Loss Of Function Variants Research Articles

Rare and population-specific functional variation across pig lines

BackgroundIt is expected that functional, mainly missense and loss-of-function (LOF), and regulatory variants are responsible for most phenotypic differences between breeds and genetic lines of livestock species that have undergone diverse selection histories. However, there is still limited knowledge about the existing missense and LOF variation in commercial livestock populations, in particular regarding population-specific variation and how it can affect applications such as across-breed genomic prediction.MethodsWe re-sequenced the whole genome of 7848 individuals from nine commercial pig lines (average sequencing coverage: 4.1×) and imputed whole-genome genotypes for 440,610 pedigree-related individuals. The called variants were categorized according to predicted functional annotation (from LOF to intergenic) and prevalence level (number of lines in which the variant segregated; from private to widespread). Variants in each category were examined in terms of their distribution along the genome, alternative allele frequency, per-site Wright’s fixation index (FST), individual load, and association to production traits.ResultsOf the 46 million called variants, 28% were private (called in only one line) and 21% were widespread (called in all nine lines). Genomic regions with a low recombination rate were enriched with private variants. Low-prevalence variants (called in one or a few lines only) were enriched for lower allele frequencies, lower FST, and putatively functional and regulatory roles (including LOF and deleterious missense variants). On average, individuals carried fewer private deleterious missense alleles than expected compared to alleles with other predicted consequences. Only a small subset of the low-prevalence variants had intermediate allele frequencies and explained small fractions of phenotypic variance (up to 3.2%) of production traits. The significant low-prevalence variants had higher per-site FST than the non-significant ones. These associated low-prevalence variants were tagged by other more widespread variants in high linkage disequilibrium, including intergenic variants.ConclusionsMost low-prevalence variants have low minor allele frequencies and only a small subset of low-prevalence variants contributed detectable fractions of phenotypic variance of production traits. Accounting for low-prevalence variants is therefore unlikely to noticeably benefit across-breed analyses, such as the prediction of genomic breeding values in a population using reference populations of a different genetic background.

Clinical features of UK Biobank subjects carrying loss of function variants in genes implicated in schizophrenia pathogenesis

IntroductionThe SCHEMA consortium has identified ten genes in which severely damaging variants substantially increase schizophrenia risk.ObjectivesTo characterise the clinical features of carriers of variants causing complete loss of function (LOF) of these genes.MethodsThis research was conducted using the UK Biobank Resource and 200,000 exome-sequenced volunteers were screened to identify carriers of LOF variants in these genes. For these subjects, data fields were extracted which reflected educational and occupational functioning as well as clinical features including diagnoses and medication.Results LOF variants in CACNA1G were commoner than in SCHEMA cases, suggesting this was not a real schizophrenia susceptibility gene. 159 subjects carried LOF variants in one of the other nine genes and overall they did not have poorer educational or occupational functioning or increased mental or physical health problems. Detailed examination revealed that one had schizophrenia, one had psychotic depression and two had a developmental disorder. Otherwise, a number of subjects had features of minor mental illness such as depression or anxiety and these rates were somewhat increased in subjects carrying LOF variants in HERC1, of whom more than half reported having consulted their GP for such problems. However the majority appeared to be entirely normal from a neuropsychiatric point of view.ConclusionsAlthough particular genetic variants can substantially increase the risk of schizophrenia, most people carrying them are entirely normal. This further supports the concept of schizophrenia as a distinct illness rather than representing the extreme of a trait which is present in the population.DisclosureNo significant relationships.

244-LB: GPR75 Ablation Protects against Diet-Induced Obesity, Inflammation, and Insulin Resistance

