F. Kutlar

Separation of normal and abnormal hemoglobin chains by reversed-phase high-performance liquid chromatography

A review is presented of the elution patterns on reversed-phase columns of the normal and abnormal globin chains of different hemoglobin types, including 16 beta-chain variants, 7 alpha-chain variants, 9 gamma-chain variants, and 4 variants with fusion or hybrid chains. Separations appear to be based primarily on differences in hydrophobicity. The method is ideally suited for the detection of abnormal globin chains, their quantitation and their isolation. Semi-quantitative data based on the calculation of the delta/non-alpha ratios allow the detection of beta-thalassemic conditions in situations where the quantitation of hemoglobin A2 by other procedures is impossible or complicated.

Fetal hemoglobin in normal adults and β-thalassemia heterozygotes

SummaryA recently developed high performance liquid chromatographic (HPLC) procedure using a weak cation exchanger (PolyCAT) in columns of different sizes was used to quantify fetal hemoglobin (Hb F) in blood of normal adults and β-thalassemia (β-thal) heterozygotes with ten different types of mutations. Preparative PolyCATHPLC greatly facilitated the characterization of isolated Hb F, i.e., the determination of the relative quantities of the Gγ and Aγ chains. The method is accurate and allows quantitation of Hb F at the 0.5% level; preparative PolyCAT-HPLC allows isolation of (nearly) pure Hb F from blood samples with low (< 1%) Hb F. Adult Hb F levels were determined in 69 normal adults (including 24 diabetics); Hb F levels fell below 1% except for subjects with abnormal -Gγ-Gγ arrangement and a C→T mutation at position -158 relative to the Cap site of both Gγ genes. The effect of the same mutation in the normal-Gγ-Aγ-arrangement was variable. Certain β-thal mutations (namely, those at positions -29;-88; IVS-I-1; IVS-II-1) were associated with high Hb F levels in heterozygotes, while those at nucleotide (nt) positions IVS-I-6; IVS-I-110; codon 24; codon 39; codons 41/42; IVS-II-745 were not. Gγ values varied and often fell into two groups (high Gγ and low Gγ); high Gγ values were not associated with high Hb F values. The chromatographic procedure is ideally suited for Hb A2 quantitation. Average values of Hb A2 in β-thal heterozygotes with any one of nine of the ten mutations were twice that of normals; the one exception was the β-thal heterozygote with the IVS-I-6 (T→C) mutation with an average low Hb A2 value of 3.6%.

β-Thalassemia in Turkey

A review is presented of the various β-thalassemia alleles observed in nearly 191 patients with β-thalassemia major and their 182 heterozygous relatives. Determination was by gene amplification and dot-blot hybridization with synthetic probes, specific for 27 different mutations. Eighteen mutations have been observed; six of these acount for nearly 83% of all thalassemia abnormalities (Table I). A new mutation, i.e. a G ↣ C mutation at the acceptor splice site of IVS-I, was found in one teenager who was homozygous for this disease. the high consanguinity among the families was considered the main reason for the high number of patients with a homozygosity for the IVS-I-110 (G ↣ A) mutation. Combinations of different mutations were present in many patients; some were mildly affected because of the specific mutation present on one chromosome. Combinations of classical β-thalassemia and an abnormal hemoglobin mainly concerned Hb S. Hbs Knossos and Lepore were rare occurrences. A comparison of hematological da...

Molecular characterization of β-globin gene mutations in Malay patients with Hb E-β-thalassaemia and thalassaemia major

Summary. This study concerned the identification of the β‐thalassaemia mutations that were present in 27 Malay patients with Hb E‐β‐thalassaemia and seven Malay patients with thalassaemia major who were from West Malaysia. Nearly 50% of all β‐thalassaemia chromosomes carried the G → C substitution at nucleotide 5 of IVS‐I; the commonly occurring Chinese anomalies such as the frameshift at codons 41 and 42, the nonsense mutation A → T at codon 17, the A → G substitution at position −28 of the promoter region, and the C → T substitution at position 654 of the second intron, were rare or absent. Two new thalassaemia mutations were discovered. The first involves a frameshift at codon 35 (‐C) that was found in two patients with Hb E‐β°‐thalassaemia and causes a β°‐thalassaemia because a stop codon is present at codon 60. The second is an AAC → AGC mutation in codon 19 that was present on six chromosomes. This substitution results in the production of an abnormal β chain (β‐Malay) that has an Asn → Ser substitution at position β19. Hb Malay is a‘Hb Knossos‐like’β+‐thalassaemia abnormality; the A → G mutation at codon 19 likely creates an alternate splicing site between codons 17 and 18, reducing the efficiency of the normal donor splice site at IVS‐I to about 60%.

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.

Haplotypes of βS chromosomes among patients with sickle cell anemia from Georgia

Fetal hemoglobin and G gamma levels have been correlated with the presence or absence of eight restriction sites within the beta globin gene cluster (haplotypes) for numerous sickle cell anemia patients from Georgia. The most common haplotypes were #19 (Benin) and #20 (CAR); all patients with haplotype combinations 19/19, 20/20, and 19/20 were severely affected with low Hb F and low G gamma levels. A modified #19 beta S chromosome with a -G gamma-G gamma- globin gene arrangement, instead of -G gamma-A gamma-, was present in SS and SC newborn babies with G gamma values above 80%. Haplotype #3 (Senegal) was present among 15% of the beta S chromosomes; the two adult patients with the 3/3 combination were mildly affected with high Hb F and G gamma values. The haplotype AT with the variant A gamma T chain was a rarity. A new haplotype was found in one 17-year-old SS patient and five of his Hb S heterozygous relatives. This haplotype is associated with an increased production of Hb F in heterozygous and homozygous Hb S individuals; this Hb F contained primarily A gamma chains. A comparison was made between the different haplotypes among SS patients and normal Black individuals, and a remarkable similarity was noted in the fetal hemoglobin data for subjects with these different chromosomes.

