Fetal hemoglobin in normal adults and β-thalassemia heterozygotes

Summary A recently developed high performance liquid chromatographic (HPLC) procedure using a weak cation exchanger (PolyCAT) in columns of different sizes was used to quantify fetal hemoglobin (Hb F) in blood of normal adults and -thalassemia (-thal) heterozygotes with ten different types of mutations. Preparative PolyCATHPLC greatly facilitated the characterization of isolated Hb F, i.e., the determination of the relative quantities of the ^G and ^A chains. The method is accurate and allows quantitation of Hb F at the 0.5% level; preparative PolyCAT-HPLC allows isolation of (nearly) pure Hb F from blood samples with low (< 1%) Hb F. Adult Hb F levels were determined in 69 normal adults (including 24 diabetics); Hb F levels fell below 1% except for subjects with abnormal -^G-^G arrangement and a CT mutation at position -158 relative to the Cap site of both ^G genes. The effect of the same mutation in the normal-^G-^A-arrangement was variable. Certain -thal mutations (namely, those at positions -29;-88; IVS-I-1; IVS-II-1) were associated with high Hb F levels in heterozygotes, while those at nucleotide (nt) positions IVS-I-6; IVS-I-110; codon 24; codon 39; codons 41/42; IVS-II-745 were not. ^G values varied and often fell into two groups (high ^G and low ^G); high ^G values were not associated with high Hb F values. The chromatographic procedure is ideally suited for Hb A₂ quantitation. Average values of Hb A₂ in -thal heterozygotes with any one of nine of the ten mutations were twice that of normals; the one exception was the -thal heterozygote with the IVS-I-6 (TC) mutation with an average low Hb A₂ value of 3.6%.     

The molecular basis of β-thalassemia in Turkey

Summary By using oligonucleotide hybridization, restriction endonuclease analysis and direct sequencing of amplified genomic DNA, we have been able to characterize 18 different mutations in the -globin genes of 161 thalassemia homozygotes and 107 -thalassemia heterozygotes from Turkey (429 -thalassemia chromosomes). Previous studies dealing with -thalassemia in Mediterranean countries have shown that, in most Mediterranean populations, only a few mutations are prevalent. In contrast, -thalassemia in Turkey does not seem to be associated with a few predominant mutations. The six most frequent alleles, IVS-I-110 (GA), IVS-I-6(TC), FSC-8 (-AA), IVS-I-1(GA), -30(TA) and FSC-5 (CT), account for only 69.3% of the disease genes; indeed, all 26 mutations assayed represent 85.8% of the disease genes, confirming the considerable molecular heterogeneity of -thalassemia among Turks, and indicating the possible presence of rare, previously undefined, mutations in the population. Two mutations observed in this study, IVS-I-116 (TG) and Cd44(-C), have not been reported in the Turkish population to date. Since preventive medical services, such as genetic counseling and prenatal diagnosis, are greatly improved by detailed knowledge of the molecular pathology of thalassemia, we strongly believe that the presented data will facilitate the intended establishment of a prenatal diagnosis center, based on DNA analysis, in Turkey.     

The spectrum of β-thalassemia mutations in northern and northeastern Thailand

Summary A total of 123 -thalassemia genes from northern (n = 113) and northeastern (n = 10) Thailand were examined. Using five oligonucleotide probes, the mutation in 108 genes (88%) was identified: 50 nonsense 17, 49 frameshift 41-42, 4-28(AG), 2IVS1 nt5(GC), 2IVS2 nt654, and 1 deletion removing the entire -globin gene. The nonsense 17 mutation (n = 39) was linked to a single haplotype, whereas the frameshift 41-42 mutation occurred with several haplotypes. The results of the present study indicate that prenatal diagnosis of clinically important -thalassemia syndromes using a limited set of oligonucleotides is feasible in approximately 80% of affected families in northern Thailand and most of the families with -thalassemia-Hb E disease in northeastern Thailand.     

