טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentMelikhan Svetlana
SubjectA Molecular Genetic Study of Congenital Lymphedema Using
Homozygosity Mapping Strategy
DepartmentDepartment of Medicine
Supervisors Clinical Professor Ruth Gershoni-Baruch
Dr. Efrat Dagan
Full Thesis text - in Hebrew Full thesis text - Hebrew Version


Abstract

Primary lymphedema results from a dysfunction of the lymphatic system. Primary lymphedema results in chronic tissue swelling, due to impaired lymphatic drainage in the presence of normal capillary filtration. Primary lymphedema is recorded to affect 1.15/100,000 individuals younger than 20 years of age. The edema is mainly restricted to the lower extremities, the genitalia and the face, and may also involve the pleura, the peritoneum and the pericardium. The etiology of the disorder varies, from hereditary or primary to acquired or secondary (e.g. trauma, radiation, surgery, infections). Primary lymphedema comprises around 10% of all lymphedema cases.  Familial lymphedema usually segregates as an autosomal dominant trait, with only one case described in line with autosomal recessive transmission. Genetic studies of families with hereditary lymphedema have yielded mutations in three lymphangiogentic genes: FLT4 (VEGFR3), FOXC2, SOX18. Our research sought to map and ultimately hunt the mutation that causes hereditary lymphedema in an extended consanguineous Muslim family consisting of four affected individuals: one female, presenting with isolated edema of the lower extremities, and three males presenting with lymphedema of the lower extremities, lymphocele and mild mental retardation.

After locating a candidate locus using whole genome linkage analysis (Illumina’s 6000 SNP array), on 12 family members, both healthy and affected, Sanger sequencing of the candidate genes was preformed.

A candidate locus of 2.3 Mb located on chromosome 5q35.3 was identified, showing homozygosity in the affected individuals. This locus has been previously linked to congenital lymphedema. Using SUPERLINK online, linkage analysis generated a two-point LOD score of 3.18. Several mutations in the candidate gene, FLT4, previously described in Muslim-Israeli families, were discarded as culprit using sequence analysis. While sequencing FLT4, a missense mutation, p.Ser1235Cys, in exon 28 has been discovered. The c.3704C>G, p.Ser1235Cys variant is not recorded in the common databases (dbSNP, 1000Genomes, Exome Variant Server). Evolutionary conservation analysis by UniProt shows that the amino acid serine in position 1235 of the protein is highly conserved in different species. Moreover, both PolyPhen-2 and MutationTaster predicted this variant to be damaging.  Unlike other mutations previously described, this mutation is located outside the tyrosine-kinase domains, and has perfect segregation within the family. A population screen for the mutation was performed, with no carriers found in 100 Muslim Israeli-Arabs. Furthermore, a whole-exome analysis of an affected individual revealed no other mutations in lymphedema-related genes.