Single-strand Conformation Polymorphism Technique Research Articles

Heterogeneous point mutations of the p53 gene in pulmonary fibrosis.

Lung cancer is a frequent complication in pulmonary fibrosis. Overexpression of p53 proteins has been demonstrated by immunostaining in bronchoepithelial cells in patients with idiopathic pulmonary fibrosis. However, it is still unclear whether this overexpressed p53 protein is wild-type or mutant. It was hypothesized that pulmonary fibrosis may be a precancerous lesion with deoxyribonucleic acid point mutations in bronchoepithelial cells. Mutations of the p53 gene were tested for by fluorescence-based single-strand conformation polymorphism (FSSCP), cloning-sequencing and immunostaining techniques. Out of 10 tissue samples that demonstrated overexpression of p53 protein by immunostaining, nine (90%) exhibited point mutations and eight (80%) exhibited heterogeneous point mutations of the p53 gene. The mutations found in pulmonary fibrosis were scattered throughout the central part of the p53 gene, and both guanine (G):cytosine (C) to adenine (A):thymine (T) and A:T to G:C transitions were frequently observed. In conclusion, frequent heterogeneous point mutations of the p53 gene were detected in pulmonary fibrosis. These mutations may have resulted from several types of deoxyribonucleic acid damage that occurred in bronchoepithelial cells and this may explain previous findings of a very high incidence of lung cancer complicating pulmonary fibrosis.

Distinguishing Oesophagostomum dentatum from Oesophagostomum quadrispinulatum developmental stages by a single-strand conformation polymorphism method

At some life-cycle stages, it is impossible to distinguish between the two species of porcine nodular worm, Oesophagostomum dentatum and Oesophagostomum quadrispinulatum, using morphological features. A PCR-based single-strand conformation polymorphism technique was established to overcome this limitation. The rDNA region spanning the second internal transcribed spacer was amplified by PCR from genomic DNA from morphologically well-defined adult worms. The PCR products were then denatured and subjected to electrophoresis in a non-denaturing gel matrix. Single-strand conformation polymorphism analysis of the products generated characteristic and reproducible patterns for each of the two species and allowed their unequivocal delineation. The single-strand conformation polymorphism was also applied effectively to assess the purity of nine laboratory-maintained cultures of infective third-stage larvae believed to be monospecific for O. dentatum or O. quadrispinulatum. The analysis showed that all six O. dentatum cultures were indeed monospecific, whereas the three cultures believed to be monospecific for O. quadrispinulatum were either a mixture of O. dentatum and O. quadrispinulatum larvae or pure O. dentatum larvae. These findings demonstrated the usefulness of the single-strand conformation polymorphism approach for the routine monitoring of the purity of parasite lines and indicated its value for studies on the population biology of porcine nodular worms.

Novel mutation of the PTEN gene in an Italian Cowden's disease kindred.

Cowden disease (CD) is an autosomal dominant multiple hamartoma syndrome with an elevated risk of thyroid and breast cancers. The CD susceptibility gene has recently been identified as the PTEN/MMAC1/TEP1 gene localized at 10q23 and coding for a dual specificity protein phosphatase. We report the mutational analysis of the PTEN gene in one Italian CD kindred. By using the single strand conformation polymorphism technique and subsequent direct DNA sequencing of the polymerase chain reaction product, we identified a novel mutation in the exon 5 of the PTEN gene. A heterozygous germline TGT-TAT transition was detected at the nucleotide 407; this causes the amino acid substitution cys136-tyr136 and the generation of a new NSI I restriction site. This mutation was not detected in the unaffected member of the family thereby indicating that it is causally linked to the disease. We ruled out that this mutation is a polymorphic variant because it was not detected in over 100 chromosomes analyzed. Using reverse trancriptase-polymerase chain reaction, we detected the expression of the mutant allele in lymphocytes and pathological tissues from an affected member of the family.