Obesity related conditions including atherosclerosis and type 2 diabetes mellitus (T2DM) have a profound effect on cardiovascular health and morbidity. Recent reports by Akbari et. al. indicate that truncated loss of function variants of the orphan G-protein coupled receptor, GPR75, are associated with 5.3 kg lower bodyweight and 54% lower odds of obesity in heterozygous individuals (Science, 2021) . Based on these findings, we hypothesized that whole body deletion of Gpr75 protects against diet-induced obesity (DIO) and the resulting insulin resistance. Wild-type and Gpr75 null mice were subjected to 14 weeks of regular chow or high-fat diet (HFD) feeding. Body weight, intraperitoneal glucose and insulin tolerance tests, and pro-inflammatory cytokine gene expressions were measured initially and at week 14. There were no significant differences in body weight, glucose homeostasis, and tumor necrosis factor alpha (TNF-α) expression between wild-type (WT) and Gpr75 -/- (KO) mice at baseline. However, WT mice obtained a diabetogenic phenotype after HFD-feeding while the KO counterparts were protected as indicated by reduced body weight, (45.32 ± 1.387 grams vs. 28.29 ± 1.47 grams) and increased sensitivity to insulin (blood glucose 30 min after insulin injection as percent change, -28.5 ± 4.41% vs. -50.68 ± 4.15%, p<0.0001) . This correlated with a 1.98-fold decrease in brown adipose tissue (BAT) and 2.31-fold decrease in subcutaneous adipose tissue (SAT) TNF-α mRNA expression in KO compared to WT mice. Furthermore, there was a 3.19-fold reduction in circulating chemokine ligand 5 (CCL5) in KO compared to WT mice. This data may provide a potential novel target for the control of obesity-driven disorders such as inflammation and insulin resistance. Disclosure S. Hossain: None. E. Dirice: None. Funding NIH123203

Allelic dosage of RB1 drives CDK4/6 inhibitor treatment resistance in metastatic breast cancer.

1010 Background: We recently reported inferior outcomes to CDK4/6 inhibitors and endocrine therapy (CDK4/6i-ET) associated with germline BRCA2 (g BRCA2) in a cohort of estrogen receptor (ER) positive breast cancers. Co-occurrence of gBRCA2 with loss of heterozygosity (LOH) of neighboring RB1 was found to portend particularly poor outcomes. Here, we sought to define the effects of pre-treatment RB1 allelic copy number status on outcomes of CDK4/6i-ET and the likelihood of developing RB1 loss-of-function (LOF) mutations on CDK4/6i through the analysis of an expanded cohort of metastatic ER+ breast cancer patients. Methods: Patients who underwent sequencing on MSK-IMPACT from April 2014 to May 2021 were included. For every sample preceding CDK4/6i-ET, we performed FACETS to infer RB1 allele specific copy number, ploidy, tumor purity and fraction genome altered (FGA). Patients were categorized based on RB1 allelic status: HetLoss (total of one allelic copy), copy neutral LOH (CNLOH), other allelic imbalance including all other aneuploidy states, and diploid. Progression free survival (PFS) was assessed using univariate and multivariate Cox proportional hazard models adjusted for ET partner and FGA. Firth penalized logistic regression was used to study association of pre-treatment RB1 status with acquired RB1 LOF variants in paired post-CDK4/6i samples. Results: Of 2,630 potentially eligible patients, 279 patients had genomic sequencing performed prior to 1st line CDK4/6i-ET. Of these, 75 (26.8%) exhibited RB1 HetLoss, 39 (14.0%) had CNLOH of RB1, 111 (39.7%) exhibited diploid RB1 state, while 54 (19.4%) had other patterns of RB1 allelic imbalance. All non-diploid RB1 states were associated with significantly shortened PFS relative to diploid (univariate HetLoss HR: 2.05, 95% CI: 1.42, 2.97; CNLOH HR: 2.08, 95% CI: 1.32, 3.25; other imbalance HR: 1.70, 95% CI: 1.11, 2.58). Only HetLoss remained significant when adjusted for FGA (HR 1.61, 95% CI: 1.09, 2.38, p = 0.017). RB1 LOF was rare in pre-CDK4/6i tumors (< 1%); excluding these cases did not change our results. Of the 176 patients with paired pre- and post-CDK4/6i samples, only RB1 HetLoss in pre-CDK4/6i sample was significantly associated with development of RB1 LOF mutations in post-CDK4/6i sample (18.4%) as compared to diploid (4.2%, OR 4.25, 95% CI 1.02, 17.7, p = 0.047). These results indicate that tumors with one functional copy of RB1 are more likely to acquire RB1 LOF on CDK4/6i to achieve biallelic RB1 loss as a mechanism of CDK4/6i resistance. Conclusions: We demonstrate that LOH and allelic imbalance of RB1 are associated with shorter PFS on CDK4/6-ET. We postulate this may occur partly as a result of more frequent acquired RB1 LOF mutations under selective pressure of CDK4/6i. These data supports the implementation of more refined allele-specific copy number methods and identifies a high-risk population for escalated monitoring and treatment approaches.

