Indirect exclusion of four candidate genes for generalized progressive retinal atrophy in several breeds of dogs

BACKGROUND: Generalized progressive retinal atrophy (gPRA) is a hereditary ocular disorder with progressive photoreceptor degeneration in dogs. Four retina-specific genes, ATP binding cassette transporter retina (ABCA4), connexin 36 (CX36), c-mer tyrosin kinase receptor (MERTK) and photoreceptor cell retinol dehydrogenase (RDH12) were investigated in order to identify mutations leading to autosomal recessive (ar) gPRA in 29 breeds of dogs. RESULTS: Mutation screening was performed initially by PCR and single strand conformation polymorphism (SSCP) analysis, representing a simple method with comparatively high reliability for identification of sequence variations in many samples. Conspicuous banding patterns were analyzed via sequence analyses in order to detect the underlying nucleotide variations. No pathogenetically relevant mutations were detected in the genes ABCA4, CX36, MERTK and RDH12 in 71 affected dogs of 29 breeds. Yet 30 new sequence variations were identified, both, in the coding regions and intronic sequences. Many of the sequence variations were in heterozygous state in affected dogs. CONCLUSION: Based on the ar transmittance of gPRA in the breeds investigated, informative sequence variations provide evidence allowing indirect exclusion of pathogenetic mutations in the genes ABCA4 (for 9 breeds), CX36 (for 12 breeds), MERTK (for all 29 breeds) and RDH12 (for 9 breeds)

Cone rod dystrophies

Cone rod dystrophies (CRDs) (prevalence 1/40,000) are inherited retinal dystrophies that belong to the group of pigmentary retinopathies. CRDs are characterized by retinal pigment deposits visible on fundus examination, predominantly localized to the macular region. In contrast to typical retinitis pigmentosa (RP), also called the rod cone dystrophies (RCDs) resulting from the primary loss in rod photoreceptors and later followed by the secondary loss in cone photoreceptors, CRDs reflect the opposite sequence of events. CRD is characterized by primary cone involvement, or, sometimes, by concomitant loss of both cones and rods that explains the predominant symptoms of CRDs: decreased visual acuity, color vision defects, photoaversion and decreased sensitivity in the central visual field, later followed by progressive loss in peripheral vision and night blindness. The clinical course of CRDs is generally more severe and rapid than that of RCDs, leading to earlier legal blindness and disability. At end stage, however, CRDs do not differ from RCDs. CRDs are most frequently non syndromic, but they may also be part of several syndromes, such as Bardet Biedl syndrome and Spinocerebellar Ataxia Type 7 (SCA7). Non syndromic CRDs are genetically heterogeneous (ten cloned genes and three loci have been identified so far). The four major causative genes involved in the pathogenesis of CRDs are ABCA4 (which causes Stargardt disease and also 30 to 60% of autosomal recessive CRDs), CRX and GUCY2D (which are responsible for many reported cases of autosomal dominant CRDs), and RPGR (which causes about 2/3 of X-linked RP and also an undetermined percentage of X-linked CRDs). It is likely that highly deleterious mutations in genes that otherwise cause RP or macular dystrophy may also lead to CRDs. The diagnosis of CRDs is based on clinical history, fundus examination and electroretinogram. Molecular diagnosis can be made for some genes, genetic counseling is always advised. Currently, there is no therapy that stops the evolution of the disease or restores the vision, and the visual prognosis is poor. Management aims at slowing down the degenerative process, treating the complications and helping patients to cope with the social and psychological impact of blindness

Severe autosomal recessive retinitis pigmentosa maps to chromosome 1p13.3–p21.2 between D1S2896 and D1S457 but outside ABCA4

A severe form of autosomal recessive retinitis pigmentosa (arRP) was identified in a large Pakistani family ascertained in the Punjab province of Pakistan. All affected individuals in the family had night blindness in early childhood, early complete loss of useful vision, and typical RP fundus changes plus macular degeneration. After exclusion of known arRP loci, a genome-wide scan was performed using microsatellite markers at about 10 cM intervals and calculating two-point lod scores. PCR cycle dideoxynucleotide sequencing was used to sequence candidate genes inside the linked region for mutations. RP in this family shows linkage to markers in a 10.5 cM (8.9 Mbp) region of chromosome 1p13.3–p21.2 between D1S2896 and D1S457. D1S485 yields the highest lod score of 6.54 at θ=0. Sequencing the exons and intron–exon boundaries of five candidate genes and six ESTs in this region, OLFM3, GNAI3, LOC126987, FLJ25070, DKFZp586G0123, AV729694, BU662869, BU656110, BU171991, BQ953690, and CA397743, did not identify any causative mutations. This novel locus lies approximately 4.9 cM (7.1 Mbp) from ABCA4, which is excluded from the linked region. Identification and study of this gene may help to elucidate the phenotypic diversity of arRP mapping to this region.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47597/1/439_2005_Article_54.pd

