DNAase I Hypersensitive Site 3′ to the β-Globin Gene Cluster Containing Two TAA Insertions and a G→A Polymorphism is Predominantly Associated with the β+-Thalassemia IVS-I-6 (T→C) Mutation

Analysis of DNA polymorphic sites is an important tool for the detection of gene flow in human evolutionary studies and to study the genetic background for gene mutations. The β-globin locus contains several single-base restriction fragment length polymorphism (RFLP) sites throughout chromosome 11. In addition to these polymorphic sequence repeats, others are being studied in order to expand our knowledge concerning the role between haplotype-genotype and phenotype associations. Far downstream of the expressed β-globin genes, there is a hypersensitive site (HS) whose function remains obscure. We sequenced this region in 27 thalassemia patients and found a new pattern in the micro-satellite-like AT-rich region of this site: a new TAA insertion in addition to the one previously described in sickle cell patients with a concomitant polymorphism (G→A). This new variation was found to be linked to the IVS-I-6 (T→C) mutation. This polymorphism may be useful for studies concerning genotype and phenotype associations.     

Differential Segregation of β+ IVS‐I‐110 (G→A), Aγ − 117 (G→A), and Gγ − 158 (C→T) Mutations in Members of an Albanian Family

Hb Bab-Saadoun which has a Leu→Pro substitution at position 48 of the β chain was detected in a young Arabian boy living in Tunisia. His parents did not have the variant which suggests that it occurred as a spontaneous mutation. The substitution is located in the interhelical CD segment; leucine at β48 is an invariable amino acid that may be important as part of a spacer sequence between the two helices and its replacement by proline may affect the stability of the hemoglobin molecule. Hb Bab-Saadoun is unstable in heat and isopropanol stability tests and its chain was best isolated by parachloromercuribenzoate precipitation. It appears unlikely that the presence of Hb Bab-Saadoun results in a hemolytic anemia.     

Role of polymorphic sequences 5′ to the Gγ gene and 5′ to the β gene on the homozygous β thalassemic phenotype

Sixty‐seven homozygous male and female thalassemic patients with different phenotypes, aged between 8 and 33 years, were divided into three groups, according to the severity of their β‐thalassemia (thal) mutations. We investigated whether some co‐inherited genetic factors could influence the phenotype. Patients with milder β‐thal defects, homozygotes or compound heterozygotes for the IVS‐I‐6 (T→C) or ?87 (C→G) mutations had a milder disease. In addition, determination of the co‐inheritance of the ?158 (C→T) ^Gγ polymorphism and the (AT)₉T₅ repeat motif in the region ?540 to ?525, 5′ to the β‐globin gene, showed that in some patients with severe or mild/severe β‐thal mutations, linked to haplotype III, there was higher Hb F expression. We conclude that in homozygous β‐thal patients, the severity of the mutations is the most important factor influencing the phenotype, but some polymorphisms such as the ?158 (C→T) ^Gγ and (AT)₉T₅ repeat motif, increasing the Hb F expression and ameliorate the clinical course of the disease.     

Influence of Xmn 1Gγ (HBG2 c.-211 C → T) Globin Gene Polymorphism on Phenotype of Thalassemia Patients of North India

Beta (β) thalassemia is the most common single gene disorder in India. It has been reported that in patients with β-thalassemia in the presence of Xmn 1^Gγ polymorphic site the level of fetal hemoglobin (HbF) is increased thereby reducing the severity of disease. To determine the prevalence of Xmn 1^Gγ polymorphic site and its effect on the clinical phenotype and HbF level in 39 β-thalassemia major and 62 thalassemia intermedia patients, along with response to hydroxyurea therapy in thalassemia intermedia cases. Status of Xmn 1^Gγ polymorphism was determined by polymerase chain reaction-restricted fragment length polymorphism procedure. The HbF level was determined using high performance liquid chromatography. Genotypes and allele frequencies of the Xmn 1^Gγ polymorphism did not vary significantly between the various thalassemia groups. HbF levels were observed to be significantly increased and age at presentation was significantly greater in presence of Xmn 1^Gγ polymorphic site on both alleles as compared to its absence in thalassemia major but not in thalassemia intermedia cases. The response of hydroxyurea in thalassemia intermedia was found only in a few patients irrespective of their Xmn 1^Gγ status. Xmn 1^Gγ polymorphisms appear to significantly influence HbF levels and age at presentation in thalassemia major but not in thalassemia intermedia patients. Small numbers precluded a definitive correlation of the polymorphism with response to hydroxyurea therapy.     

