Hypotrichosis-lymphedema-telangiectasia-renal defect associated with a truncating mutation in the SOX18 gene.

SOX18 mutations in humans are associated with both recessive and dominant hypotrichosis-lymphedema-telangiectasia syndrome (HLTS). We report two families with affected children carrying a SOX18 mutation: a living patient and his stillborn brother from Canada and a Belgian patient. The two living patients were diagnosed with HLTS and DNA analysis for the SOX18 gene showed that both had the identical heterozygous C > A transversion, resulting in a pre-mature truncation of the protein, lacking the transactivation domain. Both living patients developed renal failure with severe hypertension in childhood for which both underwent renal transplantation. To our best knowledge this is the first report of renal failure associated with heterozygous mutations in the SOX18 gene. We conclude that this specific mutation results in a new, autosomal dominant condition and propose the acronym HLT-renal defect syndrome for HLTRS.

Tandem inversion duplication within F8 Intron 1 associated with mild haemophilia A.

In approximately 90% of mild haemophilia A (HA) patients, a missense mutation can be identified using complete gene sequencing. In this study, multiplex ligation-dependent probe amplification analysis was performed as a second step in 10 French-speaking Belgian with mild HA presenting no detectable causal mutation by complete sequencing of the factor VIII (FVIII) (F8) gene's 26 exons and its 1.2 kb of contiguous promoter sequence. This gene dosage technique enabled the detection of exon 1 duplications of F8 in three apparently unrelated subjects. Using array-comparative genomic hybridization, breakpoint analysis delimited the duplication extent to 210 kb in the F8 intron 1 and VBP1 gene intragenic position. We postulated that the rearrangement responsible for this duplication, never before reported, could be attributed to a symmetrical tandem inversion duplication, resulting in a large 233 kb rearrangement of F8 intron 1. This rearranged intron should lead to the production of a small number of normal mRNA transcripts in relation to the mild HA phenotype. Our analysis of the entire F8 mRNA from index case 1, particularly the segment containing exons 1-9, revealed normal amplification and sequencing. Reduced plasma FVIII antigen levels caused by cross-reacting material is associated with a quantitative deficiency of plasma FVIII. Male patients were unresponsive to desmopressin (1-deamino-8-D-arginine vasopressin). All patients displayed identical F8 haplotypes, despite not being related, which suggests a possible founder effect caused by a 210 kb duplication involving F8 exon 1.

Capillary malformation-arteriovenous malformation syndrome: a report of 2 cases, diagnostic criteria, and management.

Capillary malformation-arteriovenous malformation syndrome is a rare type of vascular malformation first described in 2003. It is an autosomal dominant inherited disorder that has been reported in association with heterozygous mutations in the RASA1 gene, which encodes the protein RASp21. The clinical picture is characterized by multiple small capillary malformations which are associated with either arteriovenous malformations or arteriovenous fistulas in both the affected individual and other members of their family. We describe 2 new familial cases of this syndrome that were clinically and genetically diagnosed and studied in our hospital.

Prevalence and spectrum of SDHx mutations in pheochromocytoma and paraganglioma in patients from Belgium: an update.

Since the early 2000s, the prevalence and spectrum of mutations in genes encoding subunits of succinate dehydrogenase (SDHx) were reported in large cohorts of patients with pheochromocytoma (PC) and paraganglioma (PGL) from most Western countries. Unfortunately, in Belgium, no equivalent work was performed thus far. Therefore, the aim of the work was to look for mutations in SDHx genes and genotype-phenotype correlations in patients with PC and/or PGL from Belgium. Screening of the coding parts of SDHx genes and deletion search were performed in all patients with PC and/or PGL referred to the -Cliniques Universitaires Saint-Luc from 05/2003 to 05/2011. Genetic screening was performed in 59 unrelated head and neck (hn)PGLs (8 fami-lial) and 53 PCs (7 extra-adrenal; 3 metastatic). In hnPGLs, 10 different SDHD mutations (3 substitutions, 5 deletions, 2 splice site mutations) were detected in 16 patients, including 7 familial cases and 9 apparently sporadic cases. In the same subset, we found 8 different SDHB mutations (5 substitutions, 1 splice site mutation, 1 deletion, 1 duplication) in 10 patients with sporadic hnPGL without evidence of malignancy. No SDHx mutation was detected in patients harboring PCs and no SDHC mutation whatsoever. In conclusion, in our multicentric database of PC-PGLs from Belgium, (i) the prevalence of SDHx mutations was high in hnPGLs (44% in the whole subset, 37% of apparently sporadic cases); (ii) in sporadic cases, the prevalence of SDHB mutations was high (20%), similar to that of SDHD (18%); and (iii) no SDHx mutation was found in a subset of mostly adrenal, benign PCs.

