Molecular biology of alcohol dependence, a complex polygenic disorder.

Alcohol dependence, and the medical conditions which arise from prolonged excessive alcohol use, have no single cause. Like other complex diseases, they result from a combination of social, personal and genetic contributions; but within any society genetic variation has a substantial influence on individual risk. The genes presently known to affect alcohol dependence produce variation in alcohol metabolism; other genes which affect personality or susceptibility to intoxication are likely to be significant but so far reproducible evidence is scanty. Designs which include related subjects have advantages for the study of complex diseases, because any association effects can be placed in the context of overall heritability and because linkage analysis can also be included. Examples of our studies of alcohol metabolism, consumption and dependence are presented.

ADH genotypes and alcohol use and dependence in Europeans.

We have tested for effects of alcohol dehydrogenase (ADH) genotypes on self-reported alcohol consumption and symptoms of alcohol dependence, recorded on three occasions up to 15 years apart, in 377 male and female subjects of European descent. ADH2 genotype had significant effects on both consumption and dependence in the men, but not in the women. The effects of ADH3 genotype were considerably less than those of ADH2, but significant results could be demonstrated when the combined genotypes were considered. The direction of the effects on alcohol consumption and dependence risk were consistent with reports on Asian subjects, and with the in vitro properties of ADH isoenzymes. As with previous studies on the relationship between ADH type and alcohol use, population stratification cannot be excluded as a contributing factor in these results.

Fourier transform infrared spectroscopy of dysplastic, papillomavirus-positive cervicovaginal lavage specimens.

To assess the utility of a new, rapid, economical procedure that may prove valuable in cervical screening, Fourier transform infrared (ir) spectroscopy was performed on 25 cervicovaginal lavage specimens from women referred for colposcopy on the basis of a cytological abnormality detected on their Pap smear and whose lavage specimen was positive for human papillomavirus. Of the 18 classed as CIN I or less by histopathology, 11 showed band frequencies that deviated only slightly from spectra that characterize normal cervical cells and 3 of 5 "atypia" specimens had spectra identical to normal. Two of 3 classed as CIN II had spectra only slightly more abnormal to these 11. In the case of 2 graded as CIN I, several bands were similarly altered in the direction of the pattern seen for 4 CIN III specimens. A further CIN I sample gave a spectrum that was even further shifted toward the latter and the remaining CIN I sample had a pattern that matched the 4 CIN IIIs. The most obvious change in each of the CIN IIIs was an additional peak at 972 cm-1 and this has been suggested as a key indicator for malignancy. One of the 3 CIN IIs had this peak. Other characteristic spectral changes were seen as well in the CIN III samples. High-risk HPV18 was present in 3 of the CIN III samples, as well as in one specimen classed as atypia, but having an abnormal ir spectrum. Low-risk HPV 6 or 11 was seen along in samples with a normal or slightly abnormal ir spectrum, but never in those that showed an ir pattern that was abnormal. The current study has therefore shown complete concordance between ir spectral findings and histopathology result in the case of CIN III specimens, but less precise matching for other grades of CIN. The spectral differences revealed by ir spectroscopy are likely to characterize molecular abnormalities in cervical cells during progression to cancer and may therefore have potential in assisting with clinical decision making. More studies will, however, be required to establish the place of this technique in cervical screening.

Assessment of HIV status using the polymerase chain reaction in antibody-positive patients and high-risk antibody-negative haemophiliacs.

The polymerase chain reaction (PCR) was used to identify the presence of DNA sequences homologous to HIV-1 in the buffy-coat leukocytes of antibody-positive and antibody-negative individuals in a haemophiliac population. The presence of HIV sequences was demonstrated in all of the antibody-positive haemophiliacs with the exception of one patient who was repeatedly negative. None of the seronegative haemophiliacs gave an overall positive result, although there were clear differences between this population and the negative controls who were examined. We conclude that, in our hands, PCR represents a reliable test which represents a useful diagnostic advance in HIV medicine.

Automated polymerase chain reaction for papillomavirus screening of cervicovaginal lavages: comparison with dot-blot hybridization in a sexually transmitted diseases clinic population.

