Dysfunctional alpha-globin gene in hemoglobin H disease in blacks. A dinucleotide deletion produces a frameshift and a termination codon

alpha-Thalassemia trait is common in Black Americans; the (-alpha) haplotype occurs in 30% of that population. However, hemoglobin H disease (genotype:- -/-alpha) is very uncommon due to the rarity of the (- -) haplotype. A subject with HbH-HbG Philadelphia (alpha 2(68)Asn----Lys) synthesized only alpha G and no alpha A. Digestion of DNA with BamHI produced a single 10-kilobase (kb) alpha-specific fragment. Her son had alpha-thalassemia trait, did not make HbG Philadelphia, and demonstrated 14- and 10-kb alpha fragments upon BamHI digestion. Since the 14-kb fragment could not have been inherited from the mother, the son apparently received from her a chromosome bearing a single nonfunctional (alpha T) gene. Therefore, the two genotypes are: mother (-alpha G/-alpha T), son (-alpha T/alpha alpha). A 16-kb BglII fragment, containing the gene of interest from the son, was cloned into the BamHI site of phage EMBL 3 followed by subcloning of a 1.5-kb PstI alpha-specific fragment into plasmid pBR322. The mutant alpha gene demonstrated a deletion of an AG dinucleotide from the tandem repeat normally occurring in the Glu-Arg codons 30 and 31 at the junction of the first exon with intervening sequence 1. The loss of two nucleotides leads to a reading frameshift and a totally novel amino acid coding sequence in the second exon from codons 31-54 followed by the appearance of a chain termination codon (TAA) at position 55. No complete globin chain can be produced from this gene. HbH disease in this Black family is thus due to the combination of gene deletion and a nonfunctional alpha gene.     

Alpha-globin gene markers identify genetic differences between Australian aborigines and Melanesians.

Australian aborigines exhibit a number of alpha-globin cluster rearrangements involving both alpha- and zeta-globin genes. alpha+-Thalassemia (-alpha/) in this population is heterogeneous and includes the 3.7 types I, II, and III gene deletions. The alpha alpha alpha/ and zeta zeta zeta/ rearrangements are each found in association with two haplotypes, indicating origins from at least two separate DNA crossover events. Differences in alpha-globin cluster rearrangements and in haplotypes between Australian aborigines, Papua New Guinea highlanders and island Melanesians, are consistent with multiple colonizing events into Australia.     

Detection of severe nondeletional alpha-thalassemia mutations using a single-tube multiplex ARMS assay

Alpha-thalassemia is a common hereditary anemia due to decreased or absent synthesis of alpha-globin chains. The most common causes of alpha-thalassemia are deletions that remove one or both functional alpha-globin genes, with a small proportion of cases involving nondeletional mutations of the alpha2- or alpha1-globin genes. Herein, we describe a single-tube multiplex amplification refractory mutation system (ARMS) assay for rapid detection of six of the most common and severe nondeletional alpha-thalassemia mutations. These alleles are found predominantly among southeast Asian populations, and are associated with the most severe forms of hemoglobin (Hb) H disease or Hb H hydrops fetalis.     

Molecular analysis of alpha/beta-thalassemia in a southern Chinese population

Thalassemia is endemic to many regions in southern China. The screening of severe determinants of thalassemia is of critical importance in management and control of thalassemia. We designed a protocol based on microarray technology to screen for a spectrum of alpha/beta-globin gene mutations in the Chinese population. A total of 38 probes were capable of screening 98% of alpha/beta-globin gene mutations in the China population, including 16 mutations of beta-globin [beta(41-42)(-TCTT), IVSII-654(C-->T), beta17(A-->T), -28(A-->G), beta(71-72)(+A), beta(71-72)(+T), HbE26(G-->A), -29(A-->G), beta(27-28)(+C), IVSI-1(G-->T), IVSI-5(G-->C), beta(14-15)(+G), IVSII-5(G-->C), beta41(+T), 37(G-->A), and beta43(G-->T)] and five mutations of alpha/beta[three deletions of -alpha;(3.7), -alpha(4.2), and --(SEA); two nondeletions of alpha(Quong Sze) codon alpha125(T-->C) and alpha(Constant Spring) codon alpha142(T-->C)]. Multiplex PCR products were amplified from human genomic DNA and allowed to hybridize with the oligonucleotide array. alpha/beta-Globin genotypes were assigned by quantitative analysis of the hybridization results. The protocol, standardized by analysis of 100 thalassemia samples with known mutations and 13 recombinant plasmids, was 100% reliable in genotyping all mutant alleles. In subsequent screening of 2,030 Chinese with unknown mutations, the protocol was 100% accurate. This method provides unambiguous detection of complex combinations of heterozygous, compound heterozygous, and homozygous alpha/beta-thalassemia genotypes. The protocol was also flexible, detecting globin gene mutations from different population groups.     

