Cosmic Whirl: Navigating the Comet Trail in DNA: H2AX Phosphorylation and the Enigma of Uncertain Significance Variants.

Pathogenic variations in the BRCA2 gene have been detected with the development of next-generation sequencing (NGS)-based hereditary cancer panel testing technology. It also reveals an increasing number of variants of uncertain significance (VUSs). Well-established functional tests are crucial to accurately reclassifying VUSs for effective diagnosis and treatment. We retrospectively analyzed the multi-gene cancer panel results of 922 individuals and performed in silico analysis following ClinVar classification. Then, we selected five breast cancer-diagnosed patients' missense BRCA2 VUSs (T1011R, T1104P/M1168K, R2027K, G2044A, and D2819) for reclassification. The effects of VUSs on BRCA2 function were analyzed using comet and H2AX phosphorylation (γH2AX) assays before and after the treatment of peripheral blood mononuclear cells (PBMCs) of subjects with the double-strand break (DSB) agent doxorubicin (Dox). Before and after Dox-induction, the amount of DNA in the comet tails was similar in VUS carriers; however, notable variations in γH2AX were observed, and according to combined computational and functional analyses, we reclassified T1001R as VUS-intermediate, T1104P/M1168K and D2819V as VUS (+), and R2027K and G2044A as likely benign. These findings highlight the importance of the variability of VUSs in response to DNA damage before and after Dox-induction and suggest that further investigation is needed to understand the underlying mechanisms.

ADJUBIL: phase II study of adjuvant immunotherapy with STRIDE regimen with/without capecitabine in biliary tract cancers.

Biliary tract cancer is a highly heterogeneous group of gastrointestinal cancers, and the only curative treatment is surgery, which is only applicable at early stages of the malignancy. ADJUBIL, a phase II trial (NCT05239169), aims to evaluate immunotherapy with durvalumab and tremelimumab with or without capecitabine in adjuvant situations for biliary tract cancers. A total of 40 prospective patients will be randomly assigned following surgery, consisting of a two-arm feasibility pilot part with a pick-the-winner design with durvalumab and tremelimumab in combination with or without capecitabine.

This article describes the design of a phase II clinical trial called ADJUBIL, which evaluates the use of immunotherapy (durvalumab and tremelimumab) with or without classic chemotherapy (capecitabine) in biliary tract cancer patients who have undergone curative surgery. This type of treatment is also called adjuvant therapy, meaning it is used after the primary treatment. Biliary tract cancer is a rare type of liver cancer, often diagnosed late. Following surgery, patients may experience an early return of the disease, called tumor relapse. To avoid or delay tumor relapse, patients need extra treatment. Pure chemotherapy (capecitabine) is the standard after curative surgery. For patients with no option for cure, chemotherapy together with new powerful immunotherapy has become standard. This study will recruit 40 adult patients with tumor removal, who will be randomly divided into two groups. Half of them will be treated with immunotherapy only (durvalumab and tremelimumab). The other half will be treated with capecitabine together with immunotherapy. This study will continue for 12 months, but the treatment can be stopped if, for example, the tumor reoccurs or any possible side effect of the therapy is detected. The most effective treatment type will be selected. This type of selection is called pick-the winner.

Concurrent Pathogenic Variants of BRCA1, MUTYH and CHEK2 in a Hereditary Cancer Family.

Concurrent pathogenic variants (PVs) in cancer predisposition genes have been reported in 0.1-2% of hereditary cancer (HC) patients. Determining concurrent PVs is crucial for the diagnosis, treatment, and risk assessment of unaffected family members. Next generation sequencing based diagnostic tests, which are widely used in HCs, enable the evaluation of multiple genes in parallel. We have screened the family members of a patient with bilateral breast cancer who was found to have concurrent PVs in BRCA1 (NM_007294.3;c.5102_5103del, p.Leu1701Glnfs*14) and MUTYH (NM_001128425.1;c.884C>T, p.Pro295Leu). Further analysis revealed concurrent PVs in CHEK2 (NM_007194.4;c.1427C>T, p.Thr476Met) and MUTYH (NM_001128425.1;c.884C>T, p.Pro295Leu) in the maternal uncle of the index case. Eight additional family members were found to have PVs in BRCA1 and MUTYH among 26 tested relatives. The sister and the brother of the index case who were diagnosed with breast and colon cancers, respectively, presented with the same genotype as the index case. Each family member was evaluated individually for clinical care and surveillance. This is the first report describing a family with BRCA1, MUTYH and CHEK2 concurrent PVs. Our findings provide valuable information for the assessment and management considerations for families with concurrent PVs.

