PALB2 Variants Extend the Mutational Profile of Hungarian Patients with Breast and Ovarian Cancer.

BACKGROUND: The pathogenic/likely pathogenic (P/LP) variant detection rate and profile of PALB2, the third most important breast cancer gene, may vary between different populations. METHODS: PALB2 was analyzed in peripheral blood samples of three independent cohorts: prospectively between September 2021 and March 2023 (i) in 1280 consecutive patients with breast and/or ovarian cancer (HBOC), (ii) in 568 patients with other cancers (controls), and retrospectively, (iii) in 191 young breast cancer (<33 years, yBC) patients. These data were compared with data of 134,187 non-cancer individuals retrieved from the Genome Aggregation Database. RESULTS: Altogether, 235 cases (235/1280; 18.3%) carried at least one P/LP variant in one of the HBOC susceptibility genes. P/LP PALB2 variants were identified in 18 patients (1.4%; 18/1280) in the HBOC and 3 cases (1.5%; 3/191) in the yBC group. In the control group, only one patient had a disease-causing PALB2 variant (0.17%; 1/568) as a secondary finding not related to the disease, which was similar (0.15%; 205/134,187) in the non-cancer control group. The NM_024675.4:c.509_510delGA variant was the most common among our patients (33%; 6/18). We did not find a significant difference in the incidence of PALB2 disease-causing variants according to age; however, the median age of tumor onset was lower in PALB2 P/LP carriers versus wild-type patients (44 vs. 48 years). In our cohort, the odds ratio for breast cancer risk in women with PALB2 P/LP variants was between 8.1 and 9.3 compared to non-HBOC cancer patients and the non-cancer population, respectively. CONCLUSIONS: PALB2 P/LP variants are not uncommon among breast and/or ovarian cancer patients. Their incidence was the same in the two breast cancer cohorts studied but may occur rarely in patients with non-breast/ovarian cancer. The c.509_510delGA variant is particularly common in the studied Hungarian patient population.

Whole genome sequencing resolves 10 years diagnostic odyssey in familiar myxoma.

Carney complex (CNC) is an ultrarare disorder causing cutaneous and cardiac myxomas, primary pigmented nodular adrenocortical disease, hypophyseal adenoma, and gonadal tumours. Genetic alterations are often missed under routine genetic testing. Pathogenic variants in PRKAR1A are identified in most cases, while large exonic or chromosomal deletions have only been reported in a few cases. Our aim was to identify the causal genetic alteration in our kindred with a clinical diagnosis of CNC and prove its pathogenic role by functional investigation. Targeted testing of PRKAR1A gene, whole exome and whole genome sequencing (WGS) were performed in the proband, one clinically affected and one unaffected relative. WGS identified a novel, large, 10,662 bp (10.6 kbp; LRG_514t1:c.-10403_-7 + 265del; hg19, chr17:g.66498293_66508954del) deletion in the promoter of PRKAR1A in heterozygous form in the affected family members. The exact breakpoints and the increased enzyme activity in deletion carriers compared to wild type carrier were proved. Segregation analysis and functional evaluation of PKA activity confirmed the pathogenic role of this alteration. A novel deletion upstream of the PRKAR1A gene was proved to be the cause of CNC. Our study underlines the need for WGS in molecular genetic testing of patients with monogenic disorders where conventional genetic analysis fails.

Challenging interpretation of germline TP53 variants based on the experience of a national comprehensive cancer centre.

