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Development and clinical implementation of a combination deletion PCR and multiplex ligation-dependent probe amplification assay for detecting deletions involving the human ?-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 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 ?-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.

SUBMITTER: Kipp BR

PROVIDER: S-EPMC3157609 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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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.

Kipp Benjamin R BR Roellinger Samantha E SE Lundquist Patrick A PA Highsmith W Edward WE Dawson D Brian DB

The Journal of molecular diagnostics : JMD 20110625 5

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 ...[more]

PMID: 21708285

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Project description:Connective tissue diseases characterized by aortic aneurysm, such as Marfan syndrome, Loeys-Dietz syndrome and Ehlers Danlos syndrome type IV are heterogeneous and despite overlapping phenotypes, the natural history, clinical manifestations and interventional course for each diagnosis can be quite unique. The majority of mutations involved in the etiology of these disorders are missense and nonsense mutations. However, large deletions and duplications undetected by sequencing may be implicated in their pathogenesis, and may explain the apparent lack of genotype-phenotype correlation in a subset of patients. The objective of this study was to search for large pathogenic deletions and/or duplications in the FBN1, TGF?R1, and TGF?R2 genes using multiplex-ligation dependent probe amplification (MLPA) in patients with aortopathy, in whom no mutations in the FBN1, TGF?R1, and TGF?R2 genes were identified by sequencing.The study included 14 patients from 11 unrelated families with aortic aneurysm. Of those, six patients (including 3 first-degree relatives), fulfilled the revised Ghent criteria for Marfan syndrome, and eight had predominantly aortic aneurysm/dilatation with variable skeletal and craniofacial involvement. MLPA for FBN1, TGF?R1, and TGF?R2 was carried out in all patients. A 385 K chromosome 15 specific array was used in two patients with a deletion of the entire FBN1 in order to define its size and boundaries.We identified two novel large deletions in the FBN1 gene in four patients of two unrelated families who met clinical diagnostic criteria for Marfan syndrome. One patient was found to have a FBN1 deletion encompassing exons 1-5. The other three patients had a 542 Kb deletion spanning the whole FBN1 gene and five additional genes (SLC24A5, MYEF2, CTXN2, SLC12A1, DUT) in the chromosome 15.Our findings expand the number of large FBN1 deletions, and emphasize the importance of screening for large genomic deletions in connective tissue disorders featuring aortopathies, especially for those with classic Marfan phenotype.

Dataset Information

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

Publications

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.

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