Molecular characterization of β‐thalassaemia in 174 Greek patients with thalassaemia major

The mutations producing β‐thalassaemia in 174 Greek patients with thalassaemia major were investigated by dot‐blot hybridization of oligonucleotide probes to genomic DNA amplified by the polymerase chain reaction procedure, by direct sequencing of amplified DNA, and by gene mapping. β‐thalassaemia in Greeks was found to be very heterogeneous at the molecular level as 17 different mutations were observed; 86.6% of the β‐thalassaemic genes, however, could be identified with five probes: IVS‐I‐110 (G→A) (42.5%), codon 39 (C→T) (17%), IVS‐I‐1 (G→A) (13.2%), IVS‐I‐6 (T°C) (7.2%) and IVS‐II‐745 (C→G) (6→9%). Several mutations which had not previously been reported in the Greek population and which occurred at an incidence of 2% or lower were observed in this study. The information obtained will facilitate the prenatal diagnosis of β‐thalassaemia in Greece.

Sickle cell anaemia among Eti-Turks: haematological, clinical and genetic observations

Summary. Haematological and genetic observations have been made on 71 SS Eti‐Turk patients and their relatives from Çukurova (southern Turkey) and of immigrant families in The Netherlands. Similar data were collected for 2 5 Black patients and their relatives from Surinam, Netherlands Antilles, and Kenya. Haematological and clinical results were the same for both groups; the haemolytic anaemia in the Turkish patients was as severe as in the others. Haplotyping, involving nine restriction sites, identified haplotype 19 (Antonar‐akis et al, 1984) as the major type among the Eti‐Turks; this chromosome has previously primarily been observed among SS patients from West Africa. The suggestion that the βS‐chromosome among Eti‐Turks originates from that area is supported by a relatively high incidence of α‐thalassaemia‐2 (the 3.7 kb deletion), also frequently present in the Black population of West Africa, and by the absence of other major haplotypes, such as types 20 and 3, characteristic for the βS‐chromosome in the population of Central Africa and Kenya, and in Senegal, respectively. The Saudi Arabian type of βS chromosome in association with the haplotype 19 βS chromosome was present in only one Eti‐Turk patient; this 30‐year‐old female was mildly affected and exhibited a high level of fetal haemoglobin.

β-Thalassemia, HB S-β-Thalassemia and Sickle Cell Anemia Among Tunisians

We analyzed the mutations present in 19 patients with beta-thalassemia major, in 11 patients with Hb S-beta-thalassemia, and the beta S haplotypes of 34 patients with sickle cell anemia. The study included 84 relatives. Dot-blot analysis of amplified DNA with various specific oligonucleotide probes identified 11 different known beta-thalassemia mutations and frameshifts; a new frameshift at codons 25/26 (+T) was detected through sequencing of amplified DNA. The common beta-thalassemia mutations at codon 39 (C----T) and at IVS-I-110 (G----A) were also most prevalent among the Tunisian patients, while the milder T----C mutation at IVS-I-6 was not found. All mutations cause a beta 0-thalassemia or a severe beta + -thalassemia [T----A at -30; IVS-I-5 (G----A); IVS-I-110 (G----A)] which explains the need for regular blood transfusions in the thalassemia major and S-beta-thalassemia patients. Nearly all sickle cell anemia patients carried the beta S mutation on a chromosome with haplotype 19 (or Benin) and all had severe anemia with sickling complications. Identification of the beta S haplotype was through dot-blot analysis with oligonucleotide probes that detect mutations in the G gamma and A gamma promoter sequences, specific for this haplotype.

Diagnostic Approach to Hemoglobinopathies

Abnormalities of hemoglobin (Hb) synthesis are among the most common inherited disorders of man and can be quantitative (thalassemia syndromes) or qualitative (variant Hbs). Definite identification of hemoglobinopathies can be achieved by a stepwise algorithmic approach, starting with a detailed clinical history, through hematologic evaluation [complete blood count (CBC)], reticulocyte count, red blood cell (RBC) morphology], protein based analytic methods [Hb electrophoresis or isoelectric focusing (IEF), cation exchange high performance liquid chromatography (HPLC), reversed phase HPLC] to nucleic acid based methods [such as polymerase chain reaction (PCR), reverse transcribed (RT)-PCR, sequencing of genomic DNA and sequencing of RT-PCR amplified globin cDNA of the gene of interest]. When an abnormality of Hb function (increased or decreased oxygen affinity) or stability (unstable Hb variants) is suspected from the phenotype, special confirmatory tests (determination of p50, Heinz body prep and isopropanol or heat stability tests) can be useful. Family studies are also helpful in certain cases. A review of the application of these methods to the diagnosis of hemoglobinopathies at the Sickle Cell Center Laboratory in Augusta, GA, USA, is presented below.