Evaluation of pathogenic variants detected in high homology regions of the PMS2 gene. How effective is long-range PCR?

Introduction: Lynch syndrome (LS) is an inherited cancer predisposition syndrome characterized by a high risk of colorectal and extracolonic tumors. Germline pathogenic variants (GPV) in the PMS2 gene are associated with <15% of all cases. The PMS2CL pseudogene presents high homology with PMS2, challenging molecular diagnosis by next-generation sequencing (NGS). Due to the high methodological complexity required to distinguish variants between PMS2 and PMS2CL, most laboratories do not clearly report the origin of this molecular finding. Objective: The aim of this study was to confirm the GPVs detected by NGS in regions of high homology segments of the PMS2 gene in a Brazilian sample. Methods: An orthogonal and gold standard long-range PCR (LR-PCR) methodology to separate variants detected in the PMS2 gene from those detected in the pseudogene. Results: A total of 74 samples with a PMS2 GPV detected by NGS in exons with high homology with PMS2CL pseudogene were evaluated. The most common was NM_000535.6:c.2182_2184delinsG, which was previously described as deleterious mutation in a study of African-American patients with LS and has been widely reported by laboratories as a pathogenic variant associated with the LS phenotype. Of all GPVs identified, only 6.8% were confirmed by LR-PCR. Conversely, more than 90% of GPV were not confirmed after LR-PCR, and the diagnosis of LS was ruled out by molecular mechanisms associated with PMS2. Conclusion: In conclusion, the use of LR-PCR was demonstrated to be a reliable approach for accurate molecular analysis of PMS2 variants in segments with high homology with PMS2CL. We highlight that our laboratory is a pioneer in routine diagnostic complementation of the PMS2 gene in Brazil, directly contributing to a more assertive molecular diagnosis and adequate genetic counseling for these patients and their families.

Prevalence and clinical implications of germline pathogenic variants in cancer predisposing genes in young patients across sarcoma subtypes.

BACKGROUND: Sarcomas are a rare and diverse group of cancers occurring mainly in young individuals for which an underlying germline genetic cause remains unclear in most cases. METHODS: Germline DNA from 177 children, adolescents and young adults with soft tissue or bone sarcomas was tested using multigene panels with 113 or 126 cancer predisposing genes (CPGs) to describe the prevalence of germline pathogenic/likely pathogenic variants (GPVs). Subsequent testing of a subset of tumours for loss of heterozygosity (LOH) evaluation was performed to investigate the clinical and molecular significance of these variants. RESULTS: GPVs were detected in 21.5% (38/177) of the patients (15.8% in children and 21.6% in adolescents and young adults), with dominant CPGs being altered in 15.2% overall. These variants were found in genes previously associated with the risk of developing sarcomas (TP53, RB1, NF1, EXT1/2) but also in genes where that risk is still emerging/limited (ERCC2, TSC2 and BRCA2) or unknown (PALB2, RAD50, FANCM and others). The detection rates of GPVs varied from 0% to 33% across sarcoma subtypes and GPV carriers were more likely to present more than one primary tumour than non-carriers (21.1%×6.5%; p=0.012). Loss of the wild-type allele was detected in 48% of tumours from GPV carriers, mostly in genes definitively associated with sarcoma risk. CONCLUSION: Our findings reveal that a high proportion of young patients with sarcomas presented a GPV in a CPG, underscoring the urgency of establishing appropriate genetic screening strategies for these individuals and their families.

Characterization of genetic predisposition to molecular subtypes of breast cancer in Brazilian patients.

