Szilvia Kovács
email: kovacs.szilvia@aotk.szie.hu

Tibor Csörgő

Andrea Harnos

Péter Fehérvári

Krisztina Nagy

Change in migration phenology and biometrics of two conspecific species in Hungary

Keywords: garden warbler, blackcap, climate change, migration phenology, biometrics Zusammenfassung vielen Vogelarten haben sich jüngerer Zeit Verbreitung und Zugphänologie verändert, vermutlich als Folge des Klimawandels. In dieser Studie untersuchten wir die Wirkung von vorne, während er sich bei der Mönchsgrasmücke im Verlauf der Studie nicht änderte. Wir nehmen an, unterstützt durch andere Beobachtungen von Mönchsgrasmücken in Westeuropa, dass bei ihnen diese zeitliche Verschiebung schon vor den 1980er Jahren erfolgte Herbstzug um 13 Tage nach hinten, Altvögel hingegen nicht. Ihre mittlere Körpermasse verringerte sich, während sich die mittlere Flügellänge erhöhte, was auf eine Änderung in der Zusammensetzung der durchziehenden Population hindeutet. Wir nehmen an, dass sich die Brutgebiete dieser Arten auf Grund des Klimawandels nach Norden verschoben haben, wodurch mehr nördliche, langflügelige Individuen auftreten

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INTRODUCTION

One of the most intriguing questions is to what extent living organisms are able to adapt to rapid environmental changes [START_REF] Berteaux | Keeping pace with fast climate change: can arctic life count on evolution?[END_REF]). Global and regional warming play an important role in distribution shifts in many taxa including birds [START_REF] Root | Fingerprints of global warming on wild animals and plants[END_REF][START_REF] Gordo | Why are bird migration dates shifting? A review of weather and climate effects on avian migratory phenology[END_REF]. In birds specifically, climate change may alter the geographic distribution of species [START_REF] Huntley | Climatic Atlas of European Breeding Birds[END_REF], influence the timing and phenology of migration [START_REF] Lehikoinen | Arrival and departure dates[END_REF][START_REF] Tøttrup | Changes in timing of autumn migration in North European songbird populations[END_REF][START_REF] Zalakevicius | Spring arrival response to climate change in birds: a case study from Eastern Europe[END_REF], moult (e.g. [START_REF] Pulido | Correlation between timing of juvenile moult and onset of migration in the blackcap, Sylvia atricapilla[END_REF] and breeding (e.g. [START_REF] Crick | Climate change related to egg-laying trends[END_REF][START_REF] Charmantier | Adaptive phenotypic plasticity in response to climate change in a wild bird population[END_REF]). The rate and direction of changes may differ among species and between populations of the same species [START_REF] Bearhop | Assortative mating as a mechanism for rapid evolution of a migratory divide[END_REF][START_REF] Catry | Are blackcaps Sylvia atricapilla differential distance migrants by sex?[END_REF]. Even age and sex groups may react differently [START_REF] Catry | Sex differences in settlement behaviour and condition of Chiffchaffs Phylloscopus collybita at a wintering site in Portugal. Are females doing better?[END_REF].

In case of European birds, climate change may cause northward shifts in the breeding areas [START_REF] Huntley | Climatic Atlas of European Breeding Birds[END_REF]). These changes depend on the migration strategy of the species concerned. The migration of long distance migrants is predominantly under genetic control [START_REF] Gwinner | Endogenously controlled changes in migratory direction of garden warbler, Sylvia borin[END_REF] allowing a slower adaptation to rapid changes compared to residents, short-range or facultative migrants [START_REF] Both | Adjustment to climate change is constrained by arival date in a long-distance migrant bird[END_REF]. Spring migration shifted earlier, in particular in species with more generalized diet, shorter migration distance, more broods per year, and less extensive prenuptial moult [START_REF] Végvári | Life history predicts advancement of avian spring migration in response to climate change[END_REF]).

The prediction is obvious for spring migration due to the advantages of early breeding [START_REF] Crick | Climate change related to egg-laying trends[END_REF][START_REF] Dunn | Breeding dates and reproductive performance[END_REF]). The timing of spring migration shifted earlier at most of the examined species. In case of good weather conditions earlier arrival is beneficial, since earlier birds can occupy better territories and can raise their reproductive success [START_REF] Forstmeier | Benefits of early arrival at breeding grounds vary between males[END_REF].

In autumn, the causes of the shifts are more complicated as birds generally optimize for survival and could use different strategies to adapt to the changing environment. Departures may occur earlier under a warming climate if nesting is completed earlier, or even later if the extended season encourages additional nesting attempts [START_REF] Jenni | Timing of autumn bird migration under climate change: advances in long-distance migrants, delays in short-distance migrants[END_REF]. Advanced spring migration and delayed autumn migration of several passerine species have been detected at our study site [START_REF] Miholcsa | Change of timing of autumn migrarion in Acrocephalus and Locustella genus[END_REF].

Blackcaps have a large Palearctic distribution area. The southern populations are resident, the northern and eastern populations of European blackcaps are long-range migrants while the Central European populations are partial or medium-distance migrants (Cramp and Brooks 1992). Blackcaps breeding in Hungary are medium-distance migrants, their main wintering range covers the Eastern Mediterranean Basin, but birds may overwinter in any part of the Mediterranean region. Cross migrant blackcaps at our study site originate from northern populations, thus are long-distance migrants (Csörgő and Gyurácz 2009 a). In Western Europe the spring arrival of blackcaps shifted earlier according to the studies of Lungberg and Endholm (1982) and [START_REF] Tøttrup | Changes in timing of autumn migration in North European songbird populations[END_REF], similar shift have been observed in Central Eurasia by Askayev et al. (2009), while the timing of autumn migration is delayed [START_REF] Nowakowski | Terms of autumn migration of the genus Sylvia in Central Poland[END_REF].

Garden warbler has the most northern distribution area in the Palearctic region amongst the Sylvidae warblers (Cramp and Brooks 1992). According to ring recovery data, birds from our study area migrate through the Apennine Peninsula towards Africa (Csörgő and Gyurácz 2009 b). Their wintering grounds cover the Sahel region south from the Sahara (Cramp and Brooks 1992;[START_REF] Grattarola | Spring migration of the garden warbler (Sylvia borin) across the Mediterranean Sea[END_REF]. In Northern and Western Europe and Central Eurasia the timing of spring migration of the garden warblers shifted earlier [START_REF] Hüpopp | North Atlantic Oscillation and timing of spring migration in birds[END_REF][START_REF] Jonzén | Rapid advance of spring arrival dates in longdistance migratory birds[END_REF]Sparks et al. 2007, Askayev et al. 2009), the timing of autumn migration did not change in Denmark [START_REF] Tøttrup | Changes in timing of autumn migration in North European songbird populations[END_REF] and in Switzerland [START_REF] Jenni | Timing of autumn bird migration under climate change: advances in long-distance migrants, delays in short-distance migrants[END_REF].

In our study we analyzed the changes in timing of migration of two Sylvia warbler species at an Eastern European stopover site. We hypothesize that birds shift their migration earlier in spring and later in autumn. We suppose it can be a possible effect of recent climate change. We propose that the changes depend on age and moulting strategy in autumn.

Furthermore, we analyzed the change in differences in migration peaks of the two sex groups of blackcaps as we hypothesize that the two sex groups may use different migration strategy.

Additionally, we used wing length and body mass in order to examine how these biometric parameters have changed during the last 25 years. In our view the biometric changes could be explained by the changes in the composition of populations passing through the area, thus indirectly showing the possible effects of climate change.

MATERIALS AND METHODS

Location and data

Data were collected in Central Hungary (47°15'N, 19°15'E) at the Ócsa Bird Ringing Station. The station is situated within the Ócsa Landscape Protection Area, a coherent part of the Danube-Ipoly National Park. The site is at the edge of a postglacial peatbog. The surrounding is highly variable comprising all habitat types from reedbeds to closed forests.