Two distinct kerato-epithelin mutations in Reis-Bücklers corneal dystrophy

PURPOSE: Two patients were diagnosed with Reis-Bücklers corneal dystrophy (RBCD), although the pattern and severity of corneal opacification differed. To see whether there was a genetic basis for these phenotypic variations, we analyzed βig-h3, the gene that codes for kerato-epithelin and that contains a mutation (Arg555Gln) that causes RBCD. METHODS: A 30-year-old man with honeycomb-shaped subepithelial opacities in his central cornea and a 25-year-old man with progressive subepithelial geographic opacities were both considered to have RBCD. We isolated genomic DNA from leukocytes of the two patients and their family members and screened for an Arg555Gln kerato-epithelin mutation. Then we analyzed all exons of the gene using the single-strand conformation polymorphism (SSCP) technique to search for any other kerato-epithelin mutations. RESULTS: The patient with honeycomb-shaped opacities had an Arg555Gln kerato-epithelin mutation that caused his RBCD, whereas the patient with geographic opacities did not; instead, he had a new kerato-epithelin mutation (Arg124Leu), which cosegregated with his family members. CONCLUSIONS: The variant of RBCD characterized by honeycomb-shaped opacities is caused by an Arg555Gln kerato-epithelin mutation. On the other hand, a new kerato-epithelin mutation, Arg124Leu, was found to cause the RBCD variant characterized by recurrent epithelial erosions and progressive geographic subepithelial opacification. Codon 124 is a hot spot for kerato-epithelin mutations, where the mutations responsible for three autosomal dominant corneal dystrophies—lattice type I (Arg124Cys), Avellino (Arg124His), and the variant of RBCD with geographic rather than honeycomb opacities (Arg124Leu)—are located.

No relationship between identified variants in the uncoupling protein 2 gene and energy expenditure.

The uncoupling protein 2 (UCP2) uncouples respiration from the oxidative phosphorylation in most cell types, predominantly in white fat and skeletal muscle. Since a decreased basal metabolic rate (BMR) would increase the susceptibility to weight gain, genetic alterations in the UCP2 gene could contribute to the pathogenesis of obesity and the metabolic syndrome (MSDR). To test this hypothesis, we PCR amplified the introns of the UCP2 gene and sequenced the exon/intron boundaries. This information was used to construct intronic primers and to screen obese patients with low BMR for mutations in the coding regions of the UCP2 gene, using the single-strand conformational polymorphism technique. Furthermore, we examined whether there is an association between a biallelic marker in the UCP2 gene and BMR or MSDR. The UCP2 gene is composed of six coding exons, covering 5 kb of chromosome 11q13. One polymorphism, but no mutations, were identified in the coding regions of the UCP2 gene. There were no significant differences in the allele or genotype frequencies of the Ala55Val polymorphism between 55 patients with MSDR and 46 healthy controls. No association was found between the UCP2 gene and BMR in patients with MSDR or in healthy controls. Mutation screening and association studies suggest that mutations in the coding regions of the UCP2 gene do not affect BMR and do not contribute to increased susceptibility to obesity or MSDR. The results cannot, however, exclude the possibility that variants in regulatory elements of the gene could contribute to the development of obesity or MSDR.

Granular corneal dystrophy with homozygous mutations in the kerato-epithelin gene

