ABSTRACT
Beta (ß)-thalassemia is one of the most common monogenic diseases worldwide. Affected pregnancies can be avoided through preimplantation genetic diagnosis (PGD), which commonly involves customized assays to detect the different combinations of ß-globin (HBB) gene mutations present in couples, in conjunction with linkage analysis of flanking microsatellite markers. Currently, the limited number of reported closely linked markers hampers their utility in indirect linkage-based PGD for this disorder. To increase the available markers closely flanking the HBB gene, an in silico search was performed to identify all markers within 1 Mb flanking the HBB gene. Fifteen markers with potentially high polymorphism information content (PIC) and heterozygosity values were selected and optimized into a single-tube pentadecaplex PCR panel. Allele frequencies and polymorphism and heterozygosity indices of each marker were assessed in five populations. A total of 238 alleles were observed from the 15 markers. PIC was >0.7 for all markers, with expected heterozygosity and observed heterozygosity values ranging from 0.74 to 0.90 and 0.72 to 0.88, respectively. Greater than 99% of individuals were heterozygous for at least seven markers, with at least two heterozygous markers on either side of the HBB gene. The pentadecaplex marker assay also performed reliably on single cells either directly or after whole genome amplification, thus validating its use in standalone linkage-based ß-thalassemia PGD or in conjunction with HBB mutation detection.
Subject(s)
Polymerase Chain Reaction/methods , Preimplantation Diagnosis/methods , beta-Globins/genetics , beta-Thalassemia/diagnosis , Female , Gene Frequency , Humans , Microsatellite Repeats , Polymorphism, Genetic , Pregnancy , beta-Thalassemia/geneticsSubject(s)
Hemoglobins, Abnormal/genetics , Hydrops Fetalis/diagnosis , Multiplex Polymerase Chain Reaction/methods , Preimplantation Diagnosis/methods , alpha-Thalassemia/diagnosis , DNA Primers/chemical synthesis , Female , Genetic Loci , Genetic Markers , Hemoglobins, Abnormal/chemistry , Humans , Hydrops Fetalis/genetics , Hydrops Fetalis/pathology , Microsatellite Repeats , Pregnancy , alpha-Thalassemia/genetics , alpha-Thalassemia/pathologyABSTRACT
OBJECTIVE: To develop a single-tube multi-marker assay for improved preimplantation genetic diagnosis (PGD) of deletional and/or non-deletional Hb Bart's hydrops fetalis syndrome, providing haplotype confirmation of deletional status, and maximization of linkage informativity. METHODS: We performed in silico mining to identify novel microsatellites within 1 Mb flanking the alpha-globin gene cluster, and optimized a single-tube assay combining detection of α(0) -thalassemia deletions with multi-marker linkage analysis. We performed validation on 100 single cells prior to clinical PGD application. RESULTS: Of 42 markers encompassing the α-globin gene cluster that were identified in silico, 9 were highly polymorphic (0.68 ≤ polymorphism information content ≤ 0.92; 0.66 ≤ Ho ≤ 0.90; 10 ≤ alleles ≤ 35) and optimized to co-amplify directly from a single cell. A validation analysis of 100 single lymphoblasts yielded 100% amplification success for all markers, and individual marker allele drop-out (ADO) rates of 0-5%. Clinical application of the assay in PGD for Hb Bart's (2 cases/cycles) resulted in a twin pregnancy and healthy live birth of two baby girls. CONCLUSIONS: This single-tube nonaplex microsatellite PCR panel can be applied directly to PGD of most deletional Hb Bart's without the need for deletion-specific customization, and to linkage-based PGD of non-deletional Hb Bart's.