BTB domain mutations perturbing KCTD15 oligomerisation cause a distinctive frontonasal dysplasia syndrome.

INTRODUCTION: KCTD15 encodes an oligomeric BTB domain protein reported to inhibit neural crest formation through repression of Wnt/beta-catenin signalling, as well as transactivation by TFAP2. Heterozygous missense variants in the closely related paralogue KCTD1 cause scalp-ear-nipple syndrome. METHODS: Exome sequencing was performed on a two-generation family affected by a distinctive phenotype comprising a lipomatous frontonasal malformation, anosmia, cutis aplasia of the scalp and/or sparse hair, and congenital heart disease. Identification of a de novo missense substitution within KCTD15 led to targeted sequencing of DNA from a similarly affected sporadic patient, revealing a different missense mutation. Structural and biophysical analyses were performed to assess the effects of both amino acid substitutions on the KCTD15 protein. RESULTS: A heterozygous c.310G>C variant encoding p.(Asp104His) within the BTB domain of KCTD15 was identified in an affected father and daughter and segregated with the phenotype. In the sporadically affected patient, a de novo heterozygous c.263G>A variant encoding p.(Gly88Asp) was present in KCTD15. Both substitutions were found to perturb the pentameric assembly of the BTB domain. A crystal structure of the BTB domain variant p.(Gly88Asp) revealed a closed hexameric assembly, whereas biophysical analyses showed that the p.(Asp104His) substitution resulted in a monomeric BTB domain likely to be partially unfolded at physiological temperatures. CONCLUSION: BTB domain substitutions in KCTD1 and KCTD15 cause clinically overlapping phenotypes involving craniofacial abnormalities and cutis aplasia. The structural analyses demonstrate that missense substitutions act through a dominant negative mechanism by disrupting the higher order structure of the KCTD15 protein complex.

Reassessing the association: Evaluation of a polyalanine deletion variant of RUNX2 in non-syndromic sagittal and metopic craniosynostosis.

The RUNT-related transcription factor RUNX2 plays a critical role in osteoblast differentiation, and alterations to gene dosage cause distinct craniofacial anomalies. Uniquely amongst the RUNT-related family, vertebrate RUNX2 encodes a polyglutamine/polyalanine repeat (Gln23-Glu-Ala17 in humans), with the length of the polyalanine component completely conserved in great apes. Surprisingly, a frequent 6-amino acid deletion polymorphism, p.(Ala84_Ala89)del, occurs in humans (termed 11A allele), and a previous association study (Cuellar et al. Bone 137:115395;2020) reported that the 11A variant was significantly more frequent in non-syndromic sagittal craniosynostosis (nsSag; allele frequency [AF] = 0.156; 95% confidence interval [CI] 0.126-0.189) compared to non-syndromic metopic craniosynostosis (nsMet; AF = 0.068; 95% CI 0.045-0.098). However, the gnomAD v.2.1.1 control population used by Cuellar et al. did not display Hardy-Weinberg equilibrium, hampering interpretation. To re-examine this association, we genotyped the RUNX2 11A polymorphism in 225 individuals with sporadic nsSag as parent-child trios and 164 singletons with sporadic nsMet, restricting our analysis to individuals of European ancestry. We compared observed allele frequencies to the non-transmitted alleles in the parent-child trios, and to the genome sequencing data from gnomAD v.4, which display Hardy-Weinberg equilibrium. Observed AFs (and 95% CI) were 0.076 (0.053-0.104) in nsSag and 0.082 (0.055-0.118) in nsMet, compared with 0.062 (0.042-0.089) in non-transmitted parental alleles and 0.065 (0.063-0.067) in gnomAD v.4.0.0 non-Finnish European control genomes. In summary, we observed a non-significant excess, compared to gnomAD data, of 11A alleles in both nsSag (relative risk 1.18, 95% CI 0.83-1.67) and nsMet (relative risk 1.29, 95% CI 0.87-1.92), but we did not replicate the much higher excess of RUNX2 11A alleles in nsSag previously reported (p = 0.0001).

Craniosynostosis, inner ear, and renal anomalies in a child with complete loss of SPRY1 (sprouty homolog 1) function.

