Canadian Multicentric Pan-TRK (CANTRK) Immunohistochemistry Harmonization Study.

Tumor-agnostic testing for NTRK1-3 gene rearrangements is required to identify patients who may benefit from TRK inhibitor therapies. The overarching objective of this study was to establish a high-quality pan-TRK immunohistochemistry (IHC) screening assay among 18 large regional pathology laboratories across Canada using pan-TRK monoclonal antibody clone EPR17341 in a ring study design. TRK-fusion positive and negative tumor samples were collected from participating sites, with fusion status confirmed by panel next-generation sequencing assays. Each laboratory received: (1) unstained sections from 30 cases of TRK-fusion-positive or -negative tumors, (2) 2 types of reference standards: TRK calibrator slides and IHC critical assay performance controls (iCAPCs), (3) EPR17341 antibody, and (4) suggestions for developing IHC protocols. Participants were asked to optimize the IHC protocol for their instruments and detection systems by using iCAPCs, to stain the 30 study cases, and to report the percentage scores for membranous, cytoplasmic, and nuclear staining. TRK calibrators were used to assess the analytical sensitivity of IHC protocols developed by using the 2 reference standards. Fifteen of 18 laboratories achieved diagnostic sensitivity of 100% against next-generation sequencing. The diagnostic specificity ranged from 40% to 90%. The results did not differ significantly between positive scores based on the presence of any type of staining vs the presence of overall staining in ≥1% of cells. The median limit of detection measured by TRK calibrators was 76,000 molecules/cell (range 38,000 to >200,000 molecules/cell). Three different patterns of staining were observed in 19 TRK-positive cases, cytoplasmic-only in 7 samples, nuclear and cytoplasmic in 9 samples, and cytoplasmic and membranous in 3 samples. The Canadian multicentric pan-TRK study illustrates a successful strategy to accelerate the multicenter harmonization and implementation of pan-TRK immunohistochemical screening that achieves high diagnostic sensitivity by using laboratory-developed tests where laboratories used centrally developed reference materials. The measurement of analytical sensitivity by using TRK calibrators provided additional insights into IHC protocol performance.

Homozygous missense variants in YKT6 result in loss of function and are associated with developmental delay, with or without severe infantile liver disease and risk for hepatocellular carcinoma.

PURPOSE: YKT6 plays important roles in multiple intracellular vesicle trafficking events but has not been associated with Mendelian diseases. METHODS: We report 3 unrelated individuals with rare homozygous missense variants in YKT6 who exhibited neurological disease with or without a progressive infantile liver disease. We modeled the variants in Drosophila. We generated wild-type and variant genomic rescue constructs of the fly ortholog dYkt6 and compared their ability in rescuing the loss-of-function phenotypes in mutant flies. We also generated a dYkt6KozakGAL4 allele to assess the expression pattern of dYkt6. RESULTS: Two individuals are homozygous for YKT6 [NM_006555.3:c.554A>G p.(Tyr185Cys)] and exhibited normal prenatal course followed by failure to thrive, developmental delay, and progressive liver disease. Haplotype analysis identified a shared homozygous region flanking the variant, suggesting a common ancestry. The third individual is homozygous for YKT6 [NM_006555.3:c.191A>G p.(Tyr64Cys)] and exhibited neurodevelopmental disorders and optic atrophy. Fly dYkt6 is essential and is expressed in the fat body (analogous to liver) and central nervous system. Wild-type genomic rescue constructs can rescue the lethality and autophagic flux defects, whereas the variants are less efficient in rescuing the phenotypes. CONCLUSION: The YKT6 variants are partial loss-of-function alleles, and the p.(Tyr185Cys) is more severe than p.(Tyr64Cys).

Transbronchial Lung Cryobiopsies, Transbronchial Forceps Lung Biopsies, and Surgical Lung Biopsies in Mechanically Ventilated Patients with Acute Hypoxemic Respiratory Failure: A Retrospective Cohort Study.

