Nobuya Sasaki

Functional validation of tensin2 SH2-PTB domain by CRISPR/Cas9-mediated genome editing

Podocytes are terminally differentiated and highly specialized cells in the glomerulus, and they form a crucial component of the glomerular filtration barrier. The ICGN mouse is a model of glomerular dysfunction that shows gross morphological changes in the podocyte foot process, accompanied by proteinuria. Previously, we demonstrated that proteinuria in ICR-derived glomerulonephritis mouse ICGN mice might be caused by a deletion mutation in the tensin2 (Tns2) gene (designated Tns2nph). To test whether this mutation causes the mutant phenotype, we created knockout (KO) mice carrying a Tns2 protein deletion in the C-terminal Src homology and phosphotyrosine binding (SH2-PTB) domains (designated Tns2ΔC) via CRISPR/Cas9-mediated genome editing. Tns2nph/Tns2ΔC compound heterozygotes and Tns2ΔC/Tns2ΔC homozygous KO mice displayed podocyte abnormalities and massive proteinuria similar to ICGN mice, indicating that these two mutations are allelic. Further, this result suggests that the SH2-PTB domain of Tns2 is required for podocyte integrity. Tns2 knockdown in a mouse podocyte cell line significantly enhanced actin stress fiber formation and cell migration. Thus, this study provides evidence that alteration of actin remodeling resulting from Tns2 deficiency causes morphological changes in podocytes and subsequent proteinuria.

Quantitative trait Loci for resistance to the congenital nephropathy in tensin 2-deficient mice.

The ICR-derived glomerulonephritis (ICGN) mouse is a chronic kidney disease (CKD) model that is characterized histologically by glomerulosclerosis, vascular sclerosis and tubulointerstitial fibrosis, and clinically by proteinuria and anemia, which are common symptoms and pathological changes associated with a variety of kidney diseases. Previously, we performed a quantitative trait locus (QTL) analysis to identify the causative genes for proteinuria in ICGN mice, and found a deletion mutation of the tensin 2 gene (Tns2nph, MGI no: 2447990). Interestingly, the congenic strain carrying the Tns2nph mutation on a C57BL/6J (B6) genetic background exhibited milder phenotypes than did ICGN mice, indicating the presence of several modifier genes controlling the disease phenotype. In this study, to identify the modifier/resistant loci for CKD progression in Tns2-deficient mice, we performed QTL analysis using backcross progenies from susceptible ICGN and resistant B6 mice. We identified a significant locus on chromosome (Chr) 2 (LODu200a=u200a5.36; 31 cM) and two suggestive loci on Chrs 10 (LODu200a=u200a2.27; 64 cM) and X (LODu200a=u200a2.65; 67 cM) with linkage to the severity of tubulointerstitial injury. One significant locus on Chr 13 (LODu200a=u200a3.49; approximately 14 cM) and one suggestive locus on Chr 2 (LODu200a=u200a2.41; 51 cM) were identified as QTLs for the severity of glomerulosclerosis. Suggestive locus in BUN was also detected in the same Chr 2 region (LODu200a=u200a2.34; 51 cM). A locus on Chr 2 (36 cM) was significantly linked with HGB (LODu200a=u200a4.47) and HCT (LODu200a=u200a3.58). Four novel epistatic loci controlling either HGB or tubulointerstitial injury were discovered. Further genetic analysis should lead to identification of CKD modifier gene(s), aiding early diagnosis and providing novel approaches to the discovery of drugs for the treatment and possible prevention of kidney disease.

Genetic background-dependent diversity in renal failure caused by the tensin2 gene deficiency in the mouse.

Tensin2 (Tns2) is thought to be a component of the cytoskeletal structures linking actin filaments with focal adhesions and is known to play a role as an intracellular signal transduction mediator through integrin in podocytes, although the mechanism by which it functions remains unclear. A Tns2-null mutation (nph) leads to massive albuminuria following podocyte foot process effacement in the ICGN mice, the origin of the mutation, and the DBA/2J (D2) mice, but not in the C57BL/6J (B6) mice or 129(+Ter)/SvJcl (129T) mice. Elucidating the reasons for these differences in diverse genetic backgrounds could help in unraveling Tns2 function in podocytes. We produced congenic mice in which Tns2(nph) was introgressed into a FVB/NJ background (FVB-Tns2(nph)), and evaluated the progression of kidney disease. FVB-Tns2(nph) mice developed albuminuria, renal fibrosis and renal anemia as seen in ICGN mice. The FVB-Tns2(nph) mice demonstrated podocyte foot process alteration under an electron microscope by as early as 4 weeks of age. This revealed that FVB strain is susceptible to Tns2-deficiency.

