Lifestyle Factors and Breast Cancer in Females with PTEN Hamartoma Tumor Syndrome (PHTS).

Females with PTEN Hamartoma Tumor Syndrome (PHTS) have breast cancer risks up to 76%. This study assessed associations between breast cancer and lifestyle in European female adult PHTS patients. Data were collected via patient questionnaires (July 2020-March 2023) and genetic diagnoses from medical files. Associations between lifestyle and breast cancer were calculated using logistic regression corrected for age. Index patients with breast cancer before PHTS diagnosis (breast cancer index) were excluded for ascertainment bias correction. In total, 125 patients were included who completed the questionnaire at a mean age of 44 years (SD = 13). This included 21 breast cancer indexes (17%) and 39 females who developed breast cancer at 43 years (SD = 9). Breast cancer patients performed about 1.1 times less often 0-1 times/week physical activity than ≥2 times (ORtotal-adj = 0.9 (95%CI 0.3-2.6); consumed daily about 1.2-1.8 times more often ≥1 than 0-1 glasses of alcohol (ORtotal-adj = 1.2 (95%CI 0.4-4.0); ORnon-breastcancer-index-adj = 1.8 (95%CI 0.4-6.9); were about 1.04-1.3 times more often smokers than non-smokers (ORtotal-adj = 1.04 (95%CI 0.4-2.8); ORnon-breastcancer-index-adj = 1.3 (95%CI 0.4-4.2)); and overweight or obesity (72%) was about 1.02-1.3 times less common (ORtotal-adj = 0.98 (95%CI 0.4-2.6); ORnon-breastcancer-index-adj = 0.8 (95%CI 0.3-2.7)). Similar associations between lifestyle and breast cancer are suggested for PHTS and the general population. Despite not being statistically significant, results are clinically relevant and suggest that awareness of the effects of lifestyle on patients' breast cancer risk is important.

CYP19A1 mediates severe SARS-CoV-2 disease outcome in males.

Male sex represents one of the major risk factors for severe COVID-19 outcome. However, underlying mechanisms that mediate sex-dependent disease outcome are as yet unknown. Here, we identify the CYP19A1 gene encoding for the testosterone-to-estradiol metabolizing enzyme CYP19A1 (also known as aromatase) as a host factor that contributes to worsened disease outcome in SARS-CoV-2-infected males. We analyzed exome sequencing data obtained from a human COVID-19 cohort (n = 2,866) using a machine-learning approach and identify a CYP19A1-activity-increasing mutation to be associated with the development of severe disease in men but not women. We further analyzed human autopsy-derived lungs (n = 86) and detect increased pulmonary CYP19A1 expression at the time point of death in men compared with women. In the golden hamster model, we show that SARS-CoV-2 infection causes increased CYP19A1 expression in the lung that is associated with dysregulated plasma sex hormone levels and reduced long-term pulmonary function in males but not females. Treatment of SARS-CoV-2-infected hamsters with a clinically approved CYP19A1 inhibitor (letrozole) improves impaired lung function and supports recovery of imbalanced sex hormones specifically in males. Our study identifies CYP19A1 as a contributor to sex-specific SARS-CoV-2 disease outcome in males. Furthermore, inhibition of CYP19A1 by the clinically approved drug letrozole may furnish a new therapeutic strategy for individualized patient management and treatment.

Gain- and Loss-of-Function CFTR Alleles Are Associated with COVID-19 Clinical Outcomes.

Carriers of single pathogenic variants of the CFTR (cystic fibrosis transmembrane conductance regulator) gene have a higher risk of severe COVID-19 and 14-day death. The machine learning post-Mendelian model pinpointed CFTR as a bidirectional modulator of COVID-19 outcomes. Here, we demonstrate that the rare complex allele [G576V;R668C] is associated with a milder disease via a gain-of-function mechanism. Conversely, CFTR ultra-rare alleles with reduced function are associated with disease severity either alone (dominant disorder) or with another hypomorphic allele in the second chromosome (recessive disorder) with a global residual CFTR activity between 50 to 91%. Furthermore, we characterized novel CFTR complex alleles, including [A238V;F508del], [R74W;D1270N;V201M], [I1027T;F508del], [I506V;D1168G], and simple alleles, including R347C, F1052V, Y625N, I328V, K68E, A309D, A252T, G542*, V562I, R1066H, I506V, I807M, which lead to a reduced CFTR function and thus, to more severe COVID-19. In conclusion, CFTR genetic analysis is an important tool in identifying patients at risk of severe COVID-19.