Beta-globin gene haplotypes and alpha-thalassemia analysis in Babinga pygmies from Congo-Brazzaville

We analyzed beta-globin gene cluster haplotypes and deletional alpha+-thalassemia (-alpha3.7kb) in 54 Babinga pygmy subjects from Congo-Brazzaville. The beta(S)-globin gene frequency was 0.065 and that of the deletional alpha-globin gene (-alpha3.7kb) was 0.29. Eighty-five percent of the beta(S) chromosomes and 13% of the beta(A) chromosomes were associated with the Bantu haplotype, 10% of beta(A) chromosomes with the Senegal haplotype, and the remaining beta chromosomes with atypical haplotypes. None of the chromosomes were of the Benin haplotype. These results are clearly of anthropological and evolutionary interest. They also support earlier observations that alpha+-thalassemia is prevalent at a high frequency in African populations.     

Abnormal arrangements in the alpha- and gamma-globin gene clusters in a relatively large group of Japanese newborns.

Data were obtained on blood samples from a relatively large group (264) of healthy Japanese newborns, collected at hospitals in Tokyo, Kurashiki, and Ube. The studies included an evaluation of anomalies in alpha-globin gene and gamma-globin gene arrangements using gene mapping and gamma-chain composition analyses. The results confirmed the rarity of alpha-thalassemia among Japanese; only a few babies had alpha-thalassemia-2 trait (the -3.7-kilobase [kb] deletion), while others had alpha-globin gene triplications (both the alpha alpha alpha anti-3.7 and the alpha alpha alpha anti-4.2 types). Among the gamma-globin gene anomalies that were observed, a few babies had the -A gamma-A gamma- globin gene arrangement or the -G gamma A gamma- type of deletion. The gamma-chain triplication (-G gamma-A gamma G gamma-A gamma-) occurred in 10 out of 256 newborns, and its frequency exceeded that of its corresponding -G gamma A gamma- deletion by a factor of 5. The restriction endonuclease XmnI was a useful tool, in addition to the enzymes Bg1II and BclI, to evaluate and confirm the gamma-globin gene deletion and triplication. The A gamma T variant, which is the product of a mutant A gamma-globin gene, occurred at a frequency of 0.156. The chromosome carrying this mutant A gamma gene had a characteristic haplotype that was originally seen in black and Mediterranean patients with Hemoglobin (Hb) S or with beta-thalassemia.     

Implications of the genetic epidemiology of globin haplotypes linked to the sickle cell gene in southern Iran

To determine the origin of sickle cell mutation in different ethnic groups living in southern Iran, we studied the haplotype background of the betaS and betaA genes in subjects from the provinces of Fars, Khuzestan, Bushehr, Hormozgan, and Kerman and from the islands of Khark and Qeshm. beta-globin gene cluster haplotypes were determined using the PCR-RFLP technique. Detection of -alpha 3.7 deletion and beta-thalassemia mutations were defined by PCR and reverse dot blot techniques, respectively. The framework of the beta-globin gene was determined using denaturing gradient gel electrophoresis. We found that the betaS mutation in southern Iran is associated with multiple mutational events. Most of the patients were from two ethnic groups: Farsi speakers (presumably Persian in origin) from Fars province and patients of Arab origin from Khuzestan province. In both ethnic groups the Arab-Indian haplotype was the most prevalent. The frequencies of the Arab-Indian and African haplotypes in sickle cell anemia patients from the provinces of Fars and Khuzestan were similar. Among betaA chromosomes the Bantu A2 haplotype was the most prevalent. The decrease in alpha-globin production in SS patients and AS individuals appeared to be related to the reduction in mean cell volume and mean cell hemoglobin. The Arab-Indian haplotype gene flow into this region of Iran can be traced to the Sassanian Empire. It is likely that the influx of betaS genes linked to the Benin and Bantu haplotypes, of African origin, must have occurred during the Arab slave trade.     

