A Description of the Yield of Genetic Reinvestigation in Patients with Inherited Retinal Dystrophies and Previous Inconclusive Genetic Testing

Abstract

In the present era of evolving gene-based therapies for inherited retinal dystrophies (IRDs), it has become increasingly important to verify the genotype in every case, to identify all subjects eligible for treatment. Moreover, combined insight concerning phenotypes and genotypes is crucial for improved understanding of thevisual impairment, prognosis, and inheritance. The objective of this study was to investigate to what extent renewed comprehensive genetic testing of patients diagnosed with IRD but with previously inconclusive DNA test results can verify the genotype, if confirmation of the genotype has an impact on the understanding of the clinical picture, and, to describe the genetic spectrum encountered in a Swedish IRD cohort. The study included 279 patients from the retinitis pigmentosa research registry (comprising diagnosis within the whole IRD spectrum), hosted at the Department of Ophthalmology, Skåne University hospital, Sweden. The phenotypes had already been evaluated with electrophysiology and other clinical tests, e.g., visual acuity, Goldmann perimetry, and fundus imaging at the first visit, sometime between 1988–2015 and the previous—in many cases, multiple—genetic testing, performed between 1995 and 2020 had been inconclusive. All patients were aged 0–25 years at the time of their first visit. Renewed genetic testing was performed using a next generation sequencing (NGS) IRD panel including 322 genes (Blueprint Genetics). Class 5 and 4 variants, according to ACMG guidelines, were considered pathogenic. Of the 279 samples tested, a confirmed genotype was determined in 182 (65%). The cohort was genetically heterogenous, including 65 different genes. The most prevailing were ABCA4 (16.5%), RPGR (6%), CEP290 (6%), and RS1 (5.5%). Other prevalent genes were CACNA1F (3%), PROM1 (3%), CHM (3%), and NYX (3%). In 7% of the patients there was a discrepancy between the diagnosis made based on phenotypical or genotypical findings alone. To conclude, repeated DNA-analysis was beneficial also in previously tested patients and improved our ability to verify the genotype–phenotype association increasing the understanding of how visual impairment manifests, prognosis, and the inheritance pattern. Moreover, repeated testing using a widely available method could identify additional patients eligible for future gene-based therapies.

1. Introduction

Inherited retinal dystrophies (IRDs) are one of the most common causes of serious visual impairment in children and young adults in developed countries [1,2]. Until quite recently, IRDs have been untreatable, but during the last decades, extensive research concerning gene-based therapies [3,4,5,6,7] has evolved and the first gene augmentation therapy, Voretigene Neparvovec for treatment of RPE65-associated retinal dystrophies [8,9], was approved in USA in 2017 and in Europe 2018. Since the novel therapies such as gene augmentation/replacement, gene silencing, antisense oligonucleotides (AONs), and gene editing using the CRISPR/Cas9 system [3,5,6,7] are all based on correcting the specific genetic defect; verification of the genotype is essential nowadays. Moreover, there is a complicated overlap of genotypes and phenotypes in the sense that the same pathogenic genetic variant can cause several different clinical manifestations, e.g., either retinitis pigmentosa (RP), first engaging rods and, after some time, also cones, or Lebers congenital amaurosis (LCA) with early-onset rod and cone engagement, but also cone–rod dystrophy (CRD) with the cones affected primarily and rod secondarily [10,11,12]. Similarly, one phenotype, RP, can be caused by mutations in many different genes, (with over 60 currently known [13]). Concerning the whole spectrum of IRDs, over 300 causative genes [14] are known presently and they can be linked with over 50 separate phenotypes [13]. In this setting, careful mapping of the genetic cause of IRDs has become more important and lately, our ability to assess genotypes has improved significantly. Over the years, the procedure for DNA-analysis has evolved from single gene testing with the first gene associated with X-linked RP described in 1984 [15,16], via the APEX technique, to NGS panels and whole exome as well as whole genome sequencing (WES and WGS) [17]. Although modern procedures such as NGS panels, WES, and WGS are used, the diagnostic yield is not complete but ranges between 50–75% [14]. Thus, to optimize our ability to make the accurate diagnosis in each patient and thereby enable better understanding of the type of visual impairment, prognosis, and inheritance patterns, we must combine thorough clinical assessments and genetic testing. And, when it comes to finding patients eligible for gene-based therapies, genotyping is crucial, both the approved one and for therapies in clinical trials [3,4,6,7,18]. At the Department of Ophthalmology of Skåne University Hospital, we have, since the mid-1990s, had the ambition to verify the genotype in all patients, but that has not yet been fully possible. In this study, we wanted to investigate to what extent renewed comprehensive genetic testing with a widely available, broad NGS panel for IRDs, could verify the genotype in patients where previous genetic testing had been inconclusive and if confirmation of the genotype has an impact on the understanding of the clinical picture. Moreover, we aimed to describe the spectrum of genes encountered in a Swedish cohort of IRD patients.

2. Materials and Methods

2.1. Subjects

The study included 279 patients, with inconclusive previous DNA test results, from the retinitis pigmentosa research registry hosted at the Department of Ophthalmology, Skåne University Hospital, Lund, Sweden. Despite the name, the registry includes subjects with the whole spectrum of IRDs. The patients had made their first visit to the department between 1988 and 2015 and the initial appointment included a thorough clinical examination that mapped the phenotype carefully. Among the most prevalent diagnoses (based on the phenotype) were RP (94 subjects), CRD (38 subjects), Stargardt diseases (STGD) (24 subjects), X-linked juvenile retinoschisis (XLRS) (22 subjects), LCA (14 subjects), cone dystrophy (CD) (12 subjects), congenital stationary night blindness (CSNB) (11 subjects), macular dystrophy (11 subjects), and Usher syndrome (9 subjects). Previous DNA analyses were performed between 1995 and 2020 in cooperation with several collaborators, using both research laboratories and commercial facilities. Over time, the available techniques have developed from single gene tests and APEX panels to NGS panels and WES. Of the subjects, 122 had been tested with single-gene analysis, often including a range of genes on several occasions and in many different laboratories, while 157 of the patients that were investigated more recently had been tested with APEX—or NGS panels. A few cases with unsolved genotypes had also been tested with WES in addition to any of the other methods. In many cases, several DNA tests have been carried out over time. In this study, the term, inconclusive test results, means that either no pathogenic variant at all had been identified with previous tests or that only one pathogenic variant had been detected in a gene that is known to cause autosomal recessive disease. The study included 117 females and 162 males. They were all between 0 and 25 years of age at the time of their first visit (median 10 and mean 11 with standard deviation 6). Patients from widely distributed parts of Sweden are represented in the cohort, in which 60% had been referred from areas outside the department’s own region, Skåne. Hence, these results provide information about the genetic characteristics of Swedish IRD patients on a national level rather than on a regional level. The study was conducted in accordance with the Tenets of the Declaration of Helsinki and it was approved by the Ethical Committee for Medical Research at Lund University (nr 2015/602). All subjects gave their informed consent concerning the study including the DNA analysis.

2.2. Genetic Analysis

In 2021, DNA samples from all 279 patients were sent for renewed genetic testing with an NGS IRD panel including 322 genes at Blueprint Genetics, a College of American Pathologists- and Clinical Laboratory Improvement Amendments-certified laboratory. Investigated genes are listed in

Table 1. Listing the genes that were investigated in the NGS retinal dystrophy panel.