INTRODUCTION: SPRY1 encodes protein sprouty homolog 1 (Spry-1), a negative regulator of receptor tyrosine kinase signalling. Null mutant mice display kidney/urinary tract abnormalities and altered size of the skull; complete loss-of-function of Spry-1 in humans has not been reported. METHODS: Analysis of whole-genome sequencing data from individuals with craniosynostosis enrolled in the 100,000 Genomes Project identified a likely pathogenic variant within SPRY1. Reverse-transcriptase PCR and western blot analysis were used to investigate the effect of the variant on SPRY1 mRNA and protein, in lymphoblastoid cell lines from the patient and both parents. RESULTS: A nonsense variant in SPRY1, encoding p.(Leu27*), was confirmed to be heterozygous in the unaffected parents and homozygous in the child. The child's phenotype, which included sagittal craniosynostosis, subcutaneous cystic lesions overlying the lambdoid sutures, hearing loss associated with bilateral cochlear and vestibular dysplasia and a unilateral renal cyst, overlapped the features reported in Spry1-/- null mice. Functional studies supported escape from nonsense-mediated decay, but western blot analysis demonstrated complete absence of full-length protein in the affected child and a marked reduction in both parents. CONCLUSION: This is the first report of complete loss of Spry-1 function in humans, associated with abnormalities of the cranial sutures, inner ear, and kidneys.

Pathogenic variants in the paired-related homeobox 1 gene (PRRX1) cause craniosynostosis with incomplete penetrance.

PURPOSE: Studies have previously implicated PRRX1 in craniofacial development, including demonstration of murine Prrx1 expression in the preosteogenic cells of the cranial sutures. We investigated the role of heterozygous missense and loss-of-function (LoF) variants in PRRX1 associated with craniosynostosis. METHODS: Trio-based genome, exome, or targeted sequencing were used to screen PRRX1 in patients with craniosynostosis; immunofluorescence analyses were used to assess nuclear localization of wild-type and mutant proteins. RESULTS: Genome sequencing identified 2 of 9 sporadically affected individuals with syndromic/multisuture craniosynostosis, who were heterozygous for rare/undescribed variants in PRRX1. Exome or targeted sequencing of PRRX1 revealed a further 9 of 1449 patients with craniosynostosis harboring deletions or rare heterozygous variants within the homeodomain. By collaboration, 7 additional individuals (4 families) were identified with putatively pathogenic PRRX1 variants. Immunofluorescence analyses showed that missense variants within the PRRX1 homeodomain cause abnormal nuclear localization. Of patients with variants considered likely pathogenic, bicoronal or other multisuture synostosis was present in 11 of 17 cases (65%). Pathogenic variants were inherited from unaffected relatives in many instances, yielding a 12.5% penetrance estimate for craniosynostosis. CONCLUSION: This work supports a key role for PRRX1 in cranial suture development and shows that haploinsufficiency of PRRX1 is a relatively frequent cause of craniosynostosis.

Australian evidence-based guidelines for the prevention and management of diabetes-related foot disease: a guideline summary.

INTRODUCTION: Diabetes-related foot disease (DFD) - foot ulcers, infection, ischaemia - is a leading cause of hospitalisation, disability, and health care costs in Australia. The previous 2011 Australian guideline for DFD was outdated. We developed new Australian evidence-based guidelines for DFD by systematically adapting suitable international guidelines to the Australian context using the ADAPTE and GRADE approaches recommended by the NHMRC. MAIN RECOMMENDATIONS: This article summarises the most relevant of the 98 recommendations made across six new guidelines for the general medical audience, including: prevention - screening, education, self-care, footwear, and treatments to prevent DFD; classification - classifications systems for ulcers, infection, ischaemia and auditing; peripheral artery disease (PAD) - examinations and imaging for diagnosis, severity classification, and treatments; infection - examinations, cultures, imaging and inflammatory markers for diagnosis, severity classification, and treatments; offloading - pressure offloading treatments for different ulcer types and locations; and wound healing - debridement, wound dressing selection principles and wound treatments for non-healing ulcers. CHANGES IN MANAGEMENT AS A RESULT OF THE GUIDELINE: For people without DFD, key changes include using a new risk stratification system for screening, categorising risk and managing people at increased risk of DFD. For those categorised at increased risk of DFD, more specific self-monitoring, footwear prescription, surgical treatments, and activity management practices to prevent DFD have been recommended. For people with DFD, key changes include using new ulcer, infection and PAD classification systems for assessing, documenting and communicating DFD severity. These systems also inform more specific PAD, infection, pressure offloading, and wound healing management recommendations to resolve DFD.