PURPOSE: To report a family with several members affected with granular corneal dystrophy Groenouw type 1. Three members of the family were affected with a severe placoid type of corneal dystrophy. To determine the relationship between gene mutations and phenotypic variations of the disease, we analyzed the kerato-epithelin gene. METHODS: The pedigree included a consanguineous marriage of two affected individuals. The three family members affected with a severe form of corneal dystrophy were offspring of these parents. However, the phenotype of other affected family members was typical granular corneal dystrophy. We isolated genomic DNA from leukocytes of the family members. Exons of the kerato-epithelin gene were amplified by the polymerase chain reaction and were analyzed using the single-strand conformation polymorphism technique. Mutations were identified by direct sequencing method and restriction digestion analysis. RESULTS: The three severely affected family members exhibited homozygous mutations at codon 555 (arginine to tryptophan) in the kerato-epithelin gene, whereas those with typical granular corneal dystrophy had the heterozygous mutation at the same codon. Unaffected family members did not have the mutation. CONCLUSIONS: We determined that the severe phenotype of granular corneal dystrophy is caused by homozygous mutations in the kerato-epithelin gene. Clinical manifestation of the severe phenotype is a placoid type of corneal dystrophy and early recurrence after surgery. Granular corneal dystrophy appears to be the first ophthalmic disease in which homozygosity for a dominant allele has been genetically identified.

Common amino acid substitutions in insulin receptor substrate-4 are not associated with Type II diabetes mellitus or insulin resistance.

The family of insulin receptor substrates (IRS1-4) is defined by proteins with an overall similar structure. IRS-1 and IRS-2 have been shown to have key roles in cellular transmission of the action of insulin, insulin-like growth factor-1 and various cytokines. We have previously identified amino acid polymorphisms in the human IRS-1 and IRS-2 proteins. Given the documented importance of IRS-1 and -2 in insulin signalling and the implications of distribution of these genes for the pathogenesis of insulin resistance and diabetes, we decided that the most recently identified member of the IRS family, IRS-4, was a relevant candidate to examine for genetic variability which might be associated with subsets of diabetes or insulin resistance. The gene encoding IRS-4 was analysed by the single strand conformation polymorphism technique in 83 Danish Caucasians with Type II (non-insulin-dependent) diabetes mellitus. Five amino acid polymorphisms were identified: Leu34Phe, Arg411Gly, Gly584Cys, His879Asp and Lys883Thr. In an association study of 324 patients with Type II diabetes and 267 control subjects with normal glucose tolerance the polymorphism at codon 34 was found with allelic frequencies of 3.9 and 2.3 %, respectively, the variant at codon 411 with allelic frequencies of 3.9 and 5.6%, respectively, and the variant at codon 879 with frequencies of 19.2 and 18.0%, respectively. Each carrier of the codon 34 polymorphism was also a carrier of the codon 411 and codon 879 variants and similarly, carriers of the variant at codon 411 were also carriers of the polymorphism at codon 879. The variants at codon 584 and 883 were each found in only one Type II diabetic patient. The allelic frequencies of the variants at codon 411 and 879 were also determined in 380 young healthy subjects (4.6 and 18.1 %, respectively). The insulin sensitivity index as estimated by Bergman's minimal model of the young healthy subjects carrying either polymorphism was indistinguishable from the carriers of wild-type IRS-4. Moreover, none of the men were heterozygous for the IRS-4 polymorphisms indicating that the gene is located on the X-chromosome. In conclusion, amino acid polymorphisms in human IRS-4 are common in Caucasians but are not associated with Type II diabetes or with insulin resistance in young healthy subjects.

A common pentanucleotide polymorphism of the 3'-untranslated part of the leptin receptor gene generates a putative stem-loop motif in the mRNA and is associated with serum insulin levels in obese individuals.

