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Macular and cone/cone-rod dystrophies (MD/CCRD) demonstrate a broad genetic and phenotypic heterogeneity, with retinal alterations solely or predominantly involving the central retina. Targeted next-generation sequencing (NGS) is an efficient diagnostic tool for identifying mutations in patient with retinitis pigmentosa, which shows similar genetic heterogeneity. To detect the genetic causes of disease in patients with MD/CCRD, we implemented a two-tier procedure consisting of Sanger sequencing and targeted NGS including genes associated with clinically overlapping conditions. Disease-causing mutations were identified in 74% of 251 consecutive MD/CCRD patients (33% of the variants were novel). Mutations in ABCA4, PRPH2 and BEST1 accounted for 57% of disease cases. Further mutations were identified in CDHR1, GUCY2D, PROM1, CRX, GUCA1A, CERKL, MT-TL1, KIF11, RP1L1, MERTK, RDH5, CDH3, C1QTNF5, CRB1, JAG1, DRAM2, POC1B, NPHP1 and RPGR. We provide detailed illustrations of rare phenotypes, including autofluorescence and optical coherence tomography imaging. Targeted NGS also identified six potential novel genotype-phenotype correlations for FAM161A, INPP5E, MERTK, FBLN5, SEMA4A and IMPDH1. Clinical reassessment of genetically unsolved patients revealed subgroups with similar retinal phenotype, indicating a common molecular disease cause in each subgroup.

Purpose: Hearing loss is genetically extremely heterogeneous, making it suitable for next-generation sequencing (NGS). We identified a four-generation family with nonsyndromic mild to severe hearing loss of the mid-to high frequencies and onset from early childhood to second decade in seven members. Methods: NGS of 66 deafness genes, Sanger sequencing, genome-wide linkage analysis, whole-exome sequencing (WES), semiquantitative reverse-transcriptase polymerase chain reaction. Results: We identified a heterozygous nonsense mutation, c.6881G> A (p.Trp2294*), in the last coding exon of PTPRQ. PTPRQ has been linked with recessive (DFNB84A), but not dominant deafness. NGS and Sanger sequencing of all exons (including alternatively spliced 5' and N-scan-predicted exons of a putative extended transcript) did not identify a second mutation. The highest logarithm of the odds score was in the PTPRQ-containing region on chromosome 12, and p.Trp2294* cosegregated with hearing loss. WES did not identify other cosegregating candidate variants from the mapped region. PTPRQ expression in patient fibroblasts indicated that the mutant allele escapes nonsense-mediated decay (NMD). Conclusion: Known PTPRQ mutations are recessive and do not affect the C-terminal exon. In contrast to recessive loss-of-function mutations, c.6881G> A transcripts may escape NMD. PTPRQ(Trp2294*) protein would lack only six terminal residues and could exert a dominant-negative effect, a possible explanation for allelic deafness, DFNA73, clinically and genetically distinct from DFNB84A.

Retinitis pigmentosa (RP) is an inherited degenerative disease causing severe retinal dystrophy and visual impairment mainly with onset in infancy or adolescence. Targeted next-generation sequencing (NGS) has become an efficient tool to encounter the enormous genetic heterogeneity of diverse retinal dystrophies, including RP. To identify disease-causing mutations in unselected, consecutive RP patients, we conducted Sanger sequencing of genes commonly involved in the suspected genetic RP subtype, followed by targeted large-panel NGS if no mutation was identified, or NGS as primary analysis. A high (70%) detection rate of disease-causing mutations was achieved in a large cohort of 116 unrelated patients. About half (48%) of the solved RP cases were explained by mutations in four genes: RPGR, EYS, PRPF31 and USH2A. Overall, 110 different mutations distributed across 30 different genes were detected, and 46 of these mutations were novel. A molecular diagnosis was achieved in the majority (82-100%) of patients if the family history was suggestive for a particular mode of inheritance, but only in 60% in cases of sporadic RP. The diagnostic potential of extensive molecular analysis in a routine setting is also illustrated by the identification of unexpected genotype-phenotype correlations for RP patients with mutations in CRX, CEP290, RPGRIP1, MFSD8. Furthermore, we identified numerous mutations in autosomal dominant (PRPF31, PRPH2, CRX) and X-linked (RPGR) RP genes in patients with sporadic RP. Variants in RP2 and RPGR were also found in female RP patients with apparently sporadic or dominant disease. In summary, this study demonstrates that massively parallel sequencing of all known retinal dystrophy genes is a valuable diagnostic approach for RP patients.

