Diagnostic yield of targeted next generation sequencing in infantile nystagmus syndrome

Ophthalmic Genetics ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/iopg20 Diagnostic yield of targeted next-generation sequencing in infantile nystagmus syndrome Jae-Hwan Choi, Su-Jin Kim, Mervyn G Thomas, Jae-Ho Jung, Eun Hye Oh, JinHong Shin, Jae Wook Cho, Hyang-Sook Kim, Ji-Yun Park, Seo Young Choi, Hee Young Choi & Kwang-Dong Choi To cite this article: Jae-Hwan Choi, Su-Jin Kim, Mervyn G Thomas, Jae-Ho Jung, Eun Hye Oh, Jin-Hong Shin, Jae Wook Cho, Hyang-Sook Kim, Ji-Yun Park, Seo Young Choi, Hee Young Choi & Kwang-Dong Choi (2021): Diagnostic yield of targeted next-generation sequencing in infantile nystagmus syndrome, Ophthalmic Genetics, DOI: 10.1080/13816810.2021.1938138 To link to this article: https://doi.org/10.1080/13816810.2021.1938138 View supplementary material Published online: 16 Jun 2021 Submit your article to this journal Article views: 44 View related articles View Crossmark data Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=iopg20 OPHTHALMIC GENETICS https://doi.org/10.1080/13816810.2021.1938138 RESEARCH REPORTS Diagnostic yield of targeted next-generation sequencing in infantile nystagmus syndrome Jae-Hwan Choi Jae Wook Cho a , Su-Jin Kim b, Mervyn G Thomasc, Jae-Ho Jung d, Eun Hye Oh a, Jin-Hong Shina, , Hyang-Sook Kima, Ji-Yun Parke, Seo Young Choi f, Hee Young Choi g, and Kwang-Dong Choi a f a Department of Neurology, Pusan National University School of Medicine, Research Institute for Convergence of Biomedical Science and Technology, Pusan National University Yangsan Hospital, Yangsan, Korea; bDepartment of Ophthalmology, Pusan National University School of Medicine, Research Institute for Convergence of Biomedical Science and Technology, Pusan National University Yangsan Hospital, Yangsan, Korea; cUlverscroft Eye Unit, Department of Neuroscience, Psychology and Behaviour, University of Leicester, Leicester, UK; dDepartment of Ophthalmology, Seoul National University Hospital, Seoul, Korea; eDepartment of Neurology, Ulsan University Hospital, University of Ulsan College of Medicine, Ulsan, Korea; f Department of Neurology, Pusan National University Hospital, Pusan National University School of Medicine and Biomedical Research Institute, Busan, Korea; gDepartment of Ophthalmology, Pusan National University Hospital, Pusan National University School of Medicine and Biomedical Research Institute, Busan, Korea ABSTRACT ARTICLE HISTORY Background: Infantile nystagmus syndrome (INS) is a genetically heterogeneous disorder Identifying genetic causes of INS would help clinicians to facilitate clinical diagnosis and provide appropriate treatment The aim of this study was to determine the diagnostic utility of targeted next-generation sequencing (NGS) for INS Materials and methods: We recruited 37 patients who were referred to the Neuro-ophthalmology clinics for evaluations of INS NGS was performed using a targeted panel that included 98 candidate genes associated with INS We identified pathogenic variants according to guidelines of the American College of Medical Genetics and Genomics We also calculated the sensitivity and specificity of each clinical sign to assess the diagnostic yield of our gene panel Results: After variant filtering, annotation, and interpretation, the potential pathogenic variants were detected in 13 of the 37 patients, achieving a molecular diagnostic rate of 35% The identified genes were PAX6 (n = 4), FRMD7 (n = 4), GPR143 (n = 2), CACNA1F (n = 1), CNGA3 (n = 1) and GUCY2D (n = 1) In approximately 30% (n = 4) of the patients, the initial clinical diagnosis was revised after a molecular diagnosis was performed The presence of a family history had the highest predictive power for a molecular diagnosis (sensitivity = 61.5%, specificity = 91.7%), and the sensitivity increased when the family history was considered together with one of two clinical signs such as pendular nystagmus waveforms or anterior segment dysgenesis Conclusions: Our study shows that targeted NGS can be useful to determine a molecular diagnosis for patients with INS Targeted NGS also helps to confirm a clinical diagnosis in atypical phenotypes or unresolved cases Received November 11, 2020 Revised May 13, 2021 Accepted May 21, 2021 Introduction Infantile nystagmus syndrome (INS) is characterized by invo­ luntary oscillations of the eyes that are present at birth or during infancy (1) The nystagmus usually manifests as hor­ izontal conjugate oscillations that are often characterized by an accelerating slow phase It can be associated with afferent visual system disorders such as ocular albinism, anterior segment dysgenesis, Leber congenital amaurosis (LCA), and foveal hypoplasia, suggesting sensory deficits as the primary cause of INS On the other hand, about 14% of all individuals with INS have no ocular or neurological abnormalities (2) This idiopathic INS may be caused by abnormal development of the ocular motor system itself rather than disorders of the afferent visual pathway (3,4) INS is a genetically heterogeneous disorder (5) To determine the underlying cause of INS, patients often receive extensive KEYWORDS Infantile nystagmus syndrome; targeted