Genetic studies suggest a multicentric origin for Hb G-Coushatta [beta22(B4)Glu-->Ala].

Hb G-Coushatta [beta22(B4)Glu-->Ala] is found in geographically separated ethnic groups. Commonest along the Silk Road region of China but also present in the North American Coushatta, we sought to determine whether this variant had a unicentric or multicentric origin. We examined the haplotype of the beta-globin gene cluster in two Chinese families and in five Louisiana Coushatta heterozygous for this mutation. Chinese and Louisiana Coushatta had different haplotypes associated with the identical Hb G mutation. These haplotypes were defined by the presence of a HindIII restriction site in the Agamma-globin gene and AvaII restriction site in the beta-globin gene in Chinese subjects and their absence in the Louisiana Coushatta. We found a CAC at codon beta2 (beta-globin gene framework 1 or 2) linked to the Hb G-Coushatta gene in Chinese, and a CAT (framework 3) in Louisiana Coushatta, indicating different beta-globin gene frameworks. Both the Hb G-Coushatta mutation (GAA-->GCA) and the codon 2 CAC-->CAT polymorphism are normal delta-globin gene sequences, suggesting the possibility of gene conversion. We conclude that Hb G-Coushatta had at least two independent origins. This could be due to separate mutations at codon beta22 in Chinese and Louisiana Coushatta, a mutation at this codon and a beta-->delta conversion, or two beta-->delta gene conversion events.

Hb Seal Rock [(alpha 2)142 term-->Glu, codon 142 TAA-->GAA]: an extended alpha chain variant associated with anemia, microcytosis, and alpha-thalassemia-2 (-3.7 Kb).

Hb Seal Rock was first reported in a young African-American women and her 2-year-old daughter (1). It is an extended alpha chain variant which, like Hb Constant Spring, is present in small quantity and is expressed as an alpha-thalassemia. The mutation, TAA-->GAA affects codon 142 of the alpha 2 gene. In this family, the index case was a compound heterozygote for Hb Seal Rock trait and for alpha-thalassemia trait (-3.7 kb). Her hematologic expression was similar to mild Hb H disease, presumably because the Seal Rock mutation affects the alpha 2 gene that is normally responsible for approximately 70% of alpha-globin synthesis. Her daughter had only Hb Seal Rock trait, but was phenotypically alpha-thalassemia-2 trait due to the expression of the Seal Rock mutation on one of her alpha 2-globin genes, the other three alpha-globin genes being unaffected.

Two missense mutations in the beta-globin gene can cause severe beta thalassemia. Hemoglobin Medicine Lake (beta 32[B14]leucine-->glutamine; 98 [FG5] valine-->methionine).

We studied the molecular basis of transfusion-dependent hemolytic anemia in an infant who rapidly developed the phenotype of beta thalassemia major. DNA sequence of one beta-globin gene of the proband revealed two mutations, one for the moderately unstable hemoglobin (Hb) Köln and another for a novel codon 32 cytosine-thymidine-guanine-->cytosine-adenine-guanine transversion encoding a leucine-->glutamine mutation. A hydrophilic glutamine residue at beta 32 has an uncharged polar side chain that could potentially distort the B helix and provoke further molecular instability. This new hemoglobin was called Hb Medicine Lake. Biosynthesis studies showed a deficit of beta-globin synthesis with early loss of beta-globin chains. An abnormal unstable hemoglobin, globin chain, or tryptic globin peptide was not present, demonstrating the extreme lability of this novel globin. Hb Medicine Lake mRNA was present, but an aberrantly spliced message was not. Absence of an abnormal beta-globin gene in the mother makes it likely that a de novo mutation occurred in the proband. The molecular pathogenesis of Hb Medicine Lake illustrates a mechanism whereby the phenotype of a genetic disorder, like the mild hemolytic anemia associated with a hemoglobinopathy, can be modulated by a coincident mutation in the same gene.

A four base pair deletion 5' to the A gamma T gene is associated not only with decreased expression of the A gamma T-globin gene, but also of the G gamma-globin gene in cis.

A four base pair deletion 5' to A gamma T-globin gene at positions -222 to -225 has been reported to reduce the expression of this gene. To evaluate the prevalence and effect of this deletion, PCR-based methods were employed. The deletion had a gene frequency of 0.06 in a sample of African-American individuals with sickle cell trait, 0.18 in adult African-Americans with normal Hb AA, and 0.36 in caucasians. Seventy cord blood samples from African-American newborns with Hb AA were evaluated by both HPLC and PCR. The frequency of the A gamma T allele was 0.13. The A gamma T-globin chain was always present in a lower proportion than the A gamma I allele (70% of A gamma I), but the percentage of A gamma-globin was the same whether or not A gamma T was present. The total percentage of Hb F, however, was significantly lower in the group with the A gamma T allele (77.1% vs. 87.4%, P < 0.01). These results indicate that the four base pair deletion is not only associated with reduced expression of the A gamma T allele, but also of the G gamma allele in cis, further suggesting a possible role of this region in the modulation of the expression of the linked gamma-globin genes.

