Members of the genus Francisella and the species F. tularensis appear to be genetically very similar despite pronounced differences in virulence and geographic localization, and currently used typing methods do not allow discrimination of individual strains. Here we show that a number of short-sequence tandem repeat (SSTR) loci are present in F. tularensis genomes and that two of these loci, SSTR9 and SSTR16, are together highly discriminatory. Labeled PCR amplification products from the loci were identified by an automated DNA sequencer for size determination, and each allelic variant was sequenced. Simpson's index of diversity was 0.97 based on an analysis of 39 nonrelated F. tularensis isolates. The locus showing the highest discrimination, SSTR9, gave an index of diversity of 0.95. Thirty-two strains isolated from humans during five outbreaks of tularemia showed much less variation. For example, 11 of 12 strains isolated in the Ljusdal area, Sweden in 1995 and 1998 had identical allelic variants. Phenotypic variants of strains and extensively cultured replicates within strains did not differ, and, for example, the same allelic combination was present in 55 isolates of the live-vaccine strain of F. tularensis and another one was present in all 13 isolates of a strain passaged in animals. The analysis of short-sequence repeats of F. tularensis strains appears to be a powerful tool for discrimination of individual strains and may be useful for a detailed analysis of the epidemiology of this potent pathogen.

Previous studies have demonstrated that the four subspecies of the human pathogen Francisella tularensis, despite showing marked variations in their virulence for mammals and originating from different regions in the Northern Hemisphere, display a very close phylogenetic relationship. This property has hampered the development of generally applicable typing methods. To overcome this problem, we evaluated the use of PCR for discrimination of the subspecies using various forms of long arbitrary primers or primers specific for repetitive extragenic palindromic sequences (REP) or enterobacterial repetitive intragenic consensus (ERIC) sequences. Patterns generated by use of REP, ERIC, or long arbitrary primers allowed differentiation at the species level and of the four subspecies of F. tularensis. With each of these three methods, similar or identical clustering of strains was found, and groups of strains of different geographical origins or differing in virulence showed distinct patterns. The discriminatory indices of the methods varied from 0.57 to 0.65; thus, the patterns were not sufficiently discriminatory to distinguish individual strains. The sequence of a fragment generated by amplification with an arbitrary primer was determined, and a region showing interstrain heterogeneity was identified. Specific primers were designed, and a PCR was developed that distinguished strains of F. tularensis subsp.holarctica from strains of other F. tularensissubspecies, including strains of the highly virulent F. tularensis subsp. tularensis. Notably, one European isolate showed the genetic pattern typical of the highly virulentF. tularensis subsp. tularensis, generally believed to exist only in North America. It is proposed that a combination of the specific PCR together with one method generating subspecies-specific patterns is suitable as a rapid and relatively simple strategy for discrimination of Francisella species and subspecies.

Our results provide strong confirmatory evidence of linkage at 19q13.3 and 5q11.2. The lack of confirmation of linkage at several loci identified in other genome-wide scans emphasizes the need to combine linkage data between research groups.

The E-cadherin (CDH1) gene has been associated with prostate carcinogenesis. The C/A polymorphism ؊160 base pairs relative to the transcription start site has been shown to decrease gene transcription. We analyzed the association between this polymorphism and the risk of sporadic, familial (2 close relatives) and hereditary (3 or more close relatives) prostate cancer. We combined data from 3 population-based epidemiologic studies in Sweden encompassing altogether 1,036 prostate cancer cases and 669 controls that were genotyped for the short nucleotide polymorphism. Odds ratios with 95% confidence intervals were estimated through unconditional logistic regression. We found no significant association between the A-allele and sporadic (OR ‫؍‬ 1.0; 95% CI ‫؍‬ 0.8 -1.2) or familial (OR ‫؍‬ 1.4; 95% CI ‫؍‬ 0.9 -2.2) prostate cancer. In contrast, risk of hereditary cancer was increased among heterozygote CA carriers (OR ‫؍‬ 1.7; 95% CI ‫؍‬ 1.0 -2.7) and particularly among homozygote AA carriers (OR ‫؍‬ 2.6; 95% CI ‫؍‬ 1.4 -4.9). Our data indicate that the ؊160 single nucleotide polymorphism in CDH1 is a low-penetrant prostate cancer susceptibility gene that might explain a proportion of familial and notably hereditary prostate cancer.

Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominant disorder that predisposes to predominantly colorectal and endometrial cancers due to germline mutations in DNA mismatch repair genes, mainly MLH1, MSH2 and in families with excess endometrial cancer also MSH6. In this population-based study, we analysed the mutation spectrum of the MLH1, MSH2 and MSH6 genes in a cohort of patients with microsatellite unstable double primary tumours of the colorectum and the endometrium by PCR, DHPLC and sequencing. nl).A germline mutation in one of the MMR genes in HNPCC patients predisposes to an early onset colon cancer: 30% risk to age 40 and over 80% lifetime risk, 9 compared to 3-4% in the general population. In addition, affected individuals are prone to develop multiple primary cancers, both from the colorectum and extracolonic sites, of which endometrial cancers are the most common with a lifetime risk of 20 -43% in women, 10 followed by carcinoma of the ovary, stomach, small intestine and upper urinary tract. 11 When the MMR system is defective, especially when due to MLH1 and MSH2 mutations, replication errors arising during DNA replication are not corrected, which results in widespread genomic instability. Through their repetitive nature, microsatellites are particularly prone to replication errors and therefore microsatellite instability (MSI) is a hallmark of MMR deficiency. MSI is found in more than 90% of all HNPCC tumours 12 but only in 10 -15% of sporadic colorectal tumours. 13 Moreover, MMR deficiency leads to an increased mutation rate in specific cancer related genes, including BAX, 14 IGFIIR 15 and TGF-␤ type II receptor, 16 which may enhance tumour progression. While MLH1 and MSH2 defects give instability primarily in dinucleotide repeats, 13 MSH6-defective tumours show alterations in predominantly mononucleotide repeats. [17][18][19] This is in congruence with the biological function, where MLH1 and MSH2 manage repair of small insertion/deletion loops, 20,21 while MSH6 mostly is involved in repair of base-base and single nucleotide insertion/deletion mismatches. 22,23 Double primary cancers within the HNPCC spectrum is a hallmark of HNPCC and an indicator to clinically suspect HNPCC. 24 In a previous population-based study, 25 we observed an increased risk of HNPCC-associated cancers in first-degree relatives to patients diagnosed with double primary cancers of the colorectum or the colorectum/endometrium. The overall standardised incidence ratio (SIR) was 1.69 (95% CI; 1.39 -2.03). The SIR was highest among relatives to the patients with young age of onset (Ͻ50 years) and MSI positive tumours. The aim of our study was to investigate the MLH1, MSH2 and MSH6 mutation spectrum in the patients with MSI positive double primary tumours. MATERIAL AND METHODS PatientsSeventy-eight patients with double primary cancers of the colorectum or the colorectum and the endometrium (Fig. 1) were identified in a population-based cohort study previously reported. 25 The MSI status of one of the patie...