Identification of Novel Microsatellite Markers <1 Mb from the HTT CAG Repeat and Development of a Single-Tube Tridecaplex PCR Panel of Highly Polymorphic Markers for Preimplantation Genetic Diagnosis of Huntington Disease

Abstract

BACKGROUND: Preimplantation genetic diagnosis (PGD) of Huntington disease (HD) generally employs linkage analysis of flanking microsatellite markers to complement direct mutation testing, as well as for exclusion testing. Thus far, only 10 linked markers have been developed for use in HD PGD, with a maximum of 3 markers coamplified successfully. We aimed to develop a single-tube multiplex PCR panel of highly polymorphic markers to simplify HD PGD. METHODS: An in silico search was performed to identify all markers within 1 Mb flanking the huntingtin (HTT) gene. Selected markers were optimized in a single-tube PCR panel, and their polymorphism indices were determined in 2 populations. The panel was tested on 63 single cells to validate its utility in PGD. RESULTS: We identified 102 markers in silico, of which 56 satisfied the selection criteria. After initial testing, 12 markers with potentially high heterozygosity were optimized into a single-tube PCR panel together with a 13th more distally located marker. Analysis of DNA from 183 Chinese and Caucasian individuals revealed high polymorphism indices for all markers (polymorphism information content >0.5), with observed heterozygosities ranging from 0.5- 0.92. All individuals were heterozygous for at least 5 markers, with 99.5% of individuals heterozygous for at least 2 markers upstream and downstream of the HTT CAG repeat. CONCLUSIONS: The tridecaplex marker assay amplified reliably from single cells either directly or after whole genome amplification, thus validating its standalone use inHDexclusion PGD or as a complement to HTT CAG repeat expansion-mutation detection.