Hemi-nested Polymerase Chain Reaction Research Articles

Hepatitis C and hepatitis B nucleic acids are present in intrahepatic cholangiocarcinomas from the United States

Intrahepatic cholangiocarcinomas (IHCs) are rare in the United States, but the prevalence is steadily increasing and risk factors are poorly understood. Tissues were obtained at the time of surgical resection, and 11 cases of IHC were retrospectively studied for the presence of hepatitis B virus (HBV) DNA and hepatitis C virus (HCV) RNA. Hemi-nested and real-time polymerase chain reaction assays were used to detect HBV DNA, and nested reverse transcriptase-polymerase chain reaction was used to detect HCV RNA. Genotypes were determined for both HBV and HCV. The cases were predominantly from women (10/11), with an average age at surgery of 63 years and an average tumor size of 6 cm. Three cases (27%) were positive for either HBV or HCV nucleic acids: HBV alone (n = 1), HCV alone (1), coinfection with HBV and HCV (1). Both HBV-positive cases were genotype A, and both HCV cases were genotype 1a. Hepatitis B and C viral copy numbers were low in all cases. Evidence for active HBV replication was found in both HBV-positive cases, as they were positive for covalently closed circular DNA. In this study, 27% of ICC cases contained HBV and/or HCV nucleic acids, suggesting an etiologic role for these viruses in some cases of IHC.

Diagnosis of Invasive Group A Streptococcal Infections by Using Immunohistochemical and Molecular Assays

Invasive group A streptococcus (GAS) infections cause 1,100 to 1,300 deaths annually in the United States. Diagnosis is made when Streptococcus pyogenes is isolated from pus or body fluids; however, cultures are not always obtained, and antibiotic treatment can preclude bacterial growth. An immunohistochemical assay for GAS was applied to formalin-fixed tissue samples from 122 patients with suspect GAS infection. Immunohistochemical staining of well-defined cocci and small, granular antigen fragments was observed in 27 cases. S pyogenes was isolated in 18 cases, whereas in 8 cases, immunohistochemical staining was confirmed by amplification of the sepB gene of S pyogenes from paraffin-embedded samples in a heminested polymerase chain reaction (PCR) assay. A primary focus of infection (respiratory, mucocutaneous, or gynecologic) was present in 22 patients, whereas 5 had no identifiable primary focus of infection. Eighteen patients had systemic infection. Immunohistochemical analysis and PCR can be used for diagnosis of GAS infections in formalin-fixed, paraffin-embedded samples.

Diagnosis of Invasive Group A Streptococcal Infections by Using Immunohistochemical and Molecular Assays

Invasive group A streptococcus (GAS) infections cause 1,100 to 1,300 deaths annually in the United States. Diagnosis is made when Streptococcus pyogenes is isolated from pus or body fluids; however, cultures are not always obtained, and antibiotic treatment can preclude bacterial growth. An immunohistochemical assay for GAS was applied to formalin-fixed tissue samples from 122 patients with suspect GAS infection. Immunohistochemical staining of well-defined cocci and small, granular antigen fragments was observed in 27 cases. S pyogenes was isolated in 18 cases, whereas in 8 cases, immunohistochemical staining was confirmed by amplification of the sepB gene of S pyogenes from paraffin-embedded samples in a heminested polymerase chain reaction (PCR) assay. A primary focus of infection (respiratory, mucocutaneous, or gynecologic) was present in 22 patients, whereas 5 had no identifiable primary focus of infection. Eighteen patients had systemic infection. Immunohistochemical analysis and PCR can be used for diagnosis of GAS infections in formalin-fixed, paraffin-embedded samples.

Molecular diagnosis of lyssaviruses and sequence comparison of Australian bat lyssavirus samples

