patterns at the DYSF locus showed no changes in methylation levels at CpG islands and shores between samples. Our results suggest that: (1) dysferlin expression can also be regulated by factors outside of the dysferlin gene, but not related to DNA methylation; (2) carrier frequency and therefore the number of affected individuals could be higher than previously estimated; and (3) although reliable for evaluating dysferlinopathies, the monocyte assay cannot be used to determine the carrier status; for this, a molecular analysis of DYSF must be performed.

Cytidine deaminase (CDA) is the major enzyme involved in metabolism of gemcitabine, a pyrimidine analog widely used for chemotherapy of solid tumors. While only low amounts of administered gemcitabine undergo intracellular phosphorylation into active forms and involve in antineoplastic activities, majority of it is rapidly inactivated by CDA and excreted to avoid drug toxicity. Knowledge of the genetic polymorphisms mildly effecting cellular activity of the enzyme CDA is therefore crucial to understanding drug-induced toxicities associated with gemcitabine. Functional significance and allele frequencies for common SNPs including 79A>C (*2) and 208G>A (*3) have been reported in various ethnic populations including Caucasian, African, Korean and Japanese. However, such studies have not been reported in any Indian sub-population. In the present study, conventional polymerase chain reaction (PCR) based amplification using gene specific primers and Sanger sequencing were performed to identify CDA variants in 50 healthy individuals from Indian sub-population. Established common variant 79A>C known to reduce CDA activity was observed at a frequency of 0.14 in the study cohort. In addition to other known variants, one novel variant, c.325-209T>C was detected at a frequency of 0.06. Genetic variants in CDA gene and their frequencies established in our study hold value in pharmacogenetics.

New antimalarials are urgently needed. We have shown that tetrahydroquinoline (THQ) protein farnesyltransferase (PFT) inhibitors (PFTIs) are effective against the Plasmodium falciparum PFT and are effective at killing P. falciparum in vitro. Previously described THQ PFTIs had limitations of poor oral bioavailability and rapid clearance from the circulation of rodents. In this paper, we validate both the Caco-2 cell permeability model for predicting THQ intestinal absorption and the in vitro liver microsome model for predicting THQ clearance in vivo. Incremental improvements in efficacy, oral absorption, and clearance rate were monitored by in vitro tests; and these tests were followed up with in vivo absorption, distribution, metabolism, and excretion studies. One compound, PB-93, achieved cure when it was given orally to P. berghei- infected rats every 8 h for a total of 72 h. However, PB-93 was rapidly cleared, and dosing every 12 h failed to cure the rats. Thus, the in vivo results corroborate the in vitro pharmacodynamics and demonstrate that 72 h of continuous high-level exposure to PFTIs is necessary to kill plasmodia. The metabolism of PB-93 was demonstrated by a novel technique that relied on double labeling with a radiolabel and heavy isotopes combined with radiometric liquid chromatography and mass spectrometry. The major liver microsome metabolite of PB-93 has the PFT Zn-binding N -methyl-imidazole removed; this metabolite is inactive in blocking PFT function. By solving the X-ray crystal structure of PB-93 bound to rat PFT, a model of PB-93 bound to malarial PFT was constructed. This model suggests areas of the THQ PFTIs that can be modified to retain efficacy and protect the Zn-binding N -methyl-imidazole from dealkylation.

Nemaline myopathy (NM) is a congenital neuromuscular disorder often characterized by hypotonia, facial weakness, skeletal muscle weakness, and the presence of rods on muscle biopsy. A rare form of nemaline myopathy known as Amish Nemaline Myopathy has only been seen in a genetically isolated cohort of Old Order Amish patients who may additionally present with tremors in the first 2-3 months of life.We describe an Hispanic male diagnosed with nemaline myopathy histopathologically and subsequently confirmed by next generation gene sequencing.Direct sequencing revealed that he is homozygous for a pathogenic nonsense variant c.323CG (p.S108X) in exon 9 of the TNNT1 gene.This report describes a novel pathogenic variant in the TNNT1 gene and represents a nemaline myopathy-causing variant in the TNNT1 gene outside of the Old Order Amish and Dutch ancestry.

Neuromuscular diseases (NMDs) are a group of200 highly genetically as well as clinically heterogeneous inherited genetic disorders that affect the peripheral nervous and muscular systems, resulting in gross motor disability. The clinical and genetic heterogeneities of NMDs make disease diagnosis complicated and expensive, often involving multiple tests.To expedite the molecular diagnosis of NMDs, we designed and validated several next generation sequencing (NGS)-based comprehensive gene panel tests that include complementary deletion and duplication testing through comparative genomic hybridization arrays. Our validation established the targeted gene panel test to have 100% sensitivity and specificity for single nucleotide variant detection. To compare the clinical diagnostic yields of single gene (NMD-associated) tests with the various NMD NGS panel tests, we analyzed data from all clinical tests performed at the Emory Genetics Laboratory from October 2009 through May 2014. We further compared the clinical utility of the targeted NGS panel test with that of exome sequencing (ES).We found that NMD comprehensive panel testing has a 3-fold greater diagnostic yield (46%) than single gene testing (15-19%). Sanger fill-in of low-coverage exons, copy number variation analysis, and thorough in-house validation of the assay all complement panel testing and allow the detection of all types of causative pathogenic variants, some of which (about 18%) may be missed by ES.Our results strongly indicate that for molecular diagnosis of heterogeneous disorders such as NMDs, targeted panel testing has the highest clinical yield and should therefore be the preferred first-tier approach.