To find out whether genetic alterations of the leptin receptor gene underlie human forms of obesity. Among 249 morbidly obese adults (body mass index, BMI > or = 40 kg/m2), we screened 30 patients with the highest serum leptin levels for alterations of their leptin receptor gene by single-strand conformation polymorphism (SSCP) technique. 249 severely obese subjects (present or past BMI > or = 40 kg/m2) and 138 lean controls (BMI < or = 25 kg/m2). DNA analysis was carried out using SSCP technique, sequencing and polymerase chain reaction (PCR) followed by digestion with the restriction enzyme Rsal. Serum leptin, glucose, insulin and lipid concentrations were determined in obese subjects. We were able to detect a pentanucleotide insertion (CTTTA) in the 3'-untranslated region of the leptin receptor gene. The presence of this pentanucleotide insert generates a putative stem-loop structure in the mRNA. Association studies were carried out on this variant. The frequency of the insertion allele did not differ between 249 obese (12.4%) and 138 lean (12.0%) subjects. There was no association of serum leptin, glucose or lipid levels with the pentanucleotide genotype in the obese individuals. However, when subjects without medication affecting insulin or glucose levels were considered, serum insulin levels were found to be lower in the heterozygous carriers of the insertion allele (15.1 +/- 9.2 mU/l) than in the subjects homozygous for the deletion allele (21.8 +/- 13.7 mU/l, P = 0.0035). We were able to confirm the presence of a frequent insertion/deletion polymorphism close to the 3'-end of the leptin receptor gene. We also showed that serum insulin levels in morbidly obese subjects are associated with 3'-UTR variant genotype.

A mutation scanning approach for the identification of hookworm species and analysis of population variation

To overcome limitations in the morphological identification of different developmental stages of hookworms to species, we have established a polymerase chain reaction-linked single strand conformation polymorphism technique (PCR–SSCP) utilizing the internal transcribed spacers (ITS) of ribosomal (r)DNA. These spacers were specifically chosen because they provide reliable species markers for strongylid nematodes. ITS spacers were amplified by PCR from DNA derived from individual parasites of seven species of hookworm, then denatured and subjected to electrophoresis in a mutation detection enhancement (MDE) (non-denaturing) gel matrix. PCR–SSCP analysis showed that the single-strand ITS patterns produced allowed the unequivocal identification of all species. The method also allowed the direct display of sequence variation within some species where multiple individual worms were examined. These findings demonstrate the usefulness of the SSCP approach for hookworm identification, the detection of population variation and the direct display of sequence variation in rDNA.

Mutational analysis of the CCR5 and CXCR4 genes (HIV-1 co-receptors) in resistance to HIV-1 infection and AIDS development among intravenous drug users.

We analysed a group of Spanish intravenous drug users and controls to determine the role of mutations at the chemokine receptor-5/HIV-1 cofactor (CCR5), previously implicated in resistance to HIV-1 infection, and CXCR4 genes in susceptibility to HIV-1 infection. The complete coding sequence of both genes was amplified by the polymerase chain reaction from genomic DNA of 50 seropositive slow progressors and 10 long-term non-progressors, and analysed by the single-strand conformation polymorphism technique in a search for mutations. No mutation in CXCR4 was found, and delta ccr5 was the only mutation identified at the CCR5 gene. We genotyped (delta ccr5 allele) 150 HIV-1+ intravenous drug users and 250 healthy controls from the same population (Asturias, Northern Spain). Patients were divided into rapid progressors, presenting an event indicating progression to the acquired immunodeficiency syndrome (AIDS) in the 2 years after infection (100 patients), and slow progressors, remaining asymptomatic for 2-10 years (50 patients). The frequencies of the delta ccr5 allele were 0.105 and 0.040 in controls and HIV-1+ patients, respectively. Eighteen per cent of the controls (45/250) and 8% (12/150) of the patients carried the delta ccr-5 allele (P=0.013). The frequency of delta ccr5 carriers among rapid and slow disease progressors was 3 and 15%, respectively. A highly significant difference was found between rapid progressors and controls (P=0.0014). No patient (0/150) was delta ccr5 homozygous compared with 1% among controls. Thus, the delta ccr5 allele (the only CCR5 mutation found in our HIV-1 patients) was rare among seropositive intravenous drug users, suggesting that the absence of this mutation confers an advantage to the virus when infecting cells in vivo. In addition, patients carrying the delta ccr5 allele tend to show a slow progression towards HIV-1-related disease, remaining asymptomatic for longer periods of time.