To evaluate and implement molecular diagnostic tests for the detection of lyssaviruses in Australia. A published hemi-nested reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of all lyssavirus genotypes was modified to a fully nested RT-PCR format and compared with the original assay. TaqMan assays for the detection of Australian bat lyssavirus (ABLV) were compared with both the nested and hemi-nested RT-PCR assays. The sequences of RT-PCR products were determined to assess sequence variations of the target region (nucleocapsid gene) in samples of ABLV originating from different regions. The nested RT-PCR assay was highly analytically specific, and at least as analytically sensitive as the hemi-nested assay. The TaqMan assays were highly analytically specific and more analytically sensitive than either RT-PCR assay, with a detection level of approximately 10 genome equivalents per microl. Sequence of the first 544 nucleotides of the nucleocapsid protein coding sequence was obtained from all samples of ABLV received at Australian Animal Health Laboratory during the study period. The nested RT-PCR provided a means for molecular diagnosis of all tested genotypes of lyssavirus including classical rabies virus and Australian bat lyssavirus. The published TaqMan assay proved to be superior to the RT-PCR assays for the detection of ABLV in terms of analytical sensitivity. The TaqMan assay would also be faster and cross contamination is less likely. Nucleotide sequence analyses of samples of ABLV from a wide geographical range in Australia demonstrated the conserved nature of this region of the genome and therefore the suitability of this region for molecular diagnosis.

Small Anellovirus in Hepatitis C Patients and Healthy Controls

To the Editor: Torquetenovirus (TTV) and torquetenominivirus (TTMV) are characterized by a small, negative-sense, circular, single-stranded DNA genome and by an extraordinary ability to produce chronic plasma viremia. Indeed, >80% of humans harbor variably high viral loads of TTV, TTMV, or both, in plasma, regardless of geographic provenance, age, sex, and health conditions (1). Currently, TTV and TTMV are classified as distinct species in the floating (although closely linked to the family Circoviridae) genus Anellovirus, but their extreme genetic heterogeneity and some distinctive features in genomic organization have led some to suggest that they should be classified as an independent family (2,3). Most recently, after examining serum specimens from patients with symptoms of an acute viral infection by using DNase sequence-independent single-primer amplification, Jones et al. (4) identified, among other viruses, 2 novel TTV- and TTMV-like agents. Because of their even smaller genomes (≈2.4 and 2.6 kb vs. 3.6–3.8 kb for TTV and 2.8–2.9 kb for TTMV), these agents were named small anelloviruses (SAVs). Because tissue culture and serologic methods are not yet available, diagnosis of anellovirus infection relies exclusively on viral DNA detection. We tested 55 Italian hepatitis C patients (mean age 56 ± 14 years, male/female ratio 30/25, 53 TTV positive) and, for comparison, 35 healthy donors (mean age 36 ± 12 years, male/female ratio 17/18, 33 TTV positive) for SAV in plasma by using the polymerase chain reaction (PCR) primers described by Jones et al. (4), followed by direct amplicon sequencing. To increase assay sensitivity, a heminested PCR format was adopted that used a sense primer designed in a segment of the untranslated region that is highly conserved among all anelloviruses (5´-TCAAGGGGC AATTCGGGCT-3´). We found 5 positive results among the hepatitis C patients (9.1%, all of whom were TTV positive) and 3 positive results among healthy controls (8.6%); and all were confirmed by sequence data. The amino acid sequences inferred from the coding segment of the amplicon of SAV in this study and the corresponding sequences of the 10 SAV in GenBank at the time of this writing were then aligned with representative TTV and TTMV sequences (Figure A1). This method allowed us to identify the motif WX7HX3CXCX5H, which is highly characteristic of the open reading frame 2 (ORF2) of anelloviruses (5), in all SAVs. SAV sequences, as well as a large number of TTVs and all TTMVs, were then used to construct a phylogenetic tree and to calculate the extent of genetic divergence within SAV, TTV, and TTMV. Although a precise phylogenetic description will require the analysis of full-length ORF2, the SAV sequences clustered quite separately from those of TTV and TTMV, and the extent of divergence observed among SAV was huge and in the same range as among TTV or TTMV. Furthermore, SAVs obtained from hepatitis C patients and healthy participants were intermingled (Figure A2). While this study was under way, Biagini et al. reported a 12% prevalence of SAV viremia in French blood donors (6). Our results confirm the high prevalence of SAV viremia in healthy persons and extend the finding to hepatitis C patients. Our data, combined with those of Biagini et al., indicate that, since SAV clusters separately from previously identified anelloviruses, it should be considered a distinct species (or possibly genus). This would increase the already high genetic diversity of anelloviruses, further arguing for the appropriateness of creating a separate viral family. Because the clinical and viral parameters of hepatitis C in SAV-positive patients were not significantly different from those in the SAV-negative patients (data not shown), our results suggest that, similar to TTV (7), SAV has little or no effect on the course of hepatitis C. Although anelloviruses have not yet been definitely linked to any specific disease, evidence is growing that they might be involved in acute respiratory diseases in children (8,9). Furthermore, a florid TTV replication in the respiratory tract correlated with severity of lung impairment in children with asthma (10). A precise appreciation of the wide range of viruses classified within the anelloviruses is a prerequisite to understanding such disease associations and the disease-inducing potential of these viruses in general.

