Optimization of a pentaplex panel for MSI analysis without control DNA in a Brazilian population: correlation with ancestry markers

Abstract

Microsatellite instability (MSI) testing has been advocated for all newly diagnosed colorectal cancer patients. One of the most common tests is composed by a pentaplex panel of mononucleotides markers , which allows the analysis of MSI in tumors without the need of reference DNA. For that, it is fundamental to establish a quasi-monomorphic variation range (QMVR) for each marker. Herein, we aimed to establish the QMVR in a Brazilian healthy population, to evaluate the feasibility of MSI determination of tumors, without the matching normal DNA. Furthermore, we intend to assess their ancestry using specific ancestry-informative markers (AIMs) and correlate with QMVR. The QMVR was assessed in 214 individuals, through a pentaplex PCR followed by fragment analysis. The ancestry analysis was done by 46 AIMs in a single multiplex PCR followed by capillary electrophoresis. Following QMVR establishment, we observed 23 individuals with alleles outside the QMVR. Importantly, none of them exhibited more than one marker outside the range. Therefore, individuals with instability at Z2 markers would be accurately classified as MSI. The European ancestry proportion was the most frequent (67.5%), followed by the African (19.6%). The comparison of the individuals with alleles within (n ¼ 191) and outside (n ¼ 23) the QMVR showed statistical difference in the proportions of European and African alleles, confirming the higher polymorphic nature of African ancestry. In conclusion, the present study reports an accurate methodology to assess MSI status without matched-normal DNA and independently of the ethnicity, even in the highly admixed population of Brazil.

KRAS and BRAF mutations and MSI status in precursor lesions of colorectal cancer detected by colonoscopy

“…The MSI evaluation was performed using a multiplex PCR comprising five quasimonomorphic mononucleotide repeat markers (NR27, NR21, NR24, BAT 25 and BAT26), as described by our group (20). The MSI status of the lesion was analyzed using GeneMapper 4.1 software (Applied Biosystems).…”

Absence of microsatellite instability andBRAF(V600E) mutation in testicular germ cell tumors

“…126 had a microsatellite stable (MSS) genotype, 7 had MSI-L and none had MSI-H. In a previous study (Campanella et al, 2014), our group reported that in order to accurately determine MSI-L, the MSI markers of tumor tissue should be compared with the germ-line tissue of the patient. Therefore, for the seven cases with MSI-L, we isolated adjacent normal tissue and identified the presence of an identical MSI marker profile, thereby indicating a MSS phenotype.…”

“…Herein, we used a validated methodology that comprised five quasimonomorphic mononucleotide repeat markers to identify MSI (NR-27, NR-21, NR-24, (Boland et al, 1998), when an international consensus updated the recommendation and recommended that dinucleotide repeats be substituted for mononucleotide repeats because of improved sensitivity (Buhard et al, 2004). We utilized this new recommended methodology for this study -a methodology that has been recently validated by our group in the Brazilian population (Campanella et al, 2014). Other potential causes of differing results may be associated with tumor aggressiveness.…”

“…In a recent study, our group determined the quasimonomorphic variation range (QMVR) of each marker for the Brazilian population (Campanella et al, 2014). Accordingly, samples were considered MSI-H when two or more markers were altered and MSI-low (MSI-L) when only one marker was altered, with further validation by MSI analysis of normal tissue or immunohistochemistry of the mismatch repair (MMR) enzymes.…”

“…Microsatellite instability evaluation was performed using multiplex PCR for five quasimonomorphic mononucleotide repeat markers (NR-27, NR-21, NR-24, BAT-25, and BAT-26) as previously described (Viana-Pereira et al, 2011;Campanella et al, 2014;Yamane et al, 2014). Briefly, each antisense primer was end-labeled with a fluorescent dye: FAM (6-carboxyfluorescein) for BAT-26 and NR-21, VIC (2 0 -chloro-7 0 -phenyl-1,4-dichloro-6-carboxyfluorescein) for and NED (2,7,4, for NR-24.…”