Diagnostic Application of an Extensive Gene Panel for Leber Congenital Amaurosis with Severe Genetic Heterogeneity

The Journal of Molecular Diagnostics, Vol. 17, No. 1, January 2015

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Diagnostic Application of an Extensive Gene Panel for Leber Congenital Amaurosis with Severe Genetic Heterogeneity Moon-Woo Seong,* Soo Hyun Seo,* Young Suk Yu,y Jeong-Min Hwang,z Sung Im Cho,* Eun Kyung Ra,* Hyunwoong Park,* Seung Jun Lee,* Ji Yeon Kim,x and Sung Sup Park*x From the Departments of Laboratory Medicine* and Ophthalmologyy and the Biomedical Research Institute,x Seoul National University Hospital, Seoul National University College of Medicine, Seoul; and the Department of Ophthalmology,z Seoul National University Bundang Hospital, Seongnam, Republic of Korea Accepted for publication September 18, 2014. Address correspondence to Sung Sup Park, M.D., Ph.D., Department of Laboratory Medicine, Seoul National University Hospital, 28, Yongon-dong, Chongno-gu, Seoul 110-744, Korea. E-mail: [email protected].

Leber congenital amaurosis (LCA) is a genetically heterogeneous disorder and the most severe form of inherited retinal dystrophy. We report results of a diagnostic application of an extensive gene panel composed of 204 retinal dystrophyerelated genes and discuss its feasibility as a diagnostic tool. Nineteen unrelated LCA patients were included in the study: two patients for validation purposes of our gene panel, 15 previously analyzed patients with no identified mutations, and two previously unanalyzed patients. Genetic diagnosis for each patient was conducted according to whether the variants were consistent with the known inheritance pattern of each gene. We identified two heterozygous or homozygous pathogenic variants in seven of 19 patients. On the basis of mutation information, clinical features were re-reviewed, and clinical diagnoses for two patients were revised from LCA to LCA-related disorders. In addition, a coverage simulation was performed to determine the optimal depth of coverage of the gene panel. Using our gene panel, we diagnosed LCA and LCA-related disorders in 36.8% of patients and one or more deleterious variants or variants of unknown significance in 89.5% of patients. Molecular diagnosis using this extensive gene panel is expected to facilitate diagnosis of retinal dystrophy and help provide proper treatment to patients, although further analyses is needed for a complete clinical validation. (J Mol Diagn 2015, 17: 100e105; http://dx.doi.org/10.1016/j.jmoldx.2014.09.003)

Leber congenital amaurosis (LCA) is the most severe form of inherited retinal dystrophies.1,2 Its incidence is estimated at 2 to 3 per 100,000 live births, and it accounts for 5% of all inherited retinal dystrophies as well as up to 20% of children attending schools for the blind worldwide.1,2 LCA is a clinically heterogeneous disorder. In addition to early-onset blindness during the first year of life, systemic symptoms, such as neurodevelopmental delay and intellectual disability, may be associated with LCA. Some systemic diseases, such as Senior-Loken syndrome (SLS), conorenal syndrome, and Joubert syndrome (JBTS), can manifest with ocular symptoms that complicate the differential diagnosis.3,4 Alternatively, early-onset retinal dystrophies, such as retinitis pigmentosa (RP) and cone-rod dystrophy (CORD), may have clinical features that resemble LCA. Copyright ª 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jmoldx.2014.09.003

To date, mutations in 19 genes have been reported to cause LCA.5 However, LCA might be associated with many more genes because only half of the cases were molecularly diagnosed, even in large studies using whole-exome sequencing (WES).5e7 In addition, >130 genes are known to be implicated in inherited retinal diseases, and some genes related to LCA are also involved in other inherited retinal diseases, such as RP and CORD. Thus, these diseases may be considered a spectrum of genetically related diseases.8,9 Supported by the Basic Science Research Program through the National Research Foundation of Korea funded by grant NRF-2012R1A1A2006958 from the Ministry of Education, Science, and Technology (N.R.F.). Disclosures: None declared.

Gene Panel Sequencing of LCA The severe clinical and genetic heterogeneity of LCA hampers its routine molecular diagnosis and establishment of genotype-phenotype correlations. The development of a high-throughput diagnostic method would offer a means of overcoming these difficulties. We report the results of a diagnostic application of an extensive gene panel composed of 204 retinal dystrophyerelated genes and discuss its feasibility for use as a diagnostic tool.

