Characterization of two mutations associated with epimerase-deficiency galactosemia, by use of a yeast expression system for human UDP-galactose-4-epimerase

UDP-galactose-4-epimerase (GALE) is a highly conserved enzyme that catalyzes the interconversion of UDP-galactose and UDP-glucose. Impairment of this enzyme in humans results in one of two clinically distinct forms of epimerase-deficiency galactosemia-one benign, the other severe. The molecular and biochemical distinction between these disorders remains unknown. To enable structural and functional studies of both wild-type and patient-derived alleles of human GALE (hGALE), we have developed and applied a null-background yeast expression system for the human enzyme. We have demonstrated that wild-type hGALE sequences phenotypically complement a yeast gal10 deletion, and we have biochemically characterized the wild-type human enzyme isolated from these cells. Furthermore, we have expressed and characterized two mutant alleles, L183P-hGALE and N34S-hGALE, both derived from a patient with no detectable GALE activity in red blood cells but with approximately 14% activity in cultured lymphoblasts. Analyses of crude extracts of yeast expressing L183P-hGALE demonstrated 4% wild-type activity and 6% wild-type abundance. Extracts of yeast expressing N34S-hGALE demonstrated approximately 70% wild-type activity and normal abundance. However, yeast coexpressing both L183P-hGALE and N34S-hGALE exhibited only approximately 7% wild-type levels of activity, thereby confirming the functional impact of both substitutions and raising the intriguing possibility that some form of dominant-negative interaction may exist between the mutant alleles found in this patient. The results reported here establish the utility of the yeast-based hGALE-expression system and set the stage for more-detailed studies of this important enzyme and its role in epimerase-deficiency galactosemia.     

Two CPT2 mutations in three Japanese patients with carnitine palmitoyltransferase II deficiency: functional analysis and association with polymorphic haplotypes and two clinical phenotypes

Carnitine palmitoyltransferase II (CPT II) deficiency manifests as two different clinical phenotypes: a muscular form and a hepatic form. We have investigated three nonconsanguineous Japanese patients with CPT II deficiency. Molecular analysis revealed two missense mutations, a glutamate (174)-to-lysine substitution (E174K) and a phenylalanine (383)-to-tyrosine substitution (F383Y) in the CPT II cDNA. Transfection experiments in COS-1 cells demonstrated that the two mutations markedly decreased the catalytic activity of mutant CPT II. Case 1 (hepatic form) was homozygous for the F383Y mutation, whereas case 3 (muscular form) was homozygous for the E174K mutation. Case 2 and her brother, who were compound heterozygotes for E174K and F383Y, exhibited the hepatic phenotype. We also identified a novel polymorphism in the CPT2 gene, a phenylalanine (352)-to-cysteine substitution (F352C), which did not alter CPT II activity in transfected cells. It was present in 21 out of 100 normal alleles in the Japanese population, but absent in Caucasian populations. Genotyping with the F352C polymorphism and the two previously reported polymorphisms, V368I and M647V, allowed normal Japanese alleles to be classified into five haplotypes. In all three families with CPT II deficiency, the E174K mutation resided only on the F1V1M1 allele, whereas the F383Y mutation was observed on the F2V2M1 allele, suggesting a single origin for each mutation.     

Identification of missense mutations and haplotyping of carnitine palmitoyltransferase II gene

Carnitine palmitoyltransferase II(CPTII) deficiency manifests as two different clinical phenotypes: an adult form associated with muscular symptoms and an infantile form presenting with hepatocardiomuscular manifestations. We have investigated three Japanese patients with CPT II deficiency. Molecular analysis revealed two novel missense mutations, a glutamate (174)-to-lyine substitution (E174K) and a phenylalanine (383)-to-tyrosine substitution (F383Y) in the CPTII cDNA. Transfection experiments demonstrated that the two mutations reduced CPTII catalytic activity. We also identified a novel polymorphism in the CPTII gene, a phenylalanine (352)-to-cysteine substitution (F352C). According to an expression analysis this mutation did not alter CPTII activity. It was present in 21 out of 100 normal alleles in the Japanese population, but was not observed among Caucasians. Genotyping with the F352C polymorphism and the previously reported polymorphisms V368I and M647V allowed normal alleles to be classified into five haplotypes. In all three families, the E174K mutation resided only on F1V1M1 allele, while the F383Y mutation was observed on F2V2M1 allele, suggesting a single origin of each mutation.     

