Targeted massively parallel sequencing of autism spectrum disorder-associated genes in a case control cohort reveals rare loss-of-function risk variants

BACKGROUND: Autism spectrum disorder (ASD) is highly heritable, yet genome-wide association studies (GWAS), copy number variation screens, and candidate gene association studies have found no single factor accounting for a large percentage of genetic risk. ASD trio exome sequencing studies have revealed genes with recurrent de novo loss-of-function variants as strong risk factors, but there are relatively few recurrently affected genes while as many as 1000 genes are predicted to play a role. As such, it is critical to identify the remaining rare and low-frequency variants contributing to ASD. METHODS: We have utilized an approach of prioritization of genes by GWAS and follow-up with massively parallel sequencing in a case-control cohort. Using a previously reported ASD noise reduction GWAS analyses, we prioritized 837 RefSeq genes for custom targeting and sequencing. We sequenced the coding regions of those genes in 2071 ASD cases and 904 controls of European white ancestry. We applied comprehensive annotation to identify single variants which could confer ASD risk and also gene-based association analysis to identify sets of rare variants associated with ASD. RESULTS: We identified a significant over-representation of rare loss-of-function variants in genes previously associated with ASD, including a de novo premature stop variant in the well-established ASD candidate gene RBFOX1. Furthermore, ASD cases were more likely to have two damaging missense variants in candidate genes than controls. Finally, gene-based rare variant association implicates genes functioning in excitatory neurotransmission and neurite outgrowth and guidance pathways including CACNAD2, KCNH7, and NRXN1. CONCLUSIONS: We find suggestive evidence that rare variants in synaptic genes are associated with ASD and that loss-of-function mutations in ASD candidate genes are a major risk factor, and we implicate damaging mutations in glutamate signaling receptors and neuronal adhesion and guidance molecules. Furthermore, the role of de novo mutations in ASD remains to be fully investigated as we identified the first reported protein-truncating variant in RBFOX1 in ASD. Overall, this work, combined with others in the field, suggests a convergence of genes and molecular pathways underlying ASD etiology. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13229-015-0034-z) contains supplementary material, which is available to authorized users

ABCA7 frameshift deletion associated with Alzheimer disease in African Americans

Objective: To identify a causative variant(s) that may contribute to Alzheimer disease (AD) in African Americans (AA) in the ATP-binding cassette, subfamily A (ABC1), member 7 (ABCA7) gene, a known risk factor for late-onset AD. Methods: Custom capture sequencing was performed on ∼150 kb encompassing ABCA7 in 40 AA cases and 37 AA controls carrying the AA risk allele (rs115550680). Association testing was performed for an ABCA7 deletion identified in large AA data sets (discovery n = 1,068; replication n = 1,749) and whole exome sequencing of Caribbean Hispanic (CH) AD families. Results: A 44-base pair deletion (rs142076058) was identified in all 77 risk genotype carriers, which shows that the deletion is in high linkage disequilibrium with the risk allele. The deletion was assessed in a large data set (531 cases and 527 controls) and, after adjustments for age, sex, and APOE status, was significantly associated with disease (p = 0.0002, odds ratio [OR] = 2.13 [95% confidence interval (CI): 1.42–3.20]). An independent data set replicated the association (447 cases and 880 controls, p = 0.0117, OR = 1.65 [95% CI: 1.12–2.44]), and joint analysis increased the significance (p = 1.414 × 10−5, OR = 1.81 [95% CI: 1.38–2.37]). The deletion is common in AA cases (15.2%) and AA controls (9.74%), but in only 0.12% of our non-Hispanic white cohort. Whole exome sequencing of multiplex, CH families identified the deletion cosegregating with disease in a large sibship. The deleted allele produces a stable, detectable RNA strand and is predicted to result in a frameshift mutation (p.Arg578Alafs) that could interfere with protein function. Conclusions: This common ABCA7 deletion could represent an ethnic-specific pathogenic alteration in AD

Defective HNF4alpha-dependent gene expression as a driver of hepatocellular failure in alcoholic hepatitis