Materials and Methods

patient’s genomic DNA was fragmented with a median size of 300 bp. The DNA fragments were end-repaired, phosphorylated, and adenylated on the 30 ends. The index adaptors were ligated to the repaired ends, DNA fragments were PCR amplified, and fragments of 200 to 500 bp were isolated. Nineteen precapture libraries were pooled at equimolar concentrations. The capture process was conducted according to the standard manufacturer’s protocol (Roche NimbleGen). Captured libraries were sequenced on Illumina HiSeq 2000 using the paired-end (2  100 bp) program (Illumina, Inc.).

Patients A total of 19 unrelated LCA patients were recruited from the ophthalmology clinics at Seoul National University Hospital and Seoul National University Bundang Hospital from 1999 to 2011. Informed consent was obtained from all patients or their legal guardians. Validation studies with 19 patients, including two with compound heterozygous mutations (to validate the diagnostic gene panel) and 15 previously analyzed for nine LCA-related genes (AIPL1, CRB1, CRX, GUCY2D, LRAT, RDH12, RPE65, RPGRIP1, and TULP1) and the common CEP290: c.2991þ1655A>G mutation using Sanger sequencing, resulted in identification of either no mutation or only a single heterozygous mutation.10,11 The remaining two patients were previously unanalyzed. All patients had their conditions diagnosed using the following criteria: i) early-onset blindness or severe visual impairment during the first year of life, especially before six months, with oculodigital signs (eye poking, rubbing, and pressing); ii) an extinguished or severely reduced electroretinogram; and iii) the exclusion of other systemic diseases.12

Designing the Comprehensive Gene Capture Panel A gene panel was designed to include all known retinal dystrophyerelated genes or retina-specific expressed genes through review of RetNet (https://sph.uth.edu/retnet/sumdis.htm#A-genes, last accessed August 18, 2010), NEIBank (http://neibank.nei.nih.gov/cgi-bin/eyeDiseaseGenes. cgi, last accessed August 18, 2010), and RetinaCentral (http://www-app.uni-regensburg.de/Fakultaeten/Medizin/ Humangenetik/RetinaCentral/database.php, last accessed August 18, 2010) (Supplemental Table S1). The gene category was divided into LCA genes, retinal disease genes, and other candidate genes. Hybridization probes for target capture were synthesized using NimbleGen SeqCap EZ Choice Library (Roche NimbleGen, Madison, WI). The target regions were 807 kb and covered 3121 exons, spanning splice junctions of 204 genes.

Library Preparation and Targeted Sequencing Precapture libraries were constructed according to the manufacturer’s sample preparation protocol for genomic DNA (Illumina, Inc., San Diego, CA). Briefly, 1 mg of each

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Bioinformatics Analysis Sequence reads were aligned to the human hg19 reference genome using the Burrows-Wheeler Alignment version 0.6.2.13 Variant calling was conducted using the genome analysis toolkit version 1.0.5506, Samtools version 0.1.18, and Dindel version 1.01.14e16 Annovar was used to annotate discovered variants.17 Moreover, NextGENe software version 2.3 (SoftGenetics, State College, PA) was also used for analysis. NextGENe parameters were set as follows: single-end analysis, 40 base seeds, 15 base move step, and matching base percentage >85. Only variants that fulfilled the following criteria were selected for further analyses: minor read frequency 20, quality score 20, and minimum coverage >3. The 1000 Genome database, dbSNP135, and the National Heart, Lung and Blood Institute (NHLBI) Exome Sequencing database were used to filter out common variants, and the Human Gene Mutation Database professional database was used to search for known pathogenic mutations. Precomputed results of SIFT, Polyphen, and MutationTaster were based on data gathered from dbNSFP version 2.0 and used to predict functional significance of missense variants.18,19

Variant Prioritization and Sanger Validation Variants were included if i) they were not included in the 1000 Genome, dbSNP, or NHLBI Exome Sequencing Table 1

Summary of Targeted Sequencing in This Study

Variable

Finding*

Total no. of reads Reads mapped to the target region, % Target coverage per base Target base pairs covered 10, % Target base pairs covered 100, % Total no. of variants discovered Synonymous Missense Nonsense Splice site Frameshift

29,916,588 80.3 793 99.0 98.1 62.2 19.6 32.7 2.4 4.4 3.1

*The data are mean values of all patients.

