A Multiplex Kindred with Hennekam Syndrome due to Homozygosity for a CCBE1 Mutation that does not Prevent Protein Expression

Abstract

Collagen and calcium-binding EGF domain-containing protein 1 (CCBE1) bi-allelic mutations have been associated with syndromes of widespread congenital lymphatic dysplasia, including Hennekam Syndrome (HS). HS is characterized by lymphedema, lymphangiectasia, and intellectual disability. CCBE1 encodes a putative extracellular matrix protein but the HS-causing mutations have not been studied biochemically. We report two HS siblings, born to consanguineous parents of Turkish ancestry, whose clinical phenotype also includes protein losing enteropathy, painful relapsing chylous ascites, and hypogammaglobulinemia. We identified by whole exome and Sanger sequencing the homozygous CCBE1 C174Y mutation in both siblings. This mutation had been previously reported in another HS kindred from the Netherlands. In over-expression studies, we found increased intracellular expression of all forms (monomers, dimers, trimers) of the CCBE1 C174Y mutant protein, by Western blot, despite mutant mRNA levels similar to wild-type (WT). In addition, we detected increased secretion of the mutant CCBE1 protein by ELISA. We further found the mutant and WT proteins to be evenly distributed in the cytoplasm, by immunofluorescence and confocal microscopy. Finally, we found a strong decrease of lymphatic vessels, with a corresponding diminished expression of CCBE1, by immunohistochemistry of the patients’ intestinal biopsies. In contrast, mucosal blood vessels and muscularis mucosae showed normal CCBE1 staining. Our findings show that the mutant CCBE1 C174Y protein is not loss-of-function by loss-of-expression.

The clinical significance of CCBE1 expression in human colorectal cancer

“… 35 Previous studies found that CCBE1 is a secreted molecule involved in lymphangiogenesis, 14 , 36 and congenital CCBE1 mutations have been shown to cause Hennekam syndrome. 36 , 37 To confirm the relationship between CCBE1 expression and prognosis and clinicopathological features of CRC, 204 cases of CRC tissues were stained to analyze. IHC results showed that high expression of CCBE1 was observed in 55.4% (113/204) CRC patients.…”

Novel mutation in CCBE 1 as a cause of recurrent hydrops fetalis from Hennekam lymphangiectasia‐lymphedema syndrome‐1

“…Homozygosity mapping has identified CCBE1 gene variants to be a cause of HKLLS1 . So far, at least 12 different CCBE1 variants have been reported in at least seventeen probands/families …”

Expanding the genotypic spectrum of CCBE1 mutations in Hennekam syndrome

“…Of the various forms of primary lymphedema, CCBE1 mutations appear to be predominantly causative for Hennekam syndrome [Alders et al, ]. Fewer than 50 patients with Hennekam Syndrome have been reported in the literature to date, with 25–29% of those having biallelic CCBE1 coding mutations [Alders et al, ; Jackson et al, ] and 20% having biallelic FAT4 mutations. Fotiou et al [] recently reported biallelic mutations in PIEZO1 can result in generalized lymphatic dysplasia, phenotypically similar to Hennekam syndrome.…”