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. Author manuscript; available in PMC: 2013 Apr 13.
Published in final edited form as: Hum Genet. 2010 Dec 14;129(3):319–327. doi: 10.1007/s00439-010-0928-y

Potential involvement of more than one locus in trait manifestation for individuals with Leber congenital amaurosis

Wojciech Wiszniewski 1, Richard Alan Lewis 2,3,4,5, David W Stockton 6,7, Jianlan Peng 8, Graeme Mardon 9,10,11,12,13, Rui Chen 14,15,16, James R Lupski 17,18,19
PMCID: PMC3625363  NIHMSID: NIHMS457260  PMID: 21153841

Abstract

Leber congenital amaurosis (LCA) is a clinically and genetically heterogeneous retinal dystrophy. The causes of LCA have been unraveled partially at the molecular level. At least 14 genes have been reported that, when mutated, result in LCA. To understand the roles of the known genes in LCA, a group of outbred subjects from 60 apparently either recessive families, with one or more affected individuals, or isolated patients were evaluated. One affected individual from each family underwent comprehensive mutational analysis by direct DNA sequencing of all coding regions and splice junctions of 13 LCA genes. Mutations were identified in 70% of individuals. CEP290 made the largest contribution to the identified mutations, providing 43% of those mutant alleles. We identified seven families in which affected individuals with two mutant alleles, sufficient to cause disease, had an additional mutation at a second LCA locus. Our findings suggest that mutational load can be important to penetrance of the LCA phenotype.

Introduction

Leber congenital amaurosis (LCA, MIM#204000) is a clinically heterogeneous disorder of the retina that affects approximately 2–3 per 100,000 individuals in the general population and accounts for 5% of all inherited retinal dystrophies (Koenekoop 2004). The inclusion criteria for establishing a diagnosis of LCA are profound visual impairment present at birth or at an early age, infantile nystagmus, oculodigital signs, and a diminished or non-recordable electroretinographic response (ERG). Additional clinical features or later findings may include keratoconus, neural hearing deficits, developmental delay, seizures, and a variety of fundus appearances (Lewis 1988). This clinical diversity likely reflects both its genetic and allelic heterogeneity. To date, 14 genes have been identified in which mutant alleles can cause LCA (Table 1). The sequencing of known LCA genes allows identification of mutations in 65–70% of LCA patients, leaving a substantial fraction of LCA clinical cases for which the genetic etiology is not yet determined (Dharmaraj et al. 2000; Hanein et al. 2004; Lotery et al. 2000; Yzer et al. 2006, den Hollander et al. 2006, 2008).

Table 1.

LCA genes, their encoded protein product functions and reported mutational frequencies among patients with LCA

Gene Function Mutation frequency (%)
MIM#610142 CEP290 Centrosome protein 290 kDa Ciliary protein 21
MIM#600179 GUCY2D Photoreceptor-specific guanylate cyclase Signal transduction 12
MIM#604210 CRB1 Crumbs homolog 1 Photoreceptor polarity determination 10
MIM#180069 RPE65 Retinal pigment epithelium 65 kDa protein Vitamin A metabolism 6
MIM#604392 AIPL1 Aryl hydrocarbon receptor-interacting protein like 1 Chaperone protein 5
MIM#605446 RPGRIP1 Retinitis pigmentosa GTPase regulator interacting protein 1 Ciliary protein 4
MIM#608830 RDH 12 Retinal dehydrogenase 12 Vitamin A metabolism 3
MIM#611408 LCA5 Lebercilin Microtubule transport 2
MIM#602225 CRX Photoreceptor-specific homeobox gene Photoreceptor differentiation control 1
MIM#602280 TULP1 Tubby-like gene 1 Vesicular trafficking <1
MIM#604863 LRAT Lecithin retinol acyltransferase Vitamin A metabolism <1
MIM#609868 SPATA7 Spermatogenesis-associated protein 7 Unclear <1
MIM#146690 IMPDH1 Inosine monophosphate dehydrogenase 1 Purine synthesis <1
MIM#610612 RD3 Retinal degeneration 3 Unclear <1
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Literature reports based on the following studies: den Hollander et al. (2006, 2008), Dharmaraj et al. (2000), Hanein et al. (2004), Lotery et al. (2000) and Yzer et al. (2006)

The LCA genes encode proteins involved in various biological pathways that include retina development (CRB1, CRX), phototransduction (GUCY2D, AIPL1), vitamin A metabolism (RPE65, LRAT, RDH12), cellular transport (TULP1), and ciliary processes (RPGRIP1, CEP290, LCA5) (Table 1) (den Hollander et al. 2001, 2006, 2008; Sohocki et al. 2000a; Swaroop et al. 1999; Cremers et al. 2002; Perrault et al. 2004; Thompson et al. 2005). The discovery of mutations in the ciliary proteins, RPGRIP1, CEP290, and LCA5, emphasizes the emerging role of disrupted ciliary functions in the pathogenesis of selected LCA cases and places LCA in a group of disorders now classified as ciliopathies (Badano and Katsanis 2002; Badano et al. 2006a, b). Mutations of these genes are responsible for nearly a third of causally definable LCA cases (den Hollander et al. 2006). The ciliary proteins participate in transport and communication between inner and outer segments of rods and cones and thereby their dysfunction can trigger photoreceptor death (Abd-El-Barr et al. 2007).

The discovery of CEP290/NPHP6, encoding a 290 kDa centrosomal protein, highlighted a new pathogenetic mechanism underlying some LCA. The CEP290 mutations are frequent and detected in 21% of patients, thus making CEP290 a major contributor to LCA. The protein localizes to centrosomes of dividing cells and to the connecting cilium of photoreceptors. It appears that CEP290 cooperates with Rab8 to promote ciliogenesis (Tsang et al. 2008). Mutations in CEP290 are found in patients with LCA among other ciliopathies including Joubert syndrome (JBTS, MIM#610188), Meckel Syndrome (MS, MIM#610142), McKusick-Kaufman syndrome (MKKS, MIM#236700), Senior–Loken syndrome (SLSN, MIM#610189) and Bardet Biedl Syndrome (BBS, MIM#2099000), each of which has retinal dystrophy accompanied by systemic features.

