Hunting genetic diseases: exploring a multistage approach to identifying disease loci

Genomic screen data for a hypothetical disease was used in a two-stage analysis to search for disease loci. We performed both trait-model-dependent and trait-model-free analyses to test their relative power. Results of our first-stage screen in 200 families suggested 13 regions for further analysis. Second-stage follow-up in another 200 families confirmed a single region on chromosome 3 near marker D3G045 with a combined lod score across all 400 families of 6.24 and a sib-pair maximum lod score (MLS) of 4.79. The MLS were highly correlated with both the autosomal dominant and autosomal recessive lod scores in all data sets, suggesting that both trait-model-dependent and trait-model-free methods can be useful for identifying candidate regions for complex disease loci. Reanalysis of the data using alternative sampling schemes suggested that sampling variation has a significant effect on locus detection.     

Multiorgan transplacental and neonatal carcinogenicity of 3'-azido-3'-deoxythymidine in mice

The anti-HIV drug 3'-azido-3'-deoxythymidine (AZT) is used successfully for reduction of perinatal viral transmission. However toxic side effects including carcinogenesis are possible. To test this, pregnant CD-1 Swiss mice were given 25.0 or 12.5 mg AZT on gestation days 12-18. Previously we reported an increase in lung, liver, and female reproductive system tumors in offspring euthanized at 1 year (Olivero et al., J. Natl. Cancer Inst. 89, 1602-1608, 1997). Findings for all remaining offspring up to 2 years old are reported here. AZT effects were most prominent in female offspring, with a significant threefold increase in lung tumors, a reduction in lymphoblastic and follicle center cell lymphomas, and a significant increase in histiocytic sarcomas (0 in controls, 3% after low-dose AZT, and 8% after high-dose AZT, p = 0.022). Dose-dependent incidences of mammary gland, ovarian, and seminal vesicle tumors were low but significant: 0/106 controls, 3/105 low-dose, and 8/105 high-dose mice presented one of these neoplasms (p = 0.0025). Incidences of females showing any clearly AZT-related neoplasm, in lung, liver, ovary, or mammary gland or histiocytic sarcoma, in the second year, were 12/32 after the low dose and 14/27 after the high dose vs 3/23 controls (p = 0.0045). Also, the sensitivity of neonatal mice was assessed by administration of 25, 50, 100, or 200 mg/kg AZT on postnatal days 1 through 8. The effects at 2 years were similar to those seen after transplacental exposure, with significant increases in lung, liver, and mammary tumors in females. The results confirm that AZT is a moderately effective perinatal carcinogen in mice, targeting several tissue types.     

Libraries and the R&D strategy: a way forward

As guest editor for the December 1994 issue of Health Libraries Review, I chose the theme of Evidence-Based Practice. In my editorial I suggested that Evidence-based Practice offered tremendous opportunities for NHS librarians to demonstrate their skills in supporting a knowledge-based NHS, because many clinicians had complained that they did not have time, retrieval skills of knowledge of relevant information resources to be effective at finding scientific evidence. Librarians, on the other hand have advanced online searching skills, rapid document retrieval and delivery services, and up-to-date knowledge of the world's medical information resources and networks. These skills mean that librarians are not only well-placed to support clinicians in finding and sifting scientific evidence, but also in teaching clinicians how to search for and store information themselves. NHS librarians have not been slow to recognize these opportunities and innovative professional development programmes have appeared to help hone their skills, such as the Librarian of the 21st Century Programme in the Anglia and Oxford region. The success of NHS librarians in supporting evidence-based health care has led to their formal development in evidence-based medicine workshops for clinicians in a number of regions. In March 1996, as NHS Library Adviser. I was asked to prepare a paper for the R&D Board of the NHS Executive about how this library support could be formally integrated into the R&D Strategy. My paper was unanimously endorsed by the Board and later by the Central R&D Committee of the NHS Executive. It suggests principles of library provision in support of R&D and is reprinted below.     

Anionic lipid headgroups as a proton-conducting pathway along the surface of membranes: a hypothesis.

