Tomoharu Tokutomi

Circulating endothelial cells in Kawasaki disease

Recent reports have demonstrated that circulating endothelial cells (CECs) are observed in several diseases with vascular injury. Because Kawasaki disease (KD) is one type of systemic vasculitis, we hypothesized that an increased number of CECs may be associated with the appearance of complicated coronary artery lesions (CAL). In the present study we investigated the enumeration and origin of CECs in 20 patients with KD, using an immunohistochemical method with monoclonal antibodies: clone P1H12 against ECs and clone AC133 against endothelial progenitor cells (EPCs), which were derived from the bone marrow. The mean number of CECs increased significantly (P < 0·05) from the acute through the subacute phases of KD compared with both the convalescent phase of KD and healthy children. The mean number of CECs was significantly (P < 0·05) higher in six KD patients with CAL than in 14 KD patients without CAL. The population of EPCs in the total CECs in KD was 4·4 ± 1·2% (range 0–18%). The number of EPCs during the subacute phase was also significantly higher (P < 0·05) in KD patients with CAL than in those without CAL. Our findings indicate that the number of CECs increase in KD vasculitis and suggest that the increased numbers of CECs and EPCs may reflect the EC damage of this disease.

Elevated serum levels of matrix metalloproteinase-9 (MMP-9) in Kawasaki disease

Matrix metalloproteinases (MMPs) play an important role in the progression of tumour cells and the invasion of inflammatory cells by degrading the extracellular matrix. In the MMP family, MMP‐9 gelatinase is thought to contribute to the pathogenesis of inflammatory arteritis by disrupting the elastic lamina. The aim of the present study is to investigate the potential role of MMP‐9 in Kawasaki disease (KD), an acute type of systemic vasculitis in children. We studied the total levels of MMP‐9 (free proMMP‐9 and free MMP‐9) in the sera using a new assay system and the expression of MMP‐9 mRNA in the leucocytes using reverse transcription‐polymerase chain reaction in 18 patients with KD, 10 patients with sepsis and 10 healthy children (HC). The serum MMP‐9 levels were significantly higher (P < 0·01) in the acute phase of KD than in the acute phase of sepsis and HC. In the time course of KD, the serum MMP‐9 levels decreased significantly (P < 0·01) from the subacute through the convalescent phases. In the acute phase of KD, the serum MMP‐9 levels showed a significantly positive correlation (P < 0·05) with the circulating leucocyte counts, especially the neutrophil counts. Furthermore, the expression of MMP‐9 mRNA in the circulating leucocytes increased in the acute phase of KD and decreased from the subacute through the convalescent phases. These findings indicate that an excessive amount of MMP‐9 is present in the plasma during the acute phase of KD, thus suggesting that circulating leucocytes may be a source of the MMP‐9 secreted into the circulation.

Delayed apoptosis of circulating neutrophils in Kawasaki disease

Circulating polymorphonuclear neutrophils (PMNs) are known to increase in number and are functionally activated in the acute phase of Kawasaki disease (KD). In the present study, we investigated whether the apoptosis of PMNs is deregulated in KD. When the isolated PMNs were cultured in vitro, the proportions of spontaneous apoptotic PMNs (annexin V+ cells and cells with fragmented DNA) were found to be significantly lower (P < 0·01) in the patients with KD (n = 25) than in the patients with a bacterial infection (n = 20) or a viral infection (n = 20), or in healthy children (n = 20). The proportion of circulating Fas‐positive PMNs was also significantly lower (P < 0·01) in the acute KD patients than in the other groups. In the acute phase of KD, the proportion of spontaneous apoptotic PMNs showed a significant positive correlation (P < 0·01) with the proportions of circulating Fas‐positive PMNs. Furthermore, the agonistic anti‐Fas MoAb (CH‐11) induced a significant increase in the proportion of apoptotic PMNs in the patients with a viral infection and healthy children, but not in either the patients with KD or the patients with a bacterial infection. In the intracellular expression of anti‐ and pro‐apoptotic proteins, the A1/Bax ratio was significantly higher in acute KD than in the other groups. These findings indicate that PMN apoptosis is inhibited during the acute phase of KD and also suggest that both the resistance against the Fas‐mediated death signal and the down‐regulation of the mitochondrial apoptotic signalling pathway due to an altered balance of Bcl‐2 protein expression are responsible for the delayed PMN apoptosis.

