Autosomal recessive congenital cataracts linked to HSF4 in a consanguineous pedigree

Abstract

Purpose: To investigate the genetic basis of autosomal recessive congenital cataracts in a large consanguineous pedigree.

Methods: All participating members underwent a thorough ophthalmic examination. A small aliquot of blood sample was collected from all participants and genomic DNA was extracted. A genome wide scan was performed with short tandem repeat (STR) markers and logarithm of odds (LOD) scores were calculated. All coding exons and exon-intron boundaries of candidate gene were sequenced and expression of Hsf4 in embryonic and postnatal mouse lens was investigated. The N-terminal GFP tagged wild type and mutant HSF4 constructs were prepared to examine the localization pattern while luciferase transactivation assay exploiting the gammaC-crystallin promoter was completed to investigate transcriptional activity of the HSF4 proteins. Finally, HSF4 mutant knock-in human lens epithelial cell line was prepared using CRISPR technology.

Results: The ophthalmological examinations were suggestive of nuclear cataracts without any other ocular anomalies. Genome-wide linkage analyses localized the disease interval to a 20.78 cM (15.08 Mb) region of chromosome 16q with a maximum two-point LOD score of 4.46. Sanger sequencing identified a novel missense mutation: c.433G>C (p.A145P) that segregated with the disease phenotype in the family and was not present in ethnically-matched controls. Real time PCR analysis identified expression of Hsf4 in mouse lens as early as embryonic day 15 (E15) with steady level of expression thereafter. The immunofluorescence tracking confirmed that p.A145P HSF4 localized to the nucleus similar to the wild type protein. Surprisingly, the p.A145P HSF4 stimulated the human gammaC-promter driven luciferase significantly higher (1.4 fold) compared to the wild type protein in luciferase reporter assay.

Conclusions: Here, we report a novel missense mutation in HSF4 associated with autosomal recessive congenital cataracts that does not affect the nuclear localization of HSF4 but exhibits higher transcriptional activity compare to wild type protein. We are currently investigating the transcriptional targets that are differentially expressed in response to the p.A145P HSF4.