GATK hard filtering: tunable parameters to improve variant calling for next generation sequencing targeted gene panel data

BACKGROUND: NGS technology represents a powerful alternative to the standard Sanger sequencing in the context of clinical setting. The proprietary software that are generally used for variant calling often depend on preset parameters that may not fit in a satisfactory manner for different genes. GATK, which is widely used in the academic world, is rich in parameters for variant calling. However the self-adjusting parameter calibration of GATK requires data from a large number of exomes. When these are not available, which is the standard condition of a diagnostic laboratory, the parameters must be set by the operator (hard filtering). The aim of the present paper was to set up a procedure to assess the best parameters to be used in the hard filtering of GATK. This was pursued by using classification trees on true and false variants from simulated sequences of a real dataset data. RESULTS: We simulated two datasets, with different coverages, including all the sequence alterations identified in a real dataset according to their observed frequencies. Simulated sequences were aligned with standard protocols and then regression trees were built up to identify the most reliable parameters and cutoff values to discriminate true and false variant calls. Moreover, we analyzed flanking sequences of region presenting a high rate of false positive calls observing that such sequences present a low complexity make up. CONCLUSIONS: Our results showed that GATK hard filtering parameter values can be tailored through a simulation study based-on the DNA region of interest to ameliorate the accuracy of the variant calling

Neutralizing antibodies to Omicron after the fourth SARS-CoV-2 mRNA vaccine dose in immunocompromised patients highlight the need of additional boosters

IntroductionImmunocompromised patients have been shown to have an impaired immune response to COVID-19 vaccines.MethodsHere we compared the B-cell, T-cell and neutralizing antibody response to WT and Omicron BA.2 SARS-CoV-2 virus after the fourth dose of mRNA COVID-19 vaccines in patients with hematological malignancies (HM, n=71), solid tumors (ST, n=39) and immune-rheumatological (IR, n=25) diseases. The humoral and T-cell responses to SARS-CoV-2 vaccination were analyzed by quantifying the anti-RBD antibodies, their neutralization activity and the IFN-γ released after spike specific stimulation.ResultsWe show that the T-cell response is similarly boosted by the fourth dose across the different subgroups, while the antibody response is improved only in patients not receiving B-cell targeted therapies, independent on the pathology. However, 9% of patients with anti-RBD antibodies did not have neutralizing antibodies to either virus variants, while an additional 5.7% did not have neutralizing antibodies to Omicron BA.2, making these patients particularly vulnerable to SARS-CoV-2 infection. The increment of neutralizing antibodies was very similar towards Omicron BA.2 and WT virus after the third or fourth dose of vaccine, suggesting that there is no preferential skewing towards either virus variant with the booster dose. The only limited step is the amount of antibodies that are elicited after vaccination, thus increasing the probability of developing neutralizing antibodies to both variants of virus.DiscussionThese data support the recommendation of additional booster doses in frail patients to enhance the development of a B-cell response directed against Omicron and/or to enhance the T-cell response in patients treated with anti-CD20

KRAS-Driven Lung Adenocarcinoma and B Cell Infiltration: Novel Insights for Immunotherapy

Non-small-cell lung cancer, histologically classified into adenocarcinoma (AD) and squamous cell carcinoma, is one of the most deadly malignancies worldwide. Lung AD (LUAD) could benefit of a plethora of target therapies and, in the last few years, also of immunotherapies. Here we focused on a real-life cohort of LUAD and The Cancer Genome Atlas (TCGA)-LUAD dataset aiming to gain insights into the immune contexture of such a malignancy. We explored the mutational status of 41 genes and the expression of 94 genes, related to immune-checkpoint, inflammation, and stromal microenvironment. Surprisingly, we found that our cohort has a very low mutational burden if we consider our panel as its surrogate. Regarding gene expression data, we identified 31 genes significantly deregulated in tumor tissues compared with a pool of normal samples. Unsupervised hierarchical clustering of the deregulated genes is able to identify two clusters of tumor samples, differently enriched in alterations in actionable genes. In particular, we identified a cluster enriched in patients carrying KRAS alterations. In silico deconvolution, that is the inferring of tumor microenvironment composition by gene expression data, through TIMER algorithm has been performed to explore immune microenvironment. Estimation performed on our gene expression matrix showed that B cell infiltration is lower in the KRAS-mutated enriched cluster, as in the TCGA-LUAD dataset. Such a finding has been validated in situ through immunohistochemistry in an independent cohort. Moreover, cases in LUAD-TCGA with low B cell infiltration have a significantly worse overall survival than those with higher levels. In the real-life cohort we observed that cases belonging to cluster enriched in KRAS-mutated patients have a poor outcome. LUAD driven by KRAS mutation represents an unmet clinical need, being refractory to pharmacological inhibition. Our results link KRAS mutations to B cell infiltration. Thus, the present findings could be helpful in a better definition of immunotherapeutic approaches for KRAS mutated patients

