Prognostic significance of microsatellite alterations at 1p36 in cholangiocarcinoma

AIM: To investigate loss of heterozygosity (LOH) and microsatellite instability (MSI) on the chromosomal region 1p36-pter in cholangiocarcinoma (CCA) patients and determine the association between microsatellite alterations and clinicopathological parameters. METHODS: Ten polymorphic microsatellite markers were determined for LOH and MSI using GS-3000 gel scan fragment autoanalyzer. RESULTS: Sixty-eight out of 90 cases (75.6%) showed LOH in one or more loci. LOH was found most frequently at D1S199 (40.0%), D1S507 (34.6%), D1S2845 (30.5%), and D1S2734 (30.1%). MSI was found in 34 of 90 cases (37.8%) at one or more loci. Fine mapping at 1p36 showed two distinctive regions of common loss, which were D1S2845 and the 25.5-cM region between D1S507 and D1S2734, indicating the existence of putative tumor suppressor genes that is likely to play important roles in the development of CCA. Patients with LOH at D1S234 showed less lymphatic invasion (P = 0.017), whereas patients with LOH at D1S2676 exhibited more lymphatic invasion than those without (P = 0.031). LOH at D1S2845 showed a significant correlation with nerve invasion (P = 0.029). Moreover, patients who demonstrated MSI at D1S228 showed a poor prognosis (P = 0.0026). CONCLUSION: Allelic loss plays a major role in microsatellite alterations at chromosome 1p36, which may contribute to carcinogenesis and pathogenesis of liver fluke related CCA and these alterations can be used as molecular prognostic indicators for CCA patients.     

Stability of genomic DNA in dried blood spots stored on filter paper

The stability of DNA in dried blood samples obtained from the neonatal screening program in Thailand was retrospectively studied in order to determine the conditions necessary for the long term storage of samples for DNA banking. Specimens from 1991 to 2001, which had been kept in the ambient conditions at the Department of Medical Science, Ministry of Public Health, Thailand, were randomly sampled and used for the study. Genomic DNA was extracted from the samples and DNA fragments of the PAX8 and beta-globin genes were amplified by PCR to determine DNA stability. The study showed that 255-bp and 674-bp fragments of the PAX8 gene could be amplified from all the samples. The DNA fragment of 1,039 bp of the beta-globin gene could be detected in all of the samples for the years 1993 to 2001, but only in seven and five out of the ten studied samples for each of the years 1991 and 1992, respectively. Our study shows that genomic DNA is stable in dried blood stored on filter paper at ambient tropical conditions for at least 11 years. However, DNA quality for amplification of larger DNA fragments decreased when the specimens were stored for longer than 10 years.     

Neonatal screening program in Thailand

The Neonatal Screening Program for congenital hypothyroidism (CHT) and phenylketonuria (PKU) commenced in 1996 with the objective of bringing better quality of life to people throughout the country, especially in the remote areas. This involved the implementation of routine services to the public health infrastructure all over the country. The plan of action has been designed so that by the year 2000 all public health service units throughout the country may provide screening services which can cover 1.2 million babies/ annum. Implementation of the screening program has been performed through public health sectors all over the country. These involved: education of the health personnel and communities, implementation of routine specimens collection and delivery systems to the central laboratories, establishment of central laboratory screening services, routine follow up and case management. Local in-house reagents using ELISA and IRMA techniques have been developed and utilized as screening and confirmation tests for CHT. In addition, Guthrie's test has been used for PKU screening and the automated Fluorometry has been selected for PKU confirmation. All 724 community hospitals have provided newborn screening services as one of the basic requirements for newborns according to public health policy. Of 1,425,025 babies screened, 3,450 (0.24%) were above the first screening cut off for CHT (TSH > 25 mU/l) and 321 (0.02%) for PKU (PKU > 4mg/dl). With a 63.10% follow up rate, the incidences were 1:3,314 for CHT and 1:237,504 for PKU. Newborn screening has been implemented as routine practice for all public health sectors of the country for CHT and PKU. It is expected that by the year 2003, all Thai newborns will be provided with screening services resulting in a better quality of life for the next generation.     

