Toxic myopathy with multiple deletions in mitochondrial DNA associated with long-term use of oral anti-viral drugs for hepatitis B: A case study.

Oral nucleoside analogs (NAs) reduce hepatitis B virus (HBV) replication by inhibiting HBV DNA polymerase. However, NAs can also affect human mitochondrial DNA (mtDNA) polymerase, which can lead to mtDNA depletion (quantitative abnormality). Indeed, several mitochondrial myopathy cases have been reported in which a reduced mtDNA copy number was induced by oral NAs for hepatitis B. Herein, we report a case of toxic myopathy with multiple mtDNA deletions (qualitative abnormality) associated with long-term use of NAs for hepatitis B. A 68-year-old woman, who underwent long-term treatment with lamivudine and adefovir for chronic hepatitis B, developed proximal muscle weakness in the four extremities. Neurological examination showed mild proximal muscle weakness and atrophy in the four extremities. Upon admission to our hospital, her blood lactate/pyruvate ratio during an aerobic exercise test was elevated. Myogenic patterns were observed in lower limb muscles on electromyographic examination. Muscle magnetic resonance imaging revealed diffuse atrophy of proximal muscles in the four extremities with no signal changes. A biopsy from the biceps brachii muscle showed an abnormally large variation in fiber size, scattered muscle fibers with decreased cytochrome c oxidase activity, and ragged-red fibers. Analysis of mtDNA from skeletal muscle revealed no decrease in copy number but increased incidence of multiple deletions, including a deletion of 4977 base pairs (known as the common deletion) reflecting oxidative stress-induced mtDNA damage. This case study indicates that long-term oral antiviral therapy for hepatitis B can induce chronic oxidative damage to mtDNA resulting in qualitative mtDNA abnormalities and toxic myopathy.

Streptococcus pyogenes-purpura fulminans as an invasive form of group A streptococcal infection.

BACKGROUND: Streptococcus pyogenes is an uncommon pathogen of purpura fulminans, and the pathogenesis of S. pyogenes-purpura fulminans remains unclear because of paucity of cases. We reported a pediatric case of S. pyogenes-purpura fulminans with literature review of the disease. CASE PRESENTATION: A 3-year-old boy showed limping, lethargy and acral gangrene within 24 h. A diagnosis of S. pyogenes-purpura fulminans was made for bacterial isolation from throat and peripheral blood. Intensive therapy led to a survival with amputation of the left distal metatarsal bone, and normal development. The isolated M12 carried no mutation of csrS/R or rgg. Thrombophilia or immunodeficiency was excluded. DISCUSSION: Twelve-reported cases (9 pediatric and 3 elderly) of S. pyogenes-purpura fulminans started with shock and coagulopathy. Five patients age < 8 years had no underlying disease and survived. One youngest and two immunocompromised patients died. CONCLUSION: Streptococcus pyogenes-acute infectious purpura fulminans is a distinctive rare form of aggressive GAS infections.

The clinical presentation and genotype of protein C deficiency with double mutations of the protein C gene.

BACKGROUND: Severe protein C (PC) deficiency is a rare heritable thrombophilia leading to thromboembolic events during the neonatal period. It remains unclear how individuals with complete PC gene (PROC) defects develop or escape neonatal stroke or purpura fulminans (PF). PROCEDURE: We studied the onset of disease and the genotype of 22 PC-deficient patients with double mutations in PROC based on our cohort (n = 12) and the previous reports (n = 10) in Japan. RESULTS: Twenty-two patients in 20 unrelated families had 4 homozygous and 18 compound heterozygous mutations. Sixteen newborns presented with PF (n = 11, 69%), intracranial thromboembolism and hemorrhage (n = 13, 81%), or both (n = 8, 50%), with most showing a plasma PC activity of <10%. Six others first developed overt thromboembolism when they were over 15 years of age, showing a median PC activity of 31% (range: 19-52%). Fifteen of the 22 patients (68%) had the five major mutations (G423VfsX82, V339M, R211W, M406I, and F181V) or two others (E68K and K193del) that have been reported in Japan. Three of the six late-onset cases, but none of the 16 neonatal cases, had the K193del mutation, which has been reported to be the most common variant of Chinese thrombophilia. A novel mutation of A309V was determined in a family of two patients with late onset. CONCLUSIONS: The genotype of double-PROC mutants might show less diversity than heterozygous mutants in terms of the timing of the onset of thrombophilia (newborn onset or late onset).