Relationships between Sex and Adaptation to Physical Exercise in Young Athletes: A Pilot Study

The purpose of this study was to compare the redox, hormonal, metabolic, and lipid profiles of female and male basketball players during the seasonal training period, compared to their relative sedentary controls. 20 basketball players (10 female and 10 male) and 20 sedentary controls (10 female and 10 male) were enrolled in the study. Oxidative stress, adiponectin level, and metabolic profile were determined. Male and female athletes showed an increased antioxidant capacity (27% for males; 21% for females) and lactate level (389% for males; 460% for females) and reduced salivary cortisol (25% for males; 51% for females) compared to the sedentary controls. Moreover, a peculiar metabolite (in particular, amino acids and urea), hormonal, and lipidic profile were highlighted in the two groups of athletes. Female and male adaptations to training have several common traits, such as antioxidant potential enhancement, lactate increase, and activation of detoxifying processes, such as the urea cycle and arachidonic pathways as a response to inflammation. Moreover, we found different lipid and amino acid utilization related to sex. Deeper investigation could help coaches in developing training programs based on the athletes’ sex in order to reduce the drop-out rate of sporting activity by girls and fight the gender stereotypes in sport that also have repercussions in social fields

Different methylation and MicroRNA expression pattern in male and female familial breast cancer

Epigenetic regulation, has been very scarcely explored in familial breast cancer (BC). In the present study RASSF1A and RAR beta promoter methylation and miR17, miR21, miR 124, and let-7a expression were investigated to highlight possible differences of epigenetic regulation between male and female familial BC, also in comparison with sporadic BC. These epigenetic alterations were studied in 56 familial BC patients (27 males and 29 females) and in 16 female sporadic cases. RASSF1A resulted more frequently methylated in men than women (76% vs. 28%, respectively, P=0.0001), while miR17 and let-7a expression frequency was higher in women than in men (miR17: 66% in women vs. 41% in men, P<0.05; let-7a: 45% in women vs. 15% in men, P=0.015). RASSF1A methylation affected 27.6% of familial BC while 83% of familial cases showed high expression of the gene (P=0.025); on the contrary, only 17% of familial BC presented RAR beta methylation and 55% of familial cases overexpressed this gene (P=0.005). Moreover, miR17, miR21, and let-7a resulted significantly overexpressed in familial compared to sporadic BC. RASSF1A overexpression (86% vs. 65%, P=0.13) and RAR beta overexpression (57% vs. 32%, P=0.11) were higher in BRCA1/2 carriers even if not statistical significance was reached. BRCA mutation carriers also demonstrated significant overexpression of: miR17 (93% vs. 35%, P=0.0001), let-7a (64% vs. 16%, P=0.002), and of miR21 (100% vs. 65%, P=0.008). In conclusion, the present data suggest the involvement of RASSF1A in familial male BC, while miR17 and let-7a seem to be implied in familial female BC. J. Cell. Physiol. 228: 12641269, 2013. (c) 2012 Wiley Periodicals, Inc

Modulation of Plasma Proteomic Profile by Regular Training in Male and Female Basketball Players: A Preliminary Study

Monitoring fatigue and recovery during training periods contributes to identifying the best training methods to achieve sports performance. To date, little is known about sex-related differences in sports adaptations. The aim of the present study is to identify sex-related sports adaptation proteins in female basketball players and male basketball players using proteomics approach on plasma samples withdrawn from athletes during in-season training period but far from a competition. A cohort of 20 professional basketball players, 10 female (BF) and 10 male (BM), and 20 sedentary male (10 CM) and female (10 CF) as control, of comparable age and BMI, were involved in this study. Protein profiles of plasma samples obtained from BM, BF, CM, and CF were analyzed by two-dimensional electrophoresis (2-DE). Differentially expressed proteins were identified by mass spectrometry. The computational 2-DE gel image analysis pointed out 33 differentially expressed protein spots (ANOVA p-value < 0.05) and differences between male and female basketball players are more evident among the players than controls. The expression profile of 54.5% of the total proteins is affected by sports activity. Furthermore, 14 proteins are differentially expressed in basket female players in comparison with their relative controls while seven are differentially expressed in basket male players in comparison with their controls. In conclusion, we identify in female athletes a reduction in proteins related to transcription regulation, most of these modulate chronic inflammation confirming the anti-inflammatory effect of regular training in female muscle metabolism. In male and female athletes, we found a decrease in Transthyretin involved in muscle homeostasis and regeneration and Dermcidin a stress-induced myokine linked to inflammatory and it will be interesting to fully understand the role of its different isoforms in male and female skeletal muscle contraction