Experimental assessment of bioenergetic differences caused by the common European mitochondrial DNA haplogroups H and T Studies of both survival after sepsis and sperm motility in human populations have shown significant associations with common European mitochondrial DNA haplogroups , and have led to proposals that mitochondria bearing haplogroup H have different bioenergetic capacities than those bearing haplogroup T. However , the validity of such associations assumes that there are no non-random influences of nuclear genes or other factors . Here , we removed the effect of any differences in nuclear genes by constructing transmitochondrial cybrids harbouring mitochondria with either haplogroup H or haplogroup T in cultured A549 human lung carcinoma cells with identical nuclear backgrounds . We compared the bioenergetic capacities and coupling efficiencies of mitochondria isolated from these cells , and of mitochondria retained within the cells , as a critical experimental test of the hypothesis that these haplogroups affect mitochondrial bioenergetics . We found that there were no functionally-important bioenergetic differences between mitochondria bearing these haplogroups , using either isolated mitochondria or mitochondria within cells . 1 Introduction Analysis of the variation in normal human mitochondrial DNA -LRB- mtDNA -RRB- has identified many haplogroups -- specific patterns of polymorphisms that have arisen over the last 150,000 -- 200,000 years -- that have been used to study the origin , radiations and evolution of human populations -LRB- Cann et al. , 1987 ; Wallace et al. , 1999 ; Wallace , 2005 -RRB- . These mtDNA haplogroups have been less studied bioenergetically than mtDNA mutations that lead to disease -LRB- Dimauro and Davidzon , 2005 ; Taylor and Turnbull , 2005 -RRB- , but normal variation in mtDNA may affect disease susceptibility -LRB- Herrnstadt and Howell , 2004 -RRB- and longevity -LRB- Santoro et al. , 2006 -RRB- . These effects could be explained by differences in mitochondrial coupling efficiency -LRB- the percentage of oxygen consumption used for ATP synthesis rather than heat generation -RRB- or mitochondrial production of ROS -LRB- reactive oxygen species -RRB- . It has been proposed that particular haplogroups cause inefficient oxidative phosphorylation and greater heat production and were therefore selected during the radiations of humans into Arctic environments -LRB- Mishmar et al. , 2003 ; Ruiz-Pesini et al. , 2004 ; Wallace , 2005 ; Montiel-Sosa et al. , 2006 -RRB- , but this proposal is controversial -LRB- Elson et al. , 2004 ; Kivisild et al. , 2006 ; Ruiz-Pesini and Wallace , 2006 -RRB- and is not supported by direct measurement of mitochondrial coupling efficiencies in mitochondria carrying representative `` arctic '' and `` tropical '' mtDNAs -LRB- Amo and Brand , 2007 ; Elson et al. , 2007 -RRB- . Two lines of evidence suggest that common European haplogroups may affect bioenergetic function : there are associations of both survival after sepsis and sperm motility with haplogroup H . In a prospective study of intensive care patients in Newcastle-upon-Tyne , U.K. , individuals with haplogroup H -LRB- about 40 % of the population studied -RRB- were more than twice as likely to survive 180 days after sepsis than those with non-H haplogroups -LRB- primarily the closely-related haplogroups J and T ; about 30 % of the population -RRB- -LRB- Baudouin et al. , 2005 -RRB- . Mitochondrial dysfunction may be linked to sepsis-induced multiple organ failure -LRB- Protti and Singer , 2007 -RRB- , so altered mitochondrial bioenergetics might be the causal link between haplogroup and survival . Two possibilities have been proposed : haplogroup H might protect through greater heat generation -LRB- because of higher electron transport rates or looser coupling -RRB- -LRB- Baudouin et al. , 2005 -RRB- , or through greater ROS production -LRB- because of tighter coupling and raised protonmotive force -RRB- , which could reduce bacterial infection -LRB- Wallace , 2005 -RRB- . There have been no direct experimental tests of these possibilities . The second line of evidence comes from studies of sperm motility , which is correlated with mitochondrial enzymatic activities and depends on the activity of the mitochondrial electron transport chain -LRB- Ruiz-Pesini et al. , 1998 ; Ruiz-Pesini et al. , 2000 -RRB- . Haplogroup T was over-represented in men with asthenozoospermia -LRB- reduced sperm motility -RRB- , whereas haplogroup H was over-represented in men with other fertility problems . Sperm with haplogroup H performed better in a test of motility and had higher cytochrome oxidase activity than those with haplogroup T -LRB- Ruiz-Pesini et al. , 2000 -RRB- , suggesting that mitochondria carrying haplogroup H make more ATP than those with haplogroup T . This conclusion has been extended to sublineages of haplogroup U -LRB- Montiel-Sosa et al. , 2006 -RRB- , but associations between haplogroups and reduced male fertility and sperm motility have been disputed by others -LRB- Pereira et al. , 2005 ; Pereira et al. , 2007 -RRB- . Less well-coupled mitochondria make less ROS -LRB- Korshunov et al. , 1997 ; Liu , 1997 ; Lambert and Brand , 2004 -RRB- , so people having such mitochondria might suffer less from neurodegenerative diseases caused by ROS -LRB- Wallace , 2005 -RRB- . Some epidemiological studies support this prediction ; e.g. haplogroup T is under-represented in Alzheimer 's disease patients -LRB- Chagnon et al. , 1999 -RRB- . However , other studies have not replicated this finding -LRB- Elson et al. , 2006 -RRB- , and correlations between mitochondrial haplogroups and neurodegenerative diseases are controversial -LRB- Raule et al. , 2007 -RRB- . Again , there have been no direct experimental tests of possible differences in the bioenergetic properties of mitochondria that might underlie the reported associations of haplogroup and phenotype . In the present study , we analyse bioenergetic capacity and coupling efficiency in mitochondria isolated from cytoplasmic hybrids -LRB- cybrids -RRB- carrying haplogroups H and T with identical nuclear DNA . Furthermore , to investigate the effects of haplogroups H and T on the bioenergetic status of mitochondria at the cellular level , we analyse the respiratory capacities and mitochondrial coupling efficiencies of intact cybrids . 2 Materials and methods 2.1 Subjects Healthy volunteers were recruited by advertisement and written informed consent was obtained . Ethical approval was obtained from the Cambridge Research Ethics Committee . DNA was extracted from buccal swab samples using a QIAamp DNA Mini Kit -LRB- Qiagen , Hilden , Germany -RRB- . Mitochondrial haplogroups were determined by PCR-RFLP analysis according to published criteria -LRB- Wallace et al. , 1999 -RRB- . Entire mtDNAs were amplified in several overlapping fragments by PCR -LRB- Torroni et al. , 1997 -RRB- . Each fragment was digested by restriction enzymes and resolved on agarose gels . 