Identification of a novel heterozygous SOX9 variant in a Chinese family with congenital heart disease.

BACKGROUND: Previous studies of individuals with hereditary or sporadic congenital heart disease (CHD) have provided strong evidence for a genetic basis for CHD. The aim of this study was to identify novel pathogenic genes and variants in a Chinese CHD family. METHODS: Three generations of a family with CHD were recruited. We performed whole exome sequencing for the affected individuals and the proband's unaffected aunt to investigate the genetic causes of CHD in this family. Heterozygous variants carried by the proband and her maternal grandmother, but not the proband's aunt, were selected. The frequencies of the variants detected were assessed using public databases, and their influences on protein function were predicted using online prediction software. The candidate variant was further confirmed by Sanger sequencing of other members of the family. RESULTS: On the basis of the family's pedigree, the mode of inheritance was speculated to be autosomal dominant with incomplete penetrance. We identified a novel heterozygous missense variant in SOX9 in all affected individuals and one asymptomatic family member, suggesting an inheritance pattern with incomplete penetrance. The variant was not found in any public database. In addition, the variant was highly conserved among mammals, and was predicted to be deleterious by online software programs. CONCLUSIONS: We report for the first time a novel heterozygous missense variant in SOX9 (NM_000346:c.931G>T:p.Gly311Cys) in a Chinese CHD family. Our results provide further evidence supporting a causative role for SOX9 variants in CHD.

Association Between miR-143/145 rs4705343 Polymorphism and Risk of Congenital Heart Disease in a Chinese Tibetan Population.

Objective: Congenital heart disease (CHD) is the most common birth defect worldwide and is caused by both genetic and environmental factors. The microRNA (miR)-143/145 cluster is involved in various biological processes related to cardiovascular development. The functional single nucleotide polymorphism (SNP) rs4705343 of miR-143/145 may influence the expression of these miRNAs. In this study, we aimed to estimate the association between miR-143/145 rs4705343 and the risk of CHD in a Chinese Tibetan population. Methods: Matrix-assisted laser desorption ionization time-of-flight mass spectrometry assays were performed to genotype the miRNA-143/145 rs4705343 SNP in 510 CHD Tibetan patients and 681 unrelated Tibetan healthy controls. The associations between the SNP frequencies and the CHD risk were analyzed by χ2 test/Fisher's test and assessed by odds ratios (ORs) and 95% confidence intervals (95% CIs). Results: We successfully genotyped 1165 subjects with a SNP call rate of 97.8%. Under the allelic model we found that rs4705343 was not associated with the risk of CHD (p = 0.082), but under the recessive model the CC genotype at this locus was associated with a significantly increased risk of CHD compared with the other genotypes (CC vs TT+TC: OR = 1.60, 95% CI = 1.08-2.37, p = 0.017). Conclusion: The present study suggests that the rs4705343 CC genotype of miR-143/145 is associated with CHD risk in a Chinese Tibetan population.

Mutations in EPAS1 in congenital heart disease in Tibetans.

EPAS1 encodes HIF2 and is closely related to high altitude chronic hypoxia. Mutations in the EPAS1 coding sequence are associated with several kinds of human diseases, including syndromic congenital heart disease (CHD). However, whether there are rare EPAS1 coding variants related to Tibetan non-syndromic CHD have not been fully investigated. A group of 286 Tibetan patients with non-syndromic CHD and 250 unrelated Tibetan healthy controls were recruited from Qinghai, China. Sanger sequencing was performed to identify variations in the EPAS1 coding sequence. The novelty of identified variants was confirmed by the examination of 1000G and ExAC databases. Control samples were screened to establish that the rare candidate variants were specific to the Tibetan patients with non-syndromic CHD. Bioinformatics software was used to assess the conservation of the mutations and to predict their effects. The effect of EPAS1 mutations on the transcription of its target gene, VEGF, was assessed by dual-luciferase reporter assay. The mammalian two-hybrid assay was used to study the protein interactions between HIF2 and PHD2 or pVHL. We identified two novel EPAS1 mutations (NM_001430: c.607A>C, p.N203H; c.2170G>T, p.G724W) in two patients. The N203H mutation significantly affected the transcription activity of the VEGF promoter, especially in conditions of hypoxia. The N203H mutation also showed enhanced protein-protein interactions between HIF2 and PHD2, and HIF2 and pVHL, especially in conditions of hypoxia. However, the G724W mutation did not demonstrate the same effects. Our results indicate that EPAS1 mutations might have a potential causative effect on the development of Tibetan non-syndromic CHD.

