The impact of single gene and chromosomal disorders on hospital admissions of children and adolescents: a population-based study.

BACKGROUND: It is well recognized that genetic disease makes a significant contribution to childhood illness. Here, we present recent population data describing the impact of single gene and chromosomal disorders on hospital admissions of children and adolescents. METHODS: Hospital admissions for patients aged 0-19 years between 2000 and 2006, with a single gene or chromosomal disorder, were extracted from the Western Australian Hospital Morbidity Data System using 296 diagnosis codes identified from the International Statistical Classification of Diseases, Tenth Revision, Australian Modification. Data extracted for each patient included the number, length and cost of all admissions. RESULTS: Between 2000 and 2006, 14,197 admissions were identified for 3,271 patients aged 0-19 years with single gene and chromosomal disorders, representing 2.6% of admissions and 4.3% of total hospital costs in this age group. Patients with genetic disorders had more admissions and stayed longer in hospital than patients admitted for any reason. Specific disorders associated with a high demand on hospital services included cystic fibrosis, Down syndrome, osteogenesis imperfecta, thalassemia, and von Willebrand's disease. CONCLUSIONS: Children and adolescents with single gene and chromosomal disorders placed higher demands on hospital services than other patients in their age group, but were responsible for a relatively small proportion of hospital admissions and costs. These data will enable informed planning of health care services for patients with single gene and chromosomal disorders in Western Australia.

Magnetic resonance findings in women at high risk for developing breast cancer: an Australian feasibility study.

In younger women at high risk for developing breast cancer the value of mammography is limited by a higher prevalence of breast tissue density, low rate of DCIS in gene carriers, faster growing tumours and concerns over radiation exposure. We report on our experience of MR screening in high risk patients over a three year period. Women at high risk of developing breast cancer were offered an MRI scan and an Ultrasound in addition to their annual mammogram for two years. The following MR-protocol was used: pre-contrast T2 STIR sequence and pre contrast 3D FLASH sequence, post contrast axial dynamic 3D FLASH sequence. Seventy two women consented to participate in this study. One hundred thirty nine breast MRI examinations were performed. Two pre-cancerous lesions and an axillary lymph node metastasis were found, but the majority of the lesions were benign. Difficulties in screening young women at high risk are discussed in this paper. In our study three lesions of significance were detected. Two lesions were precancerous thus curable. The recall rates show the difficult nature of screening younger breasts. MRI generated more findings judged as uncertain, so short term-term follow up studies or MR-guided biopsy techniques are required.

Beta2-Adrenoceptor polymorphisms and asthma phenotypes: interactions with passive smoking.

The aim of the present study was to assess the effects of possible interactions between beta(2)-adrenoceptor gene polymorphisms and passive smoking on forced expiratory volume in one second (FEV(1)), forced vital capacity (FVC) and exhaled nitric oxide (eNO) in children aged 11 yrs. A cross-sectional analysis of the longitudinal cohort was conducted for associations between beta(2)-adrenoceptor gene polymorphisms and lung function and eNO with regard to passive smoking. Among children exposed to tobacco smoke, those with Arg16 (at least one Arg allele) exhibited lower adjusted mean FEV(1) (2.19 versus 2.38 L) and FVC (2.43 versus 2.64 L) than Gly16 homozygotes. Those with Gln27 (at least one Gln allele) also exhibited a lower adjusted mean FEV(1) relative to Glu27 homozygotes (2.24 versus 2.39 L). Among children with no exposure to smoking, those with Arg16 or Gln27 showed lower adjusted geometric mean eNO levels compared with Gly16 homozygotes (15.4 versus 30.9 ppb) and Glu27 homozygotes (18.0 versus 49.7 ppb). In conclusion, passive smoking had a significant effect on associations between beta(2)-adrenoceptor gene polymorphisms and asthma-related phenotypes, enhancing the relationship between Arg16 and lung function and removing the relationship between Arg16 or Gln27 and exhaled nitric oxide levels.

Early-onset atopy is associated with enhanced lymphocyte cytokine responses in 11-year-old children.