ABCA4 ABCC6 ABHD12 ACO2 ADAM9 ADAMTS18 ADGRV1 ADIPOR1 AGBL5 AHI1 AIPL1 ALMS1 ARHGEF18 ARL13B ARL2BP ARL3 ARL6 ARMC9 ARSG ATF6 ATOH7 B9D1 B9D2 BBIP1 BBS1 BBS10 BBS12 BBS2 BBS4 BBS5 BBS7 BBS9 BEST1 C1QTNF5 C21ORF2 C2ORF71 C5ORF42 C8ORF37 CA4 CABP4

CACNA1F CACNA2D4 CAPN5 CC2D2A CDH23 CDH3 CDHR1 CEP104 CEP120 CEP164 CEP19 CEP250 CEP290 CEP41 CEP78 CERK CHM CIB2 CISD2 CLN3 CLRN1 CNGA1 CNGA3 CNGB1 CNGB3 CNNM4 COL11A1 COL11A2 COL18A1 COL2A1 COL9A1 COL9A2 COL9A3 CPE CRB1 CRX CSPP1 CTC1 CTNNA1 CTNNB1

CWC27 CYP4V2 DFNB31 DHDDS DHX38 DRAM2 DTHD1 EFEMP1 ELOVL4 EMC1 ESPN EYS FAM161A FDXR FLVCR1 FRMD7 FZD4 GNAT1 GNAT2 GNB3 GNPTG GPR179 GRK1 GRM6 GUCA1A GUCY2D HAR HGSNAT HK1 HMX IDH3A IDH3B IFT140 IFT172 IFT27 IFT81 IMPDH1 IMPG1 IMPG2 INPP5E

INVS IQCB1 JAG1 KCNJ13 KCNV2 KIAA0556 KIAA0586 KIAA0753 KIAA154 KIF11 KIF7 KIZ KLHL7 LCA5 LRAT LRIT3 LRP2 LRP5 LZTFL1 MAK MERTK MFN2 MFRP MFSD8 MKKS MKS1 MMACHC MT-ATP6 MT-ATP8 MT-CO1 MT-CO2 MT-CO3 MT-CYB MT-ND1 MT-ND2 MT-ND3 MT-ND4 MT-ND4L MT-ND5 MT-ND6

MT-RNR1 MT-RNR2 MT-TA MT-TC MT-TD MT-TE MT-TF MT-TG MT-TH MT-TI MT-TK MT-TL1 MT-TL2 MT-TM MT-TN MT-TP MT-TQ MT-TR MT-TS1 MT-TS2 MT-TT MT-TV MT-TW MT-TY MTTP MVK MYO7A NDP NEK2 NMNAT1 NPHP1 NPHP3 NPHP4 NR2E3 NR2F1 NRL NYX OAT OFD1 OPA1

OPA3 OTX2 P3H2 PANK2 PAX2 PCDH15 PCYT1A PDE6A PDE6B PDE6C PDE6D PDE6G PDE6H PDZD7 PEX1 PEX10 PEX11B PEX12 PEX13 PEX14 PEX16 PEX19 PEX2 PEX26 PEX3 PEX5 PEX6 PEX7 PHYH PISD PITPNM3 PLA2G5 PNPLA6 POC1B POMGNT1 PRCD PRDM13 PROM1 PRPF3 PRPF31

PRPF4 PRPF6 PRPF8 PRPH2 PRPS1 RAB28 RAX2 RBP3 RBP4 RCBTB1 RD3 RDH11 RDH12 RDH5 REEP6 RGR RGS9 RGS9BP RHO RIMS1 RLBP1 ROM1 RP1 RP1L1 RP2 RPE65 RPGR RPGRIP1 RPGRIP1L RS1 RTN4IP1 SAG SAMD11 SCAPER SCLT1 SDCCAG8 SEMA4A SLC24A1 SLC25A46 SLC7A14 SNRNP200

SPATA7 SPP2 SRD5A3 TCTN1 TCTN2 TCTN3 TEAD1 TIMM8A TIMP3 TMEM107 TMEM126A TMEM138 TMEM216 TMEM231 TMEM237 TMEM67 TOPORS TRAF3IP1 TREX1 TRIM32 TRPM1 TSPAN12 TTC21B TTC8 TTLL5 TTPA TUB TUBB4B TULP1 USH1C USH1G USH2A VCAN VPS13B WDPCP WDR19 WFS1 YME1L1 ZNF408 ZNF423 ZNF513

. Class 5 and 4 variants according to ACMG guidelines were considered pathogenic. In a few cases (

Table 2. Showing demographic data as well as genotype and phenotype for the patients with conclusive genetic re-testing.