[Effect of altitude chronic hypoxia on liver enzymes and its correlation with ACE/ACE2 in yak and migrated cattle].

OBJECTIVE: To investigate the difference of liver enzyme levels and its correlation with serum ACE/ACE2 among yak and cattle on Qinghai-Tibetan plateau, and to further explore the biochemical mechanism of their liver of altitude adaptation. METHODS: The serum samples of yak were collected at 3,000 m, 3,500 m, 4,000 m and 4,300 m respectively, meanwhile the serum samples of migrated cattle on plateau (2,500 m) and lowland cattle (1,300 m) were also collected. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), cholinesterase (CHE), gamma glutamyl transferase (GGT), alkaline phosphatase (ALP), serum lipase (LPS), angiotensin converting enzyme(ACE), angiotensin converting enzyme-2 (ACE2) in serum were measured by using fully automatic blood biochemcal analyzer. We analysed the differences of the above enzymes and its correlation with ACE/ACE2. We used one way analysis of variance (ANOVA). RESULTS: The levels of ALT in 4,000 m group and 4,300 m group of yak increased significantly compared with other groups, there were no statistically significant differences in AST, CHE, GGT, ACE/ACE2 levels of yaks at different altitudes. As compared to lowland cattle, the serum levels of AST and CHE were increased, the level of LPS and ACE was decreased significantly, respectively, and especially, the ratio of ACE/ACE2 of migranted cattle reduced nearly two times. The levels of LPS were significantly correlated to the ratio of ACE/ACE2 in yak (r = 0.357, P < 0.01), and a high correlation between ALP and ACE/ACE2 in lowland cattle( r = 0.418, P < 0.05), But the biggest contribution rate of the ratio of ACE/ACE2 was only 17.5% for the changes of the levels of liver enzyme. CONCLUSION: The results indicated that with the altitude increased did not significantly influence the changes of liver enzymes' activities in mountainous yaks but not in cattle. However, all above these changes weren't actually correlated to the ratio of ACE/ACE2.

[Risk factors of pulmonary arterial hypertension in patients with atrial septal defect at high altitude area].

OBJECTIVE: To analyze risk factors of pulmonary arterial hypertension (PAH) in patients with atrial septal defect (ASD) patients living at above 2000 m high altitude area. METHODS: We retrospectively analyzed the incidence of PAH in 526 ASD patients out of 1178 congenital heart disease patients who were hospitalized in Qinghai cardiovascular hospital between January 2007 to December 2009 and explored the risk factors including gander, age, altitude, defect size and nationalities for developing PAH in ASD patients using binary logistic regression. RESULTS: PAH prevalence was 68.4% (360/526) in ASD patients. The risk factors of developing PAH in these ASD patients were defect size (OR: 1.200, 95%CI: 1.156 - 1.246, P = 0.000), age (OR: 1.027, 95%CI: 1.003 - 1.052, P = 0.025) and altitude (OR: 1.389, 95%CI: 1.001 - 1.637, P = 0.043) while gender and nationality were not risk factors for PAH. The incidence of developing PAH increased with aging (P = 0.000). The standardized ratio of PAH at ≥ 3500 m was 74.8% which was significantly higher than that at 2000 - 2499 m altitude (66.2%, P = 0.005) and at 2500 - 3499 m altitude (66.9%, P = 0.005). CONCLUSIONS: The risk for developing PAH is high in patients living at high altitude area. The risk factors of developing PAH in ASD patients living at high altitude are defect size, age and altitude.

[Susceptibility of patients with congenital heart disease to pulmonary hypertension at a high altitude].

