An Entirely Novel Form of α Thalassemia in Patients from the South Pacific Linked to Chromosome 16.

Although a great deal is known about the molecular mechanism underlying α thalassemia (thal), which is characterized by reduced or absent α-globin expression, the molecular basis of α thalassemia in Pacific Islanders is not fully understood. Previously single α gene deletion alleles (−α^3.7 and −α^4.2) were the only types of α thalassemia identified in this population, however these genotypes do not account for several cases of Hb H disease in the Melanesian Islanders of Papua New Guinea (PNG) and Vanuatu. To date, no mutations have been found in the α globin gene cluster of these individuals. To determine the cause of α thalassemia in this population, we studied 35 individuals from three unrelated families of Pacific Islanders. 16 of them had classical Hb H disease characterized by mild to moderate anemia (Hb; 10.3 ± 2.3 g/dL), microcytosis (MCV; 66 ± 3.5 fL), hypochromia (MCH; 19.8 ± 1.6 pg), positive Hb H by electrophoresis (5–18%) and numerous Hb H inclusion bodies in peripheral blood. Gene mapping revealed that six had normal genotypes (αα/αα) while the rest were heterozygotes for α⁺ thalassemia (−α/αα). Preliminary sequence analyses demonstrated that the α globin genes and their regulatory elements were entirely normal casting some doubt on whether this novel form of α thal is genuinely linked to chromosome 16. Using a combination of a long range haplotype analysis by MALDI-TOF to simultaneously genotype 80 SNPs covering 350 kb of the telomeric end of chromosome 16 (16p; 13.3) and several tandem repeat markers (VNTRs and HVRs), we demonstrated that the α thal phenotype is strongly associated with a single haplotype (IIIa;PNG) found in these cases. Consistent with the Hb H patients, individuals from these families and another 15 unrelated individuals (4.7%) identified in a population survey (n = 315) who were hypochromic (MCH < 25 pg), despite having normal α genes (αα/αα) and iron status, were also found to carry the same type of chromosome 16 linked to the affected haplotype. These findings indicate that this form of α thalassemia is unequivocally linked to chromosome 16.

Subsequent linkage disequilibrium analysis (LD) in these cases limited the chromosomal interval containing the mutation to within 169 kb from the 16p-telomere. Extensive genomic mapping using more than 10 restriction enzymes and 37 plasmid and PCR probes together with Pulse Field Gel Electrophoresis (PFGE), covering 400 kb of 16p, excluded small deletions, insertions, rearrangements and chromosomal truncations in the affected segment of chromosome 16. Direct genomic sequencing of collectively 40 kb of the α globin genes, all known multispecies conserved sequences (MCS-R, 1–4) demonstrated no apparent mutation. Interestingly, interspecific somatic cell hybrid analysis of the affected chromosome 16 in a mouse erythroid leukemia (MEL) background did not recapitulate the phenotype of α thalassemia. These results suggest a novel mechanism, either at the genetic or epigenetic level, underlying the reduced expression of the α globin genes in the Pacific Islanders and understanding such mechanism might provide more insights on the complexity of stage- and tissue-specific gene regulation in human erythroid environment.