Korean J Reprod Med.  2007 Sep;34(3):179-188.

Optimized Methods of Preimplantation Genetic Diagnosis for Trinucleotide Repeat Diseases of Huntington's Disease, Spinocerebellar Ataxia 3 and Fragile X Syndrome

• Kim MJ
• Lee HS
• Lim CK
• Cho JW
• Kim JY
• Koong MK
• Song IO
• Kang IS
• Jun JH

Abstract

OBJECTIVES
Many neurological diseases are known to be caused by expansion of trinucleotide repeats (TNRs). It is hard to diagnose the alteration of TNRs with single cell level for preimplantation genetic diagnosis (PGD). In this study, we describe methods optimized for PGD of TNRs related diseases such as Huntington's disease (HD), spinocerebellar ataxia 3 (SCA3) and fragile X syndrome (FXS).
METHODS
We performed the preclinical assays with heterozygous patient's lymphocytes by single cell PCR strategy. Fluorescent semi-nested PCR and fragment analysis using automatic genetic analyzer were applied for HD and SCA 3. Whole genome amplification with multiple displacement amplification (MDA) method and fluorescent PCR were carried out for FXS. Amplification and allele drop-out (ADO) rate were evaluated in each case.
RESULTS
The fluorescent semi-nested PCR of single lymphocyte showed 100.0% of amplification and 14.0% of ADO rate in HD, and 94.7% of amplification and 5.6% of ADO rate in SCA3, respectively. We could not detect the PCR product of CGG repeats in FXS using the fluorescent semi-nested PCR alone. After applying the MDA method in FXS, 84.2% of amplification and 31.3% of ADO rate were achieved.
CONCLUSIONS
Fluorescent semi-nested PCR is a reliable method for PGD of HD and SCA3. The advanced MDA method overcomes the problem of amplification failure in CGG repeats of FXS case. Optimization of methods for single cell analysis could improve the sensitivity and reliability of PGD for complicated single gene disorders of TNRs.

Keyword

Preimplantation genetic diagnosis (PGD); Trinucleotide repeats disease; Huntington's disease (HD); Spinocerebellar ataxia 3 (SCA3); Fragile X syndrome (FXS)

MeSH Terms

Alleles
Fragile X Syndrome*
Genome
Huntington Disease*
Lymphocytes
Machado-Joseph Disease*
Polymerase Chain Reaction
Preimplantation Diagnosis*
Prostaglandins D
Single-Cell Analysis
Spinocerebellar Ataxias*
Trinucleotide Repeats*
Prostaglandins D