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. Author manuscript; available in PMC: 2014 Mar 5.
Published in final edited form as: Hum Mutat. 2011 Sep 23;32(12):1450–1459. doi: 10.1002/humu.21587

Whole-Exome Sequencing Identifies ALMS1, IQCB1, CNGA3, and MYO7A Mutations in Patients with Leber Congenital Amaurosis

Xia Wang 1,†, Hui Wang 1,2,†, Ming Cao 2, Zhe Li 3, Xianfeng Chen 1, Claire Patenia 2, Athurva Gore 3, Emad B Abboud 4, Ali A Al-Rajhi 4, Richard A Lewis 1,5,6, James R Lupski 1,6,7, Graeme Mardon 1,7,8,9,10, Kun Zhang 3, Donna Muzny 1,2, Richard A Gibbs 1,2, Rui Chen 1,2,10,*
PMCID: PMC3943164  NIHMSID: NIHMS450228  PMID: 21901789

Abstract

It has been well documented that mutations in the same retinal disease gene can result in different clinical phenotypes due to difference in the mutant allele and/or genetic background. To evaluate this, a set of consanguineous patient families with Leber congenital amaurosis (LCA) that do not carry mutations in known LCA disease genes was characterized through homozygosity mapping followed by targeted exon/whole-exome sequencing to identify genetic variations. Among these families, a total of five putative disease-causing mutations, including four novel alleles, were found for six families. These five mutations are located in four genes, ALMS1, IQCB1, CNGA3, and MYO7A. Therefore, in our LCA collection from Saudi Arabia, three of the 37 unassigned families carry mutations in retinal disease genes ALMS1, CNGA3, and MYO7A, which have not been previously associated with LCA, and 3 of the 37 carry novel mutations in IQCB1, which has been recently associated with LCA. Together with other reports, our results emphasize that the molecular heterogeneity underlying LCA, and likely other retinal diseases, may be highly complex. Thus, to obtain accurate diagnosis and gain a complete picture of the disease, it is essential to sequence a larger set of retinal disease genes and combine the clinical phenotype with molecular diagnosis.

Keywords: Leber congenital amaurosis, LCA, whole-exome sequencing, SNP, padlock

Introduction

Leber congenital amaurosis (LCA; MIM# 204000) is an early-onset retinal dystrophy that is often diagnosed at birth or within the first year of life. The clinical features of LCA include blindness or severe vision impairment, congenital nystagmus, amaurotic pupils, and reduced or absence of signal in electroretinogram (ERG) [Franceschetti and Dieterle, 1954]. It is estimated to affect one in every 30,000~80,000 individuals and represents 5% of all retinal dystrophies [Dharmaraj et al., 2000; Kaplan et al., 1990; Leber, 1869; Stone, 2007].

LCA is a genetically heterogeneous and predominantly autosomal recessive disease. Up to now, mutations in 16 genes have been associated with LCA: aryl hydrocarbon receptor interacting protein-like 1 (AIPL1), centrosomal protein 290 kDa (CEP290), crumbs homolog 1 (Drosophila) (CRB1), cone-rod homeobox (CRX), guanylate cyclase 2D (GUCY2D), inosine 5′-monophosphate dehydrogenase 1 (IMPDH1), IQ motif containing B1 (IQCB1, also known as NPHP5), Leber congenital amaurosis 5 (LCA5), lecithin retinol acyltransferase (LRAT), orthodenticle homeobox 2 (OTX2), retinal degeneration 3 (RD3), retinol dehydrogenase 12 (RDH12), retinal pigment epithelium-specific protein 65 kDa (RPE65), retinitis pigmentosa GTPase regulator interacting protein 1 (RPGRIP1), spermatogenesis associated 7 (SPATA7), and tubby-like protein 1 (TULP1). [Bowne et al., 2006; den Hollander et al., 2001, 2007a,b, 2006; Dryja et al., 2001; Estrada-Cuzcano et al., 2011; Freund et al., 1998; Friedman et al., 2006; Henderson et al., 2009; Janecke et al., 2004; Marlhens et al., 1997; Mataftsi et al., 2007; Perrault et al., 2004, 1996; Senechal et al., 2006; Sohocki et al., 2000; Stone et al., 2011; Wang et al., 2009]. These genes are involved in various physiological pathways that are important for retinal function, including phototransduction (GUCY2D), maintenance of photoreceptor cell function (AIPL1, TULP1, RD3), visual cycle (LRAT, RPE65, RDH12), centrosomal or ciliary function (CEP290, IQCB1, LCA5, RPGRIP1), retinal development (CRB1, CRX, OTX2), and guanine nucleotide biosynthesis (IMPDH1) [Azadi et al., 2010; den Hollander et al., 2007a, 2006; Dizhoor, 2000; Escobar-Henriques and Daignan-Fornier, 2001; Furukawa et al., 1997; Mehalow et al., 2003; Murga-Zamalloa et al., 2009; Nishida et al., 2003; O’Byrne et al., 2005; Otto et al., 2005; Ramamurthy et al., 2004; Redmond et al., 1998; Thompson et al., 2005; Xi et al., 2005].

