Nature Genetics
31, 276 - 278 (2002)
Published online: 24 June 2002; | doi:10.1038/ng921
Mutant DNA-binding domain of HSF4 is associated with autosomal dominant lamellar and Marner cataractLei Bu1, 2, 9, Yiping Jin3, 9, Yuefeng Shi1, Renyuan Chu3, Airong Ban4, Hans Eiberg5, Lisa Andres6, Haisong Jiang1, Guangyong Zheng1, Meiqian Qian1, Bin Cui1, Yu Xia1, Jing Liu2, Landian Hu1, Guoping Zhao1, Michael R. Hayden6, 7
& Xiangyin Kong1, 81 Shanghai Research Center of Biotechnology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200233, People's Republic of China. 2 University of Science and Technology of China, Hefei, People's Republic of China. 3 Department of Ophthalmology, EENT Hospital, Medical College of Fudan University, People's Republic of China. 4 Department of Ophthalmology, People's Hospital of Yichuan, Luoyang, People's Republic of China. 5 University of Copenhagen/Panum Institute, Copenhagen, Denmark. 6 Xenon Genetics, Burnaby, British Columbia, Canada. 7 University of British Columbia/Centre for Molecular Medicine and Therapeutics, Vancouver, British Columbia, Canada. 8 Health Science Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and Shanghai Second Medical University, Shanghai 200025, People's Republic of China. 9 These authors contributed equally to this work.
Correspondence should be addressed to Xiangyin Kong xykong@srcb.ac.cnCongenital cataracts cause 10−30% of all blindness in children, with one-third of cases estimated to have a genetic cause1. Lamellar cataract is the most common type of infantile cataract2. We carried out whole-genome linkage analysis of Chinese individuals with lamellar cataract, and found that the disorder is associated with inheritance of a 5.11-cM locus on chromosome 16. This locus coincides with one previously described for Marner cataract3. We screened individuals of three Chinese families for mutations in HSF4 (a gene at this locus that encodes heat-shock transcription factor 4) and discovered that in each family, a distinct missense mutation, predicted to affect the DNA-binding domain of the protein, segregates with the disorder. We also discovered an association between a missense mutation and Marner cataract in an extensive Danish family. We suggest that HSF4 is critical to lens development.
We identified a large Chinese family with autosomal dominant lamellar cataract (Fig. 1a). The lens opacity is perinuclear-shaped with a transparent embryonic nucleus (Fig. 1b). The earliest age of observed onset in this family is 15 months, and the disease allele shows biased transmission (P = 0.02). In addition, we studied a Danish kindred of nine generations3,
4 affected by Marner cataract—which is characterized by zonular stellate lens opacity with an anterior polar opacity and early childhood onset.
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The gene HSF4 is expressed in eye (Fig. 2a) and regulates the expression of heat-shock proteins (HSPs)5,
6, which may be important components of lens development7,
8; we therefore believed it to be a good candidate for the 'disease' gene. Besides HSF4, there are several other candidates, including SNTB2 (syntrophin,  2) and PSMB10 (proteasome subunit, -type 10). It has at least two alternatively spliced transcripts, HSF4a and HSF4b, both lacking the suppressor domain that would normally inhibit the formation of homotrimers (required for DNA binding). These two transcripts are therefore thought to bind constitutively to DNA5,
6. Previous studies have indicated that HSF4a suppresses the constitutive expression of heat-shock genes5, whereas HSF4b stimulates constitutive and inducible transcription of heat-shock genes6. The HSF4b transcript is predominantly expressed in mouse tissues, including testis6.
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We screened all 13 exons and the flanking intronic sequences of HSF4 by direct sequencing of PCR products generated from genomic DNA (Figs 2b and 3a). We found a T C transition at nucleotide 348 in all of the affected individuals of the large Chinese family, but not in the unaffected members of this family or in 300 unrelated normal controls (Fig. 2b). This mutation is predicted to result in a Leu115Pro substitution in the DNA-binding domain of HSF4 (Fig. 3a). The leucine residue specified by codon 115 is conserved among yeast, Caenorhabditis elegans, Drosophila melanogaster, mouse, rat and human and within the mostly conserved DNA-binding domains of the heat shock factors (Fig. 3b).
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A prediction derived from the PepTool Lite program (BioTools) indicated that the Leu115Pro mutation disrupts the  -helix structure of the DNA-binding domain. Moreover, it has been reported that the Leu269Pro substitution of HSF1 in yeast, which corresponds to the Leu115Pro substitution we identified in human HSF4, severely affects the DNA-binding activity and transactivation activity of HSF1 (ref. 9). The Leu269Pro substitution probably disrupts the orientation of the DNA-binding domain9. It therefore seems likely that the Leu115Pro substitution is the mutation underlying cataract disease. We identified a CT transition at nucleotide 362 in exon 3 in affected members of the Danish family. The mutation segregates with the disease in the family and is expected to result in a substitution of a highly conserved Arg120 residue by cystine (Figs 2b and 3). This mutation was not found in 100 control chromosomes of individuals of Scandinavian origin.
