OTUD5 Variants Associated With X-Linked Intellectual Disability and Congenital Malformation.

BACKGROUND: X-linked intellectual disability (XLID), which occurs predominantly in males, is a relatively common and genetically heterogeneous disorder in which over 100 mutated genes have been reported. The OTUD5 gene at Xp11.23 encodes ovarian tumor deubiquitinase 5 protein, which is a deubiquitinating enzyme member of the ovarian tumor family. LINKage-specific-deubiquitylation-deficiency-induced embryonic defects (LINKED) syndrome, arising from pathogenic OTUD5 variants, was recently reported as a new XLID with additional congenital anomalies. METHODS: We investigated three affected males (49- and 47-year-old brothers [Individuals 1 and 2] and a 2-year-old boy [Individual 3]) from two families who showed developmental delay. Their common clinical features included developmental delay, hypotonia, short stature, and distinctive facial features, such as telecanthus and a depressed nasal bridge. Individuals 1 and 2 showed epilepsy and brain magnetic resonance imaging showed a thin corpus callosum and mild ventriculomegaly. Individual 3 showed congenital malformations, including tetralogy of Fallot, hypospadias, and bilateral cryptorchidism. To identify the genetic cause of these features, we performed whole-exome sequencing. RESULTS: A hemizygous OTUD5 missense variant, c.878A>T, p.Asn293Ile [NM_017602.4], was identified in one family with Individuals 1 and 2, and another missense variant, c.1210 C>T, p.Arg404Trp, in the other family with Individual 3, respectively. The former variant has not been registered in public databases and was predicted to be pathogenic by multiple in silico prediction tools. The latter variant p.Arg404Trp was previously reported as a pathogenic OTUD5 variant, and Individual 3 showed a typical LINKED syndrome phenotype. However, Individuals 1 and 2, with the novel variant (p.Asn293Ile), showed no cardiac or genitourinary malformations. CONCLUSIONS: Unlike previous reports of LINKED syndrome, which described early lethality with congenital cardiac anomalies, our three cases are still alive. Notably, the adult brothers with the novel missense OTUD5 variant have lived into their forties. This may be indicative of a milder phenotype as a possible genotype-phenotype correlation. These findings imply a possible long-term prognosis for individuals with this new XLID syndrome, and a wider phenotypic variation than initially thought.

Genetic architecture of laterality defects revealed by whole exome sequencing.

Aberrant left-right patterning in the developing human embryo can lead to a broad spectrum of congenital malformations. The causes of most laterality defects are not known, with variants in established genes accounting for <20% of cases. We sought to characterize the genetic spectrum of these conditions by performing whole-exome sequencing of 323 unrelated laterality cases. We investigated the role of rare, predicted-damaging variation in 1726 putative laterality candidate genes derived from model organisms, pathway analyses, and human phenotypes. We also evaluated the contribution of homo/hemizygous exon deletions and gene-based burden of rare variation. A total of 28 candidate variants (26 rare predicted-damaging variants and 2 hemizygous deletions) were identified, including variants in genes known to cause heterotaxy and primary ciliary dyskinesia (ACVR2B, NODAL, ZIC3, DNAI1, DNAH5, HYDIN, MMP21), and genes without a human phenotype association, but with prior evidence for a role in embryonic laterality or cardiac development. Sanger validation of the latter variants in probands and their parents revealed no de novo variants, but apparent transmitted heterozygous (ROCK2, ISL1, SMAD2), and hemizygous (RAI2, RIPPLY1) variant patterns. Collectively, these variants account for 7.1% of our study subjects. We also observe evidence for an excess burden of rare, predicted loss-of-function variation in PXDNL and BMS1- two genes relevant to the broader laterality phenotype. These findings highlight potential new genes in the development of laterality defects, and suggest extensive locus heterogeneity and complex genetic models in this class of birth defects.

Whole exome sequencing in 342 congenital cardiac left sided lesion cases reveals extensive genetic heterogeneity and complex inheritance patterns.

