A PMM2-CDG caused by an A108V mutation associated with a heterozygous 70 kilobases deletion case report.

BACKGROUND: Congenital Disorders of Glycosylation (CDG) are a large group of inborn errors of metabolism with more than 140 different CDG types reported to date (1). The first characterized, PMM2-CDG, with an autosomal recessive transmission, is also the most frequent. The PMM2 gene encodes a phosphomannomutase. Here, a novel genetic variation causing PMM2-CDG is reported.  CASE PRESENTATION: We report the case of a French child, from healthy and unrelated parents, presenting congenital ataxia with hypotonia, hyperlaxity, inverted nipples, as well as altered coagulation parameters and liver function. Transferrin isoelectrofocusing revealed a typical type I CDG profile. Direct Sanger sequencing and quantitative PCR of PMM2 revealed a unique and novel genotype. On one allele, the patient was heterozygote with a known missense variant NM_000303.3(PMM2):c.323C > T, p.Ala108Val in exon 4. On the second allele, whole genome sequencing (WGS) indicated the presence of a novel heterozygous 70 kb deletion. CONCLUSION: We report in the present paper the largest known heterozygous deletion of a PMM2 gene. The observation reveals the impact of a precise diagnostic on genetic counselling: by using WGS, an erroneous conclusion of homozygosity in the case of a relatively rare variant could be avoided, and an index patient with healthy and unrelated parents correctly identified.

Prevention of isoniazid toxicity by NAT2 genotyping in Senegalese tuberculosis patients.

Isoniazid (INH), recommended by WHO (World Health Organization) in the treatment of tuberculosis (TB), is metabolized primarily by the genetically polymorphic N-acetyltransferase 2 (NAT2) enzyme. The human population is divided into three different phenotypic groups according to acetylation rate: slow, intermediate, and fast acetylators. The objective of this study was to explore the relationship between NAT2 genotypes and the serum concentrations of INH. Blood samples from 96 patients with TB were taken for the analysis. NAT2 polymorphisms on coding region were examined by polymerase chain reaction (PCR) direct sequencing; the acetylation status was obtained by measuring isoniazid (INH) and its metabolite, acetylisoniazid (AcINH) in plasma was obtained by using the liquid chromatography coupled to mass spectrometry. TB patients were distributed into two groups of fast and slow acetylators according to the acetylation index calculated based on the plasma concentration of INH in the 3rd hour (T3) after an oral dose. Our PCR analysis identified several alleles, where NAT2*4, NAT2*5A, NAT2*6A, and NAT2*13A were the most important. The concentrations of INH varied between 1.10 mg/L and 13.10 mg/L at the 3rd hour and between 0.1 and 9.5 mg/L at the 6th hour. The use of the acetylating index I3 allowed the classification of tested patients into two phenotypic groups: slow acetylators (44.3% of TB patients), and rapid acetylators (55.7%). Patient's acetylation profile provides valuable information on their therapeutic, pharmacological, and toxicological responses.

[Type 1 xanthinuria: Report on three cases]. / Xanthinurie héréditaire de type 1 : à propos de trois cas.

Type 1 xanthinuria is a rare cause of urolithiasis due to xanthine dehydrogenase deficiency. Pediatric cases are exceptional. Through the genetic analysis of two cases, we discovered three mutations responsible for a loss of enzyme activity. The first one had a C.3536T>C missense mutation in the XDH gene and the other one was heterozygous for two mutations c.700+1G>T and c.31778_82delTCAT. We review the diagnostic methods, possible complications, and preventive measures for stone formation.

Study of NAT2 genetic polymorphism in West African subjects: example of an healthy non-smoker Senegalese population.

