A New A1-Crystallin Splice Junction Mutation in Autosomal Dominant

A New ␤A1-Crystallin Splice Junction Mutation in Autosomal Dominant Cataract

J. Bronwyn Bateman,1,2,3 David D. Geyer,1,3 Pamela Flodman,4 Meriam Johannes,3 James Sikela,5 Nicole Walter,5 Ana Teresa Moreira,6 Kevin Clancy,3 and M. Anne Spence4

PURPOSE. To map the locus for autosomal dominant cataracts (ADCs) in a Brazilian family using candidate gene linkage analyses, describe the clinical variability, and identify potential mutations in the human ␤A1-crystallin gene (CRYBA1), a candidate gene identiﬁed through linkage studies demonstrating cosegregation with markers on chromosome 17.

METHODS. Members of a Brazilian family with ADC were studied. Clinical examinations and linkage analyses with polymerase chain reaction (PCR) polymorphisms of 22 anonymous markers and 2 within the neuroﬁbromatosis type 1 gene were performed; two-point lod scores were calculated. DNA sequences of all 6 exons and 12 exon–intron boundaries of the ␤A1-crystallin gene, a proximal candidate gene mapped to 17q11.1-q12 in one unaffected and two affected individuals, were screened and new variants assessed for cosegregation with the disease.

RESULTS. Affected individuals exhibited variable expressivity of pulverulent opacities in the embryonal nucleus and sutures; star-shaped, shieldlike, or radial opacities in the posterior embryonal nucleus; and/or midcortical opacities. All known loci for ADC in this family on chromosomes 1 and 13 were excluded. A positive lod score on chromosome 17 was calculated. This ADC locus was mapped to two potential regions on the long arm with an intervening recombination. The only known candidate gene in these regions was ␤A1-crystallin. Three previously unreported single nucleotide variants were found in this gene, one in the donor splice junction site of intron C. This variant was found in all affected members and is presumed to be the causative mutation.

CONCLUSIONS. An ADC locus was mapped in a Brazilian family with variable expressivity to either 17q23.1-23.2 or 17q11.1-12 based on linkage analyses. Analyses of DNA sequences of the ␤A1- crystallin gene in this family revealed three new variants, one of which is within a donor splice junction and cosegregates with affected members. (Invest Ophthalmol Vis Sci. 2000;41: 3278–3285)

ediatric cataracts may be caused by intrauterine embry- within the lens, shape, size, color, and the presence or absence opathies and single gene defects or may be associated of refractivity. Some have been named for the affected family with chromosomal rearrangements. Some cataracts, par- such as the Coppock cataract6 and others for the author re- P 7 ticularly unilateral, are of unknown cause. The disease is clin- porting the family such as the Marner cataract. Despite at- ically and genetically heterogeneous. The prevalence of con- tempts to clinically categorize hereditary cataracts, there is genital cataracts has been estimated to be 2.2 per 10,000 poor correlation of phenotype with genetic loci. births,1 and the incidence in some countries has been reduced Previous studies have identified at least 13 loci for ADC by immunization programs.2 Most autosomal dominant cata- based on linkage analyses for which mutations in seven genes ract (ADC) forms are congenital and isolated. Phenotypic vari- have been implicated. Three loci have been identified on chro- ability of ADC has been documented among and within fami- mosome 17, one of which is attributed to a mutation of the 8 9,10 lies, and some forms are progressive.3–5 Generally, the ␤A1 (formerly ␤A3/A1)-crystallin gene (CRYBA1). We cataracts are bilateral and characterized on the basis of location studied a Brazilian family with ADC with variable expressivity of the embryonal, pulverulent variety and mapped the gene using linkage analysis to two potential regions, 17q11.1-12, containing the ␤A1-crystallin gene (17qcen-q23)11,12 and a From the Departments of 1Ophthalmology and 5Pharmacology, and 2The Children’s Hospital, the University of Colorado School of second in a relatively broad region in 17q22-24.1. We identified Medicine; the 3Eleanor Roosevelt Institute, Denver, Colorado; the 4De- three new variants within the ␤A1-crystallin gene in this fam- partment of Pediatrics, University of California, Irvine; and the 6De- ily, one of which is within the donor splice junction of intron partment of Ophthalmology, University of Curitiba School of Medicine, C (letter nomenclature in common usage as originally desig- Brazil. nated13; intron 3) following exon 3 and is present in all af- Supported by Grant EY 08282 National Eye Institute (JBB). Submitted for publication February 28, 2000; accepted April 11, fected family members. 2000. Commercial relationships policy: N. Corresponding author: J. Bronwyn Bateman, Department of Oph- MATERIALS AND METHODS thalmology, University of Colorado School of Medicine, 4200 East Ninth Avenue, Box B204, Denver, CO 80264. The study adhered to the tenets of the Declaration of Helsinki [email protected] for research involving human subjects. The family was ascer-

