L1 elements are mammalian retrotransposons contributing to genome evolution and causing rare mutations in human. We describe a de novo insertion of an L1 element into the dystrophin gene resulting in skipping of exon 44 and causing Duchenne muscular dystrophy in a boy. The L1 element was rearranged due to the twin-priming mechanism, but contrary to all described L1 rearrangements the 5' region of the inverted L1 sequence ended within the poly(A) tail of the element. Furthermore, the target site for the insertion was located only 87 bp from the insertion site in another patient described previously. These findings can contribute to the understanding of the mechanisms of L1 element rearrangement, and may support the notion that some subregions of the human genome could be preferred targets for retroelements using the L1 enzymatic machinery.

• MeSH
• Child MeSH
• Muscular Dystrophy, Duchenne genetics   MeSH
• Dystrophin genetics   MeSH
• Exons genetics   MeSH
• Financing, Organized MeSH
• Genetic Predisposition to Disease genetics   MeSH
• Genetic Variation immunology   MeSH
• Humans MeSH
• Chromosome Mapping MeSH
• Evidence-Based Medicine MeSH
• Molecular Sequence Data MeSH
• DNA Mutational Analysis MeSH
• Retroelements genetics   MeSH
• Base Sequence MeSH
• DNA Transposable Elements genetics   MeSH
• Bias MeSH
• Check Tag
• Child MeSH
• Humans MeSH
• Male MeSH

Molecular testing of EGFR is required to predict the response likelihood to targeted therapy in non-small cell lung cancer. Analysis of circulating tumor DNA in plasma may complement limitations of tumor tissue. This study evaluated the interlaboratory performance and reproducibility of a real-time PCR EGFR mutation test (cobas EGFR Mutation Test v2) to detect EGFR variants in plasma. Fourteen laboratories received two identical panels of 27 single-blinded plasma samples. Samples were wild type or spiked with plasmid DNA to contain seven common EGFR variants at six predefined concentrations from 50 to 5000 copies per milliliter. The circulating tumor DNA was extracted by a cell-free circulating DNA sample preparation kit (cobas cfDNA Sample Preparation Kit), followed by duplicate analysis with the real-time PCR EGFR mutation test (Roche Molecular Systems, Pleasanton, CA). Lowest sensitivities were obtained for the c.2156G>C p.(Gly719Ala) and c.2573T>G p.(Leu858Arg) variants for the lowest target copies. For all other variants, sensitivities varied between 96.3% and 100.0%. All specificities were 98.8% to 100.0%. Coefficients of variation indicated good intralaboratory and interlaboratory repeatability and reproducibility but increased for decreasing concentrations. Prediction models revealed a significant correlation for all variants between the predefined copy number and the observed semiquantitative index values, which reflect the samples' plasma mutation load. This study demonstrates an overall robust performance of the real-time PCR EGFR mutation test kit in plasma. Prediction models may be applied to estimate the plasma mutation load for diagnostic or research purposes.

• MeSH
• Algorithms MeSH
• Circulating Tumor DNA blood   genetics   MeSH
• ErbB Receptors blood   genetics   MeSH
• Gene Dosage MeSH
• Real-Time Polymerase Chain Reaction methods   MeSH
• Humans MeSH
• Models, Genetic MeSH
• Mutation genetics   MeSH
• Sensitivity and Specificity MeSH
• Bias MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Multicenter Study MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH
• Geographicals
• Europe MeSH

Mutations in human genes can be responsible for inherited genetic disorders and cancer. Mutations can arise due to environmental factors or spontaneously. It has been shown that certain DNA sequences are more prone to mutate. These sites are termed hotspots and exhibit a higher mutation frequency than expected by chance. In contrast, DNA sequences with lower mutation frequencies than expected by chance are termed coldspots. Mutation hotspots are usually derived from a mutation spectrum, which reflects particular population where an effect of a common ancestor plays a role. To detect coldspots/hotspots unaffected by population bias, we analysed the presence of germline mutations obtained from HGMD database in the 5-nucleotide segments repeatedly occurring in genes associated with common inherited disorders, in particular, the PAH, LDLR, CFTR, F8, and F9 genes. Statistically significant sequences (mutational motifs) rarely associated with mutations (coldspots) and frequently associated with mutations (hotspots) exhibited characteristic sequence patterns, e.g. coldspots contained purine tract while hotspots showed alternating purine-pyrimidine bases, often with the presence of CpG dinucleotide. Using molecular dynamics simulations and free energy calculations, we analysed the global bending properties of two selected coldspots and two hotspots with a G/T mismatch. We observed that the coldspots were inherently more flexible than the hotspots. We assume that this property might be critical for effective mismatch repair as DNA with a mutation recognized by MutSα protein is noticeably bent.