Neonatal Scn1b-null mice have sinoatrial node dysfunction, altered atrial structure, and atrial fibrillation.

Loss-of-function (LOF) variants in SCN1B, encoding the voltage-gated sodium channel β1/β1B subunits, are linked to neurological and cardiovascular diseases. Scn1b-null mice have spontaneous seizures and ventricular arrhythmias and die by approximately 21 days after birth. β1/β1B Subunits play critical roles in regulating the excitability of ventricular cardiomyocytes and maintaining ventricular rhythmicity. However, whether they also regulate atrial excitability is unknown. We used neonatal Scn1b-null mice to model the effects of SCN1B LOF on atrial physiology in pediatric patients. Scn1b deletion resulted in altered expression of genes associated with atrial dysfunction. Scn1b-null hearts had a significant accumulation of atrial collagen, increased susceptibility to pacing induced atrial fibrillation (AF), sinoatrial node (SAN) dysfunction, and increased numbers of cholinergic neurons in ganglia that innervate the SAN. Atropine reduced the incidence of AF in null animals. Action potential duration was prolonged in null atrial myocytes, with increased late sodium current density and reduced L-type calcium current density. Scn1b LOF results in altered atrial structure and AF, demonstrating the critical role played by Scn1b in atrial physiology during early postnatal mouse development. Our results suggest that SCN1B LOF variants may significantly impact the developing pediatric heart.

Phenotypic expansion and variable expressivity in individuals with JARID2-related intellectual disability: A case series.

Loss of function variants in JARID2 were recently reported in 16 patients with a neurodevelopmental disorder characterized by delays, intellectual and learning disability, autism, behavioral abnormalities, and dysmorphic features. Most cases were de novo, with only one variant inherited from an affected parent. Here, we present seven additional individuals from five families with pathogenic or likely pathogenic JARID2 variants, confirming this gene-disease association and highlighting palatal abnormalities and heart defects as part of the phenotype. In addition, we report inheritance of JARID2 variants from mildly affected parents, demonstrating the variable expressivity of the disease. We also note the high prevalence of intragenic JARID2 copy number variants, emphasizing the importance of exon-level analysis.

Loss-of-function variants in SRRM2 cause a neurodevelopmental disorder

SRRM2 encodes the SRm300 protein, a splicing factor of the SR-related protein family characterized by its serine- and arginine-enriched domains. It promotes interactions between messenger RNA and the spliceosome catalytic machinery. This gene, predicted to be highly intolerant to loss of function (LoF) and very conserved through evolution, has not been previously reported in constitutive human disease. Among the 1000 probands studied with developmental delay and intellectual disability in our database, we found 2 patients with de novo LoF variants in SRRM2. Additional families were identified through GeneMatcher. Here, we report on 22 patients with LoF variants in SRRM2 and provide a description of the phenotype. Molecular analysis identified 12 frameshift variants, 8 nonsense variants, and 2 microdeletions of 66 kb and 270 kb. The patients presented with a mild developmental delay, predominant speech delay, autistic or attention-deficit/hyperactivity disorder features, overfriendliness, generalized hypotonia, overweight, and dysmorphic facial features. Intellectual disability was variable and mild when present. We established SRRM2 as a gene responsible for a rare neurodevelopmental disease.

