Properties of permutation-based gene tests and controlling type 1 error using a summary statistic based gene test

Background: The advent of genome-wide association studies has led to many novel disease-SNP associations, opening the door to focused study on their biological underpinnings. Because of the importance of analyzing these associations, numerous statistical methods have been devoted to them. However, fewer methods have attempted to associate entire genes or genomic regions with outcomes, which is potentially more useful knowledge from a biological perspective and those methods currently implemented are often permutation-based. Results: One property of some permutation-based tests is that their power varies as a function of whether significant markers are in regions of linkage disequilibrium (LD) or not, which we show from a theoretical perspective. We therefore develop two methods for quantifying the degree of association between a genomic region and outcome, both of whose power does not vary as a function of LD structure. One method uses dimension reduction to “filter” redundant information when significant LD exists in the region, while the other, called the summary-statistic test, controls for LD by scaling marker Z-statistics using knowledge of the correlation matrix of markers. An advantage of this latter test is that it does not require the original data, but only their Z-statistics from univariate regressions and an estimate of the correlation structure of markers, and we show how to modify the test to protect the type 1 error rate when the correlation structure of markers is misspecified. We apply these methods to sequence data of oral cleft and compare our results to previously proposed gene tests, in particular permutation-based ones. We evaluate the versatility of the modification of the summary-statistic test since the specification of correlation structure between markers can be inaccurate. Conclusion: We find a significant association in the sequence data between the 8q24 region and oral cleft using our dimension reduction approach and a borderline significant association using the summary-statistic based approach. We also implement the summary-statistic test using Z-statistics from an already-published GWAS of Chronic Obstructive Pulmonary Disorder (COPD) and correlation structure obtained from HapMap. We experiment with the modification of this test because the correlation structure is assumed imperfectly known

‘Location, Location, Location’: a spatial approach for rare variant analysis and an application to a study on non-syndromic cleft lip with or without cleft palate

Motivation: For the analysis of rare variants in sequence data, numerous approaches have been suggested. Fixed and flexible threshold approaches collapse the rare variant information of a genomic region into a test statistic with reduced dimensionality. Alternatively, the rare variant information can be combined in statistical frameworks that are based on suitable regression models, machine learning, etc. Although the existing approaches provide powerful tests that can incorporate information on allele frequencies and prior biological knowledge, differences in the spatial clustering of rare variants between cases and controls cannot be incorporated. Based on the assumption that deleterious variants and protective variants cluster or occur in different parts of the genomic region of interest, we propose a testing strategy for rare variants that builds on spatial cluster methodology and that guides the identification of the biological relevant segments of the region. Our approach does not require any assumption about the directions of the genetic effects. Results: In simulation studies, we assess the power of the clustering approach and compare it with existing methodology. Our simulation results suggest that the clustering approach for rare variants is well powered, even in situations that are ideal for standard methods. The efficiency of our spatial clustering approach is not affected by the presence of rare variants that have opposite effect size directions. An application to a sequencing study for non-syndromic cleft lip with or without cleft palate (NSCL/P) demonstrates its practical relevance. The proposed testing strategy is applied to a genomic region on chromosome 15q13.3 that was implicated in NSCL/P etiology in a previous genome-wide association study, and its results are compared with standard approaches. Availability: Source code and documentation for the implementation in R will be provided online. Currently, the R-implementation only supports genotype data. We currently are working on an extension for VCF files. Contact: [email protected]

Imputation of Orofacial Clefting Data Identifies Novel Risk Loci and Sheds Light on the Genetic Background of Cleft Lip ± Cleft Palate and Cleft Palate Only.

Abstract Nonsyndromic cleft lip with or without cleft palate (nsCL/P) is among the most common human birth defects with multifactorial etiology. Here, we present results from a genome-wide imputation study of nsCL/P in which, after adding replication cohort data, four novel risk loci for nsCL/P are identified (at chromosomal regions 2p21, 14q22, 15q24 and 19p13). On a systematic level, we show that the association signalswithin this high-density datasetare enriched in functionally-relevant genomic regions that are active in both human neural crest cells (hNCC) and mouse embryonic craniofacial tissue. This enrichment is also detectable in hNCC regions primed for later activity. Using GCTA analyses, we suggest that 30% of the estimated variance in risk for nsCL/P in the European population can be attributed to common variants, with 25.5% contributed to by the 24 risk loci known to date. For each of these, we identify credible SNPs using a Bayesian refinementapproach, with two loci harbouring only one probable causal variant. Finally, we demonstrate that there is no polygenic component of nsCL/P detectable that is shared with nonsyndromic cleft palate only (nsCPO). Our data suggest that, while common variants are strongly contributing to risk for nsCL/P, they do not seem to be involved in nsCPO which might be more often caused by rare deleterious variants. Our study generates novel insights into both nsCL/P and nsCPO etiology and provides a systematic framework for research into craniofacial development and malformation

