Comprehensive Mutational Screening in a Cohort of Danish Families with Hereditary Congenital Cataract

Abstract

purpose. Identification of the causal mutations in 28 unrelated families and individuals with hereditary congenital cataract identified from a national Danish register of hereditary eye diseases. Seven families have been published previously, and the data of the remaining 21 families are presented together with an overview of the results in all families.

methods. A combined screening approach of linkage analysis and sequencing of 17 cataract genes were applied to mutation analyses of total 28 families.

results. The study revealed a disease locus in seven of eight families that were amenable to linkage analysis. All loci represented known genes, and subsequent sequencing identified the mutations. Mutations were found in eight genes, among them crystallins (36%), connexins (22%), and the transcription factors HSF4 and MAF (15%). One family carried a complex CRYBB2 allele of three DNA variants, and a gene conversion is the most likely mutational event causing this variant. Ten families had microcornea cataract, and a mutation was identified in eight of those. Most families displayed mixed phenotypes with nuclear, lamellar, and polar opacities and no apparent genotype–phenotype correlation emerged.

conclusions. In total, 28 families were analyzed, and mutations were identified in 20 (71%) of them. Despite considerable locus heterogeneity, a high mutation identification rate was achieved by sequencing a limited number of major cataract genes. Provided these results are representative of Western European populations, the applied sequencing strategy seems to be suitable for the exploration of the large group of isolated cataracts with unknown etiology.

Congenital cataract (CC) is among the most common developmental anomalies of the eye. It occurs as an isolated trait, in association with other ocular dysmorphology as well as systemic malformations. The etiology of isolated CC is unknown in approximately 50% of cases, and approximately 30% is monogenetic, with autosomal dominant transmission as the most common mode of inheritance. The knowledge of the genetic background has increased considerably during the past decennia, mainly based on linkage strategies in large families (for review, see Refs. ¹ and ² and references therein). Extensive locus heterogeneity has been documented, and more than 40 cataract-associated loci are known, of which 25 represent identified genes, and the number of mutations exceeds more than 100. ^{1 2} Mutations causing developmental cataracts mainly involve proteins with structural and chaperone functions, including α-, β-, and γ-crystallins. Another group includes the lens-specific transmembrane gap junction protein genes GJA3 and GJA8, and the membrane protein genes MIP and LIM2. A third group of genes represents the lens-associated transcription factors HSF4, PITX3, MAF, PAX6, and FOXE3. Mutations in HSF4 have mainly been associated with nonsyndromic cataract, whereas MAF mutations often involve the microcornea–cataract phenotype. Structural proteins as the lens-specific beaded filament protein genes BFSP1 and BFSP2 represent an additional group of proteins that may have mutations leading to cataract formation. For most of these genes, cataract is the only disease phenotype observed. ^{1 2}  

Dominantly inherited mutations are mainly missense mutations that lead to amino acid substitutions. Only a few examples of nonsense mutations or frame shift mutations have been described ^{1 2} (see dbCCM, http://www.wjc.ku.dk/ccmd1.html/ Congenital Cataract Mutation Database, provided in the public domain by the Panum Institute, University of Copenhagen, Copenhagen, Denmark). With a few exceptions, such as the hyperferritinemia-cataract syndrome, ^{3 4} no consistent genotype–phenotype relations have become evident to facilitate the identification of the involved gene. 

We initiated an investigation of hereditary isolated CC and CC with microcornea in the Danish population, to trace the genetic background in selected families. ^{5 6 7}  

Material and Methods

Patients

Families and patients were recruited from The National Danish Register of Hereditary Eye Diseases at the former National Eye Clinic for the Visually Impaired, now The Kennedy Center (www.kennedy.dk/). Of 97 families with congenital or infantile cataract with or without microcornea we contacted members of 11 families suited for linkage analyses. A sufficient number of participants from eight families attended the study. DNA from an additional 20 unrelated individuals belonging to smaller families collected during clinical examinations was retrieved from the DNA bank of the Eye Clinic. Except microcornea cataract, families with syndromic cataract including congenital cataract and mental retardation and cataract in aniridia syndrome were excluded. The results of mutational analysis of patients with aniridia syndrome have been reported earlier. ⁸ A single two-generation family with possible X-linked transmission and clinical signs of Nance-Horan syndrome, and two families with probable autosomal recessive inheritance did not participate in the present project. Genomic DNA was extracted from whole blood by standard procedures. Mutation controls in the background population were performed with DNA from unrelated normal individuals retrieved from the Copenhagen Family Bank. ⁹ The study adhered to the tenets of the Declaration of Helsinki and was approved by the Copenhagen Scientific Ethics Committee. After being informed, all subjects gave written consent to participate in the study. 