A novel nonsense variant in EXOC8 underlies a neurodevelopmental disorder

Human exocyst complex is an evolutionary conserved multimeric complex composed of proteins encoded by eight genes EXOC1-EXOC8. It is known that the exocyst complex plays a role in ciliogenesis, cytokinesis, cell migration, autophagy, and fusion of secretory vesicles. Recently, loss of function variants in EXOC7 and EXOC8 has been associated with abnormalities of cerebral cortical development leading to a neurodevelopmental phenotype. Neurodevelopmental disorders are a huge group of clinically and genetically heterogeneous disorders. In the present study, we recruited a large consanguineous family segregating a neurodevelopmental disorder in an autosomal recessive form. We performed clinical phenotyping by imaging the patient's brain followed by whole exome sequencing examining DNA from two affected individuals. The clinical phenotypes of the disease were suggestive of brain atrophy. Clinical examination revealed intellectual impairment with hypertonia and brisk reflexes. WES followed by Sanger sequencing revealed a novel homozygous nonsense mutation [EXOC8; NM_175876.5; c.1714G > T; p.(Glu572Ter)] in the DNA of affected individuals. Both parents of the patients were heterozygous for the identified mutation. All the pathogenicity prediction softwares predicted the identified variant as disease causing. This study reports a second protein-truncating variant in EXOC8. The findings confirm that loss of function variants in EXOC8 underlies a neurodevelopmental disorder. The identification of a protein-truncating variant in EXOC8 in the current study can be helpful in establishing genotype-phenotype correlations. Our results also provide new insights into genetic counseling and clinical management for the affected individuals.

Membrane Anchorage-Induced (MAGIC) Knockdown of Non-synonymous Point Mutations.

The promise of personalized medicine for monogenic and complex polygenic diseases depends on the availability of strategies for targeted inhibition of disease-associated polymorphic protein variants. Loss of function variants, including non-synonymous single nucleotide variants (nsSNVs) and insertion/deletion producing a frameshift, account for the vast majority of disease-related genetic changes. Because it is challenging to interpret the functional consequences of nsSNVs, they are considered a big barrier for personalized medicine. A method for inhibiting the specific expression of nsSNVs without editing the human genome will facilitate the elucidation of the biology of nsSNVs, but such a method is currently lacking. Here, I describe the phenomenon of membrane anchorage-induced (MAGIC) knockdown of allele-specific inhibition of protein and mRNA expression upon inner membrane tethering of point mutation-specific monoclonal antibodies (mAb). This phenomenon is likely mediated by a mechanism distinct from the protein degradation pathways, as the epitope-specific knockdown is replicated upon intracellular expression of a membrane-anchored single domain intrabody that lacks the Fc domain of the mAb. By harnessing the MAGIC knockdown of epitope-containing protein targets, I report a novel approach for inhibiting the expression of amino-acid-altering germline and somatic nsSNVs. As a proof-of-concept, I show the inhibition of human disease-associated variants namely, FGFR4 p.G388R, KRAS p.G12D and BRAF p.V600E protein variants. This method opens up a new avenue for not just therapeutic suppression of undruggable protein variants, but also for functional interrogation of the nsSNVs of unknown significance.

NEK1 Variants in a Cohort of Italian Patients With Amyotrophic Lateral Sclerosis.

IntroductionIn the last few years, different studies highlighted a significant enrichment of NEK1 loss of function (LoF) variants in amyotrophic lateral sclerosis (ALS), and an additional role for the p.Arg261His missense variant in the disease susceptibility. Several other missense variants have been described so far, whose pathogenic relevance remains however unclear since many of them have been reported in both patients and controls. This study aimed to investigate the presence of NEK1 variants and their correlation with phenotype in a cohort of Italian patients with ALS.MethodsWe sequenced a cohort of 350 unrelated Italian patients with ALS by next-generation sequencing (NGS) and then we analyzed the clinical features of NEK1 carriers.ResultsWe detected 20 different NEK1 rare variants (four LoF and 16 missense) in 33 unrelated patients with sporadic ALS (sALS). The four LoF variants (two frameshift and two splice-site variants) were all novel. The p.Arg261His missense variant was enriched in the patients’ cohort (p < 0.001). Excluding this variant from counting, the difference in the frequency of NEK1 rare missense variants between patients and controls was not statistically significant. NEK1 carriers had a higher frequency of flail arm (FA) phenotype compared with the other patients of the cohort (29.2% vs. 6.4%). Nine NEK1 carriers (37.5%) also harbored variants in other ALS-related genes.ConclusionThis study confirms that NEK1 LoF and p.Arg261. His missense variants are associated with ALS in an Italian ALS cohort and suggests a correlation between the presence of NEK1 variants and FA phenotype.

Comparison of the frequency of loss-of-function LZTR1 variants between schwannomatosis patients and the general population.