Most patients with LCA resulting from mutations in CEP290 have an intronic mutation c.2991+1655A>G that affects splicing and results in a putatively hypomorphic allele (den Hollander et al. 2006). The severity of the CEP290-dependent phenotypes might correlate inversely with CEP290 activity. Less severe phenotypes, including LCA or nephronophthisis, involving just one system would result from hypomorphic mutations, while multisystem disorders result from complexity similar to what has been proposed for alterations in ABCA4 residual activity for Stargardt disease, cone rod dystrophy, retinitis pigmentosa, and age-related macular degeneration (Shroyer et al. 1999, 2001a, b; Lewis et al. 1999; Wiszniewski et al. 2005). Another hypothesis suggests the existence of second-site modifiers that could directly or indirectly interact with CEP290 and thus moderate or modulate the phenotype (Khanna et al. 2009; Coppieters et al. 2010). RPGRIP1L is the first identified second-site modifier that does not cause LCA alone but is proposed to modulate the retinal phenotype in LCA patients with known pathogenic mutations at another locus (Khanna et al. 2009). Interestingly, the presence of a third mutant allele in 7% of subjects from an LCA cohort study has been reported, and interpreted to reflect a modifier effect on the LCA phenotype (Zernant et al. 2005).

The presence of mutations in more than one disease locus is observed frequently in patients with ciliopathies. In fact, the genetic concept of triallelic inheritance whereby two mutant alleles at one locus and one mutant allele at another locus are required for trait manifestation was first described in BBS (Katsanis et al. 2001; Eichers et al. 2004). In patients with BBS, mutations in more than one locus are found in approximately 10% of patients, and it has been shown that the third mutant allele may modify the severity of the disease (Hichri et al. 2005; Khanna et al. 2009). Thus, the third mutant allele may be associated with the either penetrance of the clinical phenotype or variability of expression. The ability to discern the latter depends on both the type of trait and the methods applied to assay phenotypic manifestations. Digenic triallelic inheritance has now been reported in Waardenburg syndrome, Usher syndrome, and some forms of right ventricular hypertrophy (Ebermann et al. 2010; Xu et al. 2010; Chiang et al. 2009).

Here, we report mutational analysis of 13 LCA genes in 60 unrelated North American families with a clinical diagnosis of Leber congenital amaurosis. We found mutations in 84/120 (70%) alleles in 42/60 (70%) of patients. In addition, we identified a third disease-associated mutant allele in a second locus in 7/60 (12%) patients. Our findings further support the concept that mutational load may be important to disease manifestations.

Materials and methods

Patient enrollment

After informed consent, individuals and families were enrolled for this study in a protocol approved by the Institutional Review Board for Human Subject Research at Baylor College of Medicine, Houston, TX. The inclusion requirement was a clinical diagnosis of LCA based on (1) evidence of substantial reduction of vision in the first year of life, (2) infantile nystagmus with or without oculodigital blindisms, and (3) markedly diminished ERG responses. An additional inclusion criterion of multiple affected family members, incorporated early in the recruitment process, was later relaxed to allow increased enrollment. Anticoagulated blood samples were obtained and DNA extraction done by standard methods. DNA samples on control individuals were obtained from the Baylor Human Polymorphism Resource through the Kleberg Genotyping Center at Baylor College of Medicine. Ninety-six control samples from healthy unrelated non-Hispanic white individuals were also studied.

Mutation detection

Samples of genomic DNA from an affected individual in each family were amplified by PCR with primers in the introns flanking each exon or cluster of exons for each of the genes. To detect the common CEP290 mutation: c.2991+1655A>G localized to IVS 26, the intronic region was amplified and sequenced using published primers (Li et al. 2009).

Data were analyzed with ABI Sequence Analysis version 3.2 (Applied Biosystems, Foster City, CA), Sequencher version 4.1 (GeneCodes, Ann Arbor, MI), and Mutation Surveyor v3.24 (SoftGenetics).

Each sequence variation identified in an affected family member was tested for co-segregation with the phenotype in samples from all available family members and, if the variation was not previously reported, it was tested in the control DNA sample sets and assessed with three predictive software tools: MutationTaster (http://neurocore.charite.de/ MutationTaster/), PolyPhen (http://genetics.bwh.harvard.edu/pph2/) and SIFT (http://blocks.fhcrc.org/sift/SIFT.html).

Results

Subjects with LCA

To investigate LCA genes mutations, we enrolled 60 unrelated patients and analyzed coding sequences of 13 LCA genes. The LCA families were recruited in North America and patients had various ethnic backgrounds. In all subjects, ophthalmic manifestations consistent with LCA were present in the newborn period. All subjects presented with non-syndromic retinal disease, except for Ar 187-04 who was also diagnosed with non-disjunction trisomy 21 Down syndrome. In each family, the parents of the affected individuals had no known retinal disease or visual impairment (other than refractive error). In 31/60 families, two or more siblings were diagnosed with LCA. No significant difference in severity of retinal disease was apparent between affected siblings.

Mutation analysis

Of the 60 families, 42 (70%) had one or more mutations in the 13 LCA genes tested, 34 (57%) had two or three mutations, and 7 (12%) had three mutations in two different LCA genes (Table 2). The distribution of disease alleles in the tested genes among these 60 families was: 36 (43%) with CEP290 mutations, 13 (15%) with GUCY2D mutations, 13 (15%) with AIPL1 mutations, 8 (9%) with CRB1 mutations, 6 (7%) with RPE65 mutations, 4 (5%) with RPGRIP1 mutations, 3 (4%) with LCA5 mutations and 1 (1%) with CRX mutations. No coding sequence mutations were detected in five known LCA genes: RD3, RDH12, TULP1, SPATA7, and LRAT. We detected 52 different mutant alleles including 33 reported and 19 novel (12 in CEP290, 3 in AIPL1, 3 in CRB1, and 1 in RGRIP1). None of the novel alterations was found in the 96 control samples nor was any documented in human mutation and SNP databases.

