DETECTION OF ECUADOREAN LARON SYNDROME GENE CARRIERS BY MOLECULAR GENETIC ANALYSIS OF GUTHRIE CARD BLOOD SAMPLES

Abstract

Laron syndrome's high incidence in several southern Ecuadurean villages is due to a high frequency of asymptomatic heterozygous gene carriers who are at risk of having affected offspring. As a single growth hormone receptor (GHR) gene mutation, E180splice, accounts for over 97% of Ecuadorean Laron syndrome (LS) alleles, we have developed a rapid and reliable method to detect this mutation: finger-prick blood spots are collected onto Guthrie filter paper and used directly as DNA templates for PCR amplification of a GHR gene fragment which includes codon 180. The amplification products are applied to nylon membrane and hybridized serially with digoxigenin-lahelled oligonneleotide probes complementary to the normal and mutant alleles. Probe hybridization is delected using a chemilumninescence nucleic acid detection kit. We have analyzed blood spot samples from 96 individuals. In 16 affected individuals, homozygosity for the E180splice mutation was confirmed. In 19 parents of affected individuals, heterozygosity was confirmed. Of 61 unaffected individuals al risk to be carriers, 34 were found in carry [he E180splice mutation. Accurate determination of carrier status among Ecuadorean individuals at-risk is possible using this method. The testing is simplified by using Guthrie filter blood samples which are easy in obtain, store, mail, and use directly in PCR reactions, eliminaiing the need for DNA extraction. Detection of the normal and mutant alleles by chemiluminescence eliminates the need for radioisolope use.