This document was last modified on 14-April-2008.
A list of candidate genes for a role in asthma and allergy pathogenesis can be generated for all the mediators and their receptors described earlier, the receptors characteristic of particular effector cells involved in allergic inflammation and asthma, and of course IgE and its receptors. A number of these candidates are currently under investigation. Likely ones are some of the interleukin receptors eg the IL-5 receptor (located on chromosome 3p26), IL-4 receptor (16p12.1-p11.2); the immunoglobulin genes on 14q; the high affinity IgE receptor (assigned as part of a search described below to 11q13); the HLA region; the T-cell receptor (on 14q11.2 - alpha and delta subunits, 7p15-p14 - gamma subunit, and the beta subunit on 7q35) [Genome Data Base 1993]. Other regions of recent interest are the chromosome 5q cluster of interleukin genes (including IL-4 and IL-5), close to the beta-2-adrenoceptor gene (ADRB2), and interferon-gamma on 12q.
Some readers may find the organisation of this section slightly confusing. Although some of the earliest studies in this area started in the HLA region looking for allergen specific human equivalents of mouse Immune Response genes, I have chosen to start with the positional-based search for a (generalised) atopy gene, culminating in the era of genome scans. I then return to the older candidate based approach.
Reanalysis of the pedigrees using MLINK and SIBPAL actually finds weak hints of linkage to AB0. Mean identity by descent sharing at the AB0 locus among affected sib pairs is 0.61 (SE=0.26, P=0.02), while maximum likelihood linkage analysis under an additive SML model (q=0.5, f2=0.6, f1=0.3, f0=0.0), finds a maximum lod score of 0.44 at recombination fraction r=0.00. In view of the small families and less than fully informative marker used, these results are all the more surprising.
The next study I am aware of dates to 1985. This abstract describes a preliminary linkage analysis for total sIgE level. A maximum lod score for Kidd blood group of 1.0 in males and for both sexes a lod score of 2.07 for Esterase D (recombination fraction=0.00). This would place a gene regulating overall IgE level at 13q14. This finding was not replicated by Cookson et al [1989]. More recently, Daniels et al [1996] and several other linkage studies have found evidence of linkage of atopy to this region.
The group including William Cookson and Julian Hopkin has published several papers [Cookson & Hopkin 1988; Hopkin 1988; Cookson et al 1988; Cookson et al 1989a; Cookson et al 1989b; Cookson et al 1992; Young et al 1992; Sandford et al 1993], describing a large family study of atopy including RFLP and later STRP based linkage analysis. Initially they examined the nuclear families of 20 (skin) atopic asthmatics from a respiratory outpatient clinic (age 12-15 years) and 20 control families ascertained via similarly aged children admitted to hospital for appendicectomy (12), pneumothorax (6) or pneumonia (2). A further 158 subjects from three large extended families with strong histories of atopic disease were also recruited via letters to general practitioners and newspaper publicity. This total of 394 subjects was extended to a total of over 500 subjects in a later analysis [Hopkin 1988]. No statistical allowance for ascertainment bias was made in this initial analysis.
The norms for sIgE were age-sex-standardised [Hopkin et al 1993]. The definition of skin atopy as being 1 mm or greater than the control would be lower than that used in a number of other studies, and would proportionately increase the number of subjects diagnosed as being atopic. Similarly RAST results are commonly reported in semiquantitative form; the cutoff for a positive reaction may vary from kit to kit. I note this because other workers (eg Sibbald 1988) have criticised the definition of atopy used in this study.
Within the nuclear families examined, 80% of the parents defined as being atopic fulfilled two out of three criteria, 12% had an elevated total sIgE, and 8% a positive skin test alone. While 83% of all atopic subjects met questionnaire criteria for asthma or hayfever (defined as rhinitis and conjunctivitis), only 30% specifically reported diagnoses of these syndromes. Of the nonatopic subjects, 13% met these criteria. Within the nuclear families, significantly more of the parents of cases were atopic than were those of controls (X2(2 df)=11.2, P=0.004; see Table). This gives an unadjusted odds ratio for atopic asthma versus (any) parental history of atopy of 11 (95% confidence interval 2-60), and a population prevalence estimated from the control parents of 25% (11-38%), not dissimilar from most estimates in the literature, as discussed earlier.
Among the extended families, the authors felt that the segregation of atopy was most consistent with an autosomal dominant gene of fairly high penetrance (Table). In only four nuclear families out of 31 producing atopic offspring were the parents nonatopic as defined by this study; in each case, one of the grandparents was atopic, and in three families, the linking parent had a history consistent with childhood rhinitis.
| Group of children | Number of parents atopic | ||
|---|---|---|---|
| None | One | Both | |
| Atopic asthmatics (20) | 2 | 11 | 7 |
| Control children (20) | 11 | 8 | 1 |
| Mating Type | UXU | UXA | AxA |
|---|---|---|---|
| Atopic offspring | 4/? | 31/47 | 8/12 |
| Percentage | -- | 66% | 75% |
The linkage study was performed using seven extended families, three of these being those described above, others recruited from, among other sources, the authors' respiratory outpatient clinic. A two liability class model was used: the first class contained subjects where the diagnosis of atopy or normality was clearcut; the second class included those subjects where skin prick testing and total IgE were equivocal. For example, as sIgE diminishes with age, nonatopic individuals (by the authors' criteria) aged over 60 years were assigned to this second liability class. Penetrance for the first liability class was modelled as being 99%, and 75-90% for the second class. Two particular kindreds, with a high prevalence of cigarette use, contributed the bulk of this second liability class.
The strategy used was a chromosome by chromosome search - see Table. The seventeenth probe examined, p lambda-MS.51 (D11S97), showed 8 recombinants from 53 meioses. A further 69 meioses were then recruited. The maximum lod score for all the families was 5.58 at a recombination fraction of 10.5%. Subsequently, more families were included [Cookson et al 1989]. This led to a maximum lod score of 6.39 at a recombination fraction of 10%. For the original data, four families contributed a maximum lod score of 5.24 at a recombination fraction of 6%, with the second liability class members contributing little to the lod score. The remaining families, where cigarette use was high, also suffered from poor family structure, with a small third generation and many uninformative meioses.
Hopkin [1990] summarised further work. The number of families included now numbered 60. Most of these were relatively small kindreds. The lod score remained highly significant at over 5. There was no evidence of genetic heterogeneity on testing. This test would not be very powerful if most families were small.
This work gives us a model for atopic disease in terms of a single gene, backed up by the linkage analysis. The gene frequency is between 15-25%. The penetrance for any atopic symptoms was 85%, for wheeze 60%, and for diagnosed asthma 20%. If one uses the higher figure for the prevalence of atopy in the general population, this equates to a gene frequency of 0.16; if the prevalence is 30%, the gene frequency 0.19. Using a gene frequency of 0.16 and a penetrance of 85% gives VA of 14% and for VD of 1%. Using the formulas presented by James [1971] and Risch [1990], this suggests the population relative risk (recurrence risk compared to the general population risk) for atopy in the parents or siblings of atopic children would be 2.1, and for MZ twins would be 3.4 (see also Section 11.5.2). If both parents have no symptoms of atopy, the risk to the children of developing wheeze is approximately 7%.
In the same pericentromeric region of Chromosome 11, one finds CD20 (11q12-q13.1), uteroglobin, complement component 1 inhibitor (hereditary angioedema, 11q12-13.1), Multiple Endocrine Neoplasia I (11q12-13), muscle glycogen phosphorylase (11q12- q13.2), the gene associated with McArdle syndrome, NFKB3 (the beta-3 subunit of the nuclear factor of Kappa light chain gene enhancer in B cells) and various pepsinogens (3-5, Gp I) making up pepsinogen A (11q13). In addition, this region is homologous to the mouse 7F (T50H) region, associated with parental imprinting effects [Hall 1990]. A number of human disease genes existing in other homologous areas have already been demonstrated to exhibit maternal (Wilm's tumour) or paternal (Huntingdon's disease) imprinting; this might extend to atopy.
In the period since the last paragraph was written, evidence supporting this latter prediction was published. This group published a further two papers in the Lancet, which included GM Lathrop as an author. Young et al [1992] described a separate analysis of sixty-four nuclear families. These were ascertained through children (age under 15 years) reporting eczema, hayfever or asthma. The same diagnostic criteria as before were used. The model assumed a gene frequency of 0.2, and a 95% penetrance. Genetic homogeneity was assessed using the test proposed by Smith [Ott 1991].
Under an assumption of equal recombination distance in both sexes, the maximum lod score was 3.8, but there was strong evidence for a male-female difference. Relaxing the equality constraint led to a maximum lod score of 5.2, with rM=0.18 and rF=0.001. The proportion of unlinked families was estimated at zero, with a 95% upper limit of 40%. The marked sex difference was left unexplained.
Cookson et al [1992] described sib-pair analyses that examined sharing of maternal and paternal marker alleles. All subjects genotyped up to August 1991 were included (N=723). Affected sib-pairs were significantly more likely to share a maternal Chromosome 11 than a paternal Chromosome 11, whether using an affected phenotype defined as any skin test 4 mm or more in diameter (maternal allele shared 30/43 and paternal alleles shared 24/42), a high total sIgE (maternal 51/81; paternal 28/59), or the combination of three measures described earlier (maternal 125/203; paternal 83/179). There was no significant sharing of alleles in unaffected sibling pairs in the informative families. A "hot spot" of male recombination could not be found to explain the sex difference. In families where the father was atopic, and the mother nonatopic, the sharing of maternal alleles was 19/32 (0.59, exact 95% CI 0.41- 0.76), which trends in the same direction as the previous results. The authors note that alternative explanations include phenotypic maternal effects, so a pair of sibs carrying this atopy gene are more likely to express it after transplacental or breast milk exposure to maternal IgE or other factors. Since the presence of atopy in offspring of atopic fathers is increased, they also concur that the disease must be genetically heterogenous.
Finally, this gives a possible explanation of the failure to replicate the original linkage between 11q13 markers and atopy in other studies (see Section 6.1.4 et seq). If sex of the parent is ignored, obviously the evidence for linkage will be attenuated: for atopy via three measures - the phenotype giving the strongest evidence - affected sib pairs shared 208 alleles out of 382 (0.54, exact 95% CI 0.49-0.60), not significantly different from the null expectation of 192.
The second paper [Sandford et al 1993] gives multipoint linkage information. Because of the presence of probable maternal inheritance and genetic heterogeneity, sib-pair methods of analysis were again used. Two novel STRP's in the 11q13 region were described. One CA-repeat marker was derived from FCER1B DNA, the other was derived from a cosmid clone.