Patient	Gender	Age at First Examination	Genotype	Description of Pathogenic Variants	Phenotype at Initial Examination
RP145LU	K	12	ABC4A	heterozygous for the missense variant, ABCA4 c.2915C > A, p.(Thr972Asn), which is pathogenic and heterozygous for the frameshift variant, ABCA4 c.4042del, p.(Thr1348Hisfs*41), which is likely pathogenic.	STGD
RP146LU	M	8	ABC4A	heterozygous for ABCA4 c.2915C > A, p.(Thr972Asn), which is pathogenic and heterozygous for ABCA4 c.4042del, p.(Thr1348Hisfs*41), which is likely pathogenic	STGD
RP173LU	K	14	ABCA4	heterozygous for ABCA4 c.2894A > G, p.(Asn965Ser), which is pathogenic heterozygous for ABCA4 c.768G > T, p.(Val256=), which is pathogenic.	CD
RP125LU	M	4	ABCA4	heterozygous for ABCA4 c.2588G > C, p.(Gly863Ala) classified as pathogenic and heterozygous for ABCA4 c.5603A > T, p.(Asn1868Ile) classified as a risk factor	CRD
RP135LU	M	15	ABCA4	homozygous for a deletion, ABCA4 c.2918 + 11_3522 + 86del which encompasses exons 20–23 classified as pathogenic	CRD
RP209LU	K	8	ABCA4	homozygous for ABCA4 c.768G > T, p.(Val256=), which is pathogenic	CRD
RP253LU	K	10	ABCA4	homozygous for ABCA4 c.319C > T, p.(Arg107*), which is pathogenic	CRD
RP5LU	K	7	ABCA4	homozygous for ABCA4 c.768G > T, p.(Val256=) which is pathogenic	CRD
RP161LU	K	20	ABCA4	heterozygous for ABCA4 c.4773 + 3A > G, which is pathogenic and heterozygous for ABCA4 c.768G > T, p.(Val256=), which is pathogenic	STGD
RP162LU	M	10	ABCA4	heterozygous for ABCA4 c.6286G > A, p.(Glu2096Lys), which is pathogenic and heterozygous for ABCA4 c.5461–10T > C, which is pathogenic and for ABCA4 c.5603A > T, p.(Asn1868Ile), which is risk factor	STGD
RP170LU	K	14	ABCA4	heterozygous for ABCA4 c.4139C > T, p.(Pro1380Leu), which is pathogenic. heterozygous for ABCA4 c.2894A > G, p.(Asn965Ser), which is pathogenic. heterozygous for ABCA4 c.5603A > T, p.(Asn1868Ile), which is a risk factor	STGD
RP171LU	K	15	ABCA4	heterozygous for ABCA4 c.6181_6184del, p.(Thr2061Serfs*53), which is pathogenic, heterozygous for ABCA4 c.3322C > T, p.(Arg1108Cys), which is pathogenic and heterozygous for ABCA4 c.5603A > T, p.(Asn1868Ile), which is a risk factor	STGD
RP18LU	K	12	ABCA4	heterozygous for ABCA4 c.2894A > G, p.(Asn965Ser), which is pathogenic and heterozygous for ABCA4 c.319C > T, p.(Arg107*), which is pathogenic	STGD
RP200LU	K	17	ABCA4	heterozygous for ABCA4 c.3322C > T, p.(Arg1108Cys), which is pathogenic and heterozygous for ABCA4 c.768G > T, p.(Val256=), which is pathogenic	STGD
RP206LU	K	18	ABCA4	heterozygous for ABCA4 c.4601del, p.(Leu1534Trpfs*2), which is pathogenic, heterozygous for ABCA4 c.2588G > C, p.(Gly863Ala), which is pathogenic and heterozygous for ABCA4 c.5603A > T, p.(Asn1868Ile), which is risk factor	STGD
RP215LU	K	19	ABCA4	heterozygous for ABCA4 c.5461-10T > C, which is pathogenic, heterozygous for ABCA4 c.2894A > G, p.(Asn965Ser), which is pathogenic and heterozygous for ABCA4 c.5603A > T, p.(Asn1868Ile), which is a risk factor	STGD
RP224LU	M	11	ABCA4	heterozygous for ABCA4 c.4139C > T, p.(Pro1380Leu), which is pathogenic heterozygous for ABCA4 c.2599del, p.(Thr867Profs*34), which is likely pathogenic.	STGD
RP242LU	K	13	ABCA4	heterozygous for ABCA4 c.2894A > G, p.(Asn965Ser), which is pathogenic and heterozygous for ABCA4 c.1610G > A, p.(Arg537His), which is likely pathogenic	STGD
RP261LU	M	10	ABCA4	homozygous for ABCA4 c.5584G > C, p.(Gly1862Arg), which is likely pathogenic	STGD
RP287LU	K	19	ABCA4	heterozygous for ABCA4 c.6088C > T, p.(Arg2030*), which is pathogenic and heterozygous for ABCA4 c.5882G > A, p.(Gly1961Glu), which is pathogenic	STGD
RP94LU	M	11	ABCA4	homozygous for ABCA4 c.868C > T, p.(Arg290Trp), which is pathogenic	STGD
RP48LU	K	25	ABCA4	heterozygous for ABCA4 c.3322C > T, p.(Arg1108Cys), which is pathogenic and heterozygous for ABCA4 c.2894A > G, p.(Asn965Ser), which is pathogenic	STGD
RP85LU	K	25	ABCA4	homozygous for ABCA4 c.5882G > A, p.(Gly1961Glu), which is pathogenic	STGD
RP191LU	K	16	ABCA4	heterozygous for ABCA4 c.1804C > T, p.(Arg602Trp), which is pathogenic, heterozygous for ABCA4 c.3113C > T, p.(Ala1038Val), which is pathogenic, heterozygous for ABCA4 c.1622T > C, p.(Leu541Pro), which is pathogenic and heterozygous for ABCA4 c.5603A > T, p.(Asn1868Ile), which is a risk factor	CD
RP21LU	K	18	ABCA4	homozygous ABCA4c.5882G > A, p.(Gly1961Glu pathogenic. homozygous for ABCA4 c.634C > T, p.(Arg212Cys), which is pathogenic	CD
RP22LU	K	10	ABCA4	heterozygous for ABCA4 c.4773 + 1G > A, which is pathogenic and heterozygous for ABCA4 c.53G > A, p.(Arg18Gln), which is pathogenic	CRD
RP172LU	K	8	ABCA4	homozygous for ABCA4 c.3113C > T, p.(Ala1038Val), which is pathogenic and homozygous for ABCA4 c.1622T > C, p.(Leu541Pro), which is pathogenic	CRD
RP20LU	K	14	ABCA4	heterozygous for ABCA4 c.5413A > G, p.(Asn1805Asp), which is pathogenic and heterozygous for ABCA4 c.6159G > A, p.(Trp2053*), which is likely pathogenic	STGD
RP34LU	M	18	ABCA4	heterozygous for ABCA4 c.5461-10T > C, which is pathogenic, heterozygous for ABCA4 c.5196 + 1137G > A, which is pathogenic and heterozygous for ABCA4 c.5603A > T, p.(Asn1868Ile), which is risk factor	STGD
RP41LU	M	15	ABCA4	heterozygous for ABCA4 c.6079C > T, p.(Leu2027Phe), which is pathogenic and heterozygous for ABCA4 c.4139C > T, p.(Pro1380Leu), which is pathogenic	STGD
RP273LU	M	1	AIPL1	heterozygous for AIPL1 c.834G > A, p.(Trp278*), which is pathogenic and heterozygous for AIPL1 c.537del, p.(Val180Serfs*29), which is likely pathogenic	LCA
RP181LU	K	18	BBS1	homozygous for BBS1 c.1169T > G, p.(Met390Arg), which is pathogenic	RP
RP12LU	M	3	BBS10	homozygous BBS10 c.271dup, p.(Cys91Leufs*5), which is pathogenic	Bardet–Biedl
RP154LU	K	13	BBS10	homozygous for BBS10 c.1244del, p.(His415Leufs*16), which is pathogenic	Bardet–Biedl
RP155LU	K	14	BBS10	homozygous for BBS10 c.271dup, p.(Cys91Leufs*5), which is pathogenic	Bardet–Biedl
RP190LU	K	8	BBS10	homozygous for BBS10 c.271dup, p.(Cys91Leufs*5), which is pathogenic	Bardet–Biedl
RP236LU	M	12	BBS5	homozygous for BBS5 c.790G > A, p.(Gly264Arg), which is pathogenic	achromatopsia
RP30LU	K	8	BBS5	homozygous for BBS5 c.790G > A, p.(Gly264Arg), which is pathogenic	achromatopsia
RP76LU	M	16	BBS9	homozygous for BBS9 c.1561C > T, p.(Arg521*), which is pathogenic	Bardet–Biedl
RP1LU	M	10	CACNA1F	hemizygous for CACNA1F c.4156C > T, p.(Gln1386*) which is likely pathogenic	CHM
RP205LU	M	6	CACNA1F	hemizygous for CACNA1F c.3895C > T, p.(Arg1299*), which is pathogenic	CSNB
RP195LU	M	7	CACNA1F	hemizygous for CACNA1F c.4134–1G > C, which is pathogenic	XLRS
RP23LU	M	8	CACNA1F	hemizygous for CACNA1F c.3542_3548del, p.(Tyr1181Cysfs*5), which is likely pathogenic	XLRS
RP166LU	M	8	CACNA1F	hemizygous for CACNA1F c.952_954del, p.(Phe318del), which is pathogenic	XLRS
RP50LU	M	2	CACNA1F	hemizygous for CACNA1F c.2071C > T, p.(Arg691*), which is pathogenic	XLRS
RP65LU	M	19	CACNA2D4	homozygous for CACNA2D4 c.1564C > T, p.(Arg522*), which is likely pathogenic	CD
RP123LU	K	6	CDH23	homozygous for CDH2 c.8733del, p.(Asp2911Glufs*41), which is likely pathogenic	Usher
RP108LU	M	12	CDH3	heterozygous for CDH3 c.1795 + 1G > A, which is likely pathogenic and heterozygous for CDH3 c.1643C > G, p.(Pro548Arg), which is a VUS; however, this variant is absent in control populations and predicted to be deleterious via in silico tools and NGS data strongly suggest that these variants are in trans, thus interpreted as causative	macular dystrophy
RP221LU	K	19	CDHR1	heterozygous for CDHR1 c.783G > A, p.(Pro261=), which is pathogenic and heterozygous for CDHR1 c.2522_2528del, p.(Ile841Serfs*119), which is pathogenic	RP
RP106LU	M	1	CEP290	heterozygous for CEP290 c.4661_4663del, p.(Glu1554del), which is pathogenic and heterozygous for CEP290 c.2052 + 1_2052 + 2del, which is pathogenic	LCA
RP116LU	K	3	CEP290	heterozygous for CEP290 c.4661_4663del, p.(Glu1554del), pathogenic. heterozygous for CEP290 c.955del, p.(Ser319Leufs*16), likely pathogenic	LCA
RP137LU	K	1	CEP290	heterozygous for CEP290 c.2991 + 1655A > G, which is pathogenic and heterozygous for CEP290 c.1992del, p.(Pro665Leufs*10), which is pathogenic	LCA
RP150LU	M	1	CEP290	homozygous for CEP290 c.2991 + 1655A > G, which is pathogenic	LCA
RP156LU	K	1	CEP290	heterozygous for CEP290 c.2991 + 1655A > G, which is pathogenic and heterozygous for CEP290 c.384_387del, p.(Asp128Glufs*34), which is pathogenic	LCA
RP157LU	K	0	CEP290	heterozygous for CEP290 c.2991 + 1655A > G, which is pathogenic. heterozygous for CEP290 c.170C > A, p.(Ser57*), which is likely pathogenic	LCA
RP249LU	K	7	CEP290	heterozygous for CEP290 c.3249dup, p.(Arg1084Thrfs*11), which is pathogenic and heterozygous for CEP290 c.1065 + 1G > A, which is likely pathogenic	LCA
