Primary idiopathic lymphedema has a prevalence of 1 of 100,000 persons.1 The legs are almost always affected; only 10% of patients with primary lymphedema have upper extremity disease.2 Although pathogenic germline variants in 30 genes are associated with a malformed lymphatic system causing lower extremity or generalized lymphedema, no variants have been associated with nonsyndromic, nongeneralized upper extremity disease.2 The purpose of this investigation was to identify novel causes of primary upper limb lymphedema.
A 17-year-old male presented to our lymphedema program with right upper extremity swelling since 9 months of age (Figure 1). The patient had suffered 12 episodes of cellulitis in the affected arm. At 19 years of age, he developed swelling of his right leg. He was otherwise healthy with a negative medical history. His maximum body mass index was 21 kg/m2, and he had no physical examination signs of syndromic or generalized lymphedema (e.g., distichiasis, abnormal nails, facial abnormalities, intellectual disability, intestinal lymphangiectasia, pleural effusions, ascites). His family history was significant for right lower extremity lymphedema in his mother. A diagnosis of right arm lymphedema was made by history, physical examination, and lymphoscintigraphy.3
Genomic DNA was extracted from the blood of the patient and mother using DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany). Whole-exome sequencing (WES) was performed on the patient’s DNA (Psomagen, Rockville, MD). Libraries were made with Agilent SureSelectXT Human All Exon V5 and sequenced on Illumina NovaSeq6000 S4 to generate paired-end, 150 bp reads. A mean coverage of 290× was obtained and 99.4% of exons were covered at greater than 10×. Generated reads were aligned to the human genome reference (assembly GRCh37/hg19). Variants were called and annotated using the Codified Genomics and Variant Explorer pipelines.4 Sanger sequencing was used to confirm variants identified by WES. PCR primers were designed flanking exon 3 of the human Celsr1 gene (FP: 5′-cagggtctgcgacaggctcgttg-3′; RP: 5′-cacggaggacatcaacgtcacgatcas-3′). PCR amplification of a 405 bp fragment was done with CloneAmp polymerase (Takara) using the blood extracted DNA of patient and mother. The PCR fragments were cloned using the zero-blunt-TOPO kit (Invitrogen), and the TOPO reactions were transformed in Stellar competent bacteria (Takara). Plasmids were prepared from 10 individual colonies each for the patient and his mother and sequenced with T7 (Eton Biosciences).
In the patient’s white blood cell DNA, WES identified a frameshift deletion resulting in a premature stop codon in exon 3 of the CELSR1 gene hg19: chr22:46,835,160_46,835,166del (c.4326_4332del; p.T1443Gfs*14). The variant was absent from gnomAD and ExAC databases and deemed pathogenic (PVS1, PP4, PM2) based on ACMG Standards and Guidelines.5 A second point variant (c.A4336G; p.S1446G) also was found, which was of uncertain significance. Sanger sequencing confirmed the variants in the patient and showed that they are in cis. The same variants were also identified in the mother’s white blood cell DNA using Sanger sequencing.
Almost all of the approximately 30 known lymphedema-associated variants cause lower extremity disease.2,6-8 Variants resulting in primary upper extremity lymphedema have been found in syndromic conditions or in genes causing generalized lymphatic dysplasia: 45 XO, CCBE1, GJC2, PIEZ01.7,9-13 In this report, we show that an autosomal dominant germline frameshift deletion in CELSR1 causes nonsyndromic upper and lower extremity lymphedema without generalized lymphedema.
A role for CELSR1 in the pathogenesis of primary arm lymphedema is supported by evidence that CELSR1 variants cause autosomal dominant lower extremity disease.14-16 The lymphedema phenotypes associated with CELSR1 variants reported to date only involve the lower extremity, most commonly affect females, and exhibit incomplete or delayed penetrance in males. These CELSR1 variants are loss of function and include as follows: (1) p.E290*, (2) p.N681Mfs*16, (3) p.I1708fs*44, (4) p.W1957*, (5) c.5226 + 2T>A, (6) c.5702–1G>C, and (7) c.6739 + 1G>A.14-16 It has been hypothesized that upper and lower extremity lymphedema associated with 22q13.3 deletion syndrome (Phelan-McDermid) may result from CELSR1 inactivation because this gene is located on the same chromosome.17CELSR1 has been shown to influence the development of lymphatic valves.18
We hypothesized that primary arm lymphedema in a nonsyndromic patient without generalized lymphedema might result from a variant in a unique gene. We found, however, that a gene known for lower limb lymphedema (CELSR1) also can cause upper extremity disease.14-16 Arm involvement only comprises 10% of patients with primary lymphedema and 50% of these individuals also have the disease in their legs.2 Consequently, patients with primary arm lymphedema may represent a more severe phenotype of lower extremity disease-causing variants. The legs may be more susceptible than the arms to exhibiting edema because lymph fluid has a longer distance to travel against gravity to the venous circulation at the base of the neck. Alternatively, other modifying factors such as anatomical variation or trigger events may be responsible for upper extremity disease. CELSR1 should be included in variant screening for patients with nonsyndromic primary upper limb lymphedema.