The aim of the present study was to compare the recently developed polymerase chain reaction (PCR) technique with conventional dot-blot DNA hybridization for human papillomavirus (HPV) detection. Cells were collected by cervicovaginal lavage from a study group of 109 women attending a sexually transmitted diseases clinic. Using a machine that we developed for alternation of temperature cycles, HPV was detected in 51% of patients by PCR. By dot-blot hybridization, 44% of the patients were positive. Concordance of combined positive and negative results between PCR and dot blot was 69%. The greater sensitivity of PCR may have accounted for 19% of specimens that were PCR positive but dot-blot negative. Unexpectedly, however, 12% of specimens were dot-blot positive but negative by PCR, and several specimens were discordant for type of HPV. Both HPV DNA tests agreed with cytology in 41% of women, and in 33% cytology was negative in the face of positive PCR and dot blot. Concordance of cytology with just PCR was 59%, and only with dot blot was 56%. Cervicography agreed with both HPV DNA tests in 41% of patients, with PCR alone in 55%, and with dot blot alone in 58%. Biopsy results did not reveal a strong correlation between histopathological criteria of HPV infection and detection of HPV DNA by either PCR or dot-blot hybridization. Thus the present study has shown that PCR is a slightly more sensitive indicator of HPV infection than dot-blot hybridization. Agreement of HPV DNA results with conventional screening tests was not strong, an observation consistent with many comparative studies by others. In conclusion, PCR is slightly more sensitive than DNA hybridization for detection of HPV, it can be used in conjunction with specimen collection by gentle lavage of the cervicovaginal epithelium, and the possibility remains that it may prove suitable as a screening test.

Randomised controlled trial of recombinant human interferon -alpha A for chronic active hepatitis B.

The efficacy of interferon treatment for Australian patients with chronic active hepatitis B (CAH-B) was assessed by a three-centre randomised controlled trial in Sydney and Brisbane. Thirty patients (29 with histologically-proven CAH-B with and without cirrhosis and one with chronic persistent hepatitis) were allocated to receive either thrice weekly intramuscular injections of recombinant human leucocyte interferon -alpha A (either 2.5, 5.0 or 10.0 million units/m2) for six months followed by 12 months of observation, or to be observed for 18 months without active treatment. Three of 23 treated patients but none of seven controls underwent clinical, biochemical and histological resolution of their disease with loss of HBsAg, HBeAg and HBV-DNA from serum. An additional six treated and two control patients underwent a sustained partial remission of their disease. This was characterised by resolution of symptoms and serum aminotransferase abnormalities in association with seroconversion from HBeAg positive to negative, loss of HBV-DNA from serum but persistent hepatitis B surface antigenaemia. In such patients, there was significant improvement in histological appearances but some necroinflammatory activity remained and fibrosis was unchanged. Although total response rates were similar in treated and control subjects, they appeared to occur earlier after interferon treatment. Treatment with interferon was associated with predictable but minor side effects that usually did not necessitate dose reduction and rarely compromised the patient's life style. Interferon is thus a feasible treatment for CAH-B. Complete responses occurred only in treated patients and partial responses appeared to occur earlier in treated than in untreated patients. However, differences in the partial response rate at 18 months were not significant and seroconversion from HBeAg positive to negative was not associated with complete histological resolution of disease activity. Hence, while interferon is a promising agent for treatment of CAH-B, efforts must continue to define more optimal treatment regimes and to identify those patients most likely to respond to this agent.

The effect of concurrent human immunodeficiency virus infection on chronic hepatitis B: a study of 150 homosexual men.

To determine the influence of concurrent human immunodeficiency virus (HIV) infection on chronic hepatitis B virus (HBV) infection, 150 male homosexual chronic hepatitis B surface antigen (HBsAg) carriers were studied. Of these, 82 subjects (55%) tested positive for antibodies to HIV. They were more likely to express hepatitis B "e" antigen (HBeAg) (P less than .001) and HBV-DNA (P less than .0005) in serum than were HIV-seronegative individuals. However, the degree of immune suppression did not influence HBeAg-HBV-DNA expression. In HBeAg-seropositive subjects, concurrent HIV infection was associated with lower serum alanine transferase levels (P less than .001). This effect increased with the degree of immune suppression as determined by CD4+ lymphocyte counts. Conversely, in patients negative for HBeAg, there was a weak trend towards higher alanine transferase levels with concurrent HIV. This study suggests that chronic hepatitis B may be less severe when accompanied by HIV infection; however, greater viral replication may make it more contagious and resistant to antiviral therapy. These data support an immune-mediated pathogenesis for hepatitis B and have implications for its control.

Polymerase chain reaction for fast, nonradioactive detection of high- and low-risk papillomavirus types in routine cervical specimens and in biopsies.