Schwannomatosis is a rare tumor predisposition syndrome that causes multiple schwannomas. Germline loss‐of‐function (LoF) LZTR1 variants were only recently identified as disease‐causing, so relatively few variants have been identified in patients. In addition, many LoF variants exist in Genome Aggregation Database (gnomAD) in people who do not have clinical symptoms of schwannomatosis. These factors, and the incomplete penetrance seen in this condition, hinder definitive interpretation of the clinical significance of novel LoF variants identified in schwannomatosis patients. We collated published LOF LZTR1 variants identified in schwannomatosis patients and classified them according to current American College of Medical Genetics and Genomics/Association for Molecular Pathology/Association of Clinical Genomic Science guidelines. Subsequently, pathogenic/likely pathogenic schwannomatosis‐associated LoF variants were compared with LoF LZTR1 variants reported in gnomAD data. Using current classification guidelines, 64/71 LoF LZTR1 variants reported in schwannomatosis patients in the literature were classified as pathogenic/likely pathogenic, and their frequency in probands 64/359 (17.8%) was significantly higher than the frequency of potential LoF variants identified in the general population (0.36%; p < 0.0001). The majority of published classifications of schwannomatosis‐associated LoF variants are robust. However, the high frequency of LoF LZTR1 variants in the general population suggests that LZTR1 variants confer a reduced risk of schwannomas compared to germline NF2 and SMARCB1 pathogenic variants, making classification of novel variants challenging.

Effect of Occurrence of Lamin A/C (LMNA) Genetic Variants in a Cohort of 101 Consecutive Apparent "Lone AF" Patients: Results and Insights.

ObjectiveMutations in the Lamin A/C (LMNA) gene are commonly associated with cardiac manifestations, such as dilated cardiomyopathy (DCM) and conduction system disease. However, the overall spectrum and penetrance of rare LMNA variants are unknown. The present study described the presence of LMNA variants in patients with “lone atrial fibrillation (AF)” as their sole clinical presentation.MethodsOne-hundred and one consecutive patients with “lone AF” criteria were initially screened by genetic testing. Genetic variants were classified according to the American College of Genetic and Genomic criteria. All subjects were evaluated through clinical and familial history, ECG, 24-h Holter monitoring, echocardiogram, cardiac magnetic resonance, treatment response, and the present relatives of LMNA carriers. In addition, whole-exome data from 49,960 UK Biobank (UKB) participants were analyzed to describe the overall penetrance of rare LMNA missense and loss of function (LOF) variants.ResultsThree missense variants in LMNA were identified in probands with AF as their first and unique clinical manifestation. Other five first-degree relatives, after the screening, also presented LMNA gene variants. Among 49,960 analyzed UKB participants, 331 carried rare LMNA missense or LOF variant. Participants who carried a rare LMNA variant were significantly associated with higher odds of arrhythmic events and of an abnormal ECG in the per-protocol ECG exam (p = 0.03 and p = 0.05, respectively).ConclusionAlthough a rare occurrence, our findings emphasize the possibility of an initial presentation of apparently “lone AF” in LMNA gene variant carriers.

Profiling PI3K-AKT-MTOR variants in focal brain malformations reveals new insights for diagnostic care.