15 ml of blood were taken from each of three volunteers who had haplogroup H , and three who had haplogroup T , for platelet preparation and cybrid construction . Haplogroup H -LRB- Achilli et al. , 2004 -RRB- is subdivided into at least 15 sub-haplogroups -LRB- H1-H15 -RRB- . In the Newcastle sepsis study , there were no significant differences in survival of sub-haplogroups H1 -LRB- − 3008 TaqI -RRB- , H2 -LRB- − 4769 AluI -RRB- , H3 -LRB- + 6773 NlaIII -RRB- and the remaining H sub-haplogroups combined -LRB- Baudouin et al. , 2005 -RRB- . Our haplogroup H volunteers were H1 , H3 and -LRB- H , not H1 , H2 or H3 -RRB- . Haplogroup T -LRB- Macaulay et al. , 1999 -RRB- is subdivided into at least 5 sub-haplogroups -LRB- T1 -- T5 -RRB- -LRB- Pike 2006 -RRB- . Our haplogroup T volunteers were one T -LRB- sub-haplogroup not checked -RRB- , one T1 -LRB- − 12629 AvaII -RRB- and one -LRB- T , not T1 -RRB- . No major differences in cybrid phenotypes between sub-haplogroups were observed . 2.2 Generation of cybrid cell lines Cybrid cell lines are constructed by repopulation of mtDNA-less -LRB- ρ0 -RRB- cells with exogenous mitochondria -LRB- King and Attardi , 1989 -RRB- . A549.B2 ρ0 -LRB- mtDNA-less -RRB- cells derived from human lung carcinoma A549 -LRB- originally carrying mitochondrial DNA of haplogroup H -RRB- were cultivated in Dulbecco 's modified Eagle 's medium -LRB- DMEM -RRB- containing 4.5 g/l glucose , 110 µg/ml sodium pyruvate , 10 % -LRB- v/v -RRB- fetal bovine serum -LRB- FBS -RRB- and 50 µg/ml uridine . Platelets -LRB- which have no nuclei -RRB- were isolated from the volunteers ' blood samples and fused with A549.B2 ρ0 cells as described elsewhere -LRB- Chomyn , 1996 -RRB- . The resultant cybrids had mtDNA from the different donors , but their nuclear DNA was identical . After many generations of cybrid growth -LRB- diluting platelet-derived nuclear-encoded subunits -RRB- , all nuclear-encoded mitochondrial protein subunits will be specified by the host cell , but all mitochondrial-encoded subunits will be specified by the donor mtDNA . The cybrid cell lines were constructed and routinely maintained in DMEM with 10 % dialysed FBS . Stocks of cell lines were frozen and kept at − 80 °C until required . DNA was extracted from cultured cybrid cells as described previously -LRB- Laird et al. , 1991 -RRB- to confirm mitochondrial haplogroups by PCR-RFLP analysis . 2.3 Mitochondrial respiration and membrane potential Human A549 cell mitochondria were prepared from cultured cybrids as previously described -LRB- Amo and Brand , 2007 -RRB- . Mitochondrial oxygen consumption was measured at 37 °C using a Clark electrode -LRB- Rank Brothers , Cambridge , UK -RRB- calibrated with air-saturated medium comprising 0.115 M KCl , 10 mM KH2PO4 , 3 mM Hepes -LRB- pH 7.2 -RRB- , 2 mM MgCl2 , 1 mM EGTA and 0.3 % -LRB- w/v -RRB- defatted BSA , assumed to contain 406 nmol atomic oxygen · ml − 1 -LRB- Reynafarje et al. , 1985 -RRB- . No correction was made for consumption of oxygen by the electrode , so oxygen consumption at low rates will be slightly overestimated . Mitochondrial membrane potential -LRB- Δψ -RRB- was measured simultaneously with respiratory activity using an electrode sensitive to the lipophilic cation TPMP + -LRB- triphenylmethylphosphonium -RRB- -LRB- Brand , 1995 -RRB- . Mitochondria were incubated at 1.0 mg · ml − 1 -LRB- for succinate respiration -RRB- or 1.5 mg · ml − 1 -LRB- for 2-oxoglutarate + malate respiration -RRB- in the presence of 80 ng · ml − 1 nigericin -LRB- to collapse the pH gradient so that the protonmotive force was expressed exclusively as Δψ -RRB- and 4 µM rotenone -LRB- to inhibit complex I -RRB- . The TPMP + - sensitive electrode was calibrated with sequential additions of TPMP + up to 2 µM , then 4 mM succinate or 3.2 mM 2-oxoglutarate + 0.8 mM malate -LRB- with rotenone omitted -RRB- was added to initiate respiration . Experiments were terminated with 1.6 µM FCCP -LRB- carbonyl cyanide p-trifluoromethoxyphenylhydrazone -RRB- , allowing correction for any small baseline drift . Δψ was calculated from the distribution of TPMP + across the mitochondrial inner membrane using a binding correction factor of 0.35 mg protein · µl − 1 . Respiratory rates and respiratory control ratios -LRB- the state 3 respiration rate with 0.8 mM ADP divided by the state 4 rate with oligomycin -RRB- with 2-oxoglutarate + malate as substrate were determined in the absence of nigericin . 2.4 Modular kinetics To investigate differences in oxidative phosphorylation caused by mitochondrial DNA variants , we applied a systems approach : modular kinetic analysis -LRB- Amo and Brand , 2007 -RRB- . This analyses the kinetics of the whole of oxidative phosphorylation divided into three modules connected by their common substrate or product , Δψ . The modules are -LRB- i -RRB- the reactions that produce Δψ , consisting of the substrate translocases , dehydrogenases and other enzymes and the components of the respiratory chain , called ` substrate oxidation ' , -LRB- ii -RRB- the reactions that consume Δψ and synthesize , export and dephosphorylate ATP , consisting of the ATP synthase , the phosphate and adenine nucleotide translocases and any ATPases that may be present , called the ` phosphorylating system ' , and -LRB- iii -RRB- the reactions that consume Δψ without ATP synthesis , called the ` proton leak ' -LRB- Brand , 1990 -RRB- . The analysis reports changes anywhere within oxidative phosphorylation that are functionally-important , but is unresponsive to changes that have no functional consequences . Comparison of the kinetic responses of each of the three modules to Δψ obtained using mitochondria isolated from different cybrids reveals any effects of mitochondrial haplogroup on the kinetics of oxidative phosphorylation . Oxygen consumption and Δψ were measured simultaneously using mitochondria incubated with 80 ng · ml − 1 nigericin and 4 µM rotenone . Respiration was initiated by 4 mM succinate or 3.2 mM 2-oxoglutarate + 0.8 mM malate -LRB- with rotenone omitted -RRB- . The kinetic behaviour of a ` Δψ-producer ' can be established by specific modulation of a Δψ-consumer and the kinetics of a consumer can be established by specific modulation of a Δψ-producer -LRB- Brand , 1998 -RRB- . To measure the kinetic response of proton leak to Δψ , the state 4 -LRB- non-phosphorylating -RRB- respiration of mitochondria in the presence of oligomycin -LRB- 0.8 µg · ml − 1 ; to prevent any residual ATP synthesis -RRB- , which was used solely to drive the proton leak , was titrated with malonate -LRB- up to 0.5 mM -RRB- . In a similar way , state 4 respiration was titrated by FCCP -LRB- up to 0.8 µM -RRB- for measurement of the kinetic response of substrate oxidation to Δψ . State 3 -LRB- maximal rate of ATP synthesis -RRB- was obtained by addition of excess ADP -LRB- 0.8 mM -RRB- . Titration of state 3 respiration with malonate -LRB- up to 1.15 mM -RRB- allowed measurement of the kinetics of the Δψ-consumers -LRB- the sum of the phosphorylating system and proton leak -RRB- . The coupling efficiencies of oxidative phosphorylation were calculated from the kinetic curves as the percentage of mitochondrial respiration rate at a given Δψ that was used for ATP synthesis and was therefore inhibited by oligomycin . Note that any slip reactions will appear as proton leak in this analysis -LRB- Brand et al. , 1994 -RRB- . 