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FIGURE 1. Pedigree of the ADC-affected family with haplotypes for the most relevant markers. Two regions, 17q23.1-q23.2 and 17q11.1-q12, are without recombinants. Affected individuals are shaded black; the proband is identiﬁed by an arrow. Filled boxes: disease haplotype inherited from founder (number 1); open boxes: all other haplotypes; thick lines connecting ﬁlled and open boxes: regions where inheritance cannot be determined.

ϫ ϫ tained through the Ophthalmology Clinic of the University of PCR buffer, and 1 Q-Solution (1 PCR buffer: KCl, NH4SO4, Curitiba School of Medicine in Curitiba, Brazil. The proband and Tris-HCl, [pH 8.7]; Qiagen, Santa Clarita, CA) and 1 to 3

(Fig. 1) underwent cataract extraction. On the basis of the mM MgCl2. Reactions were cycled in a twin-block system pedigree and male-to-male transmission, autosomal dominant cycler (EriComp; San Diego, CA) as follows: an initial 5-minute inheritance was demonstrated. Informed consent, with Univer- denaturation at 95°C, then 35 cycles at 1 minute at 94°C, 1 sity of Colorado Institutional Review Board approval, was ob- minute at 55°C, and 1 minute at 72°C, finishing with a 7-minute tained with translation provided by one of the authors (ATM). extension cycle at 72°C and a final hold of 4°C. PCR products Nineteen individuals participated. No other diseases aside from were mixed with a loading cocktail containing 50% (vol/vol) of age-related disorders were identified by history. Affected status deionized formamide and 0.5 ␮l of internal lane standard was determined by pupillary dilation and evaluation of lenses (ROX; Perkin–Elmer Applied Biosystems), denatured for 5 min- at the slit lamp biomicroscope or by a history of cataract utes at 95°C and immediately placed on ice. The product was extraction (JBB, ATM). loaded onto a 6% sequencing gel (Burst Pak; Owl Scientific, Blood samples were collected in EDTA and leukocyte Cambridge, MA) and run on a DNA sequencer (Prism model genomic DNA extracted.14 Sequence-tagged sites (STSs) of the microsatellite variety were amplified using polymerase chain 373; Perkin–Elmer Applied Biosystems). The data were col- reaction (PCR) with fluorescently labeled primers or by incor- lected and analyzed by computer (Genescan 672 Collection poration of fluorescent dUTPs ([F]dUTPs). For [F]dUTP ampli- Software ver. 1.1 and Genescan Analysis Software ver. 2.1; PE fication, the protocol recommended by the manufacturer was Applied Biosystems) For labeled primers (Research Genetics, modified (Perkin–Elmer Applied Biosystems, Foster City, CA) Huntsville, AL, or Molecular Resource Center, National Jewish as follows. Twenty-five nanograms of template DNA was used Medical and Research Center, Denver, CO), the process was in a 10-␮l reaction with 200 ␮m of dNTPs, 4 picomoles of identical except for the use of the following: 250 ␮M dNTPs, forward and reverse primers, 0.5 to 2.0 ␮M [F]dUTP (Perkin– 2.4 picomoles of fluorescently labeled forward and reverse Elmer Applied Biosystems), 0.125 U Taq DNA polymerase in primers, and 2.4 picomoles of TAMRA (Perkin–Elmer Applied

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TABLE 1. Lod Scores between Chromosome 17 DNA Markers and ADC Loci in a Brazilian Family