Immunohistochemical Evaluation of BAP1 Expression in Breast Cancer with Known BRCA1 and BRCA2 Mutations and Comparison with Histopathological Features.

Introduction. BRCA-mutated breast cancers have specific pathological characteristics. BAP1 is a tumor suppressor gene that is important in many cancers with different pathways. The relationship between BRCA1 mutation and BAP1 immunohistochemical staining is still unclear. Our aim is to determine whether BAP1 immunohistochemical expression indicates BRCA mutation status in breast carcinomas with specific pathological characteristics. In addition, we aim to determine the histopathological characteristics of tumors according to BRCA mutations. Methods. Histomorphology, molecular subtypes and BAP1 immunohistochemical expression patterns of the BRCA1/BRCA2 mutated and non-mutated tumors were evaluated. The BAP1 immunohistochemical stain was applied to nine tumor tissues with the BRCA1 mutation, six tumor tissues with the BRCA2 mutation, and 16 tumor tissues without any BRCA mutation. Pearson's chi square test and the Fisher Freeman Halton test were used to analyze the associations between the datas. The statistical significance was considered as P value of <.05. Results. Immunohistochemical BAP1 loss was not detected in any mutated or non-mutated tumor group. BRCA1 mutated tumors had the statistically highest histopathological grade (P = .04) and BRCA1/2 mutated tumors had significant immunohistochemical triple negative expression pattern (P = .01). Conclusions. Intrinsic and histopathological characteristics may vary between BRCA1 mutated and non-BRCA1 mutated tumors. Also, BAP1 loss was not detected in BRCA mutated breast tumors because of several effects of BAP1 that are non-related with BRCA in the cell cycle.

Determining the accuracy of next generation sequencing based copy number variation analysis in Hereditary Breast and Ovarian Cancer.

BACKGROUND: Copy number variations (CNVs) are commonly associated with malignancies, including hereditary breast and ovarian cancers. Next generation sequencing (NGS) provides solutions for CNV detection in a single run. This study aimed to compare the accuracy of CNV detection by NGS analyzing tool against Multiplex Ligation Dependent Probe Amplification (MLPA). RESEARCH DESIGN AND METHODS: In total, 1276 cases were studied by targeted NGS panels and 691 cases (61 calls in 58 NGS-CNV positive and 633 NGS-CNV negative cases) were validated by MLPA. RESULTS: Twenty-eight (46%) NGS-CNV positive calls were consistent, whereas 33 (54%) calls showed discordance with MLPA. Two cases were detected as SNV by the NGS and CNV by the MLPA analysis. In total, 2% of the cases showed an MLPA confirmed CNV region in BRCA1/2. The results of this study showed that despite the high false positive call rate of the NGS-CNV algorithm, there were no false negative calls. The cases that were determined to be negative by the NGS and positive by the MLPA were actually carrying SNVs that were located on the MLPA probe binding sites. CONCLUSION: The diagnostic performance of NGS-CNV analysis is promising; however, the need for confirmation by different methods remains.

Consistency of variant interpretations among bioinformaticians and clinical geneticists in hereditary cancer panels.