TP53 variant interpretation is still challenging, especially in patients with attenuated Li-Fraumeni syndrome (LFS). We investigated the prevalence of pathogenic/likely pathogenic (P/LP) variants and LFS disease in the Hungarian population of cancer patients. By testing 893 patients with multiplex or familial cancer, we identified and functionally characterized novel splice variants of TP53 helping accurate variant classification. The differences among various semi-automated interpretation platforms without manual curation highlight the importance of focused interpretation as the automatic classification systems do not apply the TP53-specific criteria. The predicted frequency of the TP53 P/LP variants in Hungary is 0.3 per million which most likely underestimates the real prevalence. The higher detection rate of disease-causing variants in patients with attenuated LFS phenotype compared to the control population (OR 12.5; p < 0.0001) may raise the potential benefit of the TP53 genetic testing as part of the hereditary cancer panels of patients with multiple or familial cancer even when they do not meet Chompret criteria. Tumours developed at an earlier age in phenotypic LFS patients compared to the attenuated LFS patients which complicates genetic counselling as currently there are no different recommendations in surveillance protocols for LFS, phenotypic LFS, and attenuated LFS patients.

A common genetic variation in GZMB may associate with cancer risk in patients with Lynch syndrome.

Lynch syndrome (LS), also known as hereditary nonpolyposis colorectal cancer syndrome (HNPCC) is a common genetic predisposition to cancer due to germline mutations in genes affecting DNA mismatch repair. Due to mismatch repair deficiency, developing tumors are characterized by microsatellite instability (MSI-H), high frequency of expressed neoantigens and good clinical response to immune checkpoint inhibitors. Granzyme B (GrB) is the most abundant serine protease in the granules of cytotoxic T-cells and natural killer cells, mediating anti-tumor immunity. However, recent results confirm a diverse range of physiological functions of GrB including that in extracellular matrix remodelling, inflammation and fibrosis. In the present study, our aim was to investigate whether a frequent genetic variation of GZMB, the gene encoding GrB, constituted by three missense single nucleotide polymorphisms (rs2236338, rs11539752 and rs8192917) has any association with cancer risk in individuals with LS. In silico analysis and genotype calls from whole exome sequencing data in the Hungarian population confirmed that these SNPs are closely linked. Genotyping results of rs8192917 on a cohort of 145 individuals with LS demonstrated an association of the CC genotype with lower cancer risk. In silico prediction proposed likely GrB cleavage sites in a high proportion of shared neontigens in MSI-H tumors. Our results propose the CC genotype of rs8192917 as a potential disease-modifying genetic factor in LS.

Genome sequencing-based discovery of a novel deep intronic APC pathogenic variant causing exonization.

Familial adenomatous polyposis (FAP) is a hereditary cancer syndrome that occurs as a result of germline mutations in the APC gene. Despite a clear clinical diagnosis of FAP, a certain proportion of the APC variants are not readily detectable through conventional genotyping routines. We accomplished genome sequencing in duo of the disease-affected proband and non-affected sibling followed by in silico predictions and a series of RNA-based assays clarifying variant functionality. By prioritizing variants obtained by genome sequencing, we discovered the novel deep intronic alteration APC:c.531 + 1482 A > G that was demonstrated to cause out-of-frame exonization of 56 base pairs from intron 5 of the gene. Further cDNA assays confirmed, that the aberrant splicing event was complete and its splice product was subject to nonsense-mediated decay. Co-segregation was observed between the variant carrier status and the disease phenotype. Cumulative evidence confirmed that APC:c.531 + 1482 A > G is a pathogenic variant causative of the disease.

Case Report: A Novel Pathomechanism in PEComa by the Loss of Heterozygosity of TP53.

Since the introduction of next-generation sequencing, the frequency of germline pathogenic TP53 variants and the number of cases with unusual clinical presentations have been increasing. This has led to the expansion of the classical Li-Fraumeni syndrome concept to a wider cancer predisposition syndrome designated as the Li-Fraumeni spectrum. Here, we present a case with a malignant, metastatic perivascular epithelioid cell tumor (PEComa) of the thigh muscle and a sinonasal carcinoma harboring a novel TP53 germline splice mutation (NM_000546.5:c.97-2A>C). The classical presentation of LFS in the long-since deceased mother and the presence of a germline TP53 variant in the proband suggested a possible familial TP53-related condition. Complex pathological, molecular, and clinical genetic analyses (whole exome sequencing of germline variants, multigene panel sequencing of tumor DNA, Sanger validation, an in vitro functional test on splicing effect, 3D protein modeling, p53 immunohistochemistry, and pedigree analysis) were performed. The in vitro characterization of the splice mutation supported the pathogenic effect that resulted in exon skipping. A locus-specific loss of heterozygosity in the PEComa but not in the sinonasal carcinoma was identified, suggesting the causative role of the splice mutation in the PEComa pathogenesis, because we excluded known pathogenetic pathways characteristic to PEComas (TSC1/2, TFE3, RAD51B). However, the second hit affecting TP53 in the molecular pathogenesis of the sinonasal carcinoma was not identified. Although PEComa has been reported previously in two patients with Li-Fraumeni syndrome, to the best of our knowledge, this is the first report suggesting a relationship between the aberrant TP53 variant and PEComa.