The polymorphism L412F in TLR3 inhibits autophagy and is a marker of severe COVID-19 in males.

The polymorphism L412F in TLR3 has been associated with several infectious diseases. However, the mechanism underlying this association is still unexplored. Here, we show that the L412F polymorphism in TLR3 is a marker of severity in COVID-19. This association increases in the sub-cohort of males. Impaired macroautophagy/autophagy and reduced TNF/TNFα production was demonstrated in HEK293 cells transfected with TLR3L412F-encoding plasmid and stimulated with specific agonist poly(I:C). A statistically significant reduced survival at 28 days was shown in L412F COVID-19 patients treated with the autophagy-inhibitor hydroxychloroquine (p = 0.038). An increased frequency of autoimmune disorders such as co-morbidity was found in L412F COVID-19 males with specific class II HLA haplotypes prone to autoantigen presentation. Our analyses indicate that L412F polymorphism makes males at risk of severe COVID-19 and provides a rationale for reinterpreting clinical trials considering autophagy pathways.Abbreviations: AP: autophagosome; AUC: area under the curve; BafA1: bafilomycin A1; COVID-19: coronavirus disease-2019; HCQ: hydroxychloroquine; RAP: rapamycin; ROC: receiver operating characteristic; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; TLR: toll like receptor; TNF/TNF-α: tumor necrosis factor.

13q Deletion Syndrome Involving RB1: Characterization of a New Minimal Critical Region for Psychomotor Delay.

Retinoblastoma (RB) is an ocular tumor of the pediatric age caused by biallelic inactivation of the RB1 gene (13q14). About 10% of cases are due to gross-sized molecular deletions. The deletions can involve the surrounding genes delineating a contiguous gene syndrome characterized by RB, developmental anomalies, and peculiar facial dysmorphisms. Overlapping deletions previously found by traditional and/or molecular cytogenetic analysis allowed to define some critical regions for intellectual disability (ID) and multiple congenital anomalies, with key candidate genes. In the present study, using array-CGH, we characterized seven new patients with interstitial 13q deletion involving RB1. Among these cases, three patients with medium or large 13q deletions did not present psychomotor delay. This allowed defining a minimal critical region for ID that excludes the previously suggested candidate genes (HTR2A, NUFIP1, PCDH8, and PCDH17). The region contains 36 genes including NBEA, which emerged as the candidate gene associated with developmental delay. In addition, MAB21L1, DCLK1, EXOSC8, and SPART haploinsufficiency might contribute to the observed impaired neurodevelopmental phenotype. In conclusion, this study adds important novelties to the 13q deletion syndrome, although further studies are needed to better characterize the contribution of different genes and to understand how the haploinsufficiency of this region can determine ID.

Corrigendum: Exome Sequencing in BRCA1-2 Candidate Familias: The Contribution of Other Cancer Susceptibility Genes.

[This corrects the article DOI: 10.3389/fonc.2021.649435.].

SELP Asp603Asn and severe thrombosis in COVID-19 males.

Thromboembolism is a frequent cause of severity and mortality in COVID-19. However, the etiology of this phenomenon is not well understood. A cohort of 1186 subjects, from the GEN-COVID consortium, infected by SARS-CoV-2 with different severity was stratified by sex and adjusted by age. Then, common coding variants from whole exome sequencing were mined by LASSO logistic regression. The homozygosity of the cell adhesion molecule P-selectin gene (SELP) rs6127 (c.1807G > A; p.Asp603Asn) which has been already associated with thrombotic risk is found to be associated with severity in the male subcohort of 513 subjects (odds ratio = 2.27, 95% Confidence Interval 1.54-3.36). As the SELP gene is downregulated by testosterone, the odd ratio is increased in males older than 50 (OR 2.42, 95% CI 1.53-3.82). Asn/Asn homozygotes have increased D-dimers values especially when associated with poly Q ≥ 23 in the androgen receptor (OR 3.26, 95% CI 1.41-7.52). These results provide a rationale for the repurposing of antibodies against P-selectin as adjuvant therapy in rs6127 male homozygotes especially if older than 50 or with an impaired androgen receptor.

