Molecular and clinical analysis of 27 German patients with Leber congenital amaurosis.

Leber congenital amaurosis (LCA) is the earliest and most severe form of all inherited retinal dystrophies (IRD) and the most frequent cause of inherited blindness in children. The phenotypic overlap with other early-onset and severe IRDs as well as difficulties associated with the ophthalmic examination of infants can complicate the clinical diagnosis. To date, 25 genes have been implicated in the pathogenesis of LCA. The disorder is usually inherited in an autosomal recessive fashion, although rare dominant cases have been reported. We report the mutation spectra and frequency of genes in 27 German index patients initially diagnosed with LCA. A total of 108 LCA- and other genes implicated in IRD were analysed using a cost-effective targeted next-generation sequencing procedure based on molecular inversion probes (MIPs). Sequencing and variant filtering led to the identification of putative pathogenic variants in 25 cases, thereby leading to a detection rate of 93%. The mutation spectrum comprises 34 different alleles, 17 of which are novel. In line with previous studies, the genetic results led to a revision of the initial clinical diagnosis in a substantial proportion of cases, demonstrating the importance of genetic testing in IRD. In addition, our detection rate of 93% shows that MIPs are a cost-efficient and sensitive tool for targeted next-generation sequencing in IRD.

Introduction

Leber congenital amaurosis (LCA, MIM #204000) was first described by Theodor Leber in 1869 and refers to a heterogeneous group of severe, mostly recessively inherited, early infantile-onset retinal dystrophies with typically extinguished electroretinograms (ERGs). Later, a separate group of milder disease phenotypes, with some preservation of the ERG responses, the so-called “early-onset severe retinal dystrophy” (EOSRD) or “severe early childhood onset retinal dystrophy” has been described. LCA and EOSRD together are the most severe and earliest forms of all inherited retinal diseases (IRDs). They affect 20% of blind children and account for 5% of all IRDs [1]. In Germany, the estimated number of cases is 2000 (source: Pro Retina Deutschland e. V.). To date, mutations in 25 genes have been associated with LCA (https://sph.uth.edu/retnet/). A substantial proportion of cases (10–20%) remain unsolved despite extensive molecular testing [2-4]. This is due to technical limitations as copy number variations often remain undetected in datasets derived from capture panels or whole exome sequencing, but also because of the focus on coding regions in most diagnostic settings which will not detect deep intronic variants acting on splicing or variants in regulatory sequences.

There is a considerable clinical and genetic overlap between LCA, EOSRD and other types of IRD, therefore, an accurate clinical diagnosis cannot always be made at the first visit of the young patients. Furthermore, the clinical examination of infants is challenging or limited. Hence, the initial clinical diagnosis sometimes has to be revised once genetic results are available.

For a long time the genetic heterogeneity of LCA (and IRD in general) hampered DNA-based (molecular) diagnoses, since parallel screening of all associated genes requires next generation sequencing approaches, for which reimbursement to the patient is often not guaranteed. We sought for a cost-effective and sensitive approach to obtain a molecular diagnosis for 27 patients that had been diagnosed with LCA at the University Eye Hospital Tuebingen. The present study focuses on these genetically unsolved cases, which were screened for sequence variants in 108 genes associated with non-syndromic IRD by a cost-effective targeted panel-based next-generation sequencing approach.

Materials and methods

Subjects and clinical assessment

In this study we included 27 unrelated patients of German origin with a clinical diagnosis of LCA who were not genetically pre-investigated. Their clinical diagnosis was established by standard clinical ophthalmologic examinations including patient history, psychophysical and electrophysiological examinations. Genomic DNA of patients was extracted from peripheral blood using standard protocols. Samples from all patients and family members were recruited in accordance with the principles of the Declaration of Helsinki and were obtained with written informed consent accompanying the patients´ samples. The study was approved by the institutional review board of the Ethics Committee of the University Hospital of Tuebingen.

