Evidence for a functional genetic polymorphism of the human retinoic acid-metabolizing enzyme CYP26A1, an enzyme that may be involved in spina bifida.

BACKGROUND: CYP26A1, together with CYP26B1 and CYP26C1, are key enzymes of all-trans retinoic acid (RA) inactivation and their specific and restricted expression in developing embryos participate in the fine tuning RA levels. As RA is a critical regulator of gene expression during embryonic development, the imbalance between the synthesis and degradation of RA during embryogenesis could contribute to malformations and developmental defects. METHODS: A PCR-single strand conformation polymorphism (PCR-SSCP) strategy was developed to screen for CYP26A1 sequence variations that could affect the enzyme expression and/or activity and applied to DNA samples from 80 unrelated Caucasians, comprising 40 French healthy volunteers and 40 Italian patients with spina bifida. The consequence of the 1-bp deletion identified in the coding sequence was investigated by an in vitro functional assay using COS-7 cells. RESULTS: A total of 7 polymorphisms were identified, comprising 1 nucleotide deletion in the coding sequence (g.3116delT) that results in a frameshift and consequently in the creation of a premature stop codon. The g.3116delT mutation is of particular interest because it was identified in a patient with spina bifida and likely encodes a truncated protein with no enzymatic activity, as demonstrated by our preliminary in vitro data. CONCLUSIONS: Despite the fact that our findings could not show any evidence that the CYP26A1 genetic polymorphism has implications in the pathogenesis of spina bifida, this work represents the first description of a functional genetic polymorphism affecting the coding sequence of the human CYP26A1 gene.

Evidence for a functional genetic polymorphism of the human thiosulfate sulfurtransferase (Rhodanese), a cyanide and H2S detoxification enzyme.

Rhodanese or thiosulfate sulfurtransferase (TST) is a mitochondrial matrix enzyme that plays roles in cyanide detoxification, the formation of iron-sulfur proteins and the modification of sulfur-containing enzymes. Transsulfuration reaction catalyzed by TST is also involved in H(2)S detoxification. To date, no polymorphism of the human TST gene had been reported. We developed a screening strategy based on a PCR-SSCP method to search for mutations in the 3 exons of TST and their proximal flanking regions. This strategy has been applied to DNA samples from 50 unrelated French individuals of Caucasian origin. Eleven polymorphisms consisting in seven nucleotide substitutions in non-coding regions, two silent mutations and two missense mutations were characterized. The functional consequences of the identified mutations were assessed in vivo by measurement of erythrocyte TST activity and/or in vitro using heterologous expression in Saccharomyces cerevisiae or transient transfection assay in HT29 and Caco-2 cell lines. The P(285)A variant appears to encode a protein with a 50% decrease of in vitro intrinsic clearance compared to the wild-type enzyme. Additionally, the six polymorphisms located upstream the ATG initiation codon are responsible for a significant decrease (ranging from 40% to 73%) in promoter activity of a reporter gene compared to the corresponding wild-type sequence. This work constitutes the first report of the existence of a functional genetic polymorphism affecting TST activity and should be of great help to investigate certain disorders for which impairment of CN(-) or H(2)S detoxification have been suggested to be involved.

Evidence for a functional genetic polymorphism of the human mercaptopyruvate sulfurtransferase (MPST), a cyanide detoxification enzyme.

Mercaptopyruvate sulfurtransferase (MPST) plays a central role in both cysteine degradation and cyanide detoxification. Moreover, deficiency in MPST activity has been suggested to be responsible for a rare inheritable disorder known as mercaptolactate-cysteine disulfiduria (MCDU). To date, no mutation of the human MPST gene has been reported. We developed a screening strategy to search for mutations in the MPST gene of 50 unrelated French individuals. Two intronic polymorphisms (IVS1-110C>G and IVS2+39C>T) and a nonsense mutation (Tyr(85)Stop) were identified and their functional consequences were assessed in vivo by measurement of erythrocyte MPST activity and/or in vitro using heterologous expression or transient transfection assay. The nonsense mutation likely leads to the synthesis of a severely truncated protein without enzymatic activity, as supported by our in vitro data. This work constitutes the first report of the existence of a functional genetic polymorphism affecting MPST and should be of great help to investigate certain disorders such as MCDU.

Human CYP4F12 genetic polymorphism: identification and functional characterization of seven variant allozymes.

The human cytochrome CYP4F12 has been shown to be metabolically active toward inflammatory mediators and exogenous compounds such as antihistaminic drugs. We recently identified a genetic polymorphism within the promoter region, associated with a decreased level of enzyme expression. In the present study, we report the further identification of single nucleotide polymorphisms in the coding sequence of the CYP4F12 gene. A polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis of DNA samples from 53 unrelated French Caucasians, allowed the identification of ten mutations, comprising seven missense mutations, 31C>T (Leu11Phe), 38C>T (Pro13Leu), 47C>T (Met16Thr), 4759G>A (Asp76Asn), 4801G>A (Val90Leu), 8896C>T (Arg188Cys) and 23545G>A (Gly522Ser). Their functional impact toward ebastine hydroxylation was evaluated using heterologous expression in Saccharomyces cerevisiae cells of site-directed mutated cDNA variants. Five out seven variants did not exhibit any significant difference in CYP4F12 catalytic activity, whereas two variants, Val90Ile and Arg188Cys, displayed significant changes in their Michaelis-Menten (Km, Vm) parameters. These data on CYP4F12 genetic polymorphism provide tools for further studies of association with pathological processes involving an inflammatory component and with variations in anti-histaminic drug response.

Functional characterization of genetic polymorphisms identified in the human cytochrome P450 4F12 (CYP4F12) promoter region.

The human cytochrome CYP4F12 has been shown to be active toward inflammatory mediators and exogenous compounds such as antihistaminic drugs. In the present study, we report the first investigation of polymorphisms in the human CYP4F12 gene. A screening for sequence variations in the 5'-flanking region was performed by a Polymerase Chain Reaction-Single Strand Conformational Polymorphism (PCR-SSCP) strategy, using DNA samples from 53 unrelated French individuals of Caucasian origin. Several polymorphisms were identified, comprising a large deletion located in intron 1 (CYP4F12*v1), two isolated substitutions -402G>A (CYP4F12*v3) and -188 T>C (CYP4F12*v4) and nine combined mutations, -474T>C, -279A>C, -224A>G, -173G>A, -145C>G, -140T>C, -126T>C, -56T>C, and -21T>G (CYP4F12*v2). Considering the nature and location of the polymorphisms characterizing the CYP4F12*v1 and *v2, the functional relevance of those two allelic variants was further examined by transfecting different cell lines with constructs of the related region of the CYP4F12/luciferase reporter gene. Both alleles lead to a significant decrease of CYP4F12 gene expression in HepG2 cell line and, therefore, are likely to determine interindividual differences in CYP4F12 gene expression.