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Infertility affects 10% of reproductive-age women and is extremely heterogeneous in etiology. The genetic contribution to female infertility is incompletely understood, and involves chromosomal and single-gene defects. Our aim in this study is to decipher single-gene causes in infertile women in whom endocrinological, anatomical, and chromosomal causes have been excluded. Our cohort comprises women with recurrent pregnancy loss and no offspring from spontaneous pregnancies (RPL, n = 61) and those who never achieved clinical pregnancy and were referred for in vitro fertilization [primary infertility (PI), n = 14]. Whole-exome sequencing revealed candidate variants in 14, which represents 43% of those with PI and 13% of those with RPL. These include variants in previously established female infertility-related genes (TLE6, NLRP7, FSHR, and ZP1) as well as genes with only tentative links in the literature (NLRP5). Candidate variants in genes linked to primary ciliary dyskinesia (DNAH11 and CCNO) were identified in individuals with and without systemic features of the disease. We also identified variants in genes not previously linked to female infertility. These include one homozygous variant each in CCDC68, CBX3, CENPH, PABPC1L, PIF1, PLK1, and REXO4, which we propose as candidate genes for infertility based on their established biology or compatible animal models. Our study expands the contribution of single genes to the etiology of PI and RPL, improves the precision of disease classification at the molecular level, and offers the potential for future treatment and development of human genetics-inspired fertility regulators.

Purpose Male infertility remains poorly understood at the molecular level. We aimed in this study to investigate the yield of a “genomics first” approach to male infertility. Methods Patients with severe oligospermia and nonobstructive azoospermia were investigated using exome sequencing (ES) in parallel with the standard practice of chromosomal analysis. Results In 285 patients, 10.5% ( n  = 30) had evidence of chromosomal aberrations while nearly a quarter ( n  = 69; 24.2%) had a potential monogenic form of male infertility. The latter ranged from variants in genes previously reported to cause male infertility with or without other phenotypes in humans (24 patients; 8.4%) to those in novel candidate genes reported in this study (37 patients; 12.9%). The 33 candidate genes have biological links to male germ cell development including compatible mouse knockouts, and a few ( TERB1 [ CCDC79 ], PIWIL2 , MAGEE2 , and ZSWIM7 ) were found to be independently mutated in unrelated patients in our cohort. We also found that male infertility can be the sole or major phenotypic expression of a number of genes that are known to cause multisystemic manifestations in humans ( n  = 9 patients; 3.1%). Conclusion The standard approach to male infertility overlooks the significant contribution of monogenic causes to this important clinical entity.

Purpose Genetic testing in pediatric cholestasis can be very informative but genetic causes have not been fully characterized. Methods Exome sequencing and positional mapping in seven families with cholestatic liver disease and negative clinical testing for known disease genes. Results KIF12 , which encodes a microtubule motor protein with a tentative role in cell polarity, was found to harbor three homozygous likely deleterious variants in three families with sclerosing cholangitis. KIF12 expression is dependent on HNF-1β, deficiency which is known to cause bile duct dysmorphogenesis associated with loss of KIF12 expression. In another extended family, we mapped an apparently novel syndrome of sclerosing cholangitis, short stature, hypothyroidism, and abnormal tongue pigmentation in two cousins to a homozygous variant in PPM1F ( POPX2 ), a regulator of kinesin-mediated ciliary transport. In the fifth family, a syndrome of normal gamma glutamyltransferase (GGT) cholestasis and hearing loss was found to segregate with a homozygous truncating variant in USP53 , which encodes an interactor with TJP2. In the sixth family, we mapped a novel syndrome of transient neonatal cholestasis, intellectual disability, and short stature to a homozygous variant in LSR , an important regulator of liver development. In the last family of three affected siblings, a novel syndrome of intractable itching, hypercholanemia, short stature, and intellectual disability was mapped to a single locus that contains a homozygous truncating variant in WDR83OS ( C19orf56 ), known to interact with ATP13A2 and BSEP. Conclusion Our results expand the genetic heterogeneity of pediatric cholestatic liver disease and highlight the vulnerability of bile homeostasis to a wide range of molecular perturbations.