Table 2.

Mutations in families affected with LCA

Pedigree Allele I
 Allele II
Nucleotide Translation Nucleotide Translation
GUCY2D
 Ar-039 c.2743A>G P.I915V c.2516delC p.T838RfsX27
 Ar-085 c.1343C>A p.S448X c.1343C>A p.S448X
 Ar-123 c.2303G>A p.R768Q c.2303G>A p.R768Q
 Ar-695 C.2302C>T p.R768W c.692_693insG p.K232EfsX86
 Ar-712 c.1343C>A p.S448X c.1343C>A p.S448X
 Ar-098 c.2101C>T p.P701S ? ?
 Ar-666 c.2248G>T p.E750X ? ?
 Ar-785 c.2302C>T p.R768W ? ?
RPE65
 Ar-073 c.284A>G p.E95G c.284A>G p.E95G
 Ar-089 C.271C>T p.R91W c.1102T>C p.Y368H
 Ar-782 c.1022T>C p.L341S c.355delT p.S121LfsX6
CRX
 Ar-589 c.705delC p.L236SfsX135 ? ?
AIPL1
 Ar-072 c.834G>A p.W278X c.834G>A p.W278X
 Ar-098 c.834G>A p.W278X c.809G>A p.R270H
 Ar-553 c.834G>A p.W278X c.834G>A p.W278X
 Ar-770 c.266G>A p.C89Y c.266G>A p.C89Y
 Ar-123 c.834G>A p.W278X ? ?
 Ar-164 c.971G>T p.R324L ? ?
 Ar-177 c.905G>T p.R302L ? ?
 Ar-782 c.971G>T p.R324L ? ?
CRB1
 Ar-170 c.3038A>C p.Q1013P c.2548G>A p.G850S
 Ar-597 c.2843G>A p.C948Y c.2843G>A p.C948Y
 Ar-611 c.2401A>T p.K801X c.2688T>A p.C896X
 Ar-115 c.2401A>T p.K801X ? ?
 Ar-785 c.853A>C p.S285R ? ?
RPGRIP1
 Ar-848 c.2367+1G>A Splice c.2710+1G>A splice
 Ar-021 c.3341A>G P.D1114G ? ?
 Ar-039 c.1753C>T p.P585S ? ?
CEP290
 Ar-21 c.2991+1655A>G p.C998X;WT c.5777G>C p.R1926P
 Ar-51 c.6277delG p.V2093SfsX4 ? ?
 Ar-81 c.2991+1655A>G p.C998X;WT c.829G>C p.E277Q
 Ar-86 c.2991+1655A>G p.C998X;WT c.1429C>T p.R447X
 Ar-107 c.2991+1655A>G p.C998X;WT c.3178delA p.T1060LfsX5
 Ar-110 c.2991+1655A>G p.C998X;WT c.4452_4455delAGAA p.E1484LfsX3
 Ar-113 c.2991+1655A>G p.C998X;WT c.1236delG p.L412X
 Ar-137 c.2991+1655A>G p.C998X;WT c.1066-1G>A splice
 Ar-163 c.2991+1655A>G p.C998X;WT c.1830delA p.L611FfsX5
 Ar-177 c.2991+1655A>G p.C998X;WT c.5493delA p.A1831PfsX19
 Ar-187 c.2991+1655A>G p.C998X;WT c.5813_5817delCTTTA p.T1938DfsX16
 Ar-612 c.2213delT p.L738X c.4438-3delC splice
 Ar-681 c.2991+1655A>G p.C998X;WT c.4962_4963delAA p.E1655NfsX3
 Ar-746 c.2991+1655A>G p.C998X;WT C.4882C>T p.Q1628X
 Ar-757 c.2991+1655A>G p.C998X;WT ? ?
 Ar-759 c.2991+1655A>G p.C998X;WT c.2505_2506delAG p.G836IfsX2
 Ar-863 c.2991+1655A>G p.C998X;WT c.5850delT p.F1950LfsX15
 Ar-864 c.2991+1655A>G p.C998X;WT c.3185delT p.L1062RfsX3
 Ar-889 c.2991+1655A>G p.C998X;WT c.5218C>T p.Q1740X
LCA5
 Ar-588 c.835C>T p.Q279X c.835C>T p.Q279X
 Ar-863 c.1316G>A p.G439E ? ?
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? unknown

Potential triallelic inheritance

Mutations in more than one gene were detected in seven subjects who were compound heterozygotes for mutations in one LCA gene and also carried a heterozygous mutation in another LCA gene, giving a total of three mutated LCA alleles. These patients tested positive for two mutant alleles at CEP290 with an additional mutations at either AIPL1, RPGRIP1 or LCA5; two mutant alleles at GUCY2D with an additional mutation at either AIPL1 or RPGRIP1; two AIPL1 mutations with one at GUCY2D; and two RPE65 mutations with one in AIPL1 (Table 3). No particular pattern or combination of mutated genes was observed in subjects with three independent mutated alleles. The severe retinal phenotype in these patients could not be further distinguished from other LCA patients with compound heterozygous mutations at a single locus (Table 3).

Table 3.

