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The pathophysiological basis of non-syndromic orofacial cleft (NsOFC) is still largely unclear. However, exome sequencing (ES) has led to identify several causative genes, often with reduced penetrance. Among these, the Rho GTPase activating protein 29 (ARHGAP29) has been previously implicated in 7 families with NsOFC. We investigated a cohort of 224 NsOFCs for which no genetic pathogenic variant had been identified by diagnostic testing. We used ES and bioinformatic variant filtering and identified four novel putative pathogenic variants in ARHGAP29in four families. One was a missense variant leading to the substitution of the first methionine with threonine, two were heterozygous frameshift variants leading to a premature termination codon, and one was a nonsense variant. All variants were predicted to result in loss of function, either through mRNA decay, truncated ARHGAP29, or abnormal N-terminal initiation of translation of ARHGAP29. The truncated ARHGAP29 proteins would lack the important RhoGAP domain. The variants were either absent or rare in the control population databases, and the loss of intolerance score (pLI) of ARHGAP29 is 1.0, suggesting that ARHGAP29 haploinsufficiency is not tolerated. Phenotypes ranged from microform cleft lip (CL) to complete bilateral cleft lip and palate (CLP), with one unaffected mutation carrier. These results extend the mutational spectrum of ARHGAP29and show that it is an important gene underlying variable NsOFC phenotypes. ARHGAP29should be included in diagnostic genetic testing for NsOFC, especially familial cases, as it may be mutated in ∼4% of them (4/97 in our cohort) with high penetrance (89%).

Vascular malformations, which arise from anomalies in angiogenesis, encompass capillary, lymphatic, venous, arteriovenous, and mixed malformations, each affecting specific vessel types. Historically, therapeutic options such as sclerotherapy and surgery have shown limited efficacy in complicated malformations. Most vascular malformations stem from hereditary or somatic mutations akin to oncogenic alterations, activating the PI3K-AKT-mTOR, RAS-MAPK-ERK, and G-protein coupled receptor pathways. Recognizing the parallels with oncogenic mutations, we emphasize the potential of targeted molecular inhibitors in the treatment of vascular malformations by repurposing anticancer drugs. This review delves into the recent development and future use of such agents for the management of slow- and fast-flow vascular malformations, including in more specific situations, such as prenatal treatment and the management of associated coagulopathies.

This study describes genomic findings among individuals with both orofacial clefts (OC) and microphthalmia/anophthalmia/coloboma (MAC) recorded in the Brazilian Database on Craniofacial Anomalies (BDCA). Chromosomal microarray analysis (CMA) and Whole Exome Sequencing (WES) were performed in 17 individuals with OC-MAC. Clinical interpretation of molecular findings was based on data available at the BDCA and on re-examination. No copy number variants (CNVs) classified as likely pathogenic or pathogenic were detected by CMA. WES allowed a conclusive diagnosis in six individuals (35.29%), two of them with variants in the CHD7gene, and the others with variants in the TFAP2A, POMT1, PTPN11, and TP63genes with the following syndromes: CHARGE, CHD7-spectrum, Branchiooculofacial, POMT1-spectrum, LEOPARD, and ADULT. Variants of uncertain significance (VUS) possibly associated to the phenotypes were found in six other individuals. Among the individuals with VUSes, three individuals presented variants in genes associated to defects of cilia structure and/or function, including DYNC2H1, KIAA0586, WDR34, INTU, RPGRIP1L, KIF7, and LMNA. These results show that WES was the most effective molecular approach for OC-MAC in this cohort. This study also reinforces the genetic heterogeneity of OC-MAC, and the importance of genes related to ciliopathies in this phenotype.

BackgroundPrimary 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.MethodsWe investigated 742 index patients from our PL cohort using exome sequencing.ResultsWe identified nine variants predicted to cause CELSR1loss 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 CELSR1before. CELSR1is 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, CELSR1may be the long-sought gene for the renal defects.ConclusionPL associated with a renal anomaly suggests a CELSR1-related cause.

BackgroundHydrops 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 resultsWhole 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.ConclusionPathogenic heterozygous ANGPT2missense variants were recently shown to cause autosomal dominant primary lymphoedema. ANGPT2is 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.

