Consanguinity and susceptibility to infectious diseases in humans

Studies of animal populations suggest that low genetic heterozygosity is an important risk factor for infection by a diverse range of pathogens, but relatively little research has looked to see whether similar patterns exist in humans. We have used microsatellite genome screen data for tuberculosis (TB), hepatitis and leprosy to test the hypothesis that inbreeding depression increases risk of infection. Our results indicate that inbred individuals are more common among our infected cases for TB and hepatitis, but only in populations where consanguineous marriages are common. No effect was found either for leprosy, which is thought to be oligogenic, or for hepatitis in Italy where consanguineous marriages are rare. Our results suggest that consanguinity is an important risk factor in susceptibility to infectious diseases in humans.


INTRODUCTION
Increasing numbers of papersr eportalink between genetic diversity andd iseases usceptibility, particularly in natural populations of animals (Coltman et al. 1999;Acevedo-Whitehouse et al. 2003). By implication, infection is farf rom random and relatively homozygousi ndividuals may play ak ey role in the maintenance of pathogens in ap opulation. Twon onexclusive mechanisms may be responsible:i nbreeding depression and chancel inkage between am arkera nd an immune-related gene experiencing balancing selection (Hansson&Westerberg 2002;K aeuffer et al. 2008).Recentstudies have suggestedthatbothmechanismsm ay play ar olea nd maye vena ffectd ifferent aspectso ft he same disease ( Acevedo-Whitehouse et al. 2006).
It remainsu nclear whethert hese studieso fa nimals have anyr elevance to humand isease becausem edical intervention maynegateormaskany effect.Inaddition, with respect to inbreeding depression, most human populations are large enough to ensure that inbred individuals are likely to be extremely rare (Balloux et al. 2004). However, in several cultures, cousin marriages are actively encouraged, potentially exposings uchp opulations to fitness differentialsn oted elsewhere (Jaber et al. 1997).
Complex pedigree analysis is commonly used to determine the degree of inbreeding in human populations. Witht his approach, consanguinity has been implicated in susceptibility to an umbero fh uman diseases including heartd isease, multiple sclerosis, depression and asthma (Roberts 1991;Becker et al. 2001). By contrast, infectiousd iseases have received less attention, in partb ecause of the difficulty in obtaining large numbers of deep, well-resolved pedigrees in thed eveloping worldw here them ajor infectious diseases occurm ostc ommonly. Here, in what we believe is the first study of its kind in humans,w eh aver evisited microsatellite genome scan linkage data for three infectious diseases in contrasting populations, to determine the extent to which genomewide heterozygosity is an important predictor of susceptibility to some diseases, particularlyi n populations where inbreedingi sc ommon.

MATERIAL AND METHODS
We haver eanalysed genome scan data for three important infectious diseases: tuberculosis (TB) in The Gambia (Bellamy et al. 2000);l eprosy in India ( Siddiqui et al. 2001);a nd persistent hepatitis Bi nfection both in The Gambia and Italy (Frodsham 2000;F rodsham et al.2 006); these populations differing in their rates of cousin marriages from less than 1p er cent in Italy up to 43 per cent in India. All four studies wereb ased on an affected sib-pair design, with unaffected parents acting as controls for two or more affected offspring. Sample sizes for these studies are as follows: TB in TheG ambia comprising 272 autosomal markers genotyped across 263 individualsi n7 4f amilies containing 155 affected offspring, 25 affected parents, 19 unaffectedo ffspring and6 4 affected parents; hepatitis in Gambia comprising 276 autosomal markers genotypeda cross 280 individuals in 62 families containing 152 affected offspring,2 2a ffected parents, 42 unaffected offspring and 64 unaffected parents; leprosyi nI ndia comprising 390 autosomal markers genotyped in 394 individualsi n9 6f amilies containing2 02 affected offspring, 51 affected parents and 141 unaffected parents; and hepatitis in Italy comprising 295 autosomal markers genotypeda cross 147 individuals in 32 families containing 92 affected offspring,8affected parents, 19 unaffected offspring and 28 unaffected parents.
Since we lack pedigrees for these populations, we use multilocus heterozygosity as as urrogate measure of the inbreeding coefficient, F .S pecifically,w ef ollowt he method of Balloux et al. (2004),w hich estimatest he degree to which heterozygosity is correlated across unlinked markers. Data from ap anel of markers are repeatedly divided into randomly selected groups of approximately equal size, yielding twoe stimates of heterozygosity fore ach sample. The average correlation between these paired estimates then provides a measure of consanguinity,t he argument being that when inbred individualsa re absent,h eterozygosity is uncorrelated, while increasesi ne ither the proportion of inbred individuals or the mean F -value of inbred individuals both act to create and strengthen the correlation.
The structure of the affected sib-pair datasets raises questions about how best to conduct the analysis.F irst, the sib-pairs are genetically non-independent. Second, givent he severity of these diseases, affected parents in some or many cases mayb ec onsidered to carry different forms of the disease compared with their offspring and thereforem ay not be comparable. We therefore conducted two parallel analyses, one based on all individuals classified only by disease status, regardless of whether they were parents or offspring, and am ore conservative analysis based only on unaffected parents and one randomly selected affected offspring per family.Inp ractice, these yield almost identical results (see below). In addition,i norder to assess the average level of inbreeding in each sample, we also analysed each entire dataset after scrambling affected status.