Recently, it was reported that certain mutant alleles in syndromic ocular disease genes may cause nonsyndromic LCA. For example, mutations in gene IQCB1 were previously reported to cause Senior-Løken syndrome (SLSN), which is characterized by retinal defects and nephronophthisis [Otto et al., 2005]. However, several nonsense and frameshift mutations in IQCB1 have been found in LCA patients without nephronophthisis or overt renal disease, suggesting that mutations in IQCB1 may cause LCA without having other syndromic phenotypes [Estrada-Cuzcano et al., 2011; Stone et al., 2011]. Considering that many nonsyndromic and syndromic ocular diseases, such as achromatopsia, Alstrom syndrome (ALMS), Batten disease, and SLSN, are associated with “LCA-like ocular phenotypes” [den Hollander et al., 2008], it is conceivable that some percentage of patients diagnosed with LCA are actually caused by either a specific allele or combination of disease gene alleles or mis-assignment due to the absence of syndromic features at the time of diagnosis.

Previously, we collected 37 consanguineous families with recessive LCA from Saudi Arabia. PCR and Sanger sequencing were performed for these families to screen for mutations in all known LCA genes [Li et al., 2009]. Among them, mutations have been identified in nine families. To identify the underlying mutations in the remaining consanguineous LCA disease families, homozygosity mapping using high-density SNP genotyping arrays followed by targeted or whole-exome sequencing was performed. Interestingly, mutations have been identified in four genes, ALMS1, IQCB1 (also known as NPHP5), CNGA3, and MYO7A, in six consanguineous LCA families, accounting for 16% of our collection (six of the 37) (Table 1). Recent studies have linked mutations in IQCB1 with LCA without renal failure [Estrada-Cuzcano et al., 2011; Stone et al., 2011]. Whereas mutations in ALMS1, CNGA3, and MYO7A are known to be associated with syndromic or nonsyndromic eye diseases, they have not been previously linked to LCA. Therefore, our results support an emerging theme that a significant fraction of patients diagnosed with LCA may be accounted for by mutations in syndromic and other retinal disease genes. These results highlight the importance of combining molecular diagnosis with clinical findings for diseases with high genetic heterogeneity in order to obtain accurate diagnosis and devise a proper treatment strategy.

Table 1.

Summary of Mutations Found Segregating with LCA in Six Families

Mutation # Protein change Family Affected individuals Gene OMIM accession number
c.10945G>T p.E3649X KKESH72 6 ALMS1 606844
c.1130-1G>C KKESH24,88 2 IQCB1 609237
c.1479C>A p.Y493X KKESH28 1 IQCB1 609237
c.1579C>A p.L527M KKESH2 1 CNGA3 600053
c.578C>T p.T193I KKESH34 4 MYO7A 276903
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#

ALMS1 gene sequence [NM_015120.4]. IQCB1 gene sequence [NM_001023570.2]. CNGA3 gene sequence [NM_001298.2]. MYO7A gene sequence [NM_000260.3]. Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence, according to journal guidelines (www.hgvs.org/mutnomen). The initiation codon is codon 1. None of these mutations are found in normal matching controls or the dbSNP130 or 1,000 Genome databases.