Examination of the maternal or paternal origin of marker alleles in affected sib-pairs again gave strong evidence for maternal transmission (D11S97 paternal alleles shared 45/87 and maternal alleles shared 72/100; FCER1B-ca paternal 43/84, maternal 56/67; cosmid clone marker cCl11-319ca paternal 28/60 and maternal 56/67 - in each case maternal allele sharing is markedly greater than 50%). The best ordering of the markers was D11S97, cCl11-4, cCl11-319ca, CD20, FCER1B-ca, cCl11-222. The atopy locus mapped using maternally derived alleles into the (95% support) region running from cCl11-4 (with the lowest estimated recombination fraction r=0.005) to FCER1BI-ca (r=0.03). There was no evidence for allelic association (linkage disequilibrium). Critically, the authors note that in 16 of 88 families informative for loci within this region, atopy was not linked to 11q13. They commented that linkage might be spurious in approximately this number of families again, as the phenotype was so common. Therefore approximately 60% of atopy in these families would be attributable to the 11q gene. The FCER1B gene (MS4A2)is therefore a strong candidate gene, as presumably is CD20 (later excluded), though I can find few references ascribing a function to this cell surface marker. A table summarizes the subsequent replication attempts described below.
| Genetic Probe | Chrom | Comment |
|---|---|---|
| pYNZ2 | 1 | |
| pYNH | 2 | |
| p-lambda-g3 | 7 | |
| collNJ3 | 7 | |
| p-lambda-MS.51 | ||
| D11S97 | 11q12-13 | Initially described probe linked to atopy; first to show evidence of maternal imprinting |
| FCER1B-ca | 11q13 | Probe with strong evidence of linkage to atopy; strong candidate |
| CD20 | 11q13 | Marker with strong evidence of linkage to atopy; Possible candidate (B-cell surface marker of unknown function). |
| CCl11-319 | 11q13 | Probe with strong evidence of linkage to atopy |
| CCl11-4 | 11q13 | Probe with strong evidence of linkage to atopy |
| CCl11-222 | 11q13 | Probe with strong evidence of linkage to atopy |
| InsHVR | 11p | |
| Esterase D | 13 | previous report of linkage [Eiberg et al. Cytogen Cell Gen 1985;14:622] |
| WC25 | 13 | |
| WC83 | 13 | |
| p7f12 | 13 | |
| AW101 | 14 | Excludes variation around IgE H chain locus |
| 3' a globin | 16 | |
| p79-2-23 | 16 | |
| pYNZ22 | 17 | |
| RMU3 | 17 | |
| Jeffries 33.15 | multiple | poss linkage with asthma [Brereton et al 1990] Jeffries 33.6 multiple |
| M13 gene 3 tandem repeat | multiple | |
| zeta intron HVR | multiple |
This abstract [1990] was the first published attempt at replication of the Oxford group's findings. Seventeen Portuguese families were examined (14 two generation and 3 three-generation, total N=83). Half (40/83) of the subjects were atopic. It is implied but not stated in the abstract that all these individuals were pollen sensitive. There was no evidence of linkage to the lambda-MS51 probe site on 11q. They note that "[t]he observed patterns were distinct and different from those reported in the literature". Apparently [D. Meyers, pers comm], this refers to the fact that the Pasteur Institute group could not identify the particular length restriction fragments (alleles) described by Hopkin et al [1989]. However, all the atopic individuals did carry the HLA DQA2-DQB2 haplotype (see below).
A successful replication of Cookson et al was reported by a Japanese group at HGM 11 [1991]. There were 274 atopic asthmatic probands, but only 136 families were finally selected - the remainder were excluded because of "discrepancies between measurements" of the three measures of atopy used [as per Cookson et al]. Of these, only 4 families containing more than 15 meioses each (out of the 8 families eligible) were used to examine linkage to D11S97 (N=69 meioses). One family gave a lod score of 2.98 at r=0.01, the other three 0.6 to 0.7, so that the four families gave a total lod score of 4.88. The genetic model chosen is not described, but is presumably the same autosomal dominant model of Cookson et al. Shirakawa et al [1994] revises this maximum lod score to 9.35 for D11S97 and FCER1B-ca under the assumption of unequal rates of maternal and paternal recombination, and report evidence for maternal transmission in 2 families.
These authors [1992] described a continuing study in Groningen (Netherlands). The group includes Deborah Meyers. Twenty families (N=117 typed individuals) were ascertained through a proband diagnosed as asthmatic in 1962-70. Linkage to both atopy and BHR was tested for, using both LINKAGE under two different genetic models, and sibpair analyses. The lod score was below -2 for r lt;0.12 with the PCR-based marker INT2. A sib-pair analysis found that of the (possible) 54 affected pairs, 28 shared the same INT2 allele as the affected parent, and 26 were discordant. In addition, they examined D6S105, tightly linked to the HLA-B region. In this case, a lod score of -2 was found at r=0.07.
In contrast to Shirakawa et al [1991], this Japanese group [1992] found no evidence for linkage between D11S97 and atopy in four families (N=60). A number of definitions of atopy were used, including that of Cookson et al. Using this definition in the LIPED analyses, linkage was excluded below r < 0.04. Tightening the definition led to stronger evidence for exclusion.
This is another attempted replication of the 11q linkage findings [1992a; 1992b] in nine families (2 and 3 generation, N=89). The phenotypes studied were skin atopy (a panel of 10 common allergens), positive RAST (to the same 10 allergens), as well as BHR to methacholine using the Yan protocol. This group used VNTRs with the lambda-MS51 probe (D11S97). The heterozygosity at this locus was 77% (9 alleles). No evidence for linkage (using Liped) was found for any of seven phenotypes defined. For atopy, the overall maximum lod score was -0.3 at 30% with Hinf1 and -0.04 at 30% with Taq1. Segregation analysis using REGD found atopy "not inherited in a simple mendelian fashion". They note that they were unable to reproduce the 10.8 kb allele described by Cookson et al.
Further analysis with INT2 and PGYM found no association with atopy, or linkage - for PGYM LOD<-2 for r<0.07; for INT2 for r<0.04. Altering the definition of atopy, or restricting it to BHR did significantly alter these results.
Blumenthal's group in Minnesota [1992] examined three large pedigrees (N=67 typed at D11S97). Atopy was defined as by Cookson et al, save that skin wheals had to be 5 mm greater than control. D11S97, HLA-A and HLA-B typing results, were used for linkage analysis (LINKAGE). In addition, sib-pair analysis (126 pairs) were performed using SIBPAL. Linkage with D11S97 was excluded for r<0.05, and with the HLA region out to r=0.23. Sib-pair analyses found no evidence for linkage to either region.
Further linkage analysis of 11q13 was also performed in 95 families (N=407) with at least two first-degree relatives with active atopic eczema [1993]. Atopy defined as a positive skin prick test, or elevated specific or total sIgE was present in 80% of parents and 86% of offspring, and asthma or hayfever in 70% of the eczema probands. Ignoring the possibility of maternal effects gave a multipoint lod score (D11S97, PGYM, CD20, centromere) of -7.8 for 11q13. Sib-pair analysis (101 pairs) found a nonsignificant absence of sharing of maternal alleles (D11S97 paternal 21/47, maternal 23/61 - 38% with exact 95% CI=26-51%; PGYM 30/62, 25/61 - 41% with 95%CI=29-54%; CD20 9/16, 14/28). Linkage using subclasses of parental eczema found persisting negative lod scores for families where the mother was unaffected or both parents were affected. For 12 informative families where the father was unaffected, the lod score was +0.8. The authors conclude that a maternal effect might exist, but that heterogeneity is masking it.
This sib-pair analysis appeared as a letter to the Lancet [1993]. There were 26 affected sib-pairs from 26 nuclear families, using as the disease definition elevated sIgE (>100 IU/ml for over 10 year olds), a positive RAST (0.35 PRU/ml) and two or more symptoms on a modified MRC questionnaire. Families were typed at 3 loci - D11S97, PGYM, and FCER1B-ca. The proportion of alleles shared IBD for D11S97 was 18/23 (78%, exact 95%CI=56-92%); PGYM, 33/44 (75%, 60-87%); and FCER1B-ca, 17/28 (61%, 41-78%). Breaking this down by parental origin showed a tendency for sharing to be greater for maternal than paternal alleles, but because of the small sample size, this was not significant (Cochran-MantelHaenszel statistic X2(2 df)=2.84).
This abstract [1994] describes a linkage study of 355 sib-pairs (170 individuals) from 11 (Amish) pedigrees studied by the group headed by David Marsh. Three 11q13 markers were used: FCER1B-ca, INT2 and PGYM. No evidence for linkage with any of four phenotypes (total sIgE, specific IgE to D pter, D far, and a panel of 20 common aeroallergens) in a Haseman-Elston regression analysis using SIBPAL was found.
More recently, this study was extended to include one additional pedigree, further individuals in the existing pedigrees, and total sIgE was reassayed. Reanalysis finds some evidence for linkage to INT2 (FGF3).
Holgate's group [1994] have performed linkage and segregation analysis of 131 nuclear families recruited for having three or more children, but not specifically for atopic disease. "Evidence for major loci [was] suggestive, but there was no evidence for imprinting or linkage to 11q13". In a subsequent paper [Watson et al, 1995], these authors reported more fully these combined segregation-linkage analyses under one and two trait locus models.
A number of markers on chromosomes 1, 5, 11, 12, 16 and 19 were examined: D1S104, IL2RB, IL9, TGFB, IFNA, D11S480 (3.9 cM from FCER1B), D11S527, D11S534, IGF1, D16S298, D19S112, D19S177. No maximized LOD score greater than 0.57 (or less than -1.00) was obtained.
This South Australian linkage study [1994] of 12 extended pedigrees in which atopy appeared to segregate in an autosomal dominant fashion failed to find linkage or association to 11q.
In a Victorian sample of 123 sib-pairs [1995], by contrast, evidence for linkage of FCER1B-ca to methacholine BHR and diagnosed asthma was found. This study was nested within the European Community Respiratory Health Survey as part of which a random sample of 4500 individuals aged 25-40 were screened via the IUATLD questionnaire. A subset of 757 of these underwent methacholine challenge and skin prick testing to 11 common allergens. Subjects with a history of asthma (episode in previous 12 months, use of asthma medication, or nocturnal dyspnoea), atopy (any SPT > 3 mm), or BHR (PD20<2 mg cumulative dose of methacholine) were asked if they had a sibling, who was invited to undergo testing. A total of 137 pairs were recruited. Linkage of asthma, atopy and BHR to FCER1B-ca was tested an affected sib-pair identity-by-state analysis.
There was significantly increased sharing of alleles in 67 concordant pairs concordant for asthma (observed number of shared alleles=98, expected=83.1, P=0.002), 106 pairs concordant for atopy (O=145, E=131.4, P=0.02), 53 BHR concordant pairs (O=80, E=65.7, P=0.001). Among the 17 BHR pairs where there was not concordance for atopy, the sharing was still greater than expected (O=28, E=21.7, P=0.004); this was not the case in the 70 atopy concordant pairs not concordant for BHR (O=93, E=86.8, P=0.124). No evidence for parental imprinting was found when reported family history, and its interaction with ibs sharing was examined.
In Wong et al [1997], results from another three markers is presented: D11S987, D11S1314, D11S937. In this case, no increased sharing in any subgroups was detected, but these markers are probably too distant from FCER1B to obtain significant results given the sample size.
The authors' conclusion that they have demonstrated linkage to a BHR gene rather than an atopy gene may be a little premature. I am skeptical of the ibs methods here, as these can give inappropriately low P-values, as opposed to ibd methods (which are perfectly applicable).
This Italian study [1996] describes sib-pair linkage analysis in 45 nuclear families (213 subjects) ascertained through atopic asthmatic children (median age 12 years, range 2-47), and containing at least one additional atopic member. Atopy was defined as per Cookson et al [1989]. Analysis of maternal versus paternal sharing at two markers (FCER1B-ca and CCl11-319ca) was performed on 128 affected sib pairs.
No striking evidence for linkage was detected, although the sharing of maternal alleles is in the expected direction. The maternal and paternal sharing at FCER1B-ca was 28/47 (60%, exact 95%CI=44-74%) and 20/50 (40%) respectively; for CCl11-319ca, 12/19 (63%) and 21/39 (54%). The difference between maternal and paternal sharing at FCER1B-ca is almost significant (OR=2.21, Fisher exact 95%CI=0.96-5.03).
An extension [1998] of the above study examined the second intron RsaI and E237G polymorphisms of FCER1B in a significantly increased total of 168 families (659 individuals) recruited through a child attending an allergy and respiratory medicine clinic. There were 57 sib-pairs where both exhibited BHR, 117 where both exhibited one or more positive skin prick tests, and 137 concordant for either a positive SPT or a sIgE level > 100 IU/ml.
Within the sample, the E237G allele frequency was 4%, and the int 2 RsaI B allele frequency was 56%. There was slightly increased IBD sharing at the i2 polymorphism within sib pairs (55% for BHR, P=0.05), with slightly stronger evidence from an APM test using SimIBD (empirical P=0.02 for BHR; empirical P=0.01 for a positive SPT). There was no evidence of allelic association using the TDT (number of parent-offspring trios not reported).
It seems typical that despite the large sample size, the strength of the evidence for linkage to the region remains quite low.
A similar study [1997] used 68 nuclear families (306 subjects) ascertained through atopic asthmatic children attending a Tsukuba (Japan) pediatric allergy clinic and their affected sibs (median age 10.5 years, range 1-29). The geometric mean sIgE in children was 670 U/ml.
