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The CDKN2A/ARF genes have been associated with increased risk of malignant melanoma (MM) in families with multiple members affected by disease and in families characterized by the constellation of breast cancer and MM. The exact contribution of CDKN2A/ARF to disease risk remains poorly characterized, especially in diverse populations. In this report, the contribution of CDKN2A/ARF germline mutations and large rearrangements to disease in Polish familial MM (FMM) and aggregations of breast cancer and MM were assessed using a strategy that included genomic sequencing, restriction fragment length polymorphism and multiplex ligation-dependent probe amplification. We examined 16 FMM cases (group 1), 44 MM probands with a cancer family aggregation (CFA) that included at least one breast cancer (group 2) and 22 breast cancer probands with CFA and MM (group 3). The results revealed a paucity of mutations in CDKN2A/ARF, suggesting that in the Polish population this gene does not contribute significantly to either FMM or MM within the context of CFA., (Copyright 2004 Wiley-Liss, Inc.)

Variants in the CHEK2 have been found to be associated with prostate cancer risk in the United States and Finland. We sequenced CHEK2 gene in 140 Polish patients with prostate cancer and then genotyped the three detected variants in a larger series of prostate cancer cases and controls. CHEK2 truncating mutations (IVS2 + 1G>A or 1100delC) were identified in 9 of 1921 controls (0.5%) and in 11 of 690 (1.6%) unselected patients with prostate cancer [odds ratio (OR) = 3.4; P = 0.004]. These mutations were found in 4 of 98 familial prostate cases (OR = 9.0; P = 0.0002). The missense variant I157T was also more frequent in men with prostate cancer (7.8%) than in controls (4.8%), but the relative risk was more modest (OR = 1.7; P = 0.03). I157T was identified in 16% of men with familial prostate cancer (OR = 3.8; P = 0.00002). Loss of the wild-type CHEK2 allele was not observed in any of prostate cancers from five men who carried CHEK2-truncating mutations. Our results provide evidence that the two truncating mutations of CHEK2 confer a moderate risk of prostate cancer in Polish men and that the missense change appears to confer a modest risk.

In this study the proportion of sporadic and familial malignant melanoma (MM) cases harbouring 1100delC in CHK2 was determined to assess whether this mutation is associated with the occurrence of MM. Three groups of patients were studied: (i) 101 patients with histologically confirmed sporadic MM of the skin diagnosed in the city of Szczecin, Poland; (ii) 16 MM patients with a family history of MM in their first-degree relatives; and (iii) 1024 individuals selected at random by family doctors from the city of Szczecin. Molecular examination included an allele-specific oligonucleotide polymerase chain reaction assay for the CHK2 founder mutation (1100delC), genomic sequencing, loss of heterozygosity analysis using CA-repeat microsatellite markers, and haplotype analysis. The CHK2 founder mutation was detected in one out of 101 (1%) of the sporadic MM cases, in none of the 16 familial MMs, and in two of the 1024 individuals (0.2%) from the general population. The differences between the groups of patients were not statistically significant. The MM patient with a CHK2 founder mutation was a 56-year-old female with a history of brain tumours at age 33 and 40 years, sarcoma at 41 years and finally MM at 55 years. Examination of tumorous DNA isolated from the MM and the sarcoma from this patient revealed no loss of heterozygosity in either tumour. It seems that examination of sporadic or familial MM cases for the 1100delC germline mutation in CHK2 is not justified. To evaluate whether this CHK2 founder mutation is associated with MM in patients with LFS syndrome, more MM cases from LFS families should be examined.