Fetal hydrocephalus and neonatal stroke as the first presentation of protein C deficiency.

Severe protein C-deficiency is a rare heritable thrombophilia of the newborn. Infants with biallelic PROC mutations present purpura fulminans and intracranial thromboembolism, while the prenatal onset of mutated heterozygotes remains unclear. We herewith present the first case of fetal ventriculomegaly and neonatal stroke associated with heterozygous PROC mutation. The infant was born to a healthy mother at 38 gestational weeks. The fetal growth had been normal, but the routine ultrasound screening had indicated mild hydrocephalus at 28 weeks of gestation. He developed convulsions two days after birth. Computed tomography of the brain revealed multiple hemorrhagic infarctions and ventriculomegaly. Dissociated levels of the plasma activity between protein C (21%) and protein S (42%) reached to determine the heterozygote of PROC c.574_576delAAG, a common thrombophilic predisposition in Asian ancestries. PC-mutant heterozygotes may have a limited high risk of cerebral thromboembolism during the perinatal course.

[Analysis of influenza A/H3N2 neuraminidase genes obtained from influenza patients in the 2011/12 and 2012/13 seasons in Japan].

BACKGROUND: Influenza virus has neuraminidase (NA), a surface protein with enzymatic activity that is essential for virus replication. Mutation may affect the effectiveness of NA inhibitors that are used for the treatment of influenza patients. In this study, we determined the NA gene sequences from the clinical isolates of influenza patients to examine the chronological genetic changes and the relation to drug susceptibility. METHODS: For 96 A/H3N2 virus isolates the 50% inhibitory concentration (IC50) (48 each from the 2011-12 and 12-13 influenza seasons) was measured. RT-PCR was done with extracted viral RNA, followed by nucleotide sequencing. RESULTS: One putative amino acid mutation, D151N, was found in an NA activity-related cite in five of ninety-six tested isolate. The mutation did not affect the IC50 value. The mutations identified at amino acid positions 387 and 400 were statistically correlated with an increased IC50 value, although the change was less than ten times, suggesting no significant difference in the clinical effectiveness. A small number .of isolates showed mutation in the T and/or B cell epitope region of NA. CONCLUSION: No mutation that affected the IC50 value or effectiveness of NAIs was detected. Antigenic mutations of NA, which influence the selection of epidemic strains, were not determined. Continuous observation will be necessary to further clarify the genetic features of NA.

A novel mutation in protein C gene (PROC) causing severe phenotype in neonatal period.

Homozygous protein C deficiency is among rare causes of thrombophilia. Herein, we present a neonate with purpura fulminans, disseminated intravascular coagulation and severe intracranial hemorrhage who was found to have plasma protein C level of 4%. The molecular work-up revealed a novel homozygous mutation of T903C (amino acid position Leu 270 Pro) located in a catalytic domain region of PROC gene. Asymptomatic course in patients with low but measurable levels of protein C levels has been reported, which is different than observed in our patient who had a very severe course despite plasma protein C level of 4%.

[A case of sensory ataxic neuropathy, dysarthria, and ophthalmoparesis with multiple mitochondrial DNA deletions].

