Aberrant RNA splicing as the molecular basis of some pathogenic variants.

Identification and correct classification of disease-associated mutations are essential for molecular diagnosis and clinical management of many genetic disorders. Although next-generation sequencing has greatly accelerated the detection of nucleotide changes, the biological interpretation of most variants has become a real challenge. Moreover, attention is typically paid to protein-coding changes and the potential impact of exonic variants on RNA splicing is often ignored. There is increasing evidence showing that disease-causing aberrant RNA splicing is more widespread than currently appreciated. Here, we review the major types of the variants involved in RNA splicing and the approaches used to identify and characterize these variants. We hope to provide a reference for evaluation of the effects of mutations on diseases.

Association of common polymorphisms in the LRP6 gene with sporadic coronary artery disease in a Chinese population.

BACKGROUND: Genetic factors contribute to the development of coronary artery disease (CAD). Recently, a missense mutation in the low density lipoprotein receptor related protein 6 (LRP6) gene, encoding low density lipoprotein receptor related protein 6, has been implicated in an autosomal dominant form of early-onset CAD. The aim of this study was to determine whether the common variants in LRP6 are associated with sporadic CAD in Chinese. METHODS: A total of 766 CAD patients and 806 healthy controls were included in this study. The presence of angiographic CAD was determined by coronary angiographic analysis. Six signal nucleotide polymorphisms (SNPs) were genotyped using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. RESULTS: A significant association was detected between rs11054731 in LRP6 intron 2 and CAD in our cohort (P = 0.001). The CC genotype and C allele frequency in the case group were 52% and 72%. Using a dominant model of inheritance, the C allele of rs11054731 was shown to be an independent risk factor for CAD with an OR of 1.45 (95%CI: 1.19 - 1.77, P = 0.0002). With the stratification according to the number of affected coronary arteries, an association was observed between rs11054731 and CAD (P = 0.0002). No significant association was observed between any other SNPs and the risk of CAD. CONCLUSION: The C allele of the rs11054731 within the LRP6 gene was associated with increased risk and extent of CAD in Chinese.

[Synpolydactyly in a Chinese kindred: mutation detection, prenatal ultrasonographic and molecular diagnosis].

OBJECTIVE: To identify potential mutation responsible for synpolydactyly (SPD) in a large Chinese kindred and to offer genetic counseling and prenatal diagnosis for the members of the family. METHODS: All family members were examined clinically, and blood samples were obtained for linkage analysis and mutation screening. Ultrasound examinations were conducted at 16-21 weeks. Amniotic fluid sample was obtained by ultrasound-guided amniocentesis at 18 weeks of gestation. RESULTS: A large kindred affected with SPD was identified and characterized. With two short tandem repeat (STR) markers (D2S1238 and D2S1245) flanking the HOXD13 gene, the disease was mapped to 2q31. A heterozygous 27 bp expansion within the imperfect GCN triplet-repeat of exon 1, c. 184_210dup, was identified. The mutation resulted in a gain of 9 alanine residues between the 14th and 15th alanine of the normal 15-amino-acid-long polyalanine tract. On ultrasound examination, all fingers and toes of the fetus appeared to be normal. Linkage analysis and mutation detection confirmed that the fetus did not inherit the mutant allele from his affected mother. CONCLUSION: HOXD13 gene mutation was responsible for the SPD phenotype in this family. Accurate prenatal diagnosis of SPD was achieved with combined ultrasound and molecular analysis.

[Association of the ABCG1 gene polymorphism with the susceptibility and severity of coronary atherosclerotic disease].

OBJECTIVE: To investigate the association of the ATP-binding cassette sub-family G member 1 (ABCG1) gene polymorphisms with coronary atherosclerotic disease (CAD) in Chinese Han population. METHODS: A population based case-control association study was carried out in 541 patients with CAD and 649 healthy controls from Chinese Han population. Two single nucleotide polymorphisms (SNPs) of the ABCG1 gene were genotyped using polymerase chain reaction-restriction fragment length polymorphism. Logistic regression was used to compare the genotypic and allelic frequency difference. RESULTS: The frequency of allele C of rs225374 was significantly higher in the CAD patients than that in the healthy controls (OR=1.186, 95%CI: 1.009-1.394, P=0.039), while the difference was also significant in the male subgroup (OR=1.236, 95%CI: 1.014-1.506, P=0.036). A statistically higher frequency of rs1044317 allele A was found in the CAD patients in comparison to the healthy controls (OR=1.187, 95%CI: 1.009-1.397, P=0.039). In case-only association study, rs225374 showed significant association in the high Gensini score group compared with the low Gensini score group (OR=1.303, 95%CI: 1.024-1.657, P=0.031). CONCLUSION: The two SNPs of the ABCG1 gene might be associated with the susceptibility and severity of CAD in Chinese Han population.

Two single nucleotide polymorphisms in ALOX15 are associated with risk of coronary artery disease in a Chinese Han population.

