Genomic organization of the human CYP3A locus: identification of a new, inducible CYP3A gene.

Proteins encoded by the human CYP3A genes metabolize every second drug currently in use. The activity of CYP3A gene products in the general population is highly variable and may affect the efficacy and safety of drugs metabolized by these enzymes. The mechanisms underlying this variability are poorly understood, but they include gene induction, protein inhibition and unknown genetic polymorphisms. To better understand the regulation of CYP3A expression and to provide a basis for a screen of genetic polymorphisms, we determined and analysed the sequence of the human CYP3A locus. The 231 kb locus sequence contains the three CYP3A genes described previously (CYP3A4, CYP3A5 and CYP3A7), three pseudogenes as well as a novel CYP3A gene termed CYP3A43. The gene encodes a putative protein with between 71.5% and 75.8% identity to the other CYP3A proteins. The highest expression level of CYP3A43 mRNA is observed in the prostate, an organ with extensive steroid metabolism. CYP3A43 is also expressed in several other tissues including liver, where it can be induced by rifampicin. CYP3A43 transcripts undergo extensive splicing. The identification of a new member of the CYP3A family and the characterization of the full CYP3A locus will aid efforts to identify the genetic variants underlying its variable expression. This, in turn, will lead to a better optimization of therapies involving the numerous substrates of CYP3A proteins.

The genetic determinants of the CYP3A5 polymorphism.

CYP3A proteins comprise a significant portion of the hepatic cytochrome P450 (CYP) protein and they metabolize around 50% of drugs currently in use. The dissection of the individual contributions of the four CYP3A genes identified in humans to overall hepatic CYP3A activity has been hampered by sequence and functional similarities. We have investigated the expression of CYP3A5 and its genetic determinants in a panel of 183 Caucasian liver samples. CYP3A5 expression is increased in 10% of livers in this ethnic group. Using a high density map of CYP3A5 variants, we searched for genetic markers of the increased CYP3A5 expression. In agreement with an independent, recent study, we report that a SNP within intron 3 (g.6986G>A) is the primary cause of the CYP3A5 protein polymorphism. The frequencies of the g.6986A variant which allow for normal splicing of CYP3A5 transcripts are 5% in Caucasians, 29% in Japanese, 27% in Chinese, 30% in Koreans and 73% in African-Americans. In the last ethnic group, the expression of CYP3A5 in some individuals who carry the g.6986A variant is affected adversely by a frame shift mutation (CYP3A5*7, D348., q = 0.10). In summary, these results should add to efforts to identify clinically relevant, CYP3A5-specific reactions and to further elucidate traits responsible for variable expression of the entire CYP3A family.

Identification and functional characterization of eight CYP3A4 protein variants.

The genetic component of the inter-individual variability in CYP3A4 activity has been estimated to be between 60% and 90%, but the underlying genetic factors remain largely unknown. A study of 213 Middle and Western European DNA samples resulted in the identification of 18 new CYP3A4 variants, including eight protein variants. A total of 7.5% of the population studied was found to be heterozygous for one of these variants. In a bacterial heterologous expression system, two mutants, R130Q and P416L, did not result in detectable P450 holoprotein. One mutant, T363M, expressed at significantly lower levels than wild-type CYP3A4. G56D, V170I, D174H and M445T were not significantly different when compared with wild-type CYP3A4 in expression or steroid hydroxylase activity. L373F displayed a significantly altered testosterone metabolite profile and a four-fold increase in the Km value for 1'-OH midazolam formation. The results suggest a limited contribution of CYP3A4 protein variants to the inter-individual variability of CYP3A4 activity in Caucasians. Some variants may, however, play a role in the atypical response to drugs or altered sensitivity to carcinogens.

Rbt (Rabo torcido), a new mouse skeletal mutation involved in anteroposterior patterning of the axial skeleton, maps close to the Ts (tail-short) locus and distal to the Sox9 locus on chromosome 11.

Rbt (Rabo torcido) is a new semidominant mouse mutant with a variety of skeletal abnormalities. Heterozygous Rbt mutants display homeotic anteroposterior patterning problems along the axial skeleton that resemble Polycomb group and trithorax gene mutations. In addition, the Rbt mutant displays strong similarities to the phenotype observed in Ts (Tail-short), indicating also a homeotically transformed phenotype in these mice. We have mapped the Rbt locus to an interval of approximately 6 cM on mouse Chromosome (Chr) 11 between microsatellite markers D11Mit128 and D11Mit103. The Ts locus was mapped within a shorter interval of approximately 3 cM between D11Mit128 and D11Mit203. This indicates that Rbt and Ts may be allelic mutations. Sox9, the human homolog of which is responsible for the skeletal malformation syndrome campomelic dysplasia, was mapped proximal to D11Mit128. It is, therefore, unlikely that Ts and Rbt are mouse models for this human skeletal disorder.