2.5 Cell respiration Cell respiration was measured at 37 °C using a Seahorse XF24 Extracellular Flux Analyzer -LRB- Seahorse Bioscience , Billerica , MA , USA -RRB- , which is fully described elsewhere -LRB- Wu et al. , 2007 -RRB- . Cybrids were thawed and subcultured in Petri dishes in DMEM to which had been added 4.5 g/l glucose , 110 µg/ml sodium pyruvate , 0.02 vol of penicillin/streptomycin -LRB- 5000 units/5000 µg/ml -RRB- , 0.02 vol of 200 mM l-glutamine and 0.1 vol Fetal Bovine Serum -LRB- heat inactivated -RRB- . Cybrids were seeded at 20,000 -- 50,000 cells per well in Seahorse XF24 24-well plates and grown for 3 -- 5 d in a 37 °C incubator under 95 % air/5 % CO2 , then washed and incubated for about 1 h in a 37 °C incubator under air in 700 µl of assay medium -LRB- comprising 120 mM NaCl , 3.5 mM KCl , 2 mM MgCl2 , 1.3 mM CaCl2 , 1.2 mM Na2SO4 , 0.4 mM KH2PO4 , 15 mM d-glucose and 0.4 % -LRB- w/v -RRB- BSA , pH adjusted to 7.4 with NaOH -RRB- . Experiments were carried out to a paired design : each plate was seeded half with one H-haplotype cybrid line and half with one T-haplotype cybrid line . Appropriate amounts of concentrated stocks of oligomycin , FCCP , rotenone and myxothiazol dissolved in 70 % ethanol were diluted into 75 µl of assay medium and loaded into the appropriate addition ports in a measuring cartridge . The plate and measuring cartridge -LRB- containing waveguides and fluorescent oxygen sensors together with reagents for automated pneumatic additions of inhibitors and uncouplers -RRB- was loaded into the machine and after the calibration procedure -LRB- 30 min calibration , 10 min rest -RRB- , measurement cycles of 1 min sample mixing , 2 min waiting and 3 min measurement of oxygen consumption rate were initiated . All respiration rates were calculated as a percentage of the rate in the same well for the fourth measurement point -- the basal rate just preceding addition of oligomycin -LRB- see Fig. 4 -RRB- . This corrected for differences in cell density between wells . In the range measured , absolute rates of oxygen consumption were linearly related to cell numbers seeded . Absolute basal oxygen consumption rates for the parental A549 cell line are reported to be about 1.7 nmol/min/106 cells -LRB- Wu et al. , 2007 -RRB- . Respiration rates at each time-point from three or four replicate wells were averaged . 2.6 Statistics Values are given as mean ± SEM for n cell clones -LRB- n = 15 cell clones for Figs. 1 -- 3 , five different clones for each donor and three donors for each haplogroup , and n = 6 cell clones for Fig. 4 , two different clones for each donor and three donors for each haplogroup -RRB- . To test for significant differences in modular kinetics between haplogroups , we interpolated the mean respiration rates of mitochondria from the individual clones at a range of different potentials between the highest and lowest common membrane potential . The significance of differences between means was assessed by unpaired Student 's t-test using Microsoft Excel X ; P values < 0.05 were taken to be significant . 3 Results 3.1 Modular kinetic analysis of oxidative phosphorylation Haplogroup H is a subgroup of haplogroup HV , and haplogroup T is a subgroup of haplogroup JT ; HV and JT diverged about 40,000 years ago -LRB- Kivisild et al. , 2006 -RRB- . Haplogroup H is found in about 41 % of Europeans and haplogroup T is found in about 15 % -LRB- Wallace et al. , 1999 -RRB- . Haplogroup H differs from haplogroup T by four non-synonymous mutations in proteins -LRB- one each in the complex I subunits , ND1 and ND2 , and two in the complex III subunit , cytochrome b -RRB- , two mutations in tRNAs -LRB- tRNAarg and tRNAthr -RRB- and three mutations in rRNA -LRB- two in 16S rRNA and one in 12S rRNA -RRB- -LRB- Kivisild et al. , 2006 -RRB- , so differences in oxidative phosphorylation between these haplogroups are plausible . To compare mitochondrial functions between haplogroup H and T , we measured the kinetics and coupling efficiency of oxidative phosphorylation using mitochondria isolated from cybrid cells . Fig. 1A , B and C shows the kinetics of the three modules of oxidative phosphorylation using succinate as respiratory substrate . Fig. 1A shows the kinetic response of substrate oxidation to its product , Δψ ; Fig. 1B shows the kinetic response of proton leak to its driving force , Δψ , and Fig. 1C shows the kinetic response of the ATP phosphorylating pathway to its driving force , Δψ . The three sets of kinetics curves using succinate were indistinguishable between mtDNA haplogroup H -LRB- closed symbols -RRB- and T -LRB- open symbols -RRB- . We also measured the kinetics of substrate oxidation using 2-oxoglutarate + malate as substrate instead of succinate to check for any effects of differences in the mitochondrial complex I genes . Once again , the kinetic response of the substrate oxidation system to Δψ using 2-oxoglutarate + malate as substrate was not significantly different between haplogroups H and T -LRB- Fig. 1D -RRB- . From the kinetic curves shown in Fig. 1 , we can analyse the coupling efficiency of oxidative phosphorylation . The classic measure is the respiratory control ratio : respiration at maximal rate of ATP synthesis -LRB- state 3 -RRB- divided by respiration with no ATP synthesis -LRB- state 4 -RRB- -LRB- Fig. 2A and B -RRB- . A more precise measure of coupling efficiency is given by the percentage of respiration that is coupled to ATP synthesis . This is calculated by subtracting the rate of proton leak -LRB- Fig. 1B -RRB- from the total rate of respiration -LRB- Fig. 1A -RRB- at any chosen membrane potential , and expressing the difference as a percentage of the total rate . If this percentage is lower , then a greater proportion of the redox energy is diverted to heat production . Mitochondria in cells normally operate at less than maximal rates and values of Δψ . Fig. 2C reports the coupling efficiency across the whole range of rates of ATP synthesis and values of Δψ from state 3 -LRB- maximal rate of ATP synthesis -RRB- to close to state 4 -LRB- where ATP synthesis is minimal -RRB- . As expected -LRB- Brand et al. , 1993 -RRB- , coupling efficiency decreased as mitochondria moved