A. D. Adekile

βs haplotypes in various world populations

SummaryWe have determined the βs haplotypes in 709 patients with sickle cell anemia, 30 with SC disease, 91 with S-β-thalassemia, and in 322 Hb S heterozygotes from different countries. The methodology concerned the detection of mutations in the promoter sequences of the Gγ- and Aγ-globin genes through dot blot analysis of amplified DNA with 32P-labeled probes, and an analysis of isolated Hb F by reversed phase high performance liquid chromatography to detect the presence of the AγT chain [Aγ75 (E19) Ile→Thr] that is characteristic for haplotype 17 (Cameroon). The results support previously published data obtained with conventional methodology that indicates that the βs gene arose separately in different locations. The present methodology has the advantage of being relatively inexpensive and fast, allowing the collection of a vast body of data in a short period of time. It also offers the opportunity of identifying unusual βs haplotypes that may be associated with a milder expression of the disease. The numerous blood samples obtained from many SS patients living in different countries made it possible to compare their hematological data. Such information is included (as average values) for 395 SS patients with haplotype 19/19, for 2 with haplotype 17/17, for 50 with haplotype 20/20, for 2 with haplotype 3/3, and for 37 with haplotype 31/31. Some information on haplotype characteristics of normal βA chromosomes is also presented.

Possible factors influencing the haemoglobin and fetal haemoglobin levels in patients with β‐thalassaemia due to a homozygosity for the IVS‐I‐6 (T→C) mutation

Summary. We have collected haematological, haemoglobin (Hb) and DNA sequence data for 29 patients with a homozygosity for the IVS‐I‐6 (TC) mutation with the intention of identifying factors contributing to the observed variability in the severity of the disease. None of the patients had received blood transfusion therapy for at least 6 months prior to the study. Hb levels varied from 5·0 to 9·9 g/dl. Patients with high Hb F (more than 1·5 g/dl or <20%) had high total Hb levels (7·5–9·7 g/dl) but some with low Hb F also had high total Hb levels; two had a concomitant α‐thalassaemia‐2 (α‐thal‐2) heterozygosity. An inverse correlation between the Hb F and Hb A2 levels was observed. The majority of the patients were homozygous for haplotype VI (49/58 chromosomes) but haplotypes IV (2/58) and VII (7/58) were also present. The only haplotype IV homozygote had high Hb F levels with high Gγ values and the CT mutation at position – 158 in the Gγ promoter, while both high and low Hb F levels were observed among patients with haplotypes VI and VII. Analysis of sequence variations in regulatory regions included the 5 hypersensitive sites (HS) 4, 3 and 2 of the locus control region (LCR), the Gγ and Aγ 5 flanking regions, the second intervening sequence (IVS‐II), and the 5 β‐globin gene region in two patients with high Hb F (one homozygote each for haplotypes VI and IV), and in two patients with low Hb F levels (one homozygote each for haplotypes VI and VII). Haplotype specific differences were observed in the LCR 5 HS‐2 and in the Gγ and Aγ flanking and IVS‐II regions; however, no differences were present between the low and high Hb F‐producing haplotype VI chromosomes, suggesting a major role for factors which are not linked to the β‐globin gene cluster in mediating γ‐globin gene expression in patients with this type of β‐thal.

The influence of uridine diphosphate glucuronosyl transferase 1A promoter polymorphisms, βS‐globin gene haplotype, co‐inherited α‐thalassemia trait and Hb F on steady‐state serum bilirubin levels in sickle cell anemia

Abstract:  Purpose: Homozygosity for the (AT)7 allele of uridine diphosphate glucuronosyl transferase 1A (UGT1A1) gene polymorphism is associated with increased bilirubin levels in sickle cell anemia (SCA). In the present study, in addition to UGT1A1 promoter genotype, serum bilirubin level was related to other genetic modifiers –βS‐globin gene haplotype, Hb F, co‐inherited α‐thal trait, age and gender. Methods: The patients were randomly selected from the sickle cell clinic, Medical College of Georgia. UGT1A1 promoter polymorphisms were determined using automated sequencing. Other investigations were with standard techniques. Results: There were 67 SCA patients (41 males and 26 females), aged 2–44 yr (mean of 20.6 ± 10.7). Ten (14.9%) patients were homozygous for the (AT)6 UGT1A1 allele, 35 (52.2%) were heterozygous for (AT)6 and (AT)7 alleles while 22 (32.8%) were homozygous for (AT)7. Serum bilirubin was significantly higher in the homozygous (AT)7 group (3.7 ± 1.5, 3.8 ± 2.3 and 5.6 ± 2.4 mg/dL, respectively). It was also significantly higher in males than females and in patients aged >10 yr. There was a significant negative linear correlation (r = −0.304, P = 0.016) of serum bilirubin with Hb F. The β‐globin haplotype and co‐existing α‐thal trait did not have any significant influence on serum bilirubin levels. Patients on hydroxyurea were older, had lower Hb F, but higher mean serum bilirubin. The latter also was signifcantly higher among those with UGT1A1 (AT)7 homozygosity. Conclusions: Apart from UGT1A1 (AT)7 homozygosity, Hb F, age and gender are the other factors that significantly influence serum bilirubin level in SCA.

