Hb St. Jozef, A Val-->Leu N-terminal mutation leading to retention of the methionine, and partial acetylation found in the globin gene in Cis with a -alpha3.7 thalassemia deletion.

We report a new hemoglobin (Hb) variant found in a 6-year-old girl of Moroccan origin, living in the Dutch city of Gouda. The child was referred because of microcytic and hypochromic parameters. A normal zinc protoporphyirin (ZPP) value excluded iron deficiency and gap-polymerase chain reaction (gap-PCR) revealed a heterozygosity for the common -alpha(3.7) thalassemia deletion, partially justifying the hematological picture. The Hb pattern on alkaline electrophoresis and capillary electrophoresis was normal, while a fraction of 9% preceding the Hb A peak, remained visible on different high performance liquid chromatography (HPLC) devices. This fraction, located in front of the Hb A peak, is usually considered as a Hb A derivate that becomes more expressed in older samples. However, the sample was freshly collected and the peak unusually evident. Therefore, direct sequencing of the alpha-globin genes was performed revealing a GTG-->CTG transversion at codon 1 of the alpha1-globin gene or of the hybrid gene. This point mutation induces a single amino acid substitution from valine to leucine. Electrospray-mass spectrometry (ES-MS) analysis revealed, in addition to this substitution, that the N-terminal methionine was retained and that about 20% of the variant was acetylated. As expected for an association with a -alpha(3.7)-thalassemia (thal) deletion, the non acetylated and acetylated abnormal alpha chain amounted to 32% of the total alpha chains. Family studies revealed that the mutated codon was located in cis of the deletion.

The rare Hb Showa-Yakushiji [beta110(G12)Leu-->Pro, CTG-->CCG] in combination with an alpha gene triplication found in a Dutch patient during her first pregnancy examination.

We report a semi dominant beta-thalassemia (thal) phenotype caused by the rare Hb Showa-Yakushiji [beta110(G12)Leu-->Pro, CTG-->CCG] mutation in combination with an alpha gene triplication. This combination of two rare mutations was observed during hemoglobinopathy carrier diagnostics in a 26-year-old Dutch female at 9 weeks gestation, at the first pregnancy examination in the midwives practice. The partner was promptly examined and no abnormalities were found. The beta-thal trait was diagnosed by a standard high performance liquid chromatography (HPLC) procedure showing a normal separation but an elevated Hb A(2) level of 5.9% in the presence of pronounced hypochromic microcytic parameters and mild chronic hemolysis. Direct sequencing of the beta-globin genes was subsequently performed revealing a CTG-->CCG transition at codon 110. This rare mutation was previously described as two independent events in a few Japanese and Indian individuals. The mutation induces a Leu-->Pro substitution and the gene product is highly unstable. Gap-polymerase chain reaction (gap-PCR) revealed a heterozygosity for the alpha gene triplication as well. The excess of alpha-globin chains contributed only marginally to the hematological abnormalities of the patient and did not aggravate the phenotype to an intermediate level.

Hb Bleuland [alpha108(G15)Thr-->Asn, ACC-->AAC (alpha2)]: a new abnormal hemoglobin associated with a mild alpha-thalassemia phenotype.

We report a new structural defect of the alpha2-globin chain, not detectable on high performance liquid chromatography (HPLC) or electrophoresis, characterized in a 12-year-old boy of Surinamese-Hindustani origin. The child was suspected to be a carrier of alpha-thalassemia (thal) because of microcytic hypochromic parameters in the absence of iron depletion. Gap-polymerase chain reaction (gap-PCR) revealed only normal fragments in the proband, and the pattern of a -alpha4.2 (leftward) deletion in his father and sister. Direct sequencing of the alpha-globin genes revealed an ACC-->AAC transversion at codon 108 of the alpha2-globin gene in the proband, in his mother and in a younger sister. The new mutation predicts a Thr -->Asn amino acid substitution at the corresponding residue. Threonine, a covalent binder with an R-active OH group, situated in the G helix of the alpha-globin chain, is involved in alpha1beta1 contacts. Asparagine, being an equally covalent binder but with a different R-active H2N-C=O group, could make the mutated chain less suitable for tetramer cooperation. Alternatively, an absent or reduced interaction with the alpha hemoglobin (Hb) stabilizing protein (AHSP) could lead to loss of alpha chains. Hb Bleuland is the first mutation described at codon 108 and is therefore interesting in regard to the possible effects and genetic risk. The nearest variant, Hb Suan-Dok [alpha109(G16)Leu -->Arg, CTG-->CGG (alpha2)] was originally observed in a Thai patient affected with Hb H, in combination with an alpha0-thal allele. The same Hb Suan-Dok mutation, recently described in our laboratory in a carrier of African ancestry, was also not detectable as a protein and presented with an alpha-thal phenotype similar to Hb Bleuland.

Hb Buffalo [alpha89(FG1)His-->Gln (alpha1)], observed solely and in the presence of an Hb S [beta6(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 S1c 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 alpha1 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 alpha chain is expressed at the expected rate and without thalassemic effect or instability. The mutated alpha chain seems to associate with a slight preference to the beta(A) (15%) rather than with the beta(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 alpha1 gene.

A new Hb evanston allele [alpha14(A12)Trp --> Arg] found solely, and in the presence of common alpha-thalassemia deletions, in three independent Asian cases.

Hb Evanston [alpha14(A12)Trp --> Arg] is considered to be a rare alpha chain mutant, and was originally observed in two Black families in 1982, inducing a mild Hb H disease phenotype in a homozygous state for the -alpha3.7 deletion ( -alpha(Evanston)/ -alpha). The mutant, evidently linked with one of the two -alpha3.7 thalassemia (thal) alleles, was considered to be unstable and rapidly proteolyzed. We describe Hb Evanston in three new independent Asian cases, all induced by a TGG --> CGG transition. In all cases the mutation is linked to the alpha1-globin gene, either on a wild type allele or in linkage with the common -alpha3.7 and -alpha4.2 deletion alleles. The beta/alpha ratio was balanced in the presence of the mutation only, and accordingly unbalanced in co-inheritance with the deletion defects. Although a second independent mutation event on a -alpha3.7 or a -alpha4.2 deletion allele could not be excluded, we conclude that at least one independent Hb Evanston mutation has occurred on a wild type allele in the Asian populations. Unstable Hb tetramers tend to degrade and disappear during purification. Both Hb Evanston tetramers, formed in combination with normal beta and delta chains, remain perfectly stable after extensive purification and concentration steps, suggesting an early posttranslational thalassemic effect, probably at the dimer/tetramer affinity level.