De novo mutations identified by whole-genome sequencing implicate chromatin modifications in obsessive-compulsive disorder

Trio-based whole-genome sequencing identified the role of chromatin modification in OCD pathology.

Mutation bias reflects natural selection in Arabidopsis thaliana

Since the first half of the twentieth century, evolutionary theory has been dominated by the idea that mutations occur randomly with respect to their consequences1. Here we test this assumption with large surveys of de novo mutations in the plant Arabidopsis thaliana. In contrast to expectations, we find that mutations occur less often in functionally constrained regions of the genome—mutation frequency is reduced by half inside gene bodies and by two-thirds in essential genes. With independent genomic mutation datasets, including from the largest Arabidopsis mutation accumulation experiment conducted to date, we demonstrate that epigenomic and physical features explain over 90% of variance in the genome-wide pattern of mutation bias surrounding genes. Observed mutation frequencies around genes in turn accurately predict patterns of genetic polymorphisms in natural Arabidopsis accessions (r = 0.96). That mutation bias is the primary force behind patterns of sequence evolution around genes in natural accessions is supported by analyses of allele frequencies. Finally, we find that genes subject to stronger purifying selection have a lower mutation rate. We conclude that epigenome-associated mutation bias2 reduces the occurrence of deleterious mutations in Arabidopsis, challenging the prevailing paradigm that mutation is a directionless force in evolution.

A de novo paradigm for male infertility

De novo mutations are known to play a prominent role in sporadic disorders with reduced fitness. We hypothesize that de novo mutations play an important role in severe male infertility and explain a portion of the genetic causes of this understudied disorder. To test this hypothesis, we utilize trio-based exome sequencing in a cohort of 185 infertile males and their unaffected parents. Following a systematic analysis, 29 of 145 rare (MAF < 0.1%) protein-altering de novo mutations are classified as possibly causative of the male infertility phenotype. We observed a significant enrichment of loss-of-function de novo mutations in loss-of-function-intolerant genes (p-value = 1.00 × 10−5) in infertile men compared to controls. Additionally, we detected a significant increase in predicted pathogenic de novo missense mutations affecting missense-intolerant genes (p-value = 5.01 × 10−4) in contrast to predicted benign de novo mutations. One gene we identify, RBM5, is an essential regulator of male germ cell pre-mRNA splicing and has been previously implicated in male infertility in mice. In a follow-up study, 6 rare pathogenic missense mutations affecting this gene are observed in a cohort of 2,506 infertile patients, whilst we find no such mutations in a cohort of 5,784 fertile men (p-value = 0.03). Our results provide evidence for the role of de novo mutations in severe male infertility and point to new candidate genes affecting fertility.

A young infant with complete androgen insensitivity syndrome

Complete androgen insensitivity syndrome (CAIS) is a rare X-linked recessive disorder resulting from maternally inherited or de novo mutations involving the androgen receptor (AR) gene. The AR is a vital steroid hormone receptor that has a critical role in male sexual differentiation and development and preservation of the male phenotype. The diagnosis of CAIS is based on the presence of female external genitalia in an individual with 46, XY karyotype having normally developed but undescended testes and target tissue unresponsiveness to androgen. Our case presented at the age of 2 months with asymmetric labia majora with bilateral labial mass. Ultrasonography revealed absence of female internal genital organs and presence of testes at labial folds. The child was found to have 46, XY karyotype. BIRDEM Med J 2022; 12(1): 74-77

An ensemble of toxic channel types underlies the severity of the de novo variant G375R of the human BK channel

De novo mutations play a prominent role in neurodevelopmental diseases including autism, schizophrenia, and intellectual disability. Many de novo mutations are dominant and so severe that the afflicted individuals do not reproduce, so the mutations are not passed into the general population. For multimeric proteins, such severity may result from a dominant-negative effect where mutant subunits assemble with WT to produce channels with adverse properties. Here we study the de novo variant G375R heterozygous with the WT allele for the large conductance voltage- and Ca2+-activated potassium (BK) channel, Slo1. This variant has been reported to produce devastating neurodevelopmental disorders in three unrelated children. If mutant and WT subunits assemble randomly to form tetrameric BK channels, then ~6% of the assembled channels would be wild type (WT), ~88% would be heteromeric incorporating from 1-3 mutant subunits per channel, and ~6% would be homomeric mutant channels consisting of four mutant subunits. To test this hypothesis, we analyzed the biophysical properties of single BK channels in the ensemble of channels expressed following a 1:1 injection of mutant and WT cRNA into oocytes. We found ~3% were WT channels, ~85% were heteromeric channels, and ~12% were homomeric mutant channels. All of the heteromeric channels as well as the homomeric mutant channels displayed toxic properties, indicating a dominant negative effect of the mutant subunits. The toxic channels were open at inappropriate negative voltages, even in the absence of Ca2+, which would lead to altered cellular function and decreased neuronal excitability.