BACKGROUND: Early age at onset of atopy is associated with more severe asthma and increased airway responsiveness (AR); the underlying mechanism is unclear but may involve T cell responses. OBJECTIVE: To test the hypothesis that enhanced T cell responses may be associated with early-onset atopy. METHODS: In a longitudinal study, atopy was determined in infancy and at 6 and 11 years of age. Individuals were categorized as persistent infant-onset atopy (PIOA), early childhood-onset atopy (ECOA) and later childhood-onset atopy (LCOA). At 11 years of age, peripheral blood T cell cytokine responses, AR, exhaled nitric oxide (FE(NO)) and forced expiratory volume in 1 s were determined. RESULTS: The age at onset of atopy was determined for 60 children, of whom 15 had PIOA, 24 had ECOA and 21 had LCOA. An additional 76 children who were never atopic were also included. T cell responses to house dust mite, including interleukin-5, -9, -10 and tumour necrosis factor alpha, were higher among children with PIA and ECOA, and lower in children with LCOA, P<0.05. In contrast, those children with LCOA or who were not atopic had the highest IL-10 response to PHA (P=0.014). Children with PIOA and ECOA, but not LCOA, had higher AR and FE(NO) compared with non-atopic children (P<0.05). The group with PIOA were more likely among the atopic children to be admitted to hospital for asthma (P<0.05) and also had lower %FEV(1) compared with non-atopic children (P=0.023). CONCLUSIONS: Early age at sensitization is associated with enhanced T cell cytokine responses and indices of adverse asthma outcome. T cell cytokine responses might be programmed at the time of initial atopic sensitization.

Evolution and respiratory genetics.

Evolution is a plausible explanation for between-population differences in particular allele frequencies if: the genes involved have related functions; the heterogeneous alleles involved have similar functional consequences; the involved genes are not linked chromosomally; and the patterns observed would result in a biologically plausible, survival-enhancing gene-environment interaction. However, possible evolutionary effects have to be differentiated from founder effects and random genetic drift. The current authors have noted the existence of a consistent pattern of allelic frequencies in genes related to T-helper 2 (Th2) immune responses in humans of different ancestral backgrounds, residing in climatically similar regions. Th2 responses are thought to have evolved in mammals to resist infection by parasites, particularly helminths. Modern man arose in tropical Africa where helminths thrived. Relatively recently, humans migrated to cooler or drier climates where most helminths struggled to reproduce. The genetic tendency to strong Th2 responses may have become a health liability, the reduction in risk from parasites being counterbalanced by an increased inherited propensity to atopic or allergic diseases. The pattern noted by the present authors includes specific alleles of interleukin-4 and its receptor, interleukin-13, interleukin-10, the beta chain of the high-affinity receptor for immunoglobulin E, the beta(1)-adrenergic receptor, and the alpha chain of tumour necrosis factor. These population-specific polymorphism profiles are likely to be relevant in current disease patterns. The high incidence of asthma in migrants from tropical locations to affluent temperate countries is likely to be related to these patterns. Of even more concern is the possibility that increasing westernisation among the approximately 2 billion people living in the tropics will produce rapidly increasing levels of asthma, as these populations have a high genetic predisposition to allergic disease.

Economic evaluation of the familial cancer programme in Western Australia: predictive genetic testing for familial adenomatous polyposis and hereditary non-polyposis colorectal carcinoma.

AIM: To evaluate costs and outcomes of genetic testing for familial colorectal cancer through services provided by Genetic Services of Western Australia (GSWA). METHODS: Costs and outcomes of predictive DNA-based testing for inherited colorectal cancers (CRC) were assessed, specifically for familial adenomatous polyposis (FAP) and hereditary non-polyposis CRC (HNPCC) using a decision-analysis model. Costs were assigned according to standards of care in Western Australia (WA). Cancer risks and the efficacy of surveillance on long-term outcomes were derived from the published literature. RESULTS: The cost-effectiveness of genetic testing was compared in first-degree relatives of known mutation carriers who have a 50% risk of carrying the mutated gene (intervention group) to individuals with the same risk but who do not undergo a genetic test (control subjects). Compared with control subjects undergoing the same high-level surveillance and surgery, the FAP and HNPCC intervention groups provided total savings of 13,390 US dollars and 14,783-15,460 per person (males-females), respectively. HPNCC mutation carriers also gained 1 CRC-free year. Compared to control subjects having only population surveillance, individuals in the FAP intervention group delayed the onset of CRC by 40 years for a net cost of 9,042 US dollars. Individuals in the HNPCC intervention group delayed the onset of CRC by 8 years at a net cost of 12,141 US dollars for males and 12,596 US dollars for females. CONCLUSIONS: Genetic testing for familial CRC in WA allows targeted surveillance for mutation carriers, which ensures the efficient use of resources and reduces cancer-related morbidity, if clinical recommendations for intervention are adopted.

