Background: Type 1 diabetes (T1D) shares clinical characteristics with other forms of diabetes, particularly monogenic diabetes such as maturity-onset diabetes of the young (MODY). Differential diagnosis is complicated by the existence of intermediate phenotypes. We aimed to delineate the phenotypic continuum between T1D and monogenic diabetes. Methods: The multicentric GENEPEDIAB study included patients aged 6 months to 18 years diagnosed with diabetes and treated for either T1D or monogenic diabetes. Analyses comprised glycemic variability, continuous glucose monitoring metrics, application of the DIAMODIA criteria, and genetic investigations. Results: A gradient was observed across T1D, atypical diabetes (Adia), and MODY cohorts for several glycemic parameters. T1D patients exhibited values furthest from treatment targets, whereas MODY patients showed better glycemic control. Stratification of the Adia cohort according to the number of positive DIAMODIA criteria further supported this trend, as demonstrated by glycemic measures and multiple correspondence analysis. Genetic analyses did not identify a uniform causative variant in the Adia cohort; however, several rare variants, including variants of uncertain significance and likely pathogenic variants in diabetes-related genes, were detected. Conclusions: These findings showed, in our specific cohort of pediatric patients, the existence of a phenotypic gradient between T1D and monogenic diabetes, with atypical diabetes occupying an intermediate position, including when stratified by DIAMODIA criteria.

Infantile hemangioma (IH) is the most common tumor in children and a paradigm for pathological vasculogenesis, angiogenesis, and regression. Propranolol, the mainstay of treatment, inhibits IH vessel formation via a 3-adrenergic receptor-independent off-target effect of its R(+) enantiomer on endothelial SOX18a member of the SOX (SRY-related HMG-box) family of transcription factors. Transcriptomic profiling of patient-derived hemangioma stem cells uncovered the mevalonate pathway (MVP) as a target of R(+) propranolol. Loss and gain of function of SOX18 confirmed it is both necessary and sufficient for R(+) propranolol suppression of the MVP, including regulation of sterol regulatory element-binding protein 2 (SREBP2) and the rate-limiting enzyme HMG-CoA reductase (HMGCR). A biological relevance of the endothelial SOX18MVP axis in IH patient tissue was demonstrated by nuclear colocalization of SOX18 and SREBP2. Functional validation in a preclinical IH xenograft model revealed that statins - competitive inhibitors of HMGCR - efficiently suppress IH vessel formation. We propose an endothelial SOX18-MVP axis as a central regulator of IH pathogenesis and suggest statin repurposing to treat IH. The pleiotropic effects of R(+) propranolol and statins along the SOX18-MVP axis to disable an endothelial cell-specific program may have therapeutic implications for othervascular disease entities involving pathological vasculogenesis and angiogenesis.

The genetic basis of nonsyndromic orofacial cleft (NsOFC) remains elusive, although associations have been identified with various genetic loci. NsOFC has a less pronounced genetic background than syndromic orofacial cleft (SyOFC), albeit Mendelian inheritance has been identified. Our hypothesis was that genes related to spliceosome function may contribute to NsOFC pathophysiology, as they do for some syndromic cases. Exome sequencing was conducted on 224 unrelated NsOFC probands. We performed filtering and analyses of predicted pathogenicity of rare variants using Highlander. We focused on 26 genes encoding spliceosome proteins. Subsequently, bioinformatic tools, such as AlphaFold, and PyMol, were applied to generate three-dimensional structures to interpret the effects of the identified variants on protein structure and interaction domains. We found six likely damaging variants: three heterozygous missense variants in small nuclear ribonucleoprotein U5 200 kDa subunit (SNRNP200) in three multiplex NsOFC families, and two missense and one splice site variant in splicing factor 3b subunit 1 (SF3B1), 4 (SF3B4), and 2 (SF3B2) in two posterior CP families and a complete CP patient, respectively. These results suggest that variants in the spliceosome complex genes, observed in 2.7% of NsOFC cases in our cohort, may contribute to disease susceptibility as potential risk factors.