Aerobic degradation of lindane (γ-hexachlorocyclohexane) in bacteria and its biochemical and molecular basis

γ-Hexachlorocyclohexane (γ-HCH, also called γ-BHC and lindane) is a halogenated organic insecticide that causes serious environmental problems. The aerobic degradation pathway of γ-HCH was extensively revealed in bacterial strain Sphingobium japonicum (formerly Sphingomonas paucimobilis) UT26. γ-HCH is transformed to 2,5-dichlorohydroquinone through sequential reactions catalyzed by LinA, LinB, and LinC, and then 2,5-dichlorohydroquinone is further metabolized by LinD, LinE, LinF, LinGH, and LinJ to succinyl-CoA and acetyl-CoA, which are metabolized in the citrate/tricarboxylic acid cycle. In addition to these catalytic enzymes, a putative ABC-type transporter system encoded by linKLMN is also essential for the γ-HCH utilization in UT26. Preliminary examination of the complete genome sequence of UT26 clearly demonstrated that lin genes for the γ-HCH utilization are dispersed on three large circular replicons with sizes of 3.5 Mb, 682 kb, and 191 kb. Nearly identical lin genes were also found in other HCH-degrading bacterial strains, and it has been suggested that the distribution of lin genes is mainly mediated by insertion sequence IS6100 and plasmids. Recently, it was revealed that two dehalogenases, LinA and LinB, have variants with small number of amino acid differences, and they showed dramatic functional differences for the degradation of HCH isomers, indicating these enzymes are still evolving at high speed.     

Molecular basis of β-thalassemia in Thailand: analysis of β-thalassemia mutations using the polymerase chain reaction

Summary -Thalassemia mutations in 71 chromosomes of Thai patients from the northeast, the middle and the south of the country were investigated using dot blot hybridization of PCR (polymerase chain reaction)-amplified DNA with allelespecific oligonucleotide probes. Eight different known molecular defects were detected, at different frequencies. There was an amber mutation in codon 17, a C-T transversion at position 654 of IVS-2, a frameshift mutation between codons 71 and 72, an A-G transition at nucleotide -28 within the TATA box (known as Chinese mutations), a G-T transversion at position 1 of IVS-1 (an Indian mutation), a 4bp deletion in codons 41/42 and a G-C transversion at position 5 of IVS-1 (described as both Chinese and Indian mutations) and a Thai original mutation, an ochre mutation in codon 35. Analysis of the three unknown alleles by DNA sequencing of the cloned DNA fragment amplified by PCR revealed an A-G substitution at the second position of the codon for amino acid 19 (AAC-AGC). The analytic approach used in the present study and the characteristic distribution of mutations in each region of Thailand will prove useful for setting up a prenatal diagnosis program.     

Molecular screening and fetal diagnosis of β-thalassemia in the Italian population

Summary This paper reports our experience of molecular screening and fetal diagnosis of -thalassemia in 457 at risk couples of Italian descent. Molecular screening was carried out by dot blot analysis on amplified DNA with oligonucleotide probes complementary to the eight most common mutations in Italians [39 (CT); 6 (-A); ⁺-87 (CG); ⁺ IVSI nt 110 (GA); IVSI nt 1 (GA); ⁺ IVSI nt 6 (TC); IVSII nt 1 (GA); ⁺ IVSII nt 745 (CG)]. By using this approach, we have been able to define the mutation in 92.8% of cases. The rest (all but four) were defined by direct sequencing and this led to the detection of nine rare mutations [76 (-C); ⁺ IVSI nt 5 (GA); ⁺ IVSI nt 5 (GC); ⁺ IVSI -1 (cod 30) (GC); ⁺-87 (CT), -290 bp del.; ⁺-101 (CT)], and to the characterization of a novel mutation consisting of the deletion of the G at the invariant AG of the IVSII splice acceptor site of the -globin gene ( IVSII nt 850-1 bp). In the remaining four cases, the -globin gene showed entirely normal sequences and the -globin gene cluster was intact, as indicated by Southern blot analysis. Fetal diagnosis was carried out by dot blot analysis with the oligonucleotide probes defined in the parents. The procedure is simple and reliable, and the results can be obtained within 1 week of sampling. No misdiagnosis has so far occurred. The results indicate that fetal diagnosis of -thalassemia by DNA analysis may be obtained in practically all cases (even in a population showing marked heterogeneity of -thalassemia) by the combination of dot blot analysis for detecting common mutations, and direct sequencing for defining those that are uncommon.     

