Xuan Shang

Molecular epidemiological survey of haemoglobinopathies in the Guangxi Zhuang Autonomous Region of southern China

Xiong F, Sun M, Zhang X, Cai R, Zhou Y, Lou J, Zeng L, Sun Q, Xiao Q, Shang X, Wei X, Zhang T, Chen P, Xu X. Molecular epidemiological survey of haemoglobinopathies in the Guangxi Zhuang Autonomous Region of southern China.

The molecular basis of beta-thalassemia intermedia in southern China: genotypic heterogeneity and phenotypic diversity

BackgroundThe clinical syndrome of thalassemia intermedia (TI) results from the β-globin genotypes in combination with factors to produce fetal haemoglobin (HbF) and/or co-inheritance of α-thalassemia. However, very little is currently known of the molecular basis of Chinese TI patients.MethodsWe systematically analyzed and characterized β-globin genotypes, α-thalassemia determinants, and known primary genetic modifiers linked to the production of HbF and the aggravation of α/β imbalance in 117 Chinese TI patients. Genotype-phenotype correlations were analyzed based on retrospective clinical observations.ResultsA total of 117 TI patients were divided into two major groups, namely heterozygous β-thalassemia (n = 20) in which 14 were characterized as having a mild TI with the Hb levels of 68-95 g/L except for five co-inherited αααanti-3.7 triplication and one carried a dominant mutation; and β-thalassemia homozygotes or compound heterozygotes for β-thalassemia and other β-globin defects in which the β+-thalassemia mutation was the most common (49/97), hemoglobin E (HbE) variants was second (27/97), and deletional hereditary persistence of fetal hemoglobin (HPFH) or δβ-thalassemia was third (11/97). Two novel mutations, Term CD+32(A→C) and Cap+39(C→T), have been detected.ConclusionsChinese TI patients showed considerable heterogeneity, both phenotypically and genotypically. The clinical outcomes of our TI patients were mostly explained by the genotypes linked to the β- and α-globin gene cluster. However, for a group of 14 patients (13 β0/βN and 1 β+/βN) with known heterozygous mutations of β-thalassemia and three with homozygous β-thalassemia (β0/β0), the existence of other causative genetic determinants is remaining to be molecularly defined.

Mutation analysis of 73 southern Chinese Wilson's disease patients: identification of 10 novel mutations and its clinical correlation

This study was designed to investigate the molecular basis and the correlation between genotype and phenotype in the southern Chinese patients with Wilsons disease (WD). Genotypes of the ATP7B gene in 73 WD patients were examined by denaturing high-performance liquid chromatography (DHPLC) and DNA sequencing. A total of 38 different disease-causing mutations were identified, including 10 novel mutations: missense mutations (p.Gln707Arg, p.Cys1079Phe, p.Gly1149Glu, p.Ser855Tyr, p.Ala874Pro and p.Ser921Arg), nonsense mutation (p.Arg1228Stop), splice-site mutations (2121+3A>T and 3244-2A>G) and frameshift mutation (1875_1876insAATT). We found that a pair of siblings carried the same genotype but different clinical type, and two patients were found to have three mutations. In addition, we compared the clinical data for p.Arg778Leu homozygotes and compound heterozygotes. Our research has enriched the mutation spectrum of the ATP7B gene in the Chinese population and can serve as the basis for genetic counseling and clinical/prenatal diagnosis to prevent WD in China.

Analyses of GATA4, NKX2.5, and TFAP2B genes in subjects from southern China with sporadic congenital heart disease

BACKGROUND Congenital heart disease is the most common birth defect in newborns in southern China. The germline mutations in GATA4, NKX2.5, and TFAP2B genes have been identified to be responsible for congenital heart disease. The frequency of GATA4, NKX2.5, and TFAP2B mutations in subjects with congenital heart disease in southern China and the correlation between their genotype and congenital heart disease phenotype are not known. METHODS We screened germline mutations in the coding exons and the flanking intron sequences of the GATA4, NKX2.5, and TFAP2B genes in 224 congenital heart disease patients located in southern China by denaturing high-performance liquid chromatography and DNA sequencing. RESULTS Fifteen heterozygous mutations in the GATA4 gene were identified in 30 congenital heart disease patients, including a novel heterozygous missense mutation (c.788 C>G) of GATA4 in one patient with ventricular septal defect. A novel TFAP2B mutation (c.31 A>G) in a patient with endocardial cushion defect and an unreported novel TFAP2B variant (c.1006 G>A) in six patients suffering from tetralogy of Fallot (one patient), persistent truncus arteriosus (two patients) and patent ductus arteriosus (three patients) was found. There were no reported NKX2.5 mutations except for several single nucleotide polymorphisms in the patients. CONCLUSION These results suggest that genomic GATA4 and TFAP2B missense mutations may be associated with nonfamilial congenital heart disease with diverse clinical phenotypes in patients with congenital heart disease from southern China. They also revealed that the variation of the NKX2.5 gene may not be a risk factor for sporadic patients with congenital heart disease in this population.