Several predispositions to colorectal cancer have been identified, but little is known about genetic susceptibilities to disease in older persons. Colorectal cancer is a risk in Crohn's disease and is believed to be associated with an inappropriate inflammatory response. Recently, the NOD2 gene has been associated with Crohn's disease, which further strengthens the notion that the inflammatory response plays a crucial role in this disease. Several mutations have been identified in the NOD2 gene, which appear with significantly higher frequency in patients with the disease. One such mutation (3020insC) is believed to be clearly causative because it results in a prematurely truncated protein with a predicted reduction in functional efficiency. In this report, we have examined the frequency of the 3020insC mutation in a series of 856 individuals including 556 patients with colorectal cancer. The frequency of the 3020insC mutation in a consecutive series of 250 non-hereditary nonpolyposis colorectal cancer patients >50 years of age was significantly elevated compared with the control population (odds ratio, 2.23; P = 0.0046). The results indicate that NOD2 may be a predisposing factor to colorectal cancer characterized by an older average age of disease onset in persons who do not harbor any other genetic predisposition to disease.

In this report the proportion of consecutive and familial breast cancer cases harboring the 657del5 of exon 6 of the NBS1 gene was determined to assess whether it is associated with the increased risk of breast cancer development. The study consisted of 3 groups of patients: a series of consecutive 150 patients with histologically confirmed breast cancer, diagnosed under the age of 50 in the city of Szczecin; a series of 80 breast cancer patients with a family history of breast cancer in their first-degree relatives; and a series of 530 consecutive individuals without the diagnosis of breast cancer selected at random by family doctors from the city of Szczecin. Molecular examination included allele-specific PCR assay for the common Slavic NBS1 mutation (657del5), LOH analysis using denucleotide CA repeat microsatellite markers, haplotype analysis and sequencing. The NBS1 founder mutation was detected in 2 of 150 (1.3%) consecutive breast cancer cases diagnosed under the age of 50 years; in 3 of 80 familial breast cancer cases (3.7%); and in 3 of 530 individuals (0.6%) from the general population. Examination of tumor DNA from patients with the NBS1 mutation (groups A and B) revealed loss of heterozygosity (LOH) in all cases. Additional haplotype analysis revealed that allelic loss affects specifically wild-type alleles. The majority of probands with breast cancer and the NBS1 mutation had a positive family history of breast cancer in their first-degree relatives. It appears that the 657del5 mutation in exon 6 of the NBS1 gene is responsible for the occurrence of a small but significant proportion of familial breast cancer patients., (Copyright 2003 Wiley-Liss, Inc.)

Purpose: To assess retinal function in HNPCC gene mutation carriers., Patients: 19 carriers (38 eyes) of HNPCC genes and controls., Methods: Electro-oculogram, standard flash electroretinogram and pattern electroretinogram (PERG) recordings were performed., Results: In the total group of HNPCC gene mutation carriers examined by oscillatory potentials, reduced amplitude (p < 0.0004) and increased latency (p < 0.04) of the O3 wave and increased latency (p < 0.02) of the O4 wave were found. In the subgroup of carriers with hMLH1 gene mutation, reduced amplitudes of the O3 (p < 0.0005) and O4 (p < 0.04) waves were identified. In the total group of HNPCC gene mutation carriers examined by PERG, reduced amplitudes of the P50 (p < 0.003), N95 (p < 0.02) and abnormal N95/P50 ratio (p < 0.02) were revealed. In the subgroup of hMLH1 gene mutation carriers, reduced amplitude of the P50 (p < 0.04) and abnormal N95/P50 ratio (p < 0.02) were observed, whereas in the hMSH2 gene mutation carrier subgroup, reduced amplitude (p < 0.03), shortened latency of the P50 wave (p < 0.02) and reduced amplitude of the N95 wave (p < 0.03) were found., Conclusion: Constitutional dysfunction of the inner retina appears to be a characteristic feature of HNPCC gene mutation carriers., (Copyright 2003 S. Karger AG, Basel)