Clinical presentation of LCA patients with mutations identified in two loci

Pedigree Gene Mutation Age of onset Visual Acuity ERG Clinical findings
Ar-21-04 RPGRIP1 p.D1114G Birth LPO NR Nystagmus, vitreous cells, narrow versus, severe “taporetinal dystrophy”
CEP290 c.2991+1655A>G
CEP290 p.R1926P
Ar-39-04 GUCY2D p.I915V <3 months N/A NR Nystagmus, keratoconus in early teens, hyperopia (+7.50 spherical equivalent), “retinitis pigmentosa”—like retinal dystrophy
GUCY2D p.T838RfsX27
RPGRIP1 p.P585S
Ar-98-11 GUCY2D p.P701S Birth 5/160 NR Optic atrophy, diffuse vascular attenuation and retinal atrophy with midperiheral bone spicules
AIPL1 p.W278X
AIPL1 p.R270H
Ar-123-04 GUCY2D p.R768Q Birth LPO NR KCNS at age of 12 years, diffuse retinal atrophy and optic nerve atrophy, hyperopia (+8.00 spherical equivalent), autism, consanguinity (1st cousin parents)
GUCY2D p.R768Q
AIPL1 p.W278X
Ar-177-04 AIPL1 p.R302L Birth No fixation NR Slow pupils, high hyperopia (+7.75 spherical equivalent), nystagmus, mild optic nerve atrophy, midretinal pigment degeneration
CEP290 p.A1832PfsX19
CEP290 c.2991+1655A>G
Ar-782-04 RPE65 p.L341S Birth LP NA Sandy diffuse peripheral pigmentary granularity and clumping, optic nerve atrophy
RPE65 p.S121LfsX6
AIPL1 p.R324L
Ar-863-03 CEP290 c.2991+1655A>G Birth NLP NR Sluggish pupils, high hyperopia, nystagmus
CEP290 p.F1950LfsX15
LCA5 p.G439E
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NR non-recordable, NA not available, LP light perception, LPO light perception only, NLP no light perception

Discussion

We report an analysis of 13 of 14 known LCA genes in a cohort of North American patients with a clinical diagnosis of Leber congenital amaurosis. We recruited 60 families with one or more family members affected with LCA. Patients fulfilled clinical criteria for LCA, and anamnestic data were evaluated by a single physician (R.A.L.). Our results show that mutations in known LCA genes are present in 42/60 (70%) subjects diagnosed with this condition. A similar rate of mutation detection in known LCA genes, with an average frequency of 65–70%, has been reported in other studies conducted in Europe and North America (den Hollander et al. 2008; Perrault et al. 2010).

We found a substantial predominance of CEP290 mutations in our study, affecting 19/60 (32%) patients and comprising 43% of all identified mutant alleles. We did not detect mutations in RD3, RDH12, TULP1, SPATA7, and LRAT, likely reflecting a low frequency of mutations in these genes in subjects with LCA from our unrelated North American cohort. Mutations in these genes have been reported in 1–4% of patients with LCA. We did not perform mutational analysis of the recently identified LCA gene—IMPDH1, in which mutations have been identified in only a few families. Overall, we were able to identify 52 different mutations including 33 previously reported and 19 unpublished. We studied the prevalence of new sequence variants in the general population and were unable to identify these alterations in a control population of 96 individuals, or in LCA mutation databases or SNP databases suggesting that carrier states are quite rare and these LCA-associated mutations are unlikely to represent benign variants. We were not able to perform segregation studies for a few sequence variants in affected families because DNA samples from other family members were not available.

Our studies show that 7/60 (12%) patients with Leber congenital amaurosis have three mutated alleles in two LCA loci. In 4/7 subjects with three mutated alleles all three mutations were reported previously as pathogenic (Gerber et al. 2001; Ito et al. 2004; Dharmaraj et al. 2000; Akey et al. 2002; Booij et al. 2005; Sohocki et al. 2000b; Morimura et al. 1998). In the remaining 3/7 individuals, one or two variants have been identified for the first time (Table 4). In the group of patients with three mutated alleles, a total number of 17 different alleles were detected including: two nonsense mutations and four single nucleotide deletions, that represent likely disease causing variants; seven missense mutations predicted in silico to be disease causing changes by at least one predictive tool; and four missense alleles: p.D1114G (RPGRIP1), p.P701S (GUCY2D), p.R302L (AIPL1) and p.G439E (LCA5), not classified by applied predictive tools as likely to be pathogenic. Sequence variants p.D1114G and p.P701S have been reported previously as possible modifiers of the LCA phenotype (Zernant et al. 2005). The latter variant was observed in 2% of general population and in 5% of patients with LCA. There are no functional studies assessing the physiological effects of this allele but available segregation studies for p.P701S in multiple families strongly suggest a likely pathogenic effect. Another allele, p.R302L was also not classified as pathogenic by predictive programs. This variant was originally identified as a homozygous mutation in a large consanguineous family from India with at least three individuals affected with LCA. Epidemiologic studies did not reveal the presence of the allele in control DNA samples (Sohocki et al. 2000b). The newly reported LCA5 variant, p.G439E is predicted to be benign by available bioinformatic tools but at this point we are lacking additional data from segregation studies and functional assays to asses reliably its pathogenicity. In silico analysis is a powerful tool to study the effects of sequence substitutions on protein expression and some biophysical properties of the encoded proteins; however, further studies with biological systems are required to elucidate any potential pathological consequences of sequence variants. The reliable classification of sequence variants is frequently challenging, especially for genes exclusively expressed in the retina wherein functional studies of mutant alleles in vitro are difficult.

Table 4.

Characteristics of sequence variants identified in LCA patients with triallelic inheritance

Pedigree Gene Mutation Reference Grantham scorea MutationTaster prediction PolyPhen prediction SIFT prediction
Ar-21-04 RPGRIP1 p.D1114G 1 94 B B B
CEP290 c.2991+1655A>G 2 – – – –
CEP290 p.R1926P 3 103 D D D
Ar-39-04 GUCY2D p.I915V This report 29 D B B
GUCY2D p.T838RfsX27 3 – – – –
RPGRIP1 p.P585S This report 74 B D B
Ar-98-11 GUCY2D p.P701S 4 74 B B B
AIPL1 p.W278X 5 – – – –
AIPL1 p.R270H 6 29 D D B
Ar-123-04 GUCY2D p.R768Q 7 43 D D D
GUCY2D p.R768Q 7 43 D D D
AIPL1 p.W278X 5 – – – –
Ar-177-04 AIPL1 p.R302L 5 102 B B B
CEP290 p.A1832PfsX19 8 – – – –
CEP290 c.2991+1655A>G 2 – – – –
Ar-782-04 RPE65 p.L341S 9 145 D D D
RPE65 p.S121LfsX6 This report – – – –
AIPL1 p.R324L This report 102 D B/D B
Ar-863-03 CEP290 c.2991+1655A>G 2 – – – –
CEP290 p.F1950LfsX15 10 – – – –
LCA5 p.G439E This report 85 B B B
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B benign variant, D disease causing variant, B/D variant of unknown significance

