The inflammatory nature of atherosclerosis has been well established. However, the initial steps that trigger this response in the arterial intima remain obscure. Previous studies reported a significant rate of genomic alterations in human atheromas. The accumulation of genomic rearrangements in vascular endothelium and smooth muscle cells may be important for disease development. To address this issue, 78 post-mortem obtained aortic atheromas were screened for microsatellite DNA alterations versus correspondent venous blood. To evaluate the significance of these observations, 33 additional histologically normal aortic specimens from age and sex-matched cases were examined. Loss of heterozygosity (LOH) was found in 47,4% of the cases and in 18,2% of controls in at least one locus. The LOH occurrence in aortic tissue is associated to atherosclerosis risk (OR 4,06, 95% CI 1,50 to 10,93). Significant genomic alterations were found on 1p32-p31, 1q22-q25, 2q35 and 6p21.3 where VCAM1, SELE, APEG1 and AIF1 genes have been mapped respectively. Our data implicate somatic DNA rearrangements, on loci associated to leukocyte adhesion, vascular smooth muscle cells growth, differentiation and migration, to atherosclerosis development as an inflammatory condition.

p73, a p53 homologue important for growth suppression, differentiation and induction of apoptosis, utilizes different promoters and undergoes alternative splicing to produce several isoforms differing in their ability to overlap p53 functions. Using reverse transcriptase-polymerase chain reaction (RT-PCR) to assess the mRNA levels of p53, p73 (total and isoforms specific for exons 2 and 13), MDM2, CDKN1A and beta2-microglobulin as internal control, we analyzed 35 prostate carcinomas and 44 benign prostate hyperplasias (BPH) compared to 14 normal prostates. Shift of p73 isoform mRNA levels from exon 13 lacking to exon 13 containing copies was observed in 80% of prostate cancer cases and in 52.3% of BPH specimens, and from exon 2 containing to exon 2 lacking (p73Deltaexon2) transcripts in 45.7% of cancer cases, but only in 9.1% of BPH samples. From these findings we deduce that p73 isoform balance is disrupted in prostate cancer and BPH, suggesting that this disequilibrium could play an important role in both prostate hyperplasia and malignancy.

Using PCR-based microsatellite DNA analysis with 48 markers we examined sputum and bronchial washing for genetic alterations compared with lymphocyte extracted DNA from 124 lung cancer patients and 36 healthy donors as normal control. Microsatellite alterations (MA) in at least one locus were detected in all cancer patient-derived specimens but only in 22.2% of the healthy donors. Loss of heterozygosity (LOH) was detected in bronchial washings from 101 non-small cell lung cancer (NSCLC) predominantly on 17p13.1-p13.3 (69.7%), 9p13.3-p24.1 (63.3%), 1p34.2-p36.22 (48.5%), 13q12.1-q13.1 (47.7%) and 3p22.3-p23 (42.7%). In bronchial washings from 23 small cell lung cancer (SCLC) LOH was detected mostly on 3p22.3-p23 (88.6%), 17p13.1-p13.2 (82.3%), 5q32-q33.1 (66.6%), 13q12.2-q13.1 (65.6%) and 9q22.33-q31.3 (52.9%). The different LOH patterns indicate that different genetic background may be responsible for the different physiology of NSCLC and SCLC. The fractional allele loss (FAL) mean value of all cancer specimens was 0.243+/-0.021 compared with 0.007+/-0.008 of healthy donors with a confidence interval (CI) 99.5%. Only seven out of 124 lung cancer specimens (5.2%) exhibited FAL value less than 0.083, the highest was observed in the healthy donors group. FAL appears to be a likely indicator for lung cancer detection. Microsatellite instability (MIN) was detected in 8.7% of SCLC and 4.0% of NSCLC bronchial washings in at least three loci tested. LOH and MIN detection in sputum and bronchial washing from the same patient was 77.6%. Calculation of these indexes per marker exhibits significant variations that could be attributed to diffuse lung disorders or non-cancer specific genetic alterations.

