Sporadic vestibular schwannoma: a molecular testing summary.

OBJECTIVES: Cases of sporadic vestibular schwannoma (sVS) have a low rate of association with germline pathogenic variants. However, some individuals with sVS can represent undetected cases of neurofibromatosis type 2 (NF2) or schwannomatosis. Earlier identification of patients with these syndromes can facilitate more accurate familial risk prediction and prognosis. METHODS: Cases of sVS were ascertained from a local register at the Manchester Centre for Genomic Medicine. Genetic analysis was conducted in NF2 on blood samples for all patients, and tumour DNA samples when available. LZTR1 and SMARCB1 screening was also performed in patient subgroups. RESULTS: Age at genetic testing for vestibular schwannoma (VS) presentation was younger in comparison with previous literature, a bias resulting from updated genetic testing recommendations. Mosaic or constitutional germline NF2 variants were confirmed in 2% of patients. Pathogenic germline variants in LZTR1 were found in 3% of all tested patients, with a higher rate of 5% in patients <30 years. No pathogenic SMARCB1 variants were identified within the cohort. Considering all individuals who received tumour DNA analysis, 69% of patients were found to possess two somatic pathogenic NF2 variants, including those with germline LZTR1 pathogenic variants. CONCLUSIONS: Undiagnosed schwannoma predisposition may account for a significant minority of apparently sVS cases, especially at lower presentation ages. Loss of NF2 function is a common event in VS tumours and may represent a targetable common pathway in VS tumourigenesis. These data also support the multi-hit mechanism of LZTR1-associated VS tumourigenesis.

The proportion of endometrial tumours associated with Lynch syndrome (PETALS): A prospective cross-sectional study.

BACKGROUND: Lynch syndrome (LS) predisposes to endometrial cancer (EC), colorectal cancer, and other cancers through inherited pathogenic variants affecting mismatch-repair (MMR) genes. Diagnosing LS in women with EC can reduce subsequent cancer mortality through colonoscopic surveillance and aspirin chemoprevention; it also enables cascade testing of relatives. A growing consensus supports LS screening in EC; however, the expected proportion of test positives, and optimal testing strategy is uncertain. Previous studies from insurance-based healthcare systems were limited by narrow selection criteria, failure to apply reference standard tests consistently, and poor conversion to definitive testing. The aim of this study was to establish the prevalence of LS and the diagnostic accuracy of LS testing strategies in an unselected EC population. METHODS AND FINDINGS: This was a prospective cross-sectional study carried out at a large United Kingdom gynaecological cancer centre between October 2015 and January 2017. Women diagnosed with EC or atypical hyperplasia (AH) were offered LS testing. Tumours underwent MMR immunohistochemistry (IHC), microsatellite instability (MSI), and targeted MLH1-methylation testing. Women <50 years, with strong family histories and/or indicative tumour molecular features, underwent MMR germline sequencing. Somatic MMR sequencing was performed when indicative molecular features were unexplained by LS or MLH1-hypermethylation. The main outcome measures were the prevalence of LS in an unselected EC population and the diagnostic accuracy of clinical and tumour testing strategies for risk stratifying women with EC for MMR germline sequencing. In total, 500 women participated in the study; only 2 (<1%) declined. Germline sequencing was indicated and conducted for 136 and 135 women, respectively. A total of 16/500 women (3.2%, 95% CI 1.8% to 5.1%) had LS, and 11 more (2.2%) had MMR variants of uncertain significance. Restricting testing to age <50 years, indicative family history (revised Bethesda guidelines or Amsterdam II criteria) or endometrioid histology alone would have missed 9/16 (56%), 8/13 (62%) or 9/13 (69%), and 5/16 (31%) cases of LS, respectively. In total 132/500 tumours were MMR deficient by IHC of which 83/132 (63%) had MLH1-hypermethylation, and 16/49 (33%) of the remaining patients had LS (16/132 with MMR deficiency, 12%). MMR-IHC with targeted MLH1-methylation testing was more discriminatory for LS than MSI with targeted methylation testing, with 100% versus 56.3% (16/16 versus 9/16) sensitivity (p = 0.016) and equal 97.5% (468/484) specificity; 64% MSI-H and 73% MMR deficient tumours unexplained by LS or MLH1-hypermethylation had somatic MMR mutations. The main limitation of the study was failure to conduct MMR germline sequencing for the whole study population, which means that the sensitivity and specificity of tumour triage strategies for LS detection may be overestimated, although the risk of LS in women with no clinical or tumour predictors is expected to be extremely low. CONCLUSIONS: In this study, we observed that age, family history, and histology are imprecise clinical correlates of LS-EC. IHC outperformed MSI for tumour triage and reliably identified both germline and somatic MMR mutations. The 3.2% proportion of LS-EC is similar to colorectal cancer, supporting unselected screening of EC for LS.

