Börjeson-Forssman-Lehmann syndrome: delineating the clinical and allelic spectrum in 14 new families.

Börjeson-Forssman-Lehmann syndrome (BFLS) is an X-linked intellectual disability syndrome caused by variants in the PHF6 gene. We ascertained 19 individuals from 15 families with likely pathogenic or pathogenic PHF6 variants (11 males and 8 females). One family had previously been reported. Six variants were novel. We analysed the clinical and genetic findings in our series and compared them with reported BFLS patients. Affected males had classic features of BFLS including intellectual disability, distinctive facies, large ears, gynaecomastia, hypogonadism and truncal obesity. Carrier female relatives of affected males were unaffected or had only mild symptoms. The phenotype of affected females with de novo variants overlapped with the males but included linear skin hyperpigmentation and a higher frequency of dental, retinal and cortical brain anomalies. Complications observed in our series included keloid scarring, digital fibromas, absent vaginal orifice, neuropathy, umbilical hernias, and talipes. Our analysis highlighted sex-specific differences in PHF6 variant types and locations. Affected males often have missense variants or small in-frame deletions while affected females tend to have truncating variants or large deletions/duplications. Missense variants were found in a minority of affected females and clustered in the highly constrained PHD2 domain of PHF6. We propose recommendations for the evaluation and management of BFLS patients. These results further delineate and extend the genetic and phenotypic spectrum of BFLS.

Expanding the Molecular Spectrum of ANKRD11 Gene Defects in 33 Patients with a Clinical Presentation of KBG Syndrome.

KBG syndrome (KBGS) is a neurodevelopmental disorder caused by the Ankyrin Repeat Domain 11 (ANKRD11) haploinsufficiency. Here, we report the molecular investigations performed on a cohort of 33 individuals with KBGS clinical suspicion. By using a multi-testing genomic approach, including gene sequencing, Chromosome Microarray Analysis (CMA), and RT-qPCR gene expression assay, we searched for pathogenic alterations in ANKRD11. A molecular diagnosis was obtained in 22 out of 33 patients (67%). ANKRD11 sequencing disclosed pathogenic or likely pathogenic variants in 18 out of 33 patients. CMA identified one full and one terminal ANKRD11 pathogenic deletions, and one partial duplication and one intronic microdeletion, with both possibly being pathogenic. The pathogenic effect was established by RT-qPCR, which confirmed ANKRD11 haploinsufficiency only for the three deletions. Moreover, RT-qPCR applied to six molecularly unsolved KBGS patients identified gene downregulation in a clinically typical patient with previous negative tests, and further molecular investigations revealed a cryptic deletion involving the gene promoter. In conclusion, ANKRD11 pathogenic variants could also involve the regulatory regions of the gene. Moreover, the application of a multi-test approach along with the innovative use of RT-qPCR improved the diagnostic yield in KBGS suspected patients.

A mild case of acromesomelic dysplasia, type Maroteaux with novel natriuretic peptide receptor B (NPR2) variants.

Acromesomelic dysplasia, type Maroteaux is caused by variants in NPR2. It is a severe chondrodysplasia resulting in shortening of the middle and distal segments of the limbs. Limb length at birth may be normal but decreased growth becomes obvious in the first 2 years of life. Here we present an 11-year-old male with mild but typical skeletal features of acromesomelic dysplasia, type Maroteaux. Whole exome sequencing has identified two likely pathogenic variants in NPR2 which have not previously been reported in individuals with acromesomelic dysplasia, type Maroteaux. Given these findings, a diagnosis of AMDM should be considered in individuals with characteristic radiological findings, even if stature is only modestly affected.

BRCA gene testing in women with high-grade serous ovarian carcinoma.

