L.-H. Gu

Molecular Characterization of α-Thalassemia Determinants, β-Thalassemia Alleles, and βs Haplotypes among Kuwaiti Arabs

Using amplification, allele-specific oligonucleotide (ASO) hybridization and DNA sequencing we have documented the molecular basis of 64 α- and 123 β-thalassemia (thai) chromosomes, and the haplotypes

Types of Thalassemia Among Patients Attending a Large University Clinic in Kuala Lumpur, Malaysia

We have identified the beta-thalassemia mutations in 59 patients with thalassemia major and 47 patients with Hb E-beta-thalassemia, and the deletional and nondeletional alpha-thalassemia determinants in 23 out of 24 patients with Hb H disease. All persons were attending the Haematology Clinic at the National University of Malaysia in Kuala Lumpur (Malaysia). Most patients (76) were of Malay descent, while 52 patients were Chinese, and two came from elsewhere. The most frequently occurring beta-thalassemia alleles among the Malay patients were IVS-I-5 (G----C) and G----A at codon 26 (Hb E), while a few others were present at lower frequencies. The Chinese patients carried the mutation characteristic for Chinese [mainly codons 41/42 (-TTCT) and IVS-II-654 (C----T)]; Malay mutations were not observed among Chinese and Chinese mutations were virtually absent in the Malay patients. The large group of patients with Hb E-beta-thalassemia and different beta-thalassemia alleles offered the opportunity of comparing hematological data; information obtained for patients with Hb E-beta-thalassemia living in other countries was included in this comparison. Twenty-three patients with Hb H disease carried the Southeast Asian (SEA) alpha-thalassemia-1 deletion; 13 had the alpha CS alpha (Constant Spring) nondeletional alpha-thalassemia-2 determinant, while the deletional alpha-thalassemia-2 (-3.7 or -4.2 kb) was present in 10 subjects. The --/alpha CS alpha condition appeared to be the most severe with higher Hb H values. Both deletional and nondeletional types of alpha-thalassemia-2 were seen among Malay and Chinese patients.

Molecular characterization of β-thalassemia in Azerbaijan

We have analyzed the β-thalassemia mutations in 99 chromosomes of 49 adults with β-thalassemia major and of one with Hb S-β-thalassemia, who are regular patients at a large hematology clinic in Bakü, Azerbaijan. A total of 20 different mutants were identified; three [frameshift at codon 8 (-AA); IVS-II-I (G→A); IVS-I-110 (G→A)] were present in about two-thirds of all chromosomes. Most alleles are the same as found in Mediterranean populations; a few have an Asian origin or come from Kurdistan, Lebanon, Saudi Arabia, or a black population. One mutant [frameshift at codons 82/ 83 (-G)] might be specific for the Azerbaijanian population. Nearly all patients were transfused, which made quantitation of Hb F impossible; highGγ values were present in the Hb F of those patients whose β-thalassemia chromosome carried the C → T mutation at position — 158 in the promoter of the Gγ-globin gene.

Hb H disease caused by a homozygosity for the AATAAA→AATAAG mutation in the polyadenylation site of the α2-globin gene : hematological observations

We have identified 7 patients with Hb H disease as homozygotes for a mutation in the polyadenylation site (AATAAA-->AATAAG) and have compared their hematological data with those of Hb H patients having other types of alpha-thalassemia determinants. All 7 patients exhibited moderate anemia with microcytosis and hypochromia being similar to that observed in the other patients. Relatives with a heterozygosity for this mutation are borderline microcytic and hypochromic without a significant anemia but with a low in vitro alpha/beta chain synthesis ratio. Analyses of the hemoglobin components identified low levels of Hb A2 and Hb H that were comparable to those found in other patients with Hb H disease; the level of the zeta-chain was low (average 0.14%).

γ Chain Abnormalities and γ-Globin Gene Rearrangements in Newborn Babies of Various Populations

The present review provides a summary of quantitative hemoglobin data and lists the results of gene mapping and sequencing analyses for blood samples from newborn babies of different countries. Methodology suitable for such studies is reviewed, various abnormal fetal hemoglobins are discussed, the occurrence of Hb Barts (γ4) and of the embryonic chain is evaluated, and the various types of γ-globin gene rearrangements (−Aγ. Aγ-; −Gγ. Gγ-; γ-thalassemia; γ-globin gene triplications, quadruplications, and quintuplications) are compared. The several tables list the frequencies of the common AγT variant and of the different γ gene rearrangements in various populations, while the results of quantitative analyses suggest that most anomalies are not associated with disease.

