Correction: A Genome-Wide Association Study for Diabetic Retinopathy in a Japanese Population: Potential Association with a Long Intergenic Non-Coding RNA

<p>Correction: A Genome-Wide Association Study for Diabetic Retinopathy in a Japanese Population: Potential Association with a Long Intergenic Non-Coding RNA</p

Regional association plot of the region around the SNP rs9362054.

<p>The -log₁₀P values of the Fisher's exact test under the allele model in the GWAS of diabetic retinopathy in stage 1 were plotted against relative chromosomal locations (UCSC human genome 19).</p

SNPs showing associations with diabetic retinopathy (DR) in combined stages 1 and 2 samples with <i>P</i><10⁻³ using the allele model.

<p>SNPs showing associations with diabetic retinopathy (DR) in combined stages 1 and 2 samples with <i>P</i><10⁻³ using the allele model.</p

Summary of the study strategy.

<p>Summary of the study strategy.</p

Clinical features of Japanese patients with type 2 diabetes.

<p>DR: diabetic retinopathy, PDR: proliferative diabetic retinopathy; DN: diabetic nephropathy, SMU: Saitama Medical University Hospital, SSI: Saitama Social Insurance Hospital, KU: Keio University Hospital, NCGM: National Center for Global Health and Medicine Center Hospital.</p><p>Data are % or mean +− SD.</p>a<p><i>P</i><0.05 vs patients without DR.</p><p>Clinical features of Japanese patients with type 2 diabetes.</p

Associations of rs9362054 in <i>RP1-90L14.1</i> with diabetic retinopathy (DR), proliferative diabetic retinopathy (PDR) and diabetic nephropathy (DN) in each stage and combined stages 1, 2 and 3, using logistic regression under genotype, dominant and recessive models after adjusting for sex, duration of diabetes and HbA1c.

a<p>With additive genetic effect.</p><p>Associations of rs9362054 in <i>RP1-90L14.1</i> with diabetic retinopathy (DR), proliferative diabetic retinopathy (PDR) and diabetic nephropathy (DN) in each stage and combined stages 1, 2 and 3, using logistic regression under genotype, dominant and recessive models after adjusting for sex, duration of diabetes and HbA1c.</p

Manhattan plot for the GWAS of diabetic retinopathy in stage 1.

<p><i>P</i> values of the Fisher's exact test under the allele model for SNPs in 446 patients were shown.</p

Tysnd1 Deficiency in Mice Interferes with the Peroxisomal Localization of PTS2 Enzymes, Causing Lipid Metabolic Abnormalities and Male Infertility

<div><p>Peroxisomes are subcellular organelles involved in lipid metabolic processes, including those of very-long-chain fatty acids and branched-chain fatty acids, among others. Peroxisome matrix proteins are synthesized in the cytoplasm. Targeting signals (PTS or peroxisomal targeting signal) at the C-terminus (PTS1) or N-terminus (PTS2) of peroxisomal matrix proteins mediate their import into the organelle. In the case of PTS2-containing proteins, the PTS2 signal is cleaved from the protein when transported into peroxisomes. The functional mechanism of PTS2 processing, however, is poorly understood. Previously we identified Tysnd1 (Trypsin domain containing 1) and biochemically characterized it as a peroxisomal cysteine endopeptidase that directly processes PTS2-containing prethiolase Acaa1 and PTS1-containing Acox1, Hsd17b4, and ScpX. The latter three enzymes are crucial components of the very-long-chain fatty acids β-oxidation pathway. To clarify the <em>in vivo</em> functions and physiological role of Tysnd1, we analyzed the phenotype of <em>Tysnd1^−/−</em> mice. Male <em>Tysnd1^−/−</em> mice are infertile, and the epididymal sperms lack the acrosomal cap. These phenotypic features are most likely the result of changes in the molecular species composition of choline and ethanolamine plasmalogens. <em>Tysnd1^−/−</em> mice also developed liver dysfunctions when the phytanic acid precursor phytol was orally administered. Phyh and Agps are known PTS2-containing proteins, but were identified as novel Tysnd1 substrates. Loss of <em>Tysnd1</em> interferes with the peroxisomal localization of Acaa1, Phyh, and Agps, which might cause the mild Zellweger syndrome spectrum-resembling phenotypes. Our data established that peroxisomal processing protease Tysnd1 is necessary to mediate the physiological functions of PTS2-containing substrates.</p> </div

Male <i>Tysnd1</i>^−/− mice are infertile.

