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.

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Re-evaluation of single nucleotide variants and identification of structural variants in a cohort of 45 sudden unexplained death cases

International Journal of Legal Medicine ◽

10.1007/s00414-021-02580-5 ◽

2021 ◽

Author(s):

Jacqueline Neubauer ◽

Shouyu Wang ◽

Giancarlo Russo ◽

Cordula Haas

Keyword(s):

Sudden Death ◽

Cardiac Diseases ◽

Structural Variants ◽

Single Nucleotide Variants ◽

Single Nucleotide ◽

Sudden Unexplained Death ◽

Unexplained Death ◽

Pathogenic Variants ◽

The Impact ◽

Death Cases

AbstractSudden unexplained death (SUD) takes up a considerable part in overall sudden death cases, especially in adolescents and young adults. During the past decade, many channelopathy- and cardiomyopathy-associated single nucleotide variants (SNVs) have been identified in SUD studies by means of postmortem molecular autopsy, yet the number of cases that remain inconclusive is still high. Recent studies had suggested that structural variants (SVs) might play an important role in SUD, but there is no consensus on the impact of SVs on inherited cardiac diseases. In this study, we searched for potentially pathogenic SVs in 244 genes associated with cardiac diseases. Whole-exome sequencing and appropriate data analysis were performed in 45 SUD cases. Re-analysis of the exome data according to the current ACMG guidelines identified 14 pathogenic or likely pathogenic variants in 10 (22.2%) out of the 45 SUD cases, whereof 2 (4.4%) individuals had variants with likely functional effects in the channelopathy-associated genes SCN5A and TRDN and 1 (2.2%) individual in the cardiomyopathy-associated gene DTNA. In addition, 18 structural variants (SVs) were identified in 15 out of the 45 individuals. Two SVs with likely functional impairment were found in the coding regions of PDSS2 and TRPM4 in 2 SUD cases (4.4%). Both were identified as heterozygous deletions, which were confirmed by multiplex ligation-dependent probe amplification. In conclusion, our findings support that SVs could contribute to the pathology of the sudden death event in some of the cases and therefore should be investigated on a routine basis in suspected SUD cases.

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De novo mutations in inhibitors of Wnt, BMP, and Ras/ERK signaling pathways in non-syndromic midline craniosynostosis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1709255114 ◽

2017 ◽

Vol 114 (35) ◽

pp. E7341-E7347 ◽

Cited By ~ 29

Author(s):

Andrew T. Timberlake ◽

Charuta G. Furey ◽

Jungmin Choi ◽

Carol Nelson-Williams ◽

Erin Loring ◽

...

Keyword(s):

De Novo ◽

Erk Signaling ◽

Loss Of Function ◽

De Novo Mutations ◽

Neurodevelopmental Outcomes ◽

Locus Heterogeneity ◽

Syndromic Craniosynostosis ◽

New Genes ◽

Morphogenetic Protein ◽

Nuclear Targets

Non-syndromic craniosynostosis (NSC) is a frequent congenital malformation in which one or more cranial sutures fuse prematurely. Mutations causing rare syndromic craniosynostoses in humans and engineered mouse models commonly increase signaling of the Wnt, bone morphogenetic protein (BMP), or Ras/ERK pathways, converging on shared nuclear targets that promote bone formation. In contrast, the genetics of NSC is largely unexplored. More than 95% of NSC is sporadic, suggesting a role for de novo mutations. Exome sequencing of 291 parent–offspring trios with midline NSC revealed 15 probands with heterozygous damaging de novo mutations in 12 negative regulators of Wnt, BMP, and Ras/ERK signaling (10.9-fold enrichment, P = 2.4 × 10−11). SMAD6 had 4 de novo and 14 transmitted mutations; no other gene had more than 1. Four familial NSC kindreds had mutations in genes previously implicated in syndromic disease. Collectively, these mutations contribute to 10% of probands. Mutations are predominantly loss-of-function, implicating haploinsufficiency as a frequent mechanism. A common risk variant near BMP2 increased the penetrance of SMAD6 mutations and was overtransmitted to patients with de novo mutations in other genes in these pathways, supporting a frequent two-locus pathogenesis. These findings implicate new genes in NSC and demonstrate related pathophysiology of common non-syndromic and rare syndromic craniosynostoses. These findings have implications for diagnosis, risk of recurrence, and risk of adverse neurodevelopmental outcomes. Finally, the use of pathways identified in rare syndromic disease to find genes accounting for non-syndromic cases may prove broadly relevant to understanding other congenital disorders featuring high locus heterogeneity.