De novo mutations in childhood cases of sudden unexplained death that disrupt intracellular Ca2+ regulation

Sudden unexplained death in childhood (SUDC) is an understudied problem. Whole-exome sequence data from 124 “trios” (decedent child, living parents) was used to test for excessive de novo mutations (DNMs) in genes involved in cardiac arrhythmias, epilepsy, and other disorders. Among decedents, nonsynonymous DNMs were enriched in genes associated with cardiac and seizure disorders relative to controls (odds ratio = 9.76, P = 2.15 × 10−4). We also found evidence for overtransmission of loss-of-function (LoF) or previously reported pathogenic variants in these same genes from heterozygous carrier parents (11 of 14 transmitted, P = 0.03). We identified a total of 11 SUDC proband genotypes (7 de novo, 1 transmitted parental mosaic, 2 transmitted parental heterozygous, and 1 compound heterozygous) as pathogenic and likely contributory to death, a genetic finding in 8.9% of our cohort. Two genes had recurrent missense DNMs, RYR2 and CACNA1C. Both RYR2 mutations are pathogenic (P = 1.7 × 10−7) and were previously studied in mouse models. Both CACNA1C mutations lie within a 104-nt exon (P = 1.0 × 10−7) and result in slowed L-type calcium channel inactivation and lower current density. In total, six pathogenic DNMs can alter calcium-related regulation of cardiomyocyte and neuronal excitability at a submembrane junction, suggesting a pathway conferring susceptibility to sudden death. There was a trend for excess LoF mutations in LoF intolerant genes, where ≥1 nonhealthy sample in denovo-db has a similar variant (odds ratio = 6.73, P = 0.02); additional uncharacterized genetic causes of sudden death in children might be discovered with larger cohorts.

Environmental carcinogens disproportionally mutate genes implicated in neurodevelopmental disorders

De novo mutations contribute to a large proportion of sporadic psychiatric and developmental disorders, yet the potential role of environmental carcinogens as drivers of causal de novo mutations in neurodevelopmental disorders is poorly studied. We demonstrate that several mutagens, including polycyclic aromatic hydrocarbons (PAHs), disproportionately mutate genes related to neurodevelopmental disorders including autism spectrum disorders (ASD), schizophrenia, and attention deficit hyperactivity disorder (ADHD). Other disease genes including amyotrophic lateral sclerosis (ALS), Alzheimers disease, congenital heart disease, orofacial clefts, and coronary artery disease were generally not mutated more than expected. Our findings support a new paradigm of neurodevelopmental disease etiology driven by a contribution of environmentally induced rather than random mutations.

Intermittent BRAF inhibition in advanced BRAF mutated melanoma results of a phase II randomized trial

Combination treatment with BRAF (BRAFi) plus MEK inhibitors (MEKi) has demonstrated survival benefit in patients with advanced melanoma harboring activating BRAF mutations. Previous preclinical studies suggested that an intermittent dosing of these drugs could delay the emergence of resistance. Contrary to expectations, the first published phase 2 randomized study comparing continuous versus intermittent schedule of dabrafenib (BRAFi) plus trametinib (MEKi) demonstrated a detrimental effect of the “on−off” schedule. Here we report confirmatory data from the Phase II randomized open-label clinical trial comparing the antitumoral activity of the standard schedule versus an intermittent combination of vemurafenib (BRAFi) plus cobimetinib (MEKi) in advanced BRAF mutant melanoma patients (NCT02583516). The trial did not meet its primary endpoint of progression free survival (PFS) improvement. Our results show that the antitumor activity of the experimental intermittent schedule of vemurafenib plus cobimetinib is not superior to the standard continuous schedule. Detection of BRAF mutation in cell free tumor DNA has prognostic value for survival and its dynamics has an excellent correlation with clinical response, but not with progression. NGS analysis demonstrated de novo mutations in resistant cases.

Whole-genome Sequencing Reveals De-novo Mutations Associated with Nonsyndromic Cleft Lip/Palate

The majority (85%) of nonsyndromic cleft lip with or without cleft palate (nsCL/P) cases occur sporadically, suggesting a role for de novo mutations (DNMs) in the etiology of nsCL/P. To identify high impact DNMs that contribute to the risk of nsCL/P, we conducted whole genome sequencing (WGS) analyses in 130 African case-parent trios (affected probands and unaffected parents). We identified 162 high confidence protein-altering DNMs that contribute to the risk of nsCL/P. These include novel loss-of-function DNMs in the ACTL6A, ARHGAP10, MINK1, TMEM5 and TTN genes; as well as missense variants in ACAN, DHRS3, DLX6, EPHB2, FKBP10, KMT2D, RECQL4, SEMA3C, SEMA4D, SHH, TP63, and TULP4. Experimental evidence showed that ACAN, DHRS3, DLX6, EPHB2, FKBP10, KMT2D, MINK1, RECQL4, SEMA3C, SEMA4D, SHH, TP63, and TTN genes contribute to facial development and mutations in these genes could contribute to CL/P. Association studies have identified TULP4 as a potential cleft candidate gene, while ARHGAP10 interacts with CTNNB1 to control WNT signaling. DLX6, EPHB2, SEMA3C and SEMA4D harbor novel damaging DNMs that may affect their role in neural crest migration and palatal development. This discovery of pathogenic DNMs also confirms the power of WGS analysis of trios in the discovery of potential pathogenic variants.

Drastic mutations can be hidden in short-read mapping: thousands of mutation clusters in human genome are explained by short-range template switching

Variation within human genomes is distributed unevenly and variants show spatial clustering. DNA-replication related template switching is a poorly known mutational mechanism capable of causing major chromosomal rearrangements as well as creating short inverted sequence copies that appear as local mutation clusters in sequence comparisons. We reanalyzed haplotype-resolved genome assemblies representing 25 human populations and multinucleotide variants aggregated from 140,000 human sequencing experiments. We found local template switching to explain thousands of complex mutation clusters across the human genome, the loci segregating within and between populations with a small number appearing as de novo mutations. We developed computational tools for genotyping candidate template switch loci using short-read sequencing data and for identification of template switch events using both short-read data and genotype data. These tools will enable building a catalogue of affected loci and studying the cellular mechanisms behind template switching both in healthy organisms and in disease. Strikingly, we noticed that widely-used analysis pipelines for short-read sequencing data - capable of identifying single nucleotide changes - may miss TSM-origin inversions of tens of base pairs, potentially invalidating medical genetic studies searching for causative alleles behind genetic diseases.