Julian M. Hopkin

Detection of Pneumocystis carinii with DNA amplification.

Oligonucleotide primers and probes were used in the polymerase chain reaction to amplify Pneumocystis carinii specific DNA sequences from alveolar lavage samples from 47 diagnostic bronchoscopies. No P carinii DNA was found in lavage from 10 immunocompetent patients; only low levels were found in 3 of 13 samples from immunosuppressed individuals without P carinii pneumonia (PCP), and the highest levels, readily demonstrated by simple ethidium bromide staining, were found in all of 16 samples from immunosuppressed patients with PCP confirmed by means of standard silver staining and in 4 from patients with clinical PCP but negative silver staining. DNA amplification provides a highly sensitive and specific technique for the identification of P carinii that should be valuable in epidemiological studies on this parasitic infection and in diagnosis.

Ile50Val variant of IL4R alpha upregulates IgE synthesis and associates with atopic asthma.

Atopy is an immune disorder characterized by heightened immunoglobulin E (IgE) production and leads to the clinical disorders of asthma, eczema and rhinitis1. Interleukin 4 (IL4) is a pleiotropic cytokine which plays a crucial role in IgEdependent atopic disorders2; it is central to B cells switching to IgE antibody production and to the maturation of T-helper cells to the Th2 phenotype (type-2 Thelper lymphocyte). Human IL4 operates through the IL4 receptor (IL4R) and thereby Stat6 (signal transducer and activator of transcription 6) activation3. Mice deficient in Stat6 (refs 4,5) or the IL4Rα chain6 lack IgE production and Th2 inflammatory reactions. IL4Rα is therefore a crucial component required for IL4 binding and signal transduction. A cytoplasmic variant of IL4Rα, an Arg576Gln substitution (numbering from the first ATG), has been recently identified and is found in excess in a study of a group of individuals with hyper-IgE syndrome and severe eczema7; functional assays showed impaired binding of the negative regulatory molecule, protein tyrosine phosphatase, SHP-1, and increased expression of CD23 was found on peripheral blood mononuclear cells after being challenged with IL4. Stat6 activation, however, was not upregulated and no information was provided on cellular IgE synthesis. An extracellular variant, Thr49Ile, in IL4Rα of BALB/c mice alters the ligandbinding affinity of IL4Rα (ref. 8). An Ile50Val (numbering for mature peptide) variant of human IL4Rα has also been identified9,10 and it is, to date, the only known extracellular variant of human IL4Rα (ref. 11). To test whether the Ile50Val variant promotes dysregulation of IgE synthesis, we first conducted a genetic association study for serum IgE levels in a Japanese population. There was a significant difference in Ile/Val 50 genotype frequencies between control and atopic subjects (Table 1); Ile50 associated with atopic asthma but not with non-atopic asthma; Ile50 specifically and significantly associated with raised total serum IgE levels and mite-specific IgE. The association with atopy was especially strong in children. The high frequency of Ile50 homozygotes (approximately 60%) in the childhood atopic asthma group described here, and the significant skewing from Hardy-Weinberg equilibrium (P<0.0001), suggest a largely recessive genetic effect for Ile50 on atopy; the previously reported

Maternal Inheritance of atopic IgE responsiveness on chromosome 11q

Atopy is a common familial state underlying allergic asthma and rhinitis. Lately, we have assigned a gene for atopy to chromosome 11q by linkage to the marker D11S97. Since previous studies have suggested that the risk of atopy is higher for children of atopic mothers than for those of atopic fathers, we sought differences between maternal and paternal patterns of transmission at the 11q13 locus among pairs of siblings in families affected by atopy. When we defined atopy as the presence of a positive skinprick test (greater than or equal to 2 mm) to any of a panel of common allergens, a higher than normal concentration of total serum IgE, or a positive radioallergosorbent test for a specific IgE, we found that 125 (62%) of the sibling-pairs affected by atopy shared the maternal 11q13 allele and 78 (38%) did not. This distribution differs significantly from the expected 50/50 distribution (p = 0.001). Of paternally derived alleles, 83 (46%) were shared and 96 (54%) were not (not significantly different from 50/50). The result was similar whatever definition of atopy was used and with other genetic markers on 11q. These findings show that transmission of atopy at the chromosome 11q locus is detectable only through the maternal line. The pattern of inheritance is consistent either with paternal genomic imprinting or with maternal modification of developing immune responses.

Atopy and asthma: genetic variants of IL-4 and IL-13 signalling

Abstract Recent genetic and functional studies highlight the importance of IL-4/IL-13 signalling in the development of asthma and atopy. Here, Taro Shirakawa and colleagues discuss genetic variants of IL-4/IL-13 signalling, and whether they promote asthma or atopy among different ethnic groups.

Localisation of atopy and β subunit of high-affinity IgE receptor (Fc∈RI) on chromosome 11q

Abstract Atopy, a common familial syndrome underlying allergic asthma and rhinitis, is characterised by sustained immunoglobulin E (IgE) responses to common allergens. We have previously shown genetic linkage of atopy to the chromosome 11 q13 marker D11S97 (pms51) through maternally derived alleles, but no likely candidate genes were known to lie near this marker. We have analysed maternally derived alleles from 155 sibling-pairs affected by atopy to seek evidence of linkage between the gene predisposing to atopy and other markers on chromosome 11 q13. We found that the β subunit of the high-affinity receptor for IgE (Fc∈RI-β) also lies on chromosome 11 q13, and that it is in close genetic linkage with the gene for atopy. The known roles of Fc∈RI in antigen-induced mast-cell degranulation and in the release of cytokines that enhance IgE production make the gene for its beta subunit a candidate for the chromosome 11 atopy locus.