The affected sib-pair analysis for atopy (log sIgE 1 SD above population mean, or a positive RAST to any of six allergens; 85 pairs), and asthma (recurrent wheeze and dyspnoea in the previous 12 months, spontaneous or bronchodilator reversibility; 46 pairs) supported linkage to 5q31-33 (see below) but not FCER1B-ca (mean ibd sharing 52%, P=0.43; and 51%, P=0.26). The results for a Haseman-Elston analysis of tIgE (using SIBPAL) were more encouraging for FCER1B-ca (t=-1.51, df=88, P=0.067).
This study involved 12 families (98 individuals) ascertained through a proband with atopic dermatitis. Commenges' WPC (APM) test was used to screen a set of 15 Chromosome 11 markers, and an additional parametric analysis was performed for FCER1B using MLINK and TMLINK (maximizing the lod score over 4 models). The best nonparametric result was a P=0.005 for FCER1B, while the highest lod score was 3.55 under a two-locus model: a recessive FCER1B-linked locus (r=0), and a dominant "background" locus. This lod score was contributed by only 2 families. By contrast, a lod of 0.8 was the best they coukld obtain under a single locus model.
Another Italian study examined the second intron RsaI and E237G polymorphisms of FCER1B in 168 families (659 individuals) recruited through a child attending an allergy and respiratory medicine clinic. There were 57 sib-pairs where both exhibited BHR, 117 where both exhibited one or more positive skin prick tests, and 137 concordant for either a positive SPT or a sIgE level > 100 IU/ml.
Within the sample, the E237G allele frequency was 4%, and the int 2 RsaI B allele frequency was 56%. There was slightly increased IBD sharing at the int 2 polymorphism within sib pairs (55% for BHR, P=0.05), with slightly stronger evidence from an APM test using SimIBD (empirical P=0.02 for BHR; empirical P=0.01 for a positive SPT).
This West Australian study examined 121 nuclear families: 95 (442 individuals) ascertained via a child regardless of asthma status, and 26 (134 individuals) through a child with severe symptomatic asthma Multipoint linkage analysis using Haseman-Elston sib-pair methods provided evidence of linkage between chromosome 5q markers (D5S393, D5S399) and total sIgE levels (P=0.04), specific sIgE levels (P=0.04), and eosinophil counts (P=0.03), while chromosome 11q markers (FCER1B-ca and D11S480) were weakly linked only to specific IgE (summed D. pter and mixed grass RAST scores) level (P=0.03). No linkage to BHR (Yan protocol log His DRS) was exhibited by either region. Evidence of linkage to 5q was slightly stronger for total sIgE adjusted for specific IgE level (P=0.004), but this diminished the evidence for linkage to FCER1B (P=0.3).
This paper describes both a case-control and a linkage study. In the latter, four extended pedigrees (106 individuals) in which asthma was common. All subjects underwent SPT and methacholine inhalation challenge, and 27 met the study criteria for the diagnosis of asthma. There is some evidence of linkage between FCER1B-E237G genotype and various atopic phenotypes (lods 1-2).
Two important association studies confirmed the Oxford group's original linkage findings and, to some extent, their explanations as to why replication by other groups was so difficult. One study used a sample from the original Oxford families, the other, families from Busselton, West Australia.
Shirakawa et al [1994] first sequenced FCER1B in six atopic and six nonatopic subjects. Three (6th exon) mutations were found in one atopic individual leading to substitutions of isoleucine for leucine at position 181 (I181L), and valine for leucine at position 183 (V183L). A PCR based assay for these two mutations was developed (AS-PCR - allele-specific DNA amplification).
In a "random sample" of 163 patients unselected for allergic disease (undergoing venesection for other purposes), 25 were found to carry the Ile181Leu mutation, but none, the Val183Leu. A total sIgE greater than 100 IU/ml was present in 41 (25%), of whom 11 carried the Leu181 mutation (OR=3.1, 95%CI=1.2-7.5). A similar association was found for the presence of grass pollen specific IgE (OR=2.6, 1.1-6.4).
The Leu181 mutation was also found to segregate in 10 of 60 of the atopic nuclear families described earlier. In each family, the mutation was transmitted from the mother (and was present in the proband). Among 14 nonproband offspring, four were atopic, and two carried Leu181; none of the ten nonatopic offspring carried the mutation. Furthermore, the two "sporadics" arose in bilineal atopy - that is the father was atopic, and did not carry Leu181.
In the Busselton family study [Hill et al 1994], 232 nuclear families (1020 individuals, 556 children) were typed for the Leu181 mutation in the FcERI beta-subunit gene. This was found in 28 subjects. There were 8 children carrying the gene where the parent of origin was the mother - three with asthma, the remaining five with hayfever. Specific IgE and skin prick test wheals to house dust mite (as well as a sum of RAST scores) were significantly higher than in controls on Wilcoxon test - the most appropriate in view of the clustered nature of the data [Shirley & Hickling 1981].
Subsequently [Hill & Cookson 1996], they have described an exon 7 coding polymorphism in the Busselton population, Gly237Glu (E237G, rs569108). An ASO-PCR assay was developed, and demonstrated this mutation to be present in 53 of 1004 subjects. These individuals exhibited larger wheals to mixed grass allergens and house dust mite, more asthma, as well as increased BHR to methacholine. Serum IgE levels were only slightly higher (68.0 v 46.4 IU/ml). There was no parent of origin effect for this polymorphism (17 paternal, 13 maternal).
Shirakawa et al [1996a, 1996b] have reported a case-control study of FCER1B, using 500 atopic patients (allergic - early and late onset - and nonallergic asthma, hayfever or eczema) ascertained via Osaka hospital clinics were compared to 100 controls (disease status presumably unknown) attending a "health examination company". Initially, they found no Leu181 mutations either by ASO-PCR in the entire sample, or via sequencing in 10 atopic subjects. Therefore they developed RFLPs in intron 2 of FCER1B (RsaI), CD20 and GIF. Significant differences in genotype frequencies were detected for the FCER1B polymorphism between the controls (AA: 89; AB: 10; BB: 1) and several different subgroups of the patients, most strongly for childhood onset allergic asthma (AA: 50; AB: 42; BB: 8), and not at all for the intrinsic asthma group (AA: 86; AB: 11; BB: 3). There were no such effects for CD20 or GIF.
When they tested for the Gly237Glu mutation [Shirakawa et al 1996b], this was found in 6/100 controls, but 16/100 adult atopic asthmatics (P=0.025) and 20/100 child asthmatics (P=0.005). It was not significantly increased in the nonatopic asthmatics (8/100), but was markedly increased in the subgroup with a total sIgE greater than 1000 IU/ml.
Weaker evidence for association in the region was described by Holgate et al [1996]. As part of the Southampton study described above, allelic effects of two markers were detected on different phenotypes: allele 168 of D11S527 with BHR (P=0.0003, Bonferroni corrected for 13 alleles P=0.004), and allele 235 of D11S534 with total IgE (P=0.007, Bonferroni corrected for 14 alleles P=0.09).
There was strong evidence for association with the maternally transmitted allele for two Rsa1 polymorphisms in the 2nd intron and the 7th exon. Interestingly, the E237G allele was not associated with eczema. This probably reflects low power, as it was present in only 6% of probands, but argues against it being a functional mutation.
| Paternal | Maternal | |||
|---|---|---|---|---|
| Panel A | ||||
| Tr | NT | Tr | NT | |
| In 2 | 17 | 10 | 3 | 16 |
| Ex 7 | 9 | 16 | 17 | 4 |
| Panel B | ||||
| In 2 | 24 | 17 | 10 | 23 |
| Ex 7 | 18 | 21 | 24 | 11 |
These authors [1997] tested for allelic association in a case-control design comparing 129 unrelated individuals with a total sIgE level of 200 IU/ml or more, to 266 controls with total sIgE below 200 IU/ml. The subjects were genotyped at the FCER1B-ca microsatellite. They detected no significant differences in allele frequencies.
This report [1998] describes allele frequencies for the Intron 2 RsaI and Exon 7 UTR RsaI polymorphisms in FCERB1 among 78 asthmatics and 122 nonasthmatic members of 52 Kuwaiti Arab families. The overall allele frequencies were quite different from those published for Caucasian or Aboriginal Australians. There was complete linkage disequilibrium between the 2nd intron and 7th exon polymorphisms, and no difference in allele/haplotype frequency between the asthmatics and controls. A subgroup analysis of hayfever, eczema and skin-prick test positivity (SPT+) did find a trend for SPT+ asthmatics to carry the int2 B allele more often than did SPT- asthmatics (56% v 33%, P=0.01). This effect was much weaker in nonasthmatics (Breslow-Day test comparing asthmatics' with nonasthmatics' odds ratio X2(1 df)=3.41, P=0.06).
| Group | N | RsaI int2 B frequency | |
|---|---|---|---|
| Asthmatics | SPT+ | 54 | 0.56 |
| SPT- | 24 | 0.33 | |
| Nonasthmatics | SPT+ | 30 | 0.53 |
| SPT- | 92 | 0.51 | |
This paper describes an SSCP and exon amplification based screen for variants in FCER1B among 224 atopic asthmatic children and 227 related and unrelated controls. The I181L mutation was not detected, but 3.7% of cases and 2.6% of controls carried E237G. The authors also detected nine previously unreported variants.
These authors sequenced FCER1B (all exons, some introns and part of the 5' UTR) in 71 subjects from the Australian sib pair study [van Herwerden et al 1995] described earlier. No I181L, V183L and E237G alleles were detected, and two novel noncoding polymorphisms were not associated to asthma or BHR.
This case control study compared 146 asthmatics presenting to hospital acutely to 50 controls. Half the cases had presented during the soybean epidemic (see above). Several candidate genes were tested for association. No participants were found to carry an I181L allele, and the E237G allele frequency was not increased in cases (7.6% v. 8.5%, P=0.88).
This is another Japanese case-control study, that includes 226 asthmatics (54 nonatopic) and 226 healthy controls. None of 24 cases undergoing sequencing harboured a I181L, V183L or E237G variant allele, but a -109C>T SNP in the promotor region had a frequency of 65% in cases and 69% in controls (P=0.10). There was Hardy-Weinberg disequilibrium in the cases (a heterozygote excess, P=0.003), but not the controls. Paradoxically, the T/T genotype was associated with a higher total sIgE level within the asthmatics (P=0.0015).
The authors concluded that this polymorphism does not contribute directly to risk of asthma, but was a modulator of total sIgE level in individuals with atopy.
| Asthmatics (N=226) | Nonasthmatics N=226 | |||||
|---|---|---|---|---|---|---|
| T/T | C/T | C/C | T/T | C/T | C/C | |
| Genotype frequency | 85 | 123 | 18 | 108 | 99 | 19 |
| Expected under HWE | 95.0 | 103.1 | 28.0 | 109.8 | 95.5 | 20.8 |
| total sIgE (IU/ml) | 427 | 234 | 275 | 54 | 54 | 56 |
This study was of 22 "olive-pollen allergic" families (N=88). The TDT was applied to several favourite genes: HLA DRB1, DQB1, TCR-Va 8.1, LTA and FCER1B. The FCER1B Exon 7 UTR RsaI *1 allele was associated with elevated total sIgE level (P=0.01) as well as olive pollen specific IgE.
This describes a French-Canadian case-control study of asthma and E237G, as well as a family based study (see above). There were 100 cases (total sIgE>280 ug/l plus 3 or more positive SPTs to a panel of 6 allergens, 25 asthmatics) and 100 controls (nonatopic). No L181 alleles were found in any subjects. The G237 allele was much more common among the cases (Fisher exact P=0.00001) than the controls.
| Group | E/E | E/G | G/G | G237 Frequency | HWE P-val |
|---|---|---|---|---|---|
| Controls | 98 | 1 | 1 | 1.5% | 0.01 |
| Atopic | 80 | 20 | 0 | 10.0% | 0.14 |
In the four three-generation atopy pedigrees (all ascertained through a highly atopic single proband), there was significant support for association between the G237 allele and atopy via various transmission- disequilibrium tests. The strongest individual result was for sensitisation to indoor allergens: G237 allele frequency was 36% in affecteds and 6% in unaffecteds (unified transmission-disequilibrium test P=0.002). Paternal transmission was as skewed as maternal transmission for indoor allergen sensitisation: 20/26 transmissions of G237 overall, 9/11 paternal, 10/13 maternal.
