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.

Magnetic resonance lymphangiography: Establishing normal.

BACKGROUND: Despite an increased interest in visualizing the lymphatic vessels with magnetic resonance lymphangiography (MRL), little literature is available describing their appearance in nonlymphedematous individuals. To determine lymphatic abnormalities, an understanding of how healthy lymphatic vessels appear and behave needs to be established. Therefore, in this study, MRL of individuals without a history of lymphatic disease was performed. METHODS: A total of 25 individuals (15 women) underwent MRL of their lower limbs using a 3.0 T Philips magnetic resonance imaging scanner (Philips Medical Systems). The first nine participants were recruited to establish the concentration of gadolinium-based contrast agent (GBCA) to administer, with the remainder imaged before and after interdigital forefoot GBCA injections at the optimized dose. Outcomes, including lymphatic vessel diameter, tortuosity, and frequency of drainage via particular drainage routes, were recorded. RESULTS: Healthy lymphatic vessels following the anteromedial pathway were routinely observed in post-contrast T1-weighted images (average tortuosity, 1.09 ± 0.03), with an average of 2.16 ± 0.93 lymphatic vessels with a diameter of 2.47 ± 0.50 mm crossing the anterior ankle. In six limbs, vessels following the anterolateral pathways were observed. No vessels traversing the posterior of the legs were seen. In a subset of 10 vessels, the lymphatic signal, measured at the ankle, peaked 29 minutes, 50 seconds ± 9 minutes, 29 seconds after GBCA administration. No lymphatic vessels were observed in T2-weighted images. CONCLUSIONS: Contrast-enhanced MRL reliably depicts the lymphatic vessels in the legs of healthy controls. Following interdigital contrast injection, anteromedial drainage appears dominant. Quantitative measures related to lymphatic vessel size, tortuosity, and drainage rate are readily obtainable and could be beneficial for detecting even subtle lymphatic impairment.

Profound and selective lymphopaenia in primary lymphatic anomaly patients demonstrates the significance of lymphatic-lymphocyte interactions.

Introduction: The lymphatic system has a pivotal role in immune homeostasis. To better understand this, we investigated the impact of Primary Lymphatic Anomalies (PLA) on lymphocyte numbers and phenotype. Methods: The study comprised (i) a retrospective cohort: 177 PLA subjects from the National Primary Lymphatic Anomaly Register with clinical and laboratory data, and (ii) a prospective cohort: 28 patients with PLA and 20 healthy controls. Patients were subdivided using established phenotypic diagnostic categories and grouped into simplex (localised tissue involvement only) and systemic (involvement of central lymphatics). Further grouping variables included genital involvement and the likelihood of co-existent intestinal lymphangiectasia. Haematology laboratory parameters were analysed in both cohorts. In the prospective cohort, prospective blood samples were analysed by flow cytometry for markers of proliferation, differentiation, activation, skin-homing, and for regulatory (CD4+Foxp3+) T cells (Treg). Results: In patients with PLA, lymphopaenia was frequent (22% of subjects), affected primarily the CD4+ T cell subset, and was more severe in subjects with systemic versus simplex patterns of disease (36% vs 9% for lymphopaenia; 70% vs 33% for CD4+ cells). B cells, NK cells and monocytes were better conserved (except in GATA2 deficiency characterised by monocytopaenia). Genital oedema and likelihood of concomitant intestinal lymphangiectasia independently predicted CD4+ T cell depletion. Analysing CD4+ and CD8+ T cells by differentiation markers revealed disproportionate depletion of naïve cells, with a skewing towards a more differentiated effector profile. Systemic PLA conditions were associated with: increased expression of Ki67, indicative of recent cell division, in naïve CD4+, but not CD8+ T cells; increased levels of activation in CD4+, but not CD8+ T cells; and an increased proportion of Treg. Skin-homing marker (CCR10, CLA and CCR4) expression was reduced in some patients with simplex phenotypes. Discussion: Patients with PLA who have dysfunctional lymphatics have a selective reduction in circulating lymphocytes which preferentially depletes naïve CD4+ T cells. The presence of systemic disease, genital oedema, and intestinal lymphangiectasia independently predict CD4 lymphopaenia. The association of this depletion with immune activation and increased circulating Tregs suggests lymphatic-lymphocyte interactions and local inflammatory changes are pivotal in driving immunopathology.

