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The unstable hemoglobin Montreal with a deletion of three amino acid residues (Asp, Gly, Leu) at positions 73, 74, and 75 of the β chain and an insertion of four residues (Ala, Arg, Cys, Gln) at the same location was observed in a 7-year-old Canadian boy suffering from a moderate hemolytic anemia. The introduction of an extra amino acid residue and of other changes in the crevice where the heme group is located is the likely cause of the instability of this hemoglobin variant. The above listed changes were detected through analyses of tryptic peptides of theβ-Montreal chain, sequencing of amplified DNA, and hybridization of amplified DNA with appropriate, 32P-labeled, oligonucleotide probes. It is suggested that a mispairing involving the AGTG sequences at codons 66 and 67 and at codons 72 and 73 of the normalβ gene caused a repetition of a 16-bp segment, while a deletion of 10 nucleotides due to recombination or slippage followed by a second short deletion during DNA repair resulted in the modified sequence of theβ-Montreal gene.

A novel deletion of ~27 kb with the 5’breakpoint 1.5 to 2.2 kb upstream of the β-globin gene, and the 3’breakpoint approximately 24 kb downstream of the β-globin gene, has been found in five members of two families from Southeast Asia (Vietnam and Cambodia). Six members of another family from China, previously reported from our laboratory, have also been shown to carry this deletion. The patients presented with mild hypochromia and microcytosis, a hemoglobin (Hb) A2level of approximately 4.0%, and a markedly increased, heterocellularly distributed, Hb F level (14.0 to 26.0%). In vitro globin-chain synthesis showed a mild imbalance with appreciable γ-chain compensation (α/β+ γratio of 1.46). The 3’end of this deletion includes the 3’HS-1, and we hypothesize that removal of this region results in the loss of its 7-globin gene-silencing effect, which causes a markedly elevated Hb F level with a modest increase in Hb A2levels, unlike the situation in other deletional β°-thalassemias. The possible influence of particular sequence variations in the locus control region 5 HS-2 and the Gγpromoter, present on the chromosome with this deletion, on the overall γ-globin gene should also be considered.

We have compared the sequence of the 5’ hypersensitive site-2 (5‘ ‘HS-2) of the locus control region (LCR) from a sickle cell anemia (SS) patient homozygous for haplotype 19 and with low levels of fetal hemoglobin (HbF), with the same sequence from an SS patient homozygous for haplotype 3 and with high levels of HbF. Several nucleotide variations were present in the 5’HS-2 of the haplotype 19 individual. One is the A → G at position -10905 that creates an Sp1 binding site GCCCC (A → GJCCCC. A second is the T G at position -10924 in a sequence that binds both erythroid and ubiquitous factors and exhibits high homology to the long terminal repeat of the Moloney leukemia viruses and Friend murine leukemia virus. Other differences were in the two AT-rich stretches of DNA, and an A →T substitution at position -10390. Dot-blot analyses of amplified DNA from several SS patients showed that these variations are specific for βs chromosomes with haplotype 19. We also examined the 5’HS-2 sequence from an SS patient who is homozygous for haplotype 19, but has abnormally high levels of HbF (>20%). We observed a cross-over that has placed sequences similar to the 5’HS-2 of haplotype 3 in juxtaposition to the 5’ flanking regions of haplotype 19. Thus, a βs chromosome with haplotype 19 but having a 5’HS-2 (LCR) characteristic for haplotype 3 is associated with high γ-chain expression. We postulate that factors produced under conditions of hematopoietic stress, together with genetic determinants on the haplotype 3-like LCR sequences, allow for high level expression of γ-globin genes.© 1992 by The American Society of Hematology. 0006-4971/92/7903-0024$3.00/0

We have amplified and sequenced the 5’ flanking and the second intervening sequence (IVS-II) regions of both the Gγ-and Aγ-globin genes of the βSchromosomes from sickle cell anemia (SS) patients with homozygosities for five different haplotypes. The sequencing data, compared with previously published sequences for the normal chromosomes A and B, show many similarities to chromosome B for haplotypes 19, 20, and 17, while haplotypes 3 and 31 are remarkably similar to chromosome A and also similar to each other. Several unique mutations were found in the 5’ flanking regions (Gγ and Aγ) of haplotypes 19 and 20 and in the IVS-II segments of the same genes of haplotypes 19, 20, and 17; the IVS-II of haplotypes 3 and 31 were identical to those of chromosome A. Dot-blot analyses of amplified DNA from additional SS patients with specific probes have confirmed that these mutations are unique for each haplotype. The two general patterns that have been observed among the five haplotypes have most probably arisen by gene conversion events between the A and B type chromosomes in the African population. These patterns correlate with high and low fetal hemoglobin expression, and it is speculated that these and other yet unknown gene conversions may contribute to the variations in hemoglobin F and Gγ levels observed among SS patients. In vitro expression experiments involving the ~ 1.3-kb 5’ flanking regions of the Gγ and Aγ-globin genes of the βSchromosomes with the five different haplotypes failed to detect differences between the levels of expression, suggesting that the sequence variations observed between these segments of DNA are not the primary cause of the differences in hemoglobin F levels among the SS patients.