The objective of this study was to compare the pick-up rate of pathogenic BRCA variants in those with a high-grade serous ovarian carcinoma (HGSOC) undergoing oncology-led testing with the traditional genetics family history-based testing model. With novel therapies, BRCA status can affect treatment. Welsh oncologists are now testing all women with HGSOC at diagnosis rather than referring to genetics, where family history is required for testing. The records of 332 women who underwent testing via oncology were analysed. The outcome measures were; percentage of women with a pathogenic BRCA variant and the difference in identification of pathogenic BRCA variants between the oncology-led and traditional genetics testing models. Of the 332 women, 25 women (7.5%) tested positive for a pathogenic BRCA variant. This was slightly lower than the detection rate of 9.8% for patients tested via the genetics service over the same period. Testing through genetics, using family history criteria would have identified only 19 (76%) of those with pathogenic variants in the oncology cohort. Since women with a pathogenic BRCA variant can be offered life-extending targeted treatment and a significant proportion of these women would be missed if testing was offered based on family history criteria alone, universal BRCA testing of all women with HGSOC is justified.Impact statement:What is already known on this subject? It is well established that individuals with a strong family history of breast and ovarian cancer are more likely to carry a pathogenic BRCA gene variant. With the use of tools such as the Manchester scoring system women are often invited for testing through clinical genetics services. Until recently there was no clinical impact for those already diagnosed with ovarian cancer.What do the results of this study add? Our study has shown that the diagnosis of high grade serious ovarian carcinoma alone without the need for any family history leads to a similar rate of detection of pathogenic BRCA variants as traditional methods. With the advent of targeted treatments such as olaparib, women with a pathogenic BRCA variant can access different life extending treatment options. With comparable pick-up rates to traditional family history based scoring systems, oncologists can now arrange BRCA gene testing directly.What are the implications of these findings for clinical practice and/or further research? Our study shows universal genetic testing of those with high-grade serious ovarian carcinoma by oncologists allows more women to access life extending treatment in a shorter timeframe compared to the traditional testing model used by clinical genetics services. We hope that other centres, both in the UK and beyond, will adopt this approach.

Endotoxin induced hyperlactatemia and hypoglycemia is linked to decreased mitochondrial phosphoenolpyruvate carboxykinase.

AIMS: Phosphoenolpyruvate carboxykinase (PEPCK) is the rate limiting enzyme for gluconeogenesis, and plays a key role in recycling lactate for glucose production. It is synthesized as two separate isoforms; cytosolic (PEPCK-C, gene code; PCK1) and mitochondrial (PEPCK-M, gene code; PCK2). Previous studies of gluconeogenesis in endotoxemia have focused solely on PCK1. We investigated the relative roles of the two isoforms in hepatic and renal gluconeogenesis in a rat model of endotoxic shock, and in cultured hepatocytes. MAIN METHODS: Rats were administered lipopolysaccharide (6 mg/kg; LPS) for 6 h. Cultured cells were incubated with lactate (5 mM) with or without tumor necrosis factor alpha (1 - 10 ng/ml). Rat liver and kidney samples as well as cultured cells were subjected to subcellular fractionation to produce mitochondrial and cytosolic fractions for PEPCK activity assay. PCK1 and PCK2 mRNA levels were measured using quantitative RT-PCR. KEY FINDINGS: In rat endotoxemia, hepatic PCK2 mRNA and PEPCK-M enzyme activity decreased by 53% and 38%, compared to sham controls. Hepatic PCK1 mRNA levels increased by 44%, but PEPCK-C enzyme activity remained unchanged. The changes in hepatic PEPCK-M coincided with a marked hypoglycemia and hyperlactatemia as well as elevated plasma interleukin 1 beta (IL1beta). Incubation of cultured hepatocytes with TNF-alpha inhibited lactate-induced increases in glucose production, PCK2 mRNA levels and PEPCK-M enzyme activity but had no effect on PCK1 mRNA levels or PEPCK-C activity. SIGNIFICANCE: These results indicate that decreases in hepatic PEPCK-M play a key role in the manifestation of hyperlactatemia and hypoglycemia in endotoxemia.

Sphingosylphosphorylcholine reduces the organ injury/dysfunction and inflammation caused by endotoxemia in the rat.

OBJECTIVE: Sphingosylphosphorylcholine (SPC) has been reported to activate a variety of G-protein coupled receptors, including S1P(1-5), G2A, GPR4, and OGR1 (GPR68). Interestingly, other structurally related lysophospholipid agonists of these receptors have been shown to exhibit immunomodulatory properties both in vitro and in vivo. These include prevention of tumor necrosis factor-alpha-induced monocyte adhesion to aortic endothelium in mice (sphingosine-1-phosphate via S1P(1-5) receptors) and reduction of organ injury and/or mortality in animal models of sepsis and endotoxemia (lysophosphatidylcholine via G2A). Here, we investigate the effects of SPC on the organ injury/dysfunction caused by systemic administration of lipopolysaccharide and the mechanisms underlying the observed effects of SPC. DESIGN: Prospective, randomized study. SETTING: University-based research laboratory. SUBJECTS: Sixty-one anesthetized male Wistar rats. INTERVENTIONS: Rats received either SPC (10 mg/kg intravenously) or vehicle (phosphate-buffered saline 1 mL/kg intravenously) 15 mins before or 15 mins after induction of endotoxemia with lipopolysaccharide (6 mg/kg intravenously). MEASUREMENTS AND MAIN RESULTS: Treatment with SPC significantly reduced the organ/dysfunction injury caused by lipopolysaccharide. SPC pretreatment significantly reduced the circulating levels of interleukin-1beta and interleukin-6, the expression of CD11b (ligand for intercellular adhesion molecule-1) on circulating polymorphonuclear cells, the expression of proteins of intercellular adhesion molecule-1 (Western blot and immunohistochemistry), cyclooxygenase-2 and nuclear translocation of nuclear factor-kappaB (Western blot analysis), and inducible nitric oxide synthase (immunohistochemistry) as well as the lung injury caused by endotoxemia in the rat. CONCLUSIONS: SPC reduced the organ injury/dysfunction caused by endotoxin in the rat. These beneficial effects of SPC are associated with potent anti-inflammatory effects.