Linkage Analysis

Four families were analyzed using one or several locus-specific STS markers close (<1 cM) to known cataract disease genes. Haplotypes were drawn for each locus (Cyrillic ver. 2.1.3; Cherwell Scientific, Oxford, UK) and checked by visual examination. Initial locus screening was performed with markers for 12 different known cataract loci (Table 1A) . Additional STS markers were included for fine mapping of candidate loci (Table 2)and two-point LOD scores for initial exclusion were calculated with the program LIPED. ¹⁰ The STS marker analyses were performed using P-33 radioactive labeled oligonucleotide primers followed by PCR. Endlabeling of oligonucleotides was performed according to a standard protocol with T4-DNA-polynucletide kinase (Fermentas, Vilnius, Lithuania) and γ-³³P-ATP (Hartmann Analytic, Braunschweig, Germany) and the labeled oligonucleotides were used for PCR without further purification. Taq-DNA polymerase was purchased from New England Bio Laboratories (Ipswich, MA), and PCR primers were purchased from TAG Copenhagen A/S (Copenhagen, Denmark). A complete genomic scan (data not shown) was performed with a 10K SNP array analysis in two families (CC00116 and CC00162) in which the STS marker analyses failed to show linkage either due to incomplete penetrance (CC00162) or inconclusive results (CC00116). 

Direct Genomic DNA Sequencing

Direct DNA sequencing of amplified PCR products of all exons, exon–intron borders, and parts of the 5′ and 3′ UTR was performed in 17 known cataract disease genes (Table 1B ; BigDye version 1.1 sequencing technology and 3130xl sequencing apparatus; Applied Biosystems, Inc. [ABI], Foster City, CA). PCR and sequencing primers were designed by Primer 3 and purchased from TAG Copenhagen A/S (Copenhagen, Denmark). (PCR primer sequences are found in Supplementary Table S1.) The sequence data for the coding regions were aligned to GenBank reference sequences, and genomic intron sequences were aligned to human reference assembly hg17 (NCBI Build 35 from UCSC, http://genome.ucsc.edu/ provided in the public domain by UCSC Genome Bioinformatics, University of California at Santa Cruz, Santa Cruz, CA). ¹¹ Taq DNA polymerases were purchased from three sources (New England Biolabs; HotStartTaq DNA Polymerase from Qiagen, Hilden, Germany; and Platinum Taq DNA polymerase from Invitrogen, Carlsbad, CA). PCR and sequencing were performed according to standard protocols and analyzed (Chromas software; Technelysium Pty. Ltd., Tewantin, Australia). 

Restriction Enzyme Digests

Identified mutations were analyzed by restriction enzymes digests in accordance with the manufacturer’s protocol (New England Biolabs) in a 20-μL volume with 2- to 4-μL PCR product and 5 to 10 units enzyme. The cleaved PCR products were analyzed by 2% agarose or 20% acrylamide gel electrophoresis with 1× TBE, and the DNA was visualized by staining with ethidium bromide. 

Subcloning of PCR Products

PCR products were subcloned by TA cloning (pCR-XL-TOPOII vector; Topo XL PCR cloning procedure; Invitrogen). The cloned PCR fragments were screened by direct colony PCR in conditions identical with those used for the genomic DNA PCR. 

Results

The present study includes the remaining 21 families from an investigation of 28 families of Northern European decent with hereditary congenital cataract. The results from seven of these families have been presented in previous publications. ^{5 6 7} Overall, likely pathogenic mutations were identified in 20 (71%) of the 28 families. The affirmative results implicated a total of eight genes involving four crystallin genes, CRYAA (six families), CRYBB2 (two families), and CRYBB3 and CRYGD (one family each); two connexin genes, GJA3 and GJA8 (three families each); and two transcription factors, HSF4 and MAF (two families each; Table 3 ). In addition to the identified mutations a genome-wide scan of a large family (CC00116) revealed two provisional novel loci with equal LOD scores (Z = 2.7, θ = 0). The two regions, 2q32.2-33.3 and 17q11.2-q21.2, are bordered by the SNPs rs952242 and rs1551443 for chromosome 2 and the SNPs rs952581 and rs1846043 for chromosome 17 (data not shown). The remaining seven inconclusive families underwent sequencing of the 17 examined cataract genes without identifying any pathogenic mutation. Figures 1 2 3 4 5illuminate the hitherto unpublished results according to the implicated genes. Seven novel polymorphisms were identified and are presented in Table 4 . 

Genotype–Phenotype Relations

Most patients had their cataracts surgically removed, and the phenotypes were therefore retrieved retrospectively from the files of various ophthalmology departments. Many of the notes, however, dated 30 to 50 years ago and the available information was often too insufficient for an adequate classification resulting in only fragmentary data. Cases demanding early surgical intervention were frequently accompanied by nystagmus, which persisted after surgery. 

The study included six families with CRYAA mutations, CC00105, CC00124, CC00174, CCMC0101, CCMC0106, and CCMC0106. Among these identical mutations, p.Arg21Trp, were found in three families, CC00105, CC00124, and CCMC0108. Affected members of CC00105 and CC00124 had anterior polar cataracts and various nuclear and lamellar cortical involvements with highly variable impact on visual function. Furthermore, one member of CC00105 had a congenital, unilateral, inferior iris coloboma, and another individual in the same family had reduced corneal diameters of 9 mm. A third mutation carrier had completely clear lenses when examined at 24 years of age. Four of six individuals in family CC00124 still had their lenses left at the ages of 5, 13, 45, and 59—the latter with optical iridectomies in both eyes. The cataracts were mainly of the anterior polar type with different involvement of nucleus, posterior pole, and cortex in six individuals. (See the Discussion section for further details.) Information was available for two families with a GJA3 mutation (CC00162) and a GJA8 mutation (CC00145). Three members of family CC00162 showed a lamellar cataract type with moderate opacity of the fetal nucleus and Y-shaped condensations in the anterior suture. In three members of family CC00145 the lens morphology was described as dense and star-shaped with various locations in the nucleus or the poles. One individual with an anterior polar opacity developed a nuclear opacity during early infancy. Family CC00128 harbored a HSF4 mutation. In this family, four individuals had lamellar cataracts involving faint opacities of the fetal nucleus with condensation along the anterior Y-suture and various, partly progressive opacities in the cortices. One of these patients also developed a dense anterior polar cataract. 