Introduction: BRCA1 and BRCA2 germline pathogenic variants (GPVs) account for most of the 5-10% of breast cancer (BC) that is attributable to inherited genetic variants. BRCA1 GPVs are associated with the triple negative subtype, whereas BRCA2 GPVs are likely to result in higher grade, estrogen-receptor positive BCs. The contribution of other genes of high and moderate risk for BC has not been well defined and risk estimates to specific BC subtypes is lacking, especially for an admixed population like Brazilian. Objective: The aim of this study is to evaluate the value of a multigene panel in detecting germline mutations in cancer-predisposing genes for Brazilian BC patients and its relation with molecular subtypes and the predominant molecular ancestry. Patients and methods: A total of 321 unrelated BC patients who fulfilled NCCN criteria for BRCA1/2 testing between 2016-2018 were investigated with a 94-genes panel. Molecular subtypes were retrieved from medical records and ancestry-specific variants were obtained from off-target reads obtained from the sequencing data. Results: We detected 83 GPVs in 81 patients (positivity rate of 25.2%). Among GPVs, 47% (39/83) were identified in high-risk BC genes (BRCA1/2, PALB2 and TP53) and 18% (15/83) in moderate-penetrance genes (ATM, CHEK2 and RAD51C). The remainder of the GPVs (35% - 29/83), were identified in lower-risk genes. As for the molecular subtypes, triple negative BC had a mutation frequency of 31.6% (25/79), with predominance in BRCA1 (12.6%; 10/79). Among the luminal subtypes, except Luminal B HER2-positive, 18.7% (29/155) had GPV with BRCA1/2 genes contributing 7.1% (11/155) and non-BRCA1/2 genes, 12.9% (20/155). For Luminal B HER2-positive subtype, 40% (16/40) had GPVs, with a predominance of ATM gene (15% - 6/40) and BRCA2 with only 2.5% (1/40). Finally, HER2-enriched subtype presented a mutation rate of 30.8% (4/13) with contribution of BRCA2 of 7.5% (1/13) and non-BRCA1/2 of 23% (3/13). Variants of uncertain significance (VUS) were identified in 77.6% (249/321) of the patients and the number of VUS was increased in patients with Asian and Native American ancestry. Conclusion: The multigene panel contributed to identify GPVs in genes other than BRCA1/2, increasing the positivity of the genetic test from 9.6% (BRCA1/2) to 25.2% and, considering only the most clinically relevant BC predisposing genes, to 16.2%. These results indicate that women with clinical criteria for hereditary BC may benefit from a multigene panel testing, as it allows identifying GPVs in genes that directly impact the clinical management of these patients and family members.

Exome sequencing and targeted gene panels: a simulated comparison of diagnostic yield using data from 158 patients with rare diseases.

Next-generation sequencing (NGS) has altered clinical genetic testing by widening the access to molecular diagnosis of genetically determined rare diseases. However, physicians may face difficulties selecting the best diagnostic approach. Our goal is to estimate the rate of possible molecular diagnoses missed by different targeted gene panels using data from a cohort of patients with rare genetic diseases diagnosed with exome sequencing (ES). For this purpose, we simulated a comparison between different targeted gene panels and ES: the list of genes harboring clinically relevant variants from 158 patients was used to estimate the theoretical rate of diagnoses missed by NGS panels from 53 different NGS panels from eight different laboratories. Panels presented a mean rate of missed diagnoses of 64% (range 14%-100%) compared to ES, representing an average predicted sensitivity of 36%. Metabolic abnormalities represented the group with highest mean of missed diagnoses (86%), while seizure represented the group with lowest mean (46%). Focused gene panels are restricted in covering select sets of genes implicated in specific diseases and they may miss molecular diagnoses of rare diseases compared to ES. However, their role in genetic diagnosis remains important especially for well-known genetic diseases with established genetic locus heterogeneity.

Frequency of carriers for rare recessive Mendelian diseases in a Brazilian cohort of 320 patients.

Several Mendelian disorders follow an autosomal recessive inheritance pattern. Epidemiological information on many inherited disorders may be useful to guide health policies for rare diseases, but it is often inadequate, particularly in developing countries. We aimed to calculate the carrier frequencies of rare autosomal recessive Mendelian diseases in a cohort of Brazilian patients using whole exome sequencing (WES). We reviewed the molecular findings of WES from 320 symptomatic patients who had carrier status for recessive diseases. Using the Hardy-Weinberg equation, we estimated recessive disease frequencies (q2 ) considering the respective carrier frequencies (2pq) observed in our study. We calculated the sensitivity of carrier screening tests based on lists of genes from five different clinical laboratories that offer them in Brazil. A total of 425 occurrences of 351 rare variants were reported in 278 different genes from 230 patients (71.9%). Almost half (48.8%) were carriers of at least one heterozygous pathogenic/likely pathogenic variant for rare metabolic disorders, while 25.9% of epilepsy, 18.1% of intellectual disabilities, 15.6% of skeletal disorders, 10.9% immune disorders, and 9.1% of hearing loss. We estimated that an average of 67% of the variants would not have been detected by carrier screening panels. The combined frequencies of autosomal recessive diseases were estimated to be 26.39/10,000 (or ~0.26%). This study shows the potential research utility of WES to determine carrier status, which may be a possible strategy to evaluate the clinical and social burden of recessive diseases at the population level and guide the optimization of carrier screening panels.

Diagnostic power and clinical impact of exome sequencing in a cohort of 500 patients with rare diseases.