Reclassification of Five BRCA1/2 Variants with Unknown Significance Using Complex Functional Study.

PURPOSE: While BRCA1/2 genes are commonly investigated, variants of unknown significance (VUS) and variants with potential splice effect are still being detected and they represent a substantial challenge in genetic counseling and therapy. MATERIALS AND METHODS: Out of genetically tested 3,568 hereditary breast and ovarian cancer probands five, functionally not investigated variants with potential splice-modifying effect were subjected to functional characterization. Transcript-level analysis on peripheral blood-derived RNA of the carriers was performed to test aberrant splicing. The completeness of the aberrant splicing event was also studied, existence and extent of nonsense-mediated decay was even addressed. Clinical and phenotype data, pedigree and co-segregation analyses were also done. Locus-specific loss of heterozygosity (LOH) in tumor tissues was additionally tested. RESULTS: In case of the BRCA1:c.4484+4dupA and the BRCA1:c.5407-10G>A variants functional results allowed us to reclassify them from VUS into likely pathogenic category. BRCA1:c.4358-31A>C, by producing incomplete aberrant splicing, was highlighted as strong VUS, but in lack of other supporting evidence, re-categorization was not possible. The likely pathogenic assertion of previously not reported BRCA2:c.8487G>T was reinforced based on its spliceogenic property and tumor LOH, while BRCA2:c.793G>A failed to present aberrant splicing in spite of suggestive predictions, which altered its original VUS evaluation into likely benign class. CONCLUSION: We presented molecular and clinical evidence for reclassification of four out of five BRCA1/2 variants. Both up- and down-classification harbour important clinical significance. Patients carrying re-classified pathogenic variants in the future will not be dropped out from medical surveillance, preventive measures, treatment and predictive family screening in relatives at risk.

Surprising genetic and pathological findings in a patient with giant bilateral periadrenal tumours: PEComas and mutations of PTCH1 in Gorlin-Goltz syndrome.

Gorlin-Goltz syndrome (GGS) or nevoid basal cell carcinoma syndrome is a rare tumour-overgrowth syndrome associated with multiple developmental anomalies and a wide variety of tumours. Here, we describe a case of a man aged 23 years with GGS with bilateral giant tumours adjacent to both adrenals that raised the suspicion of malignancy on imaging. Histological analysis of both surgically resected tumours revealed perivascular epitheloid cell tumours (PEComas) that were independent of the adrenals. Exome sequencing of the patient's blood sample revealed a novel germline heterozygous frameshift mutation in the PTCH1 gene. As a second hit, a somatic five nucleotide long deletion in the PTCH1 gene was demonstrated in the tumour DNA of both PEComas. To the best of our knowledge, this is the first report on PEComa in GGS, and this finding also raises the potential relevance of PTCH1 mutations and altered sonic hedgehog signalling in PEComa pathogenesis. The presence of the same somatic mutation in the bilateral tumours might indicate the possibility of a postzygotic somatic mutation that along with the germline mutation of the same gene could represent an intriguing genetic phenomenon (type 2 segmental mosaicism).

Breast and Prostate Cancer Risks for Male BRCA1 and BRCA2 Pathogenic Variant Carriers Using Polygenic Risk Scores.

