Koji Terabe

Bile salt export pump gene mutations in two Japanese patients with progressive familial intrahepatic cholestasis.

BackgroundIn recent years, progressive familial intrahepatic cholestasis has been classified into at least three types by genetic analysis: PFIC1, PFIC2, and MDR3. Liver transplantation is effective for treating patients with this intractable syndrome. Confirming the correct diagnosis is of paramount importance because prognosis after transplantation differs with the genetic type of the disease. MethodsSynthesis of cDNA was accomplished using RNA extracted from liver tissue of two Japanese patients with progressive familial intrahepatic cholestasis. Polymerase chain reaction was performed using 13 primer sets designed for amplification of the bile salt export pump cDNA. Direct sequencing was undertaken, and identified sequences were compared with the sequence for bile salt export pump gene registered with GenBank. In addition, gene sequences for nonprogressive familial intrahepatic cholestasis patients were analyzed. ResultsGenetic analysis of patient 1 revealed that substitutions in bile salt export pump protein sequences, namely R575X and E636G, might be the cause of the disease. In patient 2, V330X and R487H might fulfill the same role. Results of gene analysis in parents and cholestatic controls supported these conclusions. ConclusionsAbsence or presence of bile salt export protein gene mutations was confirmed as representing a useful prognostic marker for clinical course after liver transplantation.

Route of TT virus infection in children

TT virus (TTV) is a novel viral agent, detected recently in non‐A to E hepatitis cases. Little is known about its natural history or routes of transmission in childhood. For the detection of serum TTV DNA, semi‐nested polymerase chain reaction (PCR) was carried out using TTV‐specific primers and TTV nucleotide sequences were determined by the dideoxy chain‐mediated termination method. Five of the 70 children studied (including 20 hepatitis B virus [HBV] carriers, 40 children born to HBV carrier mothers and 10 children born to hepatitis C virus [HCV] carrier mothers) had serum TTV DNA. Three of the 5 children had siblings (4 in total), so that a total of 9 children were studied to determine the time of initial serum TTV DNA detection. In the 8 seropositive children, the time of serum TTV DNA detection ranged from 6 to 14 months after birth, and TTV DNA persisted thereafter throughout the follow‐up period. The TTV DNA‐negative child was assessed most recently at 6 months of age. TTV DNA was detected in only 2 of the 4 mothers tested (families 2 and 3). When 271‐bp TTV DNA fragments from each of the 8 children were sequenced, the degree of homology between siblings in families 1–3 was 100%, 99.5%, and 92.3%, respectively. The degree of homology between child‐mother pairs of families 2 and 3 was 99.5–100% and 62.6–63.9%, respectively. The distribution of different TTV strains was consistent within families, except for family 3. None of the TTV‐infected children had elevated levels of alanine aminotransferase or clinical signs of liver disease. J. Med. Virol. 59:204–207, 1999.

Detection rates of TT virus among children who visited a general hospital in Japan

Recently, genomic DNA of the novel TT virus (TTV) was isolated from patients suffering from posttransfusion hepatitis of unknown etiology. We examined sera from 197 children who visited the Department of Pediatrics at Toyohashi National Hospital. Sera were tested for TTV DNA by seminested polymerase chain reaction (PCR) using a set of primers synthesized according to the published TTV sequence. Ten children were found to be positive for TTV (5.1%). All positive PCR products were directly sequenced in both directions using a fluorescent dye terminator cycle sequencing system. The sequences were compared by a multiple sequence alignment and a phylogenetic tree was constructed. The phylogenetic tree showed that two of the TTV isolates found in the present experiment did not belong to any of the phylogenetic groups previously reported. J. Med. Virol. 57:405–407, 1999.

Prevalence of TTV DNA among children with a history of transfusion or liver disease.

The prevalence rates of serum TT virus (TTV) DNA among children with or without a history of transfusion or liver disease were studied by polymerase chain reaction (PCR) using either the Okamoto primer set or the Takahashi primer set developed more recently. Using Okamoto and Takahashi primer sets, the prevalence rates were 31.6% (12/38) and 78.9% (30/38), respectively, for children with a history of blood transfusion (including malignant and non‐malignant groups) and 6.7% (2/30) and 60% (18/30), respectively, for children without a history of blood transfusion. Among pregnant women, these rates were 12.9% (4/31) and 61.3% (19/31), respectively. On the other hand, the prevalence rates were 0% (0/16) and 50% (8/16), respectively, in hepatitis B patients, 21.4% (3/14) and 71.4% (10/14), respectively, for hepatitis C patients, and 20.0% (9/45) and 57.8% (26/45), respectively, for non‐A to C hepatitis patients (including 27 acute hepatitis patients, 5 fulminant patients and 13 chronic hepatitis patients). In this study, the prevalence rates determined by the Takahashi primer set tended to be 2–9 times higher than those determined using the Okamoto primer set. These results suggest that TTV infection is widespread among Japanese children. Furthermore, blood transfusion does not appear to be the major route of infection. The similar prevalence rates between control children and children with various types of hepatitis using the Takahashi primer system suggest that TTV infection does not play a direct causative role in the development of liver disease in children. J. Med. Virol. 60:172–176, 2000.

