PCR-based Microsatellite Analysis Research Articles

Assessment of Genetic Purity of Inbred Strains of Mice using Microsatellite Markers

Monitoring of genetic make-up of inbred strains of mice is one of the most essential and basic requirements to assure the continued purity and homogeneity of the strains. It further helps in maintaining phenotypic characteristics of mice strain, which in turn, ensures the reproducibility of research outcomes between the group of mice and over time. In the present study, the phenotypic and genotypic characteristics of various inbred strains of mice maintained in our Animal Facility were assessed to ascertain their purity. The mice exhibited strain-specific phenotypic characters and skin coat colours in accordance with the published profile of the specific strains. The average litter size of C57BL/6 and BALB/c mice, the most commonly used strains of mice, was calculated over different generations and found to be 5 to 6.6 and 4 to 6, respectively. For checking the genetic purity of mice, more than 1700 tissue samples (ear punched tissue) were collected from various strains of mice. Genomic DNA was isolated from the tissue samples and subjected to PCR-based microsatellite analysis. Amplified PCR products of genomic DNA of tested mice showed identical amplicon base-pair length as per the published profile in the MGI database, indicating the purity and uniformity of the mice.

Molecular alterations in gastric cancer and the surrounding intestinal metaplastic mucosa: an analysis of isolated glands.

Intestinal metaplasias (IMs) are generally regarded as pre-neoplastic gastric lesions. However, molecular alterations including genetic and epigenetic changes occurring in individual IM glands are not well defined. We sought to identify DNA methylation status, microsatellite instability (MSI) and allelic imbalance (AI) occurring in individual IM glands and non-IM glands within the same mucosa. We divided examined isolated gland obtained from GC into 4 components: isolated cancer, antral isolated intestinal metaplastic tissue, antral isolated non-metaplastic gland and isolated non-metaplastic gland derived from the greater curvature of the most distant gastric body without mucosal atrophy. We examined AI and microsatellite instability statuses using PCR-based microsatellite analysis. Next, the DNA methylation status (high methylation epigenome [HME], intermediate methylation epigenome [IME], and low methylation epigenome [LME]) was investigated. DNA methylation analysis of CDKN2A, mir34-b/c and MLHI genes was also performed. Although antral isolated IM glands were characterized by IME, isolated non-IM glands showed LME. In isolated cancer glands, HME was frequently found, compared with isolated non-IM glands. DNA methylation of mir34-b/c was common in isolated cancer and IM glands, whereas DNA methylation of CDKN2A was a rare event in isolated samples. The MLH1 gene was not methylated in isolated non-IM glands. Although multiple AIs were frequently found in isolated cancer glands, a few AIs were detected in isolated IM glands. We suggest that the DNA methylation status and the status of the mir34-b/c gene among isolated samples of IMs and isolated non-IM glands have an impact on IM development.

An APC Mutation in a Large Chinese Kindred With Familial Adenomatous Polyposis Was Identified Using Both Next Generation Sequencing and Simple STR Marker Haplotypes.

BackgroundFamilial adenomatous polyposis (FAP) is an autosomal dominant disorder characterized primarily by the development of numerous adenomatous polyps in the colon and a high risk for colorectal cancer. FAP is caused by germline mutations of the adenomatous polyposis coli (APC) gene. The proband in this family was a 39-year-old female patient with the pathologic diagnosis of adenomatous polyps, and then a five-generation kindred with FAP was characterized in the following years. This article identified an APC mutation, and demonstrated the practical use of APC-linked STR markers, which could be used to reduce misdiagnosis of prenatal diagnosis or preimplantation genetic diagnosis resulted from contamination or allele drop-out.MethodsNext-generation sequencing (NGS) was used to identify the possible APC mutations in an affected individual from a family with autosomal dominant colon cancer. Targeted sequencing then used to identify additional related individuals with the mutation. Three short tandem repeat (STR) loci, D5S299, D5S134, and D5S346, were used for PCR-based microsatellite analysis of the APC gene in the extended family.ResultsWe identified an APC: p.W553X mutation. The STR haplotype at the APC locus, A1B4C1, was shared by all clinically affected individuals with the APC: p.W553X mutation. In addition, the APC: p.D1822V variant was observed in 40% affected individuals and in two unaffected individuals.ConclusionWe described a protein truncation mutation, APC: p.W553X; demonstrated the value of APC-linked STR markers (D5S299, D5S134, and D5S346) haplotypes; and suggested the potential role of these haplotypes in detecting loss of heterozygosity of the APC gene.

