Journal of Heredity Advance Access originally published online on July 4, 2007
Journal of Heredity 2007 98(5):531-533; doi:10.1093/jhered/esm043
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Mutation in HSF4 Associated with Early but Not Late-Onset Hereditary Cataract in the Boston Terrier
From the Animal Health Trust, Lanwades Park, Newmarket, Suffolk, CB8 7UU, UK (Mellersh, McLaughlin, Pettitt, Vaudin, and Barnett); and the Department of Veterinary Science, University of Kentucky, Lexington, KY 40546-0076 (Graves and Ennis)
Address correspondence to C. S. Mellersh at the address above, or e-mail: cathryn.mellersh{at}aht.org.uk.
Primary hereditary cataract (HC) is one of the most common disorders in purebred dogs and is a leading cause of blindness. Boston Terriers suffer from 2 distinct forms of HC which occur at different ages and which are different in their appearance and progression. Early-onset hereditary cataract (EHC) affects dogs within the first few months of life, is always progressive and bilateral, and results in total blindness, whereas late-onset hereditary cataract (LHC) in general affects dogs over the age of 3 and is more variable in its clinical phenotype, age of onset, progression, and the degree to which vision is impaired. A mutation in HSF4 has recently been reported in a small number of Boston Terriers affected with EHC. In this study, we analyzed 22 additional Boston Terriers affected with early-onset cataract to confirm that the HSF4 mutation is causative for this form of cataract in this breed. In addition, we analyzed 40 Boston Terriers that were either clinically clear or affected with LHC for the presence or absence of the HSF4 mutation. By also sequencing HSF4 in dogs affected with LHC, we conclude that HSF4 is not associated with the development of the late-onset form of cataract and that the 2 forms of cataract in this breed are therefore genetically discrete conditions.
Primary hereditary cataract (HC) is one of the most common genetic disorders in purebred dogs, affecting at least 70 breeds and representing a leading cause of blindness in dogs (Helper 1989; Rubin 1989; Davidson and Nelms 1999; Slatter 2001). Hereditary forms of canine cataract are also often referred to as "primary cataracts," to distinguish them from "secondary cataracts" that follow other eye diseases, such as progressive retinal atrophy, retinal dysplasia, and glaucoma. HCs usually display marked breed specificity with respect to their ophthalmic appearance, part of the lens involved, age of onset, rate of progression, and degree of bilateral symmetry. In North America, primary HCs have the highest prevalence of any ophthalmic disease studied to date, and in a recently reported retrospective study, 58 breeds were identified as having a prevalence greater than the 1.61% observed in mixed breed dogs, although the prevalence of primary cataract differs greatly between breeds (Gelatt and Mackay 2005).
The occurrence of primary cataract in the Boston Terrier is very common in the United States, with 11.11% of all dogs of this breed presenting to North American teaching hospitals between 1964 and 2003 being diagnosed with primary HC (Gelatt and Mackay 2005). Two distinct forms of HC are known to affect the Boston Terrier. The first form, originally described in 1978 (Barnett 1978), has a very early age of onset. Cataracts occur bilaterally and can be diagnosed as early as 8–12 weeks of age but are not congenital. Opacities begin at the suture lines and involve the nucleus of the lens as well as cortical opacities. By 6 months, most of the lens are involved, and defective vision and obvious cataracts occur usually between 9 and 15 months of age with further progression and maturity of the cataract between 2–4 years of age. This type of cataract has a recessive mode of inheritance. In our study, we refer to this form of HC as "early-onset hereditary cataract" (EHC) to distinguish it from the second type of HC that occurs in the Boston Terrier (Curtis 1984) for which the age of onset is generally 3–6 years. We refer to this later form of HC as "late-onset hereditary cataract" (LHC). Late-onset cataracts may initially occur unilaterally and rarely show bilateral symmetry. However, if annual ophthalmologic examinations are performed, it is usually observed that the other eye will also develop punctate opacities, with subsequent progression to "spoke-type" cataracts. These cataracts are invariably of cortical type and primarily anterior although the equatorial and posterior cortex can also be affected (Canine Eye Registry Foundation [CERF] Boston Terrier Statistics 1996–2005.) Nuclear involvement has not been noted. Progression is slow, and visual impairment is not usually noticed until the dog is 10 years of age or older.
