Positive correlation between transcriptomic stemness and PI3K/AKT/mTOR signaling scores in breast cancer, and a counterintuitive relationship with PIK3CA genotype.

A PI3Kα-selective inhibitor has recently been approved for use in breast tumors harboring mutations in PIK3CA, the gene encoding p110α. Preclinical studies have suggested that the PI3K/AKT/mTOR signaling pathway influences stemness, a dedifferentiation-related cellular phenotype associated with aggressive cancer. However, to date, no direct evidence for such a correlation has been demonstrated in human tumors. In two independent human breast cancer cohorts, encompassing nearly 3,000 tumor samples, transcriptional footprint-based analysis uncovered a positive linear association between transcriptionally-inferred PI3K/AKT/mTOR signaling scores and stemness scores. Unexpectedly, stratification of tumors according to PIK3CA genotype revealed a "biphasic" relationship of mutant PIK3CA allele dosage with these scores. Relative to tumor samples without PIK3CA mutations, the presence of a single copy of a hotspot PIK3CA variant was associated with lower PI3K/AKT/mTOR signaling and stemness scores, whereas the presence of multiple copies of PIK3CA hotspot mutations correlated with higher PI3K/AKT/mTOR signaling and stemness scores. This observation was recapitulated in a human cell model of heterozygous and homozygous PIK3CAH1047R expression. Collectively, our analysis (1) provides evidence for a signaling strength-dependent PI3K-stemness relationship in human breast cancer; (2) supports evaluation of the potential benefit of patient stratification based on a combination of conventional PI3K pathway genetic information with transcriptomic indices of PI3K signaling activation.

Genome-wide analysis identifies gallstone-susceptibility loci including genes regulating gastrointestinal motility.

BACKGROUND AND AIMS: Genome-wide association studies (GWAS) have identified several risk loci for gallstone disease. As with most polygenic traits, it is likely that many genetic determinants are undiscovered. The aim of this study was to identify genetic variants that represent new targets for gallstone research and treatment. APPROACH AND RESULTS: We performed a GWAS of 28,627 gallstone cases and 348,373 controls in the UK Biobank, replicated findings in a Scottish cohort (1089 cases, 5228 controls), and conducted a GWA meta-analysis (43,639 cases, 506,798 controls) with the FinnGen cohort. We assessed pathway enrichment using gene-based then gene-set analysis and tissue expression of identified genes in Genotype-Tissue Expression project data. We constructed a polygenic risk score (PRS) and evaluated phenotypic traits associated with the score. Seventy-five risk loci were identified (p < 5 × 10-8 ), of which 46 were new. Pathway enrichment revealed associations with lipid homeostasis, glucuronidation, phospholipid metabolism, and gastrointestinal motility. Anoctamin 1 (ANO1) and transmembrane Protein 147 (TMEM147), both in novel, replicated loci, are expressed in the gallbladder and gastrointestinal tract. Both regulate gastrointestinal motility. The gallstone risk allele rs7599-A leads to suppression of hepatic TMEM147 expression, suggesting that the protein protects against gallstone formation. The highest decile of the PRS demonstrated a 6-fold increased odds of gallstones compared with the lowest decile. The PRS was strongly associated with increased body mass index, serum liver enzymes, and C-reactive protein concentrations, and decreased lipoprotein cholesterol concentrations. CONCLUSIONS: This GWAS demonstrates the polygenic nature of gallstone risk and identifies 46 novel susceptibility loci. We implicate genes influencing gastrointestinal motility in the pathogenesis of gallstones.

Diagnosis and treatment of anti-insulin antibody-mediated labile glycaemia in insulin-treated diabetes.

