Parallel DNA/RNA NGS Using an Identical Target Enrichment Panel in the Analysis of Hereditary Cancer Predisposition.

Germline DNA testing using the next-gene-ration sequencing (NGS) technology has become the analytical standard for the diagnostics of hereditary diseases, including cancer. Its increasing use places high demands on correct sample identification, independent confirmation of prioritized variants, and their functional and clinical interpretation. To streamline these processes, we introduced parallel DNA and RNA capture-based NGS using identical capture panel CZECANCA, which is routinely used for DNA analysis of hereditary cancer predisposition. Here, we present the analytical workflow for RNA sample processing and its analytical and diagnostic performance. Parallel DNA/RNA analysis allowed credible sample identification by calculating the kinship coefficient. The RNA capture-based approach enriched transcriptional targets for the majority of clinically relevant cancer predisposition genes to a degree that allowed analysis of the effect of identified DNA variants on mRNA processing. By comparing the panel and whole-exome RNA enrichment, we demonstrated that the tissue-specific gene expression pattern is independent of the capture panel. Moreover, technical replicates confirmed high reproducibility of the tested RNA analysis. We concluded that parallel DNA/RNA NGS using the identical gene panel is a robust and cost-effective diagnostic strategy. In our setting, it allows routine analysis of 48 DNA/RNA pairs using NextSeq 500/550 Mid Output Kit v2.5 (150 cycles) in a single run with sufficient coverage to analyse 226 cancer predisposition and candidate ge-nes. This approach can replace laborious Sanger confirmatory sequencing, increase testing turnaround, reduce analysis costs, and improve interpretation of the impact of variants by analysing their effect on mRNA processing.

Germline pathogenic variants in the MRE11, RAD50, and NBN (MRN) genes in cancer predisposition: A systematic review and meta-analysis.

The MRE11, RAD50, and NBN genes encode the MRN complex sensing DNA breaks and directing their repair. While carriers of biallelic germline pathogenic variants (gPV) develop rare chromosomal instability syndromes, the cancer risk in heterozygotes remains controversial. We performed a systematic review and meta-analysis of 53 studies in patients with different cancer diagnoses to better understand the cancer risk. We found an increased risk (odds ratio, 95% confidence interval) for gPV carriers in NBN for melanoma (7.14; 3.30-15.43), pancreatic cancer (4.03; 2.14-7.58), hematological tumors (3.42; 1.14-10.22), and prostate cancer (2.44, 1.84-3.24), but a low risk for breast cancer (1.29; 1.00-1.66) and an insignificant risk for ovarian cancer (1.53; 0.76-3.09). We found no increased breast cancer risk in carriers of gPV in RAD50 (0.93; 0.74-1.16; except of c.687del carriers) and MRE11 (0.87; 0.66-1.13). The secondary burden analysis compared the frequencies of gPV in MRN genes in patients from 150 studies with those in the gnomAD database. In NBN gPV carriers, this analysis additionally showed a high risk for brain tumors (5.06; 2.39-9.52), a low risk for colorectal (1.64; 1.26-2.10) and hepatobiliary (2.16; 1.02-4.06) cancers, and no risk for endometrial, and gastric cancer. The secondary burden analysis showed also a moderate risk for ovarian cancer (3.00; 1.27-6.08) in MRE11 gPV carriers, and no risk for ovarian and hepatobiliary cancers in RAD50 gPV carriers. These findings provide a robust clinical evidence of cancer risks to guide personalized clinical management in heterozygous carriers of gPV in the MRE11, RAD50, and NBN genes.

Population-specific validation and comparison of the performance of 77- and 313-variant polygenic risk scores for breast cancer risk prediction.

BACKGROUND: The polygenic risk score (PRS) allows the quantification of the polygenic effect of many low-penetrance alleles on the risk of breast cancer (BC). This study aimed to evaluate the performance of two sets comprising 77 or 313 low-penetrance loci (PRS77 and PRS313) in patients with BC in the Czech population. METHODS: In a retrospective case-control study, variants were genotyped from both the PRS77 and PRS313 sets in 1329 patients with BC and 1324 noncancer controls, all women without germline pathogenic variants in BC predisposition genes. Odds ratios (ORs) were calculated according to the categorical PRS in individual deciles. Weighted Cox regression analysis was used to estimate the hazard ratio (HR) per standard deviation (SD) increase in PRS. RESULTS: The distributions of standardized PRSs in patients and controls were significantly different (p < 2.2 × 10-16) with both sets. PRS313 outperformed PRS77 in categorical and continuous PRS analyses. For patients in the highest 2.5% of PRS313, the risk reached an OR of 3.05 (95% CI, 1.66-5.89; p = 1.76 × 10-4). The continuous risk was estimated as an HRper SD of 1.64 (95% CI, 1.49-1.81; p < 2.0 × 10-16), which resulted in an absolute risk of 21.03% at age 80 years for individuals in the 95th percentile of PRS313. Discordant categorization into PRS deciles was observed in 248 individuals (9.3%). CONCLUSIONS: Both PRS77 and PRS313 are able to stratify individuals according to their BC risk in the Czech population. PRS313 shows better discriminatory ability. The results support the potential clinical utility of using PRS313 in individualized BC risk prediction.

A deep intronic recurrent CHEK2 variant c.1009-118_1009-87delinsC affects pre-mRNA splicing and contributes to hereditary breast cancer predisposition.