Predicting clinical benefit from EGFR TKIs: Not all EGFR mutations are equal

7072 Background: EGFR mutations are associated with better response and in some studies (mainly Asian) also with prolonged survival after gefitinib therapy in patients with advanced NSCLC. Although a number of mutations have been reported, most frequent are exon 19 or exon 21 mutations of the EGFR tyrosine kinase domain.Whether clinical outcomes differ by subtype of EGFR mutation has not been previously reported. Methods: Mutation analysis (as previously described) was performed in 157 of 204 patients with advanced NSCLC treated with gefitinib (250 mg or 500 mg) in 2 study cohorts (Italian study of Iressa Expanded Access Program and SWOG 0126). Fifty nanograms of genomic DNA was isolated from pretreatment tumors, amplified for EGFR exons 19 and 21 by touchdown hemi-nested polymerase chain reaction and sequenced in both sense and antisense directions. Results: EGFR mutations were found in 43 pts (27%). Overall, patients with EGFR mutations had a response rate of 39% versus 7% for those without (p ≤ 0.001), disease control rate of 52% versus 37% (p = 0.14), time to progression of 3 months in both groups and median survival of 13 months versus 11 months (p = 0.14). Patients with exon 19 mutations exclusively (N = 11) had better outcome than those with exon 21 mutations exclusively (N = 31), with response rates of 67% versus 20% (p = 0.02), median time to progression of 15 months versus 2 months, and median survival of 26 months versus 10 months. There was a difference in time to progression (11 months versus 3 months) and overall survival (median 26 months versus 11 months) between patients with and without exon 19 mutations, while no difference was apparent in these outcome measures in patients with and without exon 21 mutations. Sample size provided insufficient power for significance tests of differences in survival outcomes. Conclusions: Not all EGFR mutations are created equal. Mutations in exon 19 are more predictive of response and survival after gefitinib than exon 21 mutations. [Table: see text]

Instability in the transmission of the myotonic dystrophy CTG repeat in human oocytes and preimplantation embryos

To elucidate the timing and variability of CTG repeat expansion within the human dystrophia myotonica protein kinase (DMPK) gene in early development. Triplet-primed polymerase chain reaction was used to amplify the expanded CTG repeat in oocytes and embryos obtained from myotonic dystrophy 1 (DM1) patients, and a heminested polymerase chain reaction approach was used to amplify the normal CTG repeats in supernumerary IVF embryos. University hospital laboratory. Two DM1-affected females undergoing preimplantation genetic diagnosis who carried different CTG repeats. Also, 61 IVF patients who carried a (CTG)(5-18) and (CTG)(19-37) normal DMPK repeat. None. The degree of expansion of the repeat in the oocytes and embryos compared with the DM1-affected maternal repeat and the size of the (CTG)(19-37) repeat compared with the parental size in IVF embryos. The degree of repeat expansion was greater than the DM1 maternal lymphocyte for two of four oocytes, including a germinal vesicle-stage oocyte and 17 of 20 three-cell to blastocyst stage embryos. A change in the (CTG)(19-37) repeat was seen in 7 (7%) of 95 paternal transmissions but in no maternal transmissions. Because the repeat was already expanded in the immature oocyte, the initial expansion most likely occurs during oogenesis. A variable degree of DMPK(CTG)(n) expansion in the embryo is seen from different mothers. In addition, instability in paternal transmission of normal-range (CTG)(19-37) repeats occurs at the level of the embryo.

