Coordination of canonical and noncanonical Hedgehog signalling pathways mediated by WDR11 during primordial germ cell development.

WDR11, a gene associated with Kallmann syndrome, is important in reproductive system development but molecular understanding of its action remains incomplete. We previously reported that Wdr11-deficient embryos exhibit defective ciliogenesis and developmental defects associated with Hedgehog (HH) signalling. Here we demonstrate that WDR11 is required for primordial germ cell (PGC) development, regulating canonical and noncanonical HH signalling in parallel. Loss of WDR11 disrupts PGC motility and proliferation driven by the cilia-independent, PTCH2/GAS1-dependent noncanonical HH pathway. WDR11 modulates the growth of somatic cells surrounding PGCs by regulating the cilia-dependent, PTCH1/BOC-dependent canonical HH pathway. We reveal that PTCH1/BOC or PTCH2/GAS1 receptor context dictates SMO localisation inside or outside of cilia, respectively, and loss of WDR11 affects the signalling responses of SMO in both situations. We show that GAS1 is induced by PTCH2-specific HH signalling, which is lost in the absence of WDR11. We also provide evidence supporting a role for WDR11 in ciliogenesis through regulation of anterograde intraflagellar transport potentially via its interaction with IFT20. Since WDR11 is a target of noncanonical SMO signalling, WDR11 represents a novel mechanism by which noncanonical and canonical HH signals communicate and cooperate.

The Editors' Personal Biography of Professor Robert Anderson.

Robert (Bob) Henry Anderson was born in Wellington, Shropshire, UK, in 1942 and he completed his medical training in Manchester (UK) in 1966 [...].

Assessing the prevalence of alcohol consumption in early pregnancy using blood biomarker analysis: a consistent pattern across north-east England?

BACKGROUND: We previously investigated the prevalence of alcohol consumption in early pregnancy in Northumbria Healthcare NHS Foundation Trust, a locality of north-east England. The prevalence was 1.4% based on blood sample biomarker analysis using carbohydrate deficient transferrin (CDT) and 3.5% for gamma-glutamyltransferase (GGT). AIMS: To supplement this research by investigating the prevalence of alcohol use using identical methods in a different locality of the same region. METHODS: Six-hundred random blood samples taken at the antenatal booking appointment were anonymously analysed for the presence of CDT, a validated marker of chronic alcohol exposure (normalizing 2-3 weeks from abstinence) and GGT, a liver enzyme elevated for up to 8 weeks after alcohol exposure. RESULTS: The North Tees and Hartlepool NHS Foundation Trust data revealed a CDT prevalence rate of 1.7% (95% CI: 0.7-2.9) and GGT prevalence rate of 4.2% (95% CI: 2.6-5.9). However, these measures are not sensitive to low levels of alcohol; and no overlapping cases were identified or a significant correlation demonstrated between CDT or GGT. DISCUSSION: These data support our earlier work. Prevalence rates according to CDT and GGT analysis were similar in both areas, suggesting similar patterns of sustained alcohol use in pregnancy across the region.

Clinically Significant Fibrosis Is Associated With Longitudinal Increases in Fibrosis-4 and Nonalcoholic Fatty Liver Disease Fibrosis Scores.

BACKGROUND & AIMS: There is limited knowledge regarding the longitudinal utility of biomarkers of fibrosis, such as the nonalcoholic fatty liver disease (NAFLD) fibrosis score (NFS) or the fibrosis-4 score (FIB-4) score. We examined longitudinal changes in the NFS and the FIB-4 score in patients with NAFLD, with and without clinically significant fibrosis (CSF). METHODS: We performed a retrospective study of 230 patients with NAFLD, collecting clinical and laboratory records to calculate NFS and FIB-4 scores at 6 monthly intervals for 5 years before hepatology assessment of fibrosis. Linear mixed models with random intercept and slope and adjusted for age at baseline were used to assess the progression of NFS and log-transformed FIB-4 scores over time in subjects with and without CSF, determined by liver stiffness measurements of 8.2 kPa or greater. RESULTS: Patients had a median of 11 (minimum, 10; maximum, 11) retrospective observations over a median time period of 5 years (minimum, 4.5 y; maximum, 5 y). Of patients with low baseline NFS and FIB-4 scores, 31.11% and 37.76%, respectively, had CSF at the time of hepatology assessment. There was a correlation between NFS and log10 FIB-4 over time (repeated measure r = 0.55; 95% CI, 0.52-0.59). The rate of increase in NFS and log10 FIB-4 was significantly higher in patients with than without CSF (both P < .001). Predicted NFS increased by 0.17 and 0.06 units per year in subjects with and without CSF, respectively. Predicted log10 FIB-4 score increased by 0.032 and 0.0003 units per year in subjects with and without CSF, respectively. CONCLUSIONS: Noninvasively measured fibrosis scores increase progressively in patients with NAFLD and CSF. Further studies are needed to determine whether repeated measurements can identify patients at risk for CSF.

