Alphabet of hepatitis viruses

Crigler-Najjar syndrome type II showed 6% of the activity in a similar co-transfection experiment. These results indicate that there are mutations in the coding region, with a wide variation in activity depending on the nucleotide and the location of the substitution, and that there may be no discrete boundary between the enzyme activities of Crigler-Najjar syndrome type II and Gilbert’s syndrome. Another noteworthy feature is that a homozygous mis-sense mutation was present in 11 of the 19 patients.4 This result indicates that a tiny proportion of heterozygous mutations found in Gilbert’s syndrome become Crigler-Najjar syndrome type I or type II in the homozygous state. In type I there is null enzyme activity (serum bilirubin usually 7342 amol/L). In type II there is 10% of normal enzyme activity (103-342 )J,mol/L). This may explain the difference in incidence rates between Gilbert’s syndrome with heterozygous mutations and the rare Crigler-Najjar syndromes with homozygous mutations. The TATA box mutation may contribute to a shift of bilirubin concentration in carriers of the mutations in the coding region from a low to high concentration group of Gilbert’s syndrome, and from the concentration range of Gilbert’s syndrome to that of Crigler-Najjar syndrome type II. Monaghan and Bosma based their findings on analysis of serum bilirubin concentration. For precise evaluation of the TATA box mutation, it is important to analyse the hepatic transcription level of the gene.If such analysis proves difficult, then it is necessary to analyse the TATA box of those who have been clearly diagnosed as having Gilbert’s syndrome based on hepatic enzyme activity.

substituted

bilirubin UDP-glucuronosyltransferase Japanese patients with Gilbert’s syndrome Boxes E1-E5 denote exon 1 through 5 (not precisely to scale). Large

Figure: Mutation

sites

on

gene of 19

solid black boxed

areas

denote

coding regions. Open and

solid

triangles

represent heterozygous and homozygous mutations, respectively.

Triangles with asterisks indicate patients with simultaneous heterozygous mutations both m the TATA box and in the coding regions. Triangles with crosses indicate patients whose TATA box has not been analysed.

by Monaghan and Bosma indicated the cause of the decreased activity by showing that these patients have a homozygous mutation in the TATA box (2-base insertion mutation), which results in a low level of transcription. What is the relation between the newly found mutation and mutations previously detected in the coding region of the gene?2 The generally accepted definition of Gilbert’s syndrome is a bilirubin concentration between 17 and 102 mol/L (1-6 mg/dL). Average concentrations in the mutated TATA box homozygotes, as reported by Monaghan and Bosma, are 24 and 14 jjmol/L, respectively (1-4 and 0-8 mg/dL). These concentrations seem too low to explain the condition in all patients with the syndrome. As suggested by Monaghan, there may be two groups of individuals, one with low and the other with high bilirubin concentrations, caused by different types of mutations; or, as described by Bosma, the mutation in the promoter region may not be sufficient for full manifestation of the syndrome. Our reported and ongoing gene analyses of 19 patients (age 14-52 years) revealed eight patients with no mutations in the coding region (figure) .2’ The average bilirubin concentration of the 19 patients was 44 mol/L (2-6 mg/dL). Two of the eight patients were heterozygous rather than homozygous for the mutation, indicating that the TATA box abnormality is not the sole cause of the syndrome. In view of these results, there must be other the factors that contribute to development of unconjugated hyperbilirubinaemia. Monaghan and Bosma each analysed more than ten patients and did not find any mutations in the coding region. However, our ongoing analyses showed that 11 of 19 patients have mutations in the coding region (figure). The reason for the discrepancy between these results is unclear. Diagnoses in eight of our 19 cases were based on analysis of hepatic enzyme activity and/or bile analysis. None of the patients with mutations in the coding region was homozygous for the mutated TATA box (TATA box of four patients not analysed). Three were heterozygous for the abnormal TATA box and four were homozygotes of wild-type TATA box. These results indicate that some individuals with Gilbert’s syndrome do not have the homozygous mutation in the TATA box.4 One of the defective genes found in these heterozygous patients (Pro229 Gln) was expressed in cultured cells.2,3 Co-transfection of the gene together with the same of normal gene showed 25% of the enzyme of that of the normal control. Furthermore, a activity mutation (Gln331Stop) detected in a patient with amount