RP294LU	M	19	CEP290	heterozygous for CEP290 c.2991 + 1655A > G, which is pathogenic and heterozygous for CEP290 c.384_387del, p.(Asp128Glufs*34), which is pathogenic	LCA
RP66LU	K	0	CEP290	heterozygous for CEP290 c.2991 + 1655A > G, which is pathogenic and heterozygous for CEP290 c.1681C > T, p.(Gln561*), which is likely pathogenic.	LCA
RP82LU	M	1	CEP290	heterozygous for CEP290 c.2991 + 1655A > G, which is pathogenic and heterozygous for CEP290 c.1992del, p.(Pro665Leufs*10), which is pathogenic	LCA
RP262LU	M	5	CEP290	heterozygous for CEP290 c.4438–3del, which is pathogenic and heterozygous for CEP290 c.164_167del, p.(Thr55Serfs*3), which is pathogenic	RP
RP258LU	K	5	CFAP410	homozygous for CFAP410 c.33_34insAGCTGCACAGCGTGCA, p.(Ala12Serfs*60), which is pathogenic	CD
RP59LU	K	5	CFAP410	homozygous for CFAP410 c.218G > C, p.(Arg73Pro) which is pathogenic	CD
RP64LU	K	11	CFAP410	homozygous for deletion CFAP410 c.(?_-1)_(*1_?)del, which is pathogenic	CD
RP80LU	K	14	CFAP410	homozygous for a deletion CFAP410 c.(?_-1)_(*1_?)del, which encompasses the whole CFAP410 gene and is pathogenic	CD
RP256LU	K	10	CFAP410	homozygous for CFAP410 c.218G > C, p.(Arg73Pro), which is pathogenic	RP
RP288LU	M	24	CHM	hemizygous for CHM c.1244 + 1G > C, which is likely pathogenic	CHM
RP49LU	M	16	CHM	hemizygous for CHM c.1144G > T, p.(Glu382*), which is pathogenic	CHM
RP257LU	M	18	CHM	hemizygous for a deletion CHM c.(314 + 1_315 − 1)_(1166 + 1_1167 − 1)del, which encompasses exons 5–8 of CHM, classified as pathogenic	RP
RP264LU	M	10	CHM	hemizygous for a 6 Mb deletion, seq[GRCh37] del(X)(q21.1q21.2), chrX:g.79270061–85302755del, encompassing the entire panel gene CHM and classified as pathogenic.	RP
RP129LU	K	8	CHM	heterozygous for CHM c.1411del, p.(Gln471Argfs*5), which is likely pathogenic	CHM carrier
RP42LU	K	13	CHM	heterozygous for CHM c.1144G > T, p.(Glu382*), which is pathogenic	CHM carrier
RP227LU	K	7	CLN3	homozygous for deletion CLN3 c.(460 + 1_461 − 1)_(677 + 1_678 − 1)del, which encompasses exons 8–9 of CLN3 and is classified as pathogenic	CLN3
RP234LU	M	7	CLN3	homozygous for deletion CLN3 c.(460 + 1_461 − 1)_(677 + 1_678 − 1)del, which encompasses exons 8–9 of CLN3 and is classified as pathogenic	CLN3
RP220LU	M	6	CLN3	homozygous for a deletion CLN3 c.(460 + 1_461 − 1)_(677 + 1_678 − 1)del, which encompasses exons 8–9 of CLN3 and is classified as pathogenic	RP
RP68LU	M	18	CNGB1	heterozygous for CNGB1 c.2957A > T, p.(Asn986Ile), which is pathogenic and heterozygous for CNGB1 c.2293C > T, p.(Arg765Cys), which is likely pathogenic	RP
RP26LU	M	10	CNGB3	heterozygous for CNGB3 c.1285dup, p.(Ser429Phefs*33), which is pathogenic and heterozygous for CNGB3 c.819_826del, p.(Arg274Valfs*13), which is pathogenic	achromatopsia
RP27LU	M	1	CNGB3	heterozygous for CNGB3 c.1148del, p.(Thr383Ilefs*13), which is pathogenic and heterozygous for CNGB3 c.1643G > T, p.(Gly548Val), which is VUS, however, CNGB3 c.1643G > T, p.(Gly548Val) is absent in control populations and predicted to be deleterious by insilico tools and thus compound heterozygosity of the variants would explain the phenotype	achromatopsia
RP15LU	K	6	COL18A1	homozygous for COL18A1 c.2157 + 2T > C, which is likely pathogenic	Knobloch syndrome
RP132LU	M	2	COL18A1	homozygous for COL18A1 c.3514_3515del, p.(Leu1172Valfs*72), which is pathogenic	LCA
RP244LU	K	12	COL18A1	homozygous for COL18A1 c.874del, p.(Glu292Lysfs*17), which is likely pathogenic	macular dystrophy
RP207LU	M	10	COL18A1	heterozygous for COL18A1 c.3666_3682del, p.(Ala1223Glnfs*19), which is likely pathogenic and heterozygous for COL18A1 c.3809 + 2T > C, which is likely pathogenic	vitreoretinal dystrophy
RP140LU	M	13	CRX	heterozygous for CRX c.413del, p.(Ile138Thrfs*49), which is pathogenic	CRD
RP92LU	K	5	CRX	heterozygous for CRX c.413del, p.(Ile138Thrfs*49), which is pathogenic	CRD
RP117LU	M	4	CRX	heterozygous frameshift variant CRX c.413del, p.(Ile138Thrfs*49) which is pathogenic	RP
RP114LU	K	1	GUCA1A	heterozygous for GUCA1A c.332A > T, p.(Glu111Val), which is likely pathogenic	CRD
RP134LU	M	13	GUCY2D	heterozygous for GUCY2D c.2377del, p.(Glu793Asnfs*42), which is likely pathogenic and heterozygous for GUCY2D c.1567-17T > A, which is a VUS, however, these GUCY2D variants are consistent with the patient’s phenotype, and GUCY2D c.1567-17T > A is rare in control populations and predicted to affect splicing by in silico tools, thus compound heterozygosity of the variants could explain the phenotype	CRD
RP148LU	M	3	GUCY2D	heterozygous for GUCY2D c.2944 + 1del, which is pathogenic and heterozygous for GUCY2D c.2965G > C, p.(Val989Leu), which is a VUS, however, these GUCY2D variants are consistent with the patient’s phenotype, and GUCY2D c.2965G > C, p.(Val989Leu) is absent in control populations and predicted to be deleterious by in silico tools, NGS data suggests that these variants are in trans in thispatient, which could explain the patient’s clinical presentation	CRD
RP176LU	K	18	GUCY2D	heterozygous for GUCY2D c.2944 + 1del, which is pathogenic. heterozygous for GUCY2D c.1982G > T, p.(Gly661Val), which is a VUS, however, these GUCY2D variants are consistent with the patient’s phenotype, and GUCY2D c.1982G > T, p.(Gly661Val) is absent in control populations and predicted to be deleterious by in silico tools, compound heterozygosity of the variants would explain the patient’s clinical presentation	CRD
RP24LU	M	6	GUCY2D	heterozygous for GUCY2D c.2302C > T, p.(Arg768Trp), which is pathogenic and heterozygous for GUCY2D c.1567-17T > A, which is a VUS, however, these GUCY2D variants are consistent with the patient’s phenotype, and GUCY2D c.1567-17T > A is rare in control populations and predicted to affect splicing by in silico tools, compound heterozygosity of the variants could explain the patient’s clinical presentation	CRD
RP217LU	M	16	IMPDH1	heterozygous for IMPDH1 c.931G > A, p.(Asp311Asn), which is pathogenic	RP
RP40LU	K	2	IQCB1	heterozygous for IQCB1 c.1332G > A, p.(Trp444*), which is pathogenic and heterozygous for IQCB1 c.424_425del, p.(Phe142Profs*5), which is pathogenic	Senior–Loken
RP104LU	M	16	KCNV2	homozygous for the nonsense variant KCNV2 c.427G > T, p.(Glu143*), which is pathogenic	CRD
RP112LU	K	13	KCNV2	homozygous for KCNV2 c.427G > T, p.(Glu143*), which is pathogenic	CRD
RP98LU	M	11	KCNV2	homozygous for KCNV2 c.427G > T, p.(Glu143*), which is pathogenic	CRD
RP43LU	K	1	KIF11	heterozygous for KIF11 c.1985T > A, p.(Leu662*), which is pathogenic	microcephaly and RD
RP78LU	K	20	KLHL7	heterozygous for KLHL7 c.422T > C, p.(Val141Ala) which is likely pathogenic	RP
RP91LU	K	21	LRAT	homozygous for LRAT c.470T > C, p.(Leu157Pro), which is likely pathogenic	EORD
RP266LU	M	17	LRAT	homozygous for LRAT c.470T > C, p.(Leu157Pro), which is likely pathogenic	RP
RP121LU	M	9	MERTK	homozygous for MERTK c.2302G > A, p.(Ala768Thr), which is pathogenic	RP
RP290LU	M	13	MERTK	homozygous for MERTK c.1960 + 1G > A, which is likely pathogenic	RP
RP71LU	M	12	MERTK	heterozygous for MERTK c.345C > G, p.(Cys115Trp), which is pathogenic and heterozygous for MERTK c.1377_1379delinsAGCC, p.(Arg460Alafs*15), which is likely pathogenic	RP
RP238LU	M	19	MFN2	heterozygous for deletion MFN2 c.(474 + 1_475 − 1)_(816 + 1_817 − 1)del, which encompasses exons 6–8 of MFN2. This alteration is classified as likely pathogenic	macular dystrophy
RP4LU	K	17	MFRP	homozygous for MFRP c.1090_1091del, p.(Thr364Glnfs*27), which is pathogenic	RP
RP203LU	M	4	MYO7A	heterozygous for MYO7A c.1556G > A, p.(Gly519Asp), which is pathogenic and heterozygous for MYO7A c.3719G > A, p.(Arg1240Gln), which is pathogenic	Usher
RP300LU	M	2	MYO7A	heterozygous for MYO7A c.401T > A, p.(Ile134Asn), which is pathogenic. heterozygous for MYO7A c.6558 + 1G > T, which is likely pathogenic	Usher
RP115LU	K	1	NMNAT1	heterozygous for NMNAT1 c.196C > T, p.(Arg66Trp) and heterozygous for NMNAT1 c.769G > A, p.(Glu257Lys), which are both pathogenic	LCA
RP90LU	K	9	NPHP1	homozygous for a deletion NPHP1 c.(?_-1)_(*1_?)del, which encompasses the whole NPHP1 gene, which is classified as pathogenic	RP and renal failure
RP194LU	K	11	NR2E3	heterozygous for NR2E3 c.119-2A > C and heterozygous for NR2E3 c.349 + 5G > C, which are both pathogenic	RP
RP216LU	M	5	NR2E3	heterozygous for NR2E3 c.119-2A > C, and NR2E3 c.932G > A, p.(Arg311Gln), which are both pathogenic	RP
RP136LU	M	6	NYX	hemizygous for NYX c.85_108del, p.(Arg29_Ala36del), which is pathogenic	CSNB
RP138LU	M	9	NYX	hemizygous for NYX c.559_560delinsAA, p.(Ala187Lys), which is likely pathogenic	CSNB
RP84LU	M	2	NYX	hemizygous for NYX c.559_560delinsAA, p.(Ala187Lys), which is likely pathogenic	CSNB
RP185LU	M	8	NYX	hemizygous for NYX c.559_560delinsAA, p.(Ala187Lys), which is likely pathogenic	CSNB