Next-generation sequencing (NGS) is used increasingly in hereditary cancer patients' (HCP) management. While enabling evaluation of multiple genes simultaneously, the technology brings to light the dilemma of variant interpretation. Here, we aimed to reveal the underlying reasons for the discrepancy in the evidence titles used during variant classification according to ACMG guidelines by two different bioinformatic specialists (BIs) and two different clinical geneticists (CGs). We evaluated final reports of 1920 cancer patients and 189 different variants from 285 HCP were enrolled to the study. A total of 173 of these variants were classified as pathogenic (n = 132) and likely pathogenic (n = 41) by the BI and an additional 16 variants, that were classified as VUS by at least one interpreter and their classification would change the clinical management, were compared for their evidence titles between different specialists. The attributed evidence titles and the final classification of the variants among BIs and CGs were compared. The discrepancy between P/LP final reports was 22.5%. The discordance between CGs was 30% whereas the discordance between two BIs was almost 75%. The use of PVS1, PS3, PP3, PP5, PM1, PM2, BP1, BP4 criteria markedly varied from one expert to another. This difference was particularly noticeable in PP3, PP5, and PM1 evidence and mostly in the variants affecting splice sites like BRCA1(NM_007294.4) c.4096 + 1 G > A and CHEK2(NM_007194.4) c.592 + 3 A > T. With recent advancements in precision medicine, the importance of variant interpretations is emerging. Our study shows that variant interpretation is subjective process that is in need of concrete definitions for accurate and standard interpretation.

COVID-19 PCR test performance on samples stored at ambient temperature.

The WHO-named Coronavirus Disease 2019 (COVID-19) infection had become a pandemic within a short time period since it was detected in Wuhan. The outbreak required the screening of millions of samples daily and overwhelmed diagnostic laboratories worldwide. During this pandemic, the handling of patient specimens according to the universal guidelines was extremely difficult as the WHO, CDC and ECDC required cold chain compliance during transport and storage of the swab samples. The aim of this study was to compare the effects of two different storage conditions on the COVID-19 real-time PCR assay on 30 positive nasopharyngeal and/or oropharyngeal samples stored at both ambient temperature (22 ± 2 °C) and +4 °C. The results revealed that all the samples stored at ambient temperature remain PCR positive for at least six days without any false-negative result. In conclusion, transporting and storing these types of swab samples at ambient temperature for six days under resource-limited conditions during the COVID-19 pandemics are acceptable.

Further Expansion of the Mutational Spectrum of 3MC Syndrome: A Novel MASP1 Pathogenic Variant in a Male Patient.

The 3MC syndrome is a rare autosomal recessive syndrome characterized by facial dysmorphism, multiple congenital abnormalities, and postnatal growth deficiency. Hypertelorism, blepharophimosis, blepharoptosis, high-arched eyebrows, and cleft lip/palate compose the facial gestalt, which is the key component for diagnosing the syndrome. Biallelic pathogenic variants in MASP1, COLEC11, and COLEC10 are responsible for 3MC syndrome in which both genotypic and phenotypic heterogeneity is described. To date, 16 homozygous/compound heterozygous pathogenic variations in 27 patients from 22 families have been reported in the MASP1 gene associated with 3MC syndrome. Here, we report a male patient with a novel homozygous pathogenic variant in MASP1 in whom macrocephaly, pyloric stenosis, and prenatal findings including polyhydramnios, aortic dilatation, and intracranial cysts beside the distinctive facial features were detected. Reporting detailed clinical and molecular findings in patients is pivotal in terms of enabling the phenotypic and genotypic spectrum of this rare syndrome to be delineated.

Concurrent pathogenic variations in patients with hereditary cancer syndromes.

Cancer is a multifactorial disorder; however, 5-10% of all cancers show hereditary background. In recent years many targeted next generation sequencing panels comprising cancer predisposition genes have been developed and used for diagnostic purposes in patients with increased cancer risk. Screening multiple genes at a time allows multiple variants in different genes to be detected as well. This study aims to determine the cases with concurrent mutations in different hereditary cancer predisposition genes and how they are clinically affected. Here, we screened 1090 index cases by next generation sequencing based hereditary cancer panels and evaluated the reflection of multiple variations on the phenotype. We detected 11 (1%) cases with pathogenic variants in more than one gene. These concurrent variations occurred mostly in BRCA1/2 (7/11) accompanied with MUTYH, ATM, CHECK2, NBN, and RAD50. In addition, MUTYH&ATM, NBN&MSH6, MUTYH&CHEK2 double heterozygous cases were detected. Moreover, we identified a case with three heterozygous variations in CDH1, MUTYH, and CHEK2. These patients presented malignancies that were mostly related to pathogenic variations they carried. Although they are rare, defining double heterozygous cases is important for managing appropriate therapy and accurate genetic consulting for the patients and family members.

Severe acute respiratory syndrome coronavirus (SARS-CoV-2) is not detected in the vagina: A prospective study.