AIMS: Anti-insulin antibodies in insulin-treated diabetes can derange glycaemia, but are under-recognised. Detection of significant antibodies is complicated by antigenically distinct insulin analogues. We evaluated a pragmatic biochemical approach to identifying actionable antibodies, and assessed its utility in therapeutic decision making. METHODS: Forty people with insulin-treated diabetes and combinations of insulin resistance, nocturnal/matutinal hypoglycaemia, and unexplained ketoacidosis were studied using broad-specificity insulin immunoassays, polyethylene glycol (PEG) precipitation and gel filtration chromatography (GFC) with or without ex vivo insulin preincubation. RESULTS: Twenty-seven people had insulin immunoreactivity (IIR) below 3000 pmol/L that fell less than 50% after PEG precipitation. Insulin binding by antibodies in this group was low and judged insignificant. In 8 people IIR was above 3000 pmol/L and fell by more than 50% after PEG precipitation. GFC demonstrated substantial high molecular weight (HMW) IIR in 7 of these 8. In this group antibodies were judged likely significant. In 2 people immunosuppression was introduced, with a good clinical result in one but only a biochemical response in another. In 6 people adjustment of insulin delivery was subsequently informed by knowledge of underlying antibody. In a final group of 5 participants IIR was below 3000 pmol/L but fell by more than 50% after PEG precipitation. In 4 of these GFC demonstrated low levels of HMW IIR and antibody significance was judged indeterminate. CONCLUSIONS: Anti-insulin antibodies should be considered in insulin-treated diabetes with unexplained glycaemic lability. Combining immunoassays with PEG precipitation can stratify their significance. Antibody depletion may be beneficial, but conservative measures often suffice.

p53 Regulates Mitochondrial Dynamics in Vascular Smooth Muscle Cell Calcification.

Arterial calcification is an important characteristic of cardiovascular disease. It has key parallels with skeletal mineralization; however, the underlying cellular mechanisms responsible are not fully understood. Mitochondrial dynamics regulate both bone and vascular function. In this study, we therefore examined mitochondrial function in vascular smooth muscle cell (VSMC) calcification. Phosphate (Pi)-induced VSMC calcification was associated with elongated mitochondria (1.6-fold increase, p < 0.001), increased mitochondrial reactive oxygen species (ROS) production (1.83-fold increase, p < 0.001) and reduced mitophagy (9.6-fold decrease, p < 0.01). An increase in protein expression of optic atrophy protein 1 (OPA1; 2.1-fold increase, p < 0.05) and a converse decrease in expression of dynamin-related protein 1 (DRP1; 1.5-fold decrease, p < 0.05), two crucial proteins required for the mitochondrial fusion and fission process, respectively, were noted. Furthermore, the phosphorylation of DRP1 Ser637 was increased in the cytoplasm of calcified VSMCs (5.50-fold increase), suppressing mitochondrial translocation of DRP1. Additionally, calcified VSMCs showed enhanced expression of p53 (2.5-fold increase, p < 0.05) and ß-galactosidase activity (1.8-fold increase, p < 0.001), the cellular senescence markers. siRNA-mediated p53 knockdown reduced calcium deposition (8.1-fold decrease, p < 0.01), mitochondrial length (3.0-fold decrease, p < 0.001) and ß-galactosidase activity (2.6-fold decrease, p < 0.001), with concomitant mitophagy induction (3.1-fold increase, p < 0.05). Reduced OPA1 (4.1-fold decrease, p < 0.05) and increased DRP1 protein expression (2.6-fold increase, p < 0.05) with decreased phosphorylation of DRP1 Ser637 (3.20-fold decrease, p < 0.001) was also observed upon p53 knockdown in calcifying VSMCs. In summary, we demonstrate that VSMC calcification promotes notable mitochondrial elongation and cellular senescence via DRP1 phosphorylation. Furthermore, our work indicates that p53-induced mitochondrial fusion underpins cellular senescence by reducing mitochondrial function.

Achievements, prospects and challenges in precision care for monogenic insulin-deficient and insulin-resistant diabetes.

Integration of genomic and other data has begun to stratify type 2 diabetes in prognostically meaningful ways, but this has yet to impact on mainstream diabetes practice. The subgroup of diabetes caused by single gene defects thus provides the best example to date of the vision of 'precision diabetes'. Monogenic diabetes may be divided into primary pancreatic beta cell failure, and primary insulin resistance. In both groups, clear examples of genotype-selective responses to therapy have been advanced. The benign trajectory of diabetes due to pathogenic GCK mutations, and the sulfonylurea-hyperresponsiveness conferred by activating KCNJ11 or ABCC8 mutations, or loss-of-function HNF1A or HNF4A mutations, often decisively guide clinical management. In monogenic insulin-resistant diabetes, subcutaneous leptin therapy is beneficial in some severe lipodystrophy. Increasing evidence also supports use of 'obesity therapies' in lipodystrophic people even without obesity. In beta cell diabetes the main challenge is now implementation of the precision diabetes vision at scale. In monogenic insulin-resistant diabetes genotype-specific benefits are proven in far fewer patients to date, although further genotype-targeted therapies are being evaluated. The conceptual paradigm established by the insulin-resistant subgroup with 'adipose failure' may have a wider influence on precision therapy for common type 2 diabetes, however. For all forms of monogenic diabetes, population-wide genome sequencing is currently forcing reappraisal of the importance assigned to pathogenic mutations when gene sequencing is uncoupled from prior suspicion of monogenic diabetes.