Vascular malformations are subdivided into capillary, lymphatic, venous, arteriovenous, and mixed malformations, according to the type of affected vessels. Until a few years ago, treatment options were limited to sclerotherapy and/or surgery. Since, it has been demonstrated that the majority of vascular malformations are caused by inherited or somatic mutations in various genes. These mutations lead to hyperactivity of two major signaling pathways: the RAS/mitogen-activated protein kinase and the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin pathways. These discoveries paved the way for the development and testing of targeted molecular inhibitors as therapies for vascular anomalies via repurposing of anticancer drugs.

Mutations in the type II collagen gene may increase the risk of developing osteoarthritis. Osteoarthritis is a noninflammatory degenerative joint disease occurring mainly in older persons. Collagen is the protein substance of the white fibers of skin, tendon, bone, cartilage and all other connective tissue. Among 138 Finnish individuals who were evaluated for two markers for the type II collagen gene, 41 had generalized osteoarthritis, 49 had osteoarthritis of the finger joints and 48 were healthy individuals. Four different forms of the VNTR marker were found, and two different forms of the PvuII marker were found. Mutations in the different forms of the markers were associated with an increased risk of developing osteoarthritis. The Finnish population has remained genetically isolated from other populations.

BackgroundCapillary malformation-arteriovenous malformation is an autosomal dominant disorder, characterised by capillary malformations and increased risk of fast-flow vascular malformations, caused by loss-of-function mutations in the RASA1or EPHB4genes. Around 25% of the patients do not seem to carry a germline mutation in either one of these two genes. Even if other genes could be involved, some individuals may have mutations in the known genes that escaped detection by less sensitive techniques. We tested the hypothesis that mosaic mutations could explain some of previously negative cases.MethodsDNA was extracted from peripheral blood lymphocytes, saliva or vascular malformation tissues from four patients. RASA1and EPHB4coding regions and exon/intron boundaries were analysed by targeted custom gene panel sequencing. A second panel and/or Sanger sequencing were used to confirm the identified mutations.ResultsFour distinct mosaic RASA1mutations, with an allele frequency ranging from 3% to 25%, were identified in four index patients with classical capillary malformation-arteriovenous malformation phenotype. Three mutations were known, one was novel. In one patient, a somatic second hit was also identified. One index case had three affected children, illustrating that the mosaicism was also present in the germline.ConclusionThis study shows that RASA1mosaic mutations can cause capillary malformation-arteriovenous malformation. Thus, highly sensitive sequencing techniques should be considered as diagnostic tools, especially for patients with no family history. Even low-level mosaicism can cause the classical phenotype and increased risk for offspring. In addition, our study further supports the second-hit pathophysiological mechanism to explain the multifocality of vascular lesions in this disorder.

IMPORTANCE: Myofibroma is the most frequent fibrous tumor in children. Multicentric myofibroma (referred to as infantile myofibromatosis) is a life-threatening disease. OBJECTIVE: To determine the frequency, spectrum, and clinical implications of mutations in the PDGFRB receptor tyrosine kinase found in sporadic myofibroma and myofibromatosis. DESIGN, SETTING, AND PARTICIPANTS: In this retrospective study of 69 patients with sporadic myofibroma or myofibromatosis, 85 tumor samples were obtained and analyzed by targeted deep sequencing of PDGFRB. Mutations were confirmed by an alternative method of sequencing and were experimentally characterized to confirm gain of function and sensitivity to the tyrosine kinase inhibitor imatinib. MAIN OUTCOMES AND MEASURES: Frequency of gain-of-function PDGFRB mutations in sporadic myofibroma and myofibromatosis. Sensitivity to imatinib, as assessed experimentally. RESULTS: Of the 69 patients with tumor samples (mean [SD] age, 7.8 [12.7] years), 60 were children (87%; 29 girls [48%]) and 9 were adults (13%; 4 women [44%]). Gain-of-function PDGFRB mutations were found in samples from 25 children, with no mutation found in samples from adults. Mutations were particularly associated with severe multicentric disease (13 of 19 myofibromatosis cases [68%]). Although patients had no familial history, 3 of 25 mutations (12%) were likely to be germline, suggesting de novo heritable alterations. All of the PDGFRB mutations were associated with ligand-independent receptor activation, and all but one were sensitive to imatinib at clinically relevant concentrations. CONCLUSIONS AND RELEVANCE: Gain-of-function mutations of PDGFRB in myofibromas may affect only children and be more frequent in the multicentric form of disease, albeit present in solitary pediatric myofibromas. These alterations may be sensitive to tyrosine kinase inhibitors. The PDGFRB sequencing appears to have a high value for diagnosis, prognosis, and therapy of soft-tissue tumors in children.