Severe COVID-19 in Hospitalized Carriers of Single CFTR Pathogenic Variants.

The clinical presentation of COVID-19 is extremely heterogeneous, ranging from asymptomatic to severely ill patients. Thus, host genetic factors may be involved in determining disease presentation and progression. Given that carriers of single cystic fibrosis (CF)-causing variants of the CFTR gene-CF-carriers-are more susceptible to respiratory tract infections, our aim was to determine their likelihood of undergoing severe COVID-19. We implemented a cohort study of 874 individuals diagnosed with COVID-19, during the first pandemic wave in Italy. Whole exome sequencing was performed and validated CF-causing variants were identified. Forty subjects (16 females and 24 males) were found to be CF-carriers. Among mechanically ventilated patients, CF-carriers were more represented (8.7%) and they were significantly (p < 0.05) younger (mean age 51 years) compared to noncarriers (mean age 61.42 years). Furthermore, in the whole cohort, the age of male CF-carriers was lower, compared to noncarriers (p < 0.05). CF-carriers had a relative risk of presenting an abnormal inflammatory response (CRP ≥ 20 mg/dL) of 1.69 (p < 0.05) and their hazard ratio of death at day 14 was 3.10 (p < 0.05) in a multivariate regression model, adjusted for age, sex and comorbidities. In conclusion, CF-carriers are more susceptible to the severe form of COVID-19, showing also higher risk of 14-day death.

Exome sequencing in BRCA1-2 candidate familias: the contribution of other cancer susceptibility genes

Hereditary Breast and Ovarian Cancer (HBOC) syndrome is a condition in which the risk of breast and ovarian cancer is higher than in the general population. The prevalent pathogenesis is attributable to inactivating variants of the BRCA1-2 highly penetrant genes, however, other cancer susceptibility genes may also be involved. By Exome Sequencing (WES) we analyzed a series of 200 individuals selected for genetic testing in BRCA1-2 genes according to the updated National Comprehensive Cancer Network (NCCN) guidelines. Analysis by MLPA was performed to detect large BRCA1-2 deletions/duplications. Focusing on BRCA1-2 genes, data analysis identified 11 cases with pathogenic variants (4 in BRCA1 and 7 in BRCA1-2) and 12 with uncertain variants (7 in BRCA1 and 5 in BRCA2). Only one case was found with a large BRCA1 deletion. Whole exome analysis allowed to characterize pathogenic variants in 21 additional genes: 10 genes more traditionally associated to breast and ovarian cancer (ATM, BRIP1, CDH1, PALB2, PTEN, RAD51C, and TP53) (5% diagnostic yield) and 11 in candidate cancer susceptibility genes (DPYD, ERBB3, ERCC2, MUTYH, NQO2, NTHL1, PARK2, RAD54L, and RNASEL). In conclusion, this study allowed a personalized risk assessment and clinical surveillance in an increased number of HBOC families and to broaden the spectrum of causative variants also to candidate non-canonical genes.

Protective Role of a TMPRSS2 Variant on Severe COVID-19 Outcome in Young Males and Elderly Women.

The protease encoded by the TMPRSS2 gene facilitates viral infections and has been implicated in the pathogenesis of SARS-CoV-2. We analyzed the TMPRSS2 sequence and correlated the protein variants with the clinical features of a cohort of 1177 patients affected by COVID-19 in Italy. Nine relatively common variants (allele frequency > 0.01) and six missense variants which may affect the protease activity according to PolyPhen-2 in HumVar-trained mode were identified. Among them, p.V197M (p.Val197Met) (rs12329760) emerges as a common variant that has a deleterious effect on the protease and a protective effect on the patients. Its role appears particularly relevant in two subgroups of patients-young males and elderly women-and among those affected by co-morbidities, where the variant frequency is higher among individuals who were mildly affected by the disease and did not need hospitalization or oxygen therapy than among those more severely affected, who required oxygen therapy, ventilation or intubation. This study provides useful information for the identification of patients at risk of developing a severe form of COVID-19, and encourages the usage of drugs affecting the expression of TMPRSS2 or inhibiting protein activity.

Shorter androgen receptor polyQ alleles protect against life-threatening COVID-19 disease in European males.

