Markus N. Preising

TRPM1 is mutated in patients with autosomal-recessive complete congenital stationary night blindness.

Night vision requires signaling from rod photoreceptors to adjacent bipolar cells in the retina. Mutations in the genes NYX and GRM6, expressed in ON bipolar cells, lead to a disruption of the ON bipolar cell response. This dysfunction is present in patients with complete X-linked and autosomal-recessive congenital stationary night blindness (CSNB) and can be assessed by standard full-field electroretinography (ERG), showing severely reduced rod b-wave amplitude and slightly altered cone responses. Although many cases of complete CSNB (cCSNB) are caused by mutations in NYX and GRM6, in approximately 60% of the patients the gene defect remains unknown. Animal models of human diseases are a good source for candidate genes, and we noted that a cCSNB phenotype present in homozygous Appaloosa horses is associated with downregulation of TRPM1. TRPM1, belonging to the family of transient receptor potential channels, is expressed in ON bipolar cells and therefore qualifies as an excellent candidate. Indeed, mutation analysis of 38 patients with CSNB identified ten unrelated cCSNB patients with 14 different mutations in this gene. The mutation spectrum comprises missense, splice-site, deletion, and nonsense mutations. We propose that the cCSNB phenotype in these patients is due to the absence of functional TRPM1 in retinal ON bipolar cells.

Increasing the Yield in Targeted Next-Generation Sequencing by Implicating CNV Analysis, Non-Coding Exons and the Overall Variant Load: The Example of Retinal Dystrophies

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 5′ 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 mutations may be misleading.

Whole-Exome Sequencing Identifies Mutations in GPR179 Leading to Autosomal-Recessive Complete Congenital Stationary Night Blindness

Congenital stationary night blindness (CSNB) is a heterogeneous retinal disorder characterized by visual impairment under low light conditions. This disorder is due to a signal transmission defect from rod photoreceptors to adjacent bipolar cells in the retina. Two forms can be distinguished clinically, complete CSNB (cCSNB) or incomplete CSNB; the two forms are distinguished on the basis of the affected signaling pathway. Mutations in NYX, GRM6, and TRPM1, expressed in the outer plexiform layer (OPL) lead to disruption of the ON-bipolar cell response and have been seen in patients with cCSNB. Whole-exome sequencing in cCSNB patients lacking mutations in the known genes led to the identification of a homozygous missense mutation (c.1807C>T [p.His603Tyr]) in one consanguineous autosomal-recessive cCSNB family and a homozygous frameshift mutation in GPR179 (c.278delC [p.Pro93Glnfs(∗)57]) in a simplex male cCSNB patient. Additional screening with Sanger sequencing of 40 patients identified three other cCSNB patients harboring additional allelic mutations in GPR179. Although, immunhistological studies revealed Gpr179 in the OPL in wild-type mouse retina, Gpr179 did not colocalize with specific ON-bipolar markers. Interestingly, Gpr179 was highly concentrated in horizontal cells and Müller cell endfeet. The involvement of these cells in cCSNB and the specific function of GPR179 remain to be elucidated.

Genotype-phenotype correlation and longitudinal course in ten families with Best vitelliform macular dystrophy

AimLongitudinal course and genotype-phenotype correlation in patients and carriers with heterozygous mutations in hBEST1 (bestrophin).MethodsThirteen patients and seven possible carriers were characterised by mutation analysis with SSCPA and direct sequencing, clinical examination and fundus autofluorescence (AF). Electrophysiology (EOG and mfERG) and optical coherence tomography (OCT) were additionally performed whenever possible.ResultsWe identified seven different heterozygous mutations in ten unrelated families with Best disease. I296del was the most frequent mutation. Five of nine individuals with I295del and two of three with N99K were asymptomatic carriers. One patient with I295del mutation had funduscopically unilateral Best disease. In three children (all with I295del), EOG initially showed a clearly present light peak that deteriorated during 5 years of follow-up in two of them. Increased AF corresponded well to funduscopically visible lesions. During 3–6 years of follow-up, the lesion area did not change significantly, but there were obvious changes in the inner structure of the lesion.ConclusionIn the present series I295del, the most frequent mutation in our study, and N99K showed reduced penetrance. EOG was normal in young patients even if prime signs were visible. The lesion area did not depend on the mutation and did not correlate with VA. Lower VA was associated with a more irregular AF pattern due to scarring or haemorrhage. Our results indicate a disease causing effect that is cumulative over time.