In this study we determined in what proportion of consecutive malignant melanoma (MM) cases the 657del5 mutation of exon 6 of the NBS1 gene can be detected and whether it is associated with the occurrence of MM. Two groups of patients were studied: a series of 80 consecutive patients with histologically confirmed MM of the skin diagnosed in the city of Szczecin, Poland, and a series of 530 consecutive individuals selected at random by family doctors from the city of Szczecin. Molecular examination included an allele-specific polymerase chain reaction assay for the NBS1 founder mutation (657del5), genomic sequencing, loss of heterozygosity analysis using CA-repeat microsatellite markers, and haplotype analysis. The NBS1 founder mutation was detected in two of the 80 (2.5%) MM cases and in three of the 530 individuals (0.6%) from the general population. The difference was not statistically significant. However, examination of tumorous DNA from the patients with MM and NBS1 mutation revealed loss of heterozygosity in both cases. Haplotype analysis revealed that allellic loss affects wild-type alleles. Breast cancer was found in second-degree relatives of both MM probands with NBS1 mutations. One of these probands was simultaneously affected with breast cancer. It seems that the 657del5 mutation of exon 6 of NBS1 gene may be responsible for the occurrence of a small proportion of MM patients, characterized by the occurrence of breast cancer among their relatives.

Mutation analysis of large genes, such as MSH2 and MLH1, is time-consuming and expensive. We investigated the sensitivity and specificity of DHPLC analysis for the detection of mutations within both MSH2 and MLH1. Studies included a series of 46 patients affected by colorectal cancer from HNPCC families. We confirmed 19 changes previously identified by DNA sequencing and, in a blind study, an additional 16 rare alterations including four mutations not previously described. Generally, false negative results were not observed. Elution profiles were highly characteristic for a given change and in 98.5% cases allowed the distinction between novel alterations and previously identified mutations and polymorphisms. For the detection of changes in almost all amplicons, it was sufficient to use just one denaturing temperature. DHPLC was confirmed to be highly sensitive, specific and a cost-effective technique with particularly high potential for the detection of MSH2 and MLH1 gene mutations in the diagnostic setting.

On the basis of literature data and own experience the authors review the current knowledge about the molecular basis of inherited predispositions for tumors. They hypothesize that in the near perspective 5-10 years studies using existing registry data/material and the latest novel technology will allow the identification of the molecular background for the majority of hereditary cancers which will have enormous practical consequences especially for the prevention of malignancies.

Multicenter study of the diagnostic parameters was conducted by three groups in Poland to determine if in situ fluorescence detection of human cutaneous melanoma based on digital imaging of spectrally resolved autofluorescence can be used as a tool for a preliminary selection of patients at increased risk of the disease. Fluorescence examinations were performed for 7228 pigmented lesions in 4079 subjects. Histopathologic examinations showed 56 cases of melanoma. A sensitivity of fluorescence detection of melanoma was 82.7% in agreement with 82.5% found in earlier work. Using as a reference only the results of histopathologic examinations obtained for 568 cases we found a specificity of 59.9% and a positive predictive value of 17.5% (melanomas versus all pigmented lesions) or 24% (melanomas versus common and dysplastic naevi). The specificity and positive predictive value found in this work are significantly lower than reported earlier but still comparable with those reported for typical screening programs. In conclusion, the fluorescence method of in situ detection of melanoma can be used in screening large populations of patients for a selection of patients who should be examined by specialists.

Purpose: To evaluate and compare alterations detected by Alu-PCR, microsatellite instability (MI), and absence of hMLH1 and hMSH2 protein expression measured by immunohistochemical (IHC) analyses as features characteristic of hereditary nonpolyposis colorectal cancer (HNPCC)., Methods: Alu-PCR, MI, and IHC analyses were performed in two groups of patients: (A) HNPCC diagnosed definitively or with high probability (11 patients); (B) sporadic late-onset colorectal cancers (15 patients)., Results: Quantitative alterations recorded by Alu-PCR were not characteristic for Lynch syndrome, occurring more frequently in sporadic late-onset CRC (73% in group B vs 45% in group A). Qualitative changes (occurrence of additional peaks or shifts) have been found to be associated with HNPCC with odds ratio (OR) 2.4, specificity approximately 70% and sensitivity approximately 55%. Findings in MI and IHC analyses have been recognized as features more characteristic of HNPCC suggesting Lynch syndrome with OR 4.8, specificity approximately 80%, sensitivity approximately 55% (MI) and OR 8.0, specificity approximately 93%, sensitivity approximately 36% (IHC)., Conclusion: Molecular techniques allowing identification of patients with a high probability of having HNPCC include IHC and MI analyses. Our results suggest that their replacement by Alu-PCR analysis in diagnosis of HNPCC is not justified.