BACKGROUND:Lymphedema is an incurable disease associated with lymphatic dysfunction that causes tissue swelling and fibrosis. We investigated whether lymphedema could be attenuated by interfering with uPARAP (urokinase plasminogen activator receptor-associated protein; Mrc2 gene), an endocytic receptor involved in fibrosis and lymphangiogenesis. METHODS:We generated mice with lymphatic endothelial cell (LEC)-specific uparap deficiency and compared them with constitutive knockout mice by applying a preclinical model of secondary lymphedema (SL). Computerized methods were applied for 2-dimensional and 3-dimensional image quantifications. Cellular effects of uPARAP deletion on lymphatic permeability were assessed by small interfering RNA-mediated silencing in human dermal LECs and a pharmacologic treatment targeting ROCK (Rho-associated coiled coil containing kinase), an established regulator of cell junctions. The uPARAP and vascular endothelial cadherin partnership was investigated through proximity ligation assay, coimmunoprecipitation, and immunostaining. An in silico model was generated to analyze the fluid-absorbing function of the lymphatic vasculature. To interfere with uPARAP, its downregulation was achieved in vivo through a gapmer approach. RESULTS:uparap deficiency mitigated several key pathologic features of SL, including hindlimb swelling, epidermal thickening, and the accumulation and size of adipocytes. In both global and LEC-conditional uparap-deficient mice, induction of SL led to a distinctive labyrinthine vasculature, defined herein by twisted and hyperbranched vessels with overlapping cells. This topology, mainly composed of pre-collecting vessels, correlated with reduced SL, but not with change in fibrosis, highlighting the importance of uPARAP in regulating LEC functions in a lymphedematous context. In vitro, uPARAP knockdown in LECs impaired vascular endothelial growth factor C-mediated endosomal trafficking of vascular endothelial cadherin and induced overlapping cell junctions. The pharmacologic inhibition of ROCK recapitulated cell superimposition in vitro and the labyrinthine vasculature in vivo with attenuated SL. Computational modeling of labyrinthine lymphatic vasculature supported the observation on their improved fluid-absorbing function in comparison with a normal hierarchic network. These data provide proof of concept of inducing a labyrinthine topology to treat SL. For therapeutic purposes, we validated the use of an anti-uPARAP gapmer to induce a labyrinthine vasculature and attenuate SL formation. CONCLUSIONS:Our findings provide evidence that downregulating uPARAP expression can induce a beneficial remodeling of lymphatic vasculature that attenuates lymphedema through a cell junction-based mechanism, offering a novel therapeutic pathway for lymphedema.

Common capillary malformations are red vascular skin lesions, most commonly associated with somatic activating GNAQ or GNA11 mutations. We focused on capillary malformations lacking such a mutation to identify previously unreported genetic causes. We used targeted next-generation sequencing on 82 lesions. Bioinformatic analysis allowed the identification of 9 somatic pathogenic variants in PIK3R1 and PIK3CA, encoding for the regulatory and catalytic subunits of phosphoinositide 3-kinase, respectively. Recharacterization of these lesions unraveled a common phenotype: a pale capillary malformation associated with visible dilated veins. Primary endothelial cells from 2 PIK3R1-mutated lesions were isolated, and PI3k-Akt-mTOR and RAS-RAF-MAPK signaling were assessed by western blot. This unveiled an abnormal increase in Akt phosphorylation, effectively reduced by PI3K pathway inhibitors, such as mTOR, Akt, and PIK3CA inhibitors. The effects of mutant PIK3R1 were further studied using zebrafish embryos. Endothelium-specific expression of PIK3R1 mutants resulted in abnormal development of the posterior capillary-venous plexus. In summary, capillary malformation associated with visible dilated veins emerges as a clinical entity associated with somatic pathogenic variants in PIK3R1 or PIK3CA (nonhotspot). Our findings suggest that the activated Akt signaling can be effectively reversed by PI3K pathway inhibitors. In addition, the proposed zebrafish model holds promise as a valuable tool for future drug screening aimed at developing patient-tailored treatments.