Alcoholic hepatitis (AH) is a life-threatening condition characterized by profound hepatocellular dysfunction for which targeted treatments are urgently needed. Identification of molecular drivers is hampered by the lack of suitable animal models. By performing RNA sequencing in livers from patients with different phenotypes of alcohol-related liver disease (ALD), we show that development of AH is characterized by defective activity of liver-enriched transcription factors (LETFs). TGFβ1 is a key upstream transcriptome regulator in AH and induces the use of HNF4α P2 promoter in hepatocytes, which results in defective metabolic and synthetic functions. Gene polymorphisms in LETFs including HNF4α are not associated with the development of AH. In contrast, epigenetic studies show that AH livers have profound changes in DNA methylation state and chromatin remodeling, affecting HNF4α-dependent gene expression. We conclude that targeting TGFβ1 and epigenetic drivers that modulate HNF4α-dependent gene expression could be beneficial to improve hepatocellular function in patients with AH

Absence of both MGME1 and POLG EXO abolishes mtDNA whereas absence of either creates unique mtDNA duplications

Both POLG and MGME1 are needed for mitochondrial DNA (mtDNA) maintenance in animal cells. POLG, the primary replicative polymerase of the mitochondria, has an exonuclease activity (3’→5’) that corrects for the misincorporation of bases. MGME1 serves as an exonuclease (5’→3’), producing ligatable DNA ends. Although both have a critical role in mtDNA replication and elimination of linear fragments, these mechanisms are still not fully understood. Using digital PCR to evaluate and compare mtDNA integrity, we show that Mgme1 knock out (Mgme1 KK) tissue mtDNA is more fragmented than POLG exonuclease deficient “Mutator” (Polg MM) or WT tissue. In addition, next generation sequencing of mutant hearts showed abundant duplications in/nearby the D-loop region and unique 100bp duplications evenly spaced throughout the genome only in Mgme1 KK hearts. However, despite these unique mtDNA features at steady-state, we observed a similar delay in the degradation of mtDNA after an induced double strand DNA break in both Mgme1 KK and Polg MM models. Lastly, we characterized double mutant (Polg MM/Mgme1 KK) cells and show that mtDNA cannot be maintained without at least one of these enzymatic activities. We propose a model for the generation of these genomic abnormalities which suggests a role for MGME1 outside of nascent mtDNA end ligation. Our results highlight the role of MGME1 in and outside of the D-loop region during replication, support the involvement of MGME1 in dsDNA degradation and demonstrate that POLG EXO and MGME1 can partially compensate for each other in maintaining mtDNA

A review of current advancements and limitations of artificial intelligence in genitourinary cancers.

Advances in deep learning and neural networking have allowed clinicians to understand the impact that artificial intelligence (AI) could have on improving clinical outcomes and resources expenditures. In the realm of genitourinary (GU) cancers, AI has had particular success in improving the diagnosis and treatment of prostate, renal, and bladder cancers. Numerous studies have developed methods to utilize neural networks to automate prognosis prediction, treatment plan optimization, and patient education. Furthermore, many groups have explored other techniques, including digital pathology and expert 3D modeling systems. Compared to established methods, nearly all the studies showed some level of improvement and there is evidence that AI pipelines can reduce the subjectivity in the diagnosis and management of GU malignancies. However, despite the many potential benefits of utilizing AI in urologic oncology, there are some notable limitations of AI when combating real-world data sets. Thus, it is vital that more prospective studies be conducted that will allow for a better understanding of the benefits of AI to both cancer patients and urologists

Ancestry‐Specific eQTL Associations in AA/NHW Alzheimer Disease Cohorts

Background Although the genetic etiology of Alzheimer’s Disease (AD) has been shown to vary among ancestrally diverse populations, the influence of genetic variation on the transcriptome within these populations has not yet been extensively investigated. Multi‐ancestry AD datasets featuring both genetic information and gene expression measurements are needed to support expression quantitative trait loci (eQTl) studies in historically underrepresented populations. In this work, we have compared eQTL signals in case/control AD sample populations of both African American (AA) and non‐Hispanic white (NHW) ancestries. Method We analyzed cohorts of AA and NHW ancestries (Naa = 232, Nnhw = 241) with corresponding array‐based genotypes and RNA‐seq mRNA expression levels from whole blood, along with clinical AD diagnoses (Naa_case = 115, Naa_control = 117, Nnhw_case = 121, Nnhw_control = 120). Transcriptome‐wide eQTL associations between genetic variants and gene expression levels were assessed via regression across 17,638 gene units, adjusting for age, sex, experimental factors, and genetic principal components. This was repeated across all sample set combinations of ancestry‐AD pairings (ancestry = [aa, nhw, both] * AD = [case, control, both]). Result The number of significant (p‐value < 10e‐7) eQTL signals varied by ancestry, with the AA cohort showing 76,742 raw eQTL associations, the NHW cohort 19,734, and both ancestries combined 81,763. When further sub‐setting sample sets to AD cases only, 770 and 788 significant eQTL signals were observed for AA and NHW cohorts respectively, which impacted 135 (AA) and 130 (NHW) genes. Interestingly, these ancestry‐aware, case‐only eQTLs were remarkably distinct between the two ancestries, with only 71 (∼9.1%) of the eQTLs and only 19 (∼14.3%) of the impacted genes being shared by both ancestral cohorts. Of particular interest is the appearance of WWOX (a gene of wide neurological importance, and known AD risk loci), as differentially significant in the AA cohort (P = 8.07e‐7) as compared to the NHW cohort (P = 9.06e‐3). Conclusion We have observed differences in eQTL signals relative to AD diagnosis status between AA and NHW sample populations, which may indicate the presence of novel ancestry‐specific effects impacting the genetic etiology of AD through changes in gene expression