The mutation in the ALMS1 gene is a previously reported allele and all the other mutations are novel. Mutations are validated by Sanger sequencing and segregate with the LCA phenotype within each family. Mutations are not present in normal matching controls. Mutations are within the homozygous regions that we identified for each family by SNP genotyping. All listed genes have been previously associated with syndromic or nonsyndromic eye diseases other than LCA.

Materials and Methods

Sample Collection

We obtained blood samples and pedigrees after receiving informed consent from all individuals. Approval was obtained from the Institutional Review Boards of the participating centers. LCA families KKESH24, KKESH28, KKESH34, KKESH72, and KKESH88 were obtained by Dr. Lewis through the King Khaled Eye Specialist Hospital (KKESH) in Riyadh, Saudi Arabia. Blood samples were collected from all available family members, and DNA was extracted with the Qiagen blood genomic DNA extraction kit (QIAGEN Inc., Valencia, CA) following the protocol provided by the manufacturer. The pedigrees of these families are shown in Figure 1. The number of affected and unaffected members of each family is listed in Table 2 and can be seen in Figure 1. All of the LCA patients were diagnosed with typical LCA clinical features (Table 2). Patients have experienced vision defects since birth or as early as 2 years old. No significant syndromic phenotypes were observed, except that a patient in KKESH88 showed midfacial hypoplasia and psychomotor delay.

Figure 1.

Figure 1

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Pedigrees of six consanguineous LCA KKESH families. Affected: solid symbols; unaffected: open symbols; squares: male; circles: female.

Table 2.

Summary of Six Families with LCA

Family Affected individuals Unaffected individuals Clinical features
KKESH2 1 6 At age 5 months, nystagmus was recorded “shortly after birth,” increasing in amplitude. A nonrecordable ERG was observed at age 10 months old. The patient was noted at 2 years old to have very sluggish pupils. No visual responses were elicited.
KKESH72 6 29 The patients show irregular horizontal nystagmus, anterior and posterior cuneiform spoke-like cataracts, and mild cortical haze. Patients’ fundus shows vertically oval discs, waxy orange pallor, 3+/4 vascular attenuation, pigmentary maculopathy with central ellipse of gray silvery atrophy, and coarse granular diffuse RPE peripheral atrophy.
KKESH24 1 4 The patient has nystagmus, hyperopic discs, vascular attenuation, no retinal pigmentation, diffuse RPE atrophy, and nonrecordable ERG.
KKESH28 1 4 The patient shows nystagmus and eye rubbing. The eyes also have trouble in fixation and following movements. The ERG of the patient is nonrecordable.
KKESH88 1 6 The patient shows midfacial hypoplasia, enophthalmos, horizontal and rotary nystagmus, variable LET, and LIO overaction. Furthermore, he has hypermetropic discs with hyperemia, moderate vascular attenuation, and sandy RPE throughout. The ERG is nonrecordable. In addition to eye phenotypes, he also shows psychomotor delay.
KKESH34 4 11 All patients of KKESH34 have poor vision from birth. They all show nystagmus and neuroepithelial atrophy. The ERG of all patients is nonrecordable.
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All affected individuals from these KKESH families present with typical LCA phenotypes.

LET, left esotropia; LIO, left inferior oblique.

Homozygosity Mapping of LCA Patient Families

Homozygosity mapping using high-density SNP arrays was performed on these six consanguineous LCA families (Fig. 1). SNP genotypes were obtained for multiple affected members and unaffected siblings from each pedigree. Homozygous blocks greater than 1 Mb in size were identified from each individual. Based on recessive inheritance models, candidate disease loci are defined by shared homozygous blocks among all affected individuals but those are heterozygous or absent in unaffected siblings. The homozygous regions for six families range from 2000 bp to more than 57 Mb in size (Table 3).

Table 3.