This is an update on the Southampton group's study. There are three panels of families: the "random" sample of 131 nuclear families (685 individuals) unselected for asthma; 60 "multiplex" asthma families (354 individuals) containing two or more affected members; 49 "simplex" families ascertained via a single affected child.
Results for 6 markers around FCER1B, including the coding polymorphism FCER1B*E237G, were presented. The best single-point linkage was between wheezing and FCER1B*E237G (lod=1.52), and the best multipoint result (lod=1.4) with a quantitative asthma score was close to D11S480. There was no association with any marker.
These authors chose allergic rhinitis as the phenotype to test for association with FCER1B E237G. A total of 233 cases (outpatients mainly from Chiba University Hospital, rhinitis plus positive RAST score, mean total sIgE of 641 IU/ml) and 100 controls (chronic sinusitis, hypertrophic rhinitis, parosmia etc, with negative RAST screen, mean total sIgE 56 IU/ml).
The G237 allele was more common among the cases (P=0.015), and most strikingly (30%) among the 45 cases with multiple sensitization. There was Hardy-Weinberg disequilibrium in the controls (a dearth of heterozygotes).
| Group | E/E | E/G | G/G | G237 Frequency | HWE P-val |
|---|---|---|---|---|---|
| Controls | 77 | 18 | 5 | 14.0% | 0.012 |
| Rhinitis | 150 | 76 | 7 | 19.3% | 0.468 |
Among 94 Icelandic atopic asthmatics and 94 unrelated controls the L181 and L183 alleles were not detected at all. The G237 allele was uncommon, and not increased in cases (Table 9).
| Group | E/E | E/G | G/G | G237 Frequency | HWE P-val |
|---|---|---|---|---|---|
| Controls | 93 | 1 | 0 | 1.1% | 0.942 |
| Asthmatics | 92 | 2 | 0 | 1.7% | 0.883 |
These authors describe functional studies of I181L, V183L and E237G and wild type Fc-epsilon beta chains in mouse-derived mast cells. There were no detected differences between all four variant chains in cytokine and LTC4 production, intracellular calcium mobilization and beta-hexosaminidase release after activation of the receptor. This is another line of evidence suggesting the functional variant may be in LD with E237G.
This is a survey of 461 Swedish farmers performed by a collaboration including Bill Cookson and Miriam Moffatt. There were 83 asthmatics in the sample, and 147 atopics. The 237G allele was present in only 7 subjects (0.8% allele frequency). The RsaI-in2 and RsaI-ex7 "B" allele frequencies were 62% and 39% respectively. There was no association of these polymorphisms with asthma, total sIgE level or atopy. In the case of specific IgE, there was some evidence of association (P=0.005-0.03) of RsaI-ex7 to mites, notably L. destructor, T. putrescentiae, and mixed pollens. The numbers of subjects included in these analyses are smaller (eg "all" mites uses 73 subjects).
These authors compared allele frequencies in 76 Hong-Kong Chinese asthmatic children and 70 controls at six candidate SNPs. The allele frequencies for the Intron 2 RsaI (18% and 21%) and Exon 7 UTR RsaI (4% and 4%) polymorphisms in FCERB1 did not differ between the two groups.
Human uteroglobin (gene symbol UGB, 11q13) is a possible candidate for the 11q13 atopy gene, as it has cytokine-like anti-inflammatory activities, and is expressed in almost all epithelial cells, most notably in this context by Clara cells (the nonciliated secretory cells present in respiratory epithelia), where it is known as the 16 kD (or 10 kD, presumably because it is a homodimer of two 8 kD subunits) Clara cell protein.
Zhang et al [1997] used SSCP analysis to screen 26 sib pairs affected with asthma and their parents through the 5' promotor region and the three exons of the gene. This failed to detect any variation, although a similar screen of probands with Best's disease did detect a common C to T substitution at position +33.
Laing et al [1998] reported a mutation screening and case-control study of 67 children with asthma and 46 controls. Cases either attended a respiratory outpatient clinic or were sampled from a cohort of 76 families (266 subjects) unselected for asthma. Nonasthmatic controls came solely from the latter source, had no personal or family history of asthma, atopy, and no BHR on histamine challenge.
The authors detected a common A to G substitution at +38 in the 5' UTR of exon 1. A Sau96I RFLP was then used to type the entire sample. The A>G variant was present at a frequency of 67% in the family cohort (N=266), and was in HWE (P=0.5). Among asthma cases, the allele frequency was 57.7%, and was 73.9% among the 46 unaffected controls (P=0.008). If an additional 7 children from the cohort study were included as cases, using a definition of past doctor diagnosis of asthma plus BHR,
Mao et al [1998] and Gao et al [1998] describe attempted replications using the Osaka case-control sample and a British sample using both the Sau96I RFLP and a first intron STRP. There were no significant differences in allele frequency between any case groups and the controls (see Table; tests for HWE all nonsignificant). The frequency of the A>G polymorphism is quite similar across all three studies.
| Group | N | A>G frequency | |
|---|---|---|---|
| Japanese | Controls | 100 | 0.610 |
| Allergic asthma | 100 | 0.655 | |
| Nonallergic asthma | 100 | 0.610 | |
| British | Controls | 150 | 0.620 |
| Atopic asthmatics | 125 | 0.676 | |
This abstract (also from Mukherjee's group) describes a study of 22 asthma families. The +38A>G variant was more common in asthmatics. A similar claim was made for a novel tetranucleotide SSR (-3100).
Weaker evidence for association in the region was described by Holgate et al [1996]. As part of the Southampton study described above, allelic effects of two markers were detected on different phenotypes: allele 168 of D11S527 with BHR (P=0.0003, Bonferroni corrected for 13 alleles P=0.004), and allele 235 of D11S534 with total IgE (P=0.007, Bonferroni corrected for 14 alleles P=0.09).
Martinati et al [1996] used the published AS-PCR assay to look for the Leu181 mutation in all 213 subjects. As in Shirakawa et al [1996b], no mutations were detected. Similar findings were reported by Hill [1996], a sample of 65 asthmatics, Kofler et al [1996] in 40 atopic subjects (30 asthmatics), and by the present author [Duffy et al 1996], where no Leu181 mutations were present in the total 939 subjects tested (MZ and DZ twins, available parents of DZ twins and unrelated controls). In the latter three studies, the AS-PCR results were confirmed by sequencing in some or all of the atopic subjects.
| Study | No. subjects (families) | lod Score and comment |
|---|---|---|
| Cookson et al [1989] | (7) | 6.4 to D11S97 (10 cM) |
| Young et al [1992] | 281 (64) | 5.2 to D11S97 (M 18 cM, F 0.1 cM) |
| Inacio et al [1991] | 83 (17) | No linkage to D11S97 |
| Shirakawa et al [1991,1994] | (4) | 4.88 to D11S97 Selected pedigrees: used 4 of 136. |
| Amelung et al [1992] | 117 (20) | < -2.0 within 12 cM of FGF3 Families linked to chr 5. |
| Hizawa et al [1992] | 60 (4) | < -2.0 within 4 cM of D11S97 |
| Lympany et al [1992] | 89 (9) | < -2.0 within 4 cM of FGF3 |
| Rich et al [1992] | 67 (3) | < -2.0 within 5 cM of D11S97 |
| Coleman et al [1992] | 407 (95) | -7.88 multipoint D11S97, PGYM, CD20 12 families with affected mothers and unaffected fathers gave lod 0.8 |
| Collee et al [1993] | 52 (26) | increased ASP sharing D11S97 |
| Brereton et al [1994] | (12) | negative lod |
| van Herwerden et al [1995] | 246(123) | increased ASP sharing FCER1B-ca |
| Duffy et al [1994] | 424 (212) | no increase ASP sharing FCER1B-ca Unable to detect Leu181 mutation |
| Watson et al [1995] | 560 (131) | negative lods for 3 markers Combined segregation-linkage |
| Martinati et al [1996] | 213 (45) | no increase ASP sharing FCER1B-ca Unable to detect Leu181 mutation |
| Noguchi et al [1997] | 306 (68) | weak H-E result for sIgE and FCER1B-ca Negative ASP analysis |
| Neely et al [1996] | 218 (12) | increased ASP sharing (and positive TDT) INT2, D11S1369 |
| Trabetti et al [1998] | 659 (168) | increased ASP sharing FCER1B i2 RsaI poly |
| Folster-Holst et al [1998] | 98 (12) | 3.55 to FCER1B (0 cM) under two locus model |
| Palmer et al [1998] | 576 (121) | H-E linkage to total sIgE |
| Laprise et al [2001] | 4 (106) | 2.3 for atopy to E237G |
| Gene or Marker | Location (Mbp) | Remarks |
|---|---|---|
| SLC22A4 | 131.658-131.708 | RA and Crohn's |
| IL5 | 131.905-131.907 | Especially eosinophilia |
| IL13 | 132.022-131.025 | IL13 KO mice hypoallergic |
| IL4 | 132.037-132.046 | Gene methylation important |
| D5S393 | 135.729 | |
| IL9 | 135.256-135.259 | T-cell growth factor |
| CD14 | 139.993 | Binds LPS; phagocytosis |
| NR3C1 | 142.763 | Glucocorticoid receptor |
| D5S436 | 145.184 | |
| SCGB3A2 (UGRP1) | 147.238 | Uteroglobin related protein |
| SPINK5 | 147.424 | |
| ADRB2 | 152.638 | Beta-adrenergic receptor |
| D5S487 | 155.601 | |
| HAVCR1 | 156.419 | Hepatitis-A receptor |
| CYFIP2 | 156.629 | Cytoplasmic FMR1 interacting protein 2 |
| IL12B | 158.674 | IL-12 and IL-23 p40 beta-subunit |
| D5S422 | 162.086 | |
| LTC4S | 187.999 |
In the same 11 Amish families discussed above, sib-pair linkage analysis of total sIgE, mite- and multiallergen-specific (chemiluminescent immunoassay) sIgE was performed using markers from the 5q (5q31.1-33) lymphokine gene cluster. Significant evidence of linkage between total, but not specific, sIgE and polymorphisms in the IL-4, IL-9, and Interferon Regulatory Factor-1 genes were detected in SIBPAL analyses. However, IL-3, IL-5, CD14, CSF-1 receptor, and a lymphocyte-specific glucocorticoid receptor are also close neighbours, and as such, good candidates for disease genes. If the analysis was restricted to the 128 pairs without any detectable allergen-specific IgE (from a total of 349), probability of linkage increased, while age and sex adjustment made minimal difference.
The study of asthma families from Groningen has also confirmed the presence of linkage to the 5q region of total IgE level as well as nonspecific bronchial hyperresponsiveness [1994]. There were 92 families containing 538 individuals studied, all ascertained through a hospital diagnosed asthmatic parent (in the case of nuclear families) originally studied 1962-70. Histamine challenge testing, skin testing, and measurement of total, mite- and grass-specific serum IgE levels were performed on all family members.
A preliminary Class D regressive segregation analysis of logIgE level was consistent with the presence of a common (increasing allele frequency 59%) autosomal recessive gene with a residual sib-sib correlation for IgE levels of 0.27 (general model log likelihood, -520.35; polygenic with one distribution, -534.6; environmental with three distributions, -530.8; codominant, -523.3; recessive with mean(BB,AB)=41.7 and mean(AA)=436.5, -523.8). An analysis with PAP reached similar parameter estimates P=60%, mean(BB,AB)=22.9 and mean(AA)=239.9.