Identification of differential regulation of European versus African local ancestry haplotypes surrounding ApoEε4

Background The risk for late‐onset Alzheimer disease (AD) in ApoEε4 carriers differs between ancestral groups. ApoEε4 Non‐Hispanic White (NHW) homozygotes have an odds ratio of 14.9 while the risk is much lower in Africans (AF) (OR 2.2‐5.7). Local ancestry (LA) analyses in ApoEε4 carrier populations have shown the protective effect in Africans relative to NHW is due to factors lying in the LA surrounding ApoEε4. No coding differences between genes in the LA have been observed between AF and NHW ancestries. Thus regulatory differences in LA non‐coding regions are most likely involved in the protective factor(s) lowering the risk for AF carriers of ApoEε4. Enhancers are the most common regulatory element, and thus we sought to identify if any of these variants had functional enhancer effects between the two ancestries. Little functional characterization of genetic regulation in AF ancestries has been investigated. Method We identified 56 significant sequence differences among AF and ApoEε4 haplotypes from the 1000 genomes in a topologically associated area (56kb) surrounding ApoE. None of these differences were identified to be protein coding. We applied Massively Parallel Reporter Assay (MPRA) supplemented with single variant reporter assays using Promega Dual Glo‐Luciferase System in AD relevant cell lines to identify the regulatory potential of these variants and their surrounding regions and to assess the differential effect sizes of the variant alleles on enhancer activity. Result For MPRA and complementary single variants reporter assays, we generated ∼900bp PCR fragments surrounding these variants to ensure full representation of potential regulatory elements. MPRA vector library or single reporter vectors were transfected in three cell lines (SHY‐SY5Y neuronal cells, U‐118 astrocytes and HMC3 microglia). We identified evidence for differential regulation between AF and EU variant haplotypes in intron 5 of PVRL2, TOMM40 intronic regions and a large region located 3’ of APOC1. The latter encompasses a putative enhancer identified through ENCODE analyses in brains with NHW ancestry. Conclusion Our results indicate several areas of differential regulation in this LA region on ApoEε4 haplotypes. Follow‐up of the identified regulatory regions is currently ongoing using publicly available data and in‐house iPSC derived cell lines

An Alzheimer's disease risk variant in TTC3 modifies the actin cytoskeleton organization and the PI3K-Akt signaling pathway in iPSC-derived forebrain neurons

A missense variant in the() gene (rs377155188, p.S1038C, NM_003316.4:c.3113C>G) was found to segregate with disease in a multigenerational family with late onset Alzheimer's disease. This variant was introduced into induced pluripotent stem cells (iPSCs) derived from a cognitively intact individual using CRISPR genome editing and the resulting isogenic pair of iPSC lines were differentiated into cortical neurons. Transcriptome analysis showed an enrichment for genes involved in axon guidance, regulation of actin cytoskeleton, and GABAergic synapse. Functional analysis showed that the TTC3 p.S1038C iPSC-derived neuronal progenitor cells had altered 3D morphology and increased migration, while the corresponding neurons had longer neurites, increased branch points, and altered expression levels of synaptic proteins. Pharmacological treatment with small molecules that target the actin cytoskeleton could revert many of these cellular phenotypes, suggesting a central role for actin in mediating the cellular phenotypes associated with the TTC3 p.S1038C variant.The AD risk variant TTC3 p.S1038C reduces the expression levels ofThe variant modifies the expression of AD specific genes,, andNeurons with the variant are enriched for genes in the PI3K-Akt pathwayiPSC-derived neurons with the alteration have increased neurite length and branchingThe variant interferes with actin cytoskeleton and is ameliorated by Cytochalasin D