Heterogeneity in the molecular basis of three types of hereditary persistence of fetal hemoglobin and the relative synthesis of the Gγ and Aγ types of γ chain

Restriction endonuclease analyses of DNA from one Black ^GA-HPFH homozygote and four Black and one Indian ^GA-HPFH heterozygotes have identified three different HPFH types which are the result of large deletions including the and genes. Two of the types are comparable to those characterized previously, but the third, which is present in the Indian heterozygote, shows a distinct difference in the size of the deletion. The 5 end point of the deletion in this type III ^GA-HPFH extends 0.5–1.0 kb beyond the 5 end point of one of the Black types of HPFH (type I). Each of the three types is associated with a distinct ratio between the ^G and the ^A chains, an observation supported by family data. The highest ratio is found in the heterozygote with the Indian type III ^GA-HPFH, with 69.3% ^G chains, while the averages for the other types were 50.7% ^G (type I) and 32.3% ^G (type II).This research was supported in part by USPHS Research Grants HLB-05168 and HLB-15158 and by designated research funds of the Veterans Administration. This is Contribution 0764 of the Department of Cell and Molecular Biology, Medical College of Georgia, Augusta, Georgia 30912. P. K. Sukumaran was on leave from the Bai Jerbai Wadia Hospital for Children, Parel, Bombay, India.     

Molecular basis of β-thalassemia in Turkey: detection of rare mutations by direct sequencing

Summary Using restriction endonuclease analysis, oligonucleotide hybridization, and direct sequencing of amplified genomic DNA, we characterized 11 different mutations in the DNA of 26 patients from Turkey homozygous for -thalassemia. We found that mutations IVS-1 nt110, IVS-1 nt6, and the frameshift at codon 8 were the most frequent. By direct sequencing we characterized two very rare mutations not previously reported in the Turkish population: a frameshift +1 at codons 9/10 and a nonsense mutation at codon 15.     

Detection of β-thalassemia and hemoglobin E genes in Thai by a DNA amplification technique

Summary Enzymatic DNA amplification and polyacrylamide gel electrophoresis, which demonstrate different sizes of DNA fragments, were used to detect the common mutations causing -thalassemia and hemoglobin (Hb) E in Thai people. The 4-bp deletion at codons 41 and 42 can be detected directly by polyacrylamide gel electrophoresis and ethidium bromide staining. Whereas the nonsense mutations at codon 17 (AAG TAG) and Hb E (GAGAAG at codon 26) were detected after digestion of the amplified DNA with the enzymes MaeI and MnlI, respectively.     

Adaptive response to mutagenesis and its molecular basis in a human T-cell leukemia line primed with a low dose ofγ-rays

The effect was studied of a low dose of-ray preexposure on the frequency and molecular spectrum of radiation-induced mutations at the hprt locus in a human T-cell leukemia line. When the cells were preexposed to 0.01 Gy of-rays, the yield of mutations induced by a subsequent 2-Gy challenge dose was reduced by 60%, compared with the 2 Gy of irradiation alone. The data of Southern blot analysis showed that 47% of the mutants induced by 2 Gy in the cells without low-dose preexposure were of the deletion or rearranged mutations type. In contrast, in the low-dose radioadapted cells the proportion of this type of 2-Gy-induced mutations decreased to 28%. This is close to the control level (22%) of spontaneous mutations. Our results confirm that a low dose of-ray preexposure leads to a decreased susceptibility to gene deletions and rearrangements after high-dose irradiation.     

Intrinsic potential for high fetal hemoglobin production in a Druze family with β-thalassemia is due to an unlinked genetic determinant

Summary The mechanism for elevated production of fetal hemoglobin (Hb F) in a Druze patient with °-thalassemia intermedia was investigated. Heterozygous family members exhibited normal Hb F levels, suggesting that the increase in -gene expression in the propositus may be partly due to anemic stress. Erythroid progenitors of these family members cultured in vitro [burst forming units (erythroid); (BFUe)] showed elevated synthesis of Hb F, indicating the existence of a genetically determined intrinsic capacity for high Hb F production in this family. The propositus was found to be homozygous for a IVS2-position 1 mutation, on the background of Mediterranean haplotype I, which is not known to be linked to high Hb F production. Moreover, extensive molecular studies of the -globin gene cluster, including sequence analysis of the promoter regions of the -globin genes, did not reveal any cisacting mechanism that could account for the high Hb F production in the propositus. A young niece of the propositus with °-thalassemia major was recently discovered, who was homozygous for the same -globin allele and haplotype as the propositus. However, unlike her uncle, she does not have a high Hb F level and presents with a severe clinical course. Her inability to produce high Hb F suggests that the genetic determinant for increased -gene expression in the propositus is unlinked to the -globin gene cluster.     

