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PubMed1996

Amino acid substitutions of thyroid hormone receptor-beta at codon 435 with resistance to thyroid hormone selectively alter homodimer formation.

Thyroid hormone action is mediated through its nuclear receptors (TRs), which bind to target DNA sequences [thyroid hormone response element (TRE)] as a homodimer or a heterodimer with 9-cis-retinoic acid receptors. Mutations of TR beta identified in patients with resistance to thyroid hormone (RTH) cluster primarily at two areas separated by the putative dimerization region. Two TR beta mutations were newly found in patients with RTH at codon 435 histidine (H435L and H435Q) close to the dimerization region. Recent crystallographic study suggested that H435 is critical for direct contact with T3. To study how the side-chain charge of amino acids at this position affects receptor characteristics, T3-binding activity, receptor dimerization, transcriptional activity, and dominant negative action were analyzed in two RTH mutants and two additional artificial mutants (H435R and H435E). The T3 binding affinities of all four mutants were below detection. In electrophoretic mobility shift assay using TRE-DR4 or the inverted palindrome (Lap), heterodimer formation of mutant receptors with 9-cis-retinoic acid receptor was similar to that of wild type receptors. However, homodimer formation varied among mutant receptors, especially using TRE-DR4, with a rank order of wild type = H435R > H435Q > H435L > > H435E. In the presence of a basic amino acid at codon 435, homodimer formation was preserved, whereas substitution to neutral or acidic amino acids resulted in decreased homodimer formation. In transient transfection assays using reporter genes under the control of 2xPal-thymidine kinase (TK), DR4-TK, Lap-TK, or TSH alpha promoter, these four mutants were inactive in T3-dependent transcriptional activation. Dominant negative inhibition was similar for all four mutants. These results indicate that 1) newly found TR beta mutations at codon 435 are responsible for RTH; and 2) codon 435 in TR beta is located at a position that can predominantly alter homodimer formation on certain TREs, such as DR4.

Amino Acid Sequence↗
PubMed1995

Mutations of codon 918 in the RET proto-oncogene correlate to poor prognosis in sporadic medullary thyroid carcinomas.

The hereditary multiple endocrine neoplasia syndromes types 2A and B (MEN 2A and B) were recently linked to germline mutations in the RET proto-oncogene, altering one of five cysteine residues in exon 10 or 11 (MEN 2A), or substituting a methionine for a threonine at codon 918 in exon 16 (MEN 2B). The latter mutation also occurs somatically in some sporadic medullary thyroid carcinomas (MTC), and has in a previous study been correlated with a less favorable clinical outcome. In the present study, 46 MTCs were selected for investigation of the codon 918 mutation. The mutation was found in 29 tumors (63%), and was significantly correlated with a poor outcome, with regard to distant metastasis or tumor recurrence (p < 10(-4)). Two tumors showed multifocal growth and C-cell hyperplasia, and these patients were therefore also investigated for germline mutations in exons 10, 11 and 16. The codon 918 mutation was found only in the tumors, thus of somatic origin. The RET codon 918 mutation may have prognostic impact, and therefore preoperative assessment may influence decision-making in the treatment of patients suffering from MTC.

Base Sequence↗
PubMed1993

A new point mutation in the prion protein gene at codon 210 in Creutzfeldt-Jakob disease.

We screened 16 cases of sporadic Creutzfeldt-Jakob disease (CJD) and 28 healthy control subjects to detect possible polymorphisms in their prion protein gene (PRNP). The molecular analysis of the PRNP coding sequence was performed using denaturing gradient gel electrophoresis of polymerase chain reaction products and direct sequencing. We identified (1) a silent mutation at codon 177 in a healthy individual, (2) a codon 200 glutamate-to-lysine substitution in a 48-year-old CJD-affected Libyan Jew, and (3) a G-to-A point substitution at codon 210, leading a valine-to-isoleucine change, in a 63-year-old French CJD patient. This new mutation occurs in a highly conserved part of the PRNP coding sequence, close to the known CJD-associated codon 200 mutation, and might be linked to a symptomatologic and neuropathologic pattern of typical sporadic CJD. This mutation was also present in a sister of the patient who died at the age of 67 without neurologic symptomatology.

Aged↗
PubMed1997

Hepatocellular carcinoma p53 G > T transversions at codon 249: the fingerprint of aflatoxin exposure?