Evidence has been gathering from several laboratories that protons in proton-pumping membranes move along or within the bilayer rather than exchange with the bulk phase. These experiments are typically conducted on the natural membrane in vivo or in vitro or on fragments of natural membrane. Anionic lipids are present in all proton-pumping membranes. Model studies on the protonation state of the fatty acids of liposomes containing entrapped water show that the bilayers always contain mixtures of protonated and deprotonated carboxylates. Protonated fatty acids form stable acid-anion pairs with deprotonated fatty acids through unusually strong hydrogen bonds. Such acid-anion dimers have a single negative charge, which is shared by the four negative oxygens of both headgroups. The two pK values of the resulting dimer will be significantly different from the pK of the monomeric species, so that the dimer will be stable over a wide pH range. It is proposed that anionic lipid headgroups in biological membranes share protons as acid-anion dimers and that anionic lipids thus trap and conduct protons along the headgroup domain of bilayers that contain such anionic lipids. Protons pumped from the other side of the membrane may enter and move within the headgroup sheet because the protonation rate of negatively charged proton acceptors is 5 orders of magnitude faster than that of water. Protons trapped in the acidic headgroup sheet need not leave this region in order to be utilized by a responsive proton-translocating pore (a transport protein using the proton gradient). Experiments suggest the proton concentration in the headgroup domain may vary widely and the anionic lipid headgroup sheet may therefore function as a proton buffer. Due to the Gouy-Chapman-Stern layer at polyanionic surfaces, anionic lipids will also sequester protons from the bulk solution at low and moderate ionic strengths. At high ionic strength metal cations may replace protons sequestered near the headgroups, but these cations cannot substitute for protons in the "proton-conducting pathway," which is based on hydrogen bonding.     

Increased prevalence of the HFE C282Y hemochromatosis allele in women with breast cancer.

Individuals with the major hemochromatosis (HFE) allele C282Y and iron overload develop hepatocellular and some extrahepatic malignancies at increased rates. No association has been previously reported between the C282Y allele and breast cancer. We hypothesized that due to the pro-oxidant properties of iron, altered iron metabolism in C282Y carriers may promote breast carcinogenesis. Because 1 in 10 Caucasians of Northern European ancestry carries this allele, any impact it may have on breast cancer burden is potentially great. We determined C282Y genotypes in 168 patients who underwent high-dose chemotherapy and blood cell transplantation for cancer: 41 with breast cancer and 127 with predominantly hematological cancers (transplant cohort). Demographic, clinical, and tumor characteristics were reviewed in breast cancer patients. The frequency of C282Y genotypes in breast cancers was compared with the frequency in nonbreast cancers, an outpatient sample from Tennessee (n = 169), and a published United States national sample. The frequency of at least one C282Y allele in breast cancers was higher (36.6%, 5 homozygotes/10 heterozygotes) than frequencies in Tennessee (12.7%, P < 0.001), the general population (12.4%, P < 0.001), and similarly selected nonbreast cancers (17.0%, P = 0.008). The likelihood of breast cancer in the transplant cohort increased with C282Y allele dose (P(trend) = 0.010). These results were supported by the finding in a nontransplant cohort of a higher frequency of C282Y mutations in Caucasian (18.4%, P = 0.039) and African-American (8.5%, P = 0.005) women with breast cancer than race-specific national frequency estimates. A high prevalence of C282Y alleles in women with breast cancer with and without poor risk features suggests that altered iron metabolism in C282Y carriers may promote the development of breast cancer and/or more aggressive forms of the disease.     

Outcome of children with respiratory symptoms without objective evidence of asthma: a two-year, prospective, follow-up study

This study evaluated the outcome of 33 children with asthma-like symptoms without objective evidence of asthma, and the role of certain factors in predicting the development of clinical asthma in these children. Data on symptom histories, lung functions (flow-volume spirometry, free running test and methacholine inhalation challenge test) and atopic sensitization (skin prick tests and markers of eosinophilic inflammation) were collected twice with an interval of 2 y, and the diagnoses were re-evaluated after the follow-up period. Based on the results, it was concluded that one-third of the children with prolonged or recurrent lower airway symptoms, such as cough or wheeze, either have mild asthma or will develop asthma in the near future. Children who had a significant response [≥ 10% fall in forced expiratory volume in 1 s (FEV1)] in the free running test formed a risk group for active asthma, whereas other baseline characteristics seemed not to predict the outcome.     

Randomized trial examining cerebral haemodynamics following artificial or animal surfactant

To determine the effects of animal and artificial surfactants on cerebral haemodynamics, 20 premature babies receiving mechanical ventilation were randomized to receive Curosurf or Exosurf surfactant. Anterior cerebral artery blood flow velocity (CABFV) was measured using Doppler ultrasound before and up to 2 h after treatment. Following animal surfactant there was a rapid reduction in CABFV (median -36%, range -43% to +8%, p < 0:01), whereas artificial surfactant resulted in a slower rise which was less marked (median +20%, range -7% to +62%, p < 0:05). There were no significant changes in blood pressure. Two hours after administration, the oxygenation index (OI) improved significantly only in babies receiving animal surfactant. In this group there was a significant association between the change in CABFV at 1 min and the change in OI at 2 h ( r = 0:66, p < 0:05). Animal surfactant produces rapid improvements in ventilation which are associated with marked alterations in cerebral haemodynamics.     