Objective: To investigate the possibility of genetic contribution of CYP1A1, CYP19, GSTM1, and GSTT1 polymorphisms to endometriosis.Design: Genetic polymorphism analysis.Setting: Case-control study.Patient(s): A group of 275 women with sporadic endometriosis was compared with a group of 346 fertile, endometriosis-free women.Intervention(s): Surgical, laparoscopic, and histological examination.Main Outcome Measure(s): Blood specimens were obtained from endometriosis cases and controls. Polymerase chain reaction-based assays were performed for the determination of individual's genotype.Result(s): The CYP19 VNTR, located in intron 4 (TTTA)(10) allele increases the risk for endometriosis development (odds ratio [OR], 4.99; 95% confidence interval [95% CI], 1.351 to 18.436). The combined genotype CYP1A1 wt/m1 or m1/m1 and GSTM1 null deletion adds to this risk (OR, 1.95; 95% CI, 1.266 to 2.995 and OR, 2.23; 95% CI, 0.631 to 7.906, respectively). In contrast, the CYP1A1 wt/wt genotype exhibits a protective effect, with a 38% reduction in the odds for endometriosis development (OR, 0.62; 95% CI, 0.440 to 0.883).Conclusion(s): Our data suggest that CYP19 VNTR (TTTA)(10) allele as well as the combined genotype CYP1A1 m1 polymorphism and GSTM1 null deletion associate with the endometriosis phenotype, whereas the GSTT1 null deletion does not. (Fertil Steril((R)) 2003;79(Suppl 1):702-9. (C) 2003 by American Society for Reproductive Medicine.).

Idiopathic pulmonary fibrosis (IPF) is a specific form of chronic interstitial pneumonia limited to the lung and characterized by a fibroproliferative response with only minor signs of inflammation, which almost always causes rapid fibrotic destruction of the lung. In this study, we investigated genomic instability in IPF, using microsatellite DNA analysis, aiming to detect any specific genetic alterations for this disease. We used 40 highly polymorphic microsatellite DNA markers, in multiplex PCR assays, to examine 52 sputum specimens from IPF patients versus correspondent venous blood. Loss of heterozygosity (LOH) was found in 20 (38.5%) patients in at least one locus. These alterations were found on markers previously associated with lung cancer located on 1p34.3, 3p21.32-p21.1, 5q32-q33.1, 9p21 and 17p13.1 where MYCL1, FHIT, SPARC, p16(Ink4) and TP53 genes have been mapped respectively. These data provide new insights into IPF pathogenesis and a new perspective for its correlation with lung cancer.

Objective: To investigate whether genetic polymorphisms of CYP1A1, GSTM1, and GSTT1 are associated with endometriosis.Design: Genetic polymorphism analysis. Setting: University department.Patient(s): A family with four women in two generations who had endometriosis and one member with suspected endometriosis in the third generation were compared with a group of fertile women.Intervention(s): Laparoscopic examination.Main Outcome Measure(s): Blood specimens were obtained from fertile females and available affected female family members. Multiplex polymerase chain reaction (PCR) and restriction fragment length polymorphism PCR was done to determine each participant's genotype.Result(s): All affected family members had genotype CYP1A1 wt/ml and GSTM1 null deletion. The frequency of this genotype in 54 fertile women was 13%. A 17-year-old family member with suspected endometriosis bad the same genotype. One affected member was also a carrier of a GSTT1 null deletion. This combination was not found in any of the fertile participants. The most frequent genotypes in the sample were CYP1A1 wt/wt, with GSTM1 null deletion and at least one functional allele of GSTT1, and CYP1A1 wt/wt, with at least one functional allele of GSTM1 and GSTT1 (33% and 31%, respectively).Conclusion(s): The combination of CYP1A1 ml polymorphism and GSTM1 null deletion is closely associated with penetration of the endometriosis phenotype, whereas GSTT1 null deletion may add to the penetration of this trait.

Human apolipoprotein E (apoE) exists in three major isoforms encoded by distinct alleles (APOE ε2, ε3 and ε4) and has important functions in nerve development and repair. Inheritance of the ε4 allele is a major risk factor for the development of Alzheimer's disease. To investigate the role of APOE polymorphisms in embryonic development, we analyzed the APOE genotypes of 81 spontaneously aborted embryos and 110 adult controls using a solid-phase minisequencing technique. The ε4 allele was significantly less frequent in the spontaneous abortion group than in the control group (P=0.009), while the frequency of ε3 was significantly increased (P=0.005), suggesting that ε4 may have protective effects during embryogenesis. These protective effects might counterbalance the deleterious age-related effects of the ε4 allele in natural selection.