The detection of somatic, activating genetic mutations to underlie development of vascular tumors and malformations led to a better understanding of their pathophysiology. Proteins encoded by the detected mutated genes activate the two major signaling pathways, also involved in cancer: the RAS/MAPK/ERK pathway and/or the PI3K/AKT/mTOR pathway. This gives a strong basis for studies to repurpose cancer therapeutics to patients with vascular tumors and malformations.

Venomous malformations (VMs) of the head and neck arise from deficits in the development of venous network. Clinically, VMs are highly variable, from small and asymptomatic varicosities to massive cervicofacial lesions. Therapeutic approaches include surgery; laser photocoagulation; sclerotherapy; and, more recently, systemic targeted drugs. This article discusses the natural history, diagnosis, and management of VMs.

Common capillary malformations (CMs) are red vascular skin lesions, most commonly associated with somatic activating GNAQor GNA11mutations. We focused on CMs lacking such a mutation to identify novel genetic causes. We used targeted next-generation-sequencing on 82 lesions. Bioinformatic analysis allowed the identification of 9 somatic pathogenic variants in PIK3R1and PIK3CA, encoding for the regulatory and catalytic subunits of the PI3K kinase, respectively. Re-characterization of these lesions unraveled a common phenotype: a pale Capillary Malformation associated with visible Dilated Veins (CMDV). Primary-endothelial cells from two 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 PIK3R1were further studied using zebrafish embryos. Endothelium-specific expression of PIK3R1mutants resulted in abnormal development of the posterior capillary-venous plexus. In summary, CMDV emerges as a clinical entity associated with somatic pathogenic variants in PIK3R1or PIK3CA(non-hotspot). Our findings suggest that the activated AKT signaling can be effectively reversed by PI3K-pathway inhibitors. Additionally, the proposed zebrafish model, holds promise as a valuable tool for future drug screening aimed at developing patient-tailored treatments.

Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine, usually benign, tumors. Currently, the only reliable criterion of malignancy is the presence of metastases.

BackgroundOral clefts, that is, clefts of the lip and/or cleft palate (CL/P), are the most common craniofacial birth defects with an approximate incidence of ~1/700. To date, physicians stratify patients with oral clefts into either syndromic CL/P (syCL/P) or non-syndromic CL/P (nsCL/P) depending on whether the CL/P is associated with another anomaly or not. In general, patients with syCL/P follow Mendelian inheritance, while those with nsCL/P have a complex aetiology and, as such, do not adhere to Mendelian inheritance. Genome-wide association studies have identified approximately 30 risk loci for nsCL/P, which could explain a small fraction of heritability.MethodsTo identify variants causing nsCL/P, we conducted whole exome sequencing on 84 individuals with nsCL/P, drawn from multiplex families (n=46).ResultsWe identified rare damaging variants in four genes known to be mutated in syCL/P: TP63(one family), TBX1(one family), LRP6(one family) and GRHL3(two families), and clinical reassessment confirmed the isolated nature of their CL/P.ConclusionThese data demonstrate that patients with CL/P without cardinal signs of a syndrome may still carry a mutation in a gene linked to syCL/P. Rare coding and non-coding variants in syCL/P genes could in part explain the controversial question of ‘missing heritability’ for nsCL/P. Therefore, gene panels designed for diagnostic testing of syCL/P should be used for patients with nsCL/P, especially when there is at least third-degree family history. This would allow a more precise management, follow-up and genetic counselling. Moreover, stratified cohorts would allow hunting for genetic modifiers.

Supplemental Digital Content is available in the text.