Sequencing analysis

Molecular testing was performed by targeted next-generation sequencing at a core facility (Department of Human Genetics, Radboud University Nijmegen Medical Centre). We used molecular inversion probes (MIPs) with 5-bp molecular tags to conduct targeted next generation sequencing of 108 genes associated with IRD (see S1 Table). The 1,524 coding exons and the 10 bp flanking each exon were targeted with 6,129 probes for an overall target size of 647,574 bp. On average, 4–6 MIPs cover one exon. The panel also includes the frequent LCA-associated pathogenic intronic variant c.2992+1655A>G in CEP290 [5]. Pooled and phosphorylated probes were added to the capture reactions with 100 ng of genomic DNA from each individual to produce a library for each individual. The libraries were amplified with 21 cycles of PCR, during which an 8-bp sample barcode was introduced. The barcoded libraries were then pooled and purified with AMPureXP beads (Beckman-Coulter). Sequencing was performed on an Illumina NextSeq 500 system. Demultiplexed BAM files were aligned to a human reference sequence (UCSC Genome Browser hg19) via the Burrows-Wheeler Aligner (BWA) v.0.6.2 [6]. In-house automated data analysis pipeline and variant interpretation tools were used for variant calling. Rare and potentially disease-causing variants were confirmed by Sanger sequencing using standard protocols. Sanger sequencing was also used to screen for the recurrent c.2843G>A/p.C948Y variant in the CRB1 gene because it was not covered by the MIP reads.

Variant filtering and classification

Only non-synonymous single nucleotide variants (nsSNVs), nonsense variants, putative splice site (±10 bps) variants, insertions, duplications and deletions represented by more than 20 sequence reads were considered for further analysis. In addition, variants with a minor allele frequency (MAF) >0.5% in the Genome Aggregation Database (gnomAD) Version r2.0.2 [7] were excluded from further investigation. For variant classification we applied the terminology proposed by the American College of Medical Genetics and Genomics and the Association for Molecular Pathology [8].

In silico predictions

The potential pathogenicity of the missense changes identified in this study was assessed using four online prediction software tools, namely SIFT (http://sift.bii.a-star.edu.sg/) [9], PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/) [10], Mutation Taster (www.mutationtaster.org/) [11], and Provean (http://provean.jcvi.org/) [12].

Results

Utilizing our capture panel technology, we were able to obtain an average of 1.2 million reads on target per sample, with an average coverage of 213 reads per probe. Moreover, an average of 88% of targeted regions had 10x coverage or more, which was sufficient for accurate variant calling. The pipeline initially called an average of 532 single nucleotide variants and 64 insertions/deletions for each sample. Putative pathogenic variants were identified in 25/27 index cases (Table 1), thereby achieving a detection rate of 93%. All putative disease-associated variants were validated by conventional Sanger sequencing. Homozygosity was observed for eight patients (26%): variants were seen in true homozygous state in four patients and in apparent homozygous state in four patients, respectively. Two patients were hemizygous, and compound heterozygosity was observed for four patients based on the analysis of paternal alleles. Trans configuration of variants could not be demonstrated for 11 patients because DNA of family members was not available and the respective variants were located too far apart for allelic cloning. In patient 26, a single heterozygous variant in IMPG2 was observed. In patient 27, no putative disease-causing variants were identified. The mutation spectrum comprises 34 different alleles, 17 of which are novel. All novel variants were deposited to the ClinVar database (https://www.ncbi.nlm.nih.gov/clinvar/) [13] with accession codes provided in Table 1.

Table 1

Putative pathogenic variants in 25 unrelated German patients initially diagnosed with LCA.