nextgeneration sequencing; molecular diagnosis; FRMD7; PAX6 evaluations including optical coherence tomography (OCT), elec­ troretinography (ERG), and visual evoked potential (VEP) These tests are certainly useful for the differential diagnosis of INS, but in some cases the results might be inconclusive due to poor coopera­ tion in children or the severe intensity of the nystagmus Furthermore, more than 100 genes have been reported to cause INS, and there is significant overlap in its phenotypic character­ istics (4–6) Hence, the initial clinical diagnosis is revised in some patients after a molecular diagnosis is performed (6,7) Identifying the genetic causes of INS can help clinicians to facilitate clinical diagnosis and provide appropriate treatment and genetic counseling Next-generation sequencing (NGS) is now used widely in clinical diagnostics to identify the genetic causes of various monogenic diseases Also, it is possible to sequence regions of interest on the genome using targeted gene panels (6,7) The present study applied targeted gene panel-based NGS to patients CONTACT Kwang-Dong Choi kdchoi@pusan.ac.kr Department of Neurology, Pusan National University Hospital, Pusan National University School of Medicine and Biomedical Research Institute 1-10 Ami-dong, Seo-gu, Busan 49241, Korea Supplemental data for this article can be accessed on the publisher’s website © 2021 Taylor & Francis Group, LLC J-H CHOI ET AL with INS, and investigated the utility of this approach in clinical practice Materials and methods Subjects and clinical assessment We recruited 37 unrelated patients who were referred to the Neuro-ophthalmology clinics of two university hospitals (PNUH and PNUYH) for evaluations of INS INS was defined as conjugate oscillations of the eyes with an onset within the first months of life (1) The patients included 23 males and 14 females with ages ranging from to 72 years (36.3 ± 16.9 years, mean±SD) Ten of the patients had a family history of INS, and the remaining 27 were sporadic cases The patients received detailed ophthalmic examina­ tions, including measurement of the best-corrected visual acuity using the Snellen chart, refractive error, strabismus, abnormal head posture (AHP), slit-lamp examination of the anterior and posterior segments, and a dilated fundus examination Spectral-domain OCT (Visante OCT, Carl Zeiss Meditec, Dublin, CA, USA) was used to acquire tomograms of the anterior and posterior segments, as described previously (8,9) Eye movements were recorded bino­ cularly using infrared video-oculography (SLMED, Seoul, Korea), as described previously (9,10) Types of nystagmus waveforms were classified based on the 12 waveforms described by Dell’Osso and Daroff (11) Twenty-two patients received brain magnetic reso­ nance imaging (MRI) either as an initial test or subsequent test, which only one showed abnormal result with septo-optic dysplasia Based on the clinical assessment, an initial diagnosis was made by special neuro-ophthalmologists (S.-J.K and H.Y.C.) who were blinded to the genetic diagnosis According to recent clinical guidelines suggested by the American Academy of Ophthalmology (https://www.aao.org/disease-review/clinicalguidelines-childhood-nystagmus-workup), the etiology of INS was classified into broad categories: 1) Ocular group, primar­ ily an ocular pathology with various degrees of visual depriva­ tion (n = 20); 2) Neurologic group, primarily neurological abnormalities that may or may not be associated with ocular pathology (n = 2); 3) Motor group (idiopathic), isolated ocu­ lomotor disorder without retinal, optic nerve, and central nerve system abnormalities (n = 10) Patients in whom a clinical diagnosis was not made due to incomplete or declined tests were considered “unknown” category (n = 5) All experiments followed the tenets of the Declaration of Helsinki, and informed consent was obtained after the nature and possible consequences of this study had been explained to the participants This study was approved by the Institutional Review Boards of the two participating hospitals (PNUH and PNUYH) Targeted next-generation sequencing We utilized 98 candidate genes associated with INS based on literature reviews and the OMIM (Online Mendelian Inheritance in Man) database (Supplementary TableS 1) (6,7) The INS gene panel was designed using the SureDesign Web-based application (Agilent Technologies) to cover the exons and 20 bp in the flanking regions Genomic DNA was extracted from blood samples of all patients Standard exome capture libraries were generated using the Agilent SureSelect Target Enrichment protocol for Illumina paired-end sequencing library (version B.3, June 2015) with μg of input DNA The DNA was quantified and its quality was assessed using PicoGreen and NanoDrop Each qualified genomic DNA sample was randomly fragmented using the Covaris, fol­ lowed by adapter ligation, purification, hybridization, and PCR Captured libraries were subjected to Agilent 2100 Bioanalyzer to estimate the quality and were loaded onto the Illumina HiSeq2500 (San Diego, CA, USA) in accordance with the manufacturer’s instructions Raw image files were processed for base-calling using HCS software (version 1.4.8) with default