The application of epiphenotyping approaches to DNA methylation array studies of the human placenta.

BACKGROUND: Genome-wide DNA methylation (DNAme) profiling of the placenta with Illumina Infinium Methylation bead arrays is often used to explore the connections between in utero exposures, placental pathology, and fetal development. However, many technical and biological factors can lead to signals of DNAme variation between samples and between cohorts, and understanding and accounting for these factors is essential to ensure meaningful and replicable data analysis. Recently, "epiphenotyping" approaches have been developed whereby DNAme data can be used to impute information about phenotypic variables such as gestational age, sex, cell composition, and ancestry. These epiphenotypes offer avenues to compare phenotypic data across cohorts, and to understand how phenotypic variables relate to DNAme variability. However, the relationships between placental epiphenotyping variables and other technical and biological variables, and their application to downstream epigenome analyses, have not been well studied. RESULTS: Using DNAme data from 204 placentas across three cohorts, we applied the PlaNET R package to estimate epiphenotypes gestational age, ancestry, and cell composition in these samples. PlaNET ancestry estimates were highly correlated with independent polymorphic ancestry-informative markers, and epigenetic gestational age, on average, was estimated within 4 days of reported gestational age, underscoring the accuracy of these tools. Cell composition estimates varied both within and between cohorts, as well as over very long placental processing times. Interestingly, the ratio of cytotrophoblast to syncytiotrophoblast proportion decreased with increasing gestational age, and differed slightly by both maternal ethnicity (lower in white vs. non-white) and genetic ancestry (lower in higher probability European ancestry). The cohort of origin and cytotrophoblast proportion were the largest drivers of DNAme variation in this dataset, based on their associations with the first principal component. CONCLUSIONS: This work confirms that cohort, array (technical) batch, cell type proportion, self-reported ethnicity, genetic ancestry, and biological sex are important variables to consider in any analyses of Illumina DNAme data. We further demonstrate the specific utility of epiphenotyping tools developed for use with placental DNAme data, and show that these variables (i) provide an independent check of clinically obtained data and (ii) provide a robust approach to compare variables across different datasets. Finally, we present a general framework for the processing and analysis of placental DNAme data, integrating the epiphenotype variables discussed here.

The impact of socioeconomic deprivation on congenital hand differences: A retrospective cohort study.

Socioeconomic deprivation is an established risk factor for a range of adverse perinatal and infant outcomes. The primary aim of this study was to investigate any association between socioeconomic deprivation and the prevalence of Congenital Hand Differences (CHDs). This retrospective cross-sectional study was undertaken at a single tertiary referral center over a five year period (March 2015 to February 2020). The inclusion criterion was all patients referred for a review at a CHD clinic. As a measure of socioeconomic status, patients were assigned to a deprivation quintile using the Scottish Index of Multiple Deprivation (SIMD): quintile 1 indicates the most deprived area and quintile 5 indicates the least deprived area. CHDs were classified according to the Oberg-Manske-Tonkin (OMT) Classification. During the study period 259 patients were identified. The overall prevalence of CHD was 15 per 100,000 per year, mean referral age was 2.6 years (Standard Deviation: 4 years) and 135 patients (52%) were female. Areas of greater social deprivation had a significantly higher prevalence of CHD (22 per 100,000 per year in quintile 1 vs. 13 per 100,000 per year in quintile 5; p < 0.001), surgery (75% of patients in quintile 1 vs 43% of patients in quintile 5; p = 0.003), and younger referral age (1.5 years in quintile 1 vs 4.4 years in quintile 5; p = 0.003). This study has shown a greater CHD prevalence rate amongst patients from more socially deprived areas. In the most deprived group, the patient referral age was also significantly younger and surgical intervention rate was higher.

Real-world Effectiveness and Safety of Vedolizumab for the Treatment of Inflammatory Bowel Disease: The Scottish Vedolizumab Cohort.