1. Smeltzer DM, Stickler GB, Schirger A. Primary lymphedema in children and adolescents: a follow-up study and review. Pediatrics. 1985;76:206–218.
2. Goss JA, Maclellan RA, Greene AK. Primary lymphedema of the upper extremities: clinical and lymphoscintigraphic features in 23 patients. Lymphat Res Biol. 2019;17:40–44.
3. Hassanein AH, Maclellan RA, Grant FD, Greene AK. Diagnostic accuracy of lymphoscintigraphy for lymphedema and analysis of false-negative tests. Plast Reconstr Surg Glob Open. 2017;5:e1396.
4. Schmitz-Abe K, Li Q, Rosen SM, et al. Unique bioinformatic approach and comprehensive reanalysis improve diagnostic yield of clinical exomes. Eur J Hum Genet. 2019;27:1398–1405.
5. Richards S, Aziz N, Bale S, et al.; ACMG Laboratory Quality Assurance Committee. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 2015;17:405–424.
6. Greene A, Brouillard P, Sudduth CL, Smits PJ, Konczyk DJ, Vikkula M. EPHB4 mutation causes adult and adolescent-onset primary lymphedema. Am J Med Genet A. 2021;185:3810–3813.
7. Mendola A, Schlögel MJ, Ghalamkarpour A, et al.; Lymphedema Research Group. Mutations in the VEGFR3 signaling pathway explain 36% of familial lymphedema. Mol Syndromol. 2013;4:257–266.
8. Sudduth CL, Greene AK. Primary lymphedema: update on genetic basis and management [published online ahead of print January 27, 2021]. Adv Wound Care. doi: 10.1089/wound.2020.1338.
9. Fotiou E, Martin-Almedina S, Simpson MA, et al. Novel mutations in PIEZO1 cause an autosomal recessive generalized lymphatic dysplasia with non-immune hydrops fetalis. Nat Commun. 2015;6:8085.
10. Alders M, Hogan BM, Gjini E, et al. Mutations in CCBE1 cause generalized lymph vessel dysplasia in humans. Nat Genet. 2009;41:1272–1274.
11. Ostergaard P, Simpson MA, Brice G, et al. Rapid identification of mutations in GJC2 in primary lymphoedema using whole exome sequencing combined with linkage analysis with delineation of the phenotype. J Med Genet. 2011;48:251–255.
12. Hennekam RC, Geerdink RA, Hamel BC, et al. Autosomal recessive intestinal lymphangiectasia and lymphedema, with facial anomalies and mental retardation. Am J Med Genet. 1989;34:593–600.
13. Atton G, Gordon K, Brice G, et al. The lymphatic phenotype in Turner syndrome: an evaluation of nineteen patients and literature review. Eur J Hum Genet. 2015;23:1634–1639.
14. Maltese PE, Michelini S, Ricci M, et al. Increasing evidence of hereditary lymphedema caused by CELSR1 loss-of-function variants. Am J Med Genet A. 2019;179:1718–1724.
15. Gonzalez-Garay ML, Aldrich MB, Rasmussen JC, et al. A novel mutation in CELSR1 is associated with hereditary lymphedema. Vasc Cell. 2016;8:1.
16. Erickson RP, Lai LW, Mustacich DJ, Bernas MJ, Kuo PH, Witte MH. Sex-limited penetrance of lymphedema to females with CELSR1 haploinsufficiency: a second family. Clin Genet. 2019;96:478–482.
17. Xia S, Liu Z, Yan H, et al. Lymphedema complicated by protein-losing enteropathy with a 22q13.3 deletion and the potential role of CELSR1: a case report. Medicine (Baltimore). 2021;100:e26307.
18. Tatin F, Taddei A, Weston A, et al. Planar cell polarity protein Celsr1 regulates endothelial adherens junctions and directed cell rearrangements during valve morphogenesis. Dev Cell. 2013;26:31–44.