Deafblindness is mostly due to Usher syndrome caused by recessive mutations in the known genes. Mutation-negative patients therefore either have distinct diseases, mutations in yet unknown Usher genes or in extra-exonic parts of the known genes-to date a largely unexplored possibility. In a consanguineous Saudi family segregating Usher syndrome type 1 (USH1), NGS of genes for Usher syndrome, deafness and retinal dystrophy and subsequent whole-exome sequencing each failed to identify a mutation. Genome-wide linkage analysis revealed two small candidate regions on chromosome 3, one containing the USH3A gene CLRN1, which has never been associated with Usher syndrome in Saudi Arabia. Whole-genome sequencing (WGS) identified a homozygous deep intronic mutation, c.254-649T > G, predicted to generate a novel donor splice site. CLRN1 minigenebased analysis confirmed the splicing of an aberrant exon due to usage of this novel motif, resulting in a frameshift and a premature termination codon. We identified this mutation in an additional two of seven unrelated mutation-negative Saudi USH1 patients. Locus-specific markers indicated that c.254-649T > G(CLRN1) represents a founder allele that may significantly contribute to deafblindness in this population. Our finding underlines the potential of WGS to uncover atypically localized, hidden mutations in patients who lack exonic mutations in the known disease genes.

BackgroundCombined retinal degeneration and sensorineural hearing impairment is mostly due to autosomal recessive Usher syndrome (USH1: congenital deafness, early retinitis pigmentosa (RP); USH2: progressive hearing impairment, RP). MethodsSanger sequencing and NGS of 112 genes (Usher syndrome, nonsyndromic deafness, overlapping conditions), MLPA, and array-CGH were conducted in 138 patients clinically diagnosed with Usher syndrome. ResultsA molecular diagnosis was achieved in 97% of both USH1 and USH2 patients, with biallelic mutations in 97% (USH1) and 90% (USH2), respectively. Quantitative readout reliably detected CNVs (confirmed by MLPA or array-CGH), qualifying targeted NGS as one tool for detecting point mutations and CNVs. CNVs accounted for 10% of identified USH2A alleles, often in trans to seemingly monoallelic point mutations. We demonstrate PTC124-induced read-through of the common p.Trp3955* nonsense mutation (13% of detected USH2A alleles), a potential therapy target. Usher gene mutations were found in most patients with atypical Usher syndrome, but the diagnosis was adjusted in case of double homozygosity for mutations in OTOA and NR2E3, genes implicated in isolated deafness and RP. Two patients with additional enamel dysplasia had biallelic PEX26 mutations, for the first time linking this gene to Heimler syndrome. ConclusionTargeted NGS not restricted to Usher genes proved beneficial in uncovering conditions mimicking Usher syndrome.