Currently, oncology testing includes molecular studies and cytogenetic analysis to detect genetic aberrations of clinical significance. Next-generation sequencing (NGS) allows rapid analysis of multiple genes for clinically actionable somatic variants. The WUCaMP assay uses targeted capture for NGS analysis of 25 cancer-associated genes to detect mutations at actionable loci. We present clinical validation of the assay and a detailed framework for design and validation of similar clinical assays. Deep sequencing of 78 tumor specimens (≥1000× average unique coverage across the capture region) achieved high sensitivity for detecting somatic variants at low allele fraction (AF). Validation revealed sensitivities and specificities of 100% for detection of single-nucleotide variants (SNVs) within coding regions, compared with SNP array sequence data (95% CI = 83.4–100.0 for sensitivity and 94.2–100.0 for specificity) or whole-genome sequencing (95% CI = 89.1–100.0 for sensitivity and 99.9–100.0 for specificity) of HapMap samples. Sensitivity for detecting variants at an observed 10% AF was 100% (95% CI = 93.2–100.0) in HapMap mixes. Analysis of 15 masked specimens harboring clinically reported variants yielded concordant calls for 13/13 variants at AF of ≥15%. The WUCaMP assay is a robust and sensitive method to detect somatic variants of clinical significance in molecular oncology laboratories, with reduced time and cost of genetic analysis allowing for strategic patient management.

Dysferlin deficiency causes dysferlinopathies. Among peripheral blood mononuclear cells (PBMCs), the dysferlin protein is expressed specifically in CD14(+) monocytes.We quantified dysferlin protein levels in PBMC lysates of 77 individuals suspected clinically of having a dysferlinopathy to screen for true positives. Subsequent molecular confirmation was done by Sanger sequencing and comparative genomic hybridization arrays to establish diagnosis.Of the 44 individuals who had significantly reduced dysferlin levels (≤10%), 41 underwent molecular testing. We identified at least 1 mutation in 85% (35 of 41), and 2 mutations, establishing a dysferlinopathy diagnosis, in 61% (25 of 41) of these individuals. Among those with dysferlin protein levels of10% (33 of 77), only 1 individual (of 14 who underwent molecular testing) had a detectable mutation.Our results suggest that dysferlin protein levels of ≤10% in PBMCs, are highly indicative of primary dysferlinopathies. However, this assay may not distinguish carriers from those with secondary dysferlin reduction.

Clinical heterogeneity of limb-girdle muscular dystrophies (LGMDs, 24 known subtypes), which includes overlapping phenotypes and varying ages of onset and morbidity, adds complexity to clinical and molecular diagnoses.To diagnose LGMD subtype, protein analysis, using immunohistochemistry (IHC) and immunoblotting, was followed by gene sequencing through a panel approach (simultaneous sequencing of known LGMD genes) in 9 patients from unrelated families of the Indian Agarwal community. Haplotype studies were performed by targeted SNP genotyping to establish mutation segregation.We identified 2 founder mutations in CAPN3, a missense (c.2338GC; p.D780H) and a splice-site (c.2099-1GT) mutation, on 2 different haplotype backgrounds. The patients were either heterozygous for both or homozygous for either of these mutations.Founder mutations have immediate clinical application, at least in selected population groups. Clinically available gene panels may provide a definitive molecular diagnosis for heterogeneous disorders such as LGMD.

Non-allelic homologous recombination (NAHR), non-homologous end joining (NHEJ), and microhomology-mediated replication-dependent recombination (MMRDR) have all been put forward as mechanisms to explain DNA rearrangements associated with genomic disorders. However, many nonrecurrent rearrangements in humans remain unexplained. To further investigate the mutation mechanisms of these copy number variations (CNVs), we performed breakpoint mapping analysis for 62 clinical cases with intragenic deletions in the human DMD gene (50 cases) and other known disease-causing genes (one PCCB, one IVD, one DBT, three PAH, one STK11, one HEXB, three DBT, one HRPT1, and one EMD cases). While repetitive elements were found in only four individual cases, three involving DMD and one HEXB gene, microhomologies (2–10 bp) were observed at breakpoint junctions in 56% and insertions ranging from 1 to 48 bp were seen in 16 of the total 62 cases. Among these insertions, we observed evidence for tandem repetitions of short segments (5–20 bp) of reference sequence proximal to the breakpoints in six individual DMD cases (six repeats in one, four repeats in three, two repeats in one, and one repeat in one case), strongly indicating attempts by the replication machinery to surpass the stalled replication fork. We provide evidence of a novel template slippage event during replication rescue. With a deeper insight into the complex process of replication and its rescue during origin failure, brought forward by recent studies, we propose a hypothesis based on aberrant firing of replication origins to explain intragenic nonrecurrent rearrangements within genes, including the DMD gene.

The congenital muscular dystrophies (CMDs) comprise a heterogeneous group of heritable muscle disorders with often difficult to interpret muscle pathology, making them challenging to diagnose. Serial Sanger sequencing of suspected CMD genes, while the current molecular diagnostic method of choice, can be slow and expensive. A comprehensive panel test for simultaneous screening of mutations in all known CMD-associated genes would be a more effective diagnostic strategy. Thus, the CMDs are a model disorder group for development and validation of next-generation sequencing (NGS) strategies for diagnostic and clinical care applications. Using a highly multiplexed PCR-based target enrichment method (RainDance) in conjunction with NGS, we performed mutation detection in all CMD genes of 26 samples and compared the results with Sanger sequencing. The RainDance NGS panel showed great consistency in coverage depth, on-target efficiency, versatility of mutation detection, and genotype concordance with Sanger sequencing, demonstrating the test's appropriateness for clinical use. Compared to single tests, a higher diagnostic yield was observed by panel implementation. The panel's limitation is the amplification failure of select gene-specific exons which require Sanger sequencing for test completion. Successful validation and application of the CMD NGS panel to improve the diagnostic yield in a clinical laboratory was shown.