Implementation of a congenital hypothyroidism newborn screening procedure with mutation detection on genomic DNA extracted from blood spots: the experience of the Italian northeastern reference center.

After a couple of decades from the institution of nationwide congenital hypothyroidism (CH) newborn screening program, the first generation properly treated is now displaying normal reproductive rate and the causative molecular defects are spreading from one generation to the next. In the present study we propose a method of detection of mutations in the thyrotropin receptor (TSHR) and in the paired box 8 (PAX8) genes that have been proved to be responsible for some forms of CH. The method, carried out by means of denaturing high-performance liquid chromatography (DHPLC) followed by direct sequencing, takes advantage of the CH newborn screening procedure, because genomic DNA for the analysis is extracted from the same blood spot collected for recall confirmation. Among 16 hypothyroid newborns with thyroid hypoplasia born between January 1999 and April 2005 in northeastern Italy, three heterozygous causative mutations in the TSHR gene were evidenced, whereas the analysis of the PAX8 gene revealed an unknown heterozygous substitution that could interfere with the start of transcription.