Identification of Functional Variants in Alzheimer\u27s Disease-Associated Genes

Alzheimer\u27s disease (AD) is the most common form of dementia affecting the health of more than 5 million Americans in 2013. Understanding how genetic variants contribute to AD is important to develop effective therapeutics for delaying and eventually curing the disease. Recent sequencing studies have identified rare variants p.V232M in PLD3 and p.R47H in TREM2 significantly associated with AD risk. Additionally, genome-wide association studies (GWAS) uncovered common variants in ABCA7, BIN1, CD33, CD2AP, CLU, CR1, EPHA1, MS4A4A, and PICALM that contribute to AD; however, the most-significant variants in these loci are located within non-coding regions and have no direct functional impact on AD pathogenesis. We hypothesized that if PLD3 and TREM2 are truly AD risk genes, they will carry additional functional variants that can substantially affect AD risk. Moreover, we hypothesized that GWAS genes carry additional risk alleles across the frequency spectrum so that common, low frequency or rare variants within these genes may contribute to AD risk. We undertook pooled sequencing of exonic and flanking intronic sequence for the aforementioned genes in 3,730 European Americans (EA) (2,082 AD cases and 1,648 controls) and 336 African Americans (AA) (204 AD cases and 132 controls). We found rare variants in PLD3 and TREM2 are more frequently seen in cases than in controls of EA descent. Single-variant analyses showed that p.M6R and p.A442A in PLD3 and p.R62H in TREM2 are significantly associated with AD risk besides p.V232M in PLD3 and p.R47H in TREM2. We found rare variants in PLD3 (PSKAT-O=1.44×10-11) and TREM2 (PSKAT-O=5.37×10-7) are genome-wide significantly associated with AD. Additionally, we found a significant association for PLD3 coding variants with AD risk in AA (PSKAT-O=1.40×10-3). However, we did not find evidence of association for TREM2 variants with AD risk in AA. We validated 90 coding variants in GWAS-identified genes and bioinformatic analyses implicated that a large proportion of these variants are functional. The International Genomics of Alzheimer\u27s Project recently performed meta- and gene-wide analyses and identified 23 loci associated with AD risk, of which 13 were novel. However, these loci\u27s role in affecting the molecular pathways of AD remains unknown. To determine whether these loci are also associated with cerebrospinal fluid (CSF) amyloid-beta 1-42 (Aβ42) and phosphorylated tau181 (ptau181) levels, we combined CSF biomarker datasets from several studies and performed single-variant, set-based, and conditional analyses for each locus. In the APOE locus, rs769449 is genome-wide significantly associated with CSFAβ42 and ptau181 levels independently of APOE and the association for CSF ptau181 levels was not driven by Aβ metabolism. We found rs7937331, within the CELF1 fine-mapping region, tags the same signal as the IGAP top SNP (rs62003531) and is significantly associated with CSF Aβ42 levels and AD risk. Additionally, rs62003531, located in the intronic region of FERMT2, tags the same association as the IGAP top SNP (rs17125944) and is associated with CSF Aβ42 levels. None of the SNPs within the IGAP-identified AD risk loci except the APOE locus are significantly associated with CSF ptau181 levels after multiple test correction. In investigating the potential regulatory functions associated with IGAP top SNPs and CSF top SNPs, most of GWAS top SNPs have no significant regulatory potential and are unlikely to be functional variants for AD risk. However, RegulomeDB predicts that several proxy SNPs in linkage disequilibrium (LD) with rs7937331 may be cis-acting expression quantitative trait loci (eQTLs) for nearby genes. The IGAP study also identified an intergenic polymorphism near TREML2 suggestively associated with AD risk; however, due to the study design, it was not possible to uncover the underlying functional variant or to determine whether this observed association was driven by the known AD risk allele, TREM2 p.R47H, or represented a novel locus. We performed analyses using whole-exome sequencing data, CSF biomarker analyses and meta-analyses to demonstrate that the AD risk association is likely driven by a TREML2 variant p.S144G (rs3747742) independently of TREM2 p.R47H risk for AD. Finally, we sought to functionally characterize the effects of novel TREM2 variants on TREM2 cell surface transport. We transduced a T cell hybridoma cell line with virus containing TREM2 wild type (WT) and risk variants and measured TREM2 cell surface expression with a TREM2-specific monoclonal antibody. We found cells expressing p.T66M and p.R136W have a robust effect on TREM2 cell surface expression but cells expressing p.R47H and p.R62H are similar to hTREM2 WT. Additionally, since polymorphisms in the CELF1 fine-mapping region were implicated to be eQTLs for nearby genes, we performed cis-eQTL analysis for mRNA expression levels in several brain regions using four publicly available datasets to identify genetic determinants of gene expression in human brains. We found several C1QTNF4-expression-associated SNPs which tag the same signal are in LD with rs7937331, the top CSF Aβ42 SNP in the CELF1 fine-mapping region. Additionally, we found evidence of differential expression in the C1QTNF4 transcript between AD cases and controls in human brains. These findings provide additional evidence that genes involved in the inflammatory response play an important role in AD pathogenesis