(1) Gerber et al. (2001), (2) den Hollander et al. (2006), (3) Stone (2007), (4) Dharmaraj et al. (2000), (5) Sohocki et al. (2000b), (6) Simonelli et al. (2007), (7) Lotery et al. (2000), (8) Brancati et al. (2007), (9) Morimura et al. (1998), (10) Perrault et al. (2007)

a

Substitutions determined as conservative (0–50), moderately conservative (51–100), moderately radical (101–150), radical (>150)

The significance of the third mutated allele is unknown but one potential interpretation is that the penetrance or trait manifestation can be associated with triallelic inheritance as has been observed for other ciliopathies. An alternative explanation is that this combination of three mutant alleles occurred randomly and is not associated with trait manifestations. Inconsistent with this latter hypothesis is our observation that the putative third mutant allele is often one that is in a gene infrequently found causative in this North American population studied (e.g. RPGRIP1, LCA5). Furthermore, the occurrence of a mutation in another gene appears more frequently than would be expected by chance (p = 1.9 × 10−6 by binomial test, assuming the background LCA mutant allele frequency at 0.01). If the mutation of one of the genes is responsible for causing LCA in affected family members, then the chances of having a mutation in another LCA gene should be no different than the population risk. Mutations in more than one LCA locus were reported in 22/300 (7%) patients with Leber congenital amaurosis by Zernant et al. (2005). Those authors proposed that the additional mutation might exert an additive effect on the LCA phenotype as some patients with three hits presented with an apparently more severe disease as determined by clinical examination and onset of clinical symtoms (Zernant et al. 2005).

Non-Mendelian inheritance has been described previously for a number of hereditary eye disorders. Mutations in peripherin and RDS can result in a digenic retinitis pigmentosa (RP7), wherein affected individuals are double heterozygotes (Kajiwara et al. 1994). The pleiotropic disorder Bardet–Biedl syndrome is characterized by early-onset retinal dystrophy and multisystem involvement. There are at least 14 BBS loci and mutations in two BBS loci have been described in less than 10% of families with BBS. The third mutant allele may be required for penetrance (Katsanis et al. 2001). However, it has been proposed that the third allele may modify the severity of clinical phenotype (Badano et al. 2006a).

Since the discovery of triallelic inheritance, much progress has been made to identify genes that modify the clinical outcome in patients with retinal diseases. One of these genes, RPGRIP1L, modifies the severity of retinal dystrophy in patients with LCA and other ciliopathies including SLS, BBS, and MKS, although mutations in this gene alone are not sufficient to cause LCA. The RPGRIP1L variants were found in LCA patients among whom mutation studies revealed homozygous, compound heterozygous, and heterozygous mutations in LCA-causing genes (Khanna et al. 2009). We hypothesize that the third mutated allele may contribute to the penetrance of retinal deterioration in patients with LCA. However, it is challenging to assess its clinical effect as LCA is a condition of very early onset, rapid progression, and relatively homogenous clinical presentation.

Our study provides evidence that mutational load may be important to trait manifestation in LCA. Further studies are required to better comprehend the molecular underpinnings of these genetic observations.

Acknowledgments

We are grateful for willing participation and the continuing support of families who joined these efforts. This work was supported in part by National Eye Institute (NIH) R01EY018571to R.C. R.A.L. is a Senior Scientific Investigator of Research to Prevent Blindness, New York. Some of the early work reported here was supported in part by the Foundation Fighting Blindness, Owens Mills, MD.