The role of xenobiotic metabolizing enzymes in arylamine toxicity and carcinogenesis: functional and localization studies

Familial apolipoprotein C-II (apo C-II) deficiency is an autosomal recessive genetic disorder characterized by fasting hypertriglyceridemia and accumulation of chylomicrons in the plasma. To elucidate the genetic defect, the apo C-II gene of a neonatal Japanese patient (C-IITokyo) was analyzed. Nucleotide sequence analysis showed a G+1 to C transversion at the donor splice site of intron 2 (INT2 G+1 to C). Restriction fragment length polymorphism analyses of the patient's family members with Hph I showed that the patient was homozygous and the parents were heterozygous for the INT2 G+1 to C mutation. Although consanguinity could not be demonstrated, haplotype analysis of the C-II gene revealed the identity of the patient's alleles on the mutation, suggesting that the parents had a common Japanese ancestor. Sequence analysis of the patient's cDNA isolated from peripheral blood lymphocytes revealed that the INT2 G+1 to C mutation causes skipping of exon 2, which encodes the initiation codon, and results in deficiency of apo C-II proteins. The outstanding feature of our patient was that he showed severe hypertriglyceridemia beginning in the neonatal period, a feature not reported in a case of apo C-II deficiency (C-IIHamburg) with the same mutation as our patient. A previous report of another case of apo C-II deficiency (C-IIToronto) suggested that the apo E4 isoform is associated with higher levels of plasma triglycerides in subjects heterozygous for the apo C-II mutation. Determination of the apo E isoform of our patient revealed that apo E4 was coinherited with the INT2 G+1 to C mutation, whereas the apo E isoform has been reported to be E2/3 in C-IIHamburg. We speculate that apo E4/4 aggravated the hypertriglyceridemia in our patient with apo C-II deficiency.     

Protective effect of diltiazem on myocardial ischemic injury associated with .OH generation

Two patients from two unrelated families with a history of thrombosis showed severe plasma activated protein C (APC) resistance. However, genotypic analysis demonstrated that the patients were heterozygous for factor V (FV) Leiden mutation. Coagulation studies revealed that FV clotting activity and antigen were similarly reduced at about 50% of normal in the patients. One brother of propositus A also showed the same abnormalities. Genetic analysis showed that, in addition to FV Leiden mutation in exon 10 of the FV gene (G1691A), these patients had a transition in exon 13 of the FV gene (A4070G; R2 allele) predicting His1299Arg substitution in the mature FV. Study by RT-PCR of platelet FV mRNA indicated that the mRNA produced by the FV gene, marked by the R2 allele, was reduced in amount in both pseudohomozygous patients of family A. The R2 allele has previously been demonstrated to be significantly associated with plasma FV deficiency in the Italian population. The presence of FV deficiency did not protect the propositi from thrombosis. These data confirm that genotypic analysis is mandatory in patients with phenotypic severe APC resistance before these patients are definitely classified as homozygotes for FV Leiden and that further genotypic analysis is advisable.     

