Fiona Connell

Mutations in **KIF11** cause autosomal-dominant microcephaly variably associated with congenital lymphedema and chorioretinopathy

We have identified KIF11 mutations in individuals with syndromic autosomal-dominant microcephaly associated with lymphedema and/or chorioretinopathy. Initial whole-exome sequencing revealed heterozygous KIF11 mutations in three individuals with a combination of microcephaly and lymphedema from a microcephaly-lymphedema-chorioretinal-dysplasia cohort. Subsequent Sanger sequencing of KIF11 in a further 15 unrelated microcephalic probands with lymphedema and/or chorioretinopathy identified additional heterozygous mutations in 12 of them. KIF11 encodes EG5, a homotetramer kinesin motor. The variety of mutations we have found (two nonsense, two splice site, four missense, and six indels causing frameshifts) are all predicted to have an impact on protein function. EG5 has previously been shown to play a role in spindle assembly and function, and these findings highlight the critical role of proteins necessary for spindle formation in CNS development. Moreover, identification of KIF11 mutations in patients with chorioretinopathy and lymphedema suggests that EG5 is involved in the development and maintenance of retinal and lymphatic structures.

Mutation in Vascular Endothelial Growth Factor-C, a Ligand for Vascular Endothelial Growth Factor Receptor-3, Is Associated With Autosomal Dominant Milroy-Like Primary Lymphedema

Rationale: Mutations in vascular endothelial growth factor (VEGF) receptor-3 (VEGFR3 or FLT4) cause Milroy disease, an autosomal dominant condition that presents with congenital lymphedema. Mutations in VEGFR3 are identified in only 70% of patients with classic Milroy disease, suggesting genetic heterogeneity. Objective: To investigate the underlying cause in patients with clinical signs resembling Milroy disease in whom sequencing of the coding region of VEGFR3 did not reveal any pathogenic variation. Methods and Results: Exome sequencing of 5 such patients was performed, and a novel frameshift variant, c.571_572insTT in VEGFC, a ligand for VEGFR3, was identified in 1 proband. The variant cosegregated with the affected status in the family. An assay to assess the biological function of VEGFC activity in vivo, by expressing human VEGFC in the zebrafish floorplate was established. Forced expression of wild-type human VEGFC in the floorplate of zebrafish embryos leads to excessive sprouting in neighboring vessels. However, when overexpressing the human c.571_572insTT variant in the floorplate, no sprouting of vessels was observed, indicating that the base changes have a marked effect on the activity of VEGFC. Conclusions: We propose that the mutation in VEGFC is causative for the Milroy disease-like phenotype seen in this family. This is the first time a mutation in one of the ligands of VEGFR3 has been reported to cause primary lymphedema.Rationale: Mutations in VEGFR3 (FLT4) cause Milroy Disease (MD), an autosomal dominant condition that presents with congenital lymphedema. Mutations in VEGFR3 are identified in only 70% of patients with classic MD, suggesting genetic heterogeneity. Objective: To investigate the underlying cause in patients with clinical signs resembling MD in whom sequencing of the coding region of VEGFR3 did not reveal any pathogenic variation. Methods and Results: Exome sequencing of five such patients was performed and a novel frameshift variant, c.571\_572insTT in VEGFC , a ligand for VEGFR3, was identified in one proband. The variant co-segregated with the affected status in the family. An assay to assess the biological function of VEGFC activity in vivo, by expressing human VEGFC in the zebrafish floorplate was established. Forced expression of wildtype human VEGFC in the floorplate of zebrafish embryos leads to excessive sprouting in neighbouring vessels. However, when overexpressing the human c.571\_572insTT variant in the floorplate, no sprouting of vessels was observed, indicating that the base changes have a marked effect on the activity of VEGFC. Conclusions: We propose that the mutation in VEGFC is causative for the MD-like phenotype seen in this family. This is the first time a mutation in one of the ligands of VEGFR3 has been reported to cause primary lymphedema.