Erythematous capillary-lymphatic malformations mimicking blood vascular anomalies.

Superficial erythematous cutaneous vascular malformations are assumed to be blood vascular in origin, but cutaneous lymphatic malformations can contain blood and appear red. Management may be different and so an accurate diagnosis is important. Cutaneous malformations were investigated through 2D histology and 3D whole-mount histology. Two lesions were clinically considered as port-wine birthmarks and another 3 lesions as erythematous telangiectasias. The aims were (i) to demonstrate that cutaneous erythematous malformations including telangiectasia can represent a lymphatic phenotype, (ii) to determine if lesions represent expanded but otherwise normal or malformed lymphatics, and (iii) to determine if the presence of erythrocytes explained the red color. Microscopy revealed all lesions as lymphatic structures. Port-wine birthmarks proved to be cystic lesions, with nonuniform lymphatic marker expression and a disconnected lymphatic network suggesting a lymphatic malformation. Erythematous telangiectasias represented expanded but nonmalformed lymphatics. Blood within lymphatics appeared to explain the color. Blood-lymphatic shunts could be detected in the erythematous telangiectasia. In conclusion, erythematous cutaneous capillary lesions may be lymphatic in origin but clinically indistinguishable from blood vascular malformations. Biopsy is advised for correct phenotyping and management. Erythrocytes are the likely explanation for color accessing lymphatics through lympho-venous shunts.

Genetic association analysis of 77,539 genomes reveals rare disease etiologies.

The genetic etiologies of more than half of rare diseases remain unknown. Standardized genome sequencing and phenotyping of large patient cohorts provide an opportunity for discovering the unknown etiologies, but this depends on efficient and powerful analytical methods. We built a compact database, the 'Rareservoir', containing the rare variant genotypes and phenotypes of 77,539 participants sequenced by the 100,000 Genomes Project. We then used the Bayesian genetic association method BeviMed to infer associations between genes and each of 269 rare disease classes assigned by clinicians to the participants. We identified 241 known and 19 previously unidentified associations. We validated associations with ERG, PMEPA1 and GPR156 by searching for pedigrees in other cohorts and using bioinformatic and experimental approaches. We provide evidence that (1) loss-of-function variants in the Erythroblast Transformation Specific (ETS)-family transcription factor encoding gene ERG lead to primary lymphoedema, (2) truncating variants in the last exon of transforming growth factor-ß regulator PMEPA1 result in Loeys-Dietz syndrome and (3) loss-of-function variants in GPR156 give rise to recessive congenital hearing impairment. The Rareservoir provides a lightweight, flexible and portable system for synthesizing the genetic and phenotypic data required to study rare disease cohorts with tens of thousands of participants.

Genome-wide association study of a lipedema phenotype among women in the UK Biobank identifies multiple genetic risk factors.

Lipedema is a common disorder characterized by excessive deposition of subcutaneous adipose tissue (SAT) in the legs, hips, and buttocks, mainly occurring in adult women. Although it appears to be heritable, no specific genes have yet been identified. To identify potential genetic risk factors for lipedema, we used bioelectrical impedance analysis and anthropometric data from the UK Biobank to identify women with and without a lipedema phenotype. Specifically, we identified women with both a high percentage of fat in the lower limbs and a relatively small waist, adjusting for hip circumference. We performed a genome-wide association study (GWAS) for this phenotype, and performed multiple sensitivity GWAS. In an independent case/control study of lipedema based on strict clinical criteria, we attempted to replicate our top hits. We identified 18 significant loci (p < 5 × 10-9), several of which have previously been identified in GWAS of waist-to-hip ratio with larger effects in women. Two loci (VEGFA and GRB14-COBLL1) were significantly associated with lipedema in the independent replication study. Follow-up analyses suggest an enrichment of genes expressed in blood vessels and adipose tissue, among other tissues. Our findings provide a starting point towards better understanding the genetic and physiological basis of lipedema.