Analysis of significance of age at cancer diagnosis as a factor allowing identification of a subgroup of patients with a high frequency of hMSH2 and hMLH1 mutations among families that fulfil suspected HNPCC criteria was performed. DNA from thirty-one unrelated patients affected by colorectal cancer from families matching the above criteria were studied by direct sequencing for occurrence of hMSH2 and hMLH1 gene mutations. Seven unequivocal constitutional mutations were detected: five in the hMLH1 gene and two in the hMSH2 gene. Additionally, one hMLH1 alteration of unknown significance was found. All seven mutations were found in a subgroup of 19 patients with cancer diagnosed before the age of 50 years. In a subgroup of 12 patients with cancer diagnosed at an older age only one case with hMLH1 alteration of unknown significance was detected. Our results indicate that early age at cancer diagnosis seems to be a crucial pedigree factor in discrimination of patients with hMSH2 or hMLH1 mutations among families suspected of HNPCC and matching criteria I of ICG-HNPCC.

The most sensitive technique for the detection of germline mutations is exon by exon sequencing of the gene under investigation using genomic DNA as a template for analysis. This approach, however, has cost and sensitivity limitations that can, at least in part, be overcome by RNA-based analysis. Germline mutations of MLH1 and MSH2 are the most frequent cause of the inherited susceptibility to colorectal and other epithelial cancers known as hereditary non-polyposis colorectal cancer (HNPCC). We compared the analysis of the MLH1 and MSH2 genes using mRNA and genomic DNA as starting material from 21 HNPCC patients. All samples were investigated by RT-PCR, sequencing of cDNA and simultaneous sequencing of genomic DNA. The cDNA was generated using specific primers complementary to the ends of MLH1 and MSH2 genes, respectively. Mutations in MLH1 and MSH2 were detected in 11 out of 21 unrelated patients. In 10 out of 11 cases, mutations were detected independently of the type of primers used for reverse transcription (RT). One novel missense mutation (K751R) in MLH1 was detected using this method. One nonsense mutation (E205X) in MSH2 was only detectable when RT was performed using MSH2 gene-specific primers. Shorter PCR products indicative of alternatively spliced transcripts were not observed when MLH1 or MSH2 specific cDNA RT primers were employed to generate template, except in one case where exon skipping was observed for exons 9 and 10. In this report we demonstrate that primers specific for RT of MLH1 and MSH2 are crucial for increasing the sensitivity of cDNA analysis. DNA sequencing using RNA as a basis for template construction may be a valuable and economical alternative to genomic DNA sequencing., (Copyright 2001 Wiley-Liss, Inc.)

The majority of hereditary nonpolyposis colorectal cancer (HNPCC) is caused by mutations in DNA mismatch repair genes, especially in MLH1 and MSH2. Tumours in such patients also show microsatellite instability characteristic for DNA repair defects. The FHIT gene, a candidate tumour suppressor gene located at 3p14.2 has been shown to be involved in carcinogenesis of many human tissues, including digestive tract tissues. In our study, we characterized Fhit protein expression in hereditary and sporadic colorectal cancers (CRC). Our intention was to determine if cancers with mutations in the mismatch repair genes, MSH2 and MLH1, would show more frequent inactivation of the FHIT gene. Sixteen HNPCC and 28 sporadic CRC cases were examined by standard immunohistochemical analyses. Both study groups comprised carefully and selectively chosen cases. We have observed higher frequency of loss or reduction of Fhit protein expression in hereditary CRC than in sporadic cases (44% vs. 25%). Although this difference was not statistically significant (p = 0.17), possibly due to the small number of available tumour specimens, the tendency is interesting. More extensive studies on a larger number of cases should be done in the HNPCC group to confirm statistical significance. Our results suggest that the FHIT gene plays an important role in carcinogenesis of at least one fourth of all colorectal cancers.

