临床表型-基因型关联发现Leber先天性黑矇（LCA）家系新的RDH12基因复合杂合突变

Phenotype-genotype correlation with Sanger sequencing identified retinol dehydrogenase 12 ( RDH12 ) compound heterozygous variants in a Chinese family with Leber congenital amaurosis 临床表型-基因型关联发现Leber先天性黑矇（LCA）家系新的 RDH12 基因复合杂合突变 1 first-author 2 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:guyangshun_1@hotmail.com †E-mail:guyangshun_1@hotmail.com guyangshun_1@hotmail.com Department of Ophthalmology, the First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China Department of Ophthalmology, the First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China Department of Ophthalmology, Zhejiang Provincial People’s Hospital, Hangzhou 310014, China Department of Ophthalmology, Zhejiang Provincial People’s Hospital, Hangzhou 310014, China Background: 2 Leber congenital amaurosis (LCA) is a group of clinically and genetically heterogeneous retinal dystrophy. To date, 22 genes are known to be responsible for LCA, and some specific phenotypic features could provide significant prognostic information for a potential genetic etiology. This study is to identify gene variants responsible for LCA in a Chinese family using direct Sanger sequencing, with the help of phenotype-genotype correlations. Methods: 2 A Chinese family with six members including two individuals affected with LCA was studied. All patients underwent a complete ophthalmic examination. Based on phenotype-genotype correlation, direct Sanger sequencing was performed to identify the candidate gene on all family members and normal controls. Targeted next-generation sequencing was used to exclude other known LCA genes. Results: 2 By Sanger sequencing, we identified two novel missense variants in the retinol dehydrogenase 12 ( RDH12 ) gene: a c.164C > A transversion predicting a p.T55K substitution, and a c.535C > G transversion predicting a p.H179D substitution. The two affected subjects carried both RDH12 variants, while their parents and offspring carried only one of heterozygous variants, showing complete cosegregation of the variants. The compound heterozygous variants were not present in 600 normal controls. Besides, the RDH12 variants were confirmed by targeted next-generation sequencing. Conclusions: 2 The RDH12 compound heterozygous variants might be the cause of the LCA family. Our study adds to the molecular spectrum of RDH12 -related retinopathy and offers an effective example of the power of phenotype-genotype correlations in molecular diagnosis of LCA. 目的 2 临床表型-基因型关联分析筛查Leber先天性黑矇（LCA）家系候选基因，确定其分子遗传病因。 创新点 2 成功应用临床表型-基因型关联分析鉴定LCA家系致病基因，并发现新的 RDH12 基因复合杂合突变。 方法 2 收集一个中国常染色体隐性遗传三代LCA家系，详细分析该家系眼部表型特征（图1和表1），经临床表型-基因型关联分析确定 RDH12 为候选基因。Sanger测序发现新的 RDH12 基因复合杂合突变（图2），目标序列捕获高通量测序技术排除其他已知LCA相关基因（表2）。该家系成员基因型显示完整的共分离（图3），同时在600例普通人群中未发现该突变。 结论 2 RDH12 基因复合杂合突变可能为该LCA家系的致病基因，临床表型-基因型关联分析在LCA分子遗传学诊断中有重要价值。 Leber congenital amaurosis Phenotype-genotype correlation RDH12 Compound heterozygosity Leber先天性黑矇 临床表型-基因型关联 RDH12 复合杂合突变 Introduction 1 Leber congenital amaurosis (LCA) is a group of severe hereditary retinal neurodegenerative diseases resulting in congenital blindness, and is characterized by the following clinical features: severe visual impairment, sensory nystagmus, amaurotic pupils, and unrecordable electroretinographic response (den Hollander et al., 2008 2008 ). In LCA patients, the oscillations of the eyes or the absence of fixation is present as early as the age of 6 weeks, and electroretinogram (ERG) recordings are usually severely subnormal or extinguished (Chacon-Camacho and Zenteno, 2015 2015 ). It is a rare congenital retinal dystrophy with an incidence between 1/30 000 (Koenekoop, 2004 2004 ) and 1/81 000 (Stone, 2007 2007 ), although it may be much more frequent in consanguineous populations or genetically isolated communities (Sherwin et al., 2008 2008 ). LCA is a genetically heterogeneous disorder typically transmitted as autosomal recessive inheritance, although several