Exome sequencing implicates impaired GABA signaling and neuronal ion transport in trigeminal neuralgia

Trigeminal neuralgia (TN) is a common, debilitating neuropathic face pain syndrome often resistant to therapy. The familial clustering of TN cases suggests that genetic factors play a role in disease pathogenesis. However, no unbiased, large-scale genomic study of TN has been performed to date. Analysis of 290 whole exome-sequenced TN probands, including 20 multiplex kindreds and 70 parent-offspring trios, revealed enrichment of rare, damaging variants in GABA receptor-binding genes in cases. Mice engineered with a TN-associate

Whole-exome sequencing reveals damaging gene variants associated with hypoalphalipoproteinemia

Low levels of high density lipoprotein-cholesterol (HDL-C) are associated with an elevated risk of arteriosclerotic coronary heart disease. Heritability of HDL-C levels is high. In this research discovery study, we used whole-exome sequencing to identify damaging gene variants that may play significant roles in determining HDL-C levels. We studied 204 individuals with a mean HDL-C level of 27.8 ± 6.4 mg/dl (range: 4-36 mg/dl). Data were analyzed by statistical gene burden testing and by filtering against candidate gene lists. We found 120 occurrences of probably damaging variants (116 heterozygous; four homozygous) among 45 of 104 recognized HDL candidate genes. Those with the highest prevalence of damaging variants were ABCA1 (n = 20), STAB1 (n = 9), OSBPL1A (n = 8), CPS1 (n = 8), CD36 (n = 7), LRP1 (n = 6), ABCA8 (n = 6), GOT2 (n = 5), AMPD3 (n = 5), WWOX (n = 4), and IRS1 (n = 4). Binomial analysis for damaging missense or loss-of-function variants identified the ABCA1 and LDLR genes at genome-wide significance. In conclusion, whole-exome sequencing of individuals with low HDL-C showed the burden of damaging rare variants in the ABCA1 and LDLR genes is particularly high and revealed numerous occurrences in HDL candidate genes, including many genes identified in genome-wide association study reports. Many of these genes are involved in cancer biology, which accords with epidemiologic findings of the association of HDL deficiency with increased risk of cancer, thus presenting a new area of interest in HDL genomics

The prognostic factors for locally advanced cervical cancer patients treated by intensity-modulated radiation therapy with concurrent chemotherapy