Impact of PCR-based molecular analysis in daily diagnosis for the patient with gliomas

The WHO2016 CNS update requires a combined histological and molecular assessment. To assess the major aberrations such as co-deletion of complete chromosome arms 1p and 19q (Co-del), isocitrate dehydrogenase and histone H3 mutations, direct sequencing, multiplex ligation-dependent probe amplification and/or FISH are methods considered to be "golden standard" in the community. However, these methods are expensive and complicated. The aim of this study is verification of the sensitivity of the simple PCR-based techniques for assessment of molecular information in daily diagnosis. We analyzed a total number of 80 patients with gliomas. FISH and PCR-based microsatellite analysis were compared for Co-del assessment. Direct sequencing and qPCR using hig-resolution melting (HRM) were compared for IDH and histone H3 mutations. The sensitivity and specificity of FISH were 0.71 and 0.79, respectively. FISH using a commercially available Vysis probe had a risk of high false-positive rate (0.25). For assessment of IDH1 mutations, the sensitivity and specificity of HRM were 1.0 and 0.96, respectively. For assessment of IDH2 and H3 mutations by HRM, both sensitivity and specificity were 1.0. We consider PCR-based molecular analysis to be a simple and accurate technique in daily diagnosis that is readily available for a small scientific facility.

Genetic differences stratified by PCR-based microsatellite analysis in gastric intramucosal neoplasia.

Although genetic alterations in patients with advanced gastric cancer have been extensively studied, those in patients with intramucosal neoplasia (IMN) are still poorly understood. We evaluated genetic differences in 158 IMNs, including 51 low-grade dysplasias, 58 high-grade dysplasias (HGDs), 30 intramucosal cancers (IMCs), and 19 mixed tumors (composed of IMC and HGD within the same tumor), using PCR-based microsatellite analysis [allelic imbalance (AI) and microsatellite instability (MSI)]. We classified the DNA methylation status as a hypermethylated epigenome, a moderately methylated epigenome, or a hypomethylated epigenome. In addition, p53 overexpression, β-catenin nuclear localization, and mucin expression were also examined. From cluster analysis, the IMNs examined were categorized into four subgroups as follows. Tumors in subgroup 1 were characterized by MSI-high status, a hypermethylated epigenome, and loss or reduction of expression of MLH-1. Tumors in subgroup 2 showed a mixed pattern consisting of AI and MSI. In contrast, tumors in subgroup 3, which showed accumulation of multiple AIs, were closely associated with HGD, IMC, or mixed tumor and exhibited nuclear expression of β-catenin. Tumors in subgroup 4, which were generally low-grade dysplasias, exhibited a low frequency of AIs and no MSI. Although the mucin phenotype was not correlated with any subgroup, expression of mucin was associated with some subgroups. Overexpression of p53 was common in all subgroups. The approach described herein was useful for studying genetic differences in IMNs. In addition, we suggest that stratification of genetic differences may help to identify genetic molecular profiles in IMNs.

Molecular diagnostics: techniques and recommendations for 1p/19q assessment.

Several morphology- and polymerase chain reaction (PCR)-based methods for chromosome 1p 19q deletion status assessment are available. Important prerequisites for all molecular techniques concern tissue quality and selection of regions of interest. The most common methods for diagnostic 1p 19q assessment are fluorescence in situ hybridization and PCR-based microsatellite analysis. While the latter requires the use of autologous blood samples, more advanced techniques such as array comparative genomic hybridization, multiplex ligation-dependent probe amplification or real-time PCR are independent from autologous DNA samples. However, due to high technical demand and experience required their applicability as diagnostic tests remains to be shown. On the other hand, chromogenic in situ hybridization evolves as attractive alternative to FISH. Herein, the available test methods are reviewed and outlined, their advantages and drawbacks being discussed in detail.