Redefining WILD syndrome: a primary lymphatic dysplasia with congenital multisegmental lymphoedema, cutaneous lymphovascular malformation, CD4 lymphopaenia and warts.

BACKGROUND: Primary lymphoedema (PL) syndromes are increasingly recognised as presentations of complex genetic disease, with at least 20 identified causative genes. Recognition of clinical patterns is key to diagnosis, research and therapeutics. The defining criteria for one such clinical syndrome, 'WILD syndrome' (Warts, Immunodeficiency, Lymphoedema and anogenital Dysplasia), have previously depended on a single case report. METHODS AND RESULTS: We present 21 patients (including the first described case) with similar clinical and immunological phenotypes. All had PL affecting multiple segments, with systemic involvement (intestinal lymphangiectasia/pleural or pericardial effusions) in 70% (n=14/20). Most (n=20, 95%) had a distinctive cutaneous lymphovascular malformation on the upper anterior chest wall. Some (n=10, 48%) also had hyperpigmented lesions resembling epidermal naevi (but probably lymphatic in origin). Warts were common (n=17, 81%) and often refractory. In contrast to the previous case report, anogenital dysplasia was uncommon-only found in two further cases (total n=3, 14%). Low CD4 counts and CD4:CD8 ratios typified the syndrome (17 of 19, 89%), but monocyte counts were universally normal, unlike GATA2 deficiency. CONCLUSION: WILD syndrome is a previously unrecognised, underdiagnosed generalised PL syndrome. Based on this case series, we redefine WILD as 'Warts, Immunodeficiency, andLymphatic Dysplasia' and suggest specific diagnostic criteria. The essential criterion is congenital multisegmental PL in a 'mosaic' distribution. The major diagnostic features are recurrent warts, cutaneous lymphovascular malformations, systemic involvement (lymphatic dysplasia), genital swelling and CD4 lymphopaenia with normal monocyte counts. The absence of family history suggests a sporadic condition, and the random distribution of swelling implicates mosaic postzygotic mutation as the cause.

Image registration and subtraction in dynamic magnetic resonance lymphangiography (MRL) of the legs.

Dynamic contrast-enhanced magnetic resonance lymphangiography (DCE-MRL) is regularly reported as unable to depict lymphatic vessels in healthy limbs. In this study, we aim to improve lymph vessel conspicuity with appropriate registration and subtraction of a reference baseline image. Five unaffected individuals and a single unilateral primary lymphoedema patient were recruited to undergo fat suppressed 3D T 1 weighted spoiled gradient echo imaging of the lower limbs at 3.0 T. Images were quality assessed by two physicians and a medical physicist following registration via one of six registration pipelines, and subtraction of the first post-contrast dynamic image (PC1). Wilcoxon non-parametric testing was performed to compare image quality ranking vs the unregistered images and inter-rater reliability estimated using intraclass correlation coefficient. Signal enhancement curves were also computed in lymphatic vessels for two participants. Subtraction images were considered to improve lymphatic visibility, and three registration pipelines significantly (p < 0.05) outranked those without registration. Those registered to PC1 with an affine and elastic approach were rated best quality (p = 0.006). Moderate inter-rater reliability was observed (intraclass correlation coefficient = 0.71) and signal enhancement behaviour appears affected by registration when motion is evident across the DCE-MRL series. We therefore conclude that lymphatic vessel visibility in DCE-MRL images can be improved with registration and baseline subtraction.

