A successful strategy for preimplantation genetic diagnosis of myotonic dystrophy using multiplex fluorescent PCR.

The most common form of inherited muscular dystrophy in adults is myotonic dystrophy (DM), an autosomal-dominant disease caused by the expansion of an unstable CTG repeat sequence in the 3' untranslated region of the myotonin protein kinase (DMPK) gene. Expanded (mutant) CTG repeat sequences are refractory to conventional PCR, but alleles with a number of repeats within the normal range can be readily amplified and detected. Preimplantation genetic diagnosis (PGD) of DM has been successfully applied. However, a misdiagnosis using the reported protocol was recently documented. Two new PGD protocols for DM have been developed which utilise multiplex fluorescent PCR. Ideally a linked polymorphic marker, APOC2, is amplified in addition to the normal DMPK alleles, thus providing a back-up diagnostic result. However, the two couples reported in the present study were not fully informative at the APOC2 locus and so an unlinked short tandem repeat (STR) marker, D21S1414, was substituted. The highly polymorphic nature of the D21S1414, DMPK and APOC2 loci means that a very simple genetic fingerprint can be generated by analyses of these loci. This allows most DNA contaminants to be detected. Contamination is a significant problem for PGD and is the primary reason for the inclusion of D21S1414 and APOC2 in this protocol. This paper reports the first clinical experience and pregnancies following PGD for DM using a multiplex fluorescent PCR protocol.

Detailed chromosomal and molecular genetic analysis of single cells by whole genome amplification and comparative genomic hybridisation.

Molecular genetic analysis of isolated single cells and other minute DNA samples is limited because there is insufficient DNA to perform more than one independent PCR amplification. One solution to this problem is to first amplify the entire genome, thus providing enough DNA for numerous subsequent PCRs. In this study we have investigated four different methods of whole genome amplification performed on single cells, and have identified a protocol that generates sufficient quantities of DNA for comparative genomic hybridisation (CGH) as well as more than 90 independent amplification reactions. Thus, numerous specific loci and the copy number of every chromosome can be assessed in a single cell. We report here the first reliable application of CGH to single cells from human preimplantation embryos (blastomeres) and to single fibroblasts, buccal cells and amniocytes.

Strategies for preimplantation genetic diagnosis of single gene disorders by DNA amplification.

Preimplantation genetic diagnosis (PGD) is an alternative to prenatal diagnosis for ensuring the genetic health of offspring born to families affected by inherited disease. This paper sets out to review current protocols for the diagnosis of single gene defects in human preimplantation embryos. These methods, which depend on DNA amplification using PCR, are subject to a variety of pitfalls, such as allele dropout (ADO), contamination and reduced amplification efficiency. Advances in single cell DNA amplification, such as improved multiplex PCR protocols, fluorescent-PCR and whole genome amplification (WGA), can be applied to address some of these problems. Different PGD strategies are discussed in the context of their clinical application.

Homologies between human and marmoset (Callithrix jacchus) chromosomes revealed by comparative chromosome painting.

Regions of DNA homology between human and marmoset (Callithrix jacchus) chromosomes have been demonstrated using fluorescence in situ hybridization. All 24 chromosome paints and two centromere repeat sequences from Homo sapiens (HSA) have been annealed to previously G-banded metaphase spreads of Callithrix jacchus. All human paint probes, except Y, successfully hybridized to marmoset chromosomes. Fifteen of them hybridized to one region only, seven to two regions, and paint 1 to three regions. Homologies proposed from previous banding comparisons have been confirmed for HSA 2, 4-6, 10-12, 18, 19, 21 and X and partially confirmed for HSA 1 and 3, but were not in agreement for HSA 14 and 17. Human centromere repeat sequences for X and 18 did not hybridize to marmoset chromosomes. Because, at present, there is the confusion situation of several different numbering systems for marmoset chromosomes, we propose a new simpler nomenclature based on descending order of chromosome size.