学位論文内容の要旨(Summary of dissertation)

博士の専攻分野の名称
博士（医学）(Degree conferred: Doctor of Philosophy)

氏 名 蔡 華英(Name of recipient: Cai Huaying)

学 位 論 文 題 名(Title of dissertation)
Clinical, pathological, and genetic analysis ofsporadic inclusion body myositis in Japanese people（日本人の封入体筋炎における臨床症状、筋病理所見と遺伝学的背景に関する研究）【Background and Objectives】 Inclusion body myositis (IBM) is a common adult-onset, sporadic
muscle disease with pathological findings of rimmed vacuoles, mononuclear cell invasion of
non-necrotic fibers, and amyloid deposits or 15–18 nm filaments on electron microscopy.
However, rimmed vacuoles are not specific to IBM and may be observed in inherited vacuolar
myopathies such as hereditary inclusion body myopathy (hIBM) and myofibrillar myopathy(MFM). In general, clinical features of IBM may be diverse and show some overlap with othervacuolar myopathies. Molecular genetic studies have identified several genetic loci associatedwith some inherited vacuolar myopathies: 3 genes for hIBMs (Desmin, GNE, and MYHC2A genes),
the VCP gene for inclusion body myopathy with early-onset Paget disease of the bone andfrontotemporal dementia (IBMPFD), the ZASP gene for zaspopathy (one form of MFM) and theDNAJB6 gene for limb-girdle muscular dystrophy 1D (LGMD1D). However, the extent to whichthese genes play a role in generating the phenotype of IBM is poorly understood. In order toexplore the molecular basis of IBM and to investigate genotype-phenotype correlations, we
performed a clinicopathological analysis of 23 IBM patients and screened for mutations in theabove 6 candidate genes.
【Methods】 According to the Needham and Mastaglia criteria, 23 suspect IBM patients wererecruited from a total of 692 cases with available muscle pathology from December 1993 toDecember 2011. Samples from all patients underwent a battery of conventional pathologicalstudies, including hematoxylin-eosin (HE), modified Gomori trichrome (mGT), nicotinamide
adenine dinucleotide-tetrazolium reductase (NADH-TR), adenosine triphosphatase (ATPase;
pre-incubation at pH 4.2, 4.6, and 10.4), non-specific esterase, and alkaline phosphatase, as wellas β-amyloid peptide precursor protein (βAPP) immunostaining. Cytochrome c oxidase (COX)
studies and immunostaining for CD8 + T cells and MHC-I expression could only be performed in18 patients due to insufficient specimen samples. Double immunostaining for MYHC isoforms
was performed in 3 patients with MYHC mutation. Immunohistochemical staining of Z-band
associated proteins was performed in 2 patients with a ZASP mutation or variant. After extracting
DNA from blood or frozen muscle, PCR was used to amplify all coding exons of the Desmin GNE,
MYHC2A, VCP, ZASP, and DNAJB6 genes, as well as the flanking non-coding regions of theabove. Direct sequencing of the DNAJB6 gene was performed in 20 patients due to insufficient
DNA, while sequencing of other 5 genes was performed in all patients.

【Results】 According to the Needham and Mastaglia criteria, 15 patients were diagnosed withdefinite IBM, 3 patients with probable IBM, and 5 patients with possible IBM. No cases showed
missense mutations in the Desmin, VCP, or DNAJB6 genes. Three patients with IBM carried themissense mutation p.V805A in the MYHC2A gene. The p.V805A variant was only present in 1 of160 control chromosomes. The frequency of this allele was significantly higher in the IBM
patients than in the controls (6.5% vs. 0.6%, respectively; p < 0.05). Further, the p.V805A variant
in the MYHC2A gene was associated with a significantly increased risk of IBM (Odds Ratio =11.1; 95% CI = 1.13-109.3). The immunohistochemical staining for MYHC isoforms in these 3
cases showed atrophy or loss of muscle fibers expressing MYHC IIa or IIx. One patient with IBM
harbored the missense mutation p.V566M in the ZASP gene. The mutation was not present in 160
control chromosomes. Immunohistochemical studies of Z-band associated proteins revealed strong
accumulation of desmin, dystrophin C-terminus, neural cell adhesion molecule (NCAM), andβAPP in affected fibers, with mild to moderate immunoreactivity for cell division cycle 2 (CDC2),
myotilin, α-B crystalline (α-BC), and ubiquitin at the vacuole margins. The immunohistochemicalstudies of Z-band associated proteins revealed typical Z-band abnormalities, indicating that theV566M mutation alters the functionally important ZASP protein. One patient with possible IBM
was identified to carry both the GNE missense mutations and the ZASP variant: novel compound
heterozygous missense mutations in the GNE gene (p.V421A and p.N635K) and a heterozygous
variant in the ZASP gene (p.D673N). Both the GNE mutations were not observed in 160 control
chromosomes while the ZASP p.D673N variant was identified in 2 of 160 control chromosomes.
Immunohistochemical analysis of Z-band associated proteins showed mild to moderate
immunoreactivity to βAPP, NCAM, α-BC, and ubiquitin, and no immunoreactivity to desmin,
CDC2, and myotilin, indicating atypical features of ZASP associated MFM. All of the mutations
were located in highly evolutionarily conserved domains of their respective genes.
【Discussion】 Of the 6 genes screened in the 23 IBM patients, we identified 5 IBM patients
carrying missense mutations. The 3 patients with MYHC2A p.V805A mutation displayed proximal
dominance of limb weakness with involvement of the quadriceps femoris and finger flexors, andshowed both clinical and pathological phenotypes resembling that of IBM. The diagnosis
remained considerably indistinguishable between IBM and MYHC2A associated IBM3. However,
the immunostaining results of MYHC isoforms revealed atrophy or loss of fibers expressing
MYHC IIa in 3 patients, suggesting the p.V805A mutation alters MYHC IIa protein function andp.V805A mutation is pathogenic in 3 patients. The patient with ZASP V566M mutation displayed
distal dominance of limb weakness with the tibialis anterior being more affected than thequadriceps femoris, indicating this patient did not show a classical IBM phenotype. The typicalfindings of the Z-band associated protein immunostaining were compatible with ZASP associatedMFM. It appears that the V566M mutation was responsible for this patient, and the diagnosis wasrevised as zaspopathy from IBM. The patient with GNE mutations and ZASP variant presented
with distal weakness sparing the quadriceps muscles, and showed atypical pathological findings ofIBM lacking mononuclear cell invasion of non-necrotic fibers and upregulation of MHC class Iexpression. Moreover, the atypical findings of the Z-band associated protein immunostaining did
not compatible with ZASP associated MFM. It seems that the GNE mutations are pathogenic andthe diagnosis was revised as IBM2/DMRV from possible IBM.
【Conclusion】Cumulatively, approximately 20% of the IBM patients in this study harboredmissense mutations, and 3 genes (MYHC2A, ZASP, and GNE) appeared to play a role in generating
the phenotype of IBM. These findings highlight the possibility of the non-familial cases carrying
the mutations. Meanwhile, the immunohistochemical studies and causative gene screening would
be further emphasized in the diagnostic procedure of IBM. Moreover, we suggest that typicalclinical features and typical pathological findings, including CD8 + T cell invasion of non-necroticfibers, are both required for accurately diagnosing IBM.