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Results Targeted Sequencing Summary

Figure 1 Distribution of mean coverage depth, resulting from the analyzed reads.

databases; ii) they had a frequency of 10% in our patients; and iii) they had 30% heterozygous or 80% homozygous reads. The interpretative category of each variant was determined according to the American College of Medical Genetics and Genomics (ACMG) recommendations for standards for interpretation and reporting of sequence variations: deleterious, probable deleterious, uncertain, probable benign, and benign variant.20 We considered a novel nonsense, invariant splice site and frameshift variant, or missense variant that was previously reported as pathogenic with supporting evidence (see the ACMG recommendations) in the literature as deleterious. A novel missense variant of unknown significance (UV) was further subdivided into probable deleterious or probable benign, if supportive data such as amino acid change severity, conservation, or protein domain existed. Deleterious variants or UVs were further confirmed by Sanger sequencing. Genetic diagnosis for each patient was conducted according to whether the variants of concern were consistent with the known inheritance pattern of each gene. All identified variants classified as deleterious, probable deleterious, and uncertain were submitted to the ClinVar database (http:// www.ncbi.nlm.nih.gov/clinvar, last accessed July 8, 2014). Table 2

A total of 29 million reads per patient were obtained on average, 80.3% of which were mapped to the target region and resulted in an average per-base coverage of 793 (Table 1 and Figure 1). The target regions (807 kb) had a high depth of coverage; 99.0% of the bases had coverage 10, and 98.1% of the bases had coverage 100. A total of 62.2 variants per patient were discovered on average, of which 19.6 were synonymous. Most variants leading to protein changes were missense (76.8%), followed by invariant splice site (10.3%), frameshift (7.3%), and nonsense (5.6%), respectively.

Patients with Two Deleterious Variants We identified five patients who carried two heterozygous or homozygous deleterious variants in LCA genes or retinal disease genes, as well as two patients (Ret09 and Ret13) for diagnostic validation of the gene panel (Table 2). Three patients had two heterozygous variants in LCA genes, none of which were previously reported. Ret02 and Ret14 had two heterozygous loss-of-function (LOF) variants in CEP290: c.[1711þ1G>A(;)2248_2249delTT] and c.[3904C>T(;) 6869_6870insA], respectively. Given the fact that most of known deleterious variants in CEP290 were LOF (http://www. hgmd.cf.ac.uk/ac/gene.php?geneZCEP290, last accessed July 8, 2014; login required), these variants were considered to be deleterious. Patient Ret20, who was previously unanalyzed, had two nonsense RPGRIP1 variants: c.[832C>T(;) 2356C>T]. These LOF variants were considered deleterious because these variants are predicted to lead to the formation of truncated proteins lacking the interaction domain with RP GTPase regulator (RPGR) essential for these proteins. The remaining two patients carried deleterious variants in other retinal disease genes. One patient (Ret03) was

Mutation Analysis in Seven Patients with Two Deleterious Variants

Identification

Gene

Base change

Amino acid change

Mutation type

Reference

Ret02

CEP290

c.1711þ1G>A c.2248_2249delTT c.1523_1524insGA (homozygous) c.1892A>T c.3565_3571delCGAAGGC c.2174G>A c.2598_2604delAGTGTAT c.998G>A c.1576C>T c.3904C>T c.6869_6870insA c.832C>T c.2356C>T

p.Leu750Thrfs*11 p.Ala509Lysfs*3 p.His631Leu p.Arg1189Glyfs*7 p.Trp725* p.Ile866Metfs*11 p.Gly333Asp p.Arg526* p.Gln1302* p.Asn2290Lysfs*6 p.Arg278* p.Gln786*

Splice site Frameshift Frameshift Missense Frameshift Nonsense Frameshift Missense Nonsense Nonsense Frameshift Nonsense Nonsense

Novel Novel Novel 11 11 Novel Novel 11 11 Novel Novel Novel Novel

Ret03 Ret09y

IQCB1 RPGRIP1

Ret10

AHI1

Ret13y

CRB1

Ret14

CEP290

Ret20

RPGRIP1

y

Ret09 and Ret13 were previously analyzed and included to validate this diagnostic gene panel.11 The two missense variants (p.His631Leu in RPGRIP1 and p.Gly333Asp in CRB1) were previously reported as mutation, but it might be more appropriate to classify them as probable deleterious, considering their supporting evidence.