Incidence of mosaicism in 1055 de novo NF2 cases: much higher than previous estimates with high utility of next-generation sequencing.

PURPOSE: To evaluate the incidence of mosaicism in de novo neurofibromatosis 2 (NF2). METHODS: Patients fulfilling NF2 criteria, but with no known affected family member from a previous generation (n = 1055), were tested for NF2 variants in lymphocyte DNA and where available tumor DNA. The proportion of individuals with a proven or presumed mosaic NF2 variant was assessed and allele frequencies of identified variants evaluated using next-generation sequencing. RESULTS: The rate of proven/presumed mosaicism was 232/1055 (22.0%). However, nonmosaic heterozygous pathogenic variants were only identified in 387/1055 (36.7%). When variant detection rates in second generation nonmosaics were applied to de novo cases, we assessed the overall probable mosaicism rate to be 59.7%. This rate differed by age from 21.7% in those presenting with bilateral vestibular schwannoma <20 years to 80.7% in those aged ≥60 years. A mosaic variant was detected in all parents of affected children with a single-nucleotide pathogenic NF2 variant. CONCLUSION: This study has identified a very high probable mosaicism rate in de novo NF2, probably making NF2 the condition with the highest expressed rate of mosaicism in de novo dominant disease that is nonlethal in heterozygote form. Risks to offspring are small and probably correlate with variant allele frequency detected in blood.

Identifying the deficiencies of current diagnostic criteria for neurofibromatosis 2 using databases of 2777 individuals with molecular testing.

PURPOSE: We have evaluated deficiencies in existing diagnostic criteria for neurofibromatosis 2 (NF2). METHODS: Two large databases of individuals fulfilling NF2 criteria (n = 1361) and those tested for NF2 variants with criteria short of diagnosis (n = 1416) were interrogated. We assessed the proportions meeting each diagnostic criterion with constitutional or mosaic NF2 variants and the positive predictive value (PPV) with regard to definite diagnosis. RESULTS: There was no evidence for usefulness of old criteria "glioma" or "neurofibroma." "Ependymoma" had 100% PPV and high levels of confirmed NF2 diagnosis (67.7%). Those with bilateral vestibular schwannoma (VS) alone aged ≥60 years had the lowest confirmation rate (6.6%) and reduced PPV (80%). Siblings as a first-degree relative, without an affected parent, had 0% PPV. All three individuals with unilateral VS and an affected sibling were proven not to have NF2. The biggest overlap was with LZTR1-associated schwannomatosis. In this category, seven individuals with unilateral VS plus ≥2 nondermal schwannomas reduced PPV to 67%. CONCLUSIONS: The present study confirms important deficiencies in NF2 diagnostic criteria. The term "glioma" should be dropped and replaced by "ependymoma." Similarly "neurofibroma" should be removed. Dropping "sibling" from first-degree relatives should be considered and testing of LZTR1 should be recommended for unilateral VS.

Schwannomatosis: a genetic and epidemiological study.