A study of two Chinese patients with tetrasomy and pentasomy 15q11q13 including Prader-Willi/Angelman syndrome critical region present with developmental delays and mental impairment

BackgroundThe proximal chromosome 15q is prone to unequal crossover, leading to rearrangements. Although 15q11q13 duplications are common in patients with developmental delays and mental impairment, 15q aneusomies resulting in greater or equal to 4 copies of 15q11q13 are rare and no pentasomy 15q11q13 has been reported in the literature. Thus far, all reported high copy number 15q11q13 cases are from the West populations and no such study in Chinese patients have been documented. Dosage-response pattern of high copy number 15q11q13 on clinical presentations is still a subject for further study.Case PresentationIn this study, we characterized two Han Chinese patients with high copy number 15q11q13. Using chromosome banding, high resolution SNP-based cytogenomic array, Fluorescence in situ hybridization, and PCR-based microsatellite analysis, we identified two patients with tetrasomy 15q11q13 and pentasomy 15q11q13. Both 15q11q13 aneusomies resulted from a maternally inherited supernumerary marker chromosome 15, and each was composed of two different sized 15q11q13 segments covering the Prader-Willi/Angelman critical region: one being about 10 Mb with breakpoints at BP1 and BP5 regions on 15q11 and 15q13, respectively, and another about 8 Mb in size with breakpoints at BP1 and BP4 regions on 15q. Both patients presented with similar clinical features that included neurodevelopmental delays, mental impairment, speech and autistic behavior, and mild dysmorphism. The patient with pentasomy 15q11q13 was more severely affected than the patient with tetrasomy 15q11q13. Low birth weight was noted in patient with pentasomy 15q1q13.ConclusionsTo the best of our knowledge, this is the first case of pentasomy 15q11q13 and the first study of high copy number 15q11q13 in Han Chinese patients. Our findings demonstrate that patients with tetrasomy and pentasomy of chromosome 15q11q13 share similar spectrum of phenotypes reported in other high copy number 15q11q13 patients in the West, and positive correlation between 15q11q13 copy number and degree of severity of clinical phenotypes. Low birth weight observed in the pentasomy 15q11q13 patient was not reported in other patients with high copy number 15q11q13. Additional studies would be necessary to further characterize high copy number 15q11q13 aneusomies.

Loss of Heterozygosity at Tumor Suppressor Genes Detectable on Fractionated Circulating Cell-Free Tumor DNA as Indicator of Breast Cancer Progression

LOH on circulating DNA may provide tumor-specific information on breast cancer. As identification of LOH on cell-free DNA is impeded by the prevalence of wild type DNA in blood of cancer patients, we fractionated plasma DNA, and determined the diagnostic and prognostic value of both fractions. Our cohort of 388 patients with primary breast cancer before chemotherapy was selected from a multicenter study (SUCCESS). Postoperative plasma was fractionated in low- and high-molecular weight DNA by two different column systems. In both fractions, LOH was determined by a PCR-based microsatellite analysis using a panel of 8 polymorphic markers. Circulating tumor DNA in plasma from 30 patients after chemotherapy was additionally analyzed. The significance levels were adjusted for multiple comparisons. More patients (38%) had LOH at all markers in the fraction containing short DNA fragments than in the fraction containing the long DNA molecules (28%, P = 0.0001). In both fractions 32.85% of LOH were concordant. LOH at the markers D3S1605, D10S1765, D12S1725, D13S218, and D17S855 significantly correlated with tumor stage, tumor size, and lymph node metastasis, positive progesterone, and HER2 receptor status. Most importantly, LOH at D12S1725 mapping to cyclin D2 correlated with shorter overall survival (P = 0.004). The improved detection of LOH on cell-free DNA provides important information on DNA losses of tumor suppressor genes TIG1, PTEN, cyclin D2, RB1, and BRCA1 in breast cancer. In particular, loss of the cyclin D2 gene might become an important prognostic marker easily detectable in the peripheral blood.