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Hiroshi Sato, Yukihiko Adachi, Osamu Koiwai Department of Biology, Shiga Unversity of Medical Science; Second Department of Internal Medicine, Kinki University School of Medicine; and Department of Biochemistry, Aichi Cancer Center Research Institute, Japan 1

2

3

4 5

Bosma PJ, Chowdhury RJ, Bakker C, et al. The genetic basis of the reduced expression of bilirubin UDP-glucuronosyl-transferase 1 in Gilbert’s syndrome. N Engl J Med 1995; 333: 1171-75. Aono S, Adachi Y, Uyama E, et al. Analysis of genes for bilirubin UDP-glucuronosyltransferase in Gilbert’s syndrome. Lancet 1995; 345: 958-59. Koiwai O, Nishizawa M, Hasada K, et al. Gilbert’s syndrome is caused by a heterozygous missense mutation in the gene for bilirubin UDP-glucuronosyltransferase. Hum Mol Genet 1995; 4: 1183-86. Soeda Y, Yamamoto K, Adachi Y, et al. Predicted homozygous mis-sense mutation in Gilbert’s syndrome. Lancet 1995; 346: 1494. Schmid R. Gilbert’s syndrome: a legitimate genetic anomaly? N Engl J Med 1995; 333: 1217-18.

Alphabet of hepatitis viruses The past two decades have witnessed an explosion in knowledge of viral hepatitis. There is now a bewildering array of hepatotropic viruses designated hepatitis A, B, C, D, and E, with the recent addition of three GBV virusesGBV-A, GBV-B, and GBV-C.1 In January, hepatitis X, a long-awaited agent referred to informally for several months at scientific meetings, was designated hepatitis G or HGV.2 Isolation of the GB viruses A and B as novel flaviviruslike agents in tamarins infected originally with serum from a young surgeon with acute icteric hepatitis,3 facilitated the identification of another virus, GBV-C, from a human specimen.4 GBV-C RNA was subsequently found in several patients with clinical hepatitis and in population groups at risk of hepatitis such as multitransfused individuals, intravenous drug users, and haemophiliacs.

Figure: Unrooted phylogenetic

tree based on

analysis

of the

polyproteins5 cloned and characterised and its full-length genomic sequence was described.5 The virus provisionally designated HGV was isolated independently by Linnen et al2 from the plasma of a patient with chronic hepatitis who was co-infected with hepatitis C. Extension from an initial immunoreactive cDNA clone yielded a 9392 nucleotide sequence representing the entire genome. Examination of this sequence established that the genome of this virus resembles that of members of the flavivirus family in its organisation, conserved motifs, and aminoacid sequence. The immediate issue is whether this virus is a new genotype of hepatitis C virus. However, as is the case with GBV A, B, and C, the newly diagnosed isolate is only distantly related to HCV and to GBV A and B. The intriguing question is the precise relation of HGV to GBV-C. Linnen et al report that HGV and GBV-C are very similar based on a comparison of the 331 base-pair sequences from the NS3 helicase region of the two isolates. However, as was indicated by the researchers, to establish accurately the relatedness of the two isolates it is necessary to compare the entire genome of HGV and GBV-C. This exercise was possible with the availability of the genomic sequences deposited in the GenBank database, as was reported at the November, 1995, meeting of the American Association for the Study of the Liver in Chicago. (Comparisons between aminoacid sequence identity were made with the Wisconsin Sequence Analysis Software Program GAP. GenBank accession numbers: GBV-A [U 22303]; GBV-B [U 22304]; GBV-C [U 36380]; HGV [U 44402] and HCV [M 62321].) Briefly, alignment of the encoded polyprotein sequences of GBV-C with that of HGV shows aminoacid sequence identity at 95% (85% at the nucleotide level). Thus, based on this high degree of sequence identity, we can say that GBV-C and HGV are independent isolates of the same virus. By contrast, neither GBV-C nor HGV shows identity greater than 32% with GBV-A, GBV-B, or HCV. GBV-A and B6 and the GBV-A, B, and C (HGV) and hepatitis C viruses belong to a discrete group of viruses within the Flaviviridae. This new group of viruses can be delineated further into a GB virus lineage consisting of GBV A, B, C and HGV, and the HGV lineages (figure). Within the GB virus family, GBV-A and GBV-C (HGV) are more closely related (48% aminoacid sequence identity), whereas GBV-B, which bears no more resemblance to the other GB viruses (and HGV) than it does to hepatitis C virus,