New locus underlying auriculocondylar syndrome (ARCND): 430 kb duplication involving TWIST1 regulatory elements.

BACKGROUND: Auriculocondylar syndrome (ARCND) is a rare genetic disease that affects structures derived from the first and second pharyngeal arches, mainly resulting in micrognathia and auricular malformations. To date, pathogenic variants have been identified in three genes involved in the EDN1-DLX5/6 pathway (PLCB4, GNAI3 and EDN1) and some cases remain unsolved. Here we studied a large unsolved four-generation family. METHODS: We performed linkage analysis, resequencing and Capture-C to investigate the causative variant of this family. To test the pathogenicity of the CNV found, we modelled the disease in patient craniofacial progenitor cells, including induced pluripotent cell (iPSC)-derived neural crest and mesenchymal cells. RESULTS: This study highlights a fourth locus causative of ARCND, represented by a tandem duplication of 430 kb in a candidate region on chromosome 7 defined by linkage analysis. This duplication segregates with the disease in the family (LOD score=2.88) and includes HDAC9, which is located over 200 kb telomeric to the top candidate gene TWIST1. Notably, Capture-C analysis revealed multiple cis interactions between the TWIST1 promoter and possible regulatory elements within the duplicated region. Modelling of the disease revealed an increased expression of HDAC9 and its neighbouring gene, TWIST1, in neural crest cells. We also identified decreased migration of iPSC-derived neural crest cells together with dysregulation of osteogenic differentiation in iPSC-affected mesenchymal stem cells. CONCLUSION: Our findings support the hypothesis that the 430 kb duplication is causative of the ARCND phenotype in this family and that deregulation of TWIST1 expression during craniofacial development can contribute to the phenotype.

Unexpected role of SIX1 variants in craniosynostosis: expanding the phenotype of SIX1-related disorders.

BACKGROUND: Pathogenic heterozygous SIX1 variants (predominantly missense) occur in branchio-otic syndrome (BOS), but an association with craniosynostosis has not been reported. METHODS: We investigated probands with craniosynostosis of unknown cause using whole exome/genome (n=628) or RNA (n=386) sequencing, and performed targeted resequencing of SIX1 in 615 additional patients. Expression of SIX1 protein in embryonic cranial sutures was examined in the Six1nLacZ/+ reporter mouse. RESULTS: From 1629 unrelated cases with craniosynostosis we identified seven different SIX1 variants (three missense, including two de novo mutations, and four nonsense, one of which was also present in an affected twin). Compared with population data, enrichment of SIX1 loss-of-function variants was highly significant (p=0.00003). All individuals with craniosynostosis had sagittal suture fusion; additionally four had bilambdoid synostosis. Associated BOS features were often attenuated; some carrier relatives appeared non-penetrant. SIX1 is expressed in a layer basal to the calvaria, likely corresponding to the dura mater, and in the mid-sagittal mesenchyme. CONCLUSION: Craniosynostosis is associated with heterozygous SIX1 variants, with possible enrichment of loss-of-function variants compared with classical BOS. We recommend screening of SIX1 in craniosynostosis, particularly when sagittal±lambdoid synostosis and/or any BOS phenotypes are present. These findings highlight the role of SIX1 in cranial suture homeostasis.

Blockade of High-Fat Diet Proteomic Phenotypes Using Exercise as Prevention or Treatment.