We also searched for mutations of HSF4 in DNA derived from 69 sporadic cases of individuals with cataract disease. Of these 69 individuals, at least 7 are affected with lamellar cataract. We found two missense mutations in two individuals with infantile cataracts. One mutation, a CA transversion found in exon 1 of a 20-year-old individual, results in an Ala20Asp substitution in the DNA-binding region of HSF4 (Figs 2b and 3a). This individual's parents did not have this mutation; thus, this is a de novo mutation (Fig. 2b). The second missense mutation, identified in a sporadic case of a unilateral cataract, is an AG transition resulting in an Ile87Val substitution that is also within the highly conserved DNA-binding domain of HSF4 (Figs 2b and 3). The 46-year-old father of this individual carries the same mutation and shows a mild cataract similar to senile cataracts with cortical water clefts and lamellar separation. These two mutations were not found in 300 normal controls. That all four mutations are located in the DNA-binding region (Fig. 3a) further highlights the importance of this domain.
Previously, people defined cataracts phenotypically by the location and structure of the opacities, including size, pattern, number and shape, and by the age of onset. However, opacities may change morphologically with age, even within the same family, and therefore the classification has been somewhat arbitrary.
The cataracts in the Chinese and Danish families examined in this study had some morphological differences, yet are caused by mutations in the same gene. We also observed some variability in age of onset and severity. An individual with an interstitial deletion involving 16q23 manifested with bilateral cataract has previously been described10, suggesting that loss of function of HSF4 may also underlie the cataract disease in this particular individual. Clinical heterogeneity is evident in affected individuals with mutations in HSF4, but childhood onset appears most frequently.
The HSF4 protein regulates the expression of several heat-shock proteins, including HSP70, HSP90a and HSP275,
6. In both the embryonic and adult lens, heat-shock proteins are either widely distributed or regionally specific7,
8, suggesting that they are required in lens development7,
8. Heat-shock proteins act as molecular chaperones involved in protein synthesis, assembly, translocation, folding, repair and degeneration11,
12. Abnormalities in the structure or expression of such proteins may lead to cataract, as has been shown for the small heat-shock protein B-crystallin13. This study provides for the first time evidence that mutation of HSF4 causes human cataract disease. In mouse eye, expression of Hsf4, primarily in the form of Hsf4b, is high (Fig. 2a). Thus, mutations might reduce the activator ability of HSF4 and deteriorate the expression of HSPs, resulting in lens opacity.
Various stresses can cause protein damage to the lens and eventually promote the development of age-related cataract14. With aging, the chaperone activity of HSPs is decreased8. This may be a result of the decreased DNA-binding activity15 or functional disruption of HSFs. The functional impairment of HSF4 may thus also be associated with susceptibility to age-related cataract formation.
Methods
Clinical data and sample collection.
All family members received careful examination, including tests of visual function, such as Snellen visual acuity and corrected visual acuity, in addition to slit-lamp and fundus examinations with the dilated pupil. After obtaining informed consent, we obtained 5 ml peripheral blood from each family member for linkage analysis. We extracted genomic DNA using a Qiagen blood kit.
Linkage and haplotype analyses.
We followed the described methods16 to carry out genotyping and data collection. In the linkage analysis, we modeled the disease as an autosomal dominant inheritance with complete penetrance and set the affected allele frequency as 0.00001 and the marker allele frequencies to be uniformly distributed. We carried out two-point linkage analysis using the MLINK program from the LINKAGE v. 5.10 software package17 and conducted multipoint analyses using FASTLINK v. 4.1P (refs 18,19). We carried out pedigree and haplotype construction using Cyrillic v. 2.02 software.
Mutation detection.
We designed nine pairs of primers to amplify HSF4 (primer sequences are available upon request). We amplified PCR products from genomic DNA of the family members and normal controls and sequenced the PCR products from both directions using an ABI-3100 sequencer.
Gene expression and analysis of spice variants.
We prepared total RNA from mouse eye, testis, brain and heart using a Qiagen Kit following the manufacturer's directions. We determined the expression level and splicing pattern of mouse Hsf4 by RT-PCR, using one pair of primers spanning exons 5 to 9 with actin control primers6. We ran the PCR product on a 1.0% agarose gel, cut the major band and sequenced it after purification.
Multiple-sequence alignment database.
We obtained the multiple-sequence alignment data from Incyte Genomics (http://www.incyte.com/proteome/HumanPD/HSF4.html).
GenBank accession number:
HSF4 cDNA sequence, D87673.
Received 8 March 2002; Accepted 29 May 2002; Published online: 24 June 2002.
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Acknowledgments
We are grateful to all of the individuals described here for their contribution to this study. We thank F. Francis and Z. Chen for critical reading of this manuscript, and C. Lopez-Otin and F. Hejtmancik for providing samples for mutation analysis. This work was supported by the National High Technology "863" Programs of China, the National Science Fund for Distinguished Young Scholars and Xenon Genetics.
Competing interests statement:
The authors declare that they have no competing financial interests. |