Two families with MAF mutations (CCMC0102 and CCMC0113; Table 3 ) had small corneae. One member of the latter family was reported to be unaffected but showed up to carry the mutation. An examination, however, showed microcorneae with 10-mm horizontal diameters and only scattered punctuate stromal opacities, which otherwise would not have been noted. His visual acuity was 1.0 on both eyes. Another member of the same family showed dense nuclear opacities with a clear periphery as documented by a photograph from 1967 when the patient was 26 years of age (Fig. 6) . 

Discussion

Apart from mutation screening projects in South Indian families ^{17 18 19} and Australian families, ²⁰ our study is among the first to report on the mutation spectrum in a relative large cohort of patients with CC. Although there is a sizeable and still expanding genetic heterogeneity, we show that a relative high success rate would have been obtained with a sequencing strategy involving rather few “major” genes. Although this is true of the present family collection, it is not necessarily true of new unselected families. However, it suggests that these genes may be particularly useful for screening in samples of similar origin. In family CC00162 the initial linkage analysis failed to point out a locus. A whole-genome screening, however, led to the identification of a known cataract gene, GJA3. The family investigations disclosed the presence of two instances of nonpenetrance (CC00105 and CC00162) that explained the failed linkage analysis. Without the whole-genome screening, the reduced penetrance in this family supposedly would have passed unnoticed. Many of the included families were too small to decide about the hereditary mode. Except for one family (CC00116) linkage analyses identified only the already known loci representing known genes, which implies that the same results would have been obtained by the sequencing of a single affected individual. 

SNP-array analysis in family CC00116 identified two different loci representing a 15.6-Mbp region on chromosome 2 and a 9.3-Mbp region on chromosome 17. The results suggest at least one novel cataract-associated locus. Known cataract genes are located close to both regions, on chromosome 2 the γ-crystallin cluster (CRYGA to CRYGD) is found 1.4 Mbp distal to the linkage region and on chromosome 17, the CRYBA1 gene is located 2.4 Mbp proximal to the linkage region. Both these loci are outside the mapped regions and the genes have in addition been sequenced before the genome scan. Whether one of the two linkage regions harbors a mutation in a long-distance regulatory element or a new cataract gene awaits disclosure and is under investigation. 

In total, 20 mutations were identified among the 28 families included in the study, and it is noteworthy that five of these mutations were reported earlier. We document that a founder effect seems very unlikely with one exception (CC00124 and CCMC0108). 

The DNA sequence analyses included 17 of the known at least 25 cataract-associated genes. The remaining genes were excluded due to a weighing of workload against the possible relevance for our samples. The newly identified gene EPHA2 ²¹ was published after the conclusion of this study, but will be included in future analyses. 

In our study, most of the involved genetic variants were missense mutations; only two nonsense mutations were encountered and no insertions or deletions were found, which is in accordance with the finding of other investigators. The majority of genetic analyses of congenital cataract include AD-CC families and very few cases of AR-CC forms or sporadic cases are reported. These results infer that mainly missense and nonsense mutations are found for AD-CC, whereas AR-CC also is due to frameshift mutations. 

The finding of only one mutation in the Danish families indirectly supports an assumption of autosomal dominant transmission. The present paper includes the remaining unpublished mutational results from 21 families and includes 12 mutations in 13 families. 

Mutations in the Lens-Specific Crystallins

Eight mutations were found in 10 families corresponding to 36% of the analyzed families, which is in the same magnitude as the percentage of crystallin mutations in South India (∼30%) when corrected for the share of 53% autosomal dominant families. ¹⁹  

Among 32 families with autosomal dominant inheritance and four families of uncertain inheritance from southeastern Australia, only two (5%) crystallin mutations were identified. ²⁰ We have no explanation for this difference. However, there were few tissue samples, and selection bias for larger families may influence the results. It is also noteworthy that the Australian and the Danish source populations are of similar magnitude, while the total number of ascertained families was more than twofold in the Danish population. 