The increasing consumption of high-fat foods combined with a lack of exercise is a major contributor to the burden of obesity in humans. Aerobic exercise such as running is known to provide metabolic benefits, but how the overconsumption of a high-fat diet (HFD) and exercise interact is not well characterized at the molecular level. Here, we examined the plasma proteome in mice for the effects of aerobic exercise as both a treatment and as a preventative regimen for animals on either a HFD or a healthy control diet. This analysis detected large changes in the plasma proteome induced by the HFD, such as increased abundance of SERPINA7, ALDOB, and downregulation of SERPINA1E and complement factor D (CFD; adipsin). Some of these changes were significantly reverted using exercise as a preventative measure but not as a treatment regimen. To determine if either the intensity or duration of exercise influenced the outcome, we compared high-intensity interval training and endurance running. Endurance running slightly outperformed high-intensity interval training exercise, but overall, both provided similar reversion in abundance of plasma proteins modulated by the HFD, including SERPINA7, apolipoprotein E, SERPINA1E, and CFD. Finally, we compared the changes induced by overconsumption of a HFD with previous data from mice fed on an isocaloric high-saturated fatty acid or polyunsaturated fatty acid diet. This identified several common changes, including not only increased apolipoprotein C-II and apolipoprotein E but also highlighted changes specific for overconsumption of a HFD (fructose-bisphosphate aldolase B, SERPINA7, and CFD), saturated fatty acid-based diets (SERPINA1E), or polyunsaturated fatty acid-based diets (haptoglobin). Together, these data highlight the importance of early intervention with exercise to revert HFD-induced phenotypes and suggest some of the molecular mechanisms leading to the changes in the plasma proteome generated by HFD consumption. Web-based interactive visualizations are provided for this dataset (larancelab.com/hfd-exercise), which give insight into diet and exercise phenotypic interactions on the plasma proteome.

Feasibility and acceptability of the use of flash glucose monitoring encountered by Indigenous Australians with type 2 diabetes mellitus: initial experiences from a pilot study.

BACKGROUND: Type 2 diabetes mellitus (T2DM) is highly prevalent within the Indigenous Australian community. Novel glucose monitoring technology offers an accurate approach to glycaemic management, providing real-time information on glucose levels and trends. The acceptability and feasibilility of this technology in Indigenous Australians with T2DM has not been investigated. OBJECTIVE: This feasibility phenomenological study aims to understand the experiences of Indigenous Australians with T2DM using flash glucose monitoring (FGM). METHODS: Indigenous Australians with T2DM receiving injectable therapy (n = 8) who used FGM (Abbott Freestyle Libre) for 6-months, as part of a clinical trial, participated in semi-structured interviews. Thematic analysis of the interviews was performed using NVivo12 Plus qualitative data analysis software (QSR International). RESULTS: Six major themes emerged: 1) FGM was highly acceptable to the individual; 2) FGM's convenience was its biggest benefit; 3) data from FGM was a tool to modify lifestyle choices; 4) FGM needed to be complemented with health professional support; 5) FGM can be a tool to engage communities in diabetes management; and 6) cost of the device is a barrier to future use. CONCLUSIONS: Indigenous Australians with T2DM had positive experiences with FGM. This study highlights future steps to ensure likelihood of FGM is acceptable and effective within the wider Indigenous Australian community.

Diabetic foot ulcers: weekly versus second-weekly conservative sharp wound debridement.

Diabetic foot ulcers (DFU) are a serious and costly long-term complication of diabetes, and are one of the most prevalent hard-to-heal (chronic) wound types. Conservative sharp wound debridement (CSWD) is a mainstay of care. It is performed regularly until healing is achieved (when there is adequate blood flow for healing) to support endogenous healing and improve the efficacy of advanced healing therapies. CSWD is supported by evidence-based treatment guidelines, despite a lack of prospective studies. The first prospective randomised study to compare different frequencies of CSWD-the Diabetes Debridement Study (DDS)-showed no difference in healing outcomes at 12 weeks between those ulcers debrided weekly and those debrided every second week. A DFU may require more or less frequent debridement according to individual wound characteristics; however, the new data from DDS can inform clinical decisions and service provision. The implications of weekly versus second-weekly debridement are discussed.

Do diabetes-related foot ulcer wound fluid measures have clinical utility as biomarkers for healing? A systematic review.