A sib-pair analysis (SIBPAL) of logIgE level confirmed the presence of linkage between the 5q interleukin cluster region and log IgE level (see Table). This closely paralleled findings for BHR treated as a discrete trait (PC20>32 mg/ml histamine versus PC20<32 mg/ml - see Table). When BHR was modelled as a continuous trait with log sIgE as a covariate, the evidence for linkage persisted (P<0.002). I would interpret this last finding as a comment on the repeatability/validity of each measure in isolation as a measure of atopy. A parametric linkage analysis in PAP under the preferred model gave a lod score of 3.56 at r=0.10.
| Marker | df (pairs) | P |
|---|---|---|
| IL9 | 131 | 0.07 |
| D5S393 | 248 | 0.01 |
| D5S436 | 282 | 0.0003 |
| CSF-1R | 226 | 0.03 |
| D5S410 | 239 | --- |
| D5S412 | 252 | --- |
| D5S415 | 230 | --- |
| Marker | df | P |
|---|---|---|
| IL9 | 16 | 0.14 |
| D5S393 | 22 | 0.04 |
| D5S500 | 35 | 0.09 |
| D5S658 | 35 | 0.03 |
| D5S436 | 35 | 0.009 |
| FGF1 | 25 | 0.02 |
| D5S434 | 34 | 0.08 |
| D5S470 | 35 | 0.10 |
| CSF-1R | 20 | 0.05 |
| D5S410 | 20 | 0.31 |
| D5S412 | 33 | 0.05 |
| D5S415 | 22 | 1.00 |
This group [1996] reported an absence of linkage of chromosome 5q markers to sIgE level in their four large atopic families (110 typed individuals). Maximum likelihood linkage analysis (under a common dominant gene model), and Haseman-Elston analysis using SIBPAL, were performed.
The maximum lod score among 12 markers spanning chromosome 5 was 0.06 (r=0.23), with LOD scores of -2.3 at r=0 for IL-9, and less damningly, -0.4 at r=0 for IL4-R1. Similarly, the best P-value from SIBPAL was 0.29.
This study [1997] found neither association and linkage to 5q in asthma families from Finland. Families containing at least one self-reported asthmatic were recruited by advertising within one province (Kainuu). There were 51 multiplex families and 105 uniplex families (used for TDT) where probands met the study diagnostic criteria for asthma. These required agreement between two study respiratory physicians based on medical records, and bronchial challenge. The authors found 87% of volunteers met these criteria. A total of 487 subjects underwent total and specific (eight allergens) sIgE determination and genotyping at 17 markers spanning D5S404, IL4, IL9, to D5S413.
Mapmaker-Sibs analysis of sIgE (73 pairs) did not find strong evidence for linkage to the region (t-values running from 0.9 to 1.6), and Genehunter NPL scores for asthma were consistently negative. No specific haplotypes were increased among subjects with sIgE>100 kU/ml. An IL9 polymorphism (T113M) was found in 38/253 atopics (high sIgE), 43/323 asthmatics, and 80/542 of the entire sample. The authors also present a refined (radiation hybrid based) physical map of the region.
As noted above, these authors [1997] report evidence for linkage between atopic asthma, atopy and sIgE level and several 5q31-33 markers in a relatively small sib-pair based study. The Haseman-Elston analysis (single-point, SIBPAL) of sIgE gave a best t=-2.83 (df=91, P=0.003) for IL4 (IL9 t=-1.89, df=79, P=0.03; D5S393 t=-2.28, df=89, P=0.01). The affected sib-pair (and discordant sib-pair) results for asthma and atopy were similar (Table).
| Marker | Mean ASP ibd | P-value | Mean DSP ibd |
|---|---|---|---|
| Atopy | |||
| IL4 | 0.58 | 0.006 | 0.41 |
| IL9 | 0.55 | 0.01 | 0.43 |
| D5S393 | 0.61 | <0.00001 | 0.52 |
| D5S436 | 0.51 | 0.42 | 0.41 |
| Asthma | |||
| IL4 | 0.62 | 0.0013 | 0.49 |
| IL9 | 0.60 | 0.018 | 0.48 |
| D5S393 | 0.60 | 0.007 | 0.57 |
| D5S436 | 0.49 | 1.00 | 0.51 |
This study was extended in Yokouki et al [2000]. This used the subset of families also described in Kimura et al [1999] (see below). These were 47 families containing two siblings with mite-sensitive asthma (65 affected sib pairs), typed at 398 markers. The best genome wide affected sib pair multipoint MLS (using Mapmaker-Sibs) was 4.52, peaking over D5S820, 20 cM telomeric of IL4, and between ADRB2 and IL12B. Further fine mapping increased the MLS to 5.3 between D5S487 and D5S422 [Noguchi et al 2005].
The group from Melbourne [1997] also looked at D5S399 sharing among the 119 sib pairs. The biggest subset of affected sib pairs was 105 pairs concordant for atopy. There was no increase in IBS sharing for asthma, atopy, or BHR.
Extending this sample to a total of 53 low-low pairs, a multipoint analysis obtained a lod of 2.4, with a peak around D5S658. A single-point Haseman-Elston analysis of eosinophil count (using 70-82 pairs) obtained a peak slope of -0.41 (P=0.002) at D5S500.
The full sample of Busselton families were analysed for linkage and association to the IL4 variants described by Walley & Cookson [1996]. The phenotypes chosen were those of Dizier et al [1995]: age-sex-generation adjusted log total sIgE, with and without adjustment for specific sensitization (defined as any positive RAST).
Haseman-Elston analyses were weakly suggestive for linkage to the RAST adjusted total sIgE level (IL4, P=0.01; -590C>T IL4 promotor variant, P=0.02). A lod score analysis (combined segregation-linkage using REGRESS) under the recessive model gave similar results (lods=1.17, 1.25). Adding in an association model found no evidence for allelic association between either the IL4 STR or -590C>T.
This is a Singaporean Chinese affected sib pair study of asthma and atopy linkage to 5q. There were 88 families containing 125 asthma ASPs. The multipoint NPL analysis gave a peak lod (equivalent) of 1.7, but a peak singlepoint ASP lods of 5.3 (using GAS with 89 informative pairs) was obtained for D5S412 (close to IL12B), and 5.1 for D5S2110 (96 pairs, close to IL9). Singlepoint (old) Haseman-Elston analysis obtained a best linkage of D5S2001 to total sIgE (P=0.01).
Given the number of good candidate genes in this region, there have been a large number of pure association studies.
| Study | Subjects | N | Frequency | |
|---|---|---|---|---|
| Whalley & Cookson [1996] | Busselton | Asthma | 211 | 0.30 |
| Busselton | Control | 793 | 0.26 | |
| Oxfordshire | Asthma | 124 | 0.33 | |
| Oxfordshire | Control | 59 | 0.27 | |
| Basehore et al [2004] | US Caucasian | Asthma | 233 | 0.19 |
| US Caucasian | Control | 245 | 0.14 | |
| US Hispanic | Asthma | 116 | 0.39 | |
| US Hispanic | Control | 230 | 0.35 | |
| African-Am | Asthma | 168 | 0.64 | |
| African-Am | Control | 269 | 0.67 | |
| Nagarkatti et al [2004] | North Indian | Asthma | 171 | 0.00 |
| North Indian | Control | 128 | 0.00 | |
| Korzycka-Zaborowska et al [2004] | Polish | Atopy | 98 | 0.01 |
| Polish | Control | 87 | 0.02 | |
This abstract [1994a] describes polymorphisms (SSCPs) in the IL-3, IL-4 and IL-9 genes [Borish et al 1994b] on chromosome 5q. Rosenwasser et al [1995] enlarge on this. In 20 families ascertained through an asthmatic, a -590C>T IL4 promotor polymorphism (rs2243250) was associated with high total sIgE level. In an enlarged sample [Borish et al 1996; Rosenwasser & Borish 1997], association was also found between a -571C>A variant in the IL10 promotor, PBMC production of IL-10 and high total sIgE level.
This is another analysis of the two Oxford panels (Busselton and UK), this time seeking to replicate an association between the -590C>T IL4 promotor polymorphism and atopy. There was weak evidence of association found in the Busselton sample only to wheeze and presence of anti-HDM specific IgE (P=0.03 and P=0.01, uncorrected for relatedness of subjects or for multiple comparisons).
This case-control study was mentioned earlier. It also failed to find any evidence of association to D5S436, D5S393, D5S210, IL4, and IL9.
Britton's group [1998] genotyped 89 asthmatics (symptoms plus positive methacholine challenge) and 92 controls at 11 markers spanning 5q31. This sample came from the 2633 Nottingham residents they had previously surveyed [Britton et al 1994]. There was association to BHR and sIgE for D5S404, IRF1, and D5S210 with uncorrected P values of 0.02 to 0.04 (22 tests).
This paper describes a case-control study of the -590C>T polymorphism in 84 Kuwaiti asthmatics, 53 unaffected first-degree relatives, and 47 unrelated unaffected controls. The C>T allele frequency was 79% in cases, 75% in the unaffected relatives and 76% in the other controls.
In a survey of 1333 Taiwanese undergoing health screening, there was no association between questionnaire diagnosed atopic disease or specific or total sIgE level and -590C>T.
Basehore and coworkers [2004] looked at 11 SNPs across the IL4 gene, obtaining a best individual SNP P-value of 0.0003 within their US white group for IgE level, but only P=0.01 for asthma. The commonest haplotype (74% in whites) was associated with low sIgE level (P=0.00008) in Caucasians, but not in the other two groups. There were large differences in SNP and haplotype frequency across the three ethnic groups in this study.
The 1958 UK birth cohort contributed to the control panel for the Wellcome Trust Case Control Consortium, and association results for phenotypes such as serum IgE level and FEV1 are available on the web at http://www.b58cgene.sgul.ac.uk/.
Although well short of genome-wide significance, there are effects of several SNPs on log serum IgE level.
| Position | SNP | N | Beta trend | SE | P-value |
|---|---|---|---|---|---|
| 132036543 | rs2243248 | 1436 | 0.0465 | 0.0479 | 0.331804 |
| 132036543 | rs2243248 | 1379 | 0.0406 | 0.0490 | 0.407382 |
| 132037053 | rs2243250 | 1608 | 0.0514 | 0.0324 | 0.112387 |
| 132040624 | rs2227284 | 446 | 0.1035 | 0.0495 | 0.036872 |
| 132040624 | rs2227284 | 1436 | 0.0853 | 0.0268 | 0.001467 |
| 132041078 | rs2227282 | 1436 | 0.0853 | 0.0268 | 0.001467 |
| 132042008 | rs2243270 | 1436 | 0.0996 | 0.0318 | 0.001758 |
| 132045843 | rs2243288 | 1368 | 0.0773 | 0.0335 | 0.021338 |
| 132046068 | rs2243290 | 1436 | 0.0893 | 0.0331 | 0.007141 |
| 132046068 | rs2243290 | 1381 | 0.0771 | 0.0340 | 0.023790 |
IL-13 has been implicated in asthma by several studies and variants in CD124, a subunit of both the IL-4 and IL-13 receptors, may be associated with asthma. The British/Japanese collaboration (that includes Taro Shirakawa and Julian Hopkin) therefore screened IL13 for variants, detecting a single functional polymorphism R130Q in exon 4. They then performed case-control studies in their usual UK and Japanese panels (see Table). The Q allele was significantly more common aong asthmatics than controls in both populations (for Japanese atopic asthma OR=1.85, P=0.03; for the British, OR=1.83, P=0.01). In the cohort of 290 Japanese schoolchildren previously examined for a relationship between tuberculin reactivity and atopy [Shirakawa et al 1997], there was a similar association, and that serum IL-13 levels were higher in R130Q carriers (data not shown).
Via molecular modelling, the authors went on to show that the Gln at position 130 should alter receptor binding. In a subsequent paper [Arima et al 2002], it was shown that the Q130 variant does not affect binding to the high affinity IL-13 receptor in vitro, but slightly lowers binding to the lower affinity IL-13R-alpha-2 molecule, and seems to have a longer plasma half-life.
| Group | N | R130Q frequency | |
|---|---|---|---|
| Japanese | Controls | 100 | 0.430 |
| Allergic asthma | 100 | 0.630 | |
| Nonallergic asthma | 100 | 0.620 | |
| British | Controls | 150 | 0.267 |
| Atopic asthmatics | 150 | 0.400 | |
Mutation screening of the German MAS-90 cohort also highlighted variation in the fourth exon of IL13, this time R130E. The E allele was present in 21% of 604 children, and was found to be increased in subjects with a high total sIgE level (>85th age-sex percentile, P=0.003, see Table). A weaker effect was seen for atopic dermatitis.
| IgE Percentile | Genotype counts | E allele frequency | ||
|---|---|---|---|---|
| Q/Q | Q/R | R/R | ||
| >85% | 6 | 42 | 52 | 0.27 |
| <15% | 1 | 30 | 80 | 0.14 |
This paper describes results from the Childhood Origins of Asthma (COAST) study. A total of 207 intensively phenotyped infants were genotyped at 61 SNPs in candidate genes including FCER1B, NOS2A, IL5, IL10, IL13 and IFNG. The strongest results were for the FCER1B*E237G and a NOS2A*D346D variant, associated with low IL-13 levels in cord blood following PHA stimulation (P=0.0025, 0.0062). The TGFB1*-509T allele was a risk factor for respiratory syncytial virus-related wheezing in the first year (P=0.0005).
