Deleterious mutations in the Zinc-Finger 469 gene cause brittle cornea syndrome

Abstract

Brittle cornea syndrome (BCS) is an autosomal-recessive disorder characterized by a thin cornea that tends to perforate, causing progressive visual loss and blindness. Additional systemic symptoms such as joint hypermotility, hyperlaxity of the skin, and kyphoscoliosis place BCS among the connective-tissue disorders. Previously, we assigned the disease gene to a 4.7 Mb interval on chromosome 16q24. In order to clone the BCS gene, we first narrowed the disease locus to a 2.8 Mb interval and systematically sequenced genes expressed in connective tissue in this chromosomal segment. We have identified two frameshift mutations in the Zinc-Finger 469 gene (ZNF469). In five unrelated patients of Tunisian Jewish ancestry, we found a 1 bp deletion at position 5943 (5943 delA), and in an inbred Palestinian family we detected a single-nucleotide deletion at position 9527 (9527 delG). The function of ZNF469 is unknown. However, a 30% homology to a number of collagens suggests that it could act as a transcription factor involved in the synthesis and/or organization of collagen fibers.

Figure 1

Clinical features of BCS Patients (A) Corneal opacities due to scars. (B) Keratoglobus. (C) Hyperlaxity of the joints.

Figure 2

Haplotypes of Chromosome 16q24 in Tunisian Jewish Patients Top: Ancestral chromosome. In patient no. 5, deviation from the ancestral chromosome at the markers D16S3425 and D16S3436 on the telomeric and centromeric sides, respectively, set the disease gene interval to 4 Mb in the Tunisian patients.

Figure 3

Palestinian Family Pedigree and Chromosome 16q24 Haplotypes Recombination event in D16S3422 in individual II:4 sets this marker as the centromeric boundary. Allele 4 in D16S3420 in individual II:1 and allele 7 in D16S3437 in individual III:1 reflect mutations in these markers.

Figure 4

Mutations in ZNF469 Cause BCS (A) Sequence chromatogram of a control subject (top) and a Tunisian patient with a homozygous deletion of A at position 5943 (bottom). (B) Sequence chromatogram of a control subject (top) and a Palestinian patient with a homozygous deletion of G at position 9527 (bottom). (C) SmaI restriction digests of the Tunisian Jewish mutation. In the carrier chromosome, the 533bp product is cleaved to yield 388 bp and 145 bp fragments. Molecular-weight marker (M), homozygotes (lanes 1 and 2), heterozygotes (lanes 3 and 4), and control subjects (lanes 5 and 6). (D) ABI 3100 assay for the detection of the Palestinian mutation. The 1bp difference is evident in homozygotes (1 and 2) compared to controls (5 and 6). Heterozygotes appear as two peaks (3 and 4).

Figure 5

ZNF469-Expression Studies The 559bp RT-PCR product in cornea (c), sclera (s), skin fibroblasts (f) and striated muscle (m). M denotes molecular-weight marker.

Figure 6

Partial Homology of ZNF469 to COL1A1 The Smith-Waterman algorithm was used to calculate local alignment (EMBOSS). Top line: ZNF469.