OBJECTIVE: This study aimed to identify potential biomarkers reported in wound fluid of diabetes-related foot ulcers (DRFUs), and their ability to reflect current and prospective wound healing. METHOD: A systematic search was executed following the PRISMA methodology across five chosen databases: MEDLINE, Embase, Scopus, Cochrane Clinical Trials and Cochrane Systematic Reviews. Using keywords and phrases, it yielded 5022 results. RESULTS: Based on predetermined inclusion and exclusion criteria, 19 papers were included in the final analysis, among which: seven reported serial temporal biomarker changes in wounds; six reported measures from baseline and related them to healing rate and/or final healing outcome; four papers reported both end-points, and two papers reported solely on baseline biomarker levels in a generalised diabetic foot ulcer group. Across the studies, a total of 46 distinct markers were described from the wound fluid of n=1141 participants. Biomarkers examined included proteases, protease inhibitors, growth factors, chemokines and cytokines, with proteases being the largest subcategory making up 16 (34.8%) of the markers investigated (n=7). Matrix metalloproteinase-9 (MMP-9) was the most frequently investigated protease and it currently holds the most biomarker promise (n=5). Wound bacterial profiles variably related to wound healing outcome (n=5). One study reported biophysical markers rather than biomarkers, including measurement of wound fluid pH. Study quality was generally good. Drawing quantitative comparisons between papers was not possible due to variability in experimental design including sampling and assessment methods. CONCLUSION: These studies collectively indicate several wound fluid measures that could identify DRFU status and outcomes, and that methodological standardisation in the field is needed to determine reliable predictive thresholds for healing.

Development of Erf-Mediated Craniosynostosis and Pharmacological Amelioration.

ETS2 repressor factor (ERF) insufficiency causes craniosynostosis (CRS4) in humans and mice. ERF is an ETS domain transcriptional repressor regulated by Erk1/2 phosphorylation via nucleo-cytoplasmic shuttling. Here, we analyze the onset and development of the craniosynostosis phenotype in an Erf-insufficient mouse model and evaluate the potential of the residual Erf activity augmented by pharmacological compounds to ameliorate the disease. Erf insufficiency appears to cause an initially compromised frontal bone formation and subsequent multisuture synostosis, reflecting distinct roles of Erf on the cells that give rise to skull and facial bones. We treated animals with Mek1/2 and nuclear export inhibitors, U0126 and KPT-330, respectively, to increase Erf activity by two independent pathways. We implemented both a low dosage locally over the calvaria and a systemic drug administration scheme to evaluate the possible indirect effects from other systems and minimize toxicity. The treatment of mice with either the inhibitors or the administration scheme alleviated the synostosis phenotype with minimal adverse effects. Our data suggest that the ERF level is an important regulator of cranial bone development and that pharmacological modulation of its activity may represent a valid intervention approach both in CRS4 and in other syndromic forms of craniosynostosis mediated by the FGFR-RAS-ERK-ERF pathway.

Multi-omics of a pre-clinical model of diabetic cardiomyopathy reveals increased fatty acid supply impacts mitochondrial metabolic selectivity.

The incidence of type 2 diabetes (T2D) is increasing globally, with long-term implications for human health and longevity. Heart disease is the leading cause of death in T2D patients, who display an elevated risk of an acute cardiovascular event and worse outcomes following such an insult. The underlying mechanisms that predispose the diabetic heart to this poor prognosis remain to be defined. This study developed a pre-clinical model (Rattus norvegicus) that complemented caloric excess from a high-fat diet (HFD) and pancreatic ß-cell dysfunction from streptozotocin (STZ) to produce hyperglycaemia, peripheral insulin resistance, hyperlipidaemia and elevated fat mass to mimic the clinical features of T2D. Ex vivo cardiac function was assessed using Langendorff perfusion with systolic and diastolic contractile depression observed in T2D hearts. Cohorts representing untreated, individual HFD- or STZ-treatments and the combined HFD + STZ approach were used to generate ventricular samples (n = 9 per cohort) for sequential and integrated analysis of the proteome, lipidome and metabolome by liquid chromatography-tandem mass spectrometry. This study found that in T2D hearts, HFD treatment primed the metabolome, while STZ treatment was the major driver for changes in the proteome. Both treatments equally impacted the lipidome. Our data suggest that increases in ß-oxidation and early TCA cycle intermediates promoted rerouting via 2-oxaloacetate to glutamate, γ-aminobutyric acid and glutathione. Furthermore, we suggest that the T2D heart activates networks to redistribute excess acetyl-CoA towards ketogenesis and incomplete ß-oxidation through the formation of short-chain acylcarnitine species. Multi-omics provided a global and comprehensive molecular view of the diabetic heart, which distributes substrates and products from excess ß-oxidation, reduces metabolic flexibility and impairs capacity to restore high energy reservoirs needed to respond to and prevent subsequent acute cardiovascular events.

