CORRESPONDENCE RAPID DETECTION OF A 13.4-kb DELETION CAUSING S/3 THALASSEMIA IN AN EGYPTIAN FAMILY BY POLYMERASE CHAIN REACTION

At least 35 deletions involving the p globin cluster on chromosome 1 1 have been described.’ Analysis of the phenotypes associated with the different deletions has provided much insight into the normal regulation of expression of the human /3 globin gene cluster, leading to the identification of several important regulatory elements including the major upstream locus control region (LCR) 5‘ to the c Deletions of similar size but affecting different regions ofthe p cluster may give rise to different clinical entities such as Sp thalassemia and deletional hereditary persistence of fetal hemoglobin (HPFH); the distinction between the two conditions is subtle and is determined by the degree to which HbF is elevated and the red blood cell indices! Arguments based on genotype/phenotype correlations are strengthened by the accumulation of unrelated individuals of different ethnic origin with identical deletions, presenting with very similar phenotypes. We describe here an Egyptian family with So thalassemia caused by a 13.4-kb deletion that removes the 3’ region of the 6 gene, the entire p gene, and its 3’ flanking sequences extending into the 6.4kb LI repeat element. We have used the polymerase chain reaction (PCR)5 and direct sequencing to characterize the endpoints of this deletion which proved to be identical to those of the Sicilian (Sp)” thalassemia.6 Subsequently, an Italian individual with a similar phenotype was shown to be heterozygous for this deletion. The phenotypes of both the Egyptian and the Italian individuals are strikingly similar to those of the Sicilian individuals heterozygous for the same deletion. The hematologic parameters of individuals in the Egyptian family and the Italian patient are summarized in Table I and are compared with the average values reported for the previous cases of Sicilian Sp thalassemia.’ All individuals affected have hypochromic microcytic indices with substantially elevated HbF (4.8% to 9.6%) and normal hemoglobin A2 (HbA2) levels. The @ globin complex in the propositus of the Egyptian family was analyzed with a series of restriction enzymes and Southern blot hybridizations with specific globin gene probes. Hybridization with the $p probe proved to be informative. In addition to the expected normal fragments, abnormal-sized fragments were observed when DNA digested with Xmn I, BstE 11, HindII, and HindIII was hybridized with the $@ probe, but BamHI-$P hybridization produced the normal 15.3-kb band. The probe pRK29, which is located 19 kb 3’ to the /3 gene, was used to obtain data pertaining to the 3’ end of the deletion. When pRK29 was hybridized to DNA digested with a range of restriction enzymes, no abnormal bands were observed, with the exception of HindIII, which produced a band of approximately 30 kb that appeared to be identical in size to the HindIII-$(3 fragment. On the basis of the restriction mapping data (not shown), the 5‘ end ofthe deletion was localized to between the BamHI site at coordinate 55232 (Genbank HUMHBB), and the Hind111 site at coordinate 59607. The 3’ deletion breakpoint was shown to lie downstream of the BstE I1 site at coordinate 68829 by considering the data obtained from the relevant $0 probe hybridizations. PCR primers chosen to flank the putative breakpoints were synthesized and used to amplify a deletion specific fragment (Fig 1) containing the deletion breakpoints. A PCR product of approximately 1.8 kb was obtained, which predicted a deletion size of 13.4 kb as the primers are 15,210 bp apart in normal DNA. The amplification primers were biotinylated to facilitate the separation of single-stranded DNA for direct sequencing as previously described.8 The noncoding strand was sequenced using the sequencing primer SI (Fig 1). The deletion is 13,377 bp in length the 5’ breakpoint of the deletion lies within IVS-2 of the 6 gene at coordinate 55961 (Genbank HUMHBB), and the 3’ breakpoint lies at position 69338 within the full-length 6.4-kb L1 repeat located downstream of the p gene. Two orphan nucleotides, AC (sense) or TG (antisense), of unknown origin lie at the breakpoint making it identical to the Sicilian Sp thalassemia breakpoint as determined from cloned DNA by Henthorn et aI6 (Fig 2). DNA from the Italian individual (who is adopted) was subjected to similar restriction mapping and abnormal bands of the same size as in the Egyptian family were found. PCR using the same primers and conditions as described above and in Fig 1 produced an abnormal band of similar size to that observed for the affected Egyptian individuals. Furthermore, direct sequencing of the PCR product showed changes identical to those found in the Egyptian and Sicilian cases. In addition to the two orphan nucleotides, two single-base variations