OBJECTIVES: Schwannomatosis is a dominantly inherited condition predisposing to schwannomas of mainly spinal and peripheral nerves with some diagnostic overlap with neurofibromatosis-2 (NF2), but the underlying epidemiology is poorly understood. We present the birth incidence and prevalence allowing for overlap with NF2. METHODS: Schwannomatosis and NF2 cases were ascertained from the Manchester region of England (population=4.8 million) and from across the UK. Point prevalence and birth incidence were calculated from regional birth statistics. Genetic analysis was also performed on NF2, LZTR1 and SMARCB1 on blood and tumour DNA samples when available. RESULTS: Regional prevalence for schwannomatosis and NF2 were 1 in 126 315 and 50 500, respectively, with calculated birth incidences of 1 in 68 956 and 1 in 27 956. Mosaic NF2 causes a substantial overlap with schwannomatosis resulting in the misdiagnosis of at least 9% of schwannomatosis cases. LZTR1-associated schwannomatosis also causes a small number of cases that are misdiagnosed with NF2 (1%-2%), due to the occurrence of a unilateral vestibular schwannoma. Patients with schwannomatosis had lower numbers of non-vestibular cranial schwannomas, but more peripheral and spinal nerve schwannomas with pain as a predominant presenting symptom. Life expectancy was significantly better in schwannomatosis (mean age at death 76.9) compared with NF2 (mean age at death 66.2; p=0.004). CONCLUSIONS: Within the highly ascertained North-West England population, schwannomatosis has less than half the birth incidence and prevalence of NF2.

Incidence of classical 21-hydroxylase deficiency and distribution of CYP21A2 mutations in Estonia.

AIMS: To determine the incidence of classical 21-hydroxylase deficiency (21-OHD) in Estonia from 1978 to 2004, and describe their phenotype and genotype. METHODS: All Estonian endocrinologists informed us about their patients with 21-OHD. The diagnosis was confirmed in 20 patients, who were all screened for 8 common mutations of the CYP21A2 gene. RESULTS: The 27-year period incidence was 1:25,500. The incidence from 1992 was 1:16,100, which more accurately reflects the real situation in Estonia. The salt-wasting form (SW) was diagnosed in 14 (7 males) and the simple virilizing form in 6 patients (1 male). The median age at diagnosis of the SW form was 30 days in males and 2 days in females. The investigation of 34 unrelated alleles showed that a common deletion/conversion was the most frequent mutation in our group (7/34). Six other mutations were present: p.Ile172Asn (5/34), 8-bp deletion (3/34), intron-2 splice mutation (3/34), p.Arg356Trp (3/34), p.Gln318X (3/34) and a small conversion (2/34). Mutations in 8 alleles remained uncertain. CONCLUSIONS: The incidence of classical 21-OHD in Estonia in 1992-2004 was 1:16,100. The genotype of our patients is similar to those from other Caucasian populations. The relatively late age at diagnosis and the skewed female:male ratio supports the need for newborn screening for 21-OHD.

UV-A induces persistent genomic instability in human keratinocytes through an oxidative stress mechanism.

Ultraviolet-A (UV-A, 320 to 400 nm) radiation comprises 95% of the solar ultraviolet radiation (UVR) reaching the earth's surface. It has been associated experimentally and epidemiologically with malignant melanoma. In this study we investigated whether UV-A radiation can induce a persistent, heritable hypermutability in mammalian cells similar to that observed following ionising radiation (IR). Using the immortalized human skin keratinocyte cell line HaCaT we found that UV-A radiation does lead to a continuing reduction in plating efficiency, an increased "spontaneous" mutant fraction, and an increase in micronucleus formation up to 21 d after initial exposure. Reversal of these effects using catalase may indicate a role for hydrogen peroxide in this phenomenon. These results add to the significance of UV-A radiation as a risk factor in skin carcinogenesis.

Primary fibroblasts from BRCA1 heterozygotes display an abnormal G1/S cell cycle checkpoint following UVA irradiation but show normal levels of micronuclei following oxidative stress or mitomycin C treatment.