BACKGROUND & AIMS: Vedolizumab is an anti-a4b7 monoclonal antibody that is licensed for the treatment of moderate to severe Crohn's disease and ulcerative colitis. The aims of this study were to establish the real-world effectiveness and safety of vedolizumab for the treatment of inflammatory bowel disease. METHODS: This was a retrospective study involving seven NHS health boards in Scotland between June 2015 and November 2017. Inclusion criteria included: a diagnosis of ulcerative colitis or Crohn's disease with objective evidence of active inflammation at baseline (Harvey-Bradshaw Index[HBI] ≥5/Partial Mayo ≥2 plus C-reactive protein [CRP] >5 mg/L or faecal calprotectin ≥250 µg/g or inflammation on endoscopy/magnetic resonance imaging [MRI]); completion of induction; and at least one clinical follow-up by 12 months. Kaplan-Meier survival analysis was used to establish 12-month cumulative rates of clinical remission, mucosal healing, and deep remission [clinical remission plus mucosal healing]. Rates of serious adverse events were described quantitatively. RESULTS: Our cohort consisted of 180 patients with ulcerative colitis and 260 with Crohn's disease. Combined median follow-up was 52 weeks (interquartile range [IQR] 26-52 weeks). In ulcerative colitis, 12-month cumulative rates of clinical remission, mucosal healing, and deep remission were 57.4%, 47.3%, and 38.5%, respectively. In Crohn's disease, 12-month cumulative rates of clinical remission, mucosal healing, and deep remission were 58.4%, 38.9%, and 28.3% respectively. The serious adverse event rate was 15.6 per 100 patient-years of follow-up. CONCLUSIONS: Vedolizumab is a safe and effective treatment for achieving both clinical remission and mucosal healing in ulcerative colitis and Crohn's disease.

Secondhand smoke exposure of children at home and prevalence of parental smoking following implementation of the new tobacco control law in Macao.

OBJECTIVE: To investigate secondhand smoke exposure (SHS) of children at home and the prevalence of parental smoking after implementation of the new tobacco control law in Macao. This study explored whether the smoking ban in public places in Macao has decreased the prevalence of smoking or led to increased SHS exposure of children at home. As smokers cannot smoke in public places any more, they may smoke at home more frequently; a displacement effect of smoke-free legislation. STUDY DESIGN: Cross-sectional survey. METHODS: This study surveyed 337 fathers and 538 mothers. Questions from a subset of key questions from the Global Adult Tobacco Survey (2nd edition) were applied to assess the SHS exposure of children and the prevalence of parental smoking since the smoking ban. A classification tree analysis was used to analyse the factors increasing SHS exposure of children. RESULTS: The prevalence of SHS exposure in children at home was 41.3%. The prevalence rates of paternal and maternal smoking were 43.7% and 3.8%, respectively. Compared with data reported by the Health Bureau of Macao SAR in 2011, the prevalence of parental smoking and the prevalence of SHS exposure of children at home have not decreased since the smoking ban. Analysis of the factors increasing the prevalence of SHS exposure of children indicated that fathers with an education level below high school were more likely to contribute to this increase, compared with fathers with a high school education or more (48.2% vs 32.4%, respectively). In addition, fathers represented the majority of smokers at home, accounting for 92.0% of 415 smoking parents. The prevalence of paternal smoking (82.0%) in the group of children with SHS exposure was much higher than that in the unexposed group (16.7%, Chi-squared test = 367.199, P = 0.000). The SHS exposure of children increased consistently with the decrease in paternal education level. This was consistent with the increasing prevalence of paternal smoking as paternal education level decreased. SHS exposure was most common among children whose fathers had an education level below high school and whose mothers were aged ≤29 years (75.0%). CONCLUSIONS: This study did not find any decline in the prevalence of parental smoking after the smoking ban. These parents were more likely to smoke at home after the ban, leading to more frequent SHS exposure for their children.

