Pathogenic variants in HGF give rise to childhood-to-late onset primary lymphoedema by loss of function.

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

Frequency of Participation in External Quality Assessment Programs Focused on Rare Diseases: Belgian Guidelines for Human Genetics Centers.

BACKGROUND: Participation in quality controls, also called external quality assessment (EQA) schemes, is required for the ISO15189 accreditation of the Medical Centers of Human Genetics. However, directives on the minimal frequency of participation in genetic quality control schemes are lacking or too heterogeneous, with a possible impact on health care quality. OBJECTIVE: The aim of this project is to develop Belgian guidelines on the frequency of participation in quality controls for genetic testing in the context of rare diseases. METHODS: A group of experts analyzed 90 EQA schemes offered by accredited providers and focused on analyses used for the diagnosis of rare diseases. On that basis, the experts developed practical recommendations about the minimal frequencies of participation of the Medical Centers of Human Genetics in quality controls and how to deal with poor performances and change management. These guidelines were submitted to the Belgian Accreditation Body and then reviewed and approved by the Belgian College of Human Genetics and Rare Diseases and by the National Institute for Health and Disability Insurance. RESULTS: The guidelines offer a decisional algorithm for the minimal frequency of participation in human genetics EQA schemes. This algorithm has been developed taking into account the scopes of the EQA schemes, the levels of experience, and the annual volumes of the Centers of Human Genetics in the performance of the tests considered. They include three key principles: (1) the recommended annual assessment of all genetic techniques and technological platforms, if possible through EQAs covering the technique, genotyping, and clinical interpretation; (2) the triennial assessment of the genotyping and interpretation of specific germline mutations and pharmacogenomics analyses; and (3) the documentation of actions undertaken in the case of poor performances and the participation to quality control the following year. The use of a Bayesian statistical model has been proposed to help the Centers of Human Genetics to determine the theoretical number of tests that should be annually performed to achieve a certain threshold of performance (eg, a maximal error rate of 1%). Besides, the guidelines insist on the role and responsibility of the national public health authorities in the follow-up of the quality of analyses performed by the Medical Centers of Human Genetics and in demonstrating the cost-effectiveness and rationalization of participation frequency in these quality controls. CONCLUSIONS: These guidelines have been developed based on the analysis of a large panel of EQA schemes and data collected from the Belgian Medical Centers of Human Genetics. They are applicable to other countries and will facilitate and improve the quality management and financing systems of the Medical Centers of Human Genetics.

Non-hotspot PIK3CA mutations are more frequent in CLOVES than in common or combined lymphatic malformations.

BACKGROUND: Theragnostic management, treatment according to precise pathological molecular targets, requests to unravel patients' genotypes. We used targeted next-generation sequencing (NGS) or digital droplet polymerase chain reaction (ddPCR) to screen for somatic PIK3CA mutations on DNA extracted from resected lesional tissue or lymphatic endothelial cells (LECs) isolated from lesions. Our cohort (n = 143) was composed of unrelated patients suffering from a common lymphatic malformation (LM), a combined lymphatic malformation [lymphatico-venous malformation (LVM), capillaro-lymphatic malformation (CLM), capillaro-lymphatico-venous malformation (CLVM)], or a syndrome [CLVM with hypertrophy (Klippel-Trenaunay-Weber syndrome, KTS), congenital lipomatous overgrowth-vascular malformations-epidermal nevi -syndrome (CLOVES), unclassified PIK3CA-related overgrowth syndrome (PROS) or unclassified vascular (lymphatic) anomaly syndrome (UVA)]. RESULTS: We identified a somatic PIK3CA mutation in resected lesions of 108 out of 143 patients (75.5%). The frequency of the variant allele ranged from 0.54 to 25.33% in tissues, and up to 47% in isolated endothelial cells. We detected a statistically significant difference in the distribution of mutations between patients with common and combined LM compared to the syndromes, but not with KTS. Moreover, the variant allele frequency was higher in the syndromes. CONCLUSIONS: Most patients with an common or combined lymphatic malformation with or without overgrowth harbour a somatic PIK3CA mutation. However, in about a quarter of patients, no such mutation was detected, suggesting the existence of (an)other cause(s). We detected a hotspot mutation more frequently in common and combined LMs compared to syndromic cases (CLOVES and PROS). Diagnostic genotyping should thus not be limited to PIK3CA hotspot mutations. Moreover, the higher mutant allele frequency in syndromes suggests a wider distribution in patients' tissues, facilitating detection. Clinical trials have demonstrated efficacy of Sirolimus and Alpelisib in treating patients with an LM or PROS. Genotyping might lead to an increase in efficacy, as treatments could be more targeted, and responses could vary depending on presence and type of PIK3CA-mutation.

Hypotrichosis-lymphedema-telangiectasia syndrome: Report of ileal atresia associated with a SOX18 de novo pathogenic variant and review of the phenotypic spectrum.

Hypotrichosis-lymphedema-telangiectasia syndrome (HLTS) is a rare condition caused by pathogenic variants in the SOX18 gene. SOX18 plays a key role in angio- and lymphangiogenesis due to its expression in venous endothelial cells from which the lymphatic system develops. It is also expressed in embryonic hair follicles, heart, and vascular smooth muscle cells. The main clinical symptoms of HLTS include sparse hair, alopecia totalis, lymphedema, most often affecting lower limbs, and telangiectatic lesions. Only 10 patients with a SOX18 pathogenic variant have been described that presented with additional features such as hydrocele, renal failure, arterial or pulmonary hypertension, aortic dilatation, and facial dysmorphism. Here, we summarize these phenotypic variations and report an additional HLTS patient, with a 14-nucleotide de novo duplication in SOX18 and congenital ileal atresia, a feature not previously associated with HLTS.