Phenotypes of 16 Stargardt macular dystrophy/fundus flavimaculatus patients with known ABCA4 mutations and evaluation of genotype–phenotype correlation

Abstract Purpose. To determine the phenotypic variability in patients with compound heterozygous or homozygous ABCA4 mutations, and to correlate the phenotypes with the functional properties of the altered protein. Methods. Sixteen patients from 13 families with signs of Stargardt macular dystrophy/fundus flavimaculatus and known mutations on both alleles of the ABCA4 gene (15 compound heterozygous, one homozygous) were characterized by clinical examination, fundus autofluorescence, psychophysics (color vision, kinetic and two-color dark- and light-adapted static threshold perimetry), and electrophysiology (Ganzfeld, multifocal ERG, EOG). Results. The homozygous 5917delG mutation resulted in the earliest disease manifestation (at 5 years) and a general cone–rod dysfunction, whereas the compound heterozygous mother (5917delG, G1961E) exhibited a very mild phenotype. Compound heterozygotes for the IVS40+5G→A and the C1488Y or Y362X mutation showed also an early age of onset but only a central dysfunction. The effect of the 2588G→C mutation, the G1961E mutation, and the complex mutation L541P-A1038V depended on the mutation in the second allele. Genotype–phenotype correlation appeared possible in most instances. Psychophysics revealed a simultaneous yet not necessarily congruent cone and rod dysfunction. Conclusions. The type and combination of ABCA4 mutations in compound heterozygous patients determined were compatible with the severity of the phenotype as to age of onset and the functional consequences in the majority of patients. Unexplained phenotypic differences indicate the influence of other factors. ABCA4 mutations result in cone and rod dysfunction. Different disease durations limit the power of presently available genotype–phenotype correlations.

A comprehensive clinical and biochemical functional study of a novel RPE65 hypomorphic mutation.

PURPOSE Later onset and progression of retinal dystrophy occur with some RPE65 missense mutations. The functional consequences of the novel P25L RPE65 mutation was correlated with its early-childhood phenotype and compared with other pathogenic missense mutations. METHODS In addition to typical clinical tests, fundus autofluorescence (FAF), optical coherence tomography (OCT), and two-color threshold perimetry (2CTP) were measured. RPE65 mutations were screened by SSCP and direct sequencing. Isomerase activity of mutant RPE65 was assayed in 293F cells and quantified by HPLC analysis of retinoids. RESULTS A very mild phenotype was detected in a now 7-year-old boy homozygous for the P25L mutation in RPE65. Although abnormal dark adaptation was noticed early, best corrected visual acuity was 20/20 at age 5 years and 20/30 at age 7 years. Nystagmus was absent. Cone electroretinogram (ERG) was measurable, rod ERG severely reduced, and FAF very low. 2CTP detected mainly cone-mediated responses in scotopic conditions, and light-adapted cone responses were approximately 1.5 log units below normal. High-resolution spectral domain OCT revealed morphologic changes. Isomerase activity in 293F cells transfected with RPE65/P25L was reduced to 7.7% of wild-type RPE65-transfected cells, whereas RPE65/L22P-transfected cells had 13.5%. CONCLUSIONS The mild clinical phenotype observed is consistent with the residual activity of a severely hypomorphic mutant RPE65. Reduction to <10% of wild-type RPE65 activity by homozygous P25L correlates with almost complete rod function loss and cone amplitude reduction. Functional survival of cones is possible in patients with residual RPE65 isomerase activity. This patient should profit most from gene therapy.

Rab escort protein 1 (REP1) in intracellular traffic: a functional and pathophysiological overview

The intracellular distribution of proteins, compartments, substrates, and products is an active process called intracellular traffic. Control of intracellular traffic is established by small GTP-binding proteins (Rab proteins). Rab proteins are modified by geranyl-geranyl moieties necessary for membrane association and target-protein recognition. Geranyl-geranyl groups are transferred to Rab proteins by geranyl-geranyl transferase 2 (GGTase2). GGTase2 requires Rab escort protein 1 (REP1) to bind Rab proteins. REP1null mutations underlie an X-linked retinal degeneration called choroideremia (CHM). This review summarizes the current biochemical and clinical knowledge on REP1 and CHM.

Fundus albipunctatus associated with compound heterozygous mutations in RPE65.

PURPOSE To describe a family with an 18-year-old woman with fundus albipunctatus and compound heterozygous mutations in RPE65 whose unaffected parents and 1 female sibling harbored single heterozygous RPE65 mutations. DESIGN Observational study. PARTICIPANTS Four family members. METHODS Clinical examinations included full-field electroretinogram (ffERG) after standard (30-minute) and prolonged (17-hour) dark adaptation, multifocal electroretinogram (mfERG), optical coherence tomography (OCT), and fundus autofluorescence (FAF). Molecular genetic testing included sequencing of RDH5 and RLBP1 and screening for known autosomal-recessive retinitis pigmentosa mutations by a commercially available microarray technique. RPE65 sequencing was performed after the identification of a known heterozygous splice-site mutation by array screening. MAIN OUTCOME MEASURES We recorded ffERG and mfERG amplitudes, OCT characteristics, the FAF intensity index, and the outcomes of DNA sequencing regarding RPE65 mutations. RESULTS Uniform, yellow-white dots typical of fundus albipunctatus were demonstrated in the proband. These dots corresponded with discrete, hyperreflective formations extending from the Bruchs membrane and retinal pigment epithelium (RPE) into the level of the external limiting membrane, thus spanning along the entire RPE and photoreceptor outer and inner segments. A reduced thickness of the central retina and the RPE-outer segment complex was demonstrated. The intensity of the FAF was severely reduced in the entire fundus. At age 18, ffERG-including prolonged dark adaptation-demonstrated a barely recordable rod response after standard dark adaptation and normalization (increase by more than 700%) of the response after prolonged dark adaptation. The cone 30-Hz flicker response was reduced after standard dark adaptation and increased by >50% after prolonged dark adaptation. In addition, mfERG demonstrated reduced central and peripheral responses. Molecular genetic analysis demonstrated compound heterozygous mutations (IVS1+5G>A and c.344T>C) in RPE65. No mutations were found in RDH5 or RLBP1. No significant abnormalities of retinal structure or function were detected in the parents and sister carrying single heterozygous mutations in RPE65. CONCLUSIONS This is the first reported association between compound heterozygous RPE65 mutations and fundus albipunctatus, indicative of a mutation-specific phenotypic effect in this gene. This finding, together with the reduced FAF, supports that disruption of retinoid recycling in the RPE is essential for the development of fundus albipunctatus.

