Phenotype-genotype correlation in Hirschsprung disease is illuminated by comparative analysis of the RET protein sequence.

The ability to discriminate between deleterious and neutral amino acid substitutions in the genes of patients remains a significant challenge in human genetics. The increasing availability of genomic sequence data from multiple vertebrate species allows inclusion of sequence conservation and physicochemical properties of residues to be used for functional prediction. In this study, the RET receptor tyrosine kinase serves as a model disease gene in which a broad spectrum (> or = 116) of disease-associated mutations has been identified among patients with Hirschsprung disease and multiple endocrine neoplasia type 2. We report the alignment of the human RET protein sequence with the orthologous sequences of 12 non-human vertebrates (eight mammalian, one avian, and three teleost species), their comparative analysis, the evolutionary topology of the RET protein, and predicted tolerance for all published missense mutations. We show that, although evolutionary conservation alone provides significant information to predict the effect of a RET mutation, a model that combines comparative sequence data with analysis of physiochemical properties in a quantitative framework provides far greater accuracy. Although the ability to discern the impact of a mutation is imperfect, our analyses permit substantial discrimination between predicted functional classes of RET mutations and disease severity even for a multigenic disease such as Hirschsprung disease.

A common sex-dependent mutation in a RET enhancer underlies Hirschsprung disease risk.

The identification of common variants that contribute to the genesis of human inherited disorders remains a significant challenge. Hirschsprung disease (HSCR) is a multifactorial, non-mendelian disorder in which rare high-penetrance coding sequence mutations in the receptor tyrosine kinase RET contribute to risk in combination with mutations at other genes. We have used family-based association studies to identify a disease interval, and integrated this with comparative and functional genomic analysis to prioritize conserved and functional elements within which mutations can be sought. We now show that a common non-coding RET variant within a conserved enhancer-like sequence in intron 1 is significantly associated with HSCR susceptibility and makes a 20-fold greater contribution to risk than rare alleles do. This mutation reduces in vitro enhancer activity markedly, has low penetrance, has different genetic effects in males and females, and explains several features of the complex inheritance pattern of HSCR. Thus, common low-penetrance variants, identified by association studies, can underlie both common and rare diseases.

Uptake of lead and iron by divalent metal transporter 1 in yeast and mammalian cells.

Although the divalent metal transporter (DMT1) was suggested to transport a wide range of metals in Xenopus oocytes, recent studies in other models have provided contrasting results. Here, we provide direct evidence demonstrating that DMT1 expressed in yeast mutants defective for high affinity iron transport facilitates the transport of iron with an 'apparent K(m)' of approximately 1.2 microM, and transport of lead with an 'apparent K(m)' of approximately 1.8 microM. DMT1-dependent lead transport was H(+)-dependent and was inhibited by iron. Human embryonic kidney fibroblasts (HEK293 cells) overexpressing DMT1 also showed a higher uptake of lead than HEK293 cells without overexpressing DMT1. These results show that DMT1 transports lead and iron with similar affinity in a yeast model suggesting that DMT1 is a transporter for lead.

The distinct methods by which manganese and iron regulate the Nramp transporters in yeast.

The bakers yeast Saccharomyces cerevisiae expresses three Smf metal transport proteins that are differentially regulated by metal ions. Smf1p and Smf2p are regulated at the post-translational level by manganese, whereas Smf3p is regulated by iron through a mechanism that, up until now, was unknown. Through promoter and protein-domain swapping experiments, we now demonstrate that the manganese regulation of Smf1p involves an internal protein-coding region that is separate from the N-terminal domain of this transporter. By comparison, iron regulation of Smf3p involves the upstream non-coding region of the gene. Using SMF3-lacZ reporter constructs, we identified two distinct regions of the SMF3 promoter that contribute to iron regulation: (1) approx. nt -435 to -350 that contain dual consensus recognition sites for the Aft1 iron transcription factor; and (2) nt -348 to -247 that do not contain obvious Aft1 binding sites. The -348 to -247 region by itself can confer strong iron regulation to the heterologous CYC1 core promoter, and therefore harbours a putative upstream activating sequence for iron. Iron regulation of SMF3 was dramatically reduced, but not completely eliminated, in strains lacking both the AFT1 and AFT2 iron regulatory factors. Together with the promoter mapping studies, these results suggest that both Aft-dependent and Aft-independent pathways may contribute to iron regulation of SMF3.