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 George's 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

Investigation of In Vivo skin stiffness anisotropy in breast cancer related lymphoedema.

There is a limited range of suitable measurement techniques for detecting and assessing breast cancer related lymphoedema (BCRL). This study investigated the suitability of using skin stiffness measurements, with a particular focus on the variation in stiffness with measurement direction (known as anisotropy). In addition to comparing affected tissue with the unaffected tissue on the corresponding site on the opposite limb, volunteers without BCRL were tested to establish the normal variability in stiffness anisotropy between these two corresponding regions of skin on each opposite limb. Multi-directional stiffness was measured with an Extensometer, within the higher stiffness region that skin typically displays at high applied strains, using a previously established protocol developed by the authors. Healthy volunteers showed no significant difference in anisotropy between regions of skin on opposite limbs (mean decrease of 4.7 +/-2.5% between non-dominant and dominant arms), whereas BCRL sufferers showed a significant difference between limbs (mean decrease of 51.0+/-16.3% between unaffected and affected arms). A large difference in anisotropy was apparent even for those with recent onset of the condition, indicating that the technique may have potential to be useful for early detection. This difference also appeared to increase with duration since onset. Therefore, measurement of stiffness anisotropy has potential value for the clinical assessment and diagnosis of skin conditions such as BCRL. The promising results justify a larger study with a larger number of participants

Magnetic Resonance Imaging-Based Assessment of Breast Cancer-Related Lymphoedema Tissue Composition.

OBJECTIVES: The aim of this study was to propose a magnetic resonance imaging acquisition and analysis protocol that uses image segmentation to measure and depict fluid, fat, and muscle volumes in breast cancer-related lymphoedema (BCRL). This study also aims to compare affected and control (unaffected) arms of patients with diagnosed BCRL, providing an analysis of both the volume and the distribution of the different tissue components. MATERIALS AND METHODS: The entire arm was imaged with a fluid-sensitive STIR and a 2-point 3-dimensional T1W gradient-echo-based Dixon sequences, acquired in sagittal orientation and covering the same imaging volume. An automated image postprocessing procedure was developed to simultaneously (1) contour the external volume of the arm and the muscle fascia, allowing separation of the epifacial and subfascial volumes; and to (2) separate the voxels belonging to the muscle, fat, and fluid components. The total, subfascial, epifascial, muscle (subfascial), fluid (epifascial), and fat (epifascial) volumes were measured in 13 patients with unilateral BCRL. Affected versus unaffected volumes were compared using a 2-tailed paired t test; a value of P < 0.05 was considered to be significant. Pearson correlation was used to investigate the linear relationship between fat and fluid excess volumes. The distribution of fluid, fat, and epifascial excess volumes (affected minus unaffected) along the arm was also evaluated using dedicated tissue composition maps. RESULTS: Total arm, epifascial, epifascial fluid, and epifascial fat volumes were significantly different (P < 0.0005), with greater volume in the affected arms. The increase in epifascial volume (globally, 94% of the excess volume) constituted the bulk of the lymphoedematous swelling, with fat comprising the main component. The total fat excess volume summed over all patients was 2.1 times that of fluid. Furthermore, fat and fluid excess volumes were linearly correlated (Pearson r = 0.75), with the fat excess volume being greater than the fluid in 11 subjects. Differences in muscle compartment volume between affected and unaffected arms were not statistically significant, and contributed only 6% to the total excess volume. Considering the distribution of the different tissue excess volumes, fluid accumulated prevalently around the elbow, with substantial involvement of the upper arm in only 3 cases. Fat excess volume was generally greater in the upper arm; however, the relative increase in epifascial volume, which considers the total swelling relative to the original size of the arm, was in 9 cases maximal within the forearm. CONCLUSIONS: Our measurements indicate that excess of fat within the epifascial layer was the main contributor to the swelling, even when a substantial accumulation of fluid was present. The proposed approach could be used to monitor how the internal components of BCRL evolve after presentation, to stratify patients for treatment, and to objectively assess treatment response. This methodology provides quantitative metrics not currently available during the standard clinical assessment of BCRL and shows potential for implementation in clinical practice

A Novel Splice-Site Mutation in VEGFC Is Associated with Congenital Primary Lymphoedema of Gordon.