Differentiation ofCulicoides obsoletusandCulicoides scoticus(Diptera: Ceratopogonidae) Based on Mitochondrial Cytochrome Oxidase Subunit I

Culicoides obsoletus (Meigen) and Culicoides scoticus Downes & Kettle (Diptera: Ceratopogonidae) are sibling species of the Obsoletus group. This group comprises species of biting midges that are suspect vectors of bluetongue virus (family Reoviridae, genus Orbivirus, BTV) and African horse sickness virus (family Reoviridae, genus Orbivirus, AHSV). BTV and AHSV have been isolated several times from females of this group, although it has not been possible to determine the particular species harboring the virus, because of the inability to clearly identify the females of each species based on morphology. Both sexes of C. obsoletus and C. scoticus midges from Catalonia and the Balearics (Spain) were sequenced for the mitochondrial gene cytochrome oxidase subunit I (COI), and these sequences were analyzed to determine intra- and interspecific genetic variability. Species-specific primers for C. obsoletus and C. scoticus were designed and a polymerase chain reaction (PCR)-based diagnostic assay based on the COI gene and using a hemi-nested PCR technique was developed for reliably distinguishing the females of both species. The species-specific PCR diagnostic was compared with morphological discrimination of C. obsoletus and C. scoticus females. The morphologic characters were not fully reliable.

Distribution of Melissococcus plutonius in Honeybee Colonies with and without Symptoms of European Foulbrood

A sensitive hemi-nested polymerase chain reaction (PCR) was used for detection of Melissococcus plutonius, the causative agent of European foulbrood (EFB). Sampling was made in Switzerland, where EFB is a widespread disease and incidences have increased in recent years. Larvae from brood samples with and without clinical signs of disease (n=92) and honey (n=92) from the same colonies were investigated. Individual larvae (n=60) and pupae (n=30) from diseased brood in single colonies were also investigated to study the distribution of the bacterium within the brood between larvae. M. plutonius was detected in larvae in all apiaries where symptoms of EFB could be seen, but not in all colonies judged as cases of EFB in the field, when healthy-looking larvae from such colonies were tested. The occurrence of the bacterium within the brood was not limited to larvae with symptoms only, but was mainly found in diseased larvae. The bacterium was also found in pupae. Healthy-looking larvae-even from heavily diseased combs-failed, in a number of cases, to amplify product in the PCR. M. plutonius could only be detected in 35% of the brood nest honey from clinically diseased colonies.

Polymerase chain reaction for detection ofToxoplasma gondii in human biological samples

Using the polymerase chain reaction (PCR), Toxoplasma gondii from gene TGR1E with primers TGR1E-1, TGR1E-2 (standard PCR), and from B1 gene with primers TM1, TM2, TM3 (hemi-nested PCR) was detected in biological samples from 347 individuals (441 biological materials). Of the total of 441 biological materials, T. gondii DNA was detected in 5.2 %; it was positive in the following samples: blood (n = 6), blood from newborns (2), biopsies (2) and samples of progenitor cells (2) (from candidates for bone marrow transplantation). DNA of T. gondii was also revealed in 11 samples (8.3 %) of 120 cases of pregnant women during prenatal examinations. A positive result in the blood was also found in two cases of newborn babies from mothers who were infected in later pregnancy. The positive PCR examination was confirmed by serological methods (ELISA and complement fixation test). Agreement of PCR results and the detection of antibodies against toxoplasma was found in 83.3 %. Rapid PCR examination for the confirmation of acute parasitemia T. gondii is particularly important for the patients in whom the infection may cause serious consequences (e.g., for fetus in pregnant women or for patients suffering from imunosuppression).

Anaplasma phagocytophilum, Sardinia, Italy

<i>Anaplasma phagocytophilum</i>, Sardinia, Italy

Bordetella pertussisin Adult Pneumonia Patients1

<i>Bordetella pertussis</i>in Adult Pneumonia Patients1

Development of a hemi-nested RT-PCR method for the specific determination of European Bat Lyssavirus 1: Comparison with other rabies diagnostic methods

A simplified hemi-nested reverse transcriptase polymerase chain reaction (hnRT-PCR) has been developed to determine specifically the European Bat Lyssavirus 1 (EBLV-1) nucleoprotein gene. The specificity of this method was determined by using the seven genotypes of lyssavirus by RT-PCR, Southern blot and sequence analysis. Compared to the rabies diagnostic methods, the hnRT-PCR showed a higher sensitivity for the detection of small amounts of EBLV-1 virus. In view of these results, we suggest this new hnRT-PCR should be performed for the epidemiological survey of bat colonies, also providing rapid detection and genotyping of EBLV-1 until now encountered in all naturally infected bats in France.

