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Team Uses Targeted Sequencing to Identify Functional Variants for Cleft Lip

NEW YORK (GenomeWeb) – Researchers led by the University of Iowa's Jeffrey Murray have identified potential functional variants behind the development of cleft lip.

As Murray and his colleagues reported in the American Journal of Human Genetics today, they performed targeted sequencing on more than 1,400 trios, focusing on 13 genomic regions identified through genome-wide association or candidate gene studies. From this, they identified some 1,050 coding variants, including one that disrupted the binding of a transcription factor and another that affected the activity of an enhancer.

"In aggregate, our analyses highlight the important role of non-coding regulatory elements and suggest that disruption of these regions by genetic variants is a critical aspect of the pathogenesis of [nonsyndromic cleft lip with or without cleft palate]," Mason and his colleagues wrote in their paper.

The researchers selected 13 regions, spanning 6.3 megabases, for targeted sequencing based on previous genome-wide association or linkage studies and candidate gene studies. They then performed this sequencing on a set of case-parent trios of Asian or European ancestry from China, Europe, the Philippines, or the US. The cases all had been diagnosed based on a physical exam with cleft lip or with cleft lip and palate, but didn't have a syndrome linked to cleft lip or palate.

Through this targeted sequencing, the researchers identified more than 168,000 variants, including 18,709 common variants, 145,223 rare variants, and 46 de novo variants.

Murray and his colleagues concentrated on two de novo mutations and one common variant that they considered most promising for follow-up functional studies, and noted that there are five genes at which their analyses pointed toward a role in the development of cleft lip and palate.

For instance, a non-synonymous de novo mutation found in PAX7 leads to a substitution at a conserved residue that the researchers suspected would disrupt the ability of PAX7 to bind to DNA. The PAX7 protein, the researchers noted, is involved in neural crest induction and is expressed in cranial neural crest cells.

Through an electrophoresis mobility shift assay examining wild-type PAX7 and mutant PAX7, the researchers noted that the wild-type version bound to the probe more tightly. Additionally, they found that wild-type PAX7 was more highly expressed than the mutant version in transfected HeLa cells. Further, mice lacking the protein have nasal and maxillary malformations.

All together this suggests, Murray and his colleagues said, that this de novo mutation affects PAX7 function and contributes to cleft lip pathogenesis.

Likewise, the researchers homed in on a de novo mutation in non-coding DNA a few hundred kilobases downstream of the FGFR2 transcription start site that harbors chromatin marks the researchers said are indicative of an active neural crest enhancer.

This de novo mutation, the researchers added, is predicted to disrupt a transcription factor binding site.

Indeed, in zebrafish, the reference allele acts as an enhancer in the neural keel, brain, and delaminating neural crest, and in both zebrafish and mice, FGFR2 is expressed in the brain and cranial neural folds.

The mutant form, though, had decreased enhancer activity in the animal model and in a mesenchymal cell line, indicating that the de novo mutation affects a neural crest enhancer that regulates FGFR2 expression.

Murray and his colleagues also noted potential roles for NOG, ARHGAP29, and NTN1 in cleft lip pathogenesis.

They identified multiple SNPs near NOG — whose protein is localized to the epithelium of the palate — including one downstream of its transcriptional start site that is in full linkage disequilibrium with a common variant that appears to influence enhancer function.

At the same time, they uncovered a number of rare variants, including nonsense mutations, in ARHGAP29, which is expressed during lip and palate development in mice, and common SNPs near the transcription start site of NTN1, which is expressed in the palate and, when lacking in mice, leads to a phenotype consistent with cleft palate.

"Here we demonstrated that targeted sequencing of large intervals surrounding GWAS regions is an effective approach for identifying functional rare and common variants in both coding and non-coding regions," the researchers said.

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