Objective: To evaluate the detection possibility of TSH receptor gene mutation within the third cytoplasmic loop and the sixth transmembrane domain in the cytological material obtained by means of fine needle biopsy of autonomous and non-autonomous nodules., Methods: The study has been carried out in 16 women with goitre showing no clinical signs of hyperthyroidism. According to the thyroid scintigraphy and serum level of thyrotropin (TSH) the patients were divided into two groups: 1. 6 patients with autonomous nodules; 2. 10 patients with non-autonomous nodules. Genomic DNA has been isolated from the cytological material and the peripheral blood nuclear cells in order to confirm possible somatic character of TSH receptor gene mutations. DNA has been amplified in polymerase chain reaction (PCR) with the use of a specific pair of primers. Purified PCR products have been subjected to further automatic sequencing., Results: Among 6 autonomous nodules tested one heterozygotic somatic mutation of adenine for cytosine at 1804 nucleotide of TSH receptor gene was detected. This mutation resulted in the change of threonine (codon ACC) at 632 position of TSH receptor protein for proline (codon CCC). Among the non-autonomous nodules one heterozygotic somatic mutation of adenine for cytosine at 1870 nucleotide of receptor TSH gene has been detected. From this mutation followed the change of lysine (codon AAG) at 624 position of the polypeptide chain for glutamine (codon CAG) followed as a consequence., Conclusions: We emphasize the validity of fine needle biopsy in the detection of somatic mutations in the TSH receptor gene. For the first time the somatic mutation in the TSH receptor gene in a non-autonomous nodule has been reported.

The frequency of the GSTM1 gene in patients with nonpolyposis colorectal cancer (CRC) (n = 70) and in subjects with colonic polyps (n = 27) was evaluated and compared with healthy individuals (n = 145). Patients with CRC were divided into the three groups: patients coming from the families with hereditary nonpolyposis colorectal cancer (HNPCC) (n = 17); patients with a high risk of HNPCC who were referred to as suspected of HNPCC (n = 25); patients with sporadic colorectal cancer without clinical features of hereditary tumours (n = 28). A simple polymerase chain reaction (PCR) - based assay to identify GSTM1 nulled and positive (non-nulled) genotype was used. No significant differences in frequency of nulled individuals were observed in both patients with HNPCC and patients suspected of HNPCC as well as in subjects with colonic polyps. The most interesting observation was made in the group of patients with sporadic CRC. Twenty individuals (71.4 %) of the group were GSTM 1 deficient which was significantly different from the control population (p < 0.04). The above data indicate that the absence of the GSTM1 gene is associated with a greater risk of sporadic colorectal cancer. There is an increase in the overall risk of approximately 2.5 as compared with the control population.

It has been shown that slow acetylation rate may be a factor that influences the development of allergic diseases. The influence of NAT2 genetic polymorphism on the risk of development of atopic diseases was evaluated among the white Polish population of 85 patients with atopy (62 children and 23 parents) and 181 healthy individuals (127 children and 54 adults). The NAT2 alleles (*4, *5, *6, and *7) were identified by polymerase chain reaction-restriction fragment length polymorphism methods with DNA extracted from peripheral blood. A significant predominance of homozygous slow acetylators (85%) among patients with atopic diseases was observed. There were no homozygous fast acetylators within this group of individuals. Comparison of the frequency of slow acetylators between the above group of patients and healthy subjects (54%) showed that the significant predominance of slow acetylators was observed in the first group (P < .001). The risk of development of atopic diseases was 5-fold greater for homozygous slow acetylators (odds ratio, 4.69; 95% confidence interval, 2.33-9.59) compared with healthy subjects. We therefore concluded that slow acetylation genotype may be an important factor of individual susceptibility to atopic diseases.