The transmission-disequilibrium test (FBAT) found some evidence of association in 640 asthma families in the Childhood Asthma Management Program (CAMP). The R130Q did not increase in frequency with AHR, asthma diagnosis or severity, but was correlated with overall allergy (combining eosinophil count, total sIgE and number of positive SPTs, P=0.007), as well as with eosinophil count alone (P=0.01).
These authors found no association or linkage of the R130Q variant to IgE level or lung function in families ascertained through 72 COPD patients.
This study also found weak evidence of association using a family-based design. A sample of 368 The R130Q allele increased risk of atopy and atopic dermatitis, most strongly in white children (best P=0.014).
CD14 is a 55 kD glycoprotein expressed on the surface of predominantly monocytes, and binds bacterial lipopolysaccharide (LPS, thus explaining its involvement in toxic shock syndrome) and peptidoglycan. It is central to phagocytosis of apoptotic cells. It is located within the 5q31.1 cluster. Three posters at the 1999 ASHG meeting described evidence for association between CD14 and atopy. Reijmerink et al genotyped 159 asthmatics and 159 spouse controls for a -159C>T promotor variant (probably rs778594). Although cases and controls did not differ in allele frequencies, the C/C homozygotes tended to have higher total IgE levels and more positive skin test results. Among the Hutterites [Schneider et al 1999], there was an association with (any) positive skin test (P=0.0009). In Iceland, Hakonarson et al [1999] found no association with moderate to severe atopic asthma (C frequency of 94 cases, 58.5%; 94 controls, 54.5%).
Robertson et al [2002] failed to find any evidence of association to asthma in the Williams and McNicholl 1964 Melbourne asthma cohort. For -159C/T, the genotype frequencies were C/C 0.262, C/T 0.488 and T/T 0.250 (N=257). In another TSANZ poster, Savarimuthu et al [2003] reported genotypes for 463 asthmatics: proportions were C/C 0.28, C/T 0.49 and T/T 0.23.
Heinzmann et al [2003] carried out a case-control study (182:270) of German child asthmatics. The -159C>T and 1359G>T SNPs were not associated with asthma (P=0.876), with genotype frequencies for -159 of C/C 0.293, C/T 0.481 and T/T 0.226 (N=443).
Rupp et al [2004] link the CD14-159 polymorphism with susceptibility to C. pneumoniae infection (detectable in circulating monocytes in 13% of their coronary artery disease patients). Stimulation by C. pneumoniae increases CD14 expression more in T/T homozygotes [Eng et al 2004].
John et al [2004] examined CD14-159 genotype in 442 children from a birth cohort. Overall, allergic sensitisation was not associated with CD14-159 genotype (P=0.4), but there was a strong interaction with endotoxin level in house dust samples from the childrens' homes: endotoxin exposure reduced sensitisation in C/C carriers (OR=0.70, 95%CI=0.55-0.89).
Choudry et al [2005] reported results from the GALA study. CD14-159 genotype was found to be associated with total sIgE level in Puerto Rican and Mexican asthmatics, but only in those exposed to cigarette smoke (best P-value 0.00008 in Puerto-Ricans). Low baseline FEV1 in smoke-exposed asthmatics was associated with CD14+1437 in family-based association analyses.
Yanamandra et al [2004], at the same meeting, found no association in African-American asthmatics compared to controls, with genotype frequencies for -159 of C/C 0.494, C/T 0.425 and T/T 0.081 (N=160 controls). These genotype frequencies are quite different to those reported for samples of European descent.
Gern et al [2004] describe results from the COAST study "at risk" birth cohort. There were 285 probands with a parental history of asthma or hayfever. Atopic dermatitis and food allergy in the first 12 months of life were assessed, and it was found that AD was less common in children exposed to dogs (30% versus 50%), with no protection evident for cat exposure. CD14 genotype effects interacted with dog ownership (see Table).
| Exposed to dogs | Group | Genotype counts | T allele frequency | ||
|---|---|---|---|---|---|
| C/C | C/T | T/T | |||
| AD | 7 | 14 | 1 | 0.364 | |
| Control | 11 | 25 | 16 | 0.548 | |
| AD | 15 | 38 | 13 | 0.485 | |
| Control | 23 | 19 | 17 | 0.449 | |
Eder et al [2005] describe gene by environment interactions in the Allergy and Endotoxin Study (Austria and Germany). Among 624 rural children, there was no overall association between the CD14*-260C>T polymorphism and sIgE level or atopy. However, both mattress endotoxin levels and contact with farm animals were found to interact with genotype in a complex fashion (see Table).
| Animal contact | C/C | C/T | T/T |
| None | 1 | 0.52 (0.15-1.84) | 0.69 (0.15-3.11) |
| Dogs/cats only | 1 | 0.23 (0.07-0.80) | 0.25 (0.05-1.15) |
| Stable animals | 1 | 1.92 (0.88-4.19) | 2.56 (1.07-6.15) |
This US patent describes association between a T117M variant in exon 5 of IL9 (rs2069885) and sIgE level. In an initial sample of Italian asthma families and unselected subsequent samples, total sIgE was lower in M/M homozygotes than the other genotypes. The estimated M allele frequency was 12-13%, so homozygotes are relatively infrequent.
The University of Washington-Fred Hutchison CRC Variation Discovery Resource (SeattleSNPs) found the M117 allele frequency to be 13% in African-Americans and 12% in their CEPH panel.
SPINK5 (5q32) encodes a serine protease inhibitor ("lymphoepithelial Kazal-type-related inhibitor"). Mutations in SPINK5 lead to Netherton's syndrome, a rare autosomal recessive disorder characterized by congenital ichthyosis, "bamboo" hair and severe atopy (with high total sIgE levels). It may modulate Th1/Th2 skewing via effects on NFKB (a mechanism shared by other serpins).
Moffat et al [2000] tested for association between several SNPs in the gene and total serum IgE level in the Busselton sample (1004 individuals in 230 families). Several of these were associated with IgE level, the strongest result with a silent polymorphism (118C>T), but common haplotypes combining the variants did not exhibit association.
Cookson's group [Walley et al 2001] further describe association to atopy in 148 families containing a child with atopic dermatitis (Greater Ormond Street dermatology outpatients panel) and the UK asthma family panel (73 families). A total of 32 SNPs through the gene were typed, and a significant TDT was exhibited for 2 cSNPs in exons 13 and 14 (best P-value, 0.005 for atopy versus Q420L, uncorrected for multiple testing). There were parent of origin effects, with maternal overtransmission in the both samples, and weaker paternal overtransmission in the asthma panel alone.
Kabesch et al [2004] looked at Q420L in the Munich children (1161 individuals). There was weak evidence of association with asthma (OR=1.8, P=0.04), and especially if accompanied by atopic dermatitis (OR=4.6, P=0.007). There was no assocation with atopic dermatitis per se, skin atopy or total sIgE level.
Rihs et al [2005] developed a high throughput assay for Q420L, and applied it to a case-control study of latex allergy (63 asthma, 172 other symptoms; 80 nonatopic controls). No association was found (for any allergy, P=0.46).
McIntire et al [2001] described mapping airway hyperesponsiveness in congenics from a BALB/c background (high BHR) with DBA/2 (low BHR, low TH2 responsiveness) derived chromosomal intervals. One line, carrying a small (20 cM) DBA/2 region homologous to human 5q23-35, was hyporesponsive to keyhole limpet hemocyanin (increases IL-4 production in BALB/c mice) or ovalbumin inhalation post-immunisation. This segment acted recessively in backcrosses to BALB/c.
Further (2000) backcrosses were used to perform high resolution linkage mapping. The BHR and IL4 responsiveness locus Tapr was found to be separate from the cytokine gene cluster and tightly linked to a marker within the mouse homologue to the rat Kim1 locus, homologue itself to the human Hepatitis A Virus receptor gene HAVCR1.
Three members of the newly designated TIM gene family were cloned (30-45% amino acid sequence identity with human HAVcr-1 protein), and multiple polymorphisms described in TIM1 and TIM3 that cosegregated with BHR. The HAVCR1 homologue TIM1 was shown to be expressed on CD4+ T cells and transcribed during primary antigen stimulation. Furthermore, the author cited two studies by Matricardi and coworkers showing atopic individuals being less likely to show seropositivity to Hepatitis A Virus, toxoplasma and H. pyloridis [Matricardi et al 1999, 2000], as evidence that the relationship between the polymorphisms and BHR was likely to be causative rather than due to linkage disequilibrium. HAV infection has not protected against asthma in other studies, however.
Monney et al [2002] showed that the TIM3 protein is only present on activated Th1 cells and macrophages, that such cells are common in the CNS at the onset of experimental autoimmune encephalomyelitis (EAE, a well known model for MS), and that anti-TIM3 antibodies exacerbated the course of the EAE.
Graves et al [2005] tested for association of the TIM family genes with sathma and atopy in the Tucson Children's Respiratory Study. There were 564 individuals with available DNA, SPT, and four rounds of questionnaire data (birth to age 16 years), of whom one-third gave a history of asthma. At 8 of 21 SNPs genotyped, there was significant heterogeneity in frequency between Hispanic and European descended participants. While no SNPs were associated to asthma, association to atopy (SPT) and to atopic dermatitis was observed (best P=0.002 for rs3087616 in TIM3). TIM3 haplotypes were similarly associated; for the rs3087616-rs1036199-rs470853 GGT haplotype, the relative risk for atopy was 1.24 (P=0.0007).
Noguchi et al [2005] describe association mapping in 26 genes (90 SNPs) under their linkage peak [Noguchi et al 1997; Yoyouki et al 2000] centred roughly on HAVCR1. HAVCR1 and IL12B were excluded, but there were strongly positive TDT results for CYFIP2. An additional 15 SNPs in CYFIP2 were therefore genotyped (a total of 30 SNPs in 6 LD blocks across the gene).
There were 6 SNPs (in complete LD) where 28/33 transmissions to cases were observed (individual P=7e-5, lod=3.46), including rs12654973. All were noncoding, but homozygotes haplotype carriers had 25% greater expression of CYFIP2 in lymphocytes than heterozygotes (P=0.03).
CYFIP2 is the gene coding for cytoplasmic fragile X mental retardation protein (FMRP) interacting protein 2 (also called p53 inducible protein, PIR121). The protein colocalizes with FMRP and ribosomes in the cytoplasm, is probably involved in p53-dependent apoptosis, but also in T cell fibronectin-mediated adhesion [Mayne et al 2004] in vitro as well as in multiple sclerosis. In keeping with the involvement in apoptosis and adhesion, it is also regulates the actin cytoskeleton. Its' close (88% homology) relative CYFIP1 is on 15q11.2.
Uteroglobin-related protein 1 is, as the name might suggest, similar in amino acid sequence to uteroglobin/CC16, and maps to 5q31 [Niimi et al 2002]. It is 93 residues long. The gene (secretoglobin, family 3a, number 2; SCGB3A2) spans 3 kbp (3 exons). There are no known coding variants.
Niimi et al [2002] also performed a case-control analysis of association of a -112G>A promotor variant, that they had shown to affect nuclear protein binding. The -112A allele was more common among 85 asthmatics (Hokkaido), compared to 85 controls (P=0.002).
| Group | Genotype counts | -112A allele frequency | ||
|---|---|---|---|---|
| G/G | A/G | A/A | ||
| Control | 70 | 13 | 2 | 0.10 |
| Asthma | 50 | 31 | 3 | 0.22 |
Interleukin-12 is a heterodimer, the p35 35 kD alpha chain coded by IL12A (3p), and the p40 (40 kD) beta chain by IL12B (5q13). The p40 chain is also shared with IL-23. The IL12B gene contains 7 exons and spans 12 kbp.