Highly diverse TTV population in infants and their mothers.

Infants born to serum HCV-positive 12 mothers were enrolled in the study. Nucleotide sequences amplified by primers deduced from a noncoding region were compared between mothers and their infants. The rates for detection of serum TTV in 12 mothers and their infants were 10/12 (83%) and 9/12 (75%), respectively. Serum TTV DNA was not detected in any infant at 1 month of age, but was detected for the first time between 1.5 and 8 months after birth. Positivity persisted thereafter throughout the follow-up period. In seven randomly selected mother-infant pairs, intrahost TTV heterogeneity was lower in infants than in mothers. Furthermore, one of seven mother-infant pairs showed a high degree of similarity (98.7-100%) in all clones, while in four infants, all nucleotide sequences differed by >10% from those of their mothers. However, the degree of homology in the two mother-infant pairs was 89-98.7% in family 2 and 88.1-99.4% in family 5. In the present study, with only one exception, it was shown that TTV from infants is not identical to TTV from mothers. The mechanism is discussed briefly in this paper.

Detection of congenital cytomegalovirus infection using umbilical cord blood samples in a screening survey

Easy screening and accurate diagnosis of congenital cytomegalovirus (CMV) infection are needed to predict and treat complications. We report the clinical course of two neonates with congenital CMV infection confirmed by real‐time polymerase chain reaction (PCR) for CMV DNA in umbilical cord blood. A total of 1,010 neonates born at Yonaha Clinic from July 2005 to March 2007 were investigated. Umbilical cord blood was collected at birth, and DNA was extracted to screen for CMV DNA by real‐time PCR. Head MRI and a developmental test were conducted for two cases (0.2%) in which CMV DNA was detected. Neither case showed clear abnormalities at birth, and head CT conducted at 1 month after birth revealed no abnormalities. Auditory brainstem responses were normal at both 1 and 12 months after birth in both cases. Head MRI at 12 months showed abnormalities in both cases. For both cases, development tests conducted at 12 months revealed mild developmental delays, particularly in posture and movement areas, which might have been caused by congenital CMV infection. J. Med. Virol. 81:1773–1776, 2009.

Prevalence of GB Virus C/Hepatitis G Virus Ribonucleic Acid and Anti-Hepatitis G Virus-E2 Antibodies among Japanese Children with Histories of Transfusions or with Liver Diseases

To clarify the prevalence of Japanese children thought to be at a risk for infection with GB virus-C (GBV-C)/hepatitis G virus (HGV), we investigated the detection rates of serum GBV-C/HGV ribonucleic acid (RNA) by reverse transcription-seminested PCR and serum anti-HGV-E2 antibody by ELISA in 162 children with histories of blood or plasma product transfusions or with liver diseases and performed phylogenetic analysis of the 5′ noncoding region sequences of GBV-C/HGV genomes. Children with histories of transfusions were divided into those who had been treated with antineoplastic agents for malignant diseases (malignant group) and those who had received transfusions for nonmalignant diseases (nonmalignant group). Children with liver diseases were divided into hepatitis B (HBV), hepatitis C (HCV), and non-A-C hepatitis groups. We detected GBV-C/HGV RNA in 11 of 33 (33.3%) and anti-HGV-E2 in 1 of 27 (3.7%) children in the malignant group and in 3 of 56 (5.4%) and 1 of 53 (1.9%) children, respectively, in the nonmalignant group. Neither GBV-C/HGV RNA nor anti-HGV-E2 was detected in the HBV and non-A-C hepatitis groups. GBV-C/HGV RNA and anti-HGV-E2 were detected in 7 of 23 (30.4%) and in 1 of 18 (5.6%) children, respectively, in the HCV group. All children positive for either GBV-C/HGV RNA or anti-HGV-E2, except one whose route of GBV-C/HGV infection suggested mother-to-infant transmission, had histories of transfusions. The phylogenetic analysis showed that all isolates in this study were divisible into three groups and that most of them were clustered into group 3 (Asian group).

Prevalence of GBV‐C/HGV infection in pregnant Japanese women

Recently, a novel viral agent, hepatitis G virus, was identified by independent researchers from the serum of patients with liver disease, and termed GBV-C or HGV. At present, GBV-C and HGV are considered to be separate isolates of the same virus; however, the role of this virus in acute and chronic liver disease remains uncertain. Although vertical transmission is known to be one of the routes of transmission, the prevalence of GBV-C/HGV viremia in pregnant Japanese women is unknown. Thus, we determined this prevalence using the reverse transcription polymerase chain reaction (RT-PCR).