Rapid identification of mutations in GJC2 in primary lymphoedema using whole exome sequencing combined with linkage analysis with delineation of the phenotype

Background Primary lymphoedema describes a chronic, frequently progressive, failure of lymphatic drainage. This disorder is frequently genetic in origin, and a multigenerational family in which eight individuals developed postnatal lymphoedema of all four limbs was ascertained from the joint Lymphoedema/Genetic clinic at St Georges Hospital. Methods Linkage analysis was used to determine a locus, and exome sequencing was employed to look for causative variants. Results Linkage analysis revealed cosegregation of a 16.1 Mb haplotype on chromosome 1q42 that contained 173 known or predicted genes. Whole exome sequencing in a single affected individual was undertaken, and the search for the causative variant was focused to within the linkage interval. This approach revealed two novel non-synonymous single nucleotide substitutions within the chromosome 1 locus, in NVL and GJC2. NVL and GJC2 were sequenced in an additional cohort of individuals with a similar phenotype and non-synonymous variants were found in GJC2 in four additional families. Conclusion This report demonstrates the power of exome sequencing efficiently applied to a traditional positional cloning pipeline in disease gene discovery, and suggests that the phenotype produced by GJC2 mutations is predominantly one of 4 limb lymphoedema.

The classification and diagnostic algorithm for primary lymphatic dysplasia: an update from 2010 to include molecular findings

Historically, primary lymphoedema was classified into just three categories depending on the age of onset of swelling; congenital, praecox and tarda. Developments in clinical phenotyping and identification of the genetic cause of some of these conditions have demonstrated that primary lymphoedema is highly heterogenous. In 2010, we introduced a new classification and diagnostic pathway as a clinical and research tool. This algorithm has been used to delineate specific primary lymphoedema phenotypes, facilitating the discovery of new causative genes. This article reviews the latest molecular findings and provides an updated version of the classification and diagnostic pathway based on this new knowledge.

Analysis of the coding regions of VEGFR3 and VEGFC in Milroy disease and other primary lymphoedemas

Milroy disease (hereditary lymphoedema type I, MIM 153100) is a congenital onset primary lymphoedema with autosomal dominant inheritance. Mutations in the gene, vascular endothelial growth factor receptor 3, VEGFR3 (FLT4), are known to cause Milroy disease, but there is uncertainty about the prevalence of VEGFR3 mutations in patients with primary lymphoedema and more specifically in those with a phenotype that resembles Milroy disease. This study aims to address this issue and thereby delineate the Milroy disease phenotype. Fifty-two patients with primary lymphoedema were analysed for mutations in the coding regions of VEGFR3. Patients were divided into four groups: Typical Milroy disease with family history (group I), typical Milroy disease with no family history (group II), atypical Milroy disease (group III), and complex primary lymphoedema (group IV). Results demonstrated that with rigorous phenotyping the likelihood of detecting VEGFR3 mutations is optimised. Mutation prevalence is 75% in typical Milroy patients with a family history (group I) and 68% if positive family history is not a diagnostic criterion. A positive family history is not essential in Milroy disease. The likelihood of detecting VEGFR3 mutations in patients who have a phenotype which is not typical of Milroy disease is very small (<5%). For the 22 mutation positive patients, 14 novel VEGFR3 mutations were identified, two of which were in exon 22 and one in exon 17, confirming that these exons should be included in VEGFR3 analysis. No mutations were found outside the kinase domains, showing that analysis of this part of the gene is not useful for Milroy disease patients. VEGFC, which encodes the ligand for VEGFR3, was sequenced in all patients with typical Milroy disease (groups I and II) and no mutations were identified.

A new classification system for primary lymphatic dysplasias based on phenotype.

Connell F, Brice G, Jeffery S, Keeley V, Mortimer P, Mansour S. A new classification system for primary lymphatic dysplasias based on phenotype.