The mutations in our cohort were found in four of the nine lens-expressed crystallin genes that we have analyzed (Table 3) . One previously published mutation ⁶ was found in two families (CRYAA, c.61C>T, p.Arg21Trp; Fig. 1 ) and a different mutation in the same codon (p.Arg21Leu) has been reported before in association with cataract, ²² which suggests the Arg21 residue to be of crucial importance for the protein function. Sequencing of exon 1 of CRYAA using the SNP rs872331 located 55 nt upstream of the mutant nucleotide c.61T demonstrated the haplotype c.[6T;61C] for family CC00105 and the haplotype c.[6C;61C] for family CC00124 (Fig. 1D) .This suggests that the mutation arose independently in the two families. The third family (CCMC0108) with cataract and microcornea carried the same mutation and sequence analysis of subcloned separated PCR products from one affected individual from CCMC0108 revealed the haplotype c.[6T;61C] identical with family CC00105 (Fig. 1D) . Subsequent genealogical studies confirmed a common ancestral founder for the two families. According to our knowledge, incomplete penetrance as documented in family CC00105 (Fig. 1A , IV:7) has not been reported before in families with a CRYAA mutation. 

The novel CRYBB2 mutation p.Tyr159X (Table 3 , Figs. 2A 2B 3B 3C ) presumably terminates the reading frame of exon 6 before the authentic stop codon. The mutant mRNA will presumably avoid the nonsense-mediated RNA decay pathway and be translated into a truncated protein. Another nonsense mutation (p.Gln155X, Fig. 2B 3B 3C ) has been reported in five unrelated families with dominantly inherited cataract. ^{20 23 24 25 26 27} Two of these mutations ^{25 27} has been shown to be a consequence of gene conversions between a region of 9 to 104 bp surrounding the mutation and the homologous region in the CRYBB2P1 pseudogene. The remaining three mutations seem to have occurred by point substitutions. ^{23 24 26} By alignment of the wild-type CRYBB2, the corresponding sequence from II:2-CC00156, and the CRYBB2P1 exon 6 sequence (Fig. 2B) , it is obvious that the p.Tyr159X mutation found in family CC00156 is a point mutation and not a result of gene conversion, which is further shown by the chromatogram that demonstrates the sequence [CCCCGGCTAC/A], which should have been [CCCC/TGGTAC/A] if both a point mutation and gene conversion have taken place. Identification of the two nonsense mutations in the same fourth Greek key motif of CRYBB2 suggests a crucial region for cataract-associated mutations, and the pathogenic mechanism is presumably the same for the two mutations. 

A complex CRYBB2 allele with three nucleotide changes in exon 5 was detected in both affected individuals of family CC00133 (Table 3 , Fig. 2C ). The DNA variations rs2330991, rs2330992, and rs4049504 (dbSNP, http://www.ncbi.nlm.nih.gov/, provided in the public domain by the National Center for Biotechnology Information [NCBI]National Institute of Health, Bethesda, MD) are affirmed as nonsynonymous polymorphisms. PCR products from individual I:1 were subcloned, and a probable cis position of the rare SNP variants was confirmed by sequencing of the subclones. Sequence analysis of the SNPs in CRYBB2 exon 5 for 100 normal unrelated individuals of matching ethnic background only detected the wild-type allele c.[433C; 440A; 449C] (data not shown) and none of the SNPs was found to be polymorphic in any of the other sequenced cataract family members. The SNP rs2330992 (p.Gln147Arg) is nonpolymorphic and represented only by the A-allele (p.Gln147) in the HapMap program (dbSNP, ss3282510, Build 129, NCBI, http://www.ncbi.nlm.nih.gov/). This strongly supports the unusual character of the G-allele for rs2330992 and from the above mentioned observations we consider it reasonable to assume that the occurrence of the three mutations in cis are pathogenic, possibly by transforming the secondary structure of the β-crystallin protein (Fig. 3B) . A gene conversion between wild-type CRYBB2 and the pseudogene CRYBB2P1 has been shown to be the most likely mechanism for the p.Gln155X mutation. ²⁵ A similar mechanism is the most likely cause for the complex allele, as shown by alignment of the homologous DNA sequences for the wild-type CRYBB2 and the CRYBB2P1 pseudogene with the sequence of individual II:1-CC00133 (Fig. 2D) . 

The mutation found in CRYBB3 (c.224G>A, p.Arg75His; Table 3 ) is the first report of a cataract-associated CRYBB3 mutation with a dominant effect. The mutant genotype was not detected in 238 normal individuals of matching ethnic background or among the other cataract families. The mutation is in the second Greek key motif (Fig. 3B)and destroys a highly conserved amino acid (Fig. 3D)and is therefore most likely pathogenic. Unfortunately, only a single affected family member, individual I:1 (Fig. 3A)with a microcornea cataract was available for investigation. One other CRYBB3 mutation (p.Gly165Arg) has been reported in a consanguineous Pakistani family with recessively inherited cataract. ²⁸  

Mutations in the Gap Junction Proteins

The six mutations, among which two were novel (Table 3) , represent 22% of the families in our sample (Fig. 4) . Three of the mutations were located in the first extracellular loop of the two gap junction proteins, and all three amino acid positions are highly conserved in humans (Fig. 4E) . This finding implies that the primary structure of transmembrane regions and the extracellular loops are crucial for the assembly of gap junction proteins into connexons. The GJA8 mutation affecting amino acid p.Ser259 in the carboxyl terminus was novel. The mutation segregated with the phenotype in all members of the small family (Fig. 4C) , which supports the interpretation as a pathogenic mutation, although the amino acid position is less conserved among the human gap junction proteins. All GJA3 mutations have been reported previously in association with isolated congenital cataract (Table 3) . Of interest, the mutation p.Arg76His is characterized by incomplete penetrance in family CC00162 which also was observed in the first report of the mutation. ¹⁴ A third mutation affecting the same arginine residue (p.Arg76Gly) has been described in association with a fully penetrant total cataract. ¹⁷  