Comparison of the mutation spectrum and association with pre and post treatment lipid measures of children with heterozygous familial hypercholesterolaemia (FH) from eight European countries.

BACKGROUND AND AIMS: Familial hypercholesterolaemia (FH) is commonly caused by mutations in the LDLR, APOB or PCSK9 genes, with untreated mean low density lipoprotein-cholesterol (LDL-C) concentrations being elevated in APOB mutation carriers, even higher in LDLR mutation and highest in those with a PCSK9 mutation. Here we examine this in children with FH from Norway, UK, The Netherlands, Belgium, Czech Republic, Austria, Portugal and Greece. METHODS: Differences in characteristics and pre- and post-treatment lipid concentrations in those with different molecular causes were compared by standard statistical tests. RESULTS: Data were obtained from 2866 children, of whom 2531 (88%) carried a reported LDLR/APOB/PCSK9 variant. In all countries, the most common cause of FH was an LDLR mutation (79% of children, 297 different), but the prevalence of the APOB p.(Arg3527Gln) mutation varied significantly (ranging from 0% in Greece to 39% in Czech Republic, p < 2.2 × 10-16). The prevalence of a family history of premature CHD was significantly higher in children with an LDLR vs APOB mutation (16% vs 7% p=0.0005). Compared to the LDLR mutation group, mean (±SD) concentrations of pre-treatment LDL-C were significantly lower in those with an APOB mutation (n = 2260 vs n = 264, 4.96 (1.08)mmol/l vs 5.88 (1.41)mmol/l, p < 2.2 × 10-16) and lowest in those with a PCSK9 mutation (n = 7, 4.71 (1.22)mmol/l). CONCLUSIONS: The most common cause of FH in children from eight European countries was an LDLR mutation, with the prevalence of the APOB p.(Arg3527Gln) mutation varying significantly across countries. In children, LDLR-FH is associated with higher concentrations of LDL-C and family history of CHD compared to those with APOB-FH.

Characterization of ANGPT2 mutations associated with primary lymphedema.

Primary lymphedema is caused by developmental and functional defects of the lymphatic vascular system that result in accumulation of protein-rich fluid in tissues, resulting in edema. The 28 currently known genes causing primary lymphedema can explain <30% of cases. Angiopoietin 1 (ANGPT1) and ANGPT2 function via the TIE1-TIE2 (tyrosine kinase with immunoglobulin-like and epidermal growth factor-like domains 1 and 2) receptor complex and α5ß1 integrin to form an endothelial cell signaling pathway that is critical for blood and lymphatic vessel formation and remodeling during embryonic development, as well as for homeostasis of the mature vasculature. By screening a cohort of 543 individuals affected by primary lymphedema, we identified one heterozygous de novo ANGPT2 whole-gene deletion and four heterozygous ANGPT2 missense mutations. Functional analyses revealed three missense mutations that resulted in decreased ANGPT2 secretion and inhibited the secretion of wild-type (WT)-ANGPT2, suggesting that they have a dominant-negative effect on ANGPT2 signaling. WT-ANGPT2 and soluble mutants T299M and N304K activated TIE1 and TIE2 in an autocrine assay in human lymphatic endothelial cells. Molecular modeling and biophysical studies showed that amino-terminally truncated ANGPT subunits formed asymmetrical homodimers that bound TIE2 in a 2:1 ratio. The T299M mutant, located in the dimerization interphase, showed reduced integrin α5 binding, and its expression in mouse skin promoted hyperplasia and dilation of cutaneous lymphatic vessels. These results demonstrate that primary lymphedema can be associated with ANGPT2 mutations and provide insights into TIE1 and TIE2 activation mechanisms.

RASA1 mosaic mutations in patients with capillary malformation-arteriovenous malformation.

BACKGROUND: Capillary 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 RASA1 or EPHB4 genes. 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. METHODS: DNA was extracted from peripheral blood lymphocytes, saliva or vascular malformation tissues from four patients. RASA1 and EPHB4 coding 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. RESULTS: Four distinct mosaic RASA1 mutations, 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. CONCLUSION: This study shows that RASA1 mosaic 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.

No evidence of locus heterogeneity in familial microcephaly with or without chorioretinopathy, lymphedema, or mental retardation syndrome.

BACKGROUND: Microcephaly with or without chorioretinopathy, lymphedema, or mental retardation syndrome (MCLMR) is a rare autosomal dominant disorder with variable expressivity. It is characterized by mild-to-severe microcephaly, often associated with intellectual disability, ocular defects and lymphedema. It can be sporadic or inherited. Eighty-seven patients have been described to carry a mutation in KIF11, which encodes a homotetrameric motor kinesin, EG5. METHODS: We tested 23 unreported MCLMR index patients for KIF11. We also reviewed the clinical phenotypes of all our patients as well as of those described in previously published studies. RESULTS: We identified 14 mutations, 12 of which are novel. We detected mutations in 12 affected individuals, from 6 out of 6 familial cases, and in 8 out of 17 sporadic patients. Phenotypic evaluation of patients (our 26 + 61 earlier published = 87) revealed microcephaly in 91%, eye anomalies in 72%, intellectual disability in 67% and lymphedema in 47% of the patients. Unaffected carriers were rare (4 out of 87: 5%). Family history is not a requisite for diagnosis; 31% (16 out of 52) were de novo cases. CONCLUSIONS: All inherited cases, and 50% of sporadic cases of MCLMR are due to germline KIF11 mutations. It is possible that mosaic KIF11 mutations cause the remainder of sporadic cases, which the methods employed here were not designed to detect. On the other hand, some of them might have another mimicking disorder and genetic defect, as microcephaly is highly heterogeneous. In aggregate, KIF11 mutations likely cause the majority, if not all, of MCLMR.