Lysophosphatidylcholine reduces the organ injury and dysfunction in rodent models of gram-negative and gram-positive shock.

1. Lysophosphatidylcholine (LPC) modulates the inflammatory response and reduces mortality in animal models of sepsis. Here, we investigate the effects of LPC from synthetic (sLPC) and natural, soy bean derived LPC, (nLPC) sources on the organ injury/dysfunction caused by systemic administration of lipopolysaccharide (LPS) or peptidoglycan (PepG) and lipoteichoic acid (LTA). 2. Rats were subjected to (i) endotoxaemia (LPS 6 mg kg(-1) i.v.) and treated with sLPC (1-100 mg kg(-1)), (ii) endotoxaemia and treated with nLPC (10 mg kg(-1)) or (iii) gram-positive shock (PepG 10 mg kg(-1) and LTA 3 mg kg(-1) i.v.) and treated with sLPC (10 mg kg(-1)). 3. Endotoxaemia or gram-positive shock for 6 h resulted in increases in serum makers of renal dysfunction and liver, pancreatic and neuromuscular injury. 4. Administration of sLPC, at 1 or 2 h after LPS, dose dependently (1-10 mg kg(-1)) reduced the organ injury/dysfunction. High doses of sLPC (30 and 100 mg kg(-1)) were shown to be detrimental in endotoxaemia. sLPC also afforded protection against the organ injury/dysfunction caused by gram-positive shock. nLPC was found to be protective in endotoxaemic animals. 5. The beneficial effects of sLPC were associated with an attenuation in circulating levels of interleukin-1beta (IL-1beta). 6. In conclusion, LPC dose and time dependently reduces the organ injury and circulating IL-1beta levels caused by gram-negative or gram-positive shock in the rat. Thus, we speculate that appropriate doses of LPC may be useful in reducing the degree of organ injury and dysfunction associated with shock of various aetiologies.

Glycogen synthase kinase-3beta inhibitors protect against the organ injury and dysfunction caused by hemorrhage and resuscitation.

Glycogen synthase kinase 3beta (GSK-3beta) is a serine/threonine protein kinase that has recently emerged as a key regulatory switch in the modulation of the inflammatory response. Dysregulation of GSK-3beta has been implicated in the pathogenesis of several diseases including sepsis. Here we investigate the effects of 2 chemically distinct inhibitors of GSK-3beta, TDZD-8 and SB216763, on the circulatory failure and the organ injury and dysfunction associated with hemorrhagic shock. Male Wistar rats were subjected to hemorrhage (sufficient to lower mean arterial blood pressure to 35 mmHg for 90 min) and subsequently resuscitated with shed blood for 4 h. Hemorrhage and resuscitation resulted in an increase in serum levels of (a) creatinine and, hence, renal dysfunction, and (b) alanine aminotransferase and aspartate aminotransferase and, hence, hepatic injury. Treatment of rats with either TDZD-8 (1 mg/kg, i.v.) or SB216763 (0.6 mg/kg, i.v.) 5 min before resuscitation abolished the renal dysfunction and liver injury caused by hemorrhagic shock. In addition, TDZD-8, but not SB216763, attenuated the increase caused by hemorrhage and resuscitation in plasma levels of the proinflammatory cytokine interleukin 6 and also of the anti-inflammatory cytokine interleukin 10. Neither of the GSK-3beta inhibitors however affected the delayed fall in blood pressure caused by hemorrhagic shock. Thus, we propose that inhibition of GSK-3beta may represent a novel therapeutic approach in the therapy of hemorrhagic shock.

