Identification of a Van der Woude syndrome mutation in the cleft palate 1 mutant mouse

Mutations in Interferon Regulatory Factor 6 (IRF6) have been identified in two human allelic syndromes with cleft lip and/or palate: Van der Woude (VWS) and Popliteal Pterygium syndromes (PPS). Furthermore, common IRF6 haplotypes and single nucleotide polymorphisms (SNP) alleles are strongly associated with nonsyndromic clefting defects in multiple ethnic populations. Mutations in the mouse often provide good models for the study of human diseases and developmental processes. We identified the cleft palate 1 (clft1) mouse mutant in a forward genetic screen for phenotypes modeling human congenital disease. In the clft1 mutant, we have identified a novel missense point mutation in the mouse Irf6 gene, which confers an amino acid alteration that has been found in a VWS family. Phenotypic comparison of clft1 mutants to previously reported Irf6 mutant alleles demonstrates the Irf6^clft1 allele is a hypomorphic allele. The cleft palate seen in these mutants appears to be due to abnormal adhesion between the palate and tongue. The Irf6^clft1 allele provides the first mouse model for the study of an etiologic IRF6 missense mutation observed in a human VWS family. genesis 48:303–308, 2010. © 2010 Wiley‐Liss, Inc.     

Experimental study on protection of vitamin B6 on TCDD-induced palatal cleft formation in the mice

BACKGROUND: 2, 3, 7, 8-Tetrachlorodibenzo-p-dioxin (TCDD) can cause a high percentage of cleft palate in fetuses when administered during organogenesis in certain strains of mice including the C57BL/6J. In this study, vitamin B₆ (B₆) was tested for antiteratogenic effects on TCDD-induced cleft palate in fetal mice. METHODS: The pregnant C57BL/6J mice were dosed with 24 µg TCDD/kg and/or 5, 10, 20, and 40 mg B₆/kg body weight on gestation day (GD) 10. The control group mice were dosed with 50 ml sesame oil/kg body weight on GD10. The mice were sacrificed on GD12.5, GD13.5, GD14.5, GD15.5, and GD17.5, respectively. The harvested embryos were examined to detect the incidence of cleft palate and the developing palatal shelves in a different phase were investigated morphologically and histologically among different groups. RESULTS: Total frequency of clefts is 55.56% in the TCDD group and 31.81% (5 mg), 44.44% (10 mg), 40.90% (20 mg), and 32.00% (40 mg) in the TCDD+ B₆ groups. There were no statistically significant differences among the TCDD and TCDD+ B₆ groups (p=0.743>0.05). CONCLUSIONS: It was demonstrated in this study that B6 could not antagonize 2, 3, 7, 8-TCDD-indued cleft palate. Birth Defects Res (Part B) 86:357–361, 2009. © 2009 Wiley-Liss, Inc.     

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.     

MobiSeq: De novo SNP discovery in model and non‐model species through sequencing the flanking region of transposable elements

In recent years, the availability of reduced representation library (RRL) methods has catalysed an expansion of genome‐scale studies to characterize both model and non‐model organisms. Most of these methods rely on the use of restriction enzymes to obtain DNA sequences at a genome‐wide level. These approaches have been widely used to sequence thousands of markers across individuals for many organisms at a reasonable cost, revolutionizing the field of population genomics. However, there are still some limitations associated with these methods, in particular the high molecular weight DNA required as starting material, the reduced number of common loci among investigated samples, and the short length of the sequenced site‐associated DNA. Here, we present MobiSeq, a RRL protocol exploiting simple laboratory techniques, that generates genomic data based on PCR targeted enrichment of transposable elements and the sequencing of the associated flanking region. We validate its performance across 103 DNA extracts derived from three mammalian species: grey wolf (Canis lupus), red deer complex (Cervus sp.) and brown rat (Rattus norvegicus). MobiSeq enables the sequencing of hundreds of thousands loci across the genome and performs SNP discovery with relatively low rates of clonality. Given the ease and flexibility of MobiSeq protocol, the method has the potential to be implemented for marker discovery and population genomics across a wide range of organisms—enabling the exploration of diverse evolutionary and conservation questions.     

Mapping breakpoints of a familial chromosome insertion (18,7) (q22.1; q36.2q21.11) to DPP6 and CACNA2D1 genes in an azoospermic male

It is widely accepted that the incidence of chromosomal aberration is 10–15.2% in the azoospermic male; however, the exact genetic damages are currently unknown for more than 40% of azoospermia. To elucidate the causative gene defects, we used the next generation sequencing (NGS) to map the breakpoints of a chromosome insertion from an azoospermic male who carries a balanced, maternally inherited karyotype 46, XY, inv ins (18,7) (q22.1; q36.2q21.11). The analysis revealed that the breakage in chromosome 7 disrupts two genes, dipeptidyl aminopeptidase-like protein 6 (DPP6) and contactin-associated protein-like 2 (CACNA2D1), the former participates in regulation of voltage-gated potassium channels, and the latter is one of the components in voltage-gated calcium channels. The deletion and duplication were not identified equal or beyond 100 kb, but 4 homologous DNA elements were verified proximal to the breakpoints. One of the proband's sisters inherited the same aberrant karyotype and experienced recurrent miscarriages and consecutive fetus death, while in contrast, another sister with a normal karyotype experienced normal labor and gave birth to healthy babies. The insertional translocation is confirmed with FISH and the Y-chromosome microdeletions were excluded by genetic testing. This is the first report describing chromosome insertion inv ins (18,7) and attributes DPP6 and CACNA2D1 to azoospermia.