Weizmann17 Genethon23 Marshﬁeld24 ␪ ␪ Marker Zmax male female (Mb) (cM) (cM)

D17S1308 0.6 * 0.07 2.20 1 0.63 D17S849 0.00 0.50 0.50 4.40 0.63 D17S1298 0.00 0.50 0.50 8.09 10.72 D17S796 1.82 0.00 0.00 8.74 16 14.69 D17S786 0.08 0.34 0.50 17.79 19 17.38 D17S974 0.30 * 0.00 20.66 22.24 D17S1303 0.00 0.50 0.50 20.85 23.56 D17S969 centromere 0.00 * 0.50 21.88 26–34 27.75 D17S953 0.12 0.01 0.01 31.74 46 43.01 D17S805 2.71 0.00 0.00 32.90 50 47.00 CRYBA1 splice site variant 4.80 0.00 0.00 D17S1294 2.07 * 0.00 50.07 50.74 D17S798 (NF1) 2.53 0.00 0.00 53.75 53.41 53.0A/53.0B (NF1) 1.64 0.00 0.06 D17S1293 1.45 0.00 0.15 56.24 61–63 56.48 D17S946 1.23 0.00 0.19 59.09 66 60.40 D17S1814 2.67 0.00 0.08 59.93 68 61.48 D17S800 3.61 0.00 0.00 63.12 69 62.01 D17S1299 3.48 0.00 0.00 64.58 62.01 D17S932 0.54 * 0.12 67.97 63.09 ATC6A06 0.08 * 0.34 69.05 66.85 D17S809 0.31 0.00 0.50 76.32 82 74.45 ATA43A10 1.08 0.00 0.34 89.32 D17S1301 0.90 0.00 0.50 80.04 100.02 D17S1290 1.68 0.00 0.23 83.58 82.00 D17S802 0.90 0.00 0.50 90.29 117 106.8 D17S836 0.08 0.34 0.49 91.30 123 112.92 D17S1806 0.08 0.34 0.50 91.41 114.41

Full table available upon request. * No informative meioses.

Biosystems) as internal lane standards at the same concentra- mated ﬂuorescence sequencer (model 373; Perkin–Elmer Ap- tion. plied Biosystems). For screening, one unaffected (number 16) Markers were chosen for analysis on the basis of previ- and two affected (numbers 13 and 14) individuals were com- ously reported linkage with an ADC locus or evidence of suggestive linkage in a previous family studied in our laboratory. The following 23 markers were tested initially: D1S1622, TABLE 2. PCR Primers Used for the Ampliﬁcation and Sequencing of D1S1665, D1S2130, D1S1669, D3S2398, D3S2427, D3S2436, the ␤A1-Crystallin Exons D8S592, D8S1119, D8S1128, D8S1132, D8S1179, D13S175, Fragment D13S1236, D14S606, D14S610, D14S617, D14S749, D17S796, Exon Sequence Size D19S589, D19S254, D19S601, and ATA43A10. After