Germline CHEK2 pathogenic variants confer an increased risk of female breast cancer (FBC). Here we describe a recurrent germline intronic variant c.1009-118_1009-87delinsC, which showed a splice acceptor shift in RNA analysis, introducing a premature stop codon (p.Tyr337PhefsTer37). The variant was found in 21/10,204 (0.21%) Czech FBC patients compared to 1/3250 (0.03%) controls (p = 0.04) and in 4/3639 (0.11%) FBC patients from an independent German dataset. In addition, we found this variant in 5/2966 (0.17%) Czech (but none of the 443 German) ovarian cancer patients, three of whom developed early-onset tumors. Based on these observations, we classified this variant as likely pathogenic.

Dysregulation of hypoxia-inducible factor 1α in the sympathetic nervous system accelerates diabetic cardiomyopathy.

BACKGROUND: An altered sympathetic nervous system is implicated in many cardiac pathologies, ranging from sudden infant death syndrome to common diseases of adulthood such as hypertension, myocardial ischemia, cardiac arrhythmias, myocardial infarction, and heart failure. Although the mechanisms responsible for disruption of this well-organized system are the subject of intensive investigations, the exact processes controlling the cardiac sympathetic nervous system are still not fully understood. A conditional knockout of the Hif1a gene was reported to affect the development of sympathetic ganglia and sympathetic innervation of the heart. This study characterized how the combination of HIF-1α deficiency and streptozotocin (STZ)-induced diabetes affects the cardiac sympathetic nervous system and heart function of adult animals. METHODS: Molecular characteristics of Hif1a deficient sympathetic neurons were identified by RNA sequencing. Diabetes was induced in Hif1a knockout and control mice by low doses of STZ treatment. Heart function was assessed by echocardiography. Mechanisms involved in adverse structural remodeling of the myocardium, i.e. advanced glycation end products, fibrosis, cell death, and inflammation, was assessed by immunohistological analyses. RESULTS: We demonstrated that the deletion of Hif1a alters the transcriptome of sympathetic neurons, and that diabetic mice with the Hif1a-deficient sympathetic system have significant systolic dysfunction, worsened cardiac sympathetic innervation, and structural remodeling of the myocardium. CONCLUSIONS: We provide evidence that the combination of diabetes and the Hif1a deficient sympathetic nervous system results in compromised cardiac performance and accelerated adverse myocardial remodeling, associated with the progression of diabetic cardiomyopathy.

Biogenic amines formation in high-pressure processed pike flesh (Esox lucius) during storage.

The effects of vacuum packaging followed by high pressure processing on the shelf-life of fillets of pike (Esox lucius) were examined. Samples were pressure-treated at 300 and 500 MPa and stored at 3.5 and 12 °C for up to 70 days. The content of eight biogenic amines (putrescine, cadaverine, spermidine, spermine, histamine, tyramine, tryptamine and phenylethylamine) were determined. Putrescine showed very good correspondence with the level of applied pressure and organoleptic properties. Polyamines spermidine and spermine did not show statistically significant changes with the level of applied pressure and the time of storage. Increased cadaverine and tyramine contents were found in samples with good sensory signs, stored for longer time and/or kept at 12 °C, thus indicating the loss of freshness. Tryptamine and phenylethylamine were not detected in pressure-treated samples kept at 3.5 °C. Histamine was not detected in samples of good quality.

Effect of high-pressure treatment on biogenic amines formation in vacuum-packed trout flesh (Oncorhynchus mykiss).

The effects of vacuum packaging followed by high pressure processing on the shelf-life of fillets of rainbow trout (Oncorhynchus mykiss) were examined. Samples were pressure-treated at 300 and 500 MPa and were stored at 3.5 and 12 °C for up to 28 days (control--0 MPa) and 42 or 70 days (pressure-treated; 12 and 3.5 °C resp.). The content of eight biogenic amines (putrescine, cadaverine, spermidine, spermine, histamine, tyramine, tryptamine and phenylethylamine) were determined. Putrescine, cadaverine and tyramine showed very good correspondence with the level of applied pressure and organoleptic properties. Samples of very good quality contained less than 10 mg/kg of each of these amines. Polyamines spermidine and spermine did not show statistically significant changes with the level of applied pressure and the time of storage. Tryptamine, phenylethylamine and histamine (with the single exception of a sample stored for 70 days) were not detected in pressure-treated samples kept at 3.5 °C.

Effect of low-dose irradiation on biogenic amines formation in vacuum-packed trout flesh (Oncorhynchus mykiss).

The effects of vacuum packaging followed by high-energy electron beam irradiation on the shelf-life of fillets of rainbow trout (Oncorhynchus mykiss) were examined. Samples were irradiated at doses of 0.25, 0.50, 0.75, 1.0 and 2.0kGy. The control and treated packs were stored at 3.5°C for up to 28, 42, 70 and 98days (control - 0, 0.25, 0.50 and >0.50kGy respectively). The content of seven biogenic amines (putrescine, cadaverine, spermidine, spermine, histamine, tyramine and tryptamine) were determined. Putrescine, cadaverine and tyramine showed very good correspondence with the irradiation dose and organoleptic properties. Samples of good quality contained less than 10mg/kg of each of these amines. The polyamines spermidine and spermine did not show statistically significant changes with the irradiation dose and the time of storage. With few exceptions, histamine was not detected in the samples treated with radiation. Tryptamine was not detected in any of the samples.