Focal malformations of cortical development including focal cortical dysplasia, hemimegalencephaly and megalencephaly, are a spectrum of neurodevelopmental disorders associated with brain overgrowth, cellular and architectural dysplasia, intractable epilepsy, autism and intellectual disability. Importantly, focal cortical dysplasia is the most common cause of focal intractable paediatric epilepsy. Gain and loss of function variants in the PI3K-AKT-MTOR pathway have been identified in this spectrum, with variable levels of mosaicism and tissue distribution. In this study, we performed deep molecular profiling of common PI3K-AKT-MTOR pathway variants in surgically resected tissues using droplet digital polymerase chain reaction (ddPCR), combined with analysis of key phenotype data. A total of 159 samples, including 124 brain tissue samples, were collected from 58 children with focal malformations of cortical development. We designed an ultra-sensitive and highly targeted molecular diagnostic panel using ddPCR for six mutational hotspots in three PI3K-AKT-MTOR pathway genes, namely PIK3CA (p.E542K, p.E545K, p.H1047R), AKT3 (p.E17K) and MTOR (p.S2215F, p.S2215Y). We quantified the level of mosaicism across all samples and correlated genotypes with key clinical, neuroimaging and histopathological data. Pathogenic variants were identified in 17 individuals, with an overall molecular solve rate of 29.31%. Variant allele fractions ranged from 0.14 to 22.67% across all mutation-positive samples. Our data show that pathogenic MTOR variants are mostly associated with focal cortical dysplasia, whereas pathogenic PIK3CA variants are more frequent in hemimegalencephaly. Further, the presence of one of these hotspot mutations correlated with earlier onset of epilepsy. However, levels of mosaicism did not correlate with the severity of the cortical malformation by neuroimaging or histopathology. Importantly, we could not identify these mutational hotspots in other types of surgically resected epileptic lesions (e.g. polymicrogyria or mesial temporal sclerosis) suggesting that PI3K-AKT-MTOR mutations are specifically causal in the focal cortical dysplasia-hemimegalencephaly spectrum. Finally, our data suggest that ultra-sensitive molecular profiling of the most common PI3K-AKT-MTOR mutations by targeted sequencing droplet digital polymerase chain reaction is an effective molecular approach for these disorders with a good diagnostic yield when paired with neuroimaging and histopathology.

Dominant negative effects of SCN5A missense variants

PurposeUp to 30% of patients with Brugada syndrome (BrS) carry loss-of-function (LoF) variants in the cardiac sodium channel gene SCN5A encoding for the protein NaV1.5. Recent studies suggested that NaV1.5 can dimerize, and some variants exert dominant negative effects. In this study, we sought to explore the generality of missense variant NaV1.5 dominant negative effects and their clinical severity. MethodsWe identified 35 LoF variants (<10% of wild type [WT] peak current) and 15 partial LoF variants (10%-50% of WT peak current) that we assessed for dominant negative effects. SCN5A variants were studied in HEK293T cells, alone or in heterozygous coexpression with WT SCN5A using automated patch clamp. To assess the clinical risk, we compared the prevalence of dominant negative vs putative haploinsufficient (frameshift, splice, or nonsense) variants in a BrS consortium and the Genome Aggregation Database population database. ResultsIn heterozygous expression with WT, 32 of 35 LoF and 6 of 15 partial LoF variants showed reduction to <75% of WT-alone peak current, showing a dominant negative effect. Individuals with dominant negative LoF variants had an elevated disease burden compared with the individuals with putative haploinsufficient variants (2.7-fold enrichment in BrS cases, P = .019). ConclusionMost SCN5A missense LoF variants exert a dominant negative effect. This class of variant confers an especially high burden of BrS.

De novo nonsense variant in ASXL3 in a Chinese girl causing Bainbridge-Ropers syndrome: A case report and review of literature.

BackgroundBainbridge‐Ropers syndrome (BRPS, OMIM #615485) was first identified in 2013 by Bainbridge et al. and is a neurodevelopment disorder characterized by failure to thrive, facial dysmorphism and severe developmental delay. BRPS is caused by heterozygous loss‐of‐function (LOF) variants in the additional sex combs‐like 3 (ASXL3) gene. Due to the limited specific recognizable features and overlapping symptoms with Bohring–Opitz syndrome (BOS, OMIM #612990), clinical diagnosis of BRPS is challenging.MethodsIn this study, a 2‐year‐8‐month‐old Chinese girl was referred for genetic evaluation of severe developmental delay. The reduced fetal movement was found during the antenatal period and bilateral varus deformity of feet was observed at birth. Whole‐exome sequencing and Sanger sequencing were used to detect and confirm the variant.ResultsA novel nonsense variant c.1063G>T (p.E355*) in the ASXL3 gene (NM_030632.3) was identified in the proband and the clinical symptoms were compatible with BRPS. The parents were physical and genetic normal and prenatal diagnosis was requested for her pregnant mother with a negative Sanger sequencing result.ConclusionThe study revealed a de novo LOF variant in the ASXL3 gene and expanded the mutation spectrum for this clinical condition. By performing a literature review, we summarized genetic results and the clinical phenotypes of all BPRSs reported so far. More cases study may help to elucidate the function of the ASXL3 gene may be critical to understand the genetic aetiology of this syndrome and assist in accurate genetic counselling, informed decision making and prenatal diagnosis.