Lymphedema is characterized by chronic swelling of any body part caused by malfunctioning or obstruction in the lymphatic system. Primary lymphedema is often considered genetic in origin. VEGFC, which is a gene encoding the ligand for the vascular endothelial growth factor receptor 3 (VEGFR3/FLT4) and important for lymph vessel development during lymphangiogenesis, has been associated with a specific subtype of primary lymphedema. Through Sanger sequencing of a proband with bilateral congenital pedal edema resembling Milroy disease, we identified a novel mutation (NM_005429.2; c.361+5G>A) in VEGFC. The mutation induced skipping of exon 2 of VEGFC resulting in a frameshift and the introduction of a premature stop codon (p.Ala50ValfsTer18). The mutation leads to a loss of the entire VEGF-homology domain and the C-terminus. Expression of this Vegfc variant in the zebrafish floorplate showed that the splice-site variant significantly reduces the biological activity of the protein. Our findings confirm that the splice-site variant, c.361+5G>A, causes the primary lymphedema phenotype in the proband. We examine the mutations and clinical phenotypes of the previously reported cases to review the current knowledge in this area

Constitutively Enhanced Lymphatic Pumping in the Upper Limbs of Women Who Later Develop Breast Cancer-Related Lymphedema.

BACKGROUND: It has previously been shown that the lymph drainage rate in both upper limbs is greater in women destined to develop breast cancer-related lymphedema (BCRL) than in those who do not develop BCRL, indicating a constitutive predisposition. We explored constitutive differences further by measuring the maximum lymphatic pump pressure (Ppump) and the rate of (99m)Tc-Nanocoll transport generated by the contractile upper limb lymphatics before and after breast cancer surgery in a group of women who were followed for 2 years to determine their eventual BCRL or non-BCRL status. METHODS AND RESULTS: Ppump and tracer transport rate were measured by lymphatic congestion lymphoscintigraphy in the ipsilateral upper limb in 26 women pre- and post-breast cancer surgery. BCRL occurred in 10/26 (38.5%) cases. Ppump in the women who later developed BCRL (40.0 ± 8.2 mmHg) was 1.7-fold higher than in those who did not develop BCRL (23.1 ± 10.8 mmHg, p = 0.001). Moreover, the rate of lymph tracer transport into the forearm was 2.2-fold greater in the women who later developed BCRL (p = 0.052). Surgery did not significantly reduce Ppump measured 21 weeks postsurgery, but impaired forearm tracer transport in pre-BCRL women by 58% (p = 0.047), although not in those who did not develop BCRL. CONCLUSIONS: Women destined to develop BCRL have higher pumping pressures and lymph transport, indicating harder-working lymphatics before cancer treatment. Axillary lymphatic damage from surgery appears to compromise lymph drainage in those women constitutively predisposed to higher lymphatic pressures and lymph transport

VIPAR, a quantitative approach to 3D histopathology applied to lymphatic malformations.

