Detection of NTRK fusions by RNA-based nCounter is a feasible diagnostic methodology in a real-world scenario for non-small cell lung cancer assessment.

NTRK1, 2, and 3 fusions are important therapeutic targets for NSCLC patients, but their prevalence in South American admixed populations needs to be better explored. NTRK fusion detection in small biopsies is a challenge, and distinct methodologies are used, such as RNA-based next-generation sequencing (NGS), immunohistochemistry, and RNA-based nCounter. This study aimed to evaluate the frequency and concordance of positive samples for NTRK fusions using a custom nCounter assay in a real-world scenario of a single institution in Brazil. Out of 147 NSCLC patients, 12 (8.2%) cases depicted pan-NTRK positivity by IHC. Due to the absence of biological material, RNA-based NGS and/or nCounter could be performed in six of the 12 IHC-positive cases (50%). We found one case exhibiting an NTRK1 fusion and another an NTRK3 gene fusion by both RNA-based NGS and nCounter techniques. Both NTRK fusions were detected in patients diagnosed with lung adenocarcinoma, with no history of tobacco consumption. Moreover, no concomitant EGFR, KRAS, and ALK gene alterations were detected in NTRK-positive patients. The concordance rate between IHC and RNA-based NGS was 33.4%, and between immunohistochemistry and nCounter was 40%. Our findings indicate that NTRK fusions in Brazilian NSCLC patients are relatively rare (1.3%), and RNA-based nCounter methodology is a suitable approach for NRTK fusion identification in small biopsies.

Insights on variant analysis in silico tools for pathogenicity prediction.

Molecular biology is currently a fast-advancing science. Sequencing techniques are getting cheaper, but the interpretation of genetic variants requires expertise and computational power, therefore is still a challenge. Next-generation sequencing releases thousands of variants and to classify them, researchers propose protocols with several parameters. Here we present a review of several in silico pathogenicity prediction tools involved in the variant prioritization/classification process used by some international protocols for variant analysis and studies evaluating their efficiency.

Whole-Exome Sequencing Identifies Pathogenic Germline Variants in Patients with Lynch-Like Syndrome.

Lynch syndrome (LS) is the most common hereditary colorectal cancer (CRC) syndrome, characterized by germline pathogenic variants in mismatch repair (MMR)-related genes that lead to microsatellite instability. Patients who meet the clinical criteria for LS and MMR deficiency and without any identified germline pathogenic variants are frequently considered to have Lynch-like syndrome (LLS). These patients have a higher risk of CRC and extracolonic tumors, and little is known about their underlying genetic causes. We investigated the germline spectrum of LLS patients through whole-exome sequencing (WES). A total of 20 unrelated patients with MMR deficiency who met the clinical criteria for LS and had no germline variant were subjected to germline WES. Variant classification was performed according to the American College of Medical Genetics and Genomics (ACMG) criteria. Pathogenic/likely pathogenic variants were identified in 35% of patients in known cancer genes such as MUTYH and ATM. Besides this, rare and potentially pathogenic variants were identified in the DNA repair gene POLN and other cancer-related genes such as PPARG, CTC1, DCC and ALPK1. Our study demonstrates the germline mutational status of LLS patients, a population at high risk of colorectal cancer.

New insights on familial colorectal cancer type X syndrome.

Familial colorectal cancer type X (FCCTX) is a heterogeneous colorectal cancer predisposition syndrome that, although displays a cancer pattern similar to Lynch syndrome, is mismatch repair proficient and does not exhibit microsatellite instability. Besides, its genetic etiology remains to be elucidated. In this study we performed germline exome sequencing of 39 cancer-affected patients from 34 families at risk for FCCTX. Variant classification followed the American College of Medical Genetics and Genomics (ACMG) guidelines. Pathogenic/likely pathogenic variants were identified in 17.65% of the families. Rare and potentially pathogenic alterations were identified in known hereditary cancer genes (CHEK2), in putative FCCTX candidate genes (OGG1 and FAN1) and in other cancer-related genes such as ATR, ASXL1, PARK2, SLX4 and TREX1. This study provides novel important clues that can contribute to the understanding of FCCTX genetic basis.