BACKGROUND: Recent population-based female breast cancer and prostate cancer polygenic risk scores (PRS) have been developed. We assessed the associations of these PRS with breast and prostate cancer risks for male BRCA1 and BRCA2 pathogenic variant carriers. METHODS: 483 BRCA1 and 1318 BRCA2 European ancestry male carriers were available from the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA). A 147-single nucleotide polymorphism (SNP) prostate cancer PRS (PRSPC) and a 313-SNP breast cancer PRS were evaluated. There were 3 versions of the breast cancer PRS, optimized to predict overall (PRSBC), estrogen receptor (ER)-negative (PRSER-), or ER-positive (PRSER+) breast cancer risk. RESULTS: PRSER+ yielded the strongest association with breast cancer risk. The odds ratios (ORs) per PRSER+ standard deviation estimates were 1.40 (95% confidence interval [CI] =1.07 to 1.83) for BRCA1 and 1.33 (95% CI = 1.16 to 1.52) for BRCA2 carriers. PRSPC was associated with prostate cancer risk for BRCA1 (OR = 1.73, 95% CI = 1.28 to 2.33) and BRCA2 (OR = 1.60, 95% CI = 1.34 to 1.91) carriers. The estimated breast cancer odds ratios were larger after adjusting for female relative breast cancer family history. By age 85 years, for BRCA2 carriers, the breast cancer risk varied from 7.7% to 18.4% and prostate cancer risk from 34.1% to 87.6% between the 5th and 95th percentiles of the PRS distributions. CONCLUSIONS: Population-based prostate and female breast cancer PRS are associated with a wide range of absolute breast and prostate cancer risks for male BRCA1 and BRCA2 carriers. These findings warrant further investigation aimed at providing personalized cancer risks for male carriers and informing clinical management.

Germline Structural Variations in Cancer Predisposition Genes.

In addition to single nucleotide variations and small-scale indels, structural variations (SVs) also contribute to the genetic diversity of the genome. SVs, such as deletions, duplications, amplifications, or inversions may also affect coding regions of cancer-predisposing genes. These rearrangements may abrogate the open reading frame of these genes or adversely affect their expression and may thus act as germline mutations in hereditary cancer syndromes. With the capacity of disrupting the function of tumor suppressors, structural variations confer an increased risk of cancer and account for a remarkable fraction of heritability. The development of sequencing techniques enables the discovery of a constantly growing number of SVs of various types in cancer predisposition genes (CPGs). Here, we provide a comprehensive review of the landscape of germline SV types, detection methods, pathomechanisms, and frequency in CPGs, focusing on the two most common cancer syndromes: hereditary breast- and ovarian cancer and gastrointestinal cancers. Current knowledge about the possible molecular mechanisms driving to SVs is also summarized.

Application of Multilayer Evidence for Annotation of C-Terminal BRCA2 Variants.

The clinical relevance of the BRCA2 C-terminal stop codon variants is controversial. The pathogenic role of the germline BRCA2 c.9976A>T and c.10095delinsGAATTATATCT variants in hereditary breast and ovarian cancer (HBOC) patients was evaluated. An association with clinicopathological parameters was performed in 2491 independent probands diagnosed with HBOC and in 122,209 cancer patients reported earlier. Loss-of-heterozygosity (LOH) in tumor samples and allelic imbalance in RNA extracted from peripheral blood cells were investigated. Neither c.10095delinsGAATTATATCT or c.9976A>T variants showed significant association with clinicopathological parameters or elevated risk for HBOC-associated tumors. Lung cancer was more prevalent in families carrying the c.9976A>T variant compared to pathogenic BRCA1 or BRCA2 carrier families. An increased prevalence of pancreatic cancer was found in families where c.9976A>T occurred together with other pathogenic BRCA1 variants. An increased risk for familial pancreatic, lung and upper aero-digestive tract cancers was confirmed in the validation set. Regarding BRCA2 C-terminal variants, no linkage with other pathogenic BRCA2 variants, no LOH in tumor tissue and no allelic imbalance in RNA level were confirmed. The c.9976A>T variant may be considered as a potential risk for lung cancer, and a potential modifying factor in pancreatic cancer when it occurs along with the pathogenic BRCA1 variant, although this observation should be validated in a larger sample cohort.