Arachidonate 12/15-lipoxygenase (12/15-LOX) has been implicated in the pathogenesis of atherosclerosis, but with contradicting results. The aim of this study was to investigate the association of two polymorphisms in ALOX15 and the risk of coronary artery disease (CAD) in a Chinese Han population. A total of 519 unrelated CAD patients and 608 unrelated control subjects of the Chinese Han population were recruited in the case-control study. Two tagSNPs, rs7217186:T>C and rs2619112:G>A, were selected and genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The carriers of the C allele (the CC homozygote and the CT heterozygote) of rs7217186:T>C and the carriers of the A allele (the AA homozygote and the GA heterozygote) of rs2619112:G>A displayed elevated odds ratios (ORs) for CAD compared with the TT homozygotes and GG homozygotes, respectively, after adjusting for other potential confounders including age, sex, body mass index, systolic blood pressure, diastolic blood pressure, glucose, triglyceride, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and smoking status (adjusted odds ratio [OR] = 3.2, 95% confidence interval [CI]: 1.335-7.665, P = 0.009 and adjusted OR = 3.5, 95% CI: 1.343-9.330, P = 0.011). In stratified analyses, after adjusting those aforementioned confounders, the CC and CT genotypes of rs7217186:T>C were associated with a greater risk of CAD in subjects <60 years (adjusted OR = 5.7, 95% CI: 1.557-21.097, P = 0.009) and in females (adjusted OR = 9.3, 95% CI: 1.048-82.213, P = 0.045). For rs2619112:G>A, subjects (<60 years) carrying the A allele had a greater risk of CAD than the GG homozygotes (adjusted OR = 4.9, 95% CI: 1.215-19.547, P = 0.025); the male carriers of A allele also had a greater risk (adjusted OR = 3.5, 95% CI: 1.136-11.006, P = 0.029). In summary, the present study shows that after adjustment for other confounding CAD factors, rs7217186:T>C and rs2619112:G>A of ALOX15 are associated with increased risk of CAD in this Chinese Han population.

[Association of rs2298212 polymorphism in OX40 gene with coronary atherosclerotic disease in Chinese Han population].

OBJECTIVE: To study the association of the OX40 gene rs2298212G/A polymorphism with coronary atherosclerotic disease (CAD) in Chinese Han population. METHODS: Five hundred and thirty six CAD patients and 544 age and ethnic matched controls of Chinese Han population were recruited from Qilu Hospital, Shandong University. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to genotype the selected single nucleotide polymorphism. Distributions of genotypic and allelic frequencies were analyzed by Chi-square test. RESULTS: The distribution of genotypic and allelic frequencies have no significant differences between the CAD cases and controls(P> 0.05), even after adjusting for age, gender, body mass index, systolic blood pressure, diastolic blood pressure, glucose, total cholesterol, and triglyceride. However, when substratification analysis of the involved coronary artery vessels was performed, significant difference was found between single-vessel and triple-vessel (P= 0.02, OR = 1.56, 95% CI: 1.08-2.26) involvement. CONCLUSION: The rs2298212G/A polymorphism in OX40 gene may be associated with the severity of coronary atherosclerotic disease.

Gene-expression profiles in gastric epithelial cells stimulated with spiral and coccoid Helicobacter pylori.

Human gastric epithelial immortalized GES-1 cells were infected with spiral and coccoid Helicobacter pylori. Scanning electron microscopy was used to determine the ability of the two forms of H. pylori to adhere to GES-1 cells. GES-1 cell apoptosis induced by coccoid and spiral H. pylori was analysed using flow cytometry. A cDNA microarray for 22,000 human genes was used to identify the gene-expression differences in GES-1 cells infected with the two forms of H. pylori, and the gene expression identified by the cDNA microarray was confirmed by RT-PCR. Scanning electron microscope observation showed that both coccoid and spiral bacteria can adhere to GES-1 cells. After 4 h infection, apoptosis induction was 27.4% for spiral-form infection and 10.2% for coccoid-form infection. Of 268 differentially expressed genes identified by cDNA microarray, 166 showed higher expression with the spiral H. pylori infection than with the coccoid H. pylori infection. To the best of the authors' knowledge, this is the first report that GES-1 cells infected with spiral H. pylori have higher expression of cxcl10, ccl11, ccl5, groalpha, TLR5, ATF3, fos, fosl2, gadd45a and myc. The cells infected with coccoid H. pylori had higher expression of survivin. The global profile of gene expression in GES-1 cells infected with coccoid and spiral H. pylori is described for the first time.

[Analysis on genetic polymorphism of 5 STR loci selected from X chromosome].