Ribose-enhanced synthesis of UTP, CTP, and GTP from parent nucleosides in cardiac myocytes

1. Characterization of allelic variants of the TPMT gene (TPMT) responsible for changes in TPMT activity, and elucidation of the mechanism by which these alleles act, are required because of the clinical importance of this polymorphism for patients receiving thiopurine drugs. 2. We defined the mutational and allelic spectrum of TPMT in a group of 191 Europeans. Using PCR-SSCP, we screened for mutation the entire coding sequence, the exon-intron boundaries, the promoter region and the 3'-flanking region of the gene. Six mutations were detected throughout the ten exons and seven TPMT alleles were characterized. Four of them, TPMT*2, *3A, *3C and *7, harbouring the known mutations, G238C, G460A, A719G or T681G, were nonfunctional and accounted for 0.5, 5.7, 0.8 and 0.3% of the allele totality, respectively. 3. Within the promoter region, six alleles corresponding to a variable number of tandem repeats (VNTR), were identified. VNTR*V4 and *V5a which harbour four or five repeats of a 17-18 bp unit, were the most frequent (55% and 34%, respectively). The other VNTR alleles, having from five to eight repeats, were rarer. 4. The TPMT phenotype was correctly predicted by genotyping for 87% of individuals. A clear negative correlation between the total number of repeats from both alleles and the TPMT activity level was observed, indicating that VNTRs contribute to interindividual variations of TPMT activity. Therefore, additional analysis of the promoter region of TPMT can improve the phenotype prediction rate by genotyping.     

A rural diabetes support group

To determine whether nonclassical steroid 21-hydroxylase deficiency in Japan has the same molecular basis as in western countries, we have characterized the mutations of the CYP21 gene in 7 Japanese patients with nonclassical (NC) steroid 21-hydroxylase deficiency. In the Japanese NC cases the P30L was present in one allele in 5 of the 7 patients and on both alleles in one patient. By contrast, the V281L mutation, which was present in about 60% of NC cases in western countries, was not identified in any patient. Among our 7 cases, 4 were detected through neonatal mass screening by a mild increase in serum 17-hydroxyprogesterone (without any symptoms of CAH) at birth, but the 2 cases who were diagnosed as adults were born before nationwide neonatal screening was instituted, so that the Japanese neonatal screening program does detect some cases of NC steroid 21-hydroxylase deficiency. We suggest that P30L mutation is more frequent in Japanese NC CAH than V281L and that the frequency of the mutations causing NC steroid 21-hydroxylase deficiency in Japan might be different from that in western countries.     

Evaluation of ADCI against convulsant and locomotor stimulant effects of cocaine: comparison with the structural analogs dizocilpine and carbamazepine

Molecular genetic analysis has been performed on a patient with Bernard-Soulier syndrome (BSS). The patient had characteristically giant platelets and was deficient in the glycoprotein (GP) Ib/IX/V complex, the von Willebrand factor (vWf) receptor on platelets. Previous studies with monoclonal antibodies directed against GP Ib alpha (CD 42b) and GP IX (CD 42a) demonstrated the absence of GP Ib alpha and presence of small amounts of GP IX on the surface of the patient's platelets. In this study the presence of GP V (CD 42d) is also demonstrated. This indicates a defect in the alpha-subunit of glycoprotein Ib. Therefore polymerase chain reaction (PCR)-amplification of the genomic DNA coding for GP Ib alpha was performed. Nucleotide sequence analysis of the entire coding region of GP Ib alpha revealed a homozygous single base pair mutation T-->A, leading to a single amino acid substitution cysteine-->serine at position 209 of the mature protein. We took advantage of the Mse I target site in the mutant allele, created by the T-->A mutation, to analyse all available family members. PCR-ASRA (allele-specific restriction enzyme analysis) using the restriction enzyme Mse I, revealed the heterozygosity of the mother and the two children of the patient, whereas homozygosity of the patient for the Cys209Ser mutation was confirmed. The sister of the patient was not found to be a carrier of the mutant allele. The mutation identified in the family studied, responsible for the deficiency of the GP Ib/IX/V complex, suggests that the cysteine at amino acid position 209 may be involved in disulphide bonding.     