<p>A. Semi-thin (8–10 micron) testes sections of 20 weeks old <i>Tysnd1</i>^+/+ and <i>Tysnd1^−/−</i> mice were stained with hematoxylin-eosin. Abnormal, round sperm heads are visible in the seminiferous tubules of <i>Tysnd1</i>^−/− mice. B. Abnormal morphology of <i>Tysnd1</i>^−/− sperms. Epididymal sperms of 15 weeks-old <i>Tysnd1</i>^+/+ and <i>Tysnd1</i>^−/− mice were stained with mitochondrial stains (MitoFluor Red) and nuclear staining (DAPI). Scale bar = 20 µm. C. Percentage of sperms showing normal morphology in <i>Tysnd1</i>^+/+ (n = 4), <i>Tysnd1</i>^+/− (n = 4) and <i>Tysnd1</i>^−/− (n = 5) mice. Each error bar represents the mean ± SE. D. Anti-MN9 antibody immunostaining and Hoechst nuclear staining of epididymal sperms isolated from a 20 weeks old <i>Tysnd1</i>^−/− mouse (red: acrosome and blue: nucleus). Arrow heads and arrows indicate abnormal round-headed sperms and normal sperms, respectively. Scale bar = 5 µm. E. Acrosomes of a semi-thin testis section from a 10 weeks old <i>Tysnd1</i>^−/− and a heterozygous control mouse were stained with PNA-FITC (green) and Hoechst nuclear stain (blue). F. EM image of a <i>Tysnd1</i>^−/− caudal epididymal sperm. The round-headed sperm lacks the acrosome and shows an abnormal mitochondrial sheath (M) around the nucleus (N). Scale bar = 1 µm. G. EM image showing normal spermatogenesis in <i>Tysnd1</i>^+/− male mice. S7: step 7 round spermatid; S16: step 16 elongated spermatid. Acrosomes (A) are normally formed. Scale bar = 2 µm. H. EM image of <i>Tysnd1</i>^−/− elongated spermatid. S16: step 16 spermatid. In some spermatids the acrosome (*) is detached from the nucleus (N). Scale bar = 2 µm.</p

Generation of <i>Tysnd1</i>^−/− mice.

<p>A. Map of <i>Tysnd1</i>^−/− targeting constructs. P1, P2 and P3 indicate primers used for genotyping by PCR. B. Identification of genotyping by PCR. Wild type and <i>Tysnd1</i>^−/− genotype were identified by 237 bp and 339 bp PCR products, respectively. C. Relative expression level of <i>Tysnd1</i> mRNA measured by quantitative real-time PCR in liver. D. Tysnd1 protein was absent in the liver of <i>Tysnd1</i>^−/− mice as shown by Western blotting using anti-Tysnd1 antibody. E. The expression of known Tysnd1 substrates in the liver homogenate was detected by Western blotting using anti-Acox1, -ScpX/Scp2, -Hsd17b4 and -Acaa1 antibodies. Processed forms of Tysnd1 substrates were not detected in <i>Tysnd1</i>^−/− mice. Arrows indicate the processed form of each enzyme. F. Peroxisomal β-oxidation activity was measured by [1-C¹⁴]lignoceric acids in 15 weeks old control diet-fed (CE2, Clea Japan) male mice liver homogenate. ***<i>p</i><0.001. Each error bar represents the mean ± SE in <i>n</i> = 3.</p