Birgit Lorenz

A gene (RPGR) with homology to the RCC1 guanine nucleotide exchange factor is mutated in X-linked retinitis pigmentosa (RP3).

X-linked retinitis pigmentosa (xIRP) is a severe progressive retinal degeneration which affects about 1 in 25,000 of the population. The most common form of xIRR RP3, has been localised to the interval between CYBB and OTC in Xp21.1 by linkage analysis and deletion mapping. Identification of microdeletions within this region has now led to the positional cloning of a gene, RPGR, that spans 60 kb of genomic DMA and is ubiquitously expressed. The predicted 90 kD protein contains in its N-terminal half a tandem repeat structure highly similar to RCC1 (regulator of chromosome condensation), suggesting an interaction with a small GTPase. The C-terminal half contains a domain, rich in acidic residues, and ends in a potential isoprenylation anchorage site. The two intragenic deletions, two nonsense and three missense mutations within conserved domains provide evidence that RPGR (retinitis pigmentosa GTPase regulator) is the RP3 gene.

An L-type calcium-channel gene mutated in incomplete X-linked congenital stationary night blindness

The locus for the incomplete form of X-linked congenital stationary night blindness (CSNB2) maps to a 1.1-Mb region in Xp11.23 between markers DXS722 and DXS255. We identified a retina-specific calcium channel α 1-subunit gene (CACNA1F) in this region, consisting of 48 exons encoding 1966 amino acids and showing high homology to L-type calcium channel α 1–subunits. Mutation analysis in 13 families with CSNB2 revealed nine different mutations in 10 families, including three nonsense and one frameshift mutation. These data indicate that aberrations in a voltage-gated calcium channel, presumably causing a decrease in neurotransmitter release from photoreceptor presynaptic terminals, are a frequent cause of CSNB2.

Positional cloning of the gene associated with X-linked juvenile retinoschisis.

X–linked juvenile retinoschisis (RS) is a recessively inherited vitreo-retinal degeneration characterized by macular pathology and intraretinal splitting of the retina. The RS gene has been localized to Xp22.2 to an approximately 1 Mb interval between DXS418 and DXS999/DXS7161. Mapping and expression analysis of expressed sequence tags have identified a novel transcript, designated XLRS1, within the centromeric RS locus that is exclusively expressed in retina. The predicted XLRS1 protein contains a highly conserved motif implicated in cell–cell interaction and thus may be active in cell adhesion processes during retinal development. Mutational analyses of XLRS1 in affected individuals from nine unrelated RS families revealed one nonsense, one frameshift, one splice acceptor and six missense mutations segregating with the disease phenotype in the respective families. These data provide strong evidence that the XLRS1 gene, when mutated, causes RS.

Mutation spectrum and splicing variants in the OPA1 gene

Abstract. Optic atrophy type 1 (OPA1, MIM 165500) is a dominantly inherited optic neuropathy that features low visual acuity leading in many cases to legal blindness. We have recently shown, with others, that mutations in the OPA1 gene encoding a dynamin-related mitochondrial protein, underlie the dominant form of optic atrophy. Here we report that OPA1 has eight mRNA isoforms as a result of the alternative splicing of exon 4 and two novel exons named 4b and 5b. In addition, we screened a cohort of 19 unrelated patients with dominant optic atrophy by direct sequencing of the 30 OPA1 exons (including exons 4b and 5b) and found mutations in 17 (89%) of them of which 8 were novel. A majority of these mutations were truncative (65%) and located in exons 8 to 28, but a number of them were amino acid changes predominantly found in the GTPase domain (exons 8 to 15). We hypothesize that at least two modifications of OPA1 may lead to dominant optic atrophy, that is alteration in GTPase activity and loss of the last seven C-terminal amino acids that putatively interact with other proteins.

A Comprehensive Survey of Sequence Variation in the ABCA4 (ABCR) Gene in Stargardt Disease and Age-Related Macular Degeneration

Stargardt disease (STGD) is a common autosomal recessive maculopathy of early and young-adult onset and is caused by alterations in the gene encoding the photoreceptor-specific ATP-binding cassette (ABC) transporter (ABCA4). We have studied 144 patients with STGD and 220 unaffected individuals ascertained from the German population, to complete a comprehensive, population-specific survey of the sequence variation in the ABCA4 gene. In addition, we have assessed the proposed role for ABCA4 in age-related macular degeneration (AMD), a common cause of late-onset blindness, by studying 200 affected individuals with late-stage disease. Using a screening strategy based primarily on denaturing gradient gel electrophoresis, we have identified in the three study groups a total of 127 unique alterations, of which 90 have not been previously reported, and have classified 72 as probable pathogenic mutations. Of the 288 STGD chromosomes studied, mutations were identified in 166, resulting in a detection rate of approximately 58%. Eight different alleles account for 61% of the identified disease alleles, and at least one of these, the L541P-A1038V complex allele, appears to be a founder mutation in the German population. When the group with AMD and the control group were analyzed with the same methodology, 18 patients with AMD and 12 controls were found to harbor possible disease-associated alterations. This represents no significant difference between the two groups; however, for detection of modest effects of rare alleles in complex diseases, the analysis of larger cohorts of patients may be required.