The Impact of Mediterranean Dietary Intervention on Metabolic and Hormonal Parameters According to BRCA1/2 Variant Type.

The female carriers of BRCA1/2 pathogenic variants (mutations) face a high lifetime risk of developing breast and/or ovarian cancer. However, the risk may differ depending on various genetic and non-genetic elements, including metabolic and hormonal factors. We previously showed that a 6-month Mediterranean dietary intervention trial reduced body weight and the levels of insulin-like growth factor I and other metabolic factors in BRCA mutation carriers. We also found that higher baseline levels of glucose and insulin were significantly associated with BRCA loss-of-function (LOF) variants. In this study, we evaluated whether the BRCA mutation type influences in a different way the metabolic and hormonal response to the dietary intervention in 366 female carriers. The LOF variant carriers randomized in the intervention group (IG) showed significantly higher changes in most considered parameters compared to the control group (CG). The nonsynonymous variant carriers in the IG showed similar changes, but none of them were statistically significant. Performing the “delta” analysis of differences (intention-to-treat analysis), we observed that in LOF variant carriers, the reduction of insulin levels was significantly more pronounced that in nonsynonymous variant carriers. These findings suggest that the changes in insulin levels might be modulated by a different response to the dietary intervention mediated by BRCA LOF variants.

Identification of three novel homozygous variants in COL9A3 causing autosomal recessive Stickler syndrome

BackgroundStickler syndrome (STL) is a rare, clinically and molecularly heterogeneous connective tissue disorder. Pathogenic variants occurring in a variety of genes cause STL, mainly inherited in an autosomal dominant fashion. Autosomal recessive STL is ultra-rare with only four families with biallelic COL9A3 variants reported to date.ResultsHere, we report three unrelated families clinically diagnosed with STL carrying different novel biallelic loss of function variants in COL9A3. Further, we have collected COL9A3 genotype–phenotype associations from the literature.ConclusionOur report substantially expands the molecular genetics and clinical basis of autosomal recessive STL and provides an overview about allelic COL9A3 disorders.

Loss-of-function variants in TIAM1 are associated with developmental delay, intellectual disability, and seizures

TIAM Rac1-associated GEF 1 (TIAM1) regulates RAC1 signaling pathways that affect the control of neuronal morphogenesis and neurite outgrowth by modulating the actin cytoskeletal network. To date, TIAM1 has not been associated with a Mendelian disorder. Here, we describe five individuals with bi-allelic TIAM1 missense variants who have developmental delay, intellectual disability, speech delay, and seizures. Bioinformatic analyses demonstrate that these variants are rare and likely pathogenic. We found that the Drosophila ortholog of TIAM1, still life (sif), is expressed in larval and adult central nervous system (CNS) and is mainly expressed in a subset of neurons, but not in glia. Loss of sif reduces the survival rate, and the surviving adults exhibit climbing defects, are prone to severe seizures, and have a short lifespan. The TIAM1 reference (Ref) cDNA partially rescues the sif loss-of-function (LoF) phenotypes. We also assessed the function associated with three TIAM1 variants carried by two of the probands and compared them to the TIAM1 Ref cDNA function invivo. TIAM1 p.Arg23Cys has reduced rescue ability when compared to TIAM1 Ref, suggesting that it is a partial LoF variant. In ectopic expression studies, both wild-type sif and TIAM1 Ref are toxic, whereas the three variants (p.Leu862Phe, p.Arg23Cys, and p.Gly328Val) show reduced toxicity, suggesting that they are partial LoF variants. In summary, we provide evidence that sif is important for appropriate neural function and that TIAM1 variants observed in the probands are disruptive, thus implicating loss of TIAM1 in neurological phenotypes in humans.