The molecular epidemiology of p53 mutations allows the possibility of correlating particular mutations with specific environmental carcinogens and establishing one step in the causal pathway between exposure to carcinogens and the development of cancer. A striking example is the G > T transversion at the third base pair of codon 249 observed in liver cancer patients possibly exposed to high levels of aflatoxins in their agricultural products. In this paper, we describe a systematic review of the literature and access the quality of the available data. We found methodologic limitations in the studies. In particular, the key independent variable, aflatoxin exposure, was not assessed in these studies, with the exception of one study that measured a marker of exposure. Instead, nationality, geographic residence, or geographic site of hospital were used as surrogate markers for exposure. Patients from areas with high aflatoxin levels were more likely to have p53 mutations than were patients from areas with low aflatoxin levels. In the group with p53 mutations, patients from areas with high aflatoxin levels had higher proportions of mutations with codon 249 G > T transversions. The differences in proportions with p53 mutations were significant, as were the differences in proportions of codon 249 G > T transversions among patients with p53 mutations. Aflatoxin may increase the proportion of p53 mutations by causing a single mutation, the codon 249 G > T transversion, thus explaining some of the excess liver cancer associated with aflatoxin exposure. However, it is premature to conclude that p53 mutations are established markers for environmental carcinogens.

Aflatoxins↗
PubMed2005

Relation of beta2-adrenoceptor polymorphisms at codons 16 and 27 to persistence of asthma symptoms after the onset of puberty.

BACKGROUND: It has long been recognized that many children with asthma outgrow the disease after the onset of puberty, but little is known about genetic factors influencing this outcome. OBJECTIVES: The aim of the present study was to determine whether the polymorphisms at codons 16 and 27 of the beta2-adrenoceptor are significant predictors of the persistence of asthma during adolescence. DESIGN AND PARTICIPANTS: We used data from the prospective Tucson Children's Respiratory Study. Children were genotyped for the polymorphisms at codons 16 and 27. The presence of wheezing/asthma was assessed by questionnaire from age 6 years up to the reported onset of puberty (prepubertal period) and after the onset of puberty up to age 16 years (adolescence). RESULTS: Among children who wheezed in the prepubertal period (n = 168), subjects homozygous for Gly at codon 16 were at significantly increased risk for persistent wheezing after puberty, as compared with carriers of the other genotypes (relative risk [RR], 1.43; 95% confidence interval [CI], 1.06 to 1.92; p = 0.019). This relation was present among boys (RR, 2.17; 95% CI, 1.41 to 3.36) but not girls (RR, 0.85; 95% CI, 0.55 to 1.30), and increased linearly according to the frequency of wheezing episodes after the onset of puberty. These findings persisted after adjusting for ethnicity and other potential confounders and after selecting only white children. The polymorphism at codon 27 showed no relation with risk for persistent wheezing. CONCLUSIONS: This study provides evidence for a strong gender-specific effect of the Gly16 polymorphism on the persistence of asthma after the onset of puberty.

Adolescent↗
PubMed2004

Association between the TAP1 gene codon 637 polymorphism and Graves' disease.

A total of 95 patients with Graves' disease (GD) and 105 normal healthy controls were enrolled in this study to determine how a single site polymorphism of the transporter associated with antigen processing 1 (TAP1) gene contributes to the pathogenesis of GD. The polymorphism was detected using polymerase chain reaction (PCR)-based restriction analysis. Associations between GD and the two-site polymorphisms of the TAP1 gene at codons 333 and 637 were evaluated. No significant differences were revealed comparing GD patients and normal individuals for the distributions of genotypes and allelic variants at codon 333 (p=0.253 and p=0.891, respectively). By contrast, the distributions for the AA homozygote at codon 637 were reduced and those for the GA heterozygote were increased comparing the two groups (p<0.0001). The allelic analysis also demonstrated lower A and higher G allele frequencies (p=0.0008; OR=2.745, 95% CI=1.482-5.085) comparing the GD patients with the normal healthy controls. This shows that the single-site polymorphism of the TAP1 gene at codon 637 may be an indicator for predicting development of GD.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Absence of mutations at codon 768 of the RET proto-oncogene in sporadic and hereditary pheochromocytomas.