LOH on 10p14‐p15 targets the PFKFB3 gene locus in human glioblastomas

Loss of heterozygosity (LOH) on chromosome arm 10p is very common in high-grade gliomas and is, among others, concentrated on the region 10p14-p15. Presence of multiple tumor suppressor genes is assumed, but until now only Krüpple-like transcription factor 6 (KLF6) has been suggested as possible target of LOH in this region. On the basis of the fact that the splice variant 4 (UBI2K4) of the PFKFB3 gene, located in 10p15.1, inhibits the anchorage-independent growth of U87 glioblastoma cells, we hypothesized that PFKFB3 is a target gene of LOH in glioblastomas. In this study, we analyzed 40 glioblastomas for LOH in 10p15, including the PFKFB3 and KLF6 loci, by PCR-based microsatellite analysis. We detected LOH of PFKFB3 in 55% (22/40) of glioblastomas. LOH of KLF6, mapped 2.5 cM telomerically to the PFKFB3 locus, was not stringently correlated to the PFKFB3 LOH. The allelic deletion of PFKFB3 resulted in a decrease of PFKFB3 mRNA level accompanied by a lower PFKFB3 protein level. The expression of growth-inhibiting splice variant UBI2K4 was effectively reduced in glioblastomas with PFKFB3 LOH and a positive correlation with overall PFKFFB3 expression was observed. The PFKFB3 LOH as well as the resulting low UBI2K4 expression level was associated with a poor prognosis of glioblastoma patients. We conclude that LOH on 10p14-p15 in glioblastomas targets PFKFB3 and in particular splice variant UBI2K4, a putative tumor suppressor protein in glioblastomas.

Abstract 2217: Primary tumor loss of heterozygosity proximal to M6P/IGF2R locus is predictive for the presence and persistence of disseminated tumor cells in the bone marrow of ovarian cancer patients

Abstract Introduction: Disseminated tumor cells (DTC) in the bone marrow (BM) are present in about 35% of all ovarian cancer patients before surgery and after platinum-based chemotherapy which has been associated with an adverse prognosis. Since invasive BM aspirations are less accepted and often not feasible, it would be highly desirable to unmask a molecular biomarker in the primary tumor predicting tumor cell spread and persistence. It was the purpose of the study to 1) investigate loss of heterozygosity (LOH) incidence at four ovarian cancer relevant chromosomal loci involved in inter alia apoptosis, platinum sensitivity or DNA repair in primary ovarian tumor tissue 2) assess the prognostic value of LOH after a median follow-up time of three years and 3) correlate LOH incidence with the occurrence of DTC before surgery and after platinum-based chemotherapy. Methods: In total, 88 ovarian cancer patients were recruited for this study. Primary tumor DNA was extracted from paraffin embedded tissue and analyzed for LOH status at four polymorphic microsatellite markers including D10S1765 (PTEN locus), D13S218 (BRCA1 locus), D17S855 (BRCA2 locus) and D6S1581 (proximal to M6P/IGF2R locus) using PCR-based fluorescence microsatellite analysis. BM aspirates were feasible in 53 patients before surgery and in 40 patients after chemotherapy. BM cells were isolated from heparinized BM by Ficoll-Hypaque density gradient centrifugation and analyzed for DTC by immunocytochemistry using the pan-cytokeratin antibody A45-B/B3. Results: In our cohort of 88 patients, we recorded at least one LOH in 56/88 patients (63%), two LOH in 30/88 patients (34%), three LOH in 8/88 patients (9%) and LOH at all four markers in 3/88 patients (3%), respectively. The occurrence of LOH at the entire marker set significantly correlated with tumor grading (p=0.0001) and histology (p=0.004). The frequency of LOH at the marker D10S1765 correlated with FIGO stage (p=0.046) and grading (p=0.05) whereas LOH at D17S855 was associated with grading (p=0.023) and histology (p=0.012). The occurrence did not correlate with disease free as well as overall survival. Prior to surgery, DTC were detected in 26/53 patients (49%) with a median number of 6 cells (range 1-38 cells). After chemotherapy, 20/40 patients (50 %) revealed DTC in their BM with a median number of 10 cells, ranging from 1 to 35 cells. Interestingly, the LOH incidence proximal to D6S1581 significantly correlated with DTC presence before surgery (p=0.05) as well as DTC persistence after chemotherapy (p=0.022). Conclusion: In primary ovarian tumors, allelic loss proximal to D6S1581 might serve as a potential molecular biomarker for the presence and persistence of DTC in the BM. Further investigations have to prove whether such a molecular marker could replace invasive BM aspirations in the future. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2217. doi:10.1158/1538-7445.AM2011-2217

