Identification of Novel Variants in MEN1: A Study Conducted with Four Multiple Endocrine Neoplasia Type 1 Patients.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant inherited endocrine tumor syndrome caused by inactivating variants of the MEN1 gene. The aim of this study is to explore the clinical and genetic characteristics of four MEN1 patients. We isolated genomic deoxyribonucleic acid from lymphocytes, parathyroid, and thymic tumoral tissue specimens from the MEN1 patients. All exons of the MEN1 and CDNK1B genes and adjacent exon-intron sequences were amplified by polymerase chain reaction and subsequently sequenced. Further, the splice alterations were studied by sequencing the amplified RT-PCR products for MEN1 cDNA. We identified four heterozygous MEN1 germline variants: c.564delC, c.1268G>A, IVS5+5delG, and c.1546_1547insC. Both c.564delC and IVS5+5delG were novel variants. The impact of the MEN1 splice variant, IVS5+5delG, was evaluated using bioinformatics and in vitro analyses. The analyses indicated that this variant resulted in skipping of the neighboring exon and was disease-causing. Two novel somatic variants, c.249_252delGTCT and c.313_314insC, were found. Additionally, loss of heterozygosity (LOH) for the MEN1 locus (IVS5+5delG and c.564delC) was found in tumor tissue samples from the MEN1 patients, consistent with Knudson's two-hit mechanism. We identified four MEN1 germline variants and two novel somatic variants. Early recognition of the phenotype coupled with variant screening of the MEN1 gene is the key to diagnosing and treating MEN1 effectively at an early stage.

Identification of two novel mutations in three Chinese families with Kallmann syndrome using whole exome sequencing.

Kallmann syndrome (KS) is a rare developmental disorder that manifests as congenital hypogonadotropic hypogonadism with anosmia. More than 19 genes have been found to be associated with KS. However, approximately 70% of the causes of KS remain unclear. Here, we studied seven KS patients, from three families, who had delayed puberty and olfactory bulb dysplasia. However, the families of these patients showed a range of other unique clinical features, including hearing loss, anosmia (to varying degrees) and unilateral renal agenesis. We performed whole exome sequencing and copy number variation (CNV) sequencing on samples acquired from these patients. We identified two novel mutations (c.844delC in ANOS1, c.475C>T in SOX10) and a novel trigenic pattern, PROKR2/CHD7/FEZF1 (c.337T>C in PROKR2, c.748C>G in FEZF1, c.8773G>A in CHD7). The c.844delC mutation in the ANOS1 gene was predicted to generate a truncated form of the anosmin-1 protein. SIFT and PolyPhen-2 predicted that the c.475C>T mutation in SOX10 had a damaging effect. The PROKR2 mutation (c.337T>C) was previously reported as harmful. No pathogenic copy number alterations were detected. Our study expands the genotypic and phenotypic spectrum of KS, a disease that shows considerable clinical and genetic heterogeneity. The application of whole exome sequencing could facilitate our understanding of the pathogenesis of KS.

[Effect of different glucose concentrations on the expressions of insig-1 and insig-2 mRNA during the differentiation of 3T3-L1 cells].

OBJECTIVE: To determine the effect of different concentrations of glucose on the differentiation of 3T3-L(1) and the expression of insig-1 and insig-2 mRNA, and to explore the effect of insulin-induced gene in the differentiation and formation of adipocytes and lipogenesis. METHODS: The 3T3-L(1) cells were induced to differentiate in high glucose concentration (25 mol/L G.S), low glucose concentration (5.5 mol/L G.S), and mannitol (19.5 mol/L Mannitol +5.5 mol/L G.S), respectively. The differentiation of 3T3-L(1) cells was examined by oil red "O" straining, and the expression of insig-1,insig-2 mRNA and AP2 mRNA was examined by RT-PCR and in situ hybridization. RESULTS: With the differentiation of 3T3-L(1) cells, the expression of insig-1 and insig-2 mRNA was gradually up-regulated. The expression of insig-1 and insig-2 mRNA significantly increased while AP(2) mRNA decreased in the low glucose concentration inducing group and mannitol inducing group. In the high glucose concentration inducing group, the cell differentiation was poor (P<0.05). There was no difference between the low glucose concentration and the mannitol group in the differentiation of 3T3-L(1) cells, and in the expression of insig-1 and insig-2 and AP(2) mRNA. CONCLUSION: Different concentrations of glucose may influence the cell differentiation and the low glucose concentration promotes insig-1 and insig-2 gene expression, which may lead to the inhibition of the differentiation and lipogenesis of preadipocytes.