EP404: Evidence supporting the pathogenicity of the NADSYN1 c.1717G>A (p.Ala573Thr) variant in individuals referred for clinical testing

Vertebral, cardiac, renal, and limb defects syndrome 3 (VCRL3; OMIM #618845) is an autosomal recessive multiple congenital anomaly syndrome caused by pathogenic variants in the NADSYN1 gene. NADSYN1 encodes a nicotinamide adenine dinucleotide (NAD) synthetase that is part of the de novo NAD synthesis pathway. Other genes involved in this pathway include HAAO and KYNU. Genetic variants in any of these genes result in reduced de novo NAD synthesis and cause congenital anomalies of the heart, kidney, and vertebrae. Of note, presence of these three features is also considered clinical evidence for vertebral defects, anal atresia, cardiac defects, tracheo-esophageal fistula, renal anomalies, and limb abnormalities (VACTERL) association, which is often a diagnosis of exclusion (ie, other genetic conditions with overlapping features must be ruled out before assigning this diagnosis). The recommended approach for genetic testing in possible VACTERL patients is to either pursue a comprehensive testing option, such as exome or genome sequencing, or a panel approach with the most likely candidates. The NADSYN1 gene is included on a VACTERL Association and Related Disorders panel and several patients were noted to be homozygous for the NADSYN1 c.1717G>A (p.Ala573Thr) variant, thus providing additional evidence for both the pathogenicity and high carrier frequency of this change. To date, we have identified five patients who are homozygous for the NADSYN1 (NM_018161.4) c.1717G>A (p.Ala573Thr) variant. One patient was identified through targeted testing, one through testing with the VACTERL Association and Related Disorders panel, and three through exome sequencing. The first four cases were from pregnancies evaluated for the differential diagnosis of VACTERL association via panel testing or exome sequencing. All four fetuses were reported to have cardiac malformation and segmental vertebral anomalies. Parental data was available for three of the four fetuses, and all parents tested were heterozygous for the NADSYN1 c.1717G>A (p.Ala573Thr) variant. All cases denied consanguinity. In contrast to the severe and early onset features of the other four patients identified, the targeted testing case was an adult patient who previously had the NADSYN1 c.1717G>A variant identified in a research study. This individual was noted to have cardiac defects, vertebral defects, and short stature. This variant is relatively common in non-Finnish Europeans with an allele frequency of 0.15% in the gnomAD v2 dataset. Five patients from four families with VCRL3 have been described in the literature. Of these patients, three had the c.1717G>A variant: two homozygous siblings and one compound heterozygote with a loss of function variant on the opposite allele. Parental sequencing data confirmed the biallelic variants in all reported patients. In addition, yeast model functional studies performed by Szot and colleagues demonstrated that the p.Ala573Thr change diminished NAD synthetase activity and reduced yeast viability, consistent with a loss of function mechanism. Taken together, although the NADSYN1 c.1717G>A (p.Ala573Thr) has a relatively high minor allele frequency, there is sufficient evidence for this variant to be classified as pathogenic using ACMG/AMP variant classifications and internal variant interpretation guidelines. The NADSYN1 c.1717G>A (p.Ala573Thr) variant should be interpreted as pathogenic given the 5 additional homozygous patients and parental segregation data presented here. Additionally, the diagnosis of VCRL3 in an adult patient suggests that although penetrance for NAD deficiency is complete, variable expressivity is observed and could be explained by other genetic or non-genetic factors modulating disease severity. Given the high population frequency of this variant, genetic testing for VACTERL association should always include evaluation of NADSYN1 to consider VCRL3 in the differential diagnosis.

PUS7 deficiency in human patients causes profound neurodevelopmental phenotype by dysregulating protein translation.

Protein translation is a highly regulated process involving the interaction of numerous genes on every component of the protein translation machinery. Upregulated protein translation is a hallmark of cancer and is implicated in autism spectrum disorder, but the risks of developing each disease do not appear to be correlated with one another. In this study we identified two siblings from the NIH Undiagnosed Diseases Program with loss of function variants in PUS7, a gene previously implicated in the regulation of total protein translation. These patients exhibited a neurodevelopmental phenotype including autism spectrum disorder in the proband. Both patients also had features of Lesch-Nyhan syndrome, including hyperuricemia and self-injurious behavior, but without pathogenic variants in HPRT1. Patient fibroblasts demonstrated upregulation of protein synthesis, including elevated MYC protein, but did not exhibit increased rates of cell proliferation. Interestingly, the dysregulation of protein translation also resulted in mildly decreased levels of HPRT1 protein suggesting an association between dysregulated protein translation and the LNS-like phenotypic findings. These findings strengthen the correlation between neurodevelopmental disease, particularly autism spectrum disorders, and the rate of protein translation.