PCR-based microsatellite analysis to accelerate diabetogenic genetic background acquisition in transgenic mice

The Non-obese diabetic (NOD) mice exhibit a susceptibility to spontaneous development of autoimmune diabetes and is the most widely used experimental model for the study of the disease. The NOD strain was established by inbreeding in 1980. This model has a MHC-matched diabetes resistant homologous, NOR/Lt mice, an insulitis-resistant and diabetes-free strain produced from an isolated genetic contamination within a NOD/Lt line. To evaluate the role of transgenes, transgenic mice can be generated in CD-1 mice for technical advantages and then backcrossed to inbred strains. To obtain transgenic mice in NOD or NOR background starting from CD-1, at least 20 backcrosses are required, spending more than two years in the process.Nucleotide repeats (microsatellites) mapped to specific locations on each chromosome are used to evaluate genomic polymorphism. From 23 microsatellites we selected eleven that were variant in PCR amplimer size between CD-1 colony and NOD or NOR strains. We used these microsatellites to identify individuals that were used for backcrossing, thus accelerating the acquisition of a new genetic background. Results yield a defined analysis of the genome in question and profiles were compared to detect genetic variation among individuals. After the selection of mice for backcrossing at the third generation, the 11 specific markers were acquired at the 5th generation and maintained to the 10th generation. Diabetes incidence and insulitis score correlated with the acquisition of genetic background, demonstrating that using this strategy, 5–6 crosses are enough to obtain the genotype of interest, shortening the process in more than one year and a half.

Aurora A is differentially expressed and regulated in chromosomal and microsatellite instable sporadic colorectal cancers

The centrosome-associated kinase aurora A has been shown to be involved in genetic instability and to be (over)expressed in several human carcinomas. This study investigated aurora A gene copy numbers, mRNA and protein expression as well as tumour cell proliferation and aneuploidy in chromosomal and microsatellite instable sporadic colorectal cancers. Case-matched tissues of normal (n=71) and dysplastic (n=49) colorectal epithelium and invasive carcinomas (n=71) were included in this study. PCR-based microsatellite analysis classified 14/71 (20%) of carcinomas as microsatellite instable. A stepwise increase of aurora A mRNA expression (P<0.0001; quantitative RT-PCR) and aurora A protein expressing tumour cells (P=0.0141; immunohistochemistry) occurred in the adenoma-carcinoma sequence. Within invasive carcinomas, aurora A mRNA levels (P=0.0259) and aurora A positive tumour cells (P<0.0001) were closely associated with tumour cell proliferation (Ki-67 specific immunohistochemistry). Compared with chromosomal instable carcinomas, microsatellite instable carcinomas had significantly more aurora A positive tumour cells (P=0.0043) and a higher tumour cell proliferation (P=0.0335). In contrast, only chromosomal instable carcinomas exhibited marked tumour cell aneuploidy (P=0.0004, fluorescence in situ hybridization) and significantly higher aurora A gene copy numbers (P=0.0206) as compared with microsatellite instable carcinomas. This study further supports a role of aurora A in the carcinogenesis of sporadic colorectal cancers. Moreover, it demonstrates that in a minority of predominantly microsatellite instable carcinomas the presence of aurora A positive tumour cells is merely reflecting tumour cell proliferation. In contrast, the large majority of chromosomal instable carcinomas shows additional (de)regulation of aurora A by gene amplification and concomitant tumour cell aneuploidy. Thus, sporadic colorectal cancers exhibit different mechanisms of aurora A regulation and this may impact the efficacy of aurora-targeted therapies.