A standard of care for individuals with PIK3CA-related disorders: An international expert consensus statement.

Growth promoting variants in PIK3CA cause a spectrum of developmental disorders, depending on the developmental timing of the mutation and tissues involved. These phenotypically heterogeneous entities have been grouped as PIK3CA-Related Overgrowth Spectrum disorders (PROS). Deep sequencing technologies have facilitated detection of low-level mosaic, often necessitating testing of tissues other than blood. Since clinical management practices vary considerably among healthcare professionals and services across different countries, a consensus on management guidelines is needed. Clinical heterogeneity within this spectrum leads to challenges in establishing management recommendations, which must be based on patient-specific considerations. Moreover, as most of these conditions are rare, affected families may lack access to the medical expertise that is needed to help address the multi-system and often complex medical issues seen with PROS. In March 2019, macrocephaly-capillary malformation (M-CM) patient organizations hosted an expert meeting in Manchester, United Kingdom, to help address these challenges with regards to M-CM syndrome. We have expanded the scope of this project to cover PROS and developed this consensus statement on the preferred approach for managing affected individuals based on our current knowledge.

Oncogenic PIK3CA promotes cellular stemness in an allele dose-dependent manner.

The PIK3CA gene, which encodes the p110α catalytic subunit of PI3 kinase (PI3K), is mutationally activated in cancer and in overgrowth disorders known as PIK3CA-related overgrowth spectrum (PROS). To determine the consequences of genetic PIK3CA activation in a developmental context of relevance to both PROS and cancer, we engineered isogenic human induced pluripotent stem cells (iPSCs) with heterozygous or homozygous knockin of PIK3CAH1047R While heterozygous iPSCs remained largely similar to wild-type cells, homozygosity for PIK3CAH1047R caused widespread, cancer-like transcriptional remodeling, partial loss of epithelial morphology, up-regulation of stemness markers, and impaired differentiation to all three germ layers in vitro and in vivo. Genetic analysis of PIK3CA-associated cancers revealed that 64% had multiple oncogenic PIK3CA copies (39%) or additional PI3K signaling pathway-activating "hits" (25%). This contrasts with the prevailing view that PIK3CA mutations occur heterozygously in cancer. Our findings suggest that a PI3K activity threshold determines pathological consequences of oncogenic PIK3CA activation and provide insight into the specific role of this pathway in human pluripotent stem cells.

DNA Polymerase Epsilon Deficiency Causes IMAGe Syndrome with Variable Immunodeficiency.

During genome replication, polymerase epsilon (Pol ε) acts as the major leading-strand DNA polymerase. Here we report the identification of biallelic mutations in POLE, encoding the Pol ε catalytic subunit POLE1, in 15 individuals from 12 families. Phenotypically, these individuals had clinical features closely resembling IMAGe syndrome (intrauterine growth restriction [IUGR], metaphyseal dysplasia, adrenal hypoplasia congenita, and genitourinary anomalies in males), a disorder previously associated with gain-of-function mutations in CDKN1C. POLE1-deficient individuals also exhibited distinctive facial features and variable immune dysfunction with evidence of lymphocyte deficiency. All subjects shared the same intronic variant (c.1686+32C>G) as part of a common haplotype, in combination with different loss-of-function variants in trans. The intronic variant alters splicing, and together the biallelic mutations lead to cellular deficiency of Pol ε and delayed S-phase progression. In summary, we establish POLE as a second gene in which mutations cause IMAGe syndrome. These findings add to a growing list of disorders due to mutations in DNA replication genes that manifest growth restriction alongside adrenal dysfunction and/or immunodeficiency, consolidating these as replisome phenotypes and highlighting a need for future studies to understand the tissue-specific development roles of the encoded proteins.