Developmental and functional defects in the lymphatic system are responsible for primary lymphoedema (PL). PL is a chronic debilitating disease caused by increased accumulation of interstitial fluid, predisposing to inflammation, infections and fibrosis. There is no cure, only symptomatic treatment is available. Thirty-two genes or loci have been linked to PL, and another 22 are suggested, including Hepatocyte Growth Factor (HGF). We searched for HGF variants in 770 index patients from the Brussels PL cohort. We identified ten variants predicted to cause HGF loss-of-function (six nonsense, two frameshifts, and two splice-site changes; 1.3% of our cohort), and 14 missense variants predicted to be pathogenic in 17 families (2.21%). We studied co-segregation within families, mRNA stability for non-sense variants, and in vitro functional effects of the missense variants. Analyses of the mRNA of patient cells revealed degradation of the nonsense mutant allele. Reduced protein secretion was detected for nine of the 14 missense variants expressed in COS-7 cells. Stimulation of lymphatic endothelial cells with these 14 HGF variant proteins resulted in decreased activation of the downstream targets AKT and ERK1/2 for three of them. Clinically, HGF-associated PL was diverse, but predominantly bilateral in the lower limbs with onset varying from early childhood to adulthood. Finally, aggregation study in a second independent cohort underscored that rare likely pathogenic variants in HGF explain about 2% of PL. Therefore, HGF signalling seems crucial for lymphatic development and/or maintenance in human beings and HGF should be included in diagnostic genetic screens for PL.

BackgroundRecent diabetes subclassifications have improved the differentiation between patients with type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus despite several overlapping features, yet without considering genetic forms of diabetes. We sought to facilitate the identification of monogenic diabetes by creating a new tool that we validated in a pediatric maturity-onset diabetes of the young (MODY) cohort. MethodsWe first created the DIAgnose MOnogenic DIAbetes (DIAMODIA) criteria based on the pre-existing, but incomplete, MODY calculator. This new score is composed of four strong and five weak criteria, with patients having to display at least one weak and one strong criterion. ResultsThe effectiveness of the DIAMODIA criteria was evaluated in two patient cohorts, the first consisting of patients with confirmed MODY diabetes (n=34) and the second of patients with T1DM (n=390). These DIAMODIA criteria successfully detected 100% of MODY patients. Multiple correspondence analysis performed on the MODY and T1DM cohorts enabled us to differentiate MODY patients from T1DM. The three most relevant variables to distinguish a MODY from T1DM profile were: lower insulin-dose adjusted A1c score ≤9, glycemic target-adjusted A1c score ≤4.5, and absence of three anti-islet cell autoantibodies. ConclusionWe validated the DIAMODIA criteria, as it effectively identified all monogenic diabetes patients (MODY cohort) and succeeded to differentiate T1DM from MODY patients. The creation of this new and effective tool is likely to facilitate the characterization and therapeutic management of patients with atypical diabetes, and promptly referring them for genetic testing which would markedly improve clinical care and counseling, as well.

We have updated the human chromosomal map of the location of known and candidate genes involved in primary lymphedema (PL) originally published in 2021. This should facilitate further discovery and provide a basis for understanding microdeletions which cause lymphedema.

Primary lymphedema (PL), characterized by tissue swelling, fat accumulation, and fibrosis, results from defects in lymphatic vessels or valves caused by mutations in genes involved in development, maturation, and function of the lymphatic vascular system. Pathogenic variants in various genes have been identified in about 30% of PL cases. By screening of a cohort of 755 individuals with PL, we identified two TIE1 (tyrosine kinase with immunoglobulin- and epidermal growth factor-like domains 1) missense variants and one truncating variant, all predicted to be pathogenic by bioinformatic algorithms. The TIE1 receptor, in complex with TIE2, binds angiopoietins to regulate the formation and remodeling of blood and lymphatic vessels. The premature stop codon mutant encoded an inactive truncated extracellular TIE1 fragment with decreased mRNA stability, and the amino acid substitutions led to decreased TIE1 signaling activity. By reproducing the two missense variants in mouse Tie1 via CRISPR/Cas9, we showed that both cause edema and are lethal in homozygous mice. Thus, our results indicate that TIE1 loss-of-function variants can cause lymphatic dysfunction in patients. Together with our earlier demonstration that ANGPT2 loss-of-function mutations can also cause PL, our results emphasize the important role of the ANGPT2/TIE1 pathway in lymphatic function.

