Germline Loss-of-Function Mutations in EPHB4 Cause a Second Form of Capillary Malformation-Arteriovenous Malformation (CM-AVM2) Deregulating RAS-MAPK Signaling.

BACKGROUND: Most arteriovenous malformations (AVMs) are localized and occur sporadically. However, they also can be multifocal in autosomal-dominant disorders, such as hereditary hemorrhagic telangiectasia and capillary malformation (CM)-AVM. Previously, we identified RASA1 mutations in 50% of patients with CM-AVM. Herein we studied non-RASA1 patients to further elucidate the pathogenicity of CMs and AVMs. METHODS: We conducted a genome-wide linkage study on a CM-AVM family. Whole-exome sequencing was also performed on 9 unrelated CM-AVM families. We identified a candidate gene and screened it in a large series of patients. The influence of several missense variants on protein function was also studied in vitro. RESULTS: We found evidence for linkage in 2 loci. Whole-exome sequencing data unraveled 4 distinct damaging variants in EPHB4 in 5 families that cosegregated with CM-AVM. Overall, screening of EPHB4 detected 47 distinct mutations in 54 index patients: 27 led to a premature stop codon or splice-site alteration, suggesting loss of function. The other 20 are nonsynonymous variants that result in amino acid substitutions. In vitro expression of several mutations confirmed loss of function of EPHB4. The clinical features included multifocal CMs, telangiectasias, and AVMs. CONCLUSIONS: We found EPHB4 mutations in patients with multifocal CMs associated with AVMs. The phenotype, CM-AVM2, mimics RASA1-related CM-AVM1 and also hereditary hemorrhagic telangiectasia. RASA1-encoded p120RASGAP is a direct effector of EPHB4. Our data highlight the pathogenetic importance of this interaction and indicts EPHB4-RAS-ERK signaling pathway as a major cause for AVMs.

Somatic uniparental isodisomy explains multifocality of glomuvenous malformations.

Inherited vascular malformations are commonly autosomal dominantly inherited with high, but incomplete, penetrance; they often present as multiple lesions. We hypothesized that Knudson's two-hit model could explain this multifocality and partial penetrance. We performed a systematic analysis of inherited glomuvenous malformations (GVMs) by using multiple approaches, including a sensitive allele-specific pairwise SNP-chip method. Overall, we identified 16 somatic mutations, most of which were not intragenic but were cases of acquired uniparental isodisomy (aUPID) involving chromosome 1p. The breakpoint of each aUPID is located in an A- and T-rich, high-DNA-flexibility region (1p13.1-1p12). This region corresponds to a possible new fragile site. Occurrences of these mutations render the inherited glomulin variant in 1p22.1 homozygous in the affected tissues without loss of genetic material. This finding demonstrates that a double hit is needed to trigger formation of a GVM. It also suggests that somatic UPID, only detectable by sensitive pairwise analysis in heterogeneous tissues, might be a common phenomenon in human cells. Thus, aUPID might play a role in the pathogenesis of various nonmalignant disorders and might explain local impaired function and/or clinical variability. Furthermore, these data suggest that pairwise analysis of blood and tissue, even on heterogeneous tissue, can be used for localizing double-hit mutations in disease-causing genes.

Isolated bilateral transverse agenesis of the distal segments of the lower limbs at the level of the knee joint in a human fetus.

Congenital limb anomalies occur in Europe with a prevalence of 3.81/1,000 births and can have a major impact on patients and their families. The present study concerned a female fetus aborted at 23 weeks of gestation because she was affected by non-syndromic bilateral absence of the zeugopod (leg) and autopod (foot). Autopsy of the aborted fetus, X-ray imaging, MRI, and histochemical analysis showed that the distal extremity of both femurs was continued by a cartilage-like mass, without joint cavitation. Karyotype was normal. Moreover, no damaging variant was detected by exome sequencing. The limb characteristics of the fetus, which to our knowledge have not yet been reported in humans, suggest a developmental arrest similar to anomalies described in chicks following surgical experiments on the apical ectodermal ridge of the lower limbs.

TMEM165 deficiency causes a congenital disorder of glycosylation.