BACKGROUND: While SARS-CoV-2 similarly infects men and women, COVID-19 outcome is less favorable in men. Variability in COVID-19 severity may be explained by differences in the host genome. METHODS: We compared poly-amino acids variability from WES data in severely affected COVID-19 patients versus SARS-CoV-2 PCR-positive oligo-asymptomatic subjects. FINDINGS: Shorter polyQ alleles (≤22) in the androgen receptor (AR) conferred protection against severe outcome in COVID-19 in the first tested cohort (both males and females) of 638 Italian subjects. The association between long polyQ alleles (≥23) and severe clinical outcome (p = 0.024) was also validated in an independent cohort of Spanish men <60 years of age (p = 0.014). Testosterone was higher in subjects with AR long-polyQ, possibly indicating receptor resistance (p = 0.042 Mann-Whitney U test). Inappropriately low serum testosterone level among carriers of the long-polyQ alleles (p = 0.0004 Mann-Whitney U test) predicted the need for intensive care in COVID-19 infected men. In agreement with the known anti-inflammatory action of testosterone, patients with long-polyQ and age ≥60 years had increased levels of CRP (p = 0.018, not accounting for multiple testing). INTERPRETATION: We identify the first genetic polymorphism that appears to predispose some men to develop more severe disease. Failure of the endocrine feedback to overcome AR signaling defects by increasing testosterone levels during the infection leads to the polyQ tract becoming dominant to serum testosterone levels for the clinical outcome. These results may contribute to designing reliable clinical and public health measures and provide a rationale to test testosterone as adjuvant therapy in men with COVID-19 expressing long AR polyQ repeats. FUNDING: MIUR project "Dipartimenti di Eccellenza 2018-2020" to Department of Medical Biotechnologies University of Siena, Italy (Italian D.L. n.18 March 17, 2020) and "Bando Ricerca COVID-19 Toscana" project to Azienda Ospedaliero-Universitaria Senese. Private donors for COVID-19 research and charity funds from Intesa San Paolo.

MET somatic activating mutations are responsible for lymphovenous malformation and can be identified using cell-free DNA next generation sequencing liquid biopsy.

OBJECTIVE: Germline mutations of either the endothelial cell-specific tyrosine kinase receptor TIE2 or the glomulin (GLMN) gene are responsible for rare inherited venous malformations. Both genes affect the hepatocyte growth factor receptor c-Met, inducing vascular smooth muscle cell migration. Germline mutations of hepatocyte growth factor are responsible for lymphatic malformations, leading to lymphedema. The molecular alteration leading to the abnormal mixed vascular anomaly defined as lymphovenous malformation has remained unknown. METHODS: A group of 4 patients with lymphovenous malformations were selected. Plasma was obtained from both peripheral and efferent vein samples at the vascular malformation site for cell-free DNA extraction. When possible, we analyzed tissue biopsy samples from the vascular lesion. RESULTS: We have demonstrated that in all four patients, an activating MET mutation was present. In three of the four patients, the same pathogenic activating mutation, T1010I, was identified. The mutation was found at the tissue level for the patient with tissue samples available, confirming its causative role in the lymphovenous malformations. CONCLUSIONS: In the present study, we have demonstrated that cell-free DNA next generation sequencing liquid biopsy is able to identify the MET mutations in affected tissues. Although a wider cohort of patients is necessary to confirm its causative role in lymphovenous malformations, these data suggest that lymphovenous malformations could result from postzygotic somatic mutations in genes that are key regulators of lymphatic development. The noninvasiveness of the method avoids any risk of bleeding and can be easily performed in children. We are confident that the present pioneering results have provided a viable alternative in the future for lymphovenous malformation diagnosis, allowing for subsequent therapy tailored to the genetic defect.

A pilot study of next generation sequencing-liquid biopsy on cell-free DNA as a novel non-invasive diagnostic tool for Klippel-Trenaunay syndrome.