Sixteen sporadic pheochromocytomas, 3 pheochromocytomas in neurofibromatosis 1, and 4 pheochromocytomas in multiple endocrine neoplasia (MEN) 2A or 2B were screened for mutations at codon 768 of the RET proto-oncogene by AluI digestion of polymerase chain reaction PCR products and mutations in exon 13 by PCR-single strand conformation polymorphism (SSCP) analysis. Although mutations at codon 768 (GAG --> GAC; Glu --> Asp) of the RET proto-oncogene were recently reported to be found in 40% of sporadic medullary thyroid carcinomas (MTCs), the absence of missense mutations at codon 768 was confirmed both with PCR-restriction fragment length polymorphism (RFLP) and PCR-SSCP analysis in all examined cases of pheochromocytomas. These results suggest that mutations at codon 768 of the RET proto-oncogene do not represent a frequent mechanism of tumorigenesis for both sporadic and hereditary pheochromocytomas.

Adrenal Gland Neoplasms↗
PubMed1975

Properties of Isoaccepting tRNALys, tRNAGlu and tRNAGln from rabbit Reticulocytes and liver. Multiplicity, Codon Recognition and Inactivation by Iodine.

The three tRNA species from rabbit liver and reticulocytes, each corresponding to the codons XAA and XAG(X equals A, G, C) were investigated. The elution patterns of the isoaccepting tRNA subspecies after separation by reversedphase chromatography followed by determination of amino acid acceptance have common and differing characteristics. Three subspecies of tRNALys, two of tRNAGlu and three of tRNAGln have been found in reticulocytes, wheras two subspecies of tRNALys, three of tRNAGlu and four of tRNAGln have been determined in rabbit liver. Examination of codon recognition by means of a ribosome binding assay showed that each subspecies binds exclusively in the presence of only one of the two possible triplets; i.e. wobbling was not found. It has been assumed that this high specificity, which is not to be expected according to the "wobble hypothesis", is due to a 2-thiouracil base in the first position of the anticodon. This was supported by oxidation experiments with iodine. Treatment with iodine significantly reduces the aminoacylation capacity of all subspecies that show specific binding with triplets ending in adenosine. We describe and compare for the first time the characteristics of eucaryotic tRNA species which occupy homologous positions in the codon table. Thus it can be seen that all the investigated subspecies have rigorously specific codon recognition in common, wherby "wobbling" can probably be excluded by a principle that is valid for all the investigated species.

Animals↗
PubMed2002

The paraoxonase-1 codon 192 polymorphism is associated with fasting total cholesterol and LDL-cholesterol concentrations only in postmenopausal women. The REGICOR study.

Paraoxonase-1 (PON1) is a high-density lipoprotein (HDL)-linked enzyme which appears to protect low-density lipoproteins (LDL) from oxidation. PON1 activity is associated with variation at the PON1 gene locus, specifically the common amino acid polymorphism at codon 192, for which the Q192 allele specifies low activity and the R192 allele specifies high activity. We investigated the association between the PON1 codon 192 polymorphism and fasting concentrations of glucose, lipids, lipoproteins and PON1 activity in 1380 subjects (724 men and 656 women). Several anthropometric and environmental factors were assessed in the present study. The PON1 Q192 allele frequency was 0.70 and 0.68 in men and women, respectively. In women, but not in men, significant associations were found between the PON1 codon 192 genotype and both total and LDL-cholesterol (p=0.004 and p=0.008, respectively), and subgroup analysis indicated that this relationship was predominant in postmenopausal women. Specifically, the Q192 allele was associated with increased total and LDL-cholesterol concentrations. Furthermore, these lipoprotein variables were higher among postmenopausal women with Q192/Q192 and Q192/R192 genotypes than in premenopausal women with the same genotypes (p<0.001). The findings suggest a gender-specific lipoprotein-genotype association with PON1 codon 192 genotypes in this study sample.

Adult↗
PubMed2004

Preferential codons enhancing the expression level of human beta-defensin-2 in recombinant Escherichia coli.

Human beta-defensin-2 (hBD2) is a small antimicrobial peptide with potential as a therapeutic agent. The effect of codon usage on the expression of hBD2 in Escherichia coli was studied. Two coding sequences encoding the same hBD2 precursor were both expressed as fusion protein with thioredoxin in E. coli BL21 (DE3). One is the wild-type human cDNA and the other is a gene synthesized by a PCR-based method in which rare codons were altered to those frequently used in E. coli. The expression level of recombinant hBD2 was over 50% of the total cellular protein when the synthetic gene with preferential codons was employed which was a 9-fold enhancement over the wild-type cDNA. The result shows the codon bias of the host was a major barrier in high-level expression of recombinant hBD2 and suggests a similar approach may be used in the expression of other defensins in E. coli.