families with autosomal dominant inheritance have been reported (den Hollander et al., 2008 2008 ). To date, 22 causative genes (Wang et al., 2015 2015 ; Xu et al., 2016 2016 ), namely GUCY2D , CEP290 , CRB1 , CRX , AIPL1 , RPE65 , RPGRIP1 , RDH12 , LCA5 , SPATA7 , LRAT , IQCB1 , KCNJ13 , NMNAT1 , RD3 , TULP1 , CABP4 , CLUAP1 , DTHD1 , GDF6 , IFT140 , and PRPH2 , have been identified from the RetNet (https://sph.uth.edu/retnet). Mutations in these genes have been recognized as the cause of 70% of LCA cases (Yucel-Yilmaz et al., 2014 2014 ). The phenotypic variability of LCA is striking, and clinical heterogeneity could be found in visual acuity, refractive errors, retinal appearance, photoaversion, nyctalopia, and other associated features such as keratoconus and cataracts (den Hollander et al., 2008 2008 ). Characteristic phenotypic findings may narrow the differential diagnosis and lend clues to the underlying genotypic etiology. Some studies have established phenotype-genotype correlations for several LCA genes, and gene-specific phenotypic features are useful in guiding the clinician to a potential genetic etiology (Perrault et al., 2004 2004 ; Chung and Traboulsi, 2009 2009 ; Traboulsi, 2010 2010 ; Mackay et al., 2011 2011 ; Chacon-Camacho and Zenteno, 2015 2015 ). LCA 13, caused by retinol dehydrogenase 12 ( RDH12 ) mutations, is characterized by progressive decline of visual function in early adulthood, absent rod and cone responses on the ERG, marked pigmentary retinopathy, and pronounced maculopathy (Schuster et al., 2007 2007 ). Posterior subcapsular cataracts or cortical cataracts could be present in LCA 13 patients with the absence of photophobia and hyperopia. In this work, we analyzed the ocular phenotype of LCA patients in a Chinese family, of which two showed typical clinical symptoms of LCA 13. Based on phenotype-genotype correlation, RDH12 was chosen as the candidate gene. Successfully we identified novel RDH12 compound heterozygous variants with Sanger sequencing in this LCA family, which was confirmed by targeted next-generation sequencing. Materials and methods 1 Subjects 2 The research protocol was approved by the Institutional Review Board of the First Affiliated Hospital of Zhejiang University (Hangzhou, China). Written informed consent was obtained from all the adults and the children’s parents involved in the study, and all clinical investigations were conducted in accordance with the tenets of the Declaration of Helsinki. Control subjects were recruited from the First Affiliated Hospital of Zhejiang University. Clinical evaluation 2 A third-generation Chinese family with LCA was studied. Complete ophthalmic examination of each family member was performed, including acquisition of a thorough ophthalmic history, best corrected visual acuity (BCVA), slit-lamp biomicroscopy, fundus photography (CR-2 Retinal Imaging, Canon, Japan), visual field tests (Humphrey Field Analyzer 3, Carl Zeiss Meditec, Germany), optical coherence tomography (OCT) (Spectralis OCT BluePeak, Heidelberg Engineering, Heidelberg, Germany), fluorescein fundus angiography (FFA) (Heidelberg Retina Angiograph 2 (HRA2), Heidelberg Engineering, Heidelberg, Germany), and ERG (RETI-PORT21, Roland Consult, Germany). All clinical tests were performed in the First Affiliated Hospital of Zhejiang University. Analysis of the ocular phenotype of LCA patients 2 We analyzed the ocular phenotype of LCA patients, including a thorough ophthalmic history, visual acuity, refractive errors, retinal appearance, photoaversion, nyctalopia, and cataracts. The identified gene-specific phenotypic features provided significant prognostic information for determining which gene may be responsible. DNA extraction 2 Peripheral blood samples from each family member were collected in ethylene diamine tetraacetic acid (EDTA) tubes. Genomic DNA was extracted using a blood DNA extraction kit (QIAamp DNA Blood Midi Kit, Qiagen, Hilden, Germany) following