In silico and functional studies of the regulation of the glucocerebrosidase gene.

In Gaucher disease (GD), the inherited deficiency of glucocerebrosidase results in the accumulation of glucocerebroside within lysosomes. Although almost 300 mutations in the glucocerebrosidase gene (GBA) have been identified, the ability to predict phenotype from genotype is quite limited. In this study, we sought to examine potential GBA transcriptional regulatory elements for variants that contribute to phenotypic diversity. Specifically, we generated the genomic sequence for the orthologous genomic region ( approximately 39.4kb) encompassing GBA in eight non-human mammals. Computational comparisons of the resulting sequences, using human sequence as the reference, allowed the identification of multi-species conserved sequences (MCSs). Further analyses predicted the presence of two putative clusters of transcriptional regulatory elements upstream and downstream of GBA, containing five and three transcription factor-binding sites (TFBSs), respectively. A firefly luciferase (Fluc) reporter construct containing sequence flanking the GBA gene was used to test the functional consequences of altering these conserved sequences. The predicted TFBSs were individually altered by targeted mutagenesis, resulting in enhanced Fluc expression for one site and decreased expression for seven others sites. Gel-shift assays confirmed the loss of nuclear-protein binding for several of the mutated constructs. These identified conserved non-coding sequences flanking GBA could play a role in the transcriptional regulation of the gene contributing to the complexity underlying the phenotypic diversity seen in GD.

Detection of potential GDF6 regulatory elements by multispecies sequence comparisons and identification of a skeletal joint enhancer.

The identification of noncoding functional elements within vertebrate genomes, such as those that regulate gene expression, is a major challenge. Comparisons of orthologous sequences from multiple species are effective at detecting highly conserved regions and can reveal potential regulatory sequences. The GDF6 gene controls developmental patterning of skeletal joints and is associated with numerous, distant cis-acting regulatory elements. Using sequence data from 14 vertebrate species, we performed novel multispecies comparative analyses to detect highly conserved sequences flanking GDF6. The complementary tools WebMCS and ExactPlus identified a series of multispecies conserved sequences (MCSs). Of particular interest are MCSs within noncoding regions previously shown to contain GDF6 regulatory elements. A previously reported conserved sequence at -64 kb was also detected by both WebMCS and ExactPlus. Analysis of LacZ-reporter transgenic mice revealed that a 440-bp segment from this region contains an enhancer for Gdf6 expression in developing proximal limb joints. Several other MCSs represent candidate GDF6 regulatory elements; many of these are not conserved in fish or frog, but are strongly conserved in mammals.

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.

Comparative sequence analysis of the Gdf6 locus reveals a duplicon-mediated chromosomal rearrangement in rodents and rapidly diverging coding and regulatory sequences.

Duplicated segments of genomic DNA can catalyze both gene evolution and chromosome evolution. Here we describe a rodent-specific duplication involving the Uqcrb gene, a cis-regulatory element for the Gdf6 gene, and a chromosomal rearrangement. Comparisons of Gdf6 sequences from several placental mammals and platypus revealed many strongly conserved regions flanking Gdf6 and the adjacent Uqcrb gene. However, in rat and mouse a synteny break resides approximately 70 kb upstream of Gdf6, such that Gdf6 and Uqcrb are on separate chromosomes. In rodents, Gdf6 and Uqcrb are both associated with homologous duplicons that may have catalyzed a rearrangement separating the two genes. However, the duplicon spanned both Uqcrb and a cis-regulatory element that controls Gdf6 transcription in limb skeletal joints. In mouse and rat, one duplicon now contains a degrading Uqcrb pseudogene but retains strongly conserved sequences within a Gdf6 enhancer. In contrast, the other duplicon has retained the intact Uqcrb gene and (in mouse) a copy of the Gdf6 enhancer that has acquired novel mutations. The duplicons have separately maintained distinct functions of the ancestral sequence, consistent with a "subfunction partitioning" evolutionary model. These findings also provide an example of a duplication that mobilized a tissue-specific enhancer from its cognate gene, and new evidence that duplications can be associated with chromosomal rearrangements. Furthermore, these data suggest that segmental duplications could lead to evolution of novel gene expression patterns via diversification of regulatory elements.

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.