The efficacy of microarray screening for autosomal recessive retinitis pigmentosa in routine clinical practice

Purpose: To determine the efficacy of multiple versions of a commercially available arrayed primer extension (APEX) microarray chip for autosomal recessive retinitis pigmentosa (arRP). Methods: We included 250 probands suspected of arRP who were genetically analyzed with the APEX microarray between January 2008 and November 2013. The mode of inheritance had to be autosomal recessive according to the pedigree (including isolated cases). If the microarray identified a heterozygous mutation, we performed Sanger sequencing of exons and exon-intron boundaries of that specific gene. The efficacy of this microarray chip with the additional Sanger sequencing approach was determined by the percentage of patients that received a molecular diagnosis. We also collected data from genetic tests other than the APEX analysis for arRP to provide a detailed description of the molecular diagnoses in our study cohort. Results: The APEX microarray chip for arRP identified the molecular diagnosis in 21 (8.5%) of the patients in our cohort. Additional Sanger sequencing yielded a second mutation in 17 patients (6.8%), thereby establishing the molecular diagnosis. In total, 38 patients (15.2%) received a molecular diagnosis after analysis using the microarray and additional Sanger sequencing approach. Further genetic analyses after a negative result of the arRP microarray (n = 107) resulted in a molecular diagnosis of arRP (n = 23), autosomal dominant RP (n = 5), X-linked RP (n = 2), and choroideremia (n = 1). Conclusions: The efficacy of the commercially available APEX microarray chips for arRP appears to be low, most likely caused by the limitations of this technique and the genetic and allelic heterogeneity of RP. Diagnostic yields up to 40% have been reported for next-generation sequencing (NGS) techniques that, as expected, thereby outperform targeted APEX analysis

The spectrum of retinal phenotypes caused by mutations in the ABCA4 gene.

Contains fulltext : 47685.pdf (publisher's version ) (Closed access)BACKGROUND: The majority of studies on the retina-specific ATP-binding cassette transporter (ABCA4) gene have focussed on molecular genetic analysis; comparatively few studies have described the clinical aspects of ABCA4-associated retinal disorders. In this study, we demonstrate the spectrum of retinal dystrophies associated with ABCA4 gene mutations. METHODS: Nine well-documented patients representing distinct phenotypes in the continuum of ABCA4-related disorders were selected. All patients received an extensive ophthalmologic evaluation, including kinetic perimetry, fluorescein angiography, and electroretinography (ERG). Mutation analysis had been performed previously with the genotyping microarray (ABCR400 chip) and/or single-strand conformation polymorphism analysis in combination with direct DNA sequencing. RESULTS: In all patients, at least one pathologic ABCA4 mutation was identified. Patient 10034 represented the mild end of the phenotypic spectrum, demonstrating exudative age-related macular degeneration (AMD). Patient 24481 received the diagnosis of late-onset fundus flavimaculatus (FFM), patient 15168 demonstrated the typical FFM phenotype, and patient 19504 had autosomal recessive Stargardt disease (STGD1). Patients 11302 and 7608 exhibited progression from FFM/STGD1 to cone-rod dystrophy (CRD). A more typical CRD phenotype was found in patients 15680 and 12608. Finally, the most severe ABCA4-associated phenotype was retinitis pigmentosa (RP) in patient 11366. This phenotype was characterised by extensive atrophy with almost complete loss of peripheral and central retinal functions. CONCLUSION: We describe nine patients during different stages of disease progression; together, these patients form a continuum of ABCA4-associated phenotypes. Besides characteristic disorders such as FFM/STGD1, CRD and RP, intermediate phenotypes may be encountered. Moreover, as the disease progresses, marked differences may be observed between initially comparable phenotypes. In contrast, distinctly different phenotypes may converge to a similar final stage, characterised by extensive chorioretinal atrophy and very low visual functions. The identified ABCA4 mutations in most, but not all, patients were compatible with the resulting phenotypes, as predicted by the genotype-phenotype model for ABCA4-associated disorders. With the advent of therapeutic options, recognition by the general ophthalmologist of the various retinal phenotypes associated with ABCA4 mutations is becoming increasingly important