RP201LU	M	6	NYX	hemizygous for NYX c.559_560delinsAA, p.(Ala187Lys), which is likely pathogenic	CSNB
RP233LU	M	5	NYX	hemizygous for NYX c.559_560delinsAA, p.(Ala187Lys), which is likely pathogenic	CSNB
RP160LU	M	4	OPA1	heterozygous for OPA1 c.983A > G, p.(Lys328Arg), which is pathogenic	optic atrophy
RP184LU	M	8	OPA1	heterozygous for a deletion OPA1 c.(?_-1)_(*1_?)del, which encompasses the whole OPA1 gene	optic atrophy
RP286LU	K	24	OPA1	heterozygous for OPA1 c.2497-4_2557del, which is likely pathogenic	optic atrophy
RP107LU	M	2	OPA1	heterozygous for OPA1 c.703C > T, p.(Arg235*), which is pathogenic	RP
RP149LU	M	13	OTX2	heterozygous for OTX2 c.483dup, p.(Asp162Argfs*25), which is likely pathogenic	EORD
RP131LU	K	9	PANK2	heterozygous for PANK2 c.981 + 1G > C, which is likely pathogenic and heterozygous for PANK2 c.1512dup, p.(Ala505Serfs*7), which is likely pathogenic	RP and neurological symptoms
RP44LU	M	9	PCARE	homozygous for PCARE c.1541del, p.(Pro514Hisfs*27), which is pathogenic	RP
RP83LU	K	18	PCDH15	heterozygous for PCDH15 c.310del, p.(Asp104Ilefs*6) and PCDH15 c.3761dup, p.(Asn1254Lysfs*54), which are likely pathogenic	Usher
RP99LU	M	2	PCDH15	homozygous for PCDH15 c.3441dup, p.(Phe1148Ilefs*8), which is pathogenic	Usher
RP120LU	M	12	PDE6B	heterozygous for PDE6B c.1580T > C, p.(Leu527Pro) and PDE6B c.2193 + 1G > A which are both pathogenic	RP
RP235LU	M	2	PDE6C	heterozygous for PDE6C c.826C > T, p.(Arg276*) and PDE6C c.2457T > A, p.(Tyr819*), which are both likely pathogenic	CD
RP2LU	M	8	PNPLA6	heterozygous for PNPLA6 c.(2143 + 1_2144-1)_(2351 + 1_2352 − 1)del, which is likely pathogenic and heterozygous for PNPLA6 c.3625T > C, p.(Trp1209Arg), which is a VUS; however, these PNPLA6 variants are consistent with the patient’s phenotype, and PNPLA6 c.3625T > C, p.(Trp1209Arg) is rare in control populations and predicted to be deleterious by in silico tools, compound heterozygosity of the variants would explain the patient’s clinical presentation	RP
RP37LU	K	12	POC1B	heterozygous for POC1B c.1331_1332dup, p.(Thr445Argfs*10), which is pathogenic and heterozygous for POC1B c.52A > T, p.(Lys18*), which is likely pathogenic	achromatopsia
RP177LU	M	4	POMGNT1	homozygous for POMGNT1 c.1539 + 1G > A, which is pathogenic	Muscle–Eye–Brain Disease
RP67LU	K	10	PROM1	heterozygous for a deletion PROM1 c.(?-1)_(220 + 1_221 − 1)del, which encompasses exon 1 of PROM1 and is classified as likely pathogenic	CD
RP198LU	M	12	PROM1	heterozygous for PROM1 c.2050C > T, p.(Arg684*), which is pathogenic and heterozygous for PROM1 c.1632G > T, p.(Gly544=), which is pathogenic	CRD
RP29LU	M	18	PROM1	heterozygous for a deletion PROM1 c.(?-1)_(220 + 1_221 − 1)del, which encompasses exon 1 of PROM1 and is classified as likely pathogenic	RP
RP32LU	M	23	PROM1	heterozygous for a deletion PROM1 c.(?-1)_(220 + 1_221 − 1)del, which encompasses exon 1 of PROM1 and is likely pathogenic	RP
RP31LU	K	10	PROM1	homozygous for PROM1 c.1909C > T, p.(Gln637*), which is likely pathogenic	RP
RP47LU	K	18	PROM1	homozygous for PROM1 c.1909C > T, p.(Gln637*), which is likely pathogenic	RP
RP245LU	M	15	PRPF31	heterozygous for a deletion PRPF31 c.(?_-396)_(*1_?)del, which encompasses the whole PRPF31 gene and is classified as pathogenic	RP
RP270LU	K	22	PRPF31	heterozygous for a deletion PRPF31 c.(?_-396)_(*1_?)del, which encompasses the entire PRPF31 gene and is classified as pathogenic	RP
RP103LU	M	12	PRPF8	heterozygous for PRPF8 c.5804G > A, p.(Arg1935His), which is pathogenic	RP
RP188LU	K	20	PRPF8	heterozygous for PRPF8 c.6901C > T, p.(Pro2301Ser), which is pathogenic	RP
RP248LU	M	5	PRPF8	heterozygous for PRPF8 c.6926A > T, p.(His2309Leu), which is likely pathogenic	RP
RP179LU	M	14	PRPH2	heterozygous for PRPH2 c.633C > G, p.(Phe211Leu), which is pathogenic	RP
RP8LU	M	19	RDH12	homozygous for RDH12 c.481C > T, p.(Arg161Trp), which pathogenic	CRD
RP7LU	K	9	RDH5	homozygous for RDH5 c.382G > A, p.(Asp128Asn), which is pathogenic	Fundus albipunctatus
RP11LU	M	10	RHO	heterozygous for RHO c.541G > A, p.(Glu181Lys), which is pathogenic	Aaland eye disease
RP74LU	K	19	RLBP1	Homozygous for RLBP1 c.286_297del p.(Phe96_Phe99del), which is pathogenic	RP with maculopathy
RP292LU	K	8	RP1	heterozygous for RP1 c.1498_1499del, p.(Met500Valfs*7), which is pathogenic and heterozygous for RP1 c.1601_1604del, p.(Lys534Argfs*11), which is likely pathogenic	RP
RP189LU	M	13	RP1	heterozygous for RP1 c.271del, p.(Ser91Alafs*25), and RP1 c.753C > A, p.(Tyr251*), which are both likely pathogenic	RP
RP124LU	M	13	RP1L1	heterozygous for RP1L1 c.133C > T, p.(Arg45Trp), which is pathogenic	macular dystrophy
RP219LU	M	16	RP2	hemizygous for RP2 c.400C > T, p.(Gln134*), which is pathogenic	RP
RP86LU	K	1	RPE65	heterozygous for RPE65 c.886dup, p.(Arg296Lysfs*7), which is pathogenic and heterozygous for RPE65 c.612C > A, p.(Tyr204*), which is likely pathogenic	RP
RP16LU	K	16	RPGR	heterozygous for RPGR c.2641G > T, p.(Glu881*), which is likely pathogenic	RP
RP225LU	M	15	RPGR	hemizygous for RPGR c.764C > T, p.(Thr255Ile), which is likely pathogenic	RP
RP60LU	M	20	RPGR	hemizygous for RPGR c.2730_2731del, p.(Glu911Glyfs*167), which is pathogenic	RP
RP63LU	M	16	RPGR	hemizygous for RPGR c.2405_2406del, p.(Glu802Glyfs*32), which is pathogenic	RP
RP113LU	M	8	RPGR	hemizygous for RPGR c.2252_2255del, p.(Lys751Argfs*63), which is pathogenic	RP
RP192LU	M	10	RPGR	hemizygous for the deletion RPGR c.(1572 + 1_1573 − 1)_(*1_?)del, which encompasses exons 14–19 of RPGR and is classified as pathogenic	RP
RP199LU	M	12	RPGR	hemizygous for RPGR c.1573-8A > G, which is likely pathogenic	RP
RP222LU	M	15	RPGR	hemizygous for RPGR c.2426_2427del, p.(Glu809Glyfs*25), which is pathogenic	RP
RP251LU	M	20	RPGR	hemizygous for a deletion RPGR c.(1414 + 1_1415 − 1)_(*1_?)del, which encompasses exons 12–15 of RPGR and is classified as pathogenic	RP
RP100LU	M	8	RPGR	hemizygous for a deletion RPGR c.(778 + 1_779 − 1)_(1245 + 1_1246 − 1)del, which encompasses exons 8–10 of RPGR and is classified as pathogenic.	RP
RP52LU	M	16	RPGR	hemizygous for RPGR c.3300_3301del, p.(His1100Glnfs*10), which is pathogenic	macular dystrophy
RP167LU	M	9	RS1	hemizygous for RS1 c.416del, p.(Gln139Argfs*10) which is classified as pathogenic	XLRS
RP168LU	M	5	RS1	hemizygous for a deletion RS1 c.(?_-1)_(52 + 1_53 − 1)del, encompassing exon 1 of RS1, which is classified as pathogenic	XLRS
RP193LU	M	18	RS1	hemizygous for RS1 c.214G > A, p.(Glu72Lys), which is pathogenic	XLRS
RP223LU	M	10	RS1	hemizygous for deletion RS1 c.(?_-1)_(52 + 1_53 − 1)del, which encompasses exon 1 of RS1 and is pathogenic	XLRS
RP226LU	M	19	RS1	hemizygous for a deletion RS1 c.(?_-1)_(52 + 1_53 − 1)del, encompassing exon 1 of RS1, classified as pathogenic	XLRS
RP33LU	M	10	RS1	hemizygous for RS1 c.149G > A, p.(Trp50*), which is pathogenic	XLRS
RP35LU	M	10	RS1	hemizygous for RS1 c.366G > A, p.(Trp122*), which is pathogenic	XLRS
RP36LU	M	11	RS1	hemizygous for a deletion RS1 c.(?_-1)_(52 + 1_53 − 1)del, encompassing exon 1 of RS1, which is pathogenic	XLRS
RP79LU	M	19	RS1	hemizygous for a deletion RS1 c.(?_-1)_(52 + 1_53 − 1)del, encompassing exon 1 of RS1, which pathogenic	XLRS
RP95LU	M	6	RS1	hemizygous for a deletion RS1 c.(?_-1)_(52 + 1_53 − 1)del, encompassing exon 1 of RS1, which is pathogenic	XLRS
RP232LU	K	15	TIMM8A	heterozygous for TIMM8A c.116del, p.(Met39Argfs*26), which is pathogenic	carrier of Mohr–Tranebjaerg syndrome
RP126LU	K	1	TRPM1	heterozygous for the deletion TRPM1 c.(−64 + 1_−63 − 1)_(899 + 1_900 − 1)del, encompassing exons 2 (first coding exon) to 7, which is classified as pathogenic and heterozygous for TRPM1 c.3607_3608del, p.(Glu1203Asnfs*11), which is likely pathogenic	CSNB
RP13LU	K	7	TRPM1	homozygous for TRPM1 c.2629C > T, p.(Arg877*), which is pathogenic	CSNB
RP243LU	M	8	TRPM1	homozygous for TRPM1 c.2629C > T, p.(Arg877*), which is pathogenic	CSNB
RP169LU	M	3	TULP1	homozygous for TULP1 c.148del, p.(Glu50Asnfs*59), which is pathogenic	LCA
RP75LU	K	11	TULP1	homozygous for TULP1 c.1153G > A, p.(Gly385Arg), which is pathogenic	RP
RP61LU	K	7	USH1C	heterozygous for USH1C c.496 + 1G > T, and USH1C c.238dup, p.(Arg80Profs*69), which are pathogenic	Usher
RP197LU	M	8	USH2A	heterozygous for USH2A c.10450C > T, p.(Arg3484*), and USH2A c.779T > G, p.(Leu260*), whichare pathogenic	Usher
RP291LU	M	1	USH2A	heterozygous for USH2A c.8682-9A > G, which is pathogenic and heterozygous for USH2A c.1070_1071del, p.(Asn357Serfs*9), which is likely pathogenic	Usher
RP159LU	K	13	WFS1	heterozygous for WFS1 c.1673G > A, p.(Arg558His), which is pathogenic and heterozygous for WFS1 c.2149G > A, p.(Glu717Lys), which is pathogenic	optic atrophy
RP9LU	K	17	WFS1	heterozygous for WFS1 c.1673G > A, p.(Arg558His), and WFS1 c.2149G > A, p.(Glu717Lys), which are pathogenic	optic atrophy