Background Primary lymphoedema (PL) is a chronic, debilitating disease caused by developmental and functional defects of the lymphatic system. It is marked by an accumulation of interstitial fluid, fat and tissue fibrosis. There is no cure. More than 50 genes and genetic loci have been linked to PL. We sought to study systematically cell polarity signalling protein Cadherin Epidermal Growth Factor Laminin G Seven-pass G-type Receptor 1 (CELSR1) variants linked to PL. Methods We investigated 742 index patients from our PL cohort using exome sequencing. Results We identified nine variants predicted to cause CELSR1 loss of function. Four of them were tested for nonsense-mediated mRNA decay, but none was observed. Most of the truncated CELSR1 proteins would lack the transmembrane domain, if produced. The affected individuals had puberty/late-onset PL on lower extremities. The variants had a statistically significant difference in penetrance between female patients (87%) and male patients (20%). Eight variant carriers had a kidney anomaly, mostly in the form of ureteropelvic junction obstruction, which has not been associated with CELSR1 before. CELSR1 is located in the 22q13.3 deletion locus of the Phelan-McDermid syndrome. As variable renal defects are often seen in patients with the Phelan-McDermid syndrome, CELSR1 may be the long-sought gene for the renal defects. Conclusion PL associated with a renal anomaly suggests a CELSR1-related cause.

Splenectomy improves the clinical parameters of patients with hereditary spherocytosis, but its potential benefit to red blood cell (RBC) functionality and the mechanism behind this benefit remain largely overlooked. Here, we compared 7 nonsplenectomized and 13 splenectomized patients with mutations in the β-spectrin or the ankyrin gene. We showed that hematological parameters, spherocyte abundance, osmotic fragility, intracellular calcium, and extracellular vesicle release were largely but not completely restored by splenectomy, whereas cryohemolysis was not. Affected RBCs exhibited decreases in β-spectrin and/or ankyrin contents and slight alterations in spectrin membrane distribution, depending on the mutation. These modifications were found in both splenectomized and nonsplenectomized patients and poorly correlated with RBC functionality alteration, suggesting additional impairments. Accordingly, we found an increased abundance of septins, small guanosine triphosphate-binding cytoskeletal proteins. Septins-2, -7, and -8 but not -11 were less abundant upon splenectomy and correlated with the disease severity. Septin-2 membrane association was confirmed by immunolabeling. Except for cryohemolysis, all parameters of RBC morphology and functionality correlated with septin abundance. The increased septin content might result from RBC maturation defects, as evidenced by (1) the decreased protein 4.2 and Rh-associated glycoprotein content in all patient RBCs, (2) increased endoplasmic reticulum remnants and endocytosis proteins in nonsplenectomized patients, and (3) increased lysosomal and mitochondrial remnants in splenectomized patients. Our study paves the way for a better understanding of the involvement of septins in RBC membrane biophysical properties. In addition, the lack of restoration of septin-independent cryohemolysis by splenectomy may call into question its recommendation in specific cases.