OBJECTIVES: Somatic mosaicism of PIK3CA gene is currently recognized as the molecular driver of Klippel-Trenaunay syndrome. However, given the limitation of the current technologies, PIK3CA somatic mutations are detected only in a limited proportion of Klippel-Trenaunay syndrome cases and tissue biopsy remains an invasive high risky, sometimes life-threatening, diagnostic procedure. Next generation sequencing liquid biopsy using cell-free DNA has emerged as an innovative non-invasive approach for early detection and monitoring of cancer. This approach, overcoming the space-time profile constraint of tissue biopsies, opens a new scenario also for others diseases caused by somatic mutations. METHODS: In the present study, we performed a comprehensive analysis of seven patients (four females and three males) with Klippel-Trenaunay syndrome. Blood samples from both peripheral and efferent vein from malformation were collected and cell-free DNA was extracted from plasma. Tissue biopsies from vascular lesions were also collected when available. Cell-free DNA libraries were performed using Oncomine™ Pan-Cancer Cell-Free Assay. Ion Proton for sequencing and Ion Reporter Software for analysis were used (Life Technologies, Carlsbad, CA, USA). RESULTS: Cell-free circulating DNA analysis revealed pathogenic mutations in PIK3CA gene in all patients. The mutational load was higher in plasma obtained from the efferent vein at lesional site (0.81%) than in the peripheral vein (0.64%) leading to conclude for a causative role of the identified variants. Tissue analysis, available for one amputated patient, confirmed the presence of the mutation at the malformation site at a high molecular frequency (14-25%), confirming its causative role. CONCLUSIONS: Our data prove for the first time that the cell-free DNA-next generation sequencing-liquid biopsy, which is currently used exclusively in an oncologic setting, is indeed the most effective tool for Klippel-Trenaunay syndrome diagnosis and tailored personalized treatment.

Two-point-NGS analysis of cancer genes in cell-free DNA of metastatic cancer patients.

BACKGROUND: Although the efficacy of molecularly target agents in vitro, their use in routine setting is limited mainly to the use of anti-HER2 and antiEGFR agents in vivo. Moreover, core biopsy of a single cancer site may not be representative of the whole expanding clones and cancer molecular profile at relapse may differ with respect to the primary tumor. METHODS: We assessed the status of a large panel of cancer driver genes by cell-free DNA (cfDNA) analysis in a cohort of 68 patients with 13 different solid tumors at disease progression. Whenever possible, a second cfDNA analysis was performed after a mean of 2.5 months, in order to confirm the identified clone(s) and to check the correlation with clinical evolution. RESULTS: The approach was able to identify clones plausibly involved in the disease progression mechanism in about 65% of cases. A mean of 1.4 mutated genes (range 1-3) for each tumor was found. Point mutations in TP53, PIK3CA, and KRAS and copy number variations in FGFR3 were the gene alterations more commonly observed, with a rate of 48%, 20%, 16%, and 20%, respectively. Two-points-Next-Generation Sequencing (NGS) analysis demonstrated statistically significant correlation between allele frequency variation and clinical outcome (P = .026). CONCLUSIONS: Irrespective of the primary tumor mutational burden, few mutated genes are present at disease progression. Clinical outcome is consistent with variation of allele frequency of specific clones indicating that cfDNA two-point-NGS analysis of cancer driver genes could be an efficacy tool for precision oncology.

PIK3CA-CDKN2A clonal evolution in metastatic breast cancer and multiple points cell-free DNA analysis.

BACKGROUND: Daily experience tells us that breast cancer can be controlled using standard protocols up to the advent of a relapse. Now new frontiers in precision medicine like liquid biopsy of cell free DNA (cfDNA) give us the possibility to understand cancer evolution and pick up the key mutation on specific cancer driver gene. However, tight schedule of standardized protocol may impair the use of personalized experimental drugs in a timely therapeutic window. MAIN BODY: Here, using a combination of deep next generation sequencing and cfDNA liquid biopsy, we demonstrated that it is possible to monitor cancer relapse over time. We showed for the first time the exact correspondence from the increasing clonal expansion and clinical worsening of metastatic breast cancer. CONCLUSION: Thanks to liquid biopsy may be possible to introduce new experimental drugs in the correct therapeutic window which would lead in the near future to an effective treatment which otherwise remains challenging.

Usefulness and Limitations of Comprehensive Characterization of mRNA Splicing Profiles in the Definition of the Clinical Relevance of BRCA1/2 Variants of Uncertain Significance.

