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We have investigated and compared two techniques for diagnostic detection of dystrephin deletion/duplications. Both examine all 79 exons, and are thus comprehensive, ie capable of detecting any deletion or duplication in either males or females. As such, they offer major advantages to the diagnostic laboratory in terms of streamlining testing protocols and rapidly identifying cases where point mutation analysis will be appropriate. We introduced a diagnostic service using MAPH (Multiplex Amplifiable Probe Hybridisation) one year ago, and are one of five laboratories world-wide to test a newly developed dystrophin MLPA (Multiplex Ligation-dependent Probe Amplification) assay. Technically, MLPA is preferred to MAPH. The manipulation and washing of small membranes in MAPH is time consuming, and introduces additional sample identity issues. In practice, MAPH has proved to be a powerful diagnostic tool. However, in order to obtain results of sufficient quality for diagnostic purposes, repeated analysis has often been required. Our initial experience with MLPA suggests that this technique may produce superior results. This confidence is strengthened by experience with other applications in the laboratory. In conclusion, techniques are now available for comprehensive dystrophin deletion/duplication analysis which form the core of our diagnostic. Preliminary evidence suggests that MLPA will be the preferred technique in our laboratory. [ABSTRACT FROM AUTHOR]

We describe the hydroxyl and mixed hydroxyl-water structures formed on a stepped copper surface following the reaction of adsorbed O with water at a low temperature and compare them to the structures found previously on plane copper surfaces. Thermal desorption profiles, STM, and low-energy electron diffraction show that water reacts with O at temperatures below 130 K on Cu(511). Two well-defined phases appear as the OH/H 2 O layer is heated to desorb excess water, a 1OH:1H 2 O phase and a pure OH phase. The 1OH:1H 2 O structure consists of 1D chains binding across two adjacent copper steps, with a double period along the step. Electronic structure calculations show that the structure has a zigzag chain of water along the terrace, stabilized by hydrogen bonds to OH groups adsorbed in the step bridge sites. This structure binds OH in its favored site and is similar to the structure observed on other open faces of Cu and Ni, suggesting that this structural arrangement may be common on other surfaces that have steps or rows of close packed metal atoms. The hydroxyl/water chains decompose at 210 K to leave OH adsorbed in the Cu step bridge site, with some forming H-bonded trimers that bridge between two Cu steps. Heating the surface causes hydroxyl to disproportionate near 300 K, desorbing water to leave chemisorbed O., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Systematic tumour profiling is essential for biomarker research and clinically for assessing response to therapy. Solving the challenge of delivering informative copy number (CN) profiles from formalin-fixed paraffin embedded (FFPE) material, the only likely readily available biospecimen for most cancers, involves successful processing of small quantities of degraded DNA. To investigate the potential for analysis of such lesions, whole-genome CNVseq was applied to 300 FFPE primary tumour samples, obtained from a large-scale epidemiological study of melanoma. The quality and the discriminatory power of CNVseq was assessed. Libraries were successfully generated for 93% of blocks, with input DNA quantity being the only predictor of success (success rate dropped to 65% if <20 ng available); 3% of libraries were dropped because of low sequence alignment rates. Technical replicates showed high reproducibility. Comparison with targeted CN assessment showed consistency with the Next Generation Sequencing (NGS) analysis. We were able to detect and distinguish CN changes with a resolution of ≤10 kb. To demonstrate performance, we report the spectrum of genomic CN alterations (CNAs) detected at 9p21, the major site of CN change in melanoma. This successful analysis of CN in FFPE material using NGS provides proof of principle for intensive examination of population-based samples.

To investigate the predictive and prognostic value of O(6) -methylguanine DNA methyltransferase (MGMT) inactivation by analyses of promoter methylation in pretreatment tumor biopsies from patients with cutaneous melanoma treated with dacarbazine (DTIC) or temozolomide (TMZ) were performed. The patient cohorts consisted of Belgian and Swedish disseminated melanoma patients. Patients were subdivided into those receiving single-agent treatment with DTIC/TMZ (cohort S, n = 74) and those treated with combination chemotherapy including DTIC/TMZ (cohort C, n = 79). Median follow-up was 248 and 336 days for cohort S and cohort C, respectively. MGMT promoter methylation was assessed by three methods. The methylation-related transcriptional silencing of MGMT mRNA expression was assessed by real-time RT-PCR. Response to chemotherapy and progression-free survival (PFS) and overall survival were correlated to MGMT promoter methylation status. MGMT promoter methylation was detected in tumor biopsies from 21.5 % of the patients. MGMT mRNA was found to be significantly lower in tumors positive for MGMT promoter methylation compared to tumors without methylation in both treatment cohorts (p < 0.005). DTIC/TMZ therapy response rate was found to be significantly associated with MGMT promoter methylation in cohort S (p = 0.0005), but did not reach significance in cohort C (p = 0.16). Significantly longer PFS was observed among patients with MGMT promoter-methylated tumors (p = 0.002). Multivariate Cox regression analysis identified presence of MGMT promoter methylation as an independent variable associated with longer PFS. Together, this implies that MGMT promoter methylation is associated with response to single-agent DTIC/TMZ and longer PFS in disseminated cutaneous melanoma., (© 2014 UICC.)