Haplotypes in SS patients from Nigeria; characterization of one atypical beta S haplotype no. 19 (Benin) associated with elevated HB F and high G gamma levels.

SummaryWe have determined the haplotypes of 669 βS and 109 βA chromosomes from numerous members of 297 Nigerian families of various ethnic backgrounds. Among the βS chromosomes, haplotype 19 was detected in 93.2%, haplotype 17 in 3.4%, and haplotype 20 in 0.1%, while 2.4% represented atypical haplotypes. As many as 60.6% of the βA chromosomes exhibited haplotype 19 mutations, 8.2% had haplotype 3, and 1.8% had haplotype 20. Two siblings with elevated Hb F andGγ levels were heterozygous for a βS chromosome with haplotype 19 and a second chromosome with a hybrid haplotype (termed 19B). In this hybrid chromosome, haplotype 3-like locus control region (LCR) [hypersensitive site-2 (HS-2)] sequences are in juxtaposition to those of the 5′ flanking region of theGγ promoter of a βS chromosome with haplotype 19. The presence of this hybrid chromosome is associated with highGγ values in individuals with both sickle cell anemia (SS) and sickle cell trait (AS); it closely resembles another hybrid βS chromosome, termed 19 A, observed in a previously reported Turkish SS patient who was homozygous for this chromosome and had high Hb F and highGγ values. In both instances, it is hypothesized that the haplotype 3-like sequences of the LCR HS-2 contain genetic determinants that can combine with factors produced during hematopoietic stress, resulting in increased γ-globin gene expression.

Sickle cell anemia, sickle cell β-thalassemia, and thalassemia major in Albania: characterization of mutations

We have analyzed the hemoglobin abnormalities in nearly 50 Albanian patients with a significant hemoglobinopathy and included 37 relatives in this study. Sickle cell anemia (SS) is a common disorder; all 15 sickle cell anemia patients had the complications expected for this disease. The βs haplotype was type 19 (Benin); α-thalassemia-2 was rare. Three β-thalassemia alleles (IVS-I-110, G→A; codon 39, C→T; IVS-I-6, T→C) were present in nearly 85% of the β-thalassemia alleles; their frequencies were intermediate between those observed in the populations of neighboring countries. A few rare mutations were also found, which might have originated in India, Turkey, Macedonia, and Greece. Nearly all patients with Hb S-β-thalassemia had the IVS-I-110 (G→A) mutation. The frequencies of 11 β-thalassemia mutations in 17 mostly Mediterranean countries have been reviewed.

Level of Fetal Hemoglobin in Children with Sickle Cell Anemia Influence of Gender, Haplotype and α-Thalassemia-2 Trait

We have evaluated hematological data for numerous blood samples collected from 51 sickle cell anemia (SS) patients during their first 10 years of life. Haplotypes and alpha-globin gene status were determined in all patients. A total of 482 hematological evaluations and 317 fetal hemoglobin (Hb F) determinations are presented. The data show that the Hb F levels are the highest in patients with haplotypes 3/3 and the lowest in those with haplotypes 20/20. This is reflected in differences in total hemoglobin, PCV value, and RBC counts, which are the lowest in the 20/20 group and the highest in the 3/3 group. Female SS patients with haplotypes 19/19 have higher Hb F levels than their male counterparts; the same was observed for the patients with the 19/3 haplotype combination but not for those with the 20/3 haplotype combination. A concomitant alpha-thalassemia-2 heterozygosity had little or no effect on the hemoglobin level. The data confirm that SS children, aged 1-10 years, with a homozygosity for haplotype No. 20 are most severely affected by their disease.

A new Variant, Hb Muscat [α2β232(B14)Leu→val] Observed in Association With Hb S in an Arabian Family

The silent Hb Muscat with a Leu→Val replacement at position β32 was discovered by reversed phase high performance liquid chromatography in two members of an Arabian family from Oman; in one person Hb Muscat occurred with Hb S and in the other with Hb A. Hb Muscat is slightly unstable but its presence has no apparent adverse effect on the health of its carriers. Additional hemoglobin abnormalities observed in this family were a common a-thal-assemia-2 (-3.7 kb) and Hb S. The βS haplotypes in the heterozygous carriers and the two sickle cell anemia patients were #19 (Benin) and #20 (Bantu); the latter likely originated from an East African population.

The in vivo expression of the globin genes of theβ cistron in γ-,δ-, andδβ-thalassemia heterozygotes

There is considerable evidence suggesting that the switch from γ to δ and β chain production after birth is due, in part, to silencing of the γ genes by stage-specific factors which bind to their promoters and to the competition from the adult (δ and β) genes for a common enhancer element located in the locus control region. As a consequence one can expect that the increased Hb F production in adults with hereditary persistence of fetal hemoglobin or δβ-thalassemia is directed mainly by γ-globin genes in cis to the deletion(s) responsible for these conditions. Here we review data on heterozygotes with γ-, δ-, or δβ-thalassemia, who also had anAγT mutation, in cis or in trans, which was used as a marker of γ gene expression. The results show that a deletion affecting adult β genes favors the expression of γ genes in cis, while the deletion of a single γ gene does not affect the expression of the β gene in cis but leads to a faster γ→β switch postnatally.