The study area has a typical continental climate with cold winters and warm summers. Our bird ringing database contains data of 25 years . Mist-netting and ringing were conducted at the same locations, using the methodology of [START_REF] Szentendrey | Methods applied at the Actio Hungarica Ringing Scheme[END_REF] throughout the whole study period. We used the records of 13 049 adult and 39 391 juvenile blackcaps (Sylvia atricapilla) trapped and ringed between February-November and 1 968 adult and 4 975 juvenile garden warblers (Sylvia borin) ringed between April-October within the study period. Only data from first captures were used. We distinguished two age groups by features published by [START_REF] Svensson | Identification guide to European Passerines[END_REF]. 1st calendar year birds are referred to as juveniles, older birds as adults. In blackcaps we could determine the sex of birds by cap colorization [START_REF] Svensson | Identification guide to European Passerines[END_REF]. All birds were measured by a few experts who follow the same methodology [START_REF] Szentendrey | Methods applied at the Actio Hungarica Ringing Scheme[END_REF]) to eliminate inter-observer bias. The right wing length (to the nearest millimetre using the ruler) and the body mass (to nearest 0.1 g using a digital balance) were measured for each individuals. Visual fat scores were estimated following the scale of [START_REF] Kaiser | A New Multi-Category Classification of Subcutaneous Fat Deposits of Songbirds[END_REF], ranging from zero (no visible fat) to eight (whole belly covered with fat). We used the data of birds whose fat scores fall into the linear range of the Kaiser scale in biometric analysis.

Statistical analysis

Bird migration phenology has been traditionally described by measures of central tendency (mean, median and mode) and spread (first and last observations, or 5% and 95% percentiles). We defined arrival times as the day of the year when the birds were caught at the first time and characterized the annual timing of migration by the location of the peak of the kernel smoothed arrival distribution curves (Fig. 1). Kernel smoothing was used to reduce the effects of missing observations [START_REF] Silverman | Density estimation for statistics and data analysis[END_REF][START_REF] Knudsen | Characterizing bird migration phenology using data from standardized monitoring at bird observatories[END_REF]).

Peaks of smoothed curves were identified separately for each species by season and age group and in blackcaps also by sex. To detect a shift in the timing of annual peaks during the study period, we applied linear regression analysis. The estimated shift in timing was calculated from the regression equations as the number of years times the estimated per year change (regression coefficient). Shift was regarded as significant if the corresponding linear regression was significant.

Quantile regression [START_REF] Cade | A gentle introduction to quantile regression for ecologists[END_REF] was used to estimate trends in 10%, 25%, 50%, 75% and 90% percentiles of the distribution of arrival times from 1984 to 2008. As different quantiles may represent different population segments which may have different responses to the changing environment, quantile regression can provide an insight to different trends of migration timing of early and late arrivals. Shift in timing and significance was determined same as for linear regression.

We analysed the relationship between migration peaks of the two species and of the two age groups of blackcap by Spearman's rank correlation [START_REF] Hollander | Nonparametric Statistical Methods[END_REF].

Furthermore we tested the changes in differences between migration peaks of male and female blackcaps by linear regression.

Concerning changes in body condition, we first applied a linear model to estimate body mass from fat score and wing length. We used wing length to control for body size. Extra deposited fat was defined as the difference between actual (observed) body mass and body mass belonging to 0 fat score estimated from the linear model. Changes in average extra deposited fat from 1984 to 2008 were estimated by linear regression weighted by sample size.

The changes in average wing length were determined by linear regression. Additionally in case of garden warblers we used quantile regression to explore changes in the annual distribution of this parameter. To see the differences between an early (1984)(1985)(1986)(1987)(1988)(1989)(1990) and a late (2002)(2003)(2004)(2005)(2006)(2007)(2008) period smoothed density plots were applied.

All statistical analyses were made by the R 2.9.2 program (R Development Core Team 2007).

RESULTS

Changes in timing of migration

No significant shift was found in spring migration peak of blackcaps (p = 0.5 and p = 0.08 for males and females, respectively). Peak of spring migration of garden warblers shifted 9 days earlier (p = 0.016). Quantile regression detected a shift of 8 days at the beginning of the migration period (10% and 25% quantiles) while the median passage date (50% quantile) changed by 6.5 days. For late arrivals (75 and 90% quantiles) the changes were not significant (Table 1 and Fig. 2a). In the timing of autumn migration of blackcaps the largest delay was detected in adult males (17 days, p = 0.02), whereas the smallest occurred in juvenile males (11.5 days, p = 0.02). Females of both age groups shifted their peak time 14 days later on average (adults: p = 0.035; juveniles: p = 0.025). We found similar results by quantile regression for all groups (Table 2). In autumn the time of peak migration of garden warblers did not change significantly in adults (p = 0.1), whereas it shifted 14 days later (p = 0.05) in juveniles. Juvenile garden warblers migrated later than adults in the whole study period. Therefore the difference of median passage dates between the two age groups increased from 2 to 14 days from 1984 to 2008. In juveniles about the same delay was found for all quantiles in autumn (Table 1).

There was no significant correlation in the peak of migration of the two species neither in spring, nor in autumn. Migration peaks of male and female blackcaps correlated significantly in autumn (adults: r = 0.55 p = 0.028, juveniles: r = 0.81 p < 0.0001). In autumn the differences between migration peak of adult male and female blackcaps increased significantly between 1984-2008: estimated differences between males and females were -5 days for 1984 and 7.6 days for 2008 (p = 0.006).

Changes in biometrics

The extra deposited fat decreased significantly from 1984 to 2008 in each age and sex group of both species in autumn (Table 3), however it did not change in spring. The average wing length of adult blackcaps and garden warblers showed a significant increase in both seasons (0.04 mm/year, p = 0.05 for blackcap males in both season and 0.03 mm/year, p = 0.05 for blackcap females in both season, 0.05 mm/year, p = 0.01 for garden warblers in spring and 0.06 mm/year, p = 0.005 for garden warblers in autumn). Although median wing length of adult garden warblers did not change significantly, wing length distribution shifted toward larger wing lengths (Table 4, Fig. 3,4). We have not observed remarkable size differences between sexes.

DISCUSSION

The distribution area and the timing of migration of many bird species has changed recently, presumably due to climatic factors [START_REF] Gordo | Why are bird migration dates shifting? A review of weather and climate effects on avian migratory phenology[END_REF]. In our study we pointed out some possible consequences of climate change on the migrating populations of blackcaps and garden warblers caught at the Ócsa Bird Ringing Station in Hungary.

The timing of spring migration of many long and medium distance migrants shifted earlier (e.g. [START_REF] Gordo | Why are bird migration dates shifting? A review of weather and climate effects on avian migratory phenology[END_REF]Sanz 2005, Sparks andTryjanowski 2007). The reason of this changing trend could be the optimization to reproduction. In spring birds benefit from the early arrival and could increase their reproductive success in case of favourable spring weather conditions [START_REF] Forstmeier | Benefits of early arrival at breeding grounds vary between males[END_REF][START_REF] Dunn | Breeding dates and reproductive performance[END_REF]). In spite of the possibility of prosperous consequences of advanced spring migration neither sex groups of blackcaps altered their spring arrival timing significantly during our study period. The reason may be that this species migrates very early in spring and earlier arrival could be non-adaptive as local weather is highly unfavourable preceding the observed arrival dates. In Hungary the first blackcaps arrive in late February, however a handful of recent observations of overwintering birds suggest that a minor fraction of the population may have become sedentary (Csörgő and Gyurácz. 2009 a). Nevertheless in early spring (late February, early March) the weather in Hungary can be very cold sub-zero temperatures and snow is often characteristic of this period. If these unfavourable circumstances occur permanently, the survival rate of birds is very low. We presume -similarly to the observations in Western Europe -that the spring migration of blackcaps has already shifted earlier before our study period in the previous decades. According to the observations in Northern and Western Europe e. g. in Sweden the timing of spring migration of blackcaps shifted on average 5 days earlier between 1965[START_REF] Szentendrey | Methods applied at the Actio Hungarica Ringing Scheme[END_REF]compared to the 1941-1950period (Lundberg and Endholm 1982). A Danish study pointed out that the median of arrival dates of blackcaps showed greater shift than those of its related species between 1976 and 1997 (Trottrup et al 2006). In Central Eurasia a study reported on 6 days earlier shift in first arrival date of blackcaps between 1957-2008 [START_REF] Askeyev | Spring migration timing of Sylvia warblers in Tatarstan (Russia) 1957-2008[END_REF]).

