Hemoglobin D-Los Angeles [β121(Gh4)Glu→Gln] in the Province of Liège,Belgium

Since 1975, more than 19,000 persons have been tested for hemoglobin abnormalities in Liege. Nine unrelated carriers of Hb D-Los Angeles were discovered. They were of Belgian origin. The family of the ninth index case was studied thoroughly. In six generations, 372 descendants of a couple married in 1818 were recorded. Of the 93 tested, 31 were carriers of the Hb D variant. The complete pedigree indicates that Hb D-Los Angeles was already present in Liege province in the 18th century. A connection with India is unlikely.     

The β-GLobin Gene Haplotypes Associated With Hb D-Los Angeles [β121(GH4)Glu→Gln] in Western Iran

Hb D-Los Angeles is characterized by the substitution of glutamine for glutamic acid at position 121 of the β-globin chain. The present investigation is the first study on the β-globin gene haplotypes associated with β-D-Los Angeles in Western Iran. Thirty two individuals from 11 unrelated families from Western Iran were studied. The Hb D-Los Angeles status of all cases was confirmed using polymerase chain reaction (PCR) followed by digestion with EcoRI. The haplotype of the β‐globin gene cluster was determined by a PCR-RFLP (restriction fragment length polymorphism) procedure. The haplotype background of the β^A chromosomes was also determined in 35 normal subjects from the same geographic region. The β-globin gene haplotype analysis demonstrated that all β-D-Los Angeles genes (23 genes) were in linkage disequilibrium with haplotype I [+????++]. Among the 70 β^A chromosomes, 30 chromosomes (42.9%) were associated with haplotype I. The present study indicates the unicentric origin of the β-D-Los Angeles gene in Western Iran. It seems that this mutation may have occurred at the same chromosomal background common in the local population.     

Genetic Origin of Hb D-Los Angeles [β121(GH4)Glu→Gln,GAA→CAA] According to the β-Globin Gene Cluster Haplotypes

Hb D-Los Angeles (also known as D-Punjab, D-North Carolina, D-Portugal, D-Chicago and Oak Ridge) is an abnormal hemoglobin (Hb) with an amino acid substitution of glutamine for glutamic acid at codon 121 of the β-globin gene. The origin and spread of Hb D-Los Angeles is not known. This is due to lack of information and remains to be elucidated. According to published reports, the Hb D-Los Angeles mutation is mostly linked with Mediterranean haplotype I [+ ? ? ? ? + +]. Besides the Mediterranean haplotype, a novel haplotype was also reported from Thailand [? ? + + ? ? + + +]. Here we report a new haplotype from Turkey [? + ?? + + +] that has not been described before. These results suggest that the Hb D-Los Angeles mutation has at least three different genetic origins.     

MOLECULAR CHARACTERIZATION OF Hb D-PUNJAB [β121(GH4)Glu→Gln] IN THAILAND

We describe the hematological and DNA characterization of Hb D-Punjab [β121(GH4)Glu→Gln] in Thailand. Nine patients from five unrelated families were studied; four patients were simple carriers of Hb D-Punjab, two were compound heterozygotes for Hb D-Punjab/β⁺-thalassemia;another two patients were double heterozygotes for Hb D-Punjab and α-thalassemia-2, and one patient was a compound heterozygote for Hb D-Punjab and Hb E [β26(B8)Glu→Lys]. Typical thalassemic indices with hypochromic microcytosis were observed in compound Hb D-Punjab/β⁺-thalassemia and Hb D-Punjab/Hb E but normal hematological profiles were observed in the remaining cases. DNA sequencing of the β-globin gene identified the G AA→C AA substitution at codon 121 causing Hb D-Punjab in all cases, and the ?28 (A→G) mutation for the β⁺-thalassemia alleles. β-Globin gene haplotype analysis demonstrated, for the first time, that all these Asian β^D-Punjab globin genes were associated with haplotype [?++?+++], previously undescribed in other populations. The finding of Hb D-Punjab in Thailand is compatible either with an independent origin of this abnormal hemoglobin or a spread of the Hb D-Punjab gene with a single origin among Asians.     