De Novo Missense Substitutions in the Gene Encoding CDK8, a Regulator of the Mediator Complex, Cause a Syndromic Developmental Disorder.

The Mediator is an evolutionarily conserved, multi-subunit complex that regulates multiple steps of transcription. Mediator activity is regulated by the reversible association of a four-subunit module comprising CDK8 or CDK19 kinases, together with cyclin C, MED12 or MED12L, and MED13 or MED13L. Mutations in MED12, MED13, and MED13L were previously identified in syndromic developmental disorders with overlapping phenotypes. Here, we report CDK8 mutations (located at 13q12.13) that cause a phenotypically related disorder. Using whole-exome or whole-genome sequencing, and by international collaboration, we identified eight different heterozygous missense CDK8 substitutions, including 10 shown to have arisen de novo, in 12 unrelated subjects; a recurrent mutation, c.185C>T (p.Ser62Leu), was present in five individuals. All predicted substitutions localize to the ATP-binding pocket of the kinase domain. Affected individuals have overlapping phenotypes characterized by hypotonia, mild to moderate intellectual disability, behavioral disorders, and variable facial dysmorphism. Congenital heart disease occurred in six subjects; additional features present in multiple individuals included agenesis of the corpus callosum, ano-rectal malformations, seizures, and hearing or visual impairments. To evaluate the functional impact of the mutations, we measured phosphorylation at STAT1-Ser727, a known CDK8 substrate, in a CDK8 and CDK19 CRISPR double-knockout cell line transfected with wild-type (WT) or mutant CDK8 constructs. These experiments demonstrated a reduction in STAT1 phosphorylation by all mutants, in most cases to a similar extent as in a kinase-dead control. We conclude that missense mutations in CDK8 cause a developmental disorder that has phenotypic similarity to syndromes associated with mutations in other subunits of the Mediator kinase module, indicating probable overlap in pathogenic mechanisms.

Testing the Feasibility and Dietary Impact of a "Produce Prescription" Program for Adults with Undermanaged Type 2 Diabetes and Food Insecurity in Australia.

BACKGROUND: There is growing interest in Food is Medicine programs that incorporate food-based interventions into health care for patients with diet-related conditions. OBJECTIVES: We aimed to test the feasibility of a "produce prescription" program and its impact on diet quality for people with type 2 diabetes (T2D) experiencing food insecurity in Australia. METHODS: We conducted a pre-post intervention study in n = 50 adults experiencing food insecurity with T2D and glycated hemoglobin (HbA1c) ≥8%. Once enrolled, participants received healthy food boxes weekly free of charge, with the contents sufficient to create 2 meals/d, 5 d/wk for the entire household, over 12 wk. Participants were also provided with tailored recipes and behavioral change support. The primary outcome was change in diet quality assessed by 24-h diet recalls. Secondary outcomes included differences in cardiovascular disease risk factors; blood micronutrients; and feasibility indicators. Differences in the baseline and 12-wk mean primary and secondary outcomes were assessed by paired t tests. RESULTS: Participants were older adults with mean ± SD age 63 ± 9 y (range: 40-87 y), HbA1c 9.8% ± 1.5%, and 46% were female. Overall, 92% completed the final study follow-up for the primary outcome. Compared with baseline, diet quality improved at week 12, with an increase in the mean overall diet quality (Alternate Healthy Eating Index score) of 12.9 (95% CI: 8.7, 17.1; P < 0.001), driven by significant improvements in vegetables, fruits, whole grains, red/processed meat, trans fat, sodium, and alcohol consumption. Blood lipids also improved (total:HDL cholesterol: -0.48; 95% CI: -0.72, -0.24; P < 0.001), and there was significant weight loss (-1.74 kg; 95% CI: -2.80, -0.68 kg, P = 0.002), but no changes in other clinical outcomes. Participants reported high levels of satisfaction with the program. CONCLUSIONS: These findings provide strong support for an adequately powered randomized trial to assess effects of produce prescription as an innovative approach to improve clinical management among individuals with T2D experiencing food insecurity. This trial was registered at https://anzctr.org.au/ as ACTRN12621000404820.