Delta beta-thalassemia in an African-American: identification of the deletion endpoints and PCR-based diagnosis.

We describe an African-American child with beta-thalassemia intermedia. Molecular studies revealed that the proband is a compound heterozygote for the -29 (A-->G) beta (+)-thalassemia mutation and an extensive deletion involving the delta- and beta-globin genes. The proband's mother is a simple carrier of the deletion and exhibits the phenotype of delta beta-thalassemia rather than hereditary persistence of fetal hemoglobin. The deletion spans 11,767 bp, with the 5' deletion endpoint located 2,455 bp upstream of the delta-globin gene mRNA Cap site and the 3' endpoint located 441 bp downstream of the termination codon of the beta-globin gene. Based on this information, we have developed a polymerase chain reaction strategy for the rapid detection of this delta beta-thalassemia deletion.

Beta-thalassemia in southwestern Iran.

Seventeen unrelated beta-thalassemia patients or carriers from Southwestern Iran were examined for the beta-globin gene mutations by polymerase chain reaction amplification of the beta-globin gene and direct genomic sequencing, or by the method of allele-specific oligonucleotide hybridization. Their clinical and hematological characteristics were also recorded. Of 26 potential thalassemia-causing chromosomes examined, 10 different mutations were found. The IVS-II-1 (G-->A) mutation was the most frequent (31%) followed by the IVS-I-6 (T-->C) mutation (15%). Eight mutations were initially described in Mediterranean populations and two were of Kurdish origin. Four of these mutations, both initially described in the Mediterranean region, are reported here for the first time in Iranians. The unexpectedly high number of different mutations that account for beta-thalassemia in this region of Iran suggest migration of chromosomes from distant places and genetic admixture.

G gamma A gamma (beta+) hereditary persistence of fetal hemoglobin: the G gamma -158 C-->T mutation in cis to the -175 T-->C mutation of the A gamma-globin gene results in increased G gamma-globin synthesis.

Hereditary persistence of fetal hemoglobin (HPFH) can be generally classified into deletional and nondeletional forms. The family described in the present study has characteristics of both types of HPFH. The proband is a healthy 30-year-old black woman. Analysis of her hemoglobin revealed 40.4% HbS, 40.9% HbF (G gamma/A gamma ratio 0.53), 16.8% HbA, and 1.9% HbA2. All of her hematologic indices were normal, and the distribution of HbF in her red cells was pancellular. Family studies demonstrated that the proband has one chromosome 11 bearing the beta s-globin gene and the other bearing a G gamma A gamma (beta+) HPFH determinant in cis to the beta A-globin gene. Gene mapping studies of the region between the G gamma- and beta-globin genes were normal. However, when the A gamma and G gamma promoters were amplified by polymerase chain reaction (PCR) and sequenced, the A gamma promoter was found to have the T-->C mutation at -175, and the G gamma promoter region was found to have the C-->T mutation at -158. The -158 C-->T mutation has been associated with elevated G gamma levels and high HbF in hemolysis, although its role in causing these effects is unclear. The present study suggests that this mutation can also enhance G gamma-globin expression in cis to the -175 T-->C mutation in the absence of hemolysis. We suggest that the alteration of the A gamma gene octamer binding site by the -175 mutation, as well as the loss of a putative G gamma "silencer" caused by the -158 mutation may account for this phenotype. We propose calling these linked mutations the G gamma A gamma(beta+) HPFH.

Beta-thalassemia intermedia with exceptionally high hemoglobin A2: relationship to mutations in the beta-gene promoter.

Small deletions of the 5' portion of the beta-globin gene that remove the promoters but stop 3' to the delta-globin gene are recognized as the sole cause of beta-thalassemia with exceptionally high hemoglobin A2 (HbA2) levels. Two patients with beta-thalassemia intermedia and exceptionally high levels of HbA2 (10.4 and 12.0%) were examined. One patient was a combined heterozygote for the -88 C----T and a novel -87 C----A mutation, while the other was homozygous for the -29 A----G beta(+)-thalassemia mutation. The remainder of the beta genes were normal. There was no evidence for deletions involving the 5' portion of the beta gene or the region between the beta and delta genes. Gene mapping studies excluded the possibility of a beta delta-anti-Lepore hemoglobin gene with beta promoters and delta coding sequences. There were no mutations in the promoters of the G gamma or A gamma-globin genes that have been associated with the hereditary persistence of HbF phenotype. The delta-globin gene promoters were normal from codon 17 to position -145 relative to the mRNA capping site. There appears to be considerable heterogeneity of HbA2 and HbF levels in patients who are homozygous or mixed heterozygotes for mutations in the TATA box and other promoter elements of the beta-globin gene. The capacity for proteolysis within the erythrocyte may vary among individuals. The authors hypothesize that in the exceptionally high HbA2 beta-thalassemia intermedia phenotype, proteolysis of superfluous alpha-globin chains is less efficient than in patients with customary levels of HbA2.(ABSTRACT TRUNCATED AT 250 WORDS)

Hb S/beta zero-thalassemia due to the approximately 1.4-kb deletion is associated with a relatively mild phenotype.