Factor XIII Val 34 Leu: a novel association with primary intracerebral hemorrhage.

A common G-to-T point mutation (Val 34 Leu) in exon 2 of the alpha-subunit of the factor XIII is strongly negatively associated with the development of myocardial infarction. This result suggests that factor XIII Val 34 Leu is interfering with the formation of cross-linked fibrin. The role of factor XIII Val 34 Leu in the pathogenesis of cerebral infarction and primary intracerebral hemorrhage is unknown. Six hundred twelve patients with acute stroke, defined by World Health Organization criteria and cranial CT, and 436 age-matched control subjects free of cerebrovascular disease were genotyped for the factor XIII Val 34 Leu mutation. Venous blood was drawn for the determination of hemostatic variables and lipids. Factor XIII genotype was determined through a single-stranded conformational polymorphism technique and plasminogen activator inhibitor (PAI)-1 4G/5G promoter genotype by allele-specific polymerase chain reaction. The mutation was more frequent in patients with primary intracerebral hemorrhage (n=62) (54.8%; P=.05) than in control subjects (41.7%) or in patients with cerebral infarction (n=529) (46.5%; P=.22). There was no relationship between PAI-1 levels and the PAI-1 4G/5G genotype. There was a slightly higher incidence of factor XIII Val 34 Leu in patients with PICH. This may be related to impaired cross-linking of fibrin and/or coagulation proteins.

Genetic complexity of the hypervariable region 1 (HVR1) of hepatitis C virus (HCV): Influence on the characteristics of the infection and responses to interferon alfa therapy in patients with chronic hepatitis C

HCV exists within its host as pools of related genetic variants referred to as quasispecies. The hypervariable region 1 (HVR1) of the E2 envelope gene is subjected to strong selective pressure from neutralizing antibodies. The genetic complexity of this region is defined as the total number of genetic variants within the quasispecies population. The genetic complexity of the HVR1 region was examined in patients with chronic hepatitis C and its relationship with the epidemiology of HCV infection, and its influence on liver disease and the response to interferon treatment were determined in 114 patients with chronic hepatitis C. The genetic complexity of the HVR1 major variants was measured before treatment by using a polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) technique, and was compared with epidemiological, clinical, virological and histological features. The patients were treated with 3 megaunits of interferon (IFN) alfa for 3 to 6 months and the response to treatment was assessed at 3, 6 and 12 months. The HVR1 could be studied in 101 of the 114 patients (89%). Genetic complexity was significantly higher in patients infected through blood transfusion than intravenous drug use (mean complexity index: 5.7 +/- 2.3 vs. 4.7 +/- 1.5, respectively; P = 0.04). This relationship was independent of age and the estimated time since infection. No significant relationship was found with other parameters of infection or liver disease. In univariate analysis, the genetic complexity of HVR1 major variants did not affect the rates of ALT normalization at months 3 and 6 of IFN treatment. HVR1 genetic complexity was lower in patients with a sustained virological response than in non-responders (4.0 +/- 1.7 vs. 5.4 +/- 2.0, respectively; P = 0.07). In multivariate analysis of pretreatment parameters associated with a sustained virological response to treatment, three parameters appeared to be independent predictors of such a response: a low viral load (P < 0.04), a low anti-HCV core IgM titer (P = 0.03) and a low genetic complexity of HVR1 major variants (P < 0.04). In conclusion, the HVR1 of HCV has a quasispecies distribution in infected individuals. Its genetic complexity is significantly higher in transfusion recipients than in intravenous drug users, suggesting that the size of the initial inoculum affects the later emergence and development of viral quasispecies. The genetic complexity of HVR1, together with viral load and the anti-HCV IgM titer, are independent predictors of a sustained virological response to IFN alfa in patients with chronic hepatitis.