Comparison of cytogenetic damage in cultured cells from cobalt-60 gamma-radiation and the Auger emitter zinc-65

PURPOSE: To assess the ability of the Auger-emitting nuclide, zinc-65 (65Zn), relative to gamma-irradiation, to cause chromosomal aberrations in cultured rat prostate cells. MATERIALS AND METHODS: Rat prostate adenocarcinoma cells in culture were exposed to doses of 1, 2, 3 or 5 Gy of external gamma-irradiation for 24h or incubated with 0.7, 1.5, 1.8 or 2.8 MBq of 65Zn for 24 h. The uptake by and clearance from cells of 65Zn was measured. Metaphase spreads prepared from washed cells were scored for chromatid- and chromosome-type aberrations. RESULTS: Following exposure to 65Zn or gamma-irradiation, chromatid-type damage was more commonly observed than chromosome-type aberrations. The relationship between induced chromatid damage and gamma dose (to 3 Gy) was best fitted by a second-order polynomial function, while the activity response relationship for chromatid damage caused by 65Zn appeared to be best fitted by a straight line. Measurements of the uptake of 65Zn by cells showed that average concentrations within cells were about 100 times the concentration in the culture medium. Assuming uniform distribution of 65Zn within cells, with 36% in the nucleus, the dose was estimated as 0.70 Gy per MBq added 65Zn, with Auger electrons contributing most (93%) of the dose. Assuming that 20% of cellular zinc was localized in the nucleus, based on previous measurements, the dose to the nucleus was calculated as 0.44 Gy per MBq added 65Zn. RBE values for chromatid damage induced by 65Zn compared to gamma-radiation range from about 1 to 3 based on a uniform dose throughout the cell and from about 2 to 5 based on 20% of 65Zn in the cell nucleus. CONCLUSION: The observed radiotoxicity of 65Zn is consistent with its behaviour as an Auger-emitting radionuclide that is localized to some extent in the nucleus.     

Immunohistochemical examination of the INK4 and Cip inhibitors in the rat neonatal cerebellum: cellular localization and the impact of protein calorie malnutrition

Expression of the cyclin-dependent kinase inhibitors (CKIs) has been linked to the inhibition of cellular proliferation and the induction of differentiation. Based on structure function analysis, two distinct families of CDKIs, the INK4 and the Cip/Kip family have been identified. The INK4 family member p16(Ink4), and the Cip/Kip protein p27(Kip1) have been implicated in normal development of the CNS and cerebellum. Recent studies have suggested a functional inter-dependence between the CKI and the abundance of cyclin D1 in orchestrating growth factor-induced cellular proliferation. The neonatal rat cerebellum undergoes proliferative growth and differentiation, localized to distinct topographical regions and cell types. The cell type and the temporal profile of CKI expression during postnatal cerebellar development had not been described. The current studies determined the specific cerebellar cell types in which the CKIs were expressed during post natal development by co-staining for cell-type specific markers. p16(Ink4a) and p27(Kip1) immunostaining was identified in both neurons and glial cells, increasing progressively between postnatal days 6 to 13 into adulthood. By contrast, neuronal and glial cell p21(Cip1) staining was prominent at days 6-11 and decreased thereafter. Cyclin D1 was expressed in the proliferating external granular cells, with occassional staining in the molecular, and internal granular layers. Dual immunostaining demonstrated cyclin D1 within cells expressing CKI (p16(Ink4a), p21(Cip1),p27(Kip1)). Cerebellar cellular growth arrest, induced by protein-calorie malnutrition, inhibited cyclin D1 protein levels without affecting CKI immunostaining suggesting CKI do not mediate the developmental arrest. These results demonstrate that the CKIs are induced by differentiation cues in specific cell types with distinct kinetics in the developing cerebellum in vivo.     

The treatment of lateral clavicle fractures

This study assesses the results of surgical treatment of 15 displaced Neer type II fractures of the lateral clavicle in 15 patients, which occurred between November 1988 and March 1995 and which were followed up for a mean period of 4.6 years (range 2-9 years). The patients fell into two groups, one 'acute group' and one 'non-union' group.Patients treated initially by a non-operative approach had suffered prolonged morbidity and time off work prior to and after surgery. The ultimate result was good. The fixation used was a Dacron arterial graft as a sling around the clavicle and coracoid process. Delayed (non-union) cases were augmented with bone graft and inter-fragmentary screw fixation. All fractures eventually united.We question the place of prolonged non-operative management in the treatment of displaced Neer type II fractures of the lateral clavicle.