parameters, and the sequences for each individual were generated as 100 bp paired-end reads Sequence reads were aligned to the human reference genome sequence (GRCh37.3, hg19) using the BurrowsWheeler Aligner (version 0.7.12) PCR duplicate reads were marked and removed using Picard tools (version 1.92) The Genome Analysis Toolkit (version 2.3–9) was used for indel realignment and base recalibration Variation annotation and interpretation analysis were performed using SnpEff (version 4.2) Variant filtering and interpretation To identify the possible pathogenic variants, we first filtered out synonymous and noncoding variants, and extracted the variants causing non-synonymous amino acid changes, stop codons, inframe insertions/deletions in coding regions, or changes to splice site sequences in exon/intron boundaries Then, common variants with a minor allele frequency of >0.01 in the dbSNP147, Exome Aggregation Consortium (ExAC), gnomAD, 1000 Genomes Project, and Korean Reference Genome Database (KRGDB) were excluded The pathogenicity of the nonsynonymous variants was analyzed using the following tools: Sorting Intolerant From Tolerant (SIFT), Likelihood Ratio Test (LRT), PolyPhen-2, and MutationTaster The strength of ectopic splicing sites created by intronic variants was evaluated using the MaxEntScan and Human Splicing Finder programs The filtered variants were interpreted according to the stan­ dards and guidelines recommended by the American College of Medical Genetics and Genomics (ACMG) (12) The ACMG scoring system uses a series of criteria that are based on infor­ mation about the variant, such as the effect of the protein, its position in the transcript, literature information, functional assays, public database, and prediction software Each variant was classified into one of the following five categories: patho­ genic, likely pathogenic, uncertain significance, likely benign, or benign In this study, we considered pathogenic or likely patho­ genic variants as disease-causing variants, all of which were confirmed using Sanger DNA sequencing The compound het­ erozygous mutations were analyzed using clone sequencing to confirm that two variants were located on different alleles Statistical analysis Pearson’s χ2 or Fisher’s exact tests were used to compare cate­ gorical variables between patients with and without a molecular diagnosis To assess the diagnostic yield of our gene panel, we also calculated the sensitivity and specificity for each of the following clinical signs: family history, strabismus, AHP, OPHTHALMIC GENETICS anterior segment dysgenesis, nystagmus waveform, and foveal hypoplasia Receiver operating characteristic (ROC) analysis and area under the curve (AUC) calculations were performed using SPSS software (version 22, SPSS, Chicago, IL, USA) Results The average read depth for the targeted regions was 4902.3 fold, and 99.9% of the targeted regions were covered by 20 or more reads, indicating that the sequencing was of high quality After variant filtering, annotation, and interpretation, the pathogenic or likely pathogenic variants were detected in 13 of 37 patients, leading to a molecular diagnostic rate of 35% The initial clinical diagnosis was revised after performing a genetic test in four patients (two in Ocular group and two in unknown group) Eight of 13 patients had a family history of INS (Figure 1) The identified genes were PAX6 (n = 4), FRMD7 (n = 4), GPR143 (n = 2), CACNA1F (n = 1), CNGA3 (n = 1) and GUCY2D (n = 1) Genotype-phenotype correlation The clinical characteristics and genetic results of the 13 patients are summarized in Tables and PAX6 (MIM #607108) variants were identified in four patients According to the ACMG guidelines, heterozygous splice site (c.142–14 C > G in patient 1) and missense (c.259 G > A p Gly87Ser in patient 2) variants were classified as likely pathogenic, and two heterozygous frameshift variants (c.889delA, p Ser297Valfs*82 in patient and c.1079_1080delCA, p Pro360Argfs*24 in patient 4) were pathogenic (Figure 2) The splice site variant has been previously reported in a de novo case with aniridia (13), while our patient (patient 1) had a family history showing the segregation of this variant (Figure 1) The other three variants were novel All had cataracts and foveal hypoplasia Two patients (patient and 2) had both pendular and jerk type of nystagmus waveforms, while the remaining two had only unidirectional jerk nystagmus Since the clinical features were consistent with PAX6-related phenotypes such as aniridia (patient and 4) or anterior segment dysgenesis (patient and 3), they all were initially considered Ocular group (Figure 2) Three likely pathogenic missense variants of FRMD7 (MIM #300628) were detected in four patients (Figure 3a) The heterozygous (c.234 G > A, p.Met78Ile in patient 5) and hemi­ zygous (c.661A>C, p.Asn221His in patient 6) variants were novel, while the remaining one (c.875 T > C, p.Leu292Pro in patient and 8) was a known mutation that was previously found to be common in a Korea INS cohort (10) Two patients carrying c.875 T > C mutation also had two common singlenucleotide polymorphisms (SNPs) at exon 12: c.1403 G > A (rs6637934) and c.1533 T > C (rs5977623) Three patients had