IL-12 is another cytokine with a complex pattern of action. It is secreted by macrophages and other antigen presenting cells. Administration of IL-12 reduces circulating eosinophil count (it induces eosinophil apoptosis via stimulation of IL-12 receptors on that cell), while treatment of RSV-infected mice with anti-IL-12 results in BHR, mucus production, and eosinophilia [Tekkanat et al 2001]. However, it failed to reduce the bronchoconstriction or the late phase response to allergen challenge in human asthmatics [Bryan et al 2000]. Natural Killer cells exposed to IL-12 tend to secrete gamma-interferon, and in individuals with IL-12 deficiency due to IL12B deletion, impairment of gamma-interferon production and bacterial immunity have been observed. In the skin IL-12 induces DNA repair and so reduces UV-induced apoptosis.
Morahan et al [2001] reported an association between a 3'UTR SNP in IL12B and IDDM. This has not replicated in a number of studies.
Using SSCP, Noguchi et al [2001] identified four variants in IL12B, two rare (1/100, 1/200). The other SNPs were tested for association with asthma and rhinitis via a TDT using the families described in Yokouki et al [2000]. Neither the S226N (allele frequency 4%) nor 1188A>C (50%) variants were overtransmitted.
More recently, Morahan et al [2002] report association between asthma and an IL12B promotor polymorphism, that they note is not in strong linkage disequilibrium with the 3'UTR polymorphism. A total of 844 children from the West Australian Pregnancy Cohort Study were genotyped at IL12B SNPs. This included 203 asthmatic children, of whom 39 were classified as severely affected.
In the first 411 children genotyped, there was a weakly significant overall chi-square for association, and Hardy-Weinberg disequilibrium in some of the asthmatics (Table). The authors interpreted this as suggesting overdominance in the severely affected subgroup.
| Group | Genotype counts | 1 allele frequency | HWE P-value | ||
|---|---|---|---|---|---|
| 1/1 | 1/2 | 2/2 | |||
| Nonatopic Control | 26 | 67 | 31 | 0.48 | 0.358 |
| Atopic Control | 43 | 81 | 45 | 0.49 | 0.591 |
| Mild Asthma | 10 | 20 | 12 | 0.48 | 0.768 |
| Moderate Asthma | 20 | 17 | 18 | 0.52 | 0.004 |
| Severe Asthma | 2 | 16 | 3 | 0.52 | 0.013 |
This specific hypothesis was then tested in the remaining 85 asthmatics, where again there was H-W disequilbrium in the "severe" group due to an excess number of heterozygotes (16/18). Functional studies of the three genotypes found suggestive decreases in IL12B mRNA expression and in IL-12 (p70) production in heterozygote compared to either type of homozygote cultured PBMCs.
There was no association with the 3'UTR polymorphism. Data on haplotypes was not presented: according to Huang et al [2000] and Morahan et al [2001], the distance between these polymorphisms is 20 kbp.
Khoo et al [2004] looked at a subsample of 244 subjects from the 1957 UK birth cohort enriched for a history of childhood asthma followed over 7 examinations. There was no association between IL12B promoter genotype and asthma or atopy, with a possible association with low (standardised) FEV1 at age 7 only. Noguchi et al [2001] also failed to find association within their panel of 47 families linked to the region.
This seems a natural candidate for steroid resistant asthma.
In the Hutterite study, linkage of BHR to D5S1470 (5p13.3) was observed, with a lod=2.1. In an ASHG meeting abstract, Kurz et al describe overtransmission of the 177 bp allele at this marker, which was followed up SNP genotyping in the neighbouring genes: the prostaglandin E4 receptor (PTGER4, deletion of which impairs contact hypersensitivity and skin APC migration in mice [Kabashima et al 2003]), leukemia inhibitory factor receptor (LIFR, Stuve-Wiedemann syndrome), atrial natriuretic peptide clearance receptor (NPR3), and ADAMTS12. Multiple SNPs exhibited overtransmission, but most of the linkage signal could be abolished by excluding individuals carrying variants at LIFR and PTGER4.
The association was replicated in a German case-control panel of 231 asthmatics, 196 atopics and 270 controls. PTGER4 was associated with asthma (P=0.008), while LIFR and NPR SNPs were associated with atopy.
The prostaglandin D2 receptor gene (PTGDR) has also exhibited association to asthma (see below). The knock-out phenotypes for both PTGDR and PTGER4 seem quite similar.
As noted earlier, Szentivanyi [Szentivanyi 1968] proposed the unifying hypothesis that asthma represents an underresponsiveness of the lungs to sympathetic neurotransmitters, suggesting the beta adrenergic receptor as the most likely site for this. A number of recent studies have looked at functional mutations in the beta-2-adrenergic receptor gene ADRB2 (5q31-32).
| Polymorphism | Study | Mutation frequency (No. subjects) | |
|---|---|---|---|
| Asthmatics | Controls | ||
| RFLP | Potter et al [1993] | (42) | (30) |
| R16G | Reishaus et al [1993] | 71.6% (51) | 73.2% (56) |
| Turki et al [1995] | 66.7% (45) | --- | |
| Kowalski et al [1997]* | 31.7% (60) | 41.4% (47) | |
| Martinez et al [1997]** | 66.7% (78) | 59.7% (191) | |
| Dewar et al [1997]*** | 67% (321) | ||
| Hakonarson et al [1999] | 55.8% (94) | 55.2% (94) | |
| Wang et al [2001] | 38.3% (128) | 48.5% (136) | |
| Leung et al [2002] | 42.1% (76) | 42.1% (70) | |
| Hermann et al [2002] | --- | 38.0% (2010) | |
| Horvath et al [2004] | 39.4% | --- | |
| Ng et al [2004] China | --- | 62.1% (207) | |
| Ng et al [2004] France | --- | 37.9% (858) | |
| Ng et al [2004] Spain | --- | 39.7% (395) | |
| Cho et al [2005] Korea | 51.3% (37) | 47.8% (157) | |
| Q27E | Reishaus et al [1993] | 49.0% (51) | 49.1% (56) |
| Hall et al [1995] | 55.0% (65) | --- | |
| Turki et al [1995] | 51.1% (45) | --- | |
| Kowalski et al [1997]* | 43.3% (60) | 51.1% (47) | |
| Martinez et al [1997]** | 38.5% (78) | 35.3% (191) | |
| Dewar et al [1997]*** | 48% (322) | ||
| Hakonarson et al [1999] | 39.5% (94) | 37.5% (94) | |
| Wang et al [2001] | 8.2% (128) | 8.8% (136) | |
| Leung et al [2002] | 7.9% (76) | 10.7% (70) | |
| Hermann et al [2002] | --- | 42.0% (2010) | |
| Horvath et al [2004] | 35.5% | --- | |
| Ng et al [2004] China | --- | 11.8% (207) | |
| Ng et al [2004] France | --- | 39.6% (858) | |
| Ng et al [2004] Spain | --- | 38.7% (395) | |
| Cho et al [2005] Korea | 10.5% (38) | 8.6% (157) | |
* Seasonal asthma and/or rhinitis.
** Wheeze in the previous 12 months.
*** All members of family ascertained through multiple asthmatic
probands.
By contrast, nine point mutations of the beta-2-adrenoceptor among 107 subjects (51 asthmatic). No excess of a particular polymorphism was found in the asthma group compared to controls, but an R16G mutation (56% of the overall sample) was found more commonly in steroid-dependent asthmatics compared to less severe asthmatics.
This paper [1995] describes a similar study of the beta-2- adrenoceptor polymorphism in 65 asthmatics. These authors concentrated on the R16G (Arg16Gly, rs1042713) and another, Q27E (Gln27Glu, rs1042714), mutation found to be common by Reihaus et al [1993], although the frequency of the latter did not differ between asthmatics and controls. However, in vitro studies have suggested that the Q27E mutation might be associated with diminished downregulation after agonist exposure [Green et al 1994].
The frequency of the E27 mutation was 55% in this sample. E27/E27 homozygotes were more likely to be atopic (trend X2(1 df)=3.1, P=0.08), and exhibited a significantly higher PD20 to a methacholine Yan challenge (P=0.03) than Q27/Q27 homozygotes. No effects of R16G were detectable. The absence of controls and small sample means replication is needed.
This report [Tan et al 1997] reanalysed three previous studies (total N=22) including Hall et al [1995] to conclude that the G16/G16 carriers became more desensitised to formoterol than asthmatics carrying R16/R16.
This study [1995] compared 23 nocturnal asthmatics (who tended to have a lower mean diurnal FEV1, and to be more likely to be steroid dependent) to 23 "normal" asthmatics. The R16G frequency was 80.4 percent in the nocturnal group, and 50.0 percent in the nonnocturnal group (P=0.004).
This abstract [1997] compares the frequencies of the two polymorphisms in 60 atopics ("seasonal asthma and/or rhinitis") and 47 history and SPT negative controls. The table they give is hard to interpret, but suggests a severe dearth of Q27/Q27 heterozygotes among the cases (HWE X2(1 df)=37.4; cases versus controls X2(2 df)=27.4).
The Southhampton group [1997] compared the frequencies of the two polymorphisms in 324 members of 60 families ascertained for asthma. In the combined segregation-association analysis using COMDS, asthma and atopy were not correlated with either polymorphism. However, sIgE was (P=0.009). There was also weak evidence for linkage between sIgE and the position 27 ADRB2 polymorphism (with NOPAR, P=0.04).
A subset (N=269) of participants in the Tucson Children's Respiratory Study were typed at these two ADRB2 polymorphisms. As before, there was no relationship between genotype and presence of a history of asthma (N=37) or wheeze in the previous 12 months (N=78). However, there was evidence of a relationship with a significant bronchodilator response (here defined as a 15% improvement in FEV1 as a fraction of predicted FEV1). The direction of the relationship was in keeping with Hall et al [1995] and Tan et al [1997] in that the G16/G16 homozygotes were less likely to exhibit a bronchodilator response.
These authors [1998] report more evidence of an association between ADRB2 polymorphisms and asthma. They examined 78 cases of fatal or near-fatal asthma, 82 cases of nonfatal asthma, and 84 controls, all presumably ascertained through the University of British Columbia. They also performed subgroup analyses among the "nonfatal" asthmatics, dividing them into moderate and mild severity groups based on therapy (all but 9 of 41 the mild group did not use inhaled corticosteroids). The sample was also stratified on ethnicity.
Importantly, the authors found significant differences in allele frequencies across Whites, Blacks and Asians. For example, while the frequency of the Gly16 allele was 61% in both Causasian asthmatics and Caucasian controls, it was 40% in the Asian asthmatics.
Although no differences were found between asthmatics and nonasthmatics as a whole, or between fatal asthma and controls for either G16 or Q27, the moderate asthmatics (N=33) had a higher frequency of the Q27 than mild asthmatics. The lack of an obvious dose-response relationship leaves open the possibility of a Type I error.
| Subgroup | N | Q27 frequency |
|---|---|---|
| Severe Asthma | 69 | 0.54 |
| Mod Asthma | 33 | 0.67 |
| Mild Asthma | 41 | 0.48 |
| Nonasthmatics | 84 | 0.60 |
This Italian study documents a relationship between nonspecific BHR and the G16/Q27 haplotype that persisted after including total and specific IgE as a covariate in the analysis.
This abstract includes an author from deCode and describes a case-control study of 94 Icelandic "moderate to severe" asthmatics and 94 controls. There were no association with either the R16G or Q27E variant. This study was presented in more in detail in Hakonarson et al [2001].
The relationship between response of isolated airway smooth muscle preparations to isoproterenol was examined in 15 individuals of differing ADRB2 genotype. The G16/Q27 haplotype was associated with greater desensitisations. They comment that E27 was in strong LD with the R19 allele in the 5' leader peptide, which was more strongly associated with the phenotype.
Anqing (China) is the site of a large genetic epidemiological study of asthma and lung diseases. Over 2000 families containing two or more doctor-diagnosed asthmatics have been ascertained, and 7100 of the 10000 subjects have undergone methacholine inhalation challenge.