Amino Acid Sequence↗
PubMed1993

Mutational status of codons 12 and 13 of the N- and K-ras genes in tissue and cell lines derived from primary and metastatic prostate carcinomas.

N- and K-ras mutations at codons 12 and 13 were investigated using oligonucleotide hybridization analysis after PCR amplification and subsequent sequence analysis of the amplified DNA from the region of interest in the following prostatic primary and metastatic (met) carcinoma-derived cell lines: 1013L (primary), PC3 (bone met), DU145 (brain met), and LNCaP (lymph node met). We also examined fresh and archival primary and metastatic prostate tumor tissue and benign prostatic hypertrophy specimens. All prostatic cells and tissues examined contain at least one wild-type N- and K-ras allele with respect to codons 12 and 13. No mutations were found at N-ras codon 13. The only mutation seen in the prostatic cell lines and tissues was a K-ras codon 12 position II G-to-T transversion. Since these are established nonclonal cell lines that have adapted to tissue culture, it is possible that this mutation does not represent the mutational state of prostatic carcinoma in vivo. However, the lack of consistent mutation in the ras genes amplified directly from tumors suggests that when ras mutations occur during the progression of prostatic carcinoma, they are late-stage events not directly involved in the initial development of disease. Immunoprecipitation studies using pan-ras antibodies revealed no evidence of altered expression of Ras proteins.

Base Sequence↗
PubMed1997

Isoniazid resistance and the point mutation of codon 463 of katG gene of Mycobacterium tuberculosis.

It has long been known that almost all isoniazid (INH) resistant mycobacteria lose the catalase and peroxidase activities along with reduced or no virulence for guinea pigs. Recently resistance to INH has become known to be associated with mutations of katG gene encoding the HPI (Hydroperoxidase I) type catalase and peroxidase. Among these mutations, the point mutation of codon 463 of katG gene is found frequently, and is suggested as being associated with INH resistance. Therefore we performed this study in order to confirm the correlation between the point mutation of codon 463 of the katG gene and INH resistance of M. tuberculosis in Korea. Fifty isolates, 32 of which were resistant to INH, and 18 of which were sensitive to INH, were selected for this study. We used PCR-SSCP and RFLP analysis to detect the point mutation of the codon 463 of katG gene and confirmed the CGG (arginine) to CTG (leucine) mutation by direct sequencing analysis. Among 32 resistant isolates, 7 isolates (22%) had the same restriction pattern compared with that of the reference strain (H37Rv), and 25 isolates (78%) showed a different restriction pattern. Among 18 sensitive isolates, 7 isolates (39%) had the same restriction pattern compared with that of H37Rv, and 11 isolates (61%) showed a different restriction pattern. These results suggest that the CGG to CTG change of codon 463 of katG gene of M. tuberculosis may be a polymorphism not related with INH resistance.

Antitubercular Agents↗
PubMed2000

Reduced or absent expression and codon 201Gly/Arg polymorphism of DCC gene in rhabdomyosarcoma and Ewing's sarcoma/PNET family.

Genetic alterations occurring in various chromosomes have been described in many human tumors. The DCC gene was originally identified in colorectal cancer and was reported as a tumor suppressor gene that might be related to tumor metastasis. We investigated 10 cell lines and 15 fresh tumors of childhood rhabdomyosarcoma, 7 cell lines of Ewing's sarcoma, and 4 cell lines of primitive neuroectodermal tumor (PNET) for the expression and mutation of DCC gene by RT-PCR analysis and PCR-single stranded conformation polymorphism (SSCP) analysis. Twenty-five pairs of primers were used for PCR-SSCP. Six of ten (60%) cell lines of rhabdomyosarcoma and 3 of 7 (43%) cell lines of Ewing's sarcoma showed reduced or absent expression of DCC gene. There was no mobility shift within 24 exons by SSCP analysis, although 3 types of polymorphism were found at codon 201 in exon 3. Direct sequencing of different bands showed types I, II, and I/II representative of codon 201Gly, codon 201Arg, and codon 201Gly/Arg, respectively. The proportion of type I between fresh rhabdomyosarcoma and normal controls was not significant. Our results suggested that the inactivation of DCC gene may play a role in the pathogenesis of a subset of rhabdomyosarcoma and Ewing's sarcoma.

Adolescent↗
PubMed1995

Association of the core clustering mutations (codon 21-34) and the severity of chronic hepatitis B in Korean patients.

