A polymerase-chain-reaction (PCR) assay was used to amplify all coding exons and intron–exon junctions of all genes mapping within the lod – 2 interval that were then sequenced (Table S2 in the Supplementary Appendix). A heterozygous G-to-A transition at nucleotide position 951, resulting in a premature termination codon (W317X), was identified in exon 9 of HDC. The mutation was present in the affected father and all affected offspring and absent in the unaffected mother (Fig. 2C). In addition, reverse-transcriptase PCR was performed on messenger RNA samples from transformed lymphoblastoid cell lines. All affected family members had the substitution, providing independent confirmation of the mutation and showing that it may escape nonsense-mediated decay. No other rare nonsense, splice-site, or missense mutations (those with an allele frequency <5%) were identified in the lod – 2 interval. Three previously reported missense substitutions, all with allele frequencies greater than 5%, were found on the linked haplotype (Table S3 in the Supplementary Appendix).
