Ritu Khanna

A variant of mitochondrial protein LOC387715/ARMS2, not HTRA1, is strongly associated with age-related macular degeneration

Genetic variants at chromosomes 1q31-32 and 10q26 are strongly associated with susceptibility to age-related macular degeneration (AMD), a common blinding disease of the elderly. We demonstrate, by evaluating 45 tag SNPs spanning HTRA1, PLEKHA1, and predicted gene LOC387715/ARMS2, that rs10490924 SNP alone, or a variant in strong linkage disequilibrium, can explain the bulk of association between the 10q26 chromosomal region and AMD. A previously suggested causal SNP, rs11200638, and other examined SNPs in the region are only indirectly associated with the disease. Contrary to previous reports, we show that rs11200638 SNP has no significant impact on HTRA1 promoter activity in three different cell lines, and HTRA1 mRNA expression exhibits no significant change between control and AMD retinas. However, SNP rs10490924 shows the strongest association with AMD (P = 5.3 × 10−30), revealing an estimated relative risk of 2.66 for GT heterozygotes and 7.05 for TT homozygotes. The rs10490924 SNP results in nonsynonymous A69S alteration in the predicted protein LOC387715/ARMS2, which has a highly conserved ortholog in chimpanzee, but not in other vertebrate sequences. We demonstrate that LOC387715/ARMS2 mRNA is detected in the human retina and various cell lines and encodes a 12-kDa protein, which localizes to the mitochondrial outer membrane when expressed in mammalian cells. We propose that rs10490924 represents a major susceptibility variant for AMD at 10q26. A likely biological mechanism is that the A69S change in the LOC387715/ARMS2 protein affects its presumptive function in mitochondria.

CFH haplotypes without the Y402H coding variant show strong association with susceptibility to age-related macular degeneration

In developed countries, age-related macular degeneration is a common cause of blindness in the elderly. A common polymorphism, encoding the sequence variation Y402H in complement factor H (CFH), has been strongly associated with disease susceptibility. Here, we examined 84 polymorphisms in and around CFH in 726 affected individuals (including 544 unrelated individuals) and 268 unrelated controls. In this sample, 20 of these polymorphisms showed stronger association with disease susceptibility than the Y402H variant. Further, no single polymorphism could account for the contribution of the CFH locus to disease susceptibility. Instead, multiple polymorphisms defined a set of four common haplotypes (of which two were associated with disease susceptibility and two seemed to be protective) and multiple rare haplotypes (associated with increased susceptibility in aggregate). Our results suggest that there are multiple disease susceptibility alleles in the region and that noncoding CFH variants play a role in disease susceptibility.

Mutations in a BTB-Kelch Protein, KLHL7, Cause Autosomal-Dominant Retinitis Pigmentosa

Retinitis pigmentosa (RP) refers to a genetically heterogeneous group of progressive neurodegenerative diseases that result in dysfunction and/or death of rod and cone photoreceptors in the retina. So far, 18 genes have been identified for autosomal-dominant (ad) RP. Here, we describe an adRP locus (RP42) at chromosome 7p15 through linkage analysis in a six-generation Scandinavian family and identify a disease-causing mutation, c.449G-->A (p.S150N), in exon 6 of the KLHL7 gene. Mutation screening of KLHL7 in 502 retinopathy probands has revealed three different missense mutations in six independent families. KLHL7 is widely expressed, including expression in rod photoreceptors, and encodes a 75 kDa protein of the BTB-Kelch subfamily within the BTB superfamily. BTB-Kelch proteins have been implicated in ubiquitination through Cullin E3 ligases. Notably, all three putative disease-causing KLHL7 mutations are within a conserved BACK domain; homology modeling suggests that mutant amino acid side chains can potentially fill the cleft between two helices, thereby affecting the ubiquitination complexes. Mutations in an identical region of another BTB-Kelch protein, gigaxonin, have previously been associated with giant axonal neuropathy. Our studies suggest an additional role of the ubiquitin-proteasome protein-degradation pathway in maintaining neuronal health and in disease.

RDH12 Activity and Effects on Retinoid Processing in the Murine Retina

RDH12 mutations are responsible for early-onset autosomal recessive retinal dystrophy, which results in profound retinal pathology and severe visual handicap in patients. To investigate the function of RDH12 within the network of retinoid dehydrogenases/reductases (RDHs) present in retina, we studied the retinal phenotype of Rdh12-deficient mice. In vivo rates of all-trans-retinal reduction and 11-cis-retinal formation during recovery from bleaching were similar in Rdh12-deficient and wild-type mice matched for an Rpe65 polymorphism that impacts visual cycle efficiency. However, retinal homogenates from Rdh12-deficient mice exhibited markedly decreased capacity to reduce exogenous retinaldehydes in vitro. Furthermore, in vivo levels of the bisretinoid compound diretinoid-pyridinium-ethanolamine (A2E) were increased in Rdh12-deficient mice of various genetic backgrounds. Conversely, in vivo levels of retinoic acid and total retinol were significantly decreased. Rdh12 transcript levels in wild-type mice homozygous for the Rpe65-Leu450 polymorphism were greater than in Rpe65-Met450 mice and increased during postnatal development in wild-type mice and Nrl-deficient mice having an all-cone retina. Rdh12-deficient mice did not exhibit increased retinal degeneration relative to wild-type mice at advanced ages, when bred on the light-sensitive BALB/c background, or when heterozygous for a null allele of superoxide dismutase 2 (Sod2+/−). Our findings suggest that a critical function of RDH12 is the reduction of all-trans-retinal that exceeds the reductive capacity of the photoreceptor outer segments.