The first garden warblers arrive to Hungary in the second half of April, the peak of migration is in the middle of May (Csörgő and Gyurácz 2009 b). We found that the spring migration of garden warblers shifted 6.5 days earlier in Ócsa. Similarly to our results, [START_REF] Jonzén | Rapid advance of spring arrival dates in longdistance migratory birds[END_REF] showed a 7 days average shift of spring migration of garden warblers in Sweden (Falsterbo, Ottenby) between 1980-2004. In Germany (Helgoland) the mostly Scandinavian originated cross migrant garden warblers shifted their spring migration on average 7.4 days earlier between 1960 and 2000 [START_REF] Hüpopp | North Atlantic Oscillation and timing of spring migration in birds[END_REF]. In England (Sussex) the first arrival date of garden warblers occurred on average 16.8 days earlier during 42 years long study period (Sparks et al. 2007), however, it only shifted 4 days earlier during 51 years in Central Eurasia (Kazan) [START_REF] Askeyev | Spring migration timing of Sylvia warblers in Tatarstan (Russia) 1957-2008[END_REF]) and did not change significantly in Lithuania (Vilnius) between 1971[START_REF] Dunn | Breeding dates and reproductive performance[END_REF][START_REF] Zalakevicius | Spring arrival response to climate change in birds: a case study from Eastern Europe[END_REF].

Contrary to the observations in Central Eurasia [START_REF] Askeyev | Is earlier spring migration of Tatarstan warblers expected under climate warming?[END_REF][START_REF] Askeyev | Spring migration timing of Sylvia warblers in Tatarstan (Russia) 1957-2008[END_REF], there was no significant correlation in the peak of migration of the two sister species neither in spring, nor in autumn at our study site. However, the lack of correlation in our data is not surprising as blackcaps in Central Eurasian are long-distance migrants, while they are medium distance migrants in Eastern Europe. Blackcaps in Central Eurasia arrive on average at the same time with garden warblers [START_REF] Askeyev | Spring migration timing of Sylvia warblers in Tatarstan (Russia) 1957-2008[END_REF], while there is a marked difference in arrival dates in Europe.

After the dispersion period of early August the autumn migration of blackcaps starts in late August and the peak of migration is in the first half of September at our study site (Csörgő and Gyurácz. 2009 a). The autumn passage of blackcaps takes two months, thus the detected approximately two weeks shift is remarkable in all sex and age groups.

Both species alter their foraging strategy before autumn migration, they switch from insects to fruits e.g. elderberry at our study site. The change of main food source may cause inexperienced juveniles to spend more time with food deposition than adults. Adult blackcaps contrary to juveniles and garden warblershave postnuptial moult, therefore they stay longer, like the inexperienced juveniles. Presumably this is the reason of the approximately two weeks shift in both age groups. Another observation on the timing of autumn migration of blackcaps was a 3 days delay in North Poland in the period between 1985-1998(Nowakovski 1999)). Blackcaps favour late migration as a consequence of increasing temperature and improved food availability on the breeding grounds [START_REF] Coppack | Photoperiodic response to early hatching in a migratory bird species[END_REF].

In small bird species, energy-demanding life cycle stages, such as breeding, moult and migration are separated in time. Thus, there is a strong correlation between the timing of postjuvenile moult and migration of blackcaps [START_REF] Pulido | Correlation between timing of juvenile moult and onset of migration in the blackcap, Sylvia atricapilla[END_REF]. The longer stopover time may be beneficial as it provides sufficient time to complete life cycle components such as moult and allows to prepare for migration [START_REF] Pulido | Correlation between timing of juvenile moult and onset of migration in the blackcap, Sylvia atricapilla[END_REF].

We found significant correlation between autumn migration peaks of the two sexes. We hypothesize that it may be due to similar responses of the sexes to annual changes of climatic parameters. Interestingly, the sign of the differences between the timing of autumn passage of adult males and females changed significantly. It means that in the early years of study period the males had earlier migration peaks compared to females and recently females leave earlier the study site and males staying longer. These results may imply that the two sex groups of blackcaps could use slightly different migration and refuelling strategies. Possibly, females place more importance on energy-maximization,in order to ensure successful reproduction [START_REF] Tryjanowski | Should avian egg increase as a result of global warming? A case study using the red-backed shrike (Lanius collurio)[END_REF]). On the other hand males are the territory defenders and that they generally arrive ahead of females to the breeding territories (Izhazi 1998[START_REF] Gustin | Relationship between biometry and migration time among blackcaps (Sylvia atricapilla) in Central Italy[END_REF]. [START_REF] Catry | Sex differences in settlement behaviour and condition of Chiffchaffs Phylloscopus collybita at a wintering site in Portugal. Are females doing better?[END_REF] have presented convincing evidence on differential distance migration of sexes in chiffchaffs, with females moving further than males. In blackcaps was found a significantly larger proportion of females in samples of British birds that wintered in Africa [START_REF] Catry | Are blackcaps Sylvia atricapilla differential distance migrants by sex?[END_REF]. If this sexual difference in sex-ratios on wintering grounds occurs, males can leave the breeding ground later, than females. In case weather is highly favourable on stopover site males can stay even longer and the differences between sexes increase.

In contrast to blackcaps, winter moulting adult garden warblers must fit this highly energy consuming life-cycle component into a relatively short winter, hindering delayed departure from the breeding grounds [START_REF] Hedenström | Migration speed and scheduling of annual events by migrating birds in relation to climate change[END_REF]. They gain weight faster and hence migrate earlier than juveniles. In this species, the timing of autumn migration of adults did not change during the past 25 years, while juveniles shifted their timing 13 days later. The autumn migration commences in early August at our study site. The peak of autumn passage is in early September and the last individuals leave the ringing site at the beginning of October (Csörgő and Gyurácz 2009 b). Considering that the duration of autumn passage of garden warblers at our study site is two months, our results of a 13 day shift in the migration peak of juvenile garden warblers are non-negligible.

In contrast to our results the passage times of the last 5% of garden warblers in autumn shifted earlier in Denmark (Christiansø) [START_REF] Tøttrup | Changes in timing of autumn migration in North European songbird populations[END_REF]. The last passage date also shifted earlier in England (Oxfordshire) between 1971-2000 [START_REF] Cotton | Avian migratory phenology and global climate change[END_REF]. However these studies cannot be compared with our results, as they have not handled the age groups separately during their analysis, while we have found large differences in the timing of adults and juveniles.

Both species deposit large amounts of fat before autumn migration. The average body mass of blackcaps is 18.7 grams in Sweden and 21 grams on Cyprus [START_REF] Fransson | Fuelling in front of the Sahara desert in autumnan overview of Swedish field studies of migratory birds in the eastern Mediterranean[END_REF].

Around 16% of body mass of garden warblers from South Sweden consist of deposited fat (Ellergen and Fransson 1992). In Greece the average body mass of juveniles is approximately 20 grams [START_REF] Fransson | When and where to fuel before crossing the Sahara desert-extended stopover and migratory fuelling in first-year garden warblers Sylvia borin[END_REF], while south from the Sahara it is around 17 grams [START_REF] Ottosson | Garden warbler Sylvia borin migration in sub-Saharan West Africa: phenology and body mass changes[END_REF].

As the published data shows that there could be large differences in mean body mass and in average deposited fat -depending on the distances between two stopover sites and the distance between their current stopover site and the wintering grounds-among migrating populations. On the other hand the pattern of refueling is strictly defined in a population. Therefore, the change in average deposited fat of species at a stopover site could indicate the changes in population composition of cross migrating birds. Remarkable decrease was found in extra deposited fat in all age and sex groups of blackcaps and in adult and juvenile garden warblers at the Ócsa stopover site (Table 3). This decrease could mean a drop with more than one fat score on average. As we have only analysed the first capture data of every individual, and as arriving migrants are predominantly caught shortly after arrival, our data represents the condition of newly arrived birds. Additionally the amount and availability of food for these species could not have changed in this rate at our study area between 1984-2008, thus, it signifies a change in the population composition of cross migrating birds.