Patient Nr.	Final diagnosis	Gene	Allele 1	Reference	ClinVar accession no.	ACMG category	Allele 2	Reference	ClinVar accession no.	ACMG category	Segregation performed
Solved by putative pathogenic mutations in known LCA genes
1	LCA	AIPL1	c.857A>T/p.D286V	this study	SCV000845180	VUS	c.857A>T/p.D286V	this study	pending	VUS	yes
2	LCA	AIPL1	c.834G>A/p.W278*	PMID: 10615133	SCV000086966.1	LP	c.276+6T>C/p.?	this study	SCV000845181	VUS	no
3	LCA	CEP290	c.2991+1655A>G/p.[C998*, = ]	PMID: 16909394PMID: 27151457	SCV000021550.2	LP	c.2991+1655A>G/p.[C998*, = ]	PMID: 16909394PMID: 27151457	SCV000021550.2	LP	yes
4	EOSRD	CRB1	c.2798G>A/p.C933Y	this study	SCV000845184	VUS	c.2843G>A/p.C948Y	PMID: 10508521	SCV000056582.2	VUS	yes
5	LCA	CRB1	c.4039del/p.T1347Lfs*5	this study	SCV000845185	LP	c.2843G>A/p.C948Y	PMID: 10508521	SCV000056582.2	VUS	no
6	LCA	CRB1	c.410del/p.P137Lfs*11	this study	SCV000845186	LP	c.2843G>A/p.C948Y	PMID: 10508521	SCV000056582.2	VUS	no
7	LCA	CRB1	c.70+1G>A/p.?	this study	SCV000845187	LP	c.2042G>A/p.C681Y	PMID: 11231775	SCV000118458.1	VUS	yes
8	EOSRD	CRB1	c.2308G>A/p.G770S	PMID: 27113771	SCV000282584.1	VUS	c.2843G>A/p.C948Y	PMID: 10508521	SCV000056582.2	VUS	no
9	LCA	CRB1	c.2072G>A/p.W691*	this study	SCV000845188	LP	c.2843G>A/p.C948Y	PMID: 10508521	SCV000056582.2	VUS	no
10	LCA	NMNAT1	c.12dup/p.E5Rfs*4	PMID: 24940029	n.a.	LP	c.769G>A/p.E257K	PMID: 22842231	SCV000053426.1	VUS	no
11	EOSRD	RD3	c.180C>A/p.Y60*	PMID: 22531706	SCV000222653.1	LP	c.180C>A/p.Y60*	PMID: 22531706	SCV000222653.1	LP	no
12	LCA	RPE65	c.110G>C/p.W37S	this study	SCV000845190	VUS	c.722A>G/p.H241R	this study	SCV000845191	VUS	no
13	LCA	RPE65	c.203A>C/p.H68P	this study	SCV000845192	VUS	c.825C>G/p.Y275*	this study	SCV000845193	LP	no
14	LCA	RPGRIP1	c.2440C>T/p.R814*	this study	SCV000845194	LP	c.2440C>T/p.R814*	this study	SCV000845194	LP	no
15	LCA	RPGRIP1	c.1303A>T/p.K435*	PMID: 27208204	SCV000282616.1	LP	c.801-25_c.843del	this study	SCV000845195	LP	yes
16	LCA	RPGRIP1	c.2941C>T/p.R981*	PMID: 28041643	SCV000599101.1	LP	c.2941C>T/p.R981*	PMID: 28041643	SCV000599101.1	LP	no
17	LCA	RPGRIP1	c.800+1G>A/p.?	PMID: 16123401	n.a.	LP	c.2718dup/p.N907*	PMID: 28714225	n.a.	LP	yes
Solved by putative pathogenic mutations in IRD genes not typically associated with LCA
18	CRD	ABCA4	c.1765del/p.W589Gfs*60	this study	SCV000845179	LP	c.1765del/p.W589Gfs*60	this study	SCV000845179	LP	no
19	CRD	ABCA4	c.5461-10T>C/ p.[T1821Vfs*13, T1821Dfs*6]	PMID: 15614537PMID: 26976702	SCV000028574.2	LP	c.3377T>C/p.L1126P	PMID: 25066811	SCV000281867.2	VUS	no
20	CRD	ABCA4	c.3259G>A/E1087K	PMID: 9054934	SCV000598963.1	VUS	c.5917del/p.V1973*	PMID: 10958763	SCV000599004.1	LP	no
21	CRD	ABCA4	c.5917del/p.V1973*	PMID: 10958763	SCV000599004.1	LP	c.5917del/p.V1973*	PMID: 10958763	SCV000599004.1	LP	yes
22	CSNB	CACNA1F	c.4504C>T/p.R1502*	this study	SCV000845182	LP	-				no
23	CRD	CDHR1	c.634G>A/p.A212T	PMID: 16288196	n.a.	VUS	c.1132C>T/p.R378W	this study	SCV000845183	VUS	no
24	RP	PROM1	c.1209_1229/p.Q403_S410delinsH	PMID: 24265693	SCV000575406.4	VUS	c.1209_1229/p.Q403_S410delinsH	PMID: 24265693	SCV000575406.4	VUS	yes
25	XRP	RP2	c.314G>A/p.C105Y	this study	SCV000845189	VUS	-				no