CD14 C-159T and early infection with Pseudomonas aeruginosa in children with cystic fibrosis.

Early acquisition of Pseudomonas aeruginosa is associated with a poorer prognosis in patients with cystic fibrosis. We investigated whether polymorphisms in CD14, the lipopolysaccharide receptor, increase the risk of early infection. Forty-five children with cystic fibrosis were investigated with annual bronchoalveolar lavage (BAL) and plasma sCD14 levels. Plasma sCD14 levels were significantly lower in children from whom P.aeruginosa was subsequently isolated (492.75 microg/ml vs. 1339.43 microg/ml, p = 0.018). Those with the CD14 -159CC genotype had a significantly increased risk of early infection with P.aeruginosa suggesting that CD14 C-159T plays a role in determining the risk of early infection with P.aeruginosa.

Determinants of airway responsiveness to histamine in children.

Increased airway responsiveness (AR) is associated with asthma, but not all individuals with increased AR have asthma. The aim of this study was to identify factors, other than physician-diagnosed asthma (PDA), which are associated with increased AR. In a longitudinal study, data were collected on atopy and lower respiratory tract illness (LRTI) in infancy, and AR (expressed as dose-response slope (DRS)), atopy, tobacco-smoke exposure and PDA in childhood. At age 6 yrs, DRS was assessed in 102 children, of whom 22 (22%) had PDA; the corresponding figures at 11 yrs of age were 176 and 29 (15%). At age 6 yrs, DRS was significantly associated with PDA, current atopy and parental smoking (n = 83). At age 11 yrs, DRS was significantly associated with PDA, current atopy and LRTI in the first six months (n = 75). There was a significant positive interaction between atopy at age 12 months and PDA age 11 yrs. In conclusion, these data suggest that factors other than asthma or atopy may determine the level of airway responsiveness in children. In children with asthma, airway responsiveness may be influenced by the early onset of atopy. The current findings may explain the inconsistent relationship between airway responsiveness and asthma.

An immunoepidemiological approach to asthma: identification of in-vitro T-cell response patterns associated with different wheezing phenotypes in children.

BACKGROUND: Increasing evidence suggests that patterns of T-cell immunity to inhalant allergens in genetically diverse human populations are more heterogeneous than previously assumed, and that covert differences in expression patterns might underlie variations in airway disease phenotypes. We tested this proposition in a community sample of children. METHODS: We analysed data from 172 individuals who had been recruited antenatally to a longitudinal birth cohort study. Of the 194 birth cohort participants, data from the 147 probands (age range 8.6-13.5 years) who consented to blood collection were included along with data from 25 consenting siblings (mean age 11 years [range 7.4-17.4]). We ascertained clinical phenotypes related to asthma and allergy. We measured T-cell responses to allergens and mitogens, together with blood eosinophils and IgE/IgG antibodies, and assessed associations between these indices and clinical phenotypes. FINDINGS: Atopy was associated with allergen-specific T-helper (Th)2 responses dominated by interleukin 4, interleukin 5, interleukin 9, interleukin 13, whereas interleukin 10, tumour necrosis factor alpha, and interferon gamma responses were common to both atopics and non-atopics. The wheal size from skin prick with allergen was positively associated with in-vitro interleukin 5 and interferon gamma responses, and negatively associated with interleukin 10. Asthma, especially in atopics, was strongly associated with eosinophilia/interleukin 5, and bronchial hyper-responsiveness (BHR) was associated with eosinophilia plus polyclonal interferon gamma production. BHR in non-atopics was associated with elevated allergen-specific and polyclonal interleukin 10 production. INTERPRETATION: Parallel immunological and clinical profiling of children identified distinctive immune response patterns related to asthma and wheeze compared with BHR, in atopics non-atopics. Immunological hyper-responsiveness, including within the Th1 cytokine compartment, is identified as a hallmark of BHR. RELEVANCE TO PRACTICE: These findings highlight the heterogeneity of immune response patterns in asthmatic children, including those with seemingly homogeneous Th2-driven atopic asthma. Further elucidation of the covert relationships between wheezing phenotypes and underlying immunophenotypes in this age group will potentially lead to more effective treatments for what is an unexpectedly heterogeneous collection of disease subtypes.