Biochemical evaluation of kidney disease.

Different biochemical markers exist in both blood and urine for assessing renal function. Most of these biomarkers have advantages and limitations associated with their use, which is important to consider when ordering and utilising them in the clinical setting. The ideal marker should be able to detect acute kidney injury (AKI) at the onset and be used for the diagnosis and ongoing monitoring and management of kidney disease. The search for such a marker is ongoing, as all potential candidates thus far are associated with certain limitations. This article will attempt to compare and contrast established and emerging kidney disease markers.

A Pragmatic Approach Identifies a High Rate of Nonalcoholic Fatty Liver Disease With Advanced Fibrosis in Diabetes Clinics and At-Risk Populations in Primary Care.

Noninvasive serum biomarkers (nonalcoholic fatty liver disease fibrosis score [NFS], fibrosis 4 score [FIB-4], or enhanced liver fibrosis [ELF] test) are recommended as first-line tools to determine the risk of advanced fibrosis in nonalcoholic fatty liver disease. We aimed to assess the utility of a pragmatic approach to screening for clinically significant fibrosis in primary care and diabetes clinics. We recruited 252 patients from an endocrine clinic or primary care facility. Anthropometric measurements, ELF test, ultrasound, and liver stiffness measurements (LSMs) were performed. Clinically significant fibrosis was defined as LSM ≥8.2 kPa or ELF ≥9.8. A subgroup of patients underwent liver biopsy (n = 48) or had imaging diagnostic of cirrhosis (n = 14). Patients were 57.3 ± 12.3 years old with a high prevalence of metabolic syndrome (84.5%), type 2 diabetes (82.5%), and body mass index (BMI) ≥40 kg/m2 (21.8%). LSM met quality criteria in 230 (91.3%) patients. NFS and FIB-4 combined had a high negative predictive value (90.0%) for excluding LSM ≥8.2 kPa. However, 84.1% of patients had indeterminate or high NFS or FIB-4 scores requiring further assessment. LSM ≥8.2 kPa and ELF ≥9.8 were present in 31.3% and 28.6% of patients, respectively. Following adjustment for age, BMI, sex, and presence of advanced fibrosis, older age was independently associated with ELF ≥9.8 (adjusted odds ratio, 1.14; 95% confidence interval, 1.06-1.24), whereas increasing BMI was independently associated with LSM ≥8.2 kPa (adjusted odds ratio, 1.15; 95% confidence interval, 1.01-1.30). Concordant LSM <8.2 kPa and ELF <9.8 and concordant LSM ≥8.2 kPa and ELF ≥9.8 had a high negative predictive value (91.7%) and positive predictive value (95.8%) for excluding and identifying clinically significant fibrosis, respectively. Conclusion: Simple scoring tools alone lack accuracy. LSM accuracy is influenced by severe obesity, whereas age impacts the ELF test. Further studies are required to confirm whether combining LSM and ELF may enhance accuracy and confidence in identifying clinically significant fibrosis. (Hepatology Communications 2018; 00:000-000).

Controlled attenuation parameter in NAFLD identifies risk of suboptimal glycaemic and metabolic control.