Allelic Imbalances of the egfr Gene as Key Events in Breast Cancer Progression – the Concept of Committed Progenitor Cells

The introduction of concepts proposing multiple cellular subgroups in the normal female breast leads to the hypothesis that distinct cellular phenotypes in the female breast give rise to different subtypes of breast carcinomas e.g. expressing ER, HER2 and EGFR differentially. Therefore, origin of breast carcinoma types may be based on the formation of a cancer prone field in which the committed progenitor cells pass mutations to their progenies, glandular as well as myoepithelial cells. The existence of such field within the human breast was inferred from the results on primary breast cancer obtained by PCR-based microsatellite analysis of allelic imbalance (AI) of the EGF receptor gene. Here, normal breast tissue shows egfr AI adjacent to breast cancer tissue also harboring egfr gene AI. The therapeutic implications of such a model are fundamental, as tumors may display different phenotypes which arise from transformation of different progenitor cells as well as from transformation of more differentiated progenies within a cancer prone field. Thereby they may show up with different clinical courses of the disease, higher rates of metastases and responses to therapy. In this review, we discuss this mechanism focusing on the EGF receptor as an example for regulators of progenitor cell growth in many tissues. Phylloides tumors serve as a putative model for embryonic differentiation stage ruled by EGFR signaling and give insights into the tumor-host-interaction. The inhibition of the EGF receptor by specific monoclonal antibodies (e.g. Erbitux) will give an answer in as far EGFR-signaling is decisive for the development of an invasive breast cancer. For this purpose new models have been inaugurated which vary in the EGF receptor gene dosage and protein expression. Moreover, we discuss the EGF receptor as a target for the treatment of pre-malignant lesions with a high risk for malignant growth, e.g. DCIS, which certainly will be detected more frequently by mammography screening programs soon.

Tissue Inhibitor of Metalloproteinases-3 Promoter Methylation is an Independent Prognostic Factor for Bladder Cancer

TIMP-3 (tissue inhibitor of metalloproteinases-3) is 1 of 4 members of a family of proteins that were originally classified according to their ability to inhibit matrix metalloproteinases. We analyzed TIMP-3 methylation in 175 urine sediment DNA samples from patients with bladder cancer with well characterized clinicopathological parameters, including patient outcome. We examined urine sediment DNA for aberrant methylation of 9 genes, including TIMP-3, by quantitative fluorogenic real-time polymerase chain reaction. Using an optimal cutoff value by TaqMan(R) quantitation we found that the risk of death was statistically significantly higher in patients with higher TIMP-3 and ARF methylation (HR 1.99, 95% CI 1.12 to 3.27, p = 0.01 and HR 1.66, 95% CI 1.00 to 2.76, p = 0.05, respectively) than in patients without/lower TIMP3 and ARF methylation in urine. A significant correlation was also seen between the risk of death and stage 3 tumor (HR 2.73, 95% CI 1.58 to 4.72, p = 0.003) and metastasis (HR 3.32, 95% CI 1.98 to 5.57, p = 0.0001). Multivariate analysis subsequently revealed that TIMP-3 methylation was an independent prognostic factor for bladder cancer survival with stage and metastasis (p = 0.001 and 0.02, respectively). These results suggest that TIMP-3 promoter methylation could be a clinically applicable marker for bladder cancer progression.

Asynchronous Growth of Prostate Cancer Is Reflected by Circulating Tumor Cells Delivered from Distinct, Even Small Foci, Harboring Loss of Heterozygosity of the PTEN Gene

The clinical value of prostate-specific antigen (PSA)-positive circulating tumor cells (CTCs) is still a matter of debate and it is also still unclear if these CTCs actually represent the primary tumor. Therefore, we isolated PSA-positive CTCs from the peripheral blood of patients suffering from multifocal cancers and did genetic profiling of each cancer focus by a multiplex PCR-based microsatellite analysis (D7S522, D8S522, NEFL, D10S541, D13S153, D16S400, D16S402, D16S422, and D17S855). In 17 of 20 prostate cancer cases, the loss of heterozygosity (LOH) pattern of the CTCs was identical with only one focus of the primary tumor. Moreover, in six cases, the LOH pattern suggested that smaller foci, down to 0.2 cm3, might deliver CTCs. Interestingly, the highest number of LOHs was observed at the marker D10S541 (85%), the PTEN gene, which was observed much less frequently in unifocal prostate cancer (48%). Furthermore, the infrequently occurring LOH in the BRCA1 gene (38%) was found in four of the five cases where a biochemical recurrence was seen within 3 years after prostatectomy. Therefore, the data might support the assumption that CTCs in prostate cancer are derived from distinct foci of a primary tumor. The size of the tumor focus is not related to the delivery of cells. Although the number of cases that were investigated in this study was small, it might be suggested that the LOH at distinct markers such as D10S541 and D17S855 represent the genes PTEN and BRCA1, which might be associated with the occurrence of CTCs in the peripheral blood of patients as well as an early biochemical recurrence.