LCA, Leber congenital amaurosis; EOSRD, early-onset severe retinal dystrophy; CRD, cone-rod dystrophy; CSNB, congenital stationary nightblindness; RP, retinitis pigmentosa; XRP, X-linked RP; VUS, variant of uncertain significance; LP, likely pathogenic; n.a., not available.

The variants comprise 14 missense variants, eight nonsense variants, seven deletions or duplications leading to a frame-shift, three canonical splice site variants, two non-canonical splice site variants and one in-frame deletion. Pathogenicity was interpreted in accordance with the American College of Medical Genetics guidelines [8]. The respective categories are given in Table 1. Missense variants that have never been reported before were analysed using different in silico prediction algorithms. These scores, together with the MAFs sourced from the gnomAD browser are shown in Table 2.

Table 2

Assessment of pathogenicity of missense variants identified in this study.

Gene	Variant	gnomAD MAF	Mutation Taster	Polyphen	SIFT	Provean	phyloP	Grantham Score
ABCA4	c.3377T>C/p.L1126P	4.061e-6	Disease causing (0.99)	Probably damaging (1.0)	Damaging (0.0)	Deleterious (-6.51)	3.60	98
ABCA4	c.3259G>A/E1087K	1.624e-5	Disease causing (0.99)	Probably damaging (1.0)	Damaging (0.0)	Deleterious (-3.84)	6.22	56
AIPL1	c.857A>T/p.D286V	none	Disease causing (0.99)	Probably damaging (1.0)	Damaging (0.0)	Deleterious (-8.31)	4.09	152
CDHR1	c.634G>A/p.A212T	0.0001312	Disease causing (0.99)	Probably damaging (0.99)	Damaging (0.0)	Deleterious (-3.22)	4.93	58
CDHR1	c.1132C>T/p.R378W	7.584e-5	Disease causing (0.99)	Probably damaging (1.0)	Damaging (0.02)	Deleterious (-3.84)	1.25	101
CRB1	c.2798G>A/p.C933Y	none	Disease causing (0.99)	Probably damaging (0.99)	Damaging (0.0)	Deleterious (-9.66)	5.69	194
CRB1	c.2308G>A/p.G770S	2.036e-5	Disease causing (0.99)	Probably damaging (1.0)	Tolerated (0.06)	Deleterious (-5.48)	5.69	56
CRB1	c.2843G>A/p.C948Y	0.0002027	Disease causing (0.99)	Probably damaging (0.99)	Damaging (0.0)	Deleterious (-9.66)	5.31	194
CRB1	c.2042G>A/p.C681Y	4.067e-6	Disease causing (0.99)	Probably damaging (1.0)	Damaging (0.0)	Deleterious (-10.74)	5.74	194
NMNAT1	c.769G>A/p.E257K	0.0006968	Disease causing (0.99)	Benign (0.09)	Tolerated (0.52)	Neutral (-2.31)	3.87	56
RP2	c.314G>A/p.C105Y	none	Disease causing (0.99)	Probably damaging (1.0)	Damaging (0.0)	Deleterious (-8.6)	5.50	194
RPE65	c.110G>C/p.W37S	none	Disease causing (0.99)	Probably damaging (1.0)	Damaging (0.02)	Deleterious (-12.62)	5.78	177
RPE65	c.722A>G/p.H241R	none	Disease causing (0.99)	Probably damaging (1.0)	Damaging (0.0)	Deleterious (-7.58)	4.74	29
RPE65	c.203A>C/p.H68P	none	Disease causing (0.99)	Probably damaging (1.0)	Tolerated (0.06)	Deleterious (-9.34)	4.78	77

MAF, minor allele frequency.