IMPORTANCE: Lung biopsies are sometimes performed in mechanically ventilated patients with acute hypoxemic respiratory failure (AHRF) of unknown etiology to guide patient management. While surgical lung biopsies (SLB) offer high diagnostic rates, they may also cause significant complications. Transbronchial forceps lung biopsies (TBLB) are less invasive but often produce non-contributive specimens. Transbronchial lung cryobiopsies (TBLC) yield specimens of potentially better quality than TBLB, but due to their novel implementation in the intensive care unit (ICU), their accuracy and safety are still unclear. OBJECTIVES: Our main objective was to evaluate the risk of adverse events in patients with AHRF following the three biopsy techniques. Our secondary objectives were to assess the diagnostic yield and associated modifications of patient management of each technique. DESIGN, SETTINGS AND PARTICIPANTS: We conducted a retrospective cohort study comparing TBLC, TBLB, and SLB in mechanically ventilated patients with AHRF. MAIN OUTCOMES AND MEASURES: The primary outcome was the proportion of patients with at least one complication, and secondary outcomes included complication rates, diagnostic yields, treatment modifications, and mortality. RESULTS: Of the 26 patients who underwent lung biopsies from 2018 to 2022, all TBLC and SLB patients and 60% of TBLB patients had at least one complication. TBLC patients had higher unadjusted numbers of total and severe complications, but also worse Sequential Organ Failure Assessment scores and P/F ratios. A total of 25 biopsies (25/26, 96%) provided histopathological diagnoses, 88% (22/25) of which contributed to patient management. ICU mortality was high for all modalities (63% for TBLC, 60% for TBLB and 50% for SLB). CONCLUSIONS AND RELEVANCE: All biopsy methods had high diagnostic yields and the great majority contributed to patient management; however, complication rates were elevated. Further research is needed to determine which patients may benefit from lung biopsies and to determine the best biopsy modality.

Transbronchial Lung Cryobiopsy and Surgical Lung Biopsy: A Prospective Multi-Centre Agreement Clinical Trial (CAN-ICE).

Rationale: Transbronchial cryobiopsy (TBCB) for the diagnosis of interstitial lung disease (ILD) has shown promising results, but prospective studies with matched surgical lung biopsy (SLB) have yielded conflicting results. Objectives: We aimed to assess within- and between-center diagnostic agreement between TBCB and SLB at both the histopathologic and multidisciplinary discussion (MDD) levels in patients with diffuse ILD. Methods: In a multicenter prospective study, we performed matched TBCB and SLB in patients referred for SLB. After a blinded review by three pulmonary pathologists, all cases were reviewed by three independent ILD teams in an MDD. MDD was performed first with TBCB, then with SLB in a second session. Within-center and between-center diagnostic agreement was evaluated using percentages and correlation coefficients. Measurements and Main Results: Twenty patients were recruited and underwent contemporaneous TBCB and SLB. Within-center diagnostic agreement between TBCB-MDD and SLB-MDD was reached in 37 of the 60 (61.7%) paired observations, resulting in a Cohen's κ value of 0.46 (95% confidence interval [CI], 0.29-0.63). Diagnostic agreement increased among high-confidence or definitive diagnoses on TBCB-MDD (21 of 29 [72.4%]), but not significantly, and was more likely among cases with SLB-MDD diagnoses of idiopathic pulmonary fibrosis than fibrotic hypersensitivity pneumonitis (13 of 16 [81.2%] vs. 16 of 31 [51.6%]; P = 0.047). Between-center agreement for cases was markedly higher for SLB-MDD (κ = 0.71 [95% CI, 0.52-0.89]) than TBCB-MDD (κ = 0.29 [95% CI, 0.09-0.49]). Conclusions: This study demonstrated moderate TBCB-MDD and SLB-MDD diagnostic agreement for ILD, while between-center agreement was fair for TBCB-MDD and substantial for SLB-MDD. Clinical trial registered with www.clinicaltrials.gov (NCT02235779).

