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We have identified the beta-thalassemia alleles in 47 pediatric patients with transfusion-dependent thalassemia major from a clinic in New Delhi and in 105 heterozygous relatives. Surprisingly, only five mutations were present in 94 beta-thalassemia chromosomes with frequencies from 10 to 32%. This observation greatly facilitated the initiation of a prenatal diagnostic program. Similar studies were conducted for seven Asian Indian patients from Calgary, Canada, seven Asian Indian patients from Durban, South Africa, and from heterozygous relatives, and persons with a beta-thalassemia trait who were not related. Besides beta-thalassemia alleles, common to Asian Indian beta-thalassemia patients, some unexpected alleles were observed in the patients from South Africa, including a newly discovered frameshift at codon 15 (-T).

We have analyzed the quantities of alpha x chain-containing hemoglobins (alpha 2 x beta 2 and alpha 2 x delta 2) in 14 heterozygotes for Hb Q-India [alpha 64(E13)Asp-->His] or Hb Q-Thailand [alpha 74(EF3)Asp-->His]; both amino acid replacements are the result of mutations in the alpha 1-globin gene. Five of these persons (three with Hb Q-India and two with Hb Q-Thailand) had an additional beta(0)-thalassemia heterozygosity. The average quantities for Hb Q + Hb Q2 in the four groups were 17.2% (alpha alpha Q/alpha alpha; beta A/beta A), 9.5% (alpha alpha Q/alpha alpha; beta A/beta(0) Th), 26.8% (-alpha Q/alpha alpha; beta A/beta A), and 16.95% (-alpha Q/alpha alpha; beta A/beta(0) Th). These variations can best be explained by a posttranslational control mechanism; an imbalance in the alpha A, alpha Q, and beta A chain ratio will favor the alpha 2 Q beta 2 formation when an alpha-thalassemia is present and will reduce its formation in the presence of a beta-thalassemia heterozygosity.

In 1991 we reported the identification of two deletional alpha-thalassaemia-2 determinants (-3.7 kb and -4.2 kb) and one nondeletional alpha-thalassaemia-2 determinant (Hb Koya Dora alpha 2 codon 142, TAA-->TCA) in a tribal population in Central India (Gupta et al, 1991). Evidence was obtained at that time for the possible presence of an additional nondeletional alpha-thalassaemia-2 because of low levels of Hb S (< 28%) in some Hb S heterozygotes with a simple alpha-thalassaemia-2 heterozygosity (-alpha/alpha alpha). This abnormality has now been identified as a G-->A mutation at IVS-I-117 of the alpha 1-globin gene (acceptor splice site) which makes this gene nonfunctional. Its frequency was established at approximately 6% which raises the total frequency of alpha-thalassaemia determinants in this population to approximately 60%. Subjects with a deletional alpha-thalassaemia-2 and the newly discovered alpha 1 acceptor splice junction mutation in trans appear to have an alpha chain deficiency similar to that of an alpha-thalassaemia-2 homozygote (-alpha/-alpha). An additional change (C-->G) at the Cap -4 site was observed in six alpha 1- and one alpha 2-globin genes; this polymorphism is not associated with a decrease in alpha chain synthesis and is not linked to the IVS-I-117 (G-->A) mutation.

We have identified three types of alpha-thalassemia in 28 members of an Indian family from Durban, South Africa. The rare South African (SA) type of alpha-thalassemia-1, which is characterized by an approximately 23-kb deletion involving the psi zeta, psi alpha 2, psi alpha 1, alpha 2, alpha 1, and theta 1 genes, was present in 13 members [6 simple heterozygotes, 5 with Hb H disease of the --(SA)/-alpha(-3.7 kb) type, and 2 with Hb H disease of the --(SA)/-alpha(-4.2 kb) type]. Seven others were heterozygotes for alpha-thalassemia-2 (-3.7 kb), 1 was homozygous for this deletion, and 1 was a compound heterozygote [-alpha(-3.7 kb/-alpha(-4.2 kb)]. Hematological and hemoglobin composition data indicated a moderate anemia in all 7 patients with Hb H disease with severe microcytosis and hypochromia, no elevation of gamma-chain synthesis, low levels of Hb A2 (0.3-0.7%), and low levels of Hb H. The most severe disease was present in 2 teenagers with the --(SA)/-alpha(-4.2 kb) combination.