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Gene Panel Sequencing of LCA Table 3

Mutation Analysis in Three Patients with a Single Heterozygous Deleterious Variant

Identification

Gene

Base change

Amino acid change

Mutation type

Related disorders

Reference

Ret04 Ret05 Ret06

RBP1 RIMS1 FSCN2

c.387_400del14 c.3139delA c.72delG

p.Lys131Glyfs*7 p.Thr1047Hisfs*31 p.Thr25Glnfs*120

Frameshift Frameshift Frameshift

None CORD7 adRP30

Novel Novel 23

adRP, autosomal dominant retinitis pigmentosa; CORD, cone-rod dystrophy.

homozygous for IQCB1: c.1523_1524insGA (p.Ala509Lysfs*3), which is a frameshift mutation. Because IQCB1 is a genetic cause of SLS, we reevaluated his clinical features. This patient was diagnosed as having LCA at 1 year of age, and no other abnormal findings were observed. However, at 9 years of age, chronic renal disease developed, and he was diagnosed as having juvenile nephronophthisis (NPHP).21,22 These clinical features are consistent with SLS. The other patient (Ret10) had two heterozygous variants in the AHI1 gene, which is a genetic cause of JBTS: c.[2174G>A(;)2598_2604delAGTGTAT]. He was diagnosed as having LCA at 4 months of age but otherwise developed normally. After 6 months, brain magnetic resonance image was performed because of weak response in both eyes on the visual evoked potential testing. Magnetic resonance imaging revealed hypoplastic cerebellar vermis, which is consistent with JBTS.

Patients with One Deleterious Variant or One or More UVs We identified nine patients with single deleterious variants (Table 3) or UVs (Table 4). A single heterozygous deleterious variant was found in three patients: RIMS1: c.3139delA (p.Thr1047Hisfs*31) in Ret05, FSCN2: c.72delG (p.Thr25Glnfs*120) in Ret06, and RBP1: c.387_400del14 (p.Lys131Glyfs*7) in Ret04. RIMS1 and FSCN2 variants were previously reported in autosomal dominant CORD7 and autosomal dominant RP30, respectively, which are known to be adult-onset diseases.23,24 On the contrary, Ret05 and Ret06 were diagnosed with severe visual impairment before 1 year of age, for which he had no family history. These variants are Table 4

therefore likely to be insufficient to establish a definitive genetic diagnosis in these two patients. No RBP1 mutation has previously been reported in retinal disease, although an RBP3 mutation has been reported in autosomal recessive RP. RBP3 encodes the interphotoreceptor retinoid-binding protein, which shuttles all trans-retinol from photoreceptors to the retinal pigment epithelium. RBP1 also encodes a retinoid-binding protein, but this protein is mainly involved in the transport of retinol from the liver storage site to the peripheral tissue. Therefore, the clinical significance of the RBP1 frameshift mutation found in the Ret04 patient is unclear. Several UVs were identified in six patients: NPHP3: c.1735A>C (p.Thr579Pro) and USH2A: c.2414G>C (p.Gly805Ala) in Ret01, LRP5: c.1697G>A (p.Arg566His) in Ret07, NPHP1: c.625-3_625-2insT and USH2A: c.4758þ3A>G in Ret08, GUCA1B: c.103G>A (p.Gly35Ser) and NPHP4: c.2360T>A (p.Val787Glu) in Ret11, NPHP4: c.2198G>A (p.Gly733Asp) in Ret17, and USH2A: c.14243C>T (p.Ser4748Phe) in Ret19 (Table 4). All these genes, excluding GUCA1B and LRP5, have been reported in autosomal recessive retinal dystrophy.25e27 GUCA1B mutations were reported in an adult-onset case of RP with autosomal dominant inheritance,28 and LRP5 mutations are known to be a genetic cause of autosomal dominant familial exudative vitreoretinopathy.

Discussion We developed a targeted sequencingebased diagnostic gene panel for retinal dystrophy. Using this panel, we were able to both detect one or more deleterious variants or UVs in 17 of the 19 patients (89.5%) tested and perform genetic

Mutation Analysis in the Six Patients with One or More Variants of Unknown Significance

Identification Gene

Base change

Amino acid change Mutation type

SIFT/polyphen

Ret01

c.1735A>C c.2414G>C c.1697G>A c.625-3_625-2insT c.4758þ3A>G c.103G>A c.2360T>A c.2198G>A c.14243C>T

p.Thr579Pro p.Gly805Ala p.Arg566His

Damaging/probably NPHP, SLS Damaging/probably RP, US Affect/possibly FEVR JBTS, NPHP, SLS US Damaging/probably RP Damaging/possibly NPHP, SLS Damaging/probably NPHP, SLS Affect/probably RP, US

Ret07 Ret08 Ret11 Ret17 Ret19

NPHP3 USH2A LRP5 NPHP1 USH2A GUCA1B NPHP4 NPHP4 USH2A

p.Gly35Ser p.Val787Glu p.Gly733Asp p.Ser4748Phe

Missense Missense Missense Possibly splicing Possibly splicing Missense Missense Missense Missense

Related disorders Reference Novel Novel Novel Novel Novel Novel Novel Novel Novel

FEVR, familial exudative vitreoretinopathy; JBTS, Joubert syndrome; NPHP, nephronophthisis; PolyPhen, probably damaging, possibly damaging, benign; RP, retinitis pigmentosa; SIFT, damaging, affect protein function, benign; SLS, Senior-Loken syndrome; US, Usher syndrome.