), a class 3 variant was upgraded to a class 4 by the geneticists at Blueprint Genetics. The analysis also included assessment copy number variations (CNVs) as well as evaluation of the maternally inherited mitochondrial genome. In addition to the coding regions, the panel targeted 20 base pairs at the intron/exon boundaries and noncoding variants previously reported as disease-causing in association with IRD.

Bioinformatics and quality control were performed as follows. Base called raw sequencing data was transformed into FASTQ format using Illumina’s software (bcl2fastq) v2.20. Sequence reads of each sample were mapped to the human reference genome (GRCh37/hg19). Burrows–Wheeler Aligner (BWA-MEM) software was used for read alignment. Duplicate read marking, local realignment around indels, base quality v0.7.12 score recalibration and variant calling were performed using GATK algorithms (Sentieon) for nDNA. Variant data was annotated using a collection of tools (VcfAnno and VEP) with a variety of public variant databases including, but not limited to, gnomAD, ClinVar and HGMD. The median sequencing depth and coverage across the target regions for the tested sample were calculated based on MQ0 aligned reads. The sequencing run was included in process reference sample(s) for quality control, which passed our thresholds for sensitivity and specificity. The patient’s sample was subjected to thorough quality control measures including assessments for contamination and sample mix-up. Copy number variations (CNVs), defined as single exon or larger deletions or duplications (Del/Dups), were detected from the sequence analysis data using a commercially available bioinformatic pipeline CNVkit and a proprietary, in-house-developed deletion caller based on read depth to improve the detection of small CNVs. The difference between observed and expected sequencing depth at the targeted genomic regions was calculated and regions were divided into segments with variable DNA copy number. The expected sequencing depth was obtained by using other samples processed in the same sequence analysis as a guiding reference. The sequence data were adjusted to account for the effects of varying guanine and cytosine content.