Endocrine and behavioural features of Lowe syndrome and their potential molecular mechanisms.

BACKGROUND: Lowe syndrome (LS) is an X linked disease caused by pathogenic variants in the OCRL gene that impacts approximately 1 in 500 000 children. Classic features include congenital cataract, cognitive/behavioural impairment and renal tubulopathy. METHODS: This study is a retrospective review of clinical features reported by family based survey conducted by Lowe Syndrome Association. Frequency of non-ocular clinical feature(s) of LS and their age of onset was summarised. An LS-specific therapy effectiveness scale was used to assess the response to the administered treatment. Expression of OCRL and relevant neuropeptides was measured in postmortem human brain by qPCR. Gene expression in the mouse brain was determined by reanalysis of publicly available bulk and single cell RNA sequencing. RESULTS: A total of 137 individuals (1 female, 89.1% white, median age 14 years (range 0.8-56)) were included in the study. Short stature (height <3rd percentile) was noted in 81% (n=111) individuals, and 15% (n=20) received growth hormone therapy. Undescended testis was reported in 47% (n=64), and median age of onset of puberty was 15 years. Additional features were dental problems (n=77, 56%), bone fractures (n=63, 46%), hypophosphataemia (n=60, 44%), developmental delay and behavioural issues. OCRL is expressed in human and mouse hypothalami, and in hypothalamic cell clusters expressing Ghrh, Sst, Oxt, Pomc and pituitary cells expressing Gh and Prl. CONCLUSIONS: There is a wide spectrum of the clinical phenotype of LS. Some of the features may be partly driven by the loss of function of OCRL in the hypothalamus and the pituitary.

Biallelic Loss-of-Function Variants in BICD1 Are Associated with Peripheral Neuropathy and Hearing Loss.

Hearing loss and peripheral neuropathy are two clinical entities that are genetically and phenotypically heterogeneous and sometimes co-occurring. Using exome sequencing and targeted segregation analysis, we investigated the genetic etiology of peripheral neuropathy and hearing loss in a large Ashkenazi Jewish family. Moreover, we assessed the production of the candidate protein via western blotting of lysates from fibroblasts from an affected individual and an unaffected control. Pathogenic variants in known disease genes associated with hearing loss and peripheral neuropathy were excluded. A homozygous frameshift variant in the BICD1 gene, c.1683dup (p.(Arg562Thrfs*18)), was identified in the proband and segregated with hearing loss and peripheral neuropathy in the family. The BIDC1 RNA analysis from patient fibroblasts showed a modest reduction in gene transcripts compared to the controls. In contrast, protein could not be detected in fibroblasts from a homozygous c.1683dup individual, whereas BICD1 was detected in an unaffected individual. Our findings indicate that bi-allelic loss-of-function variants in BICD1 are associated with hearing loss and peripheral neuropathy. Definitive evidence that bi-allelic loss-of-function variants in BICD1 cause peripheral neuropathy and hearing loss will require the identification of other families and individuals with similar variants with the same phenotype.

Recessive Rare Variants in Deoxyhypusine Synthase, an Enzyme Involved in the Synthesis of Hypusine, Are Associated with a Neurodevelopmental Disorder.