We have investigated the frequencies and types of alpha-thal, beta-thal, and Hb variants among nearly 200 inhabitants of villages in the Mandla and Jabalpur districts of Madhya Pradesh in Central India. Over 85% were tribals of the Gond group. alpha-Thal, as -alpha 3.7/and -alpha 4.2/, and the nondeletional Koya Dora mutation were present at the combined frequency of 0.54. There were indications for the presence of other nondeletional types of alpha-thal. alpha-Globin gene triplications were not observed. Four of the six beta-thal alleles observed were in the tribal groups; two (G----C at codon 30 and G----A at IVS-I-1) were found for the first time. The simultaneous presence of an alpha-thal (-alpha/alpha alpha or -alpha/-alpha) greatly improved the clinical and hematological condition of the patients with Hb S-beta(+)-thal (IVS-I-5; G----C). The lower frequency of alpha-thal among the beta-thal heterozygotes (f = 0.32) may indicate that some of the beta-thal alleles in the tribal populations originated from an outside source. Forty-one subjects had SS; all but one had beta S with haplotype #31, while one chromosome had haplotype #17. The presence of an alpha-thal-2 (f = 0.53) in the SS patients did not affect hematological data. The Hb F levels varied between 7.5% and 42.5% with high G gamma values. No difference in Hb F level between males and females was observed. Lower Hb F levels were present in 10 SS patients with an alpha-thal-2 homozygosity (average 16% versus 23.5% for eight SS patients with alpha alpha/alpha alpha) suggesting a decreased formation of alpha gamma dimers in severe alpha chain deficiency. Several younger SS patients (less than 10 years) also had high Hb F levels (32-42%). Variations in the sequence at -530 of the beta-globin gene; i.e. in the so-called silencer sequence, were present in all beta S chromosomes with haplotype #31, but were not considered important for understanding the variability in the Hb F level. gamma-Globin gene deletions (gamma-thal) and triplications were not observed.

A severe hemolytic anemia with microcytosis and hypochromia was present in a young adopted Indian patient. Reversed phase high performance liquid chromatographic methodology and heat stability tests detected an unstable alpha chain which was present in 3 to 5% of the total hemoglobin. A larger quantity of the alpha X chain was obtained by preparative reversed phase high performance liquid chromatography. Structural analyses identified an Ala----Pro replacement at position 130 of the alpha chain. The instability of the variant, named Hb Sun Prairie, is comparable to that of Hb Bibba [alpha 136 (H19)Leu----Pro]. Gene mapping failed to detect an alpha-thalassemia deletion (alpha alpha/alpha alpha), while dot-blot analysis of amplified DNA with synthetic probes localized a G----C mutation in codon 130 (resulting in the Ala----Pro mutation) of the alpha 2-globin genes of both chromosomes. These results suggest a homozygosity for the G----C mutation and the condition alpha 2(G----C)alpha 1/alpha 2(G----C)alpha 1 adequately explains the rather severe clinical status of this child, including the marked microcytosis and hypochromia. Unfortunately, family studies to exclude the presence of a large deletion involving all zeta- and alpha-globin genes were not possible.

Two sons of a previously reported Ghanaian homozygote for the hereditary persistence of fetal hemoglobin (HPFH) (Ringelhann et al., 1970) also are HPFH homozygotes. In addition, another unrelated adult Ghanaian homozygote has been detected. All of these Ghanaian homozygotes as well as three American Black HPFH homozygotes have the G gamma A gamma type of HPFH with a G gamma to A gamma ratio of about 3:2, in contrast to an Asiatic Indian homozygote who has the G gamma type. Globin chain synthesis in HPFH homozygotes is unbalanced, with a gamma/alpha ratio of 0.6 or less, whereas it is balanced in heterozygotes according to most reports.