Microsurgical skill acquisition in a one-day introductory course with performance evaluation using software-assisted scoring system.

BACKGROUND: Recent emphasis on microsurgical skill acquisition at an earlier stage of plastic surgery training has seen a shift toward objective competence-based assessment. Yet no objective measures of spacing or alignment exist, with few validated models that assess ability. The authors propose a novel software analysis scoring system to objectively measure spacing, alignment and the overall improvement in a 1-day, introductory course setting. METHODS: Images of standard 4-mm latex strips that had been sutured by participants using the Microtrainer system were uploaded onto calibrated, online software. Sutures were analysed with regard to spacing, alignment and density. From these measurements, a total score was calculated, one on initial assessment at the course beginning (Score 1) and another on final assessment at the course end (Score 2), thereby facilitating measurement of the overall improvement. RESULTS: A total of 38 microsurgical anastomoses from 19 participants ranging from postgraduate years 1-7 were analysed. Seventeen participants had no previous experience of microsurgery. The mean average Score 1 of participants was -2 (range -12 to +22) and Score 2 was 22 (range +12 to +32), thus showing a significant improvement in candidate ability throughout the course of the day (p < 0.0001). CONCLUSIONS: Microtrainer system software analysis provides a novel, reliable, and consistent objective assessment for surgical trainees at all stages of training, without risk to patients. It has an associated cost for the initial setup, yet is timely, repeatable and can efficiently demonstrate progress in a 1-day course setting.

Management of infraclavicular (Chuang Level IV) brachial plexus injuries: A single surgeon experience with 75 cases.

UNLABELLED: Infraclavicular brachial plexus injuries (Level IV in Chuang's classification) have special characteristics, including high incidences of associated scapular fractures, glenohumeral dislocations, and vascular injuries. In addition, there are specific difficulties in surgical dissection and nerve repairs, especially if surgery is delayed (>3 months). A total of 153 patients with Level IV brachial plexus injuries underwent surgery between 1987 and 2008 with 75 patients (average age 29 years) available for a minimum of 4 years follow-up. Accompanying fractures/dislocations were suffered by 48 (64%) patients, and 17 (23%) had associated vascular injuries. The most common nerves to be injured were the axillary and musculocutaneous nerves. Nerve grafts to the axillary, musculocutaneous, and radial nerves achieved impressive results, but less reliable outcomes were achieved with the median and ulnar nerves. Decompression and/or external neurolysis were also beneficial for nerve recovery. Some surgical tips are presented, and the use of the C-loop vascularized ulnar nerve graft and functioning muscle transfers are discussed. LEVEL OF EVIDENCE: IV.

Coiled-coil domain containing 68 (CCDC68) demonstrates a tumor-suppressive role in pancreatic ductal adenocarcinoma.

Using integrative genomics and functional screening, we identified coiled-coil domain containing 68 (CCDC68) as a novel putative tumor suppressor gene (TSG) in pancreatic ductal adenocarcinoma (PDAC). CCDC68 allelic losses were documented in 48% of primary PDAC patient tumors, 50% of PDAC cell lines and 30% of primary patient derived xenografts. We also discovered a single nucleotide polymorphism (SNP) variant (SNP rs1344011) that leads to exon skipping and generation of an unstable protein isoform CCDC68Δ(69-114) in 31% of PDAC patients. Overexpression of full length CCDC68 (CCDC68(wt)) in PANC-1 and Hs.766T PDAC cell lines lacking CDCC68 expression decreased proliferation and tumorigenicity in scid mice. In contrast, the downregulation of endogenous CCDC68 in MIAPaca-2 cells increased tumor growth rate. These effects were not observed with the deletion-containing isoform, CCDC68Δ(69-114).

Genotranscriptomic meta-analysis of the Polycomb gene CBX2 in human cancers: initial evidence of an oncogenic role.