Molecular Evolution of the VP1 Gene in Human Norovirus GII.4 Variants in 1974–2015

Human norovirus (HuNoV) is a leading cause of viral gastroenteritis worldwide, of which GII.4 is the most predominant genotype. Unlike other genotypes, GII.4 has created various variants that escaped from previously acquired immunity of the host and caused repeated epidemics. However, the molecular evolutionary differences among all GII.4 variants, including recently discovered strains, have not been elucidated. Thus, we conducted a series of bioinformatic analyses using numerous, globally collected, full-length GII.4 major capsid (VP1) gene sequences (466 strains) to compare the evolutionary patterns among GII.4 variants. The time-scaled phylogenetic tree constructed using the Bayesian Markov chain Monte Carlo (MCMC) method showed that the common ancestor of the GII.4 VP1 gene diverged from GII.20 in 1840. The GII.4 genotype emerged in 1932, and then formed seven clusters including 14 known variants after 1980. The evolutionary rate of GII.4 strains was estimated to be 7.68 × 10−3 substitutions/site/year. The evolutionary rates probably differed among variants as well as domains [protruding 1 (P1), shell, and P2 domains]. The Osaka 2007 variant strains probably contained more nucleotide substitutions than any other variant. Few conformational epitopes were located in the shell and P1 domains, although most were contained in the P2 domain, which, as previously established, is associated with attachment to host factors and antigenicity. We found that positive selection sites for the whole GII.4 genotype existed in the shell and P1 domains, while Den Haag 2006b, New Orleans 2009, and Sydney 2012 variants were under positive selection in the P2 domain. Amino acid substitutions overlapped with putative epitopes or were located around the epitopes in the P2 domain. The effective population sizes of the present strains increased stepwise for Den Haag 2006b, New Orleans 2009, and Sydney 2012 variants. These results suggest that HuNoV GII.4 rapidly evolved in a few decades, created various variants, and altered its evolutionary rate and antigenicity.

Identification of genetic loci affecting the severity of symptoms of Hirschsprung disease in rats carrying Ednrbsl mutations by quantitative trait locus analysis.

Hirschsprung’s disease (HSCR) is a congenital disease in neonates characterized by the absence of the enteric ganglia in a variable length of the distal colon. This disease results from multiple genetic interactions that modulate the ability of enteric neural crest cells to populate developing gut. We previously reported that three rat strains with different backgrounds (susceptible AGH-Ednrbsl/sl, resistant F344-Ednrbsl/sl, and LEH-Ednrbsl/sl) but the same null mutation of Ednrb show varying severity degrees of aganglionosis. This finding suggests that strain-specific genetic factors affect the severity of HSCR. Consistent with this finding, a quantitative trait locus (QTL) for the severity of HSCR on chromosome (Chr) 2 was identified using an F2 intercross between AGH and F344 strains. In the present study, we performed QTL analysis using an F2 intercross between the susceptible AGH and resistant LEH strains to identify the modifier/resistant loci for HSCR in Ednrb-deficient rats. A significant locus affecting the severity of HSCR was also detected within the Chr 2 region. These findings strongly suggest that a modifier gene of aganglionosis exists on Chr 2. In addition, two potentially causative SNPs (or mutations) were detected upstream of a known HSCR susceptibility gene, Gdnf. These SNPs were possibly responsible for the varied length of gut affected by aganglionosis.

The high prevalence of hepatitis E virus infection in wild boars in Ibaraki Prefecture, Japan

Hepatitis E virus (HEV) is known as a causative agent of zoonosis and food poisoning. Pigs and some species of wild animals, including wild boar, are known to be a reservoir of HEV. In this study, we investigated the situation regarding HEV infection in wild boars in Ibaraki Prefecture, Japan. Serum, liver and feces samples from 68 animals were collected, and the presence or absence of HEV genomic RNA and HEV antibodies were analyzed. The viral genome was detected in samples from 7 (10.3%) animals, with all HEVs classified as genotype 3, subtype 3b. HEV antibodies were detected in samples from 28 (41%) animals. This report demonstrates for the first time the high prevalence of HEV infection in wild boars in Ibaraki Prefecture.

