COMPETITIVE POLYMERASE CHAIN-REACTION QUANTITATION OF C-ERBA-BETA-1, C-ERBA-ALPHA-1, AND C-ERBA-ALPHA-2 MESSENGER-RIBONUCLEIC-ACID LEVELS IN NORMAL, HETEROZYGOUS, AND HOMOZYGOUS FIBROBLASTS OF KINDRED-S WITH THYROID-HORMONE RESISTANCE

Mutations in the T3-binding domain of the thyroid hormone receptor gen e c-erbAbeta result in dominant negative proteins and thyroid hormone resistance syndromes. Variable clinical manifestations of resistance t o thyroid hormones have been reported, including short stature and neu ropsychological abnormalities. The molecular bases for heterogeneity o f phenotype among and within kindreds have not been fully elucidated. Recent investigations have considered differential expression of mutan t and wild-type beta1-receptor alleles and the regulation thereof as a mechanism to explain differential sensitivity to thyroid hormones. We used reverse transcription-competitive polymerase chain reaction (PCR ) to measure c-erbAbeta1, c-erbAalpha1, and c-erbAalpha2 mRNAs in skin fibroblasts cultured from normal subjects, heterozygotes, and a sever ely affected homozygous mutant of kindred S. The homozygous mutant of kindred S had severe growth and mental retardation. After reverse tran scription with primers specific for each of the c-erbA mRNAs, first st rand cDNAs were amplified by PCR using subtype-specific amplimers. Pri mer design allowed simultaneous detection of wild-type and mutant mess ages in heterozygous fibroblasts and showed an approximately 1:1 ratio of these mRNAs in three patients. Inclusion of competitive standard c DNAs of known concentration in the PCR reactions allowed quantitation of the absolute levels of the beta1-, alpha1-, and alpha2 mRNAs by com parison of products on ethidium bromide-stained agarose gels. These st udies showed no effect of the presence of the mutant beta1-allele, as fibroblast RNA from normal subjects, heterozygotes, and the homozygote gave values of 56-184, 2.8-12, and 23-40 attomol/5 mug total RNA for beta1-, alpha1, and alpha2 mRNAs, respectively. We conclude that these sensitive methods allow the detection of molecular species present at levels as low as 10 molecules/cell, and that this potent dominant neg ative receptor does not disrupt c-erbA expression at the level of mRNA . The neuropsychological sequelae of the kindred S mutation are not du e to relative overexpression of the mutant allele.