Molecular genetic analyses of β-thalassemia in South India reveals rare mutations in the β-globin gene

-thalassemia is the most prevalent single-gene disorder. Since no viable forms of treatment are available, the best course is prevention through prenatal diagnosis. In the present study, the prevalence of -thalassemia was extensively investigated in the South Indian population, especially from the state of Andhra Pradesh. Screening for causal mutations was carried out on genomic DNA isolated from patient blood samples by using the routine reverse dot blot (RDB) and amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) techniques. DNA sequencing was performed wherever necessary. Among the nine mutations identified, four, including IVS-1-5 (G-C) (IVS1+5G>T), codon 41/42 (-TTCT) (c.124_127delTTCT), codon 15 (G-A) (c.47G>A), and HbS (sickle mutation) (c.20A>T) mutations, accounted for about 98% of the total positive cases. Two mutations viz. codon 8/9 (+G) (c.27_28insG) and HbE (codon 26 G-A) (c.79G>A) exhibited a very low frequency of occurrence, whereas the IVS-1-1 (G-T) (IVS1+1G>T) and the 619 bp deletion (c.366_494del) mutations were absent. We also identified certain rare mutations during the diagnostic evaluation. Gene sequencing confirmed the codon 30 (G-C) (c.92G>C) mutation and the rare codon 5 (-CT) (c.17_18delCT) and IVS-II-837 (T-G) (IVSII-14T>G) mutations. This is the first report of the IVS II 837 mutation in the Indian population. We also report a novel diagnostic application during RDB-based screening for the detection of the (c.92G>C) mutations. Such a comprehensive mutation screening is essential for prenatal diagnosis of -thalassemia and control of this highly prevalent monogenic disorder in the Indian population.     

Hemoglobin variants,hematological parameters and β-globin gene cluster haplotypes in an isolated Amerindian group from the Orinoco River Delta

Background: Several previous studies reported that the Venezuelan Warao Indians presented unusual genetic characteristics.

Aim: The present study checked previous reports of a high frequency of hereditary persistence of fetal hemoglobin (HPFH) and examined other hematological traits.

Subjects and methods: Standard hematology, electrophoresis on cellulose acetate, fetal hemoglobin alkali denaturation, γ-globin chain, DNA amplification and sequencing, and denaturing gradient gel electrophoresis determinations were performed in 269 individuals living in two localities of the Orinoco River Delta.

Results: Two β^s genes, in apparently non-related individuals, were found. HPFH, detected in this same population of Warao Indians 25 years ago, was present in heterozygous form in five individuals from a large kindred, with hemoglobin F levels ranging from 3.7% to 8%, and with a pancellular distribution. The HPFH mutation was of the deletional type. β-globin gene haplotypes were determined by direct counting (through family studies) in 150 chromosomes; 26% of the 150 examined cluster presented haplotype 2, 22% haplotype 6, and 13% a new, Warao haplotype. Haplotype 3, of probable African origin, was also found with a frequency of 5%.

Conclusions: The presence of the HPFH mutation was confirmed, and the new β-globin gene haplotype together with the presence of other rare variants indicates that the Warao are very distinctive in relation to other Native Americans. Evidence was also found of a slight admixture from Africa-derived subjects (Layrisse et al. 1988).     

Genetic polymorphism at codon 10 of the transforming growth factor-β1 gene in patients with alcoholic liver cirrhosis

Background/Aims

Transforming growth factor beta1 (TGF-β1) is a key cytokine in the production of extracellular matrix. A genetic polymorphism at codon 10 of the TGF-β1 gene is associated with liver fibrosis. We investigated the effect of genetic polymorphisms at codon 10 on the development of alcoholic liver cirrhosis (ALC).

Methods

In total, 119 controls and 182 patients with ALC, were enrolled in the study. Clinical and laboratory data including total lifetime alcohol intake were collected at enrollment. The genotype at codon 10 was determined for each patient by single-strand conformation polymorphism.