OBJECTIVE: To explore the incidence of congenital heart disease (CHD) complicated with pulmonary arterial hypertension (PAH) at an altitude of over 2000 meters above sea level. METHODS: We retrospectively analyzed the profiles of 688 PAH patients selected from 1178 CHD patients at our hospital during January 2007 to December 2009. The risk factors of CHD-PAH were analyzed by age, altitude, etiologies and nationalities. RESULTS: Among 1178 CHD patients, 688 (58.4%) suffered PAH. And 239 PAH cases (54.8%) were found in 436 male patients and 449 PAH cases (60.5%) in 742 female patients (χ(2) = 3.667, P = 0.058). The difference was not significant. The incidences of PAH in CHD patients varied at different altitudes (χ(2) = 10.507, P = 0.005). And 55.2%, 57.2% and 68.7% were found in < 2500 m, 2500 - 3500 m and > 3500 m groups respectively. And the > 3500 m group was statistically higher than those of the < 2500 m and 2500 - 3500 m groups (χ(2) = 9.924, P = 0.005) (χ(2) = 8.007, P = 0.005). The mean pulmonary arterial systolic pressure (PASP) of < 2500 m group was (58 ± 18) mm Hg, 2500 - 3500 m was (63 ± 17) mm Hg and > 3500 m was (64 ± 19) mm Hg. There were significant differences among them. In age composition of CHD-PAH, the highest ratio appeared before the age of 18 years old for PDA (patent ductus arteriosus)-PAH and VSD (ventricular septal defect)-PAH while that of ASD-PAH at the ages of 30 - 39 years old. There were not statistical differences among all nationalities (χ(2) = 5.404, P = 0.248). CONCLUSION: The incidence of CHD-PAH at a high altitude is significantly higher than those at a low altitude. Hypoxia may promote the occurrence and development of PAH in CHD patients.

[Epidemiological survey of congenital heart disease among people aged from 4 to 18 in Haidong area of Qinghai province].

OBJECTIVE: To investigate the epidemiological characteristics of congenital heart disease (COHD) among 4 to 17 years old children in Haidong area of Qinghai province. METHODS: All 97 718 children were surveyed with the following 3 steps: prescreening, countershock and confirmation with color Doppler. The distribution patterns were analyzed by national groups, ages and genders respectively. RESULTS: There were 496 COHD cases detected. The total incidence was 5.076 per thousand (496/97 718). The incidences of male and female were 5.046 per thousand (256/50 730) and 5.108 per thousand (240/46 988) (chi(2) = 0.018, P > 0.05). There was a significant difference between Pingan county and the others (chi(2) = 10.62, P < 0.01). The highest incidence was in Ledu (5.46 per thousand), the incidences of Huzhu and Pingan county were 5.45 per thousand and 3.64 per thousand respectively. There was no significant difference among different national groups (chi(2) = 0.33, P > 0.05). Among 496 COHD cases, the ratio of atrial septal defect (ASD), ventricular septal defect (VSD), patent ductus arteriosus (PDA) were 37.30%, 35.69% and 22.18% respectively. CONCLUSION: Total incidence of COHD was 5.076 per thousand in Haidong area of Qinghai province. The incidence was not different in both genders and national groups. The constitution of COHD in different counties were different.

[A cross-sectional study of congenital heart disease among children aged from 4 to 18 years at different altitudes in Qinghai province, China].