a family history with a suspicion of X-linked inheritance (Figure 1) Three patients (patient 5, 6, and 8) had pendular type of nystagmus waveforms, while the remaining one had only unidirectional jerk nystagmus The clinical features in three patients were consistent with idiopathic INS (Motor group) due to a good vision and an absence of afferent visual system anomalies However, the remaining one (patient 6) was initially classified as Ocular group due to relatively poor visual acuity and foveal hypoplasia in OCT, which are unusual find­ ings in FRMD7-related INS (Figure 3b) (14) Hemizygous pathogenic variants of GPR143 (MIM #300808) were identified in patient (c.1A>G, p.Met1Val) and patient 10 (c.231_249dupCGACCTTCTCGGCTGCCTG, p.Gly84Argfs*3) Patient had unidirectional jerk nystagmus, while patient 10 had pendular nystagmus Both were initially considered Ocular group because the clinical diagnosis was compatible with ocular albinism based on iris transillumina­ tion defects, the hypopigmentation of the iris and fundus, infantile nystagmus, and foveal hypoplasia (Figure 4a and b) A hemizygous nonsense variant of CACNA1F (MIM #300110) was detected in patient 11 (c.2932 C > T, p Arg978*) That patient exhibited high refractive errors, unidir­ ectional jerk nystagmus, and myopic fundus (Figure 4c) The patient was initially classified as unknown group due to denial of ERG test, but a diagnosis of congenital stationary night blindness was made after performing a genetic test A compound heterozygous variant of CNGA3 (MIM #600053) was identified in patient 12 (c.1112 C > T, p Pro371Leu and c.1642 G > A, p.Gly548Arg) (6) Clone sequen­ cing confirmed that these two variants were located on differ­ ent alleles (Figure 5a) The patient had exotropia, pendular nystagmus, AHP, and foveal hypoplasia A presumptive diag­ nosis of LCA was initially applied to this patient based on the decreased scotopic and photopic wave in ERG, but this was later revised to achromatopsia after performing a genetic test Figure Pedigree of genetically confirmed eight patients who had a family history of infantile nystagmus Clinical diagnosis Aniridia ASGD ASGD Aniridia IIN FH IIN IIN OA OA Unknown LCA Unknown Molecular diagnosis PAX6 PAX6 PAX6 PAX6 FRMD7 FRMD7 FRMD7 FRMD7 GPR143 GPR143 CACNA1F CNGA3 GUCY2D Revised diagnosis Aniridia ASGD ASGD Aniridia IIN IIN IIN IIN OA OA CSNB ACHM LCA Family history (+) (+) (-) (+) (+) (+) (+) (-) (+) (-) (-) (+) (-) OS 0.82 0.70 1.70 0.30 0.20 1.40 −0.10 0.70 0.70 0.52 0.52 1.00 NLP BCVA OD 0.70 0.70 1.70 0.40 0.40 1.00 −0.10 0.60 0.70 0.82 0.52 0.22 NLP OS UC −5.50 6.50 NP −4.75 3.50 −2.87 −0.75 −3.25 −3.50 −13.25 1.50 NA Refraction OD −0.25 −6.75 6.50 NP −5.00 2.25 −3.12 −0.25 −3.75 −3.25 −13.50 1.50 NA Strabismus (-) (-) XT (-) (-) (-) (-) (-) (-) XT (-) XT (-) AHP (-) (-) (-) (+) (-) (-) (-) (+) (-) (-) (-) (+) (-) Nystagmus P,AP,DJ Pfs,J,Jef Jef J P AP,Pfs Jef P,AP J,Jef P,AP J AP J Fundus FR (-) FR (-) FR (-) FR (-) N N N N FH, FR(-) FH, FR (-) MF N N OCT FH FH FH FH N FH N N FH FH N FH N Additional characteristics Cataract, corneal opacity Cataract, eccentric pupil Cataract, corneal opacity Cataract (-) (-) (-) (-) Iris hypopigmentation Iris hypopigmentation High myopia (-) (-) 13 CACNA1F CNGA3 GUCY2D Compound hetero Zygosity Hetero Hetero Hetero Hetero Hetero Hemi Hemi Hemi Hemi Hemi Hemi Compound hetero mRNA c.142–14 C > G c.259 G > A c.889delA c.1079_1080delCA c.234 G > A c.661A>C c.875 T > C c.875 T > C c.1A>G c.231_249dupCGACCTTCTCGGCTGCCTG c.2932 C > T c.1112 C > T c.1642 G > A c.1978 C > T c.2576 + G > A Protein (-) p.Gly87Ser p.Ser297Valfs*82 p.Pro360Argfs*24 p.Met78Ile p.Asn221His p.Leu292Pro p.Leu292Pro p.Met1Val p.Gly84Argfs*3 p.Arg978* p.Pro371Leu p.Gly548Arg p.Arg660* (-) Variant effect Aberrant splicing Missense Deletion Deletion Missense Missense Missense Missense Start codon Insertion Nonsense Missense Missense Nonsense Aberrant splicing ExAC (-) (-) (-) (-) (-) (-) 3x10−5 3x10−5 (-) (-) (-) 1x10−5 2.5x10−5 (-) 1x10−5 Pathogenic (PVS1,PM2,PM3,PP3,PP4,PP5) ACMG variant classification Likely pathogenic (PM2,PP1,PP3,PP4,PP5) Likely pathogenic (PM1,PM2,PP1,PP3,PP4) Pathogenic (PVS1,PM2,PP4) Pathogenic (PVS1,PM2,PP4) Likely pathogenic (PM1,PM2,PP2,PP4,BP4) Likely pathogenic (PM1,PM2,PM5,PP2,PP3,PP4) Likely pathogenic (PM1,PP2,PP3,PP4,PP5) Likely pathogenic (PM1,PP2,PP3,PP4,PP5) Pathogenic (PVS1,PM2,PP4) Pathogenic (PVS1,PM2,PP4) Pathogenic (PVS1,PM2,PP4) Likely pathogenic (PM2,PM3,PP1,PP4,PP5) Transcript ID: CACNA1F, NM_005183.3; CNGA3, NM_001298.2; FRMD7, NM_194277.2; GPR143, NM_000273.3; GUCY2D, NM_00180.3; PAX6, NM_001258462.1 ACMG = American College of Medical Genetics and Genomics; ExAC = Exome Aggregation Consortium Evidence code descriptions according to the ACMG classification: BP, benign supporting; PM, pathogenic moderate; PP, pathogenic supporting; PS, pathogenic strong; PVS, pathogenic very strong GPR143 FRMD7 Patient No 10 11 12 Gene PAX6 Table Potential pathogenic mutations identified in the 13 patients Previous literature [10] Novel Novel Novel Novel Novel [11] [11] Novel Novel Novel Novel [5] [13] Novel ACHM = achromatopsia; AHP = abnormal head posture; AP = asymmetric pendular; ASGD = anterior segment dysgenesis; BCVA = best-corrected visual acuity; CSNB = congenital stationary night blindness; DJ = dual jerk; F = female; FH = foveal hypopigmentation (fundus column); FH = foveal hypoplasia (Clinical diagnosis and OCT column); FR = foveal reflex; IIN = idiopathic infantile nystagmus; J = pure jerk; Jef = jerk with extended foveation; LCA = Leber congenital amaurosis; M = male; N = normal; NA = not available; NLP = no light perception; NP = not performed; OA = ocular albinism; OCT = optic coherence tomography; P = pure pendular; Pfs = pendular with foveating saccades; UC = uncheckable; XT = exotropia Patient no./ sex/age, years 1/F/34 2/M/18 3/F/34 4/M/47 5/F/34 6/M/58 7/M/38 8/M/13 9/M/49 10/M/12 11/M/44 12/F/10 13/F/39 Table Clinical characteristics of the 13 patients with genetically confirmed infantile nystagmus syndrome J-H CHOI ET AL OPHTHALMIC GENETICS Figure Localization of PAX6 mutations and phenotypic characteristics of the anterior segment The PAX6 protein consists of four domains: paired-box domain (PD), linker region (LNK), homeodomain (HD), and proline/serine/threonine-rich region (PST) Exon 5a is an alternatively spliced exon in the PD Two nontruncating mutations (c.142–14 C > G and c.259 G > A) are located within the PD, while the two truncating mutations (c.889delA and c.1079_1080delCA) are within the PST Two patients (patients and 4) exhibit total aniridia with infantile nystagmus, while the remaining two show the nonaniridia phenotypes of corectopia (patient 2) and corneal opacity (patient 3) One patient (patient 13) visited our clinic for the evaluation of migrainous headache On initial examinations the patient showed periodic alternating nystagmus with a jerk waveform, but further ophthalmic examinations were impossible due to the presence of severe visual loss Targeted NGS revealed a compound heterozygous variant of GUCY2D (MIM #600179) (c.1978 C > T, p.Arg660* and c.2576 + G > A), as confirmed by clone sequencing (Figure 5b) (15) At the initial visit, the patient was classified as unknown group due to incomplete ophthalmic examinations The ERG which was subsequently performed after molecular diagnosis, revealed the absence of photopic wave and the decreased scotopic wave, consistent with the findings seen in LCA Clinical signs for a molecular diagnosis Table shows the comparison of clinical characteristics between patients with and without a molecular diagnosis A family his­ tory and pendular-related waveforms were more commonly associated with genetically confirmed INS group, while jerkrelated waveforms were frequently observed in patients without a molecular diagnosis No significant differences were observed between two groups with respect to the sex, strabismus, AHP, anterior segment dysgenesis, foveal hypoplasia, planes of nystag­ mus oscillations, the presence of periodic alternating nystagmus, and nystagmus change in darkness Table lists the sensitivity and specificity of each clinical sign for a molecular diagnosis The sensitivity was not high for most signs when they were consid­ ered in isolation Although foveal hypoplasia showed the highest sensitivity (69.2%), the specificity was the lowest (47.4%) The presence of a family history had the highest predictive power for a molecular diagnosis using our gene panel (sensitivity = 61.5%, specificity = 91.7%, and AUC = 0.766) Consideration of the family history together with the nystagmus waveform (pendularrelated) or anterior segment dysgenesis increased the sensitivity to 76.9% Discussion This study analyzed 37 patients with INS to determine a molecular diagnosis using targeted gene panel-based NGS, which achieved a molecular diagnostic yield of 35% This yield is lower than those found in previous two studies that performed gene panel analysis for INS (58.3% (6) and 80% (7)) Although these differences can be attributed to a heterogeneous cohort composition and diverse gene panels, our finding suggests that the application of targeted NGS can help establishing a final diagnosis of INS Furthermore, the presence of a family history had the highest predictive power for a molecular diagnosis, increasing the detection rate of familial cases to 80% (8 of 10 patients) This rate is consistent with those for familial INS found in previous two studies (88% (6) and 80% (7)) Clinical ophthalmic examinations are the most important first step in a diagnosis of INS, and some clinical signs may provide a clue for a molecular diagnosis (2) For example, anterior segment dysgenesis such as aniridia may be associated with PAX6 variants, and the presence of asymmetric VEPs and transillumination defects of the iris lead to a comprehensive analysis of albinism-related genes However, in some case the clinical tests might be inconclusive due to poor cooperation in children or the severe intensity of the nystagmus Even specific clinical signs might not ensure a molecular diagnosis in J-H CHOI ET AL Figure (a) Localization of FRMD7 mutations The FRMD7 protein contains an N-terminal FERM domain (F1, F2, and F3 lobes) and a FERM-adjacent (FA) domain The FRMD7 mutations are located in the FERM (F2 and F3 lobes) and FA domains of the protein (b) Foveal morphology and nystagmus waveform of patient carrying the c.875 T > C mutation