from state 3 towards state 4 . There were no significance differences in rates , respiratory control ratio or coupling efficiencies between haplogroups H and T . We also independently measured the respiratory control ratio using 2-oxoglutarate + malate as substrate and calculated coupling efficiency from the data in Fig. 1 -LRB- Fig. 3 -RRB- . Once again , there was no clear difference between haplogroup H and T , either in respiratory rates -LRB- Fig. 3A -RRB- , respiratory control ratio -LRB- Fig. 3B -RRB- or coupling efficiencies across a range of rates and potentials -LRB- Fig. 3C -RRB- . In summary , at the mitochondrial level we observed no significant functional differences between mitochondria isolated from cybrids of haplogroup H and haplogroup T. 3.2 Coupling efficiency of respiration in intact cybrid cells To check whether differences in mitochondrial function between haplogroups were lost when mitochondria were isolated from the cells and measured at a standardised protein concentration in an artificial incubation medium , we measured the respiration rates and coupling efficiencies of mitochondria in intact cybrids . Cybrids were grown on 24-well plates and oxygen consumption was measured using a Seahorse XF24 Extracellular Flux Analyzer . Fig. 4A shows that oxygen consumption was inhibited when ATP synthesis was prevented by addition of oligomycin , stimulated when oxidation was uncoupled from phosphorylation by addition of the uncoupler FCCP , and inhibited again when mitochondrial electron transport was abolished by addition of rotenone -LRB- to inhibit complex I of the electron transport chain -RRB- and myxothiazol -LRB- to inhibit complex III -RRB- . The residual rate in the presence of rotenone and myxothiazol represents non-mitochondrial oxygen consumption . This comprised 16.7 + 2 % of basal respiration rate in H-haplotype cybrids and 18.2 + 2.7 % in T-haplotype cybrids . These values were not significantly different . Subtraction of this non-mitochondrial respiration allowed assessment of the rates and coupling efficiency of the remaining mitochondrial respiration . Oligomycin inhibits mitochondrial ATP synthesis , so mitochondrial respiration that is insensitive to oligomycin is due to uncoupled respiration , i.e. to proton leak pathways through the mitochondrial inner membrane , and respiration that is sensitive to oligomycin is due to ATP synthesis occurring in the cells . Because oligomycin prevents the energy-utilizing pathway of ATP synthesis , the mitochondrial proton motive force will rise after oligomycin addition , and the proton leak rate will increase , leading to an overestimate of the proton leak rate and an underestimate of the ATP synthesis rate that occurred before oligomycin was added -LRB- Brand , 1990 -RRB- . With this proviso , Fig. 4B shows the estimates of the percentages of basal respiration used for ATP synthesis and proton leak within the cells . There was no difference between the haplogroups . The percentage of respiration used for ATP synthesis is the coupling efficiency , and , with a value of 85 % of mitochondrial respiration , it was comparable to the coupling efficiency at intermediate respiration rates found in mitochondria isolated from the cells -LRB- Figs. 2 and 3 -RRB- , similar to the value of 80 % measured previously using the parental cell line -LRB- Wu et al. , 2007 -RRB- , and similar to values found in a range of other cell types -LRB- Brand , 2005 -RRB- . Respiration rates in the presence of FCCP are not directly limited by ATP synthesis , and represent the maximal capacity of substrate oxidation by the cells under the conditions of measurement . In some cells this rate is equivalent to the maximum rate of the electron transport chain , but in others there is strong rate-limitation imposed by the supply of substrates to the mitochondria , particularly when ATP levels drop as a secondary result of uncoupling and substrates like glucose and fatty acids can not be activated to glucose-6-phosphate and fatty acyl CoA . Fig. 4A shows that the FCCP-uncoupled respiration rates of A549 cybrids were initially high and rather variable , but then diminished with time , consistent with increasing limitation by substrate availability . There was no significant difference in uncoupled rates between haplogroups -LRB- Fig. 4B -RRB- . In summary , at the intact cell level we observed no significant functional differences between mitochondria within cybrids of haplogroup H and haplogroup T. 4 Discussion There are published associations between mitochondrial haplogroups H and T and phenotypes that might be expected to have a strong bioenergetic component . However , our results show no significant bioenergetic differences in mitochondria with H or T-haplogroup mtDNAs in a constant nuclear background , at either the mitochondrial or the cellular level . We have previously shown that our methodology can pick up 10 % differences in respiratory chain activity -LRB- Amo and Brand , 2007 -RRB- , so if haplogroup does affect mitochondrial bioenergetics , any effects must be very small . The lack of effect of haplogroup on mitochondrial coupling efficiency in intact cybrids shows that even if haplogroup affects mitochondrial proliferation or retrograde signalling , such changes do not affect coupling efficiency in the cells under basal conditions . The strengths of our approach are -LRB- i -RRB- we used a direct empirical measurement , rather than inference from genetic and physiological traits , -LRB- ii -RRB- we separated the effects of mitochondrial haplogroup from any confounding effects of nuclear DNA background and -LRB- iii -RRB- we used a simple in vitro system with the powerful modular kinetic approach where the relevant variables could be tightly controlled and manipulated . However , our approach also has some weaknesses . -LRB- i -RRB- A small effect of mitochondrial haplogroup operating over hours or days might still be too small to be detected by biochemical experiments . -LRB- ii -RRB- We measured the bioenergetic capacity using isolated mitochondria and cybrid cells in simple defined media , but effects of mitochondrial haplogroups might only emerge under special conditions -LRB- for example , they might require triggers such as sepsis or energetic stress or might require particular nuclear genes to be expressed , and so be tissue-specific or suppressed in the lung carcinoma cell line we employed -RRB- . Our results fail to provide support for the hypothesis that the common European mitochondrial haplogroups H and T have significant effects on bioenergetics that cause the associations with survival after sepsis and sperm motility that have been reported . If such associations stand up to further scrutiny , they presumably operate through more subtle mechanisms than respiratory rates , mitochondrial coupling efficiencies or ATP supply .