Changes in hematological parameters in α-thalassemia individuals co-inherited with erythroid Krüppel-like factor mutations

Phenotypic variations in α‐thalassemia mainly depend on the defective α‐globin gene number. Genetic modifiers of the phenotype of Hemoglobin H (HbH) disease were poorly reported, apart from β‐thalassemia allele that was identified ameliorating the severity of α‐thalassemia. Because erythroid Krüppel‐like factor (KLF1) mutations can modulate the red blood phenotype, we evaluated its effect on the α‐thalassemia phenotype. Overall, we identified 72 subjects with five different KLF1 heterozygous mutations in 1468 individuals, including 65 out of 432 α‐thalassemia carriers with fetal hemoglobin (HbF) levels ≥1%, 0 out of 310 carriers with HbF levels <1% and 7 out of 726 HbH disease patients. We firstly established the link between KLF1 mutations and relatively elevated hemoglobin A2 (HbA2) and HbF levels, along with lower mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) values in a group of α‐thalassemia carriers. However, we concluded that KLF1 mutations were not significantly linked to HbH disease severity. On the basis of HBA or HBB genotype and gender, clinical severity of patients with HbH disease was correctly predicted in 73.3% cases. It may improve the screening and diagnostic assessment of α‐thalassemia.

Analysis of α-hemoglobin-stabilizing protein (AHSP) gene as a genetic modifier to the phenotype of β-thalassemia in Southern China

alpha-Hemoglobin-stabilizing protein (AHSP) is a molecular chaperon binding specifically to free alpha-globin. It is considered to be a potential modifier of beta-thalassemia. In order to find out if AHSP affects the phenotype of beta-thalassemia carriers in southern China, we analyzed AHSP gene in 365 beta-thalassemia subjects which was identified in 5789 consecutive blood samples from southern China. 8 SNPs were detected including two rare SNPs which were reported by us for the first time and two novel missense mutations. One missense mutation, A to T transversion at gene position 12750, substituting aspartic acid for valine at amino acid position 29 (AHSP D29V), was detected in three beta-thalassemia carriers respectively. The other AHSP missense mutation, 12831 A>T, which substitutes valine for glycine at amino acid position 56 (AHSP V56G), was identified in only one sample. Neither of the two missense mutations leads to obvious phenotypic change to the beta-thalassemia carries. A genetic association study between AHSP gene and the phenotype of beta-thalassemia subjects was conducted simultaneously. No significant association has been found between specific AHSP alleles or haplotypes and the disease severity of beta-thalassemia. Our study suggested that AHSP is not a significant genetic modifier of beta-thalassemia in southern China.

Compound heterozygosity for KLF1 mutations is associated with microcytic hypochromic anemia and increased fetal hemoglobin.

Krüppel-like factor 1 (KLF1) regulates erythroid lineage commitment, globin switching, and the terminal maturation of red blood cells. Variants in human KLF1 have been identified as an important causative factor in a wide spectrum of phenotypes. This study investigated two unrelated male children in China who had refractory anemia associated with poikilocythemia. These were accompanied by an upregulation of biochemical markers of hemolysis, along with abnormal hemoglobin (Hb) level and elevated reticulocyte counts. Next-generation sequencing revealed that the patients were compound heterozygotes for a KLF1 frameshift mutation c.525_526insCGGCGCC (p.(Gly176ArgfsTer179)) and one of two missense variants, c.892 G>C (p.(Ala298Pro)) and c.1012C>T (p.(Pro338Ser)). The subjects had microcytic hypochromic anemia, and their healthy parents had single mutation. The two missense mutations affected a highly conserved codon in the zinc finger DNA-binding domain of KLF1, but the protein stability was unaffected in K-562 cells. A KLF1-targeted promoter–reporter assay showed that the two mutations reduce the expression of the HBB, BCL11A, and CD44 genes involved in erythropoiesis, with consequent dyserythropoiesis and an α/non-α chain imbalance. A systematic analysis was performed of the phenotypes associated with the KLF1 mutations in the two families, and the clinical characteristics and differential diagnoses of the disease are presented. This is the first report of an autosomal recessive anemia presenting with microcytic hypochromia, abnormal Hb profile, and other distinctive erythrocyte phenotypes, and provides insight into the multiple roles of KLF1 during erythropoiesis.