Study of alpha-thalassemia in northeastern Thailand at the DNA level

The frequency of alpha-thalassemias in a general population sample from northeastern Thailand and in an Austroasiatic group with high frequencies of hemoglobin E and beta-thalassemia, the So, was estimated using DNA techniques. Among 64 healthy adult subjects from the Khonkaen and Ubol areas, the following haplotype frequencies were determined: alpha alpha, 0.742; -alpha 3.7 (subtype I), 0.148; -alpha 4.2, 0.016; -alpha del, 0.008; alpha Constant Spring alpha, 0.055; --SEA, 0.023, and alpha alpha alpha (triplicated alpha-globin gene), 0.008. In the So group, the combined frequency of alpha-thalassemia chromosomes was 0.525.     

Types of alpha+ thalassemia in Southeast Asia refugees

We have determined alpha+ deletional thalassemia among 143 Southeast Asia refugees (Cambodians, Laotians, and Vietnameses). Gene frequency of alpha+ deletional thalassemia in Vietnameses (0.035) was found lower than in Cambodians and Laotians (0.11). Bam H1 and Bg1 II analysis indicated that both rightward and leftward thalassemias are encountered, the -alpha 3,7 form is being by far more frequent than the -alpha 4.2 one. Only type I cross-over was found by Apa I digestion on -alpha 3.7 chromosomes. The Rsa I polymorphism, 5' to Z alpha 2 block, is associated with -alpha 3.7 type I haplotype and the site is present in 12 out of 23 chromosomes. All these data suggest at least three origins of alpha+-thalassemia in Cambodia and Laos.     

Genotyping of alpha-thalassemia in microcytic hypochromic anemia patients from North India

Microcytic hypochromic anemia is a common condition in clinical practice and alpha-thalassemia has to be considered as a differential diagnosis. Molecular diagnosis of alpha-thalassemia is possible by polymerase chain reaction. The aim of this study was to evaluate the frequency of alpha-gene numbers in subjects with microcytosis. In total, 276 subjects with microcytic hypochromic anemia [MCV<80fl; MCH<27pg] were studied. These include 125 with thalassemia trait, 48 with thalassemia major, 26 with sickle-cell thalassemia, 15 with E beta-thalassemia, 40 with iron-deficiency anemia, 8 with another hemolytic anemia, and 14 patients with no definite diagnosis. Genotyping for -alpha3.7 deletion, -alpha4.2 deletion, Hb Constant Spring, and a-triplications was done with polymerase chain reaction. The overall frequency of -alpha3.7 deletion in 276 individuals is 12.7%. The calculated allele frequency for a-thalassemia is 0.09. The subgroup analysis showed that co-inheritance of a-deletion is more frequent with the sickle-cell mutation than in other groups. We were able to diagnose 1/3 of unexplained cases of microcytosis as a-thalassemia carriers. The a-gene mutation is quite common in the Indian subcontinent. Molecular genotyping of a-thalassemia helps to diagnose unexplained microcytosis, and thus prevents unnecessary iron supplementation.     

Alpha thalassemia and alpha-MRE haplotypes in Uruguayan patients with microcytosis and hypochromia without anemia

Alpha thalassemia is the most common genetic disorder across the world, being the α-^3.7 deletion the most frequent mutation. In order to analyze the spectrum and origin of alpha thalassemia mutations in Uruguay, we obtained a sample of 168 unrelated outpatients with normal hemoglobin levels with microcytosis and hypochromia from two cities: Montevideo and Salto. The presence of α-thalassemia mutations was investigated by gap-PCR, restriction endonucleases analysis and HBA2 and HBA1 genes sequencing, whereas the alpha-MRE haplotypes were investigated by sequencing. We found 55 individuals (32.7%) with α-thalassemia mutations, 51(30.4%) carrying the -α^3.7 deletion, one with the -α^4.2 deletion and three having the rare punctual mutation HBA2:c.-59C>T. Regarding alpha-MRE analysis, we observed a significant higher frequency of haplotype D, characteristic of African populations, in the sample with the -α^3.7 deletion. These results show that α-thalassemia mutations are an important determinant of microcytosis and hypochromia in Uruguayan patients with microcytosis and hypochromia without anemia, mainly due to the -α^3.7 deletion. The alpha-MRE haplotypes and the α-thalassemia mutations spectrum suggest a predominant, but not exclusive, African origin of these mutations in Uruguay.     

Extremely high frequencies of alpha-globin gene deletion in Madang and on Kar Kar Island, Papua New Guinea.