Human chromosome map of lymphedema-lymphangiogenesis genes: Template for current and future discovery.

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.

Recessive primary congenital lymphoedema caused by a VEGFR3 mutation.

BACKGROUND: Heterozygous mutations in VEGFR3 have been identified in some familial cases with dominantly inherited primary congenital lymphoedema, known as Nonne-Milroy disease. Recessive cases of primary lymphoedema with a genetic cause are not known, except for two families with syndromic hypotrichosis-lymphoedema-telangiectasia, with a SOX18 mutation. METHODS AND RESULTS: In this study, we present the first case of isolated primary congenital lymphoedema with recessive inheritance, caused by a homozygous mutation in VEGFR3. The novel mutation is a transition from alanine-to-threonine in amino acid 855, located in the ATP binding domain of the VEGFR3 receptor. Assessment of receptor function showed impaired ligand induced internalisation and ERK1/2 activity. Moreover, receptor phosphorylation was reduced, although less so than for a kinase-dead VEGFR3 mutation, which causes Nonne-Milroy disease. CONCLUSION: A hypomorphic VEGFR3 mutation, with moderate effect on receptor function, in a homozygous state can result in insufficient lymphatic functioning. Thus, in addition to Nonne-Milroy disease with dominant inheritance, VEGFR3 alterations can cause isolated recessive primary congenital lymphoedema. These data expand our understanding of the aetiology of congenital lymphoedema and suggest that large scale screening of VEGFR3 in all primary lymphoedema patients is necessary.

Prevalence of pathogenic variants and variants of unknown significance in patients at high risk of breast cancer: A systematic review and meta-analysis of gene-panel data.

BACKGROUND: Gene-panels are used to assess predisposition to breast cancer by simultaneous testing of multiple susceptibility genes. This approach increases the identification of variants of unknown significance (VUS) that cannot be used in clinical decision-making. We performed a systematic review of published studies to calculate the prevalence of VUS and pathogenic variants (PV) in routinely tested breast cancer susceptibility genes in patients at high risk of breast cancer. METHODS: We comprehensively searched the literature using Medline through May 23, 2017 for studies conducting gene-panel testing on germline DNA of women with familial breast cancer and reporting on both PVs and VUSs. A meta-analysis of the collected data was carried out to obtain pooled VUS and PV prevalence estimates per gene using a generalized linear mixed model with logit link for binomial distribution. RESULTS: Of 602 publications, 4 were eligible and included 1870 patients. The panels encompassed 4-27 considered genes. Overall, the estimated probability per gene of a PV and VUS was 55% (95% confidence interval (CI) 26%-81%) and 91% (95% CI 78%-97%), respectively (p = 0.0066). The estimated probability per patient of a PV and VUS was 8% (95% CI 1%-34%) and 23% (95% CI 7%-52%), respectively (p = 0.0052). The ratio of VUS to PV was highest in the mismatch repair genes MLH1, MSH2, MSH6, PMS2 (18.7), CDH1 (13.4) and ATM (9.5). Amongst the 1468 patients tested for BRCA1 and BRCA2, only these two genes had a VUS to PV ratio of less than one (0.2 and 0.6, respectively). CONCLUSION: With the current panels, the probability of detecting a VUS is significantly higher than the probability of detecting a PV. Better classification of VUSs is therefore critical and requires gene-specific VUS-assessment in every future study of gene-panel testing in patients at high risk of breast cancer.