We report a relatively mild phenotype associated with two siblings who are compound heterozygotes for Hb S and a beta zero-thalassemia mutation due to a approximately 1.4-kb deletion of the 5' region of the beta-globin gene. Each is found to have unusually high levels of Hb A2 and Hb F, accounting for more than 20% of the total hemoglobin. These may interfere with intracellular Hb S polymerization, thus leading to a mild clinical course.

Hemoglobin Terre Haute arginine beta 106. A posthumous correction to the original structure of hemoglobin Indianapolis.

The initial report of Hb Indianapolis described two affected individuals with the phenotype of severe beta-thalassemia that was dominantly inherited. The structure of this variant could not be deduced by standard techniques because of its extreme instability. Because of this limitation, the structure was ascertained by analysis of the abnormal globin chain, which had been radioactively labeled. These studies strongly suggested that the structure of this variant was cysteine beta 112 to arginine. Subsequent to this report, two additional families with Hb Indianapolis were found. The carriers were minimally affected and the abnormal hemoglobin was only mildly unstable. This major difference in phenotypic expression suggested that further investigation of the original family should be carried out. Unfortunately, both of the original carriers of the variant succumbed to their severe anemia prior to the subsequent reports. However, by the use of the polymerase chain reaction, enough DNA was obtained to sequence the third exon of the beta-globin gene in the original family from the DNA scraped off a 10-year-old bone marrow microscope slide. These studies revealed a substitution of leucine to arginine at position 106 of the beta-globin chain. The polymerase chain reaction results may be consistent with the original protein structural data, if incomplete tryptic cleavage of this arginine residue occurred in the original sample. We have renamed this variant Hb Terre Haute in an attempt to avoid confusion with the Cys beta 112----Arg substitution.

Molecular basis for alpha-thalassemia associated with the structural mutant hemoglobin Suan-Dok (alpha 2 109leu----arg)

Hemoglobin (Hb) Suan-Dok (alpha 109Arg) is a rare alpha-globin structural mutation that is linked to an alpha-thalassemia (alpha-thal) determinant. When inherited in trans to an alpha-thal-1 mutation (-), it results in Hb H disease associated with low levels (9%) of the Suan-Dok Hb. The nature of the thalassemic defect associated with the alpha SD mutation has been investigated by structural and functional studies. Sequence analysis of the cloned Suan-Dok allele showed a missense mutation (T----G) at codon 109 in an otherwise normal alpha 2-globin gene. When the alpha 2SD-globin gene was introduced into mouse erythroleukemia cells, the steady state alpha-globin messenger RNA (mRNA) level was equivalent to the alpha A-globin gene control. Although in vitro translation of a synthetic alpha 2SD-globin mRNA generated levels of alpha globin equivalent to alpha 2A-globin mRNA at early time points, the ratio of alpha SD to alpha A globin decreased markedly at later time points. These data suggest that the thalassemic defect associated with the Suan-Dok mutation results from a significant instability of the alpha SD globin.

Molecular basis for nondeletion alpha-thalassemia in American blacks. Alpha 2(116GAG----UAG).

An American black woman was found to have the phenotype of moderately severe alpha-thalassemia normally associated with the loss of two to three alpha-globin genes despite an alpha-globin gene map that demonstrated the loss of only a single alpha-globin gene (-alpha/alpha alpha). Several individuals in her kindred with normal alpha-globin gene mapping studies (alpha alpha/alpha alpha) had mild alpha-thalassemia hematologic values consistent with the loss of one to two alpha-globin genes. These data suggested the presence of a nondeletion alpha-thalassemia defect in this family which segregates with the intact alpha alpha gene cluster. An abnormally migrating and highly unstable alpha-globin gene product was demonstrated by in vitro translation of the reticulocyte mRNA from the proposita and this mutant alpha-globin protein was mapped to the alpha 2-globin gene by hybrid-selected translation. The abnormal alpha 2-globin gene was cloned and sequenced. A single base mutation that results in a premature termination codon was identified at codon 116 (GAG----UAG). The defined alpha-globin genotype of the proposita (-alpha/alpha 116UAG alpha) and the positioning of this nonsense mutation at the alpha 2-globin gene locus are fully consistent with the observed alpha-thalassemia phenotype.

A new gene deletion in the alpha-like globin gene cluster as the molecular basis for the rare alpha-thalassemia-1(--/alpha alpha) in blacks: HbH disease in sickle cell trait.

A novel deletion of at least 26 kilobase of DNA, including both alpha-globin genes, the psi alpha- and psi zeta-globin genes, but sparing the functional zeta-gene was found in a 10-year-old black boy with HbH disease and sickle cell trait. This particular deletion has not previously been described in blacks. Its existence makes it likely that the absence of Hb Barts hydrops fetalis in blacks is due to the rarity of the chromosome lacking two alpha-globin genes rather than a result of early embryonic death due to the failure to synthesize embryonic hemoglobins because of deletion of functional zeta-globin genes.