An adenosine deaminase (ADA) allele contains two newly identified deleterious mutations (Y97C and L106V) that interact to abolish enzyme activity

Genetic deficiency of the purine salvage enzyme adenosine deaminase (ADA) results in varying degrees of immunodeficiency, ranging from neonatal onset Severe Combined Immunodeficiency (SCID) to an adult onset immunodeficiency disorder. Multiple different mutations have now been identified in these immunodeficient patients. Additional mutations, initially identified in healthy individuals, abolish ADA in erythrocytes but retain 10-80% of activity in non-erythroid cells ('partial deficiency mutations'). In general, severity of disease correlates inversely with the amount of residual ADA expressed by the mutant enzymes and directly with the accumulation of the toxic metabolites deoxyATP and deoxyadenosine. We report two newly identified mutations (Y97C and L106V), both carried on the same allele of an immunodeficient patient who was diagnosed prenatally and successfully transplanted with haploidentical bone marrow. Based on the ability of mutant cDNAs to express ADA in vitro , the L106V mutation resulted in activity similar to 'partial' mutations (30% of normal) while the Y97C mutation resulted in detectable but markedly reduced activity (1.5% of normal). However, the presence of both mutations on the same allele virtually abolished detectable enzyme activity. Analysis of the crystallographic structure of ADA to understand the marked deleterious effect of the Y97C mutation suggested a previously unappreciated role of salt bridges in the catalytic mechanism of ADA. The patient was also heteroallelic for a previously described deletion of the promoter and exon 1. Testing of additional patients in whom we had not identified a mutation on the second allele revealed presence of this deletion in three of four patients tested. This deletion is therefore relatively common, accounting for 10% of almost 100 chromosomes studied by this and other laboratories, but is easily missed by currently used methods of mutation detection. Lastly, the finding of two mutations on the same allele that interact to reduce residual enzyme function emphasizes hazards in evaluating potential genotype-phenotype correlations in individuals analyzed only for the presence of single specific mutations.     

Allele-dropout using PCR-based diagnosis for the splicing mutation in intron-2 of the CYP21B-gene: successful amplification with a Taq/Pwo-polymerase mixture

The splicing mutation in intron 2 (nucleotide 656) of the 21-hydroxylase gene (CYP21B) is the most common mutation causing congenital adrenal hyperplasia (CAH). Homozygosity for nt656G is associated with the classical phenotype of CAH. In several studies, a number of clinically asymptomatic relatives of CAH-patients were genotyped as nt656G homozygotes. We have proposed that the putative asymptomatic nt656G/G individuals are incorrectly typed due to dropout of one allele (in most cases nt656C) during PCR amplification. Here, we report the successful amplification of all alleles at nt656 with a Taq/Pwo DNA polymerase mixture in the primary PCR reaction. The results were independent from the type of polymerase used for sequencing reactions as the second step in mutation analysis.     

Pseudodeficiency of arylsulphatase A: strategy for clarification of genotype in families of subjects with low ASA activity and neurological symptoms

A benign deficiency (pseudodeficiency) of the lysosomal enzyme arylsulphatase A (ASA) (EC 3.1.6.8) towards synthetic substrates complicates the diagnosis of metachromatic leukodystrophy (MLD). The pseudodeficiency is due to a single base substitution in the 3'-untranslated region of the ASA gene (1524+95 A-->G) and it has been reported that this mutation (PD2) always occurs on a chromosome carrying a second mutation in the ASA gene (PD1), which abolishes an N-glycosylation site (N350S). Analysis of the two PD mutations in the ASA gene separately was carried out in a large group of subjects with neurological symptoms and low ASA activity, including close relatives and MLD patients. The relationship between ASA enzyme activity and the different genotypes identified is presented. Evidence for the existence of an allele containing the PD2 mutation alone is presented. A strategy for cases with low ASA activity and neurological symptoms in families carrying a PD allele or both PD and MLD alleles is proposed.     

