Real-time PCR and linkage studies to identify carriers presenting HPRT deleted gene

Abstract

Lesch-Nyhan syndrome (LNS) is an X-linked genetic disorder resulting in hyperuricemia, choreoathetosis, mental retardation, and self-injurious behavior. It is caused by loss of activity of the ubiquitous enzyme hypoxanthine-guanine-phosphoribosyltransferase (HPRT). The biochemical analysis of residual HPRT activity in patients' red blood cells is the first step in LNS diagnosis, and it precedes molecular study to discover the specific mutation. Unfortunately, biochemical diagnosis of healthy carriers is difficult because HPRT enzymatic activity in blood cells is similar in LNS carriers and in healthy people; genetic tests can help reveal mutations at the genomic or cDNA level, whereas gross deletions involving the first or last exons of HPRT gene are not detectable. Until now, a test based on 6-thioguanine-resistant phenotype of HPRT mutant cells from LNS patients is the only method accepted for the diagnosis of any kind of mutation in carriers. In this work, we introduce a new approach to identify carriers of large deletions in HPRT gene using real-time PCR. Results were validated in a blinded manner with a linkage study and with results obtained in Italian families previously analyzed with selective medium test. Real-time PCR analysis clearly confirmed the results obtained by selective medium; linkage data strengthened real time results, allowing us to follow the allele with the mutated HPRT through the family pedigree. We hope that the real-time PCR approach will provide a useful and reliable method to diagnose LNS carriers of large deletions in HPRT gene.

Figure 1

Family 1 pedigree and haplotype. Microsatellites and SNPs are listed in order of map position. II.3 is the proband, carrier of the deletion from exon 1 to 3 in HPRT gene. With the use of 3 microsatellite markers and 4 SNPs, the affected allele segregation can be followed; this affected allele is present only in the proband’s sister and mother.

Figure 2

Family 2 pedigree and haplotype. Microsatellites and SNPs are listed in order of map position. II.3 and II.4 are the probands, carriers of deletion of HPRT exon 9. With the use of 3 microsatellite markers and 4 SNPs, the affected allele segregation can be followed; this affected allele is present only in the probands’ mother.

Figure 3

Semiquantitative real-time PCR results. Bars represents 2^-ΔΔCt value calculated by ΔCt method. Data are normalized to a control DNA. Control samples were divided in male and female controls (CN-M and CN-F); each bar of male or female control is the result obtained by 3 different control patients. Male and female controls present the correct number of copies of HPRT in the region analyzed; on the other hand, LNS-positive controls (CP) present no copies of HPRT. (A) Real-time semiquantitative PCR results of HPRT exon 2 analysis. II.3 is the male proband, carrier of the deletion between exons 1 and 3; II.2 and I.3 are the proband’s sister and mother, both carriers of the deletion; I.4 is the proband’s father, showing a pattern similar to that of normal controls. On the mother’s side, the female cousin, the aunt, and the aunt’s husband (II.1, I.2, and I.1, respectively) show a pattern typical of nonaffected individuals. (B) Real-time semiquantitative PCR results of HPRT exon 9 analysis. II.3 and II.4 are the 2 affected brothers; II.2 is their sister, who presents a pattern similar to that of normal controls. I.3 is the probands’ mother, who is a carrier of the deletion. The probands’ relatives (the female cousin, the maternal aunt, and uncle by marriage, II.1, I.2, and I.1, respectively) show a normal pattern.