Protein glycosylation is a complex process that depends not only on the activities of several enzymes and transporters but also on a subtle balance between vesicular Golgi trafficking, compartmental pH, and ion homeostasis. Through a combination of autozygosity mapping and expression analysis in two siblings with an abnormal serum-transferrin isoelectric focusing test (type 2) and a peculiar skeletal phenotype with epiphyseal, metaphyseal, and diaphyseal dysplasia, we identified TMEM165 (also named TPARL) as a gene involved in congenital disorders of glycosylation (CDG). The affected individuals are homozygous for a deep intronic splice mutation in TMEM165. In our cohort of unsolved CDG-II cases, we found another individual with the same mutation and two unrelated individuals with missense mutations in TMEM165. TMEM165 encodes a putative transmembrane 324 amino acid protein whose cellular functions are unknown. Using a siRNA strategy, we showed that TMEM165 deficiency causes Golgi glycosylation defects in HEK cells.

DMP1 mutations in autosomal recessive hypophosphatemia implicate a bone matrix protein in the regulation of phosphate homeostasis.

Hypophosphatemia is a genetically heterogeneous disease. Here, we mapped an autosomal recessive form (designated ARHP) to chromosome 4q21 and identified homozygous mutations in DMP1 (dentin matrix protein 1), which encodes a non-collagenous bone matrix protein expressed in osteoblasts and osteocytes. Intact plasma levels of the phosphaturic protein FGF23 were clearly elevated in two of four affected individuals, providing a possible explanation for the phosphaturia and inappropriately normal 1,25(OH)2D levels and suggesting that DMP1 may regulate FGF23 expression.

Autosomal-dominant microtia linked to five tandem copies of a copy-number-variable region at chromosome 4p16.

Recently, large-scale benign copy-number variations (CNVs)--encompassing over 12% of the genome and containing genes considered to be dosage tolerant for human development--were uncovered in the human population. Here we present a family with a novel autosomal-dominantly inherited syndrome characterized by microtia, eye coloboma, and imperforation of the nasolacrimal duct. This phenotype is linked to a cytogenetically visible alteration at 4pter consisting of five copies of a copy-number-variable region, encompassing a low-copy repeat (LCR)-rich sequence. We demonstrate that the approximately 750 kb amplicon occurs in exact tandem copies. This is the first example of an amplified CNV associated with a Mendelian disorder, a discovery that implies that genome screens for genetic disorders should include the analysis of so-called benign CNVs and LCRs.

Molecular identification of aspartate N-acetyltransferase and its mutation in hypoacetylaspartia.

The brain-specific compound NAA (N-acetylaspartate) occurs almost exclusively in neurons, where its concentration reaches approx. 20 mM. Its abundance is determined in patients by MRS (magnetic resonance spectroscopy) to assess neuronal density and health. The molecular identity of the NAT (N-acetyltransferase) that catalyses NAA synthesis has remained unknown, because the enzyme is membrane-bound and difficult to purify. Database searches indicated that among putative NATs (i.e. proteins homologous with known NATs, but with uncharacterized catalytic activity) encoded by the human and mouse genomes two were almost exclusively expressed in brain, NAT8L and NAT14. Transfection studies in HEK-293T [human embryonic kidney-293 cells expressing the large T-antigen of SV40 (simian virus 40)] indicated that NAT8L, but not NAT14, catalysed the synthesis of NAA from L-aspartate and acetyl-CoA. The specificity of NAT8L, its Km for aspartate and its sensitivity to detergents are similar to those described for brain Asp-NAT. Confocal microscopy analysis of CHO (Chinese-hamster ovary) cells and neurons expressing recombinant NAT8L indicates that it is associated with the ER (endoplasmic reticulum), but not with mitochondria. A mutation search in the NAT8L gene of the only patient known to be deficient in NAA disclosed the presence of a homozygous 19 bp deletion, resulting in a change in reading frame and the absence of production of a functional protein. We conclude that NAT8L, a neuron-specific protein, is responsible for NAA synthesis and is mutated in primary NAA deficiency (hypoacetylaspartia). The molecular identification of this enzyme will lead to new perspectives in the clarification of the function of this most abundant amino acid derivative in neurons and for the diagnosis of hypoacetylaspartia in other patients.

Rare presentation of familial paraganglioma without evidence of mutation in the SDH, RET and VHL genes: towards further genetic heterogeneity.