Hypusine is formed post-translationally from lysine and is found in a single cellular protein, eukaryotic translation initiation factor-5A (eIF5A), and its homolog eIF5A2. Biosynthesis of hypusine is a two-step reaction involving the enzymes deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH). eIF5A is highly conserved throughout eukaryotic evolution and plays a role in mRNA translation, cellular proliferation, cellular differentiation, and inflammation. DHPS is also highly conserved and is essential for life, as Dhps-null mice are embryonic lethal. Using exome sequencing, we identified rare biallelic, recurrent, predicted likely pathogenic variants in DHPS segregating with disease in five affected individuals from four unrelated families. These individuals have similar neurodevelopmental features that include global developmental delay and seizures. Two of four affected females have short stature. All five affected individuals share a recurrent missense variant (c.518A>G [p.Asn173Ser]) in trans with a likely gene disrupting variant (c.1014+1G>A, c.912_917delTTACAT [p.Tyr305_Ile306del], or c.1A>G [p.Met1?]). cDNA studies demonstrated that the c.1014+1G>A variant causes aberrant splicing. Recombinant DHPS enzyme harboring either the p.Asn173Ser or p.Tyr305_Ile306del variant showed reduced (20%) or absent in vitro activity, respectively. We co-transfected constructs overexpressing HA-tagged DHPS (wild-type or mutant) and GFP-tagged eIF5A into HEK293T cells to determine the effect of these variants on hypusine biosynthesis and observed that the p.Tyr305_Ile306del and p.Asn173Ser variants resulted in reduced hypusination of eIF5A compared to wild-type DHPS enzyme. Our data suggest that rare biallelic variants in DHPS result in reduced enzyme activity that limits the hypusination of eIF5A and are associated with a neurodevelopmental disorder.

The postnatal leptin surge in mice is variable in both time and intensity and reflects nutritional status.

BACKGROUND/OBJECTIVES: The murine postnatal leptin surge occurs within the first 4 weeks of life and is critical for neuronal projection development within hypothalamic feeding circuits. Here we describe the influence of nutritional status on the timing and magnitude of the postnatal leptin surge in mice. METHODS: Plasma leptin concentrations were measured 1-3 times per week for the first 4 weeks of life in C57BL/6J pups reared in litters adjusted to 3 (small), 7-8 (normal), or 11-12 (large) pups per dam fed breeder chow or raised in litters of 7-8 by dams fed high-fat diet (HFD) ad libitum starting either prior to conception or at parturition. RESULTS: Mice raised in small litters become fatter than pups raised in either normal or large litters. The leptin surge in small litter pups starts earlier, lasts longer, and is dramatically larger in magnitude compared to normal litter pups, even when leptin concentrations are normalized to fat mass. In mice reared in large litters, weight gain is diminished and the surge is both significantly delayed and lower in magnitude compared to control pups. Pups reared by HFD-fed dams (starting preconception or at parturition) are fatter and have augmented leptin surge magnitude compared to pups suckled by chow-fed dams. Surge timing varies depending upon nutritional status of the pup; the source of the surge is primarily subcutaneous adipose tissue. At peak leptin surge, within each group, fat mass and plasma leptin are uncorrelated; in comparison with adults, pups overproduce leptin relative to fat mass. Plasma leptin elevation persists longer than previously described; at postnatal day 27 mice continue overproducing leptin relative to fat mass. CONCLUSIONS: In mice, small litter size and maternal HFD feeding during the perinatal period augment the plasma leptin surge whereas large litter size is associated with a delayed surge of reduced magnitude.

Bi-allelic PAGR1 variants are associated with microcephaly and a severe neurodevelopmental disorder: Genetic evidence from two families.

Exome and genome sequencing were used to identify the genetic etiology of a severe neurodevelopmental disorder in two unrelated Ashkenazi Jewish families with three affected individuals. The clinical findings included a prenatal presentation of microcephaly, polyhydramnios and clenched hands while postnatal findings included microcephaly, severe developmental delay, dysmorphism, neurologic deficits, and death in infancy. A shared rare homozygous, missense variant (c.274A > G; p.Ser92Gly, NM_024516.4) was identified in PAGR1, a gene currently not associated with a Mendelian disease. PAGR1 encodes a component of the histone methyltransferase MLL2/MLL3 complex and may function in the DNA damage response pathway. Complete knockout of the murine Pagr1a is embryonic-lethal. Given the available evidence, PAGR1 is a strong candidate gene for a novel autosomal recessive severe syndromic neurodevelopmental disorder.