The first causative mutation for HC was recently identified in exon 9 of HSF4 (Mellersh et al. 2006) in the Staffordshire Bull Terrier. The condition is simply referred to as HC in this breed and is clinically very similar to EHC in the Boston Terrier. The mutation is an insertion that results in a premature stop codon and truncated protein. Because of the similarity between HCs in the Staffordshire Bull Terrier and the Boston Terrier, 5 Boston Terriers were also tested for the presence of this mutation as part of the initial study. Two affected dogs were homozygous for the mutation and 3 carriers were heterozygous, indicating that the causative mutation for EHC in Boston Terriers might be identical to the one found in Staffordshire Bull Terriers.
The object of this study was to confirm the association of the exon 9 HSF4 mutation with EHC in the Boston Terrier by analyzing additional samples from Boston Terriers affected with EHC, known carriers of EHC, unaffected siblings, dogs affected with LHC, and normal older dogs. For the purposes of this study, EHCs are defined as those occurring before 1 year of age, LHCs as those occurring between 3–6 years of age, and a normal dog is defined as one that is clear of cataracts at age 8 years or older. Because a different mutation, specifically a deletion, at the same location in HSF4 was identified in Australian Shepherds affected with cataracts, we also evaluated this gene as a candidate gene for LHC in the Boston Terrier.
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Genomic DNA Isolation
Genomic DNA was isolated from frozen whole blood collected in ethylenediaminetetraacetic acid (EDTA) or from buccal swabs using the Puregene DNA isolation kits (Gentra Systems, Minneapolis, MN) according to manufacturer's directions. DNA was resuspended in Tris–EDTA buffer at a concentration of 25 ng/µl.
Clinical Diagnosis
Blood samples or cheek swabs were submitted by owners accompanied by statements from veterinarians or CERF forms from American College of Veterinary Ophthalmology Diplomates to verify the clinical characteristics of the cataracts. Sample submission forms requested confirmation that the animal was not diabetic.
Genotyping for Presence of HSF4 Mutation
Genomic DNA from 72 Boston Terriers was genotyped with primers (forward primer: Vic-CGAGTGTGACTTCTGCGTGA, reverse primer: GTTCAGGCTGTTGGGCATT) flanking the previously reported HSF4 mutation, as previously described (Mellersh et al. 2006).
Sequencing of HSF4 Exons from Genomic DNA
Coding exons of HSF4 were sequenced in 5 Boston Terriers affected with LHC and 2 Boston Terriers over the age of 8 that were clinically clear of cataracts. Details of the 5 affected dogs are as follows:
- Sample 5: Bilateral cataract, diagnosed by regular veterinarian at 6 years of age.
- Sample 6: Bilateral, cortical cataract, diagnosed at 4 years.
- Sample 8: Bilateral, cortical cataract, diagnosed at 6 years. Dam of Sample 6.
- Sample 46: Bilateral, regional cortical cataract, diagnosed at 5 years. Obligate carrier of EHC.
- Sample 47: Bilateral, cortical cataract, diagnosed at 5 years. Obligate carrier of EHC.
- Sample 6: Bilateral, cortical cataract, diagnosed at 4 years.
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HSF4 Exon 9 Mutation
DNA samples from 72 Boston Terriers were genotyped for the previously reported insertion in exon 9 of HSF4 (CFA5 g.85286582_85286583insC) (Mellersh et al. 2006). Results could not be obtained for 10 of these samples. Results for the remainder are shown in Table 1.
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In all, 22 of the dogs had been diagnosed with HC before the age of 1 and were therefore defined as affected with EHC. Sixteen dogs had been diagnosed with cataract over the age of 3 and were therefore defined as affected with LHC, and 8 dogs were clinically clear of cataract above the age of 8. Twenty of the 22 EHC-affected dogs were homozygous for the insertion in HSF4 exon 9, which is the same mutation that has been shown to be causative for HCs in the Staffordshire Bull Terrier. One affected dog was homozygous for the wild-type allele, and one was heterozygous. No other relatives of these 2 dogs were available. Of the 19 dogs that were found to be heterozygous for the mutation, 9 of these were known carriers of EHC. None of the known EHC carriers were homozygous for either the wild-type or mutated HSF4 allele. Eight older dogs clear of cataracts were genotyped, 4 of them were homozygous for the wild-type allele, and 4 were heterozygous for the mutation.