Atrial natriuretic peptide (ANP) is involved in the pathogenesis of sodium-sensitive hypertension. The loss of Sca I restriction site in the ANP precursor gene abolishes the regular stop codon. The aim of our study was the analysis of the Sca I gene polymorphism in 23 patients with sodium-sensitive hypertension, the molecular characteristic of the mutation and the comparison of the blood pressure values, plasma renin activity, plasma ANP and aldosterone concentration between patients with or without mutation. Applying the polymerase chain reaction (PCR), followed by digestion with Sca I, the heterozygous mutation has been found in 9 (39%) patients. The sequencing of PCR products indicated that the loss of Sca I restriction site is caused by T2238-->C transition leading to the translation of ANP with two additional arginines. The higher concentration of ANP in plasma has been found in T2238-->C transition patients on normal and high sodium diet as compared with patients without mutation. These preliminary results suggest that the heterogeneity of sodium-sensitive hypertension is associated with the T2238-->C mutation of the ANP precursor gene.

Deletions of the short arm of chromosome 3 (3p) have been recognized as characteristic features of clear cell renal cell carcinomas (clear cell RCC). We analysed 55 clear-cell RCCs and 30 non-clear-cell kidney tumours (10 papillary and 7 chromophobic RCCs, 11 oncocytomas and 2 collecting duct carcinomas) in loss of heterozygosity (LOH) studies using microsatellite markers for previously observed regions of common deletions on 3p in kidney tumours (3p25, 3p21.3, 3p14.2 and 3p12-13). Alterations were found in all 55 cases of clear-cell RCCs at two to four of the 3p regions. Extensive losses were not found in non-clear-cell tumours except for collecting duct carcinomas; 1 of 10 papillary RCCs showed interstitial deletion limited to a single 3p21.3 locus. LOH analyses using microsatellite markers for regions of common deletions at 3p may be of value in differential diagnosis of kidney tumours.

To study the mechanism by which sulfated polysaccharides with 1,3-beta-D-glucan as a main chain exert anti-HIV-1 activity, we analyzed the effects of curdlan sulfate (CRDS) on HIV-1 infection of SupT-1 cells and peripheral blood mononuclear cells. CRDS had no effect on virions inhibited weakly HIV-1 attachment to cells, and had to be present for 24 hr to achieve protection. Lack of HIV-1 DNA corresponding to the gag region in cells incubated with the virus and CRDS and inhibition of infection after addition of 2',3'-dideoxyinosine to cells treated with CRDS and HIV-1 for less than 24 hr suggest that CRDS delays events that precede and/or include reverse transcription. Analysis of the effect of CRDS on binding of HIV-1 neutralizing antibodies to gp 120 demonstrated that both the continuous epitopes on the V3 loop and the discontinuous CD4 binding site of gp 120 represent targets for CDRS. This interaction of CRDS with functional gp 120 domains suggests that CRDS interferes with the membrane fusion process during HIV-1 infection. Concentrations of CRDS that were protective against infection with T cell- and macrophage-tropic HIV-1 isolates had less suppressive effects on T cell function in comparison with the related compound, dextran sulfate.

The present study examined CD8 antigen expression and variable (V) gene segment usage by T cell receptor (TCR)-gamma delta+ lymphocytes in peripheral blood of symptomatic children with perinatal HIV infection. The relative number of gamma delta+, CD8+ T cells in most of the infected children was higher than that in uninfected children from HIV+ or HIV- mothers and correlated with the immunodeficiency status of the patients. Infected infants and children over 1 year old also showed an increased proportion of V delta 1-J delta 1+ T lymphocytes. CD8 expression on those cells was higher in infected than in uninfected infants and children. Sequence analysis of the delta gene rearrangement of the predominant V delta 1 family in peripheral blood of three HIV+ donors revealed extensive junctional diversity. These results suggest that the V delta skewing in the majority of HIV+ children reflects peripheral expansion of V delta 1-J delta 1+ T lymphocytes early in life, which might be involved in the mechanisms of HIV-induced immunodeficiency.