Somatic PIK3R1 variation as a cause of vascular malformations and overgrowth.

PURPOSE: Somatic activating variants in the PI3K-AKT pathway cause vascular malformations with and without overgrowth. We previously reported an individual with capillary and lymphatic malformation harboring a pathogenic somatic variant in PIK3R1, which encodes three PI3K complex regulatory subunits. Here, we investigate PIK3R1 in a large cohort with vascular anomalies and identify an additional 16 individuals with somatic mosaic variants in PIK3R1. METHODS: Affected tissue from individuals with vascular lesions and overgrowth recruited from a multisite collaborative network was studied. Next-generation sequencing targeting coding regions of cell-signaling and cancer-associated genes was performed followed by assessment of variant pathogenicity. RESULTS: The phenotypic and variant spectrum associated with somatic variation in PIK3R1 is reported herein. Variants occurred in the inter-SH2 or N-terminal SH2 domains of all three PIK3R1 protein products. Phenotypic features overlapped those of the PIK3CA-related overgrowth spectrum (PROS). These overlapping features included mixed vascular malformations, sandal toe gap deformity with macrodactyly, lymphatic malformations, venous ectasias, and overgrowth of soft tissue or bone. CONCLUSION: Somatic PIK3R1 variants sharing attributes with cancer-associated variants cause complex vascular malformations and overgrowth. The PIK3R1-associated phenotypic spectrum overlaps with PROS. These data extend understanding of the diverse phenotypic spectrum attributable to genetic variation in the PI3K-AKT pathway.

Clinical spectrum of MTOR-related hypomelanosis of Ito with neurodevelopmental abnormalities.

PURPOSE: Hypomelanosis of Ito (HI) is a skin marker of somatic mosaicism. Mosaic MTOR pathogenic variants have been reported in HI with brain overgrowth. We sought to delineate further the pigmentary skin phenotype and clinical spectrum of neurodevelopmental manifestations of MTOR-related HI. METHODS: From two cohorts totaling 71 patients with pigmentary mosaicism, we identified 14 patients with Blaschko-linear and one with flag-like pigmentation abnormalities, psychomotor impairment or seizures, and a postzygotic MTOR variant in skin. Patient records, including brain magnetic resonance image (MRI) were reviewed. Immunostaining (n = 3) for melanocyte markers and ultrastructural studies (n = 2) were performed on skin biopsies. RESULTS: MTOR variants were present in skin, but absent from blood in half of cases. In a patient (p.[Glu2419Lys] variant), phosphorylation of p70S6K was constitutively increased. In hypopigmented skin of two patients, we found a decrease in stage 4 melanosomes in melanocytes and keratinocytes. Most patients (80%) had macrocephaly or (hemi)megalencephaly on MRI. CONCLUSION: MTOR-related HI is a recognizable neurocutaneous phenotype of patterned dyspigmentation, epilepsy, intellectual deficiency, and brain overgrowth, and a distinct subtype of hypomelanosis related to somatic mosaicism. Hypopigmentation may be due to a defect in melanogenesis, through mTORC1 activation, similar to hypochromic patches in tuberous sclerosis complex.

Increased C-Peptide Immunoreactivity in Insulin Autoimmune Syndrome (Hirata Disease) Due to High Molecular Weight Proinsulin.

BACKGROUND: Determination of C-peptide is important in the investigation of unexplained hyperinsulinemic hypoglycemia because a high C-peptide concentration usually indicates endogenous insulin hypersecretion. Insulin autoimmune syndrome (IAS) denotes hyperinsulinemic hypoglycemia due to insulin-binding antibodies that prolong insulin half-life. C-peptide clearance is considered to be unaffected, and although a marked C-peptide immunoreactivity in hypoglycemic samples has been reported, it has been suspected to be artifactual. High-resolution mass spectrometry enables examination of the basis of C-peptide-immunoreactivity in IAS. METHODS: Precipitation of plasma with polyethylene glycol was followed by C-peptide immunoassay. Plasma peptides extracted by solvent precipitation were characterized by nano-LC-MS/MS and analyzed using an untargeted data-dependent method. Peptides related to proinsulin, in amino acid sequence, were identified using proprietary bioinformatics software and confirmed by repeat LC-MS/MS analysis. Gel filtration chromatography coupled to LC-MS/MS was used to identify proinsulin-related peptides present in IAS immunocomplexes. Results were compared with those from C-peptide immunoassay. RESULTS: Polyethylene glycol precipitation of IAS plasma, but not control plasma, depleted C-peptide immunoreactivity consistent with immunoglobulin-bound C-peptide immunoreactivity. LC-MS/MS detected proinsulin and des 31,32 proinsulin at higher abundance in IAS plasma compared with control plasma. Analysis by gel filtration chromatography coupled to LC-MS/MS demonstrated proinsulin and des 31,32 proinsulin, but no C-peptide, in plasma immunocomplexes. CONCLUSIONS: Antibody binding can enrich proinsulin and des 31,32 proinsulin in IAS immunocomplexes. Proinsulin cross-reactivity in some C-peptide immunoassays can lead to artifactually increased C-peptide results.