Prevalence, causes and associated mortality of hypercalcaemia in modern hospital care.

BACKGROUND: Studies examining hypercalcaemia in inpatients were largely published over 20 years ago, and it is likely the epidemiology of hypercalcaemia has changed related to increased lifespan and changes in the prevalence of the underlying causes such as malignancy. AIM: To explore the epidemiology of hypercalcaemia in a modern tertiary hospital setting in Australia and evaluate the risk of mortality associated with hypercalcaemia. METHODS: A retrospective study was performed in all inpatients with elevated blood calcium levels admitted from July 2013 to June 2018. ICD coding data identified primary diagnoses and mortality. Electronic medical records were reviewed in n = 292 patients admitted across 12 months from January to December 2017, to determine the causes of hypercalcaemia. RESULTS: Hypercalcaemia occurred in 1819 admissions (0.93% of all hospital admissions), during the 5-year period. The admission primary diagnoses were: malignancy (20% of cases), cardiovascular disease (17%) and gastrointestinal disease (11%). The top causes of hypercalcaemia among the 292 cases where electronic records were reviewed were malignancy (26%), primary hyperparathyroidism (25%) and hyperparathyroidism in the setting of chronic kidney disease (12%). Mortality occurred in 17% of these admissions. Non-survivors had significantly higher calcium levels, phosphate and white cell count, and had lower haemoglobin and albumin levels. CONCLUSION: Hypercalcaemia occurred in ~1% of admissions with main causes being malignancy and primary hyperparathyroidism, similar to historical studies. Hypercalcaemia in hospitalised patients is associated with high mortality and higher levels may be a marker for more severe underlying disease.

Sitagliptin Is More Effective Than Gliclazide in Preventing Pro-Fibrotic and Pro-Inflammatory Changes in a Rodent Model of Diet-Induced Non-Alcoholic Fatty Liver Disease.

A diet-induced non-alcoholic fatty liver disease (NAFLD) model causing obesity in rodents was used to examine whether sitagliptin and gliclazide therapies have similar protective effects on pathological liver change. METHODS: Male mice were fed a high-fat diet (HFD) or standard chow (Chow) ad libitum for 25 weeks and randomly allocated to oral sitagliptin or gliclazide treatment for the final 10 weeks. Fasting blood glucose and circulating insulin were measured. Inflammatory and fibrotic liver markers were assessed by qPCR. The second messenger ERK and autophagy markers were examined by Western immunoblot. F4/80, collagens and CCN2 were assessed by immunohistochemistry (IHC). RESULTS: At termination, HFD mice were obese, hyperinsulinemic and insulin-resistant but non-diabetic. The DPP4 inhibitor sitagliptin prevented intrahepatic induction of pro-fibrotic markers collagen-IV, collagen-VI, CCN2 and TGF-ß1 and pro-inflammatory markers TNF-α and IL-1ß more effectively than sulfonylurea gliclazide. By IHC, liver collagen-VI and CCN2 induction by HFD were inhibited only by sitagliptin. Sitagliptin had a greater ability than gliclazide to normalise ERK-protein liver dysregulation. CONCLUSION: These data indicate that sitagliptin, compared with gliclazide, exhibits greater inhibition of pro-fibrotic and pro-inflammatory changes in an HFD-induced NAFLD model. Sitagliptin therapy, even in the absence of diabetes, may have specific benefits in diet-induced NAFLD.