Nine newly identified individuals refine the phenotype associated with MYT1L mutations

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Extending the allelic spectrum at noncoding risk loci of orofacial clefting

Genome-wide association studies (GWAS) have generated unprecedented insights into the genetic etiology of orofacial clefting (OFC). The moderate effect sizes of associated noncoding risk variants and limited access to disease-relevant tissue represent considerable challenges for biological interpretation of genetic findings. As rare variants with stronger effect sizes are likely to also contribute to OFC, an alternative approach to delineate pathogenic mechanisms is to identify private mutations and/or an increased burden of rare variants in associated regions. This report describes a framework for targeted resequencing at selected noncoding risk loci contributing to nonsyndromic cleft lip with/without cleft palate (nsCL/P), the most frequent OFC subtype. Based on GWAS data, we selected three risk loci and identified candidate regulatory regions (CRRs) through the integration of credible SNP information, epigenetic data from relevant cells/tissues, and conservation scores. The CRRs (total 57 kb) were resequenced in a multiethnic study population (1061 patients; 1591 controls), using single-molecule molecular inversion probe technology. Combining evidence from in silico variant annotation, pedigree- and burden analyses, we identified 16 likely deleterious rare variants that represent new candidates for functional studies in nsCL/P. Our framework is scalable and represents a promising approach to the investigation of additional congenital malformations with multifactorial etiology

Identification of de novo variants in nonsyndromic cleft lip with/without cleft palate patients with low polygenic risk scores

[Background]: Nonsyndromic cleft lip with/without cleft palate (nsCL/P) is a congenital malformation of multifactorial etiology. Research has identified >40 genome-wide significant risk loci, which explain less than 40% of nsCL/P heritability. Studies show that some of the hidden heritability is explained by rare penetrant variants. [Methods]: To identify new candidate genes, we searched for highly penetrant de novo variants (DNVs) in 50 nsCL/P patient/parent-trios with a low polygenic risk for the phenotype (discovery). We prioritized DNV-carrying candidate genes from the discovery for resequencing in independent cohorts of 1010 nsCL/P patients of diverse ethnicities and 1574 population-matched controls (replication). Segregation analyses and rare variant association in the replication cohort, in combination with additional data (genome-wide association data, expression, protein–protein-interactions), were used for final prioritization. [Conclusion]: In the discovery step, 60 DNVs were identified in 60 genes, including a variant in the established nsCL/P risk gene CDH1. Re-sequencing of 32 prioritized genes led to the identification of 373 rare, likely pathogenic variants. Finally, MDN1 and PAXIP1 were prioritized as top candidates. Our findings demonstrate that DNV detection, including polygenic risk score analysis, is a powerful tool for identifying nsCL/P candidate genes, which can also be applied to other multifactorial congenital malformations.The present study was supported by the German Research Foundation (DFG)-Grants BE 3828/8-1, LU 1944/2-1, MA 2546/5-1, and LU1944/3-1

Meta-analysis Reveals Genome-Wide Significance at 15q13 for Nonsyndromic Clefting of Both the Lip and the Palate, and Functional Analyses Implicate GREM1 As a Plausible Causative Gene

Nonsyndromic orofacial clefts are common birth defects with multifactorial etiology. The most common type is cleft lip, which occurs with or without cleft palate (nsCLP and nsCLO, respectively). Although genetic components play an important role in nsCLP, the genetic factors that predispose to palate involvement are largely unknown. In this study, we carried out a meta-analysis on genetic and clinical data from three large cohorts and identified strong association between a region on chromosome 15q13 and nsCLP (P = 8.13×10−14 for rs1258763; relative risk (RR): 1.46, 95% confidence interval (CI): 1.32–1.61)) but not nsCLO (P = 0.27; RR: 1.09 (0.94–1.27)). The 5 kb region of strongest association maps downstream of Gremlin-1 (GREM1), which encodes a secreted antagonist of the BMP4 pathway. We show during mouse embryogenesis, Grem1 is expressed in the developing lip and soft palate but not in the hard palate. This is consistent with genotype-phenotype correlations between rs1258763 and a specific nsCLP subphenotype, since a more than two-fold increase in risk was observed in patients displaying clefts of both the lip and soft palate but who had an intact hard palate (RR: 3.76, CI: 1.47–9.61, Pdiff<0.05). While we did not find lip or palate defects in Grem1-deficient mice, wild type embryonic palatal shelves developed divergent shapes when cultured in the presence of ectopic Grem1 protein (P = 0.0014). The present study identified a non-coding region at 15q13 as the second, genome-wide significant locus specific for nsCLP, after 13q31. Moreover, our data suggest that the closely located GREM1 gene contributes to a rare clinical nsCLP entity. This entity specifically involves abnormalities of the lip and soft palate, which develop at different time-points and in separate anatomical regions

Overlapping expression patterns and differential transcript levels of phosphate transporter genes in arbuscular mycorrhizal, Pi-fertilised and phytohormone-treated Medicago truncatula roots