BACKGROUND: Polycomb group (PcG) proteins are histone modifiers known to transcriptionally silence key tumour suppressor genes in multiple human cancers. The chromobox proteins (CBX2, 4, 6, 7, and 8) are critical components of PcG-mediated repression. Four of them have been associated with tumour biology, but the role of CBX2 in cancer remains largely uncharacterised. METHODS: Addressing this issue, we conducted a comprehensive and unbiased genotranscriptomic meta-analysis of CBX2 in human cancers using the COSMIC and Oncomine databases. RESULTS: We discovered changes in gene expression that are suggestive of a widespread oncogenic role for CBX2. Our genetic analysis of 8013 tumours spanning 29 tissue types revealed no inactivating chromosomal aberrations and only 40 point mutations at the CBX2 locus. In contrast, the overall rate of CBX2 amplification averaged 10% in all combined neoplasms but exceeded 30% in ovarian, breast, and lung tumours. In addition, transcriptomic analyses revealed a strong tendency for increased CBX2 mRNA levels in many cancers compared with normal tissues, independently of CDKN2A/B silencing. Furthermore, CBX2 upregulation and amplification significantly correlated with metastatic progression and lower overall survival in many cancer types, particularly those of the breast. CONCLUSIONS: Overall, we report that the molecular profile of CBX2 is suggestive of an oncogenic role. As CBX2 has never been studied in human neoplasms, our results provide the rationale to further investigate the function of CBX2 in the context of cancer cells.

EYA4 is inactivated biallelically at a high frequency in sporadic lung cancer and is associated with familial lung cancer risk.

In an effort to identify novel biallelically inactivated tumor suppressor genes (TSGs) in sporadic invasive and preinvasive non-small-cell lung cancer (NSCLC) genomes, we applied a comprehensive integrated multiple 'omics' approach to investigate patient-matched, paired NSCLC tumor and non-malignant parenchymal tissues. By surveying lung tumor genomes for genes concomitantly inactivated within individual tumors by multiple mechanisms, and by the frequency of disruption in tumors across multiple cohorts, we have identified a putative lung cancer TSG, Eyes Absent 4 (EYA4). EYA4 is frequently and concomitantly deleted, hypermethylated and underexpressed in multiple independent lung tumor data sets, in both major NSCLC subtypes and in the earliest stages of lung cancer. We found that decreased EYA4 expression is not only associated with poor survival in sporadic lung cancers but also that EYA4 single-nucleotide polymorphisms are associated with increased familial cancer risk, consistent with EYA4s proximity to the previously reported lung cancer susceptibility locus on 6q. Functionally, we found that EYA4 displays TSG-like properties with a role in modulating apoptosis and DNA repair. Cross-examination of EYA4 expression across multiple tumor types suggests a cell-type-specific tumorigenic role for EYA4, consistent with a tumor suppressor function in cancers of epithelial origin. This work shows a clear role for EYA4 as a putative TSG in NSCLC.

SOX15 is a candidate tumor suppressor in pancreatic cancer with a potential role in Wnt/ß-catenin signaling.

Pancreatic cancer is among the top five deadliest cancers in developed countries. Better knowledge of the molecular mechanisms contributing to its tumorigenesis is imperative to improve patient prognosis. Identification of novel tumor suppressor genes (TSGs) in pancreatic cancer will reveal new mechanisms of pathway deregulation and will ultimately help improve our understanding of this aggressive disease. According to Knudson's two-hit model, TSGs are classically disrupted by two concerted genetic events. In this study, we combined DNA methylation profiling with copy number and mRNA expression profiling to identify novel TSGs in a set of 20 pancreatic cancer cell lines. These data sets were integrated for each of ∼12 000 genes in each cell line enabling the elucidation of those genes that undergo DNA hypermethylation, copy-number loss and mRNA downregulation simultaneously in multiple cell lines. Using this integrative genomics strategy, we identified SOX15 (sex determining region Y-box 15) as a candidate TSG in pancreatic cancer. Expression of SOX15 in pancreatic cancer cell lines with undetectable expression resulted in reduced viability of cancer cells both in vitro and in vivo demonstrating its tumor suppressive capability. We also found reduced expression, homozygous deletion and aberrant DNA methylation of SOX15 in clinical pancreatic tumor data sets. Furthermore, we deduced a novel role for SOX15 in suppressing the Wnt/ß-catenin signaling pathway, which we hypothesize is a pathway through which SOX15 may exert its tumor suppressive effects in pancreatic cancer.