Results

There were three types of genetic polymorphism at codon 10: homozygous proline (P/P), heterozygous proline/leucine (P/L), and homozygous leucine (L/L). Among the controls, the proportions of P/P, P/L, and L/L were 26.1%, 44.5%, and 29.4%, respectively in the ALC group, these proportions were 23.1%, 43.4%, and 33.5%, respectively. The genotype distribution did not differ between the controls and the ALC group. In the ALC group, age, total lifetime alcohol intake, and distribution of Child-Pugh class did not differ with the genotype. Of the male patients with ALC (n=164), the proportions of P/P, P/L, and L/L were 20.1%, 44.5%, and 35.4%, respectively the genotype distribution did not differ between the male controls and the male ALC patients.

Conclusions

The genotype at codon 10 in TGF-β1 does not appear to influence the development of ALC. Further study is needed to investigate other genetic factors that influence the development of ALC in patients with chronic alcohol intake.     

Strong linkage disequilibrium of a HbE variant with the (AT)9(T)5 repeat in the BP1 binding site upstream of the β-globin gene in the Thai population

A binding site for the repressor protein BP1, which contains a tandem (AT)_x(T)_y repeat, is located approximately 530 bp 5 to the human -globin gene (HBB). There is accumulating evidence that BP1 binds to the (AT)₉(T)₅ allele more strongly than to other alleles, thereby reducing the expression of HBB. In this study, we investigated polymorphisms in the (AT)_x(T)_y repeat in 57 individuals living in Thailand, including three homozygotes for the hemoglobin E variant (HbE; 26Glu->Lys), 22 heterozygotes, and 32 normal homozygotes. We found that (AT)₉(T)₅ and (AT)₇(T)₇ alleles were predominant in the studied population and that the HbE variant is in strong linkage disequilibrium with the (AT)₉(T)₅ allele, which can explain why the ^E chain is inefficiently synthesized compared to the normal ^A chain. Moreover, the mildness of the HbE disease compared to other hemoglobinopathies in Thai may be due, in part, to the presence of the (AT)₉(T)₅ repeat on the HbE chromosome. In addition, a novel (AC)_n polymorphism adjacent to the (AT)_x(T)_y repeat (i.e., (AC)₃(AT)₇(T)₅) was found through the variation screening in this study.MIM and accession numbers and URLs for data presented herein are as follows: Online Mendelian Inheritance of Man (OMIM), (for HBB [MIM 141900]). GenBank, (accession number [NG_000007.2] reference sequence information).     

The C825T polymorphism in the G-protein β3 subunit gene and diabetic complications in IDDM patients

Complications of insulin-dependent diabetes mellitus (IDDM) are a major cause of morbidity and mortality; however, the mechanisms of their development are still to be elucidated. Genetic susceptibility contributes to the pathogenesis of nephropathy in IDDM. Enhanced G-protein activation, a cellular phenotype observed in cultured cells from patients with essential hypertension, was recently documented in IDDM subjects with nephropathy. A C825T polymorphism was recently described in GNB3, the gene encoding the beta 3 subunit of heterotrimeric G-proteins. This genetic variant has been associated with enhanced G-protein activation. The 825T allele was observed more frequently in a group with essential hypertension. We analyzed the role of the C825T polymorphism in the predisposition to diabetic complications in IDDM. In this study, we investigated the frequency of this polymorphism in a large case-control study and found no association of the 825T allele with diabetic nephropathy, retinopathy, and neuropathy. Received: July 14, 2000 / Accepted: January 4, 2001     

Regulatory sequences clustered at the 5′ end of the first intron of the human thymidylate synthase gene function in cooperation with the promoter region

A human thymidylate synthase (TS) minigene containing 5- and 3-flanking sequences, all the exons, and only intron 1 showed a normal frequency of stable transformation when transfected into TS-negative mutant cells, whereas minigenes in which intron 1 was replaced by intron 2 or deleted in the above construct showed only a few percent of the above frequency. Introduction of intron 1 into the above intronless or intron 2 minigene restored the transforming activities regardless of its position and orientation. Deletion analysis revealed two positive and one negative regulatory sequences in the 5 end of intron 1, each of which seemed to bind specific proteins as shown by gel shift analysis. Intron 1 also stimulated expression of a TS promoter-CAT gene construct but not that of an SV40 promoter-CAT gene construct. These results indicate that the multiple regulatory sequences clustered in intron 1 stimulate TS gene expression in concert with the 5-flanking sequences.     