We report the case of a 62-year-old man with sensory ataxic neuropathy, dysarthria, and ophthalmoparesis (SANDO). He developed gait disturbance at 54 years of age, muscle weakness at 56 years, and difficulty hearing at 58 years. His brother had muscle weakness in both legs from age 20 years, and was diagnosed with Charcot-Marie-Tooth disease because he had muscle weakness of the four extremities, decreased CMAP and SNAP amplitudes on peripheral nerve conduction tests, and loss of large myelinated fibers and onion-bulb formations on sural nerve biopsy. His brother died aged 46 years, but no accurate cause of death was identified. Neurological examination of the present patient revealed bilateral ptosis, external ophthalmoparesis, dysarthria, dysphagia, sensorineural hearing loss, mild weakness and atrophy of proximal muscles in all four limbs, severe sensory ataxia, and disturbance of deep sensation in his legs. He showed elevation of lactate and pyruvate levels in cerebrospinal fluid and serum. An aerobic exercise test disclosed a marked increase in lactate and pyruvate levels in serum. On nerve conduction study, amplitudes of CMAP and SNAP, and F wave-evoked frequency were decreased. Needle electromyography showed chronic neurogenic patterns with fibrillation potentials in the extremity muscles. Head MRI demonstrated T2 prolonged lesions in the bilateral basal ganglia, while brain MRS revealed a small lactate peak. Biopsy of his left lateral vastus muscle showed ragged-red fibers and group atrophy, and some muscle fibers had decreased cytochrome c activity. Left sural nerve biopsy revealed a marked loss of large myelinated fibers, and some onion-bulb formations. Genetic testing disclosed a large mtDNA deletion in the biopsied muscle. Among nuclear genes, we found point mutations in ANT-1 (exon 1 c.105G>A, 5' untranslated region) and POLG-1 (exon 4, c.1218G>A, p. and exon 23 c.3920C>T, p.A1217V). We diagnosed SANDO. This is the first case of SANDO with large mitochondrial DNA deletions in Japanese.

Fulminant sepsis/meningitis due to Haemophilus influenzae in a protein C-deficient heterozygote treated with activated protein C therapy.

A 13-month-old Japanese female with Haemophilus influenzae type b meningitis presented with unusually severe septic shock and cerebral infarction in half a day of fever. The initial therapy of plasma-derived activated protein C (Anact C) led to an impressive effect on the aggressive condition. However, purpura fulminans and the consistent decline of plasma protein C activity (<20%) required prolonged activated protein C therapy and gene analysis. The patient carried a novel heterozygous mutation of PROC (exon 4; 335 GAC>TAC, Asp46Tyr). This is the first report of infectious purpura fulminans in a protein C-deficient heterozygote. The clinical onset and treatment course adequately corroborated the aggravated immune/hemostatic reactions and the cytoprotective effects of activated protein C replacement in human heterozygous protein C deficiency. The monitoring of plasma protein C activity and sufficient administration of activated protein C product could improve the outcome of severe sepsis in children.

Platelet contamination causes large variation as well as overestimation of mitochondrial DNA content of peripheral blood mononuclear cells.

BACKGROUND: Alterations in the copy number of mitochondrial DNA (mtDNA) play a role in the pathogenesis of mitochondrial diseases and other many common diseases. Recently, the copy number of leukocyte mtDNA has been considered to serve as a biomarker to monitor or chase such diseases. Therefore, reproducible mtDNA measurement is required. METHODS: Peripheral blood mononuclear cells were prepared by a density-based method. The mtDNA/cell was measured by quantitative realtime polymerase chain reaction. RESULTS: The degree of platelet contamination varied to a large extent among preparations. The mtDNA copy numbers per mononuclear cell were 269 +/- 51 and 146 +/- 14 in the samples before and after the platelet depletion, respectively. CONCLUSION: A density-based mononuclear cell preparation causes heavy platelet contamination. The platelet depletion from a sample is particularly important for comparing the mtDNA contents between different dates or between different patients.

Successful therapy with argatroban for superior mesenteric vein thrombosis in a patient with congenital antithrombin deficiency.