O O O O O O O O O O O O O O O O O O O O O B O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O B I O B I O O O O B O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

oxidative phosphorylation
coupling efficiency
sepsis
sperm motility
cybrid

fccp, carbonyl cyanide p-trifluoromethoxyphenylhydrazone


Chitotriosidase

deficiency

is

not

associated

with

human

hookworm

infection

in

a

Papua

New

Guinean

population

Human

chitotriosidase

-LRB-

-RRB-

is

a

chitinolytic

enzyme

with

suggested

anti-fungal

properties

Previous

studies

have

suggested

that

chitotriosidase

may

also

protect

individuals

against

filarial

nematode

infections

and

malaria

A

mutant

allele

which

renders

chitotriosidase

unstable

and

enzymatically

inactive

is

found

at

a

frequency

of

in

Caucasians

and

other

populations

This

allele

is

found

at

much

lower

frequency

in

parts

of

West

Africa

where

malarial

and

intestinal

helminth

infections

are

endemic

Here

we

investigate

whether

there

is

a

significant

association

between

chitotriosidase

genotype

and

the

intensity

of

hookworm

infection

in

individuals

from

five

villages

in

Papua

New

Guinea

Individuals

were

genotyped

for

chitotriosidase

using

a

PCR-based

assay

There

was

no

association

between

genotype

and

the

intensity

of

hookworm

infection

as

determined

by

faecal

egg

counts

The

frequency

of

the

mutant

allele

was

very

similar

to

that

found

in

non-endemic

countries

The

extent

of

geographical

variation

in

allele

frequencies

across

worldwide

populations

was

not

high

-LRB-

Fst

-RRB-

and

does

not

provide

evidence

for

directional

selection

at

this

locus

between

different

geographical

areas

We

conclude

that

the

genotype

does

not

play

a

crucial

role

in

protection

against

hookworm

infection

This

does

not

correlate

with

a

previous

study

that

linked

the

mutant

genotype

to

filariasis

susceptibility

The

possible

reasons

for

this

discrepancy

are

discussed

Introduction

Chitotriosidase

is

a

chitinolytic

enzyme

which

was

recently

identified

in

man

and

is

synthesised

in

large

quantities

by

activated

macrophages

-LRB-

Hollak

et

al.

-RRB-

As

humans

lack

endogenous

chitin

a

role

for

chitotriosidase

is

not

fully

understood

However

the

anti-fungal

action

of

homologous

plant

chitinases

has

led

to

the

suggestion

that

chitotriosidase

may

degrade

chitin

containing

pathogens

-LRB-

Boot

et

al.

-RRB-

The

chitotriosidase

gene

-LRB-

-RRB-

consists

of

exons

located

on

chromosome

-LRB-

Boot

et

al.

-RRB-

A

base

pair

duplication

in

exon

leads

to

the

activation

of

a

cryptic

splice

site

that

results

in

an

abnormally

spliced

mRNA

with

an

inframe

nucleotide

deletion

-LRB-

Boot

et

al.

-RRB-

The

mutant

protein

lacks

amino

acids

that

are

required

for

the

formation

of

the

TIM-barrel

catalytic

core

-LRB-

Boot

et

al.

Fusetti

et

al.

-RRB-

Macrophages

from

chitotriosidase

deficient

individuals

express

only

small

amounts

of

mRNA

and

secrete

virtually

no

chitotriosidase

protein

-LRB-

Boot

et

al.

-RRB-

In

Dutch

and

Ashkenazi

Jewish

populations

of

individuals

are

homozygous

for

the

mutant

allele

whereas

and

respectively

are

heterozygous

carriers

-LRB-

Boot

et

al.

-RRB-

with

a

mutant

allele

frequency

of

A

similar

allelic

frequency

is

present

in

Portugal

-LRB-

-RRB-

Sicily

-LRB-

-RRB-

and

Sardinia

-LRB-

-RRB-

-LRB-

Malaguarnera

et

al.

-RRB-

However

in

two

West

African

countries

Benin

and

Burkina

Faso

that

are

mesoendemic

for

Plasmodium

falciparum

malaria

and

endemic

for

gastrointestinal

helminths

a

total

absence

of

the

homozygous

mutation

and

significant

reduction

in

heterozygous

individuals

has

been

reported

with

mutant

allele

frequency

and

respectively

-LRB-

Malaguarnera

et

al.