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Re-analysis of whole-exome sequencing data uncovers novel diagnostic variants and improves molecular diagnostic yields for sudden death and idiopathic diseases

Genome Medicine ◽

10.1186/s13073-019-0702-2 ◽

2019 ◽

Vol 11 (1) ◽

Cited By ~ 3

Author(s):

Elias L. Salfati ◽

Emily G. Spencer ◽

Sarah E. Topol ◽

Evan D. Muse ◽

Manuel Rueda ◽

...

Keyword(s):

Sudden Death ◽

Rare Disease ◽

Exome Sequencing ◽

Whole Exome Sequencing ◽

Diagnostic Yield ◽

Molecular Diagnostic ◽

Sudden Unexplained Death ◽

Unexplained Death ◽

Pathogenic Variants ◽

Whole Exome

Abstract Background Whole-exome sequencing (WES) has become an efficient diagnostic test for patients with likely monogenic conditions such as rare idiopathic diseases or sudden unexplained death. Yet, many cases remain undiagnosed. Here, we report the added diagnostic yield achieved for 101 WES cases re-analyzed 1 to 7 years after initial analysis. Methods Of the 101 WES cases, 51 were rare idiopathic disease cases and 50 were postmortem “molecular autopsy” cases of early sudden unexplained death. Variants considered for reporting were prioritized and classified into three groups: (1) diagnostic variants, pathogenic and likely pathogenic variants in genes known to cause the phenotype of interest; (2) possibly diagnostic variants, possibly pathogenic variants in genes known to cause the phenotype of interest or pathogenic variants in genes possibly causing the phenotype of interest; and (3) variants of uncertain diagnostic significance, potentially deleterious variants in genes possibly causing the phenotype of interest. Results Initial analysis revealed diagnostic variants in 13 rare disease cases (25.4%) and 5 sudden death cases (10%). Re-analysis resulted in the identification of additional diagnostic variants in 3 rare disease cases (5.9%) and 1 sudden unexplained death case (2%), which increased our molecular diagnostic yield to 31.4% and 12%, respectively. Conclusions The basis of new findings ranged from improvement in variant classification tools, updated genetic databases, and updated clinical phenotypes. Our findings highlight the potential for re-analysis to reveal diagnostic variants in cases that remain undiagnosed after initial WES.

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A de novo BRPF1 variant in a case of Sudden Unexplained Death in Childhood

European Journal of Medical Genetics ◽

10.1016/j.ejmg.2020.104002 ◽

2020 ◽

Vol 63 (9) ◽

pp. 104002

Author(s):

Christine Keywan ◽

Ingrid A. Holm ◽

Annapurna Poduri ◽

Catherine A. Brownstein ◽

Sanda Alexandrescu ◽

...

Keyword(s):

De Novo ◽

Sudden Unexplained Death ◽

Unexplained Death

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Molecular Dissection of Neurodevelopmental Disorder-Causing Mutations in CYFIP2

Cells ◽

10.3390/cells9061355 ◽

2020 ◽

Vol 9 (6) ◽

pp. 1355

Author(s):

Matthias Schaks ◽

Michael Reinke ◽

Walter Witke ◽

Klemens Rottner

Keyword(s):

De Novo ◽

Neurodevelopmental Disorder ◽

Small Gtpases ◽

Normal Brain ◽

Loss Of Function ◽

De Novo Mutations ◽

Neuronal Morphogenesis ◽

Variable Extent ◽

Regulatory Complex ◽

Almost All

Actin remodeling is frequently regulated by antagonistic activities driving protrusion and contraction downstream of Rac and Rho small GTPases, respectively. WAVE regulatory complex (WRC), which primarily operates downstream of Rac, plays pivotal roles in neuronal morphogenesis. Recently, two independent studies described de novo mutations in the CYFIP2 subunit of WRC, which caused intellectual disability (ID) in humans. Although mutations had been proposed to effect WRC activation, no experimental evidence for this was provided. Here, we made use of CRISPR/Cas9-engineered B16-F1 cell lines that were reconstituted with ID-causing CYFIP variants in different experimental contexts. Almost all CYFIP2-derived mutations (7 out of 8) promoted WRC activation, but to variable extent and with at least two independent mechanisms. The majority of mutations occurs in a conserved WAVE-binding region, required for WRC transinhibition. One mutation is positioned closely adjacent to the Rac-binding A site and appears to ease Rac-mediated WRC activation. As opposed to these gain-of-function mutations, a truncating mutant represented a loss-of-function variant and failed to interact with WRC components. Collectively, our data show that explored CYFIP2 mutations frequently, but not always, coincide with WRC activation and suggest that normal brain development requires a delicate and precisely tuned balance of neuronal WRC activity.