Investigation of clinical characteristics and genome associations in the 'UK Lipoedema' cohort.

Lipoedema is a chronic adipose tissue disorder mainly affecting women, causing excess subcutaneous fat deposition on the lower limbs with pain and tenderness. There is often a family history of lipoedema, suggesting a genetic origin, but the contribution of genetics is currently unclear. A tightly phenotyped cohort of 200 lipoedema patients was recruited from two UK specialist clinics. Objective clinical characteristics and measures of quality of life data were obtained. In an attempt to understand the genetic architecture of the disease better, genome-wide single nucleotide polymorphism (SNP) genotype data were obtained, and a genome wide association study (GWAS) was performed on 130 of the recruits. The analysis revealed genetic loci suggestively associated with the lipoedema phenotype, with further support provided by an independent cohort taken from the 100,000 Genomes Project. The top SNP rs1409440 (ORmeta ≈ 2.01, Pmeta ≈ 4 x 10-6) is located upstream of LHFPL6, which is thought to be involved with lipoma formation. Exactly how this relates to lipoedema is not yet understood. This first GWAS of a UK lipoedema cohort has identified genetic regions of suggestive association with the disease. Further replication of these findings in different populations is warranted.

Mutations in EPHB4 cause human venous valve aplasia.

Venous valve (VV) failure causes chronic venous insufficiency, but the molecular regulation of valve development is poorly understood. A primary lymphatic anomaly, caused by mutations in the receptor tyrosine kinase EPHB4, was recently described, with these patients also presenting with venous insufficiency. Whether the venous anomalies are the result of an effect on VVs is not known. VV formation requires complex "organization" of valve-forming endothelial cells, including their reorientation perpendicular to the direction of blood flow. Using quantitative ultrasound, we identified substantial VV aplasia and deep venous reflux in patients with mutations in EPHB4. We used a GFP reporter in mice to study expression of its ligand, ephrinB2, and analyzed developmental phenotypes after conditional deletion of floxed Ephb4 and Efnb2 alleles. EphB4 and ephrinB2 expression patterns were dynamically regulated around organizing valve-forming cells. Efnb2 deletion disrupted the normal endothelial expression patterns of the gap junction proteins connexin37 and connexin43 (both required for normal valve development) around reorientating valve-forming cells and produced deficient valve-forming cell elongation, reorientation, polarity, and proliferation. Ephb4 was also required for valve-forming cell organization and subsequent growth of the valve leaflets. These results uncover a potentially novel cause of primary human VV aplasia.

Janus-faced EPHB4-associated disorders: novel pathogenic variants and unreported intrafamilial overlapping phenotypes.

PURPOSE: Several clinical phenotypes including fetal hydrops, central conducting lymphatic anomaly or capillary malformations with arteriovenous malformations 2 (CM-AVM2) have been associated with EPHB4 (Ephrin type B receptor 4) variants, demanding new approaches for deciphering pathogenesis of novel variants of uncertain significance (VUS) identified in EPHB4, and for the identification of differentiated disease mechanisms at the molecular level. METHODS: Ten index cases with various phenotypes, either fetal hydrops, CM-AVM2, or peripheral lower limb lymphedema, whose distinct clinical phenotypes are described in detail in this study, presented with a variant in EPHB4. In vitro functional studies were performed to confirm pathogenicity. RESULTS: Pathogenicity was demonstrated for six of the seven novel EPHB4 VUS investigated. A heterogeneity of molecular disease mechanisms was identified, from loss of protein production or aberrant subcellular localization to total reduction of the phosphorylation capability of the receptor. There was some phenotype-genotype correlation; however, previously unreported intrafamilial overlapping phenotypes such as lymphatic-related fetal hydrops (LRFH) and CM-AVM2 in the same family were observed. CONCLUSION: This study highlights the usefulness of protein expression and subcellular localization studies to predict EPHB4 variant pathogenesis. Our accurate clinical phenotyping expands our interpretation of the Janus-faced spectrum of EPHB4-related disorders, introducing the discovery of cases with overlapping phenotypes.