Features of colorectal cancers with high-level microsatellite instability occurring in familial and sporadic settings: parallel pathways of tumorigenesis.

High-level microsatellite instability (MSI-H) is demonstrated in 10 to 15% of sporadic colorectal cancers and in most cancers presenting in the inherited condition hereditary nonpolyposis colorectal cancer (HNPCC). Distinction between these categories of MSI-H cancer is of clinical importance and the aim of this study was to assess clinical, pathological, and molecular features that might be discriminatory. One hundred and twelve MSI-H colorectal cancers from families fulfilling the Bethesda criteria were compared with 57 sporadic MSI-H colorectal cancers. HNPCC cancers presented at a lower age (P < 0.001) with no sporadic MSI-H cancer being diagnosed before the age of 57 years. MSI was less extensive in HNPCC cancers with 72% microsatellite markers showing band shifts compared with 87% in sporadic tumors (P < 0.001). Absent immunostaining for hMSH2 was only found in HNPCC tumors. Methylation of hMLH1 was observed in 87% of sporadic cancers but also in 55% of HNPCC tumors that showed loss of expression of hMLH1 (P = 0.02). HNPCC cancers were more frequently characterized by aberrant beta-catenin immunostaining as evidenced by nuclear positivity (P < 0.001). Aberrant p53 immunostaining was infrequent in both groups. There were no differences with respect to 5q loss of heterozygosity or codon 12 K-ras mutation, which were infrequent in both groups. Sporadic MSI-H cancers were more frequently heterogeneous (P < 0.001), poorly differentiated (P = 0.02), mucinous (P = 0.02), and proximally located (P = 0.04) than HNPCC tumors. In sporadic MSI-H cancers, contiguous adenomas were likely to be serrated whereas traditional adenomas were dominant in HNPCC. Lymphocytic infiltration was more pronounced in HNPCC but the results did not reach statistical significance. Overall, HNPCC cancers were more like common colorectal cancer in terms of morphology and expression of beta-catenin whereas sporadic MSI-H cancers displayed features consistent with a different morphogenesis. No individual feature was discriminatory for all HNPCC cancers. However, a model based on four features was able to classify 94.5% of tumors as sporadic or HNPCC. The finding of multiple differences between sporadic and familial MSI-H colorectal cancer with respect to both genotype and phenotype is consistent with tumorigenesis through parallel evolutionary pathways and emphasizes the importance of studying the two groups separately.

Intron-8 polythymidine sequence in Australasian individuals with CF mutations R117H and R117C.

Compound heterozygotes for a severe cystic fibrosis transmembrane conductance regulator (CFTR) mutation and the R117H or R117C mutation (R117H/C) have clinical presentations that vary from classic cystic fibrosis (CF) to an incidental genetic finding. The aim of this study was to assess the influence of the intron-8 polythvmidine sequence (IVS8) on the relationship between genotype and phenotype of individuals with R117H/C. All individuals with R117H/C known to CF clinics in Australia and New Zealand were retrospectively studied by collecting information on genotype, age, pancreatic status, sweat electrolytes, sputum microbiology and pulmonary function. Forty-one individuals (39 with R117H and two with R117C), 16 on an IVS8-5T background and 25 on an IVS8-7T background were identified. Twelve individuals presented clinically, four were siblings of known R117H/C compound heterozygotes and 25 were detected by newborn screening. Eleven of 14 of the IVS8-5T group (78%) with sweat chloride results available had sweat CI > 60 mmol x L(-1) compared to 5 (20%) of the R117H/7T group (Chi-squared=10.4, p=0.001). Two were pancreatic insufficient, both IVS8-5T. Two IVS8-5T individuals have recently died (aged 43 and 19) and of the 14 surviving IVS8-5T group, 11 (79%) are symptomatic compared to eight (32%) of the IVS8-7T individuals (Chi-squared=6.1, p=0.01). In conclusion, most individuals with R117H/C on a IVS8-5T background have an elevated sweat chloride and clinical cystic fibrosis, which in some cases is severe. Most individuals with R117H/C on an IVS8-7T background do not have clinical cystic fibrosis but should be followed for the development of clinical disease.

