A common allele in RPGRIP1L is a modifier of retinal degeneration in ciliopathies

Despite rapid advances in the identification of genes involved in disease, the predictive power of the genotype remains limited, in part owing to poorly understood effects of second-site modifiers. Here we demonstrate that a polymorphic coding variant of RPGRIP1L (retinitis pigmentosa GTPase regulator-interacting protein-1 like), a ciliary gene mutated in Meckel-Gruber (MKS) and Joubert (JBTS) syndromes, is associated with the development of retinal degeneration in individuals with ciliopathies caused by mutations in other genes. As part of our resequencing efforts of the ciliary proteome, we identified several putative loss-of-function RPGRIP1L mutations, including one common variant, A229T. Multiple genetic lines of evidence showed this allele to be associated with photoreceptor loss in ciliopathies. Moreover, we show that RPGRIP1L interacts biochemically with RPGR, loss of which causes retinal degeneration, and that the Thr229-encoded protein significantly compromises this interaction. Our data represent an example of modification of a discrete phenotype of syndromic disease and highlight the importance of a multifaceted approach for the discovery of modifier alleles of intermediate frequency and effect.This work was supported by grants R01EY007961 from the National Eye Institute (H.K. and A.S.), R01HD04260 from the National Institute of Child Health and Development (N.K.), R01DK072301, R01DK075972 (N.K.), R01DK068306, R01DK064614, R01DK069274 (F.H.), NRSA fellowship F32 DK079541 (E.E.D.) from the National Institute of Diabetes, Digestive and Kidney disorders, Intramural program of NEI (A.S.), the Macular Vision Research Foundation (N.K.), the Foundation for Fighting Blindness (H.K., S.S.B., A.S. and N.K.), the Foundation for Fighting Blindness Canada (R.K.K.), Le Fonds de la recherche en sante du Québec (FRSQ) (R.K.K.), Research to Prevent Blindness (A.S.), Harold Falls Collegiate Professorship (A.S.), the Midwest Eye Banks and Transplantation Center (H.K.), the Searle Scholars Program (M.A.B.), the Deutsche Forschungsgemeinschaft (DFG grant BE 3910/4-1; C.B.) the UK Medical Research Council (grant number G0700073; C.A.J.), NIHR Biomedical Research Centre for Ophthalmology (S.S.B.) and EU-GENORET Grant LSHG-CT-2005-512036 (S.S.B.). F.H. is an investigator of the Howard Hughes Medical Institute (HHMI) and a Doris Duke Distinguished Clinical Scientist (DDCF)

The shortening of mean telomere length during each treatment.

<p>Panel A shows the shortening of mean telomere length over the time of culture in days and Panel B the shortening of mean telomere length over the number of cell divisions occurred during the experiment, as reflected by the cumulative population doublings (CPD).</p

The Effect of Pro-Inflammatory Conditioning and/or High Glucose on Telomere Shortening of Aging Fibroblasts

<div><p>Cardiovascular disease and diabetes have been linked to shorter telomeres, but it is not yet clear which risk factors contribute to shorter telomeres in patients. Our aim was to examine whether pro-inflammatory conditioning, in combination or not with high glucose, result in a higher rate of telomere shortening during <i>in vitro</i> cellular ageing. Human fibroblasts from four donors were cultured for 90 days in: 1) medium lacking ascorbic acid only, 2) 10 mM buthionine sulphoximine (BSO) (pro-oxidant), 3) 25 mM D-glucose, 4) 1 ng/ml IL1B and 5) 25 mM D-glucose+1 ng/ml IL1B. Telomere length was measured with qPCR and intracellular reactive oxygen species (ROS) content and cell death with flow cytometry. Cultures treated with high glucose and BSO displayed a significantly lower growth rate, and cultures treated with IL1B showed a trend towards a higher growth rate, compared to the control [Glucose:0.14 PD/day, p<0.001, BSO: 0.11 PD/day, p = 0.006 and IL1B: 0.19 PD/day, p = 0.093 vs. Control:0.16 PD/day]. Telomere shortening with time was significantly accelerated in cultures treated with IL1B compared to the control [IL1B:−0.8%/day (95%CI:−1.1, −0.5) vs. Control:−0.6%/day (95%CI:−0.8, −0.3), p = 0.012]. The hastening of telomere shortening by IL1B was only in part attenuated after adjustment for the number of cell divisions [IL1B:−4.1%/PD (95%CI:−5.7, −2.4) vs. Control:−2.5%/PD (95%CI:−4.4, −0.7), p = 0.067]. The intracellular ROS content displayed 69% increase (p = 0.033) in BSO compared to the control. In aging fibroblasts, pro-inflammatory conditioning aggravates the shortening of telomeres, an effect which was only in part driven by increased cell turnover. High glucose alone did not result in greater production of ROS or telomere shortening.</p></div