Blue rubber bleb nevus syndrome (Bean syndrome) is a rare, severe disorder of unknown cause, characterized by numerous cutaneous and internal venous malformations; gastrointestinal lesions are pathognomonic. We discovered somatic mutations in TEK, the gene encoding TIE2, in 15 of 17 individuals with blue rubber bleb nevus syndrome. Somatic mutations were also identified in five of six individuals with sporadically occurring multifocal venous malformations. In contrast to common unifocal venous malformation, which is most often caused by the somatic L914F TIE2 mutation, multifocal forms are predominantly caused by double (cis) mutations, that is, two somatic mutations on the same allele of the gene. Mutations are identical in all lesions from a given individual. T1105N-T1106P is recurrent in blue rubber bleb nevus, whereas Y897C-R915C is recurrent in sporadically occurring multifocal venous malformation: both cause ligand-independent activation of TIE2, and increase survival, invasion, and colony formation when expressed in human umbilical vein endothelial cells.

In recent years, genotypic characterization of congenital vascular malformations (CVMs) has gained attention; however, the spectrum of clinical phenotype remains difficult to attribute to a genetic cause and is rarely described in the adult population. The aim of this study is to describe a consecutive series of adolescent and adult patients in a tertiary center, where a multimodal phenotypic approach was used for diagnosis.

Despite the established role of SDHB/SDHA immunohistochemistry as a valuable tool to identify patients at risk for familial succinate dehydrogenase-related pheochromocytoma/paraganglioma syndromes, the reproducibility of the assessment methods has not as yet been determined. The aim of this study was to investigate interobserver variability among seven expert endocrine pathologists using a web-based virtual microscopy approach in a large multicenter pheochromocytoma/paraganglioma cohort (n=351): (1) 73 SDH mutated, (2) 105 non-SDH mutated, (3) 128 samples without identified SDH-x mutations, and (4) 45 with incomplete SDH molecular genetic analysis. Substantial agreement among all the reviewers was observed either with a two-tiered classification (SDHB κ=0.7338; SDHA κ=0.6707) or a three-tiered classification approach (SDHB κ=0.6543; SDHA κ=0.7516). Consensus was achieved in 315 cases (89.74%) for SDHB immunohistochemistry and in 348 cases (99.15%) for SDHA immunohistochemistry. Among the concordant cases, 62 of 69 (~90%) SDHB-/C-/D-/AF2-mutated cases displayed SDHB immunonegativity and SDHA immunopositivity, 3 of 4 (75%) with SDHA mutations showed loss of SDHA/SDHB protein expression, whereas 98 of 105 (93%) non-SDH-x-mutated counterparts demonstrated retention of SDHA/SDHB protein expression. Two SDHD-mutated extra-adrenal paragangliomas were scored as SDHB immunopositive, whereas 9 of 128 (7%) tumors without identified SDH-x mutations, 6 of 37 (~16%) VHL-mutated, as well as 1 of 21 (~5%) NF1-mutated tumors were evaluated as SDHB immunonegative. Although 14 out of those 16 SDHB-immunonegative cases were nonmetastatic, an overall significant correlation between SDHB immunonegativity and malignancy was observed (P=0.00019). We conclude that SDHB/SDHA immunohistochemistry is a reliable tool to identify patients with SDH-x mutations with an additional value in the assessment of genetic variants of unknown significance. If SDH molecular genetic analysis fails to detect a mutation in SDHB-immunonegative tumor, SDHC promoter methylation and/or VHL/NF1 testing with the use of targeted next-generation sequencing is advisable.

AbstractMaffucci syndrome (MS) is a rare congenital disorder characterized by multiple central cartilaginous tumors (enchondromas) in association with cutaneous spindle cell hemangiomas. These patients have a high incidence of malignant transformation. No familial case is known and the etiopathogenic cause remains unknown. In enchondromatosis (Ollier disease, OD), which is comprised of enchondromas only, 4 mutations in the PTHR1gene have been identified in 4 patients; 3 were somatic and 1 was germline. No PTHR1mutations have been detected in MS, whereas somatic IDH1and, more rarely, IDH2mutations have been observed in 77 of patients with MS and 81 of patients with OD. These genetic alterations are shared with other tumors, including glioma, leukemia and carcinoma. To search for underlying somatic genomic causes, we screened MS tissues using Affymetrix SNP-chips. We looked for CNVs, LOH and uniparental isodisomy (UPID) by performing pairwise analyses between allelic intensities in tumoral DNA versus the corresponding blood-extracted DNA. While common chromosomal anomalies were absent in constitutional DNA, several shared CNVs were identified in MS-associated tumors. The most frequently encountered somatic alterations were localized in 2p22.3, 2q24.3 and 14q11.2, implicating these chromosomal rearrangements in the formation of enchondromas and spindle cell hemangiomas in MS. In one chondrosarcoma specimen, large amplifications and/or deletions were observed in chromosomes 3, 6, 9, 10, 12, 13, and 19. Some of these genetic changes have been reported in other chondrosarcomas suggesting an etiopathogenic role. No LOH/UPID was observed in any Maffucci tissue. Our findings identify frequent somatic chromosomal rearrangements on 2p22.3, 2q24.3 and 14q11.2, which may unmask mutations leading to the lesions pathognomonic of MS.© 2014 S. Karger AG, Basel