Molecular characterization and clinical presentation of HKαα and anti‐HKαα alleles in southern Chinese subjects

The HKαα allele is a rearrangement occurring in the α‐globin gene cluster containing both the ‐α3.7 and αααanti4.2 unequal crossover junctions. The anti‐HKαα allele is the reciprocal product containing both the ‐α4.2 and αααanti3.7 unequal crossover junctions, which had been predicted but had not been detected previously. The phenotypic feature and population frequency of these two unusual alleles were not described. We report the identification of nine individuals carrying the HKαα allele and two individuals carrying the anti‐HKαα allele in southern China and describe their phenotype and haplotype data. The molecular structures of HKαα allele and anti‐HKαα allele were confirmed by two‐round nested polymerase chain reaction assay. The mechanism of origin of both alleles is related to probably simultaneous double crossover. Heterozygotes of HKαα or anti‐HKαα allele show a normal hematological phenotype. Finally, we report the carrier rates of these both alleles in the Guangxi Zhuang Autonomous Region of southern China, namely, ∼0.07% for the HKαα allele and ∼0.02% for the anti‐HKαα allele.

Hemoglobin H disease due to a de novo mutation at the α2-globin gene and an inherited common α-thalassemia deletion found in a Chinese boy

Hemoglobin (Hb) H disease is a moderate form of α-thalassemia resulting from various genetic defects. A novel frameshift mutation cd 43/44(-C) at the α2-globin gene was identified in a Chinese boy with hemoglobin H disease by sequencing. The probands mother carries a common α-thalassemia deletion while his father was normal both in the hematological phenotype and α-globin genotype, which suggested that it occurred as a de novo mutation. Molecular studies revealed a compound heterozygote for the Southeast Asian α-thalassemia deletion and this novel spontaneous mutation (-/α(T)α) and the patient exhibited the clinical manifestation of classic hemoglobin H disease. Based on the results of excluding the possibility of a somatic mosaicism of a point mutation in the α2-globin gene, we progress that this de novo single-base deletion should have arisen during the spermatogenic process or earlier embryonic stage. The present study provides information in determining a supplementary model of inheritance for α-thalassemia, which should be useful in genetic counseling.

Mutation in SSUH2 Causes Autosomal Dominant Dentin Dysplasia Type I

Dentin dysplasia type I (DDI) is an autosomal‐dominant genetic disorder resulting from dentin defects. The molecular basis of DDI remains unclear. DDI exhibits unique characteristics with phenotypes featuring obliteration of pulp chambers and diminutive root, thus providing a useful model for understanding the genetics of tooth formation. Using a large Chinese family with 14 DDI patients, we mapped the gene locus responsible for DDI to 3p26.1–3p24.3 and further identified a missense mutation, c.353C>A (p.P118Q) in the SSUH2 gene on 3p26.1, which co‐segregated with DDI. We showed that SSUH2 (p.P118Q) perturbed the structure and significantly reduced levels of mutant (MT) protein and mRNA compared with wild‐type SSUH2. Furthermore, MT P141Q knock‐in mice (+/− and −/−) had a unique partial obliteration of the pulp cavity and upregulation or downregulation of six major genes involved in odontogenesis: Dspp, Dmp1, Runx2, Pax9, Bmp2, and Dlx2. The phenotype of missing teeth was determined in zebrafish with morpholino gene knockdowns and rescued by injection of normal human mRNA. Taken together, our observations demonstrate that SSUH2 disrupts dental formation and that this novel gene, together with other odontogenesis genes, is involved in tooth development.