Background/PurposeTo identify the prognostic factors for locally advanced cervical cancer patients treated by intensity-modulated radiotherapy (IMRT) and concurrent cisplatin-based chemotherapy.MethodsA total of 125 patients with stage IB2–III cervical carcinoma were treated with IMRT and concurrent cisplatin-based chemotherapy, plus high dose rate (HDR) brachytherapy between January 2004 and November 2010, in our institution. All patients received external irradiation of 45–54 Gy with the IMRT technique and concurrent cisplatin-based chemotherapy monthly or weekly. HDR brachytherapy of 20–30.5 Gy was prescribed to point A, as a local boost. Prognostic factors including age, histology, stage, lymph nodes metastasis, pretreatment hemoglobin level, serum squamous cell carcinoma antigen (serum SCC-Ag), chemotherapy regimens and the cumulative dose of weekly cisplatin, were analyzed. The endpoints were overall survival (OS), local failure-free survival (LFFS) and disease-free survival (DFS).ResultsThe median follow-up time was 42 months. The 4-year OS, LFFS and DFS were 73.8%, 77.9% and 67.2%, respectively. Four (3.2%) patients developed ≥grade 3 acute gastrointestinal (GI) toxicity and 29 (23.2%) patients developed ≥grade 3 acute hematological toxicity. Five (4.0%) patients developed ≥grade 3 late GI toxicity and seven (5.6%) patients developed ≥grade 3 late genitourinary system toxicity. On univariate analysis, adenocarcinoma was a poor prognostic factor for OS (p = 0.05), LFFS (p = 0.01) and DFS (p = 0.006). Patients with lymph nodes metastasis at diagnosis had worse OS (p = 0.02). The high cumulative dose of cisplatin (>180 mg/m2) had better OS (p = 0.03) and tended to have better survival on LFFS (p = 0.13) and DFS (p = 0.10). On multivariate analysis, adenocarcinoma was a significant independent prognostic factor for OS (p = 0.001), LFFS (p = 0.005) and DFS (p < 0.001). Initial lymph nodes metastasis was an independent predictor of OS (p = 0.013). Cumulative dose of weekly cisplatin significantly affected OS (p = 0.041), and high cumulative dose of cisplatin tended to have better LFFS (p = 0.083). Higher pretreatment hemoglobin level had better LFFS (p = 0.034).ConclusionAdenocarcinoma and lymph nodes metastases were poor prognostic factors for patients with locally advanced cervical cancer. Lower pretreatment hemoglobin level had poorer local control. Chemotherapy with a high cumulative dose of cisplatin tended to result in better survival

Comparison of clinical outcomes and toxicity in endometrial cancer patients treated with adjuvant intensity-modulated radiation therapy or conventional radiotherapy

PurposeTo evaluate the treatment outcomes and toxicity in endometrial cancer patients treated with hysterectomy and adjuvant intensity-modulated radiation therapy (IMRT) or conventional radiotherapy (CRT).MethodsThere were 101 patients with stage IA-IIIC2 endometrial carcinoma treated with hysterectomy and adjuvant radiotherapy. In total, 36 patients received adjuvant CRT and 65 were treated with adjuvant IMRT. The endpoints were overall survival, local failure-free survival, and disease-free survival. Patients were assessed for acute toxicity weekly according to the Common Terminology Criteria for Adverse Events version 3.0. Late toxicity was evaluated according to the Radiation Therapy Oncology Group and the European Organization for Research and Treatment of Cancer Late Radiation Morbidity Scoring Schema.ResultsThe 5-year overall survival, local failure-free survival, and disease-free survival for the CRT group and the IMRT group were 82.9% versus 93.5% (p = 0.26), 93.7% versus 89.3% (p = 0.68), and 88.0% versus 82.8% (p = 0.83), respectively. Four (11.1%) patients had Grade 3 or greater acute gastrointestinal (GI) toxicity and three (8.3%) patients had Grade 3 or greater acute genitourinary (GU) toxicity in the CRT group, whereas four (6.2%) patients had Grade 3 or greater acute GI toxicity in the IMRT group and no patient had severe GU toxicity. There was one (2.8%) patient who had Grade 3 or greater late GI toxicity and one (2.8%) patient had Grade 3 or greater late GU toxicity in the CRT group, whereas no patient had severe GI or GU toxicity in the IMRT group.ConclusionAdjuvant IMRT for endometrial cancer patients had comparable clinical outcomes with CRT and had less acute and late toxicity

Molecular diagnostic yield of exome sequencing in patients with congenital hydrocephalus: A systematic review and meta-analysis