Cerebral cavernous malformation: new molecular and clinical insights.

Cerebral cavernous malformation (CCM) is a vascular malformation causing neurological problems, such as headaches, seizures, focal neurological deficits, and cerebral haemorrhages. CCMs can occur sporadically or as an autosomal dominant condition with variable expression and incomplete penetrance. Familial forms have been linked to three chromosomal loci, and loss of function mutations have been identified in the KRIT1/CCM1, MGC4607/CCM2, and PDCD10/CCM3 genes. Recently, many new pieces of data have been added to the CCM puzzle. It has been shown that the three CCM genes are expressed in neurones rather than in blood vessels. The interaction between CCM1 and CCM2, which was expected on the basis of their structure, has also been proven, suggesting a common functional pathway. Finally, in a large series of KRIT1 mutation carriers, clinical and neuroradiological features have been characterised. These data should lead to more appropriate follow up, treatment, and genetic counselling. The recent developments will also help to elucidate the precise pathogenic mechanisms leading to CCM, contributing to a better understanding of normal and pathological angiogenesis and to the development of targeted treatment.

Four common glomulin mutations cause two thirds of glomuvenous malformations ("familial glomangiomas"): evidence for a founder effect.

BACKGROUND: Glomuvenous malformation (GVM) ("familial glomangioma") is a localised cutaneous vascular lesion histologically characterised by abnormal smooth muscle-like "glomus cells" in the walls of distended endothelium lined channels. Inheritable GVM has been linked to chromosome 1p21-22 and is caused by truncating mutations in glomulin. A double hit mutation was identified in one lesion. This finding suggests that GVM results from complete localised loss of function and explains the paradominant mode of inheritance. OBJECTIVE: To report on the identification of a mutation in glomulin in 23 additional families with GVM. RESULTS: Three mutations are new; the others have been described previously. Among the 17 different inherited mutations in glomulin known up to now in 43 families, the 157delAAGAA mutation is the most common and was present in 21 families (48.8%). Mutation 108C-->A was found in five families (11.8%), and the mutations 554delA+556delCCT and 1179delCAA were present together in two families (4.7% each). Polymorphic markers suggested a founder effect for all four mutations. CONCLUSIONS: Screening for these mutations should lead to a genetic diagnosis in about 70% of patients with inherited GVM. So far, a mutation in glomulin has been found in all GVM families tested, thus demonstrating locus homogeneity.

Orofacial clefting: update on the role of genetics.

INTRODUCTION: Cleft lip with or without cleft palate (CL/P) is one of the most common birth defects in the world. Prevalence varies between populations, with an average of 1/700. CL/P has a major clinical impact, requiring surgical, dental, orthodontic, speech, hearing and psychological management throughout childhood. The aetiology of CL/P is mostly unknown, and it is thought that both genetic and environmental factors play a role. Several causative genes for inherited syndromic forms of CL/P have been identified, and some recent studies have shown that these genes also contribute to the occurrence of isolated forms. Van der Woude syndrome (VWS) is one of the best models for non-syndromic CLP. It is an autosomal dominant disorder characterised by the presence of pits on the lower lip in addition to CL/P. Pits are the only feature distinguishing VWS from isolated clefts. Interestingly, in numerous VWS patients, the lip pits are very small and not readily diagnosed, thus mimicking isolated CL/P. Mutations in the IRF6 gene were shown to be the major genetic cause of VWS.' RESULTS: We performed direct sequence analysis of IRF6 on samples from a large European cohort and identified mutations in 27 (80%) families. This shows that IRF6 is the major causative gene of VWS in Europe also. Moreover, it is the gene to study when a seemingly isolated CL/P patient has minor signs, such as lip pits, since the identification of a mutation in IRF6 is associated with an increase in the risk of having a child with CL/P from 4-6%, the risk of transmission of an isolated cleft, to 50%, the risk of transmission of a dominant Mendelian disorder like VWS. Moreover, we studied the association of isolated CL/P with the IRF6 locus using two variants in a set of 195 patients from Belgium. As in an American study, a clear association was observed. This suggests that IRF6 also contributes to the occurrence of sporadic, isolated CL/P, even if no mutation in the gene can be identified in such patients. CONCLUSION: In conclusion, genes that are mutated in familial syndromic forms of CL/P may be predisposing genetic factors to sporadic isolated CL/P. Due to technological advances and the availability of the human genome sequence, we have now the opportunity to try and unravel the genetic factors behind the various forms of CL/P.

