Successful Linkage Analysis in Classical Phenylketonuria Families Followed by Direct Sequencing and Mutation Detection.

Abstract

Phenylketonuria (PKU) is the most common disorder of inborn errors of metabolism. Prevalence of PKU is about 1:10000 live births; however, due to high rate of consanguinity in the Middle East and North of Africa the prevalence of PKU is more than in other areas. It is estimated between 1:2600 in Turkey and 1:3672 in Iran. The best way to identify carriers in PKU families is studying causative mutations, but this approach could be costly and time consuming. As a result, linkage analysis can be considered as a reliable way to detect carriers. Ten non-related classical PKU families from Iran-Fars province were enrolled. Linkage analysis was performed through application of highly linked genetic markers to the PAH gene (VNTR, PAHSTR, and XmnI marker) with new designed primers for polymerase chain reaction (PCR). Reliability of approach was assessed by Sanger sequencing, mutation detection, and capillary electrophoresis (CE). Through application of linkage analysis, nine out of ten families were genotyped successfully. Heterozygosity of chromosome 12 was not detected in any of the enrolled PKU patients. Specificity of new designed primers for linkage analysis was confirmed by Sanger dideoxy sequencing. Results obtained from linkage analysis were confirmed by direct sequencing and detecting causative mutations in half of the genotyped families. All the results were the same as the linkage analysis results. Labeled primers were capable and linkage analysis by CE was successful. Linkage analysis is a powerful and reliable approach for detecting carriers in PKU families which have not been previously screened for causative mutations. We suggest studying the feasibility of the approach in preliminary diagnosis of PKU and confirming autozygosity of chromosome 12, prenatal diagnosis, and preimplantation genetic testing. Also, we recommend using labeled primers for constructing faithful local PKU associated haplotype databases to provide fast, cheap, and reliable detection of causative mutations in new cases of hyperphenylalaninemia.