OBJECTIVE: To determine whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is present in the vagina of women diagnosed with coronavirus disease-19 (COVID-19) pneumonia. STUDY DESIGN: The study was conducted prospectively in a university affiliated hospital. Forty-one women of reproductive age whose nasopharyngeal PCR test were positive for SARS-CoV-2 and clinically diagnosed with pneumonia were included in the study. Vaginal swabs were obtained for SARS-CoV-2 PCR tests when the patients were admitted to the inpatient service before pneumonia treatment was initiated. RESULTS: Vaginal swab samples of 38 patients were analysed with SARS-CoV-2 PCR tests. None of the vaginal swabs were positive for SARS-CoV-2. CONCLUSIONS: SARS-CoV-2 does not infect the vagina of women diagnosed with SARS-CoV-2 pneumonia.

Seroprevalence of coronavirus disease 2019 (COVID-19) among health care workers from three pandemic hospitals of Turkey.

COVID-19 is a global threat with an increasing number of infections. Research on IgG seroprevalence among health care workers (HCWs) is needed to re-evaluate health policies. This study was performed in three pandemic hospitals in Istanbul and Kocaeli. Different clusters of HCWs were screened for SARS-CoV-2 infection. Seropositivity rate among participants was evaluated by chemiluminescent microparticle immunoassay. We recruited 813 non-infected and 119 PCR-confirmed infected HCWs. Of the previously undiagnosed HCWs, 22 (2.7%) were seropositive. Seropositivity rates were highest for cleaning staff (6%), physicians (4%), nurses (2.2%) and radiology technicians (1%). Non-pandemic clinic (6.4%) and ICU (4.3%) had the highest prevalence. HCWs in "high risk" group had similar seropositivity rate with "no risk" group (2.9 vs 3.5 p = 0.7). These findings might lead to the re-evaluation of infection control and transmission dynamics in hospitals.

Does sampling saliva increase detection of SARS-CoV-2 by RT-PCR? Comparing saliva with oro-nasopharyngeal swabs.

The gold standard method in the diagnosis of SARS-CoV-2 infection is the detection of viral RNA in the nasopharyngeal sample by RT-PCR. Recently, saliva samples have been suggested as an alternative sample. In the present study, we aimed to compare RT-PCR results in nasopharyngeal, oro-nasopharyngeal and saliva samples of COVID-19 patients. 98 of 200 patients were positive in RT-PCR analysis performed before the hospitalization. On day 0, at least one sample was positive in 67 % of 98 patients. The positivity rate was 83 % for both oro-nasopharyngeal and nasopharyngeal samples, while it was 63 % for saliva samples (p < 0.001). On day 5, RT-PCR was performed in 59 patients, 34 % had at least one positive result. The positivity rate was 55 % for both saliva and nasopharyngeal samples, while it was 60 % for oro-nasopharyngeal samples. Our study shows that the sampling saliva does not increase the sensitivity of RT-PCR tests at the early stages of infection. However, on the 5th day, viral RNA detection rates in saliva were similar to nasopharyngeal and oro-nasopharyngeal samples. In conclusion, we suggest that, in patients receiving treatment, RT-PCR in saliva, in addition to the standard samples, is important to determine the isolation period and control transmission.

Germline pathogenic variant spectrum in 25 cancer susceptibility genes in Turkish breast and colorectal cancer patients and elderly controls.

Inherited pathogenic variants account for 5% to 10% of all breast cancer (BC) and colorectal cancer (CRC) cases. Here, we sought to profile the pathogenic variants in 25 cancer susceptibility genes in Turkish population. Germline pathogenic variants were screened in 732 BC patients, 189 CRC patients and 490 cancer-free elderly controls, using next-generation sequencing-based multigene panel testing and multiplex ligation-dependent probe amplification testing. Pathogenic variants were detected in 17.2% of high-risk BC patients and 26.4% of high-risk CRC patients. More than 95% of these variants were clinically actionable. BRCA1/2 and mismatch repair genes (MLH1, MSH2 and MSH6) accounted for two-thirds of all pathogenic variants detected in high-risk BC and CRC patients, respectively. Pathogenic variants in PALB2, CHEK2, ATM and TP53 were also prevalent in high-risk BC patients (4.5%). BRCA1 exons 17-18 deletion and CHEK2 c.592+3A>T were the most common variants predisposing to BC, and they are likely to be founder variants. Three frequent MUTYH pathogenic variants (c.884C>T, c.1437_1439delGGA and c.1187G>A) were responsible for all MUTYH biallelic cases (4.4% of high-risk CRC patients). The total pathogenic variant frequency was very low in controls (2.4%) and in low-risk BC (3.9%) and CRC (6.1%) patients. Our study depicts the pathogenic variant spectrum and prevalence in Turkish BC and CRC patients, guiding clinicians and health authorities for genetic testing applications and variant classification in Turkish population.