A homozygous splice variant in ATP5PO, disrupts mitochondrial complex V function and causes Leigh syndrome in two unrelated families.

Mitochondrial complex V plays an important role in oxidative phosphorylation by catalyzing the generation of ATP. Most complex V subunits are nuclear encoded and not yet associated with recognized Mendelian disorders. Using exome sequencing, we identified a rare homozygous splice variant (c.87+3A>G) in ATP5PO, the complex V subunit which encodes the oligomycin sensitivity conferring protein, in three individuals from two unrelated families, with clinical suspicion of a mitochondrial disorder. These individuals had a similar, severe infantile and often lethal multi-systemic disorder that included hypotonia, developmental delay, hypertrophic cardiomyopathy, progressive epileptic encephalopathy, progressive cerebral atrophy, and white matter abnormalities on brain MRI consistent with Leigh syndrome. cDNA studies showed a predominant shortened transcript with skipping of exon 2 and low levels of the normal full-length transcript. Fibroblasts from the affected individuals demonstrated decreased ATP5PO protein, defective assembly of complex V with markedly reduced amounts of peripheral stalk proteins, and complex V hydrolytic activity. Further, expression of human ATP5PO cDNA without exon 2 (hATP5PO-∆ex2) in yeast cells deleted for yATP5 (ATP5PO homolog) was unable to rescue growth on media which requires oxidative phosphorylation when compared to the wild type construct (hATP5PO-WT), indicating that exon 2 deletion leads to a non-functional protein. Collectively, our findings support the pathogenicity of the ATP5PO c.87+3A>G variant, which significantly reduces but does not eliminate complex V activity. These data along with the recent report of an affected individual with ATP5PO variants, add to the evidence that rare biallelic variants in ATP5PO result in defective complex V assembly, function and are associated with Leigh syndrome.

Bi-allelic Mutations in Phe-tRNA Synthetase Associated with a Multi-system Pulmonary Disease Support Non-translational Function.

The tRNA synthetases catalyze the first step of protein synthesis and have increasingly been studied for their nuclear and extra-cellular ex-translational activities. Human genetic conditions such as Charcot-Marie-Tooth have been attributed to dominant gain-of-function mutations in some tRNA synthetases. Unlike dominantly inherited gain-of-function mutations, recessive loss-of-function mutations can potentially elucidate ex-translational activities. We present here five individuals from four families with a multi-system disease associated with bi-allelic mutations in FARSB that encodes the beta chain of the alpha2beta2 phenylalanine-tRNA synthetase (FARS). Collectively, the mutant alleles encompass a 5'-splice junction non-coding variant (SJV) and six missense variants, one of which is shared by unrelated individuals. The clinical condition is characterized by interstitial lung disease, cerebral aneurysms and brain calcifications, and cirrhosis. For the SJV, we confirmed exon skipping leading to a frameshift associated with noncatalytic activity. While the bi-allelic combination of the SJV with a p.Arg305Gln missense mutation in two individuals led to severe disease, cells from neither the asymptomatic heterozygous carriers nor the compound heterozygous affected individual had any defect in protein synthesis. These results support a disease mechanism independent of tRNA synthetase activities in protein translation and suggest that this FARS activity is essential for normal function in multiple organs.

Weight-loss response to naltrexone/bupropion is modulated by the Taq1A genetic variant near DRD2 (rs1800497): A pilot study.