-RRB-

This

observation

led

to

the

hypothesis

that

chitotriosidase

may

be

involved

in

resistance

to

protozoan

or

helminth

infections

common

in

tropical

countries

-LRB-

Malaguarnera

et

al.

-RRB-

Parasitic

nematodes

are

known

to

contain

chitin

and

are

thus

a

potential

target

of

human

chitotriosidase

Chitin

is

found

in

the

egg-shell

of

both

free-living

and

parasitic

nematodes

and

in

the

microfilarial

sheath

surrounding

the

first-stage

larvae

of

filarial

nematodes

-LRB-

Fuhrman

and

Piessens

-RRB-

More

recently

chitin

has

been

demonstrated

in

the

pharynx

of

both

the

free-living

Caenorhabditis

elegans

-LRB-

Zhang

et

al.

-RRB-

and

the

gastrointestinal

parasite

Oesophagostomum

dentatum

-LRB-

Neuhaus

et

al.

-RRB-

There

are

two

chitin

synthase

genes

in

C.

elegans

and

encoding

eggshell

and

pharynx

chitin

respectively

and

knockdown

leads

to

lack

of

pharyngeal

function

and

starvation

-LRB-

Zhang

et

al.

-RRB-

This

suggests

that

host

chitotriosidase

could

potentially

interfere

with

parasitic

nematode

feeding

Two

studies

to

date

have

investigated

associations

between

genotype

and

filarial

nematode

infection

Choi

et

al.

-LRB-

-RRB-

found

significant

association

between

susceptibility

to

lymphatic

filariasis

-LRB-

Wuchereria

bancrofti

-RRB-

and

homozygosity

for

the

mutant

allele

in

an

Indian

population

but

in

a

similar

study

in

Papua

New

Guinea

no

association

between

genotype

and

lymphatic

filariasis

was

observed

-LRB-

Hise

et

al.

-RRB-

There

have

been

no

previous

studies

investigating

genotype

and

gastrointestinal

nematode

infection

Human

hookworm

infection

is

an

abundant

chronic

gastrointestinal

nematode

infection

in

sub-tropical

and

tropical

countries

causing

significant

morbidity

principally

due

to

iron-deficiency

anaemia

-LRB-

Brooker

et

al.

-RRB-

Human

hookworm

burden

-LRB-

as

assessed

by

faecal

egg

count

-RRB-

is

known

to

be

under

host

genetic

control

-LRB-

Williams-Blangero

et

al.

-RRB-

but

the

genes

responsible

have

not

been

identified

The

aims

of

the

current

study

were

-LRB-

-RRB-

to

investigate

associations

between

genotype

and

human

hookworm

infection

in

an

endemic

population

from

Madang

Province

Papua

New

Guinea

and

-LRB-

-RRB-

to

investigate

evidence

for

directional

selection

at

the

locus

by

comparing

worldwide

allele

frequency

variation

at

the

locus

to

that

observed

at

other

loci

across

the

genome

Materials

and

methods

Geographical

location

and

study

population

The

study

population

consisted

of

five

villages

in

lowland

Madang

Province

Papua

New

Guinea

where

Necator

americanus

is

the

only

hookworm

species

-LRB-

Pritchard

et

al.

-RRB-

Villages

were

censused

and

pedigree

information

collected

in

and

sample

containers

for

faecal

collection

offered

to

all

individuals

aged

years

and

above

Faecal

egg

counts

were

performed

using

a

modified

McMaster

salt

flotation

method

and

results

were

expressed

as

eggs

per

gram

-LRB-

epg

-RRB-

of

faeces

-LRB-

Quinnell

et

al.

-RRB-

Venous

blood

was

then

collected

and

all

individuals

were

offered

treatment

with

albendazole

or

pyrantel

pamoate

DNA

was

extracted

from

buffy

coats

using

phenol/chloroform

In

September

a

second

faecal

sample

was

taken

from

three

of

the

five

villages

and

reinfection

hookworm

burden

determined

some

previously

untreated

individuals

provided

samples

in

Hookworm

epg

has

been

shown

to

be

a

heritable

phenotype

in

the

study

population

-LRB-

Breitling

and

Quinnell

unpublished

results

-RRB-

The

study

was

approved

by

the

Medical

Research

Advisory

Committee

of

Papua

New

Guinea

and

informed

consent

was

received

from

all

subjects

or

their

parents

Chitotriosidase

genotyping

by

PCR

Thermal

cycling

reactions

consisted

of

Reddy

Mix

PCR

master

mix

-LRB-

ABgene

-RRB-

pmol

forward

and

pmol

reverse

chitotriosidase

primers

-LRB-

forward

AGCTATCTGAAGCAGAAG

reverse

GGAGAAGCCGGAAAGTC

-RRB-

-LRB-

Sigma

-RRB-

and

μl

genomic

DNA

in

a

final

volume

of

μl

Samples

were

run

in

well

PCR

plates

-LRB-

ABgene

-RRB-

on

a

Peltier

Thermal

Cycler

-LRB-

MJ

Research

-RRB-

The

thermal

cycling

protocol

was

°C

for

min

cycles

of

°C

for

s

°C

for

s

and

°C

for

s

and

°C

for

min

Thermal

cycling

products

with

base

pair

markers

-LRB-

Promega

-RRB-

were

separated

on

high

resolution

agarose

-LRB-

Sigma

-RRB-

gels

in

TBE

buffer

and

ethidium

bromide

Gels

were

viewed

using

the

Gene

Genius

Bio-imaging

System

and

Gene

Snap

software

-LRB-

Syngene

-RRB-

The

size

of

wild

type

product

is

base

pairs

whereas

the

mutant

product

is

base

pairs

due

to

the

mutant

allele

containing

a

base

pair

duplication

in

exon

-LRB-

Boot

et

al.

-RRB-

Statistical

analysis

Variation

in

faecal

egg

counts

by

genotype

was

analysed

under

both

a

codominant

and

recessive

model

Faecal

egg

counts

were

highly

overdispersed

so

analysis

was

performed

using

a

generalised

linear

model

with

a

negative

binomial

error

structure

in

Stata

This

method

has

been

shown

to

be

more

accurate

than

analysis

of

log-transformed

parasite

burden

data

-LRB-

Wilson

et

al.