Extremely high frequencies of the deletion form of alpha(+)-thalassemia (-alpha/), as studied by the DNA mapping technique, were found in the population of Madang, a coastal province in the north of Papua New Guinea (PNG) and in the population of Kar Kar, an island situated near Madang. Ninety-seven percent of the population tested from Madang and 89% of that from Kar Kar Island were either alpha(+)-thalassemia heterozygotes or homozygotes. By contrast, no examples of the deletion form were detected in the Eastern Highlands of PNG. The haplotype frequencies of alpha(+)-thalassemia (-alpha/) in Madang and Kar Kar Island were found to be 81.33% and 66.67%, respectively. A more detailed analysis of the gene deletion revealed that in both populations 96% were of the 4.2 kilobase (kb) type and 4% were of the 3.7-kb type. Thus, this group is the only example in which the 4.2-kb deletion is predominant over 3.7-kb defect. The presence in high frequencies of alpha(+)-thalassemia in the coastal area of Madang and on the neighboring island, where malaria has long been holoendemic or hyperendemic, and its virtual absence from the nonmalarious highlands of PNG suggest the role of malaria as the selective factor in maintaining alpha(+)-thalassemia. If this selective pressure is still operating, and since alpha(+)-thalassemia has no apparent homozygous disadvantage, the abnormal haplotype (-alpha/) will be in the process of fixation in this population.     

中国人α珠蛋白基因-α3.7缺失亚型的研究

-α3.7是中国人常见的缺失型α-地中海贫血-2。根据重组位点的不同,-α3.7可分为-α3.7Ⅰ型、-α3.7Ⅱ型和-α3.7Ⅲ型,并且亚型的种类和频率具有种族差异性。本研究在中国人群中用PCR基因分析方法检出具有α珠蛋白基因-α3.7缺失的患者56例,然后用ApalⅠ和BalⅠ限制性内切酶进行分型。 结果表明,在这56例具有-α3.7缺失的患者中,有54例是-α3.7Ⅰ型,有2例是-α3.7Ⅱ型,尚未发现-α3.7Ⅲ型。此结果丰富了我国α地贫基因型谱的资料。 Abstract:-α3.7 is a common deletional α-thalassemia-2 in China.According to different recombination sites,-α3.7 can be divided into -α3.7Ⅰ、-α3.7Ⅱand -α3.7Ⅲ.The frequency and population distribution of these -α3.7 are quite different.In this study,we detected 56 patients among Chinese population of -α3.7 defect in alpha globin gene by PCR method,then the PCR product was digested by the restriction enzyme ApalⅠand BalⅠ.The sub-typing result shows that in the 56 cases of -α3.7 defect,54 out of 56 is -α3.7Ⅰ,2 out of 56 is -α3.7Ⅱ and none of -α3.7Ⅲ is detected.This result enriches the data about the alpha thalassemia genotypes of Chinese people.     

Melanesians and Polynesians share a unique alpha-thalassemia mutation.

Several genetic markers that provide information on population migrations and affinities have been detected by studies of proteins and cellular antigens in blood. Analysis of DNA polymorphisms promises to yield many further population markers, and we report here the distribution of a new alpha-globin gene deletion (-alpha 3.7 III) detected by a restriction enzyme mapping. This is found frequently in Melanesians and Polynesians but not in five other populations in which alpha-thalassemia is prevalent. We used restriction enzyme haplotype analysis to support a single origin for this mutation and propose that it is a useful population marker. Its geographical distribution supports a route through Island Melanesia for the colonizers of Polynesia.     

Characterization of the Mlsf system. I. A novel "polymorphism" of endogenous superantigens.

In addition to Mlsa (Mls-1a) and Mlsc (Mls-2a, Mls-3a), we and others have recently described a third set of stimulatory minor lymphocyte stimulating (Mls) determinants, which are ligands for "I-E related" V beta, V beta 5, V beta 11, and V beta 12. Although all V beta associated with the recognition of the conventional Mls determinants are, in general, uniformly deleted in those animals expressing relevant Mls, expression of Mlsf-related V beta reveals various deletion patterns among different strains. Here we describe extensive genetic studies to evaluate the relationship among the self-Ag responsible for clonal deletion of T cells bearing Mlsf-related V beta by using antibodies specific for TCR V beta chain. In addition, a panel of T cell clones specific for the Mlsf determinant were generated and employed to analyze the determinant specificity, which is recognized by Mlsf-reactive T cells in vitro as well as the role of class II molecules in T cell recognition of the Mlsf determinants. The results of these two independent approaches provide evidence that the Mlsf system is composed of a set of gene products that reveal a unique polymorphism in the induction of clonal deletion in vivo and in T cell activation in vitro. One of these gene products causes almost complete deletion of the self-Mlsf reactive T cell repertoire in vivo and elicits a strong proliferative response to Mlsf-specific T cell clones. Expression of the other gene products results in the clonal deletion of only part of the Mlsf-reactive T cell repertoire. Furthermore, the response pattern of Mlsf-specific clones to intra-MHC recombinant inbred strains and the inhibition pattern of these clones by anti-class II antibody suggested that although expression of the I-E molecule is essential for T cell recognition of Mlsf determinants, the A beta gene may also contribute to the efficient presentation of Mlsf determinants by forming unique class II E alpha A beta molecules.     

