Colorectal cancer (CRC) is one of the most common cancer worldwide with approximately 2 to 5% of all colon cancers are associated with well-defined hereditary factors. Hereditary nonpolyposis colorectal cancer (Hereditary Nonpolyposis Colorectal Cancer), also known as Lynch syndrome (LS), is the most common form of hereditary CRC characterized by an early age of onset and follows the autosomal dominant inheritance pattern. HNPCC is caused by the alteration in four mismatch repair (MMR) genes. Immunohistochemistry (IHC) and microsatellite instability (MSI) testing, followed by conventional Sanger sequencing reliably identify the majority of mutations. However, methods to identify other underlying variants or genomic rearrangements of HNPCC have emerged. In addition to the clinical characterization and evaluation of HNPCC patients, the implementation of screening strategies for both affected and unaffected CRC patients together with the accelerated advancement in molecular testing methods will shed light on a more comprehensive detection of HNPCC. In this review, the approaches for the selection of high-risk HNPCC and molecular testing performed over the past few years are discussed.
The present study aimed to determine the best polymerase chain reaction (PCR) conditions for
amplification of odontoblast markers; alkaline phosphatase (ALP), dentin matrix protein 1 (DMP1), dentin
sialophosphoprotein (DSPP) and osteopontin (OPN). Informed consent was obtained from the individuals
prior to tooth extraction. RNA was extracted from odontoblasts obtained from extracted teeth using
innuPREP RNA Mini kit (Analytik Jena, Germany). Five selected target factors in enhancing PCR: primer
concentration, extension time, number of cycles, annealing time, and annealing temperature were
manipulated to yield the correct size of amplicons. One step reverse transcriptase PCR reactions were
performed using MyTaq One-Step RT-PCR kit (Bioline, USA) with a C1000 Thermal Cycler (Bio-Rad, USA)
in a 25 µL reaction, keeping the amount of 2 ng/µL RNA, 0.25 µL reverse transcriptase, 0.5 µL RiboSafe
Rnase inhibitor and 1X MyTaq One-Step Mix, constant. The optimal conditions were determined to be
400nM of primers for DMP1 and DSPP, 200 nM for ALP and OPN; 30 seconds of extension time and 35
PCR cycles for all genes; 10 seconds of annealing time for ALP, DMP1 and DSPP, 7 seconds for OPN. The
annealing temperature were 56.4°C for ALP, 58.6°C for DMP1, 52.7°C for DSPP, and 56.3°C for OPN,
respectively. The optimized PCR protocols produced the correct size of odontoblast markers.
Wan Khairunnisa Wan Juhari, Khairul Bariah Ahmad Amin Noordin, Wan Faiziah Wan Abdul Rahman, Andee Dzulkarnaen Zakaria, Ahmad Shanwani Mohd Sidek, Muhammad Radzi Abu Hassan, et al.
Background: Hereditary nonpolyposis colorectal cancer (HNPCC) also known as Lynch syndrome is commonly caused by genetic alterations in any of the four mismatch repair (MMR) genes; MLH1, MSH2, MSH6 and PMS2. This is the first study aimed to investigate genetic variants in Malay HNPCC families. Methods: Six Malay HNPCC families who fulfilled any of the Bethesda criteria were recruited into this study. A total of 3 ml of blood was withdrawn from each patient in the families. The samples were further analyzed using polymerase chain reaction and direct sequencing of the selected exons of MLH1 and MSH2 genes. Results: Two missense mutations and four single nucleotide polymorphisms (SNPs) were identified in six patients. These variants in the MLH1 and MSH2 genes were identified in four families who met the revised Bethesda guidelines. In two families, no mutation and polymorphism was identified in both the exon and intron of the respective genes. Of the mutations and polymorphisms identified, five have never been reported in Malay HNPCC families before. A missense mutation was detected in exon 5 of the MLH1 gene, c.394G>C (p.Asp132His) and four mutations and polymorphisms were detected in the MSH2 gene; heterozygous c.211+98T>C and c.211+9C>G and homozygous c.211+98T>C and c.211+9C>G, c.367-86A>C and c.382C>G. Conclusion: The results represented a new spectrum of mutations and polymorphisms in the Malay HNPCC families. However, a larger study involving additional families and analysis is required to determine the impact and nature of the identified mutations and polymorphisms.