PURPOSE: There is evidence to suggest that the breast cancer predisposing gene, BRCA1, is involved in cell cycle control and the response to damage but mouse brca1+/- heterozygotes have no distinctive phenotype. Here the response to the three forms of cellular stress was examined in primary human fibroblasts from individuals with a +/+ or +/- genotype for BRCA1. METHODS AND MATERIALS: Fibroblasts from individuals carrying mutations in the BRCA1 gene were compared with those from those wild-type for BRCA1 in their response to long wavelength uv (UVA), hydrogen peroxide, and mitomycin C (MMC). Cell cycle progression and micronucleus formation (MN) were used as end points. RESULTS: After UVA treatment there was no difference between +/- and +/+ cells in the initial fall in DNA synthetic activity (G(1) arrest) but the reentry into S-phase was restored at a faster rate in the BRCA1+/- cells after UVA exposure. Thus, for three normal (+/+) cell lines irradiated in monolayer, S-phase values averaged 15 +/- 3.7% 14 h post-UVA (1 x 10(5) J/m(2)), as compared with 35.7 +/- 1.9 (range) for two BRCA1(+/-) strains. Because a defective G(1)/S checkpoint in BRCA1 heterozygotes could lead to a greater proportion of S-phase cells with unrepaired DNA damage (strand breaks) and a resultant increase in chromosomal instability, the frequency of micronuclei induced by UVA was examined. Three normal (+/+) and three mutant (+/-) strains (two of which were used in the cell cycle experiments) produced mean micronuclei frequencies of 0.077 +/- 0.016 and 0.094 +/- 0.04/binucleate cell respectively (not statistically significant), 48 h after UVA exposure. No differences were found between BRCA1+/+ and +/- cells in MN formation after treatment with MMC or hydrogen peroxide. CONCLUSION: Our data suggest a defective G(1)/S checkpoint in cells from BRCA1 heterozygotes in response to UVA although this is not reflected in genomic instability as measured by micronuclei induction after oxidative stress or MMC treatment.

No evidence of an increase in early infant mortality from congenital adrenal hyperplasia in the absence of screening.

OBJECTIVE: Congenital adrenal hyperplasia (CAH) is not currently included in the UK newborn screening programme. We investigated the hypothesis that, owing to non-specificity of symptoms, a proportion of males affected by salt-wasting (SW) CAH have died in infancy without being diagnosed. DESIGN: Stored newborn screening blood spot samples were analysed for 17α-hydroxyprogesterone (17-OHP) in the following groups: Infants born in the North West of England, 1994 to 2006, who had died by 6 months age; (n=1198), a neonatal reference group (full-term n=100; preterm n=100) and a CAH positive control group. A newborn blood spot sample collected before diagnosis was available in 29/61 CAH patients recruited. SW CAH was present in 18/29 patients (16 males and 2 females). Samples from the deceased group with elevated 17-OHP were analysed for 8 common mutations in the 21-hydroxylase gene (CYP21A2). SETTING: North West of England. RESULTS: Grouped by gestational age, mean (maximum) blood spot 17-OHP in nmol/L was as follows. Deceased full-term n=279, 6 (107); deceased premature n=365, 28 (251); deceased unknown gestational age n=553, 13 (>394). In the SW positive control group, the lowest level of 17-OHP was 179 nmol/L and 14 had levels greater than the highest standard (>268 to >420 nmol/L). All samples from the deceased group with 17-OHP results >179 nmol/L (n=6) and a further 13 samples underwent mutation analysis. No mutations were identified. CONCLUSIONS: Our findings do not support the hypothesis that, in our unscreened population, males affected by SW CAH are dying prior to diagnosis.

Establishment of the first WHO international genetic reference panel for Prader Willi and Angelman syndromes.