BACKGROUND: Left-sided lesions (LSLs) account for an important fraction of severe congenital cardiovascular malformations (CVMs). The genetic contributions to LSLs are complex, and the mutations that cause these malformations span several diverse biological signaling pathways: TGFB, NOTCH, SHH, and more. Here, we use whole exome sequence data generated in 342 LSL cases to identify likely damaging variants in putative candidate CVM genes. METHODS: Using a series of bioinformatics filters, we focused on genes harboring population-rare, putative loss-of-function (LOF), and predicted damaging variants in 1760 CVM candidate genes constructed a priori from the literature and model organism databases. Gene variants that were not observed in a comparably sequenced control dataset of 5492 samples without severe CVM were then subjected to targeted validation in cases and parents. Whole exome sequencing data from 4593 individuals referred for clinical sequencing were used to bolster evidence for the role of candidate genes in CVMs and LSLs. RESULTS: Our analyses revealed 28 candidate variants in 27 genes, including 17 genes not previously associated with a human CVM disorder, and revealed diverse patterns of inheritance among LOF carriers, including 9 confirmed de novo variants in both novel and newly described human CVM candidate genes (ACVR1, JARID2, NR2F2, PLRG1, SMURF1) as well as established syndromic CVM genes (KMT2D, NF1, TBX20, ZEB2). We also identified two genes (DNAH5, OFD1) with evidence of recessive and hemizygous inheritance patterns, respectively. Within our clinical cohort, we also observed heterozygous LOF variants in JARID2 and SMAD1 in individuals with cardiac phenotypes, and collectively, carriers of LOF variants in our candidate genes had a four times higher odds of having CVM (odds ratio = 4.0, 95% confidence interval 2.5-6.5). CONCLUSIONS: Our analytical strategy highlights the utility of bioinformatic resources, including human disease records and model organism phenotyping, in novel gene discovery for rare human disease. The results underscore the extensive genetic heterogeneity underlying non-syndromic LSLs, and posit potential novel candidate genes and complex modes of inheritance in this important group of birth defects.

Long-lasting neurotoxicity of prenatal benzene acute exposure in rats.

Benzene is a common element of environmental pollution. Although this substance is not recognized as a teratogenic agent, it is not known whether prenatal exposure to benzene may induce neurobehavioral changes in the progeny. Benzene 0.1mg/kg was injected subcutaneously (s.c.) acutely at day 15 of gestation into pregnant female rats of the Sprague-Dawley strain and neurotoxicity of the substance was studied in pups and male adult animals of the same progeny. No change was found in total number of neonates, body weight and eye opening time between benzene-exposed animals and controls. No malformations were observed. At birth, neonatal reflexes (cliff aversion, forelimb placing, bar holding, forelimb grasping, startle) were scored in benzene-exposed pups and their percent appearance was found to be anticipated (more benzene-exposed pups exhibited reflexes each day) in comparison to that of control animals. Also, the completion (maximum appearance, i.e. 100% of the brood was found to exhibit each reflex) of neonatal reflexes in benzene-exposed animals preceded that of controls. Starting 2 months after birth, cognitive and motor performance was assessed only in male animals of the prenatally benzene-exposed progeny. The overall evaluation of motor activity in benzene-exposed animals in the open-field test revealed reduced ambulation in these rats as compared to control animals. Acquisition of active avoidance responses in the shuttle-box test, as assessed by the number of conditioned avoidance responses and the percent of learners, was impaired in benzene-exposed rats as compared to control animals. Prenatal exposure to benzene was also followed by reduced retention latency in a step-through passive avoidance task in two retention tests. These results suggest that acute exposure to benzene during gestational organogenesis may cause long-lasting changes in motor behavior and cognitive processes. This may be relevant for the assessment of benzene toxic profile for the progeny of pregnant subjects, although teratogenic effects are not observed.

Long-lasting behavioral changes induced by pre- or neonatal exposure to diazepam in rats.

Prenatal treatment with small doses of diazepam may counteract the effect of physical stress in rats, normalizing the time course of neonatal reflexes and the behavioral responses in adulthood. However, prenatal administration of diazepam in greater doses may induce desensitization of gamma-aminobutyric acid A (GABA(A)) receptors and induce hypersensitivity to convulsants. This study was designed to examine in rats the influence of prenatal or neonatal diazepam treatment on development of neonatal reflexes, as index of brain maturation, and susceptibility to pentylenetetrazol-induced convulsions in adulthood. Pregnant Wistar rats were injected with diazepam 2.5 mg/kg/day, intraperitoneally (i.p.) on days 14-21 of pregnancy. Offspring showed a delay in the appearing of neonatal reflexes (cliff aversion, forelimb placing, forelimb grasping and bar holding) except for righting and startle reflexes. At 50 days of age, male rats showed a greater sensitivity to pentylenetetrazol compared to controls. In contrast, females showed a decreased susceptibility to convulsions. The appearance of reflexes in pups exposed to diazepam during neonatal life appeared to be similar to that of controls. Only the appearance of cliff aversion and startle reflexes appeared to be delayed in animals exposed neonatally to diazepam as compared to controls. In adulthood, female rats exposed in early neonatal life to diazepam again showed a resistance to pentylenetetrazol-induced convulsions as compared to male animals. These data suggest that prenatal exposure to diazepam induces long-lasting behavioral changes, which may be influenced by sex-dependent factors.