Exon Scan of HSF4 in Boston Terriers Affected with LHC
Five dogs affected with LHC were selected for an exon scan to identify other mutations in HSF4 that might be causative for the LHCs in the Boston Terrier.
We also sequenced the coding exons of HSF4 in their entirety in 2 Boston Terriers over the age of 8 that were clinically clear of HC and a miniature long-haired dachshund, a breed not known to be affected with HC. The sequences were compared with those of 1) the dog (a Boxer) whose genome was used to determine the whole canine genome sequence (http://www.ensembl.org/Canis_familiaris/index.html) and is presumed to be clear of HC and 2) a Staffordshire bull Terrier clear of HC that had been analyzed previously (Mellersh et al. 2006). We identified 2 single-nucleotide polymorphisms within introns of HSF4, neither of which have been identified in the whole-genome sequence (http://www.ensembl.org/Canis_familiaris/geneseqview?db=core;gene=ENSCAFG0000002037). The first is an A/G polymorphism located 54 nucleotides from the start of intron 4 (CFA5 g.85,288,164) and the second is a C/T polymorphism located 71 nucleotides from the start of intron 9 (CFA5 g.85286051). Neither polymorphism was located within the coding region of HSF4, and both were identified in both affected and unaffected dogs.
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By analyzing Boston Terriers affected with EHC, we have confirmed that a previously reported insertion in exon 9 of HSF4, that is associated with HC in the Staffordshire bull Terrier, is also causative for EHC in the Boston Terrier. Twenty out of 22 (91%) Boston Terriers affected with HC before the age of 1 were homozygous for the mutation, providing compelling evidence that the mutation is responsible for the early-onset form of cataract in this breed, which has a recessive mode of inheritance. The HSF4 mutation is an insertion of a single C nucleotide within a homopolymer tract of 10 C nucleotides that exist in the wild-type gene.
Two dogs that were diagnosed by a veterinarian as being affected with HCs before the age of 1 were not homozygous for the mutation as we would have expected; one was heterozygous and the other was homozygous for the wild-type HSF4 allele. The cause of these dogs' cataracts is unexplained. We did not resequence the whole of HSF4 in these dogs, so cannot rule out the existence of another mutation within the gene that is responsible for their disease, or indeed a mutation in a gene other than HSF4 that is causing a genetically distinct form of HC in these dogs. Neither dog was examined with a slit lamp biomicroscope, so confirmation that their cataracts were of the same type as those observed in the other dogs was not available.
We examined 16 dogs affected with LHC, 11 of which were homozygous for the wild-type allele of HSF4. This result provides the first evidence that LHC in the Boston Terrier is not caused by the same mutation as EHC. The absence of alternative pathogenic mutations within any of the coding exons of HSF4 in 5 dogs affected with LHC confirms that HSF4 is not involved in the development of LHC in the Boston Terrier and that the two forms of HC in this breed are genetically as well as clinically distinct. Clarification that the 2 forms of cataract are not related is important for breeders, who may have both forms segregating in their lines. Confirmation that EHC in the Boston Terrier is caused by the same mutation as HC in the Staffordshire bull Terrier means that Boston Terrier breeders can take advantage of diagnostic tests that are available to identify their breeding animals as affected, carrier, or clear of EHC and use the information to successfully breed dogs that are unaffected by the disease.
The late-onset form of HC in the Boston Terrier is quite variable, in terms of its clinical phenotype, age of onset, and speed of progression although pedigree information provides compelling evidence that LHC is an inherited condition, and the availability of samples from both affected males and females (data not shown) indicates the trait is autosomal. This study confirms that LHC is not a variant of EHC, caused by, for example, a dosage effect of a single mutation. We cannot, however, exclude the possibility that genetically distinct forms of LHC exist within the Boston Terrier population that explain the clinical variation that is observed. A genome scan with samples from pedigrees segregating LHC would be expected to reveal regions of the genome linked to this form of the disease and hopefully lead to identification of causative mutations.
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This work was funded by the Kennel Club Charitable Trust, the Boston Terrier Club of America, and individual Boston Terrier owners.
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This paper was delivered at the 3rd International Conference on the Advances in Canine and Feline Genomics, School of Veterinary Medicine, University of California, Davis, CA, August 3–5, 2006.
Corresponding Editor: Elaine Ostrander
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