Association of matrix Gla protein gene allelic polymorphisms (G?7→A,T?138→C and Thr83→Ala) with acute coronary syndrome in the Ukrainian population

BACKGROUND:

Several allelic variants of matrix γ-carboxyglutamic acid protein (MGP) can differentially affect the development of certain forms of ischemic heart disease depending on specific characteristics of each population.

OBJECTIVE:

To study the distribution of allelic variants of MGP promoter T⁻¹³⁸→C (rs1800802) and G⁻⁷→A (rs1800801), and Thr₈₃→Ala exon 4 (rs4236) polymorphisms in a Ukrainian population of patients with acute coronary syndrome (ACS).

METHODS:

Polymerase chain reaction and restriction fragment length polymorphism (RFLP) analysis were used to detect the above-mentioned variants of the MGP gene in 115 patients with ACS and in 140 essentially healthy individuals.

RESULTS:

The distribution of homozygous carriers of a major allelic variant, and heterozygous and homozygous minor allele variants of the T⁻¹³⁸→C MGP promoter polymorphism in patients with ACS were 59.8%, 32.7% and 7.5%, respectively. The corresponding distributions of variants in the control group were 54.0%, 41.0% and 5.0%, respectively (P>0.05 [χ² test]). With respect to the G⁻⁷→A polymorphism, the respective distributions were 42.1%, 45.6% and 12.3%, compared with 50.7%, 45.0% and 4.3% in the control group, respectively (P<0.05). Finally, the respective distributions according to the Thr₈₃→Ala exon 4 polymorphism were 42.6%, 43.5% and 13.9%, respectively, compared with 45.3%, 43.0% and 11.7% in the control group. Using logistic regression analysis, it was estimated that the A/A genotype (G⁻⁷→A polymorphism) was significantly (P=0.02) associated with ACS (OR 4.302 [95% CI 1.262 to 14.673]).

CONCLUSION:

The allelic A/A promoter variant of MGP G⁻⁷→A polymorphism can be considered a risk factor for ACS in the Ukrainian population.     

Developmental Effect of the XmnI Site on Gγ-Globin Gene Expression Among Newborn Hb F-Malta-I [Gγ117(G19)His→Arg,CAT→CGT] Heterozygotes and Adult β+-Thalassemia Homozygotes

Hb F-Malta-I [^Gγ117(19)His→Arg, CAT→CGT] is a stable and benign variant of Hb F found in 1.8% of Maltese newborn. We studied 120 Hb F-Malta-I heterozygotes and four Hb F‐Malta-I homozygotes. The mean proportion of ^Gγ-F-Malta-I in Hb F was 0.26 ± 0.03 for the Hb F-Malta-I heterozygotes and 0.58 ± 0.06 for the Hb F-Malta-I homozygotes. The Hb F-Malta-I allele was shown to occur on a background of the common Mediterranean haplotype Va [+ + ? ? ? ? ? + + ?]. Furthermore, the common Mediterranean haplotypes Va, IIIb [? + + + ? + + + + ?], I [+ + ? ? ? ? ? + + +] and II [? + ? + + ? + + + +] accounted for most (66.2%) of the wild-type alleles among the tested Hb F-Malta-I heterozygotes.

Different genotypes at the 5′ ? HincII, ^Gγ and ^Aγ HindIII, and 3′ψβ HincII sites (but not at the 5′ ^Gγ XmnI site) were found to be linked to significant variations in the proportion of ^Gγ-F-Malta-I and ^Gγ-globins in the Hb F of newborn Hb F-Malta-I heterozygotes. Moreover, the 5′ ^Gγ XmnI site was found to be associated with variations in Hb F and ^Gγ-globin levels in a population of adult Maltese β-thalassemia (thal) homozygotes. This implies that a determinant linked to the XmnI site which effects ^Gγ-globin gene expression is active in anemic adults but not in normal infants.     

The Mutation of Hb Turriff [α99(G6)Lys→Glu (AAG→GAG)] Is Carried by the α1‐Globin Gene in a Japanese (Hb Turriff‐I)

The forms and severity of cardiac complications were investigated in patients with asymptomatic thalassemia intermedia and thalassemia major by M‐mode, bi‐dimensional echocardiography (ECHO) and echo‐Doppler. Twenty‐eight patients of both sexes with β‐thalassemia intermedia (β‐TI), mean age 23.2 ± 6.3 years, untransfused or minimally transfused, were compared to 42 age‐ and sex‐matched subjects with thalassemia major, who were regularly treated with hemotransfusive therapy [pre‐transfusion hemoglobin (Hb) values 9.5 ± 0.9 g/dL] and iron chelation. All patients were splenectomized. Age and sex matched healthy control subjects were randomly selected. β‐Thalassemia major (β‐TM) patients showed a marked reduction in contractile state and a milder left ventricular (LV) enlargement than β‐TI patients. Cardiac output (CO) and cardiac index (CI) were increased in both groups of patients but appeared significantly higher in β‐TI patients with consequent altered LV diastolic function indices. In addition, β‐TI patients had reduced indices of pulmonary artery flow related to long‐term chronic anemia rather than iron overload. The progressive rise in CO and CI casts doubts on the current management of β‐TI syndromes.     