Summary of Homozygous Blocks Identified in Six Families with LCA

Family Chr Start End Length
KKESH2 10 4,692,299 28,687,145 23,994,846
10 95,392,963 119,610,549 24,217,586
2 67,335,826 87,175,553 19,839,727
2 94,759,484 100,814,358 6,054,874
2 237,371,678 242,692,820 5,321,142
13 74,774,390 97,207,720 22,433,330
4 844,712 6,374,355 5,529,643
4 13,417,458 48,967,354 35,549,896
6 5,767,191 13,849,594 8,082,403
6 22,303,479 42,929,919 20,626,440
16 13,703,368 21,234,774 7,531,406
7 102,176,469 130,176,130 27,999,661
8 103,136,803 121,252,831 18,116,028
KKESH72 2 71,564,649 74,339,404 2,774,755
KKESH24 1 27,535,253 43,222,769 15,687,516
20 53,239,401 59,936,745 6,697,344
3 104,855,337 104,877,189 21,852
3 122,757,953 129,806,851 7,048,898
7 106,234,136 126,577,422 20,343,286
KKESH28 1 185,439,838 200,728,605 15,288,767
1 200,745,815 206,592,720 5,846,905
11 134,433,812 134,435,899 2,087
3 118,133,470 169,824,936 51,691,466
12 5,363,096 19,757,396 14,394,300
21 28,327,299 28,339,633 12,334
21 41,134,108 46,919,231 5,785,123
13 75,079,282 89,305,858 14,226,576
6 23,674,393 41,934,989 18,260,596
16 6,564,364 8,538,170 1,973,806
8 111,858,228 140,521,054 28,662,826
17 46,238,130 60,493,874 14,255,744
KKESH88 1 155,091,425 178,231,760 23,140,335
20 11,799 5,655,548 5,643,749
21 31,374,803 40,419,896 9,045,093
3 97,652,153 129,099,032 31,446,879
13 46,193,917 71,963,849 25,769,932
5 107,956,758 112,004,899 4,048,141
7 146,181,172 151,027,186 4,846,014
KKESH34 11 54,631,614 91,827,532 37,195,918
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The homozygous regions are shared by affected members and are either heterozygous or are not present in unaffected members. The coordinates are based on Human March 2006 (NCBI36/hg18) Assembly.

Whole-Exome Sequencing

Whole-exome capture sequencing provides an alternative way to sequence large numbers of coding regions at a lower price than traditional PCR and Sanger sequencing. One affected member from each family was chosen for NimbleGen whole-exome capture followed by Illumina HiSeq paired-end sequencing. For each patient, a total of about 4.4 to 100 million reads were uniquely mapped to the targeted gene-coding regions with an average coverage of 9× to 467×. SNPs were identified based on filtering criteria (posterior probability cutoff = 0.9, minimum coverage = 3). A total of 52,188–370,000 SNPs were identified for further analysis (Supp. Tables S1–S6).

PCR and Direct Sequencing

Direct PCR and sequencing were used to validate the mutations found in human ALMS1 (RefSeq: NM_015120.4; MIM# 606844), IQCB1 (RefSeq: NM_001023570.2; MIM# 609237), CNGA3 (RefSeq: NM_001298.2; MIM# 600053), and MYO7A (Ref-Seq: NM_000260.3; MIM# 276903). Primers were designed using primer design tool Primer3 (http://biotools.umassmed.edu/bioapps/primer3_www.cgi) [Rozen and Skaletsky, 2000]. Each amplicon was 300–500 base pairs in length and was sequenced directly with an ABI3730 machine in both forward and reverse directions. Each read was aligned to the reference sequence and base changes were identified with the Sequencher program.

For mutations, nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence, according to journal guidelines (www.hgvs.org/mutnomen). The initiation codon is codon 1.

All mutations identified in this study have been submitted into The WayStation (http://www.centralmutations.org/) database or LOVD v 2.0 database [Fokkema et al., 2011].

Results

Candidate disease loci in consanguineous families were determined based on the property of identity-by-descent (IBD). To identify underlying mutations in the LCA family collection from Saudi Arabia, homozygosity mapping and targeted exon/whole-exome sequencing were performed. The pedigrees of the six families, KKESH2, KKESH24, KKESH28, KKESH34, KKESH72, and KKESH88, reported here are shown in Figure 1. The clinical phenotypes of affected members of these families are listed in Table 2.