A correlation was observed between length of migration route and wing length and wing morphology of migrating birds [START_REF] Lockwood | Avian wing shape reconsidered: wingtip shape indices and morphological adaptation to migration[END_REF]. The wing of northern breeders having longer migration route until the wintering ground is longer and more pointed than that of southern breeders with shorter migration route. This theory is supported by the data of blackcaps on the Iberian Peninsula, where the wing length of cross migrants is larger on average than that of residents [START_REF] Perez-Tris | Age-related variation in wing shape of migratory and sedentary blackcaps Sylvia atricapilla[END_REF]. According to the data of Western Mediterranean ringing stations there are differences in wing length and body mass among garden warbler populations [START_REF] Grattarola | Spring migration of the garden warbler (Sylvia borin) across the Mediterranean Sea[END_REF]. Our study indicates that the wing length of blackcaps increased on average and the distribution of wing length shifted toward longer winged birds regarding garden warblers (Table 4,Fig 3,Fig 4). We detected a large change in wing length (average shift of 0.8-1.3 mm), which cannot be explained by local adaptations.

There are many studies, which have been reported morphological changes hypothesised as related to climatic variables [START_REF] Kaňuščák | Does climate at different scales influence the phenology and phenotype of the River Warbler Locustella fluviatilis?[END_REF], Tryjanowski et al. 2004). In accordance with our results long-term decrease in body weight and increase in wing length was found for ringed blackcaps in Britain between 1968and 2003[START_REF] Yom-Tov | Recent changes in body weight and wing length among some British passerine birds[END_REF].

We detected that the average body mass decreased and the average wing length increased in the migrating population during the study period. In our view this is explained by the change in the composition of individuals originating from different populations, by the increasing rate of northern originated birds among the trapped birds. Therefore a northward shift in the distribution or a larger reproductive success of these species in northern breeding territories, as the effect of climate change, is probable [START_REF] Huntley | Climatic Atlas of European Breeding Birds[END_REF]. Our theory is supported by the positive correlation between warming weather conditions and the reproductive success in the populations of other northern breeder species [START_REF] Møller | North Atlantic Oscillation (NAO) effects of climate on the relative importance of first and second clutches in a migratory passerine bird[END_REF]. The population sizes in northern breeding grounds of blackcaps and garden warblers have increased in the previous decades [START_REF] Jiguet | Bird population trends are linearly affected by climate change along species thermal ranges[END_REF]. According to our results there is a remarkable shift in the timing of migration in autumn and spring in case of two species.

Garden warblers advanced their spring migration and the autumn migration of garden warbler juveniles and blackcaps was delayed.

In addition, our results may indirectly show the effects of climate change on extending breeding range northwards as on the predicted distribution map for blackcaps in the Climatic Atlas of European Breeding Birds, or effects on increasing reproductive success in northern breeding territories like in case of the prediction for garden warblers [START_REF] Huntley | Climatic Atlas of European Breeding Birds[END_REF].

Tables

FiguresFig. 1 Fig. 2 Fig. 3 Fig. 4

 1234 Figures

Table 1 :

 1 Shift in timing of migration of garden warblers (Sylvia borin) estimated by quantile regression. Estimated number of shifted days (d) from 1984 to 2008 and p-values (p) are represented. Negative numbers represent earlier arrivals.

		spring	autumn adult autumn juvenile
	quantiles	d	p	d	p	d	p
	10%	-8.0 *	0.005	8 <0.001	16 <0.001
	25%	-8.0	0.001	3	0.311	9 <0.001
	50%	-6.5	0.096	1	0.142	13 <0.001
	75%	-7.5	0.052	0	0.922	16 <0.001
	90%	1.5	1.000	2	0.901	7 <0.001

*e.g. -8.0 (upper left cell in the table) means that the shift of the 10% quantile of the distribution was 8 days earlier on average in 2008 than in 1984.

Table 2 :

 2 Shift in timing of migration of blackcaps (Sylvia atricapilla) estimated by quantile regression. Number of days shifted (d) from 1984 to 2008 and p-values (p) are represented.

		autumn adult		autumn juvenile
		male		female		male		female
	quantiles	d	p	d	p	d	p	d	p
	10%	17.0 <0.001 12.5 <0.001 10.5 <0.001	8.0 <0.001
	25%	13.5 <0.001 12.5 <0.001	9.5 <0.001	7.5 <0.001
	50%	12.5 <0.001 14.0 <0.001 11.5 <0.001 12.5 <0.001
	75%	12.5 <0.001 12.5 <0.001 11.0 <0.001 15.5 <0.001
	90%	9.0 <0.001 14.5 <0.001 10.0 <0.001 16.0 <0.001

Table 3 :

 3 Change in annual average extra deposited fat in autumn in each group of garden warblers (Sylvia borin) and blackcaps (Sylvia atricapilla) from 1984 to 2008 estimated by linear regression. The changes in grams, slopes (grams/year) and p-values are represented. e.g. -0.061 in the table means that the decrease of extra deposited fat was 0.061 per year on average from 1984 to 2008.

		garden warbler		blackcap
		autumn	autumn	autumn adult	autumn juvenile
		adult	juvenile	male	female	male	female
	change (g)	-1.5	-1.1	-0.7	-0.8	-1.0	-0.9
	slope	-0.061*	-0.046	-0.029	-0.032	-0.042	-0.036
	p-value	<0.001	0.026	0.006	<0.001	<0.001	<0.001

*

Table 4 :

 4 Changes (d) of wing length (mm) of adult garden warblers (Sylvia borin) from 1984 to 2008 in spring and in autumn estimated by quantile regression. The changes (mm), slopes (mm/year) and p-values are represented.

			spring adult			autumn adult
	quantiles	slope	d p-value	slope	d p-value
	10%	0.08	2.0	0.012	0.07	1.8	<0.001
	25%	0.05	1.3	0.024	0.06	1.5	<0.001
	50%	0.00	0.0	1.000	0.00	1.0	1.000
	75%	0.00	0.0	1.000	0.06	1.5	<0.001
	90%	0.06	1.5	0.037	0.06	1.5	<0.001

Acknowledgement

The authors express their gratitude for the hard work of the volunteers at the Ócsa Bird Ringing Station. We are grateful to Dr. Jenő Reiczigel for his valuable advices in the course of our studies.


Christian Delles

Martin W Mcbride

Delyth Graham

Sandosh Pad- Manabhan

Anna F Dominiczak

Sandosh Padmanabhan

Prof F Anna

Dominiczak

Genetics of hypertension: From experimental animals to humans

Keywords: Hypertension, genetics, rodents, human

Essential hypertension affects 20 to 30 % of the population worldwide and contributes significantly to cardiovascular mortality and morbidity. Heridability of blood pressure is around 15 to 40 % but there are also substantial environmental factors affecting blood pressure variability. It is assumed that blood pressure is under the control of a large number of genes each of which has only relatively mild effects. It has therefore been difficult to discover the genes that contribute to blood pressure variation using traditional approaches including candidate gene studies and linkage studies. Animal models of hypertension, particularly in the rat, have led to the discovery of quantitative trait loci harbouring one or several hypertension related genes, but translation of these findings into human essential hypertension remains challenging. Recent development of genotyping technology made large scale genome wide association studies possible. This approach and the study of monogenic forms of hypertension has led to the discovery of novel and robust candidate genes for human essential hypertension, many of which require functional analysis in experimental models.

Please cite this article as: Christian Delles, Martin W. McBride, Delyth Graham, Sandosh Padmanabhan, Anna F. Dominiczak, Genetics of hypertension: From experimental animals to humans, BBA -Molecular Basis of Disease (2009), doi: 10.1016/j.bbadis.2009.12.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Introduction

Hypertension is characterised by chronic elevation of blood pressure and affects 20 to 30 % of the population worldwide [START_REF] Kearney | Global burden of hypertension: analysis of worldwide data[END_REF]. Hypertension contributes significantly to the global burden of cardiovascular morbidity and mortality. A recent study by Lawes et al. [START_REF] Lawes | Global burden of blood-pressure-related disease[END_REF] concluded that 13.5% of premature deaths and 54% of stroke and 47% of ischaemic heart disease worldwide are attributable to high blood pressure. Alarmingly, about 80% of the attributable burden occurred in low-income and middle-income economies, and over half occurred in people aged 45 to 69 years. Blood pressure is a continuous, quantitative trait whereas the dichotomous definition of hypertension (i.e. blood pressure above a certain threshold) is to some degree arbitrary and has been modified with expanding knowledge over the years. This has been acknowledged by recent guidelines where borderline or pre-hypertension states have been introduced and recommendations for blood pressure cut-offs and targets are dependent on comorbidities such as diabetes or renal disease [START_REF] Mancia | Guidelines for the Management of Arterial Hypertension: The Task Force for the Management of Arterial Hypertension of the European Society of Hypertension (ESH) and of the European Society of Cardiology (ESC)[END_REF][START_REF] Chobanian | Seventh report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure[END_REF]. Blood pressure in the upper range of normal is also associated with increased cardiovascular morbidity and mortality. Only half of the burden in the study by Lawes et al. [START_REF] Lawes | Global burden of blood-pressure-related disease[END_REF] was in people with hypertension, whereas the remainder was in subjects with lesser degrees of high blood pressure (≥115 mmHg but <140 mmHg systolic). It appears that there is a continuous relationship between blood pressure and cardiovascular risk without evidence of a threshold down to at least 115/75 mm Hg [START_REF] Lewington | Age-specific relevance of usual blood pressure to vascular mortality: a meta-analysis of individual data for one million adults in 61 prospective studies[END_REF].