Hb Lepore-Baltimore (δ68Leu-β84Thr) and Hb Lepore-Washington-Boston (δ87Gln-βIVS-II-8) in Central Portugal and Spanish Alta Extremadura

Abstract Hb Lepore is one of the most common abnormal haemoglobins in Caucasians in Central Portugal and in the Spanish Alta Extremadura (0.28% in a survey of school children). A group of 19 Portuguese and 14 Spanish Hb Lepore carriers (all unrelated) was characterised at the molecular level by the polymerase chain reaction, sequencing and restriction enzyme analysis. The Portuguese and one Spanish carrier were heterozygous for Hb Lepore-Baltimore, whereas all other Spanish subjects were Hb Lepore-Washington-Boston carriers. Sequencing of the Hb Lepore-Baltimore gene further established the crossover at δ68-β84, a region two codons (CDs) shorter than that previously described and easily confirmed by digestion with MaeI and BanI. Data from haplotype analysis suggest that this crossover occurred as an independent event on the lberian Peninsula. The haematological data were similar in both groups except for the levels of Hb F and the Gγ chain, which were significantly higher in the Hb Lepore-Baltimore heterozygotes. Quantification of the globin chains and the mRNA transcripts showed that the δβ gene is transcribed at a higher level than the δ gene with levels of translation giving rise to 10%–15% of Hb Lepore. The different levels of Hb F observed in the two groups are the results of the higher transcription rate of the γ genes in Hb Lepore-Baltimore heterozygotes and an apparently less efficient translation of Gγ genes in Hb Lepore-Washington-Boston heterozygotes.

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.

The relative levels of βA and βS mRNAs in Hb S heterozygotes and in patients with Hb S-β+ -thalassaemia or Hb S-β+ -HPFH combinations

SUMMARY. We have used a quantitative reverse transcription/ polymerase chain reaction (RT/PCR) procedure to evaluate the relative amounts of βA and βS mRNA transcripts in eight subjects with a simple Hb S heterozygosity, in six with Hb S‐β+ ‐thalassaemia (thal), and in three individuals with Hb S‐β+ ‐HPFH [hereditary persistence of fetal haemoglobin (Hb)] [two with the Atlanta type and one with the Gγ‐202 (C ± G) substitution]. A balanced synthesis of βA and βS mRNAs was observed in all Hb S heterozygotes, whereas the βA mRNA levels were reduced to ˜ 16% of that of the βS mRNA in the six Hb S‐β+ ‐thal compound heterozygotes, to ˜43% in the two subjects with Hb S‐β+ ‐HPFH (Atlanta type), and to 23.8% in the one individual with Hb S‐β+ ‐HPFH [Gγ‐202 (C ± G) substitution]. The higher Hb A versus Hb S levels observed in all groups of the patients studied, further confirm a post‐translational control mechanism in determining the levels of Hb A and Hb S in the peripheral blood of these individuals. The procedure described here provides an accurate and easy method for studying the relative expression of particular globin genes at the transcriptional level in patients with various haemoglobinopathies.

A Second Observation of the Fetal Methehoglobin Variant HB F-M-Fort Ripley or α2Gγ292(F8)HIS→TYR

We have identified a second baby with the fetal methemoglobin F-M-Fort Ripley. It was observed in a Caucasian infant from Canada; at least eleven additional members of that family were known to have had a neonatal cyanosis similar to that seen in the propositus and in a previously described baby (2). Sequencing of amplified DNA that included (part of) the Gγ gene greatly facilitated the characterization. The Gγx chain was readily isolated by reversed phase high performance liquid chromatography; its quantity was ∼12.5% of total γ. Interestingly, the baby also carried the AγT mutation on one chromosome, either in cis or in trans to the Gγx mutation. Hb F-M-Fort Ripley could be isolated in reasonably pure form by DEAE-cellulose chromatography. The isolated Hb Fx was unstable, had spectral changes characteristic for the M-hemoglobins, while its methemqglobin derivative reacted rapidly with cyanide. Oxygen affinity data could not be obtained. It is suggested that the formation of a rather large amount (∼25%) ...

Hb Graz or α2β22(Na2)His→Leu; a new β Chain Variant Observed in four Families from Southern Austria

Two abnormal hemoglobins were accidentally detected by cation exchange high performance liquid chromatography with the Diamat system of Bio-Rad Laboratories; the variants eluted together with the fast-moving Hb A1c. Structural analysis of isolated β chains and sequence analysis of amplified DNA identified a new variant, i.e. Hb Graz that has a His→Leu replacement at position 2 of the β chain, in four healthy, apparently unrelated, adults. The second variant was identical to Hb Sherwood Forest or α2β2104(G6)Arg→Thr; it is believed that this may be the second observation of this abnormal hemoglobin.