Aberrations in the Fragile Histidine Triad(FHIT) Gene May Be Involved in Lung Carcinogenesis in Patients with Chronic Pulmonary Tuberculosis

It has been suggested that pulmonary tuberculosis (PT) is associated with lung carcinogenesis. To identify the genetic characteristics of precancerous PT lesions in lung, 20 patients were selected with a 1-month to 36-year history of PT and 20 lung cancer patients with a 2- to 53-year history of previous PT that had undergone lung resection at the Nanfang Hospital from 1999 to 2003; PCR-based microsatellite analysis with DNA extracted from microdissected tissues and immunohistochemical analysis of FHIT protein expression in samples of hyperplasia and cancer obtained from 40 patients were performed. Three microsatellite markers of the FHIT gene for loss of heterozygosity (LOH) analysis were used. LOH of the FHIT locus was frequently found among the lesions of hyperplasia and atypical hyperplasia obtained from 6 patients with a 1- to 36-year history of PT (12 of 30 informative lesions, 40%); none of 70 hyperplastic lesions obtained from 14 patients with a 1- to 11-month history of PT showed LOH at the FHIT gene; 17 of 20 (85%) cancer lesions obtained from 20 lung cancer patients with a 2- to 53-year history of previous PT showed LOH at the FHIT gene, which was significantly higher than hyperplasia and atypical hyperplasia obtained from patients with a 1- to 36-year history of PT in FHIT LOH (Fisher’s exact test p = 0.003). Additionally, the level of FHIT protein expression was frequently reduced in the hyperplastic lung epithelial cells of PT with a 1- to 36-year history and cancer tissue. Our findings suggest that allelic loss of the FHIT gene may be involved in carcinogenesis in the lung of patients with PT.

Which genetic marker for which conservation genetics issue?

Conservation genetics focuses on the effects of contemporary genetic structuring on long-term survival of a species. It helps wildlife managers protect biodiversity by identifying a series of conservation units, which include species, evolutionarily significant units (ESUs), management units (MUs), action units (AUs), and family nets (FNs). Although mitochondrial DNA (mtDNA) evolves 5-10 times faster than single-copy nuclear DNA (scnDNA), it records few traces of contemporary events. Thus, mtDNA can be used to resolve taxonomic uncertainties and ESUs. Variable number of tandem repeats (VNTRs) evolve 100-1000 times faster than scnDNA and provide a powerful tool for analyzing recent and contemporary events. VNTR analysis techniques include polymerase chain reaction (PCR)-based microsatellite assays and oligonucleotide probing. Size homoplasy problems in PCR-based microsatellite assays can strongly affect the inference of recent population history. The high homozygosity in endangered species is reflected in a relatively low number and level of variability in microsatellite loci. This combined with "allelic dropout" and "misprinting" errors contributes to the generation of highly biased genetic data following analyses of natural populations. Thus, in conservation genetics, microsatellites are of limited use for identifying ESUs, MUs, and AUs. In contrast to PCR-based microsatellite analysis, oligonucleotide probing avoids errors resulting from PCR amplification. It is particularly suitable for inferring recent population history and contemporary gene flow between fragmented subpopulations. Oligonucleotide fingerprinting generates individual-specific DNA banding patterns and thus provides a highly precise tool for monitoring demography of natural populations. Hence, DNA fingerprinting is powerful for distinguishing ESUs, MUs, AUs, and FNs. The use of oligonucleotide fingerprinting and fecal DNA is opening new areas for conservation genetics.

Allelic loss but absence of mutations in the polyspecific transporter gene BWR1A on 11p15.5 in hepatoblastoma.