Insulin reduces the multiple organ injury and dysfunction caused by coadministration of lipopolysaccharide and peptidoglycan independently of blood glucose: role of glycogen synthase kinase-3beta inhibition.

OBJECTIVE: Insulin reduces morbidity and mortality among critically ill patients, but the molecular mechanisms of its effect remain unknown. Insulin is a well-known inhibitor of glycogen synthase kinase-3, which may play an important role in systemic inflammation and shock. Here we investigate the role of blood glucose and glycogen synthase kinase-3beta inhibition in the protective effect of insulin on the organ injury/dysfunction associated with excessive systemic inflammation. DESIGN: Prospective, randomized study. SETTING: University-based research laboratory. SUBJECTS: Eighty-five anesthetized Wistar rats. INTERVENTIONS: Rats received Escherichia coli lipopolysaccharide (1 mg/kg) and Staphylococcus aureus peptidoglycan (0.3 mg/kg) or vehicle intravenously. Insulin (1.4 units/kg intravenously) was administered in the absence or presence of continuous glucose administration (4.5 mg/kg/hr intravenously) either prophylactically or therapeutically. The potent and selective glycogen synthase kinase-3beta inhibitor TDZD-8 (1 mg/kg intravenously) or vehicle (10% dimethyl sulfoxide) was administered either prophylactically or therapeutically. MEASUREMENTS AND MAIN RESULTS: Coadministration of lipopolysaccharide and peptidoglycan resulted in increases in the serum levels of creatinine (indicator of renal dysfunction), alanine aminotransferase, and aspartate aminotransferase (indicators of liver injury) at 6 hrs. Insulin or TDZD-8 similarly attenuated the organ injury/dysfunction caused by lipopolysaccharide and peptidoglycan when given either prophylactically or therapeutically. Continuous glucose administration had no effect on blood glucose levels or organ injury/dysfunction at 6 hrs. Treatment with insulin or TDZD-8 reduced the plasma levels of the proinflammatory cytokine interleukin-1beta. In vitro, insulin or TDZD-8 caused similar reductions in the nuclear factor-kappaB p65 activity and similar increases in the phosphorylation of Ser9 of glycogen synthase kinase-3beta. CONCLUSIONS: Therapy with insulin or the potent and selective glycogen synthase kinase-3beta inhibitor TDZD-8 reduced the organ injury/dysfunction caused by lipopolysaccharide and peptidoglycan in the rat. We propose that the inhibitory effect of insulin on the activity of glycogen synthase kinase-3beta contributes to the protective effect of insulin against the organ injury/dysfunction caused by excessive systemic inflammation independently of any effects on blood glucose.

Inhibition of endogenous hydrogen sulfide formation reduces the organ injury caused by endotoxemia.

Hydrogen sulfide (H2S) is a naturally occurring gaseous transmitter, which may play important roles in normal physiology and disease. Here, we investigated the role of H2S in the organ injury caused by severe endotoxemia in the rat. Male Wistar rats were subjected to acute endotoxemia (Escherichia coli lipopolysaccharide (LPS) 6 mg kg(-1) intravenously (i.v.) for 6 h) and treated with vehicle (saline, 1 ml kg(-1) i.v.) or DL-propargylglycine (PAG, 10-100 mg kg(-1) i.v.), an inhibitor of the H2S-synthesizing enzyme cystathionine-gamma-lyase (CSE). PAG was administered either 30 min prior to or 60 min after the induction of endotoxemia. Endotoxemia resulted in circulatory failure (hypotension and tachycardia) and an increase in serum levels of alanine aminotransferase and aspartate aminotransferase (markers for hepatic injury), lipase (indicator of pancreatic injury) and creatine kinase (indicator of neuromuscular injury). In the liver, endotoxemia induced a significant increase in the myeloperoxidase (MPO) activity, and in the expression and activity of the H2S-synthesizing enzymes CSE and cystathionine-beta-synthase. Administration of PAG either prior to or after the injection of LPS dose-dependently reduced the hepatocellular, pancreatic and neuromuscular injury caused by endotoxemia, but not the circulatory failure. Pretreatment of rats with PAG abolished the LPS-induced increase in the MPO activity and in the formation of H2S and in the liver. These findings support the view that an enhanced formation of H2S contributes to the pathophysiology of the organ injury in endotoxemia. We propose that inhibition of H2S synthesis may be a useful therapeutic strategy against the organ injury associated with sepsis and shock.