Background Hydrops fetalis, a pathological fluid accumulation in two or more body compartments, is aetiologically heterogeneous. We investigated a consanguineous family with recurrent pregnancy loss due to severe early-onset non-immune hydrops fetalis. Methods and results Whole exome sequencing in four fetuses with hydrops fetalis revealed that they were homozygous for the angiopoietin-2 ( ANGPT2 ) variant Chr8 (GRCh37/Hg19): 6385085T>C, NM_001147.2:c.557A>G. The substitution introduces a cryptic, exonic splice site predicted to result in loss of 10 nucleotides with subsequent shift in reading frame, leading to a premature stop codon. RNA analysis in the heterozygous parents demonstrated loss of detectable mutant allele, indicative of loss-of-function via nonsense-mediated mRNA decay. Serum ANGPT2 levels were reduced in the parents. In a pregnancy with a healthy, heterozygous child, transiently increased fetal nuchal translucency was noted. Conclusion Pathogenic heterozygous ANGPT2 missense variants were recently shown to cause autosomal dominant primary lymphoedema. ANGPT2 is a ligand of the TIE1-TIE2 (tyrosine kinase with immunoglobulin-like and epidermal growth factor-like domains 1 and 2) pathway. It is critical to the formation and remodelling of blood and lymphatic vessels and is involved in vessel maintenance. ANGPT2 knockout mice die from generalised lymphatic dysfunction. We show here that a homozygous pathogenic variant causes loss-of-function and results in severe early-onset hydrops fetalis. This is the first report of an autosomal recessive ANGPT2 -related disorder in humans.

BACKGROUNDSlow-flow vascular malformations frequently harbor activating mutations in the PI3K/AKT/mTOR cascade. Phase II trials pinpointed sirolimus effectiveness as a drug therapy. Efficacy and safety of sirolimus thus need to be evaluated in large prospective phase III trials.METHODSThe Vascular Anomaly-Sirolimus-Europe (VASE) trial, initiated in 2016, is a large multicentric prospective phase III trial (EudraCT 2015-001703-32), which evaluates efficacy and safety of sirolimus for 2 years in pediatric and adult patients with symptomatic slow-flow vascular malformations. In this interim analysis, we studied all patients enrolled up to October 2021 who received sirolimus for 12 or more months or who prematurely stopped the treatment.RESULTSThirty-one pediatric and 101 adult patients were included in this analysis; 107 completed 12 or more months of sirolimus, including 61 who were treated for the whole 2-year period. Sirolimus resulted in a clinical improvement in 85% of patients. The efficacy appeared within the first month for the majority of them. Grade 3-4 adverse events were observed in 24 (18%) patients; all resolved after treatment interruption/arrest. Sirolimus increased feasibility of surgery or sclerotherapy in 20 (15%) patients initially deemed unsuitable for intervention. Among the 61 patients who completed the 2-year treatment, 33 (54%) reported a recurrence of symptoms after a median follow-up of 13 months after sirolimus arrest. While there was no difference in efficacy, clinical improvement was faster but subsided more rapidly in PIK3CA-mutated (n = 24) compared with TIE2-mutated (n = 19) patients.CONCLUSIONSirolimus has a high efficacy and good tolerance in treatment of slow-flow vascular malformations in children and adults.TRIAL REGISTRATIONClinicalTrials.gov NCT02638389 and EudraCT 2015-001703-32.FUNDINGThe Fonds de la Recherche Scientifique (FNRS grants T.0247.19, P.C005.22, T.0146.16, and P.C013.20), the Fund Generet managed by the King Baudouin Foundation (grant 2018-J1810250-211305), the Walloon Region through the FRFS-WELBIO strategic research programme (WELBIO-CR-2019C-06), the MSCA-ITN network V.A. Cure no. 814316, the Leducq Foundation Networks of Excellence Program grant "ReVAMP" (LFCR grant 21CVD03), the European Union's Horizon 2020 research and innovation programme under grant agreement no. 874708 (Theralymph), the Swiss National Science Foundation under the Sinergia project no. CRSII5_193694, and a Pierre M. fellowship.