PURPOSE. This study sought to characterize the ophthalmic and extraocular phenotype in patients with known and novel KIF11 mutations. METHODS. Four patients (3, 5, 36, and 38 years of age, on father-daughter constellation) from three unrelated families were characterized by retinal examination including multimodal retinal imaging, investigation for syndromic disease manifestations, and targeted next-generation sequencing. The subcellular localization of Kif11 in the retina was analyzed by light and electron microcopy. RESULTS. There was considerable interindividual and intrafamilial phenotypic heterogeneity of KIF11-related retinopathy. Two patients presented with a progressive retinal dystrophy, one with chorioretinal dysplasia and one with familial exudative vitreoretinopathy (FEVR) in one eye and thinning of the photoreceptor layer in the fellow eye. Obvious syndromic disease manifestations were present only in the youngest patient, but minor signs (e.g. reduced head circumference) were present in the three other individuals. Immunohistochemistry results demonstrated Kif11 localization in the inner segment and ciliary compartments of photoreceptor cells and in the retinal pigment epithelium. CONCLUSIONS. Progressive retinal degeneration in KIF11-related retinopathy indicates a role for KIF11 not only in ocular development but also in maintaining retinal morphology and function. The remarkable variability of the ocular phenotype suggests four different types of retinopathy which may overlap. KIF11 should be considered in the screening of patients with retinal dystrophies because other syndromic manifestations may be subtle. Evaluation of head circumference may be considered as a potential shortcut to the genetic diagnosis. The localization of Kif11 in photoreceptor cells indicates a retinal ciliopathy.

Deafblindness is mostly due to Usher syndrome caused by recessive mutations in the known genes. Mutation-negative patients therefore either have distinct diseases, mutations in yet unknown Usher genes or in extra-exonic parts of the known genes – to date a largely unexplored possibility. In a consanguineous Saudi family segregating Usher syndrome type 1 (USH1), NGS of genes for Usher syndrome, deafness and retinal dystrophy and subsequent whole-exome sequencing each failed to identify a mutation. Genome-wide linkage analysis revealed two small candidate regions on chromosome 3, one containing the USH3A gene CLRN1, which has never been associated with Usher syndrome in Saudi Arabia. Whole-genome sequencing (WGS) identified a homozygous deep intronic mutation, c.254–649T > G, predicted to generate a novel donor splice site. CLRN1 minigene-based analysis confirmed the splicing of an aberrant exon due to usage of this novel motif, resulting in a frameshift and a premature termination codon. We identified this mutation in an additional two of seven unrelated mutation-negative Saudi USH1 patients. Locus-specific markers indicated that c.254–649T > G$_{CLRN1}$ represents a founder allele that may significantly contribute to deafblindness in this population. Our finding underlines the potential of WGS to uncover atypically localized, hidden mutations in patients who lack exonic mutations in the known disease genes.

Background Dominant mutations in peripherin (PRPH2) are associated with a spectrum of retinal dystrophy phenotypes, many of which are adult onset and involve the macula. Recessive PRPH2 mutations cause retinal dystrophy associated with prominent maculopathy in adulthood; however, the presenting childhood phenotype has not been defined. We characterise this phenotype. Methods Retrospective case series of families harbouring bi-allelic PRPH2 mutations (2010-2014). Results Three children (two families; assessed at 2 years old) and two adults (one family; assessed at 24 and 35 years old) with homozygous PRPH2 mutations (c.497G>A (p.Cys166Tyr) or c.136C>T (p.Arg46(star)))all had infantile nystagmus and decreased vision noted soon after birth and a history of staring at lights during infancy (photophilia). The three children had high hyperopia, a normal or near normal fundus, and non-recordable electroretinographies (ERGs). The two adults had slight myopia, macular and peripheral retinal changes, and non-recordable ERGs. All five available carrier parents had macular+/-peripheral retinal findings, although they considered themselves asymptomatic except for one mother who had developed visual loss in one eye at 48 years old and had an associated subfoveal lesion. Conclusions Bi-allelic PRPH2 mutations cause a distinct Leber congenital amaurosis phenotype in infancy; affected adults have prominent maculopathy. Heterozygous parents can be asymptomatic but have clinically obvious macular phenotypes with or without peripheral retinal findings, which can be helpful in making the genetic diagnosis in affected children. The difference between the heterozygous and homozygous phenotypes is likely related to gene product dosage effect.