Optical coherence tomography reveals a rudimentary foveal pit (left panel, asterisk), and an eye-movement recording shows a pendular nystagmus waveform with foveating saccades (arrows) on the horizontal plane (right panel) H = horizontal position of the eye; V = vertical position of the eye; T = torsional position of the eye genetically heterogeneous disorders such as LCA and ocular albinism More than 100 genes have been reported to cause INS, and there is phenotypic overlap among genetically differ­ ent disorders (4–6) Consequently, each clinical sign did not show high predictive power for a molecular diagnosis in the present study, and the initial clinical diagnosis was revised or resolved after a molecular diagnosis in approximately 30% of the patients This is similar to two previous studies finding that the revision rate of the initial clinical diagnosis ranged from 21% to 25% (6,7) Therefore, despite the high cost and poor accessibility, targeted NGS can be an ancillary diagnostic tool for patients with INS by facilitating a clinical diagnosis While a positive family history is extremely informative for a molecular diagnosis of monogenic disorders, approximately 10–20% of familial INS not exhibit any significant variants in known INS-related genes (6,7) It may be possible that these families carry variants in cis-regulatory regions of noncoding DNA or in novel genes associated with INS (7,16–18) Meanwhile, the sensitivity of a positive family history alone was not so high (61.5%) in our study, suggesting that its presence is not necessarily required for a molecular diagnosis However, we found that assessing one of two clinical signs (pendular-related nystagmus or anterior segment dysgenesis) together increased the sensitivity for identifying the genetic causes of INS This means that even sporadic cases may have significant variants within known INS-related genes if they exhibit pendular nystagmus or anterior segment dysgenesis Indeed, pendular nystagmus waveforms have frequently been observed in FRMD7-related INS (19), yet diagnosis cannot be made on the basis of waveform only considering examples like our two patients with GPR143 mutations, one of whom had pendular nystagmus and the other jerk Although foveal hypo­ plasia was the most sensitive sign for a molecular diagnosis, it showed the lowest specificity due to a high rate of false-positive results, indicating that further studies are needed to identify novel genes associated with foveal hypoplasia A recent study using NGS proposed AHR as a novel disease gene for auto­ somal recessive INS and foveal hypoplasia (20) The present study found that PAX6 and FRMD7 were common genetic causes of INS PAX6 encodes a transcriptional regulator that plays an important role in eye development (21,22) In gen­ eral, the phenotypes of PAX6 mutations may depend on the mutation spectrums and distributions: truncating mutations cause total aniridia, while missense mutations result in nonaniridia phenotypes such as foveal hypoplasia, keratitis, and optic nerve malformations (23) Thus, PAX6 mutations can be OPHTHALMIC GENETICS Figure Sequencing results and phenotypic characteristics of two patients with GPR143 mutation and one patient with CACNA1F mutation (a) Patient with hemizygous c.1A>G mutation has an irregular ring depigmentation in the peripheral iris and an entire loss of retinal pigments (b) Patient 10 with hemizygous c.231_249dupCGACCTTCTCGGCTGCCTG has relatively mild hypopigmentation in the iris and fundus (c) Patient 11 with hemizygous c.2932 C > T mutation has myopic fundus with peripapillary atrophy Figure Clone sequencing results of two patients with compound heterozygous mutations Clone sequencings reveal that two different variants are located on different alleles; (a) c.1112 C > T and c.1642 G > A of CNGA3 in patient 12, (b) c.1978 C > T and c.2576 + G > A of GUCY2D in patient 13 misdiagnosed as a genetic cause of idiopathic INS when the con­ dition is not accompanied with aniridia or presenile cataract (6,24) However, abnormal foveal morphology is more common in PAX6 mutations, and normal-appearing iris can show abnormal changes in the architecture of an iris architecture in ASOCT (25) FRMD7 mutations are known to be linked to idiopathic INS without any abnormalities in the visual system (5,14,26–28) 8 J-H CHOI ET AL Table Comparisons of clinical characteristics between patients with and without molecular diagnosis Sex, male, n (%) Family history, n (%) Strabismus, n (%) Abnormal head posture, n (%) Anterior segment dysgenesis, n (%) Foveal hypoplasia, n (%) Plane of nystagmus oscillations, n (%) Pure horizontal Mixed horizontal/vertical/torsional Periodic alternating nystagmus, n (%) Waveform of nystagmus, n (%) Pendular Unidirectional jerk Bidirectional jerk Dual jerk Nystagmus change in darkness, n (%) Augmentation Suppression Directional change No change Patients with molecular diagnosis (n = 13) (54) (62) (23) (31) (46) (70) Patients without molecular diagnosis (n = 24) 14 (58) (8) (43) (35) (17) 10 (53) p-value 1.000 0.001 0.292 1.000 0.118 0.471 (54) (46) (8) 13 (54) 11 (46) (30) 1.000 1.000 0.216 (54) (54) (0) (8) (17) 22 (92) (8) (0) 0.028 0.032 0.543 0.351 (15) (39) (15) (31) (29) (38) (4) (29) 