Safety and efficacy of low-dose sirolimus in the PIK3CA-related overgrowth spectrum.

PURPOSE: PIK3CA-related overgrowth spectrum (PROS) encompasses a range of debilitating conditions defined by asymmetric overgrowth caused by mosaic activating PIK3CA variants. PIK3CA encodes the p110α catalytic subunit of phosphatidylinositol-3-kinase (PI3K), a critical transducer of growth factor signaling. As mTOR mediates the growth-promoting actions of PI3K, we hypothesized that the mTOR inhibitor sirolimus would slow pathological overgrowth. METHODS: Thirty-nine participants with PROS and progressive overgrowth were enrolled into open-label studies across three centers, and results were pooled. For the primary outcome, tissue volumes at affected and unaffected sites were measured by dual energy X-ray absorptiometry during 26 weeks of untreated run-in and 26 weeks of sirolimus therapy. RESULTS: Thirty participants completed the study. Sirolimus led to a change in mean percentage total tissue volume of -7.2% (SD 16.0, p = 0.04) at affected sites, but not at unaffected sites (+1.7%, SD 11.5, p = 0.48) (n = 23 evaluable). Twenty-eight of 39 (72%) participants had ≥1 adverse event related to sirolimus of which 37% were grade 3 or 4 in severity and 7/39 (18%) participants were withdrawn consequently. CONCLUSION: This study suggests that low-dose sirolimus can modestly reduce overgrowth, but cautions that the side-effect profile is significant, mandating individualized risk-benefit evaluations for sirolimus treatment in PROS.

Secretome Analysis of Hypoxia-Induced 3T3-L1 Adipocytes Uncovers Novel Proteins Potentially Involved in Obesity.

In the obese state, as adipose tissue expands, adipocytes become hypoxic and dysfunctional, leading to changes in the pattern of adipocyte-secreted proteins. To better understand the role of hypoxia in the mechanisms linked to obesity, we comparatively analyzed the secretome of murine differentiated 3T3-L1 adipocytes exposed to normoxia or hypoxia for 24 h. Proteins secreted into the culture media were precipitated by trichloroacetic acid and then digested with trypsin. The peptides were labeled with dimethyl labeling and analyzed by reversed phase nanoscale liquid chromatography coupled to a quadrupole Orbitrap mass spectrometer. From a total of 1508 identified proteins, 109 were differentially regulated, of which 108 were genuinely secreted. Factors significantly downregulated in hypoxic conditions included adiponectin, a known adipokine implicated in metabolic processes, as well as thrombospondin-1 and -2, and matrix metalloproteinase-11, all multifunctional proteins involved in extracellular matrix (ECM) homeostasis. Findings were validated by Western blot analysis. Expression studies of the relative genes were performed in parallel experiments in vitro, in differentiated 3T3-L1 adipocytes, and in vivo, in fat tissues from obese versus lean mice. Our observations are compatible with the concept that hypoxia may be an early trigger for both adipose cell dysfunction and ECM remodeling.

Evaluation of anti-insulin receptor antibodies as potential novel therapies for human insulin receptoropathy using cell culture models.