2.3. DNA Extraction

DNA was extracted from venous blood drawn from the precubital vein. Buffy coats of nucleated cells obtained from anticoagulated blood (EDTA) were resuspended in 15 mL polypropylene centrifugation tubes with 3 mL of nuclei lysis buffer (10 mM Tris-HCl, 400 mM NaCl and 2 mM Na2EDTA, pH 8.2). The cell lysates were digested overnight at 37 °C with 0.2 mL of 10% SDS and 0.5 mL of a protease K solution (1 mg protease K in 1% SDS and 2 mM Na2EDTA). After digestion was complete, 1 mL of saturated NaCl (approximately 6 M) was added to each tube and shaken vigorously for 15 s, followed by centrifugation at 2500 rpm for 15 min. The precipitated protein pellet was left at the bottom of the tube and the supernatant containing the DNA was transferred to another 15 mL polypropylene tube. Exactly 2 volumes of room temperature absolute ethanol were added, and the tubes inverted numerous times until the DNA precipitated. The precipitated DNA strands were removed with a plastic spatula or pipette and transferred to a 1.5 mL microcentrifuge tube containing 100–200 pl TE buffer (10 mM Tris-HCl, 0.2 mM Na2EDTA, pH 7.5). The DNA was allowed to dissolve for 2 h at 37 °C before quantitating.

2.4. Ophthalmological Examinations

For assessment of overall retinal function, full-field electroretinograms (ffERG) according to the ISCEV standards at the time [19,20] were recorded in all of the patients. In subjects that had their appointment after the multifocal electroretinography (mfERG) technique had been introduced (from 2002), macular function was measured with mfERG according to the ISCEV standards of the time [21,22]. Best corrected visual acuity (BCVA) was tested monocularly on a decimal letter chart at 5 or 3 m (m) and visual fields were mapped with a Goldmann perimeter, likewise monocularly, with standardized objects V4e, I4e, 04e, 03e, and 02e. For structural analysis, fundus color and red free photographs, and during later years, optical coherence tomography (OCT) and autofluorescence (FAF) images were also obtained. Moreover, slit lamp and fundus examinations were conducted.

3. Results

Pathogenic class 4 or 5 genetic variants explaining the phenotype were found in 182 of the 279 (65%) samples that were re-analyzed with the NGS retinal dystrophy panel. A description of the pathogenic variants as well as data concerning age at first examination, gender, genotype, and phenotype at first examination are presented in Table 2. The cohort was genetically heterogenous showing disease -causing variants in 65 different genes (Figure 1 and

Table 3. Showing the spectrum of mutated genes that were found in the Swedish cohort of IRDs patients and the number of patients with pathogen variants in each specific gene.

Gene	Number of Patients
ABCA4	30
AIPL1	1
BBS1	1
BBS10	4
BBS5	2
BBS9	1
CACNA1F	6
CACNA2D4	1
CDH23	1
CDH3	1
CDHR1	1
CEP290	11
CFAP410	5
CHM	6
CLN3	3
CNGB1	1
CNGB3	2
COL18A1	4
CRX	3
GUCA1A	1
GUCY2D	4
IMPDH1	1
IQCB1	1
KCNV2	3
KIF11	1
KLHL7	1
LRAT	2
MERTK	3
MFN2	1
MFRP	1
MYO7A	2
NMNAT1	1
NPHP1	1
NR2E3	2
NYX	6
OPA1	4
OTX2	1
PANK2	1
PCARE	1
PCDH15	2
PDE6B	1
PDE6C	1
PNPLA6	1
POC1B	1
POMGNT1	1
PROM1	6
PRPF31	2
PRPF8	3
PRPH2	1
RDH12	1
RDH5	1
RHO	1
RLBP1	1
RP1	2
RP1L1	1
RP2	1
RPE65	1
RPGR	11
RS1	10
TIMM8A	1
TRPM1	3
TULP1	2
USH1C	1
USH2A	2
WFS1	2

). The most frequently mutated gene was the ABCA4 gene with pathogen variants in 30 of the 182 (16.5%) cases with a verified genotype. Other prevalent causative genes in this Swedish cohort were CEP290 (11 out of 182, 6%), RPGR (11 out of 182, 6%), RS1 (10 out of 182, 5.5%), CACNA1F (6 out of 182, 3%), CHM (6 out of 182, 3%), NYX (6 out of 182, 3%), and PROM1 (6 out of 182, 3%).