Standard-dose and Short-term Corticosteroid Therapy in Immunoglobulin-resistant Kawasaki Disease

Six patients with Kawasaki disease (KD) were treated with prednisolone (1 to 2 mg/kg/day) for 3 days (from days 10 to 12 after the onset of the illness) after apparently unsuccessful treatment with intravenous immunoglobulin (IVIG, 2 g/kg/dose and additional 1 g/kg/dose). Five patients responded immediately to the first course of prednisolone infusion. One patient failed to respond to the first course of prednisolone therapy, but he did respond to the second 3-day course of therapy. None of the patients demonstrated a further progression of coronary artery dilatation or any adverse effects. Standard-dose and short-term corticosteroid therapy therefore appears to be a safe and effective treatment for patients with IVIG-resistant KD.

A non‐obese boy with Prader‐Willi syndrome shows cardiopulmonary impairment due to severe kyphoscoliosis

Prader-Willi syndrome (PWS, OMIM #176270) is a genetic disorder characterized by hypotonia, obesity, characteristic facial appearance, hypogonadism, short stature, and behavioral abnormalities. It results from deletion or disruption of one or more genes on the proximal long arm of the paternally derived chromosome 15, or from maternal uniparental disomy 15. Musculoskeletal abnormalities are common in these individuals. Scoliosis is very common in PWS being variously reported in 62–86% of patients [Holm and Laurnen, 1981; Laurance et al., 1981]. Here, we describe a non-obese boy with PWS who also presented with cardiopulmonary impairment due to severe kyphoscoliosis. This secondary manifestation was described by Guilleminault et al. [1981]. The male patient was born to healthy and nonconsanguineous parents at 41 weeks of gestation, with a birth weight of 2,430 g ( 1.9 SD) and length of 47.0 cm ( 1.4 SD). Other family members were healthy. He showed hypotonia with poor suck in infancy. He held his head at age 9 months, sat at 1 year, and walked at 41⁄2years. He had a normal male karyotype (46,XY) and therewasno SNRPN signal on FISH. Kyphoscoliosis that was recognized at 1 year of age had progressively deteriorated (Fig. 1A,B). He had received growth hormone (GH) therapy (0.175 mg/kg/week) from age 10 years. He had a restrictive deficit on spirometry at age 1011=12 years: the median forced expiratory volume in 1 sec (FEV1) and forced vital capacity (FVC) were 1.03 L and 1.15 L, respectively. Although he had no respiratory symptoms, he underwent combined anterior fusion with posterior corrective fusion surgery using spinal instrumentation for kyphoscoliosis at age 11 years. However, the instrumentation had to be removed due to deep postoperative infection, which did not resolve until age 116=12 years. His weight management had been controlled well without any obesity from birth. At age 1510=12 years, he was admitted because of a 1-month history of shortness of breath and edema of the legs. On admission, he measured 133.0 cm and weighed 41.2 kg. Under normal conditions, his weight was 33.0 kg and his calculated body mass index (BMI) was 18.7 kg/m. He had a characteristic facial appearance, narrow hands with a straight ulnar border, and chilblain scars on the legs. Physical examination revealed tachypnea, tachycardia, cyanosis, a systolic heart murmur and gallop rhythm, distended abdomen, and edema of the whole body, especially of the legs. Neurological examination showed normal deep tendon reflexes and mild hypotonia. Laboratory tests showed respiratory acidosis, hypercapnia, and heart failure: pH, 7.285; pCO2, 70.6 mmHg in room air; hANP, 557 pg/ml; and BNP, 862 pg/ml. Echocardiogram showed the interventricular septum curved toward the left

