A novel mutation in CELSR1 is associated with hereditary lymphedema
© Gonzalez-Garay et al. 2016
Received: 8 October 2015
Accepted: 29 January 2016
Published: 5 February 2016
Biological evidence reported in the literature supports the role of CELSR1 as being essential for valvular function in murine lymphatics. Yet thus far, there have been no variants in CELSR1 associated with lymphatic dysfunction in humans.
In this report, a rare early inactivating mutation in CELSR1 is found to be causal for non-syndromic, lower extremity lymphedema in a family across three generations. Near-infrared fluorescence lymphatic imaging shows that instead of being propelled within the lumen of well-defined lymphatic vessels, lymph moved in regions of both legs in an unusual fashion and within sheet-like structures.
CELSRI may be responsible for primary, non-syndromic lymphedema in humans.
KeywordsPrimary lymphedema Whole exome sequencing Near-infrared fluorescence imaging CELSR1 Planar polarity
Lymphedema (LE) is a condition caused by a defective lymphatic system where an excess of fluid accumulates and generates chronic swelling of tissues. Primary LE is a rare genetic disorder that can develop at birth (congenital), at puberty (praecox), or after 35 years of age (tarda) . FLT4 was the first pathogenic gene identified in primary LE , but to date, there is only a handful of genes (FOXC2, CCBE1, GCJ2, SOX18, GATA2, PTPN14) associated with hereditary LE, with the majority associated with patients in which LE is part of a genetic syndrome . Mendola et al. recently argued that the genetic cause(s) of the majority of families harboring hereditary LE has not yet been identified. In their group of 78 index patients with inherited LE who were screened for mutations in the top seven LE genes, they found explanations for only 36 % of the cases .
In this report, we present the unusual lymphatic phenotype obtained by near-infrared fluorescence lymphatic imaging (NIRFLI) of a woman diagnosed non-syndromic, lower extremity LE and compare it with that of her asymptomatic mother. From clinical diagnoses and whole exome sequencing (WES) of family members across three generations, we found no gene variants known to be responsible for LE, but instead found a rare, inactivating mutation in CELSR1 that could be causative for the inherited condition and the lymphatic phenotype imaged in the index case.
Family case report
A 62 yo female previously diagnosed with primary, lower extremity LE (bilateral, Stage II by International Society of Lymphology staging system) and her 84 yo asymptomatic mother enrolled in an ongoing, institutional review board (IRB) and FDA-approved (IND# 102,827) clinical study of lymphatic disorders (Clinical Trials No. NCT00833599: “Imaging lymphatic function in normal subjects and in persons with lymphatic disorders” www.clinicaltrials.gov). The index case self-reported onset of lower extremity swelling at age 10, with diagnosis of LE at age 39 at which time she began compression treatment. In addition, she was also previously diagnosed with type 2 diabetes, high cholesterol, and high blood pressure. Following consent, a clinical examination was first performed followed by near-infrared fluorescence lymphatic imaging (NIRFLI)  to assess lymphatic anatomy and function in the legs. Blood was collected for WES of DNA.
Upon questioning, we further found a family history of lower extremity LE and traveled to the family’s hometown to consent, conduct standard clinical examinations (including measurements of leg volumes and assessment of Stemmer’s sign), and to collect blood from family members spanning three generations. We did not perform NIRFLI on these family members. Other than stage I and II bilateral lower extremity, non-syndromic LE, we found no remarkable evidence of disease among the family members.
Genetic sequencing analyses
Genetic sequencing analysis was conducted as follows. Each pair of fastq files was aligned to human genome (hg19) using Novoalign (Novocraft Technologies; www.novocraft.com), keeping parameters at the default settings, as recommended by Novocraft Technologies. SAMtools (http://samtools.sourceforge.net) was used to sort the SAM files, create BAM files, and generate their index files. Picard (SourceForge; http://broadinstitute.github.io/picard) was used to remove all of the PCR duplicates from the BAM files. For local realignments, base quality recalibration, and variant calling, we used Genome Analysis Toolkit (GATK) Version 3.1-1 . Finally, for variant annotation, we used SnpEff (http://snpeff.sourceforge.net/) , variant tools and ANNOVAR  using multiple databases from UCSC Genome bioinformatics. Functional effects for each non-synonymous coding variant was evaluated using three different functional prediction algorithms: (1) Polyphen 2.0 Prediction of functional effects of human nsSNPs (http://genetics.bwh.harvard.edu/pph2) , (2) SIFT  and (3) (www.mutationtaster.org)  using the dbNSFP database . Filtration of common polymorphisms was accomplished using frequencies from the NHLBI Exome sequencing project (ESP) (http://evs.gs.washington.edu/EVS)  and the 1,000 Genomes Project (http://www.1000genomes.org/data) .
CELSR1 is a member of the cadherin superfamily, conserved during evolution with orthologs in invertebrate and vertebrates . In addition, CELSR1 is an atypical cadherin involved in planar cell polarity. This protein has nine cadherin repeats, eight EGF-like domains, and seven transmembrane segments (Uniprot_id Q9NYQ6). CELSR1 is located at the plasma membrane with the cadherin domains acting as homophilic binding regions and the EGF-like domains involved in cell adhesion and receptor-ligand interactions. We postulate that when the mutated allele is expressed, the protein lacks the entire cadherin domain, the five EGF domains, and the LamG domains (Fig. 3b).
CELRS1 has an ortholog in Drosophila, the flamingo (Fmi). Fmi is localized at cell–cell boundaries in the wing. In the absence of Fmi, planar polarity was distorted . In humans and rodents, there are three genes CELSR1–3 and Celsr1–3. Celsr1 and Celsr2 expression is observed during gastrulation and within the developing nervous system. Celsr3 transcripts are found only at sites of active neurogenesis  and mutations in CELSR1 are associated with neural tube defects in humans [29, 30].
Recent biological evidence reported in the literature supports the causal role of CELSR1 for LE in the family pedigree. Tatin et al. recently showed that CELSR1 and VANGL2 play critical roles in the complex morphogenetic process of intraluminal valve formation in murine lymphatic vessels. In their work, they demonstrated that during valve leaflet formation, endothelial cells recruit CELSR1 and VANGL2 from filopodia to discrete membrane domains at cell-cell contacts. Furthermore, mouse mutants Crsh (Celsr1) and Looptail (Vangl2) show valve aplasia .
The proband displayed a common lymphatic phenotype consisting of extensive dermal backflow, tortuous vessels, as well as unique “sheet-like flow” in both legs indicating a defect in the valvular mechanism of lymph propulsion in collecting vessels. While we have not conducted functional studies of the CELSR1 mutation identified in this family, the phenotype is consistent with the phenotype described in the mouse mutant Crsh .
In this family, the phenotype was not apparent in any living males, but obviously was passed to daughters through male parents, both now deceased and not available for clinical diagnosis or DNA collection. Three living males in the third and fourth generations were not reported to have lymphedema, but unfortunately not available for clinical diagnosis and genotyping. As a result, we are unable to determine whether penetrance is limited to females.
This report provides the first evidence that defective planar cell polarity signaling pathway may participate as the cause of primary, non-syndromic lymphedema in humans.
Written informed consent for images contained in this report was obtained from the patient's parents in accordance with the guidelines of the University of Texas Health Science Center Institutional Review Board.
near-infrared fluorescence lymphatic imaging
Sources of funding
Funded by the National Institutes of Health grants R01 HL092923 and U54 CA136404 (EMS-M).
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