-RRB-

Significant

covariates

included

in

the

analysis

were

age

village

faecal

consistency

anthelmintic

treatment

and

the

age

treatment

and

village

treatment

interaction

terms

Egg

counts

from

both

years

were

analysed

together

since

some

individuals

were

sampled

both

pre

and

post-treatment

standard

errors

were

adjusted

for

the

non-independence

of

samples

from

the

same

individual

using

the

cluster

option

To

control

for

non-independence

of

individuals

due

to

genetic

relatedness

further

analysis

was

performed

with

the

total

test

of

association

in

a

variance

components

framework

using

the

programme

QTDT

-LRB-

Abecasis

et

al.

-RRB-

Finally

to

control

for

potential

population

stratification

transmission

disequilibrium

testing

was

carried

out

using

the

orthogonal

association

model

in

QTDT

The

phenotypic

variable

used

for

these

analyses

was

the

residuals

from

a

negative

binomial

regression

of

egg

counts

against

significant

covariates

where

two

samples

were

taken

from

the

same

individual

the

mean

residual

for

that

individual

was

calculated

To

quantify

geographic

variation

in

allele

frequencies

Fst

values

were

calculated

according

to

Cockerham

and

Weir

-LRB-

-RRB-

using

the

programme

FSTAT

-LRB-

Goudet

-RRB-

Allele

frequencies

were

compared

across

five

broad

geographical

areas

using

the

weighted

mean

allele

frequency

for

each

geographical

area

-LRB-

Europe/Mediterranean

Africa

South

Asia

East

Asia

and

Papua

New

Guinea

-RRB-

To

compare

observed

values

of

Fst

to

those

expected

for

neutral

markers

Fst

was

also

calculated

across

comparable

populations

selected

from

the

Human

Genome

Diversity

Panel

using

published

data

for

genome-wide

short

insertion/deletion

polymorphisms

genotyped

by

the

Mammalian

Genotyping

Service

-LRB-

data

available

from

-RRB-

-LRB-

Soranzo

et

al.

-RRB-

The

populations

selected

were

European

-LRB-

French/Italian

-RRB-

African

-LRB-

Yoruba

-RRB-

South

Asia

-LRB-

Sindhi

-RRB-

East

Asia

-LRB-

Chinese

Han

-RRB-

and

Papuan

Results

In

total

individuals

from

Papua

New

Guinea

were

genotyped

for

the

chitotriosidase

mutation

The

allelic

frequency

of

the

bp

duplication

in

unrelated

individuals

determined

by

PCR

genotyping

was

The

percentage

of

individuals

who

were

homozygous

mutant

was

-LRB-

-RRB-

heterozygous

-LRB-

-RRB-

and

homozygous

wild

type

-LRB-

-RRB-

Genotype

frequencies

did

not

differ

from

those

expected

under

Hardy

Weinberg

equilibrium

-LRB-

χ2

d.f.

P

-RRB-

Both

egg

counts

and

genotypes

were

available

for

a

total

of

individuals

-LRB-

sampled

pre-treatment

and

sampled

post-treatment

-RRB-

The

prevalence

and

intensity

of

infection

-LRB-

mean

egg

count

-RRB-

in

these

individuals

were

-LRB-

epg

-RRB-

pre-treatment

and

-LRB-

epg

-RRB-

post-treatment

Egg

counts

were

very

similar

across

genotypes

both

pre-treatment

and

after

reinfection

-LRB-

Table

-RRB-

Combined

analysis

of

both

years

data

revealed

no

significant

relationship

between

egg

count

and

genotype

under

either

a

codominant

-LRB-

χ2

d.f.

p

-RRB-

or

recessive

model

-LRB-

χ2

d.f.

p

-RRB-

Further

analysis

using

the

total

test

of

association

in

QTDT

controlling

for

non-independence

of

individuals

due

to

genetic

relatedness

again

showed

no

significant

association

between

genotype

and

egg

count

-LRB-

χ2

d.f.

p

-RRB-

Similarly

transmission

disequilibrium

testing

which

controls

for

population

stratification

was

not

significant

-LRB-

χ2

d.f.

p

-RRB-

Published

allele

frequencies

for

in

different

geographical

areas

are

shown

in

Table

Using

these

data

and

data

from

the

current

study

Fst

values

were

calculated

to

measure

variation

in

allele

frequency

across

five

broad

geographical

areas

-LRB-

Europe

Africa

South

Asia

East

Asia

and

Papua

New

Guinea

-RRB-

The

Fst

across

all

populations

for

was

This

was

comparable

to

the

median

Fst

of

for

indels

typed

in

populations

from

similar

geographical

areas

Pairwise

comparisons

showed

higher

Fst

values

between

Africa

and

East

Asia

-LRB-

Fst

-RRB-

and

Africa

and

South

Asia

-LRB-

Fst

-RRB-

than

those

between

other

areas

-LRB-

Fst

-RRB-

In

each

case

the

observed

Fst

was

greater

than

that

for

all

but

of

indels

giving

an

empirical

P

-LRB-

-RRB-

Discussion

The

results

from

the

present

study

in

Papua

New

Guinea

strongly

suggest

that

chitotriosidase

does

not

have

a

critical

role

in

protection

against

N.

americanus

The

relative

number

of

people

who

are

wild

type

homozygous

for

chitotriosidase

heterozygous

and

homozygous

mutant

is

similar

to

that

seen

in

countries

where

this

infection

is

not

endemic

suggesting

no

advantage

for

the

wild

type

allele

in

endemic

areas

Moreover

there

was

no

evidence

for

an

association

between

genotype

and

hookworm

burden

in

our

study

population

The

mutant

chitotriosidase

genotype

has

been

associated

with

an

increased

susceptibility

to

W.

bancrofti

infection

in

a

study

of

individuals

from

South

India

-LRB-

Choi

et

al.

-RRB-

Previously

it

has

been

demonstrated

that

the

microfilariae

oocytes

and

zygotes

of

the

filarial

nematode

Brugia

malayi

contain

chitin

-LRB-

Fuhrman

and

Piessens

Schraermeyer

et

al.

-RRB-

In

the

rodent

filarial

nematode

Acanthocheilonema

vitae

there

are

distinctive

chito-oligomeric

N-glycans

that

contain

projections

of

up

to

six

GlcNAc

residues

that

may

be

involved

in

parasite-host

interactions

-LRB-

Haslam

et

al.