A 38-year-old woman was admitted with superior mesenteric vein (SMV) thrombosis, which was refractory to anticoagulation therapy. The plasma antithrombin activity was decreased and hardly compensated by concentrated antithrombin preparation due to high consumption rate. However, successful anticoagulation was achieved by administration of direct thrombin inhibitor, argatroban. Family studies of antithrombin activity revealed that she had type I congenital antithrombin deficiency. A novel heterozygous mutation in the gene for antithrombin (single nucleotide T insertion at 7916 and 7917, Glu 272 to stop in exon 4) was identified. Argatroban administration would be effective in the treatment of congenital antithrombin deficiency with SMV thrombosis.

High-sensitivity detection of the A3243G mutation of mitochondrial DNA by a combination of allele-specific PCR and peptide nucleic acid-directed PCR clamping.

BACKGROUND: The A3243G mutation of mitochondrial DNA (mtDNA) is involved in many common diseases, including diabetes mellitus and mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS). For detection of this mutation, allele-specific PCR is highly sensitive but requires strict control of PCR conditions; it thus is not adequate for a routine clinical test. We aimed to develop a routinely available PCR method for quantitative detection of low-level heteroplasmy of the A3243G mutation. METHODS: Quantitative allele-specific PCR for the A3243G mutation was performed in the presence of peptide nucleic acid (PNA), in which PNA is complementary to the wild-type mtDNA, with one primer having a 3' end matched to nucleotide position 3243 of the mutant. RESULTS: With our method, amplification of wild-type mtDNA was suppressed 7000-fold compared with amplification of the mutant mtDNA under a broad range of conditions: DNA, 5-100 ng; annealing temperature, 61-66 degrees C; and PNA, 1.5-3.5 micromol/L. Hence, 0.1% heteroplasmy of the A3243G mutation can be reliably quantified by this method. Blood samples form 40 healthy volunteers showed <0.06% heteroplasmy, suggesting that 0.1% is diagnostically significant. CONCLUSIONS: PNA maintains the specificity of allele-specific PCR over a wide range of conditions, which is important for routine clinical testing.

[A basic study of determination matrix metalloproteinase-3 and carbohydrate in rheumatoid factor in serum for diagnosis of rheumatoid arthritis].

The examination of rheumatoid factor (RF), one of the diagnostic marker of rheumatoid arthritis (RA), showed negative about 25% of patients with RA. We analyzed a matrix metalloproteinase-3 (MMP-3) and a carbohydrate in rheumatoid factor (CA.RF) for diagnosis of RA: the former is used the kit "Panaclear MMP-3[Plate]" and the latter is used the kit "Picolumi CA.RF". The basic study of these reagents showed satisfactory results. In 73.3% of seronegative RA showed positive on both MMP-3 and CA.RF levels in serum, respectively. We found that these examinations might be useful for diagnosis of RA, especially during seronegative RA.

Four missense mutations identified in the protein S gene of thrombosis patients with protein S deficiency: effects on secretion and anticoagulant activity of protein S.

Four missense mutations, G54R, T589I, K155E, and Y595C, were identified in the protein S (PS) gene of the patients with PS deficiency and venous thrombosis. Three patients were heterozygous for the novel mutations, G54R, T589I, and Y595C, while a remaining one patient was homozygous for the K155E mutation, which is known to be a polymorphism in the Japanese population. A family study revealed that the Y595C mutation was associated with a Type I PS deficiency and the K155E mutation with a Type II PS deficiency, while no family study was performed for the patients with the G54R and T589I mutations. To determine whether these four mutations play a causative role in PS deficiency, the four PS mutants and wild-type PS were stably expressed in human embryo kidney (HEK) 293 cells. Pulse-chase experiments showed intracellular degradation and decreased secretion of the Y595C mutant. In the activated protein C (APC) cofactor assays, the specific activity of the K155E mutant decreased to 58% of that of wild-type PS. The APC cofactor activity of the three mutants, G54R, K155E, and T589I, were inhibited by C4b-binding protein (C4BP) with a dose dependency similar to that of wild-type PS. These results indicate that the Y595C and the K155E mutations are responsible for a secretion defect and a decreased anticoagulant activity of PS, respectively. The remaining two mutations, G54R and T589I, however, did not produce any definite abnormality leading to a low plasma PS activity.