GBV-C

was

seems to be the sole representative of its own subgroup. Thus the newly identified virus2 may be designated provisionally as GBV-C or HGV until the Committee on Viral Taxonomy and Nomenclature has placed them into their appropriate family and genus. GBV-C/HGV RNA has been found in some patients with acute or chronic hepatitis, fulminant hepatitis patients in Japan,7 intravenous drug users, haemophiliacs, patients treated by maintenance haemodialysis, multiply transfused patients, and blood donors. These observations indicate that this virus can be transmitted by blood and blood products. Extensive epidemiological studies based on immunoassays, which are under development, and probe-based assays are required to define more clearly whether GBV-C/HGV is a true hepatitis virus and to establish its clinical significance.

Arie J Zuckerman WHO Collaborating Centre for Reference and Research on Viral Diseases, Royal Free Hospital School of Medicine, London, UK 1 2

3

4

Zuckerman AJ. The new GB hepatitis viruses. Lancet 1995; 345: 1453-54. Linnen J, Wages J, Zhang-Keck Z-Y, et al. Molecular cloning and disease association of hepatitis G virus: a transfusion-transmissible agent. Science 1996; 271: 505-09. Simons JN, Pilot-Matias TJ, Leary TP, et al. Identification of two flavivirus-like genomes in the GB hepatitis agent. Proc Natl Acad Sci USA 1995; 92: 3401-05. Simons JN, Leary TP, Dawson GJ, et al. Isolation of novel virus-like sequences associated with human hepatitis. Nature Med 1995; 1: 564-69.

5

6

7

Leary TP, Muerhoff AS, Simons JN, et al. Sequence and genomic organization of GBV-C: a novel member of the Flaviviridae associated with human non-A-E hepatitis. J Med Virol 1996; 48: 60-67. Muerhoff AS, Leary TP, Simons JN, et al. Genomic organization of GB viruses A and B: two new members of the Flaviviridae associated with GB agent hepatitis. J Virol 1995; 69: 5621-30. Yoshiba M, Okamoto H, Mishiro S. Detection of the GBV-C hepatitis virus in serum from patients with fulminant hepatitis of unknown origin. Lancet 1995; 346: 1131-32.

Do whatever works in your hands A report can capture one’s attention for all sorts of reasons. When I came across the article by Wilson and colleagues entitled "Subjective effects of double gloves on surgical performance"’ I had just tried a double-glove system myself for a thoracotomy. I agree with the researchers’ conclusion that "double gloves often protect the surgeon against needle perforation, but ... impair comfort, sensitivity and dexterity". However, once the paper had caught my eye, I saw more in it than that. I found myself pondering on the fact that, after generations of health workers have tried to protect patients from infection caught in hospital,2 we now seek to protect ourselves from the patients. Then I was reminded of the eye surgeon who operated without gloves to retain the sensibility in his fingers, and of the thoracic surgeon, in the early days of closed mitral valvotomy, who had the right index finger cut off his gloves so that he could better feel the valve. More important to me was how this research into what we put "on" our hands contrasts with the old adage "do whatever works in your hands"-a piece of advice often given to young surgeons. I have always found that slogan shallow to the point of being facile. It might apply to the choice of instruments, or how to tie a knot, but I have heard it used for the choice of prosthetic heart valves, when very few of the factors that determine the short medium, and long term results of valve surgery come

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