LCA / EOSRD patients

A summary of clinical findings is shown in Table 3 including all 27 index patients. In 19 of 27 patients, the initial diagnosis of LCA/EOSRD was confirmed by the molecular genetic analysis. In all of these cases, disease onset was typically at birth or within the first months of life. Nystagmus and strabismus were common features, indicating the lack of visual development. Visual acuity was severely reduced in all cases, ranging from 0.2 (decimal) to no light perception (NLP). Where visual field testing was possible, only small residual visual islands could be detected. Fullfield ERGs were extinguished in each case at time of recording. Morphological findings included typical salt & pepper pigmentary changes of the retina, pale optic disks and attenuated retinal vessels. Patients showed a progressive disease history with severe visual impairment from the beginning. In the following, the LCA-associated genes that were found to be mutated in these patients are listed in detail.

Table 3

Summary of clinical findings.

PatientNr.	Current age (y) Gender	Genetic findings	Revised diagnosis	Disease onset	BCVAODOS	Nystagmus	Cataract	Strabism	Fundus pigmentary changes	Other findings
1	50 / m	AIPL1c.857A>T/p.D286V homozygous	LCA	6 months	1/351/35	yes	yes	yes	S&P	no
2	31 / m	AIPL1c.834G>A/p.W278* heterozygousc.277+6T>C/p.? heterozygous	LCA	n.a.	CFCF	yes	no	yes	S&P	no
3	8 / f	CEP290c.2991+1655A>G/p.[Cys998*, = ] homozygous	LCA	birth	n.a.	yes	no	yes	n.a.	no
4	45 / f	CRB1c.2798G>A/p.C933Y heterozygousc.2843G>A/p.C948Y heterozygous	EOSRD	childhood	LPHM	yes	no	yes	S&P	n.a.
5	61 / m	CRB1c.4039del/p.T1347Lfs*5 heterozygousc.2843G>A/p.C948Y heterozygous	LCA	birth	n.a.	yes	yes	n.a.	n.a.	n.a.
6	25 / f	CRB1c.410del/p.P137Lfs*11 heterozygousc.2843G>A/p.C948Y heterozygous	LCA	6 months	n.a.	yes	no	yes	S&P	no
7	27 / m	CRB1c.70+1G>A/p.? heterozygousc.2042G>A/p.C681Y heterozygous	LCA	9 months	1/351/35	yes	no	yes	S&P	no
8	55 / f	CRB1c.2308G>A/p.G770S heterozygousc.2843G>A/p.C948Y heterozygous	EOSRD	3 years	LPLP	n.a.	yes	n.a.	S&P	anti-phospholipid syndrome, asthma
9	28 / m	CRB1c.2072G>A/p.W691* heterozygousc.2843G>A/p.C948Y heterozygous	LCA	birth	1/51/5	yes	no	yes	n.a.	n.a.
10	48 / f	NMNAT1c.12dup/p.E5Rfs*4 heterozygous c.769G>A/p.E257K heterozygous	LCA	birth	LPLP	yes	yes	n.a.	n.a.	n.a.
11	20 / f	RD3c.180C>A/p.Y60* homozygous	EOSRD	2 years	LPLP	yes	no	yes	n.a.	no
12	36/ m	RPE65c.110G>C/p.W37S heterozygousc.722A>G/p.H241R heterozygous	LCA	birth	1/501/35	yes	no	yes	S&P	n.a.
13	48 / f	RPE65c.203A>C/p.H68P heterozygousc.825C>G/p.Y275* heterozygous	LCA	birth	HM1/50	yes	no	yes	n.a.	n.a.
14	26 / f	RPGRIP1c.2440C>T/p.R814* homozygous	LCA	birth	NLPNLP	yes	no	yes	S&P	no
15	36 / m	RPGRIP1c.1303A>T/p.K435* heterozygousc.801-25_c.843del heterozygous	LCA	6 months	HMHM	yes	no	yes	n.a.	no
16	47 / m	RPGRIP1c.2941C>T/p.R981* homozygous	LCA	n.a.	LPLP	yes	yes	yes	S&P	no
17	17 / f	RPGRIP1c.800+1G>A/p.? heterozygousc.2718dup/p.N907* heterozygous	LCA	birth	1/101/10	yes	no	yes	S&P	no
18	33 / f	ABCA4c.1765del/p.W589Gfs*60 homozygous	CRD	3 years	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.
19	47 / f	ABCA4c.5461-10T>C/p.[T1821Vfs*13, T1821Dfs*6] heterozygousc.3377T>C/p.L1126P heterozygous	CRD	7 years	1/351/35	yes	no	yes	S&P	n.a.
20	44 / m	ABCA4c.3259G>A/E1087K heterozygousc.5917del/p.V1973* heterozygous	CRD	n.a.	LPLP	n.a.	yes	n.a.	dense	no
21	18 / f	ABCA4c.5917del/p.V1973* homozygous	CRD	childhood	LPLP	n.a.	no	n.a.	dense	no
22	20 / m	CACNA1Fc.4504C>T/p.R1502* hemizygous	CSNB	birth	1/61/10	yes	no	no	no	no
23	35 / f	CDHR1c.634G>A/p.A212T heterozygousc.1132C>T/p.R378W heterozygous	CRD	n.a.	HMHM	yes	no	yes	S&P	renal insufficiency, hyper-parathyroidism, obesity
24	50 / m	PROM1c.1209_1229/p.Q403_S410delinsH homozygous	RP	16 years	LPLP	yes	yes	yes	S&P	no
25	45 / m	RP2c.314G>A/p.C105Y hemizygous	XRP	childhood	1/351/35	yes	no	yes	n.a.	no
26	39/ m	IMPG2c.370T>C/p.F124L single heterozygous	LCA	4 years	n.a.	yes	no	yes	n.a.	no
27	12 / m	nothing of immediate interest	EOSRD	3 years	1/201/20	no	no	yes	S&P	no