Naltrexone/bupropion (NB) is a US Food and Drug Administration-approved antiobesity medication. Clinical trials have shown variable weight loss, with responders and non-responders. NB is believed to act on central dopaminergic pathways to suppress appetite. The Taq1A polymorphism near DRD2 (rs1800497) is associated with the density of striatal dopamine D2 receptors, with individuals carrying the A allele (AA or AG; termed A1+) having 30%-40% fewer dopamine binding sites than those who do not carry the A allele (GG; termed A1-). We performed a pilot study to assess the association of the rs1800497 ANKK1 c.2137G > A (p.Glu713Lys) variant with weight loss with NB treatment in 33 subjects. Mean (SD) weight loss was 5.9% (3.2%) for the A1+ genotype group (n = 15) and 4.2% (4.2%) for the A1- genotype group (n = 18). The mean weight loss for the A1+ genotype group was significantly greater than the predefined clinically significant 4% weight-loss target (one-sample t-test, P = .035), whereas the mean weight loss for the A1- genotype group was not (P = .85). Individuals with the A1+ genotype appear to respond better to NB than A1- individuals.

Tepotinib Efficacy in a Patient with Non-Small Cell Lung Cancer with Brain Metastasis Harboring an HLA-DRB1-MET Gene Fusion.

Alterations in c-MET, a tyrosine kinase receptor encoded by the MET gene, have been reported in approximately 3% of non-small cell lung cancer (NSCLC) cases and carry important treatment implications. The best studied genetic alterations are exon 14 skipping and gene amplification; however, gene rearrangement has also been described, and multiple fusion partners have been reported. Recently, in METex14-mutated NSCLC, multitarget tyrosine kinase inhibitors (TKIs), such as crizotinib and cabozantinib, as well as MET-selective TKIs, such as tepotinib and capmatinib, have demonstrated durable responses. In this study, we present the case of a 41-year-old woman with advanced NSCLC harboring an HLA-DRB1-MET gene fusion. The patient was offered successively two different MET multikinase inhibitors, crizotinib and cabozantinib, and the selective inhibitor tepotinib. Each time, including under tepotinib, the patient experienced rapid and complete responses associated with a tremendous improvement in her physical function. KEY POINTS: To our knowledge, this is the first report of a patient with non-small cell lung cancer harboring an HLA-DRB1-MET gene fusion demonstrating a clinical response to multiple MET inhibitors, including tepotinib. This finding illustrates the efficacy and rationale to targeting MET regardless of fusion partner and gives insight to pooling of patients with different MET fusion products in trials assessing safety and efficacy of novel molecules.

Loss of the imprinted, non-coding Snord116 gene cluster in the interval deleted in the Prader Willi syndrome results in murine neuronal and endocrine pancreatic developmental phenotypes.

Global neurodevelopmental delay is a prominent characteristic of individuals with Prader-Willi syndrome (PWS). The neuromolecular bases for these delays are unknown. We identified neuroanatomical changes in the brains of mice deficient for a gene in the minimal critical deletion region for PWS (Snord116p-/m+). In Snord116p-/m+ mice, reduced primary forebrain neuron cell body size is apparent in embryonic day 15.5 fetuses, and persists until postnatal day 30 in cerebellar Purkinje neurons. Snord116 is a snoRNA gene cluster of unknown function that can localize to the nucleolus. In cerebellar Purkinje neurons from postnatal day 30 Snord116p-/m+ mice the reduction in neuronal cell body size was associated with decreased neuronal nucleolar size. We also identified developmental changes in the endocrine pancreas of Snord116p-/m+ animals that persist into adulthood. Mice lacking Snord116 have smaller pancreatic islets; within the islet the percentage of δ-cells is increased, while the percentage of α-cells is reduced. The α-cell markers, Sst and Hhex, are upregulated in Snord116p-/m+ isolated islets while Ins1, Ins2, Pdx1, Nkx6-1, and Pax6 are downregulated. There is a 3-fold increase in the percentage of polyhormonal cells in the neonatal pancreata of Snord116p-/m+ mice, due primarily to an increase in cells co-positive with somatostatin. Snord116 may play a role in islet cell lineage specification. The Snord116 gene cluster is important for developmental processes in the brain as well as the endocrine pancreas.

Monoallelic and Biallelic Variants in EMC1 Identified in Individuals with Global Developmental Delay, Hypotonia, Scoliosis, and Cerebellar Atrophy.