• MeSH
• DNA chemistry   genetics   MeSH
• Factor VIII genetics   MeSH
• Genetic Predisposition to Disease MeSH
• Nucleic Acid Conformation MeSH
• Receptors, LDL genetics   MeSH
• Humans MeSH
• Models, Molecular MeSH
• Nucleotide Motifs MeSH
• Cystic Fibrosis Transmembrane Conductance Regulator genetics   MeSH
• Molecular Dynamics Simulation MeSH
• Germ-Line Mutation * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Germline GATA2 mutations cause cellular deficiencies with high propensity for myeloid disease. We investigated 426 children and adolescents with primary myelodysplastic syndrome (MDS) and 82 cases with secondary MDS enrolled in 2 consecutive prospective studies of the European Working Group of MDS in Childhood (EWOG-MDS) conducted in Germany over a period of 15 years. Germline GATA2 mutations accounted for 15% of advanced and 7% of all primary MDS cases, but were absent in children with MDS secondary to therapy or acquired aplastic anemia. Mutation carriers were older at diagnosis and more likely to present with monosomy 7 and advanced disease compared with wild-type cases. For stratified analysis according to karyotype, 108 additional primary MDS patients registered with EWOG-MDS were studied. Overall, we identified 57 MDS patients with germline GATA2 mutations. GATA2 mutations were highly prevalent among patients with monosomy 7 (37%, all ages) reaching its peak in adolescence (72% of adolescents with monosomy 7). Unexpectedly, monocytosis was more frequent in GATA2-mutated patients. However, when adjusted for the selection bias from monosomy 7, mutational status had no effect on the hematologic phenotype. Finally, overall survival and outcome of hematopoietic stem cell transplantation (HSCT) were not influenced by mutational status. This study identifies GATA2 mutations as the most common germline defect predisposing to pediatric MDS with a very high prevalence in adolescents with monosomy 7. GATA2 mutations do not confer poor prognosis in childhood MDS. However, the high risk for progression to advanced disease must guide decision-making toward timely HSCT.

• MeSH
• Chromosome Aberrations MeSH
• Child MeSH
• Phenotype MeSH
• Genetic Predisposition to Disease MeSH
• Deafness genetics   MeSH
• Kaplan-Meier Estimate MeSH
• Clinical Trials, Phase III as Topic MeSH
• Humans MeSH
• Chromosomes, Human, Pair 1 genetics   MeSH
• Chromosomes, Human, Pair 7 genetics   MeSH
• Chromosomes, Human, Pair 8 genetics   MeSH
• Adolescent MeSH
• Young Adult MeSH
• DNA Mutational Analysis MeSH
• Myelodysplastic Syndromes epidemiology   etiology   genetics   pathology   MeSH
• Child, Preschool MeSH
• Prevalence MeSH
• Prognosis MeSH
• Prospective Studies MeSH
• Immunologic Deficiency Syndromes genetics   MeSH
• GATA2 Transcription Factor deficiency   genetics   MeSH
• Age of Onset MeSH
• Selection Bias MeSH
• Germ-Line Mutation MeSH
• Check Tag
• Child MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Male MeSH
• Child, Preschool MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

• MeSH
• Diabetes Mellitus, Type 2 etiology   genetics   MeSH
• Genetic Heterogeneity MeSH
• Insulin Resistance genetics   MeSH
• Humans MeSH
• Mutation MeSH
• Polymorphism, Genetic MeSH
• Risk Factors MeSH
• Bias MeSH
• Check Tag
• Humans MeSH
• Publication type
• Review MeSH