Rare diseases comprise a diverse group of conditions, most of which involve genetic causes. We describe the variable spectrum of findings and clinical impacts of exome sequencing (ES) in a cohort of 500 patients with rare diseases. In total, 164 primary findings were reported in 158 patients, representing an overall diagnostic yield of 31.6%. Most of the findings (61.6%) corresponded to autosomal dominant conditions, followed by autosomal recessive (25.6%) and X-linked (12.8%) conditions. These patients harbored 195 variants, among which 43.6% are novel in the literature. The rate of molecular diagnosis was considerably higher for prenatal samples (67%; 4/6), younger children (44%; 24/55), consanguinity (50%; 3/6), gastrointestinal/liver disease (44%; 16/36) and syndromic/malformative conditions (41%; 72/175). For 15.6% of the cohort patients, we observed a direct potential for the redirection of care with targeted therapy, tumor screening, medication adjustment and monitoring for disease-specific complications. Secondary findings were reported in 37 patients (7.4%). Based on cost-effectiveness studies in the literature, we speculate that the reports of secondary findings may influence an increase of 123.2 years in the life expectancy for our cohort, or 0.246 years/cohort patient. ES is a powerful method to identify the molecular bases of monogenic disorders and redirect clinical care.

Prevalence of BRCA1 and BRCA2 pathogenic and likely pathogenic variants in non-selected ovarian carcinoma patients in Brazil.

BACKGROUND: BRCA1/2 pathogenic (P) and likely pathogenic (LP) germline variants are frequent among patients with ovarian carcinoma. However, these variants have not been extensively characterized in patients with ovarian cancer in Brazil. METHODS: In this retrospective study we evaluated clinical characteristics and BRCA1/2 genetic test results from patients with ovarian carcinoma who underwent genetic counseling at A.C.Camargo Cancer Center (Brazil) between 2015 and 2017 and had performed germline genetic testing of BRCA1/2 genes. RESULTS: Among 158 patients, 33 P and LP variants and were found (20.8%), 27 in BRCA1 and six in BRCA2, and six variants of unknown clinical significance (VUS). Thirteen percent of the patients did not have Multiplex Ligation-dependent Probe Amplification (MLPA) results. Three P variants in BRCA1 were found in more than one patient: c.5266dupC (p.Gln1756Profs*74), c.3331_3334delCAAG (p.Gln1111Asnfs5*), and c.211A > G (p.Arg71Gly). One LP variant in BRCA1 had not been previously described, c.4153_4154delCT (p.Leu1385Ilefs*5). Patients with previous diagnosis of breast cancer were carriers of P or LP variant in 8 of 12 cases (66.7%), and patients with a family history of ovarian or breast cancer in first- or second-degree relatives were carriers of P or LP variant in 26.7% of cases compared to 16.9% for patients without family history (p = 0.166). CONCLUSION: Prevalence of BRCA1/2 germline P and LP variants is slightly higher than previously described by the largest occidental studies, with a high prevalence of variant c.5266dupC (p.Gln1756Profs*74) in BRCA1 observed. Moreover, we identified a new LP variant.

Whole-body magnetic resonance imaging of Li-Fraumeni syndrome patients: observations from a two rounds screening of Brazilian patients.

BACKGROUND: Li-Fraumeni syndrome (LFS) is an autosomal dominant disease that is associated with germline TP53 mutations and it predisposes affected individuals to a high risk of developing multiple tumors. In Brazil, LFS is characterized by a different pattern of TP53 variants, with the founder TP53 p.R337H mutation being predominant. The adoption of screening strategies to diagnose LFS in its early stages is a major challenge due to the diverse spectrum of tumors that LFS patients can develop. The purpose of this study was to evaluate two rounds of whole-body magnetic resonance imaging (WB-MRI) which were conducted as a screening strategy for LFS patients. METHODS: Over a 4-year period, 59 LFS patients underwent two rounds of WB-MRI. Each MRI was characterized as positive or negative, and positive cases were further investigated to establish a diagnosis. The parameters used to evaluate the WB-MRI results included: positive rate, number of invasive investigations of positive results, and cancer detection rate. RESULTS: A total of 118 WB-MRI scans were performed. Positive results were associated with 11 patients (9.3%). Seven of these patients (11.8%) were identified in the first round of screening and 4 patients (6.7%) were identified in the second round of screening. Biopsies were performed in three cases (2.5%), two (3.4%) after the first round of screening and one (1.7%) after the second round of screening. The histopathological results confirmed a diagnosis of cancer for all three cases. There was no indication of unnecessary invasive procedures. CONCLUSIONS: WB-MRI screening of LFS carriers diagnosed cancers in their early stages. When needed, positive results were further examined with non-invasive imaging techniques. False positive results were less frequent after the first round of WB-MRI screening.