Mutations in HSF4

Both HSF4 mutations have previously been described by Bu et al. ¹⁵ The mutation p.Leu114Pro was reported in a large Chinese family with cataract and p.Arg119Cys in the large Danish family with cataract first described by Marner in 1949 (Table 3 , Fig. 5 , MIM 116800). ^{29 30 31} The repetition of the latter mutation in another Danish family suggests a common founder, which could not be documented by genealogical studies. The nomenclature for the two mutations has been corrected (Fig. 5C)according to the recommendation from the Human Genome Variation Society (http://www.hgvs.org/). ¹⁶  

MAF Mutations

Previously, two MAF mutations have been published in association with microcornea cataract. ^{32 33} The recurrent mutation p.Arg299Ser was ascertained in family CCMC0112 (Table 3)and is predicted to modify the conserved DNA-binding region (Fig. 5D) . ⁷ The novel mutation p.Lys320Glu detected in family CCMC0113 affects the leucine zipper region and is the first pathogenic cataract MAF mutation outside the DNA binding domain. Of note, an apparent case of incomplete expression was observed in individual III:2 who had microcornea, but no cataract. This shows that isolated microcornea may be caused by a mutation in a cataract-associated transcription factor gene. This observation points toward a common regulatory mechanism of corneal and lens crystallins in humans. Experimental evidence in mice seems to support the presence of such a mechanism. Recently Davis et al. ³⁴ showed activation of the specific mouse corneal crystallin Aldh3a1 by different transcription factors as Pax6, Oct1, and p300. Confirmation of a possible dual mechanism involving corneal and lens development induced by the novel MAF zipper domain mutation awaits experimental elucidation. 

Polymorphisms

Several DNA variations of the major cataract-associated genes were found by the sequence analyses. Among eight novel polymorphisms, we encountered one synonymous base-exchange, five missense mutations, one intronic substitution, and one promoter deletion (Table 4) . All variations except two were present in normal control samples. The nonsynonymous HSF4 change p.Met212Ile in the C-terminal part of the protein (Table 4)was initially considered to be causal in family CC00109, because of the absence among 170 normal unrelated individual of Danish origin. The variation, however, was also absent in two affected relatives and therefore was classified as a rare polymorphism. A CRYGD promoter deletion was detected in one affected individual of family CC00805 (Table 4) . Analyses of 170 unrelated normal individuals of same ethnic background failed to identify the deletion, but it was not found in an affected daughter of the proband and therefore considered to be a rare DNA variant without pathogenic effects. A nonsynonymous GJA8 mutation was found in family CC00159 (Table 4)in which no pathogenic mutation has been identified so far (Table 3) . The mutation, p.Asn220Asp involves an amino acid position that is highly conserved among human and mammalian gap junction proteins (data not shown). Surprisingly, the mutation was found in one allele among 170 normal persons with same ethnic background, which led to a classification as nonpathogenic. 

Phenotypes

Most of our patients showed a composite morphology with regard to size, density, and localization of the lens opacities showing mixtures of nuclear, cortical, and polar cataracts. This finding was further accentuated by considerable intrafamilial differences in phenotypes and additional congenital dysmorphology (microcornea and coloboma). In addition, some cataracts were progressive, leading to changing morphology during infancy. The highly heterogenic phenotypes preclude sound genotype–phenotype predictions based on this study. It should be kept in mind, however, that our phenotype data were historical. The descriptive terminology probably differed among clinicians, and centers and routine examination before surgery may have been cursory. A qualified phenotype description should rely on photographic documentation and be based on a descriptive standard for infantile cataracts. 

Conclusion

A mutational analysis strategy involving direct sequencing of 17 cataract genes identified nearly three fourths of the mutations in a cohort of hereditary congenital cataracts of Northern European descent. We propose the application of the strategy to investigate cases of isolated congenital cataract and unknown etiology. 

 Supported by grants from The Danish Association of the Blind and The Danish Eye Health Society. RC-Link is supported by The Danish Medical Research Council. The Wilhelm Johannsen Centre for Functional Genomics and the Genome Group/RC-LINK hosted the project; the Danish National Research Foundation funds the Wilhelm Johannsen Centre for Functional Genomics.

 Submitted for publication November 12, 2008; revised January 1, 2009; accepted April 16, 2009.

 Disclosure: L. Hansen, None; A. Mikkelsen, None; P. Nürnberg, None; G. Nürnberg, None; I. Anjum, None; H. Eiberg, None; T. Rosenberg, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Table 1.