Micrometric membrane lipid segregation is controversial. We addressed this issue in attached erythrocytes and found that fluorescent boron dipyrromethene (BODIPY) analogs of glycosphingolipids (GSLs) [glucosylceramide (BODIPY-GlcCer) and monosialotetrahexosylganglioside (GM1BODIPY)], sphingomyelin (BODIPY-SM), and phosphatidylcholine (BODIPY-PC inserted into the plasma membrane spontaneously gathered into distinct submicrometric domains. GM1BODIPY domains colocalized with endogenous GM1 labeled by cholera toxin. All BODIPY-lipid domains disappeared upon erythrocyte stretching, indicating control by membrane tension. Minor cholesterol depletion suppressed BODIPY-SM and BODIPY-PC but preserved BODIPY-GlcCer domains. Each type of domain exchanged constituents but assumed fixed positions, suggesting self-clustering and anchorage to spectrin. Domains showed differential association with 4.1R versus ankyrin complexes upon antibody patching. BODIPY-lipid domains also responded differentially to uncoupling at 4.1R complexes [protein kinase C (PKC) activation] and ankyrin complexes (in spherocytosis, a membrane fragility disease). These data point to micrometric compartmentation of polar BODIPY-lipids modulated by membrane tension, cholesterol, and differential association to the two nonredundant membrane:spectrin anchorage complexes. Micrometric compartmentation might play a role in erythrocyte membrane deformability and fragility.

Purpose: Interferon regulatory factor 6 encodes a member of the IRF family of transcription factors. Mutations in interferon regulatory factor 6 cause Van der Woude and popliteal pterygium syndrome, two related orofacial clefting disorders. Here, we compared and contrasted the frequency and distribution of exonic mutations in interferon regulatory factor 6 between two large geographically distinct collections of families with Van der Woude and between one collection of families with popliteal pterygium syndrome.Methods: We performed direct sequence analysis of interferon regulatory factor 6 exons on samples from three collections, two with Van der Woude and one with popliteal pterygium syndrome.Results: We identified mutations in interferon regulatory factor 6 exons in 68% of families in both Van der Woude collections and in 97% of families with popliteal pterygium syndrome. In sum, 106 novel disease-causing variants were found. The distribution of mutations in the interferon regulatory factor 6 exons in each collection was not random; exons 3, 4, 7, and 9 accounted for 80%. In the Van der Woude collections, the mutations were evenly divided between protein truncation and missense, whereas most mutations identified in the popliteal pterygium syndrome collection were missense. Further, the missense mutations associated with popliteal pterygium syndrome were localized significantly to exon 4, at residues that are predicted to bind directly to DNA.Conclusion: The nonrandom distribution of mutations in the interferon regulatory factor 6 exons suggests a two-tier approach for efficient mutation screens for interferon regulatory factor 6. The type and distribution of mutations are consistent with the hypothesis that Van der Woude is caused by haploinsufficiency of interferon regulatory factor 6. On the other hand, the distribution of popliteal pterygium syndrome-associated mutations suggests a different, though not mutually exclusive, effect on interferon regulatory factor 6 function.

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 MDFICtruncation variants revealed that homozygous mutant mice died perinatally exhibiting chylothorax. The lymphatic vasculature of homozygous Mdficmutant 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 β1activation and the interaction between lymphatic endothelial cells and their surrounding extracellular matrix. Our work identifies MDFICvariants 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.

Cutaneous venous anomalies are common. They are blue and vary in size, number, and location and account for most consultations at specialized interdisciplinary clinics for vascular anomalies. Venous lesions are clinically important because they cause pain, dysfunction, destruction of adjacent tissues, and esthetic concern. Only resection and sclerotherapy are helpful, although not always curative. Understanding etiopathogenesis could help design animal models and develop novel therapeutic approaches.