AIMS: To examine the relationship between steatosis quantified by controlled attenuation parameter (CAP) values and glycaemic/metabolic control. METHODS: 230 patients, recruited from an Endocrine clinic or primary care underwent routine Hepatology assessment, with liver stiffness measurements and simultaneous CAP. Multivariable logistic regression was performed to identify potential predictors of Metabolic Syndrome (MetS), HbA1c ≥ 7%, use of insulin, hypertriglyceridaemia and CAP ≥ 300 dB/m. RESULTS: Patients were 56.7 ±â€¯12.3 years of age with a high prevalence of MetS (83.5%), T2DM (81.3%), and BMI ≥ 40 kg/m2 (18%). Median CAP score was 344 dB/m, ranging from 128 to 400 dB/m. BMI (aOR 1.140 95% CI 1.068-1.216), requirement for insulin (aOR 2.599 95% CI 1.212-5.575), and serum ALT (aOR 1.018 95% CI 1.004-1.033) were independently associated with CAP ≥ 300 dB/m. Patients with CAP interquartile range < 40 (68%) had a higher median serum ALT level (p = 0.029), greater prevalence of BMI ≥ 40 kg/m2 (p = 0.020) and higher median CAP score (p < 0.001). Patients with higher CAP scores were more likely to have MetS (aOR 1.011 95% CI 1.003-1.019), HBA1c ≥ 7 (aOR 1.010 95% CI 1.003-1.016), requirement for insulin (aOR 1.007 95% CI 1.002-1.013) and hypertriglyceridemia (aOR 1.007 95% CI 1.002-1.013). CONCLUSIONS: Our data demonstrate that an elevated CAP reflects suboptimal metabolic control. In diabetic patients with NAFLD, CAP may be a useful point-of-care test to identify patients at risk of poorly controlled metabolic comorbidities or advanced diabetes.

Assessing prevalence of alcohol consumption in early pregnancy: Self-report compared to blood biomarker analysis.

Providing appropriate antenatal and postnatal care for women who drink alcohol in pregnancy is only possible if those at risk can be identified. We aimed to compare the prevalence of alcohol consumption in the first trimester of pregnancy using self-report and blood biomarker analysis. Six-hundred routine blood samples from 2014, taken at the antenatal booking appointment, in the first trimester of pregnancy, were anonymously analysed for the presence of Carbohydrate Deficient Transferrin (CDT), a validated marker of chronic alcohol exposure (normalising 2-3 weeks from abstinence) and Gamma-glutamyltransferase (GGT), a liver enzyme elevated for up to 8 weeks after alcohol exposure. In a separate sample of women, from 2015, data taken during the antenatal visit, documenting women's self-reported alcohol consumption, were collected. The percentage of women who reported alcohol intake in the first trimester was 0.8%. This compared to 74.1% of women who reported consuming alcohol before pregnancy. CDT analysis revealed a prevalence rate of 1.4% and GGT a prevalence rate of 3.5% in the first trimester of pregnancy. Although those with elevated CDT generally had high levels of GGT, only one person was positive for CDT and GGT. Results from CDT analysis and self-report may underestimate prevalence for different reasons. GGT appeared to lack specificity, but it may have value in supporting findings from CDT analysis. Further studies using additional blood biomarkers, or a combination of blood biomarkers and self-report, may be beneficial in accurately detecting alcohol drinking history in pregnancy.

Characterization of the serum and salivary cortisol response to the intravenous 250 µg ACTH1-24 stimulation test.

PURPOSE: The ACTH1-24 stimulation test is commonly used to assess the hypothalamic-pituitary-adrenal (HPA) axis. Given variations in CBG concentration and binding affinity, serum total cortisol may misclassify some patients. Salivary cortisol correlates well with serum free cortisol but is easier to measure and widely available in commercial laboratories. The aim of this study was to investigate the utility of measuring salivary cortisol during the ACTH1-24 stimulation test. DESIGN AND METHODS: Case-control study in a clinical research facility. Eighty-seven patients with suspected cortisol deficiency, twenty-four healthy controls, and ten healthy women on the oral contraceptive (OC) underwent an intravenous 250 µg ACTH1-24 stimulation test. Concordance of ACTH1-24 stimulated serum and salivary cortisol was evaluated. RESULTS: There was a significant difference in serum cortisol between the healthy volunteers and the women on the OC (P < 0.001) but no difference in salivary cortisol. The lower limit of the reference interval for salivary cortisol at 60 min was 26 nmol/L. 27/89 (30%) of tests with suspected HPA axis disorder failed the 60 min serum cortisol cut-off of 500 nmol/L. Of these, 24/27 (89%) had a salivary cortisol of <26 nmol/L. In contrast, 12/19 (63%) tests and 5/43 (12%) tests where the 60 min serum cortisol was 500-599 and ≥600 nmol/L, respectively had a salivary cortisol of <26 nmol/L. CONCLUSIONS: Salivary cortisol provides additional diagnostic value during the 250 µg ACTH1-24 stimulation test in patients with proven or suspected alterations in CBG and potentially those with a borderline 60 min serum cortisol 500-599 nmol/L.