BCVA, best corrected visual acuity; OD, right eye; OS, left eye; m, male; f, female; LCA, Leber congenital amaurosis; EOSRD, early-onset severe retinal dystrophy; CRD, cone-rod dystrophy; CSNB, congenital stationary nightblindness; RP, retinitis pigmentosa; XRP, X-linked RP; CF, counting fingers; HM, hand movement; LP, light perception; NLP, no light perception; S&P, salt and pepper.

CRB1

CRB1 variants were detected in six patients (22.2%; 6/27). In total, eight variants were identified, including one novel nonsense, two novel frame-shifting deletions, one novel canonical splice site variant and one novel missense variant. Compound heterozygosity could only be demonstrated in two patients. Five patients were heterozygous for the recurrent c.2843G>A/p.C948Y variant, which has been reported to represent 23–31% of all CRB1 disease-associated alleles [14-15]. Of note, this particular variant was not covered by the MIPs in our assay, but we screened all patients by conventional Sanger Sequencing for this variant, because of its known high frequency and relevance (MAF 0.0002027).

RPGRIP1

Of the six potentially disease-causing variants in RPGRIP1 detected in four patients (14.8%; 4/27), all represent likely null alleles and three were novel. Compound heterozygosity of a reported nonsense variant and a novel 68-bp deletion was demonstrated for one patient. One patient harbored a reported canonical splice site variant on one allele and a novel frame-shifting duplication on the other allele. Two patients were homozygous for two different nonsense variants, one of them novel.

RPE65

A total of four novel variants in RPE65 were identified in two patients (7.4%; 2/27), including one nonsense and three missense. Biallelism could not be formally proven in both cases.

AIPL1

Of the three variants detected in two affected individuals in AIPL1 (7.4%; 2/27), there was one novel missense variant found in homozygous state in one patient. Another patient harbored a nonsense variant and a non-canonical splice site change. Whether the variants are in trans configuration in this patient could not be established.

RD3

One patient was found to be homozygous for a known nonsense variant in RD3 (3.7%; 1/27).

NMNAT1

NMNAT1 variants were detected in one patient (3.7%; 1/27) who possessed one reported frame-shifting duplication and the known hypomorphic variant c.769G>A/p.E257K [16]. Biallelism could not be confirmed due to lack of additional family DNA samples.

CEP290

One patient was found to be homozygous for the common c.2991+1655A>G/p.C998* allele which causes insertion of a cryptic exon and subsequent truncation [5,17].