These considerations have major implications on studies into the genetics of hypertension. A qualitative trait of "hypertension" could be subject to classic Mendelian laws of inheritance whereas a continuous "blood pressure" trait would constitute a non-Mendelian complex trait [START_REF] Padmanabhan | Genetic factors in hypertension[END_REF]. The normal unimodal distribution of blood pressure in the general population supports the latter [START_REF] Carretero | Essential hypertension. Part I: definition and etiology[END_REF]. However, there are rare monogenic Mendelian forms of hypertension which demonstrate that at least some forms of hypertension and possibly even some components of blood pressure could be explained by classic Mendelian inheritance [START_REF] Padmanabhan | Genetic factors in hypertension[END_REF]. From family and twin studies heritabilities of systolic and diastolic blood pressure are generally estimated in the range of 15 to 40 % and 15 to 30 %, respectively [START_REF] Feinleib | The NHLBI twin study of cardiovascular disease risk factors: methodology and summary of results[END_REF][START_REF] Mongeau | The influence of genetics and household environment upon the variability of normal blood pressure: the Montreal Adoption Survey[END_REF][START_REF] Staessen | Essential hypertension[END_REF]. The sibling recurrent risk of hypertension is in the range of 1.2 to 1.5 [START_REF] Caulfield | Genomewide mapping of human loci for essential hypertension[END_REF].

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However, these figures are influenced by non genetic factors including shared environment and measurement errors so that the magnitude of the genetic effect could be different.

Another fundamental problem in the genetics of hypertension is the definition of the ancestral phenotype. Hypertension is a disease of modern civilisation and heavily depends on environmental and particularly dietary factors of modern society. For example, members of the Luo tribe had lower blood pressure in their traditional rural environment than in the urban environment of Nairobi where their urinary sodium concentration was higher and urinary potassium concentration was lower [START_REF] Poulter | Blood pressure and its correlates in an African tribe in urban and rural environments[END_REF]. It has been hypothesised that in a sodium-deprived environment the default genotype is a sodium conserving one. Likewise, the renin-angiotensin-aldosterone system may have initially been adapted for sodium conservation but may play an important role in the pathogenesis of hypertension in modern societies with high dietary salt intake [START_REF] Brunner | Is the renin system necessary?[END_REF]. These considerations highlight that for genes associated with hypertension the disease allele does not necessarily have to be the minor (rare) allele but could be the major (for examples sodium conserving) allele as well, and that studies into genes associated with low blood pressure could be equally important for the genetics of hypertension.

Our attempts to unravel the genetic basis of human essential hypertension are complicated by the above considerations and other challenges we will refer to later. We have previously reviewed this topic [START_REF] Lee | Genetics of hypertension: from experimental models to clinical applications[END_REF] and concluded that a multifaceted approach including and integrating human and animal studies is a most promising approach. Ten years later we take the opportunity to update our previous review. There has been major progress in some areas, mainly driven by the advances in technology and statistics, but it is fair to say that we are still not much closer to a complete understanding of the genetics of hypertension than a decade ago.

The rat as a physiological model of hypertension

One strategy to overcome the limitations of functional genetic studies in humans is to take advantage of specific hypertensive rat strains that have been selectively bred over many generations

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and are inherently simpler paradigms, but remain under complex control [START_REF] Rapp | Genetic analysis of inherited hypertension in the rat[END_REF][START_REF] Cowley | Application of chromosomal substitution techniques in gene-function discovery[END_REF]. This is especially relevant, as recent developments in genome sequencing [17] and software development [START_REF] Harris | Inferring Meta-Covariates in Classification[END_REF][START_REF] Hunt | The visual language of synteny[END_REF] have accelerated conserved genome analysis and translational studies between rat and human.

Numerous genome-wide studies in rat models have revealed candidate quantitative trait locus (QTL) regions on almost every rat chromosome, as well as some important interactions between loci [START_REF] Rapp | Genetic analysis of inherited hypertension in the rat[END_REF][START_REF] Monti | Interaction between blood pressure quantitative trait loci in rats in which trait variation at chromosome 1 is conditional upon a specific allele at chromosome 10[END_REF]. As with humans, however, the critical rate-determining step is no longer the identification of QTLs, but the identification of the genetic determinant underlying the QTL.

Quantitative trait loci influencing blood pressure

The principal strategy in the rat for the search of genes involved in the development of hypertension has been the identification of quantitative trait loci (QTL) responsible for blood pressure regulation by genome wide scanning [START_REF] Rapp | Genetic analysis of inherited hypertension in the rat[END_REF][START_REF] Cowley | The genetic dissection of essential hypertension[END_REF]. The difficulties in identifying QTL include epistasis, and a limited statistical power due to the number of hypotheses being tested. Moreover, any single QTL is responsible for only a fraction of the phenotypic variation and thus the phenotype-genotype correlation is low. Improvements in genomic resources and statistical analysis have led to the identification of a large number of QTL. However, despite the numerous complex traits that have been analysed by genome-wide linkage studies, few of the underlying genes have been identified [START_REF] Glazier | Finding genes that underlie complex traits[END_REF].

QTL mapping is a phenotype driven approach that does not require prior knowledge of either causative genes or their function and can lead to the identification of novel genes involved in disease. Furthermore, in animal models there is also the distinct advantage of the construction of congenic strains to first verify the existence of the QTL, and also to allow production of congenic substrains to enable genetic dissection and fine mapping in order to reduce QTL intervals (Figure 1). This congenic strategy was considered ideal, since initial estimates suggested the involvement of a relatively few major effect genes, thus requiring the construction of essentially Mendelian-like strains to facilitate gene identification. However, recent congenic studies have demonstrated the

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underlying genetic complexity of large QTL regions identified in genome wide scans.

Comprehensive congenic coverage on various rat chromosomes has revealed multiple QTLs, with evidence for loci with opposing effects on blood pressure [START_REF] Ariyarajah | Dissecting quantitative trait loci into opposite blood pressure effects on Dahl rat chromosome 8 by congenic strains[END_REF][START_REF] Dutil | Multiple quantitative trait loci for blood pressure interacting epistatically and additively on Dahl rat chromosome 2[END_REF][START_REF] Eliopoulos | Severe hypertension caused by alleles from normotensive Lewis for a quantitative trait locus on chromosome 2[END_REF][START_REF] Grondin | Complete and overlapping congenics proving the existence of a quantitative trait locus for blood pressure on Dahl rat chromosome 17[END_REF][START_REF] Palijan | Comprehensive congenic coverage revealing multiple blood pressure quantitative trait loci on Dahl rat chromosome 10[END_REF][START_REF] Palijan | Quantitative trait loci with opposing blood pressure effects demonstrating epistasis on Dahl rat chromosome 3[END_REF]. While congenic fine mapping can restrict the number of genes in a QTL interval to a minimum to facilitate gene identification [START_REF] Tripodi | Effect of Add1 gene transfer on blood pressure in reciprocal congenic strains of Milan rats[END_REF], frequent loss of the hypertensive phenotype suggests the existence of multiple genes.

Furthermore, potential epistatic interactions (i.e. the masking of a gene by the effect of another unrelated gene) may underlie many QTL peaks and congenic intervals [START_REF] Dutil | Multiple quantitative trait loci for blood pressure interacting epistatically and additively on Dahl rat chromosome 2[END_REF][START_REF] Tripodi | Effect of Add1 gene transfer on blood pressure in reciprocal congenic strains of Milan rats[END_REF], and this has hampered progress towards the identification of causative genetic elements.