A microarray carrying 5,648 probes of Medicago truncatula root-expressed genes was screened in order to identify those that are specifically regulated by the arbuscular mycorrhizal (AM) fungus Gigaspora rosea, by Pi fertilisation or by the phytohormones abscisic acid and jasmonic acid. Amongst the identified genes, 21% showed a common induction and 31% a common repression between roots fertilised with Pi or inoculated with the AM fungus G. rosea, while there was no obvious overlap in the expression patterns between mycorrhizal and phytohormone-treated roots. Expression patterns were further studied by comparing the results with published data obtained from roots colonised by the AM fungi Glomus mosseae and Glomus intraradices, but only very few genes were identified as being commonly regulated by all three AM fungi. Analysis of Pi concentrations in plants colonised by either of the three AM fungi revealed that this could be due to the higher Pi levels in plants inoculated by G. rosea compared with the other two fungi, explaining that numerous genes are commonly regulated by the interaction with G. rosea and by phosphate. Differential gene expression in roots inoculated with the three AM fungi was further studied by expression analyses of six genes from the phosphate transporter gene family in M. truncatula. While MtPT4 was induced by all three fungi, the other five genes showed different degrees of repression mirroring the functional differences in phosphate nutrition by G. rosea, G. mosseae and G. intraradices

Genome-wide association scan identifies new variants associated with a cognitive predictor of dyslexia

Developmental dyslexia (DD) is one of the most prevalent learning disorders, with high impact on school and psychosocial development and high comorbidity with conditions like attention-deficit hyperactivity disorder (ADHD), depression, and anxiety. DD is characterized by deficits in different cognitive skills, including word reading, spelling, rapid naming, and phonology. To investigate the genetic basis of DD, we conducted a genome-wide association study (GWAS) of these skills within one of the largest studies available, including nine cohorts of reading-impaired and typically developing children of European ancestry (N = 2562-3468). We observed a genome-wide significant effect (p <1 x 10(-8)) on rapid automatized naming of letters (RANlet) for variants on 18q12.2, within MIR924HG (micro-RNA 924 host gene; rs17663182 p = 4.73 x 10(-9)), and a suggestive association on 8q12.3 within NKAIN3 (encoding a cation transporter; rs16928927, p = 2.25 x 10(-8)). rs17663182 (18q12.2) also showed genome-wide significant multivariate associations with RAN measures (p = 1.15 x 10(-8)) and with all the cognitive traits tested (p = 3.07 x 10(-8)), suggesting (relational) pleiotropic effects of this variant. A polygenic risk score (PRS) analysis revealed significant genetic overlaps of some of the DD-related traits with educational attainment (EDUyears) and ADHD. Reading and spelling abilities were positively associated with EDUyears (p similar to [10(-5)-10(-7)]) and negatively associated with ADHD PRS (p similar to [10(-8)-10(-17)]). This corroborates a long-standing hypothesis on the partly shared genetic etiology of DD and ADHD, at the genome-wide level. Our findings suggest new candidate DD susceptibility genes and provide new insights into the genetics of dyslexia and its comorbities.Peer reviewe

Genome-wide association scan identifies new variants associated with a cognitive predictor of dyslexia

Developmental dyslexia (DD) is one of the most prevalent learning disorders, with high impact on school and psychosocial development and high comorbidity with conditions like attention-deficit hyperactivity disorder (ADHD), depression, and anxiety. DD is characterized by deficits in different cognitive skills, including word reading, spelling, rapid naming, and phonology. To investigate the genetic basis of DD, we conducted a genome-wide association study (GWAS) of these skills within one of the largest studies available, including nine cohorts of reading-impaired and typically developing children of European ancestry (N = 2562-3468). We observed a genome-wide significant effect (p < 1 x 10(-8)) on rapid automatized naming of letters (RANlet) for variants on 18q12.2, within MIR924HG (micro-RNA 924 host gene;rs17663182 p = 4.73 x 10(-9)), and a suggestive association on 8q12.3 within NKAIN3 (encoding a cation transporter;rs16928927, p = 2.25 x 10(-8)). rs17663182 (18q12.2) also showed genome-wide significant multivariate associations with RAN measures (p = 1.15 x 10(-8)) and with all the cognitive traits tested (p = 3.07 x 10(-8)), suggesting (relational) pleiotropic effects of this variant. A polygenic risk score (PRS) analysis revealed significant genetic overlaps of some of the DD-related traits with educational attainment (EDUyears) and ADHD. Reading and spelling abilities were positively associated with EDUyears (p similar to [10(-5)-10(-7)]) and negatively associated with ADHD PRS (p similar to [10(-8)-10(-17)]). This corroborates a long-standing hypothesis on the partly shared genetic etiology of DD and ADHD, at the genome-wide level. Our findings suggest new candidate DD susceptibility genes and provide new insights into the genetics of dyslexia and its comorbities