Functional analysis of BARD1 missense variants in homology-directed repair and damage sensitivity

The BARD1 protein, which heterodimerizes with BRCA1, is encoded by a known breast cancer susceptibility gene. While several BARD1 variants have been identified as pathogenic, many more missense variants exist that do not occur frequently enough to assign a clinical risk. In this paper, whole exome sequencing of over 10,000 cancer samples from 33 cancer types identified from somatic mutations and loss of heterozygosity in tumors 76 potentially cancer-associated BARD1 missense and truncation variants. These variants were tested in a functional assay for homology-directed repair (HDR), as HDR deficiencies have been shown to correlate with clinical pathogenicity for BRCA1 variants. From these 76 variants, 4 in the ankyrin repeat domain and 5 in the BRCT domain were found to be non-functional in HDR. Two known benign variants were found to be functional in HDR, and three known pathogenic variants were non-functional, supporting the notion that the HDR assay can be used to predict the clinical risk of BARD1 variants. The identification of HDR-deficient variants in the ankyrin repeat domain indicates there are DNA repair functions associated with this domain that have not been closely examined. In order to examine whether BARD1-associated loss of HDR function results in DNA damage sensitivity, cells expressing non-functional BARD1 variants were treated with ionizing radiation or cisplatin. These cells were found to be more sensitive to DNA damage, and variations in the residual HDR function of non-functional variants did not correlate with variations in sensitivity. These findings improve the understanding of BARD1 functional domains in DNA repair and support that this functional assay is useful for predicting the cancer association of BARD1 variants.</div

De novo variants disturbing the transactivation capacity of POU3F3 cause a characteristic neurodevelopmental disorder

POU3F3, also referred to as Brain-1, is a well-known transcription factor involved in the development of the central nervous system, but it has not previously been associated with a neurodevelopmental disorder. Here, we report the identification of 19 individuals with heterozygous POU3F3 disruptions, most of which are de novo variants. All individuals had developmental delays and/or intellectual disability and impairments in speech and language skills. Thirteen individuals had characteristic low-set, prominent, and/or cupped ears. Brain abnormalities were observed in seven of eleven MRI reports. POU3F3 is an intronless gene, insensitive to nonsense-mediated decay, and 13 individuals carried protein-truncating variants. All truncating variants that we tested in cellular models led to aberrant subcellular localization of the encoded protein. Luciferase assays demonstrated negative effects of these alleles on transcriptional activation of a reporter with a FOXP2-derived binding motif. In addition to the loss-of-function variants, five individuals had missense variants that clustered at specific positions within the functional domains, and one small in-frame deletion was identified. Two missense variants showed reduced transactivation capacity in our assays, whereas one variant displayed gain-of-function effects, suggesting a distinct pathophysiological mechanism. In bioluminescence resonance energy transfer (BRET) interaction assays, all the truncated POU3F3 versions that we tested had significantly impaired dimerization capacities, whereas all missense variants showed unaffected dimerization with wild-type POU3F3. Taken together, our identification and functional cell-based analyses of pathogenic variants in POU3F3, coupled with a clinical characterization, implicate disruptions of this gene in a characteristic neurodevelopmental disorder

Protein structure and phenotypic analysis of pathogenic and population missense variants in STXBP1

Background: Syntaxin-binding protein 1, encoded by STXBP1, is highly expressed in the brain and involved in fusing synaptic vesicles with the plasma membrane. Studies have shown that pathogenic loss-of-function variants in this gene result in various types of epilepsies, mostly beginning early in life. We were interested to model pathogenic missense variants on the protein structure to investigate the mechanism of pathogenicity and genotype–phenotype correlations. Methods: We report 11 patients with pathogenic de novo mutations in STXBP1 identified in the first 4293 trios of the Deciphering Developmental Disorder (DDD) study, including six missense variants. We analyzed the structural locations of the pathogenic missense variants from this study and the literature, as well as population missense variants extracted from Exome Aggregation Consortium (ExAC). Results: Pathogenic variants are significantly more likely to occur at highly conserved locations than population variants, and be buried inside the protein domain. Pathogenic mutations are also more likely to destabilize the domain structure compared with population variants, increasing the proportion of (partially) unfolded domains that are prone to aggregation or degradation. We were unable to detect any genotype–phenotype correlation, but unlike previously reported cases, most of the DDD patients with STXBP1 pathogenic variants did not present with very early-onset or severe epilepsy and encephalopathy, though all have developmental delay with intellectual disability and most display behavioral problems and suffered seizures in later childhood. Conclusion: Variants across STXBP1 that cause loss of function can result in severe intellectual disability with or without seizures, consistent with a haploinsufficiency mechanism. Pathogenic missense mutations act through destabilization of the protein domain, making it prone to aggregation or degradation. The presence or absence of early seizures may reflect ascertainment bias in the literature as well as the broad recruitment strategy of the DDD study

Whole exome sequencing in an Italian family with isolated maxillary canine agenesis and canine eruption anomalies

Objective: The aim of this study was the clinical and molecular characterization of a family segregating a trait consisting of a phenotype specifically involving the maxillary canines, including agenesis, impaction and ectopic eruption, characterized by incomplete penetrance and variable expressivity. Design: Clinical standardized assessment of 14 family members and a whole-exome sequencing (WES) of three affected subjects were performed. WES data analyses (sequence alignment, variant calling, annotation and prioritization) were carried out using an in-house implemented pipeline. Variant filtering retained coding and splice-site high quality private and rare variants. Variant prioritization was performed taking into account both the disruptive impact and the biological relevance of individual variants and genes. Sanger sequencing was performed to validate the variants of interest and to carry out segregation analysis. Results: Prioritization of variants “by function” allowed the identification of multiple variants contributing to the trait, including two concomitant heterozygous variants in EDARADD (c.308C&gt;T, p.Ser103Phe) and COL5A1 (c.1588G&gt;A, p.Gly530Ser), specifically associated with a more severe phenotype (i.e. canine agenesis). Differently, heterozygous variants in genes encoding proteins with a role in the WNT pathway were shared by subjects showing a phenotype of impacted/ectopic erupted canines. Conclusions: This study characterized the genetic contribution underlying a complex trait consisting of isolated canine anomalies in a medium-sized family, highlighting the role of WNT and EDA cell signaling pathways in tooth development

Towards Better Separation between Deterministic and Randomized Query Complexity

We show that there exists a Boolean function $F$ which observes the following separations among deterministic query complexity $(D(F))$, randomized zero error query complexity $(R_0(F))$ and randomized one-sided error query complexity $(R_1(F))$: $R_1(F) = \widetilde{O}(\sqrt{D(F)})$ and $R_0(F)=\widetilde{O}(D(F))^{3/4}$. This refutes the conjecture made by Saks and Wigderson that for any Boolean function $f$, $R_0(f)=\Omega({D(f)})^{0.753..}$. This also shows widest separation between $R_1(f)$ and $D(f)$ for any Boolean function. The function $F$ was defined by G{\"{o}}{\"{o}}s, Pitassi and Watson who studied it for showing a separation between deterministic decision tree complexity and unambiguous non-deterministic decision tree complexity. Independently of us, Ambainis et al proved that different variants of the function $F$ certify optimal (quadratic) separation between $D(f)$ and $R_0(f)$, and polynomial separation between $R_0(f)$ and $R_1(f)$. Viewed as separation results, our results are subsumed by those of Ambainis et al. However, while the functions considerd in the work of Ambainis et al are different variants of $F$, we work with the original function $F$ itself.Comment: Reference adde