Interaction of α-thalassemia genes with each other and with HbC in an American black family

The relative rates of in vitro synthesis of hemoglobin chains have been studied in an American black family in which the mother is doubly heterozygous for alpha-thalassemia and HbC and the father is heterozygous for alpha-thalassemia. The alpha/non-alpha synthetic ratio was equally unbalanced in both the bone marrow and the peripheral blood of the mother. Although HbC comprised 35% of her hemoglobin (compared to 42.2 +/- 2.2 in individuals with HbC trait and balanced globin synthesis), synthetic data showed that the newly synthesized beta C chain was 44% of the total newly synthesized beta chains. Isolated membranes contained more newly synthesized beta C than beta A chains. Three of the offspring were within the normal range, and the remaining three had alpha-thalassemia. There were two spontaneous abortions during the second trimester of pregnancy. Hydrops fetalis did not occur, and none of the children had HbH disease or HbC trait.     

Independent recombination events between the duplicated human alpha globin genes; implications for their concerted evolution.

We have examined the molecular structure of the human alpha globin gene complex from individuals with a common form of alpha thalassaemia in which one of the duplicated pair of alpha genes (alpha alpha) has been deleted (-alpha 3-7). Restriction mapping and DNA sequence analysis of the mutants indicate that different -alpha 3.7 chromosomes are the result of at least three independent events. In each case the genetic crossover has occurred within a region of complete homology between the alpha 1 and alpha 2 genes. Since the -alpha chromosomes may reflect the processes of crossover fixation and gene conversion between the two genes, their structures may provide some insight into the mechanism by which the concerted evolution of the human alpha globin genes occurs.     

Alpha-thalassemia in northern Thailand. Frequency of deletional types characterized at the DNA level

The frequency of alpha-thalassemias in northern Thailand was estimated using DNA techniques. Among 106 healthy adult Thais from the Chiangmai area, 28 were shown to carry alpha-globin gene anomalies. There were 19 heterozygotes and 1 homozygote for alpha-thalassemia-2. One of the alpha-thalassemia-2 deletions was of the -alpha 4.2 type and the remaining 20 of the -alpha 3.7 type (subtype I). Deletions of both alpha-globin genes on one chromosome (alpha-thalassemia-1) of the Southeast Asian type were observed in 5 cases, and 3 alpha-globin gene triplications were identified. Compared with a previous report on alpha-thalassemia in northern Thailand which was based on the determination of hemoglobin Bart's in cord blood, the present DNA study reveals a similar frequency of alpha-thalassemia-2 but a considerably lower frequency of alpha-thalassemia-1.     

Haplotypes in tribal Indians bearing the sickle gene: evidence for the unicentric origin of the beta S mutation and the unicentric origin of the tribal populations of India

To determine the origin of sickle cell anemia (SS) in India, we analyzed haplotypes of the beta gene cluster in beta S-carrying individuals belonging to tribal populations living in the Nilgiris region of southern India and complemented the available data on tribes of east-central India. We found that in the Nilgiris tribes chromosomes bearing the beta S gene are linked in 91% of the cases to the "Asian" (Arab-Indian) haplotype (although 25% of the haplotypes had the epsilon polymorphic site negative, making the 5' portion of the haplotype identical with the African Senegal haplotype). These XmnI (+) chromosomes were associated with high G gamma expression (67.2 +/- 5.9%) and a high percentage of Hb F (15.5 +/- 7.9%; range, 6-25.3%). We have similar findings for tribal groups from west-central India (Gujarat). In east-central India we have confirmed the data of others, finding the same haplotype linked to beta S in tribes living in the east (Orissa, Andhra Pradesh). We conclude that the beta S gene in presently isolated and disperse tribal populations in India is associated with one predominant typical haplotype, suggesting a unicentric origin of the mutation in India. In addition, this finding implies a unicentric origin of the tribal populations themselves: The gene must have arisen and spread before tribal dispersion. Furthermore, we find extremely high frequencies of the (-alpha) haplotype in the Nilgiris (0.89) and in Gujarat (0.95). The beta S gene linkage to a high Hb F-expressing haplotype and the high incidence of alpha-thalassemia predict a mild phenotypical expression of sickle cell anemia in India.