Distinct Signature of Altered Homeostasis in Aging Rod Photoreceptors: Implications for Retinal Diseases

Background Advanced age contributes to clinical manifestations of many retinopathies and represents a major risk factor for age-related macular degeneration, a leading cause of visual impairment and blindness in the elderly. Rod photoreceptors are especially vulnerable to genetic defects and changes in microenvironment, and are among the first neurons to die in normal aging and in many retinal degenerative diseases. The molecular mechanisms underlying rod photoreceptor vulnerability and potential biomarkers of the aging process in this highly specialized cell type are unknown. Methodology/Principal Findings To discover aging-associated adaptations that may influence rod function, we have generated gene expression profiles of purified rod photoreceptors from mouse retina at young adult to early stages of aging (1.5, 5, and 12 month old mice). We identified 375 genes that showed differential expression in rods from 5 and 12 month old mouse retina compared to that of 1.5 month old retina. Quantitative RT-PCR experiments validated expression change for a majority of the 25 genes that were examined. Macroanalysis of differentially expressed genes using gene class testing and protein interaction networks revealed overrepresentation of cellular pathways that are potentially photoreceptor-specific (angiogenesis and lipid/retinoid metabolism), in addition to age-related pathways previously described in several tissue types (oxidative phosphorylation, stress and immune response). Conclusions/Significance Our study suggests a progressive shift in cellular homeostasis that may underlie aging-associated functional decline in rod photoreceptors and contribute to a more permissive state for pathological processes involved in retinal diseases.

A role for prenylated rab acceptor 1 in vertebrate photoreceptor development

BackgroundThe rd1 mouse retina is a well-studied model of retinal degeneration where rod photoreceptors undergo cell death beginning at postnatal day (P) 10 until P21. This period coincides with photoreceptor terminal differentiation in a normal retina. We have used the rd1 retina as a model to investigate early molecular defects in developing rod photoreceptors prior to the onset of degeneration.ResultsUsing a microarray approach, we performed gene profiling comparing rd1 and wild type (wt) retinas at four time points starting at P2, prior to any obvious biochemical or morphological differences, and concluding at P8, prior to the initiation of cell death. Of the 143 identified differentially expressed genes, we focused on Rab acceptor 1 (Rabac1), which codes for the protein Prenylated rab acceptor 1 (PRA1) and plays an important role in vesicular trafficking. Quantitative RT-PCR analysis confirmed reduced expression of PRA1 in rd1 retina at all time points examined. Immunohistochemical observation showed that PRA1-like immunoreactivity (LIR) co-localized with the cis-Golgi marker GM-130 in the photoreceptor as the Golgi translocated from the perikarya to the inner segment during photoreceptor differentiation in wt retinas. Diffuse PRA1-LIR, distinct from the Golgi marker, was seen in the distal inner segment of wt photoreceptors starting at P8. Both plexiform layers contained PRA1 positive punctae independent of GM-130 staining during postnatal development. In the inner retina, PRA1-LIR also colocalized with the Golgi marker in the perinuclear region of most cells. A similar pattern was seen in the rd1 mouse inner retina. However, punctate and significantly reduced PRA1-LIR was present throughout the developing rd1 inner segment, consistent with delayed photoreceptor development and abnormalities in Golgi sorting and vesicular trafficking.ConclusionsWe have identified genes that are differentially regulated in the rd1 retina at early time points, which may give insights into developmental defects that precede photoreceptor cell death. This is the first report of PRA1 expression in the retina. Our data support the hypothesis that PRA1 plays an important role in vesicular trafficking between the Golgi and cilia in differentiating and mature rod photoreceptors.

Global expression profiling of peripheral Qa-1-restricted CD8αα+TCRαβ+ regulatory T cells reveals innate-like features: implications for immune-regulatory repertoire.

Among peripheral regulatory T cells, CD8(+) T cells also play an important role in the maintenance of immune homeostasis. A subset of CD8(+) Treg that express αβ T cell receptor (TCR) and CD8αα homodimers can recognize TCR-derived peptides in the context of the class Ib MHC molecule Qa-1. To gain a better understanding of the nature and phenotype of CD8αα(+)TCRαβ+ Treg, a global gene expression profiling using microarray, real-time quantitative polymerase chain reaction, and flow-cytometric analysis was performed using functional Treg clones and lines. The study findings show that CD8(+) Treg shared gene profile expressed by innate-like lymphocytes, including murine intraepithelial lymphocytes and thymic CD8αα(+)TCRαβ+ T-cell populations. In addition, this subset displays differential expression of several key regulatory molecules, including CD200. CD8αα(+) Treg expressed higher levels of a number of natural killer cell-related receptors and molecules belonging to the TNF superfamily. Collectively, peripheral class Ib-reactive CD8αα(+)TCRαβ+ T cells represent a unique regulatory population different from class Ia major histocompatibility complex-restricted conventional T cells. These studies have important implications for the regulatory mechanisms mediated by the CD8(+) Treg population in general.