Whole genome amplification replicates the entire DNA content of a sample and can thus help to circumvent material limitations when insufficient DNA is available for planned genetic analyses. However, there are conflicting data in the literature whether whole genome amplification introduces bias or reflects precisely the spectrum of starting DNA. We analyzed the origins of discrepancies in KRAS (Kirsten rat sarcoma viral oncogene homolog gene) mutation detection in six of ten samples amplified using the GenomePlex® Tissue Whole Genome Amplification kit 5 (WGA5; Sigma-Aldrich, St. Louis, MO, USA) and KRAS StripAssay® (KRAS SA; ViennaLab Diagnostics, Vienna, Austria). We undertook reextraction, reamplification, retyping, authentication, reanalysis, and reinterpretation to determine whether the discrepancies originated during the preanalytical, analytical, and/or interpretative phase of genotyping. We conclude that a combination of glass slide/sample heterogeneity and biased amplification due to stochastic effects in the early phases of whole genome amplification (WGA) may have adversely affected the results obtained. Our findings are relevant for both forensic genetics testing and massively parallel sequencing using preamplification.

• MeSH
• Genome, Human genetics   MeSH
• Genomics methods   MeSH
• Genotyping Techniques methods   MeSH
• Genes, ras genetics   MeSH
• Colorectal Neoplasms genetics   MeSH
• Humans MeSH
• Sequence Analysis, DNA MeSH
• Nucleic Acid Amplification Techniques methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

BACKGROUND: Ovarian cancer risk in BRCA1 and BRCA2 mutation carriers has been shown to decrease with longer duration of oral contraceptive use. Although the effects of using oral contraceptives in the general population are well established (approximately 50% risk reduction in ovarian cancer), the estimated risk reduction in mutation carriers is much less precise because of potential bias and small sample sizes. In addition, only a few studies on oral contraceptive use have examined the associations of duration of use, time since last use, starting age, and calendar year of start with risk of ovarian cancer. OBJECTIVE: This study aimed to investigate in more detail the associations of various characteristics of oral contraceptive use and risk of ovarian cancer, to provide healthcare providers and carriers with better risk estimates. STUDY DESIGN: In this international retrospective study, ovarian cancer risk associations were assessed using oral contraceptives data on 3989 BRCA1 and 2445 BRCA2 mutation carriers. Age-dependent-weighted Cox regression analyses were stratified by study and birth cohort and included breast cancer diagnosis as a covariate. To minimize survival bias, analyses were left truncated at 5 years before baseline questionnaire. Separate analyses were conducted for each aspect of oral contraceptive use and in a multivariate analysis, including all these aspects. In addition, the analysis of duration of oral contraceptive use was stratified by recency of use. RESULTS: Oral contraceptives were less often used by mutation carriers who were diagnosed with ovarian cancer (ever use: 58.6% for BRCA1 and 53.5% BRCA2) than by unaffected carriers (ever use: 88.9% for BRCA1 and 80.7% for BRCA2). The median duration of use was 7 years for both BRCA1 and BRCA2 carriers who developed ovarian cancer and 9 and 8 years for unaffected BRCA1 and BRCA2 carriers with ovarian cancer, respectively. For BRCA1 mutation carriers, univariate analyses have shown that both a longer duration of oral contraceptive use and more recent oral contraceptive use were associated with a reduction in the risk of ovarian cancer. However, in multivariate analyses, including duration of use, age at first use, and time since last use, duration of oral contraceptive use proved to be the prominent protective factor (compared with <5 years: 5-9 years [hazard ratio, 0.67; 95% confidence interval, 0.40-1.12]; >10 years [hazard ratio, 0.37; 95% confidence interval, 0.19-0.73]; Ptrend=.008). The inverse association between duration of use and ovarian cancer risk persisted for more than 15 years (duration of ≥10 years; BRCA1 <15 years since last use [hazard ratio, 0.24; 95% confidence interval, 0.14-0.43]; BRCA1 >15 years since last use [hazard ratio, 0.56; 95% confidence interval, 0.18-0.59]). Univariate results for BRCA2 mutation carriers were similar but were inconclusive because of limited sample size. CONCLUSION: For BRCA1 mutation carriers, longer duration of oral contraceptive use is associated with a greater reduction in ovarian cancer risk, and the protection is long term.