The new antiarrhythmic substance AWD 23-111 inhibits the delayed rectifier potassium current (IK) in guinea pig ventricular myocytes

In a family with three siblings, one developed classical late infantile metachromatic leukodystrophy (MLD), fatal at age 5 years, with deficient arylsulfatase A (ARSA) activity and increased galactosylsulfatide (GS) excretion. The two other siblings, apparently healthy at 12(1/2) and 15 years, respectively, and their father, apparently healthy as well, presented ARSA and GS values within the range of MLD patients. Mutation screening and sequence analysis disclosed the involvement of three different ARSA mutations being the molecular basis of intrafamilial phenotypic heterogeneity. The late infantile patient inherited from his mother the frequent 0-type mutation 459+1G>A, and from his father a novel, single basepair microdeletion of guanine at nucleotide 7 in exon 1 (7delG). The two clinically unaffected siblings carried the maternal mutation 459+1G>A and, on their paternal allele, a novel cytosine to thymidine transition at nucleotide 2435 in exon 8, resulting in substitution of alanine 464 by valine (A464V). The fathers genotype thus was 7delG/A464V. Mutation A464V was not found in 18 unrelated MLD patients and 50 controls. A464V, although clearly modifying ARSA and GS levels, apparently bears little significance for clinical manifestation of MLD, mimicking the frequent ARSA pseudodeficiency allele. Our results demonstrate that in certain genetic conditions MLD-like ARSA and GS values need not be paralleled by clinical disease, a finding with serious diagnostic and prognostic implications. Moreover, further ARSA alleles functionally similar to A464V might exist which, together with 0-type mutations, may cause pathological ARSA and GS levels, but not clinical outbreak of the disease.     

Structures that delineate orphanin FQ and dynorphin A pharmacological selectivities

A genetic polymorphism in the metabolism of the anticonvulsant drug S-mephenytoin has been attributed to defective CYP2C19 alleles. This genetic polymorphism displays large interracial differences with the poor metabolizer (PM) phenotype representing 2-5% of Caucasian and 13-23% of Oriental populations. In the present study, we identified two new mutations in CYP2C19 in a single Swiss Caucasian PM outlier (JOB 1) whose apparent genotype (CYP2C19*1/CYP2C19*2) did not agree with his PM phenotype. These mutations consisted of a single base pair mutation (G395A) in exon 3 resulting in an Arg132-->Gln coding change and a (G276C) mutation in exon 2 resulting in a coding change Glu92-->Asp. However, the G276C mutation and the G395A mutation resided on separate alleles. Genotyping tests of a family study of JOB1 showed that the exon 2 change occurred on the CYP2C19*2 allele, which also contained the known splice mutation in exon 5 (this variant is termed CYP2C19*2B to distinguish it from the original splice variant now termed CYP2C19*2A). The exon 3 mutation resided on a separate allele (termed CYP2C19*6). In all other respects this allele was identical to one of two wild-type alleles, CYP2C19*1B. The incidence of CYP2C19*6 in a European Caucasian population phenotyped for mephenytoin metabolism was 0/344 (99% confidence limits of 0 to 0.9%). Seven of 46 Caucasian CYP2C19*2 alleles were CYP2C19*2B(15%) and 85% were CYP2C19*2A. The Arg132Gln mutation was produced by site-directed mutatgenesis and the recombinant protein expressed in a bacterial cDNA expression system. Recombinant CYP2C19 6 had negligible catalytic activity toward S-mephenytoin compared with CYP2C19 1B, which is consistent with the conclusion that CYP2C19*6 represents a PM allele. Thus, the new CYP2C19*6 allele contributes to the PM phenotype in Caucasians.     

Deleterious missense mutations and silent polymorphism in the human 17beta-hydroxysteroid dehydrogenase 3 gene (HSD17B3)