Allozyme analysis of the temporal populations of Echinostoma revolutum collected from domestic ducks in Khon Kaen Province, Thailand

Four temporal populations of Echinostoma revolutum (ER1, ER2, ER3, ER4) were collected from domestic ducks in Khon Kaen Province, Thailand during February-October 2008. The ER1, ER2, ER3 and ER4 were collected in February, April, June and October, respectively. The 12 enzymes encoding 15 loci were examined. Two loci were found in each of 3 enzymes, namely glucose-6-phosphate dehydrogenase (G6PD), malic enzyme (ME) and peptidase valine-leucine (PEPA). Of these, three loci, namely, G6pd-1, Me-1 and PepA-2, were polymorphic. Genotypes were assigned for the specific allelic profiles detected at these three polymorphic loci. Twenty-eight genotypes were observed in the 4 temporal populations of E. revolutum. Three genotypes, Er22, Er23 and Er25, were found in all populations. The Er6 genotype occurred had the highest frequency of all the populations. These 28 genotypes were clustered into 3 groups with genetic differences of 2-12% among the loci. A cluster of genotypes (Er1, Er3, Er9 and Er12) showed the greatest genetic difference among the genotypes (12% difference). These results show intraspecific variation exists in E. revolutum populations in domestic ducks from Khon Kaen Province, Thailand.     

A simple method for extraction and purification of genomic DNA from dried blood spots on filter paper

We have developed an efficient and simple method for extracting and purifying genomic DNA from dried blood stored on filter paper. The quality of the genomic DNA extracted is tested by PCR amplification of a 255-bp fragment of the PAX8 gene sequence and the PCR products are determined for further genetic studies by single strand conformation polymorphism (SSCP) analysis. Larger DNA sequences of the 674-bp of the PAX8 gene and the 1,039-bp of the human beta-globin gene, a housekeeping gene, have also been amplified from the extracted DNA, thus indicating the high quality of the genomic DNA extracted by the developed method for subsequent genetic studies of any gene of interest. The method developed can also be used for the purification of genomic DNA from dried blood specimens stored under different conditions. Moreover, the genomic DNA products can be stored for long-term use due to the highly purified procedure. Therefore, the method is efficient and appropriate for the extraction and purification of genomic DNA from dried blood specimens, which has become an increasingly important tool for genetic and epidemiological studies.     

Prognostic significance of microsatellite alterations at tp36 in cholangiocarcinoma

AIM: To investigate loss of heterozygosity (LOH) and microsatellite instability (MSI) on the chromosomal region 1p36-pter in cholangiocarcinoma (CCA) patients and determine the association between microsatellite alterations and clinicopathological parameters.METHODS: Ten polymorphic microsatellite markers were determined for LOH and MSI using GS-3000 gel scan fragment autoanalyzer.RESULTS: Sixty-eight out of 90 cases (75.6%) showed LOH in one or more loci. LOH was found most frequently at D1S199 (40.0%), D1S507 (34.6%), D1S2845 (30.5%),and D1S2734 (30.1%). MSI was found in 34 of 90 cases (37.8%) at one or more loci. Fine mapping at 1p36 showed two distinctive regions of common loss, which were D1S2845 and the 25.5-cM region between D1S507and D1S2734, indicating the existence of putative tumor suppressor genes that is likely to play important roles in the development of CCA. Patients with LOH at D1S234 showed less lymphatic invasion (P = 0.017),whereas patients with LOH at D1S2676 exhibited more lymphatic invasion than those without (P = 0.031).LOH at D1S2845 showed a significant correlation with nerve invasion (P = 0.029). Moreover, patients who demonstrated MSI at D1S228 showed a poor prognosis (P = 0.0026).CONCLUSION: Allelic loss plays a major role in microsatellite alterations at chromosome 1p36, which may contribute to carcinogenesis and pathogenesis of liver fluke related CCA and these alterations can be used as molecular prognostic indicators for CCA patients.     

Multilocus enzyme electrophoresis analysis of Echinostoma revolutum and Echinostoma malayanum (Trematoda: Echinostomatidae) isolated from Khon Kaen Province,Thailand

ObjectiveTo explore the genetic variation and differentiation of 2 echinostomes from genus Echinostoma, i.e. Echinostoma revolutum (E. revolutum) and Echinostoma malayanum (E. malayanum) from Khon Kaen Province, Thailand.MethodsThese parasites were compared at 22 enzymes encoding a presumptive 30 loci by using multilocus enzyme electrophoresis (MEE) technique.ResultsTwenty-two loci can be used as diagnostic markers to differentiate these 2 species. E. revolutum and E. malayanum had fixed genetic differences at 70% of loci, whereas both species had fixed genetic differences from the liver fluke, Opisthorchis viverrini at 91% of loci. Intraspecific variation within a population of E. revolutum was observed at 5 polymorphic loci.ConclusionsMEE is a powerful technique to investigate genetic variation and differentiation of E. revolutum and E. malayanum.