Using Co-segregation and Loss of Heterozygosity Analysis to Define the Pathogenicity of Unclassified Variants in Hereditary Breast Cancer Patients.

The use of gene panels introduces a new dilemma in the genetics field due to the high frequency of variants of uncertain significance (VUS). The objective of this study was to provide evidence that may help in the classification of these germline variants in terms of their clinical impact and association with the disease in question. A total of 52 unrelated women at-risk for HBOC and negative for BRCA1/BRCA2 pathogenic variants were evaluated through a gene panel comprising 14 breast and/or ovarian cancer susceptibility genes. Of the 453 germline variants identified, 15 variants (classes 3, 4, and 5) in the ATM, BRIP1, CHEK2, MRE11A, MUTHY, PALB2, RAD50, and RAD51C genes were evaluated via databases, co-segregation studies and loss of heterozygosity in the tumor. The co-segregation analysis allowed the establishment of an association with the presence of variants and the risk of cancer for variant c.316C>T in the BRIP1 gene. Four variants of uncertain significance showed loss of heterozygosity in the tumor (ATM c.4709T>C; CHEK2 c.1036C>T; PALB2 c.1001A>G, and RAD50 c.281T>C), which is an indication of pathogenicity. Thus, the present study provides novel evidence that favors the association of variants in moderate-risk genes with the development of hereditary breast cancer.

Haplotypes from the SLC45A2 gene are associated with the presence of freckles and eye, hair and skin pigmentation in Brazil.

The Solute Carrier Family 45, Member 2 (SLC45A2) gene encodes the Membrane-Associated Transporter Protein (MATP), which mediates melanin synthesis by tyrosinase trafficking and proton transportation to melanosomes. At least two SLC45A2 coding SNPs [E272K (rs26722) and L374F (rs16891982)] were reported influencing normal variation of human pigmentation. Here we aimed at evaluating the influence of haplotypes of 12 SNPs within SLC45A2 in the determination of eye, hair and skin pigmentation in a highly admixed population sample and comparing their frequencies with the ones found in data retrieved from the 1000 Genomes Project. To achieve this goal, 12 SLC45A2 SNPs were evaluated in 288 unrelated individuals from the Ribeirão Preto city area, Southeastern Brazil. SNPs were genotyped by PCR-RFLP or Allele-specific PCR, followed by polyacrylamide gel electrophoresis. Haplotypes of each individual were inferred by two independent computational methods, PHASE and Partition-Ligation-Expectation-Maximization (PL-EM) algorithms, and 34 different haplotypes were identified. The hp9 haplotype was the most frequent (58.3%) and was associated with the presence of blond/red hair, pale skin, blue eyes and freckles. All haplotypes significantly associated with dark or light pigmentation features harbor the 374L and 374F alleles, respectively. These results emphasize the role played by haplotypes at SLC45A2 in the determination of pigmentation aspects of human populations and reinforce the relevance of SNP L374F in human pigmentation.

Mutation rates at 14 STR loci in the population from Pernambuco, Northeast Brazil.

Definition about mutation rates of short tandem repeats (STRs) loci used in forensic analysis are useful for the correct interpretation of resulting genetic profiles and the definition of criterions for exclusion in paternity testing. Germline mutation of 14 STR loci was studied for 54,105 parent-child allelic transfers from 2575 paternity testing cases carried out during 2000-2007 from the Pernambuco State, Northeast Brazil. The parenthood in each of these cases was highly validated (probability > 99.99%). We identified 43 mutations at 12 loci. Locus-specific mutation rate estimates varied between 2 x 10(-4) and 2 x 10(-3), and the overall mutation rate estimate was 8 x 10(-4). Mutation events in the male germline were more frequent than in the female germline. The majority of the mutations could be explained by losses or gains of one repeat unit and there was no evidence for selection between insertion or deletion changes. Our data were compared with those of Portuguese and North-American populations for CSF1PO, D18S51, D21S11, D7S820, TH01, TPOX and demonstrated, despite the great difference in the size of the sample, that mutation rates of STR loci in a mixed population do not differ from that encountered in different populations.