Accumulation of rare coding variants in genes implicated in risk of human cleft lip with or without cleft palate.

Cleft lip with/without cleft palate (CLP) is a common craniofacial malformation with complex etiologies, reflecting both genetic and environmental factors. Most of the suspected genetic risk for CLP has yet to be identified. To further classify risk loci and estimate the contribution of rare variants, we sequenced the exons in 49 candidate genes in 323 CLP cases and 211 nonmalformed controls. Our findings indicated that rare, protein-altering variants displayed markedly higher burdens in CLP cases at relevant loci. First, putative loss-of-function mutations (nonsense, frameshift) were significantly enriched among cases: 13 of 323 cases (~4%) harbored such alleles within these 49 genes, versus one such change in controls (p = 0.01). Second, in gene-level analyses, the burden of rare alleles showed greater case-association for several genes previously implicated in cleft risk. For example, BHMT displayed a 10-fold increase in protein-altering variants in CLP cases (p = .03), including multiple case occurrences of a rare frameshift mutation (K400 fs). Other loci with greater rare, coding allele burdens in cases were in signaling pathways relevant to craniofacial development (WNT9B, BMP4, BMPR1B) as well as the methionine cycle (MTRR). We conclude that rare coding variants may confer risk for isolated CLP.

Epigenomic profiling of newborns with isolated orofacial clefts reveals widespread DNA methylation changes and implicates metastable epiallele regions in disease risk.

Cleft lip with or without cleft palate (CL/P) is a common human birth defect whose etiologies remain largely unknown. Several studies have demonstrated that periconceptional supplementation of folic acid can reduce risk of CL/P in offspring. In this study, we tested the hypothesis that the preventive effect of folic acid is manifested through epigenetic modifications by determining whether DNA methylation changes are associated with CL/P. To more readily observe the potential effects of maternal folate on the offspring epigenome, we focused on births prior to mandatory dietary folate fortification in the United States (i.e. birth year 1997 or earlier). Genomic DNA methylation levels were assessed from archived newborn bloodspots in a 182-member case-control study using the Illumina® Human Beadchip 450K array. CL/P cases displayed striking epigenome-wide hypomethylation relative to controls: 63% of CpGs interrogated had lower methylation levels in case newborns, a trend which held up in racially stratified sub-groups. 28 CpG sites reached epigenome-wide significance and all were case-hypomethylated. The most significant CL/P-associated differentially methylated region encompassed the VTRNA2-1 gene, which was also hypomethylated in cases (FWER p = 0.014). This region has been previously characterized as a nutritionally-responsive, metastable epiallele and CL/P-associated methylation changes, in general, were greater at or near putative metastable epiallelic regions. Gene Set Enrichment Analysis of CL/P-associated DMRs showed an over-representation of genes involved in palate development such as WNT9B, MIR140 and LHX8. CL/P-associated DNA methylation changes may partly explain the mechanism by which orofacial clefts are responsive to maternal folate levels.

DNA Methylome Profiling on the Infinium HumanMethylation450 Array from Limiting Quantities of Genomic DNA from a Single, Small Archived Bloodspot.

AIMS: Archived newborn bloodspots are valuable sample collections for genetic and epigenetic disease research. However, they have often been stored for long periods of time, under less than ideal circumstances, and nucleic acid yields can be low, particularly when samples become limiting. We wished to determine whether the quantity and quality of genomic DNA (gDNA) isolated from a single, surgical bloodspot punch (2 mm dia.) was adequate for accurate and reliable DNA methylome profiling on the Illumina HumanMethylation450 array. METHODS: A total of 25-750 ng of archived bloodspot or Jurkat cell gDNA were bisulfite converted and analyzed on the array without any additional DNA amplification steps. Methylation profiles were assessed for call rate, call confidence (detection p-value), and reproducibility. RESULTS: Using 25 ng gDNA from either Jurkat cells or dried bloodspots, array-wide call rates (∼99.9%) and detection p-values (99.9% with p < 5 × 10-6) were excellent. There was good agreement between methylation profiles generated from 25 ng gDNA and those generated from 750 ng (ρ > 0.98), although a fraction of CpG sites (2-8% depending on experiment) exhibited quantitative differences. Genome-wide methylation levels were strikingly reproducible from 25 ng DNA in both replicate and interindividual samples (ρ > 0.98). CONCLUSIONS: Twenty-five nanograms of gDNA, isolated from a single, surgical punch (2 mm dia.) of an archived newborn bloodspot, generate a genome-wide methylation profile on the Illumina HumanMethylation450 array that is robust, reproducible, and suitable for differential methylation studies.

Sequence variation in folate pathway genes and risks of human cleft lip with or without cleft palate.

In an effort to comprehensively interrogate genetic variation in the folate pathway for risk of cleft lip with or without cleft palate (CLP), we evaluated 504 common and rare variants in 35 folate-related genes in a panel of 330 infants with CLP and 367 non-malformed controls. Odds ratios (OR) with 95% confidence intervals were computed for common genotypes. A Case-Control Difference metric was calculated for rare variants to highlight differentially occurring alleles. Interactions between variants and a maternal folate intake variable were also evaluated. In gene-only results, significant odds ratios were observed for multiple variants in the BHMT/BHMT2/DMGDH gene cluster, particularly in Hispanic infants. Also in this cluster, rare variant analysis highlighted a substantial case-control difference in BHMT rs60340837 (synonymous Y284Y). In Hispanics, the ALDH1L1 I812V variant (rs4646750) was the most significant risk allele: OR = 3.8 (95%CI = 1.6-9.2) when heterozygous. In non-Hispanic white infants, we observed significant risk for AHCYL2 rs1095423 (homozygous OR = 3.0, 95%CI 1.1-7.8) and the 68 bp CBS insertion (c.844ins68; heterozygous OR = 2.4, 95%CI = 1.1-5.3). Rare variant analysis in this group revealed case-control differences in MTRR and several other methionine cycle genes, a process implicated previously in clefting risk. In women with low folate intake specifically, increased risks were observed for CBS rs2851391 (OR = 3.6, 95%CI = 1.3-9.6) and the R259P nonsynonymous variant of TCN2 (rs1801198; OR = 2.8, 95%CI = 1.2-6.3). This comprehensive study provides further direction on candidate loci to help disentangle the folate-related developmental phenomena in human clefting risk. © 2016 Wiley Periodicals, Inc.