RESULTS
There is as tronga ssociation between consanguinity and human susceptibilityt ob oth TB and persistent hepatitis Bv irus infection in We st Africans (figure 1). The strongest association occursi nG ambians who have am oderately high (approx. 30%) frequency of first-cousin marriages. No significant association was found for persistent hepatitis in the Italian genome scan, probably due to the low levels of consanguinity andt he resultingl ow powero ft he test in this population. An association was also lacking in the leprosy dataset featuring populations from Andhra Pradesh andT amil Nadu,w here the heterozygosityheterozygosity correlations indicate similar levelso f inbreedingi nb oth casesand controls.

DISCUSSION
In two of threei nstances where ap opulation has high levels of consanguineous marriages,w efi nd that affected individuals reveal significantly more evidence of inbreeding comparedw ith unaffectedc ontrols.T he exception is for leprosy in India, which mayr eflect its unusual genetic architecture.W hile most infectious diseases are probably highly polygenic, susceptibility to leprosy is strongly associated with two major effect loci, suggesting oligogenicity (Siddiqui et al. 2001;Mira et al. 2004). Alternatively, it mayb et hat persistent, strongi nbreedingi nt he Indianp opulations has led to genetic purging. Importantly,t he affected sib-pair design should effectively control for biases duet op opulation stratificationt hatm ight confoundasimple case -control study.
Although difficult to comparef ormally,t he r 2 -values of the heterozygosity-heterozygosity correlations suggest as tronger impacto fc onsanguinity on hepatitis than on TB.O ur simulations suggest that, in our data set, appreciablen umbers of affected offspring,b ut many fewer unaffectedp arents, were born to first-cousin marriages. However, direct interpretation of r 2 -values is not so straightforward. High valuesc an arise if just one or two individuals have extreme F -values (whetherh igh or low) since such data pointsh aveh igh leverage on the correlation. Alternatively,h igh valuesm ight reflect the cumulative impacto fs everal generations of cousin marriages. By contrast,the average r 2 -valuesof1-2%inour Gambian controls appear in line with expectations basedo nt he knownfrequencies of second-cousinmarriages.
Our data comprise microsatellite genome screen data collected several years ago, before the current voguef or using single nucleotide polymorphisms (SNPs).A ss uch, thed ataa re similart ot hose ** * * Figure 1. Correlations in heterozygosity amongm arkers for affecteda nd unaffected individuals, usingd ataf romf our genome screens for infectious disease. Fore ach disease, resultsa re presentedf or both thee ntired ataset (suffix '-A') andaconservatively restricted datasete xcluding allb ut one affected offspring perf amilya nd alla ffectedp arents (suffix '-R'; see textf or more details). Estimated percentage of consanguinityineachpopulationisinbrackets, obtained from http://www.consang.net (Italy andI ndia)a nd Bennett et al. 2002 (The Gambia). Te stso fs ignificance foradifference betweenu naffecteda nd affected individualsa re expressedi n termso ft he proportion of 10 000r eplicate randomizations that foru naffectedi ndividuals yieldedahigher correlation than fora ffectedi ndividuals ( p ! 0.05, p Z 0.0009). hep, persistenthepatitis B; tb,tuberculosis; lep, leprosy. E .J .Lyons et al. Consanguinity andinfectiousdisease collected in the numerous studies of natural populations of animals, and benefit from the high levels of polymorphism shown by individual markers, allowing smaller numberso fm ore widely spaced markerst ob e used. Inferences based on SNP data require much larger numbers of markers, which are therefore more tightly linkeda nd,a lthoughm ethods areb eing developed for inferring individual inbreedingc oefficients, these tend to suffer from ah igh variance of the estimate andr equire 'known' allele frequencies (Leutenegger et al. 2003;C arothers et al. 2006). Nonetheless, work is now underway to put approximately onem illion SNP markersa cross this dataset.
In conclusion, consanguinitya ppearss ignificantly to increase the risk of two major infectious causes of death in humans.R ates of consanguinity aret he highest in populations that are subjectt ot he greatest burden of infectious disease mortality andm any traditional human societies may have had even higher rates. Additionally, increased susceptibility to lethal infectionsi nc onsanguineous individuals may have had am ajor impact on the evolutionarys election of pathogenresistance loci.