Purpose Ciliopathies are highly heterogeneous clinical disorders of the primary cilium. We aim to characterize a large cohort of ciliopathies phenotypically and molecularly. Methods Detailed phenotypic and genomic analysis of patients with ciliopathies, and functional characterization of novel candidate genes. Results In this study, we describe 125 families with ciliopathies and show that deleterious variants in previously reported genes, including cryptic splicing variants, account for 87% of cases. Additionally, we further support a number of previously reported candidate genes ( BBIP1 , MAPKBP1 , PDE6D , and WDPCP ), and propose nine novel candidate genes ( CCDC67 , CCDC96 , CCDC172 , CEP295 , FAM166B , LRRC34 , TMEM17 , TTC6 , and TTC23 ), three of which ( LRRC34 , TTC6 , and TTC23 ) are supported by functional assays that we performed on available patient-derived fibroblasts. From a phenotypic perspective, we expand the phenomenon of allelism that characterizes ciliopathies by describing novel associations including WDR19 -related Stargardt disease and SCLT1 - and CEP164 -related Bardet–Biedl syndrome. Conclusion In this cohort of phenotypically and molecularly characterized ciliopathies, we draw important lessons that inform the clinical management and the diagnostics of this class of disorders as well as their basic biology.

Background At least 50% of patients with suspected Mendelian disorders remain undiagnosed after whole-exome sequencing (WES), and the extent to which non-coding variants that are not captured by WES contribute to this fraction is unclear. Whole transcriptome sequencing is a promising supplement to WES, although empirical data on the contribution of RNA analysis to the diagnosis of Mendelian diseases on a large scale are scarce. Results Here, we describe our experience with transcript-deleterious variants (TDVs) based on a cohort of 5647 families with suspected Mendelian diseases. We first interrogate all families for which the respective Mendelian phenotype could be mapped to a single locus to obtain an unbiased estimate of the contribution of TDVs at 18.9%. We examine the entire cohort and find that TDVs account for 15% of all “solved” cases. We compare the results of RT-PCR to in silico prediction. Definitive results from RT-PCR are obtained from blood-derived RNA for the overwhelming majority of variants (84.1%), and only a small minority (2.6%) fail analysis on all available RNA sources (blood-, skin fibroblast-, and urine renal epithelial cells-derived), which has important implications for the clinical application of RNA-seq. We also show that RNA analysis can establish the diagnosis in 13.5% of 155 patients who had received “negative” clinical WES reports. Finally, our data suggest a role for TDVs in modulating penetrance even in otherwise highly penetrant Mendelian disorders. Conclusions Our results provide much needed empirical data for the impending implementation of diagnostic RNA-seq in conjunction with genome sequencing.

Background: Clinical exome sequencing (CES) has greatly improved the diagnostic process for individuals with suspected genetic disorders. However, the majority remains undiagnosed after CES. Although understanding potential reasons for this limited sensitivity is critical for improving the delivery of clinical genomics, research in this area has been limited. Materials and Methods: We first calculated the theoretical maximum sensitivity of CES by analyzing >100 families in whom a Mendelian phenotype is mapped to a single locus. We then tested the hypothesis that positional mapping can limit the search space and thereby facilitate variant interpretation by reanalyzing 33 families with “negative” CES and applying positional mapping. Results: We found that >95% of families who map to a single locus harbored genic (as opposed to intergenic) variants that are potentially identifiable by CES. Our reanalysis of “negative” CES revealed likely causal variants in the majority (88%). Several of these solved cases have undergone negative whole-genome sequencing. Conclusion: The discrepancy between the theoretical maximum and the actual clinical sensitivity of CES is primarily in the variant filtration rather than the variant capture and sequencing phase. The solution to negative CES is not necessarily in expanding the coverage but rather in devising approaches that improve variant filtration. We suggest that positional mapping is one such approach. Genet Med advance online publication 06 October 2016