Relationship between SIRT1 gene expression level and disease in age-related cataract cases

Background/aim: Age-related cataract is the most important visual impairment all over the world. Epigenetic modifications, especially overexpression of histone deacetylases, have become the focus of interest for cataract development in recent years. Sirtuin 1 (SIRT1), a class II histone deacetylase and a member of the sirtuin family, is one of the best-characterized histone deacetylases and has a pivotal role in age-related diseases. However, the association of SIRT1 with age-related cataracts has not yet been fully elucidated. Therefore, we aimed to determine the expression of SIRT1 in age-related cataract patients. Materials and methods: Expressions of SIRT1 were evaluated by quantitative polymerase chain reaction (qPCR) in patients and healthy controls. RNA samples were collected from the anterior capsule and peripheral blood samples of age-related cataract patients. Human lens epithelial cell line B3 and peripheral blood samples of healthy subjects were used as controls. Results: We determined that the expression of SIRT1 in blood and anterior capsule samples increased significantly compared to the control group (P < 0.05). Conclusion: The expression level of SIRT1 plays a vital role in the development of age-related cataract and it can be used as a biomarker. Thus, SIRT1 inhibitors can be used in the treatment of age-related cataract disease.

Comparison of anthropometric indices in predicting metabolic syndrome components in children.

BACKGROUND: The International Diabetes Federation (IDF) defines metabolic syndrome among children. In addition to the abdominal obesity (waist circumference ≥90 percentile of locally representative sample), the risk criteria for metabolic syndrome includes elevated blood pressure, hypertriglyceridemia, low high-density lipoprotein cholesterol (HDL-C) levels, and elevated fasting glucose. In this study, we compared the association of anthropometric indices with metabolic syndrome risk criteria in 9-year-old children. METHODS: A cross-sectional study of 9-year-old children (n=1,194) was performed in 2007-2008. Using the international cutoff points and percentiles, we determined subjects with elevated blood pressure, hypertriglyceridemia (≥90 percentile), low HDL-C (≤ 10 percentile), and elevated fasting glucose (≥100 mg/dL). We compared several anthropometric indices [triceps skin-fold thickness (SFT), waist circumference, hip circumference, waist-to-hip ratio, body mass index (BMI) and waist-to-height ratio] and their association with metabolic syndrome risk criteria other than waist circumference among 9-year-old children. RESULTS: Metabolic syndrome risk criteria were commonly observed among 9-year-old children: 244 students (20.4%) had hypertriglyceridemia and/or low HDL-C, 362 students (30.3%) had elevated blood pressure, and 254 (21.3%) students were overweight or obese. Among covariates of SFT, BMI, waist circumference, waist-to-hip, or waist-to-height ratio categories, the BMI category was the only significant predictor of having two or more metabolic syndrome risk variables [odds ratio (OR)=3.5, 95% confidence interval (CI) 1.69-7.41, P=0.001 for boys and OR=4.7, 95% CI 1.61-13.55, P=0.005 for girls]. CONCLUSIONS: Assessing anthropometric indices is crucial for early detection and prevention of metabolic syndrome among children and adolescents. Age- and sex-specific cutoff points of BMI can be used to screen for the metabolic syndrome and related risk criteria among 9-year-old children.

Sex hormones, insulin resistance and high-density lipoprotein cholesterol levels in children.