Mutational analysis of Polycomb genes in solid tumours identifies PHC3 amplification as a possible cancer-driving genetic alteration.

BACKGROUND: Polycomb group genes (PcGs) are epigenetic effectors implicated in most cancer hallmarks. The mutational status of all PcGs has never been systematically assessed in solid tumours. METHODS: We conducted a multi-step analysis on publically available databases and patient samples to identify somatic aberrations of PcGs. RESULTS: Data from more than 1000 cancer patients show for the first time that the PcG member PHC3 is amplified in three epithelial neoplasms (rate: 8-35%). This aberration predicts poorer prognosis in lung and uterine carcinomas (P<0.01). Gene amplification correlates with mRNA overexpression (P<0.01), suggesting a functional role of this aberration. CONCLUSION: PHC3 amplification may emerge as a biomarker and potential therapeutic target in a relevant fraction of epithelial tumours.

The hedgehog processing pathway is required for NSCLC growth and survival.

Considerable interest has been generated from the results of recent clinical trials using smoothened (SMO) antagonists to inhibit the growth of hedgehog (HH) signaling-dependent tumors. This interest is tempered by the discovery of SMO mutations mediating resistance, underscoring the rationale for developing therapeutic strategies that interrupt HH signaling at levels distinct from those inhibiting SMO function. Here, we demonstrate that HH-dependent non-small cell lung carcinoma (NSCLC) growth is sensitive to blockade of the HH pathway upstream of SMO, at the level of HH ligand processing. Individually, the use of different lentivirally delivered shRNA constructs targeting two functionally distinct HH-processing proteins, skinny hedgehog (SKN) or dispatched-1 (DISP-1), in NSCLC cell lines produced similar decreases in cell proliferation and increased cell death. Further, providing either an exogenous source of processed HH or a SMO agonist reverses these effects. The attenuation of HH processing, by knocking down either of these gene products, also abrogated tumor growth in mouse xenografts. Finally, we extended these findings to primary clinical specimens, showing that SKN is frequently overexpressed in NSCLC and that higher DISP-1 expression is associated with an unfavorable clinical outcome. Our results show a critical role for HH processing in HH-dependent tumors, identifies two potential druggable targets in the HH pathway, and suggest that similar therapeutic strategies could be explored to treat patients harboring HH ligand-dependent cancers.

The physiology, microcirculation and clinical application of the shunt-restricted arterialized venous flaps for the reconstruction of digital defects.

Reconstruction of digital defects using the venous flap offer several advantages but remained unpopular owing to levels of venous congestion rates. We performed animal studies to test the hypothesis that an arterio-venous shunt increases pressure for peripheral flap perfusion and decreases venous congestion. Using an abdominal adipofascial flap model in six male Sprague-Dawley rats, microcirculation was modified as follows: type I - arterial flap; type II - flow-through arterio-venous flap (AVF); and type III - shunt-restricted AVF. In type I flaps, blood flow was observed to be unidirectional in both arterioles and venules. In type I flaps, blood flow was observed to be unidirectional in both arterioles and venules. In type II flaps, blood flow oscillated without a dominant direction and came to a standstill. In type III flaps, blood flowed proximally in a reverse direction whereas distally, flow was similar to type I flaps. In a clinical series, 21 patients received a total of 22 shunt-restricted AVFs. All 22 clinical flaps survived; four flaps suffered epidermolysis but recovered without full thickness loss.