Absence of messenger RNA and gene DNA for β-globin chains in hereditary persistence of fetal hemoglobin

The relative amounts of α- and β-globin mRNA and globin gene DNA were measured in reticulocyte RNA and lymphocyte DNA of an individual with homozygous hereditary persistence of fetal hemoglobin whose red blood cells contain 100% fetal hemoglobin (Hb F: α₂γ₂). Molecular hybridization assays used as probes full-length DNA copies of human α- and β-globin messenger RNA. The results of these hybridization assays demonstrated the expected amounts of α-globin mRNA and gene DNA, but absence of β-globin mRNA and absence of β-globin gene DNA. In the individual studied, hereditary persistence of fetal hemoglobin is associated with total deletion of the β-globin structural gene.     

Organization of α-chain genes among Hb G-Philadelphia heterozygotes in association with Hb S, β-thalassemia,and α-thalassemia-2

The percentages of the -chain variant Hb G-Philadelphia (Hb G) or ₂ 68 AsnLys₂ were evaluated in 84 adult and 18 newborn heterozygotes. These included members of three families who were studied in more detail by nucleic acid hybridization techniques. The adult heterozygotes fell in two categories, one with a higher proportion of Hb G [46.5±1.0% (SD), N=21] and another with lower values (33.9±3.4%, N=63). Among the newborn heterozygotes, two babies fell in the category with the higher proportion of Hb G while 16 babies gave values between 25 and 34%. Studies of -chain gene organization on the parents of one neonate with a Hb G level of 27% at birth and 37% at 8 months excluded the presence of chromosomes with triplicated -chain genes which could lead to the ^0G/ genotype. Rather, these studies on five Hb G heterozygotes from three families confirmed the linkage between Hb G and a specific type of -thalassemia-2 associated with the presence of a 16-kbp Bgl II fragment which most probably carries the ^G locus since it has been found in 19 Hb G heterozygotes studied to date. The presence of an -thal-2 heterozygosity and three -chain genes (^0G/) was confirmed among Hb G heterozygotes with lower proportions of this variant. It is likely that the even lower values found in some newborn could arise through defective assembly of ^G- dimers. The presence of an -thal-2 homozygosity and two active -chain genes, one on each chromosome (^0G/⁰), was confirmed among heterozygotes with the higher proportion of Hb G. One of each of these categories was present in each of the three families investigated. This type of variability in the number of active -chain genes due to a heterozygosity or a homozygosity for -thalassemia-2 explains the trimodality of Hb S percentages among heterozygotes and the atypical hematological or biosynthetic features among patients with -thalassemia and sickle-cell syndromes.This research was supported by USPHS Research Grants HLB-05168 and HLB-15158 and by designated research funds of the Veterans Administration. This is Contribution No. 0693 of the Department of Cell and Molecular Biology, Medical College of Georgia, Augusta.     

A mutation in the β-myosin rod associated with hypertrophic cardiomyopathy has an unexpected molecular phenotype

Hypertrophic cardiomyopathy (HCM) is a common, autosomal dominant disorder primarily characterized by left ventricular hypertrophy and is the leading cause of sudden cardiac death in youth. HCM is caused by mutations in several sarcomeric proteins, with mutations in MYH7, encoding β-MyHC, being the most common. While many mutations in the globular head region of the protein have been reported and studied, analysis of HCM-causing mutations in the β-MyHC rod domain has not yet been reported. To address this question, we performed an array of biochemical and biophysical assays to determine how the HCM-causing E1356K mutation affects the structure, stability, and function of the β-MyHC rod. Surprisingly, the E1356K mutation appears to thermodynamically destabilize the protein, rather than alter the charge profile know to be essential for muscle filament assembly. This thermodynamic instability appears to be responsible for the decreased ability of the protein to form filaments and may be responsible for the HCM phenotype seen in patients.     