Purpose: To document a distinct vitreo-retinal dystrophy with early-onset cataract as related to recessive KCNJ13 mutations. Methods: A retrospective case series (two patients from two families) Results: A 12-year-old Saudi Arabian girl with nystagmus since birth was referred because of recent decreased vision. Parents were first cousins and a younger sister had been diagnosed with retinal dystrophy. Examination revealed total white cataract in the right eye. In the left eye, there were posterior cortical lenticular opacities and an unusual retina fundus dystrophic appearance notable for fibrosis over the optic disc and clumped pigmentation. After right eye cataract surgery, the posterior pole of the left eye was seen as similar to that of the right eye and electroretinography revealed severe cone-rod dysfunction, with only subnormal scotopic tracings recordable in both eyes. Next-generation sequencing of retinal dystrophy genes revealed homozygosity for a novel missense mutation in KCNJ13 (c.359T>C; p.Ile120Thr [NM_002242.4]), which co-segregated with the disease. Direct KCNJ13 sequencing for an unrelated 33-year-old Saudi Arabian male with similar clinical findings but early-adult-onset rather than juvenile cataract revealed the same homozygous mutation. Conclusions: Juvenile or early-adult-onset cataract in the setting of a congenital vitreo-retinal dystrophy notable for fibrosis over the disc and clumped pigmentation in the posterior pole is a unique phenotype that suggests recessive KCNJ13 mutations.

Background:Fifty to 60% of patients with dominant optic atrophy (DOA) have mutations of the OPA1 gene, which encodes dynamin-related GTPase, a protein of the internal mitochondrial membrane. To date, more than 200 OPA1 mutations in the OPA1 gene have been described. However, DOA is genetically heterogeneous with certain families linked to other chromosomal loci, that is, OPA3, OPA4, OPA5, and OPA7.Methods:This study describes a clinical series of 40 patients from Saudi Arabia with a positive DOA phenotype (i.e., decreased visual acuity during the first 2 decades of life, temporal or global optic disc pallor, and absence of other neurological or ophthalmological diseases that could explain the optic neuropathy) who underwent molecular genetic testing for OPA1 (and, in some cases, for OPA3).Results:This study describes for the first time 4 OPA1 mutations in DOA patients from Saudi Arabia, including 2 novel OPA1 mutations in 2 different patients.Conclusion:The question remains whether certain patients in Saudi Arabia with a clearly defined DOA phenotype may be due to mutations in chromosomal loci other than OPA1 and OPA3. It is likely that genetic alterations associated with different loci will be discovered in the future.

Retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) are major causes of blindness. They result from mutations in many genes which has long hampered comprehensive genetic analysis. Recently, targeted next-generation sequencing (NGS) has proven useful to overcome this limitation. To uncover hidden mutations such as copy number variations (CNVs) and mutations in non-coding regions, we extended the use of NGS data by quantitative readout for the exons of 55 RP and LCA genes in 126 patients, and by including non-coding 59 exons. We detected several causative CNVs which were key to the diagnosis in hitherto unsolved constellations, e. g. hemizygous point mutations in consanguineous families, and CNVs complemented apparently monoallelic recessive alleles. Mutations of non-coding exon 1 of EYS revealed its contribution to disease. In view of the high carrier frequency for retinal disease gene mutations in the general population, we considered the overall variant load in each patient to assess if a mutation was causative or reflected accidental carriership in patients with mutations in several genes or with single recessive alleles. For example, truncating mutations in RP1, a gene implicated in both recessive and dominant RP, were causative in biallelic constellations, unrelated to disease when heterozygous on a biallelic mutation background of another gene, or even non-pathogenic if close to the C-terminus. Patients with mutations in several loci were common, but without evidence for di- or oligogenic inheritance. Although the number of targeted genes was low compared to previous studies, the mutation detection rate was highest (70%) which likely results from completeness and depth of coverage, and quantitative data analysis. CNV analysis should routinely be applied in targeted NGS, and mutations in non-coding exons give reason to systematically include 5'-UTRs in disease gene or exome panels. Consideration of all variants is indispensable because even truncating