A mouse model for valproate teratogenicity: parental effects, homeotic transformations, and altered HOX expression.

Valproate (VPA) is one of several effective anti-epileptic and mood-stabilizing drugs, many of which are also potent teratogens in humans and several other mammalian species. Variable teratogenicity among inbred strains of laboratory mice suggests that genetic factors influence susceptibility. While studying the genetic basis for VPA teratogenicity in mice, we discovered that parental factors influence fetal susceptibility to induced malformations. Detailed examination of these malformations revealed that many were homeotic transformations. To test whether VPA, like retinoic acid (RA), alters HOX expression, pluripotent human embryonal carcinoma cells were treated with VPA or RA and Hox expression assessed. Altered expression of specific Hox genes may thus account for the homeotic transformations and other malformations found in VPA-treated fetuses.

The SOX10/Sox10 gene from human and mouse: sequence, expression, and transactivation by the encoded HMG domain transcription factor.

The SOX genes form a gene family related by homology to the high-mobility group (HMG) box region of the testis-determining gene SRY. We have cloned and sequenced the SOX10 and Sox10 genes from human and mouse, respectively. Both genes encode proteins of 466 amino acids with 98% sequence identity. Significant expression of the 2.9-kb human SOX10 mRNA is observed in fetal brain and in adult brain, heart, small intestine and colon. Strong expression of Sox10 occurs throughout the peripheral nervous system during mouse embryonic development. SOX10 shows an overall amino acid sequence identity of 59% to SOX9. Like SOX9, SOX10 has a potent transcription activation domain at its C-terminus and is therefore likely to function as a transcription factor. Whereas SOX9 maps to 17q, a SOX10 cosmid has previously been mapped by us to the region 22q13.1. Mutations in SOX10 have recently been identified as one cause of Waardenburg-Hirschsprung disease in humans, while a Sox10 mutation underlies the mouse mutant Dom, a murine Hirschsprung model.

Natural protein variants of pregnane X receptor with altered transactivation activity toward CYP3A4.

Between 45 and 60% of all drugs currently used are metabolized by the CYP3A4 protein. CYP3A4 expression in liver varies up to 60-fold in the general population, which can lead to ineffective drug therapy (high CYP3A4) or, on the other hand, to harmful drug reactions (low CYP3A4). Most of this variability has been attributed to genetic factors, but to date their identity remains unknown. Recently, it was shown that CYP3A expression is largely controlled by the pregnane X receptor (PXR). We, therefore, hypothesized that polymorphisms in PXR may contribute to CYP3A4 variability. The presence of PXR variants was investigated in two ethnic groups, Caucasians and Africans. Six missense mutations leading to variant PXR proteins were identified, and their consequences on CYP3A4 expression were analyzed. Expressed in LS174T cells, three protein variants, V140M, D163G, and A370T, exhibited altered basal and/or induced transactivation of CYP3A promoter reporter genes. Thus, these natural PXR protein variants may play a role in the observed interindividual variability of CYP3A4 expression and may be involved in rare, atypical responses to drugs or altered sensitivities to carcinogens.

Autosomal sex reversal and campomelic dysplasia are caused by mutations in and around the SRY-related gene SOX9.

A human autosomal XY sex reversal locus, SRA1, associated with the skeletal malformation syndrome campomelic dysplasia (CMPD1), has been placed at distal 17q. The SOX9 gene, a positional candidate from the chromosomal location and expression pattern reported for mouse Sox9, was isolated and characterized. SOX9 encodes a putative transcription factor structurally related to the testis-determining factor SRY and is expressed in many adult tissues, and in fetal testis and skeletal tissue. Inactivating mutations on one SOX9 allele identified in nontranslocation CMPD1-SRA1 cases point to haploinsufficiency for SOX9 as the cause for both campomelic dysplasia and autosomal XY sex reversal. The 17q breakpoints in three CMPD1 translocation cases map 50 kb or more from SOX9.