Background: The shelterin complex protects chromosomal ends by regulating how the telomerase complex interacts with telomeres. Following the recent finding in familial melanoma of inactivating germline mutations in POT1, encoding a member of the shelterin complex, we searched for mutations in the other five components of the shelterin complex in melanoma families., Methods: Next-generation sequencing techniques were used to screen 510 melanoma families (with unknown genetic etiology) and control cohorts for mutations in shelterin complex encoding genes: ACD, TERF2IP, TERF1, TERF2, and TINF 2. Maximum likelihood and LOD [logarithm (base 10) of odds] analyses were used. Mutation clustering was assessed with χ(2) and Fisher's exact tests. P values under .05 were considered statistically significant (one-tailed with Yates' correction)., Results: Six families had mutations in ACD and four families carried TERF2IP variants, which included nonsense mutations in both genes (p.Q320X and p.R364X, respectively) and point mutations that cosegregated with melanoma. Of five distinct mutations in ACD, four clustered in the POT1 binding domain, including p.Q320X. This clustering of novel mutations in the POT1 binding domain of ACD was statistically higher (P = .005) in melanoma probands compared with population control individuals (n = 6785), as were all novel and rare variants in both ACD (P = .040) and TERF2IP (P = .022). Families carrying ACD and TERF2IP mutations were also enriched with other cancer types, suggesting that these variants also predispose to a broader spectrum of cancers than just melanoma. Novel mutations were also observed in TERF1, TERF2, and TINF2, but these were not convincingly associated with melanoma., Conclusions: Our findings add to the growing support for telomere dysregulation as a key process associated with melanoma susceptibility., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

We report the association of an inherited variant located upstream of the poly(adenosine diphosphate-ribose) polymerase 1 (PARP1) gene (rs2249844), with survival in 11 BioGenoMEL melanoma cohorts. The gene encodes a protein involved in a number of cellular processes including single-strand DNA repair. Survival analysis was conducted for each cohort using proportional hazards regression adjusting for factors known to be associated with survival. Survival was measured as overall survival (OS) and, where available, melanoma-specific survival (MSS). Results were combined using random effects meta-analysis. Evidence for a role of the PARP1 protein in melanoma ulceration and survival was investigated by testing gene expression levels taken from formalin-fixed paraffin-embedded tumors. A significant association was seen for inheritance of the rarer variant of PARP1, rs2249844 with OS (hazard ratio (HR) = 1.16 per allele, 95% confidence interval (CI) 1.04-1.28, p = 0.005, eleven cohorts) and MSS (HR = 1.20 per allele, 95% CI 1.01-1.39, p = 0.03, eight cohorts). We report bioinformatic data supportive of a functional effect for rs2249844. Higher levels of PARP1 gene expression in tumors were shown to be associated with tumor ulceration and poorer OS., (© 2014 UICC. Published by Wiley Periodicals, Inc. on behalf of UICC.)

Deleterious germline variants in CDKN2A account for around 40% of familial melanoma cases, and rare variants in CDK4, BRCA2, BAP1 and the promoter of TERT have also been linked to the disease. Here we set out to identify new high-penetrance susceptibility genes by sequencing 184 melanoma cases from 105 pedigrees recruited in the UK, The Netherlands and Australia that were negative for variants in known predisposition genes. We identified families where melanoma cosegregates with loss-of-function variants in the protection of telomeres 1 gene (POT1), with a proportion of family members presenting with an early age of onset and multiple primary tumors. We show that these variants either affect POT1 mRNA splicing or alter key residues in the highly conserved oligonucleotide/oligosaccharide-binding (OB) domains of POT1, disrupting protein-telomere binding and leading to increased telomere length. These findings suggest that POT1 variants predispose to melanoma formation via a direct effect on telomeres.