Oxygen transport in blood at high altitude: role of the hemoglobin–oxygen affinity and impact of the phenomena related to hemoglobin allosterism and red cell function

Altitude hypoxia is a major challenge to the blood O₂ transport system, and adjustments of the blood–O₂ affinity might contribute significantly to hypoxia adaptation. In principle, lowering the blood–O₂ affinity is advantageous because it lowers the circulatory load required to assure adequate tissue oxygenation up to a threshold corresponding to about 5,000 m altitude, whereas at higher altitudes an increased blood–O₂ affinity appears more advantageous. However, the rather contradictory experimental evidence raises the question whether other factors superimpose on the apparent changes of the blood–O₂ affinity. The most important of these are as follows: (1) absolute temperature and temperature gradients within the body; (2) the intracapillary Bohr effect; (3) the red cell population heterogeneity in terms of O₂ affinity; (4) control of altitude alkalosis; (5) the possible role of hemoglobin as a carrier of the vasodilator nitric oxide; (6) the effect of varied red cell transit times through the capillaries.     

The important role for βVLDLs binding at the fourth cysteine of first ligand-binding domain in the low-density lipoprotein receptor

The low-density lipoprotein (LDL) receptor (LDLR) is a crucial role for binding and uptaking apolipoprotein (apo) B-containing lipoproteins, such as very-low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), and LDL. The defect function of the LDLR causes familial hypercholesterolemia (FH), the phenotype of which is elevated plasma cholesterol and premature coronary heart disease (CHD). In the present study, we characterize the role of the cysteine residue of the ligand-binding domain of the LDLR. The mutant LDLR protein of cysteine for serine at codon 25 (25S-LDLR) was expressed in Chinese hamster ovary (CHO) cell line, ldl-A7. By Western blot analysis, the 25S-LDLR was detected with monoclonal antibody IgG-12D10, which reacts with the linker site of the LDLR but not with IgG-C7, which reacts with the NH₂ terminus of the receptor. The 25S-LDLR bound LDL similarly to the wild-type LDLR, but the rate of uptake of LDL by the mutant receptor was only about half of that by the wild-type receptor. In contrast, the 25S-LDLR bound and internalized VLDL more avidly than LDL. These results suggest that the fourth cysteine residue of the first ligand-binding domain of the LDLR might be important for the internalization of atherogenic lipoproteins by vascular cells despite reduced LDL uptake, leading to atherosclerosis and premature cardiovascular disease.     

Association of promoter region single nucleotide polymorphisms at positions ?819C/T and ?592C/A of interleukin 10 gene with ischemic heart disease

Objective

Ischemic heart disease (IHD) is a disease characterized by ischemia of the heart muscle, usually due to coronary artery disease. Interleukin-10 (IL10) is a proinflammatory cytokine known to protect endothelial function. In this study, we investigated the association of promoter region polymorphisms of the IL10 gene with IHD.

Methods

We recruited 313 control and 173 IHD patients. The selected SNPs in IL10 were genotyped using pyrosequencing.

Results

SNPs at positions ?592C/A and ?819C/T were statistically associated with IHD (P?=?0.014 and P?=?0.037). Similarly, the mean value of C-reactive protein in the C allele at ?592C/A and ?819C/T was significantly higher than that in the A allele at ?592C/A (P?=?0.026) and T allele at ?819C/T (P?=?0.026). The presence of hypertension in the C allele at ?592C/A and ?819C/T was significantly more frequent than that in the A allele at ?592C/A (P?=?0.044) and T allele at ?819C/T (P?=?0.044). In the haplotype of two SNPs (?592C/A and ?819C/T), one haplotype (CC) presented an association with IHD (P?=?0.012).

Conclusions

These results indicate that the C allele with SNPs at position ?592C/A and ?819C/T of IL10 gene may be associated with IHD in the Korean population.     