References

  1. Abd-El-Barr MM, Sykoudis K, Andrabi S, Eichers ER, Pennesi ME, Tan PL, Wilson JH, Katsanis N, Lupski JR, Wu SM. Impaired photoreceptor protein transport and synaptic transmission in a mouse model of Bardet–Biedl syndrome. Vision Res. 2007;47(27):3394–3407. doi: 10.1016/j.visres.2007.09.016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Akey DT, Zhu X, Dyer M, Li A, Sorensen A, Blackshaw S, Fukuda-Kamitani T, Daiger SP, Craft CM, Kamitani T, Sohocki MM. The inherited blindness associated protein AIPL1 interacts with the cell cycle regulator protein NUB1. Hum Mol Genet. 2002;11(22):2723–2733. doi: 10.1093/hmg/11.22.2723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Badano JL, Katsanis N. Beyond Mendel: an evolving view of human genetic disease transmission. Nat Rev Genet. 2002;3(10):779–789. doi: 10.1038/nrg910. [DOI] [PubMed] [Google Scholar]
  4. Badano JL, Leitch CC, Ansley SJ, May-Simera H, Lawson S, Lewis RA, Beales PL, Dietz HC, Fisher S, Katsanis N. Dissection of epistasis in oligogenic Bardet–Biedl syndrome. Nature. 2006a;439(7074):326–330. doi: 10.1038/nature04370. [DOI] [PubMed] [Google Scholar]
  5. Badano JL, Mitsuma N, Beales PL, Katsanis N. The ciliopathies: an emerging class of human genetic disorders. Annu Rev Genomics Hum Genet. 2006b;7:125–148. doi: 10.1146/annurev.genom.7.080505.115610. [DOI] [PubMed] [Google Scholar]
  6. Booij JC, Florijn RJ, ten Brink JB, Loves W, Meire F, van Schooneveld MJ, de Jong PT, Bergen AA. Identification of mutations in the AIPL1, CRB1, GUCY2D, RPE65, and RPGRIP1 genes in patients with juvenile retinitis pigmentosa. J Med Genet. 2005;42(11):e67. doi: 10.1136/jmg.2005.035121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brancati F, Barrano G, Silhavy JL, Marsh SE, Travaglini L, Bielas SL, Amorini M, Zablocka D, Kayserili H, Al-Gazali L, Bertini E, Boltshauser E, D'Hooghe M, Fazzi E, Fenerci EY, Hennekam RC, Kiss A, Lees MM, Marco E, Phadke SR, Rigoli L, Romano S, Salpietro CD, Sherr EH, Signorini S, Stromme P, Stuart B, Sztriha L, Viskochil DH, Yuksel A, Dallapiccola B, Valente EM, Gleeson JG. CEP290 mutations are frequently identified in the oculo-renal form of Joubert syndrome-related disorders. Am J Hum Genet. 2007;81(1):104–113. doi: 10.1086/519026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chiang PW, Spector E, McGregor TL. Evidence suggesting digenic inheritance of Waardenburg syndrome type II with ocular albinism. Am J Med Genet A. 2009;149(12):2739–2744. doi: 10.1002/ajmg.a.33128. [DOI] [PubMed] [Google Scholar]
  9. Coppieters F, Casteels I, Meire F, De Jaegere S, Hooghe S, van Regemorter N, Van Esch H, Matuleviciene A, Nunes L, Meersschaut V, Walraedt S, Standaert L, Coucke P, Hoeben H, Kroes HY, Vande Walle J, de Ravel T, Leroy BP, De Baere E. Genetic screening of LCA in Belgium: predominance of CEP290 and identification of potential modifier alleles in AHI1 of CEP290-related phenotypes. Hum Mutat Aug. 2010;5 doi: 10.1002/humu.21336. [Epub ahead of print] [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cremers FP, van den Hurk JA, den Hollander AI. Molecular genetics of Leber congenital amaurosis. Hum Mol Genet. 2002;11(10):1169–1176. doi: 10.1093/hmg/11.10.1169. [DOI] [PubMed] [Google Scholar]
  11. den Hollander AI, Johnson K, de Kok YJ, Klebes A, Brunner HG, Knust E, Cremers FP. CRB1 has a cytoplasmic domain that is functionally conserved between human and drosophila. Hum Mol Genet. 2001;10(24):2767–2773. doi: 10.1093/hmg/10.24.2767. [DOI] [PubMed] [Google Scholar]
  12. den Hollander AI, Koenekoop RK, Yzer S, Lopez I, Arends ML, Voesenek KE, Zonneveld MN, Strom TM, Meitinger T, Brunner HG, Hoyng CB, van den Born LI, Rohrschneider K, Cremers FP. Mutations in the CEP290 (NPHP6) gene are a frequent cause of Leber congenital amaurosis. Am J Hum Genet. 2006;79(3):556–561. doi: 10.1086/507318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. den Hollander AI, Roepman R, Koenekoop RK, Cremers FP. Leber congenital amaurosis: genes, proteins and disease mechanisms. Prog Retin Eye Res. 2008;27(4):391–419. doi: 10.1016/j.preteyeres.2008.05.003. [DOI] [PubMed] [Google Scholar]
  14. Dharmaraj SR, Silva ER, Pina AL, Li YY, Yang JM, Carter CR, Loyer MK, El-Hilali HK, Traboulsi EK, Sundin OK, Zhu DK, Koenekoop RK, Maumenee IH. Mutational analysis and clinical correlation in Leber congenital amaurosis. Ophthalmic Genet. 2000;21(3):135–150. [PubMed] [Google Scholar]
  15. Ebermann I, Phillips JB, Liebau MC, Koenekoop RK, Schermer B, Lopez I, Schafer E, Roux AF, Dafinger C, Bernd A, Zrenner E, Claustres M, Blanco B, Nurnberg G, Nurnberg P, Ruland R, Westerfield M, Benzing T, Bolz HJ. PDZD7 is a modifier of retinal disease and a contributor to digenic Usher syndrome. J Clin Invest. 2010;120(6):1812–1823. doi: 10.1172/JCI39715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Eichers ER, Lewis RA, Katsanis N, Lupski JR. Triallelic inheritance: a bridge between Mendelian and multifactorial traits. Ann Med. 2004;36(4):262–272. doi: 10.1080/07853890410026214. [DOI] [PubMed] [Google Scholar]