Wang et al [2001] describe a nested case-control study within these families: 128 BHR+ asthmatics and 136 BHR- nonasthmatic controls. Cases were twice as likely to have ever smoked (30% versus 17%). The R16 allele was more common among the asthmatics (OR=1.04, P=0.04), but since the effect was stronger among the ever smokers (see Table), the authors interpreted the data as suggestive of a recessive gene by smoking interaction.
| Smoking | Asthma | G/G | R/G | R/R | R16 Frequency | HWE P-val |
|---|---|---|---|---|---|---|
| Ever | Yes | 6 | 11 | 22 | 70.5% | 0.045 |
| Ever | No | 6 | 12 | 5 | 47.8% | 0.827 |
| Never | Yes | 16 | 43 | 30 | 57.9% | 0.931 |
| Never | No | 28 | 52 | 33 | 52.2% | 0.408 |
This Hong-Kong study contrasted 76 asthmatic children attending outpatients and 70 controls. The allele frequencies did not differ between the groups and are close to those from Wang et al [2001].
This is a Mexican case-control study (N=907). The E27 and G16/E27 haplotype were found to be protective against asthma, especially in women. The G16 allele was associated with nocturnal asthma.
These papers describes one of the first outings for the haplotypic FBAT. The CAMP study provided 652 nuclear families (707 asthmatics, 2011 individuals) who were genotyped at seven polymorphic SNPs in ADRB2. There were no significant associations between any single SNP and measures of bronchodilator response or asthma symptom score. The 20 asthmatics with an FEV1 below 80% predicted did give significant results for 4 SNPs including R16G and Q27E.
By contrast, highly significant results were obtained for 7-SNP haplotypes and asthma (X29=35.6, P=4.10-5). These results do seem to be driven by less common haplotypes, so I do worry about breakdown of the chi-square approximation to the score statistic.
| Haplotype | 16/27 | Proportion | Z-score |
|---|---|---|---|
| ATACGCC | RQ | 0.357 | +0.54 |
| GCGGGCC | GE | 0.355 | +0.63 |
| GTGCACA | GQ | 0.180 | -0.75 |
| GTACGCC | RQ | 0.036 | +2.46 |
| GTGCACC | GQ | 0.029 | +0.41 |
| Other | -- | 0.024 | -- |
The Genetics of Asthma in Latino Americans (GALA) study includes both families and case-control samples from the US, Mexico and Puerto Rico. Among Puerto Ricans but not Mexicans, the R16 allele strongly predicted bronchodilator response, especially in subjects with a baseline FEV1 < 80% predicted. Burchard et al [2004] note that in the US, Puerto Ricans and Mexicans have the highest and lowest prevalences of asthma respectively, and Puerto Rican asthmatics are characterised by the poorest bronchodilator response.
In a West Australian cohort of children (N=253), the R16/R16 homozygotes demonstrated persistent differences compared to the G16/G16 group: higher AHR at 1 month old, a lower FEV1 at 11 years old, and a higher admission hospital admission rate for asthma (OR=3.2, 0.9-12.6), though not for the diagnosis of asthma per se.
A nested case-control study within the Normative Aging Study cohort looked at AHR cases (methacholine challenge, N=152) versus nonresponsive nonasthmatic controls (N=391). There was weak association of AHR risk to R16 (P=0.10; test for smoking by genotype interaction, P=0.37), and more so to the G16-Q27 haplotype (P=0.02), especially in the relatively small nonsmoking group.
| Smoking | AHR | G/G | R/G | R/R | R16 Frequency | HWE P-val |
|---|---|---|---|---|---|---|
| Ever | Yes | 42 | 59 | 17 | 39.4% | 0.702 |
| Ever | No | 104 | 114 | 36 | 36.6% | 0.591 |
| Never | Yes | 9 | 17 | 8 | 48.5% | 1.000 |
| Never | No | 58 | 62 | 17 | 35.0% | 1.000 |
This is a double-blind randomized controlled trial of regular salbutamol in R16/R16 and G16/G16 carriers matched on genotype and baseline FEV1. After 16 weeks on active therapy, morning peak expiratory flow rate had increased by 14 l/min in G16/G16 homozygotes, while by only 2 l/min in R16/R16 homozygotes. Most interestingly, placebo had no effect on G16/G16, but lead to a 12 l/min rise in PEFR in R16/R16. In the run-in for the study, when all subjects were salbutamol-free, R16/R16 had risen 23 l/min, and G16/G16 only 2 l/min (pretrial salbutamol use in these mild asthmatics was 1 puff/day).
1159 children from the Korean island of Jeju completed the ISAAC questionnaire and underwent methacholine inhalation challenge. A history of wheezing was reported by 15% of the sample, and 18% exhibited AHR. Genotyping at the codon 16 and 27 polymorphisms was undertaken in 195 AHR+ subjects, none of whom had taken bronchodilators in the previouis two weeks.
There was no association between genotype and history of wheeze. The R16 homozygotes did exhibit a greater response to bronchodilator.
| Genotype | N | Mean BD Response (95%CI) |
|---|---|---|
| RQ/RQ | 52 | 62.8 (53.8-71.8) |
| RQ/GE | 21 | 61.6 (47.3-75.9) |
| RQ/GQ | 76 | 53.3 (46.9-59.7) |
| GQ/GE | 13 | 52.1 (31.0-73.2) |
| GQ/GQ | 32 | 43.2 (33.0-53.4) |
Gamma interferon (IFNG, 12q13) plays an important role in T-cell regulation that makes it another good candidate gene for involvement in allergic disease. The first published examination of this region was that of Watson and coworkers [Watson 1995]. The maximal lod score for IGF1 (tightly linked to D12S318 and PAH and approximately 30 cM distal to IFNG) was 0.09 at 25 percent recombination distance.
Another candidate that has been examined is NOS1 (12q24.2, 116.1 Mbp from 12pter). The mouse knockouts for NOS1 exhibit less nonspecific BHR after OVA sensitization [DeSanctis et al 1999]. Variation in this gene has been tested for association to a huge variety of phenotypes, ranging from lupus nephritis, cluster headache, schizophrenia, COPD and asthma.
More recently, Dillon et al [2004] described the IL31 gene (12q24.31, 121.1 Mbp from 12pter), that codes for a novel four-helix bundle cytokine. Il-31 is largely produced in activated CD4+ T-cells, and especially Th2 cells. The IL-31 receptor is upregulated in IFN-gamma treated monocytes. IL-31 levels rise in the lung after allergen challenge in the ovalbumin BHR mouse model, and more so in BALB/c mice. A transgenic mouse overexpressing IL-31 developed pruritic alopecia and conjunctivitis, with histopathology reminiscent of atopic dermatitis.
The closest microsatellite markers to IL31 are D12S1349 and D12S1578. At approximately 142 cM on the Decode map of chromosome 12, this is 25-40 cM distal to the region showing strongest evidence for linkage to asthma (see below), though some linkage peaks have included this locus.
Variation in the Vitamin D receptor (VDR, 12q12) is suspected to modulate a variety of immune processes, and association with asthma was examined in three datasets in 2004. Wittke et al [2004] have shown that VDR knockout mice do not develop airway inflammation or BHR following allergen sensitisation.
| Gene or Marker | Location (Mbp) | Remarks |
|---|---|---|
| D12S1048 | 39.313 | |
| IRAK4 | 42.45 | IL1R-assoc kinase |
| D12S85 | 45.623 | "IBD2" |
| VDR | 46.523 | |
| D12S398 | 51.483 | "RA21" |
| ITGB7 | 51.86 | Integrin beta 7 |
| STAT6 | 55.775 | IL-4 Stat |
| IFNG | 66.835 | |
| IL22 | 66.928 | |
| D12S379 | 83.461 | |
| KITLG | 87.393 | c-Kit ligand |
| IGF1 | 101.292 | Somatomedin C |
| PAH | 101.735 | |
| NOS1 | 116.111 | |
| IL31 | 121.1 |
Barnes and coworkers [Barnes 1996] have reported evidence of linkage and association between asthma and tIgE over a region stretching from close to IGF1 up to IFNG in two different populations -- the Amish families originally used to detect linkage to 5q, and a sample of 29 Barbadian pedigrees (693 individuals) ascertained through a proband with a history of asthma.
Asthmatic sibpairs exhibited increased ibd sharing at D12S379 (61.8 percent), D12S95 (67.2 percent), PAH (58.5 percent) and D12S360 (58.6 percent). Similarly, Haseman-Elston regression analyses were significant for PAH and D12S360. In the Amish sample, where a diagnosis of asthma was not available on any family members, there were significant Haseman-Elston results for D12S360, IGF1, as well as PLA2. Several replications of this finding have been recently presented.
The Southampton group performed further analysis of their 240 families for linkage to 12q markers under their nonparametric BETA model [Morton 1996]. The best single point lod score was 3.3 for wheezing and D12S366, and the multipoint peak of 2.3 was near D12S97.
The German Multicentre Atopy Study has followed a cohort of 1314 children born in 1990. In this paper [1997], the authors describe results from the TDT for chromosome 12 markers in a subset of children whose total sIgE level exceeds the 85th percentile in the cohort - 214 IU/ml (N=52).
Significant global TDT results were obtained for D12S379 and D12S351 (P=0.001). The most extreme transmission imbalance was for D12S351*161, which was transmitted to the proband 31/38 times.
These authors have screened two groups ascertained to have high rates of asthma: a subset of families from the Busselton study, and individuals from a genetic semi-isolate (a Venezualuan island population). They performed SSCP on all 265 subjects, and heteroduplex analysis on a random 10% subsample. Sequencing of the first exon in a small number of subjects found no variants from that previously reported. The authors concede that intronic or upstream polymorphisms, possibly of (transcriptional) regulatory importance, might remain to be discovered.
| Allele | Cases | Students | PHS |
|---|---|---|---|
| 17 | 0.828 | 0.763 | 0.774 |
| 18 | 0.061 | 0.119 | 0.101 |
| Other | 0.111 | 0.118 | 0.125 |
This abstract describes fine mapping around several peaks in their scan of 117 Italian families (also see below). On chromosome 12q, their best P-value was 6x10-4 was for D12S270 using Genehunter's NPL statistic (equivalent lod=2.1). This is approximately 30 Mb proximal to IFNG.
The 14 repeat allele at the IFNG CA-repeat was found via combined segregation-linkage analysis to be associated with low sIgE level in EGEA families.
This paper describes linkage analysis of 12q markers in 112 asthmatic children from 55 families in the CAMP study. There was weak evidence of linkage to asthma (lod=0.6) and airway responsiveness (lod=1.91).
Shao and coworkers describe a small linkage study (39 asthmatic children in 19 nuclear families) and a case-control association study of 115 asthmatics and 184 controls, using chromosome 12 markers. All subjects came from Osaka and Sendai. The best multipoint lod score was 2.9, for asthma at 150 cM. Of the candidate polymorphisms tested (2 in STAT6; 1 in NOS1, IFNG, AICDA respectively), the best results were a lod of 1.9 for a STAT6 exon 1 GT repeat polymorphism, and 2.1 for the NOS1 intron 2 GT repeat.
Using the collection of Quebecois asthma families described earlier, Poon and coworkers tested for association of 12 Vitamin D Receptor SNPs with asthma and allergy. There were 223 families (N=1139 individuals) containing 570 asthmatics.
There was transmission distortion for the SNPs to both asthmatics, and those with high tIgE levels (FBAT test), with the best P-value being for atopy and rs2239185 at P=0.002. One common haplotype involving this SNP (45% frequency) was overtransmitted, giving a uncorrected P=0.0004.
The CAMP study looked at 7 candidate genes across 12q, IFNG, STAT6, CPM, KITLG, IL22, IRAK3 and VDR. They genotyped at 7 VDR SNPs, including rs2239185. There was undertransmission of rs2239185 to asthmatic children, and the best single SNP result was for the neighbouring (5.7 kbp distant) rs7975232 at P=0.01.
Replication was looked for using an asthma case-control panel (517 cases, 519 matched controls) from the Nurses Health Study. In this case, the minor (C) allele at rs2239185 was increased in cases (P=0.02), in keeping with the Canadian results, and contrary to those from the CAMP transmission tests.