The NAT2 genetic polymorphism determines the individual acetylator status and, consequently, the capacity to metabolize, or not, drugs and xenobiotics which are substrates of NAT2. As the nature and frequency of the NAT2 polymorphisms vary remarkably between populations of different ethnic origins, genotyping strategies used to predict the acetylation phenotype need to be adapted for each particular population regarding their genetic backgrounds at this locus. As few data on the genetic polymorphism of NAT2 are available in the Senegalese population, we performed an extensive identification of NAT2 variants in 105 healthy non-smoker Senegalese subjects by direct PCR sequencing of the coding region. Eleven previously described SNPs were identified in this Senegalese population. Upon allele analysis, the four most frequent alleles were of the NAT2*5- (35.7 %), NAT2*6- (21.0 %), NAT2*12- (16.7 %) and NAT2*14- (10.0 %) type, the remaining alleles, including the wild-type NAT2*4, having each a frequency lower than 10 %. According to the observed genotypes, 51 and 50 subjects were predicted to be of the rapid (48.6 %) and slow (47.6 %) acetylator phenotype, respectively, while four individuals (3.8 %) were considered of unknown phenotype as they carry at least one allele with a yet unknown functional effect. These baseline data would be of particular interest to set up an efficient genotyping strategy to predict the acetylation status of Senegalese patients with tuberculosis and, thus, to optimize their isoniazid treatment.

[L-carnitine treatment and fish odor syndrome: an unwaited adverse effect]. / Traitement par L-carnitine et mauvaise odeur corporelle: un effet secondaire à connaître.

INTRODUCTION: Levocarnitine treatment is usually well tolerated, with essentially dose-dependent diarrhea as the main induced adverse effect. CASE REPORT: We report a case of fish odor syndrome during levocarnitine treatment which resolved after levocarnitine discontinuation. CONCLUSION: This adverse effect seems to be correlated with excedent carnitine intake and might be expressed when the elimination pathway becomes saturated or in a situation of deficiency enzymatic metabolism.

Characterisation of novel defective thiopurine S-methyltransferase allelic variants.

Human thiopurine S-methyltransferase (TPMT, EC 2.1.1.67) is a key enzyme in the detoxification of thiopurine drugs widely used in the treatment of various diseases, such as inflammatory bowel diseases, acute lymphoblastic leukaemia and rheumatic diseases. The TPMT gene is genetically polymorphic and the inverse relationship between TPMT activity and the risk of developing severe hematopoietic toxicity is well known. In this study, the entire coding sequence of TPMT, together with its 5'-flanking promoter region, was analysed in patients with an intermediate phenotype for thiopurine drug methylation. Four polymorphisms were identified, two previously described, c.356A>C (p.Lys(119)Thr, TPMT*9) and c.205C>G (p.Leu(69)Val, TPMT*21), and two novel missense mutations, c.537G>T (p.Gln(179)His, TPMT*24) and c.634T>C (p.Cys(212)Arg, TPMT*25). Structural investigations, using molecular modeling, were undertaken in an attempt to explain the potential impact of the amino acid substitutions on the structure and activity of the variant proteins. Additionally, in order to determine kinetic parameters (K(m) and V(max)) of 6-thioguanine (6-TG) methylation, the four variants were expressed in a recombinant yeast expression system. Assays were performed by HPLC and the results were compared with those of wild-type TPMT. The p.Leu(69)Val and the p.Cys(212)Arg substitutions encode recombinant enzymes with a significantly decreased intrinsic clearance compared to that of the wild-type protein, and, consequently, characterise non-functional alleles of TPMT. The p.Lys(119)Thr and the p.Gln(179)His substitutions do not affect significantly the catalytic activity of the corresponding variant proteins, which prevents to unambiguously describe these latter alleles as defective TPMT variants.

ABCB1 allele polymorphism is associated with virological efficacy in naïve HIV-infected patients on HAART containing nonboosted PIs but not boosted PIs.

OBJECTIVE: To assess the effect of the multidrug resistance-1 single nucleotide polymorphism (ABCB1 SNP) C3435T in exon 26 on the virological responses to first-line protease inhibitor (PI)-containing HAART regimens. METHOD: A cohort of 182 HIV-infected patients with a PI-containing HAART regimen initiated from 1997 to 2004 was enrolled. Time to the first indetectable viral load (VL) was determined in patients with the CC, CT, or TT genotype. RESULTS: There were 37%, 44%, and 19% of patients who had the CC, CT and TT genotypes, respectively. The median estimated times to VL indetectability in the CC, CT, and TT groups were respectively 5.9, 3.9, and 4.8 months (p= .06). In patients on a non-boosted PI regimen, ABCB1 genotype was associated with time to VL indetectability that was shorter in patients with the CT than CC genotype (CT vs. CC, hazard ratio [HR]=0.62, p= .02; TT vs. CC, HR= 0.72, p= .21). This association was not found in patients with first-generation boosted PI-containing regimens and especially not with second-generation boosted PI-containing regimens. CONCLUSION: Our results show that the ABCB1 SNP in exon 26 is associated with virological efficacy in HIV-infected patients treated with non-boosted PI-containing regimens but not with those containing boosted PIs, particularly of the second generation.