Detection and sequencing of the microcystin LR-degrading gene, mlrA, from new bacteria isolated from Japanese lakes

mlrA is the only microcystin-degrading gene detected in Sphingomonas sp. MJ-PV. The gene has an extremely rare nucleotide sequence and homologous genes have not yet been discovered in the DNA database. We discovered the existence of a gene homologous to mlrA in new microcystin-degrading bacteria, MD-1 and Y2. These strains possessed mlrA homologues, and the identities of the genes of MD-1 and Y2 with the corresponding MJ-PV exceeded 98% and 84%, respectively. On the other hand, the mlrA gene was not detected in laboratory strains of the closely related Sphingomonas spp. strains employing hemi-nested polymerase chain reaction detection using two primer sets. Although the microcystin-degrading bacteria were closely related strains, they did not cluster together as the same species. We can conclude that the mlrA gene is conserved in three different bacterial species, and it is unique to microcystin degraders but not to the genus Sphingomonas.

Expression of high-mobility-group-protein HMGI-C mRNA in the peripheral blood is an independent poor prognostic indicator for survival in metastatic breast cancer.

HMGI-C belongs to the high-mobility-group-protein (HMG) family of architectural transcription factors and considerable interest has recently been shown in its expression in neoplastic tissues and apparent involvement in tumorigenesis. We could previously demonstrate an expression of HMGI-C mRNA in the peripheral blood of breast cancer patients for the first time. In this prospective study, we evaluated the independent prognostic power of HMGI-C mRNA expression in the peripheral blood of an unselected cohort of 69 patients with metastatic breast cancer using a hemi-nested reverse transcriptase polymerase chain reaction (RT–PCR) followed by sequence analysis of the resulting PCR products. Multivariate analysis was performed using the Cox regression model. HMGI-C mRNA was detected in peripheral blood from 21 out of 69 (30%) patients with metastatic breast cancer. Median survival was 15.9 months in patients expressing HMGI-C, while in the group of patients without HMGI-C expression the median survival had not been reached yet after a median follow-up of 24.7 months and 85.4% were still alive in this group. Disease-specific survival was significantly worse for patients positive for HMGI-C in comparison to those not expressing HMGI-C (P=0.0001). In a multivariate regression analysis, HMGI-C remained an independent prognostic factor for overall survival (P=0.001) besides oestrogen receptor status (P=0.024) and presence of metastases in liver and lungs (P=0.029). HMGI-C expression in the peripheral blood of patients with metastatic breast cancer is a powerful independent indicator for poor overall survival and this is the first study to demonstrate its prognostic relevance in univariate and multivariate analysis.

Incidence and characteristics of endemic Norwalk-like virus-associated gastroenteritis.

Endemic gastroenteritis associated with the Norwalk-like viruses (NLVs) is little understood. This study tested for NLV in gastroenteritis cases in 257 households in Melbourne, Australia, for the period September 1997 to February 1999 by a reverse transcription hemi-nested polymerase chain reaction. Positive samples were studied by nucleotide sequencing and phylogenetic analysis. NLV was detected in 73 (11.4%) of 638 faecal specimens tested. Twelve (1.9%) were NLV genogroup 1 (G1) and 61 (9.6%) NLV genogroup 2 (G2). Gastroenteritis symptoms associated with NLV G2/no other pathogens were significantly more severe than where no NLV was detected. NLV G1 and NLV G2 were detected in adults and children, males and females. NLV G2 incidence showed a marked seasonal periodicity with significant peaks in the Australian late spring/early summer periods. NLV G1 seasonality was significantly different from that of NLV G2. Seven major NLV clusters were identified by phylogenetic analysis.

The detection of Melissococcus pluton in honey bees (Apis mellifera) and their products using a hemi-nested PCR

A hemi-nested polymerase chain reaction (PCR) was further developed for the detection of Melissococcus pluton in adult bees and honey bee products. A chloroform:isoamyl alcohol DNA extraction method was used to provide template from 154 samples of adult bee tissues, honey, pollen, whole larvae and comb cells. All 36 honey bee samples tested from a diseased colony were shown to contain M. pluton and sub-clinical infections were detected in adult bee tissues, larvae and honey (49/98; 50.0%) collected from all 9 healthy colonies from areas where EFB was endemic. All 20 adult bee tissue samples from a healthy colony from Western Australia where EFB has never been reported were negative. Of 80 bulk honey samples from six Australian states, 55 of 80 (68.8%) samples were shown to contain M. pluton whereas culture techniques detected M. pluton in 22 of 80 (27.5%) of these samples. M. pluton was detected in honey from all Australian states except Western Australia. polymerase chain reaction / Melissococcus pluton / european foulbrood / honey bees / Australia

An unusual outbreak of malignant catarrhal fever in a beef herd in Israel.