Highly penetrant variants of BRCA1/2 genes are involved in hereditary predisposition to breast and ovarian cancer. The detection of pathogenic BRCA variants has a considerable clinical impact, allowing appropriate cancer-risk management. However, a major drawback is represented by the identification of variants of uncertain significance (VUS). Many VUS potentially affect mRNA splicing, making transcript analysis an essential step for the definition of their pathogenicity. Here, we characterize the impact on splicing of ten BRCA1/2 variants. Aberrant splicing patterns were demonstrated for eight variants whose alternative transcripts were fully characterized. Different events were observed, including exon skipping, intron retention, and usage of de novo and cryptic splice sites. Transcripts with premature stop codons or in-frame loss of functionally important residues were generated. Partial/complete splicing effect and quantitative contribution of different isoforms were assessed, leading to variant classification according to Evidence-based Network for the Interpretation of Mutant Alleles (ENIGMA) consortium guidelines. Two variants could be classified as pathogenic and two as likely benign, while due to a partial splicing effect, six variants remained of uncertain significance. The association with an undefined tumor risk justifies caution in recommending aggressive risk-reduction treatments, but prevents the possibility of receiving personalized therapies with potential beneficial effect. This indicates the need for applying additional approaches for the analysis of variants resistant to classification by gene transcript analyses.

Omic Approach in Non-smoker Female with Lung Squamous Cell Carcinoma Pinpoints to Germline Susceptibility and Personalized Medicine.

PURPOSE: Lung cancer is strongly associated to tobacco smoking. However, global statistics estimate that in females the proportion of lung cancer cases that is unrelated to tobacco smoking reaches fifty percent, making questionable the etiology of the disease. MATERIALS AND METHODS: A never-smoker female with primary EGFR/KRAS/ALK-negative squamous cell carcinoma of the lung and their normal sibswere subjected to a novel integrative "omic" approach using a pedigree-based model for discovering genetic factors leading to cancer in the absence of well-known environmental trigger. A first-stepwhole-exome sequencing on tumor and normal tissue did not identify mutations in known driver genes. Building on the idea of a germline oligogenic origin of lung cancer, we performed whole-exome sequencing of DNA from patients' peripheral blood and their unaffected sibs. Finally, RNA-sequencing analysis in tumoral and matched non-tumoral tissues was carried out in order to investigate the clonal profile and the pathogenic role of the identified variants. RESULTS: Filtering for rare variants with Combined Annotation Dependent Depletion (CADD) > 25 and potentially damaging effect, we identified rare/private germline deleterious variants in 11 cancer-associated genes, none ofwhich, except one, sharedwith the healthy sib, pinpointing to a "private" oligogenic germline signature. Noteworthy, among these, two mutated genes, namely ACACA and DEPTOR, turned to be potential targets for therapy because related to known drivers, such as BRCA1 and EGFR. CONCLUSION: In the era of precision medicine, this report emphasizes the importance of an "omic" approach to uncover oligogenic germline signature underlying cancer development and to identify suitable therapeutic targets as well.

FANCM c.5791C>T nonsense mutation (rs144567652) induces exon skipping, affects DNA repair activity and is a familial breast cancer risk factor.

Numerous genetic factors that influence breast cancer risk are known. However, approximately two-thirds of the overall familial risk remain unexplained. To determine whether some of the missing heritability is due to rare variants conferring high to moderate risk, we tested for an association between the c.5791C>T nonsense mutation (p.Arg1931*; rs144567652) in exon 22 of FANCM gene and breast cancer. An analysis of genotyping data from 8635 familial breast cancer cases and 6625 controls from different countries yielded an association between the c.5791C>T mutation and breast cancer risk [odds ratio (OR) = 3.93 (95% confidence interval (CI) = 1.28-12.11; P = 0.017)]. Moreover, we performed two meta-analyses of studies from countries with carriers in both cases and controls and of all available data. These analyses showed breast cancer associations with OR = 3.67 (95% CI = 1.04-12.87; P = 0.043) and OR = 3.33 (95% CI = 1.09-13.62; P = 0.032), respectively. Based on information theory-based prediction, we established that the mutation caused an out-of-frame deletion of exon 22, due to the creation of a binding site for the pre-mRNA processing protein hnRNP A1. Furthermore, genetic complementation analyses showed that the mutation influenced the DNA repair activity of the FANCM protein. In summary, we provide evidence for the first time showing that the common p.Arg1931* loss-of-function variant in FANCM is a risk factor for familial breast cancer.