Isozymes of 17beta-hydroxysteroid dehydrogenase (17betaHSD) regulate levels of bioactive androgens and estrogens in a variety of tissues. For example, the 17betaHSD type 3 isozyme catalyzes the conversion of the inactive C19-steroid androstenedione to the biologically active androgen, testosterone, in the testis. Testosterone is essential for the correct development of male internal and external genitalia; hence, deleterious mutations in the HSD17B3 gene give rise to a rare form of male pseudohermaphroditism termed 17betaHSD deficiency. Here, 2 additional missense mutations in the HSD17B3 gene in subjects with 17betaHSD deficiency are described. One mutation (A56T) impairs enzyme function by affecting NADPH cofactor binding. A second mutation (N130S) led to complete loss of enzyme activity. Also, a single base pair polymorphism in exon 11 of the HSD17B3 gene is described. The polymorphic A allele encodes a protein with a serine rather than a glycine at position 289 (GGT --> AGT). The frequency of the G allele (Gly) was 0.94, and that of the A allele (Ser) was 0.06. No difference in the frequencies of the G and A alleles was detected in 32 apparently normal women and 46 women with polycystic ovary syndrome. Enzymes bearing either glycine or serine at this position have similar substrate specificities and kinetic constants. The current findings boost to 16 the number of mutations in the HSD17B3 gene that impair testosterone synthesis and cause male pseudohermaphroditism, and add 1 apparently silent polymorphism to this tally.     

Genetic susceptibility to pregnancy-related venous thromboembolism: roles of factor V Leiden, prothrombin G20210A, and methylenetetrahydrofolate reductase C677T mutations

OBJECTIVE: This study's objective was to evaluate the association between venous thromboembolism during pregnancy and the postpartum period and the factor V Arg 506 Gln (factor V Leiden), the prothrombin G20210A, and methylenetetrahydrofolate reductase C677T polymorphisms. STUDY DESIGN: In this case-control study 42 case patients and 213 control subjects (parous age-matched women without history of thrombosis) were genotyped for all the polymorphisms. Moreover, antiphospholipid antibodies and protein C, protein S, and antithrombin III deficiencies were investigated in each case. RESULTS: Ten case patients (23.8%) and 4 control subjects (1.9%; odds ratio 16.3, 95% confidence interval 4.8-54.9) carried the factor V Leiden mutation; 13 case patients (31.0%) and 9 control subjects (4.2%; odds ratio 10.2, 95% confidence interval 4.0-25.9) were carriers of the prothrombin G20210A allele. Finally, 12 case patients (28.6%) and 34 control subjects (16.0%; odds ratio 2.1, 95% confidence interval 1.0-4.5) were homozygotes for methylenetetrahydrofolate reductase C677T. Overall, mutations were found in 25 case patients (59.5%) and 47 control patients (22.2%; odds ratio 5.2, 95% confidence interval 4.9-19.6). One patient carried the antithrombin III deficiency and 1 the protein S deficiency, whereas 2 women had a primary antiphospholipid syndrome. CONCLUSIONS: The significant risk estimates of having a pregnancy-related venous thromboembolism in the presence of the prothrombotic genetic risk factors analyzed suggest to screen for these mutations women with a personal history of thromboembolic events during pregnancy or the postpartum period.     

A novel type II complement C2 deficiency allele in an African-American family

A 9-yr-old African-American male presenting with severe recurrent pyogenic infections was found to have C2 deficiency (C2D). Analysis of his genomic DNA demonstrated that he carried one type I C2D allele associated with the HLA-A25, B18, DR15 haplotype. Screening all 18 exons of the C2 gene by exon-specific PCR/single-strand conformation polymorphism indicated abnormal bands in exons 3, 7, and 6, the latter apparently caused by the 28-bp deletion of the typical type I C2D allele. Nucleotide (nt) sequencing of the PCR-amplified exons 3 and 7 revealed a heterozygous G to A transition at nt 392, causing a C111Y mutation, and a heterozygous G to C transversion at nt 954, causing a E298D mutation and a polymorphic MaeII site. Cys111 is the invariable third half-cystine of the second complement control protein module of C2. Pulse-chase biosynthetic labeling experiments indicated that the C111Y mutant C2 was retained by transfected COS cells and secreted only in minimal amounts. Therefore, this mutation causes a type II C2D. In contrast, the E298D mutation affected neither the secretion of C2 from transfected cells nor its specific hemolytic activity. Analysis of genomic DNA from members of the patient's family indicated that 1) the proband as well as one of his sisters inherited the type I C2D allele from their father and the novel type II C2D allele from their mother; 2) the polymorphic MaeII site caused by the G954C transversion is associated with the type I C2D allele; and 3) the novel C111Y mutation is associated in this family with the haplotype HLA-A28, B58, DR12.     