Pulmonary sarcoidosis shares certain features with immune disease and neoplasia, and microsatellite DNA alterations are detectable in sputum specimens of pulmonary sarcoidosis patients. The biological basis and significance of these findings remain obscure, while information regarding the genetic basis of the disease is limited. Using multiplex PCR-based microsatellite analysis, we investigated 40 markers located on 1p, 1q, 2p, 2q, 3p, 5q, 6p, 7p, 9p, 11q, 14q and 17p in 38 sputum specimens of pulmonary sarcoidosis patients. Loss of heterozygosity (LOH) was found in 13 of 38 (34.2%) patients in at least one locus. These alterations occurred in the subset of markers located in or close to DNA mismatch repair (MMR) genes, hMSH2 (2p22.3-p16.1) and hMLH1 (3p2l.32-p21.1), as well as in CD48 (1q21-q23) and IRF4 (6p23-p25), genes associated with lymphocyte activation. Microsatellite instability (MIN) was observed in five cases (13.2%) in at least one locus. Our data suggest that genomic instability in pulmonary sarcoidosis could be due to MMR defects, while alterations of lymphocyte-specific agents could account for granuloma formation.

Effectiveness of radiotherapy is influenced by several genetic properties of the targeted cells. The aim of this study was the identification of prognostic indicators of tumor response to radiation in cervical and endometrial cancer. Using microsatellite DNA analysis, we investigated 31 markers, located on 1p, 2p, 2q, 3p, 9p, 9q, 13q, 17p and 17q for genomic alterations in 37 cervical and 21 endometrial cancer cases, with complete follow-up data. Genetic alterations of the initial tumor genotypes were observed after radiation in 86.5% of cervical and 81.0% of endometrial cases. Reversions to the original normal genotype were observed in 40.5 and 28.6% respectively, predominantly in cured patients rather than in recurred cases. Survival curves by the Kaplan-Meier method showed a worse prognosis for cervical cancer patients whose tumors harbor allelic imbalance (AI) on 3p or 13q, and for endometrial cancer patients whose tumors harbor AI on 13q. Our data suggest a possible association of the hMLH1 or BRCA2 genes, implicated in distinct DNA repair pathways and located on 3p and 13q respectively, with response of cervical and endometrial cancer to radiotherapy. Moreover, microsatellite DNA analysis before and after radiation treatment could be used as a marker of the clinical outcome of patients.

BACKGROUNDThe remethylation cycle of methionine is folate and vitamin B(12) (cobalamin) dependent and appears to be crucial for embryonic development, probably through effects on synthesis of DNA, proteins and polyamines. Transcobalamin (TC) transports vitamin B(12) to the tissues. The objective of the present investigation was to explore the putative association between the major TC genetic polymorphism (Pro259Arg) and human spontaneous abortion.METHODSThe prevalence of the TC Pro259Arg polymorphism was determined in DNA samples from embryos that had been spontaneously aborted between the 6th and 20th week after conception, and adult controls using solid-phase minisequencing technique.RESULTSThe 259-Pro allele was significantly less frequent in the spontaneous abortion group than in the control group (42.2 and 57.0% respectively; P = 0.005), while the frequency of 259-Arg was significantly increased. There was a lower prevalence of 259-Pro homozygotes in the spontaneous abortion group compared with the control group (9.1 and 32.2% respectively; P < 0.001).CONCLUSIONSThe 259-Pro allele seems to have beneficial influences during embryogenesis, conceivably through its positive effect on vitamin B(12) intracellular bioavailability. Our results warrant additional investigations addressing the question if vitamin B(12) supplementation in addition to folic acid supplementation may prevent spontaneous abortion in women planning a pregnancy.