A 3' splice site mutation of IDS gene in a Chinese family with mucopolysaccharidosis type II.

The purpose of this study was to identify the underlying genetic cause in a four generation Chinese family diagnosed with mucopolysaccharidosis type II. Peripheral blood samples were collected from family members and the iduronate-2-sulfatase (IDS) gene was analyzed by DNA sequencing. The impact of IDS mutations on mRNA transcription was determined by quantitative real-time RT-PCR (qRT-PCR) in both patients as well as in healthy control samples. In addition, RT-PCR was performed to confirm the characteristics of a found mutation located in non-canonical splicing site. A 3' splice site mutation c.880-8A>G (IVS 6-8A>G) was identified in two members of the analyzed MPS II family and sequencing of RT-PCR products showed that this mutation activates an upstream cryptic splice-site in intron 6, leads to the 7 nucleotide insertion in exon 7, which in turn results in an exon 7 frameshift introducing a premature stop codon and shorter peptide chain. In addition, qRT-PCR products from the two patients showed a reduced IDS mRNA expression (43.9% and 71.2%, respectively), when compared with the average IDS mRNA expression in healthy control samples, possibly as a result of nonsense-mediated mRNA decay. In conclusion, in this study, we have identified an IDS gene splice mutation which is associated with clinically attenuated MPS II phenotype. In addition, our study accentuates the importance of cDNA analysis in the detection of intronic mutations, since in the studies examining only gDNA, the link between genotype and phenotype may have been misinterpreted.

[Construction of eukaryotic expression vector of human TSARG4 and establishment of its stable transfected HeLa cell line].

AIM: To construction of eukaryotic expression vector of human TSARG4 and establishment of its stable transfected Hela cell line. METHODS: The open reading frame (ORF) of TSARG4 was amplified from human testis by RT-PCR. The PCR products were cloned into pUCm-T vectors and sequenced. Then the cDNA fragment was subcloned into pcDNA3.1(+), a eukaryotic expression vector. The recombined plasmid pcDNA3.1(+)/TSARG4 was sequenced and transfected into Hela cell by lipofectamine 2000. After screening culture by G418, stable transfected HeLa cell line was established, and the expression of TSARG4 was identified by RT-PCR and in situ hybridization. RESULTS: The eukaryotic expression plasmid of pcDNA3.1(+)/TSARG4 was successfully constructed and stable transfected HeLa cell line was established. RT-PCR and in situ hybridization result revealed that TSARG4 was expressed successfully in HeLa cells. CONCLUSION: The recombinant eukaryotic expression vector of pcDNA3.1(+)/TSARG4 has been constructed successfully and stably expressed in HeLa cell line, providing a foundation for further studies on the function of TSARG4 in vitro.

[Construction and expression of fusion gene eukaryotic expression plasmid of pEGFP-C(3)-insig2 and its influence to downstream genes].

AIM: To construct the eukaryotic expression plasmid of insig2 gene and detect the expression of downstream gene adiponectin and adipocyte fatty acid-binding protein 2 (AP2) after the transfection of 3T3-L1 cells. METHODS: Insig2 gene of the mouse was amplified by RT-PCR and then cloned into the eukaryon expression vector pEGFP-C(3), After confirmed by double restriction enzyme digestion analysis and DNA sequencing, pEGFP-C(3)-insig2 was transfected into 3T3-L1 cells by lipofectamine 2000. The expression of insig2 and downstream gene in the 3T3-L1 cells were detected by RT-PCR and fluorescence microscope. RESULTS: The eukaryotic expression plasmid of pEGFP-C(3)-insig2 was constructed. The expression of fusion protein in the endochylema was confirmed. The transcription of adiponectin mRNA and AP2 mRNA was down-regulated after transfection for 24 h and 72 h. CONCLUSION: The eukaryotic expression plasmid of pEGFP-C(3)-insig2 is successfully constructed. The transfected insig2 may have an effect on fat metabolism of 3T3-L1 cells.