The paradigm of a single gene associated with one specific phenotype and mode of inheritance has been repeatedly challenged. Genotype-phenotype correlations can often be traced to different mutation types, localization of the variants in distinct protein domains, or the trigger of or escape from nonsense-mediated decay. Using whole-exome sequencing, we identified homozygous variants in EMC1 that segregated with a phenotype of developmental delay, hypotonia, scoliosis, and cerebellar atrophy in three families. In addition, a de novo heterozygous EMC1 variant was seen in an individual with a similar clinical and MRI imaging phenotype. EMC1 encodes a member of the endoplasmic reticulum (ER)-membrane protein complex (EMC), an evolutionarily conserved complex that has been proposed to have multiple roles in ER-associated degradation, ER-mitochondria tethering, and proper assembly of multi-pass transmembrane proteins. Perturbations of protein folding and organelle crosstalk have been implicated in neurodegenerative processes including cerebellar atrophy. We propose EMC1 as a gene in which either biallelic or monoallelic variants might lead to a syndrome including intellectual disability and preferential degeneration of the cerebellum.

Open staged repair of splenic and extensive superior mesenteric artery aneurysms.

A 31-year-old man with a history of multiple strokes of unknown etiology presented with multiple asymptomatic visceral artery aneurysms including a 3.3-cm bilobed splenic artery aneurysm and a 3.1-cm fusiform superior mesenteric artery aneurysm. He had no family history of aneurysms, and there was no evidence of collagen disease. He underwent open splenic artery aneurysm resection without splenectomy and had an uneventful recovery. Seven months later, he had open repair of his 14.6-cm-long fusiform superior mesenteric artery aneurysm with a 15-cm-long collagen-coated polyester interposition graft with multiple visceral branch reimplantations into the graft. The postoperative course was uneventful, and computed tomography confirmed patent graft and side branches. Two years later, the patient remained free of abdominal symptoms, although he suffered another episode of a mild stroke.

The role of Rpgrip1l, a component of the primary cilium, in adipocyte development and function.

Genetic variants within the FTO (α-ketoglutarate-dependent dioxygenase) gene have been strongly associated with a modest increase in adiposity as a result of increased food intake. These risk alleles are associated with decreased expression of both FTO and neighboring RPGRIP1L (retinitis pigmentosa GTPase regulator-interacting protein 1 like). RPGRIP1L encodes a protein that is critical to the function of the primary cilium, which conveys extracellular information to the cell. Rpgrip1l+/- mice exhibit increased adiposity, in part, as a result of hyperphagia. Here, we describe the effects of Rpgrip1l in adipocytes that may contribute to the adiposity phenotype observed in these animals and possibly in humans who segregate for FTO risk alleles. Loss of Rpgrip1l in 3T3-L1 preadipocytes increased the number of cells that are capable of differentiating into mature adipocytes. Knockout of Rpgrip1l in mature adipocytes using Adipoq-Cre did not increase adiposity in mice that were fed chow or a high-fat diet. We also did not observe any effects of Rpgrip1l knockdown in mature 3T3-L1 adipocytes. Thus, to the extent that Rpgrip1l affects cell-autonomous adipose tissue function, it may do so as a result of the effects conveyed in preadipocytes in which the primary cilium is functionally important. We propose that decreased RPGRIP1L expression in preadipocytes in humans who segregate for FTO obesity risk alleles may increase the storage capacity of adipose tissue.-Martin Carli, J. F., LeDuc, C. A., Zhang, Y., Stratigopoulos, G., Leibel, R. L. The role of Rpgrip1l, a component of the primary cilium, in adipocyte development and function.

FTO mediates cell-autonomous effects on adipogenesis and adipocyte lipid content by regulating gene expression via 6mA DNA modifications.