Emberger syndrome—Primary lymphedema with myelodysplasia: Report of seven new cases

Four reports have been published on an association between acute myeloid leukaemia (AML) and primary lymphedema, with or without congenital deafness. We report seven new cases, including one extended family, confirming this entity as a genetic syndrome. The lymphedema typically presents in one or both lower limbs, before the hematological abnormalities, with onset between infancy and puberty and frequently affecting the genitalia. The AML is often preceded by pancytopenia or myelodysplasia with a high incidence of monosomy 7 in the bone marrow (five propositi and two relatives). Associated anomalies included hypotelorism, epicanthic folds, long tapering fingers and/or neck webbing (four patients), recurrent cellulitis in the affected limb (four patients), generalized warts (two patients), and congenital, high frequency sensorineural deafness (one patient). Children with lower limb and genital lymphedema should be screened for hematological abnormalities and immunodeficiency.

FLT4/VEGFR3 and Milroy disease: novel mutations, a review of published variants and database update.

Milroy disease (MD) is an autosomal dominantly inherited primary lymphedema. In 1998, the gene locus for MD was mapped to 5q35.3 and variants in the VEGFR3 (FLT4) gene, encoding vascular endothelial growth factor receptor 3 (VEGFR3), were identified as being responsible for the majority of MD cases. Several reports have since been published detailing pathogenic FLT4 mutations. To date, a total of 58 different variants in FLT4, 20 of which are unpublished, have been observed in 95 families with MD. A review of published mutations is presented in this update. Furthermore, the unpublished variants are presented including clinical data. Comparison of clinical features in patients and their families with the same mutations reveals incomplete penetrance and variable expression, making genotype–phenotype correlations difficult. Most mutations are missense, but a few deletions and one splicing variant have also been reported. Several animal models have confirmed the role of VEGFR3 in lymphangiogenesis and studies show mutant VEGFR3 receptors are not phosphorylated. Here, an MD patient with the same p.Ile1053Phe change as seen in the Chy mouse is presented for the first time. This finding confirms that this mouse lineage is an excellent model for MD. All the data reviewed here has been submitted to a database based on the Leiden Open (source) Variation Database (LOVD) and is accessible online at www.lovd.nl/flt4.

Phenotypic Characterization of Primary Lymphedema

The phenotypic entities of primary lymphedema vary in age of onset, site of edema, associated features, inheritance patterns, and underlying genetic cause. Determining the representative phenotype for different types of genetically determined primary lymphedema has been successfully achieved with Milroys disease and the lymphedema–distichiasis syndrome. Here we describe and illustrate their well‐delineated phenotypes. Phenotype characterization facilitates the identification of causative genes, as has been demonstrated with VEGFR3 and FOXC2, in Milroys disease and lymphedema–distichiasis respectively. Other forms of primary lymphedema are discussed.

Microcephaly with or without chorioretinopathy, lymphoedema, or mental retardation (MCLMR): review of phenotype associated with KIF11 mutations

Microcephaly with or without chorioretinopathy, lymphoedema, or mental retardation (MCLMR) (MIM No.152950) is a rare autosomal dominant condition for which a causative gene has recently been identified. Mutations in the kinesin family member 11 (KIF11) gene have now been described in 16 families worldwide. This is a review of the condition based on the clinical features of 37 individuals from 22 families. This report includes nine previously unreported families and additional information for some of those reported previously. The condition arose de novo in 8/20 families (40%). The parental results were not available for two probands. The mutations were varied and include missense, nonsense, frameshift, and splice site and are distributed evenly throughout the KIF11 gene. In our cohort, 86% had microcephaly, 78% had an ocular abnormality consistent with the diagnosis, 46% had lymphoedema, 73% had mild-moderate learning difficulties, 8% had epilepsy, and 8% had a cardiac anomaly. We identified three individuals with KIF11 mutations but no clinical features of MCLMR demonstrating reduced penetrance. The variable expression of the phenotype and the presence of mildly affected individuals indicates that the prevalence may be higher than expected, and we would therefore recommend a low threshold for genetic testing.