The analytical algorithm of Lynch syndrome (LS) is increasingly complex. BRAF V600E mutation and MLH1 promoter hypermethylation have been proposed as a screening tool for the identification of LS. The aim of this study was to assess the clinical usefulness and cost-effectiveness of both somatic alterations to improve the yield of the diagnostic algorithm of LS. A total of 122 colorectal tumors from individuals with family history of colorectal cancer that showed microsatellite instability and/or loss of mismatch repair (MMR) protein expression were studied. MMR germline mutations were detected in 57 cases (40 MLH1, 15 MSH2 and 2 MSH6). BRAF V600E mutation was assessed by single-nucleotide primer extension. MLH1 promoter hypermethylation was assessed by methylation-specific multiplex ligation-dependent probe amplification in a subset of 71 cases with loss of MLH1 protein. A decision model was developed to estimate the incremental costs of alternative case-finding methods for detecting MLH1 mutation carriers. One-way sensitivity analysis was performed to assess robustness of estimations. Sensitivity of the absence of BRAF mutations for depiction of LS patients was 96% (23/24) and specificity was 28% (13/47). Specificity of MLH1 promoter hypermethylation for depiction of sporadic tumors was 66% (31/47) and sensitivity of 96% (23/24). The cost per additional mutation detected when using hypermethylation analysis was lower when compared with BRAF study and germinal MLH1 mutation study. Somatic hypermethylation of MLH1 is an accurate and cost-effective pre-screening method in the selection of patients that are candidates for MLH1 germline analysis when LS is suspected and MLH1 protein expression is absent.

Dermal neurofibromas (dNFs) are benign tumors of the peripheral nervous system typically associated with Neurofibromatosis type 1 (NF1) patients. Genes controlling the integrity of the DNA are likely to influence the number of neurofibromas developed because dNFs are caused by somatic mutational inactivation of the NF1 gene, frequently evidenced by loss of heterozygosity (LOH). We performed a comprehensive analysis of the prevalence and mechanisms of LOH in dNFs. Our study included 518 dNFs from 113 patients. LOH was detected in 25% of the dNFs (N = 129). The most frequent mechanism causing LOH was mitotic recombination, which was observed in 62% of LOH-tumors (N = 80), and which does not reduce the number of NF1 gene copies. All events were generated by a single crossover located between the centromere and the NF1 gene, resulting in isodisomy of 17q. LOH due to the loss of the NF1 gene accounted for a 38% of dNFs with LOH (N = 49), with deletions ranging in size from ∼80 kb to ∼8 Mb within 17q. In one tumor we identified the first example of a neurofibroma-associated second-hit type-2 NF1 deletion. Analysis of the prevalence of mechanisms causing LOH in dNFs in individual patients (possibly under genetic control) will elucidate whether there exist interindividual variation. Hum Mutat 32:78–90, 2011.

From the beginning, the COVID-19 pandemic increased ICU workloads and created exceptionally difficult ethical dilemmas. ICU staff around the world have been subject to high levels of moral stress, potentially leading to mental health problems. There is only limited evidence on moral distress levels and coping styles among Spanish ICU staff, and how they influenced health professionals’ mental health during the pandemic. To assess moral distress, related mental health problems (anxiety and depression), and coping styles among ICU staff during the first wave of the COVID-19 pandemic in Spain. Cross-sectional. The study setting consisted of intensive care unit and areas converted into intensive care units in public and private hospitals. A total of 434 permanent and temporary intensive care staff (reassigned due to the pandemic from other departments to units) answered an online questionnaire between March and June 2020. Sociodemographic and job variables, moral distress, anxiety, depression, and coping mechanisms were anonymously evaluated through a self-reported questionnaire. Descriptive and correlation analyses were conducted and multivariate linear regression models were developed to explore the predictive ability of moral distress and coping on anxiety and depression. Moral distress during the pandemic is determined by situations related to the patient and family, the intensive care unit, and resource management of the organisations themselves. intensive care unit staff already reached moderate levels of moral distress, anxiety, and depression during the first wave of the pandemic. Temporary staff (redeployed from other units) obtained higher scores in these variables (p = 0.04, p = 0.038, and p = 0.009, respectively) than permanent staff, as well as in greater intention to leave their current position (p = 0.03). This intention was also stronger in health staff working in areas converted into intensive care units (45.2%) than in normal intensive care units (40.2%) (p = 0.02). Moral distress, coupled with primarily avoidance-oriented coping styles, explains 37% (AdR²) of the variance in anxiety and 38% (AdR²) of the variance in depression. Our study reveals that the emotional well-being of intensive care unit staff was already at risk during the first wave of the pandemic. The moral distress they experienced was related to anxiety and depression issues, as well as the desire to leave the profession, and should be addressed, not only in permanent staff, but also in temporary staff, redeployed to these units as reinforcement workers.

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder affecting 1:3,500 individuals. Disease expression is highly variable and complications are diverse. However, currently there is no specific treatment for the disease. NF1 is caused by mutations in the NF1 gene, approximately 2.1% of constitutional mutations identified in our population are deep intronic mutations producing the insertion of a cryptic exon into the mature mRNA. We used antisense morpholino oligomers (AMOs) to restore normal splicing in primary fibroblast and lymphocyte cell lines derived from six NF1 patients bearing three deep intronic mutations in the NF1 gene (c.288+2025T>G, c.5749+332A>G, and c.7908‐321C>G). AMOs were designed to target the newly created 5′ splice sites to prevent the incorporation of cryptic exons. Our results demonstrate that AMO treatment effectively restored normal NF1 splicing at the mRNA level for the three mutations studied in the different cell lines analyzed. We also found that AMOs had a rapid effect that lasted for several days, acting in a sequence‐specific manner and interfering with the splicing mechanism. Finally, to test whether the correction of aberrant NF1 splicing also restored neurofibromin function to wild‐type levels, we measured the amount of Ras‐GTP after AMO treatment in primary fibroblasts. The results clearly show an AMO‐dependent decrease in Ras‐GTP levels, which is consistent with the restoration of neurofibromin function. To our knowledge this is the first time that an antisense technique has been usedsuccessfully to correct NF1 mutations opening the possibility of a therapeutic strategy for this type of mutation not only for NF1 but for other genetic disorders. Hum Mutat 30, 454–462, 2009.