A Nonsense Mutation in ALMS1 in the KKESH72 Family

As shown in Figure 1, one of the biggest families in our collection is KKESH72. It is a four generation family containing six affected and 29 unaffected members. Four of the affected and one of the unaffected members were genotyped using SNP arrays. Although multiple homozygous blocks are present in each individual, only one homozygous region located on chromosome 2 from 71.5 to 74.3 Mb is shared among all four affected members and is either heterozygous or absent in the unaffected individual (Table 3). Consistent with previous data, no known LCA gene is located within this region, indicating that this region must contain a novel LCA disease gene. A custom-designed padlock probe set covering all exons in this region was used for capture sequencing as described in the methods section. A total of 4.45 million reads were generated, with an average sequencing coverage of 467×. For individual KKESH72#13, a total of 15 homozygous rare variants were detected that cause amino acid (aa) changes and reside within the homozygous block. Among them, only one nonsense mutation was identified. This mutation is located in the sixteenth exon of ALMS1 (c.10945G>T, p. E3649X) (Fig. 2A and B and Supp. Table S1). Interestingly, this nonsense mutation has been previously reported to be associated with ALMS [Marshall et al., 2007]. This mutation was confirmed by direct Sanger sequencing (Fig. 2B) and segregates with the disease in this family where affected individuals are homozygous for this mutation and other unaffected individuals are either heterozygous or do not carry the mutation. ALMS1 encodes a protein of 4,169 aa that localizes to centrosomes and ciliary basal bodies, and is important for normal cilium function [Li et al., 2007]. This nonsense mutation in exon 16 will either give rise to a protein with a truncated C-terminus missing 520 aa that is likely to have partial function or cause complete loss of function due to nonsense-mediated mRNA decay [Li et al., 2007]. To further check the potential effect of this mutation in splicing, ESEfinder 3.0 [Cartegni et al., 2003; Smith et al., 2006] was used to identify potential exonic splicing enhancers (ESE) around this variant. Interestingly, three potential ESEs are identified that might be affected by the mutation (Supp. Table S7). In the case that the 16th exon is skipped due to the mutation, it will lead to shift of the open reading frame and truncation of the protein. Multiple mutations in human ALMS1 have been reported to cause the ALMS, which is characterized by cone–rod retinal dystrophy, cardiomyopathy, type 2 diabetes mellitus, obesity, and hearing loss [Bond et al., 2005; Collin et al., 2002; Hearn et al., 2002; Marshall et al., 2007]. Patients in the KKESH72 family are reported to have nystagmus, cataracts, fundus defects, and other retinal defects (Table 2). In contrast to the phenotype in ALMS, no other syndromic phenotypes such as hearing loss or obesity were observed.

Figure 2.

Figure 2

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Nonsense mutations and splicing changes are identified in ALSM1 and IQCB1. A: Exon–intron structure of ALSM1. Exons are shown as black boxes. The nonsense mutation p.E3649X is located in the sixteenth exon (red arrow). B: Sequence traces of control and affected members. A homozygous nonsense mutation from G to T in c.10945 is identified in affected member KKESH72#13 (blue arrow). C: Exon–intron structure of IQCB1. Exons are shown as black boxes. The splice acceptor site change is located at the 5′ boundary of the 12th exon and the nonsense mutation p.Y493X is located in the fourteenth exon (red arrows). D: Sequence traces of control and affected members. A homozygous splicing change in c.1130-1 is identified in affected members KKESH24#5 and KKESH88#3, and a homozygous nonsense mutation in c.1479 is identified in affected member KKESH28#5 (blue arrows).