WDR11-mediated Hedgehog signalling defects underlie a new ciliopathy related to Kallmann syndrome.

WDR11 has been implicated in congenital hypogonadotropic hypogonadism (CHH) and Kallmann syndrome (KS), human developmental genetic disorders defined by delayed puberty and infertility. However, WDR11's role in development is poorly understood. Here, we report that WDR11 modulates the Hedgehog (Hh) signalling pathway and is essential for ciliogenesis. Disruption of WDR11 expression in mouse and zebrafish results in phenotypic characteristics associated with defective Hh signalling, accompanied by dysgenesis of ciliated tissues. Wdr11-null mice also exhibit early-onset obesity. We find that WDR11 shuttles from the cilium to the nucleus in response to Hh signalling. WDR11 regulates the proteolytic processing of GLI3 and cooperates with the transcription factor EMX1 in the induction of downstream Hh pathway gene expression and gonadotrophin-releasing hormone production. The CHH/KS-associated human mutations result in loss of function of WDR11. Treatment with the Hh agonist purmorphamine partially rescues the WDR11 haploinsufficiency phenotypes. Our study reveals a novel class of ciliopathy caused by WDR11 mutations and suggests that CHH/KS may be a part of the human ciliopathy spectrum.

Underappreciation of non-alcoholic fatty liver disease by primary care clinicians: limited awareness of surrogate markers of fibrosis.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is a common cause of incidental liver test abnormalities. General practitioners (GP) have a key role in identifying people with NAFLD at risk of significant liver disease. Recent specialist guidelines emphasise the use of fibrosis algorithms or serum biomarkers rather than routine liver tests, to assess advanced fibrosis. AIM: To evaluate primary care clinicians' current approach to diagnosis, management and referral of NAFLD. METHODS: A cross-sectional survey of primary care clinicians was undertaken through a structured questionnaire about NAFLD. A convenience sample of general practice clinics and general practice conferences in Metropolitan Brisbane and regional south east Queensland was selected. RESULTS: A total of 108 primary care clinicians completed the survey (participation rate 100%). Fifty-one percent of respondents considered the prevalence of NAFLD in the general population to be ≤10%. Twenty-four percent of respondents felt that liver enzymes were sufficiently sensitive to detect underlying NAFLD. Most respondents were unsure whether the Fibrosis 4 score (62.7% unsure) or Enhanced Liver Fibrosis score (63.7% unsure) could help to identify advanced fibrosis or cirrhosis. Although 47% of respondents said they would refer a patient to a Gastroenterologist/Hepatologist if they suspect the patient has NAFLD, 44.1% do not make any referrals. Of concern, 70.6% of clinicians said they were unlikely to refer a patient to Hepatology unless liver function tests are abnormal. CONCLUSION: Our findings demonstrate that many primary care clinicians underestimate the prevalence of NAFLD and under-recognise the clinical spectrum of NAFLD and how this is assessed.

Alcohol Consumption in Diabetic Patients with Nonalcoholic Fatty Liver Disease.