Chromosomal region 11p15.5 shows frequent maternal allelic loss in embryonal tumors, including rhabdomyosarcoma (RMS), Wilms' tumor (WT) and hepatoblastoma (HB), consistent with the presence of at least one tumor suppressor gene in this region, which should be paternally imprinted, i.e., expressed from the maternal allele only. The BWR1A gene encodes a polyspecific transmembrane transporter and is located on 11p15.5. It is highly expressed in liver, paternally imprinted and was found to be mutated in an RMS cell line, making it a plausible tumor suppressor gene for HB. We therefore screened 62 HBs, 3 HB cell lines and 1 pediatric hepatocellular carcinoma for BWR1A mutations using single-strand conformation polymorphism analysis. Allelic loss on 11p15.5 was assessed by PCR-based microsatellite analysis in 56 of the cases for which constitutional DNA was available. BWR1A mRNA expression was determined in 14 HBs by differential RT-PCR of matched cDNA samples from tumor and normal liver. Western blot analysis was performed on 4 tumors and matching normal liver tissue. Except for sequence polymorphisms (in exons 2, 3 and 10 as well as in introns 6 and 7), no mutations were found. Thirteen HBs (23%) had allelic loss on 11p15.5; this included BWR1A in 12 but it was telomeric to BWR1A in 1. Expression of BWR1A mRNA was reduced in 11 out of 14 cases by 19-92%, independent from allelic loss of 11p15.5. By Western blot analysis, all 4 tumors and matching liver samples displayed a 48-51 kd band corresponding to BWR1A. These results make it unlikely that BWR1A is the target of the allelic deletions in HB. However, similar to the putative 11p15.5 tumor suppressor H19, BWR1A appears to be reduced in expression. Reduced expression in the absence of mutations may contribute to HB development; however, to understand the significance of this finding will require further studies on the function of BWR1A, specifically its role in liver development.

Evidence of a secondary grapevine domestication centre detected by SSR analysis.

The origin of the grapevine was investigated with archaeobotanical, cultural and historical data. A primary domestication centre was located in the Near East region but there is no agreement on the existence or role of secondary domestication centres. In this work, PCR-based microsatellite analysis has been applied to study the origin of some Italian cultivated grapevines from in situ direct domestication of the wild autoctonous grapevine. Three different Italian locations in Grosseto, Cosenza and Nuoro were identified for this study, and domesticated grapevine as well as wild local accessions growing in these location, were analysed by SSR markers. Cluster analysis performed on Cosenza and Grosseto samples showed a high value of genetic distance between domesticated and wild accessions. On the contrary two cultivars (Bovale Murru and Bovale Muristellu) recovered in Nuoro (in the Sardinia island) were very close to some wild varieties. This suggests that the latter two cultivars may have originated from wild grapevines and consequently that in this location a secondary grapevine domestication event occurred. Six Lambrusco varieties were also included in this analysis as ancient putative ancestors of the cultivated grapevines. The molecular analysis excluded this hypothesis and suggest Lambrusco as an independent Vitis taxon.

Microsatellite analysis of synchronous and metachronous tumors: a tool for double primary tumor and metastasis assessment.

Despite well-established histopathological features and the development of immunostaining of human neoplasms, there are a number of cases in which surgical pathologists cannot assure the origin of synchronous and metachronous tumors. In many cases, the classification of these lesions as either two separate primary tumors or as a single primary tumor with a metastasis has significant implications with respect to patient prognosis and recommendations for therapy. To establish the origin of tumors, we assessed tumor cell clonality using PCR-based microsatellite analysis on microdissected archival tissues for loss of heterozygosity (LOH) and microsatellite instability (MSI) in a series of 19 paired synchronous and metachronous tumors from several organs. As a control group, 15 autopsy cases with an unequivocally recognizable primary tumor and associated metastases were also examined. Based on LOH and MSI findings, and using a panel of 4 to 12 (median 7) microsatellite markers, we were able to establish the clonal pattern of microsatellite changes in 17 out of 19 (89%) biopsy cases and thus determine if they were either double primary tumors (41%) or metastases (59%). Of interest, identical or similar pattern of microsatellite abnormalities were detected in 15 primary tumors and corresponding metastasis from autopsies. Our results indicate that microsatellite analysis for LOH and MSI, as an expression of clonality, provides a useful tool to distinguish double primary neoplasms and metastases in synchronous and metachronous tumors.