The cell death and intracellular ROS content as measured by flow cytometry after 7 days of culture in each treatment.

<p>The percentage of dying and dead cells is shown in panel A and the intracellular ROS content of viable cells in panel B. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073756#pone-0073756-g004" target="_blank"><i>Figure 4</i></a><i>footnote</i>: Due to the small number of measurements, normal distribution cannot be inferred. Thus, the graph represents median values, with inter-quartile range as error bars. *P value is obtained from Kruskal-Wallis test. **P value is obtained from Mann-Whitney test. Mann-Whitney tests between the percentages of dying and dead cells in each of the treatments compared with the control were non-significant (p = 0.121).</p

Percentage changes in mean telomere length and mtDNA copies per nucleus over the time of culture (days) or cell divisions occurred during the experiment (CPD).

<p>%: percentage change, CPD: cumulative population doublings, mtDNA: mitochondrial DNA, IL1B: interleukin 1B, BSO: buthionine sulphoximine.</p><p>Percentage changes (%) in telomere length or mtDNA with days or CPD are obtained from separate regression models for each treatment adjusted for donor.</p><p>CPD per days are also obtained from separate regression models for each treatment adjusted for donor.</p><p>P values for the percentage changes (%) over days or CPD are obtained from regression models including all treatments as dummy variables compared to the control, adjusting for donor.</p

The change in the number of mitochondria per cell, as reflected by the copy number of mtDNA per nucleus, during each treatment.

<p>Panel A shows the change in the number of mitochondria per cell over the time of culture in days and Panel B the change in the number of mitochondria per cell over the number of cell divisions occurred during the experiment [i.e. cumulative population doublings (CPD)].</p

The cumulative population doublings (CPD) over the time of culture in each treatment (growth rate).

<p>The cumulative population doublings (CPD) over the time of culture in each treatment (growth rate).</p

Mutations in TOPORS Cause Autosomal Dominant Retinitis Pigmentosa with Perivascular Retinal Pigment Epithelium Atrophy

We report mutations in the gene for topoisomerase I–binding RS protein (TOPORS) in patients with autosomal dominant retinitis pigmentosa (adRP) linked to chromosome 9p21.1 (locus RP31). A positional-cloning approach, together with the use of bioinformatics, identified TOPORS (comprising three exons and encoding a protein of 1,045 aa) as the gene responsible for adRP. Mutations that include an insertion and a deletion have been identified in two adRP-affected families—one French Canadian and one German family, respectively. Interestingly, a distinct phenotype is noted at the earlier stages of the disease, with an unusual perivascular cuff of retinal pigment epithelium atrophy, which was found surrounding the superior and inferior arcades in the retina. TOPORS is a RING domain–containing E3 ubiquitin ligase and localizes in the nucleus in speckled loci that are associated with promyelocytic leukemia bodies. The ubiquitous nature of TOPORS expression and a lack of mutant protein in patients are highly suggestive of haploinsufficiency, rather than a dominant negative effect, as the molecular mechanism of the disease and make rescue of the clinical phenotype amenable to somatic gene therapy