The role of the sensory nerve response in ultrasound accelerated fracture repair.

This study was designed to test the hypothesis that the sensory innervation of bone might play an important role in sensing and responding to low-intensity pulsed ultrasound and explain its effect in promoting fracture healing. In 112 rats a standardised mid-shaft tibial fracture was created, supported with an intramedullary needle and divided into four groups of 28. These either had a sciatic neurectomy or a patellar tendon resection as control, and received the ultrasound or not as a sham treatment. Fracture union, callus mineralisation and remodelling were assessed using plain radiography, peripheral quantitative computed tomography and histomorphology. Daily ultrasound treatment significantly increased the rate of union and the volumetric bone mineral density in the fracture callus in the neurally intact rats (p = 0.025), but this stimulating effect was absent in the rats with sciatic neurectomy. Histomorphology demonstrated faster maturation of the callus in the group treated with ultrasound when compared with the control group. The results supported the hypothesis that intact innervation plays an important role in allowing low-intensity pulsed ultrasound to promote fracture healing.

Arsenic-related DNA copy-number alterations in lung squamous cell carcinomas.

BACKGROUND: Lung squamous cell carcinomas (SqCCs) occur at higher rates following arsenic exposure. Somatic DNA copy-number alterations (CNAs) are understood to be critical drivers in several tumour types. We have assembled a rare panel of lung tumours from a population with chronic arsenic exposure, including SqCC tumours from patients with no smoking history. METHODS: Fifty-two lung SqCCs were analysed by whole-genome tiling-set array comparative genomic hybridisation. Twenty-two were derived from arsenic-exposed patients from Northern Chile (10 never smokers and 12 smokers). Thirty additional cases were obtained for comparison from North American smokers without arsenic exposure. Twenty-two blood samples from healthy individuals from Northern Chile were examined to identify germline DNA copy-number variations (CNVs) that could be excluded from analysis. RESULTS: We identified multiple CNAs associated with arsenic exposure. These alterations were not attributable to either smoking status or CNVs. DNA losses at chromosomes 1q21.1, 7p22.3, 9q12, and 19q13.31 represented the most recurrent events. An arsenic-associated gain at 19q13.33 contains genes previously identified as oncogene candidates. CONCLUSIONS: Our results provide a comprehensive approach to molecular characteristics of the arsenic-exposed lung cancer genome and the non-smoking lung SqCC genome. The distinct and recurrent arsenic-related alterations suggest that this group of tumours may be considered as a separate disease subclass.

Epigenetic inactivation of the miR-34a in hematological malignancies.

miR-34a is a transcriptional target of p53 and implicated in carcinogenesis. We studied the role of miR-34a methylation in a panel of hematological malignancies including acute leukemia [acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL)], chronic leukemia [chronic lymphocytic leukemia (CLL) and chronic myeloid leukemia (CML)], multiple myeloma (MM) and non-Hodgkin's lymphoma (NHL). The methylation status of miR-34a promoter was studied in 12 cell lines and 188 diagnostic samples by methylation-specific polymerase chain reaction. miR-34a promoter was unmethylated in normal controls but methylated in 75% lymphoma and 37% myeloma cell lines. Hypomethylating treatment led to re-expression of pri-miR-34a transcript in lymphoma cells with homozygous miR-34a methylation. In primary samples at diagnosis, miR-34a methylation was detected in 4% CLL, 5.5% MM samples and 18.8% of NHL at diagnosis but none of ALL, AML and CML (P = 0.011). In MM patients with paired samples, miR-34a methylation status remained unchanged at progression. Amongst lymphoid malignancies, miR-34a was preferentially methylated in NHL (P = 0.018), in particular natural killer (NK)/T-cell lymphoma. In conclusion, amongst hematological malignancies, miR-34a methylation is preferentially hypermethylated in NHL, in particular NK/T-cell lymphoma, in a tumor-specific manner, therefore the role of miR-34a in lymphomagenesis warrants further study.