Genetic Background Strongly Modifies the Severity of Symptoms of Hirschsprung Disease, but Not Hearing Loss in Rats Carrying Ednrbsl Mutations

Hirschsprung disease (HSCR) is thought to result as a consequence of multiple gene interactions that modulate the ability of enteric neural crest cells to populate the developing gut. However, it remains unknown whether the single complete deletion of important HSCR-associated genes is sufficient to result in HSCR disease. In this study, we found that the null mutation of the Ednrb gene, thought indispensable for enteric neuron development, is insufficient to result in HSCR disease when bred onto a different genetic background in rats carrying Ednrbsl mutations. Moreover, we found that this mutation results in serious congenital sensorineural deafness, and these strains may be used as ideal models of Waardenburg Syndrome Type 4 (WS4). Furthermore, we evaluated how the same changed genetic background modifies three features of WS4 syndrome, aganglionosis, hearing loss, and pigment disorder in these congenic strains. We found that the same genetic background markedly changed the aganglionosis, but resulted in only slight changes to hearing loss and pigment disorder. This provided the important evidence, in support of previous studies, that different lineages of neural crest-derived cells migrating along with various pathways are regulated by different signal molecules. This study will help us to better understand complicated diseases such as HSCR and WS4 syndrome

ラット頭巾斑表現型の遺伝学的解析

The hooded phenotype showing non-pigmented hairs in the abdominal skin is one of the coat color phenotypes seen peculiarly in the laboratory rat. The hooded locus showing autosomal recessive inheritance has been mapped on Chr 14 and that the hooded phenotype receives modification by hooded-modifier gene showing linkage to the hooded locus. Thus, I have conducted genetic studies to identify a gene responsible for the hooded locus and genes responsible for hooded-modifiers. In part 1 of this study, I narrowed critical region of the hooded locus and revealed that only Kit gene, known as a marker of melanocyte and one of coat color genes, exists in this region through genetic fine mapping using backcrosses from feral rat-derived inbred strain IS and hooded phenotype strain, LEA. Although a G to C transversion was observed in exon 2 of the Kit gene, it was synonymous substitution. Further, the expressions of Kit mRNA were not different in fetal neural tubes and neonatal and adult skins between IS and LEA rats. Furthermore, Kit-positive cells were observed in LEA rat abdominal skin in spite of the absence of melanin in this region. These results suggest that the synthesis of melanin is impaired possibly due to the malfunction of Kit-expressed melanocytes residing in the non-pigmented hair follicles of hooded phenotype rats. However, substantial mutation of the Kit gene and the mechanisms by which Kit impairs the function of melanocytes in non-pigmented hair follicle remain unknown. In part 2 of this study, I carried out genetic linkage studies using BN and LEA rats to clarify genetic control in the extent of the hooded phenotype. A genome-wide scan was conducted on 152 F2 rats for linkage with ratio of pigmented coat area for the dorsal, ventral, and total regions. The result indicated that a major QTL was mapped to D14Got40, which is the microsatellite marker closely present to the hooded locus. In addition, another QTL, D17Rat2 showing highly significant linkage was also detected on Chr 17 in dorsal region phenotype as well as a QTL showing suggestive linkage on Chr15 in ventral region phenotype. I further investigated a genome-wide scan for epistatic interactions and detected significant interactions between D14Got40 and D20Mit1, and between D14Got40 and D17Rat2 in dorsal region phenotype. These results suggest that the hooded locus regulates the extent of the hooded phenotype with some modifier genes. This study proposes that Kit is a strong candidate gene responsible for the hooded locus and some genetic loci modify the extent of the hooded phenotype, although the precise mechanisms of them are still unclear. Thus, further study is necessary to understand the mechanisms by which the hooded phenotype appears with various extent of pigmented ratio in the rat.げっ歯類における被毛色研究は、その明らかな表現型故に古くから研究がなされており、現在までに約150個の遺伝子座と約1,000個の対立遺伝子の存在が明らかとなっている。一般的に被毛色は、発生初期段階において神経堤より派生する神経堤細胞から分化、誘導されるメラノサイトが産生するメラニンによって規定される。神経堤細胞はメラノサイト以外にも骨細胞や軟骨細胞、末梢神経細胞、内分泌細胞など様々な系列に分化できる多能性を持っており、ワーデンブルグ症候群に代表される神経堤細胞の分化異常の疾患では、被毛色異常と同時に難聴や腸管神経節欠損など、皮膚以外の臓器にも異常が見られることが報告されている。頭巾斑表現型はラット特有の被毛色表現型で、その遺伝子座は第14染色体に座位し常染色体劣性遺伝様式を示すことが明らかとなっている。頭巾斑遺伝子座には複数の対立遺伝子が存在することや、頭巾斑の表現型を修飾する遺伝子座が存在することが報告されているが、それらの責任遺伝子は未だに明らかとなっていない。また、頭巾斑表現型は被毛色のみに現れ、その他の神経堤細胞由来の細胞系列は正常であると考えられる。従って、頭巾斑遺伝子座の解析は被毛色異常のメカニズムを明らかにするだけでなく、神経堤細胞の分化、遊走のメカニズム解明にも有用であると考えら58れる。よって本研究では、頭巾斑表現型ならびに頭巾斑表現型修飾因子の遺伝学的解析を試みた。第一章では野生型のISラットと頭巾斑表現型のLEAラットの戻し交配個体795匹を用いて詳細マッピングを行った。その結果、頭巾斑遺伝子座はマイクロサテライトマーカーD14Hok1と強く連鎖し、D14Rat84からD14Got40までの約0.4 Mbpに存在することを明らかにした。この領域に含まれるタンパク質をコードする遺伝子はKitのみであり、ゼブラフィッシュからヒトに至るまで多くの脊椎動物においてKit遺伝子の変異により色素産生異常を呈することが報告されている。このことからKitが頭巾斑遺伝子座の有力な原因遺伝子であると考えられた。しかしながらKitのコーディング領域には野生型と頭巾斑表現型の間で1つの同義変異が存在するのみで、胎齢期から新生子期、成体までKitのmRNAの発現量に有意な差は見られなかった。さらに、免疫染色によるとメラニンが存在しないLEAラットの白色被毛部位においてもメラノサイトマーカーであるKit陽性の細胞が検出された。このことから頭巾斑表現型はKitの単純な発現量の変化ではなく、何らかのメカニズムによるメラノサイトの機能異常が原因であると推測された。次に、頭巾斑表現型の修飾遺伝子座を同定するために、野生型に類似した表現型を示す頭巾斑の対立遺伝子hiを保持するBNラットとLEAラットを用いて遺伝解析59を行った。F2個体において体表面積に占める有色被毛で覆われた面積を背側、腹側、全体についてそれぞれ調べたところ、その値は全て明らかな2群に分離せず連続的な値を示したことから複数の遺伝子座による制御が示唆された。そこでQTL解析を行ったところ、全ての場合においてD14Got40近傍に非常に強いQTLが検出された。また背側における第17染色体に強いQTLが、ならびに腹側における第15染色体に弱いQTLが検出された。次に遺伝子座間の相互作用、エピスタシス解析を行ったところ、背側の表現型に関してD14Got40とD20Mit1間、およびD14Got40とD17Rat2間において強い相互作用があることが明らかとなった。D14Got40は頭巾斑遺伝子座のまさに近傍であり、これらの結果から頭巾斑遺伝子座そのものがその他の遺伝子座と相互作用して頭巾斑表現型を調節している事、つまりKitと他の遺伝子座にコードされるタンパク質の相互作用が頭巾斑表現型の調節に重要である事が示唆された