Systematic Review of Magnetic Resonance Lymphangiography From a Technical Perspective.

BACKGROUND: Clinical examination and lymphoscintigraphy are the current standard for investigating lymphatic function. Magnetic resonance imaging (MRI) facilitates three-dimensional (3D), nonionizing imaging of the lymphatic vasculature, including functional assessments of lymphatic flow, and may improve diagnosis and treatment planning in disease states such as lymphedema. PURPOSE: To summarize the role of MRI as a noninvasive technique to assess lymphatic drainage and highlight areas in need of further study. STUDY TYPE: Systematic review. POPULATION: In October 2019, a systematic literature search (PubMed) was performed to identify articles on magnetic resonance lymphangiography (MRL). FIELD STRENGTH/SEQUENCE: No field strength or sequence restrictions. ASSESSMENT: Article quality assessment was conducted using a bespoke protocol, designed with heavy reliance on the National Institutes of Health quality assessment tool for case series studies and Downs and Blacks quality checklist for health care intervention studies. STATISTICAL TESTS: The results of the original research articles are summarized. RESULTS: From 612 identified articles, 43 articles were included and their protocols and results summarized. Field strength was 1.5 or 3.0 T in all studies, with 25/43 (58%) employing 3.0 T imaging. Most commonly, imaging of the peripheries, upper and lower limbs including the pelvis (32/43, 74%), and the trunk (10/43, 23%) is performed, including two studies covering both regions. Imaging protocols were heterogenous; however, T2 -weighted and contrast-enhanced T1 -weighted images are routinely acquired and demonstrate the lymphatic vasculature. Edema, vessel, quantity and morphology, and contrast uptake characteristics are commonly reported indicators of lymphatic dysfunction. DATA CONCLUSION: MRL is uniquely placed to yield large field of view, qualitative and quantitative, 3D imaging of the lymphatic vasculature. Despite study heterogeneity, consensus is emerging regarding MRL protocol design. MRL has the potential to dramatically improve understanding of the lymphatics and detect disease, but further optimization, and research into the influence of study protocol differences, is required before this is fully realized. LEVEL OF EVIDENCE: 2 TECHNICAL EFFICACY: Stage 2.

The St George's Classification Algorithm of Primary Lymphatic Anomalies.

Clinicians and scientists at St George's University Hospital have collaborated to develop a classification algorithm for primary lymphatic anomalies. Instruction is offered on how to apply the algorithm in clinical practice to refine the diagnosis of primary lymphedema and guide on genetic testing and management. It can also be used to interpret mutation testing results of uncertain significance. The algorithm has evolved as more genes have been discovered, and it remains a "work in progress" as further discoveries are made. This transformational approach has revolutionized the understanding and classification of primary lymphatic anomalies.

Development and physiological functions of the lymphatic system: insights from human genetic studies of primary lymphedema.

Primary lymphedema is a long-term (chronic) condition characterized by tissue lymph retention and swelling that can affect any part of the body, although it usually develops in the arms or legs. Due to the relevant contribution of the lymphatic system to human physiology, while this review mainly focuses on the clinical and physiological aspects related to the regulation of fluid homeostasis and edema, clinicians need to know that the impact of lymphatic dysfunction with a genetic origin can be wide ranging. Lymphatic dysfunction can affect immune function so leading to infection; it can influence cancer development and spread, and it can determine fat transport so impacting on nutrition and obesity. Genetic studies and the development of imaging techniques for the assessment of lymphatic function have enabled the recognition of primary lymphedema as a heterogenic condition in terms of genetic causes and disease mechanisms. In this review, the known biological functions of several genes crucial to the development and function of the lymphatic system are used as a basis for understanding normal lymphatic biology. The disease conditions originating from mutations in these genes are discussed together with a detailed clinical description of the phenotype and the up-to-date knowledge in terms of disease mechanisms acquired from in vitro and in vivo research models.