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SOX2 Plays a Critical Role in the Pituitary, Forebrain, and Eye during Human Embryonic Development

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jc.2007-2337 ◽

2008 ◽

Vol 93 (5) ◽

pp. 1865-1873 ◽

Cited By ~ 92

Author(s):

Daniel Kelberman ◽

Sandra C. P. de Castro ◽

Shuwen Huang ◽

John A. Crolla ◽

Rodger Palmer ◽

...

Keyword(s):

Embryonic Development ◽

Anterior Pituitary ◽

De Novo ◽

Hypogonadotropic Hypogonadism ◽

Expression Patterns ◽

Critical Role ◽

Loss Of Function ◽

De Novo Mutations ◽

Direct Role

Abstract Context: Heterozygous, de novo mutations in the transcription factor SOX2 are associated with bilateral anophthalmia or severe microphthalmia and hypopituitarism. Variable additional abnormalities include defects of the corpus callosum and hippocampus. Objective: We have ascertained a further three patients with severe eye defects and pituitary abnormalities who were screened for mutations in SOX2. To provide further evidence of a direct role for SOX2 in hypothalamo-pituitary development, we have studied the expression of the gene in human embryonic tissues. Results: All three patients harbored heterozygous SOX2 mutations: a deletion encompassing the entire gene, an intragenic deletion (c.70_89del), and a novel nonsense mutation (p.Q61X) within the DNA binding domain that results in impaired transactivation. We also show that human SOX2 can inhibit β-catenin-driven reporter gene expression in vitro, whereas mutant SOX2 proteins are unable to repress efficiently this activity. Furthermore, we show that SOX2 is expressed throughout the human brain, including the developing hypothalamus, as well as Rathke’s pouch, the developing anterior pituitary, and the eye. Conclusions: Patients with SOX2 mutations often manifest the unusual phenotype of hypogonadotropic hypogonadism, with sparing of other pituitary hormones despite anterior pituitary hypoplasia. SOX2 expression patterns in human embryonic development support a direct involvement of the protein during development of tissues affected in these individuals. Given the critical role of Wnt-signaling in the development of most of these tissues, our data suggest that a failure to repress the Wnt-β-catenin pathway could be one of the underlying pathogenic mechanisms associated with loss-of-function mutations in SOX2.

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A de novo paradigm for male infertility

10.1101/2021.02.27.433155 ◽

2021 ◽

Author(s):

MS Oud ◽

RM Smits ◽

HE Smith ◽

FK Mastrorosa ◽

GS Holt ◽

...

Keyword(s):

Male Infertility ◽

De Novo ◽

P Value ◽

Missense Mutations ◽

Loss Of Function ◽

De Novo Mutations ◽

Systematic Analysis ◽

Significant Enrichment

IntroductionDe novo mutations (DNMs) are known to play a prominent role in sporadic disorders with reduced fitness1. We hypothesize that DNMs play an important role in male infertility and explain a significant fraction of the genetic causes of this understudied disorder. To test this hypothesis, we performed trio-based exome-sequencing in a unique cohort of 185 infertile males and their unaffected parents. Following a systematic analysis, 29 of 145 rare protein altering DNMs were classified as possibly causative of the male infertility phenotype. We observed a significant enrichment of Loss-of-Function (LoF) DNMs in LoF-intolerant genes (p-value=1.00×10-5) as well as predicted pathogenic missense DNMs in missense-intolerant genes (p-value=5.01×10-4). One DNM gene identified, RBM5, is an essential regulator of male germ cell pre-mRNA splicing2. In a follow-up study, 5 rare pathogenic missense mutations affecting this gene were observed in a cohort of 2,279 infertile patients, with no such mutations found in a cohort of 5,784 fertile men (p-value=0.009). Our results provide the first evidence for the role of DNMs in severe male infertility and point to many new candidate genes affecting fertility.

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Abstract 15841: Whole Exome Sequencing and Analysis for Sudden Unexplained Death in the Young: A Case Series

Circulation ◽

10.1161/circ.130.suppl_2.15841 ◽

2014 ◽

Vol 130 (suppl_2) ◽

Author(s):

Nupoor Narula ◽

David J Tester ◽

Anna Paulmichl ◽

Joseph J Maleszewski ◽

Michael J Ackerman

Keyword(s):

