Equine chronic lower airway disease, in its severe form known as recurrent airway obstruction (RAO) or heaves, is likely a polygenic disorder caused by both genetic and environmental factors. Affected horses show increased breathing effort, cholinergic bronchospasm, coughing, airway hyperreactivity, and neutrophil and mucus accumulation in the airways. Previous work showed that the relative risk (RR, Mantel-Haenzel statistics) of developing RAO is significantly increased in offspring with one (RR = 3.2; p < 0.05) and, even more, with two affected parents (RR = 4.6; p < 0.05). The tendency to develop the disease is inherited equally from dams or sires and the inheritance mode does not appear to follow a simple Mendelian pattern.
We have undertaken extensive sampling from Warmblood horses consisting of two half-sibling families from two RAO-affected stallions (S1 and S2). A standardized questionnaire was used to gather information on the offspring history of chronic (more than three months), environment-induced (hayfeeding, with reversibility of symptoms when hay removed) coughing, respiratory distress, increased breathing effort after exercise and muco-purulent nasal discharge. This information was compounded into a Horse Owner Assessed Respiratory Signs Index (HOARSI) of 1-4: healthy (no symptoms observed), mildly affected (some nasal discharge and/or occasional cough), moderately affected (frequent coughing), severely affected (moderate to severe coughing and increased respiratory effort). In addition to blood samples for DNA isolation, serum samples, RNA from BALF and epithelial cells, and faeces samples are collected. Based on a comprehensive examination (clinical breathing score; endoscopic scoring of mucus accumulation; TBS and BALF cytology; airway reactivity to metacholine chloride) in a subset of horses (n=71), we could show that the questionnaire data are fully consistent with the RAO-phenotype and there was no difference in clinical phenotype between S1 and S2 offspring. Multivariate regression analysis (S1 n=172; S2 n= 135), showed that the increased risk of S1 and S2 offspring developing RAO was 4.1 and 5.5 fold, respectively (Ramseyer et al., 2007). Segregation analysis (ongoing) showed high heritabilities from 0.63 to 1 and suggested several major genes to be involved.
Association and segregation analysis of markers for epithelial signaling factors (CLCA1, EGFR and BCL2) and other microsatellites located near atopy candidate genes (IL4RA, IL10, IL9, CMA1 and NOS1) indicated that the region near IL4RA (localised to ECA13q13 by FISH) is of interest. We analysed microsatellite haplotypes (AHT133- LEX04- VHL47-ASB 37) spanning the equine IL4R gene in Warmblood horses. A haplotype association with the RAO phenotype was significant in the S1 offspring, but not significant in the offspring of S2. The data therefore suggest locus heterogeneity for equine RAO and indicated that the IL4R genotype could be important for the development of the RAO phenotype in horses. Single nucleotide polymorphisms (SNPs) in IL4RA have also demonstrated strong linkage with RAO in S1 offspring. We then performed RT-PCR on broncho-alveolar lavage fluid cells in a subset of RAO-affected and unaffected horses from both families. S2 offspring (n = 33) showed a Th2-type bias (high IL13 / IFNγ ratio) compared to S1 (n = 28). Expression of IL4RA (normalized to 18s rRNA) was increased in RAO-affected S1 offspring in exacerbation compared to RAO-affected S2 offspring in exacerbation and to all other groups (RAO in remission and healthy). These results suggest that IL4RA is an interesting candidate gene in RAO in some Warmblood families. Furthermore, SNPs in the IL4R gene had previously been reported to be associated with RAO in Quarter Horses and with atopy and asthma in humans. IL4RA is also important in parasite immunity. Intriguingly, we have preliminary results which suggest that S1 off-spring have markedly reduced intestinal parasite egg counts compared to their pasture mates.
We are currently performing a whole genome scan (S1 n=132; S2 n=98), using a panel of 252 microsatellite markers spaced at 10cM on average across the 31 autosomes. The genotyping data is being analysed using a regression interval QTL mapping method for out-bred half-sib families. HOARSI, nasal discharge and coughing are included in the analysis as separate phenotypic traits. Preliminary analysis shows that there are seven chromosomal regions significant at a chromosome-wide level, of which two will probably be significant at a genome-wide level. Additional microsatellite markers will be genotyped across the significant regions to refine the linkage confidence intervals as much as possible. Further on-going efforts include the collection of unrelated Warmblood horses, and horses from other breeds in order to investigate which of the regions segregate in the general population. We are also investigating possible genetic links of susceptibility for RAO with resistance against intestinal parasites in these horses.
There is now good evidence that genetic factors influence the susceptibility to RAO. Identification of the causative genes or genetic markers for RAO could allow: 1) reduction of the prevalence through selective breeding; 2) identification of individuals with a higher risk and subsequent early preventative measures to decrease clinical severity (i.e., dust/allergen poor environment; and, maybe most importantly, 3) a better understanding of the pathogenesis of RAO.
Horse genetics is expected to progress rapidly in the near future as the National Human Genome Research Institute completed the sequencing of the whole horse genome during 2007. In addition, a map of horse genetic variation using DNA samples from a variety of modern and ancestral breeds has been established. This map, comprised of 1 million SNPs, will greatly help to identify genetic contributions to disease susceptibility.
1. Deichmann KA, Heinzmann A, Forster J, Dischinger S, Mehl C, Brueggenolte E, Hildebrandt F, Moseler M, Kuehr J. (1998) Linkage and allelic association of atopy and markers flanking the IL4-receptor gene. Clin. Exp. Allergy 28, 151-55.
2. Gerber V, Straub R, Marti E, Hauptman J, Herholz C, King M, Imhof A, Tahon L, Robinson NE. (2004) Endoscopic scoring of mucus quantity and quality: observer and horse variance and relationship to inflammation, mucus viscoelasticity and volume. Equine Vet. J., 36, 576-82.
3. Isidoro-García M, Dávila I, Laffond E, Moreno E, Lorente F, González-Sarmiento R. (2005) Interleukin-4 (IL4) and Interleukin-4 receptor (IL4RA) polymorphisms in asthma: a case control study. Clin. Mol. Allergy 3:15.
4. Jost U, Klukowska-Rötzler J, Dolf G, Swinburne JE, Ramseyer A, Bugno M, Burger D, Gerber V. (2007) Association of microsatellite markers near interleukin-4 receptor α chain gene (IL4Rα) with chronic lower airway disease--locus heterogeneity in two high-prevalence horse families. Equine Veterinary Journal 39: 236-41.
5. Marti E, Gerber H, Essich G, Oulehla J, Lazary S. (1991) The genetic basis of equine allergic diseases. 1. Chronic hypersensitivity bronchitis. Equine Vet. J. 23, 457-60.
6. Ramseyer A, Gaillard C, Burger D, Straub R, Jost U, Boog C, Marti E, Gerber V. (2007) Effects of genetic and environmental factors on Horse Owner Assessed Respiratory Signs Index (HOARSI). J Vet Intern Med 21, 149-56.
7. Robinson NE, Derksen FJ, Olszewski MA, Buechner-Maxwell VA. (1995) The pathogenesis of chronic obstructive pulmonary disease of horses. Br Vet J. 152, 283-6.
8. Watson JL, et al. (2003) IL4R in the horse: SNPs of the tale. 5th International Equine Gene Mapping Workshop conducted by the Dorothy Russell Havemeyer Foundation at Kruger National Park, Pretoria- South Africa, August 10-14, 2003.