Endothelial receptor tyrosine kinases activate the STAT signaling pathway: mutant Tie-2 causing venous malformations signals a distinct STAT activation response.

Endothelial receptor tyrosine kinases (RTKs) and their signaling mechanisms are of interest because they may control tumor angiogenesis and thereby tumor growth. In this report we have examined activation of the signal transducers and activators of transcription (STATs) by the three known vascular endothelial growth factor receptors (VEGFR1-3), as well as by the endothelial Tie-1 and -2 receptors. We also studied signaling by the R849W mutant of Tie-2 (MTie-2), which has been shown to cause venous malformations. When overexpressed in 293T cells, MTie-2 activated STAT1 while the other endothelial RTKs failed to do so. In contrast, the three VEGFRs were strong activators of STAT3 and STAT5, suggesting that they activate only a specific subset of these signal transducers. STAT3 and STAT5 were also activated by Tie-2 and, more so, by MTie-2. Tyrosine phosphorylation and DNA binding of STATs correlated with their ability to activate transcription as judged by luciferase assays. When co-expressed with STAT5, VEGFR-1 as well as both the Tie-2 receptor forms increased expression of the cell cycle inhibitor p21. Interestingly, co-expression of the Tie-2 receptors with STAT1 resulted in appearance of a novel, p21 related transcript. Taken together, these findings identify STAT proteins as novel targets for signal transduction by the endothelial RTKs, suggesting that they may be involved in the regulation of endothelial function.

Identification of eight novel 5'-exons in cerebral capillary malformation gene-1 (CCM1) encoding KRIT1.

Truncating mutations in the CCM1 gene encoding KRIT1 were recently found in patients affected by inherited cerebral capillary malformations, lesions that cause a wide variety of neurologic problems. However, CCM1 mutations have not been identified in all the families linked to CCM1. Here we demonstrate that the CCM1 gene contains eight additional exons which may thus encompass the missing mutations.

Molecular basis of vascular anomalies.

Vascular anomalies comprise a heterogeneous group of disorders that are divided into tumors (hemangiomas) and malformations. Recent advances in biomedical research provide insights into the molecular basis of these disorders and a deeper understanding of vascular morphogenesis. In the future, this emerging knowledge will contribute to novel ways to treat vascular anomalies and to regulate pathologic angiogenesis.

[Identification of implicated factors in angiogenesis for the study of vascular anomalies]. / L'identification des facteurs impliqués dans l'angiogenèse par l'etude des anomalies vasculaires.

Angiogenesis, the development of the vascular network, and lymphangiogenesis, the development of the lymphatic system, are two processes that are tightly linked and pivotal for the development and growth of a human being. Therefore, it is not surprising that lymph/angiogenesis plays a important role for health and is implicated in many diseases such as arteriosclerosis, arthritis, diabetes and cancer, both in children and in adults. To better understand lymph/angiogenesis and the factors that are implied in man, I became interested in the etiopathogenesis of vascular and lymphatic anomalies. These lesions are often congenital, very diversified, and occur with variable frequency, but in general affect about 0,3% of the population. Most frequently, they are well demarcated and localized, and histologically constituted of enlarged, tortuous vessels. By using a genetic approach on the familial forms, we have been able to identify several genes and causative mutations. These discoveries serve as a starting point for more precise clinical diagnosis and identification of the precise underlying pathophysiological mechanisms. These genes are also important for the search of novel therapies for these anomalies, as well as for all other disorders in which lymph/angiogenesis is implicated.