Our recent report (1Arvanitis D.A. Goumenou A.G. Matalliotakis I.M. Koumantakis E.E. Spandidos D.A. Low-penetrance genes are associated with increased susceptibility to endometriosis.Fertil Steril. 2001; 76: 1202-1206Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) was based on the observation that CYP1A1 wt/ml and GSTM1[−] genotypes co-occurred with an endometriosis phenotype and early onset of the disease in four female members of the same family. The number of affected family members is now five, as the 18-year-old suspected endometriosis case (assigned as case 6) was confirmed to have stage I endometriosis on laparoscopy. In our study, the mother and three daughters with a CYP1A1 wt/wt, GSTM1[+] genotype (assigned as cases 2 through 5) were compared to the endometriosis-free greatgrandmother (case 1, the only family member available who was older than the controls in the same pedigree). Family clustering of endometriosis is severe, with a value of 8.3. Also noteworthy, in this family is that the three affected daughters and granddaughter spend their childhood in a different environment (urban) from the one in which their mother was raised (rural); as adults, all live in different places from one another.Because the small sample size precluded any statistical test, we investigated the frequencies of these genotypes in a group of 54 fertile women to estimate the chance of the co-finding of the same genotype in five female members in the same family. We found that the CYP1A1 wt/ml, GSTM1[−] genotype is found in 13% of the general population of fertile women, which made such a co-finding very rare. In addition, endometriosis is by nature a complex trait that affects females, and many genes are involved under the strong influence of environment. Thus, large, multigeneration pedigrees are rarely or never seen (2Thomson G. Esposito M.S. The genetics of complex diseases.Trends Cell Biol. 1999; 9: M17-M20Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar).A recent association study that compared the same polymorphisms between endometriosis cases and controls is in agreement with our observation that CYP1A1 wt/ml, GSTM1[−] may increase the risk for endometriosis development (3Hadfield R.M. Manek S. Weeks D.E. Mardon H.J. Barlow D.H. Kennedy S.H. Linkage and association studies of the relationship between endometriosis and genes encoding the detoxification enzymes GSTM1, GSTT1 and CYP1A1.Mol Hum Reprod. 2001; 7: 1073-1078Crossref PubMed Scopus (103) Google Scholar). To the best of our knowledge, our report was the first to describe a striking accumulation of endometriosis in three generations. Our study was designed to eliminate intrinsic drawbacks and draw attention to what we feel is an important observation. Our recent report (1Arvanitis D.A. Goumenou A.G. Matalliotakis I.M. Koumantakis E.E. Spandidos D.A. Low-penetrance genes are associated with increased susceptibility to endometriosis.Fertil Steril. 2001; 76: 1202-1206Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) was based on the observation that CYP1A1 wt/ml and GSTM1[−] genotypes co-occurred with an endometriosis phenotype and early onset of the disease in four female members of the same family. The number of affected family members is now five, as the 18-year-old suspected endometriosis case (assigned as case 6) was confirmed to have stage I endometriosis on laparoscopy. In our study, the mother and three daughters with a CYP1A1 wt/wt, GSTM1[+] genotype (assigned as cases 2 through 5) were compared to the endometriosis-free greatgrandmother (case 1, the only family member available who was older than the controls in the same pedigree). Family clustering of endometriosis is severe, with a value of 8.3. Also noteworthy, in this family is that the three affected daughters and granddaughter spend their childhood in a different environment (urban) from the one in which their mother was raised (rural); as adults, all live in different places from one another. Because the small sample size precluded any statistical test, we investigated the frequencies of these genotypes in a group of 54 fertile women to estimate the chance of the co-finding of the same genotype in five female members in the same family. We found that the CYP1A1 wt/ml, GSTM1[−] genotype is found in 13% of the general population of fertile women, which made such a co-finding very rare. In addition, endometriosis is by nature a complex trait that affects females, and many genes are involved under the strong influence of environment. Thus, large, multigeneration pedigrees are rarely or never seen (2Thomson G. Esposito M.S. The genetics of complex diseases.Trends Cell Biol. 1999; 9: M17-M20Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). A recent association study that compared the same polymorphisms between endometriosis cases and controls is in agreement with our observation that CYP1A1 wt/ml, GSTM1[−] may increase the risk for endometriosis development (3Hadfield R.M. Manek S. Weeks D.E. Mardon H.J. Barlow D.H. Kennedy S.H. Linkage and association studies of the relationship between endometriosis and genes encoding the detoxification enzymes GSTM1, GSTT1 and CYP1A1.Mol Hum Reprod. 2001; 7: 1073-1078Crossref PubMed Scopus (103) Google Scholar). To the best of our knowledge, our report was the first to describe a striking accumulation of endometriosis in three generations. Our study was designed to eliminate intrinsic drawbacks and draw attention to what we feel is an important observation.

Using polymerase chain reaction-based microsatellite analysis we examined 40 cases, tumor biopsies and cytological material, of early stage cervical cancer and 20 healthy donors. Loss of heterozygosity (LOH) was detected in 35 out of 40 cases (87.5%), located on 9pter-p13 (67.5%), 9q32-34 (17.5%), 13q12 (32.5%), 17p13 (0%) and 17q11-q22 (12.5%). Microsatellite instability (MIN) phenotype was found in three out of 40 cases (7.5%). The accuracy in LOH and MIN detection in cytological material compared to tumor biopsies was 91.5 and 86.0%, respectively. None of the specimens of healthy donors exhibited any genetic alteration. Our data suggest that microsatellite analysis in cytological material could be used for the early detection of cervical cancer.