A Nonsense Mutation and a Splicing Change in IQCB1 in Three KKESH Families

It has been recently reported that some of the mutant alleles in IQCB1 cause nonsyndromic LCA [Estrada-Cuzcano et al., 2011; Stone et al., 2011]. KKESH24, KKESH88, and KKESH28 are three consanguineous LCA families with one affected member in each family and four, six, and four unaffected members, respectively (Fig. 1). Based on homozygosity mapping, multiple homozygous blocks were identified for each family (Table 3). Whole-exome sequencing was conducted for one affected member from each family: KKESH24#5, KKESH88#5, and KKESH28#5. For the three KKESH-affected individuals, a total of 107, 117, and 81 million reads were generated, resulting average sequence coverage of 40×, 21×, and 30×, respectively. A total of 6, 4, and 27 homozygous rare variants that cause aa changes reside in each family’s homozygous block (Supp. Tables S2–S4). Among these candidate SNPs, only one obvious loss-of-function mutation is found for each family, all of which locate in IQCB1. One mutation is a novel splice acceptor site change (c.1130-1G>C) in KKESH24#5 and KKESH88#5, and the other is a novel nonsense mutation (c.1479C>A, p.Y493X) in KKESH28#5 (Fig. 2C and D). Both mutations are confirmed by direct Sanger sequencing (Fig. 2D) and are likely to be pathogenic. First, both mutations segregate with the disease in each family, with affected members being homozygous for the mutations and other unaffected individuals as either heterozygous or not carrying the mutation. Second, these mutations are rare as they are not found in 200 matching controls, the dbSNP130 database, or the 1,000 Genome database. Third, IQCB1 encodes an IQ-domain protein, which is important for cilium function and colocalizes with RPGR at the connecting cilia of photoreceptors [Otto et al., 2005]. Human mutations in IQCB1 are associated with LCA [Estrada-Cuzcano et al., 2011; Stone et al., 2011] and SLSN [Otto et al., 2005], which is characterized by nephronophthisis and retinal degeneration. The patients in these three KKESH families show typical LCA phenotypes such as nystagmus, nonrecordable ERGs, and other visual defects. However, no kidney defects were observed at the time of diagnosis.

A Missense Mutation in CNGA3 in the KKESH2 Family

KKESH2 is a four generation family with one affected and six unaffected members (Fig. 1). One unaffected and one affected member were genotyped by SNP arrays. Ten homozygous blocks ranging from 5.3 to 35.5 Mb were identified in the affected member and are not present in the unaffected member (Table 3). Whole-exome sequencing was conducted for one affected individual, KKESH2#4. A total of 100 million reads were generated with 36× coverage. SNP calling reveals 11 homozygous rare variants that lead to aa changes and reside within the homozygous region (Supp. Table S5). Based on gene annotation, we focus on a missense change (c.1579C>A, p.L527M) in the CNGA3 gene. This variant was confirmed by direct Sanger sequencing (Fig. 3A and B) and is likely to be pathogenic. First, it is located within the homozygous region chr2: 94.7–100.8 Mb and segregates with the disease in this family, given the affected member is homozygous for this mutation and other unaffected individuals are either heterozygous or do not carry the mutation. Second, this variant is rare as it is absent in all 200 normal matching controls. In addition, it is not recorded in the dbSNP130 database and the 1,000 Genome database. Third, CNGA3 encodes a member of the family of cyclic nucleotide-gated channel alpha 3 proteins, which are important for normal vision and olfactory signaling transduction. The p.L527M mutation resides in the cGMP-binding domain of CNGA3, which is important for the cyclic nucleotide gating mechanism (Fig. 3C). This mutated leucine is conserved across vertebrate species, from human to stickleback, further suggesting functional importance of this residue (Fig. 3D). Human mutations in this gene are reported in hereditary cone photoreceptor disorder, which is characterized by cone photoreceptor dysfunction, and achromatopsia, which leads to early vision loss, nystagmus, photophobia, color blindness, but has normal scotopic responses [Johnson et al., 2004; Wissinger et al., 2001]. Affected members of the KKESH2 family show early-onset nystagmus (at 5 months old), sluggish pupils, no visual response, and nonrecordable ERG at 10 months of age (Table 2). Taken together, these data suggest that the p.L527M mutation in CNGA3 is associated with the LCA phenotype in the KKESH2 family.

Figure 3.

Figure 3

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A missense mutation is identified in CNGA3. A: Exon–intron structure of CNGA3. Exons are shown as black boxes. The nonsense mutation p.L527M is located in the eighth exon (red arrow). B: Sequence traces of control and affected members. A homozygous missense mutation in c.1579 is identified in affected member KKESH2#4 (blue arrow). C: Schematic view of the CNGA3 protein structure. The p.L527M mutation is located within the intracellular cGMP-binding site. D: Amino acid alignment among 11 vertebrate species. The mutated leucine is located within this region and is conserved across all species.