OBJECTIVE: Mutations in genes encoding succinate dehydrogenase and its anchoring subunits (SDH genes) are at the origin of hereditary head and neck paraganglioma (PGL) and a subset of apparently sporadic pheochromocytoma. METHODS: We describe a family including three patients harbouring bilateral head and neck PGL diagnosed before 25 years of age. Multiple hypervascular hepatic lesions were subsequently discovered in two of them. In both, liver biopsy confirmed the diagnosis of PGL. In addition, in one patient, MRI disclosed multiple target-like lesions of the spine, highly suggestive of metastatic PGL. Family history was compatible with autosomal dominant inheritance with possible maternal imprinting. RESULTS: Combined single-strand conformation polymorphism and heteroduplex analysis followed by sequencing did not show any mutation of the coding parts of SDHB, SDHC, SDHD, RET or VHL genes. Screening of copy number alterations and loss of heterozygosity in the three affected family members showed no deletion or amplification of the SDH, RET and VHL genes. Furthermore, succinate dehydrogenase activity measured in a liver PGL sample was not significantly decreased in the affected patient as compared with controls, underscoring the exclusion of the SDH genes. CONCLUSIONS: To our knowledge, this is the first reported family of hereditary head and neck PGL with metastatic dissemination in the liver and the spine. A large body of evidence supports the absence of mutations in SDH, RET and VHL genes, which suggests the existence of a yet unknown gene at the origin of this particular form of familial PGL.

Identification of candidate regions for a novel Usher syndrome type II locus.

PURPOSE: Chronic diseases affecting the inner ear and the retina cause severe impairments to our communication systems. In more than half of the cases, Usher syndrome (USH) is the origin of these double defects. Patients with USH type II (USH2) have retinitis pigmentosa (RP) that develops during puberty, moderate to severe hearing impairment with downsloping pure-tone audiogram, and normal vestibular function. Four loci and three genes are known for USH2. In this study, we proposed to localize the gene responsible for USH2 in a consanguineous family of Tunisian origin. METHODS: Affected members underwent detailed ocular and audiologic characterization. One Tunisian family with USH2 and 45 healthy controls unrelated to the family were recruited. Two affected and six unaffected family members attended our study. DNA samples of eight family members were genotyped with polymorphic markers. Two-point and multipoint LOD scores were calculated using Genehunter software v2.1. Sequencing was used to investigate candidate genes. RESULTS: Haplotype analysis showed no significant linkage to any known USH gene or locus. A genome-wide screen, using microsatellite markers, was performed, allowing the identification of three homozygous regions in chromosomes 2, 4, and 15. We further confirmed and refined these three regions using microsatellite and single-nucleotide polymorphisms. With recessive mode of inheritance, the highest multipoint LOD score of 1.765 was identified for the candidate regions on chromosomes 4 and 15. The chromosome 15 locus is large (55 Mb), underscoring the limited number of meioses in the consanguineous pedigree. Moreover, the linked, homozygous chromosome 15q alleles, unlike those of the chromosome 2 and 4 loci, are infrequent in the local population. Thus, the data strongly suggest that the novel locus for USH2 is likely to reside on 15q. CONCLUSIONS: Our data provide a basis for the localization and the identification of a novel gene implicated in USH2, most likely localized on 15q.

Localization of candidate regions for a novel gene for Kartagener syndrome.

Asymmetric positioning of internal organs is a characteristics of vertebrates. The normal left-right anatomic positioning, situs solitus, sometimes does not occur normaly, leading to laterality defects. Studies in animal models have shown that laterality decisions are mediated by a cascade of genes that lead to the asymmetric expression of Nodal, LEFTA, LEFTB and PITX2 in the lateral plate mesoderm. A search for mutations in genes implicated in left-right patterning in animal models allowed genes associated with heterotaxia defects in humans to be identified. However, these genes explain only a small percentage of human situs defects, suggesting that other genes must play a role. In this study, we report a consanguineous family of Turkish origin, composed of two unaffected parents and three children, two of whom presented Kartagener syndrome. On the basis of their family history, we hypothesize autosomal recessive mode of inheritance. A genotype analysis with polymorphic markers did not show linkage with any known genes or loci causing laterality disorders. Array CGH did not detect a duplication or microdeletion greater than 1 Mb as a possible cause. Genome wide screening using 10 K Affymetrix SNP chips was performed, allowing the identification of two regions of autozygosity, one in chromosome 1 and the other on chromosome 7. In the chromosome 1 locus, a strong candidate gene, encoding the kinesin-associated protein 3 (KIF3AP) was not mutated, based on SSCP/heteroduplex analysis and direct sequencing. These data provide a basis for the identification of a novel gene implicated in Kartagener syndrome.

Common somatic alterations identified in maffucci syndrome by molecular karyotyping.

Maffucci 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 PTHR1 gene have been identified in 4 patients; 3 were somatic and 1 was germline. No PTHR1 mutations have been detected in MS, whereas somatic IDH1 and, more rarely, IDH2 mutations 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.

Giant cell tumor occurring in familial Paget's disease of bone: report of clinical characteristics and linkage analysis of a large pedigree.