 

Cataract Disease Loci and Genes

A. Disease Loci
Chromosomal Band	STS Marker	Locus
1pter-p36.13	D1S243	CCV, Volkmann cataract
1q21.1	D1S2612	GJA8
2q33.3	D2S2208	CRYGA, CRYGB, CRYGC, CRYGD
3q22.1	D3S1290	BFSP2
10q24.32	D10S1697	PITX3
11q23.1	D11S4192	CRYAB
12q13.3	D12S1691	MIP
13q12.11	D13S175	GJA3
16q22.1	D16S3086	HSF4
17q11.2	D17S841	CRYBA1
21q22.3	D21S1890	CRYAA
22q11.23-12.1	D22S421	CRYBB2, CRYBA4, CRYBB3, CRYBB1

B. Genes
Gene Symbol	GenBank Ref. Seq.	Gene Name
CRYAA	NM_000394.2	Crystallin α-A
CRYAB	NM_001885.1	Crystallin α-B
CRYBB1	NM_001887.3	Crystallin β-B1
CRYBB2	NM_000496.2	Crystallin β-B2
CRYBB3	NM_004076.3	Crystallin β-B3
CRYBA4	NM_001886.1	Crystallin β-A4
CRYBA1	NM_005208.3	Crystallin β-A1
CRYGC	NM_020989.2	Crystallin γ-C
CRYGD	NM_006891.2	Crystallin γ-D
GJA3	NM_021954.3	Gap junction protein, α3
GJA8	NM_005267.3	Gap junction protein, α8
HSF4	NM_001538.2	Heat shock factor 4
MIP	NM_012064.2	Major intrinsic protein of lens fiber
BFSP1	NM_001195.2	Beaded filament structural protein 1, Filensin
BFSP2	NM_003571.2	Beaded filament structural protein 2, Phakinin
MAF	NM_005360.3	v-Maf musculoaponeurotic fibrosarcoma
PITX3	NM_005029.3	Paired-like homeodomain transcription factor

Table 2.

 

Two-Point LOD Score Calculations

	Mb*	Z(θ)
				0.00	0.01	0.05	0.10	0.20	0.30	0.40
CC00105—CRYAA , †
D21S1411	43.03	1.73	1.77	1.82	1.76	1.41	0.91	0.36
c.61C>T	43.46	3.38	3.38	3.29	3.07	2.46	1.69	0.79
D21S1890	43.67	1.29	1.32	1.37	1.35	1.13	0.76	0.29
CC00124—CRYAA , ‡
c.61C>T	43.46	3.01	2.96	2.74	2.46	1.85	1.16	0.43
D21S1890	43.67	0.36	0.34	0.30	0.26	0.22	0.17	0.09
CC00128—HSF4 , ‡
D16S3086	65.49	2.77	2.70	2.43	2.09	1.44	0.83	0.29
c.341T>C	65.75	5.61	5.52	5.14	4.66	3.62	2.50	1.28
D16S3095	68.50	3.86	3.78	3.43	2.99	2.10	1.25	0.46
CC00145—GJA8 , ‡
D1S442	143.12	2.25	2.21	2.04	1.82	1.39	0.95	0.50
c.218C>T	144.61	5.34	4.99	4.55	3.58	2.51	1.32	5.42
D1S3466	147.00	3.43	3.39	3.22	2.98	2.40	1.72	0.93
CC00162—GJA3 , †
c.227G>A		2.50	2.51	2.47	2.32	1.88	1.32	0.69

*  Physical distances in mega base pairs according to UCSC Marts 2006, NCBI Build 36.1.

†  Penetrance in the calculations for Z(θ) is 0.95.

‡  Penetrance in the calculations for Z(θ) is 1.00.

Table 3.

 

Accumulated Results of the Danish Congenital Cataract Mutation Study

Family	Gene	Nucleotide Change*	Amino Acid Change*	Number of Analyzed Control Persons	References
CCMC0101	CRYAA	c.34C>T	p.Arg12Cys	170	This study, 6
CC00105, CC00124, CCMC0108	CRYAA	c.61C>T	p.Arg21Trp	170	This study, 6
CC00174	CRYAA	c.155C>T	p.Arg49Cys	None	This study, 12
CCMC0106	CRYAA	c.337G>A	p.Arg116His	170	This study, 6
CC00156	CRYBB2	c.498C>A	p.Tyr159X	None	Novel
CC00133	CRYBB2	c.[433C>T; 440A>G; 449C>T]	p.[Arg145Trp; Gln147Arg; Thr150Met]	100	Novel (rs2330991, rs2330992, rs4049504)
CCMC0102	CRYBB3	c.224G>A	p.Arg75His	238	Novel
CCMC0109	CRYGD	c.418C>A	p.Tyr134X	None	This study, 6
CC00103	GJA3	c.32T>C	p.Leu11Ser	60	This study, 5
CC00129	GJA3	c.176C>T	p.Pro59Leu	None	This study, 13
CC00162	GJA3	c.227G>A	p.Arg76His	None	This study, 14
CC00145	GJA8	c.218C>T	p.Ser73Phe	60	Novel
CCMC0103	GJA8	c.565C>T	p.Pro189Leu	170	This study, 6
CC00110	GJA8	c.836C>A	p.Ser259Tyr	170	Novel
CC00128	HSF4	c.341T>C	p.Leu114Pro	None	This study, 15
CC00171	HSF4	c.355C>T	p.Arg119Cys	None	This study, 15
CCMC0112	MAF	c.895C>A	p.Arg299Ser	52	This study, 7
CCMC0113	MAF	c.958A>G	p.Lys320Glu	173	Novel
CCMC0107	—	No mutation	—	—	—
CCMC0110	—	No mutation	—	—	—
CC00109	—	No mutation	—	—	—
CC00117	—	No mutation	—	—	—
CC00155	—	No mutation	—	—	—
CC00159	—	No mutation	—	—	—
CC00805	—	No mutation	—	—	—
CC00116	Locus	chr2q32.2–33.3 or chr17q11.2–q21.2	—	—	Novel

*  DNA and protein variation nomenclature is according to Human Genome Variation Society recommendations (http://www.hgvs.org/). ¹⁶

Figure 1.