Mutations in genes encoding succinate dehydrogenase and its anchoring subunits (SDHgenes) are at the origin of hereditary head and neck paraganglioma (PGL) and a subset of apparently sporadic pheochromocytoma.

Recent reports have found genetic mutations in up to one quarter of patients harbouring pheochromocytoma and/or paraganglioma. This high prevalence was mainly due to the discovery of the role of SDHgenes. While SDHDhas been more frequently associated with the pathogenesis of head and neck paragangliomas, SDHBmutations were mainly associated with malignant and/or extra-adrenal pheochromocytoma/paraganglioma.

Vascular anomalies affect up to 10% of newborns, largely because of the high incidence of hemangioma of infancy. Vascular anomalies also frequently occur in adults; there is high prevalence of capillary malformations (0.3%). These cutaneous stains often cause psychosocial problems related to their visibility. Venous malformations occur in the skin and in internal organs and may cause destruction. Primary lymphedema causes lifelong morbidity, and arteriovenous malformations, in addition to causing distortion, obstruction, and pain, can be life endangering. The pathophysiology of these anomalies has stayed largely unknown, but genetic studies have revealed clues to their etiology. Genetic defects cause hereditary types of venous malformation, cutaneous and mucosal (VMCM); glomuvenous malformation (GVM); primary congenital lymphedema (Milroy disease); lymphedema-distichiasis syndrome; hypotrichosis-lymphedema-telangiectasia (HLT) syndrome; hereditary hemorrhagic telangiectasia (HHT); cerebral cavernous malformation (CCM); and a newly recognized disorder, capillary malformation-arteriovenous malformation (CM-AVM). These seminal discoveries have not only permitted a more precise clinical classification and diagnosis (a prerequisite for corrective measures for prevention, treatment, and follow-up) but also pointed the way to the identification of factors that play an important role in vasculogenesis or angiogenesis, or both.

The four SDH genes encode succinate dehydrogenase (SDHA and SDHB) and its anchoring subunits (SDHC and SDHD), which constitute complex II of the mitochondrial membrane, involved both in the respiratory chain and the Krebs cycle. Mutations of SDHD and less frequently SDHB and SDHC are at the origin of rare hereditary forms of head and neck paraganglioma. Interestingly, SDHB mutations are also a major cause of sporadic and familial pheochromocytomas, which have the same embryonic origin as head and neck paragangliomas. Patients with SDHB mutations are at higher risk of developing malignant and recurrent forms of pheochromocytoma. SDHB mutations have also been found in pedigrees harbouring both paragangliomas and renal cell carcinomas. This association reminds us of the Von Hippel-Lindau syndrome caused by mutations in the VHL gene. It should be pointed out that several angiogenic factors (HIFa, VEGF) are overexpressed both in tumours associated with paraganglioma and Von Hippel-Lindau syndrome. Though the different steps leading from SDH mutations to paraganglioma remain obscure, it is tempting to hypothesise that SDH gene products, VHL and angiogenic factors belong to the same functional pathway. The story of SDH genes is an impressive example of how the unravelling of the genetic basis of a rare disease i.e. familial head and neck paraganglioma can provide new insights into mechanisms involved in more frequent conditions such as endocrine hypertension, tumourigenesis, angiogenesis and adaptation to hypoxia.

Early detection of paragangliomas (PGs) has been linked to low morbidity after surgical resection. Recent identification of causative genes (SDHB, SDHC, and SDHD) has made it possible to detect individuals at high risk for tumors. We identified a three-generation family, with four individuals affected with PGs. Because pedigree analysis suggested maternal imprinting (the phenotype is present only if inherited through the paternal line), the SDHD gene (PGL1) was screened. A novel mutation that causes skipping of exon 3 was identified. Ten of the seventeen tested individuals carried the mutation. All six clinically unaffected individuals inherited the mutation from their mother. Five of them are men, with a 50% risk for affected progeny. To allow early treatment with low morbidity, genetic counseling is needed when familial paraganglioma is suspected. Asymptomatic carriers should be followed by cervical MRI. In addition, because pheochromocytomas may occur, catecholamine excretion can be performed. This screening should probably be proposed at 5 to 10 years of age. © 2003 Wiley Periodicals, Inc. Head Neck 25: 146–151, 2003