Refinement of the chromosome 5p locus for craniometaphyseal dysplasia.

Craniometaphyseal dysplasia--Jackson type (CMDJ) is an autosomal dominant bone dysplasia with hyperostosis and sclerosis of the skull and abnormal modelling of the metaphyses. In a large German pedigree, a locus for CMDJ has been mapped previously to the short arm of chromosome 5 (5p15.2-p14.1), defining a 19-cM disease interval between markers D5S2004 and D5S502. Analysis of a large Australian pedigree together with a second German family confirms linkage to the same region. Obligate recombinations in the new families and confirmation of a supposed recombination in the previously reported German kindred have enabled us to narrow the critical region down to approximately 4 cM between markers D5S1987 and D5S1991.

Clinical utility of serum HER-2/neu testing on the Bayer Immuno 1 automated system in breast cancer.

UNLABELLED: The clinical utility of automated serum HER-2/neu measurements in breast cancer run on the Bayer random analyzer Immuno 1 was analyzed in several steps: [a] The reference interval was determined for 242 normal healthy pre- and postmenopausal females. [b] The clinical specificity of serum HER-2/neu to separate healthy controls from 210 patients with non-malignant breast--and non-breast diseases was calculated. [c] The clinical sensitivity of cross-sectional serum HER-2/neu values for 204 patients (pts) with stage I-IV breast cancer was established. [d] Specimens from 103 stage IV breast cancer pts were tested for their parallel between serial serum HER-2/neu results and disease course. RESULTS: [a] The value of 13.03 ng/ml exceeded 95% of the results from the healthy female population. Based on the mean +2 standard deviations value of 14.7 ng/dl, the upper limit of normal was established at 15 ng/ml. [b] The specificity for benign breast diseases and other benign non-breast diseases was 98.0% and 94.6%, respectively. [c] The correlation of increased serum HER-2/neu levels and stage of breast cancer revealed the best sensitivity of 40% for stage IV disease. [4] Thirty-eight (36.9%) of 103 stage IV patients had initial HER-2/neu values > 15 ng/ml, 33 of whom showed longitudinal HER-2/neu concentrations which paralleled the clinical course of the disease giving a sensitivity of 86.8%.

Heterozygous mutations in ANKH, the human ortholog of the mouse progressive ankylosis gene, result in craniometaphyseal dysplasia.

Craniometaphyseal dysplasia (CMD) is a bone dysplasia characterized by overgrowth and sclerosis of the craniofacial bones and abnormal modeling of the metaphyses of the tubular bones. Hyperostosis and sclerosis of the skull may lead to cranial nerve compressions resulting in hearing loss and facial palsy. An autosomal dominant form of the disorder (MIM 123000) was linked to chromosome 5p15.2-p14.1 (ref. 3) within a region harboring the human homolog (ANKH) of the mouse progressive ankylosis (ank) gene. The ANK protein spans the outer cell membrane and shuttles inorganic pyrophosphate (PPi), a major inhibitor of physiologic and pathologic calcification, bone mineralization and bone resorption. Here we carry out mutation analysis of ANKH, revealing six different mutations in eight of nine families. The mutations predict single amino acid substitutions, deletions or insertions. Using a helix prediction program, we propose for the ANK molecule 12 membrane-spanning helices with an alternate inside/out orientation and a central channel permitting the passage of PPi. The mutations occur at highly conserved amino acid residues presumed to be located in the cytosolic portion of the protein. Our results link the PPi channel ANK with bone formation and remodeling.

Population screening for cystic fibrosis in Western Australia: community response.

We measured acceptance of carrier testing for cystic fibrosis in the community when offered in a primary care setting, determined variables influencing acceptance, and assessed knowledge of cystic fibrosis 3-6 months later. A total of 5,102 individuals age 18-50 years attending general practices or a family planning clinic in Western Australia completed questionnaires about knowledge of cystic fibrosis and the State Anxiety Inventory. Testing for the delta F508 gene was offered. After 3-6 months, carriers, a sample of consenting participants who were not tested, and a sample of test-negative participants were sent a further questionnaire; 43.5% of participants chose to be tested for cystic fibrosis carrier status. Women, younger people, people with higher education, people without children, and people planning to have children were more likely to be tested. After 3-6 months, carriers gave correct responses to questions about cystic fibrosis more frequently than those who tested negative or were not tested; 82.2% of carriers knew that they were definitely a carrier and 31.1% of test-negative individuals believed they were definitely not carriers. Thus, population carrier screening for cystic fibrosis offered in a community setting in Western Australia was acceptable to almost half of those offered testing, particularly younger people and those planning to have children, for whom knowledge of carrier status could be useful in making reproductive decisions. There was evidence that tested individuals recalled information in a way that minimised their risk of being a carrier.