We investigated the role of interleukin (IL)-4 receptor (IL-4R) signalling during mouse carcinogen-induced colorectal carcinogenesis and in a case-control genetic epidemiological study of IL-4Rα single nucleotide polymorphisms (SNPs). Azoxymethane-induced aberrant crypt focus (ACF; 6 weeks) and tumours (32 weeks) were analysed in wild-type (WT) BALB/c mice, as well as in IL-4Rα (-) (/-) , IL-13 (-/-) and 'double-knockout' (DKO) animals. Colorectal cancer (CRC) cases (1502) and controls (584) were genotyped for six coding IL-4Rα SNPs. The association with CRC risk and CRC-specific mortality was analysed by logistic regression. Lack of IL-4Rα expression was associated with increased ACFs [median 8.5 ACFs per mouse (IL-4Rα (-/-) ) versus 3 (WT); P = 0.007], but no difference in the number of colorectal tumours [mean 1.4 per mouse (IL-4Rα (-/-) ) versus 2 (WT)], which were smaller and demonstrated reduced nuclear/cytoplasmic β-catenin translocation compared with WT tumours. Tumour-bearing IL-4Rα (-/-) mice had fewer CD11b(+)/Gr1(+) myeloid-derived suppressor splenocytes than WT animals. IL-13 (-/-) mice developed a similar number of ACFs to IL-4Rα (-/-) and DKO mice. There was a significant increase in CRC risk associated with the functional SNP Q576R [odds ratio 1.54 (95% confidence interval 0.94-2.54), P trend 0.03 for the minor G allele]. There was no effect of IL-4Rα genotype on either CRC-specific or all-cause mortality. These combined pre-clinical and human data together demonstrate that reduced IL-4R signalling has stage-specific effects on colorectal carcinogenesis (increased CRC initiation and risk but reduced tumour progression and no effect on CRC mortality). These results should prompt evaluation of the effect of pharmacological manipulation of IL-4R signalling on future CRC risk and for CRC treatment.

We report the results of an association study of melanoma that is based on the genome-wide imputation of the genotypes of 1,353 cases and 3,566 controls of European origin conducted by the GenoMEL consortium. This revealed an association between several SNPs in intron 8 of the FTO gene, including rs16953002, which replicated using 12,313 cases and 55,667 controls of European ancestry from Europe, the USA and Australia (combined P = 3.6 × 10(-12), per-allele odds ratio for allele A = 1.16). In addition to identifying a new melanoma-susceptibility locus, this is to our knowledge the first study to identify and replicate an association with SNPs in FTO not related to body mass index (BMI). These SNPs are not in intron 1 (the BMI-related region) and exhibit no association with BMI. This suggests FTO's function may be broader than the existing paradigm that FTO variants influence multiple traits only through their associations with BMI and obesity.

We report a genome-wide association study for melanoma that was conducted by the GenoMEL Consortium. Our discovery phase included 2,981 individuals with melanoma and 1,982 study-specific control individuals of European ancestry, as well as an additional 6,426 control subjects from French or British populations, all of whom were genotyped for 317,000 or 610,000 single-nucleotide polymorphisms (SNPs). Our analysis replicated previously known melanoma susceptibility loci. Seven new regions with at least one SNP with P < 10(-5) and further local imputed or genotyped support were selected for replication using two other genome-wide studies (from Australia and Texas, USA). Additional replication came from case-control series from the UK and The Netherlands. Variants at three of the seven loci replicated at P < 10(-3): an SNP in ATM (rs1801516, overall P = 3.4 × 10(-9)), an SNP in MX2 (rs45430, P = 2.9 × 10(-9)) and an SNP adjacent to CASP8 (rs13016963, P = 8.6 × 10(-10)). A fourth locus near CCND1 remains of potential interest, showing suggestive but inconclusive evidence of replication (rs1485993, overall P = 4.6 × 10(-7) under a fixed-effects model and P = 1.2 × 10(-3) under a random-effects model). These newly associated variants showed no association with nevus or pigmentation phenotypes in a large British case-control series.