Vascular anomalies are congenital lesions that usually occur sporadically, but can be inherited. Previously, we have described that venous malformations, localized bluish-purple skin lesions, are caused by an activating mutation in the TIE2/TEK receptor. Moreover, we mapped another locus to chromosome 1p21–p22, for venous malformations with glomus cells (VMGLOM). Here we report a physical map, based on 18 overlapping YAC clones, spanning this 5-Mb VMGLOMlocus, from marker GATA63C06to D1S2664.In addition, we report a sequence-ready PAC map of 46 clones covering 1.48 Mb within the YAC contig, a region to which we have restricted VMGLOM.We describe 21 new STSs and nine novel CA repeats, seven of which are polymorphic. These data will enable positional cloning of genes for diseases mapped to this locus, including the VMGLOMgene, likely a currently unknown regulator of vasculogenesis and/or angiogenesis.

Cartilage diseases include a wide variety of clinical phenotypes from common osteoarthrosis to several different types of chondrodysplasias, i.e. 'disorders of cartilage', of which more than 100 different have been described. Patients frequently suffer from various symptoms affecting their joints and/or the growth of their long bones. The amount of hyaline cartilage at articular surfaces is often diminished and structurally abnormal. The surface of the cartilage may have an irregular appearance with defects extending into the subchondral bone. The major constituents of this hyaline cartilage are collagens and proteoglycans, the most abundant protein being type II collagen. It is a homotrimer of three identical α-chains, which are encoded by a single gene on human chromosome 12. The gene for type II collagen therefore became a likely candidate for some forms of chondrodysplasias and cartilage degeneration. Recently, both linkages and exclusions between this gene and various cartilage diseases have been reported and a growing number of mutations within the gene have also been identified.

Type II collagen is coded by a large gene (COL2A1) consisting of 54 exons on chromosome 12. During the past few years several cartilage disorders have been linked to this gene, and some specific nucleotide changes have been identified in patients. In a spondyloepiphyseal form of chondrodysplasia, the three mutations found so far are all in exon 48 in the region coding for the carboxyl-terminal end of the triple helix. Since folding of the type II collagen polypeptides to the triple helix is initiated from the carboxyl-terminal end, it has been suggested that mutations in this region typically result in severe cartilage diseases. Here we report a novel mutation located in the area coding for the amino-terminal part of the triple helix in a sporadic patient with spondyloepiphyseal dysplasia (SED). The mutation, a substitution of G1063to A, which results in the conversion of Gly154to Arg, was identified using exon-specific amplification of genomic DNA and subsequent analyses with denaturing gradient gel electrophoresis and single-strand conformation polymorphism. The substitution was not found in any other SED patient in Finland. This novel mutation demonstrates that amino acid substitutions in the amino-terminal part of the type II collagen triple helix can also result in SED.

The structure of type II collagen gene is extremely well conserved but contains a cluster of high frequency polymorphisms in a 2.2 kb area. Here we report the nucleotide sequence of this DNA area, essential for the PCR‐facilitated RFLP‐analyses of this gene. In the structural analyses we found four differences in the deduced human amino acid sequence when compared to the published bovine amino acid sequence. The donor and acceptor signals and branch point signals required for the splicing events were in agreement with mammalian consensus sequences. The frequency of inverted repeats which could provoke the DNA strand to loop formation and consequently to deletion mutations did not differ from that found in other sequenced genes coding for fibrillar collagens.

Osteoarthrosis (OA) is a major component of many osteochondrodysplastic syndromes which follow mendelian modes of inheritance. The use of molecular genetic techniques to unravel the molecular background of OA has therefore become possible. Linkage analysis, positional cloning, and candidate gene analysis have been used to identify genes which, when altered, can lead to syndromic OA. Allele association studies and sib-pair analysis have also made it possible to study the importance of a particular gene or locus in OA at the population level. Furthermore, the creation of transgenic mice and analyses of naturally occurring and chemically or radiologically induced mutations in mice are providing in vivo models for human osteochondrodysplasias and OA.