A Missense Mutation in MYO7A in the KKESH34 Family

KKESH34 is a large consanguineous family containing four affected and 11 unaffected individuals (Fig. 1). All the affected and three unaffected members were genotyped by SNP arrays. The only homozygous region that was shared by affected members and not present in unaffected members resides on chr11: 5463161491827532, with a size of 37.2 Mb (Table 3). Since the size of the candidate region is large, whole-exome sequencing was performed for the patient KKESH34#6. A total of 7.7 million reads were generated, with coverage of 27×. A total of six homozygous rare variants that lead to aa changes were identified within the candidate region (Supp. Table S6). Based on conservation score (data not shown) and gene annotations, we focus on a novel missense variant (c.578C>T, p.T193I) in the MYO7A gene (Fig. 4A and B). This variant is rare, as it is absent from 200 matching controls, the dbSNP database, and 1,000 Genome database. It segregates with the disease in this family, given that all the affected members are homozygous for this mutation and other unaffected individuals are either heterozygous or do not carry the mutation. MYO7A encodes an unconventional myosin, myosin VIIA, which is involved in the transportation of opsin in photoreceptor cilia [Liu et al., 1999]. Also, it was recently demonstrated that myosin VIIA protein may affect the localization and function of the visual retinoid cycle enzyme, RPE65, which has been associated with LCA [Lopes et al., 2011]. The mutation identified (p.T193I) is a novel allele residing in the motor domain, which is responsible for binding filamentous actin and hydrolyzing ATP (Fig. 4C). Human mutations in MYO7A are associated with Usher syndrome, characterized by deafness and progressive vision loss. All patients of KKESH34 have had poor vision since birth. They all also have nystagmus, neuroepithelial atrophy, and nonrecordable ERGs (Table 2). However, patients in the KKESH34 family do not exhibit hearing loss. Therefore, it is likely that this mutation in MYO7A will only partially affect protein function, resulting in retinal specific clinical phenotype.

Figure 4.

Figure 4

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A missense mutation is identified in MYO7A. A: Exon–intron structure of MYO7A. Exons are shown as black boxes. The missense mutation p.T193I is located in the fifth exon (red arrow). B: Sequence traces of control and affected members. A homozygous missense mutation in c.578 is identified in affected member KKESH34#3 (blue arrow). C: Schematic view of the MYO7A protein structure. The p.T193I mutation is located within the motor domain.

Discussion

We performed homozygosity mapping and whole-exome sequencing to identify mutations affecting a collection of consanguineous LCA families. Our experiments show that homozygosity mapping coupled with whole-exome sequencing is a very powerful tool for identifying mutations, even for families with a small number of affected members. For example, in the KKESH28 family, there was only one affected member and four unaffected members (Fig. 1 and Table 2). Homozygosity mapping alone discovered 12 homozygous blocks whose widths range from 2087 bp to 51.7 Mb, making the approach of PCR and Sanger sequencing impractical (Table 3). By using whole-exome sequencing, we were able to discover 27 rare SNPs that lead to aa changes and reside within the homozygous regions (Supp. Table S4). Among these candidate SNPs, a nonsense mutation (c.1479C>A, p.Y493X) was identified, suggesting this approach can be effective in identifying mutations in families with a small number of affected members.

Interestingly, we have identified families carrying mutations in ALMS1, IQCB1, CNGA3, and MYO7A genes that have been previously associated with other syndromic or nonsyndromic retinal diseases whose phenotypes overlap with that of LCA. The mutations include a previously reported nonsense mutation (c.10945G>T, p. E3649X) in ALMS1 in family KKESH72, a novel splicing change (c.1130-1G>C) in IQCB1 in two families, KKESH24 and KKESH88, a novel nonsense mutation (c.1479C>A, p.Y493X) of IQCB1 in family KKESH28, a novel missense mutation (c.1579C>A, p.L527M) in CNGA3 in family KKESH2, and a novel missense mutation (c.578C>T, p.T193I) in MYO7A in family KKESH34 (Table 1). Based on the phenotype of the patients, it is likely that in each case these missense mutations result in partial loss-of-function of the gene. These alleles are not only useful for molecular diagnoses, but further study of the functional consequence of these mutations will likely provide additional insight into the molecular mechanisms by which these proteins act.