OBJECTIVES: There are regions in the core gene of hepatitis B virus (HBV) where missense mutations are clustered, and mutations in that region are related to severe liver disease. However, there were some differences of the major regions for mutation clustering among ethnic groups. To explore the phenomenon of clustering mutations in Korean patients with chronic HBV infection and to elucidate the correlation between clustering mutation region of the core gene and the severity of liver damage, we analyzed the precore/core gene sequence of HBV in the sera from fifteen chronic hepatitis B (CH-B) patients. METHODS: We analysed the HBV precore and core sequences in the sera obtained from fifteen patients (14 males and 1 female, mean age 30.0 years) with biopsy-proven CH-B. The patients were divided into two groups according to the pathological severity of CH-B; namely, group I consisted of 8 patients with chronic persistent hepatitis (CPH), and group II included 7 patients with chronic active hepatitis (CAH). After extraction of HBV DNA from each serum by proteinase K and phenol-chloroform solution, the entire precore and core region of HBV was amplified by PCR, and then the PCR products were subjected to direct sequencing using thermostable DNA polymerase. Fisher's exact test and Mann-Whitney U test were used for statistical analysis. RESULTS: A total of 181 nucleotide substitutions were found in the HBV core gene from the 15 CH-B patients, of which 23 were missense and 158 were silent. The nucleotide and amino acid substitution rates were not significantly different between the two groups (p > 0.05). Two mutational hot spots (MHS), codons 21-34 (MHS1) and codons 85-100 (MHS2), were found in the deduced amino acid alignment of the core gene. The alteration rate of amino acid residue in these regions were 2.857 x 10(-2) and 5.000 x 10(-2), respectively. Of 8 CPH patients, 5 showed missense mutations only in MHS2. In comparison, of 7 CAH patients, 3 showed them both in MHS1 and MHS2, 1 only in MHS1, and 1 only in MHS2; thus, missense mutation in MHS1 was exclusively found in patient with CAH. CONCLUSIONS: There were two mutation clusterings in the core region of adr subtype of HBV from Korean CH-B patients. Mutations in MHS1 (codon 21-34), but not in MHS2 (codon 85-100), are more likely to be related to the severity of CH-B. A longitudinal study using sequential samples is warranted to further clarify the role of MHS1 in the pathogenesis of more severe CH-B.

Adult↗
PubMedDate not supplied

Ki-ras codon 12 point mutation and p53 mutation in pancreatic diseases.

BACKGROUND/AIMS: The Ki-ras gene located at 12p, encodes the GTP binding protein involving the signal transduction system and concerns cell proliferation and differentiation. METHODOLOGY: Pancreatic tissues were obtained from 37 patients with various pancreatic diseases. Ki-ras codon 12 point mutation and p53 (exon 5-8) mutation were examined in 3 patients with chronic pancreatitis, 9 mucinous adenoma of the pancreas (2 with mucinous cystadenoma and 7 with intraductal papillary-mucinous adenoma), 22 pancreatic ductal carcinoma, and 3 serous cystadenoma. RESULTS: On usual pancreatic exocrine ductal lesions, Ki-ras point mutation was evident in 0% (0/3) of chronic pancreatitis, in 56% (5/9) of mucinous adenoma, and in 57% (12/21) of ductal carcinoma, the mutation being located in the second letter in 18 and in the 1st letter in 2. One Ki-ras codon 12 positive pancreatic cancer showed Ki-ras codon 12 point mutation in the surrounding pancreas (2nd letter mutation in both areas). p53 mutation was present in 0% (0/1) of chronic pancreatitis, in 0% (0/8) of mucinous adenoma, while it was evident in 29% (6/21) of pancreatic ductal carcinoma, the mutation being situated in exon 5 in 3, in exon 6 in 1, and in exon 7 in 2. In 3 patients with serous cystadenoma, there was no mutation in Ki-ras codon 12 or p53 (exon 5-8). CONCLUSIONS: These findings suggest that Ki-ras point mutation is involved in the early events of pancreatic ductal carcinoma, while p53 mutation is intricated in the late phase of pancreatic ductal carcinogenesis and the histogenesis of serous cystadenoma is different from that of pancreatic exocrine ductal lesions including mucinous adenoma and ductal carcinoma.

Carcinoma↗
PubMed2000

Role of polymorphisms in codons 143 and 160 of the O6-alkylguanine DNA alkyltransferase gene in lung cancer risk.