Decreased frequency of the 3′UTR T>G single nucleotide polymorphism of thrombospondin-2 gene in sudden death due to plaque erosion

ObjectiveClinical studies have suggested a potentially “protective” variant of the thrombospondin gene 2 (THBS2, MIM*188061). Autopsy studies investigating the frequency of THBS2 polymorphisms in different coronary substrates have not been done.MethodsWe evaluated the frequency of the T>G THBS2 (SNP ID G5755e5) polymorphism in 439 cases of sudden unexpected death, including acute thrombosis occurring on plaque erosion (n=60), acute thrombosis occurring on plaque rupture (n=54), severe coronary artery disease without acute thrombus (n=76), and 249 sudden unexpected deaths without significant coronary disease. Allele types were determined by polymerase chain reaction of DNA extracted from autopsy frozen tissues.ResultsIn controls, there were 60.2% wild-type, 35.3 % TG, and 4.4% GG phenotypes. The frequency of G allele carriers (TG or GG) was 23.3% in erosions vs. 39.8% in controls (P=.02). There was no difference in controls vs. plaque ruptures (40.7%) or severe CAD without thrombus (38.2%). By multivariate analysis, lack of G allele (wild-type TT genotype) was associated with plaque erosion [odds ratio (OR) 2.2, P=.02], independent of age, gender, and cigarette smoking. When combined with a history of cigarette smoking, the association between TT genotype and plaque erosion was greater (OR 3.5, P≤.0001).ConclusionThe T>G THBS2 is decreased only in plaque erosion, with no difference in frequency between other coronary disease and controls.     

Amino-acid alterations in the β-tubilin gene of Neurospora crassa that confer resistance to carbendazim and diethofencarb

We have previously shown that increased sensitivity to diethofencarb in the carbendazim(MBC)-resistant F914 strain of Neurospora crassa is caused by a single amino-acid change in -tubulin, ¹⁹⁸Glu to Gly. Three diethofencarb-resistant mutants that are also resistant to MBC were isolated from strain F914. They contained single base-pair-substitution mutations in the -tubulin gene. The amino acid changes in -tubulin, Phe from ²⁵⁰Leu, Val from ¹⁶⁵Ala, and Ala from ²³⁷Thr, were responsible for diethofencarb-resistance in the mutant strains FR511, FR513, and FR421, respectively. The amino acid at position 198 of -tubulin in these mutants was Gly, which is the same as in strain F914. -tubulin genes with ¹⁹⁸Glu were constructed by site-directed mutagenesis. The altered -tubulin genes derived from FR511 and FR421 transformed the wild-type strain to resistance to MBC, indicating that ²⁵⁰Phe and ²³⁷Ala in -tubulin are responsible for resistance not only to diethofencarb but also to MBC.     

Relationship between single nucleotide polymorphisms in the 3’-untranslated region of the vascular endothelial growth factor gene and susceptibility to diabetic peripheral neuropathy in China

Introduction

This study is to elucidate the relationship between a 936C/T mutation at the 3’-untranslated region of the human vascular endothelial growth factor (VEGF) gene and diabetic peripheral neuropathy (DPN).

Material and methods

All subjects recruited in this study were divided into DM (diabetes without neuropathy, retinopathy or nephropathy), DPN (diabetes with peripheral neuropathy only) and healthy control groups. The gene polymorphism was determined by polymerase chain reaction-restriction fragment length polymorphism, as well as other clinical methods including serum VEGF by ELISA.

Results

The C allele frequency and CC genotype frequency in the DPN group were higher than those in the NC group and DM group. The T allele frequency and CT+TT genotype (carrying the T allele) frequency in the DPN group were lower than those in the NC group (χ² = 19.051 and 18.533, both p < 0.001) and DM group (χ² = 11.117 and 11.156, both p = 0.001). However, there was no statistically significant difference in the three genotype (CC/CT+TT) frequencies and allele (C/T) frequencies between the DM group and the NC group. The multivariate logistic regression analysis showed that the levels of glycated hemoglobin (HbA_1c), total cholesterol (TC), low density lipoprotein cholesterol (LDL-C) and plasma VEGF positively correlated with DPN, while the 936C/T gene polymorphism of VEGF negatively correlated with DPN.

Conclusions

Allele 936C of VEGF may serve as a genetic marker susceptible to DPN, while allele 936T may be a protective genetic marker of DPN.     