Case 2: Fever, rash and pulmonary involvement

A previously healthy 2-y-old girl was admitted to our hospital with fever persisting for 5 d, bilateral bulbar conjunctival injection, cracked lips, cervical lymphadenopathy and polymorphous exanthema, although no respiratory symptoms were observed. Even though she was treated with antibiotics for several days before the current visit, the fever did not subside. The laboratory findings were as follows: white blood cell (WBC) count 13 000/ml, red blood cell (RBC) count 376 /10/ml, haemoglobin (Hb) 9.6 g/dl, haematocrit (Ht) 29.5%, platelet (Plt) count 32.1 /10/ml, aspartate aminotransferase (AST) 21 IU/l, alanine aminotransferase (ALT) 41 IU/l, lactate dehydrogenase (LDH) 356 IU/l, C-reactive protein (CRP) 12.6 mg/ dl, immunoglobulin (Ig) G 1350 mg/dl, erythrocyte sedimentation rate (ESR) 102 mm/h, and antibody titre for Mycoplasma pneumoniae (phytohaemagglutinin, PHA) was 1280 /. Chest radiographs (Figure 1) were obtained to evaluate the source of her fever on day 5 of the disease. Case 2

A de novo direct duplication of 16q22.1 → q23.1 in a boy with midface hypoplasia and mental retardation

A de novo Direct Duplication of 16q22.1! q23.1 in a Boy With Midface Hypoplasia and Mental Retardation Tomoharu Tokutomi,* Takahito Wada, Eiji Nakagawa, Shinji Saitoh, and Masayuki Sasaki Department of Child Neurology, National Center Hospital of Neurology and Psychiatry, Kodaira, Tokyo, Japan Department of Pathology and Laboratory Medicine, National Defense Medical College, Tokorozawa, Saitama, Japan Department of Preventive Medicine and Public Health, Shinshu University School of Medicine, Matsumoto, Nagano, Japan Department of Medical Genetics, Shinshu University School of Medicine, Matsumoto, Nagano, Japan Department of Pediatrics, Graduate School of Medicine, Hokkaido University School of Medicine, Sapporo, Hokkaido, Japan

dup(8p)/del(8q) recombinant chromosome in a girl with hepatic focal nodular hyperplasia†

A 15‐year‐old girl had exertion dyspnea, focal nodular hyperplasia of the liver, portal vein hypoplasia, portopulmonary hypertension, mental retardation, and minor facial abnormalities. Cytogenetic analysis demonstrated an abnormal chromosome 8 with 8p22‐pter duplication and 8q24.3‐qter deletion, with the duplicated 8p segment attached to band 8q24.3. Her mother had a pericentric inversion of chromosome 8, inv(8)(p22q24.3). Therefore, the girls abnormal chromosome 8 was a recombinant of maternal inversion chromosome: 46,XX,rec(8)dup(8p)inv(8)(p22q24.3)mat. Further characterization of the recombinant chromosome, using array CGH and regional FISH analyses, defined 15 Mb distal 8p duplication and 0.5 Mb 8q deletion. Possible correlation of the recombinant chromosome and hepatic focal nodular hyperplasia in the patient is discussed.

Real-time PCR method for identification of Asian populations in forensic casework

The priorities for mass disaster victim identification are rapid investigation turn-around time and low cost. We describe a DNA typing procedure to selectively identify members of Asian populations by a real-time PCR method using polymorphisms of the alpha2 chain of the type I collagen gene (COL1A2) and mitochondrial DNA (mtDNA). Among the 50 members of the Asian population included in the present study, 37 harbored a deleted allele in intron 33 of COL1A2 (26822-26823del) or the 10400C>T substitution mutation in mtDNA to give a probability of 0.740 for these SNPs in the Asian population.

Application of SNPs in forensic casework: Identification of pathological and autoptical specimens due to sample mix-up

The analysis of single nucleotide polymorphisms (SNPs) together with conventional short tandem repeat (STR) and mitochondrial DNA (mtDNA) typing provide a forensic genetic approach for the identification of pathological and autoptical specimens in cases where the average length of DNA fragments is shorter than 150 bp in highly degraded samples. We applied a forensic genetic approach to digesta accidentally left after a training autopsy. PCR products were not amplified from samples containing the STR loci or common sequences used for mtDNA typing. The application of SNPs and deletion polymorphisms provides an alternative approach for DNA typing analysis.