The role of G protein β3 subunit polymorphisms C825T, C1429T, and G5177A in Turkish subjects with essential hypertension

Hypertension is a multifactorial disorder that constitutes a major risk factor for the cardiovascular system. Heterotrimeric G-proteins, which couple receptors for diverse extracellular enzymes or ion channels, are correlated with disease mechanisms. Several studies have demonstrated an association between G protein polymorphisms and essential hypertension in some populations, although contradictive results also exist. In this study, we have investigated the potential role of the C825T, C1429T, and G5177A polymorphisms of the β3 subunit of G-proteins in essential hypertension in a group of Turkish subjects. Genomic DNA from 106 normotensive individuals (117.4 ± 13.1, 75.2 ± 10.5; systolic blood pressure (SBP) and diastolic blood pressure (DBP) levels, respectively) and 101 hypertensive subjects (152.3 ± 18.0, 92.5 ± 11.6; SBP and DBP levels, respectively) were studied by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and direct sequencing methods for these polymorphisms. Allele frequencies of the polymorphisms were consistent with Hardy Weinberg equilibrium, except for the C825T polymorphism (χ(2) = 7.8). The frequencies of the 825T and 1429T variants were higher in hypertensive subjects compared to those of controls. Differences between hypertensives and controls were not statistically significant, though difference was very close to significance for C825T (p = 0.056 and 0.099 for 825T and 1429T, respectively). T allele frequency in overall population showed significant association with hypertension for C825T (0.0134). The prevalence of the 5177A-variant was very low and all subjects carrying it were heterozygotes in both groups.     

Correlation of thymidylate synthase,thymidine phosphorylase and dihydropyrimidine dehydrogenase with sensitivity of gastrointestinal cancer cells to 5-fluorouracil and 5-fluoro-2′-deoxyuridine

AIM:To determine the expression levels of three metabolic enzymes of fluoropyrimidines:thymidylate synthase (TS),thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) in seven human gastrointestinal cancer cell lines,and to compare the enzyme levels with the sensitivity to 5-fluorouracil (5-FU) and 5-fluoro-2′-deoxyuridine (FdUrd).METHODS:TS,TP and DPD mRNA levels were assessed by semi-quantitative RT-PCR,TP and DPD protein contents were measured by ELISA. Fifty percent inhibitory concentrations of growth (IC50),representing the sensitivity to drugs,were determined by MTT assay.RESULTS:IC50 values ranged from 1.28 to 12.26μM for 5-FU,and from 5.02 to 24.21μM for FdUrd,respectively.Cell lines with lower DPD mRNA and protein levels tended to be more sensitive to 5-FU (P&lt;0.05), but neither TS nor TP correlated with 5-FU IC50 (P&gt;0.05).Only TS mRNA level was sharply related with FdUrd sensitivity (P&lt;0.05),but TP and DPD were not (P&gt;0.05).A correlation was found between mRNA and protein levels of DPD (P&lt;0.05),but not TP (P&lt;0.05).CONCLUSION:DPD and TS enzyme levels may be useful indicators in predicting the antitumor activity of 5-FU or FdUrd,respectively.     

Correlation of thymidylate synthase,thymidine phosphorylase and dihydropyrimidine dehydrogenase with sensitivity of gastrointestinal cancer cells to 5-fluorouracil and 5-fluoro-2′-deoxyuridine

AIM:To determine the expression levels of three metabolicenzymes of fluoropyrimidines:thymidylate synthase (TS),thymidine phosphorylase (TP) and dihydropyrimidinedehydrogenase (DPD) in seven human gastrointestinalcancer cell lines,and to compare the enzyme levels withthe sensitivity to 5-fluorouracil (5-FU) and 5-fluoro-2'-deoxyuridine (FdUrd).METHODS:TS,TP and DPD mRNA levels were assessedby semi-quantitative RT-PCR,TP and DPD protein contentswere measured by ELISA.Fifty percent inhibitoryconcentrations of growth (IC50),representing the sensitivityto drugs,were determined by MTT assay.RESULTS:IC50 values ranged from 1.28 to 12.26 uM for5-FU,and from 5.02 to 24.21 uM for FdUrd,respectively.Cell lines with lower DPD mRNA and protein levels tendedto be more sensitive to 5-FU (P<0.05),but neither IS norTP correlated with 5-FU IC50 (P>0.05).Only TS mRNA levelwas sharply related with FdUrd sensitivity (P<0.05),but TPand DPD were not (P>0.05).A correlation was foundbetween mRNA and protein levels of DPD (P<0.05),but notTP (P<0.05).CONCLUSION:DPD and TS enzyme levels may be usefulindicators in predicting the antitumor activity of 5-FU orFdUrd,respectively.     