Cleft lip and/or palate are a split in the lip, the palate or both. This results from the inability of lip buds and palatal shelves to properly migrate and assemble during embryogenesis. By extracting primary cells from a cleft patient, we aimed at offering a better understanding of the signaling mechanisms and interacting molecules involved in the lip and palate formation and fusion. With Rho GTPases being indirectly associated with cleft occurrence, we investigated the role of the latter in both. First, whole exome sequencing was conducted in a patient with cleft lip and palate. Primary fibroblastic cells originating from the upper right gingiva region were extracted and distinct cellular populations from two individuals were obtained: a control with no cleft phenotype and a patient with a cleft lip and palate. The genetic data showed three candidate variables in ARHGEF18, EPDR1, and CUL7. Next, the molecular data showed no significant change in proliferation rates between healthy patient cells and CL/P patient cells. However, CL/P patient cells showed decreased migration, increased adhesion and presented with a more elongated phenotype. Additionally, RhoA activity was upregulated in these cells, whereas Cdc42 activity was downregulated, resulting in loss of polarity. Our results are suggestive of a possible correlation between a dysregulation of Rho GTPases and the observed phenotype of cleft lip and palate patient cells. This insight into the intramolecular aspect of this disorder helps link the genetic defect with the observed phenotype and offers a possible mechanism by which CL/P occurs.

Loss-of-function mutations in the TIE2-ligand angiopoietin 2 are associated with primary lymphedema in humans.

Chromosome instability frequently occurs in human malignant solid tumors and is a hallmark of tumorigenesis, but has not been identified in other biological processes. A large number of studies have reported mosaic whole-chromosome imbalances, and there have been sporadic reports on rare segmental aneuploidies in human in vitro–fertilized embryos; these findings suggest that chromosome instability could also occur during human embryogenesis. There are few data on chromosomal imbalances in embryos from normal fertile women. No comprehensive studies have investigated genomic imbalances during embryogenesis.

Burkitt lymphomas/leukemias (BL) are highly aggressive mature B-cell malignancies characterized by a constant MYC rearrangement. MYC deregulation is an oncogenic event known to be necessary but not sufficient in BL pathogenesis. Additional (epi)genetic alterations should cooperate, such as inactivation of the p14arf-MDM2-TP53 pathway which is frequently detected. We recently demonstrated the independent negative prognostic impact of specific chromosomal alterations, del(13q) and +7q, in childhood BL. We also showed by Multi-FISH that 13q abnormalities are more heterogeneous than expected and that13q gains are underestimated by conventional cytogenetics (CC). DNA from 30 BL (15 adults/15 children) was analyzed with 50K Xba single nucleotide polymorphisms (SNP) arrays (Affymetrix), in order to characterize additional allelic imbalances and losses of heterozygosity. 47 gains and 30 losses were found in 22 BL; 57 chromosomal imbalances were cryptic (not detected by CC). A recurring 13q amplification was detected in 6 BL. The minimal amplified region (MAR) of 4.35 Mb includes 3 candidate genes: C13orf25, GPC5 and GPC6. One C13orf25 transcripts contains a miRNA 17–92 cluster. The amplicon was unmasked with BAC probes in 2/8 other screened BL with 13q abnormalities. This amplification was associated with a terminal deletion (minimal size: 5Mb) in half cases. A recurring 11q abnormality was also assessed by FISH with a MAR of 3.2 Mb containing candidate genes such as CBL, BLR1. Acquired partial uniparental disomies (pUPD) are characterized by loss of heterozygosity without chromosomal deletion. 73 pUPD (>5 Mb) were found in 15 BL; 14 were telomeric and probably resulted from one mitotic recombination. The pathogenic role of pUPD remains unclear. Some of them may be polymorphisms also detected in healthy population. Other may reflect genomic instability but in our series, no higher incidence of pUPD was detected in BL with a complex karyotype. Thirdly, non random pUPD may render the neoplastic cell homozygous for a preexisting mutation leading to the activation of an oncogene or inactivation of a tumor suppressor gene (TSG). Four telomeric pUPD on 1p, 9p, 17p and 17q were found in at least 2 BL. We confirmed homozygous mutations of 2 TSG (TP53 and CDKN2A) located on 17p and 9p pUPD, respectively. These pUPD may be another way of p14arf-MDM2-TP53 inactivation. Moreover, these results suggest an involvement of the miR 17–92 cluster known to interact with the MYC oncogene in BL pathogenesis. The expression pattern of the different mature miR 17–92 in BL with and without 13q amplification will be presented at the meeting.