  17. Gerber S, Perrault I, Hanein S, Barbet F, Ducroq D, Ghazi I, Martin-Coignard D, Leowski C, Homfray T, Dufier JL, Munnich A, Kaplan J, Rozet JM. Complete exon-intron structure of the RPGR-interacting protein (RPGRIP1) gene allows the identification of mutations underlying Leber congenital amaurosis. Eur J Hum Genet. 2001;9(8):561–571. doi: 10.1038/sj.ejhg.5200689. [DOI] [PubMed] [Google Scholar]
  18. Hanein S, Perrault I, Gerber S, Tanguy G, Barbet F, Ducroq D, Calvas P, Dollfus H, Hamel C, Lopponen T, Munier F, Santos L, Shalev S, Zafeiriou D, Dufier JL, Munnich A, Rozet JM, Kaplan J. Leber congenital amaurosis: comprehensive survey of the genetic heterogeneity, refinement of the clinical definition, and genotype-phenotype correlations as a strategy for molecular diagnosis. Hum Mutat. 2004;23(4):306–317. doi: 10.1002/humu.20010. [DOI] [PubMed] [Google Scholar]
  19. Hichri H, Stoetzel C, Laurier V, Caron S, Sigaudy S, Sarda P, Hamel C, Martin-Coignard D, Gilles M, Leheup B, Holder M, Kaplan J, Bitoun P, Lacombe D, Verloes A, Bonneau D, Perrin-Schmitt F, Brandt C, Besancon AF, Mandel JL, Cossee M, Dollfus H. Testing for triallelism: analysis of six BBS genes in a Bardet–Biedl syndrome family cohort. Eur J Hum Genet. 2005;13(5):607–616. doi: 10.1038/sj.ejhg.5201372. [DOI] [PubMed] [Google Scholar]
  20. Ito S, Nakamura M, Nuno Y, Ohnishi Y, Nishida T, Miyake Y. Novel complex GUCY2D mutation in Japanese family with cone-rod dystrophy. Invest Ophthalmol Vis Sci. 2004;45(5):1480–1485. doi: 10.1167/iovs.03-0315. [DOI] [PubMed] [Google Scholar]
  21. Kajiwara K, Berson EL, Dryja TP. Digenic retinitis pigmentosa due to mutations at the unlinked peripherin/RDS and ROM1 loci. Science. 1994;264(5165):1604–1608. doi: 10.1126/science.8202715. [DOI] [PubMed] [Google Scholar]
  22. Katsanis N, Ansley SJ, Badano JL, Eichers ER, Lewis RA, Hoskins BE, Scambler PJ, Davidson WS, Beales PL, Lupski JR. Triallelic inheritance in Bardet–Biedl syndrome, a Mendelian recessive disorder. Science. 2001;293(5538):2256–2259. doi: 10.1126/science.1063525. [DOI] [PubMed] [Google Scholar]
  23. Khanna H, Davis EE, Murga-Zamalloa CA, Estrada-Cuzcano A, Lopez I, den Hollander AI, Zonneveld MN, Othman MI, Waseem N, Chakarova CF, Maubaret C, Diaz-Font A, Macdonald I, Muzny DM, Wheeler DA, Morgan M, Lewis LR, Logan CV, Tan PL, Beer MA, Inglehearn CF, Lewis RA, Jacobson SG, Bergmann C, Beales PL, Attie-Bitach T, Johnson CA, Otto EA, Bhattacharya SS, Hildebrandt F, Gibbs RA, Koenekoop RK, Swaroop A, Katsanis N. A common allele in RPGRIP1L is a modifier of retinal degeneration in ciliopathies. Nat Genet. 2009;41(6):739–745. doi: 10.1038/ng.366. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Koenekoop RK. An overview of Leber congenital amaurosis: a model to understand human retinal development. Surv Ophthalmol. 2004;49(4):379–398. doi: 10.1016/j.survophthal.2004.04.003. [DOI] [PubMed] [Google Scholar]
  25. Lewis RA, editor. Retinal dystrophies and degenerations. Raven Press; New York: 1988. Juvenile hereditary macular dystrophies. [Google Scholar]
  26. Lewis RA, Shroyer NF, Singh N, Allikmets R, Hutchinson A, Li Y, Lupski JR, Leppert M, Dean M. Genotype/phenotype analysis of a photoreceptor-specific ATP-binding cassette transporter gene, ABCR, in Stargardt disease. Am J Hum Genet. 1999;64(2):422–434. doi: 10.1086/302251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Li Y, Wang H, Peng J, Gibbs RA, Lewis RA, Lupski JR, Mardon G, Chen R. Mutation survey of known LCA genes and loci in the Saudi Arabian population. Invest Ophthalmol Vis Sci. 2009;50(3):1336–1343. doi: 10.1167/iovs.08-2589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lotery AJ, Namperumalsamy P, Jacobson SG, Weleber RG, Fishman GA, Musarella MA, Hoyt CS, Heon E, Levin A, Jan J, Lam B, Carr RE, Franklin A, Radha S, Andorf JL, Sheffield VC, Stone EM. Mutation analysis of 3 genes in patients with Leber congenital amaurosis. Arch Ophthalmol. 2000;118(4):538–543. doi: 10.1001/archopht.118.4.538. [DOI] [PubMed] [Google Scholar]
  29. Morimura H, Fishman GA, Grover SA, Fulton AB, Berson EL, Dryja TP. Mutations in the RPE65 gene in patients with autosomal recessive retinitis pigmentosa or Leber congenital amaurosis. Proc Natl Acad Sci USA. 1998;95(6):3088–3093. doi: 10.1073/pnas.95.6.3088. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Perrault I, Hanein S, Gerber S, Barbet F, Ducroq D, Dollfus H, Hamel C, Dufier JL, Munnich A, Kaplan J, Rozet JM. Retinal dehydrogenase 12 (RDH12) mutations in Leber congenital amaurosis. Am J Hum Genet. 2004;75(4):639–646. doi: 10.1086/424889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Perrault I, Delphin N, Hanein S, Gerber S, Dufier JL, Roche O, Defoort-Dhellemmes S, Dollfus H, Fazzi E, Munnich A, Kaplan J, Rozet JM. Spectrum of NPHP6/CEP290 mutations in Leber congenital amaurosis and delineation of the associated phenotype. Hum Mutat. 2007;28(4):416. doi: 10.1002/humu.9485. [DOI] [PubMed] [Google Scholar]
  32. Perrault I, Hanein S, Gerard X, Delphin N, Fares-Taie L, Gerber S, Pelletier V, Merce E, Dollfus H, Puech B, Defoort-Dhellemmes S, Petersen MD, Zafeiriou D, Munnich A, Kaplan J, Roche O, Rozet JM. Spectrum of SPATA7 mutations in Leber congenital amaurosis and delineation of the associated phenotype. Hum Mutat. 2010;31(3):E1241–E1250. doi: 10.1002/humu.21203. [DOI] [PubMed] [Google Scholar]