 

Pedigrees and restriction digests of three families with CRYAA mutations. (A) The MspI restriction enzyme digest of exon 1 identified all affected individuals as carriers of the mutation c.61C>T in family CC00105. Note that person IV:7^a was a healthy carrier. Wild-type allele, 205, 117, and 107 bp; mutant allele, 321 bp. (B) The mutation and the sizes of the MspI restriction enzyme digest in family CC00124 were identical with the digest of family CC00105. (C) The pedigree and the AciI restriction enzyme digest of family CC00174 showed the two affected individuals to be carriers of the mutation CRYAA c.155C>T. Wild-type allele: 15, 98, 124, and 191 bp; mutant allele: 98, 124, and 206 bp (only the 191- and 206-bp fragments are shown). Filled symbols: affected individuals; open symbols: unaffected individuals; circles: females; squares: males; M: 50-bp DNA ladder; (C) Digest of a normal unrelated individual. U: uncut PCR products. (D) The DNA sequence and the translation of the first 70 nucleotides of the CRYAA coding region show the SNP rs872331 (c.6C>T) and the CRYAA mutation (c.61C>T) together with the haplotypes of the families CC00105 and CC00124 and family CCMC0108. ⁶ All three families carried the CRYAA mutation c.61C>T, and the haplotypes excluded a common founder for CC00105 and CC00124.

Figure 2.

 

Pedigrees and analyses of two families with CRYBB2 mutations. (A) In family CC00156, only individual II:2 was available for analysis. The DNA chromatogram shows the nonsense mutation CRYBB2 c.498C>A, which changes the tyrosine codon TAC into TAA. (B) Alignment of the DNA sequence for exon 6 for the wild-type CRYBB2 (NM_000496) and the corresponding sequence for individual II:2 and for the homologous pseudogene CRYBB2P1 (BC037884) shows variation for c.475C (green), for position c.483C (blue), and position c.489C (yellow). The most abundant CRYBB2 mutation c.475C>T may be due to a gene conversion, whereas the alignment of positions c.483 and c.489 excludes a gene conversion for the novel mutation c.489C>A. Redundant nucleotide M: C and A. (C) The pedigree and the haplotypes of the two affected individuals and the inferred haplotype of one healthy individual of family CC00133. The DNA chromatogram shows the three DNA polymorphisms for individual I:1. (D) Alignment of the genomic sequences of exon 5 and the border to intron 5 shows wild-type CRYBB2 (top), the analyzed sequence of individual II:1 (middle), and the CRYBB2P1 pseudogene (bottom) suggesting that the three nonsynonymous changes in the disease allele was a result of a gene conversion. The converted region was a minimum of 80 bp long (green); the maximum length (gray) could not be predicted due to missing sequence information. Redundant nucleotides Y: C and T; R: A and G; and S: C and G. Exons are shown in capital letters, introns in lower case; the SNP rs57112959 (G>A) refers to the wild-type CRYBB2 gene.

Figure 3.

 

Sequence analyses of families carrying CRYBB3 mutations. (A) The CRYBB3 mutation c.224G>A was found in individual II:1 in family CCMC0102 and confirmed by a SacII restriction enzyme digest. Only individual II:1 was available for analyses. Wild-type allele (C): 130 and 270 bp; mutant allele and uncut (U): 400 bp; M: 100-bp DNA ladder. (B) The β-B crystallin proteins share a common secondary and tertiary structure of two crystallin domains, each composed of two Greek key motifs. A Greek key motif is inserted in the top left corner. The mutations found in the families CC00133 and CC00156 are denoted. The amino acid numbers for intron positions are shown. (C) The protein sequence alignment of the third and the fourth Greek key motif for the human β-B crystallin-1, −2, and −3 showed conservation or semiconservation for p.Arg145, p.Gln147, p.Thr150, p.Gln155, and p.Tyr159 (positions highlighted in yellow). Protein Ref. Seq.: NP_001878, NP_000487, and NP_004067. (D) The CRYBB3 c.224G>A position was highly conserved among several mammalian genomes as shown by the DNA sequence alignment (UCSC Human genome browser). ¹¹

Figure 4.