OBJECTIVE: To select short tandem repeats(STR) from X chromosome. METHODS: STR is a universal genetic marker that has changeable polymorphism and stable heredity in human genome. It is a specific DNA segment composed of 2-6 base pairs as its core sequence. It is an ideal DNA marker used in linkage analysis and gene mapping. In this study, 8 short tandem repeats were selected from two genomic clones on X chromosome by using BCM Search Launcher. Primers amplifying the STR loci were designed by using Primer 3.0 according to the unique sequence flanking the STRs. Polymorphisms of the short tandem repeats in Chinese population were evaluated by PCR amplification and PAGE. RESULTS: Five of these STRs were polymorphic. Chi-square test indicated that the distribution of genotypes agreed with Hardy-Weinberg equilibrium (P>0.05). CONCLUSION: Five polymorphic short tandem repeats have been identified on chromosome X and will be useful for linkage analysis and gene mapping.

[Characterization of genomic structure and mutation analysis of SMARCA1 gene in a Smith-Fineman-Myers syndrome family].

The study is to determine the genomic structure and the role of SMARCA1 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member1, SMARCA1) in the etiology of Smith-Fineman-Myers syndrome (SFMS). By comparing the cDNA sequence of SMARCA1 with the genomic sequences, genomic structure of SMARCA1 was determined, and conformed by amplifying and sequencing the sequences of exons and splicing junction. The results show that the genomic sequence of SMARCA1 gene exceeds 71.7 kb in length, and contains 24 exons and 23 introns. All the exon/intron boundaries follow the GT-AG rule and are in good agreement with the exon/intron consensus sequence. The characterization of genomic structure of SMARCA1 gene allows us to detect disease-causing mutation within the gene and further study its biological function. The open reading frame of SMARCA1 was detected for mutation by PCR amplification and direct sequencing in affected males from SFMS family in Shandong China. The disease in SFMS family from Shandong is not caused by the mutation within open reading frame of SMARCA1 gene.

[Fine mapping of Smith-Fineman-Myers syndrome and exclusion of GPC3, GPCR2 MST4 and GLUD2 as candidate genes].

OBJECTIVE: Smith-Fineman-Myers syndrome (SFMS) is an X-linked mental retardation syndrome. The authors had ascertained a large Chinese family with SFMS from Shandong and had mapped the disease locus to an interval of 19.8 Mb on Xq25 flanked by markers DXS8064 and DXS8050. Further investigation suggested that SFMS exhibited locus heterogeneity. In this study for facilitating the identification of the gene responsible for SFMS, the additional markers were analyzed to narrow down the candidate region, and four candidate genes (GPC3, MST4,GPCR2 and GLUD2) were chosen and screened for disease-causing mutation. METHODS: PCR and denaturing polyacrylamide gel electrophoresis were used to genotype 13 new polymorphic markers distributed within the candidate region. Mutation detection was accomplished by sequencing the exons and intron-exon junctions of the candidate genes. RESULTS: By analyzing 13 additional polymorphic markers, SFMS candidate region can be reduced to an interval of 10.18 Mb bounded by XSTR3 and XSTR4, and no disease-causing mutation was identified in the coding regions of four candidate genes. CONCLUSION: GPCR2 GPC3, MST4 and GLUD2 were excluded as pathogenic genes for SFMS. The refined SFMS locus will assist in the identification and characterization of other candidate genes for SFMS.

[Application of homozygosity mapping to the fine mapping of the osteoporosis-pseudoglioma syndrome locus].

OBJECTIVE: To evaluate the role of homozygosity mapping in the fine mapping of the genes responsible for the rare autosomal recessive diseases. METHODS: Polymerase chain reaction-single sequence length polymorphism was used to genotype the family members from 8 families with osteoporosis-pseudoglioma syndrome(OPS) for 14 polymorphic loci within candidate region. The OPS candidate region was narrowed by searching for homozygous region in affected. RESULTS: The OPS candidate region was narrowed to a 1 cM interval between D11S1296 and D11S4136. CONCLUSION: Homozygosity mapping is a powerful method for mapping and narrowing the candidate region of the genes responsible for the rare autosomal recessive diseases.

[Polymorphic loci and polymorphism analysis of short tandem repeats within XNP gene].

To select polymorphic short tandem repeat markers within X-linked nuclear protein (XNP) gene, genomic clones which contain XNP gene were recognized by homologous analysis with XNP cDNA. By comparing the cDNA with genomic DNA, non-exonic sequences were identified, and short tandem repeats were selected from non-exonic sequences by using BCM search Launcher. Polymorphisms of the short tandem repeats in Chinese population were evaluated by PCR amplification and PAGE. Five short tandem repeats were identified from XNP gene, two of which were polymorphic. Four and 11 alleles were observed in Chinese population for XNPSTR1 and XNPSTR4, respectively. Heterozygosities were 47% for XNPSTR1 and 70% for XNPSTR4. XNPSTR1 and XNPSTR4 localized within 3' end and intron 10, respectively. Two polymorphic short tandem repeats have been identified within XNP gene and will be useful for linkage analysis and gene diagnosis of XNP gene.