Identifying genetic determinants

Combining congenic fine mapping, comparative genomic tools, gene expression array resources, transgenic rescue and knockout technology in the rat provides a powerful tool for the discovery of novel genes underlying complex cardiovascular traits. The first successful example of this combined strategy was the identification of the gene encoding Cd36 as a major contributor to insulin resistance and dyslipidaemia in the spontaneously hypertensive rat (SHR) [START_REF] Aitman | Identification of Cd36 (Fat) as an insulin-resistance gene causing defective fatty acid and glucose metabolism in hypertensive rats[END_REF], confirmed by transgenic rescue [START_REF] Pravenec | Transgenic expression of CD36 in the spontaneously hypertensive rat is associated with amelioration of metabolic disturbances but has no effect on hypertension[END_REF] and subsequent translation to human populations [START_REF] Lepretre | Genetic study of the CD36 gene in a French diabetic population[END_REF][START_REF] Miyaoka | CD36 deficiency associated with insulin resistance[END_REF]. However, there was an issue concerning the contribution of Cd36 to hypertension. These issues have only recently been resolved by implicating Cd36 as a determinant of blood pressure and risk for hypertension by specifically identifying deficient renal expression of Cd36 [START_REF] Pravenec | Identification of renal Cd36 as a determinant of blood pressure and risk for hypertension[END_REF].

Our own research has focused on the stroke-prone spontaneously hypertensive rat (SHRSP), where the genetic determination of hypertension is due to multiple gene-gene and gene-environment interactions. Although exceptionally high blood pressure is clearly a factor for end-organ damage in the SHRSP, genetic factors are also likely to contribute. In particular, two blood pressure QTL have been mapped to rat chromosome 2 [START_REF] Clark | Quantitative trait loci in genetically hypertensive rats. Possible sex specificity[END_REF]. This region of chromosome 2 is a classic example of a common or overlapping QTL because it has been implicated in several rat crosses. These two QTL, therefore, have become a focus for further congenic strategies in our laboratory [START_REF] Jeffs | Applicability of a "speed" congenic strategy to dissect blood pressure quantitative trait loci on rat chromosome 2[END_REF] and other

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laboratories [START_REF] Dutil | Further chromosomal mapping of a blood pressure QTL in Dahl rats on chromosome 2 using congenic strains[END_REF]. In combination with congenic strain construction in the SP.WKYGla2a and SP.WKYGla2c* strains, genome-wide microarray expression profiling identified glutathione Stransferase mu type 1 (Gstm1) as a positional candidate gene for spontaneous hypertension [START_REF] Aitman | Progress and prospects in rat genetics: a community view[END_REF][START_REF] Mcbride | Microarray analysis of rat chromosome 2 congenic strains[END_REF][START_REF] Mcbride | Reduction of Gstm1 expression in the stroke-prone spontaneously hypertension rat contributes to increased oxidative stress[END_REF],

and endothelial differentiation gene 1 (Edg1) and vascular cell adhesion molecule 1 (Vcam1) for salt sensitivity [START_REF] Graham | Candidate genes that determine response to salt in the stroke-prone spontaneously hypertensive rat: congenic analysis[END_REF].

It is notable that there are still relatively few examples where this strategy has led to the identification of positional and functional hypertension candidate genes that can be translated and tested in human populations [START_REF] Garrett | Defining the blood pressure QTL on chromosome 7 in Dahl rats by a 177-kb congenic segment containing Cyp11b1[END_REF][START_REF] Yagil | Identification of hypertension-related genes through an integrated genomic-transcriptomic approach[END_REF]. It is also important to note that narrowing the QTL interval by substitution mapping remains a critical part of this combined strategy, as demonstrated recently by the subsequent elimination of the differentially expressed candidate gene, Resp18, with the production of the smallest reported rat blood pressure QTL interval of 117 kb [START_REF] Garrett | Locating a blood pressure quantitative trait locus within 117 kb on the rat genome: substitution mapping and renal expression analysis[END_REF]. This highlights that, even with the onset of extensive genome sequencing, our ability to identify a full complement of genes and other genetic elements continues to be a challenge. However, recent evidence suggests that once again, this locus is more complex than originally believed as further transgenic-congenic rats confirm the eligibility of Resp18 as a hypertension candidate gene [START_REF] Toland | Mapping Genetic Determinants of Blood Pressure That are Different Between Dahl Salt-Sensitive and Spontaneously Hypertensive Rats[END_REF]. This highlights the requirement to modulate the levels of target genes in the appropriate genetic background with either transgenic over expression or knockout rats by embryo microinjection of zinc-finger nucleases [START_REF] Geurts | Knockout rats via embryo microinjection of zinc-finger nucleases[END_REF].

It should be noted, however, that the efficiency of candidate gene identification is very much dependent on the complexity of the phenotype under investigation. There are many examples of other disease phenotypes in the rat that have proved more amenable to candidate gene discovery via congenic strategies including pristane-induced arthritis [START_REF] Olofsson | Positional identification of Ncf1 as a gene that regulates arthritis severity in rats[END_REF] and cresentric glomerulonephritis [START_REF] Aitman | Copy number polymorphism in Fcgr3 predisposes to glomerulonephritis in rats and humans[END_REF][START_REF] Behmoaras | Jund is a determinant of macrophage activation and is associated with glomerulonephritis susceptibility[END_REF].

Consomic, heterogeneous rat and recombinant inbred strains

As well as advances in genome resources there are a number of large scale animal resources available for exploitation (Figure 1).

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Consomic rat strains are generated by introgressing an entire chromosome from one inbred strain into the isogenic background of another inbred strain using marker-assisted selection. The PhysGen (http://pga.mcw.edu) Program for Genomic Application at the Medical College of Wisconsin developed two panels of consomic rats using the SS/JrHsdMcwi, the FHH/EurMcwi and the BN/NHsdMcwi strains. Characterization of these consomic strains, each carrying a chromosome from the sequenced Brown Norway strain, allows for immediate mapping of traits to a particular chromosome without the need for genetic crosses. This ongoing work, but gene expression [START_REF] Liang | Molecular networks in Dahl saltsensitive hypertension based on transcriptome analysis of a panel of consomic rats[END_REF] and proteomic [START_REF] Tian | Renal regional proteomes in young Dahl salt-sensitive rats[END_REF] analysis has been undertaken in a number of these consomic strains. From the consomic strains, congenic strains can be rapidly bred within 6 months to narrow the region on a specific chromosome to a region that can be targeted for gene identification [START_REF] Cowley | Consomic rat model systems for physiological genomics[END_REF].

A further strategy to improve the efficiency of gene identification is to utilise the rat heterogeneous (HS) stock panels. HS rats are generated by intercrossing several inbred progenitor strains followed by cross breeding for more than 50 generations. Each chromosome is a fine-grained mosaic of the progenitor strains: the average distance between recombinants is small (less than 2 centimorgans) so that the HS provide high resolution mapping of multiple QTLs across the genome. This allows finemapping of QTLs to sub-centimorgan intervals by exploiting recombinants that have accumulated over many generations of out breeding in genetically HS that are derived from inbred strains (Figure 1). This significantly reduces the number of candidate genes found within each QTL. The best characterized rat heterogeneous stock panel was established at the NIH in 1984 and derives from eight genetically distinct and phenotypically diverse inbred strains (ACI/N, BN/SsN, BUF/N, F344/N, M520/N, MR/N, WKY/N and WN/N). Pilot studies in 786 Rat HS [START_REF] Johannesson | A resource for the simultaneous high-resolution mapping of multiple quantitative trait loci in rats: the NIH heterogeneous stock[END_REF] strains support the findings from the mouse HS [START_REF] Valdar | Genome-wide genetic association of complex traits in heterogeneous stock mice[END_REF] indicating refinement of a behaviour QTL identification from 40cM to 13Mbp.