0.446 1.000 0.278 1.000 Table Test properties of clinical signs for a molecular diagnosis Clinical sign Strabismus Abnormal head posture Anterior segment dysgenesis Pendular-related nystagmus Family history Foveal hypoplasia Combined Family history or anterior segment dysgenesis Family history or pendular-related nystagmus Family history or foveal hypoplasia Sensitivity (gene +) 23.1% 30.8% 46.2% 53.8% 61.5% 69.2% Specificity (gene -) 57.1% 65.0% 93.3% 93.3% 91.7% 47.4% AUC 0.401 0.479 0.647 0.686 0.766 0.583 p-value 0.339 0.839 0.143 0.065 0.008 0.432 76.9% 76.9% 84.6% 79.2% 75.0% 47.4% 0.780 0.760 0.660 0.005 0.010 0.130 This leads to the assumption that FRMD7 mutations cause a primary defect in regions of the brain responsible for ocular motor control (29) However, OCT revealed had definite foveal hypoplasia in one of our patients, which is unusual finding in FRMD7-related INS (14) It has been reported that FRMD7 mutations may be associated with abnormal development of the afferent visual system including optic nerve head dysplasia and the loss of horizontal direction selectivity in the retina (9,10,30,31) These findings suggest that FRMD7 mutations have a wider phenotypic spectrum Most FRMD7 mutations are located in the FERM and FERM-adjacent domains of the FRMD7 protein, without any consistent hot spots (9,27) However, we previously found that one missense mutation (c.875 T > C) accounted for more than 50% of Korean patients carrying FRMD7 mutations, which was attributed to the foun­ der effect based on the geographic distribution and the shared haplotype (10) Two patients in our study also had the same missense mutation with two common SNPs (rs6637934 and rs5977623), which demonstrating once again that the c.875 T > C variant may be a recurrent FRMD7 mutation with a founder effect in the Korean population with idiopathic INS This study was subject to some potential limitations Most patients with suspected retinal dystrophies were not included in our cohort Because retinal disorders are known to be the most common causes of INS (2,6), the molecular diagnostic rate in this study was relatively low compared with previous studies Furthermore, our gene panel did not include AHR, which has recently been proposed to cause autosomal recessive INS and foveal hypoplasia (20) Even though AHR mutations are likely to be very rare, they are still worth considering Finally, targeted NGS has difficulty in detecting copy number variations (CNVs) CNV analysis can aid the detection of large deletions or duplications of targeted genes, but whole-genome sequencing may be more sui­ table than whole-exome sequencing for CNV analysis (6,7) In conclusion, this study has demonstrated that targeted NGS can be useful to determine a molecular diagnosis for patients with INS, and is also helpful in confirming the clinical diagnosis in atypical phenotypes or unresolved cases Since many INS patients not have identified genetic causes, further assessments to identify novel genes associated with INS are needed Declaration of interest The authors declare no conflicts of interest Funding This research was supported by Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of OPHTHALMIC GENETICS Education (NRF-2017R1D1A3B03033237);Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education [NRF-2017R1D1A3B03033237]; 15 ORCID 16 Jae-Hwan Choi http://orcid.org/0000-0002-4120-9228 Su-Jin Kim http://orcid.org/0000-0001-9693-2993 http://orcid.org/0000-0003-4742-4903 Jae-Ho Jung Eun Hye Oh http://orcid.org/0000-0001-7858-8930 Jae Wook Cho http://orcid.org/0000-0002-2742-9136 Seo Young Choi http://orcid.org/0000-0002-5320-7828 Hee Young Choi http://orcid.org/0000-0002-6984-0994 Kwang-Dong Choi http://orcid.org/0000-0002-9373-4710 References Leigh RJ, Zee DS The neurology of eye movements 5th ed New York: Oxford University Press; 2015 Bertsch M, Floyd M, Kehoe T, Pfeifer W, Drack AV.The clinical evaluation of infantile nystagmus: what to first and why Ophthalmic Genet 2017;38(1):22–33 Brodsky MC, Dell’Osso LF.A unifying neurologic mechanism for infantile nystagmus JAMA Ophthalmol 2014;132(6):761–68 Richards MD, Wong A.Infantile nystagmus syndrome: clinical characteristics, current theories of pathogenesis, diagnosis, and management Can J Ophthalmol 2015;50(6):400–08 Gottlob I, Proudlock FA.Aetiology of infantile nystagmus Curr Opin Neurol 2014;27(1):83–91 Rim JH, Lee ST, Gee HY, Lee BJ, Choi JR, Park HW, Han SH, Han J.Accuracy of next-generation sequencing for molecular diag­ nosis in patients with infantile nystagmus syndrome JAMA Ophthalmol 2017;135(12):1376–85 Thomas MG, Maconachie G, Sheth V, McLean RJ, Gottlob I Development and clinical utility of a novel diagnostic nystagmus gene panel using targeted next-generation sequencing Eur J Hum Genet 2017;25(6):725–34 Kim YC, Sung MS, Heo H, Park SW.Anterior segment configuration as a predictive factor for refractive outcome after cataract surgery in patients with glaucoma BMC Ophthalmol 2016;16(1):179 Choi JH, Shin JH, Seo JH, Jung JH, Choi KD A start codon mutation of the FRMD7 gene in two Korean families with idiopathic infantile nystagmus Sci Rep 2015;5:13003 10 Choi J-H, Jung J-H, Oh EH, Shin J-H, Kim H-S, Seo JH, Choi SY, Kim M-J, Choi