Dissection of contiguous gene effects for deletions around ERF on chromosome 19.

Heterozygous intragenic loss-of-function mutations of ERF, encoding an ETS transcription factor, were previously reported to cause a novel craniosynostosis syndrome, suggesting that ERF is haploinsufficient. We describe six families harboring heterozygous deletions including, or near to, ERF, of which four were characterized by whole-genome sequencing and two by chromosomal microarray. Based on the severity of associated intellectual disability (ID), we identify three categories of ERF-associated deletions. The smallest (32 kb) and only inherited deletion included two additional centromeric genes and was not associated with ID. Three larger deletions (264-314 kb) that included at least five further centromeric genes were associated with moderate ID, suggesting that deletion of one or more of these five genes causes ID. The individual with the most severe ID had a more telomerically extending deletion, including CIC, a known ID gene. Children found to harbor ERF deletions should be referred for craniofacial assessment, to exclude occult raised intracranial pressure.

A de novo substitution in BCL11B leads to loss of interaction with transcriptional complexes and craniosynostosis.

Craniosynostosis, the premature ossification of cranial sutures, is a developmental disorder of the skull vault, occurring in approximately 1 in 2250 births. The causes are heterogeneous, with a monogenic basis identified in ~25% of patients. Using whole-genome sequencing, we identified a novel, de novo variant in BCL11B, c.7C>A, encoding an R3S substitution (p.R3S), in a male patient with coronal suture synostosis. BCL11B is a transcription factor that interacts directly with the nucleosome remodelling and deacetylation complex (NuRD) and polycomb-related complex 2 (PRC2) through the invariant proteins RBBP4 and RBBP7. The p.R3S substitution occurs within a conserved amino-terminal motif (RRKQxxP) of BCL11B and reduces interaction with both transcriptional complexes. Equilibrium binding studies and molecular dynamics simulations show that the p.R3S substitution disrupts ionic coordination between BCL11B and the RBBP4-MTA1 complex, a subassembly of the NuRD complex, and increases the conformational flexibility of Arg-4, Lys-5 and Gln-6 of BCL11B. These alterations collectively reduce the affinity of BCL11B p.R3S for the RBBP4-MTA1 complex by nearly an order of magnitude. We generated a mouse model of the BCL11B p.R3S substitution using a CRISPR-Cas9-based approach, and we report herein that these mice exhibit craniosynostosis of the coronal suture, as well as other cranial sutures. This finding provides strong evidence that the BCL11B p.R3S substitution is causally associated with craniosynostosis and confirms an important role for BCL11B in the maintenance of cranial suture patency.

Evaluating the performance of a clinical genome sequencing program for diagnosis of rare genetic disease, seen through the lens of craniosynostosis.

PURPOSE: Genome sequencing (GS) for diagnosis of rare genetic disease is being introduced into the clinic, but the complexity of the data poses challenges for developing pipelines with high diagnostic sensitivity. We evaluated the performance of the Genomics England 100,000 Genomes Project (100kGP) panel-based pipelines, using craniosynostosis as a test disease. METHODS: GS data from 114 probands with craniosynostosis and their relatives (314 samples), negative on routine genetic testing, were scrutinized by a specialized research team, and diagnoses compared with those made by 100kGP. RESULTS: Sixteen likely pathogenic/pathogenic variants were identified by 100kGP. Eighteen additional likely pathogenic/pathogenic variants were identified by the research team, indicating that for craniosynostosis, 100kGP panels had a diagnostic sensitivity of only 47%. Measures that could have augmented diagnoses were improved calling of existing panel genes (+18% sensitivity), review of updated panels (+12%), comprehensive analysis of de novo small variants (+29%), and copy-number/structural variants (+9%). Recent NHS England recommendations that partially incorporate these measures should achieve 85% overall sensitivity (+38%). CONCLUSION: GS identified likely pathogenic/pathogenic variants in 29.8% of previously undiagnosed patients with craniosynostosis. This demonstrates the value of research analysis and the importance of continually improving algorithms to maximize the potential of clinical GS.