O6-Alkylguanine DNA alkyltransferase (AGT) plays an important role in the repair of alkylating agent-induced DNA damage and protection from the carcinogenic effects of environmental agents. To examine the importance of the AGT codon 143 and codon 160 polymorphisms in risk for lung cancer and to assess the prevalence of these polymorphisms in different racial groups, we performed genotype analysis of lung cancer patients and matched controls. The prevalence of the AGT143Val allele in controls was 0.07 in Caucasians and 0.03 in African Americans. The AGT143Val allele was not detected in an unmatched Asian control cohort. The prevalence of the AGT160Arg variant allele was 0.01 in Caucasians, 0.02 in African Americans, and 0.03 in Asians. A marginally significant association was observed between the AGT codon 143 (isoleucine/valine) genotype and risk for lung cancer (odds ratio = 2.1; 95% confidence interval = 1.01-4.7). The prevalence of the AGT160Arg variant allele was similar in lung cancer cases versus matched controls. These results suggest that the AGT codon 143 polymorphism may play an important role in risk for lung cancer.

Adult↗
PubMed2000

Both (+/-)syn- and (+/-)anti-7,12-dimethylbenz[a]anthracene-3,4-diol-1,2-epoxides initiate tumors in mouse skin that possess -CAA- to -CTA- mutations at Codon 61 of c-H-ras.

We have determined the tumor-initiating activity of (+/-)syn- and (+/-)anti-7,12-dimethylbenz[a]anthracene-3,4-diol-1,2-epoxide (syn- and anti-DMBADE), the two metabolically formed bay-region diol epoxides of DMBA, and we have also analyzed mutations in the H-ras gene from tumors induced by these compounds. Using a two-stage, initiation-promotion protocol for tumorigenesis in mouse skin, we have found that both syn- and anti-DMBADE are active tumor initiators, and that the occurrence of papillomas is carcinogen dose dependent. All of the papillomas induced by syn-DMBADE (a total of 40 mice), 96% of those induced by anti-DMBADE (a total of 25 mice), and 94% of those induced by DMBA (a total of 16 mice) possessed a -CAA- to -CTA- mutation at codon 61 of H-ras. No mutations in codons 12 or 13 were detected in any tumor. Topical application of syn- and anti-DMBADE produced stable adducts in mouse epidermal DNA, most of which comigrated with stable DNA adducts formed after topical application of DMBA. Further analysis of the data showed that levels of the major syn- and anti-DMBADE-deoxyadenosine adducts formed after topical application of DMBA are sufficient to account for the tumor-initiating activity of this carcinogen on mouse skin. Previously, we showed that both the syn- and anti-DMBADE bind to the adenine (A182) at codon 61 of H-ras. Collectively, these results indicate that the adenine adducts induced by both bay-region diol epoxides of DMBA lead to the mutation at codon 61 of H-ras and, consequently, initiate tumorigenesis in mouse skin.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed2002

Rapid typing of the codon 129 polymorphism of the human prion protein gene by combined real-time PCR and melting curve analysis.

Homozygosity of methionine (m/m) at amino acid residue 129 (codon 129) of the human prion protein (PrP) has been reported for all so far analyzed cases of the new variant Creutzfeldt-Jakob disease (vCJD). This contrasts with its general distribution in the healthy Caucasian population of only about 43%. For this reason a predisposition for carriers of the corresponding genotype to develop vCJD after infection with the bovine spongiform encephalopathy (BSE) agent is assumed, and PCR based methods such as allele-specific oligonucleotide hybridization or restriction analysis and sequencing have been developed for codon 129 genotyping. These methods are cumbersome and time-consuming and the need for extensive post-amplification manipulations increases the risk of carry-over contamination with amplified products. To overcome these shortcomings, the authors developed a real-time PCR assay on the LightCycler (LC) instrument combining PCR and temperature melting curve analysis (Tm) in a closed vessel format. Forty-six swabs and blood samples from healthy donors were tested. Of these 23 (50%) were heterozygous at codon 129, 4 (8.7%) homozygous for valine and 19 (41.3%) homozygous for methionine. Accuracy of LC-genotyping was confirmed by automated sequencing of the amplified products. Taken together, genotyping of the codon 129 polymorphism by combined LC-PCR and melting-curve analysis with the LC-instrument is a reliable and easy to perform method even in a screening context with numerous samples. Results can be obtained within 2 hours, including sample preparation.

Codon↗