CNGA3 Mutations in Hereditary Cone Photoreceptor Disorders

We recently showed that mutations in the CNGA3 gene encoding the alpha-subunit of the cone photoreceptor cGMP-gated channel cause autosomal recessive complete achromatopsia linked to chromosome 2q11. We now report the results of a first comprehensive screening for CNGA3 mutations in a cohort of 258 additional independent families with hereditary cone photoreceptor disorders. CNGA3 mutations were detected not only in patients with the complete form of achromatopsia but also in incomplete achromats with residual cone photoreceptor function and (rarely) in patients with evidence for severe progressive cone dystrophy. In total, mutations were identified in 53 independent families comprising 38 new CNGA3 mutations, in addition to the 8 mutations reported elsewhere. Apparently, both mutant alleles were identified in 47 families, including 16 families with presumed homozygous mutations and 31 families with two heterozygous mutations. Single heterozygous mutations were identified in six additional families. The majority of all known CNGA3 mutations (39/46) are amino acid substitutions compared with only four stop-codon mutations, two 1-bp insertions and one 3-bp in-frame deletion. The missense mutations mostly affect amino acids conserved among the members of the cyclic nucleotide gated (CNG) channel family and cluster at the cytoplasmic face of transmembrane domains (TM) S1 and S2, in TM S4, and in the cGMP-binding domain. Several mutations were identified recurrently (e.g., R277C, R283W, R436W, and F547L). These four mutations account for 41.8% of all detected mutant CNGA3 alleles. Haplotype analysis suggests that the R436W and F547L mutant alleles have multiple origins, whereas we found evidence that the R283W alleles, which are particularly frequent among patients from Scandinavia and northern Italy, have a common origin.

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.

CNGB3 mutations account for 50% of all cases with autosomal recessive achromatopsia

Achromatopsia is a congenital, autosomal recessively inherited disorder characterized by a lack of color discrimination, low visual acuity (<0.2), photophobia, and nystagmus. Mutations in the genes for CNGA3, CNGB3, and GNAT2 have been associated with this disorder. Here, we analyzed the spectrum and prevalence of CNGB3 gene mutations in a cohort of 341 independent patients with achromatopsia. In 163 patients, CNGB3 mutations could be identified. A total of 105 achromats carried apparent homozygous mutations, 44 were compound (double) heterozygotes, and 14 patients had only a single mutant allele. The derived CNGB3 mutation spectrum comprises 28 different mutations including 12 nonsense mutations, eight insertions and/or deletions, five putative splice site mutations, and three missense mutations. Thus, the majority of mutations in the CNGB3 gene result in significantly altered and/or truncated polypeptides. Several mutations were found recurrently, in particular a 1 bp deletion, c.1148delC, which accounts for over 70% of all CNGB3 mutant alleles. In conclusion, mutations in the CNGB3 gene are responsible for approximately 50% of all patients with achromatopsia. This indicates that the CNGB3/ACHM3 locus on chromosome 8q21 is the major locus for achromatopsia in patients of European origin or descent.

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.

Constitutional RB1-Gene Mutations in Patients with Isolated Unilateral Retinoblastoma

In most patients with isolated unilateral retinoblastoma, tumor development is initiated by somatic inactivation of both alleles of the RB1 gene. However, some of these patients can transmit retinoblastoma predisposition to their offspring. To determine the frequency and nature of constitutional RB1-gene mutations in patients with isolated unilateral retinoblastoma, we analyzed DNA from peripheral blood and from tumor tissue. The analysis of tumors from 54 (71%) of 76 informative patients showed loss of constitutional heterozygosity (LOH) at intragenic loci. Three of 13 uninformative patients had constitutional deletions. For 39 randomly selected tumors, SSCP, hetero-duplex analysis, sequencing, and Southern blot analysis were used to identify mutations. Mutations were detected in 21 (91%) of 23 tumors with LOH. In 6 (38%) of 16 tumors without LOH, one mutation was detected, and in 9 (56%) of the tumors without LOH, both mutations were found. Thus, a total of 45 mutations were identified in tumors of 36 patients. Thirty-nine of the mutations-including 34 small mutations, 2 large structural alterations, and hypermethylation in 3 tumors-were not detected in the corresponding peripheral blood DNA. In 6 (17%) of the 36 patients, a mutation was detected in constitutional DNA, and 1 of these mutations is known to be associated with reduced expressivity. The presence of a constitutional mutation was not associated with an early age at treatment. In 1 patient, somatic mosaicism was demonstrated by molecular analysis of DNA and RNA from peripheral blood. In 2 patients without a detectable mutation in peripheral blood, mosaicism was suggested because 1 of the patients showed multifocal tumors and the other later developed bilateral retinoblastoma. In conclusion, our results emphasize that the manifestation and transmissibility of retinoblastoma depend on the nature of the first mutation, its time in development, and the number and types of cells that are affected.