• MeSH
• Adult MeSH
• Genetic Predisposition to Disease MeSH
• Cohort Studies MeSH
• Contraceptives, Oral administration & dosage   MeSH
• Middle Aged MeSH
• Humans MeSH
• Mutation * MeSH
• Ovarian Neoplasms epidemiology   genetics   prevention & control   MeSH
• Follow-Up Studies MeSH
• Proportional Hazards Models MeSH
• BRCA1 Protein genetics   MeSH
• BRCA2 Protein genetics   MeSH
• Retrospective Studies MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Europe MeSH

Cardio-facio-cutaneous (CFC) and Costello syndrome (CS) are congenital disorders with a significant clinical overlap. The recent discovery of heterozygous mutations in genes encoding components of the RAS-RAF-MAPK pathway in both CFC and CS suggested a similar underlying pathogenesis of these two disorders. While CFC is heterogeneous with mutations in BRAF, MAP2K1, MAP2K2 and KRAS, HRAS alterations are almost exclusively associated with CS. We carried out a comprehensive mutation analysis in 51 CFC-affected patients and 31 individuals with CS. Twelve different BRAF alterations were found in twenty-four patients with CFC (47.0%), two MAP2K1 mutations in five (9.8%) and two MAP2K2 sequence variations in three CFC-affected individuals (5.9%), whereas three patients had a KRAS alteration (5.9%). We identified four different missense mutations of HRAS in twenty-eight cases with CS (90.3%), while KRAS mutations were detected in two infants with a phenotype meeting criteria for CS (6.5%). In 14 informative families, we traced the parental origin of HRAS alterations and demonstrated inheritance of the mutated allele exclusively from the father, further confirming a paternal bias in the parental origin of HRAS mutations in CS. Careful clinical evaluation of patients with BRAF and MAP2K1/2 alterations revealed the presence of slight phenotypic differences regarding craniofacial features in MAP2K1- and MAP2K2-mutation positive individuals, suggesting possible genotype-phenotype correlations.

• MeSH
• Child MeSH
• Adult MeSH
• Facies * MeSH
• Phenotype MeSH
• Skin Abnormalities * genetics   MeSH
• Humans MeSH
• MAP Kinase Kinase 1 genetics   MeSH
• MAP Kinase Kinase 2 genetics   MeSH
• Intellectual Disability MeSH
• Abnormalities, Multiple * genetics   MeSH
• Mutation * MeSH
• DNA Mutational Analysis MeSH
• Proto-Oncogene Proteins B-raf genetics   MeSH
• Proto-Oncogene Proteins genetics   MeSH
• ras Proteins genetics   MeSH
• Syndrome MeSH
• Heart Defects, Congenital * genetics   MeSH
• Developmental Disabilities MeSH
• Check Tag
• Child MeSH
• Adult MeSH
• Humans MeSH
• Publication type
• Research Support, Non-U.S. Gov't MeSH