One of the great successes in the rat over the past few years has been the exploitation of the rat recombinant inbred panel (Figure 1). A comprehensive genetic screen of the rat recombinant inbred strains has recently been undertaken in which gene expression values were considered as expression

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quantitative traits [START_REF] Hubner | Integrated transcriptional profiling and linkage analysis for identification of genes underlying disease[END_REF]. This integration of genome-wide expression profiling with linkage analysis is a new approach to identifying genetic determinants underlying complex traits. The expression profiling of kidney and fat RNA from all individuals allows the profile of each gene, treated as an intermediate phenotype, to be considered as an expression quantitative trait. Furthermore, by conserved genome analysis, a data set of 73 candidate genes for hypertension, including Cd36 and Gstm1, have been identified that merit translational studies in human populations [START_REF] Hubner | Integrated transcriptional profiling and linkage analysis for identification of genes underlying disease[END_REF]. Other notable successes with this strategy include candidate genes for heart failure [START_REF] Monti | Soluble epoxide hydrolase is a susceptibility factor for heart failure in a rat model of human disease[END_REF] and left ventricular hypertrophy [START_REF] Petretto | Integrated genomic approaches implicate osteoglycin (Ogn) in the regulation of left ventricular mass[END_REF].

Translation from animal to man

In our own studies in the SHRSP we have identified the glutathione S-transferase mu type 1 gene (Gstm1) as a positional candidate for hypertension in the SHRSP [START_REF] Mcbride | Microarray analysis of rat chromosome 2 congenic strains[END_REF]. Glutathione S-transferases are involved in the defences against oxidative stress and thereby also constitute functional candidates for hypertension [START_REF] Hayes | Glutathione S-transferase polymorphisms and their biological consequences[END_REF]. We and others have confirmed reduced Gstm1 gene [START_REF] Mcbride | Microarray analysis of rat chromosome 2 congenic strains[END_REF][START_REF] Hubner | Integrated transcriptional profiling and linkage analysis for identification of genes underlying disease[END_REF][START_REF] Friese | Common genetic mechanisms of blood pressure elevation in two independent rodent models of human essential hypertension[END_REF] and Gstm1 protein expression [START_REF] Mcbride | Reduction of Gstm1 expression in the stroke-prone spontaneously hypertension rat contributes to increased oxidative stress[END_REF] in SHRSP. The human orthologues of Gstm1 are therefore promising candidate genes for essential hypertension. However, when we performed a definite association study of GSTM genes in hypertension involving sequencing of GSTM genes, genotyping for the GSTM1 deletion and studies in three different and independent cohorts, we were unable to show a significant association between any of the human GSTM genes and hypertension [START_REF] Delles | Glutathione S-transferase variants and hypertension[END_REF].

This apparently negative example illustrates that direct translation of findings in experimental animal models to human disease is not without challenges. To the best of our knowledge there are no examples of a direct translation of findings in rodents to man for the genetics of hypertension.

Whilst this may in part be due to differences in the genetics of hypertension between animals and humans it also illustrates the general challenges we are facing in candidate gene studies of human essential hypertension.

Candidate gene studies

Candidate genes are chosen for their assumed role in the pathogenesis of hypertension and can derive from experimental models or knowledge of the pathophysiology. The most plausible candidate genes are components of the renin-angiotensin-aldosterone system and genes involved in signal transduction (e.g. the G-protein subunit-β3 gene GNB3), salt and water handling (e.g. the adducin-α genes ADD1), regulation of vascular tone (e.g. the endothelial nitric oxide synthase gene NOS3) and production of or defences against oxidative stress. However, despite numerous reports on association in small and medium sized cohorts since publication of our previous review [START_REF] Lee | Genetics of hypertension: from experimental models to clinical applications[END_REF] there has been little evidence for a substantial association of any of these genes or single nucleotide polymorphisms (SNPs) within these genes with hypertension. The main reasons for this lack of positive results are (1) the individual contribution of genetic variants to the blood pressure phenotype is small; (2) most of the study designs lacked power to detect these small effects; (3) inconsistency of phenotyping did not allow results to be compared or combined; (4) selection of candidate genes depended on known pathways of blood pressure regulation precluding undiscovered pathways; (5) most of the candidate gene studies tended to be single SNP studies which was not completely informative of the gene variations and additional SNP identification needed sequencing resources which were not readily accessible; (6) studies of rare frequency variants were not done; and (7) the study design issues mentioned above precluded any meaningful study of gene-gene or gene-environment interactions. Nevertheless, the candidate gene approach remains one of the cornerstones of studies into the genetics of hypertension. We refer to recent reviews on the genetics of the renin-angiotensin-aldosterone system [START_REF] Jeunemaitre | Genetics of the human renin angiotensin system[END_REF][START_REF] Rudnicki | Significance of genetic polymorphisms of the reninangiotensin-aldosterone system in cardiovascular and renal disease[END_REF][START_REF] Pereira | Metaanalysis of the association of 4 angiotensinogen polymorphisms with essential hypertension: a role beyond M235T?[END_REF], G-proteins [START_REF] Zhu | Update on G-protein polymorphisms in hypertension[END_REF],

adducin [START_REF] Bianchi | Adducin polymorphism: detection and impact on hypertension and related disorders[END_REF], and oxidative stress [START_REF] Delles | Functional Genomics of the Oxidative Stress Pathway[END_REF][START_REF] Jose | NADPH oxidase CYBA polymorphisms, oxidative stress and cardiovascular diseases[END_REF] in hypertension, but will discuss the association of the aldosterone synthase gene (CYP11B2) with hypertension as an exemplar.

The physiology of aldosterone is relatively well understood. There is considerable epidemiological evidence that aldosterone has a role in the development of hypertension and other cardiovascular diseases. Even within the physiological range, increased plasma aldosterone levels predispose to [START_REF] Vasan | Serum aldosterone and the incidence of hypertension in nonhypertensive persons[END_REF]. Increased aldosterone levels in adulthood may be determined by conditions early in life such as foetal growth [START_REF] Reynolds | Programming of hypertension: associations of plasma aldosterone in adult men and women with birthweight, cortisol, and blood pressure[END_REF]. While Plasma aldosterone and aldosterone to renin ratio were found to be heritable phenotypes, common polymorphisms in the CYP11B2 gene explained only partially variations in aldosterone levels [START_REF] Davies | Altered corticosteroid biosynthesis in essential hypertension: A digenic phenomenon[END_REF][START_REF] Holloway | Effects of genetic variation in the aldosterone synthase (CYP11B2) gene on enzyme function[END_REF]. This is akin to the previous experience with ACE and AGT levels and like before a large number of studies have been performed in recent years and these were meta-analysed by Sookoian et al. [START_REF] Sookoian | Association of the C-344T aldosterone synthase gene variant with essential hypertension: a meta-analysis[END_REF]. The evidence from the meta-analysis was borderline for association between hypertension (qualitative trait) and the C-344T variant of CYP11B2 and no association seen with systolic and diastolic blood pressure (quantitative trait). In any meta-analysis, the results depend on the constituent studies, and this is true of most of the studies included here and all the reasons mentioned above are applicable. Like other candidate genes, aldosterone synthase is an attractive target, and it is likely that evidence for association may come from studies of rare or structural variants in this locus.

A C C E P T E D M A N U S C R I P T development of hypertension
With the advances of technology and reduced costs for genotyping we will see more large-scale candidate gene projects in the future. An example for this approach is the ITMAT/Broad/CARE Vascular Disease 50k SNP Array (IBC Chip; http://bmic.upenn.edu/cvdsnp/). This chip allows genotyping of about 50,000 SNPs in 2,100 cardiovascular candidate genes at relatively low costs.

Studies into hypertension have been conducted but reports are still outstanding. The relatively large scale of these projects necessitates correction for multiple tests and will therefore have an unfavourable impact on sample size and power of such studies. Extracting gene specific data from genome wide SNP chips are not very fruitful. A recent example of a study into 160 candidate genes for blood pressure covering 2,411 SNPs in 1,644 subjects of the KORA S3 cohort is a case in point [START_REF] Sober | Targeting 160 candidate genes for blood pressure regulation with a genome-wide genotyping array[END_REF]. None of the SNPs remained significantly associated with blood pressure after Bonferroni correction. The top twelve hits in seven genes for association with blood pressure and/or hypertension (P<10 -3 ) failed to be replicated in independent cohorts.

Linkage studies

In contrast to candidate gene studies we have seen significant developments in the area of genome wide linkage and association studies since publication of our previous review [START_REF] Lee | Genetics of hypertension: from experimental models to clinical applications[END_REF]. Historically, linkage studies were the first technically feasible genome wide studies of human hypertension.

