Development and clinical implementation of a combination deletion PCR and multiplex ligation-dependent probe amplification assay for detecting deletions involving the human α-globin gene cluster

Abstract

The α-thalassemias are a group of hereditary disorders caused by reduced synthesis of the α-chain of hemoglobin. We have developed and tested an α-thalassemia assay that uses both multiplex ligation-dependent probe amplification (MLPA) with Luminex-based detection and deletion PCR technologies. The MLPA assay consisted of 20 probes, 15 of which hybridized to the α-globin gene cluster and 5 that served as control probes. A PCR assay was developed to confirm the presence of heterozygous/homozygous 3.7-kb and 4.2-kb deletions. MLPA and PCR results were compared to Southern blot (SB) results from 758 and 133 specimens, respectively. Lastly, MLPA and PCR results were reviewed and summarized from 5386 clinically tested specimens. SB and MLPA results were concordant in 678/687 (99%) specimens. PCR detected all deletions detected by SB with no false positives. No deletions or duplications were identified in 2630 (49%) clinically tested specimens. Extra α-globin copies were identified in 76 patients. A deletion of one or two α-globin genes was identified in 1251 (23%) and 1349 (25%) specimens, respectively, including 15 different genotypes. A deletion of three (hemoglobin H) and four α-globin genes (Hb Bart's) was observed in 65 or 3 specimens, respectively. Six patients had a deletion within the α-globin regulatory region MCS-R2. Thus, MLPA plus deletion PCR identify multiple α-globin gene deletions/duplications in patients being tested for α-thalassemia.

Figure 1

Location of MLPA probes (probes 6 to 20) and corresponding different-sized deletions that were observed during clinical testing within our laboratory. Probes 1 to 5 are excluded from this figure because they represent control/gender probes specific for other chromosomal regions that are not located within the α-globin gene cluster.

Figure 2

Matched PCR (top) and MLPA results (middle) from nine specimens labeled A–I. Probes 1 to 4 (in small boxes, in panels A–I) represent control probes targeting chromosomes 1p22, 3p22, 12p13, and 22q11, respectively. Probe 5 (circled) represents the gender probe targeting Xp21. The remaining 15 probes (probes 6 to 20) target the α-globin gene cluster. MLPA probes 6 to 20 and 3.7-kb/4.2-kb PCR primer targets are shown at the bottom of the figure. Het, heterozygous; Hom, homozygous.

Figure 3

MLPA results representing eight unique deletions. Probes 1 to 4 (in small boxes, in panels A–H) represent control probes targeting chromosomes 1p22, 3p22, 12p13, and 22q11, respectively. Probe 5 (circled) represents the gender probe targeting Xp21. The remaining 15 probes (probes 6 to 20) target the α-globin gene cluster as shown at the bottom of the figure. *αα/(αα)^T = deletion in MCS-R2 regulatory region.