The physiological and pathological functions of VEGFR3 in cardiac and lymphatic development and related diseases.

Vascular endothelial growth factor receptors (VEGFRs) are part of the evolutionarily conserved VEGF signalling pathways that regulate the development and maintenance of the body's cardiovascular and lymphovascular systems. VEGFR3, encoded by the FLT4 gene, has an indispensable and well-characterized function in development and establishment of the lymphatic system. Autosomal dominant VEGFR3 mutations, that prevent the receptor functioning as a homodimer, cause one of the major forms of hereditary primary lymphoedema; Milroy disease. Recently, we and others have shown that FLT4 variants, distinct to those observed in Milroy disease cases, predispose individuals to Tetralogy of Fallot, the most common cyanotic congenital heart disease, demonstrating a novel function for VEGFR3 in early cardiac development. Here, we examine the familiar and emerging roles of VEGFR3 in the development of both lymphovascular and cardiovascular systems, respectively, compare how distinct genetic variants in FLT4 lead to two disparate human conditions, and highlight the research still required to fully understand this multifaceted receptor.

Update and audit of the St George's classification algorithm of primary lymphatic anomalies: a clinical and molecular approach to diagnosis.

Primary lymphatic anomalies may present in a myriad of ways and are highly heterogenous. Careful consideration of the presentation can lead to an accurate clinical and/or molecular diagnosis which will assist with management. The most common presentation is lymphoedema, swelling resulting from failure of the peripheral lymphatic system. However, there may be internal lymphatic dysfunction, for example, chylous reflux, or lymphatic malformations, including the thorax or abdomen. A number of causal germline or postzygotic gene mutations have been discovered. Some through careful phenotyping and categorisation of the patients based on the St George's classification pathway/algorithm. The St George's classification algorithm is aimed at providing an accurate diagnosis for patients with lymphoedema based on age of onset, areas affected by swelling and associated clinical features. This has enabled the identification of new causative genes. This update brings the classification of primary lymphatic disorders in line with the International Society for the Study of Vascular Anomalies 2018 classification for vascular anomalies. The St George's algorithm considers combined vascular malformations and primary lymphatic anomalies. It divides the types of primary lymphatic anomalies into lymphatic malformations and primary lymphoedema. It further divides the primary lymphoedema into syndromic, generalised lymphatic dysplasia with internal/systemic involvement, congenital-onset lymphoedema and late-onset lymphoedema. An audit and update of the algorithm has revealed where new genes have been discovered and that a molecular diagnosis was possible in 26% of all patients overall and 41% of those tested.

Mutations in SPATA13/ASEF2 cause primary angle closure glaucoma.

Current estimates suggest 50% of glaucoma blindness worldwide is caused by primary angle-closure glaucoma (PACG) but the causative gene is not known. We used genetic linkage and whole genome sequencing to identify Spermatogenesis Associated Protein 13, SPATA13 (NM_001166271; NP_001159743, SPATA13 isoform I), also known as ASEF2 (Adenomatous polyposis coli-stimulated guanine nucleotide exchange factor 2), as the causal gene for PACG in a large seven-generation white British family showing variable expression and incomplete penetrance. The 9 bp deletion, c.1432_1440del; p.478_480del was present in all affected individuals with angle-closure disease. We show ubiquitous expression of this transcript in cell lines derived from human tissues and in iris, retina, retinal pigment and ciliary epithelia, cornea and lens. We also identified eight additional mutations in SPATA13 in a cohort of 189 unrelated PACS/PAC/PACG samples. This gene encodes a 1277 residue protein which localises to the nucleus with partial co-localisation with nuclear speckles. In cells undergoing mitosis SPATA13 isoform I becomes part of the kinetochore complex co-localising with two kinetochore markers, polo like kinase 1 (PLK-1) and centrosome-associated protein E (CENP-E). The 9 bp deletion reported in this study increases the RAC1-dependent guanine nucleotide exchange factors (GEF) activity. The increase in GEF activity was also observed in three other variants identified in this study. Taken together, our data suggest that SPATA13 is involved in the regulation of mitosis and the mutations dysregulate GEF activity affecting homeostasis in tissues where it is highly expressed, influencing PACG pathogenesis.