Multi Centric Origin of Hb D-Punjab [β121(GH4)Glu→Gln,GAA>CAA]

Hb D-Punjab [β121(GH4)Glu→Gln, GAA>CAA], common in the northern Indian province, is often unexpectedly found in other populations. To study the multi centric origin of this variant which is causing sickle cell disease in association with Hb S [β6(A3)Glu→Val, GAG>GTG], we have examined the haplotype of the Hb D allele in different populations. We studied 43 alleles from south Iran (Hormozgan and Fars provinces) and 14 from Holland and Belgium using high performance liquid chromatography (HPLC), capillary electrophoresis, direct sequencing and/or restriction enzyme analysis. In Iranians, four haplotypes were observed at different frequencies: haplotype I [+ ? ? – ?,+ +] at 67.5%, subhaplotype I' [+ – – – –,– +] at 17.5%, haplotype V [– + – – +,+ +] at 10.0% and haplotype III [– + – + +,+ +] at 5.0%. All European cases were on haplotype I. The occurrence of high Hb D frequencies on a single haplotype in specific regions can be expected if we consider founder effect and genetic drift mechanisms. However, considering that haplotype I is the most common haplotype worldwide, that Hb D-Punjab is reported in different populations on different haplotypes, and that codon β121 is a site on which six different mutations are reported, we may expect to observe Hb D-Punjab in different populations, possibly because of a relatively higher occurrence of de novo mutations, generating unexpected risk from mixtures of allochtonous Hb S and indigenous Hb D-Punjab or vice versa.     

Absolute Identification of Hb D-Los Angeles (β121 GLU → GLN)

This paper describes a method which permits the absolute identification of Hb D-Los Angeles Bithout the need for N-terminal or amino acid analysis on peptide β T-13. The procedure involves the formation of the 5-oxo-2-pyrrolidinecarboxylyl derivative of the N-terminal glutamine residue of β ^DT-13. This method, together with the use of cadmium ncetate/ninhydrin staining, allows the identification of Hb D-Los Angeles, and other possible mutntions of p121, by peptide mapping.     

Hb Beograd [β121(GH4)Glu→Val,GAA→GTA] in the Turkish Population

Hb Beograd [β121(GH4)Glu→Val, GAA→GTA] is a rare variant first reported in Yugoslavia and then in Turkey, Australia and New Zealand. We report two further unrelated cases from Turkey. The importance of identifying Hb Beograd at the molecular level, especially in regions where Hb D-Los Angeles [β121(GH4)Glu→Gln, GAA→CAA] is prevalent, is emphasized.     

β-globin gene haplotypes linked with the Hb D-Punjab [β121(GH4)Glu→Gln, GAA>CAA] mutation in eastern India

Hb D-Punjab [β121(GH4)Glu→Gln] is prevalent in the northern states of the Indian subcontinent. Due to inadequate data from Asian countries, the origin and spread of the Hb D-Punjab mutation are uncertain. In a study of sickle cell hemoglobinopathies, we detected the Hb D-Punjab mutation in 25 subjects from 11 unrelated Agharia families. Twelve cases were Hb S [β6(A3)Glu→Val]/Hb D-Punjab compound heterozygotes and 13 were Hb D trait carriers. In 76.0% of the cases, the β(D) gene was linked to haplotype I, whereas 24.0% had a novel haplotype. None of the haplotypes matched the β(A) haplotype of the local population. In view of the ancestral origin of the subjects and the high prevalence of the β(D) gene in the states of northern India, we suggest a North Indian origin for the β(D) mutation in our population. The finding of a novel haplotype in eastern India supports the hypothesis of a multicentric origin of this mutation.     