QTL Analysis Identifies a Modifier Locus of Aganglionosis in the Rat Model of Hirschsprung Disease Carrying Ednrb^[sl] Mutations

Hirschsprung disease (HSCR) exhibits complex genetics with incomplete penetrance and variable severity thought to result as a consequence of multiple gene interactions that modulate the ability of enteric neural crest cells to populate the developing gut. As reported previously, when the same null mutation of the Ednrb gene, Ednrb^[sl], was introgressed into the F344 strain, almost 60% of F344-Ednrb^[sl/sl] pups did not show any symptoms of aganglionosis, appearing healthy and normally fertile. These findings strongly suggested that the severity of HSCR was affected by strain-specific genetic factor (s). In this study, the genetic basis of such large strain differences in the severity of aganglionosis in the rat model was studied by whole-genome scanning for quantitative trait loci (QTLs) using an intercross of (AGH-Ednrb^[sl] x F344-Ednrb^[sl]) F1 with the varying severity of aganglionosis. Genome linkage analysis identified one significant QTL on chromosome 2 for the severity of aganglionosis. Our QTL analyses using rat models of HSCR revealed that multiple genetic factors regulated the severity of aganglionosis. Moreover, a known HSCR susceptibility gene, Gdnf, was found in QTL that suggested a novel non-coding sequence mutation in GDNF that modifies the penetrance and severity of the aganglionosis phenotype in EDNRB-deficient rats. A further identification and analysis of responsible genes located on the identified QTL could lead to the richer understanding of the genetic basis of HSCR development

Identification of antigenic peptides derived from B-cell epitopes of nucleocapsid protein of mouse hepatitis virus for serological diagnosis