Neoplastic degeneration represents a rare but serious complication of Paget's disease of bone (PDB). Although osteosarcomas have been described in up to 1% of PDB cases, giant cell tumors are less frequent and mainly occur in patients with polyostotic disease. We recently characterized a large pedigree with 14 affected members of whom four developed giant cell tumors at pagetic sites. The high number of affected subjects across multiple generations allowed us to better characterize the clinical phenotype and look for possible susceptibility loci. Of interest, all the affected members had polyostotic PDB, but subjects developing giant cell tumors showed an increased disease severity with a reduced clinical response to bisphosphonate treatment and an increased prevalence of bone pain, deformities, and fractures. Together with an increased occurrence of common pagetic complications, affected patients of this pedigree also evidenced a fivefold higher prevalence of coronary artery disease with respect to either the unaffected family members or a comparative cohort of 150 unrelated PDB cases from the same geographical area. This association was further enhanced in the four cases with PDB and giant cell tumors, all of them developing coronary artery disease before 60 years of age. Despite the early onset and the severe phenotype, PDB patients from this pedigree were negative for the presence of SQSTM1 or TNFRSF11A mutations, previously associated with enhanced disease severity. Genome-wide linkage analysis identified six possible candidate regions on chromosomes 1, 5, 6, 8, 10, and 20. Because the chromosome 8 and 10 loci were next to the TNFRSF11B and OPTN genes, we extended the genetic screening to these two genes, but we failed to identify any causative mutation at both the genomic and transcription level, suggesting that a different genetic defect is associated with PDB and potentially giant cell tumor of bone in this pedigree.

KITLG mutations cause familial progressive hyper- and hypopigmentation.

Familial progressive hyper- and hypopigmentation (FPHH) is thought to be an autosomal dominant disorder with reduced penetrance. Clinical signs consist of progressive diffuse, partly blotchy hyperpigmented lesions, multiple café-au-lait spots, intermingled with scattered hypopigmented-appearing maculae, and lentigines. FPHH is distinct from familial progressive hyperpigmentation (FPH), in which no hypopigmented features are present, and which is phenotypically and histologically closer to Dyschromatosis Universalis Hereditaria 2 (DUH2). It also differs from the Legius syndrome, characterized by familial café-au-lait spots and skin fold freckling, caused by mutations in SPRED1. We performed a genome-wide linkage analysis in seven families with FPHH, and identified linkage on 12q21.12-q22, which overlaps with the DUH2 locus. We investigated whether KITLG in the locus is mutated in FPHH. We discovered three different mutations in four families. A reported FPH substitution was observed in two FPHH families, and two, to our knowledge, previously unreported substitutions, p.Val33Ala and p.Thr34Pro, cosegregated with FPHH in two separate families. All three mutations were located in a conserved ß-strand in KITLG, suggesting its important role in the activation of the KITLG receptor c-Kit. In aggregate, mutations in a single gene cause various pigmentation disorders: FPH, FPHH, and likely DUH2. Therefore, KITLG is an important modulator of skin pigmentation.

Chromosome instability is common in human cleavage-stage embryos.

Chromosome instability is a hallmark of tumorigenesis. This study establishes that chromosome instability is also common during early human embryogenesis. A new array-based method allowed screening of genome-wide copy number and loss of heterozygosity in single cells. This revealed not only mosaicism for whole-chromosome aneuploidies and uniparental disomies in most cleavage-stage embryos but also frequent segmental deletions, duplications and amplifications that were reciprocal in sister blastomeres, implying the occurrence of breakage-fusion-bridge cycles. This explains the low human fecundity and identifies post-zygotic chromosome instability as a leading cause of constitutional chromosomal disorders.

Src triggers circular ruffling and macropinocytosis at the apical surface of polarized MDCK cells.

We addressed the role of Src on cortical actin dynamics and polarized endocytosis in MDCK cells harboring a thermosensitive v-src mutant. Shifting monolayers established at 40 degrees C (non-permissive temperature) to 34 degrees C (permissive temperature) rapidly reactivated v-Src kinase, but tight junctions and cell polarity resisted for >6 h. At this interval, activated v-src was recruited on apical vesicles, induced cortactin-associated apical circular ruffles productive of macropinosomes, thereby accelerating apical pinocytosis by approximately fivefold. Ruffling and macropinosome formation were selectively abrogated by inhibitors of actin polymerization, phosphoinositide 3-kinase, phospholipase C, and phospholipase D, which all returned apical pinocytosis to the level observed at 40 degrees C, underscoring the distinct control of apical micropinocytosis and macropinocytosis. Src promoted microtubule-dependent fusion of macropinosomes to the apical recycling endosome (ARE), causing its strong vacuolation. However, preservation of tubulation and apical polarity indicated that its function was not affected. The ARE was labeled for v-src, Rab11, and rabankyrin-5 but not early endosome antigen 1, thus distinguishing two separate Rab5-dependent apical pathways. The mechanisms of Src-induced apical ruffling and macropinocytosis could shed light on the triggered apical enteroinvasive pathogens entry and on the apical differentiation of osteoclasts.

v-Src accelerates spontaneous motility via phosphoinositide 3-kinase, phospholipase C and phospholipase D, but abrogates chemotaxis in Rat-1 and MDCK cells.