Sudden Death ◽

Exome Sequencing ◽

Whole Exome Sequencing ◽

Effective Means ◽

Susceptibility Gene ◽

Case Series ◽

Susceptibility Genes ◽

Sudden Unexplained Death ◽

Unexplained Death ◽

Whole Exome

Introduction: Annually, thousands of sudden deaths in individuals under the age of 35 years remain unexplained following a medico-legal autopsy and are termed autopsy negative sudden unexplained death in the young (SUDY). Cardiomyopathies, channelopathies, and metabolic disorders may underlie a significant number of SUDY cases. Previously, we demonstrated that 25% of autopsy-negative SUDY cases had mutations in the 4 major cardiac ion channel genes ( KCNQ1, KCNH2, SCN5A , and RYR2 ). However, over 100 sudden death-susceptibility genes have been discovered and may be implicated in SUDY. Objective: We explored the utility of whole exome sequencing (WES) followed by gene-specific surveillance as an efficient and effective means of performing post-mortem genetic testing in SUDY. Methods: Postmortem WES was performed on 14 consecutively-referred white SUDY victims (57% men; average age at death 17.4 ± 8.6 years) using the Agilent SureSelect Human All Exon V4+UTR capture kit and an Illumina HiSeq 2000 sequencer. Following variant alignment (hg19) and annotation, 117 cardiac channelopathy-, cardiomyopathy-, and metabolic disorder-susceptibility genes were surveyed to identify putative SUDY-associated mutations. Potentially pathogenic variants had to be non-synonymous and ultra-rare [i.e. absent in all 3 evaluated exome databases (1,000 Genome Project, the NHLBI GO Exome Sequencing Project, and Exome Chip Design)]. Results: On average, each SUDY case had 12,758 ± 2016 non-synonymous variants, of which 79 ± 15 localized to the 117 evaluated genes. Overall, 8 unique, ultra-rare variants (7 missense, 1 in-frame insertion) identified in 6 genes (3 in TTN ; 1 each in CACNA1C, JPH2, MYH7, VCL, RYR2 ) were detected in 7 of 14 cases (50%). Of the 7 missense alterations, 2 (T171M- CACNA1C , I22160T- TTN ) were predicted damaging by 3 in-silico tools. Conclusions: Although WES and gene-specific surveillance is an efficient and effective strategy to detect rare, potentially lethal, genetic variants, the accurate interpretation of each variant is daunting. Importantly, rarity, even ultra-rarity, does not equal pathogenicity even when the ultra-rare variant resides within a so-called sudden death-susceptibility gene.

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Loss-of-Function GRHL3 Variants Detected in African Patients with Isolated Cleft Palate

Journal of Dental Research ◽

10.1177/0022034517729819 ◽

2017 ◽

Vol 97 (1) ◽

pp. 41-48 ◽

Cited By ~ 11

Author(s):

M.A. Eshete ◽

H. Liu ◽

M. Li ◽

W.L. Adeyemo ◽

L.J.J Gowans ◽

...

Keyword(s):

Cleft Palate ◽

Cleft Lip ◽

De Novo ◽

Phosphorylation Site ◽

Dominant Negative ◽

Loss Of Function ◽

De Novo Mutations ◽

Sumoylation Site ◽

Genetic Etiology

In contrast to the progress that has been made toward understanding the genetic etiology of cleft lip with or without cleft palate, relatively little is known about the genetic etiology for cleft palate only (CPO). A common coding variant of grainyhead like transcription factor 3 ( GRHL3) was recently shown to be associated with risk for CPO in Europeans. Mutations in this gene were also reported in families with Van der Woude syndrome. To identify rare mutations in GRHL3 that might explain the missing heritability for CPO, we sequenced GRHL3 in cases of CPO from Africa. We recruited participants from Ghana, Ethiopia, and Nigeria. This cohort included case-parent trios, cases and other family members, as well as controls. We sequenced exons of this gene in DNA from a total of 134 nonsyndromic cases. When possible, we sequenced them in parents to identify de novo mutations. Five novel mutations were identified: 2 missense (c.497C>A; p.Pro166His and c.1229A>G; p.Asp410Gly), 1 splice site (c.1282A>C p.Ser428Arg), 1 frameshift (c.470delC; p.Gly158Alafster55), and 1 nonsense (c.1677C>A; p.Tyr559Ter). These mutations were absent from 270 sequenced controls and from all public exome and whole genome databases, including the 1000 Genomes database (which includes data from Africa). However, 4 of the 5 mutations were present in unaffected mothers, indicating that their penetrance is incomplete. Interestingly, 1 mutation damaged a predicted sumoylation site, and another disrupted a predicted CK1 phosphorylation site. Overexpression assays in zebrafish and reporter assays in vitro indicated that 4 variants were functionally null or hypomorphic, while 1 was dominant negative. This study provides evidence that, as in Caucasian populations, mutations in GRHL3 contribute to the risk of nonsyndromic CPO in the African population.

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