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Technical Briefs |
-Globin Gene Cluster Containing Both the –3.7 and anti-4.2 Crossover Junctions: Clinical Diagnostic Implications and Possible Mechanisms
1 Department of Pediatrics, National University of Singapore, Singapore; 2 Division of Hematology, Department of Pathology, The University of Hong Kong and Queen Mary Hospital, Hong Kong, People’s Republic of China; 3 Molecular Diagnosis Center, National University Hospital, Singapore;
aaddress correspondence to this author at: Department of Pediatrics, National University of Singapore, Level 4, National University Hospital, 5 Lower Kent Ridge Road, Singapore 119074, Singapore; fax 65-6779-7486, e-mail paecs{at}nus.edu.sg
Misalignment of the homologous regions of the 
-globin gene cluster and unequal crossover during meiosis produce single -globin gene deletions (–) and reciprocal -globin gene triplications (). Further unequal crossover of such recombinant alleles with wild-type alleles may produce more complex derivative alleles, such as quadruplicated alleles (1)(2)(3). Complex crossover events producing "patchwork" genes have also been reported at the human - and ß-globin gene cluster (4)(5)(6). In this report, we describe the identification of a novel rearrangement of the -globin gene cluster containing both the –3.7 and anti-4.2 crossover junctions. This allele was identified in 2 unrelated individuals and a parent in the course of screening by Southern analysis of patients with ß-thalassemia major and minor for -globin gene deletions (Table 1
). For patient 1, a routine -globin gene configuration Southern analysis was performed to screen for the presence of the –\-SEA -thalassemia deletion, a common amelioration factor of severe ß-thalassemia (7). In the case of patient 2, Southern analysis was performed to rule out the presence of the –\-SEA -thalassemia deletion, because hemoglobin H inclusion bodies typically present in -thalassemia are absent when there is concurrent ß-thalassemia (8).
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Southern analysis was performed by hybridizing [32P]dATP-labeled - or 
-globin gene probes to BamHI- or BglII-digested genomic DNA. With an -globin probe, an anti-4.2 triplication contributes an 18.2-kb hybridizing BamHI band and 16.8- and 7.4-kb BglII bands, whereas a –3.7 deletion contributes a 10.3-kb BamHI band and a 16.3-kb BglII band. With a -globin probe, both the anti-4.2 and –3.7 alleles contribute 5.9-kb and 10.8/11.3-kb BamHI bands, whereas anti-4.2 contributes 11.3/12.6-kb and 16.8-kb BglII bands, and –3.7 contributes 11.3/12.6-kb and 16.3-kb bands.
Southern analysis of the DNA of both patients revealed, instead, an unusual 
20-kb BglII band when hybridized with either the - or -globin probe (Fig. 1A
). This anomalous fragment was not consistent with any known deletion or triplication of the -globin locus and was attributed initially to a polymorphism on one allele that abolished the recognition sequence at an internal BglII site located between the 2- and 1-globin genes.
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The DNA of both patients was also analyzed with multiplex-PCR assays to detect common -globin gene deletions and triplications, as described previously (9)(10). Surprisingly, the PCR results showed that they were positive for the –3.7 and anti-4.2 alleles (Fig. 1B
). The mother of patient 1, however, showed identical Southern and multiplex-PCR results; she was positive for the presence of the anomalous 20-kb BglII fragment by Southern analysis (data not shown) as well as the –3.7 and anti-4.2 fragments by PCR (Fig. 1B
). Furthermore, analysis of the father of patient 1 showed that he had a completely normal -globin genotype. On the basis of this pedigree analysis, compound heterozygosity for –3.7 and anti-4.2 was thus excluded in both the affected patient and her mother. Parental genomic DNA was unavailable for patient 2.
To account for the discordant Southern and PCR results in patient 1 and her mother, as well as in patient 2, we hypothesized that all 3 individuals are in fact heterozygous for a novel rearrangement of the -globin gene cluster containing both the –3.7 and anti-4.2 unequal crossover junctions. This novel allele, which we refer to as the HK allele, contains neither a single gene deletion nor a triplication (see Fig. 1
in the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol51/issue11).
We developed a 2-round nested PCR strategy to confirm the presence of the novel HK allele. The first-round PCR was designed to amplify a DNA segment flanked by the X1 and Z1 boxes. Each reaction was performed in a 50-µL volume and contained the following: 200 ng of genomic DNA; 0.2 µM each of the primers L-anti4.2-F (5'-CCTTGCACCGGCCCTTCCTGGTC-3'; HSGG1 34525
34547) and L-3.7-R (5'-CCTCAAAGCACTCTAGGGTCCAGCG-3'; HSGG1 3858638562); 200 µM each deoxynucleoside triphosphate; 1.1 mM Mg(OAc)2; 1 M GC-Melt; and 1x AdvantageTM-GC Genome polymerase in 1x supplied PCR buffer (BD Biosciences). Thermal cycling was performed in a T3 instrument (Biometra). Initial denaturation at 95 °C for 1 min was followed by 35 step-cycles of incubation at 94 °C for 30 s, 68 °C for 6 min, and a final extension at 60 °C for 3 min. A 10-µL portion of each PCR product was analyzed by electrophoresis through a 1% agarose gel in 1x Tris-borate-EDTA buffer at 15 V/cm for 1 h.