Fundus autofluorescence in carriers of X-linked recessive retinitis pigmentosa associated with mutations in RPGR, and correlation with electrophysiological and psychophysical data.

PurposeTo describe fundus autofluorescence (AF) in carriers of X-linked retinitis pigmentosa (XLRP) associated with mutations in RPGR (RP3), and to compare the findings on AF with ophthalmoscopy and with electrophysiological and psychophysical data.MethodsEleven carriers from two families with XLRP and mutations in RPGR underwent clinical examination including fundus photography, AF, full-field electroretinography, Goldmann kinetic perimetry and two-colour threshold perimetry (2CT perimetry).ResultsAn abnormal AF pattern was found in 9 of 11 carriers, with a radial pattern in 6 of 11. In 2CT perimetry patchy rod and cone sensitivity losses were seen in 7 of 8 carriers. Rods tended to be more affected than cones. The areas of sensitivity loss showed some correspondence with the abnormalities seen on AF.ConclusionAF had a specific pattern in 9 of 11 carriers from two families with mutations in RPGR. The result was independent of the family investigated. The radial pattern may be explained by random X-inactivation early during embryogenesis subsequently preserved in all daughter cells and the centrifugal radial growth pattern of the developing neuroretina. AF may prove to be a rapid and easy clinical test to identify carriers of RP3.

Deletion in the OA1 gene in a family with congenital X linked nystagmus

AIMS To elucidate the molecular genetic defect of X linked congenital nystagmus associated with macular hypoplasia in three white males of a three generation family with clear features of ocular albinism in only one of them. METHODS A three generation family with congenital nystagmus following X linked inheritance, and associated with macular hypoplasia was clinically examined (three males and two obligate carriers). Flash VEP was performed to look for albino misrouting. DNA samples were subjected to PCR and subsequent analysis using SSCP for all exons of theOA1 gene. RT-PCR was performed on a mRNA preparation from a naevus from one patient. PCR products presenting divergent banding patterns in SSCP and from the RT-PCR were sequenced directly using cycle sequencing with fluorescent chain termination nucleotides and electrophoresis in a capillary sequencer. RESULTS The index case (patient 1, IV.1) was diagnosed with X linked OA1 at the age of 3 months because of typical clinical features: congenital nystagmus, iris translucency, macular hypoplasia, fundus hypopigmentation, normal pigmentation of skin and hair, and typical carrier signs of OA1 in his mother and maternal grandmother. Pigmentation of the iris and fundus had increased at the last examination at age 4 years. Albino misrouting was present at this age. In the maternal uncle (III.3, 51 years) who also suffered from congenital nystagmus there was clear macular hypoplasia and stromal focal hypopigmentation of the iris but no iris translucency or fundus hypopigmentation. Patient 3 (II.3, 79 years, maternal uncle of patient III.3) had congenital nystagmus and was highly myopic. The fundus appearance was typical for excessive myopia including macular changes. The iris did not show any translucency. Molecular genetic analysis revealed a novel 14 bp deletion of theOA1 gene at nt816 in exon 6. The mutation abolishes four amino acids (Leu 253-Ile-Ile-Cys) and covers the splice site. Nucleotides 814/815 are used as a new splice donor thus producing a frame shift in codon 252 and a new stop codon at codon 259. CONCLUSIONS Macular hypoplasia without clinically detectable hypopigmentation as the only sign of X linked OA1 has been reported occasionally in African-American, Japanese, and white patients. The present family shows absent hypopigmentation in two patients of a white family with a deletion in the OA1 gene. We propose a model of OA1 that allows increase of pigmentation with age. We hypothesise that macular hypoplasia in all forms of albinism depends on the extracellular DOPA level during embryogenesis, and that in OA1 postnatal normalisation of the extracellular DOPA level due to delayed distribution and membrane budding/fusion of melanosomes in melanocytes results in increasing pigmentation.