the manufacturer’s protocol. DNA samples were stored at −20 °C until use. Sanger sequencing and familial segregation analysis of RDH12 variants 2 The proband of this family (II:1) was initially analyzed with all the seven coding exons and intron-exon boundaries of RDH12 by Sanger sequencing provided by Sangon Biotech (Shanghai, China). The identified RDH12 variants were further confirmed bidirectionally and then checked in family members for segregation by direct Sanger sequencing. Novel variants were checked in 600 unrelated healthy Chinese controls with normal corrected visual acuity and no symptoms or family history of retinal degeneration. Primers flanking the variant were designed based on genomic sequences of Human Genome Database and synthesized by Sangon Biotech (Shanghai, China): exon 4 Fwd: 5'-GCTGGTGGAG TGTGTAGAACA-3', Rev: 5'-TTGGAGGCCACAG TGTAAGTT-3'; and exon 7 Fwd: 5'-AACACTGAA GTCCTCTTGGCTC-3', Rev: 5'-GGGCAAGCACT CTGTTTTTATTC-3'. All polymerase chain reactions (PCRs) were performed with a PCR amplification kit (TaKaRa) according to the manufacturer’s instructions. PCR products were analyzed by 2% (0.02 g/ml) agarose gel electrophoresis and subsequently purified with a gel extraction kit (QIAEX II Gel Extraction Kit; Qiagen, Hilden, Germany). Direct automated sequencing was performed with the ABI BigDye Terminator V3.1 Cycle Sequencing Kit. All samples were analyzed on an ABI 3730XL Genetic Analyzer (Applied Biosystems). Wild-type and mutated RDH12 sequences were compared manually. Target gene sequence capture and next generation sequencing analysis 2 Targeted next-generation sequencing was performed on the two affected siblings by BGI Health (Shenzhen, China) with a capture panel that enriches the exonic DNA of 146 known genes associated with genetic retinal disease including 22 known LCA-causative genes. Genomic DNA was sheared into fragments of 200–300 bp and added an A-residue, and then amplified by ligation-mediated PCR. Captured libraries were quantified and sequenced on Illumina HiSeq 2000 platform as 90 bp paired-end reads. Sequence reads were aligned to the Human Genome Reference version hg by Burrows Wheeler Aligner (Li and Durbin, 2009 2009 ). The Genome Analysis Toolkit (GATK) was used for base quality score recalibration and local realignment (McKenna et al., 2010 2010 ). The SAMtools ( http://samtools.sourceforge.net http://samtools.sourceforge.net ) was used for single nucleotide polymorphism (SNP) calling (Li, 2011 2011 ), while the Pindel ( http://gmt.genome.wustl.edu/ http://gmt.genome.wustl.edu/ packages/pindel) was used for Indel calling (Ye et al., 2009 2009 ). All changes were filtered against 1000 Genomes database, dbSNP, HapMap database, ESP6500, and ExAC. Analysis of novel variants 2 Novel variants were analyzed in silico using the SIFT (Sorting Intolerant from Tolerant, http://sift.jcvi.org http://sift.jcvi.org ) (Ng and Henikoff, 2003 2003 ) and PolyPhen-2 (Polymorphism Phenotyping v2, http://genetics.bwh.harvard.edu/pph2 http://genetics.bwh.harvard.edu/pph2 .) (Adzhubei et al., 2010 2010 ) programs to predict their possible impact on the structure and function of the protein. Detected variants were also checked in the 1000 Genomes database and Exome Variant Server (EVS) for excluding them as common polymorphisms (Hong et al., 2014 2014 ; Lin et al., 2016). Results 1 Clinical assessment of the family 2 A three-generation family from Zhejiang Province of China was recruited (Table 1 1 ). Since the parents and offspring of two affected subjects had no apparent LCA symptoms, the disease exhibited autosomal recessive inheritance pattern in this family. The affected subjects exhibited similar clinical features (Fig. 1 1 ). Patients shared poor, yet useful visual function in early childhood followed by a progressive and severe decline before the second decade. They had nyctalopia, visual field constriction, sensory nystagmus, but photophobia and hypermetropia. Anterior