Overrepresentation of missense mutations in mild hemophilia A patients from Belgium: founder effect or independent occurrence?

Roughly 40% of observed mutations responsible for hemophilia A (HA) are novel and present in either a single family or a limited number of unrelated families. During routine diagnostic analysis of 73 unrelated Belgian patients with mild HA, 4 out of 43 different mutations (p.Ser2030Asn, p.Arg2178Cys, p.Arg2178His, and p.Pro2311His) were detected in more than one family, representing 35% of total identified mutations. To discriminate between an independent recurrence or a founder effect, an analysis of intra- and -extragenic single nucleotide polymorphisms (SNPs) and short tandem repeats (STRs) flanking the F8 gene was conducted. SNP haplotype and microsatellite analysis revealed strong evidence that p.Ser2030Asn and p.Pro2311His mutations were probably associated with a founder effect. The two other mutations localized in an F8 cytosine-phosphate-guanine (CpG) site likely resulted from recurrent de novo events. This study suggests that missense mutations producing C-to-T or G-to-A substitutions in CpG dinucleotide can occur de novo with more repetition than other causal substitutions that do not affect the CpG site. Analysis of F8 database implied that CpG sites throughout the F8 gene are not all mutated with the same frequency. Causes are still unknown and remain to be identified.

Antenatal presentation of hereditary lymphedema type I.

Fetal edema can present as limited subcutaneous edema, fluid accumulation in body cavities or hydrops fetalis. Hydrops fetalis is the end stage of a variety of fetal/maternal disorders and nonimmune etiology represents more than 3/4 of cases. Lymphatic dysplasia may account for a subset of patients with nonimmune and "idiopathic" hydrops fetalis, fetal chylous ascites or chylothorax. We present two unrelated patients with antenatal features of hereditary lymphedema syndrome, in whom Milroy disease was diagnosed after birth. At least, 20 genes have been identified to cause primary lymphedema, with sometimes antenatal features. Hereditary lymphedema syndrome should be considered in cases of nonimmune hydrops fetalis/fetal edema after ruling out the more common etiologies.

Molecular genetics of vascular malformations.

Vascular malformations are localized errors of angiogenic development. Most are cutaneous and are called vascular 'birthmarks'. These anomalies are usually obvious in the newborn, grow commensurately with the child, and gradually expand in adulthood (Mulliken and Glowacki, 1982). Vascular malformations also occur in visceral organs, such as the respiratory and gastrointestinal tract, but are more common in the brain (Mulliken and Young, 1988). These anomalies are composed of tortuous vascular channels of varying size and shape, lined by a continuous endothelium and surrounded by abnormal complement of mural cells. Vascular malformation can be life threatening due to obstruction, bleeding or congestive heart failure. Most anomalies occur sporadically, but there are families exhibiting autosomal dominant inheritance. Genetic studies of such families have resulted in the identification of mutated genes, directly giving proof of their important role in the regulation of angiogenesis.

Linkage disequilibrium narrows locus for venous malformation with glomus cells (VMGLOM) to a single 1.48 Mbp YAC.

Venous malformations with glomus cells are localised cutaneous lesions of vascular dysmorphogenesis. They are usually sporadic, but sometimes familial. Using five families, we mapped the locus, VMGLOM, to chromosome 1p21-p22. In order to refine this locus, spanning 4-6 Mbp, we then studied seven additional families. They exhibited linkage to VMGLOM and the combined lod score for all 12 families was 18.41 at theta = 0.0 for marker D1S188. We found a distinct haplotype shared by seven families, comprising seven alleles which are rare in the general population (P < 0.01). This indicates that the haplotype is identical by descent in all seven families, and hence the locus can be refined by inferring ancestral crossovers. Using this approach, we position the causative gene between two markers on the same non-chimeric YAC of 1.48 Mbp, a feasible size for positional cloning. As there is no known gene involved in vasculogenesis and/or angiogenesis in this YAC, the identification of the causative gene is likely to reveal a novel regulator or vascular development.