Density heterogeneities of hepatitis C virus in human sera due to the binding of β-lipoproteins and immunoglobulins

Heterogeneities in the density of hepatitis C virus RNA-carrying material (HCV-RNA-CM) found in human sera (1.03–1.20 g/cm³) are attributed to the binding of low-density lipoproteins and/or of IgG. In some sera HCV-RNA-CM seems to be nearly totally bound to -lipoproteins and cannot be precipitated by anti-IgG (); in others more than 95% of HCV-RNA-CM is bound to IgG and cannot be precipitated by anti--lipoprotein. Furthermore, there are sera from which HCV-RNA-CM can be completely be precipitated by either anti--lipoprotein or anti-IgG (), pointing to a binding of the two serum proteins to the same HCV-RNA-CM. There are other sera from which HCV-RNA-CM can be partially precipitated by the one or the other antiserum, leaving behind fractions, which are bound to -lipoprotein or to IgG. HCV-RNA-CM cannot be precipitated from some sera either by anti--lipoprotein or by anti-IgG ().     

Distribution of C→T and T→C polymorphisms of the urokinase-type plasminogen activator gene in children with type 1 diabetes mellitus and insulin resistance

Abstract We analysed the distribution of genotypes and frequency of alleles of two polymorphisms in the urokinase-type plasminogen activator (uPA) gene: a CT substitution in exon 6 and a TC substitution in intron 7 in 89 children with type 1 diabetes mellitus and insulin resistance compared with 120 non-diabetic control subjects. All genotypes were determined by the allele-specific polymerase chain reaction. We found that the frequency of the T/T homozygote (15%) in the patient group was significantly (P<0.05) higher than in the controls (7%). There were no differences in the distribution of the TC polymorphism between patients and controls, which suggests that this genetic change is probably phenotypically silent. In conclusion, our results indicate that the higher percentage of T/T homozygotes in patients might be associated with T1DM coexisting with insulin resistance.     

Do the mutations of C1GALT1C1 gene play important roles in the genetic susceptibility to Chinese IgA nephropathy?

Background  

The deficiency of β1,3 galactose in hinge region of IgA1 molecule played a pivotal role in pathogenesis of IgA nephropathy (IgAN). Cosmc, encoded by C1GALT1C1 gene, was indispensable to β1,3 galactosylation of IgA1. We designed a serial study to investigate the relationship between the mutations of C1GALT1C1 gene and the genetic susceptibility to IgAN.     

Association of the C–509→T polymorphism, alone or in combination with the T869→C polymorphism, of the transforming growth factor-β1 gene with bone mineral density and genetic susceptibility to osteoporosis in Japanese women

Transforming growth factor-beta1 is an important local regulator of bone metabolism, acting downstream of estrogen and cooperatively with vitamin D. The possible association of a C 509-->T polymorphism in the promoter region of the transforming growth factor-beta1 gene, alone or in combination with a T869-->C (Leu10-->Pro) polymorphism, with bone mineral density and genetic susceptibility to osteoporosis was investigated in 625 postmenopausal Japanese women. The frequencies of the CC, CT, and TT genotypes of the C-509-->T polymorphism in the study population were 24%, 49%, and 27%, respectively. A significant association of C-509-->T genotype with bone mineral density was detected: lumbar spine (L2-L4) and total body bone mineral density values were 7% and 5% lower, respectively, in individuals with the TT genotype than in those with the CT or CC genotype. The serum concentration of transforming growth factor-beta1 did not vary with C-509-->T genotype. Multivariable logistic regression analysis, with adjustment for age, height, body weight, time since menopause, smoking status, body fat mass, and lean mass, revealed a significantly higher frequency of the TT genotype of the C-509-->T polymorphism in 286 individuals with osteoporosis than in 170 normal controls. Analysis of combined C-509-->T and T869-->C genotypes showed that L2-L4 bone mineral density decreases and the prevalence of osteoporosis increases with the number of T alleles. These results suggest that the C-509-->T polymorphism, alone or in combination with the T869-->C polymorphism, of the transforming growth factor-beta1 gene is a genetic determinant of bone mass, and that the number of T alleles in the combined genotype is a risk factor for the genetic susceptibility to osteoporosis in postmenopausal Japanese women.