Prader Willi and Angelman syndromes are clinically distinct genetic disorders both mapping to chromosome region 15q11-q13, which are caused by a loss of function of paternally or maternally inherited genes in the region, respectively. With clinical diagnosis often being difficult, particularly in infancy, confirmatory genetic diagnosis is essential to enable clinical intervention. However, the latter is challenged by the complex genetics behind both disorders and the unmet need for characterised reference materials to aid accurate molecular diagnosis. With this in mind, a panel of six genotyping reference materials for Prader Willi and Angelman syndromes was developed, which should be stable for many years and available to all diagnostic laboratories. The panel comprises three Prader Willi syndrome materials (two with different paternal deletions, and one with maternal uniparental disomy (UPD)) and three Angelman syndrome materials (one with a maternal deletion, one with paternal UPD or an epigenetic imprinting centre defect, and one with a UBE3A point mutation). Genomic DNA was bulk-extracted from Epstein-Barr virus-transformed lymphoblastoid cell lines established from consenting patients, and freeze-dried as aliquots in glass ampoules. In total, 37 laboratories from 26 countries participated in a collaborative study to assess the suitability of the panel. Participants evaluated the blinded, triplicate materials using their routine diagnostic methods against in-house controls or externally sourced uncertified reference materials. The panel was established by the Expert Committee on Biological Standardization of the World Health Organization as the first International Genetic Reference Panel for Prader Willi and Angelman syndromes.

UGT1A1*28 genotype predicts gastrointestinal toxicity in patients treated with intermediate-dose irinotecan.

AIMS: Variants in UGT1A1 have previously been associated with toxicity from irinotecan chemotherapy. We conducted a pragmatic prospective cohort study to establish the relevance of UGT1A1 variants in the prediction of severe diarrhea and neutropenia in patients with colorectal cancer receiving irinotecan in a routine clinical setting. MATERIALS & METHODS: Genotyping of UGT1A1*28 and c.-3156G>A was undertaken in an unselected, prospective cohort of 96 individuals treated with irinotecan at a single major UK oncology centre. Data on cytotoxic drugs received, and toxicity for all irinotecan treatment cycles were collected from case notes. Over 95% (92/96) of patients received an intermediate dose of irinotecan (180 mg/m(2), twice weekly). Irinotecan was given in combination with other cytotoxic drugs in 93/96 subjects and Grade 3 or 4 toxicity occurred in 23% of subjects. RESULTS: No association was found between UGT1A1*28 or c.-3156G>A and neutropenia. However, individuals carrying two copies of UGT1A1*28 (p = 0.04; OR: 14; 95% CI: 1.1-185) or c.-3156G>A (p = 0.03) had a significantly increased risk of diarrhea over all cycles. CONCLUSION: Our findings indicate that UGT1A1 genotyping is not a good predictor of hematological toxicity in patients treated with intermediate irinotecan doses. However, it may be useful in the identification of patients at risk of severe diarrhea.

The green tea polyphenol, epigallocatechin-3-gallate, protects against the oxidative cellular and genotoxic damage of UVA radiation.

A number of biological activities have been ascribed to the major green tea polyphenol epigallocatechin-3-gallate (EGCG) to explain its chemopreventive properties. Its antioxidant properties emerge as a potentially important mode of action. We have examined the effect of EGCG treatment on the damaging oxidative effects of UVA radiation in a human keratinocyte line (HaCaT). Using the ROS-sensitive probes dihydrorhodamine 123 (DHR) and 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA), we detected a reduction in fluorescence in UVA-irradiated (100 kJ/m(2)) cells in the case of the former but not the latter probe after a 24-hr treatment with EGCG (e.g., 14%, [p < 0.05] after 10 microM EGCG). In the absence of UVA, however, both DHR and DCFH detected a pro-oxidant effect of EGCG at the highest concentration used of 50 microM. Measurements of DNA damage in UVA-exposed cells using the single cell gel electrophoresis assay (comet assay) also showed the protective effects of EGCG. A concentration of 10 microM EGCG decreased the level of DNA single strand breaks and alkali-labile sites to 62% of the level observed in non-EGCG, irradiated cells (p < 0.001) with a 5-fold higher concentration producing little further effect. Correspondingly, EGCG ablated the mutagenic effects of UVA (500 kJ/m(2)) reducing an induced hypoxanthine-guanine phosphoribosyl transferase (HPRT) mutant frequency of (3.39 +/- 0.73) x 10(-6) to spontaneous levels (1.09 +/- 0.19) x 10(-6). Despite having an antiproliferative effect in the absence of UVA, EGCG also served to protect against the cytotoxic effects of UVA radiation. Our data demonstrate the ability of EGCG to modify endpoints directly relevant to the carcinogenic process in skin.