suggestive ϭ ␪ ϭ ␪ ϭ 1F5Ј-GAGGCCGCAGGGCTATAAAG-3Ј 135 bp linkage with D17S796 (Zmax 1.82; m f 0; where Zmax ␪ ␪ R 5Ј-TGGGCTGCAGGGCAAGGAG-3Ј represents maximum lod score, and m and f represent male recombination fraction value and female recombination frac- 2F5Ј-CCAAGAGGCCACATCATTCG-3Ј 161 bp tion value, respectively), additional markers in the region were R 5Ј-CGTGTGTGCAGCATTGTCAC-3Ј tested (Table 1). The marker loci were localized to chromo- 3F5Ј-TGATGTTCTAGCTCTCTTGCG-3Ј 189 bp somal regions based on data from the Cooperative Human R 5Ј-CTGCGGTTCTGCGGAAGTC-3Ј Ј Ј Linkage Center,15 the Genome Database (GDB),16 and the F (int) 5 -ATGGAGTTCACCAGCTCCTG-3 Sequencing 17 18 only Weizmann Institute and Whitehead Institute databases. 4F5Ј-GAACACCATGAACAAACACTAC-3Ј 205 bp For linkage analyses, pedigree and genotype data were R 5Ј-ACGGAAGTGGAAATTTCAGAG-3Ј analyzed with LIPED (freely available from Jurg Ott at http:// 5F5Ј-GAATGATAGCCATAGCACTGG-3Ј 127 bp 19 linkage.rockefeller.edu). Lod scores were calculated using R 5Ј-CAGAGATCTCCCACTGGCG-3Ј 16 published allele frequencies or estimates from 30 unrelated 5F5Ј-CTTGCAAGCCATGGGCTGG-3Ј 157 bp individuals outside this family. A gene frequency of 0.0001 and R 5Ј-ATCCGATACGTATGGATATCTG-3Ј full penetrance were assumed for the cataract locus; two-point 6F5Ј-CAACTACCACTTGGTTGCAG-3Јϳ335 bp ␪ ␪ lod scores were calculated for a full range of m and f values. R 5Ј-CAAGATATACTGATACCCACG-3Ј PCR primers (Only DNA, Midland, TX) were designed20 6F5Ј-CAACTACCACTTGGTTGCAG-3Јϳ711 bp (Table 2) to amplify all six exons and intron–exon junctions in R 5Ј-CGTGGCGAACAAGATAGCG-3Ј ␤ the A1-crystallin gene (CRYBA1), a candidate gene (GenBank Ј Ј 13,21,22 6F5 -CATCTCATACCATTGTGTTGAG-3 315 bp accession M14301-6). PCR products were sequenced R 5Ј-ACTTTCTAGAGTGCTTAGCAAG-3Ј from both directions using a dye terminator cycle sequencing kit (Prism FS; Perkin–Elmer Applied Biosystems) and an auto- F, forward; R, reverse; int, internal.

