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 ␣-thalassemias are a group of hereditary disorders caused by reduced synthesis of the ␣-chain of hemoglobin. We have developed and tested an ␣-thalassemia assay that uses both multiplex ligationdependent probe amplification (MLPA) with Luminex-based detection and deletion PCR technologies. The MLPA assay consisted of 20 probes, 15 of which hybridized to the ␣-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 ␣-globin copies were identified in 76 patients. A deletion of one or two ␣-globin genes was identified in 1251 (23%) and 1349 (25%) specimens, respectively, including 15 different genotypes. A deletion of three (hemoglobin H) and four ␣-globin genes (Hb Bart's) was observed in 65 or 3 specimens, respectively. Six patients had a deletion within the ␣-globin regulatory region MCS-R2. Thus, MLPA plus deletion PCR identify multiple ␣-globin gene deletions/duplications in patients being tested for ␣-thalassemia. The ␣-thalassemias are a group of recessively inherited disorders caused by reduced or absent synthesis of the ␣-chain of hemoglobin (Hb). Approximately 5% of the world's population is thought to be affected by ␣-thalassemia, making it one of the most common genetic disorders.1 Because of its selective advantage, the carrier rate of ␣-thalassemia is extremely high (Ͼ90%) in tropical and subtropical regions with a history of endemic malaria. 2The phenotype of patients with ␣-thalassemia is variable depending on the number of dysfunctional or absent ␣-globin genes. Patients with one dysfunctional ␣-globin gene (silent carrier) are often asymptomatic and unaware they have the genetic disorder unless they have a complete blood count examined. Similarly, patients with a deletion of two of the four ␣-globin genes (␣-thalassemia trait) may be asymptomatic and require no specific treatment. Hemoglobin H (three ␣-globin genes deleted/dysfunctional) is often more serious and is characterized by microcytic hypochromic hemolytic anemia, splenomegaly, and jaundice. All four of the ␣-globin alleles are dysfunctional in patients with Hb Bart's, the most severe and often fatal form of ␣-thalassemia. 2,3The two genes responsible for synthesis of the ␣-chain of Hb, HBA1 and HBA2, are located in the telomeric region on chromosome 16 (16p13.3) and encode ␣ 1 -globin and ␣ 2 -globin, respectively.2 These two genes are embedded within two highly homologous 4-kb duplication units that can be subdivided into separate homology blocks, written X, Y, and Z.2 Misalignment and recombination within ...

Rapid and reliable detection of α-globin copy number variations by quantitative real-time PCR

“…Several methods have been developed to identify unknown deletions and duplications in the α-globin genes, using quantitative real-time PCR (real-time qPCR) assays [ 13 - 15 ] or multiplex ligation-dependent probe amplification (MLPA) assays [ 16 , 17 ]. Here, we describe a fast, robust and specific method, designated HBA-CNV, that measures copy number variation using real-time qPCR.…”

A novel perspective on MOL-PCR optimization and MAGPIX analysis of in-house multiplex foodborne pathogens detection assay

“…In comparison to the closely related MLPA, which requires about 20 hours to complete, the MOL-PCR assay can be performed very rapidly 4 . MLPA remains, even in the version adapted to the microsphere array and when using the same detection platform as MOL-PCR, tedious due to the number of steps or necessity for overnight hybridization 34 . The MOL-PCR assay is designed to be performed in 96-well plates, while simultaneously allowing each well to be screened for up to 50 markers, and provides high-confidence results within 6 h 6,12 .…”

Detection of α-Thalassemia by Using Multiplex Ligation-Dependent Probe Amplification as an Additional Method for Rare Mutations in Southern Turkey

“…2 The MLPA results revealed that the deleted regions expanded from 3.5 kb upstream HBZ to 0.2 kb downstream HBA1 (20 probe) in the two unrelated patients P6 and P7. Whereas the deleted regions expanded from 3.5 kb upstream HBZ to 0.5 kb downstream HBA1 (21 probe) in patient 16 (a, b) et al [10] reported a study comparing MLPA and SB Analysis in a-thalassemia and emphasized the importance of MLPA in defining patogenic mutations as a complimentary method to SB, rather than a stand alone technique. Colosimo et al [8] also identified MLPA as an effective method for screening a-thalassemia mutations.…”

“…A wide range of testing strategies have been used to detect deletions and mutations within the a-globin gene cluster, including Reverse Dot-Blot Hybridisation (RDB), Southern blot (SB), Gap-PCR, multiplex ligation dependent probe amplification (MLPA) and sequence analysis [8][9][10]. Since the MLPA technique was first described by Schouten et al [14], many centers have used this method to identify the a-globin gene cluster deletions [11].…”