A Novel CDH1 Mutation Causing Reduced E-Cadherin Dimerization Is Associated with Nonsyndromic Cleft Lip With or Without Cleft Palate.

Aims: Cleft lip with or without cleft palate (CL/P) is a common birth defect with an average prevalence of 1/700 to 1/1000. Almost 70% of CL/P cases are nonsyndromic CL/P (NSCL/P). The aim of this study was to identify the underlying cause of a four-generation Chinese family with autosomal dominant NSCL/P. Methods: Genomic DNA was extracted from peripheral blood leukocytes, and whole-exome sequencing was carried out to identify the underlying genetic cause of the disorder. The mutation was confirmed by Sanger sequencing and polymerase chain reaction-restriction fragment length polymorphism methods. Western blotting and coimmunoprecipitation were used to analyze the protein expression level and adhesive dimerization of the CDH1 mutants. Slow aggregation assays were conducted to investigate the cell-cell adhesion ability. Results: A novel missense mutation (c.468G>C/p.Trp156Cys) of CDH1 was identified in the proband and the mutation was shown to cosegregate with the phenotype in the family. Furthermore, we found that the p.Trp156Cys mutation led to decreased E-cadherin dimerization and cell-cell adhesion ability. Conclusions: Our findings identified a novel CDH1 variant (c.468G>C/p.Trp156Cys) responsible for NSCL/P in a Chinese family, which expanded the mutational spectrum of the CDH1 gene and may contribute to understanding the molecular basis of NSCL/P.

Mutation analysis of COL4A3 and COL4A4 genes in a Chinese autosomal-dominant Alport syndrome family.

Autosomal dominant Alport syndrome (ADAS) accounts for 5% of all cases of Alport syndrome (AS), a primary basement membrane disorder arising from mutations in genes encoding the type IV collagen protein family.Mutations in COL4A3 and COL4A4 genes were reported to be associated with ADAS. In this study, clinical data in a large consanguineous family with seven affected members were reviewed, and genomic DNA was extracted. For mutation screening, all exons of COL4A3 and COL4A4 genes were polymerase chain reaction-amplified and direct sequenced from genomic DNA, and the mutations were analyzed by comparing with members in this family, 100 ethnicitymatched controls and the sequence of COL4A3 and COL4A4 genes from GenBank. A novel mutation determining a nucleotide change was found, i.e. c.4195 A>T (p.Met1399Leu) at 44th exon of COL4A4 gene, and this mutation showed heterozygous in all patients of this family. Also a novel intron mutation (c.4127+11 C>T) was observed at COL4A4 gene. Thus the novel missense mutation c.4195 A>T (p.Met1399Leu) and the intron mutation (c.4127+11 C>T) at COL4A4 gene might be responsible for ADAS of this family. Our results broadened the spectrum of mutations in COL4A4 and had important implications in the diagnosis, prognosis, and genetic counselling of ADAS.

[Analysis of COL1A1 gene mutation in an ethnic Han Chinese family from Henan affected with osteogenesis imperfecta].

OBJECTIVE: To identify potential mutation of COL1A1 gene in an ethnic Han Chinese family from Henan affected with osteogenesis imperfecta (OI). METHODS: Peripheral blood samples were collected from all 11 members of the family and 50 healthy adults for the extraction of genomic DNA. All exons and introns of the COL1A1 gene were amplified by polymerase chain reaction and subjected to direct sequencing. Mutations found in the proband were analyzed through comparison with other members of the family, 50 healthy individuals and sequence from the GenBank. RESULTS: Fifteen sequence variants were discovered, which included 1 missense mutation, 1 synonymous mutation and 13 intronic mutations. All of the 4 patients from the family were detected as having carried a novel heterozygous missense mutation (c.4193T>G, p.I1398S) in exon 50 of the COL1A1 gene. The father of the proband has carried the same mutation but had a normal phenotype. The same mutation was not found in other healthy members of the family. CONCLUSION: The OI type of this family may have been autosomal dominant with incomplete penetrance or autosomal recessive associated with COL1A1 gene mutations.

Screening of a protein that interacts with the matrix attachment region-binding protein from Dunaliella salina.

We isolated the matrix attachment region-binding protein (MBP) DMBP-1 from Dunaliella salina in our previous studies. MBPs are part of the cis-acting protein family cluster. The regulatory function possibly works through the interaction of the MBPs with each other. In the present study, DMBP-1 was used as the bait in screening the D. salina cDNA library for DMBP-1 interactors that could potentially mediate the DMBP-1-regulated functions. A novel MBP, namely, DMBP-2, was identified as a DMBP-1 binding partner. The cDNA of DMBP-1 was 823 bp long and contained a 573 bp open reading frame, which encoded a polypeptide of 191 amino acids. The interaction between DMBP-2 and DMBP-1 was further confirmed through glutathione S-transferase pull-down assays.

Molecular characterization and functional analysis of a protease-related protein in Chang-liver cells.

In this study, the cDNA library of Chang-liver cells was immunoscreened using common ADAMs antibody to obtain ADAM related genes. We found one positive clone that was confirmed as a new gene by Blast, which is an uncharacterized helical and coil protein and processes protease activity, and named protease-related protein 1 (ARP1). The submitted GenBank accession number is AY078070. Molecular characterizations of ARP1 were analyzed with appropriate bioinformatics software. To analyse its expression and function, ARP1 was subcloned into glutathione S-transferase fusion plasmid pGEX-2T and expressed by E. coli system. The in vitro expression product of ARP1 was recognized by common ADAMs antibody with western blot. Interestingly, ARP1 cleaves gelatine at pH9.5, which suggests it is an alkaline protease. Semi-quantitative RT-PCR result indicates that ARP1 mRNA is strongly transcribed in the liver and the treated Chang-liver cells.

Increased expression of transgene in stably transformed cells of Dunaliella salina by matrix attachment regions.

Nuclear matrix attachment regions (MARs) are known to bind specifically to the nuclear scaffold and are thought to influence expression of the transgenes. In our previous studies, a new deoxyribonucleic acid fragment isolated from Dunaliella salina could bind to the nuclear matrix in vitro and had the typical characteristics of MARs. In this study, to investigate effects of MARs on expression of transgenes in the stably transformed cells of D. salina, expression vectors with and without MARs, which contained chloramphenicol acetyltransferase (CAT) reporter gene driven by D. salina ribulose 1,5-bisphosphate carboxylase/oxygenase promoter, were constructed and delivered, respectively, into cells of D. salina by electroporation. Twenty stably transformed colonies of D. salina were randomly picked out, and CAT gene expression was assayed. The results showed that the CAT enzyme of the colonies of D. salina transformed with the expression vector containing MARs averaged out about 4.5-fold higher than those without MARs, while the transgene expression variation among individuals of transformants decreased threefold. The CAT enzyme in the stably transformed lines was not significantly proportional to the gene copy numbers, suggesting that the effects of MARs on transgene expression may not be through increasing the transgene copy numbers.