Complexed prostate specific antigen provides significant enhancement of specificity compared with total prostate specific antigen for detecting prostate cancer.

PURPOSE: Determining serum total prostate specific antigen (PSA) has proved to be a valuable diagnostic aid for detecting prostatic carcinoma, although the lack of specificity has limited its usefulness. Studies indicate that the use of percent free PSA would improve specificity while maintaining sensitivity. Since complexed PSA represents the major proportion of measurable PSA in serum, we determined whether it represents a single test alternative to the use of percent free PSA for the early detection of prostate cancer. MATERIALS AND METHODS: Archival serum was obtained from 385 men with no evidence of malignancy on biopsy and 272 with biopsy confirmed prostate cancer. We determined the concentration and proportion of total, complexed and free PSA. RESULTS: Receiver operating characteristics analysis using total PSA results from all samples (range 0.32 to 117 ng./ml.) indicated that the areas under the curve for complexed PSA alone as well as the free-to-total and complexed-to-total PSA ratios were similar and significantly greater than those for total PSA alone. Within the range of 85% to 95% sensitivity receiver operating characteristics analysis revealed that the specificity of complexed PSA was higher than that of total PSA and equivalent to that of the free-to-total PSA ratio. We noted a similar improvement in specificity in the 4 to 10 ng./ml. total PSA range. Using published cutoff values for complexed, total and percent free PSA when total PSA was in the 4 to 10 ng./ml. range the sensitivity and specificity of complexed and percent free PSA were similar. Within the 4 to 10 ng./ml. total PSA range the population of patients with no evidence of malignancy and complexed PSA below the upper limit was different with respect to total PSA from that with no evidence of malignancy and free PSA greater than 25%. CONCLUSIONS: The measurement of complexed PSA represents an alternative to the use of percent free PSA, although the patient populations identified by the 2 tests are different.

Multicenter evaluation of the performance and clinical utility in longitudinal monitoring of the Bayer Immuno 1 complexed PSA assay.

We conducted a multicenter evaluation of the analytical and clinical performance of the automated Bayer Immuno 1 complexed PSA (cPSA) assay, and compared assay performance to the Bayer Immuno 1 PSA assay. We sought to determine whether measurements of cPSA could be of clinical utility in the management of patients with prostate cancer. Results of the 10-day imprecision across three evaluation sites produced total CV < 2.50% and an analytical sensitivity of 0.02 microgram/L. There was an increased trend in clinical sensitivity for prostate cancer with increasing stage of disease (71-86%). Clinical specificity for patients with benign urogenital disease was 74.8%, and for other nonprostate diseases ranged from 91.1-100%. Retrospective serial monitoring of 155 patients with prostate cancer demonstrated concordance of cPSA measurements to clinical status for 97% of the patients analyzed. Results from the clinical studies using the Bayer Immuno 1 cPSA assay were comparable to results obtained with the Bayer Immuno 1 PSA assay. The Bayer Immuno 1 cPSA assay demonstrates analytical performance and clinical effectiveness in the management of prostate cancer patients during the course of disease and therapy.

Efficacy of gene testing for von Hippel-Lindau disease.

OBJECTIVE: To determine the efficacy of genetic testing of individuals presenting with features possibly indicative of von Hippel-Lindau (VHL) disease, regardless of other relevant family and clinical details. SETTING AND PARTICIPANTS: Between September 1994 and December 1997, 16 unrelated individuals were referred to Genetic Services of Western Australia by local clinicians and by similar genetic services in other States, for VHL gene mutation analysis because of clinical manifestations suggestive of the diagnosis. METHODS: The subjects were investigated by screening for mutations in the polymerase chain reaction products of the three VHL gene exons using single-stranded conformational polymorphism analysis (SSCP). If no mutations were detected the exons were sequenced, and if no variations were found DNA was examined by Southern analysis for germinal rearrangements. RESULTS: Mutations in the VHL gene were detected in eight of 16 individuals (50%), including 3 individuals with no family history suggestive of VHL disease. Five mutations were detected by SSCP, two by gene sequencing and one by Southern analysis. Each mutation occurred only in a single family and three had not been previously reported. CONCLUSION: Genetic screening of individuals presenting with clinical features suggestive of VHL facilitates confirmation of the diagnosis, accurate genetic counselling and surveillance of at-risk family members. The necessity for costly and time-consuming screening programs can be reduced and screening directed at those carrying the mutation. Our low stringency criteria are justified for screening for VHL mutations.