BACKGROUND: Lack of investigatory and diagnostic tools has been a major contributing factor to the failure to mechanistically understand lymphedema and other lymphatic disorders in order to develop effective drug and surgical therapies. One difficulty has been understanding the true changes in lymph vessel pathology from standard 2D tissue sections. METHODS: VIPAR (volume information-based histopathological analysis by 3D reconstruction and data extraction), a light-sheet microscopy-based approach for the analysis of tissue biopsies, is based on digital reconstruction and visualization of microscopic image stacks. VIPAR allows semiautomated segmentation of the vasculature and subsequent nonbiased extraction of characteristic vessel shape and connectivity parameters. We applied VIPAR to analyze biopsies from healthy lymphedematous and lymphangiomatous skin. RESULTS: Digital 3D reconstruction provided a directly visually interpretable, comprehensive representation of the lymphatic and blood vessels in the analyzed tissue volumes. The most conspicuous features were disrupted lymphatic vessels in lymphedematous skin and a hyperplasia (4.36-fold lymphatic vessel volume increase) in the lymphangiomatous skin. Both abnormalities were detected by the connectivity analysis based on extracted vessel shape and structure data. The quantitative evaluation of extracted data revealed a significant reduction of lymphatic segment length (51.3% and 54.2%) and straightness (89.2% and 83.7%) for lymphedematous and lymphangiomatous skin, respectively. Blood vessel length was significantly increased in the lymphangiomatous sample (239.3%). CONCLUSION: VIPAR is a volume-based tissue reconstruction data extraction and analysis approach that successfully distinguished healthy from lymphedematous and lymphangiomatous skin. Its application is not limited to the vascular systems or skin. FUNDING: Max Planck Society, DFG (SFB 656), and Cells-in-Motion Cluster of Excellence EXC 1003

Case Report: Progressive central conducting lymphatic abnormalities in the RASopathies. Two case reports, including successful treatment by MEK inhibition

The RASopathies are a group of genetic conditions resulting from mutations within the RAS/mitogen-activated protein kinase (RAS-MAPK) pathway. Lymphatic abnormalities are commonly associated with these conditions, however central conducting lymphatic abnormalities (CCLA) have only recently been described. CCLAs may be progressive and can result in devastating systemic sequelae, such as recurrent chylothoraces, chylopericardium and chylous ascites which can cause significant morbidity and even mortality. Improvements in imaging modalities of the central lymphatics have enhanced our understanding of these complex abnormalities. Management is challenging and have mainly consisted of diuretics and invasive mechanical drainages. We describe two adult males with Noonan syndrome with a severe and progressive CCLA. In one patient we report the therapeutic role of targeted molecular therapy with the MEK inhibitor ‘Trametinib’, which has resulted in dramatic, and sustained, clinical improvement. The successful use of MEK inhibition highlights the importance of understanding the molecular cause of lymphatic abnormalities and utilising targeted therapies to improve quality of life and potentially life expectancy

Manual lymphatic drainage therapy in patients with breast cancer related lymphoedema

<p>Abstract</p> <p>Background</p> <p>Lymphoedema is a common and troublesome condition that develops following breast cancer treatment. The aim of this study is to analyze the effectiveness of Manual Lymphatic Drainage in the treatment of postmastectomy lymphoedema in order to reduce the volume of lymphoedema and evaluate the improvement of the concomitant symptomatology.</p> <p>Methods</p> <p>A randomized, controlled clinical trial in 58 women with post-mastectomy lymphoedema. The control group includes 29 patients with standard treatment (skin care, exercise and compression measures, bandages for one month and, subsequently, compression garnments). The experimental group includes 29 patients with standard treatment plus Manual Lymphatic Drainage. The therapy will be administered daily for four weeks and the patient's condition will be assessed one, three and six months after treatment.</p> <p>The primary outcome parameter is volume reduction of the affected arm after treatment, expressed as a percentage. Secondary outcome parameters include: duration of lymphoedema reduction and improvement of the concomitant symptomatology (degree of pain, sensation of swelling and functional limitation in the affected extremity, subjective feeling of being physically less atractive and less feminine, difficulty looking at oneself naked and dissatisfaction with the corporal image).</p> <p>Discussion</p> <p>The results of this study will provide information on the effectiveness of Manual Lymphatic Drainage and its impact on the quality of life and physical limitations of these patients.</p> <p>Trial registration</p> <p>ClinicalTrials (NCT): <a href="http://www.clinicaltrials.gov/ct2/show/NCT01152099">NCT01152099</a></p