Congenital cataracts show considerable clinical and locus heterogeneity and represent one of the major causes of vision loss world-wide 12. Cataracts can be isolated or can occur in association with a large number of different metabolic diseases or genetic syndromes 3. Nonsyndromic congenital cataracts tend to be highly penetrant as Mendelian traits with autosomal dominant more common than autosomal recessive forms. Nonsyndromic congenital cataract has an estimated frequency of 1–6 per 100,000 live births 4. To date, loci for 28 autosomal dominant and 12 autosomal recessive forms of congenital cataracts have been mapped. Out of 12 autosomal recessive loci, mutations in eight genes have been identified 5678910111213141516. Of those families with inherited cataracts for whom the mutant gene is known, about half show mutations in genes encoding for crystallins, about a quarter have mutations in genes coding for connexins, with the remainder divided among the genes encoding the heat shock transcription factor-4 (HSF4), aquaporin-0 (AQP0, MIP), and beaded filament structural protein-2 (BFSP2) 3.

Here, we report a large consanguineous family from Pakistan with eight affected individuals affected by autosomal recessive cataracts. Consanguineous marriages are a common practice in the Pakistani society and 60% of marriages are reported to be within families 17. Such families have been instrumental for mapping disease loci and for identification of causative genes and mutations in especially autosomal recessive disorders. During linkage analysis for known recessive cataracts loci, we showed co-segregation of the disease phenotype with markers D16S397 (85.94 cM corresponding to position 65,295,710 bp in NCBI build 36.1), D16S3086 (85.94 cM; 65,493,383 bp) and D16S421 (85.94 cM; 65,853,130 bp). A maximum two-point LOD score of 5.6 at recombination fraction θ = 0 was obtained for marker D16S421. As the critical interval contained the HSF4 gene (65,754,789 – 65,761,349 bp) previously reported to cause autosomal dominant or autosomal recessive cataracts, sequence analysis of HSF4 was performed which identified a novel nonsense mutation (p.R405X) in exon 11 as the cause of the disease in this family.

This family (Fig. 1) originated from Afghanistan and migrated to Quetta-Pakistan in the late 1980s. Cataracts in affected individuals were either present at birth or developed during infancy. By locus-specific linkage analysis, the disease phenotype was shown to perfectly co-segregate with flanking markers to the already known causative gene HSF4 (OMIM 602438). A highly significant maximum two-point LOD score was obtained at D16S421 (Zmax = 5.6 at θ = 0) (table 1). PCR amplification and subsequent direct sequencing of the coding exons of HSF4 (based on the largest coding Genbank entry with the accession number NM_001040667.2) revealed the novel nucleotide exchange c.1213C > T in exon 11 of HSF4 in the family BUIT-CA01 (Fig. 2). This mutation was not present in any of the unaffected individuals neither of this family nor in altogether 334 control alleles of Pakistani and German healthy control samples. This transition is predicted to change the amino acid arginine into a stop codon (p.Arg405X in isoform B [NP_001035757.1] corresponding to p.Arg375X in isoform A [NP_001529.2]) (Fig 2). A mutation-specific restriction enzyme (HphI) digest confirmed homozygosity for the mutant allele in all eight affected individuals (IV:1, IV:2, IV:3, IV:4, IV:5, IV:6, IV:7 and IV:8; bands of 403 bp, 245 bp and 148 bp, Fig. 1) whereas homozygous wild type alleles were shown in individual II:4 and III:3 (bands of 648 bp and 148 bp; Fig. 1). The four parents (III:1, III:2, III:4 and III:5) showed heterozygosity for one WT allele (648 bp and 148 bp) and one mutant allele (403 bp, 245 bp and 148 bp; Fig. 1).

HSF4 (OMIM 602438) belongs to the family of heat shock transcription factors that regulate the expression of heat shock proteins in response to different cellular stresses, such as oxidants, heavy metals, elevated temperature, and bacterial or viral infections. Two isoforms, HSF4a and HSF4b, resulting from two alternative spliced regions in exons 8 and 9 have been reported 2021. HSF4a actively represses transcription of other heat shock factor genes by directly binding to the heat shock elements. HSF4b, which has 30 additional amino acids, acts as an activator of transcription. It has been demonstrated that the additional 30 amino acids are responsible for this activity 2223.

Here, we report the first HSF4 nonsense mutation in a large Afghani/Pakistani family. DNA sequencing revealed the transition c.1213C > T (p.Arg405X) in exon 11. This mutation is predicted to cause a premature termination, presumably resulting in a complete loss of function of the aberrant HSF4 protein in affected homozygotes. This causes a severe phenotype with autosomal recessive congenital cataracts. Indeed, the HSF4 gene that has been reported responsible both for autosomal dominant and autosomal recessive cataracts. The association of the HSF4 gene with two different modes of inheritance for congenital cataracts can at least in part be explained by the location and severity of the mutations. Interestingly, all known dominant mutations in HSF4 lie within the α-helical DNA binding domain, whereas the recessive mutations lie outside this highly conserved functional domain. To-date, only eight different mutations in HSF4 have been reported, three of them are causing autosomal dominant cataracts, three are causing autosomal recessive cataracts, and two mutations were found in sporadic cases. In addition to this novel nonsense mutation in exon 11, the already known mutations in HSF4 comprise six missense alterations, one deletion, and one splice site mutation (Human Mutation Gene Database, http://www.hgmd.cf.ac.uk/). Exon 11 is encoding the so called DHR domain of the HSF4 protein, the importance of which was already demonstrated by identifying a 5'splice site mutation in intron 12 causing skipping of exon 12 in a large Tunisian family 8. Two previously identified mutations (p.Arg175Pro and c.595_599delGGGcc) in Pakistani families showed recessive cataracts and are located in the HR-A and HR-B domains of the HSF4 protein again demonstrating the extreme genetic heterogeneity of the Pakistani population 24. Historically, Pakistan has experienced not only internal migrations across the Indus valley but also successive waves of migrations from northwest. In more recent times, the region experienced massive migrations of refugees from Afghanistan. So, the Pakistani society is composed of many ethnic groups and many of these are common to populations of India, Afghanistan, Bangladesh and Iran. Concerning the clinical phenotype caused by mutations in this transcription factor, it is unclear, why mutations in HSF4 that is also expressed in tissues including the heart, muscle, lung and brain should cause only nonsyndromic cataracts 38 and more comprehensive studies will be needed to answer this question.

As a conclusion, we have shown the first nonsense mutation in HSF4 causing autosomal recessive cataracts in a large consanguineous family from Pakistan. The identification of this and additional mutations enables proper genetic diagnostics and counseling in affected families and may lead to a better understanding of the structure and function of HSF4 in health and disease.

The authors declare that they have no competing interests.

NS performed the clinical ascertainment and linkage analysis, IG performed the mutation screening, NK helped in linkage analysis and clinical evaluation, AMC provided funds for this study, CK analyzed and supervised the mutation identification, JA designed, and supervised the study, CK and JA drafted the manuscript. All authors read and approved the final manuscript.