A polymerase chain reaction (PCR) procedure capable of amplifying specific DNA sequences by up to a millionfold was developed for detection of infection by human papillomaviruses (HPVs) of low (HPV6, HPV11) or high (HPV16, HPV18, HPV33) oncogenic potential. For high-risk HPVs the region chosen was within the E6 open reading frame, which can become integrated into genomic DNA. A region corresponding to this was chosen for low-risk HPVs. After repeated cycles of specific oligonucleotide-primed extension of viral DNA with Klenow or thermophilic DNA polymerase, the type of HPV present was then determined on the basis of the size of the ethidium-bromide-stained band visible after polyacrylamide gel electrophoresis: for HPV6 or 11 the band was approximately 120 bp, for HPV 16 or 33 it was approximately 200 bp, and for HPV18 it was approximately 100 bp. Specific hybridization to the relevant band was seen using radioactive or nonradioactive (alkaline-phosphatase-linked) target oligonucleotide probes. Using the PCR method, we have determined, within as little as a few hours, the infection status of a variety of clinical specimens, including cervical scrapes and lavages, anal scrapes, and anogenital biopsies. The PCR steps can be automated, adding to the potential of PCR for widespread use in the detection of HPV, which is becoming increasingly popular in cervical screening.

Papillomavirus and cervical cancer: a clinical and laboratory study.

It is now widely accepted that HPV types 16, 18, 31, and 33 are associated with the development of high grade intraepithelial neoplasia and malignant lesions in the cervix. On this basis, the identification of HPV types in cervical scrape samples has been advocated as a supplement to cytological screening tests. However, little is known of the distribution of the virus at different sites in the lower female genital tract or of how this distribution may change during the natural course of HPV infection. In this survey, HPV DNA dot hybridizations and, in some instances, Southern blot hybridizations with mixed HPV 6/11 and 16/18 probes were undertaken to detect HPV DNA in cervical scrapes and biopsies of the cervix, vagina, and vulva. A total of 92 women attending a Sydney hospital were screened: 59 of these patients had cervical disease, either invasive cervical carcinoma (CaCx) or cervical intraepithelial neoplasia (CIN), grades I-III. A group of 33 women who lacked evidence of cervical abnormalities served as controls. HPV DNA, predominantly type 16/18, was detected in the cervical biopsies of 96% of the CaCx patients, 80% of the CIN III patients, and 65% of the CIN I-II patients. In contrast only 9% of the cervical biopsies from the control group contained detectable HPV 6, 11, 16, or 18 DNA. A high proportion of the women with cervical abnormalities had evidence of concurrent vaginal and/or vulval papillomavirus involvement. The significance of these findings for routine screening and subsequent management of patients with HPV-associated cervical disease is discussed.

Are specimens with "at risk" biochemical profiles more likely to be infectious for hepatitis B virus?

The potential infectivity of 640 plasma specimens with various biochemical profiles was directly assessed by hepatitis B virus (HBV) DNA dot-hybridization. We found that specimens with "at risk" biochemical profiles typical of various forms of liver disease were not significantly more likely to carry HBV than the general patient population. Specimens containing HBV cannot be distinguished from non-infectious specimens by any simple biochemical tests, including aminotransferases and bilirubin. The only predictive feature of HBV-positive samples was that they were more likely to be labeled as "biohazardous" by the medical staff, but even this was not always the case.

False-positive results with hepatitis B virus DNA dot-hybridization in hepatitis B surface antigen-negative specimens.

Three serum samples derived from healthy hepatitis B surface antigen-negative subjects were found to be reactive for hepatitis B virus (HBV) DNA sequences when assayed by DNA dot-hybridization. All three results were shown to be due to the presence of sequences which reacted with residual bacterial plasmid vector sequences in the DNA probe, and no evidence of HBV markers was demonstrated in the sera. This is the first report of a false-positive result with the HBV DNA dot-hybridization assay.

Relationship between hepatitis B virus DNA in blood and serological markers of hepatitis B infection.

The hepatitis B virus (HBV) DNA dot-hybridization assay has been introduced into clinical practice in Australia and its behaviour compared with that of the classic markers of HBV infection. A good correlation exists between the presence of HBV DNA and that of hepatitis B e antigen but the degree of dissociation between HBV DNA and the e:anti-e system was smaller than earlier studies would suggest. Patients who are seronegative for hepatitis B surface antigen (HBsAg), whether they possess anti-HBs, anti-HBc HBc (antibody to the core antigen), or neither antibody, gave uniformly negative results for the presence of HBV DNA. Theses results are different from those that were obtained from earlier studies of patients with chronic liver disease.

Can potentially infectious specimens containing hepatitis B virus be identified on the basis of their biochemical profile?

The potential infectivity of 1129 randomly selected plasma specimens was directly assayed by hepatitis B virus (HBV) DNA dot-hybridization. Presence or absence of HBV was then correlated with a biochemical profile of 20 common analytes obtained on these same specimens. We found that potentially infectious specimens could not be identified on the basis of any combination of simple biochemical tests; indeed, the infectious specimens were more "normal" in some tests of liver function than were the non-infectious specimens.