Other patients

In addition to the cases described above, we identified eight patients (30%) who harbored pathogenic variants in genes not typically associated with LCA. Clinical re-evaluation of these cases led to a revision of the initial clinical diagnosis in all of them. Within this group, ABCA4 was the most frequently mutated gene, as biallelic variants were seen in four patients. In these cases, a later onset of disease and a dense pigmentation of the retina were observed (Table 3). After genetic testing and re-evaluation of clinical data, the diagnosis was corrected to cone-rod dystrophy (CRD), demonstrating severe morphological and functional damage in all cases.

In addition, we found a male patient to be hemizygous for a pathogenic variant in RP2. He had been initially diagnosed in adult age with severely progressed retinal degeneration. Consequently, his diagnosis was corrected to X-linked retinitis pigmentosa.

Another male patient was shown to be hemizygous for a pathogenic variant in CACNA1F. He was suffering from nystagmus, night blindness, photophobia and very poor vision since birth. His fullfield ERGs showed residual photopic and scotopic responses. Morphologically, slight attenuation of the retinal vessels, changes in the macular reflexes and only minimal peripheral pigmentary changes could be observed. In this case, the diagnosis was changed to X-linked congenital stationary night blindness (CSNB).

One patient harbored pathogenic variants in CDHR1. The revised clinical diagnosis in this case was CRD, but interestingly, this female patient also suffered from renal insufficiency, secondary hyperparathyroidism and obesity. Whether these symptoms can be considered as a unique disease identity or syndrome remains unexplained. So far, such extra-ocular symptoms have not been described as a feature of CDHR1-related disease but would be typical features of a ciliopathy to which CDHR1-associated IRD does not belong to.

The last male patient presented in our clinic with a severe retinal degeneration at the age of 50 years and was found to be homozygous for an in-frame insertion/deletion in PROM1. On the basis of patient history, clinical findings and genetic results, the clinical diagnosis was changed to autosomal recessive retinitis pigmentosa.

Discussion

In a cohort of 27 German patients initially diagnosed with LCA, we were able to identify sequence variants likely explaining the disease phenotype in 25 cases (93%) by applying a cost-efficient targeted next-generation sequencing approach designed at the Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands. The MIP panel targets 108 known IRD genes, including 22 genes that are associated with LCA, that were reported in October 2013.

Undoubtedly, those LCA genes with the highest disease-causing variant load have already been discovered. However, the fact that half of the variants (17/34) we identified are novel suggests that the mutation spectrum of LCA and other IRD genes is far from being saturated and confirms the known genetic heterogeneity of IRD in an outbred European population.

The most frequently mutated LCA genes in our cohort were CRB1 (6 cases, 22%) and RPGRIP1 (4 cases, 15%). Among the six patients with CRB1 mutations, five carried the recurrent p.C948Y variant on one allele, which is known to be a founder mutation [18]. We only identified one patient with a CEP290 variant in our cohort, despite CEP290 being one of the most frequently mutated LCA genes in different populations [5, 19], but this is due to the fact that most patients in the present study had already been pre-screened for the recurrent pathogenic intronic variant c.2992+1655A>G.

Several criteria were considered to evaluate the potential pathogenicity of variants: (1) variants have previously been reported to be pathogenic, (2) variants are observed only in few heterozygous cases or are absent among 277,264 general population alleles sourced from gnomAD browser; (3) variants represent likely null alleles (nonsense, canonical splice site and frame-shift variants), and (4) in the case of missense variants they are predicted to be damaging by in silico prediction algorithms. In addition, all variants were classified according to their pathogenicity based on the American College of Medical Genetics and Genomics (ACMG) guidelines [8]. With nonsense, canonical splice site and frame-shifting variants having a strong weight in the ACMG scoring system, this class of variants are consequently classified either as likely pathogenic or pathogenic, whereas missense variants that lack segregation data and functional analyses to support a damaging effect are always classified as variants of uncertain significance (VUS). To compensate for this simplistic categorization of the ACMG classification system, we provide in silico predictions from four algorithms for all missense variants identified in this study, regardless of having been reported previously or not, along with phyloP scores, Grantham differences and MAFs sourced from the gnomAD browser (Table 2). The extremely low MAF or even the absence in the gnomAD browser, the evolutionary conservation as well as the type of the respective amino acid substitution are strong indicators that all missense variants we identified and reported are indeed pathogenic. One missense variant that is predicted to be benign by the majority of algorithms is the recurrent c.769G>A/p.E257K variant in NMNAT1, but it has been shown previously that this is a hypomorphic variant and almost always causes LCA in combination with more severe alleles [16].

