<i>De Novo</i> and Rare Variants at Multiple Loci Support the Oligogenic Origins of Atrioventricular Septal Heart Defects

Congenital heart disease (CHD) has a complex genetic etiology, and recent studies suggest that high penetrance de novo mutations may account for only a small fraction of disease. In a multi-institutional cohort surveyed by exome sequencing, combining analysis of 987 individuals (discovery cohort of 59 affected trios and 59 control trios, and a replication cohort of 100 affected singletons and 533 unaffected singletons) we observe variation at novel and known loci related to a specific cardiac malformation the atrioventricular septal defect (AVSD). In a primary analysis, by combining developmental coexpression networks with inheritance modeling, we identify a de novo mutation in the DNA binding domain of NR1D2 (p.R175W). We show that p.R175W changes the transcriptional activity of Nr1d2 using an in vitro transactivation model in HUVEC cells. Finally, we demonstrate previously unrecognized cardiovascular malformations in the Nr1d2tm1-Dgen knockout mouse. In secondary analyses we map genetic variation to protein-interaction networks suggesting a role for two collagen genes in AVSD, which we corroborate by burden testing in a second replication cohort of 100 AVSDs and 533 controls (p = 8.37e-08). Finally, we apply a rare-disease inheritance model to identify variation in genes previously associated with CHD (ZFPM2, NSD1, NOTCH1, VCAN, and MYH6), cardiac malformations in mouse models (ADAM17, CHRD, IFT140, PTPRJ, RYR1 and ATE1), and hypomorphic alleles of genes causing syndromic CHD (EHMT1, SRCAP, BBS2, NOTCH2, and KMT2D) in 14 of 59 trios, greatly exceeding variation in control trios without CHD (p = 9.60e-06). In total, 32% of trios carried at least one putatively disease-associated variant across 19 loci,suggesting that inherited and de novo variation across a heterogeneous group of loci may contribute to disease risk

Kidney Biopsy in Autosomal Dominant Polycystic Kidney Disease

Proteinuria is an easily quantified biomarker of kidney disease and often a sign of glomerular pathology. Significant proteinuria is uncommon in cystic kidney diseases and should be further evaluated to exclude the presence of another simultaneous kidney disease. While renal biopsy is a valuable part of the diagnostic evaluation of proteinuria, careful consideration of risks and benefits is necessary before proceeding in a patient with bilateral renal cysts. We report the case of a man with Polycystic Kidney Disease (PKD) who was found to have nephrotic-range proteinuria. An ultrasound-guided kidney biopsy revealed evidence of Focal Segmental Glomerulosclerosis (FSGS), which was attributed to hyperfiltration-related injury in the context of extensive kidney cysts. Genetic testing did not reveal a cause of FSGS and showed a variant of uncertain significance in PKD1. We use this case to highlight three important issues that are applicable to patients with PKD: the role of diagnostic evaluation for proteinuria in cystic kidney disease, the feasibility of kidney biopsy despite the presence of bilateral renal cysts, and the roles and limitations of genetic testing in cystic kidney disease and FSGS

Apolipoprotein E Epsilon 4 Genotype, Mild Traumatic Brain Injury, and the Development of Chronic Traumatic Encephalopathy.

The annual incidence of mild traumatic brain injury (MTBI) is 3.8 million in the USA with 10⁻15% experiencing persistent morbidity beyond one year. Chronic traumatic encephalopathy (CTE), a neurodegenerative disease characterized by accumulation of hyperphosphorylated tau, can occur with repetitive MTBI. Risk factors for CTE are challenging to identify because injury mechanisms of MTBI are heterogeneous, clinical manifestations and management vary, and CTE is a postmortem diagnosis, making prospective studies difficult. There is growing interest in the genetic influence on head trauma and development of CTE. Apolipoprotein epsilon 4 (APOE-ε4) associates with many neurologic diseases, and consensus on the ε4 allele as a risk factor is lacking. This review investigates the influence of APOE-ε4 on MTBI and CTE. A comprehensive PubMed literature search (1966 to 12 June 2018) identified 24 unique reports on the topic (19 MTBI studies: 8 athletic, 5 military, 6 population-based; 5 CTE studies: 4 athletic and military, 1 leucotomy group). APOE-ε4 genotype is found to associate with outcomes in 4/8 athletic reports, 3/5 military reports, and 5/6 population-based reports following MTBI. Evidence on the association between APOE-ε4 and CTE from case series is equivocal. Refining modalities to aid CTE diagnosis in larger samples is needed in MTBI

Diacylglycerol Kinase K Variants Impact Hypospadias in a California Study Population