A polymorphism of the CC16 gene is associated with an increased risk of asthma.

Several quantitative traits associated with the asthma phenotype have been linked to markers on chromosome 11q13, although the gene responsible has yet to be well established. The gene for Clara cell secretory protein (CC16) is an ideal candidate for involvement in an inherited predisposition to asthma because of its chromosomal location, the role of the CC16 protein in controlling airway inflammation, and differences in levels of the protein between asthmatics and healthy controls. All three CC16 exons were screened in an unselected population of 266 subjects from 76 families and a cohort of 52 severely asthmatic children. A combination of single strand conformational polymorphism (SSCP) analysis, heteroduplex analysis, DNA sequencing, and restriction digestion was used. Mutation detection methods identified an adenine to guanine substitution in the CC16 gene at position 38 (A38G) downstream from the transcription initiation site within the non-coding region of exon 1. In the unselected population, 43.6% were homozygous for the polymorphic sequence (38GG) and 46.2% were heterozygous (38AG). All the asthmatic and unaffected children from both populations were selected for an unmatched case control analysis consisting of 67 asthmatic and 46 unaffected subjects. Those homozygous for the published sequence (38AA) had a 6.9-fold increased risk of developing asthma (p=0.049) and heterozygotes (38AG) a 4.2-fold increased risk (p=0.028). Modelling of genotype as a continuous covariate indicated evidence of a significant linear trend across the three genotypes (odds ratio=2.84 per unit increase in genotype code, p=0.018). These associations were independent of age, gender, and tobacco smoke exposure. These data and the known anti-inflammatory role of CC16 in the respiratory tract suggest that alteration to the gene at position 38 may contribute to asthma.

Association of polymorphisms within the tumour necrosis factor (TNF) genes and childhood asthma.

BACKGROUND: Tumour necrosis factor alpha (TNFalpha) is a potent modulator of immune and inflammatory responses, and has been implicated in a variety of autoimmune diseases, including asthma. Increased levels of TNFalpha have been detected in both sputa and bronchoalveolar lavage fluid of asthmatic subjects during acute attacks. Interindividual variation in TNFalpha levels may be genetically determined and polymorphisms within the TNF genes and nearby HLA Class II region have been associated with differences in TNFalpha production. OBJECTIVE: To investigate the association of differences in asthma-related phenotypes with two biallelic polymorphisms: a G to A substitution at position - 308 of the TNFalpha gene promoter (TNF1 and TNF2 alleles) and an NcoI polymorphism in the first intron of the lymphotoxin alpha gene (LT-alpha*1 and LT-alpha*2 alleles). METHODS: The regions of interest were amplified from genomic DNA using specific primers and PCR. Dot blot analysis was used for genotyping individuals for the TNFalpha - 308 polymorphism, while restriction enzyme digestion was used for genotyping individuals for the LT-alpha gene NcoI polymorphism. A case-control analysis was then performed on 74 asthmatic and 50 non-asthmatic unrelated children for each polymorphism. RESULTS: The TNFalpha - 308 TNF1 allele was present at a significantly higher frequency in cases than controls (OR= 2.4, P=0.003), and homozygosity for the TNF1 allele was associated with a fivefold increased risk of physician diagnosed asthma relative to the other genotypes (OR = 5.23, P = 0.004). The LT-alpha*2 allele showed similar associations, including an approximately fivefold higher risk of physician diagnosed asthma for LT-alpha*2 homozygotes (OR = 4.89, P = 0.019). Evidence of a significant linear trend in asthma risk across the three genotypes was found for both polymorphisms. CONCLUSION: These results suggest an important role for the TNFalpha gene or a linked locus in an inherited asthma diathesis.