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pared. Variants of potential biologic significance were assessed in all members of the family. After identification of several variants and a probable mutation, the newly identified sequences were studied in five normal and unrelated Brazilians (10 chromosomes).

RESULTS

Adults in the family reported that cataracts were present from early in life. Historical information was limited, and there were no previous records aside from those of the proband. Most affected individuals who had not undergone cataract extraction had opacities that appeared to be congenital, including clustering of pulverulent opacities in the embryonal nucleus and sutures and star-shaped, shieldlike, or radial opacities in the posterior embryonal nucleus. Midcortical opacities appeared to be postnatal. None of the affected individuals had nystagmus. Markers on chromosomes 1, 3, 8, 13, 14, and 19 were FIGURE 2. Forward sequence analysis of the donor splice junction of ␤ recombinant with the disease locus, and two-point lod scores intron C (following exon 3) of A1-crystallin (GenBank accession M1430322) in our ADC-affected Brazilian family. The unaffected indi- did not reveal any regions suggestive of linkage (data not vidual (A) was homozygous with a G at position 474 (arbitrarily shown). For chromosome 17q loci, scores are summarized position 61) and the affected person (B) was heterozygous, with a (Table 1) and haplotypes depicted (Fig. 1). Significant lod G-to-C transversion. scores mapped the ADC locus to 17q23.1-q23.2 (markers D17S800 and D17S1299), and suggestive scores mapped the locus to 17q11.1-12 (markers D17S805, D17S1294, and before the AG acceptor splice junction of intron E was found D17S798). Recombination had occurred in individual 7 be- in all. tween the two clusters. The lod scores differed in the two regions due to degrees of informativeness. ␤ By sequencing, three new variants in the A1-crystallin DISCUSSION gene were identified compared with the original, nearly com- plete13 and the revised exonic sequences21 (GenBank, acces- Transparency of the lens is predicated on the tertiary structure sion numbers M14301-622). For exon 1, sequences of the of the crystallins,25 and any disruption of oligomerization or affected and unaffected individuals were identical with the destabilization could result in cataract formation. With the 13 original published sequence. For exons 2 and 3, sequences in exception of the iron-responsive element of the L-ferritin gene, one affected ( number 13) and one unaffected individual were mutations in eight genes have been identified as causative of identical with the original published sequence.13 For exon 4, ADC, and most are in crystallins. In the ␥-crystallin cluster on the sequences of all three individuals agreed with the recent 2q33-q35, three alterations in the CRYGD26–28 and a missense sequence revision.21 For exon 5, the sequence of the unaf- mutation of CRYGC,26 a chain termination mutation in the fected individual was normal.13,21 For exon 6, one affected ␤-crystallin gene (CRYBB2) on chromosome 22 (␤B2-crystal- (number 13) family member was normal.13,21 lin),29,30 a missense mutation in the connexin50 (gap junction We identified three new and unreported variants. The protein ␣-8; MP70) gene (GJA8)31,32 on 1q21.1,33 a missense final nucleotide of the second glycine codon in exon 5 was a G mutation in the human ␣A-crystallin gene (CRYAA)34 on chro- instead of a C13 at position 316 (GenBank accession M1430522) mosome 21, and two mutations,35 one missense and one frame in our affected individuals and did not result in an amino acid shift, in connexin46 (gap junction protein ␣-3; GJA3)on change. Additionally, there was a C-to-T transition at position 6 chromosome 13 have been shown to cause ADC. The previ- before the AG acceptor splice junction at the 3Ј end of intron ously reported activation of a ␥E-crystallin pseudogene36 has E (intron 5; GenBank accession M1430622) in both the affected been found to be an error.26 Although mutations in the ho- and unaffected individuals. This results in a TAG compared meobox DNA-binding PAX6 gene usually cause aniridia and/or with the published CAG,13 which, as an intronic sequence, anterior segment dysgenesis, isolated cataracts have been doc- would not be expected to alter the protein product. We found umented.37,38 Hyperferritinemia with congenital cataracts and a probable mutation at position 474 in the donor splice junc- without other signs and symptoms is inherited as an autosomal tion of intron C (intron 3) following exon 3 (GenBank acces- dominant disease.39,40 Multiple mutations have been reported sion M1430322). A G-to-C transversion was identified in indi- since 1995.39 Recently, a mutation was identified by Kannabi- viduals 13 and 14 (Figs. 2, 3). This variant was found in all 13 ran et al.9,10 in a donor splice junction of the ␤A1-crystallin affected individuals and was not evident in the 6 unaffected gene (position 474 of exon 3 at the 5Ј donor splice junction) as members of the family (numbers 4, 7, 15, 16, 17, and 19). In the basis for ADC in a family41 that has demonstrated linkage the five normal Brazilians, no donor splice site mutations were with the marker D17S805 on 17q11.2-q12.42 found in the ␤A1-crystallin gene. In four of the unrelated Three loci for ADC43–45 (Fig. 4) and numerous retinal Brazilians, position 316 was a C, as originally reported,13 and degenerations and dystrophies have been localized to chromo- one individual had a G. Our new variant (TAG) at position 6 some 17.45–53 In 1996, Berry et al.43 localized an anterior polar

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FIGURE 3. Exon 3 and ﬂanking intronic sequences (GenBank accession M1430322). Exons are in upper- case, introns in lowercase, and primers for PCR and sequencing un- derlined; position 474, the ﬁrst base of the 5Ј donor splice site junction, is in bold.