Germline Genetic Variants of Viral Entry and Innate Immunity May Influence Susceptibility to SARS-CoV-2 Infection: Toward a Polygenic Risk Score for Risk Stratification.

The ongoing COVID-19 pandemic caused by the novel coronavirus, SARS-CoV-2 has affected all aspects of human society with a special focus on healthcare. Although older patients with preexisting chronic illnesses are more prone to develop severe complications, younger, healthy individuals might also exhibit serious manifestations. Previous studies directed to detect genetic susceptibility factors for earlier epidemics have provided evidence of certain protective variations. Following SARS-CoV-2 exposure, viral entry into cells followed by recognition and response by the innate immunity are key determinants of COVID-19 development. In the present review our aim was to conduct a thorough review of the literature on the role of single nucleotide polymorphisms (SNPs) as key agents affecting the viral entry of SARS-CoV-2 and innate immunity. Several SNPs within the scope of our approach were found to alter susceptibility to various bacterial and viral infections. Additionally, a multitude of studies confirmed genetic associations between the analyzed genes and autoimmune diseases, underlining the versatile immune consequences of these variants. Based on confirmed associations it is highly plausible that the SNPs affecting viral entry and innate immunity might confer altered susceptibility to SARS-CoV-2 infection and its complex clinical consequences. Anticipating several COVID-19 genomic susceptibility loci based on the ongoing genome wide association studies, our review also proposes that a well-established polygenic risk score would be able to clinically leverage the acquired knowledge.

Complex Characterization of Germline Large Genomic Rearrangements of the BRCA1 and BRCA2 Genes in High-Risk Breast Cancer Patients-Novel Variants from a Large National Center.

Large genomic rearrangements (LGRs) affecting one or more exons of BRCA1 and BRCA2 constitute a significant part of the mutation spectrum of these genes. Since 2004, the National Institute of Oncology, Hungary, has been involved in screening for LGRs of breast or ovarian cancer families enrolled for genetic testing. LGRs were detected by multiplex ligation probe amplification method, or next-generation sequencing. Where it was possible, transcript-level characterization of LGRs was performed. Phenotype data were collected and analyzed too. Altogether 28 different types of LGRs in 51 probands were detected. Sixteen LGRs were novel. Forty-nine cases were deletions or duplications in BRCA1 and two affected BRCA2. Rearrangements accounted for 10% of the BRCA1 mutations. Three exon copy gains, two complex rearrangements, and 23 exon losses were characterized by exact breakpoint determinations. The inferred mechanisms for LGR formation were mainly end-joining repairs utilizing short direct homologies. Comparing phenotype features of the LGR-carriers to that of the non-LGR BRCA1 mutation carriers, revealed no significant differences. Our study is the largest comprehensive report of LGRs of BRCA1/2 in familial breast and ovarian cancer patients in the Middle and Eastern European region. Our data add novel insights to genetic interpretation associated to the LGRs.

Polygenic risk scores and breast and epithelial ovarian cancer risks for carriers of BRCA1 and BRCA2 pathogenic variants.