Mouse hepatitis virus (MHV) infection is found commonly in laboratory mice and this virus has been known to cause various diseases such as subclinical infection, enteritis, hepatitis, and encephalitis. Serological tests are used commonly to diagnose MHV infection. Complete MHV virions have been used primarily as antigens for serological diagnosis to date. To develop an antigen that is more specific, more sensitive, and easier to prepare for serological diagnosis, the antigenic sites in the MHV-nucleocapsid (N) protein were screened in this study. Sixteen antigenic linear sequences in the N protein were found using antisera obtained from mice infected naturally with MHV and a peptide array containing overlapping 10-mer peptides covering the entire N protein. From these antigenic sequences, two synthesized peptides, ILKKTTWADQTERGL and RFDSTLPGFETIMKVL, which were consistent with positions 24-38 and 357-372 of the N protein respectively, were used as antigens in ELISA. Evaluation of ELISA with these peptides revealed that both peptides were specific to anti-MHV antisera. Furthermore, ELISA performed using these peptides was more sensitive than commercial ELISA used for a screening sera from mice infected accidentally to MHV maintained in cages, suggesting that these peptides are useful for serological diagnosis of MHV infection

Genetic analysis of modifiers for the hooded phenotype in the rat

The hooded phenotype is one of the coat color phenotype seen peculiarly in the rat. The hooded locus showing autosomal recessive inheritance is mapped to chromosome (Chr) 14 and that the hooded phenotype receives modification by hooded-modifier gene showing the linkage to the hooded locus. However, a gene responsible for either the hooded or hooded-modifier gene is not yet identified. To clarify genetic control of hooded phenotype, we carried out genetic linkage studies using BN and LEA rats. For determination of phenotypic variation, we measured ratio of pigmented coat area in parental and their F1 and F2 rats. We, then, conducted a genome-wide scan on 152 F2 rats for linkage with ratio of pigmented coat area for the dorsal, ventral, and total regions. A major quantitative trait locus (QTL), D14Got40, showing highly significant linkage contributing 70-90% of the variance for hooded phenotype was detected on Chr 14, which may be correspondent to the hooded locus. In addition, another QTL, D17Rat2, showing highly significant linkage was also detected on Chr 17 in dorsal region phenotype as well as a QTL showing suggestive linkage on Chr15 in ventral region phenotype. We, further, investigated a genome-wide scan for epistatic interactions and detected significant interactions between D14Got40 and D20Mit1, and between D14Got40 and D17Rat2 in the dorsal region phenotype. These results suggest that a major QTL in Chr 14, which is possibly correspondent to the hooded locus, mainly regulates the hooded phenotype with some modifier loci, two of which show epistatic interactions with the hooded locus

Analysis of the Relationship Between Enzymatic and Antiviral Activities of the Chicken Oligoadenylate Synthetase-Like

The oligoadenylate synthetase (OAS) is well known as an antiviral factor against the flavivirus infection in mammals. It is known that the oligoadenylate synthetase-like (ChOAS-L) gene is only present in the chicken genome. It has been shown in the previous report that the ChOAS-L possesses enzymatic activity to convert ATP into 2'-5'-linked oligoadenylates and antiviral activity against West Nile virus (WNV) replicon. Therefore, this study aimed to investigate the relationship between enzymatic and antiviral activities of ChOAS-L. Eight mutated ChOAS-L proteins were generated using either the site-directed mutagenesis or standard polymerase chain reaction protocol. The wild-type and mutated proteins were ectopically expressed in 293FT cells to analyze the enzymatic activity and in BHK-21 and BALB/3T3 cells to analyze the antiviral activity using WNV replicon. The results revealed that all mutated proteins showed no enzymatic activity except for ChOAS-L-A Delta UbL2. However, all mutated proteins showed antiviral activity to inhibit the replication of the WNV replicon except for ChOAS-L-A Delta UbL1/UbL2, which showed a partial inhibition compared to the wild-type ChOAS-L-A or other mutated proteins. These results suggest that the ChOAS-L expresses the antiflavivirus activity in a manner independent of enzymatic activity. Our results propose reconsideration of the mechanism of antiviral activity against the flavivirus replication of ChOAS-L

Does the routine handling affect the phenotype of disease model mice?

The three different mouse handling methods, picking up by tails, tunnels, and open hands were performed using the ICGN glomerulonephritis mouse and the severity of symptoms was evaluated. The handling groups exhibited a tendency of more severe symptoms than the non-handling control group. Female mice handled by their tails showed significantly more severe symptoms than the control group. In addition, we subjected the normal laboratory mice, C57BL/6 and BALB/c mice to tail and tunnel handling to assess the stress conditions. The plasma corticosterone level in the tail-handled mice was higher than that in control mice. These results indicate that handling causes stress and may affect the phenotype of disease model mice