Compound Heterozygosity for Two New Mutations in the β‐Globin Gene [Codon 9 (+ TA) and Polyadenylation Site (AATAAA→AAAAAA)] Leads to Thalassemia Intermedia in a Tunisian Patient

More than 200 mutations that are associated with β‐thalassemia (thal) have been found. In most cases, studies to detect a mutation in a patient is made easier because of the existence of geographical sets of mutations that allow the use of a dedicated mutation detection kit. We describe here a patient who originated from Tunisia, in whom we found two as yet unreported mutations, showing that even in a well‐studied population a full gene study might be needed to characterize mutation(s).     

Associations of the TNF-alpha -308 G/A, IL6 -174 G/C and AdipoQ 45 T/G polymorphisms with inflammatory and metabolic responses to lifestyle intervention in Brazilians at high cardiometabolic risk

Background

Obesity contributes to Type 2 diabetes by promoting systemic insulin resistance. Obesity causes features of metabolic dysfunction in the adipose tissue that may contribute to later impairments of insulin action in skeletal muscle and liver; these include reduced insulin-stimulated glucose transport, reduced expression of GLUT4, altered expression of adipokines, and adipocyte hypertrophy. Animal studies have shown that expansion of adipose tissue alone is not sufficient to cause systemic insulin resistance in the absence of adipose tissue metabolic dysfunction. To determine if this holds true for humans, we studied the relationship between insulin resistance and markers of adipose tissue dysfunction in non-obese individuals.

Method

32 non-obese first-degree relatives of Type 2 diabetic patients were recruited. Glucose tolerance was determined by an oral glucose tolerance test and insulin sensitivity was measured with the hyperinsulinaemic-euglycaemic clamp. Blood samples were collected and subcutaneous abdominal adipose tissue biopsies obtained for gene/protein expression and adipocyte cell size measurements.

Results

Our findings show that also in non-obese individuals low insulin sensitivity is associated with signs of adipose tissue metabolic dysfunction characterized by low expression of GLUT4, altered adipokine profile and enlarged adipocyte cell size. In this group, insulin sensitivity is positively correlated to GLUT4 mRNA (R?=?0.49, p?=?0.011) and protein (R?=?0.51, p?=?0.004) expression, as well as with circulating adiponectin levels (R?=?0.46, 0?=?0.009). In addition, insulin sensitivity is inversely correlated to circulating RBP4 (R?=??0.61, 0?=?0.003) and adipocyte cell size (R?=??0.40, p?=?0.022). Furthermore, these features are inter-correlated and also associated with other clinical features of the metabolic syndrome in the absence of obesity. No association could be found between the hypertrophy-associated adipocyte dysregulation and HIF-1alpha in this group of non-obese individuals.

Conclusions

In conclusion, these findings support the concept that it is not obesity per se, but rather metabolic dysfunction of adipose tissue that is associated with systemic insulin resistance and the metabolic syndrome.     

Gγ −37 (A→T): A New Nondeletional Hereditary Persistence of Fetal Hemoglobin Determinant Associated with the Rare Codon 91 (+T) δ0-Thalassemia

We recently described a rare frameshift mutation in the δ-globin gene in a Dutch patient, in association with a new mutation of the ^Gγ-globin gene promoter [^Gγ ?37 (A→T)] with a moderately elevated Hb F level of 2.3%. The δ mutation at codon 91 (+T) has been described once before in our laboratory in 1989, in a complex Belgian family with ^Gγ (^Aγδβ)⁰-thalassemia (thal) and moderately elevated Hb F levels, without the ^Gγ (^Aγδβ)⁰-thal deletion in some individuals. Analysis of the patients from 1989 revealed the presence of the same ^Gγ-globin gene mutation and moderately elevated Hb F in all patients, who were also carriers of the δ-globin gene frameshift. Further analysis demonstrated that the two mutations were in linkage with the same haplotype in both the Belgian family and the recently found patient, confirming the association of the elevated Hb F expression with the new ^Gγ-globin gene mutation.