Cardiovascular risk starts early in life; therefore, it is of interest to clarify the relation between puberty, sex hormones, insulin resistance and lipid levels in children. This is a cross sectional study of 365 school students (8-18 years of age). We analyzed the associations of sex hormones (testosterone, free androgen index, estradiol, free estradiol index) and sex hormone-binding globulin (SHBG) with insulin resistance and lipid levels. Analyses were performed in prepubertal versus adolescent girls and boys. Among prepubertal boys, estradiol was significantly associated with increased log homeostasis model assessment-estimated insulin resistance (HOMA-IR; B = 0.9, model R(2) = 0.62, p < 0.001) and insulin levels (B = 0.8, model R(2) = 0.58, p < 0.001). Testosterone was associated with increased high-density lipoprotein cholesterol (HDL-C) levels among prepubertal boys (B = 10, model R(2) = 0.42, p = 0.04). Among adolescent girls, SHBG was significantly associated with decreased HOMA-IR (B = -0.8, model R(2) = 0.34, p = 0.01) and insulin levels (B = -0.7, model R(2) = 0.34, p = 0.01). SHBG was also related to increased HDL-C levels among prepubertal (B = 24, model R(2) = 0.42, p = 0.047) and adolescent girls (B = 21, model R(2) = 0.44, p = 0.002). In conclusion, sex hormone levels and SHBG have important effects on HDL-C and insulin resistance among children and adolescents.

Sex hormones and metabolic syndrome in children and adolescents.

Cardiovascular risk starts early in life, yet the patterns of changes in metabolic syndrome (MS) during puberty and normal development have not been completely defined. Sex hormones are shown to play a pivotal role in the modulation of insulin resistance and MS. Our aim is to clarify the relation between sex hormones and MS in normal children and adolescents. This is a cross-sectional study of 365 (8-12 and 14-18 years old) school students. We analyzed the associations of sex hormones (testosterone, free androgen index, estradiol, free estradiol index [FEI], and sex hormone-binding globulin [SHBG]) with cardiovascular risk factors and MS. Prevalence of MS varied depending on the definition, and 33 (9%) students had MS based on at least 1 definition of MS. Frequency of MS doubled among 14- to 18-year-old adolescents compared with 8- to 12-year-old children (12.4% vs 5.6%, P = .02). Adolescent boys and girls with MS had significantly lower SHBG levels compared with controls. Adolescent boys with MS also had significantly higher FEI levels compared with controls. Logistic regression analysis was performed to find the predictors of MS. Among covariates of age, estradiol, testosterone, free androgen index, and FEI, SHBG was the only significant predictor of MS (B = -0.3, odds ratio = 0.8, 95% confidence interval for odds ratio are 0.64 and 0.92, P = .005, Nagelkarke R(2) = 0.48) in adolescent boys. In conclusion, sex hormone levels and androgen/estrogen balance may play an important role in determining MS and future cardiovascular risk among children and adolescents.

Blood pressure and anthropometric measurements in Albanian versus Turkish children and adolescents.

OBJECTIVE: Understanding intercultural, regional and interracial differences in blood pressure and anthropometric indices may help to determine the contributors of mortality from coronary artery disease in different populations. DESIGN AND SETTING: In this article we used data collected from two different countries: (a) Survey on school children from Istanbul, Turkey, (b) Cross Sectional Study of Albanian school children. RESULTS: We compared age- and gender-matched Albanian and Turkish children and adolescents (age 11-12 y and 15-17 y) living in 2 different countries with regard to blood pressure, weight, height, body mass index (BMI), waist circumference, hip circumference, waist-to-hip (WHpR) and waist-to-height (WHtR) ratio. We observed significant differences in blood pressure, BMI and other anthropometric indices among age- and gender-matched subjects from the two countries. Birthweight was lower among Turkish children and adolescents compared to Albanian subjects (3258 vs. 3510 g, P < 0.01). Turkish children had lower BMI and WHtR compared to Albanian children, while adolescents displayed similar BMI and WHtR. Paradoxically, Turkish children and adolescents displayed higher systolic blood pressures compared to Albanian children and adolescents. Significant differences were observed in the dietary intake and physical activity, which may partially explain the differences in blood pressure and anthropometric indices. CONCLUSIONS: The socio-economic, dietary and physical activity status may account for significant differences in blood pressure and anthropometric indices of children and adolescents living in two different countries. Future studies should focus on the ethnic differences in the definition and prevention of cardiovascular risks among children and adolescents.