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The nondeletional types of hereditary persistence of fetal hemoglobin (ndHPFH) concern the continued synthesis of hemoglobin (Hb) F with either Gγor Aγchains in amounts varying from 5% to 30%. Several mutations have been identified in either the Aγor Gγpromoter which are considered causative to the continued production of one of the two 7 chains because the substitutions occur in sequence motifs essential for the expression characteristics of the γ-globin gene in the 3’ position. We report the discovery of a T → C mutation at position - 175 in the Aγpromoter which was associated with a greatly increased level of Hb F (with mainly Aγ) and a decreased level of Hb A in the one (Black) heterozygote who had a βcgene in trans. The same mutation has been observed in the Gγpromoter of a Black heterozygote who had high levels of Hb F with Gγchains only. A detailed comparison between these two individuals indicated significant differences in the levels of Hb F and Hb A which may result from an additional mutation at position - 158 in the Gγpromoter. © 1989 by Grune & Stratton, Inc.

Sequence analyses and dot-blot analyses with synthetic oligonucleotide probes have identified eight individuals in three Turkish families and one Bulgarian family with one chromosome having a C → T mutation at nucleotide position -101 relative to the Cap site of the β-globin gene. This nucleotide is part of one of the conserved blocks of nucleotides within the promoter region; in vitro expression analyses with the chloramphenicol acetyltransferase system showed that this substitution will decrease the effectiveness of transcription. Five subjects had a thalassemia intermedia due to the additional presence of a known classical high hemoglobin (Hb) A2β-thalassemia mutation on the second chromosome; their hematologic condition was relatively mild. The three persons with a heterozygosity for the -101 C -→ T mutation had normal hematologic data without microcytosis but with high-normal levels of Hb A2and a mild imbalance in chain synthesis. The newly discovered mutation is considered one of the silent types of β-thalassemia. It is relatively rare because it was absent among several hundred normal and β-thalassemia chromosomes.

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

The presence of various substitutions and deletions resulting in β-thalassemia was studied in 19 black patients with homozygous β-thalassemia and in numerous relatives; all patients were from Georgia, South Carolina, and Alabama. Methodology included gene mapping, amplification of genomic DNA with Taq polymerase, identification of known nucleotide substitutions or a single nucleotide deletion through hybridization with synthetic oligonucleotides, cloning and sequencing of a β-globin gene, and sequencing of amplified genomic DNA. Of the 38 chromosomes tested, 21 (55%) had the A →G substitution at nt -29, eight (21%) had the C → T substitution at nt —88, three (8%) had the substitution at codon 24, while one each of the following abnormalities were also detected: frameshift at codon 6, a C → A mutation at nt 848 of the βIVS-II (new), an A→ T mutation at codon 61 (new), a deletion of 1.35 kilobases including the 5’ end of β a Gγ(Aγδβ)°-thalassemia, and one thalassemia determinant that remained unidentified. The C → A mutation at nt 848 of IVS-II occurred at a position 3 nucleotides 5’ to the third exon, adjacent to the invariant AG dinucleotide of the acceptor sequence. The A →T mutation in codon 61 (AAG → TAG) resulted in the creation of a stop codon and thus in β°-thalassemia. The various mutations occurred on chromosomes with different haplotypes; however, chromosomes with a specific mutation but with different haplotypes belonged to one specific framework, which suggested that crossovers were responsible for these different types. Hemoglobin (Hb) F levels were generally high (55% to 75% with 98.5% in one patient with β°/β°); a few patients with specific haplotypes and an a-thalassemia-2 heterozygosity had a lower Hb F level. The Gγ in the Hb F was consistently high when the C →T mutation occurred at nt — 158 to the Cap site of the Gγ-globin gene; seven patients with + / + at this site had an average Gγof 73.8%, eight patients with + /- had 64.8%, and one patient with — / — had 34.2%. Variations in hematologic values and in Hb F, Gγ, and Hb A2 levels of relatives with a β-thalassemia heterozygosity depended to some extent on the types of mutations or deletions and on the haplotypes of the chromosomes with the β-thalasse-mia determinant.© 1988 by Grune & Stratton, Inc. 0006-4971/88/7203-0014$3.00/0

We have determined the nucleotide sequence of the 2,360-bp long EcoRlfragment from four chromosomes; this fragment is located 3’ to the Aγ globin gene and is considered to contain the enhancer element identified by Bodine and Ley.2The chromosomes were from an Arabian sickle cell anemia patient with high Hb F and a homozygosity for haplotype No 31 and from a black sickle cell anemia patient with low Hb F and a homozygosity for haplotype No 19. A third chromosome carried the determinant for a nondele-tional hereditary persistence of fetal hemoglobin seen in a Chinese subject, and the fourth was a normal chromosome from a Yugoslavian subject. Twenty-one differences were observed when a comparison was made with the published sequence; no differences were seen between the sequences of the four different samples except for an additional mutation in the Chinese. These data make it unlikely that specific mutations within this sequence are associated with increases in Gγ and Aγ production.

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.