Functional characterization of N-terminal TIMP3 mutation underlying Sorsby fundus dystrophy in Belgian and French pedigrees.

Abstract

Purpose : Sorsby fundus dystrophy (SFD) is an autosomal dominant retinal dystrophy, caused by mutations in TIMP3. In 2000, Assink et al. examined a five-generation Belgian family with SFD. Although linkage was shown with region 22q12.1-q13.2, no TIMP3 mutation was found. Here, it was our aim to elucidate the genetic cause of SFD in this family.

Methods : Microsatellite analysis with additional markers was performed in 31 members of the Belgian family, followed by Sanger sequencing of the coding region of TIMP3 (NM_000362). A French and second Belgian family with the same TIMP3 mutation, c.113C>G p.(Ser38Cys), were identified. Haplotype reconstruction was done in order to assess the origin of this mutation. Western blot analysis and enzymatic assays with MMP2, a target of TIMP3, were conducted on patient-derived fibroblasts. Molecular modeling of the mutated protein was done to test protein structure and predict interaction patterns. Mass spectrometry analysis was performed using the Oxicat method.

Results : A heterozygous TIMP3 variant, c.113C>G p.(Ser38Cys) was found to co-segregate with disease in 63 affected individuals. Haplotype analysis in three families segregating this mutation revealed a common haplotype of 6,47 Mb. So far this is the only known missense mutation located in the N-terminal domain of TIMP3, adding a cysteine to six disulfide bonds. In 2003, Arris et al. proposed a mechanism whereby mutations adding an extra cysteine create proteins that form disulfide-bonded dimers. Western blot analysis on fibroblasts could not confirm the proposed hypothesis on protein dimer formation however. The N-terminal domain of TIMP3 alone is sufficient to mediate its metalloproteinase inhibitory activities. Both enzymatic assays and molecular modeling could not identify an effect on folding of the protein and on interaction patterns. To evaluate possible intramolecular disturbances in disulfide bonding, mass spectrometry analysis is ongoing.

Conclusions : In conclusion, we pinpointed the molecular cause underlying SFD in a historical large Belgian pedigree to TIMP3, excluding a noncoding TIMP3 mutation or genetic heterogeneity of SFD. A founder effect of mutation c.113C>G p.(Ser38Cys) was shown in 2 Belgian and 1 French pedigree. Further functional characterization of this unique N-terminal mutation is ongoing.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.