In 13 out of the 182 (7%) patients, there was a discrepancy between the diagnosis based on phenotypical or genotypical findings alone. The most common error was that CSNB initially was considered to be XLRS or choroideremia or that early choroideremia was mistaken for RP. In two cases, Bardet–Biedl syndrome initially was interpreted as achromatopsia before more general symptoms such as obesity and renal problems were apparent.

4. Discussion

Since gene-based treatments like gene augmentation/replacement [8,9,23,24,25,26,27,28,29,30,31], gene silencing, AONs [32,33], and gene editing using the CRISPR/Cas9 system [3,5,6,7] may be the future for patients with IRDs, confirmation of the genotype has become even more crucial during the last years. In our department, we have, since the 1990s, strived to both perform careful phenotyping and to verify the genotype in all our IRD patients. However, we have failed to identify the causative genetic background in quite a few of them and therefore, we wanted to investigate if it is beneficial to perform genetic re-testing with a widely available broad NGS panel for IRDs. WES or WGS could possibly have revealed more pathogenic variants, but in this study, we wanted to test a method that is affordable in a clinical setting and for the health care systems in different countries. In these patients, that had previously been investigated with various techniques such as single-gene analysis, APEX panels, NGS panels, and WES with inconclusive results, the renewed testing with a comprehensive NGS panel revealed the presence of the genotype in 182 individuals (65%). Thus, the success rate was approximately the same as the general yield described for first time-testing using NGS (50–71%) [34,35] although our subjects were selected unsolved cases. This means that it is of great value to re-test IRDs patients with unsolved genotypes using a broad NGS panel for IRDs. When it comes to the cases with compound heterozygosity it would, of course, be ideal to perform segregation analyses for all of them, but in this study, it was not possible to make contact with and test relatives of all of the patients. In many cases, NGS data could confirm that the variants were in trans and in all cases we were very careful in the interpretation of the genetic data only considering the genotype as causative if it was completely consistent with the phenotype. It is difficult to set a proper interval for DNA re-testing. In our study, the positive yield of testing for the patients with the shortest re-test interval (previously tested between 2016–2020 with APEX or NGS panels) was 32 out of 49 samples (65%), which means a similar positive success rate as for the whole group, indicating the usefulness of re-testing with quite short intervals.

When it comes to the prevalence of different causative genes in this Swedish cohort, which to our knowledge is the first larger cohort investigated concerning the genetic spectrum in Sweden, the ABCA4 gene was the most common gene, encountered in 16.5% of the patients. This is in line with both an international estimate by Schneider et.al., 2022 called the Global Retinal Inherited Disease (GRID) dataset [13], and with reports from separate countries, although the absolute percentage varies slightly: GRID 25%, USA 14% [36], Canada 20% [36], Brazil 21% [37], Taiwan 15% [38], and Italy 26% [39]. Our second-most common genes were RPGR and CEP290, which were found in 6% of the patients, respectively. RGPR is also among the most prevalent genes in other studies; fourth-most common in the GRID dataset (3.4%) [13], in USA and Canada it was the third-most common gene (10% and 4%) [36], the fourth-most common in Brazil (5%) [37], fifth-most common in Taiwan (5%) [38], and in the Italian cohort, it was the third-most common gene (5%) [39]. CEP 290, on the other hand, is only represented to the same extent in the Brazilian cohort (5.5%) [37], while it is less common in the other cohorts (1–3%) [13,36,38,39]. Another difference is that USH2A is quite common in the other studies, being the second-most prevalent gene in the GRID dataset (15%) [13] as well as in the Italian (11%) [39] and the Canadian (6%) [36] cohorts, the third-most prevalent gene in Taiwan and Brazil (10% and 5%, respectively) [37,38], and found in 3% of American IRD patients [36], while it was found in only two of our 182 patients (1.1%) with a verified genotype. It is well known that genes have different prevalences in various countries and geographic areas, but most of the difference concerning the USH2A gene in our study can be explained by the fact that the patients with Usher syndrome type 2A are referred to us at an older age (mean age 39 at genotypic diagnosis in our registry) than the investigated group, since their visual decline becomes evident somewhat later in life. EYS was also among the more common genes in some of the other cohorts; e.g., second-most common among the Taiwanese subjects (12%) [38], third-most common in the GRID dataset (4.4%) [13,36], and was found in 4% of Brazilian IRD patients [37], while it was actually absent from our study as well as from the American and Canadian cohorts [36]. Concerning RS1, the setting was the opposite. It was among the more common genes in our cohort, verified in 5.5% of the subjects, but less prevalent in the other studies, in which it was described in only 0.5–2% of the patients [37,39,40] or was not specified at all [13,36]. Thus, these data indicate that to an extent, the same genes are the most prevalent across different cohorts with the exception of certain genes, e.g., RS1, CEP290, and EYS, that show more inconsistent distribution. This is of special interest when it comes to introducing gene-based therapies, since particular genes have a more urgent need to be dealt with in some populations than in others. Figure 2 shows the genotypic pattern of the 201 patients with established genotypes belonging to the same age group in the RP registry. In this group, 45 different genotypes were demonstrated. It can be noted from Figure 1 and Figure 2 that some genes such as CNGB3, RHO, CLN3, BEST1, BCM, and GUCY2D were quite well covered in the former analyses and not many new cases were encountered in the re-analysis. For ABCA4, RPGR, and RS1, new variants were discovered in rather many subjects although these genes were among the most prevalent causative genotypes also in the registry cohort and thus the coverage of those genes has improved. It is also noteworthy that CEP290, CACNA1F, CHM, and NYX are much better covered in the newer genetic work-up identifying many more subjects than in the registry cohort.

In the re-analyzed material, the gender distribution was slightly skewed, which can be explained by the occurrence of X-linked disorders that were encountered in 40 of the subjects (22% of the subjects with a verified genotype).

The basis for the choice of age range of 25 years or younger in the study was that younger patients are more suitable for future treatments, since many of the IRDs are progressive and thus, early detection is essential for enough viable retinal cells to be left for decent treatment results. Moreover, it is very important for young patients to obtain a correct diagnosis as early as possible, in order to enable adequate visual habilitation including visual aids, as well as fair expectations concerning the course of the visual impairment. In line with this, we can confirm the importance of a combined phenotypic and genotypic work-up, since in 7% of the patients, the result of genetic testing or clinical examinations alone led to different diagnoses, delaying correct counselling. For instance, in some early cases, X-linked congenital stationary night blindness (CSNB) due to CACNA1F variants was diagnosed as X-linked RP with the risk of giving the family incorrect information concerning the progression of the disease over time, since CSNB is a stationary and XLRP a progressive disorder. In some cases, the genetic result was also important for the confirmation of the inheritance pattern.

To conclude, renewed DNA-analysis was also beneficial in previously tested patients with inconclusive genetic test results, and it improved our ability to verify the genotype–phenotype association increasing the understanding of visual impairment, disease prognosis, and sometimes the inheritance pattern. Thus, repeated testing using a widely available method may identify additional patients eligible for future gene-based therapies.