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Seong et al diagnosis of LCA and LCA-related disorders in 7 of the 19 patients (36.8%). Notably, LCA-related disorders, including SLS or JBTS, were also diagnosed by this comprehensive gene panel. LCA typically presents as an isolated ocular anomaly without systemic involvement, but systemic disorders, such as SLS, JBTS, or conorenal syndrome, can also present with similar retinal findings. Therefore, molecular diagnosis using similar comprehensive gene panels can facilitate differentiation of LCA versus LCA-related disorders and help provide proper treatment to patients. In this study, approximately 63% of patients were genetically undiagnosed, although a considerable number of patients had one or more deleterious variants or UVs in retinal disease genes. WES is the most comprehensive targeted sequencing strategy to date. In a recent study, WES detected deleterious variants in 48% of previously unanalyzed LCA patients.5 Surprisingly, the patients had deleterious variants only in known LCA-related genes, and no other genes were detected, even using WES.5 A comparable rate of reported undiagnosed cases5,29 suggests that the lack of diagnosis may result from the mutations that are not covered by targeted sequencing: variants in deep introns or regulatory regions, which are not covered by coding region-oriented strategies, and large deletion or structural variants that were not detected by sequencing-based technology. Thus, WES may not be much better to produce a well-designed, comprehensive gene panel for diagnosis of LCA and LCA-related disorders. To apply gene panel sequencing as a diagnostic tool, lowcoverage or low-quality regions need to be identified and validated by Sanger sequencing. Therefore, the cost of diagnostic gene panel sequencing includes Sanger validation and nextgeneration sequencing (NGS). In general, the cost of NGS is inversely proportional to the number of multiplexing, whereas the cost of Sanger validation is proportional to the number of multiplexing. This occurs because more multiplexed samples increase the low-coverage region in each sample, as our coverage simulation indicated (Supplemental Figure S1). Therefore, additional costs for Sanger sequencing of this lowcoverage region and costs used in gene panel sequencing should be considered to determine the optimal depth of coverage for this diagnostic gene panel. We combined our results with those of a previous report to determine the mutation spectrum of LCA-related genes in this population.11 Eight patients among 22 unrelated LCA patients had compound heterozygous or homozygous mutations in the following genes: two mutations each in CEP290 and RPGRIP1 and one each in AHI1, CRB1, IQCB1, and RPE65. The prevalence of LCA-related genes varies, depending on the study population. Among 17 genes, CEP290, GUCY2D, RPE65, and RPGRIP1 are commonly identified, but marked genetic heterogeneity is generally found.6,9,31,32 In a recent large cohort of LCA patients, CEP290 was identified in 23.4% of patients tested. However, this might be an overestimate because this study included only patients for whom genetic causes were previously unidentified.29 Although our study included a limited number of patients, CEP290 and RPGRIP1

mutations are prevalent genetic causes of LCA in this population, contributing to the disease in half of the patients for whom genetic diagnosis was made. Our study indicates that special caution is needed for interpretation of targeted gene panels because nonsynonymous pathogenic variants are found in a considerable number of patients. A recent whole-exome study revealed that there were >300 variants that passed quality and frequency filters in normal human genomes.33 These results indicate that at least one possible pathogenic variant per patient in the case of our gene panel composed of 204 genes can be identified by chance. Therefore, evidence-based algorithms should be established to provide definitive genetic diagnoses for genetic diseases with marked locus heterogeneity such as LCA-related disorders. Because NGS technology has challenges for clinical testing, NGS-based gene panels must be extensively validated with a larger number of samples to cover sufficient numbers of all variant types before clinical application.34 In this study, diagnostic validation of our gene panel was performed to a limited extent. Therefore, full validation is needed before its clinical application. In summary, LCA and LCA-related disorders could be diagnosed in 36.8% and one or more pathogenic variants or UVs could be detected in 89.5% of patients using our gene panel, respectively. Molecular diagnosis using this extensive gene panel is expected to facilitate diagnosis of retinal dystrophy and help provide proper treatment to patients, although further analyses is needed for a complete clinical validation.

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Acknowledgments We thank Jin Young Lee and Hyun Kyung Kim for technical assistance.

Supplemental Data Supplemental material for this article can be found at http://dx.doi.org/10.1016/j.jmoldx.2014.09.003.

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