[Molecular pharmacogenetics in hospital laboratories in France: current data and future prospects]. / La pharmacogénétique moléculaire hospitalière en France: données actuelles et perspectives.

Molecular pharmacogenetic units have recently been established in several hospital laboratories in France. The clinical impact of these units is still limited and numerous problems of organizational, ethical, legal, technical, social and economical nature remain to be resolved. However, an increasing number of these units, a rise in their activities and an enlargement of their scope of application are foreseeable in the future. Ultimately, these units would significantly contribute to limit the public health problem caused by interindividual variabilities in drug effects. In view of these prospects, it seems essential that such hospital activity should be quickly recognised by the authorities and the various health sectors in France. It is also essential that the problems that arise from such pharmacogenetic activities should be considered by the authorities and would profit from the organization of a national network and from financial guarantees.

CYP2F1 genetic polymorphism: identification of interethnic variations.

Since human cytochrome P450 2F1 (CYP2F1) is predominantly expressed in lung tissue and is involved in the metabolism of various pneumotoxicants with potential carcinogenic effects, variations in the nucleotidic sequence of its gene may contribute to interindividual and interethnic differences in the susceptibility to lung tumorigenesis. The aim of the current study was to compare the frequency of a previously reported frameshift mutation, namely c.14_15insC, responsible for the synthesis of a severely truncated protein, between several populations of different ethnic origins. The frequencies of this polymorphism were 26.1, 51.6, 42.7 and 22.9% in French, Gabonese, Senegalese, and Tunisian population samples, respectively, thereby representing a substantial inter ethnic variation in the CYP2F1 gene. These findings provide data for further studies that investigate the potential association of CYP2F1 haplotypes with an incidence of lung cancer genesis in respect of ethnicity.

Ethnic differences in the distribution of CYP3A5 gene polymorphisms.

The genetic polymorphism affecting the CYP3A5 enzyme is responsible for interindividual and interethnic variability in the metabolism of CYP3A5 substrates. The full extent of the CYP3A5 genetic polymorphism was analysed in French Caucasian, Gabonese and Tunisian populations using a polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) strategy. In the three populations, eight, 17 and ten single nucleotide polymorphisms (SNPs), respectively, were identified, among which nine correspond to rare new mutations. Also identified were 16 alleles including eight new allelic variants. Significant differences were observed in the distribution of these alleles. Particularly, the frequency of the CYP3A5*3C null allele in French Caucasians (81.3%) and in Tunisians (80.0%) is higher than in the Gabonese population (12.5%) (p < 0.001). Considering the CYP3A5 genotypes of the tested individuals, only 10.4% of French Caucasians and 30.0% of Tunisians were identified as CYP3A5 expressors. In contrast, 90.0% of Gabonese subjects appear to express the CYP3A5 protein.

[Therapeutic failure: importance of genes?]. / Echec thérapeutique: peut-être une affaire de gènes?

Drug management can be a difficult task in certain situations because of the variable response observed from one patient to another. Genetic factors affecting the pharmacokinetics and pharmacodynamics of drug reactions could explain the interindividual variability in drug response. Pharmacogenetic analysis provides insight into the molecular mechanisms involved in drug response, with the ultimate goal of achieving optimal drug efficacy and safety. Numerous polymorphisms have been described in genes encoding drug-metabolising enzymes, transporters, and receptors. For some drugs, the impact on drug bioavailability and effect has been elucidated. We review here the molecular basis of interindividual variation in drug response and the methods used to identify individual risk of drug failure or toxicity. Clinical applications, concerning enzymes metabolising drugs (cytochrome P4502D6, thiopurine S-methyltransferase and N-acetyltransferase) provide an illustrative demonstration of the usefulness of pharmacogenetic tests in improving patient management. Clinical validation of these tests and new technologies (real-time PCR, DNA chips) should, in the future promote pharmacogenetics in clinical practice and may be lead to more individualized drug therapy.