AIM: To examine the association between lifetime alcohol consumption and significant liver disease in type 2 diabetic patients with NAFLD. METHODS: A cross-sectional study assessing 151 patients with NAFLD at risk of clinically significant liver disease. NAFLD fibrosis severity was classified by transient elastography; liver stiffness measurements ≥8.2 kPa defined significant fibrosis. Lifetime drinking history classified patients into nondrinkers, light drinkers (always ≤20 g/day), and moderate drinkers (any period with intake >20 g/day). RESULT: Compared with lifetime nondrinkers, light and moderate drinkers were more likely to be male (p = 0.008) and to be Caucasian (p = 0.007) and to have a history of cigarette smoking (p = 0.000), obstructive sleep apnea (p = 0.003), and self-reported depression (p = 0.003). Moderate drinkers required ≥3 hypoglycemic agents to maintain diabetic control (p = 0.041) and fibrate medication to lower blood triglyceride levels (p = 0.044). Compared to lifetime nondrinkers, light drinkers had 1.79 (95% CI: 0.67-4.82; p = 0.247) and moderate drinkers had 0.91 (95% CI: 0.27-3.10; p = 0.881) times the odds of having liver stiffness measurements ≥8.2 kPa (adjusted for age, gender, and body mass index). CONCLUSIONS: In diabetic patients with NAFLD, light or moderate lifetime alcohol consumption was not significantly associated with liver fibrosis. The impact of lifetime alcohol intake on fibrosis progression and diabetic comorbidities, in particular obstructive sleep apnea and hypertriglyceridemia, requires further investigation.

Human venous valve disease caused by mutations in FOXC2 and GJC2.

Venous valves (VVs) prevent venous hypertension and ulceration. We report that FOXC2 and GJC2 mutations are associated with reduced VV number and length. In mice, early VV formation is marked by elongation and reorientation ("organization") of Prox1hi endothelial cells by postnatal day 0. The expression of the transcription factors Foxc2 and Nfatc1 and the gap junction proteins Gjc2, Gja1, and Gja4 were temporospatially regulated during this process. Foxc2 and Nfatc1 were coexpressed at P0, and combined Foxc2 deletion with calcineurin-Nfat inhibition disrupted early Prox1hi endothelial organization, suggesting cooperative Foxc2-Nfatc1 patterning of these events. Genetic deletion of Gjc2, Gja4, or Gja1 also disrupted early VV Prox1hi endothelial organization at postnatal day 0, and this likely underlies the VV defects seen in patients with GJC2 mutations. Knockout of Gja4 or Gjc2 resulted in reduced proliferation of Prox1hi valve-forming cells. At later stages of blood flow, Foxc2 and calcineurin-Nfat signaling are each required for growth of the valve leaflets, whereas Foxc2 is not required for VV maintenance.

Multimorbidity and polypharmacy in diabetic patients with NAFLD: Implications for disease severity and management.

An observational study describing the number and type of chronic conditions and medications taken by diabetic patients with NAFLD and identifying characteristics that may impact liver disease severity or clinical management.Adults with type 2 diabetes have a high prevalence of nonalcoholic fatty liver disease (NAFLD) and increased risk of developing advanced liver disease. Appropriate management should consider the characteristics of the diabetic NAFLD population, as comorbid conditions and medications may increase the complexity of treatment strategies.Diabetic patients with NAFLD at risk of clinically significant liver disease (as assessed by the FIB-4 or NAFLD fibrosis scores) were recruited consecutively from the Endocrine clinic or primary care. Medical conditions, medication history, anthropometric measurements, and laboratory tests were obtained during assessment. NAFLD severity was classified by transient elastography and liver ultrasound into "no advanced disease" (LSM < 8.2 kPa) or "clinically significant liver disease" (LSM ≥ 8.2 kPa).The most common coexistent chronic conditions were metabolic syndrome (94%), self-reported "depression" (44%), ischaemic heart disease (32%), and obstructive sleep apnoea (32%). Polypharmacy or hyperpolypharmacy was present in 59% and 31% of patients respectively. Elevated LSM (≥ 8.2 kPa) suggesting significant liver disease was present in 37% of this at-risk cohort. Increasing obesity and abdominal girth were both independently associated with likelihood of having significant liver disease.There is a high burden of multimorbidity and polypharmacy in diabetic NAFLD patients, highlighting the importance of multidisciplinary management to address their complex health care needs and ensure optimal medical treatment.

The independence of the infundibular building blocks in the setting of double-outlet right ventricle.