Association between genetic variants of mast-cell chymase and eczema

BACKGROUND Atopy is a common syndrome underlying asthma, rhinitis, and eczema, and is characterised by high immunoglobulin E (IgE) responses to common antigens. IgE and mast-cell chymase (MCC-a serine protease secreted by skin mast cells) have a key role in atopic or allergic inflammation of the skin. The gene for MCC is located within a cluster of genes for cellular proteases on chromosome 14q11.2. We aimed to identify variants of MCC and another gene within this complex, and assess whether there is a genetic association between variants of MCC and atopic disorders-particularly eczema. METHODS We randomly selected 100 controls and recruited patients-100 in each group-with atopic asthma, non-atopic asthma, atopic rhinitis, and atopic eczema. PCR amplification was used to test genomic DNA for an association between allelic polymorphisms in MCC and a flanking gene (CGL1, for the cathepsin-G-like protein) on chromosome 14q11 and asthma, rhinitis, and eczema. FINDINGS We found a significant association between a BstXI polymorphism in MCC and eczema (odds ratio 2.17 [95% CI 1.21-3.88], p = 0.009), but no association with atopic asthma, rhinitis, or non-atopic asthma. There was no association between an Mboll polymorphism in CGL1 and any of the atopic disorders. INTERPRETATION These findings suggest that variants of MCC may be one source of genetic risk for eczema.

Amplification of mitochondrial ribosomal RNA sequences from Pneumocystis carinii DNA of rat and human origin

Pneumocystis carinii specific DNA sequences have been cloned from the experimental rat model. The sequence of the gene coding for the large subunit of mitochondrial ribosomal RNA has been used to construct P. carinii specific oligonucleotide primers for the polymerase chain reaction. These oligonucleotides produced amplification of specific sequences from both P. carinii infected rat and human lung samplings, but none from a range of other organisms including potential pulmonary pathogens. Comparison of the sequence of amplified products from the infected rats and humans demonstrated limited but consistent differences between P. carinii from these two hosts and allowed for the construction of a human specific internal oligonucleotide. The application of the specific oligonucleotides for DNA amplification and subsequent Southern hybridisation affords extremely sensitive and specific detection of P. carinii in human samples, which may be applicable to both epidemiological research and clinical studies.

Confirmation of genetic linkage between atopic IgE responses and chromosome 11q13.

Genetic linkage between atopic IgE responses and chromosome 11q13 (D11S97) has been previously reported in a limited number of extended families. Difficulties of phenotyping in the older family members, poor family structure in some families, and genetic heterogeneity were proposed as possible explanations for the variability in lod scores. To test this finding a second linkage study of 64 young nuclear families was undertaken and gave a two point lod score of 3.8 at theta = 0.07 (assuming theta m = theta f). A test of genetic heterogeneity in the nuclear families shows that atopic IgE responses are linked to this locus in 60 to 100% of families (approximate 95% confidence limits).

Genetic polymorphisms and lung cancer susceptibility: a review.

Lung cancer is a major cause of cancer-related death in the developed countries and the overall survival rate has still an extremely poor. Cigarette smoking is an established risk factor for lung cancer although a possible role for genetic susceptibility in the development of lung cancer has been inferred from familial clustering of the disease and segregation analyzes. Everyone may have a unique combination of polymorphic traits that modify genetic susceptibility and response to drugs, chemicals and carcinogens. Developments in molecular biology have led to growing interest in investigation of biological markers, which may increase predisposition to lung carcinogenesis. Therefore, the high-risk genotype of an individual could be determined easily. As there are the great number of carcinogen-activating and -detoxifying enzymes, the variation in their expression and the complexity of exposures to tobacco carcinogens, the existence of multiple alleles at loci of those enzymes may result in differential susceptibilities of individuals. This review summarize data addressing the relationships of lung cancer to markers of genetic susceptibility genes, including metabolic polymorphisms other than well-investigated cytochrome P450s or glutathione S-transferases, DNA repair genes and the p53 tumor suppressor gene. Among genetic polymorphisms reviewed here, myeloperoxidase gene (a G to A mutation) and microsomal epoxide hydrolase exon 4 polymorphism (substitution of Arg for His) were significantly associated with lung cancer risk. As lung cancer is a multifactorial disease, an improved understanding of the interplay of environmental and genetic polymorphisms at multiple loci may help identify individuals who are at increased risk for lung cancer. Hopefully, in the future we will be able to screen for lung cancer susceptibility by using specific biomarkers.

Dominant inheritance of atopic immunoglobulin-E responsiveness.

The familial occurrence of atopy, defined by skin prick test responses and serum immunoglobulin-E (IgE) titres to common inhaled allergens, was studied in 239 members of 40 nuclear and 3 extended families. 90% of the atopic children in the nuclear families had at least one demonstrably atopic parent. In each extended family, atopy was vertically transmitted, and 31 of 47 (66%) offspring of marriages between atopic and unaffected parents were atopic. Of the designated atopic subjects, 83% admitted to symptoms suggesting atopic disease but only 30% regarded themselves as having any such disorder. It is suggested that atopy, the propensity to produce IgE in response to common, usually inhaled, allergens, is inherited as an autosomal dominant character but that its clinical expression depends on interaction with other factors.