PurposeA recent genome wide association study demonstrated the novel finding that variants in DGKK are associated with hypospadias. Our objectives were to determine whether this finding could be replicated in a more racially/ethnically diverse study population of California births and to provide a more comprehensive investigation of variants.Materials and methodsWe examined the association of 27 DGKK single nucleotide polymorphisms with hypospadias relative to population based nonmalformed controls born in selected California counties from 1990 to 2003. Analyses included a maximum of 928 controls and 665 cases (mild in 91, moderate in 336, severe in 221 and undetermined in 17). Results for mild and moderate cases were similar, so they were grouped together.ResultsFor mild and moderate cases OR for 15 of the 27 single nucleotide polymorphisms had p values less than 0.05, with 2 less than 1 and the others ranging from 1.3 to 1.8. Among severe cases ORs tended to be closer to 1, and none of the p values were less than 0.05. Due to high linkage disequilibrium across the single nucleotide polymorphisms, haplotype analyses were conducted and 2 blocks were generated. These analyses identified a set of 8 variants associated with a threefold to fourfold increased risk relative to the most common haplotype, regardless of severity of the phenotype (OR 4.1, p &lt;10(-4) for mild to moderate cases and 3.3, p = 0.001 for severe cases).ConclusionsThis study confirms that DGKK variants are associated with hypospadias. Additional studies are needed to allow a more thorough investigation of DGKK variability and to delineate the mechanism by which DGKK contributes to urethral development

Protein interaction networks identify collagen genes putatively associated with atrioventricular septal defects.

<p><b>(a)</b> A network diagram displaying protein-protein interactions between genes. A greater number of networks (n = 86) and genes (n = 231) are detected by protein-interaction analysis in AVSD-trio subnetworks (green nodes) relative to the networks (n = 26) and genes (n = 60) detected in the Control-trio subnetworks (yellow nodes). A median <i>p</i>-value from a protein interaction network permutation procedure is reported for each group of subnetworks, which shows an enrichment of true protein-protein interactions in the AVSD-trio subnetworks (<i>p</i> = 0.01) that is not observed in the control trio subnetworks (<i>p</i> = 1). The black lines indicate a protein-protein interaction between two nodes in the subnetwork and the weight of the line corresponds to “heat” value output by the Hotnet2 algorithm. The number of protein interactions for each observed gene corresponds to the size of the node. The two collagen genes in the AVSD-trio subnetworks (<i>COL2A1</i> and <i>COL9A1</i>) are labeled and the nodes highlighted in red. The networks from each group of trios are arranged in a circular layout. <b>(b)</b> A Venn diagram comparing genes/variants in the AVSD-trio subnetworks (green circle) and Control-trio subnetworks (yellow circle) to genes expressed during early mouse cardiac development (grey circle). The number of genes in each dataset and their overlap with other datasets is indicated and the <i>p</i>-value from a hypergeometric test is reported. A greater proportion of genes in the AVSD-trio subnetworks (60 of 231 genes, <i>p</i> = 9e-09) are found to be expressed in the early cardiac development dataset, compared to the control-trio subnetworks (10 of 60 genes, <i>p</i> = 0.34). <b>(c)</b> The subnetwork comprised of <i>COL2A1</i> and <i>COL9A1</i> are highlighted in a Manhattan plot of test statistics from the SKAT linear weight test for each of the 86 AVSD-trio subnetworks identifies an elevated burden of variation in a replication cohort of 100 AVSD subjects and 533 control subjects. The <i>p</i>-value is plotted for the chromosomal position of each gene within the subnetwork. A significance cutoff of 5.81e-04 was derived from the Bonferroni correction of 86 tests performed.</p

An illustration of discovery of protein interaction networks for AVSD and validation by burden testing in separate a replication cohort.

<p>Filtered SNPs and CNVs from the AVSD trio probands are mapped to a protein interaction network (represented by grey dots and black lines), and the network is pruned to yield subnetworks (green dots and black lines). The subnetworks represent variants in genes which have verified interactions at the protein level, often constituting a portion of a signaling pathway or enzymatic complex [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005963#pgen.1005963.ref022" target="_blank">22</a>]. To validate the disease association of the individual subnetworks discovered in the trios, we performed burden testing for each subnetwork in a replication cohort of 100 AVSD cases originating largely from a separate study of AVSDs [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005963#pgen.1005963.ref010" target="_blank">10</a>] along with 533 controls without congenital heart disease.</p

AVSD trio probands display <i>de novo</i> and inherited variation in genes related to human and mouse cardiac malformations.

<p>AVSD trio probands display <i>de novo</i> and inherited variation in genes related to human and mouse cardiac malformations.</p

Discovered CNVs associated with AVSDs and other cardiac malformations.

<p>Discovered CNVs associated with AVSDs and other cardiac malformations.</p

Analytical approach for disease gene discovery in a cohort of AVSD trios.

<p>Protein altering variants meeting allele frequency, quality, and read depth cutoffs from 59 trios were sorted with an inheritance model consistent with rare disease which combines <i>de novo</i>, compound heterozygous, and rare homozygous variants to collate variants into a list of genes [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005963#pgen.1005963.ref016" target="_blank">16</a>]. Three analytical approaches were applied to the final dataset. Primary Analysis. <i>De novo</i> variants in the AVSD trios were cross-referenced with the genes in a module highly enriched for CHD and cardiac development obtained from unsupervised weighted-gene coexpression network analysis to identify a novel AVSD gene. Secondary Analyses. Variants from the AVSD trios were mapped onto a protein interaction network followed by burden testing in a replication cohort (details in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005963#pgen.1005963.g003" target="_blank">Fig 3</a>). In a final analysis employing inheritance modeling, <i>de novo</i>, compound heterozygous, and rare homozygous loci observed in the AVSD trios were compared with a predefined list of genes associated with human or mouse cardiac malformations. Statistical comparisons were performed in a control group of 59 control trios without CHD.</p