IMPORTANCE: Exome sequencing (ES) has been established as the preferred first line of diagnostic testing for certain neurodevelopmental disorders, such as global developmental delay and autism spectrum disorder; however, current recommendations are not specific to or inclusive of congenital hydrocephalus (CH). OBJECTIVE: To determine the diagnostic yield of ES in CH and whether ES should be considered as a first line diagnostic test for CH. DATA SOURCES: PubMed, Cochrane Library, and Google Scholar were used to identify studies published in English between January 1, 2010, and April 10, 2023. The following search terms were used to identify studies: congenital hydrocephalus, ventriculomegaly, cerebral ventriculomegaly, primary ventriculomegaly, fetal ventriculomegaly, prenatal ventriculomegaly, molecular analysis, genetic cause, genetic etiology, genetic testing, exome sequencing, whole exome sequencing, genome sequencing, microarray, microarray analysis, and copy number variants. STUDY SELECTION: Eligible studies included those with at least 10 probands with the defining feature of CH and/or severe cerebral ventriculomegaly that had undergone ES. Studies with fewer than 10 probands, studies of mild or moderate ventriculomegaly, and studies using genetic tests other than ES were excluded. A full-text review of 68 studies was conducted by 2 reviewers. Discrepancies were resolved by consensus. DATA EXTRACTION AND SYNTHESIS: Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines and Meta-Analysis of Observational Studies in Epidemiology guidelines were used by 2 reviewers to extract data. Data were synthesized using a random-effects model of single proportions. Data analysis occurred in April 2023. MAIN OUTCOMES AND MEASURES: The primary outcome was pooled diagnostic yield. Additional diagnostic yields were estimated for specific subgroups on the basis of clinical features, syndromic presentation, and parental consanguinity. For each outcome, a 95% CI and estimate of interstudy heterogeneity (I2 statistic) was reported. RESULTS: From 498 deduplicated and screened records, 9 studies with a total of 538 CH probands were selected for final inclusion. The overall diagnostic yield was 37.9% (95% CI, 20.0%-57.4%; I2 = 90.1). The yield was lower for isolated and/or nonsyndromic cases (21.3%; 95% CI, 12.8%-31.0%; I2 = 55.7). The yield was higher for probands with reported consanguinity (76.3%; 95% CI, 65.1%-86.1%; I2 = 0) than those without (16.2%; 95% CI, 12.2%-20.5%; I2 = 0). CONCLUSIONS AND RELEVANCE: In this systematic review and meta-analysis of the diagnostic yield of ES in CH, the diagnostic yield was concordant with that of previous recommendations for other neurodevelopmental disorders, suggesting that ES should also be recommended as a routine diagnostic adjunct for patients with CH

Analysis workflow to assess de novo genetic variants from human whole-exome sequencing

Here, we present a protocol to analyz

Insights from genetic studies of cerebral palsy

Cohort-based whole exome and whole genome sequencing and copy number variant (CNV) studies have identified genetic etiologies for a sizable proportion of patients with cerebral palsy (CP). These findings indicate that genetic mutations collectively comprise an important cause of CP. We review findings in CP genomics and propose criteria for CP-associated genes at the level of gene discovery, research study, and clinical application. We review the published literature and report 18 genes and 5 CNVs from genomics studies with strong evidence of for the pathophysiology of CP. CP-associated genes often disrupt early brain developmental programming or predispose individuals to known environmental risk factors. We discuss the overlap of CP-associated genes with other neurodevelopmental disorders and related movement disorders. We revisit diagnostic criteria for CP and discuss how identification of genetic etiologies does not preclude CP as an appropriate diagnosis. The identification of genetic etiologies improves our understanding of the neurobiology of CP, providing opportunities to study CP pathogenesis and develop mechanism-based interventions

Quantifying concordant genetic effects of de novo mutations on multiple disorders

Exome sequencing on tens of thousands of parent-proband trios has identified numerous deleterious de novo mutations (DNMs) and implicated risk genes for many disorders. Recent studies have suggested shared genes and pathways are enriched for DNMs across multiple disorders. However, existing analytic strategies only focus on genes that reach statistical significance for multiple disorders and require large trio samples in each study. As a result, these methods are not able to characterize the full landscape of genetic sharing due to polygenicity and incomplete penetrance. In this work, we introduce EncoreDNM, a novel statistical framework to quantify shared genetic effects between two disorders characterized by concordant enrichment of DNMs in the exome. EncoreDNM makes use of exome-wide, summary-level DNM data, including genes that do not reach statistical significance in single-disorder analysis, to evaluate the overall and annotation-partitioned genetic sharing between two disorders. Applying EncoreDNM to DNM data of nine disorders, we identified abundant pairwise enrichment correlations, especially in genes intolerant to pathogenic mutations and genes highly expressed in fetal tissues. These results suggest that EncoreDNM improves current analytic approaches and may have broad applications in DNM studies