Mechanism of Bracken Fern Carcinogenesis: Evidence for H-Ras Activation Via Initial Adenine Alkylation by Ptaquiloside

Bracken fern (Pteridium spp.) causes cancer of the oesophagus and the urinary bladder in cattle and sheep. Ptaquiloside (PT) is believed to be the carcinogenic principle which alkylates DNA when activated to its unstable dienone form (APT) under alkaline conditions. In this report we present evidence for the presence of PT-DNA adducts in the ileum of bracken fem-fed calves using the 32P-postlabelling assay. H-ras mutations were also observed in the ileum using single strand conformation polymorphism (SSCP) technique. Mutations corresponding to adenine to pyrimidine transversions in the codon 61 of H-ras were identified by the cycle sequencing method. In vitro DNA alkylation studies showed that APT alkylated H-ras primarily at the adenines. In addition, the rate of depurination of alkylated adenine was sequence dependent. Investigation of DNA template activity using a plasmid DNA showed that DNA synthesis by T7 DNA polymerase was terminated by the presence of all alkylated bases but certain apurinic sites allowed the DNA synthesis to continue. These results suggest that initial alkylation of adenine by PT in codon 61 followed by depurination and error in DNA synthesis lead to activation of H-ras proto-oncogene.

P53 expression in Langerhans cell histiocytosis.

Langerhans cell histiocytosis (LCH) is a disorder of unknown etiology involving the proliferation and accumulation of cells with the phenotype of a bone marrow-derived antigen-presenting cell of the skin, the Langerhans cell. We have studied p53 expression, an element in the control of cell proliferation, to determine whether it plays a role in the pathogenesis of LCH. LCH lesions from 10 patients with either localized (n = 5) or multisystem disease (n = 5) were studied. p53 protein expression was assessed by immunohistochemistry, and p53 gene mutation by the single strand conformation polymorphism (SSCP) technique. p53 protein expression was detected in all 10 LCH biopsy specimens examined. It was restricted to Langerhans cells (LCH cells), absent from adjacent cells, and localized to the cell nuclei. No mutations of the p53 gene were detected, nor was there abnormal expression of the p53 binding protein, mdm2. p53 is readily detectable in LCH cells but not in normal cells. This is either caused by an unusual mechanism (given the absence of mutations in the p53 gene and of mdm2 expression in LCH cells) or by overexpression or posttranslational changes of normal p53 in response to an as yet unidentified cellular stress. Stabilization and inactivation of p53 could lead to the uncontrolled proliferation of LCH cells, or the abnormality could lead to the induction of programmed cell death.

PCR single strand conformational polymorphism can be used to detect the gene encoding SHV-7 extended-spectrum beta-lactamase and to identify different SHV genes within the same strain.

The PCR single strand conformational polymorphism (PCR-SSCP) technique described to identify mutants of the SHV beta-lactamases was extended to identify an SHV-7 type beta-lactamase. This was found in a strain of Klebsiella pneumoniae, the first recorded isolate in the UK to produce this type of enzyme. We also demonstrate that PCR-SSCP can be used to identify more than one SHV beta-lactamase gene in a single strain.

Intrafamilial variability of the ocular phenotype in a Polish family with a missense mutation (A63D) in the Norrie disease gene

Purpose: To describe the phenotypic variability in a Polish Norrie disease (ND) family associated with the missense mutation A63D. Methods: A patient with spared vision from a Polish ND family underwent detailed ophthalmological examinations including slit-lamp biomicroscopy, ultrasound (USG), angiography, Goldmann kinetic visual field, and electroretinography (ERG). Mutation screening was carried out using the single-strand conformation polymorphism (SSCP) technique and subsequent DNA sequencing of the coding part of the ND gene. Results: A mutation was detected (exon 3, A63D) in a large Polish family with 12 affected males, all but one presenting with classical ND symptoms. In one male, partially preserved vision was observed up to 40 years of age (distance acuity of the right eye 1/50 and left eye 2/50). Slit-lamp examination revealed remnants of a persistent primary vitreous and hyaloid artery. Upon angiography, the retina was vascularized within the posterior pole but not in the periphery. The ERG revealed pathological changes characteristic for chorioretinal degenerations. Conclusion: Within one family, individuals with identical sequence alterations in the ND gene can show remarkable phenotypic variablity of the ocular symptoms. These findings indicate the involvement of additional factors (epigenetic or genetic) in ocular pathogenesis of ND.