Hb Hofu, alpha 2 beta 2 [126 (H4) Va1 leads to Glu], was found in 10 members of 2 apparently unrelated Valmiki families in central India. None showed evidence of hemolysis and hemoglobin levels were normal in most. In two individuals, Hb Hofu occurred in combination with Hg S, but neither had clinical manifestations of sickle cell disease. In samples containing Hb Hofu, the isopropanol precipitation test was positive. Quantitation of the hemoglobin fractions by DEAE-cellulose chromatography showed that Hb Hofu constituted a mean of 23--25% of the total whether in combination with Hb A or Hb S.

An Indian (Asian) patient with compound heterozygosity for Hb Riyadh and beta 0-thalassemia is described. Hb Riyadh forms about 95% of the hemoglobin present. The clinico-pathological picture is identical to that of simple beta-thalassemia trait confirming the harmless nature of the substitution beta 120(GH3) Lys leads to Asn.

Restriction endonuclease analyses of DNA from one Black G gamma A gamma-HPFH homozygote and four Black and one Indian G gamma A gamma-HPFH heterozygotes have identified three different HPFH types which are the result of large deletions including the delta and beta 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 G gamma A gamma-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 gamma and the A gamma chains, an observation supported by family data. The highest ratio is found in the heterozygote with the Indian type III G gamma A gamma-HPFH, with 69.3% G gamma chains, while the averages for the other types were 50.7% G gamma (type I) and 32.3% G gamma (type II).

The gamma chain compositions of the fetal hemoglobins of 2453 newborn babies from East Asian countries (1350 babies), from Italy, Yugoslavia, Bulgaria, and Georgia (417 Caucasian babies), and 686 black babies from Georgia were determined by high pressure liquid chromatography. Unusual results for a limited number of babies were confirmed by chemical analyses, and were evaluated further by family studies. Statistical analyses indicated high gene frequencies for the A gamma T chain in Italian (f = 0.237), Yugoslavian and Bulgarian (f = 0.238), and white Georgia babies (f = 0.224), a lower frequency in Japan (f = 0.178), and India (f = 0.173), and particularly in mainland China (f = 0.079). The A gamma T gene frequency in normal (AA) Black babies was 0.102. When a beta S or beta C mutation was also present this frequency was greatly decreased, particularly in babies with the AC condition (f = 0.036). These results suggest the near absence of the A gamma T mutation on the chromosome also carrying the beta C determinant. Most babies had the expected G gamma values which vary between 60 and 80%, but several (mainly black) babies had higher values (between 80 and 90%), while one normal black baby had a G gamma value of (nearly) 100%. This condition may be a form of A gamma +1-thalassemia and has been discussed in detail elsewhere (Blood 58:491-500, 1981). Thirty-five clinically normal (mainly Chinese, Indian, and Japanese) babies had G gamma values of about 40%. Twenty-six babies had A gamma I values of about 60%, while the remaining nine babies had A gamma T and A gamma I chains in a ratio of either 1 to 2 or 1 to 1. Two additional newborns did not produce any G gamma chains, but had only A gamma I chains or A gamma T chains. Family studies failed to indicate a specific hematological abnormality. These unusual ratios between the G gamma and A gamma (either A gamma I or A gamma T) chains have led to speculations regarding possible genetic abnormalities present in these infants.

Population surveys and family studies among 568 members of nine ethnic groups in southern India identified 15 homozygotes for sickle hemoglobin (HbS)who had mild clinical and hematological manifestations with high levels of fetal hemoglobin (mean=20%, range 8-36%) in a heterogeneous red cell distribution. In one family, the heterozygous mother had a hemoglobin pattern consistent with a form of the heterocellular hereditary persistence of fetal hemoglobin. Sickle cell trait was found in 153(27%) of those studied. Chromatographic quantitation of the hemoglobin fractions in these heterozygotes showed a trimodal distribution of the proportion of HB Sexplicable by a genetic model postulating the presence of genotypes with two (-alpha/-alpha), three (-alpha/alpha alpha) and four (alpha alpha/alpha alpha) active alpha-globin genes. Globin synthesis studies in four heterozygotes believed to have two active alpha-globin genes demonstrated an alpha/non-alpha total activity ratio (0.57) consistent with this model.