Malignant catarrhal fever (MCF. corrizza contagiosa) is an invariably fatal communicable disease in cattle, whose causative agent is the ovine herpes virus-2, or the alcelaphine herpes virus-1. In one feed-lot family farm, 34 calves out of 100 became ill at the rate of one to four calves per week, and all of them subsequently died over a period of 4 months. Most of the initial cases were manifested clinically as the head and eye form, but most of the entire clinical spectrum of forms (the respiratory, intestinal and nervous forms) characteristic for MCF were observed as this epidemic progressed. Very few calves died without showing any specific signs of MCF. Pathological examinations revealed characteristic obliterative arteriovasculitis in the brain of calves with nervous signs, typical of MCF. Polymerase chain reaction (PCR) testing revealed 100% homology between the 238 bp hemi-nested PCR fragment and the ovine herpes virus-2 sequences. Based on the clinical signs, epidemiological data, pathological, and histopathological findings, and the PCR results, it was concluded that MCF occurred on the farm. The fact that sheep and goats were housed in close proximity on the same farm reinforced this diagnosis.

Common occurrence of multiple K-RAS mutations in pancreatic cancers with associated precursor lesions and in biliary cancers.

Recent studies on small series of pancreatic cancer (PC) with foci of pancreatic intraepithelial neoplasia (PanIN), a putative precursor lesion, have shown that multiple K-RAS mutations may coexist in the same neoplastic pancreas. To see whether mutant-K-RAS polyclonality is a common and specific feature of pancreatic carcinogenesis, we investigated a unselected series of periampullary cancers (41 pancreatic, 13 biliary and two ampullary adenocarcinomas). After hemi-nested polymerase chain reaction (PCR), mutations identified with single strand conformation polymorphism (SSCP) were confirmed by allele-specific PCR and sequencing. K-RAS codon 12 was mutated in 34 (83%) pancreatic cancers and in 11 (85%) biliary cancers. Multiple distinct K-RAS mutations were found in 16 PC (39% of all cases, 47% of those with mutated K-RAS) and in eight biliary cancers (62 and 72%, respectively). In PC, multiple K-RAS mutations were more frequent (P<0.001) in cancers with (nine of 12, 75%) than in those without detectable PanIN (seven of 29, 24%). Individual precursor lesions of the same neoplastic pancreas were found to harbor distinct mutations. Results show that multiple K-RAS mutations are frequent both in PC with associated PanIN and in biliary cancers, and indicate that clonally distinct precursor lesions of PC might variably contribute to tumor development.

Polymorphism of the vitamin D3 receptor in patients with psoriasis

1,25-Dihydroxyvitamin D is the biologically active form of vitamin D for the treatment of skin eruptions in patients with psoriasis. 1,25-(OH)(2)D(3) elicits its action on skin eruptions through the vitamin D receptor (VDR). Allelic frequencies of VDR were studied in 86 normal subjects and 50 patients with psoriasis. Genomic DNA was extracted from peripheral blood leukocytes and the VDR gene was amplified using a heminested polymerase chain reaction (PCR). The products were digested with respective restriction enzymes ApaI, TaqI and BsmI. The restriction fragment length polymorphisms (RFLP) were coded as Aa, Tt or Bb. The frequencies of ApaI, BsmI and TaqI RFLP genotypes in psoriasis patients showed no significant differences compared with normal controls. The frequency of the AA genotype was significantly higher in pustulosis palmaris et plantaris patients than in psoriasis vulgaris patients ( P<0.05), and in psoriasis vulgaris patients than in psoriasis pustulosa patients ( P<0.01). In patients with psoriasis, the levels of serum alanine 2-oxoglutarate aminotransferase (ALT) were significantly higher in patients with the AA genotype (54.0+/-22.0 IU/l, n=4) than in those with the aa genotype (24.0+/-15.9 IU/l, n=27; P<0.02). The distribution of ApaI, BsmI, TaqI RFLP VDR genotypes showed no significant relationship to the PASI score, serum aspartate 2-oxoglutarate aminotransferase or triglyceride levels, or age at onset. These results show that the VDR genotype contributes to the liver dysfunction in patients with psoriasis, although no correlation was found between VDR genotype and the skin eruptions of psoriasis.