BACKGROUND: Various factors-including age, family history, inflammation, reproductive factors, and tubal ligation-modulate the risk of ovarian cancer. In this study, our aim was to establish whether women with, or at risk of developing, ovarian cancer have an imbalanced cervicovaginal microbiome. METHODS: We did a case-control study in two sets of women aged 18-87 years in the Czech Republic, Germany, Italy, Norway, and the UK. The ovarian cancer set comprised women with epithelial ovarian cancer and controls (both healthy controls and those diagnosed with benign gynaecological conditions). The BRCA set comprised women with a BRCA1 mutation but without ovarian cancer and controls who were wild type for BRCA1 and BRCA2 (both healthy controls and those with benign gynaecological conditions). Cervicovaginal samples were gathered from all participants with the ThinPrep system and then underwent 16S rRNA gene sequencing. For each sample, we calculated the proportion of lactobacilli species (ie, Lactobacillus crispatus, Lactobacillus iners, Lactobacillus gasseri, and Lactobacillus jensenii), which are essential for the generation of a protective low vaginal pH, in the cervicovaginal microbiota. We grouped samples into those in which lactobacilli accounted for at least 50% of the species present (community type L) and those in which lactobacilli accounted for less than 50% of the species present (community type O). We assessed the adjusted association between BRCA1 status and ovarian cancer status and cervicovaginal microbiota community type, using a logistic regression model with a bias reduction method. FINDINGS: Participants were recruited between Jan 2, 2016, and July 21, 2018. The ovarian cancer set (n=360) comprised 176 women with epithelial ovarian cancer, 115 healthy controls and 69 controls with benign gynaecological conditions. The BRCA set (n=220) included 109 women with BRCA1 mutations, 97 healthy controls wild type for BRCA1 and BRCA2 and 14 controls with a benign gynaecological condition wild type for BRCA1 and BRCA2. On the basis of two-dimensional density plots, receiver-operating characteristic curve analysis, and age thresholds used previously, we divided the cohort into those younger than 50 years and those aged 50 years or older. In the ovarian cancer set, women aged 50 years or older had a higher prevalence of community type O microbiota (81 [61%] of 133 ovarian cancer cases and 84 [59%] of 142 healthy controls) than those younger than 50 years (23 [53%] of 43 cases and 12 [29%] of 42 controls). In the ovarian cancer set, women younger than 50 years with ovarian cancer had a significantly higher prevalence of community type O microbiota than did age-matched controls under a logistic regression model with bias correction (odds ratio [OR] 2·80 [95% CI 1·17-6·94]; p=0·020). In the BRCA set, women with BRCA1 mutations younger than 50 years were also more likely to have community type O microbiota than age-matched controls (OR 2·79 [95% CI 1·25-6·68]; p=0·012), after adjustment for pregnancy (ever). This risk was increased further if more than one first-degree family member was affected by any cancer (OR 5·26 [95% CI 1·83-15·30]; p=0·0022). In both sets, we noted that the younger the participants, the stronger the association between community type O microbiota and ovarian cancer or BRCA1 mutation status (eg, OR for community type O for cases aged <40 years in the ovarian cancer set 7·00 [95% CI 1·27-51·44], p=0·025; OR for community type O for BRCA1 mutation carriers aged <35 years in the BRCA set 4·40 [1·14-24·36], p=0·031). INTERPRETATION: The presence of ovarian cancer, or factors known to affect risk for the disease (ie, age and BRCA1 germline mutations), were significantly associated with having a community type O cervicovaginal microbiota. Whether re-instatement of a community type L microbiome by using, for example, vaginal suppositories containing live lactobacilli, would alter the microbiomial composition higher up in the female genital tract and in the fallopian tubes (the site of origin of high-grade serous ovarian cancer), and whether such changes could translate into a reduced incidence of ovarian cancer, needs to be investigated. FUNDING: EU Horizon 2020 Research and Innovation Programme, EU Horizon 2020 European Research Council Programme, and The Eve Appeal.

• MeSH
• Cervix Uteri microbiology   MeSH
• Adult MeSH
• Carcinoma, Ovarian Epithelial genetics   microbiology   MeSH
• Genetic Predisposition to Disease MeSH
• Middle Aged MeSH
• Humans MeSH
• Microbiota MeSH
• Adolescent MeSH
• Young Adult MeSH
• Ovarian Neoplasms genetics   microbiology   MeSH
• BRCA1 Protein genetics   MeSH
• Risk Factors MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Case-Control Studies MeSH
• Vagina microbiology   MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Abnormalities of the TP53 gene are associated with a particularly severe prognosis in patients with B-cell chronic lymphocytic leukemia (B-CLL). This tumor-suppressor is mostly inactivated by the deletion of one and point mutation of the other allele and has not been previously shown to be hypermutated in B-CLL. We identified two patients whose lymphocytes showed repeatedly an extensive proportion of TP53 mutated cells by FASAY analysis (the yeast functional assay) and harbored various TP53 mutations, mostly single-base substitutions, in individual cells. The mutation targeting exhibited characteristic traits of the somatic hypermutation process. In the first patient (harboring the unmutated IgVH locus) a significant bias to point mutations at CG pairs (21/25; P=0.009), their remarkable preference for the RGYW/WRCY motives (28%) and the highest expression of the activation-induced cytidine deaminase (AID) mRNA among the 34 tested B-CLL samples. In the second patient no CG bias was observed but the targeting of point mutations into the RGYW/WRCY motives was even more prominent here (7/16; 44%). Moreover, six out of eight point mutations affecting AT pairs were localized in the WA/TW motives, which are also characteristic for the somatic hypermutations. This patient, who was IgVH-mutated, already did not express any significant amount of the AID transcript. Our findings add a new aspect to the mosaic of the p53 mutability in B-CLL.

• MeSH
• Point Mutation MeSH
• Leukemia, Lymphocytic, Chronic, B-Cell genetics   MeSH
• Cytidine Deaminase genetics   MeSH
• Financing, Organized MeSH
• Genes, p53 MeSH
• Humans MeSH
• Lymphocytes pathology   MeSH
• Mutation MeSH
• Somatic Hypermutation, Immunoglobulin MeSH
• Check Tag
• Humans MeSH