Development of a molecular typing method for Pneumocystis carinii sp.f. hominis.

We are developing a typing method for Pneumocystis carinii sp.f. hominis consisting in the PCR amplification of four variable regions of the genome from bronchoalveolar lavages, followed by the detection of their variation by the single-strand conformation polymorphism technique (SSCP). Most samples of each region from eleven unrelated patients showed two SSCP bands. Six patients were probably infected by a single strain since they showed a single sequence morph of each region. The combinations of the four sequence morphs of these patients were all different so that six different strains were distinguished. Other samples generated three or four SSCP bands which were found to correspond to the presence of two sequence morphs, possibly due to co-infections. Five patients could have been co-infected since they showed two sequence morphs of at least one of the regions. The combinations of sequence morphs of the possible co-infecting strains were different from all other combinations, except that three possible strains in two patients might have been present in other patients. Two BALs from the same patient, which were collected at an interval of 2.5 weeks, gave the same pattern for each of the four regions suggesting that these regions of the genome are stable. These results suggest a great diversity of P. carinii sp.f. hominis strains. Thus, the method should be suitable for epidemiological studies.

PCR-based genotyping of MNSs blood group: subtyping of M allele to MG and MT.

PCR-based genotyping of MNSs blood group system was investigated in combination with restriction fragment length polymorphism (RFLP), single-stand conformation polymorphism (SSCP) and allele-specific PCR amplification (ASPA) techniques. M and N alleles are based on three nucleotide substitutions in exon 2 and one base change (G or T) in an intron of glycophorin A locus. The latter single base change was also found among M alleles analyzed in this study, so that M allele appeared to be subdivided into MG and MT. All three alleles, MG, MT and N were identified clearly by RFLP or SSCP analysis following a single amplification. S and s alleles are based on one nucleotide substitution in exon 3 of glycophorin B gene. Genotyping of Ss blood group system was also explored by PCR-SSCP or ASPA analysis, and problems in the methods were discussed.

Typing of Pneumocystis carinii f. sp. hominis by single-strand conformation polymorphism of four genomic regions.

To better investigate Pneumocystis carinii f. sp. hominis epidemiology, we have developed a molecular typing method. Because of the limited genetic variability of the P. carinii hominis genome, a multitarget approach was used. Four variable regions of the genome were amplified by PCR, polymorphism in each region was assessed by the single-strand conformation polymorphism (SSCP) technique, and the results for the four regions of each patient were combined. Bronchoalveolar lavage specimens collected from 11 patients were examined. Four patients were probably infected by a single strain, since their specimens yielded simple SSCP patterns (two bands corresponding to one allele). The combinations of these patterns were unique, suggesting that the strains which infected these patients were different. For the other seven patients, complex patterns were found (three or four bands corresponding to two alleles). The presence of more than one allele of a region in a patient is likely to be due to coinfection. Polymorphism was also assessed by sequencing, which revealed variations at nucleotide positions previously reported to vary. About half of the observed alleles had already been reported by laboratories in different countries. Multitarget typing of P. carinii hominis by PCR-SSCP should allow investigation of strain diversity and thus be useful for future epidemiological studies.

An automated fluorescent single-strand conformation polymorphism technique for screening mutations in the hepatocyte nuclear factor-1alpha gene (maturity-onset diabetes of the young).

An automated fluorescent single-strand conformation polymorphism technique for screening mutations in the hepatocyte nuclear factor-1alpha gene (maturity-onset diabetes of the young).