  33. Shroyer NF, Lewis RA, Allikmets R, Singh N, Dean M, Leppert M, Lupski JR. The rod photoreceptor ATP-binding cassette transporter gene, ABCR, and retinal disease: from monogenic to multifactorial. Vis Res. 1999;39(15):2537–2544. doi: 10.1016/s0042-6989(99)00037-1. [DOI] [PubMed] [Google Scholar]
  34. Shroyer NF, Lewis RA, Yatsenko AN, Lupski JR. Null missense ABCR (ABCA4) mutations in a family with Stargardt disease and retinitis pigmentosa. Invest Ophthalmol Vis Sci. 2001a;42(12):2757–2761. [PubMed] [Google Scholar]
  35. Shroyer NF, Lewis RA, Yatsenko AN, Wensel TG, Lupski JR. Cosegregation and functional analysis of mutant ABCR (ABCA4) alleles in families that manifest both Stargardt disease and age-related macular degeneration. Hum Mol Genet. 2001b;10(23):2671–2678. doi: 10.1093/hmg/10.23.2671. [DOI] [PubMed] [Google Scholar]
  36. Simonelli F, Ziviello C, Testa F, Rossi S, Fazzi E, Bianchi PE, Fossarello M, Signorini S, Bertone C, Galantuomo S, Brancati F, Valente EM, Ciccodicola A, Rinaldi E, Auricchio A, Banfi S. Clinical and molecular genetics of Leber's congenital amaurosis: A multicenter study of Italian patients. Invest Ophthalmol Vis Sci. 2007;48(9):4284–4290. doi: 10.1167/iovs.07-0068. [DOI] [PubMed] [Google Scholar]
  37. Sohocki MM, Browne SJ, Sullivan LS, Blackshaw S, Cepko CL, Payne AM, Bhattacharya SS, Khaliq S, Mehdi SQ, Birch DG, Harrison WR, Elder FF, Heckenlively JR, Daiger SP. Mutations in a new photoreceptor-pineal gene on 17p cause Leber congenital amaurosis. Am J Ophthalmol. 2000a;129(6):834–835. doi: 10.1016/s0002-9394(00)00517-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sohocki MM, Perrault I, Leroy BP, Payne AM, Dharmaraj S, Bhattacharya SS, Kaplan J, Maumenee IH, Koenekoop R, Meire FM, Birch DG, Heckenlively JR, Daiger SP. Prevalence of AIPL1 mutations in inherited retinal degenerative disease. Mol Genet Metab. 2000b;70(2):142–150. doi: 10.1006/mgme.2000.3001. [DOI] [PubMed] [Google Scholar]
  39. Stone EM. Leber congenital amaurosis—a model for efficient genetic testing of heterogeneous disorders: LXIV Edward Jackson memorial lecture. Am J Ophthalmol. 2007;144(6):791–811. doi: 10.1016/j.ajo.2007.08.022. [DOI] [PubMed] [Google Scholar]
  40. Swaroop A, Wang QL, Wu W, Cook J, Coats C, Xu S, Chen S, Zack DJ, Sieving PA. Leber congenital amaurosis caused by a homozygous mutation (R90W) in the homeodomain of the retinal transcription factor CRX: direct evidence for the involvement of CRX in the development of photoreceptor function. Hum Mol Genet. 1999;8(2):299–305. doi: 10.1093/hmg/8.2.299. [DOI] [PubMed] [Google Scholar]
  41. Thompson DA, Janecke AR, Lange J, Feathers KL, Hubner CA, McHenry CL, Stockton DW, Rammesmayer G, Lupski JR, Antinolo G, Ayuso C, Baiget M, Gouras P, Heckenlively JR, den Hollander A, Jacobson SG, Lewis RA, Sieving PA, Wissinger B, Yzer S, Zrenner E, Utermann G, Gal A. Retinal degeneration associated with RDH12 mutations results from decreased 11-cis retinal synthesis due to disruption of the visual cycle. Hum Mol Genet. 2005;14(24):3865–3875. doi: 10.1093/hmg/ddi411. [DOI] [PubMed] [Google Scholar]
  42. Tsang WY, Bossard C, Khanna H, Peranen J, Swaroop A, Malhotra V, Dynlacht BD. CP110 suppresses primary cilia formation through its interaction with CEP290, a protein deficient in human ciliary disease. Dev Cell. 2008;15(2):187–197. doi: 10.1016/j.devcel.2008.07.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wiszniewski W, Zaremba CM, Yatsenko AN, Jamrich M, Wensel TG, Lewis RA, Lupski JR. ABCA4 mutations causing mislocalization are found frequently in patients with severe retinal dystrophies. Hum Mol Genet. 2005;14(19):2769–2778. doi: 10.1093/hmg/ddi310. [DOI] [PubMed] [Google Scholar]
  44. Xu T, Yang Z, Vatta M, Rampazzo A, Beffagna G, Pilichou K, Scherer SE, Saffitz J, Kravitz J, Zareba W, Danieli GA, Lorenzon A, Nava A, Bauce B, Thiene G, Basso C, Calkins H, Gear K, Marcus F, Towbin JA. Compound and digenic heterozygosity contributes to arrhythmogenic right ventricular cardiomyopathy. J Am Coll Cardiol. 2010;55(6):587–597. doi: 10.1016/j.jacc.2009.11.020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Yzer S, Leroy BP, De Baere E, de Ravel TJ, Zonneveld MN, Voesenek K, Kellner U, Ciriano JP, de Faber JT, Rohrschneider K, Roepman R, den Hollander AI, Cruysberg JR, Meire F, Casteels I, van Moll-Ramirez NG, Allikmets R, van den Born LI, Cremers FP. Microarray-based mutation detection and phenotypic characterization of patients with Leber congenital amaurosis. Invest Ophthalmol Vis Sci. 2006;47(3):1167–1176. doi: 10.1167/iovs.05-0848. [DOI] [PubMed] [Google Scholar]
  46. Zernant J, Kulm M, Dharmaraj S, den Hollander AI, Perrault I, Preising MN, Lorenz B, Kaplan J, Cremers FP, Maumenee I, Koenekoop RK, Allikmets R. Genotyping microarray (disease chip) for Leber congenital amaurosis: detection of modifier alleles. Invest Ophthalmol Vis Sci. 2005;46(9):3052–3059. doi: 10.1167/iovs.05-0111. [DOI] [PubMed] [Google Scholar]

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