Objectives: Capillary malformation-arteriovenous malformation (CM-AVM) is a rare disorder characterized by multifocal CMs associated with AVMs. While the phenotype of CMAVM1 has been well established in the literature, there are limited published reports documenting the clinical features of CMAVM2. Our aim is to highlight this phenotype, and the distinctive features of CMAVM2 compared with CMAVM1 and hereditary hemorrhagic telangiectasia, as prompt recognition is crucial to screen for fast-flow vascular malformations, which, if left untreated, can sometimes be life-threatening. Methods: This is a retrospective review of cases seen at multiple tertiary care centers in the United States and Argentina. Results and Conclusions: We report 6 pediatric patients with CMAVM2 presenting with diffuse multifocal cutaneous CMs, telangiectases favoring the lips, perioral area and upper trunk, and Bier spots. Three patients had epistaxis. One patient had an extracranial AVM and 1 patient had Parkes Weber syndrome. EPHB4 pathogenic variants were identified in all patients. CMAVM2 remains an underrecognized condition. Genetic testing and imaging studies are recommended to guide management.

Central conducting lymphatic anomaly (CCLA), characterized by the dysfunction of core collecting lymphatic vessels including the thoracic duct and cisterna chyli, and presenting as chylothorax, pleural effusions, chylous ascites, and lymphedema, is a severe disorder often resulting in fetal or perinatal demise. Although pathogenic variants in RAS/mitogen activated protein kinase (MAPK) signaling pathway components have been documented in some patients with CCLA, the genetic etiology of the disorder remains uncharacterized in most cases. Here, we identified biallelic pathogenic variants in MDFIC , encoding the MyoD family inhibitor domain containing protein, in seven individuals with CCLA from six independent families. Clinical manifestations of affected fetuses and children included nonimmune hydrops fetalis (NIHF), pleural and pericardial effusions, and lymphedema. Generation of a mouse model of human MDFIC truncation variants revealed that homozygous mutant mice died perinatally exhibiting chylothorax. The lymphatic vasculature of homozygous Mdfic mutant mice was profoundly mispatterned and exhibited major defects in lymphatic vessel valve development. Mechanistically, we determined that MDFIC controls collective cell migration, an important early event during the formation of lymphatic vessel valves, by regulating integrin β 1 activation and the interaction between lymphatic endothelial cells and their surrounding extracellular matrix. Our work identifies MDFIC variants underlying human lymphatic disease and reveals a crucial, previously unrecognized role for MDFIC in the lymphatic vasculature. Ultimately, understanding the genetic and mechanistic basis of CCLA will facilitate the development and implementation of new therapeutic approaches to effectively treat this complex disease.

We have created a human chromosomal map of the location of known and candidate genes involved in primary lymphedema (PLE). This should facilitate further discovery and provide a basis for understanding microdeletions which cause lymphedema.

Gorham-Stout disease (GSD) is a sporadically occurring lymphatic disorder. Patients with GSD develop ectopic lymphatics in bone, gradually lose bone, and can have life-threatening complications, such as chylothorax. The etiology of GSD is poorly understood, and current treatments for this disease are inadequate for most patients. To explore the pathogenesis of GSD, we performed targeted high-throughput sequencing with samples from a patient with GSD and identified an activating somatic mutation in KRAS (p.G12V). To characterize the effect of hyperactive KRAS signaling on lymphatic development, we expressed an active form of KRAS (p.G12D) in murine lymphatics (iLECKras mice). We found that iLECKras mice developed lymphatics in bone, which is a hallmark of GSD. We also found that lymphatic valve development and maintenance was altered in iLECKras mice. Because most iLECKras mice developed chylothorax and died before they had significant bone disease, we analyzed the effect of trametinib (an FDA-approved MEK1/2 inhibitor) on lymphatic valve regression in iLECKras mice. Notably, we found that trametinib suppressed this phenotype in iLECKras mice. Together, our results demonstrate that somatic activating mutations in KRAS can be associated with GSD and reveal that hyperactive KRAS signaling stimulates the formation of lymphatics in bone and impairs the development of lymphatic valves. These findings provide insight into the pathogenesis of GSD and suggest that trametinib could be an effective treatment for GSD.