 

Pedigrees and restriction digests of four families with mutations in the gap junction proteins. (A) Pedigree of family CC00145 shows the haplotypes for all analyzed persons. The STS marker D1SGJA5-GJA8 was located between two genes, GJA5 and GJA8. The EarII restriction enzyme digest showed cosegregation of the mutation GJA8 c.218C>T with the disease trait in the family. Wild-type allele: 199 bp; mutant allele: 165 bp. (B) Pedigree of family CC00162. The AciI restriction enzyme digest illustrated the segregation of the mutation GJA3 c.227G>A. Note the healthy carrier V:2^a. Wild-type allele: 151, 91, and 79 bp; mutant allele: 191, 91, and 79 bp; M: 50 bp DNA ladder. (C) Pedigree of family CC00110. The BseRI restriction enzyme digest showed segregation of the mutation GJA8 c.836C>A in both affected individuals. Wild-type allele (C): 114, 135, 154, and 225 bp; mutant allele: 114, 225, and 289 bp. (D) The pedigree of family CC00129. The AluI restriction enzyme digest illustrates the mutation in individual II:1. Wild-type allele (C): 129 and 328 bp; mutant allele: 129, 160, and 168 bp; U: undigested PCR products; M: 50 bp DNA ladder. (E) Graphic representation of the two lens-specific gap junction proteins and the mutations found in the Danish cohort. CP, cytoplasmic domain; TM, transmembrane domain; EC, extracellular domain. Alignment of the α group of human gap junction proteins demonstrated conservation of the mutant positions except for the C-terminal mutation. Protein Ref. Seq.: Gja1, NP_000156; Gja3, NP_068773; Gja4, NP_002051; Gja5, NP_005257; Gja7, NP_005488; Gja8, P_005258; Gja10, NP_115991; and Gja12, NP_065168.

Figure 5.

 

Pedigrees and restriction digests of families with HSF4 and MAF mutations. (A) Pedigree with haplotypes for family CC00128. The BsrI restriction enzyme digests showed that the mutation HSF4 c.341T>C cosegregates with the disease. Wild-type allele: 49, 57, 60, 74, and 252 bp; mutant allele: 49, 57, 74, and 312 bp; U: uncut PCR products; M: 50 bp DNA ladder. (B) Pedigree of family CC00171. The mutation HSF4 c.355C>T was confirmed by HpyCH4V restriction enzyme digest in both affected individuals. Wild-type allele: 492 bp; mutant allele: 48 bp and 444 bp; U: uncut PCR products; M: 100 bp DNA ladder. (C) The mutations are identical with two mutations described in a Chinese and a Danish family. Bu et al. ¹⁵ named the mutations c.348T>C L115P and c.362C>T, R120C, respectively, according to the GenBank cDNA clone, accession number D87673, that starts at the −4 position from the first ATG codon. The translation of the D87673 results in a HSF4 protein that includes an additional valine residue at position 2. This results in discrepancies between the mutation names published by Bu et al. and the nomenclature used herein (Fig. 4C) . The HSF4 isoform A (NM_001538) has been used for the systematic nomenclature (http://www.hgvs.org/mutnomen/). ¹⁶ (D) Pedigree of family CCMC0113. The half-filled symbol of individual III:3 refers to a case of no cataract with microcornea. The restriction enzyme MboII digest showed segregation of the mutation in the family. Wild-type allele: 84, 92 (seen as one band), and 231 bp; mutant allele: 92 and 315 bp; U: uncut PCR products; M: 100-bp DNA ladder. The graphic representation of the MAF protein shows the known mutations ^{6 32 33} and the novel cataract mutation.

Table 4.

 

Novel Polymorphisms Identified in the Danish Cataract Study

Gene	Exon	Variation	Amino Acid Change	Allele Frequency*	Family (dbLaH)
CRYBA1	Ex2	c.74C>T	p.Pro25Leu	6/110 (1/38)	CC00159 (391)
CRYBA1	Ivs3	c.215+16C>T	—	(2/30)	CC00805 (395)
CRYGD	Promoter	c.−16_37del	—	0/340 (1/34)	CC00805 (395)
CRYGD	Ex3	c.376G>A	p.Val126Met	2/240 (1/34)	CC00171 (393)
HSF4	Ex7	c.636G>T	p.Met212Ile	0/340 (1/34)	CC00109 (387)
GJA8	Ex2	c.658A>G	p.Asn220Asp	1/340 (1/38)	CC00159 (391)
MIP	Ex1	c.141A>G	p.Ala45Ala	(4/30)	Several families
MIP	Ex1	c.319G>A	p.Val107Ile	5/76 (1/30)	CC00110 (388)

*  Allele frequencies are given for number of chromosomes analyzed in the normal persons and in brackets the number chromosomes representing persons from the cataract cohort.

Figure 6.

 

Photograph of the right eye of an individual with nuclear cataract. The 26 year-old patient III:5 belonged to family CCMC0113 with microcornea cataract and a MAF p.Lys320Glu mutation (Table 3) . The corneal diameter was not measured but she had steep corneas (K-readings 6.7 × 6.85 mm of curvature), which indirectly indicates a reduced overall corneal size. The cataract consisted of a circular dense nuclear opacity with condensations in a triangular configuration according to the fetal Y-suture. Faintly seen triangular extensions outside the central opacity were present. The cortex and the anterior polar zones were clear. The left eye had identical findings. The patient underwent surgery with insertion of artificial lenses at the age of 43 years.

Supplementary Materials

Supplementary Table - (PDF) 

The authors thank the families for their participation, Jeanette Andreasen and Maria Jørgensen for excellent technical assistance, and Erik Kann for the genealogical studies.