AIMS/HYPOTHESIS: Bi-allelic loss-of-function mutations in the INSR gene (encoding the insulin receptor [INSR]) commonly cause extreme insulin resistance and early mortality. Therapeutic options are limited, but anti-INSR antibodies have been shown to activate two mutant receptors, S323L and F382V. This study evaluates four well-characterised murine anti-INSR monoclonal antibodies recognising distinct epitopes (83-7, 83-14, 18-44, 18-146) as surrogate agonists for potential targeted treatment of severe insulin resistance arising from insulin receptoropathies. METHODS: Ten naturally occurring mutant human INSRs with defects affecting different aspects of receptor function were modelled and assessed for response to insulin and anti-INSR antibodies. A novel 3T3-L1 adipocyte model of insulin receptoropathy was generated, permitting conditional knockdown of endogenous mouse Insr by lentiviral expression of species-specific short hairpin (sh)RNAs with simultaneous expression of human mutant INSR transgenes. RESULTS: All expressed mutant INSR bound to all antibodies tested. Eight mutants showed antibody-induced autophosphorylation, while co-treatment with antibody and insulin increased maximal phosphorylation compared with insulin alone. After knockdown of mouse Insr and expression of mutant INSR in 3T3-L1 adipocytes, two antibodies (83-7 and 83-14) activated signalling via protein kinase B (Akt) preferentially over signalling via extracellular signal-regulated kinase 1/2 (ERK1/2) for seven mutants. These antibodies stimulated glucose uptake via P193L, S323L, F382V and D707A mutant INSRs, with antibody response greater than insulin response for D707A. CONCLUSIONS/INTERPRETATION: Anti-INSR monoclonal antibodies can activate selected naturally occurring mutant human insulin receptors, bringing closer the prospect of novel therapy for severe insulin resistance caused by recessive mutations.

Mutations disrupting the Kennedy phosphatidylcholine pathway in humans with congenital lipodystrophy and fatty liver disease.

Phosphatidylcholine (PC) is the major glycerophospholipid in eukaryotic cells and is an essential component in all cellular membranes. The biochemistry of de novo PC synthesis by the Kennedy pathway is well established, but less is known about the physiological functions of PC. We identified two unrelated patients with defects in the Kennedy pathway due to biallellic loss-of-function mutations in phosphate cytidylyltransferase 1 alpha (PCYT1A), the rate-limiting enzyme in this pathway. The mutations lead to a marked reduction in PCYT1A expression and PC synthesis. The phenotypic consequences include some features, such as severe fatty liver and low HDL cholesterol levels, that are predicted by the results of previously reported liver-specific deletion of murine Pcyt1a. Both patients also had lipodystrophy, severe insulin resistance, and diabetes, providing evidence for an additional and essential role for PCYT1A-generated PC in the normal function of white adipose tissue and insulin action.

Phosphoinositide 3-kinase-related overgrowth: cellular phenotype and future therapeutic options.

BACKGROUND: Somatic activating mutations in PIK3CA, which encodes the p110α catalytic subunit of phosphoinositide-3-kinase (PI3K) are frequently found in cancers and have been identified in a spectrum of mosaic overgrowth disorders ranging from isolated digit enlargement to more extensive overgrowth of the body, brain, or vasculature. We aimed to study affected dermal fibroblasts with a view to inform therapeutic studies, and to observe cancer-associated mutations in isolation. METHODS: We measured PIP3 concentrations in dermal fibroblasts with endogenous PIK3CA mutations and in wild type fibroblasts using mass spectrometry, and we measured downstream signalling events with ELISA and immunoblotting. Cellular proliferation was evaluated with 5-bromo-2'-deoxyuridine incorporation, and cell size assessed by fluorescence-activated cell sorting (FACS). Glycolysis and mitochondrial tests were performed with an extracellular flux analyser (Seahorse Bioscience, Billerica, MA, USA), and mitochondrial potential was measured by FACS-based JC1 staining. Experiments were repeated after exposure to 5 nmol everolimus for 72 h. FINDINGS: Mutant fibroblasts had two times higher basal PIP3 concentrations than wild-type fibroblasts (p=0·0017), with concomitant AKT and p70S6 activation downstream. The rate of cellular proliferation was higher in mutant cells under low serum conditions, but median cell size was not statistically different. Glycolytic capacity was similar between mutant and wild type fibroblasts, but subtle differences in mitochondrial function were detected with blunted responses to uncoupling agents and reduced membrane potentials. Treatment with everolimus reversed aberrant AKT(ser473) and p70S6 signalling, slowed cellular proliferation, and reversed mitochondrial abnormalities, but was associated, paradoxically, with increases in PIP3 concentrations. INTERPRETATION: These experiments demonstrate activation of the PI3K-AKT pathway in affected fibroblasts with increased proliferation, but no hypertrophy. Moreover, we identified changes in mitochondrial function in keeping with the known propensity of AKT to modulate elements of the Warburg effect. These results suggest that inhibitors of the mammalian target of rapamycin (mTOR) might be beneficial, but these inhibitors will require formal evaluation in clinical trials. More targeted therapy with p110α inhibitors is an enticing future option. FUNDING: Wellcome Trust, Sackler Fund, National Instititute for Health Research.