β-Globin Gene Haplotypes Linked with the Hb D-Punjab [β121(GH4)Glu→Gln,GAA>CAA] Mutation in Eastern India

Hb D-Punjab [β121(GH4)Glu→Gln] is prevalent in the northern states of the Indian subcontinent. Due to inadequate data from Asian countries, the origin and spread of the Hb D-Punjab mutation are uncertain. In a study of sickle cell hemoglobinopathies, we detected the Hb D-Punjab mutation in 25 subjects from 11 unrelated Agharia families. Twelve cases were Hb S [β6(A3)Glu→Val]/Hb D-Punjab compound heterozygotes and 13 were Hb D trait carriers. In 76.0% of the cases, the β^D gene was linked to haplotype I, whereas 24.0% had a novel haplotype. None of the haplotypes matched the β^A haplotype of the local population. In view of the ancestral origin of the subjects and the high prevalence of the β^D gene in the states of northern India, we suggest a North Indian origin for the β^D mutation in our population. The finding of a novel haplotype in eastern India supports the hypothesis of a multicentric origin of this mutation.     

Hb Sarrebourg [β131(H9)Gln→Arg, CAG>CGG] in Turkey

We describe Hb Sarrebourg [β131(H9)Gln→Arg, CAG>CGG] in four heterozygous members of a Turkish family. It was associated with iron deficiency in the proband.     

The Thermal Stability of Hb O-Indonesia [α116(GH4)Glu→Lys]

Hb O-Indonesia was initially found in an Indonesian woman (1) and later reported in patients from South Africa and Italy (2). The mutation is α116 (GH4)Glu←Lys (3). There was no evidence of abnormal clinical properties associated with Hb O-Indonesia (1,2,3).     

Hb Beograd (α2β2 121 (GH4) Glu → Val) Observed In A New Yugoslavian Family

Hb Beograd was first discovered in a Yugoslavian family (1) while the same variant, under the name of Hb D Camperdown, was found in a Yugoslavian woman living in Australia (2). Recently, Wilkinson et al (3,4) reported two more Yugoslavian families with this variant. The present report describes the same variant in a new Yugoslavian family. The methodology used in these studies followed procedures routinely in use in our laboratories (5).     

Hb Buffalo [α89(FG1)His→Gln (α1)], Observed Solely and in the Presence of an Hb S [β6(A3)Glu→Val] Heterozygosity

The hemoglobin (Hb) pattern of a 32‐year‐old Somali male living in The Netherlands, during routine diabetes mellitus monitoring, showed two more peaks in addition to the characteristic heterozygous Hb A/S pattern. A major peak of 15% faster than Hb A, and a minor one of 10.8%, overlapping Hb A₂ and the glycated Hb S_1c fraction were present. The patient was not anemic or microcytic but had a low haptoglobin level, possibly indicating a slightly elevated red blood cell (RBC) turnover. Hb S was confirmed by a sickle test and at the DNA level. The DNA sequence of the α1 gene revealed a C→G transversion at position 89, changing the local positively charged histidine to a neutral glutamine. This mutant has been previously described in a Yemenite woman and two apparently unrelated Somali males. Our case is the first showing Hb Buffalo in combination with Hb S and a G6PD deficiency, and is again observed in a Somali. No functional abnormalities associated with mutations at this amino acid residue are reported in the literature. Also, in this case no sign of any hematological abnormalities that could not be explained by the Hb S heterozygosity G6PD deficiency was found. The abnormal α chain is expressed at the expected rate and without thalassemic effect or instability. The mutated α chain seems to associate with a slight preference to the β^A (15%) rather than with the β^S counterpart. The sum of both Hb A^Buffalo and Hb S^Buffalo results in about 19–20% of total Hb. This figure is in agreement with a stable mutant of the α1 gene.     