Background: We report the determinants of serum levels of vitamin D in a U.K. melanoma case-control study benefitting from detailed exposure and genotyping data., Methods: Sun exposure, supplemental vitamin D, and SNPs reported to be associated with serum levels were assessed as predictors of a single serum 25-hydroxyvitamin D3 measurement adjusted for season, age, sex, and body mass index., Results: Adjusted analyses showed that vitamin D levels were sub-optimal especially in the sun-sensitive individuals (-2.61 nmol/L, p = 0.03) and for inheritance of a genetic variant in the GC gene coding for the vitamin D-binding protein (-5.79 for heterozygotes versus wild type, p = <0.0001). Higher levels were associated with sun exposure at the weekend in summer (+4.71 nmol/L per tertile, p = <0.0001), and on hot holidays (+4.17 nmol/L per tertile, p = <0.0001). In smoothed scatter plots, vitamin D levels of 60 nmol/L in the non-sun-sensitive individuals were achieved after an average 6 h/day summer weekend sun exposure but not in the sun-sensitive individuals. Users of supplements had levels on average 11.0 nmol/L higher, p = <0.0001, and achieved optimal levels irrespective of sun exposure., Conclusions: Sun exposure was associated with increased vitamin D levels, but levels more than 60 nmol/L were reached on average only in individuals reporting lengthy exposure (≥12 h/weekend). The sun-sensitive individuals did not achieve optimal levels without supplementation, which therefore should be considered for the majority of populations living in a temperate climate and melanoma patients in particular. Inherited variation in genes such as GC is a strong factor, and carriers of variant alleles may therefore require higher levels of supplementation.

Background: National surveys have shown that painful breasts are the second most common reason for giving up breastfeeding in the first two weeks after birth in the UK. One factor contributing to such pain can be breast engorgement. Views differ as to how engorgement arises, although restrictive feeding patterns in hospital are likely to have contributed in the past. These differing views are reflected in the range of solutions offered to treat engorgement in breastfeeding mothers and these treatments are assessed in this review., Objectives: To determine the effects of any proposed intervention to relieve symptoms of breast engorgement among breastfeeding women., Search Strategy: The register of clinical trials maintained and updated by the Cochrane Pregnancy and Childbirth Group. CINAHL and MEDLINE were also searched. Date of last search: December 2000., Selection Criteria: All randomised and 'quasi-randomised' controlled trials, with or without blinding, that assess the effectiveness of treatments for the alleviation of symptoms in breastfeeding women experiencing engorgement ., Data Collection and Analysis: Data were extracted by one reviewer and verified by a second reviewer., Main Results: Eight trials, involving 424 women, were included. Three different studies were identified which used cabbage leaves or cabbage leaf extracts;. no overall benefit was found. Ultrasound treatment and placebo were equally effective. Use of Danzen (an anti-inflammatory agent) significantly improved the total symptoms of engorgement when compared to placebo (odds ratio (OR) 3.6, 95% confidence interval (CI) 1.3 - 10.3) as did bromelain/trypsin complex (OR 8.02, 95% CI 2.8-23.3). Oxytocin and cold packs had no demonstrable effect on engorgement symptoms., Authors' Conclusions: Cabbage leaves and gel packs were equally effective in the treatment of engorgement. Since both cabbage extract and placebo cream were equally effective, the alleviation in symptoms may be brought about by other factors, such as breast massage. Ultrasound treatment is equally effective with or without the ultra-wave emitting crystal, therefore its effectiveness is more likely to be due to the effect of radiant heat or massage. Pharmacologically, oxytocin was not an effective engorgement treatment while Danzen and bromelain/trypsin complex significantly improved the symptoms of engorgement. Initial prevention of breast engorgement should remain the key priority.

Background: Exclusive breastfeeding until around six months of age, followed by the introduction of solids with continued breastfeeding, is considered to be the optimal nutritional start for newborn infants., Objectives: To determine whether the exclusivity and duration of breastfeeding is affected by giving mothers commercial discharge packs in hospital which contain artificial formula or promotional material for artificial formula. These packs are those which are commonly given to mothers on leaving hospital after giving birth (thus discharge packs)., Search Strategy: Comprehensive electronic search of the register of clinical trials maintained and updated by the Cochrane Pregnancy and Childbirth Group and CINAHL and MEDLINE., Selection Criteria: All randomised controlled trials with or without blinding to examine the effects of commercial discharge packs on breastfeeding., Participants: Consenting postpartum women who initiate breastfeeding while in hospital or immediately upon discharge., Interventions: Commercial discharge packs which contain free samples of infant formula or promotional material versus non commercial discharge packs (specifically those from which free samples of infant formula have been removed or have been replaced with e.g. breast pads) or no pack., Main Outcome Measures: The proportion of women breastfeeding at six weeks and 3 months (13 weeks) postpartum.Other outcomes: Rates of breastfeeding at other fixed time points between 0 and 6 months postpartum., Data Collection and Analysis: Data were extracted by one reviewer and checked by a second reviewer., Main Results: Nine randomised controlled trials involving a total of 3730 women were analysed. The studies only included women from North America. The meta-analysis showed that when comparing commercial discharge packs with any of the controls (no intervention, non-commercial pack and combinations of these), exclusive breastfeeding was reduced at all time points in the presence of commercial hospital discharge packs. There was no evidence to support the conjecture that use of hospital discharge packs causes the early termination of non-exclusive breastfeeding. Where the introduction of solid food was measured, giving a commercial pack (with or without formula) reduced the time before solid food was introduced., Authors' Conclusions: The giving of commercial hospital discharge packs (with or without formula) appears to reduce the number of women exclusively breastfeeding at all times but has no significant effect upon the earlier termination of non-exclusive breastfeeding.