In Rat-1 fibroblasts, v-Src causes a profound remodelling of cortical actin cytoskeleton. This transformation includes membrane ruffling, a hallmark of the leading edge in migrating cells, and results from activation of phosphoinositide 3-kinase (PI 3-kinase), phospholipase C (PLC) and phospholipase D (PLD). We therefore reexamined whether motility is constitutively triggered by v-Src and studied whether this response is controlled by the same signalling pathway. The study was performed using Rat-1/tsLA29 and MDCK/tsLA31 cells, each harbouring a different thermosensitive v-Src kinase, active at 34 degrees C but inactivated at 40 degrees C. In both cell lines, overnight v-Src activation induced transformation and accelerated spontaneous motility by approximately twofold, as evidenced by wound-healing assay and by single-cell track, time-lapse recording in Dunn chambers. Inhibitors of PI 3-kinase, PLC and PLD selectively abrogated acceleration of motility by v-Src. Since mechanisms that co-ordinate spontaneous, as distinct from oriented, cell migration are separable, we further analysed in Dunn chambers chemotactic response of Rat-1/tsLA29 cells to PDGF and of MDCK/tsLA31 cells to EGF. In both cases, v-Src decreased the steady-state level of growth factor receptors at the cell surface twofold, and abrogated movement directionality at comparable level of occupancy as in non-transformed cells. The burst of pinocytosis in response to growth factors was also abolished by v-Src. Altogether, these results indicate that v-Src triggers motility in a PI 3-kinase-, PLC- and PLD-dependent manner, but abrogates directionality by suppressing polarised signalling downstream of growth factor receptors.

Origin, originality, functions, subversions and molecular signalling of macropinocytosis.

Macropinocytosis refers to the formation of primary large endocytic vesicles of irregular size and shape, generated by actin-driven evaginations of the plasma membrane, whereby cells avidly incorporate extracellular fluid. Macropinosomes resemble "empty" phagosomes and show no difference with the "spacious phagosomes" triggered by the enteropathogenic bacteria Salmonella and Shigella. Macropinosomes may fuse with lysosomes or regurgitate their content back to the extracellular space. In multiple cell types, macropinocytosis is a transient response to growth factors. When amoebas are cultured under axenic conditions, macropinocytosis is induced so as to fulfil nutritional requirements. In immature dendritic cells, macropinocytosis allows for extensive sampling of soluble antigens; after a few days of maturation, this activity vanishes as processed peptides are being presented. Macropinosomes are also formed at the leading edge of motile leukocytes or neurons. In all these examples, macropinocytosis appears tightly regulated. Transformation of fibroblasts by Src or Ras also results in constitutive formation of macropinosomes at "ruffling" zones, that could be related to accelerated cell motility. Like phagocytosis, macropinocytosis depends on signalling to the actin cytoskeleton. We have explored this signalling in transformed cells. v-Src and K-Ras activate PI3K and PLC, as demonstrated by in situ production of the corresponding lipid products. Pharmacological inhibitors of PI3K and PLC and stable transfection leading to a dominant-negative PI3-kinase construct in transformed fibroblasts abolish macropinocytosis, demonstrating that both enzyme activities are essential. Conversely, stable transfection leading to a dominant-positive P13K in non-transformed fibroblasts is sufficient to induce macropinocytosis. Combination of experiments allows to conclude that P13K and PLC act in sequential order. In non-polarized cells expressing a thermosensitive v-Src mutant, v-Src kinase activation accelerates fluid-phase endocytosis. In polarized MDCK cells, this stimulation occurs selectively at the apical domain and the response is selectively abrogated by pharmacological inhibitors of P13K and PLC. Thus, two paradigmatic oncogenes cause constitutive macropinocytosis. For v-Src, this response is polarized at the apical membrane. It is suggested that, in enterocytes that do not normally phagocytose, the P13K-PLC signalling pathway leading to selective induction of macropinocytosis at the luminal surface has been subverted by enteropathogenic bacteria to penetrate via "spacious phagosomes".