Lymphoscintigraphic Abnormalities Associated with Milroy Disease and Lymphedema-Distichiasis Syndrome.

Background: Primary lymphedema is genetically heterogeneous. Two of the most common forms of primary lymphedema are Milroy disease (MD) and lymphedema-distichiasis syndrome (LDS). This study aims to look further into the pathogenesis of the two conditions by analyzing the lymphoscintigram images from affected individuals to ascertain if it is a useful diagnostic tool. Methods and Results: The lymphoscintigrams of patients with MD and LDS were analyzed, comparing the images and transport parameters of the two genotypes against a control population. Lymphoscintigrams were available for 12 MD and 16 LDS patients (all genetically proven diagnoses). Eight of the 12 (67%) lymph scans performed on patients with MD demonstrated little or no uptake from the initial lymphatics and poor visualization of the inguinal lymph nodes. These changes were consistent with a "functional aplasia," that is, the lymphatic vessels were present but appeared to be ineffective in absorbing the interstitial fluid into the lymphatic system. In patients with LDS the lymphoscintigraphic appearances were different. In 12 of the 16 scans (75%), the lymph scans were highly suggestive of lymphatic collector reflux. Quantification revealed a significantly reduced uptake of tracer within the inguinal lymph nodes and a higher residual activity in the feet at 2 hours in MD compared with LDS and compared with controls. Conclusion: Lymphoscintigraphic imaging and quantification can be characteristic in specific genetic forms of primary lymphedema and may be useful as an additional tool for in-depth phenotyping, leading to a more accurate diagnosis and providing insight into the underlying mechanism of disease.

Clinical and Molecular Characterization of Familial Exudative Vitreoretinopathy Associated With Microcephaly.

PURPOSE: Familial exudative vitreoretinopathy (FEVR) is a rare finding in patients with genetic forms of microcephaly. This study documents the detailed phenotype and expands the range of genetic heterogeneity. DESIGN: Retrospective case series. METHODS: Twelve patients (10 families) with a diagnosis of FEVR and microcephaly were ascertained from pediatric genetic eye clinics and underwent full clinical assessment including retinal imaging. Molecular investigations included candidate gene Sanger sequencing, whole-exome sequencing (WES), and whole-genome sequencing (WGS). RESULTS: All patients had reduced vision and nystagmus. Six were legally blind. Two probands carried bi-allelic LRP5 variants, both presenting with bilateral retinal folds. A novel homozygous splice variant, and 2 missense variants were identified. Subsequent bone density measurement identified osteoporosis in one proband. Four families had heterozygous KIF11 variants. Two probands had a retinal fold in one eye and chorioretinal atrophy in the other; the other 2 had bilateral retinal folds. Four heterozygous variants were found, including 2 large deletions not identified on Sanger sequencing or WES. Finally, a family of 2 children with learning difficulties, abnormal peripheral retinal vasculogenesis, and rod-cone dystrophy were investigated. They were found to have bi-allelic splicing variants in TUBGCP6. Three families remain unsolved following WES and WGS. CONCLUSIONS: Molecular diagnosis has been achieved in 7 of 10 families investigated, including a previously unrecognized association with LRP5. WGS enabled molecular diagnosis in 3 families after prior negative Sanger sequencing of the causative gene. This has enabled patient-specific care with targeted investigations and accurate family counseling.