Our findings support the emerging theme that a significant fraction of patients diagnosed with LCA may carry mutations in other syndromic or nonsyndromic retinal disease genes. This is likely due to several reasons. First, it is possible that patients diagnosed as having LCA are too young at the time of diagnosis to present with other syndromic phenotypes. For example, patients who carry mutations in IQCB1 may not show kidney defects until they are in their teens. Second, it is possible that different alleles in the same gene can result in different clinical presentations. For example, patients in family KKESH34 carry a missense mutation (c.578C>T, p.T193I) in MYO7A and show typical LCA phenotypes without hearing problems. It is possible that this mutation results in a partial loss-of-function allele that is only critical for function in the eye. Indeed, similar phenomena have been observed for many retinal disease genes. For example, partial loss-of-function mutations in CEP290 lead to LCA [Coppieters et al., 2010; den Hollander et al., 2006], while null alleles are also associated with recessive Meckel syndrome [Baala et al., 2007; Frank et al., 2008], Bardet-Biedl syndrome [Leitch et al., 2008], and Joubert syndrome [Sayer et al., 2006; Valente et al., 2006]. This phenomenon is also observed in other LCA causal genes, such as CRX [Freund et al., 1997; Swain et al., 1997] and RDH12 [Fingert et al., 2008; Janecke et al., 2004]. Third, it is possible that the same mutation can lead to a different clinical presentation, presumably due to modifications from the genetic background and/or the environment. In our report, the nonsense mutation (c.10945G>T, p. E3649X) in the ALMS1 gene identified in family KKESH72 has been previously reported to cause ALMS [Marshall et al., 2007]. However, none of the patients in this family show other syndromic phenotypes, such as hearing loss or obesity, which are typical for ALMS. It is possible that the different genetic background in the LCA patients from family KKESH72 lead to the different clinical presentations. Indeed, it has been reported recently that a common allele in RPGIP1L can modify the retinal degeneration phenotype in ciliopathies [Katsanis et al., 2001; Khanna et al., 2009]. In addition, in this report, the same mutation in IQCB1 has been identified in both KKESH24#5 and KKESH88#3 patients. It is interesting to note that while the eye phenotype in both patients is quite similar, KKESH88#3 also shows additional neural defects such as midfacial hypoplasia and psychomotor delay. With the whole-exome sequencing data, it is possible to check if KKESH88#3 carries potential genetic modifiers that can potentially explain the clinic phenotype. Indeed, in KKESH88#3, rare variants have been found in gene ACAT1 and FGFR2, which is associated with psychomotor delay and midfacial hypoplasia, respectively (Supp. Table S8) [Korman, 2006; Tartaglia et al., 1997].

In summary, it is evident that in addition to the 16 known LCA genes, mutations in several other syndromic or nonsyndromic eye disease genes may also lead to the LCA phenotype. Due to the high genetic heterogeneity of LCA, it is likely to be informative to sequence a larger set of retinal genes along with the known LCA genes. Combining accurate molecular diagnoses with the clinical phenotypes of LCA patients will be an essential step to proper treatment of this disease in the future.

Supplementary Material

supplement

Acknowledgments

Contract grant sponsor: Retina Research Foundation and the National Eye Institute (R01EY018571 to R.C.).

We are indebted to John Cavender, M.D., the Research Director of the King Khalid Eye Specialist Hospital at the time of this study, to the Research Council of KKESH for financial support, and to the staff of the KKESH Research Department for their diligent commitment to this program. In addition, we thank the family reported here for their willing cooperation in this study. We would like to thank Dr. Molly Bray for the SNP genotyping. Dr. Lewis is a Senior Scientific Investigator of Research to Prevent Blindness, New York. HW was supported by postdoctoral fellowship F32EY19430.

Footnotes

Additional Supporting Information may be found in the online version of this article.

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