Whole genome sequencing of orofacial cleft trios from the Gabriella Miller Kids First Pediatric Research Consortium identifies a new locus on chromosome 21.

Orofacial clefts (OFCs) are among the most prevalent craniofacial birth defects worldwide and create a significant public health burden. The majority of OFCs are non-syndromic, and the genetic etiology of non-syndromic OFCs is only partially determined. Here, we analyze whole genome sequence (WGS) data for association with risk of OFCs in European and Colombian families selected from a multicenter family-based OFC study. This is the first large-scale WGS study of OFC in parent-offspring trios, and a part of the Gabriella Miller Kids First Pediatric Research Program created for the study of childhood cancers and structural birth defects. WGS provides deeper and more specific genetic data than using imputation on present-day single nucleotide polymorphic (SNP) marker panels. Genotypes of case-parent trios at single nucleotide variants (SNV) and short insertions and deletions (indels) spanning the entire genome were called from their sequences using human GRCh38 genome assembly, and analyzed for association using the transmission disequilibrium test. Among genome-wide significant associations, we identified a new locus on chromosome 21 in Colombian families, not previously observed in other larger OFC samples of Latin American ancestry. This locus is situated within a region known to be expressed during craniofacial development. Based on deeper investigation of this locus, we concluded that it contributed risk for OFCs exclusively in the Colombians. This study reinforces the ancestry differences seen in the genetic etiology of OFCs, and underscores the need for larger samples when studying for OFCs and other birth defects in populations with diverse ancestry.

Mutations in COL27A1 cause Steel syndrome and suggest a founder mutation effect in the Puerto Rican population.

Osteochondrodysplasias represent a large group of developmental structural disorders that can be caused by mutations in a variety of genes responsible for chondrocyte development, differentiation, mineralization and early ossification. The application of whole-exome sequencing to disorders apparently segregating as Mendelian traits has proven to be an effective approach to disease gene identification for conditions with unknown molecular etiology. We identified a homozygous missense variant p.(Gly697Arg) in COL27A1, in a family with Steel syndrome and no consanguinity. Interestingly, the identified variant seems to have arisen as a founder mutation in the Puerto Rican population.

Family-based study shows heterogeneity of a susceptibility locus on chromosome 8q24 for nonsyndromic cleft lip and palate.

BACKGROUND: Nonsyndromic cleft lip with or without cleft palate is a common birth defect. Although a number of susceptibility loci have been reported, replication has often been lacking. This is likely due, in part, to the heterogeneity of datasets and methodologies. Two independent genome-wide association studies of individuals of largely western European extraction have identified a possible susceptibility locus on 8q24.21. METHODS: To determine the overall effect of this locus, we genotyped six of the previously associated single nucleotide polymorphisms in our Hispanic and non-Hispanic white family-based datasets and evaluated them for linkage and association. In addition, we genotyped a large African American family with nonsyndromic cleft lip with or without cleft palate that we had previously mapped to the 8q21.3-24.12 region to test for linkage. RESULTS: There was no evidence for linkage to this region in any of the three ethnic groups. Nevertheless, strong evidence for association was noted in the non-Hispanic white group, whereas none was detected in the Hispanic dataset. CONCLUSION: These results confirm the previously reported association and provide evidence suggesting that there is ethnically based heterogeneity for this locus.

Mutation and association analysis of the PVR and PVRL2 genes in patients with non-syndromic cleft lip and palate.

Orofacial clefts (OFC; MIM 119530) are among the most common major birth defects. Here, we carried out mutation screening of the PVR and PVRL2 genes, which are both located at an OFC linkage region at 19q13 (OFC3) and are closely related to PVRL1, which has been associated with both syndromic and non-syndromic cleft lip and palate (nsCLP). We screened a total of 73 nsCLP patients and 105 non-cleft controls from the USA for variants in PVR and PVRL2, including all exons and encompassing all isoforms. We identified four variants in PVR and five in PVRL2. One non-synonymous PVR variant, A67T, was more frequent among nsCLP patients than among normal controls, but this difference did not achieve statistical significance.

Mutation and association analysis of the PVR and PVRL2 genes in patients with non-syndromic cleft lip and palate

Orofacial clefts (OFC; MIM 119530) are among the most common major birth defects. Here, we carried out mutation screening of the PVR and PVRL2 genes, which are both located at an OFC linkage region at 19q13 (OFC3) and are closely related to PVRL1, which has been associated with both syndromic and non-syndromic cleft lip and palate (nsCLP). We screened a total of 73 nsCLP patients and 105 non-cleft controls from the USA for variants in PVR and PVRL2, including all exons and encompassing all isoforms. We identified four variants in PVR and five in PVRL2. One non-synonymous PVR variant, A67T, was more frequent among nsCLP patients than among normal controls, but this difference did not achieve statistical significance.

Lack of mutations in the PVRL3 gene in North American caucasians with non-syndromic cleft lip/palate

Cleft lip with or without cleft palate (CLP) is one of the most common birth defects. In about 70 percent of cases, CLP occurs as an isolated anomaly, denoted non-syndromic CLP (nsCLP). Genetic linkage and association studies have implicated many loci in susceptibility to nsCLP, including some members of the nectin gene family. We performed mutation screening of the PVRL3 gene that encodes nectin-3 in 73 unrelated Caucasian nsCLP patients and 105 unrelated controls from North America. We detected no sequence variants in the PVRL3 gene in either the nsCLP patients or the controls. These data suggest that PVRL3 is not an important susceptibility gene for nsCLP in the North American Caucasian population.

CRISPLD2: a novel NSCLP candidate gene.

Non-syndromic cleft lip with or without cleft palate (NSCLP) results from the complex interaction between genes and environmental factors. Candidate gene analysis and genome scans have been employed to identify the genes contributing to NSCLP. In this study, we evaluated the 16q24.1 chromosomal region, which has been identified by multiple genome scans as an NSCLP region of interest. Two candidate genes were found in the region: interferon regulatory factor 8 (IRF8) and cysteine-rich secretory protein LCCL domain containing 2 (CRISPLD2). Initially, Caucasian and Hispanic NSCLP multiplex families and simplex parent-child trios were genotyped for single nucleotide polymorphisms (SNPs) in both IRF8 and CRISPLD2. CRISPLD2 was subsequently genotyped in a data set comprised of NSCLP families from Colombia, South America. Linkage disequilibrium analysis identified a significant association between CRISPLD2 and NSCLP in both our Caucasian and Hispanic NSCLP cohorts. SNP rs1546124 and haplotypes between rs1546124 and either rs4783099 or rs16974880 were significant in the Caucasian multiplex population (P=0.01, P=0.002 and P=0.001, respectively). An altered transmission of CRISPLD2 SNPs rs8061351 (P=0.02) and rs2326398 (P=0.06) was detected in the Hispanic population. No association was found between CRISPLD2 and our Colombian population or IRF8 and NSCLP. In situ hybridization showed that CRISPLD2 is expressed in the mandible, palate and nasopharynx regions during craniofacial development at E13.5-E17.5, respectively. Altogether, these data suggest that genetic variation in CRISPLD2 has a role in the etiology of NSCLP.