Unlike disorders of primary cilium, primary ciliary dyskinesia (PCD) has a much narrower clinical spectrum consistent with the limited tissue distribution of motile cilia. Nonetheless, PCD diagnosis can be challenging due to the overlapping features with other disorders and the requirement for sophisticated tests that are only available in specialized centers. We performed exome sequencing on all patients with a clinical suspicion of PCD but for whom no nasal nitric oxide test or ciliary functional assessment could be ordered. Among 81 patients (56 families), in whom PCD was suspected, 68% had pathogenic or likely pathogenic variants in established PCD-related genes that fully explain the phenotype (20 variants in 11 genes). The major clinical presentations were sinopulmonary infections (SPI) (n = 58), neonatal respiratory distress (NRD) (n = 2), laterality defect (LD) (n = 6), and combined LD/SPI (n = 15). Biallelic likely deleterious variants were also encountered in AKNA and GOLGA3, which we propose as novel candidates in a lung phenotype that overlaps clinically with PCD. We also encountered a PCD phenocopy caused by a pathogenic variant in ITCH, and a pathogenic variant in CEP164 causing Bardet–Biedl syndrome and PCD presentation as a very rare example of the dual presentation of these two disorders of the primary and motile cilia. Exome sequencing is a powerful tool that can help “democratize” the diagnosis of PCD, which is currently limited to highly specialized centers.

Purpose: The purpose of this study is to describe recessive alleles in strictly dominant genes. Identifying recessive mutations in genes for which only dominant disease or risk alleles have been reported can expand our understanding of the medical relevance of these genes both phenotypically and mechanistically. The Saudi population is enriched for autozygosity, which enhances the homozygous occurrence of alleles, including pathogenic alleles in genes that have been associated only with a dominant inheritance pattern. Methods: Exome sequencing of patients from consanguineous families with likely recessive phenotypes was performed. In one family, the genotype of the deceased children was inferred from their parents due to lack of available samples. Results: We describe the identification of 11 recessive variants (5 of which are reported here for the first time) in 11 genes for which only dominant disease or risk alleles have been reported. The observed phenotypes for these recessive variants were novel (e.g., FBN2 -related myopathy and CSF1R -related brain malformation and osteopetrosis), typical (e.g., ACTG2 -related visceral myopathy), or an apparently healthy state (e.g., PDE11A ), consistent with the corresponding mouse knockout phenotypes. Conclusion: Our results show that, in the era of genomic sequencing and “reverse phenotyping,” recessive variants in dominant genes should not be dismissed based on perceived “incompatibility” with the patient’s phenotype before careful consideration. Genet Med advance online publication 06 April 2017

Myelin pathologies are an important cause of multifactorial, e.g., multiple sclerosis, and Mendelian, e.g., leukodystrophy, neurological disorders. CNP encodes a major component of myelin and its CNS expression is exclusive to myelin-forming oligodendrocytes. Deficiency of CNP in mouse causes a lethal white matter neurodegenerative phenotype. However, a corresponding human phenotype has not been described to date. Here, we describe a multiplex consanguineous family from Oman in which multiple affected members display a remarkably consistent phenotype of neuroregression with profound brain white matter loss. A novel homozygous missense variant in CNP was identified by combined autozygome/exome analysis. Immunoblot analysis suggests that this is a null allele in patient fibroblasts, which display abnormal F-actin organization. Our results suggest the establishment of a novel CNP-related hypomyelinating leukodystrophy in humans.

Arthrogryposis multiplex congenita (AMC) is an important birth defect with a significant genetic contribution. Many syndromic forms of AMC have been described, but remain unsolved at the molecular level. In this report, we describe a novel syndromic form of AMC in two multiplex consanguineous families from Saudi Arabia and Oman. The phenotype is highly consistent, and comprises neurogenic arthrogryposis, microcephaly, brain malformation (absent corpus callosum), optic atrophy, limb fractures, profound global developmental delay, and early lethality. Whole-exome sequencing revealed a different homozygous truncating variant in SCYL2 in each of the two families. SCYL2 is a component of clathrin-coated vesicles, and deficiency of its mouse ortholog results in a severe neurological phenotype that largely recapitulates the phenotype observed in our patients. Our results suggest that severe neurogenic arthrogryposis with brain malformation is the human phenotypic consequence of SCYL2 loss of function mutations.