cataract (CTAA2) to 17p with a maximum lod score of 4.17 ulation of expression as well as posttranslational modifica- between marker D17S796 (␪ ϭ 0.05) and the disease locus. A tions.57 The first two exons of the ␤A1-crystallin gene encode score of 4.01 (␪ ϭ 0.05) was calculated with D17S849. Based the amino terminal arm, and exons 3 through 6 encode the on current localization,15–17 this ADC locus is unchanged at 14 Greek key motifs.13 Our sequence data of all six exons and six Mb from the pter at 17p13.3-11.2. A second locus, a progres- intron–exon boundaries for ␤A1-crystallin gene identified sive early-onset cerulean cataract (CCA1) described by Armit- three new variants, two of which do not alter the amino acid age et al.44 maps more distally on the long arm of chromosome sequence. The third variant is a G-to-C transversion in the 17 than originally reported in 1995. They calculated maximum conserved donor splice junction of intron C (following exon 3) lod scores with the disease (all at ␪ Ͼ0) of 9.46, 5.26, and 7.11 at position 474 (GenBank accession M1430322) and probably for markers D17S802, D17S836, and AFMa238yb5 (D17S1806), represents a mutation. respectively, placing the gene in 17q24. Currently, these mark- Position 474 is the first nucleotide in the invariant GT ers are 90.29, 91.3, and 91.41 Mb from 17pter, respectively, dinucleotide of the 5Ј splice junction consensus sequence. which localizes this ADC locus to the more distal 17q25.2 Krawczak et al.58 reviewed the literature and found that 15% of region.15–17 all point mutations alter pre-mRNA and 60% of the 5Ј splice Padma et al.42 mapped a third ADC locus41 to 17q11.2-12 mutations involve this dinucleotide. This variant is at the iden- based on a maximum lod score of 3.91 (␪ ϭ 0) with D17S805; tical site that Kannabiran et al.9,10 found in affected members additionally, they found linkage with a neurofibromatosis-1 of their Indian family with ADC; however, their variant was a ϭ ␪ ϭ (NF1) marker (Zmax 3.85 at 0) confirming localization at G-to-A transition and cosegregated with the disease. There are 17q11.2 (GDB). Kannabiran et al.9,10 found a variant in the multiple reports of mutations at this site in human disease.58,59 ␤A1-crystallin gene in affected members of this family,41,42 Because the variants, although different, cosegregate with the further confirming the mapping and identifying the presumed disease in both the Indian family9,10 and our Brazilian family mutation. The G-to-A transition in the donor splice junction of and occur in a conserved region of the donor splice junction, the ␤A1-crystallin gene cosegregated with the cataract in their we believe that it represents the causative mutation. Indian family and resulted in interruption of the donor splice We expect that all forms of the ␤A3- and the ␤A1- junction. crystallins would be disrupted by the mutation. Kannabiran The ␤A1-crystallin gene encodes both the ␤A3- and ␤A1- et al.9,10 speculated that the effect would be a skipping of crystallins, which differ by the addition of 17 amino acids in the donor splice junction or the recruitment of a cryptic the ␤A3-crystallin terminus.54 An intermediate form of the splice site. They postulated an alteration of the reading ␤A3-crystallin gene has only nine additional amino acids.55 frame if splicing of exon 2 to exon 4 occurred; after addition The ␤A1-crystallin aggregates range from dimers to octamers56 of four amino acids to the 32 amino acids of exon 2, a and further complexity is related to temporal and spatial reg- premature termination would be encountered. If splicing of

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form in another has been mapped to 17q25.2 (formerly 17q24).44 Explanations for this variability are speculative and probably relate to expression patterns and tertiary structure of the crystallins. The clinical characteristics were varied among the affected members of this Brazilian family. The affected individuals who were examined had pulverulent opacities in the embryonal nucleus and sutures; star-shaped, shieldlike, or radial opacities in the posterior embryonal nucleus, and/or midcortical opacities that appeared to be postnatal. None of the affected individuals had nystagmus. Such clinical variability of ADC within families has been documented previously.3,5,6,41 The clinical features in our family are similar to those in the family with a mutation of the ␤A1-crystallin described by Padma et al.42 The marked variability of the cataract in members of our Brazilian family appears to be greater than the family described by Basti et al.41 and may be related to the alternative effects of the mutation at the protein product level, skipping of the donor splice junction, or recruitment of several cryptic splice sites. Thus, based on previous reports of mutations at this donor splice site causing human disease and the identification of ADC in the Indian family reported by Kannabiran et al.,9,10 we believe that we have identified the basis of ADC in our Brazilian family. Although the specific mutation, a FIGURE 4. Ideogram of chromosome 17 with loci of anterior polar G-to-C transversion, found in the affected members our 43 44 (CTAA2), cerulean or blue-dot (CCA1), and zonular-sutural cata- family is different from the G-to-A variant found in the Indian racts (CCZS)9,10 in an Indian family and our Brazilian family. family,9,10 the donor splice junction locus is identical. Be- cause the clinical features of the cataract in our family were exons 3 to 5 occurred, the coding frame would be main- highly variable, the type of the gene defect would not be tained and a truncated protein predicted. Splicing to exon 6 suggested by the phenotype. would result in a frame shift with preservation of the amino terminal arm followed by 18 additional amino acids. Using a References WALKER representation (a graphical method to display how binding proteins interact with DNA or RNA sequences), they 1. 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