Advanced non-alcoholic fatty liver disease and adipose tissue fibrosis in patients with Alström syndrome.

BACKGROUND AND AIMS: Alström syndrome (AS) is a recessive monogenic syndrome characterized by obesity, extreme insulin resistance and multi-organ fibrosis. Despite phenotypically being high risk of non-alcoholic fatty liver disease (NAFLD), there is a lack of data on the extent of fibrosis in the liver and its close links to adipose in patients with AS. Our aim was to characterize the hepatic and adipose phenotype in patients with AS. METHODS: Observational cohort study with comprehensive assessment of metabolic liver phenotype including liver elastography (Fibroscan® ), serum Enhanced Liver Fibrosis (ELF) Panel and liver histology. In addition, abdominal adipose histology and gene expression was assessed. We recruited 30 patients from the UK national AS clinic. A subset of six patients underwent adipose biopsies which was compared with control tissue from nine healthy participants. RESULTS: Patients were overweight/obese (BMI 29.3 (25.95-34.05) kg/m2 ). A total of 80% (24/30) were diabetic; 74% (20/27) had liver ultrasound scanning suggestive of NAFLD. As judged by the ELF panel, 96% (24/25) were categorized as having fibrosis and 10/21 (48%) had liver elastography consistent with advanced liver fibrosis/cirrhosis. In 7/8 selected cases, there was evidence of advanced NAFLD on liver histology. Adipose tissue histology showed marked fibrosis as well as disordered pro-inflammatory and fibrotic gene expression profiles. CONCLUSIONS: NAFLD and adipose dysfunction are common in patients with AS. The severity of liver disease in our cohort supports the need for screening of liver fibrosis in AS.

PIK3CA-related overgrowth spectrum (PROS): diagnostic and testing eligibility criteria, differential diagnosis, and evaluation.

Somatic activating mutations in the phosphatidylinositol-3-kinase/AKT/mTOR pathway underlie heterogeneous segmental overgrowth phenotypes. Because of the extreme differences among patients, we sought to characterize the phenotypic spectrum associated with different genotypes and mutation burdens, including a better understanding of associated complications and natural history. Historically, the clinical diagnoses in patients with PIK3CA activating mutations have included Fibroadipose hyperplasia or Overgrowth (FAO), Hemihyperplasia Multiple Lipomatosis (HHML), Congenital Lipomatous Overgrowth, Vascular Malformations, Epidermal Nevi, Scoliosis/Skeletal and Spinal (CLOVES) syndrome, macrodactyly, Fibroadipose Infiltrating Lipomatosis, and the related megalencephaly syndromes, Megalencephaly-Capillary Malformation (MCAP or M-CM) and Dysplastic Megalencephaly (DMEG). A workshop was convened at the National Institutes of Health (NIH) to discuss and develop a consensus document regarding diagnosis and treatment of patients with PIK3CA-associated somatic overgrowth disorders. Participants in the workshop included a group of researchers from several institutions who have been studying these disorders and have published their findings, as well as representatives from patient-advocacy and support groups. The umbrella term of "PIK3CA-Related Overgrowth Spectrum (PROS)" was agreed upon to encompass both the known and emerging clinical entities associated with somatic PIK3CA mutations including, macrodactyly, FAO, HHML, CLOVES, and related megalencephaly conditions. Key clinical diagnostic features and criteria for testing were proposed, and testing approaches summarized. Preliminary recommendations for a uniform approach to assessment of overgrowth and molecular diagnostic testing were determined. Future areas to address include the surgical management of overgrowth tissue and vascular anomalies, the optimal approach to thrombosis risk, and the testing of potential pharmacologic therapies.