Genetic variation in the GDNF promoter affects its expression and modifies the severity of Hirschsprung's disease (HSCR) in rats carrying Ednrb(sl) mutations.

Glial cell line-derived neurotrophic factor (GDNF) is necessary for the migration of neural crest stem cells in the gut. However, mutations in GDNF per se are deemed neither necessary nor sufficient to cause Hirschsprungs disease (HSCR). In a previous study, a modifier locus on chromosome 2 in rats carrying Ednrb(sl) mutations was identified, and several mutations in the putative regulatory region of the Gdnf gene in AGH-Ednrb(sl) rats were detected. Specifically, the mutation -232C>T has been shown to be strongly associated with the severity of HSCR. In the present study, the influence of genetic variations on the transcription of the Gdnf gene was tested using dual-luciferase assay. Results showed that the mutation -613C>T, located near the mutation -232C>T in AGH-Ednrb(sl) rats, decreased Gdnf transcription in an in vitro dual-luciferase expression assay. These data suggested an important role of -613C in Gdnf transcription. Expression levels of the Gdnf gene may modify the severity of HSCR in rats carrying Ednrb(sl) mutations.

Novel murine model of congenital diabetes: The insulin hyposecretion mouse

Diabetic animal models have made an enormous contribution to our understanding of the etiology of diabetes and the development of new medications. The aim of the present study was to develop and characterize a novel, non‐obese murine strain with spontaneous diabetes – the insulin hyposecretion (ihs) mouse.

Genetic loci for resistance to podocyte injury caused by the tensin2 gene deficiency in mice

BackgroundTensin2 is a focal adhesion-localized multidomain protein expressed in various tissues, and its dysfunction leads to alterations in podocytes. However, these podocyte-related manifestations are dependent on murine strain. Tensin2 dysfunction results in susceptible strains developing podocyte foot process effacement and massive albuminuria, whereas podocytes in resistant strains remain almost intact. In our previous studies, quantitative trait loci analysis and congenic analysis using resistant C57BL/6J and susceptible ICGN mice identified a modifier locus associated with podocyte injury caused by tensin2 dysfunction on chromosome 2. However, the effect of this modifier locus on chromosome 2 is insufficient to explain the resistance of C57BL/6J mice to tensin2 dysfunction, indicating the existence of other modifier genes.ResultsWhereas previous studies focused on the severity of chronic kidney disease, the present study focused on podocyte injury. We performed a genome-wide linkage analysis of backcrosses between two tensin2-deficient mouse strains, B6.ICGN-Tns2nph and FVB.ICGN-Tns2nph, and detected a novel major modifier locus on chromosome 10. The combined effect of the C57BL/6J alleles of the two loci on chromosomes 2 and 10 reduced the urinary albumin excretion caused by tensin2 dysfunction to a level comparable to that of C57BL/6J mice.ConclusionsThese data indicate that the resistance to podocyte injury caused by tensin2 dysfunction is mainly produced by the effects of the modifier genes on the two loci. The identification of these modifier genes is expected to help elucidate the mechanism underlying podocyte injury.

QTL analysis of modifiers for pigmentary disorder in rats carrying Ednrb sl mutations.

Pigmentary variation in animals has been studied because of its application in genetics, evolution, and developmental biology. The large number of known color loci provides rich resource to elucidate the functional pigmentary system. Nonetheless, more color loci remain to be identified. In our previous study, we revealed that two different strains, namely, AGH rats and LEH rats, but which had the same null mutation of the Ednrb gene (Ednrbsl) showed markedly different pigmented coat ratio. This result strongly suggested that the severity of pigment abnormality was modified by genetic factor(s) in each strain. To elucidate the modifier locus of pigment disorder, we carried out whole-genome scanning for quantitative trait loci (QTLs) on 149 F2 (AGH-Ednrbslu2009×u2009LEH-Ednrbsl) rats. A highly significant QTL, constituting 26% of the total pigmentation phenotype variance, was identified in a region around D7Got23 on chromosome (Chr) 7. In addition, investigation on epistatic interaction revealed significant interactions between D7Got23 and D3Rat78 and between D7Got23 and D14Mit4. Results suggested that a modified locus on Chr 7 was mainly responsible for the variance of pigmentary disorder between AGH-Ednrbsl rats and LEH-Ednrbsl rats, and two modifier loci showing epistatic interaction may, in part, influence pigment phenotype.