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

Symptom-based screening tool for asthma syndrome among young children in Uganda

Under-diagnosis of asthma in ‘under-fives’ may be alleviated by improved inquiry into disease history. We assessed a questionnaire-based screening tool for asthma among 614 ‘under-fives’ with severe respiratory illness in Uganda. The questionnaire responses were compared to post hoc consensus diagnoses by three pediatricians who were guided by study definitions that were based on medical history, physical examination findings, laboratory and radiological tests, and response to bronchodilators. Children with asthma or bronchiolitis were categorized as “asthma syndrome”. Using this approach, 253 (41.2%) had asthma syndrome. History of and present breathing difficulties and present cough and wheezing was the best performing combination of four questionnaire items [sensitivity 80.8% (95% CI 77.6–84.0); specificity 84.7% (95% CI 81.8–87.6)]. The screening tool for asthma syndrome in ‘under-fives’ may provide a simple, cheap and quick method of identifying children with possible asthma. The validity and reliability of this tool in primary care settings should be tested

Linkage and sequence analysis indicate that CCBE1 is mutated in recessively inherited generalised lymphatic dysplasia

Generalised lymphatic dysplasia (GLD) is characterised by extensive peripheral lymphoedema with visceral involvement. In some cases, it presents in utero with hydrops fetalis. Autosomal dominant and recessive inheritance has been reported. A large, non-consanguineous family with three affected siblings with generalised lymphatic dysplasia is presented. One child died aged 5 months, one spontaneously miscarried at 17 weeks gestation, and the third has survived with extensive lymphoedema. All three presented with hydrops fetalis. There are seven other siblings who are clinically unaffected. Linkage analysis produced two loci on chromosome 18, covering 22 Mb and containing 150 genes, one of which is CCBE1. A homozygous cysteine to serine change in CCBE1 has been identified in the proband, in a residue that is conserved across species. High density SNP analysis revealed homozygosity (a region of 900 kb) around the locus for CCBE1 in all three affected cases. This indicates a likely ancestral mutation that is common to both parents; an example of a homozygous mutation representing Identity by Descent (IBD) in this pedigree. Recent studies in zebrafish have shown this gene to be required for lymphangiogenesis and venous sprouting and are therefore supportive of our findings. In view of the conserved nature of the cysteine, the nature of the amino acid change, the occurrence of a homozygous region around the locus, the segregation within the family, and the evidence from zebrafish, we propose that this mutation is causative for the generalised lymphatic dysplasia in this family, and may be of relevance in cases of non-immune hydrops fetalis

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

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

The Healthy Start project: a randomized, controlled intervention to prevent overweight among normal weight, preschool children at high risk of future overweight

BACKGROUND: Research shows that obesity prevention has to start early. Targeting interventions towards subgroups of individuals who are predisposed, but yet normal weight, may prove more effective in preventing overweight than interventions towards unselected normal weight subsets. Finally, interventions focused on other factors than diet and activity are lacking. The objectives were to perform a randomized, controlled intervention aiming at preventing overweight in children aged 2–6 years, who are yet normal weight, but have high predisposition for future overweight, and to intervene not only by improving diet and physical activity, but also reduce stress and improve sleep quality and quantity. METHODS/DESIGN: Based on information from the Danish National Birth Registry and administrative birth forms, children were selected based on having either a high birth weight, a mother who was overweight prior to pregnancy, or a familial low socioeconomic status. Selected children (n = 5,902) were randomized into three groups; an intervention group, a shadow control group followed in registers exclusively, and a control group examined at the beginning and at the end of the intervention. Approximately 21% agreed to participate. Children who presented as overweight prior to the intervention were excluded from this study (n = 92). In the intervention group, 271 children were included, and in the control group 272 were included. Information obtained from the shadow control group is on-going, but it is estimated that 394 children will be included. The intervention took place over on average 1½ year between 2009 and 2011, and consisted of optional individual guidance in optimizing diet and physical activity habits, reducing chronic stress and stressful events and improving sleep quality and quantity. The intervention also included participation in cooking classes and play arrangements. Information on dietary intake, meal habits, physical activity, sleep habits, and overall stress level was obtained by 4–7 day questionnaire diaries and objective measurements. DISCUSSION: If the Healthy Start project is effective in preventing excessive weight gain, it will provide valuable information on new determinants of obesity which should be considered in future interventions, and on new strategies to prevent development of overweight and obesity at an early age. TRIAL REGISTRATION: ClinicalTrials.gov, ID NCT01583335

Striatal interneurons in dissociated cell culture

In addition to the well-characterized direct and indirect projection neurons there are four major interneuron types in the striatum. Three contain GABA and either parvalbumin, calretinin or NOS/NPY/somatostatin. The fourth is cholinergic. It might be assumed that dissociated cell cultures of striatum (typically from embryonic day E18.5 in rat and E14.5 for mouse) contain each of these neuronal types. However, in dissociated rat striatal (caudate/putamen, CPu) cultures arguably the most important interneuron, the giant aspiny cholinergic neuron, is not present. When dissociated striatal neurons from E14.5 Sprague–Dawley rats were mixed with those from E18.5 rats, combined cultures from these two gestational periods yielded surviving cholinergic interneurons and representative populations of the other interneuron types at 5 weeks in vitro. Neurons from E12.5 CD-1 mice were combined with CPu neurons from E14.5 mice and the characteristics of striatal interneurons after 5 weeks in vitro were determined. All four major classes of interneurons were identified in these cultures as well as rare tyrosine hydroxylase positive interneurons. However, E14.5 mouse CPu cultures contained relatively few cholinergic interneurons rather than the nearly total absence seen in the rat. A later dissection day (E16.5) was required to obtain mouse CPu cultures totally lacking the cholinergic interneuron. We show that these cultures generated from two gestational age cells have much more nearly normal proportions of interneurons than the more common organotypic cultures of striatum. Interneurons are generated from both ages of embryos except for the cholinergic interneurons that originate from the medial ganglionic eminence of younger embryos. Study of these cultures should more accurately reflect neuronal processing as it occurs in the striatum in vivo. Furthermore, these results reveal a procedure for parallel culture of striatum and cholinergic depleted striatum that can be used to examine the function of the cholinergic interneuron in striatal networks

A multicenter study on Leigh syndrome: Disease course and predictors of survival

Background: Leigh syndrome is a progressive neurodegenerative disorder, associated with primary or secondary dysfunction of the mitochondrial oxidative phosphorylation. Despite the fact that Leigh syndrome is the most common phenotype of mitochondrial disorders in children, longitudinal natural history data is missing. This study was undertaken to assess the phenotypic and genotypic spectrum of patients with Leigh syndrome, characterise the clinical course and identify predictors of survival in a large cohort of patients. Methods. This is a retrospective study of patients with Leigh syndrome that have been followed at eight centers specialising in mitochondrial diseases in Europe; Gothenburg, Rotterdam, Helsinki, Copenhagen, Stockholm, Brussels, Bergen and Oulu. Results: A total of 130 patients were included (78 males; 52 females), of whom 77 patients had identified pathogenic mutations. The median age of disease onset was 7 months, w