CYP2A13 genetic polymorphism in French Caucasian, Gabonese and Tunisian populations.

Since human CYP2A13 is expressed in the respiratory tract and is involved in the activation of tobacco-specific nitrosamines, some of the previously reported sequence variations may contribute to inter-individual and inter-ethnic differences in the susceptibility of tobacco-related tumorigenesis. The aim was to compare the frequencies of the 578C > T (Arg101Stop), 3375C > T (Arg257Cys) and 7520C > G (3'-untranslated region) mutations in several populations. The frequencies of the 578C > T polymorphism were 3.8, 0 and 1.0% in French Caucasians, Gabonese and Tunisians, respectively. In the same populations, the frequencies of the 3375C > T mutation were 0, 15.3 and 4.2%, respectively, whereas the frequencies of the 7520C > G mutation were 1.0, 20.8 and 7.3%, respectively. Marked inter-ethnic variations in CYP2A13 were identified and confirmed. These findings provide data for further studies that associate CYP2A13 haplotypes with an incidence of smoking-related tumours in respect of ethnicity.

Clomipramine, fluoxetine and CYP2D6 metabolic capacity in depressed patients.

Cytochrome P450-2D6 may be involved in the metabolism of many drugs such as psychotropic drugs and its genetic polymorphism is responsible for inter-individual differences in the therapeutic effect and toxicity of these drugs. Moreover with the same genetic basis, CYP2D6 metabolic capacity variations are observed. Different factors of variation may be involved, among them the prescribed drugs. The aim of this study was to compare the influence of two types of antidepressants, tricyclic (clomipramine) and serotonergic specific recapture inhibitor (SSRI) (fluoxetine), on the CYP2D6 metabolic capacity of depressed inpatients. The CYP2D6 phenotype (dextromethorphan test) was determined in 56 genotyped (PCR-SSCP) depressed caucasian inpatients with a heterozygous genotype. Forty-five subjects were treated with clomipramine and eleven received fluoxetine. The dextromethorphan metabolic ratio (MR) median was significantly higher in the fluoxetine group (0.255) than in the clomipramine group (0.083, p < 0.014). In this study, fluoxetine involved a greater decrease of CYP2D6 metabolic capacity than clomipramine. Clinical implications and the possible connection between a decreased CYP2D6 activity and adverse drug effects were discussed. Caution should be taken when drugs with a low therapeutic index must be coprescribed in such patients.

N-acetyltransferase 2 acetylation polymorphism: prevalence of slow acetylators does not differ between atopic dermatitis patients and healthy subjects.

The genetic polymorphism of human N-acetyltransferase 2 (NAT2) divides the human population into groups with rapid, intermediate and slow acetylator status. Slow acetylator status has been considered a predisposing factor for allergic diseases, lupus erythematosus, toxic epidermal necrolysis or Stevens-Johnson syndrome. The aim of this study was to investigate whether Caucasian patients suffering from atopic dermatitis differed from healthy individuals with regard to the genotype and phenotype of NAT2. Twenty unrelated healthy Caucasian volunteers (9 females and 11 males, aged from 22 to 59 years) and twenty unrelated Caucasian patients suffering from atopic dermatitis (9 females and 11 males, aged between 20 and 54 years) participated in this study. For each one, the NAT2 genotype was determined by polymerase chain reaction with DNA extracted from peripheral blood, using specific primers for the wild-type allele (wt) and the 3 most frequent mutated alleles of NAT2 (C481-->T, G590-->A and G857-->A). The NAT2 phenotype was evaluated with dapsone as a test substrate using high-pressure liquid chromatography. Statistical analysis was performed using the chi(2) test. Phenotype and genotype were distributed as follows: (1) of the healthy subjects, 60% were rapid acetylators (RA) and 40% were slow acetylators (SA); 10% of the RA and 15% of the SA were homozygous, 50% of the RA and 25% of the SA were heterozygous; (2) of the patients, 55% were RA, 40% were SA and 5% were intermediate acetylators (IA); 10% of the RA and 10% of the SA were homozygous, 45% of the RA and 35% of the SA were heterozygous. No significant statistical difference was found between the two groups for genotypes (p = 0.75) or phenotypes (p = 0.60). The phenotyping and genotyping results of healthy subjects were comparable to those found in previous studies. The absence of a significant statistical difference between healthy subjects and atopic dermatitis patients is in contrast to the results of previous studies. Some authors considered that allergic patients are mostly SAs. This could be explained by the fact that we only considered patients suffering from atopic dermatitis whereas, in other studies, patients suffered from different (one or several associated) allergic diseases. NAT2 polymorphism does not differ between patients suffering from atopic dermatitis and healthy subjects. The importance attributed to the SA status, which was previously considered a predisposing factor for allergic diseases such as atopic dermatitis, should be reviewed.