It has long been contentious as to whether the presence of bilateral infundibulums, or conuses, is a prerequisite for the diagnosis of double-outlet right ventricle. As the use of such a criterion would abrogate the so-called "morphological method", which correctly states that one variable entity should not be defined on the basis of another entity that is itself variable, it is now accepted that double outlet can exist in the setting of fibrous continuity between the leaflets of the atrioventricular and arterial valves. Although this debate has now been resolved, there are other contentious areas still requiring clarification in the setting of hearts unified because of the presence of this particular ventriculo-arterial connection - for example, it is questionable whether the channel between the ventricles should be described as a "ventricular septal defect", whereas it is equally arguable that the mere presence of fibrous continuity between the leaflets of the arterial valves does not necessarily place the channel in a doubly committed location. In this review, we describe a series of autopsied hearts in which the anatomical features serve to illuminate these various topics. We then discuss recent findings regarding cardiac development that point to the individuality of the building blocks of the ventricular outflow tracts, specifically the outlet septum, the inner heart curvature, or ventriculo-infundibular fold, and the septomarginal trabeculation, or septal band.

How strong is the evidence for using blood biomarkers alone to screen for alcohol consumption during pregnancy? A systematic review.

Accurate and early identification of women at risk from alcohol consumption during pregnancy allows education and support programmes to be targeted at those most in need. We aimed to conduct a systematic review to compare the efficacy of blood analysis and maternal self-report in detecting at risk women during pregnancy. This review investigated diagnostic accuracy. We searched four databases (Medline, Embase, Psychinfo and CINAHL) for relevant articles and conducted hand searches of recent issues of key journals in the field. No restriction was placed on inclusion in terms of publication date or language. Studies were deemed eligible if they were original research and included a direct comparison of the results of blood biomarker analysis and self-reported alcohol use for the detection of alcohol consumption in pregnant women. Quality appraisal of included studies was conducted using the QUADAS II tool. Eight studies met the inclusion criteria. Gamma-glutamyltransferase (GGT) was investigated in five studies, mean corpuscular volume (MCV) and phosphatidylethanol (PEth) in three studies and carbohydrate deficient transferrin (CDT), aspartate aminotransferase (AST), alanine aminotransferase (ALT) and whole blood associated acetaldehyde assay (WBAA) were each investigated in two studies. Although all of the studies were rated of good methodological quality, none of the biomarkers had both high sensitivity and specificity when compared to self-report. There was some evidence that a combination of biomarkers, or combining biomarkers with self-report, increases accuracy. In summary, the blood biomarkers examined were of limited use in screening for low and moderate alcohol consumption in pregnancy when compared to self-report. However, certain biomarkers, such and CDT and PEth may complement self-report and help improve the accuracy of diagnosis.

Evaluation of the Greiner Bio-One serum separator BCA Fast Clot tube.

BACKGROUND: Current commercial tubes have difficulties in producing "true" serum from all blood samples even within the recommended clotting times. Hence, Becton Dickinson (BD) and now Greiner have produced tubes containing thrombin as the procoagulant to reduce the clotting time and increase the possibility of producing serum from anticoagulated blood samples. METHODS: The Greiner BCA Fast Clot (GBBCAFC) tube was evaluated in a hospital environment using 40 participants, (30 healthy and 10 undergoing renal dialysis) for 32 analytes against the Greiner lithium heparin tube and the BD Rapid Serum Tubes (BD RST) tube measured on Beckman DxC 800 and DxI 800 analyzers. Clotting strength was also examined using thromboelastography (TEG). RESULTS: The analytes results showed there was a very close agreement between the BD RST tube and GBBCAFC tube in comparison with lithium heparin plasma. The result comparison data showed equivalent performance with lower levels of hemolysis. The prolonged storage study also showed very similar agreement between the BD RST and the GBBCAFC tubes. Likewise, the TEG data showed there was very little difference in clotting ability between the tubes, and neither was capable of producing true serum from blood spiked with 2 U heparin/mL of blood. CONCLUSIONS: The study showed the GBBCAFC tube with the combination of the two procoagulants blood clotting activator and thrombin produced comparable performance with the lithium heparin plasma and the BD RST serum samples.