Prenatal and post-natal screening of β-thalassemia and hemoglobin E genes in Thailand using denaturing high performance liquid chromatography

We have developed methods based on PCR and denaturing high performance liquid chromatography (DHPLC) for rapid identifications of common β-thalassemia mutations found in Thailand. The β-globin gene was separately amplified by PCR on four different fragments covering eight most common β-thalassemia mutations including nucleotide ?28 A-G, codon 17 (A-T), IVSI-1 (G-T), IVSI-5 (G-C), codon 26 (G-A or Hb E), codons 41/42 (–TTCT), codons 71/72 (+A) and IVSII-654 (C-T). After PCR amplification, heteroduplex was generated by denaturation at 95 °C for 5 min followed by a slow reduction in temperature to 25 °C at 0.03 °C/s. Analysis of heteroduplex was done on an automated WAVE Nucleic Acid Fragment Analysis System. Specific DHPLC profile for each mutation was demonstrated which could be used in screening for all eight β-thalassemia mutations. Further validation was done on 42 pre- and post-natal DNA samples which demonstrated 100 % accuracy as compared to the result obtained with conventional PCR assays. In a remaining case with an unknown mutation, a different DHPLC profile was noted on one of the amplified fragment. Further DNA sequencing of this fragment revealed a T-G transversion at the IVSI-116, a previously un-described mutation in Thai population. The DHPLC assay developed should prove useful for rapid screening of known and unknown β-thalassemia mutations during carrier screening and pre-natal diagnosis which would facilitate an ongoing prevention and control program of thalassemia.     

Structure of soybean β-cyanoalanine synthase and the molecular basis for cyanide detoxification in plants

Plants produce cyanide (CN-) during ethylene biosynthesis in the mitochondria and require β-cyanoalanine synthase (CAS) for CN- detoxification. Recent studies show that CAS is a member of the β-substituted alanine synthase (BSAS) family, which also includes the Cys biosynthesis enzyme O-acetylserine sulfhydrylase (OASS), but how the BSAS evolved distinct metabolic functions is not understood. Here we show that soybean (Glycine max) CAS and OASS form α-aminoacrylate reaction intermediates from Cys and O-acetylserine, respectively. To understand the molecular evolution of CAS and OASS in the BSAS enzyme family, the crystal structures of Gm-CAS and the Gm-CAS K95A mutant with a linked pyridoxal phosphate (PLP)-Cys molecule in the active site were determined. These structures establish a common fold for the plant BSAS family and reveal a substrate-induced conformational change that encloses the active site for catalysis. Comparison of CAS and OASS identified residues that covary in the PLP binding site. The Gm-OASS T81M, S181M, and T185S mutants altered the ratio of OASS:CAS activity but did not convert substrate preference to that of a CAS. Generation of a triple mutant Gm-OASS successfully switched reaction chemistry to that of a CAS. This study provides new molecular insight into the evolution of diverse enzyme functions across the BSAS family in plants.     

Atrial fibrillation in β-thalassemia patients with a focus on the role of iron-overload and oxidative stress: A review

Cardiac complications including arrhythmia and especially atrial fibrillation (AF) are common causes of death in β-thalassemia patients. The main factor in the etiopathogenesis of these complications is iron overload, which results in increased oxidative stress. Although there is a known association between cardiac complications and iron overload in β-thalassemia patients, there is no comprehensive review on AF and excessive iron with a focus on oxidative stress in these patients. The aim of this article was to review the different aspects of AF in β-thalassemia patients with a focus on the prevention and treatment of AF by using iron chelators and/or anti-oxidants. AF in β-thalassemia patients is more common than in the general population. One of the most important causes of AF is cardiac iron overload and the harmful effects of increased oxidative stress. Iron-induced AF can be reversed by using an intensive iron chelation regimen. Based on a few experimental studies, the combination of iron chelators with some anti-oxidants, including NAC, vitamin C, and acetaminophen, can lead to improved cardiac protection. However, the effect of such combinations on cardiac arrhythmias should be further evaluated with animal and human studies.     

A comprehensive molecular analysis and genotype–phenotype correlation in patients with familial mediterranean fever

Molecular Biology Reports - Familial Mediterranean fever is an auto inflammatory genetic disease involving especially Turks, Armenians, Arabs and non-Ashkenazi Jews and caused by variants in the...