In-vitro analysis of the contribution of CYP2D6.35 to ultra-rapid metabolism.

From 10 to 30% of CYP2D6 ultra-rapid metabolizers of Caucasian origin harbor alleles with duplicated or amplified functional CYP2D6 genes. Recently, the CYP2D6*35 allele has been reported to be more frequent in ultra-rapid metabolizing subjects than in extensive metabolizers, suggesting a possible role of this variant in CYP2D6 duplication-negative ultra-rapid metabolizing subjects. In this study, we examined the functional consequences of the Val11Met, Arg296Cys and Ser486Thr amino acid substitutions associated with the CYP2D6*35 on the expression and catalytic activity of the variant enzyme, heterologously expressed in yeast. Our results indicate that the functional activity and level of expression of recombinant CYP2D6.35 are comparable with those of the wild-type enzyme, thus precluding the hypothesis that the high level of enzyme activity in CYP2D6 duplication-negative ultra-rapid metabolizing subjects is a consequence of the expression of a more catalytically effective CYP2D6.35 enzyme.

Identification of genetic variants in the human thromboxane synthase gene (CYP5A1).

Thromboxane synthase (CYP5A1) catalyzes the conversion of prostaglandin H2 to thromboxane A2, a potent mediator of platelet aggregation, vasoconstriction and bronchoconstriction. It has been implicated in the patho-physiological process of a variety of diseases, such as atherosclerosis, myocardial infarction, stroke and asthma. On the basis of the hypothesis that variations of the CYP5A1 gene may play an important role in human diseases, we performed a screening for variations in the human CYP5A1 gene sequence. We examined genomic DNA from 200 individuals, for mutations in the promoter region, the protein encoding sequences and the 3'-untranslated region of the CYP5A1. Eleven polymorphisms have been identified in the CYP5A1 gene including eight missense mutations R61H, D161E, N246S, L357V, Q417E, E450K, T451N and R466Q. This is the first report of genetic variants in the human CYP5A1 altering the protein sequence. The effect of these variants on the metabolic activity of CYP5A1 remains to be further evaluated.

Characterization of new mutations in the coding sequence and 5'-untranslated region of the human prostacylcin synthase gene (CYP8A1).

Inheritable interindividual differences in prostacyclin production may be implicated in the pathogenesis of several human vascular diseases. Using a polymerase chain reaction/single-strand conformation polymorphism strategy, we screened for mutations in the gene encoding cytochrome P450 prostacyclin synthase (CYP8A1). DNA samples from healthy French volunteers (n = 130) of Caucasian origin were examined. Five mutations, comprising two previously reported silent mutations and three novel rare missense mutations (P38L, S118R, and R379S), were identified in the coding sequence of the gene. In the 5'-proximal region, we also found a variable number of tandem repeats (VNTR) polymorphism that consisted of four different alleles with 4-6 tandem repeats of a 9-bp unit containing a putative Spl transcriptional factor binding site. One of these (R6), a frequent allele (23.6% of alleles tested) harboring six repeats, is novel, whereas the other three are known. In vitro analysis of the effect of each VNTR allele on promoter activity of a reporter gene was performed by a transient transfection assay. Data confirmed the modulator effect of the VNTR polymorphism on reporter gene transcription. Furthermore, the data demonstrate that allele R6 has the most potent inducing effects in the A549 cell line and, after IL-6 stimulation, in human pulmonary artery endothelial cells. Overall, the data demonstrate that CYP8A1 is polymorphic in Caucasians, and that a polymorphism affecting the 5'-proximal region may result in interindividual differences in CYP8A1 transcriptional regulation in vivo. Additional factors, such as the presence of inflammatory mediators, may be required to modulate transcription of the CYP8A1 gene.