Background: National surveys have shown that painful breasts are the second most common reason for giving up breastfeeding in the first two weeks after birth in the UK. One factor contributing to such pain can be breast engorgement. Views differ as to how engorgement arises, although restrictive feeding patterns in hospital are likely to have contributed in the past. These differing views are reflected in the range of solutions offered to treat engorgement in breastfeeding mothers and these treatments are assessed in this review., Objectives: To determine the effects of any proposed intervention to relieve symptoms of breast engorgement among breastfeeding women., Search Strategy: The register of clinical trials maintained and updated by the Cochrane Pregnancy and Childbirth Group. CINAHL and MEDLINE were also searched. Date of last search: December 2000., Selection Criteria: All randomised and 'quasi-randomised' controlled trials, with or without blinding, that assess the effectiveness of treatments for the alleviation of symptoms in breastfeeding women experiencing engorgement., Data Collection and Analysis: Data were extracted by one reviewer and verified by a second reviewer., Main Results: Eight trials, involving 424 women, were included. Three different studies were identified which used cabbage leaves or cabbage leaf extracts;. no overall benefit was found. Ultrasound treatment and placebo were equally effective. Use of Danzen (an anti-inflammatory agent) significantly improved the total symptoms of engorgement when compared to placebo (odds ratio (OR) 3.6, 95% confidence interval (CI) 1.3 - 10.3) as did bromelain/trypsin complex (OR 8.02, 95% CI 2.8-23.3). Oxytocin and cold packs had no demonstrable effect on engorgement symptoms., Reviewer's Conclusions: Cabbage leaves and gel packs were equally effective in the treatment of engorgement. Since both cabbage extract and placebo cream were equally effective, the alleviation in symptoms may be brought about by other factors, such as breast massage. Ultrasound treatment is equally effective with or without the ultra-wave emitting crystal, therefore its effectiveness is more likely to be due to the effect of radiant heat or massage. Pharmacologically, oxytocin was not an effective engorgement treatment while Danzen and bromelain/trypsin complex significantly improved the symptoms of engorgement. Initial prevention of breast engorgement should remain the key priority.

Background: Exclusive breastfeeding until around six months of age, followed by the introduction of solids with continued breastfeeding, is considered to be the optimal nutritional start for newborn infants., Objectives: To determine whether the exclusivity and duration of breastfeeding is affected by giving mothers commercial discharge packs in hospital which contain artificial formula or promotional material for artificial formula. These packs are those which are commonly given to mothers on leaving hospital after giving birth (thus discharge packs)., Search Strategy: Comprehensive electronic search of the register of clinical trials maintained and updated by the Cochrane Pregnancy and Childbirth Group and CINAHL and MEDLINE., Selection Criteria: All randomised controlled trials with or without blinding to examine the effects of commercial discharge packs on breastfeeding., Participants: Consenting postpartum women who initiate breastfeeding while in hospital or immediately upon discharge., Interventions: Commercial discharge packs which contain free samples of infant formula or promotional material versus non commercial discharge packs (specifically those from which free samples of infant formula have been removed or have been replaced with e.g. breast pads) or no pack., Main Outcome Measures: The proportion of women breastfeeding at six weeks and 3 months (13 weeks) postpartum. Other outcomes: Rates of breastfeeding at other fixed time points between 0 and 6 months postpartum., Data Collection and Analysis: Data were extracted by one reviewer and checked by a second reviewer., Main Results: Nine randomised controlled trials involving a total of 3730 women were analysed. The studies only included women from North America. The meta-analysis showed that when comparing commercial discharge packs with any of the controls (no intervention, non-commercial pack and combinations of these), exclusive breastfeeding was reduced at all time points in the presence of commercial hospital discharge packs. There was no evidence to support the conjecture that use of hospital discharge packs causes the early termination of non-exclusive breastfeeding. Where the introduction of solid food was measured, giving a commercial pack (with or without formula) reduced the time before solid food was introduced., Reviewer's Conclusions: The giving of commercial hospital discharge packs (with or without formula) appears to reduce the number of women exclusively breastfeeding at all times but has no significant effect upon the earlier termination of non-exclusive breastfeeding.