Hb I-High Wycombe [β59(E3)Lys→Glu]: The First Instance in Japan

15,661 cord bloods from Jamaican infants were examined for abnormal hemoglobins wine alkaline cellulose acetate electro-phoresis for the initial screening, supplemented by acid agar gel electrophoresis for samples exhibiting abnormal hemoglobin bands. Of the 16 electrophoretic variants which were detected, six were fully characterized and found to be: four Hb F Port Royal (α₂^Gγ₂125 Glu→Ala) and two Hb F Victoria Jubilee (α₂^Aγ₂80 Asp→Tyr). The Hb F Port Royal samples each constituted about one eighth of the total Hb F as did seven additional samples presumed to be Hb F Port Royal. The infants with this variant exhibited no special hematological characteristics or other consistent associations. Both Hb F Victoria Jubilee samples occurred in somewhat lower proportions of the total Hb F compared with Hb F Port Royal and exhibited an apparent increase of free alpha chains in the whole hemolysate. The data available on detectable gamma chain variants suggest that a specific point mutation may occur in either a Hb_Gγ or a Hb_Aγ locus.     

Incidence of the Hb E [β26(B8)Glu→Lys,GAG>AAG] Variant In Totos,One of the Smallest Primitive Tribes in the World

Toto is one of the smallest tribes in the world. This primitive sub Himalayan, endogamous tribe lives in a small, isolated village called Totopara in the Jalpaiguri district of West Bengal in India. The tribal communities of West Bengal are vulnerable to various genetic disorders such as β-thalassemia (β-thal). We have studied 443 Totos to define their Hb E [β26(B8)Glu→Lys, GAG>AAG] status. Awareness and screening camps have been organized in various parts of Totopara during the last 2 years. We collected 3 mL peripheral blood from each individual aseptically on which to use the naked eye single tube red cell osmotic fragility test (NESTROFT); complete hemogram and high performance liquid chromatography (HPLC) were done to detect their carrier status. The Hb E variant had been found to be prevalent among the Totos. To confirm the codon 26 (GAG>AAG) mutation in the β-globin gene, amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) was performed. Restriction fragment length polymorphism (RFLP)-PCR was carried out with 44 Hb E alleles to construct the haplotype(s) of the Totos. Our extensive studies have revealed that 49.21% of Totos are Hb E heterozygotes and 19.19% Totos are Hb E homozygotes. The most prevalent haplotype linked with the codon 26 mutation in the Totos is [+ ? ? ? ? ?] (HincII 5′?, HindIII ^Gγ, HindIII ^Aγ, HincII 5′ψβ, HincII 3′ψβ and HinfI 3′β). Consanguineous marriages have resulted in a significant increase of the percentages of heterozygotes and homozygotes of Hb E in the Totos. Genetic counseling is essential and important to prevent the spread of this mutation and hence to save them from having any kind of clinically significant hemoglobinopathy in the future.     

Complex Interaction of Hb E [β26(B8)Glu→Lys], Hb Korle-Bu [β73(E17)Asp→Asn] and a Deletional α-thalassemia-1 in Pregnancy

A pregnant Thai woman with mild hypochromic microcytic anemia caused by α- and β- globin defects is described. The proband was a 26-year-old pregnant woman discovered through our ongoing thalassemia screening program. Initial hemoglobin (Hb) high performance liquid chromatography (HPLC) analysis revealed a homozygosity for an unknown variant at the D window, inconsistent with results of family analyses. Further Hb analysis using automated capillary zone electrophoresis identified that the proband was in fact a compound heterozygote for Hb E [β26(B8)Glu→Lys, GAG>AAG] and another β chain variant. DNA analysis demonstrated that she carried the Hb Korle-Bu mutation [β73(E17)Asp→Asn (GAT>AAT)] in trans to the Hb E and an α-thalassemia-1 (α-thal-1) with the Southeast Asian (? ?^SEA) deletion. Family studies identified that her father and sister were double heterozygotes for Hb Korle-Bu and α-thal-1, whereas her mother was a double heterozygote for Hb E/Hb Constant Spring [Hb CS; α142, Term→Gln (TAA>CAA in α2)]. The genotype-phenotype relationship observed in this Thai family with complex hemoglobinopathies and methods for characterization are presented.