Objective: To detect allelic imbalance on specific genetic loci occurring in endometriosis.Design: Microsatellite analysis.Setting: Paraffin-embedded tissues histologically confirmed as endometriotic or normal endometrium.Patient(s): Premenopausal women undergoing laparoscopy for suspected endometriosis.Intervention(s): Laparoscopic excision of specimens.Main Outcome Measure(s): Allelic imbalance and alterations of intensity of microsatellite alleles.Result(s): Five of 17 microsatellite DNA markers (29.4%) showed allelic imbalance. Eight samples (36.4%) showed allelic imbalance in at least one locus. Loci 9p21, 1q21, and 17p13.1 exhibited imbalance in 27.3%, 4.5%, and 4.5%, respectively. A 3-fold increase of the fractional allelic loss was observed from disease stage II to III and IV, whereas only 1.3-fold was found between patients of 41-50 and 20-40 years.Conclusion(s): We found that loss of heterozygosity on p16(Ink4), GALT, and p53, as well as on APOA2, a region frequently lost in ovarian cancer, occurs in endometriosis, even in stage II of the disease. The occurrence of such genomic alterations may represent important events in the development of endometriosis. The 9p21 locus may contain a gene associated with the pathogenesis of the disease, and therefore its loss may be a prognostic marker of the disease. (C) 2001 by American Society for Reproductive Medicine.

In order to detect the contribution of cytochrome P450 1A1 (CYP1A1), aryl hydrocarbon receptor (AhR), glutathione S-transferases M1 (GSTM1), P1 (GSTP1), and T1 (GSTT1) genes in breast cancer, genetic analysis was performed, as well as transcriptional analysis in sporadic primary tumours and corresponding adjacent normal tissues from the same patient. CYP1A1 3'-untranslated region (3'-UTR) termed as m1 (MspI) polymorphism and the null(-) deletions of both GSTM1 and GSTT1 genes were examined in genomic DNA from blood samples of 207 female breast cancer patients and 171 age and sex matched controls. The frequencies of the m1 genotype of the CYP1A1 gene in cases and controls were 0.13 and 0.15, while the frequencies of homozygotes with GSTM1(-) were 0.52, in each, and for homozygotes with GSTT1(-) were 0.14 and 0.10, respectively. Statistical analysis of these genotypes in combinations did not reveal any significant difference between the breast cancer population and the control group. Expression of mRNA levels of CYP1A1, GSTM1, GSTP1, GSTT1 and AhR genes in 31 breast cancer patients, revealed inter-individual variation in an independent manner to patient age, genotype, or tumour stage. Eighty-seven percent of the tumour specimens tested were deregulated, compared to their normal counterparts, in at least one locus. Up-regulation of CYP1A1 was observed only when one of the GSTM1 or GSTP1 was down-regulated while the other remained constant. Genotyping analysis did not show any correlation to breast cancer risk. However, RT-PCR analysis provided evidence that CYP1A1, AhR, GSTM1, GSTP1 and GSTT1 genes are frequently deregulated in breast cancer and could be used as molecular biomarkers for better clinical management of such patients, with respect to chemotherapy.

Adenomyosis is a gynecological condition in which tissue histologically similar to that in endometrium is found within the myometrium in the uterus. Although, lesions of both adenomyosis and endometriosis are identical to their sources with respect to structure and function, they are generally regarded as separate and distinct nosologic processes. In this study, we used 17 microsatellite markers, in four tetraplex and one single PCR assay, to determine the incidence of loss of heterozygosity (LOH) in 31 cases of adenomyosis. The markers used are located close to tumor suppressor genes, DNA repair genes, and genes which are thought to be involved in endometriosis. Moreover, the markers were involved in regions frequently lost in ovarian cancer, on chromosomal arms 1p, 1q, 2p, 2q, 3p, 9p, 9q, 17p and 17q. Nine samples (29.0%) showed LOH in at least one locus. Loci 2p22.3-p16.1, 3p24.2-p22 and 9p21 exhibited imbalance (19.4%, 9.7% and 6.5% respectively). This is the first report, that LOH occurs in adenomyosis. The regional chromosomal losses were detectable early during the development of this condition. In addition, hMSH2, hMLH1, p16Ink4 and GALT genes were associated for the first time with adenomyosis and its pathogenesis.

Laryngeal cancer is a rare type of neoplasia, constituting approximately 2% of all human cancers. Mutations of the ras gene family is one of the main activating mechanisms in human cancer. Their involvement in head and neck cancer has been mainly demonstrated at the level of the overexpression whereas ras mutations in these cancers are rare in the Western world. In the present study we explored the incidence of codon 12-point mutation in the H-, K- and N-ras genes, in 41 laryngeal cytological specimens. These specimens corresponded to 19 benign and 22 malignant lesions of the larynx. Only two specimens carried a codon 12-point mutation in the K-ras gene (4.8%) while no mutation was detected in the H- and N-ras genes. K-ras mutations were detected in one benign and one malignant specimen. These results indicate low incidence of ras oncogene mutations in laryngeal cytological specimens.