HY, Lee C, et al Genotype and phenotype spec­ trum of FRMD7-associated infantile nystagmus syndrome Invest Ophthalmol Vis Sci 2018;59(7):3181–88.doi:10.1167/iovs.1824207 11 Dell’Osso LF, Daroff RB.Congenital nystagmus waveforms and foveation strategy Doc Ophthalmol 1975;39(1):155–82.doi:10.1007/BF00578761 12 Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, Grody WW, Hegde M, Lyon E, Spector E, et al Standards and guidelines for the interpretation of sequence variants: a joint con­ sensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology Genet Med 2015;17:405–24 13 Vasilyeva TA, Voskresenskaya AA, Käsmann-Kellner B, Khlebnikova OV, Pozdeyeva NA, Bayazutdinova GM, Kutsev SI, Ginter EK, Semina EV, Marakhonov AV, et al Molecular analysis of patients with aniridia in Russian Federation broadens the spec­ trum of PAX6 mutations Clin Genet 2017;92:639–44 14 Thomas MG, Maconachie G, Hisaund M, Gottlob I FRMD7related infantile nystagmus In: Adam MP, Ardinger HH, Pagon RA, Wallace SE, Bean LJH, Stephens K, Amemiya A, editors 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 GeneReviews® [Internet] Seattle: University of Washington p 1993–2020 2018 Lotery AJ, Namperumalsamy P, Jacobson SG, Weleber RG, Fishman GA, Musarella MA, Hoyt CS, Héon E, Levin A, Jan J, et al Mutation analysis of genes in patients with Leber congenital amaurosis Arch Ophthalmol 2000;118:538–43 Plaisancié J, Tarilonte M, Ramos P, Jeanton-Scaramouche C, Gaston V, Dollfus H, Aguilera D, Kaplan J, Fares-Taie L, BlancoKelly F, et al Implication of non-coding PAX6 mutations in aniridia Hum Genet 2018;137:831–46 Ray K, Chaki M, Sengupta M.Tyrosinase and ocular diseases: some novel thoughts on the molecular basis of oculocutaneous albinism type Prog Retin Eye Res 2007;26(4):323–58 Bhatia S, Bengani H, Fish M, Brown A, Divizia MT, De Marco R, Damante G, Grainger R, Van Heyningen V, Kleinjan DA.Disruption of autoregulatory feedback by a mutation in a remote, ultraconserved PAX6 enhancer causes aniridia Am J Hum Genet 2013;93(6):1126–34 Thomas S, Proudlock FA, Sarvananthan N, Roberts EO, Awan M, McLean R, Surendran M, Kumar AS, Farooq SJ, Degg C, et al Phenotypical characteristics of idiopathic infantile nystagmus with and without mutations in FRMD7 Brain 2008;131:1259–67 Mayer AK, Mahajnah M, Thomas MG, Cohen Y, Habib A, Schulze M, Maconachie GDE, AlMoallem B, De Baere E, Lorenz B, et al Homozygous stop mutation in AHR causes auto­ somal recessive foveal hypoplasia and infantile nystagmus Brain 2019;142:1528–34 Lang D, Powell SK, Plummer RS, Young KP, Ruggeri BA.PAX genes: roles in development, pathophysiology, and cancer Biochem Pharmacol 2007;73(1):1–14 Kokotas H, Petersen MB.Clinical and molecular aspects of aniridia Clin Genet 2010;77(5):409–20 Tzoulaki I, White IM, Hanson IM PAX6 mutations: genotype-phenotype correlations BMC Genet 2005;6:27 Thomas S, Thomas MG, Andrews C, Chan WM, Proudlock FA, McLean RJ, Pradeep A, Engle EC, Gottlob I.Autosomal-dominant nystagmus, foveal hypoplasia and presenile cataract associated with a novel PAX6 mutation Eur J Hum Genet 2014;22(3):344–49 Lee S, Lee SH, Heo H, Oh EH, Shin JH, Kim HS, Jung JH, Choi SY, Choi KD, Lee H, et al Impaired DNA-binding affinity of novel PAX6 mutations Sci Rep 2020;10:3062 Tarpey P, Thomas S, Sarvananthan N, Mallya U, Lisgo S, Talbot CJ, Roberts EO, Awan M, Surendran M, McLean RJ, et al Mutations in FRMD7, a newly identified member of the FERM family, cause X-linked idiopathic congenital nystagmus Nat Genet 2006;38:1242–44 Watkins RJ, Thomas MG, Talbot CJ, Gottlob I, Shackleton S The role of FRMD7 in idiopathic infantile nystagmus J Ophthalmol 2012;2012:460956 AlMoallem B, Bauwens M, Walraedt S, Delbeke P, De Zaeytijd J, Kestelyn P, Meire F, Janssens S, Van Cauwenbergh C, Verdin H, et al Novel FRMD7 mutations and genomic rearrangement expand the molecular pathogenesis of X-linked idiopathic infantile nystagmus Invest Ophthalmol Vis Sci 2015;56:1701–10 Thomas MG, Crosier M, Lindsay S, Kumar A, Thomas S, Araki M, Talbot CJ, McLean RJ, Surendran M, Taylor K, et al The clinical and molecular genetic features of idiopathic infantile periodic alternating nystagmus Brain 2011;134:892–902 Thomas MG, Crosier M, Lindsay S, Kumar A, Araki M, Leroy BP, McLean RJ, Sheth V, Maconachie G, Thomas S, et al Abnormal retinal development associated with FRMD7 mutations Hum Mol Genet 2014;23:4086–93 Yonehara K, Fiscella M, Drinnenberg A, Esposti F, Trenholm S, Krol J, Franke F, Scherf BG, Kusnyerik A, Müller J, et al Congenital nystagmus gene FRMD7 is necessary for establishing a neuronal circuit asymmetry for direction selectivity Neuron 2016;89:177–93  ... REPORTS Diagnostic yield of targeted next- generation sequencing in infantile nystagmus syndrome Jae-Hwan Choi Jae Wook Cho a , Su-Jin Kim b, Mervyn G Thomasc, Jae-Ho Jung d, Eun Hye Oh a, Jin-Hong... cause of INS, patients often receive extensive KEYWORDS Infantile nystagmus syndrome; targeted nextgeneration sequencing; molecular diagnosis; FRMD7; PAX6 evaluations including optical coherence... Accepted May 21, 2021 Introduction Infantile nystagmus syndrome (INS) is characterized by invo­ luntary oscillations of the eyes that are present at birth or during infancy (1) The nystagmus usually