-RRB-

Additionally

chitin

synthase

is

required

for

sheath

development

in

microfilarial

progeny

Chitin

has

also

been

identified

in

the

cuticle

of

mature

W.

bancrofti

-LRB-

Araujo

et

al.

-RRB-

The

presence

of

chitin

in

the

sheath

and

cuticle

of

filarial

nematodes

and

the

requirement

for

chitin

synthase

in

reproduction

show

that

chitin

has

an

important

role

in

the

lifecycle

of

these

species

If

host-derived

chitotriosidase

can

access

this

chitin

thus

interfering

with

the

parasite

's

growth

and

development

then

it

may

explain

why

susceptibility

to

W.

bancrofti

is

increased

in

individuals

with

the

mutant

genotype

However

a

second

study

by

Hise

and

coauthors

-LRB-

Hise

et

al.

-RRB-

in

individuals

in

a

region

endemic

for

bancroftian

filariasis

in

Papua

New

Guinea

did

not

find

any

significant

correlation

with

infection

status

or

disease

phenotype

Thus

the

issue

is

in

further

need

of

clarification

In

contrast

to

filarial

worms

gastrointestinal

nematodes

such

as

hookworm

do

not

have

a

microfilarial

stage

and

the

chitin-containing

eggs

are

not

accessible

to

host

chitotriosidase

However

chitin

has

recently

been

demonstrated

in

the

pharynx

of

the

free-living

nematode

C.

elegans

-LRB-

Zhang

et

al.

-RRB-

It

is

not

known

whether

chitin

occurs

in

the

pharynx

of

N.

americanus

but

it

has

been

demonstrated

in

the

related

strongyloid

parasite

O.

dentatum

-LRB-

Neuhaus

et

al.

-RRB-

Pharyngeal

chitin

provides

a

potential

target

for

host

chitotriosidase

both

in

tissue-dwelling

larval

stages

which

may

be

accessible

for

macrophage

or

neutrophil-derived

chitotriosidase

and

blood-feeding

adults

Chitotriosidase

levels

can

be

elevated

in

serum

e.g.

in

acute

falciparum

malaria

-LRB-

Barone

et

al.

-RRB-

and

as

adult

hookworms

are

thought

to

ingest

ml

of

blood

daily

they

may

be

exposed

to

high

levels

of

chitotriosidase

However

the

lack

of

an

association

between

genotype

and

hookworm

burden

seen

in

the

present

study

suggests

that

chitotriosidase

is

not

an

important

effector

mechanism

against

hookworm

infection

Further

studies

would

be

useful

to

examine

the

bioavailability

of

nematode

chitin

its

accessibility

for

exogenous

chitinases

or

their

effect

on

parasite

viability

The

mutant

allele

frequency

observed

in

the

current

study

was

higher

than

that

in

another

Papuan

population

from

East

Sepik

Province

-LRB-

Hise

et

al.

-RRB-

Such

variation

within

Papua

New

Guinea

is

not

surprising

and

differences

between

these

populations

have

been

observed

at

other

loci

-LRB-

Main

et

al.

-RRB-

Analysis

of

worldwide

variation

in

allele

frequencies

showed

that

the

degree

of

population

differentiation

at

the

locus

-LRB-

Fst

-RRB-

was

very

similar

to

the

median

value

for

indels

across

the

genome

Though

the

populations

sampled

were

not

identical

this

suggests

that

there

has

not

been

directional

selection

at

this

locus

across

different

geographical

areas

and

that

the

worldwide

variation

in

allele

frequencies

is

consistent

with

genetic

drift

-LRB-

Akey

et

al.

-RRB-

There

was

some

evidence

for

higher

than

average

population

differentiation

at

the

locus

between

African

and

Asian

populations

which

may

suggest

some

local

adaptation

though

this

would

not

be

significant

after

correction

for

multiple

testing

Malaguarnera

et

al.

-LRB-

-RRB-

suggest

that

an

intact

genotype

may

be

advantageous

in

protection

against

malaria

based

on

the

very

low

frequency

of

the

mutant

genotype

in

Burkina

Faso

and

Benin

where

malaria

is

meso-endemic

However

our

results

are

not

consistent

with

the

hypothesis

that

chitotriosidase

is

involved

in

protection

against

malaria

or

any

tropical

disease

There

is

no

evidence

for

directional

selection

worldwide

and

the

greatest

difference

in

allele

frequencies

is

observed

between

Africa

and

other

tropical

countries

also

endemic

for

malaria

-LRB-

Choi

et

al.

Chien

et

al.

-RRB-

Although

Barone

et

al.

-LRB-

-RRB-

found

that

chitotriosidase

levels

are

elevated

in

the

serum

in

acute

P.

falciparum

malaria

there

is

no

evidence

that

elevated

serum

chitotriosidase

levels

have

an

impact

on

the

severity

and

outcome

of

malaria

and

no

study

has

compared

genotypes

with

disease

status

Indeed

increased

serum

chitotriosidase

may

actually

increase

malaria

transmission

by

inhibiting

the

formation

of

the

peritrophic

membrane

in

the

anopheline

gut

-LRB-

Di

Luca

et

al.

-RRB-

As

the

study

area

in

Papua

New

Guinea

is

endemic

for

malaria

we

have

performed

an

association

analysis

which

showed

no

significant

difference

between

chitotriosidase

genotype

and

the

presence

of

P.

falciparum

and

P.

vivax

malaria

infection

-LRB-

data

not

shown

-RRB-

The

caveat

of

this

analysis

is

that

it

was

based

on

a

single

time

point

assessment

of

parasitaemia

rather

than

repeated

measurements

As

parasitaemia

is

very

variable

we

can

not

make

a

reliable

statement

regarding

the

effect

of

the

genotype

on

malarial

infection

and

further

studies

are

needed

Taken

together

our

data

suggest

that

the

chitotriosidase

genotype

does

not

play

a

major

role

in

protection

against

hookworm

infection

but

this

may

be

different

in

the

case

of

lymphatic

or

cutaneous

filariasis

However

there

is

no

evidence

for

selection

at

this

locus

mediated

by

malaria

or

another

tropical

disease

B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B I I O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O B O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O

chitotriosidase
necator americanus
malaria
papua new guinea