SNPs in the first intron of α-ketoglutarate-dependent dioxygenase (FTO) convey effects on adiposity by mechanisms that remain unclear, but appear to include modulation of expression of FTO itself, as well as other genes in cisFTO expression is lower in fibroblasts and iPSC-derived neurons of individuals segregating for FTO obesity risk alleles. We employed in vitro adipogenesis models to investigate the molecular mechanisms by which Fto affects adipocyte development and function. Fto expression was upregulated during adipogenesis, and was required for the maintenance of CEBPB and Cebpd/CEBPD expression in murine and human adipocytes in vitro. Fto knockdown decreased the number of 3T3-L1 cells that differentiated into adipocytes as well as the amount of lipid per mature adipocyte. This effect on adipocyte programming was conveyed, in part, by modulation of CCAAT enhancer binding protein (C/ebp)ß-regulated transcription. We found that Fto also affected Cebpd transcription by demethylating DNA N6-methyldeoxyadenosine in the Cebpd promoter. Fto is permissive for adipogenesis and promotes maintenance of lipid content in mature adipocytes by enabling C/ebpß-driven transcription and expression of Cebpd These findings are consistent with the loss of fat mass in mice segregating for a dominant-negative Fto allele.

The subgingival microbiome, systemic inflammation and insulin resistance: The Oral Infections, Glucose Intolerance and Insulin Resistance Study.

BACKGROUND: Inflammation might link microbial exposures to insulin resistance. We investigated the cross-sectional association between periodontal microbiota, inflammation and insulin resistance. METHODS: The Oral Infections, Glucose Intolerance and Insulin Resistance Study (ORIGINS) enrolled 152 diabetes-free adults (77% female) aged 20-55 years (mean = 34 ± 10). Three hundred and four subgingival plaque samples were analysed using the Human Oral Microbe Identification Microarray to measure the relative abundances of 379 taxa. C-reactive protein, interleukin-6, tumour necrosis factor-α and adiponectin were assessed from venous blood and their z-scores were summed to create an inflammatory score (IS). Insulin resistance was defined via the HOMA-IR. Associations between the microbiota and both inflammation and HOMA-IR were explored using multivariable linear regressions; mediation analyses assessed the proportion of the association explained by inflammation. RESULTS: The IS was inversely associated with Actinobacteria and Proteobacteria and positively associated with Firmicutes and TM7 (p-values < 0.05). Proteobacteria levels were associated with insulin resistance (p < 0.05). Inflammation explained 30-98% of the observed associations between levels of Actinobacteria, Proteobacteria or Firmicutes and insulin resistance (p-values < 0.05). Eighteen individual taxa were associated with inflammation (p < 0.05) and 22 with insulin resistance (p < 0.05). No findings for individual taxa met Bonferroni-adjusted statistical significance. CONCLUSION: Bacterial measures were related to inflammation and insulin resistance among diabetes-free adults.

A recurrent PDGFRB mutation causes familial infantile myofibromatosis.

Infantile myofibromatosis (IM) is the most common benign fibrous tumor of soft tissues affecting young children. By using whole-exome sequencing, RNA sequencing, and targeted sequencing, we investigated germline and tumor DNA in individuals from four distinct families with the familial form of IM and in five simplex IM cases with no previous family history of this disease. We identified a germline mutation c.1681C>T (p.Arg561Cys) in platelet-derived growth factor receptor ß (PDGFRB) in all 11 affected individuals with familial IM, although none of the five individuals with nonfamilial IM had mutations in this gene. We further identified a second heterozygous mutation in PDGFRB in two myofibromas from one of the affected familial cases, indicative of a potential second hit in this gene in the tumor. PDGFR-ß promotes growth of mesenchymal cells, including blood vessels and smooth muscles, which are affected in IM. Our findings indicate p.Arg561Cys substitution in PDGFR-ß as a cause of the dominant form of this disease. They provide a rationale for further investigations of this specific mutation and gene to assess the benefits of targeted therapies against PDGFR-ß in aggressive life-threatening familial forms of the disease.