segment examination found delayed pupillary light reflex and mild posterior subcapsular or a spot of peripheral cortical lens opacity. Fundus examination showed pronounced maculopathy, peripheral hyperpigmentation, and retinal vascular attenuation. OCT imaging revealed a thick retina with loss of the foveal laminar architecture. ERG showed undetectable responses under scotopic and photopic conditions, indicating severe generalized loss of rod and cone photoreceptor function. All these clinical characteristics were consistent with LCA 13, as reported earlier (Schuster et al., 2007 2007 ; Mackay et al., 2011 2011 ). Based on phenotype-genotype correlation, RDH12 was chosen as the first candidate gene. (a–f) Funduscopic features include pronounced maculopathy, peripheral hyperpigmentation, and retinal vascular attenuation. (g, h) Optical coherence tomography (OCT) demonstrates thick retina with loss of the foveal laminar architecture. (i) Electroretinogram (ERG) shows undetectable rod and cone responses. II:1 and II:2: family members; OD: oculus dexter; OS: oculus sinister BCVA: best corrected visual acuity; OD: oculus dexter; OS: oculus sinister; OPM: ocular pursuit movements; PM: pronounced maculopathy; PH: peripheral hyperpigmentation; RVA: retinal vascular attenuation; ERG: electroretinogram Family member Age (year)/sex Onset age (year) BCVA (OD/OS) Nystagmus and nyctalopia Fundus appearance ERG  I:1 50/M 1.00/1.00 − Normal Normal  I:2 46/F 1.00/1.00 − Normal Normal  II:1 24/M 2 0.30/0.06 + PM, PH, RVA Extinguished  II:2 25/F 2 0.04/0.12 + PM, PH, RVA Extinguished  III:1 1/M OPM − Normal Normal Sanger sequencing results and verification of familial segregation of RDH12 variants 2 By Sanger sequencing of the proband (II:1), we identified novel RDH12 compound heterozygous variants (Fig. 2 2 ), which were further confirmed on patient II:2. The first RDH12 variant was a c.164C > A transversion at exon 4, predicting a novel p.T55K substitution; the second variant was a c.535C > G transversion at exon 7, which predicts a novel p.H179D substitution. Other family members were then screened for the two RDH12 variants. We found that the proband’s father (I:1) was unaffected heterozygous carrier of c.535C > G and his mother (I:2) as well as his sister’s offspring (III:1) were unaffected heterozygous carriers of c.164C > A, showing complete cosegregation of the variants with LCA (Fig. 3 3 ). Neither of the RDH12 variants was detected in family member II:3 or in the 600 normal controls by direct Sanger sequencing. (a) A novel heterozygous c.164C > A transversion (arrow) was demonstrated in RDH12 exon 4 in DNA from two LCA patients (II:1, II:2) and two unaffected family members (I:2, III:1). (b) A novel heterozygous c.535C > G transition (arrow) was identified in exon 7 in DNA from two LCA patients (II:1, II:2) and their father (I:1). (c, d) Normal RDH12 sequences are shown for comparison. (e) Protein sequence alignment of RDH12 from different species Solid symbols designate affected individuals. Open symbols indicate unaffected individuals. Square indicates male, and circle indicates female. Arrow indicates the proband. WT indicates wild-type sequence Targeted next-generation sequencing 2 About 218 SNPs and Indels were identified by targeted next-generation sequencing. Since LCA is a rare disease, all common variants were removed by filtering against the following variation databases: 1000 Genomes database, dbSNP, HapMap database, ESP6500, and ExAC. As a result, five rare variants that may result in protein coding change were identified as candidate variants (Table 2 2 ). Given that the disease exhibited autosomal recessive inheritance pattern in this family, a homozygous variant or compound heterozygous variants should be shared by both affected siblings, and the heterozygous variant should be verified on their unaffected parents respectively. Finally, only the compound heterozygous variants c.164C > A and c.535C > G in