Clinical value of Doppler ultrasound controlled puncture of the inguinal vessels with the "Smart Needle" within the scope of heart catheter examination]

BACKGROUND: Thiopurine S-methyltransferase (TPMT) catalyzes the S-methylation (that is, inactivation) of mercaptopurine, azathioprine, and thioguanine and exhibits genetic polymorphism. About 10% of patients have intermediate TPMT activity because of heterozygosity, and about 1 in 300 inherit TPMT deficiency as an autosomal recessive trait. If they receive standard doses of thiopurine medications (for example, 75 mg/m2 body surface area per day), TPMT-deficient patients accumulate excessive thioguanine nucleotides in hematopoietic tissues, which leads to severe and possibly fatal myelosuppression. OBJECTIVE: To elucidate the genetic basis and develop molecular methods for the diagnosis of TPMT deficiency and heterozygosity. DESIGN: Diagnostic test evaluation. SETTING: Research hospital. PATIENTS: The TPMT phenotype was determined in 282 unrelated white persons, and TPMT genotype was determined in all persons who had intermediate TPMT activity (heterozygotes) and a randomly selected, equal number of persons who had high activity. In addition, genotype was determined in 6 TPMT-deficient patients. MEASUREMENTS: Polymerase chain reaction (PCR) assays were developed to detect the G238C transversion in TPMT*2 and the G460A and A719G transitions in TPMT*3 alleles. Radiochemical assay was used to measure TPMT activity. Mutations of TPMT were identified in genomic DNA, and the concordance of TPMT genotype and phenotype was determined. RESULTS: 21 patients who had a heterozygous phenotype were identified (7.4% of sample [95% CI, 4.7% to 11.2%]). TPMT*3A was the most prevalent mutant allele (18 of 21 mutant alleles in heterozygotes; 85%); TPMT*2 and TPMT*3C were more rare (about 5% each). All 6 patients who had TPMT deficiency had two mutant alleles, 20 of 21 patients (95% [CI, 76% to 99.9%]) who had intermediate TPMT activity had one mutant allele, and 21 of 21 patients (100% [CI, 83% to 100%]) who had high activity had no known TPMT mutation. Detection of TPMT mutations in genomic DNA by PCR coincided perfectly with genotypes detected by complementary DNA sequencing. CONCLUSIONS: The major inactivating mutations at the human TPMT locus have been identified and can be reliably detected by PCR-based methods, which show an excellent concordance between genotype and phenotype. The detection of TPMT mutations provides a molecular diagnostic method for prospectively identifying TPMT-deficient and heterozygous patients.     

Molecular characterization of homozygous variegate porphyria

Variegate porphyria (VP) is a low penetrance, autosomal dominant disorder that results from partial deficiency of protoporphyrinogen oxidase (PPOX) activity caused by mutation in the PPOX gene. The rare homozygous variant of VP is characterized by severe PPOX deficiency, onset of photosensitization by porphyrins in early childhood, skeletal abnormalities of the hand and, less constantly, short stature, mental retardation and convulsions. We have identified PPOX mutations on both alleles of five of the 11 unrelated patients with homozygous VP reported to date. Two patients were homoallelic for missense mutations (D349A and A433P), while three were heteroallelic. Functional analysis by prokaryotic expression showed that the D349A and A433P and one missense mutation in each of the three heteroallelic patients (G358R in two patients and A219KANA) preserved some PPOX activity (9.5-25% of wild-type). Mutations on the other allele of the heteroallelic patients abolished or markedly decreased activity. There was no relation between genotype assessed by functional analysis and the presence or severity of non-cutaneous manifestations. The mutations were absent from 104 unrelated patients with autosomal dominant VP. Our findings define the molecular pathology of homozygous VP and suggest that mild PPOX mutations occur in the general population but have very low or no clinical penetrance in heterozygotes.