PURPOSE: We assessed the associations between population-based polygenic risk scores (PRS) for breast (BC) or epithelial ovarian cancer (EOC) with cancer risks for BRCA1 and BRCA2 pathogenic variant carriers. METHODS: Retrospective cohort data on 18,935 BRCA1 and 12,339 BRCA2 female pathogenic variant carriers of European ancestry were available. Three versions of a 313 single-nucleotide polymorphism (SNP) BC PRS were evaluated based on whether they predict overall, estrogen receptor (ER)-negative, or ER-positive BC, and two PRS for overall or high-grade serous EOC. Associations were validated in a prospective cohort. RESULTS: The ER-negative PRS showed the strongest association with BC risk for BRCA1 carriers (hazard ratio [HR] per standard deviation = 1.29 [95% CI 1.25-1.33], P = 3×10-72). For BRCA2, the strongest association was with overall BC PRS (HR = 1.31 [95% CI 1.27-1.36], P = 7×10-50). HR estimates decreased significantly with age and there was evidence for differences in associations by predicted variant effects on protein expression. The HR estimates were smaller than general population estimates. The high-grade serous PRS yielded the strongest associations with EOC risk for BRCA1 (HR = 1.32 [95% CI 1.25-1.40], P = 3×10-22) and BRCA2 (HR = 1.44 [95% CI 1.30-1.60], P = 4×10-12) carriers. The associations in the prospective cohort were similar. CONCLUSION: Population-based PRS are strongly associated with BC and EOC risks for BRCA1/2 carriers and predict substantial absolute risk differences for women at PRS distribution extremes.

Molecular mechanisms for the antitumor activity of inositol hexakisphosphate (IP6).

BACKGROUND: Inositol hexakisphosphate (IP6), a naturally occurring polyphosphorylated carbohydrate, has been reported to have significant in vivo and in vitro anticancer activity against numerous tumors. However, the molecular mechanism of the anticancer effect of IP6 has not been fully elucidated. MATERIALS AND METHODS: Using K-562 human leukemia cells we analysed the induction of the erythroid differentiation program, as well as modulation of the gene expression profile of K-562 leukemia cells treated with IP6. RESULTS: A single treatment with IP6 (0.75 or 5.0 mM) resulted in a time- and dose-dependent growth inhibition of K-562 cells and also activation of the erythroid differentiation program. K-562 cells expressed a concomitant differentiation after 12 hours of exposure. Possible molecular mechanisms and key signaling pathways, as well as gene expression behind this anticancer effect were examined using oligonucleotide microarrays and quantitative real-time PCR. Treatment with IP6 (750 microM, 5 mM) had a marked impact, resulting in early (60 min) and late (12 h) modulation of expression of about 1800 and 1200 transcripts (at p<0.05). Through microarray analysis, the anticancer effect of IP6 in K-562 was found to be associated with the modulation of multiple genes involved in immunity, Wnt and IGF pathways, PI3 kinase signaling and apoptosis. Using selected subsets of genes, the microarray hits could be validated by Q-PCR. A 2-fold upregulation of the apoptosis pathway, measured using the BAX/BCL-2 ratio was observed for 12 hours. IP6 (5 mM) induced up to 6-fold increases in differentiation measured by hemoglobin synthesis, yielding up to 70% of benzidine-positive cells at 120 hours. CONCLUSION: The results of this study show that IP6 is a strong inducer of differentiation (cytostatic effect) and a moderately strong inducer of apoptosis (cytotoxic effect). Evidence has been provided to show that the growth inhibitory effects of IP6 are mediated through the modulation of key signaling pathways.

Marker construction and cloning of a cut1-like sequence with ARS activity in the fission yeast Schizosaccharomyces japonicus.

The dimorphic fission yeast Schizosaccharomyces japonicus has proved to be an excellent experimental model for the investigation of the eukaryotic cell. Here we show that it has a haplontic life cycle, in which the diploid phase is confined to the zygote. To make it amenable to genetic and molecular analysis, we generated genetic markers and cloned a genomic sequence which acts as ars when integrated into a plasmid. Diploids suitable for testing complementation and recombination between markers can be formed by protoplast fusion. The complementation tests and the recombination frequencies determined in octads of spores identified 28 non-allelic groups (genes) of mutations of the auxotrophic and mycelium-negative mutants. Two groups of linked markers were also identified. The cloned fragment, which expresses ars activity, encodes a putative amino acid sequence highly similar to a conserved domain of proteins Cut1 (Schizosaccharomyces pombe), BimB (Aspergillus nidulans) and Esp1 (Saccharomyces cerevisiae).