RDH12 were identified. The result of targeted next-generation sequencing was consistent with that of candidate gene sequencing indicated by phenotype-genotype correlation. Het: heterozygous mutation; Hom: homozygous mutation; EX: exon; II:1 and II:2: family members Chromosome Gene NM Variants Position Het/Hom II:1 II:2  Chr14 RDH12 NM_152443 p.T55K EX4 Het + +  Chr14 RDH12 NM_152443 p.H179D EX7 Het + +  Chr3 CLRN1 NM_174878 p.G136E EX2 Het + +  Chr3 MITF NM_198159 p.D491Y EX10 Het + +  Chr4 PROM1 NM_001145848 p.E728K EX20 Het − + Novel RDH12 variant analysis 2 The novel p.T55K RDH12 variant was predicted to be probably damaging by both SIFT and PolyPhen-2 programs with scores of 0 and 1, respectively. The novel p.H179D RDH12 variant was predicted to be benign by both SIFT and PolyPhen-2 programs with scores of 0.36 and 0.188, respectively. Both novel variants were not annotated at 1000 Genomes or EVS databases. Discussion 1 LCA is a group of congenital retinal dystrophy with clinical and genetic heterogeneity. The number of identified LCA-associated genes has been increasing, and now 22 genes are known to harbor mutations responsible for LCA. These genes encode proteins involved in a wide variety of retinal functions such as phototransduction, photoreceptor morphogenesis, retinoid cycling, ciliary transport, and guanine synthesis (den Hollander et al., 2006 2006 ; 2008 2008 ; Estrada-Cuzcano et al., 2011 2011 ; Sergouniotis et al., 2011 2011 ; Perrault et al., 2012 2012 ; Kolandaivelu and Ramamurthy, 2014 2014 ; Yucel-Yilmaz et al., 2014 2014 ). Although phenotypic variability could be found in the complete ophthalmic examinations of LCA patients, some specific clinical features could provide significant prognostic information for determining which gene may be responsible and thereby significantly reducing the involved cost. LCA 13, the characteristic phenotype associated with RDH12 mutations, exhibits severe and homogeneous clinical features characterized by onset of symptoms in early childhood and progression to legal blindness in early adulthood due to significant loss of the function of both rods and cones (Janecke et al., 2004 2004 ), with fundi showing marked pigmentary retinopathy with bone spicules in the peripheral retina and pronounced maculopathy (Jacobson et al., 2007 2007 ; Valverde et al., 2009 2009 ; Sodi et al., 2010 2010 ; Kuniyoshi et al., 2014 2014 ). Nyctalopia is the predominant symptom of LCA 13. Most LCA 13 patients have no photophobia, hyperopia, or keratoconus, while mild posterior subcapsular cataract can occur. OCT shows thick retinas with loss of the foveal laminar architecture (Jacobson et al., 2007 2007 ). ERG is extinguished under scotopic and photopic conditions. In our study, the clinical examinations of two affected subjects were consistent with the characteristics mentioned above, showing typical clinical symptoms of LCA 13. For the ophthalmologist, the clinical presentation of LCA represents a complex dilemma, as the clinical overlap among different genetic phenotypes could be striking and confusing. Similar phenotypic trends could be observed in other LCA genes, such as RPE65 , GUCY2D , AIPL1 , CRB1 , and CEP290 . The following clinical differences could be useful to separate these overlapping phenotypes from LCA 13. Patients with RPE65 mutations show preservation of visual acuity in early life followed by severe yet progressive decline in adulthood, which is similar to patients with RDH12 mutations. However, transient visual function improvement in patients with RPE65 mutations could last through teenage years and visual acuity deteriorates in their third to fifth decade (Traboulsi, 2010 2010 ; Chacon-Camacho and Zenteno, 2015 2015 ), while visual function in patients with RDH12 mutations could progress to legal blindness in early adulthood (18–25 years) (Janecke et al., 2004 2004 ). OCT scan shows that the retina is significantly thinner in patients with RPE65 mutations (Pasadhika et al., 2010 2010 ). Patients with GUCY2D mutations have a severe, congenital stationary visual function with a relatively preserved retinal appearance. Meanwhile, cataract and nyctalopia usually do not occur (Dharmaraj et al., 2000 2000 ; Milam et al., 2003 2003 ; Jacobson et al., 2013 2013 ). The phenotype of LCA patients with AIPL1 mutations is characterized by nystagmus, nyctalopia, maculopathy, and pigmentary retinopathy from young ages (Tan et al., 2012 2012 ). In addition, there is a high frequency of keratoconus, hyperopia, and photophobia (Testa et al., 2011 2011 ). In patients with CRB1 mutations, the retina exhibits nummular pigment clumps at early ages, and most patients have high hypermetropia and keratoconus (Bujakowska et al., 2012 2012 ). Patients carrying CEP290 mutations exhibit a severe yet progressive visual function. Moreover, photophobia, hyperopia, enophthalmos, and keratoconus are frequently observed (Yzer et al., 2012 2012 ), which are usually not present in patients with RDH12 mutations. In our study, with great awareness of potential correlations with clinical characteristics and specific genotypes, we successfully identified novel RDH12 compound heterozygous variants in LCA patients by Sanger sequencing: c.164C > A (p.T55K) at exon 4 and c.535C > G (p.H179D) at exon 7. Their unaffected parents and offspring carried only one of heterozygous variants, showing complete cosegregation of the variants. The variant p.T55K was predicted to be probably damaging to protein function, and the variant p.H179D was predicted to be benign by both SIFT and PolyPhen-2. The two heterozygous variants were absent in 600 normal controls and public databases such as 1000 Genomes or EVS, excluding them as common polymorphisms. These findings indicated that the RDH12 compound heterozygous variants confirmed by targeted next-generation sequencing might be responsible for LCA in the two affected patients. Mutations in RDH12 were first described as a cause of LCA in 2004 (Janecke et al., 2004 2004 ; Perrault et al., 2004 2004 ). To date, over 70 different RDH12 mutations have been reported predominantly in LCA patients (Chacon-Camacho and Zenteno, 2015 2015 ; Gong et al., 2015 2015 ). RDH12 is located at human chromosome 14q24.1 (Kiser et al., 2012 2012 ). The protein product, a member of the short-chain dehydrogenase/reductase (SDR) family, is predominantly localized to the photoreceptor inner segments and the outer nuclear layer (Haeseleer et al., 2002 2002 ; Kurth et al., 2007 2007 ), playing a crucial role in the interconversion of vitamin A (all- trans retinal) to 11- cis retinal (Schuster et al., 2007 2007 ; Chrispell et al., 2009 2009 ). Deletion of the RDH12 in mice increases the susceptibility to light-induced retinal apoptosis in both cone and rod photoreceptors (Maeda et al., 2006 2006 ). Another study suggests that RDH12 may be the key component in clearance of free all- trans retinal, thereby preventing accumulation of N -retinylidene- N -retinylethanolamine (A2E), a toxic substance responsible for the retinal degeneration (Maeda et al., 2007 2007 ). To date, the mechanism of retinal degeneration associated with mutations in RDH12 is unclear, and the specific role of RDH12 in the visual cycle remains to be studied further. 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Ocular and extra-ocular features of patients with Leber congenital amaurosis and mutations in CEP290 Compliance with ethics guidelines Yun LI, Qing PAN, and Yang-shun GU declare that they have no conflict of interest. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2008 (5). Informed consent was obtained from all patients for being included in the study. 10.1631/jzus.B1600156 Project supported by the National Natural Science Foundation of China (No. 81300741) 2016-04-08 2017-04-18 2016-08-26 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2017 2017 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 05 18