Apart from the fact that we lack segregation data for several patients, the only case that is left with some level of uncertainty is patient LCA 108 who carries a nonsense variant and a non-canonical splice site variant in AIPL1. The latter is a transition of T to C at position +6 of the splice donor of exon 2. It is absent in the gnomAD browser, but since the +6 position is not invariable, we performed an in silico prediction. The bioinformatic tool Human Splicing Finder [20] predicts that the c.277+6T>C variant breaks the natural splice donor site, since the mutant score is reduced by 41% compared to the wildtype score when using maximum entropy as the algorithm type. However, since AIPL1 is not expressed in accessible tissues like blood or skin fibroblasts, mRNA analyses to confirm the in silico prediction are not feasible. Sanger sequencing of the entire coding region of AIPL1 in this patient revealed no other variants than c.834G>A/p.W278* and c.277+6T>C. While most cases with mutations in AIPL1 are biallelic, certain mutations may result in dominant cone-rod dystrophy or juvenile retinitis pigmentosa [21], however, this most probably is not the case for loss of function alleles like the c.834G>A/p.W278* variant in our patient. Of course, we cannot rule out that the phenotype of our patient might not be related to AIPL1 at all.

The different forms of IRD may present with considerable clinical overlap [22]. This often precludes the assessment of a diagnosis on the basis of the disease phenotype alone, no matter how experienced and meticulous the clinician might be. Hence, we were not surprised that eight patients in our cohort (30%) were found to carry pathogenic variants in genes not typically associated with LCA. We reassessed the clinical data of these patients and revisited the initial diagnosis in all of them. A recently published study on Brazilian patients with LCA found the same proportion (i.e. 30%) of patients that were solved by identifying variants in non-LCA genes [23]. This impressively demonstrates how a molecular diagnosis can help to refine a clinical diagnosis.

The underlying variants in two patients remained unresolved (7.4%; 2/27). One of these patients was found to be heterozygous for a known missense variant in IMPG2. Biallelic mutations in IMPG2 are a known cause for RP [24]. All exons and adjacent intronic regions of this gene were sufficiently covered which excludes the existence of a second variant in the coding region. Whether non-coding deep-intronic variants or large deletions in the IMPG2 gene account for the second pathogenic allele in this patient remains unknown.

Supposing that all patients in whom we could not confirm trans configuration of variants are indeed biallelic, our detection rate is 93%. This is in line with recent studies for LCA which achieved 80–90% in panel-based approaches [2-3] and 89% by whole exome/genome sequencing [4]. Analysis of our sequencing data revealed several regions with low or no coverage, as for instance for parts of exon 6 of CRB1. We would have missed several patients carrying the recurrent c.2843G>A/p.C948Y variant in this gene, had we not re-sequenced this exon in all patients with conventional Sanger sequencing.

Several studies have shown that whole exome sequencing (WES) and whole genome sequencing (WGS) can outperform targeted sequencing approaches in terms of variant detection [4, 25–27]. In fact, NHS England is already planning to commission WGS into routine clinical care pathways [28]. However, targeted sequencing approaches have several benefits, including a higher coverage rate for targeted regions and higher throughput in terms of patient numbers. What is more important, they are associated with considerable lower costs, which is relevant for those patients who cannot expect reimbursement from their health care provider or have no health insurance at all. The MIP technology we used can be as low as € 80 per sample per gene panel, which is 10 to 20 times lower than the price tag for other NGS-based sequencing procedures. Reaching a detection rate of 93%, we could demonstrate that MIPs are a cost-efficient and sensitive tool for targeted next-generation sequencing in IRD.

List of genes included in the MIP panel.

Genes associated with LCA according to RetNet (https://sph.uth.edu/Retnet/) are shown in bold.

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