In the presence of a wild-type allele () or a triplicated anti-4.2 allele, an 4-kb fragment containing the X1, Y1, and Z1 boxes will be amplified, whereas a slightly larger 4.5-kb fragment containing the X1/X2 hybrid box, the Y2 box, and the Z2/Z1 hybrid box will be amplified when the HK allele is present (see Fig. 1
in the online Data Supplement). It was not possible, however, to unambiguously distinguish between the similar 4-kb and 4.5-kb fragments after the first-round PCR (Fig. 1C
, top panel).
The second-round PCR involved 2 separate reactions: a –3.7 PCR and an anti-4.2 nested PCR. Each reaction used 2 µL of the first-round PCR-amplified product as template. The –3.7 nested PCR reaction was performed as described (9), except that the reaction contained only the –3.7 primer pair. In the presence of the Z2/Z1 hybrid box (i.e., the –3.7 junction fragment), an 2-kb amplification fragment is detected. The anti-4.2 nested PCR reaction was performed as described (10), except that the reaction contained only the anti-4.2 primer pair. In the presence of the X1/X2 hybrid box (i.e., the anti-4.2 junction fragment), an 1.7-kb amplification fragment is detected. We performed agarose gel electrophoresis using 10 µL of each nested PCR product, as described above.
Under our hypothesis, the 4.5-kb first-round PCR amplicon of the HK allele contains both the –3.7 and anti-4.2 crossover junctions (see Fig. 1
in the online Data Supplement) and thus should yield positive fragments of the correct size after both second-round PCR reactions. Conversely, no nested PCR fragments would be generated from the 4-kb first-round PCR amplicons of wild-type or anti-4.2 triplicated alleles. As predicted, an 2-kb –3.7 junction fragment and an 1.7-kb anti-4.2 junction fragment were observed only in patient 1 and her mother, as well as in patient 2, but not in the father of patient 1, a wild-type control, and a –3.7/anti-4.2 compound heterozygous control (Fig. 1C
, middle and bottom panels). These results thus confirm that patient 1, her mother, and patient 2 are heterozygous for the novel HK allele, fully explaining the previous Southern, multiplex-PCR, and pedigree analysis (Fig. 1D
) results.
Because the HK allele contains neither deletion nor triplication, carriers of this novel allele are unlikely to suffer any deleterious effects; however, the existence of such a rearrangement in individuals has important implications for a PCR-based -thalassemia molecular diagnosis. Most PCR-based assays for -globin single gene deletions and triplications detect unequal crossover junctions; therefore, for DNA samples that are positive for both the –3.7 and anti-4.2 junction fragments by PCR analysis, it is no longer possible to definitively make a conclusive diagnosis of compound heterozygous –3.7 and anti-4.2. Further confirmation is necessary, either by Southern analysis or the 2-round nested PCR analysis described in this report, or by pedigree analysis of parents and siblings, if available.
Further confirmatory analysis is required only when the PCR results are positive for both the –3.7 and anti-4.2 junction fragments. Given the rarity of the –3.7/anti-4.2 compound heterozygous genotype, as well as the presumed rarity of the HK allele, such additional analyses are unlikely to be necessary on most occasions, and PCR-based testing for single gene deletions and triplications are likely to continue being widely used in diagnostic laboratories.
The novel HK allele documented in this report could have originated through one of several mechanisms. The first involves a nonreciprocal gene conversion event (see Fig. 2 in the online Data Supplement). The second involves a simultaneous double crossover between misaligned X and Z boxes (see Fig. 3 in the online Data Supplement). More likely, we believe, the HK allele originated via an intermediate recombinant allele such as the –3.7 or anti-4.2 allele, or both. Three possibilities are likely under this assumption. The first involves unequal crossover between the Z1 box of a wild-type allele and the Z2 box of an anti-4.2 allele to generate the HK derivative and its reciprocal anti3.7 derivative (see Fig. 4 in the online Data Supplement). The second involves unequal crossover between the X1 box of a wild-type allele and the X2 box of a –3.7 allele, giving rise to the novel HK derivative and its reciprocal –4.2 derivative (see Fig. 5 in the online Data Supplement). This is a likely mechanism of origin of the HK allele involving an intermediate allele, because the –3.7 carrier state is quite common in the population. The third involves unequal crossover between a –3.7 allele and an anti-4.2 allele, occurring in the X2 to Z2 region, leading to the HK derivative and a reciprocal (wild-type) derivative (see Fig. 6 in the online Data Supplement). This is a low-probability mechanism, given the rarity of concurrence of the –3.7 and anti-4.2 alleles.
Acknowledgments
This study was supported in part by Grant NMRC/0732/2003 from the National Medical Research Council to S.S.C.
References
Anti-3.7 type II: a new -globin gene rearrangement suggesting that the -globin gene duplication could be caused by intrachromosomal recombination. Hum Genet 1992;89:37-41.[CrossRef][Web of Science][Medline]
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globin gene arrangements. Br J Haematol 1987;66:245-250.[Web of Science][Medline]
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2 globin gene among American blacks. Hum Genet 1988;79:68-72.[CrossRef][Web of Science][Medline]
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-thalassemia (SEA) deletion ameliorates the clinical phenotype of ß(0)/ß(+) but not necessarily ß(0)/ß(0) thalassemia. Haematologica 2002;87:443-444.-globin gene deletion in ß-thalassemia carriers and prevention of hydrops fetalis. Haematologica 2000;85:991-993.-thalassemia. Blood 2001;98:250-251.-globin gene triplications. Clin Chem 2003;49:1679-1682.
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