OBJECTIVE: The epidemiological characteristics of congenital heart disease (CHD) in children aged from 4 to 18 years were investigated in Qinghai province. METHODS: Altogether 288 066 children inhabiting at 6 prefectures and 3 counties were examined by the following three steps: pre-screening, re-examination and diagnosis with color Doppler. And the entity distribution was analyzed while the differences were compared by age, gender, altitudes and nationalities respectively. RESULTS: Altogether 1633 cases of CHD were discovered. The total prevalence of CHD was 5.71 per thousand. The prevalence of CHD was found to increase with the increase of altitude by 4.89 per thousand at the altitude of 2535 m, 5.71 per thousand at 3600 m, and 8.74 per thousand at 4200 m respectively. There were significant differences among different altitude (chi(2) = 54.696, P < 0.001). chi(2) trend analysis showed the increase with chi(2) = 41.826(P < 0.001). The total incidence of CHD in females was 6.95 per thousand, which was significantly higher than that in males with 4.54 per thousand (chi(2) = 73.79, P < 0.001). There were significant differences between males and females at the altitude of 3000 m (chi(2) = 84.733, P < 0.001) and 4000 m (chi(2) = 16.313, P < 0.001) except at the altitude of 2000 m (chi(2) = 0.807, P > 0.05). The prevalence of CHD in different age groups was statistically significant at the every altitude of 2000 m (chi(2) = 18.138, P < 0.001), 3000 m (chi(2) = 18.544, P < 0.001) and 4000 m (chi(2) = 27.535 P < 0.001). The prevalence of CHD was increasing with the increase of age groups at the altitude of 3000 m (chi(2) = 19.230, P < 0.001) and 4000 m (chi(2) = 26.894, P < 0.001) except at the altitude of 2000 m. Within the prevalence of CHD of different nationalities, there was a significant difference with chi(2) = 24.456 (P < 0.001). Within the constituent rate of CHD, the prevalence of atrial septal defect (ASD) was as high as 37.42%, followed by the prevalence of patent ductus arteriosus (PDA) as 28.47% and ventricular septal defect (VSD) as 26.01%. Regarding the four categories of CHD, the constituent rate varied at different altitudes. For example, the prevalence rate of ASD constituted 37% at the altitude of 2000 m and 3000 m, and that of PDA accounted for 46.36% at the altitude of 4200 m. CONCLUSION: The epidemiological characteristics of CHD in Qinghai children were possibly associated with altitude levels.

Cross-sectional study of congenital heart disease among Tibetan children aged from 4 to 18 years at different altitudes in Qinghai Province.

BACKGROUND: Congenital heart disease (CHD) is a common heart condition which does considerable harm to the health of children and adolescents. CHD epidemiological characteristics of Tibetan children whose ages ranged from 4 to 18 years were investigated in Qinghai Province. METHODS: A total of 32 578 Tibetan children, living at altitudes of 2535 m, 3600 m and 4200 m, were examined using a three-stage protocol: prescreening, rechecking and diagnosis using a color Doppler. The distribution of CHD at different altitudes was analyzed together with differences in occurrence according to age and gender. RESULTS: A total of 235 CHD cases were discovered. The total prevalence of CHD was 7.21 per thousand. Prevalence of CHD has been shown to increase along with increase in altitude with 5.45 per thousand at an altitude of 2535 m, 6.80 per thousand at 3600 m and 9.79 per thousand at 4200 m. There were no statistically significant differences between the prevalence at 2535 m and 3600 m (chi(2) = 1.594, P > 0.05). However, there was a significant difference between the prevalence at 2535 m and 4200 m (chi(2) = 7.002, P < 0.01). Also, apparent differences existed between the prevalence at 3600 m and at 4200 m (chi(2) = 5.540, P < 0.05). There was no statistically significant difference in prevalence according to age at an altitude of 2535 m, but the rate of CHD increased significantly along with increasing age at 3600 m and 4200 m. The total prevalence ratio of children aged from 16 to 18 years was significantly higher than that of children from 4 to 7, and from 8 to 12 with chi(2) values of 10.79 (P < 0.005), and 5.60 (P < 0.05) respectively. Within the constituent ratio of CHD, the prevalence of atrial septal defect (ASD) was the highest at 39.10%, followed by the prevalence of ventricular septal defect (VSD) at 32.8% and patent ductus arteriosus (PDA) at 24.7%. Furthermore the proportion of the four categories of CHD varied at different altitude levels: at 2535 m, of those diagnosed with CHD, the prevalence rate of VSD was the highest at 43.5%, at 3600 m ASD was the highest at 42.8% and at 4200 m, PDA was the highest at 50.8%. CONCLUSION: The epidemiological characteristics of CHD in Tibetan children may be associated with altitude levels.