Linkage studies examine transmission of disease loci from parents to offspring, but modifications of this concept allowed studies complex traits in the form of affected sib-pair studies [START_REF] Blackwelder | A comparison of sib-pair linkage tests for disease susceptibility loci[END_REF]. The latter is a powerful method of performing linkage studies with small family sizes, but there are practical issues in recruitment of families with a mobile adult population worldwide, although we have recently demonstrated that family-based recruitment is still possible [START_REF] Neisius | Recruitment of Families for A Study Into the Genetics of Hypertension (Ingenious Hypercare). A Single Centre Experience[END_REF].

The British Genetics of Hypertension (BRIGHT) study is probably the best example for a linkage study into essential hypertension [START_REF] Caulfield | Genomewide mapping of human loci for essential hypertension[END_REF]. The consortium genotyped 2,010 affected sibling pairs drawn from 1,599 severely hypertensive families, and completed a 10 centimorgan genome-wide scan.

Linkage analysis identified a principle locus on chromosome 6q, with a lod score of 3.21 that attained genome-wide significance (P=0.042). Three further loci with lod scores higher than 1.57 (2q, 5q, and 9q) also showed genome-wide significance (P=0.017) when assessed under a locuscounting analysis. Genes or functional genetic variants underlying the linkage peaks have still not been identified. The authors originally interpreted these results as evidence for an oligogenic element of human essential hypertension. However, insufficient marker density and insufficient sample size to detect genetic variants with small effects on blood pressure were thought to be limitations of the BRIGHT study. These limitations have recently been addressed by regenotyping hypertensive probands from the BRIGHT study as part of SNP based genome wide association studies (GWAS) into common disease [START_REF]Genome-wide association study of 14,000 cases of seven common diseases and 3,000 shared controls[END_REF].

There have been a number of similar genome wide linkage studies. QTLs for blood pressure have been found on virtually all human chromosomes in linkage studies [START_REF] Binder | A review of the genetics of essential hypertension[END_REF]. Koivukoski et al. [START_REF] Koivukoski | Meta-analysis of genome-wide scans for hypertension and blood pressure in Caucasians shows evidence of susceptibility regions on chromosomes 2 and 3[END_REF] meta-analysed 9 genome wide scans of blood pressure or hypertension using the genome-search meta-analysis method and found susceptibility loci on chromosomes 2 (2p12-q22.1) and 3 (3p14.1-

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3). Their study demonstrated that sample size is a crucial factor detection of markers with genome wide significance. Subsequently, we demonstrated a successful attempt at reducing heterogeneity by using antihypertensive drug response to partition different pathways of hypertension [START_REF] Padmanabhan | Chromosome 2p shows significant linkage to antihypertensive response in the British Genetics of Hypertension Study[END_REF]. In the BRIGHT population, hypertensive sib-pairs who were non-responsive to ACE inhibitors, ARBs or beta-blockers showed significant linkage on chromosome 2p (LOD = 4.84 at 90.68 Kosambi cM). This susceptibility locus co-localises to a region found in African-American hypertensives in the Family Blood Pressure Program who showed evidence of linkage with hypertension status at 93 cM with a LOD score of 2.84 [START_REF] Barkley | Positional identification of hypertension susceptibility genes on chromosome 2[END_REF]. Thus the chromosomal 2p locus independently identified in different populations may contain a gene or genes for the salt-sensitive form of hypertension which is common among Africans, and the same mechanism may be operative in a subset of white European hypertensives identified by unresponsiveness to beta blockers and ACE inhibitors. Another approach was taken by the BRIGHT investigators who performed linkage analysis of covariate data including serum and urine biochemistry, biometric measurements and additional blood pressure phenotypes [START_REF] Wallace | Linkage analysis using co-phenotypes in the BRIGHT study reveals novel potential susceptibility loci for hypertension[END_REF]. Genome-wide significant results were obtained for body mass and renal function related traits on chromosomes 20 and 5, respectively. Although these results need to be interpreted with caution as the analysis has not been adjusted for tretmant and was performed in a selected population of hypertensive subjects, additional phenotypic information may help to reduce genetic heterogeneity and identify novel loci for hypertension.

Genome wide association studies

Linkage studies are extremely attractive tools in human genetics but they can only identify highly penetrant loci and have been notably successful in the identification of monogenic forms of hypertension, The recent developments in genotyping technology with the introduction of chipbased genotyping arrays [START_REF] Matsuzaki | Genotyping over 100,000 SNPs on a pair of oligonucleotide arrays[END_REF][START_REF] Gunderson | A genome-wide scalable SNP genotyping assay using microarray technology[END_REF] has paved the way for GWAS that genotype for a large number of genetic markers usually SNPs (for example about 500,000 in the Wellcome Trust Case Control Consortium (WTCCC) study [START_REF]Genome-wide association study of 14,000 cases of seven common diseases and 3,000 shared controls[END_REF]) as opposed to the highly polymorphic microsatellite markers that

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have been used in classic genome wide linkage studies. With the resources from Hapmap, it is possible to impute up to 2.5 million SNPs in LD with the tag SNPs present in these GWAS SNP arrays.

In 2007 two major GWAS into hypertension have been reported. Levy et al. [START_REF] Levy | Framingham Heart Study 100K Project: genome-wide associations for blood pressure and arterial stiffness[END_REF] conducted a study in Framingham Heart Study families and associated blood pressures at two different time points and long-term averaged blood pressure with 100,000 polymorphic markers using the Affymetrix 100k chip. In the primary analyses, none of the associations attained genome-wide significance. The other study was reported by the WTCCC and examined association with 500,000 genetic markers in 2,000 patients with hypertension (derived from the BRIGHT cohort) and 3,000 control subjects using the Affymetrix 500k chip [START_REF]Genome-wide association study of 14,000 cases of seven common diseases and 3,000 shared controls[END_REF]. Also the WTCCC study did not find a SNP for hypertension that achieved genome wide statistical significance. At the same time, the WTCCC examined other common diseases against the same common control subjects and successfully identified loci for coronary artery disease, bipolar disorder, rheumatoid arthritis, Crohn's disease and type 1 and 2

diabetes. Whilst the WTCCC study was characterised by better marker density compared to the Framingham Heart Study, there have been a number of criticisms about the study design that may have caused underperformance of hypertension compared to the other common diseases in the WTCCC study. The common controls were not selected and in part not even phenotyped for blood pressure. With a prevalence of hypertension of 20 to 30% in the general population [START_REF] Kearney | Global burden of hypertension: analysis of worldwide data[END_REF] is very likely that the presence of cases in apparent controls has reduced the power to identify disease specific associations [START_REF]Genome-wide association study of 14,000 cases of seven common diseases and 3,000 shared controls[END_REF]. This concern has been partly addressed by the selection of cases from the top 5% of blood pressure distribution in the UK population thereby reducing significantly the number of misclassified controls with the same phenotype as the cases. Nevertheless, due to the results of these two GWAS there has been disappointment and concern that the effect sizes of genes related to hypertension could be lower than those observed for other common diseases where odds ratios in the range of 1.2 to 1.5 were found [START_REF] O'shaughnessy | Dissecting complex traits: recent advances in hypertension genomics[END_REF]. Most recently a study in 8,842 samples from a Korean general population with replication in 7,861 independent samples revealed an association of

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rs17249754 near the ATP2B1 gene at genome wide significance level [START_REF] Cho | A large-scale genome-wide association study of Asian populations uncovers genetic factors influencing eight quantitative traits[END_REF]. The effect size of this SNP was relatively small (-1.309±0.266 mmHg for systolic and -0.882±0.181 mmHg for diastolic blood pressure), but certainly in a realistic range for a multigenic disorder.

The major breakthrough came with the report of two studies that examined associations of a large number of SNPs with blood pressure and hypertension. The Global BPgen consortium tested 2.5 million genotyped or imputed SNPs for association with systolic or diastolic blood pressure in 34,433 subjects of European ancestry [89] and identified eight regions with genome wide significance. The variants were near the CYP17A1, CYP1A2, FGF5, SH2B3, MTHFR, ZNF652 and PLCD3 genes and chromosome 10 open reading frame 107 (c10orf107). These loci were also associated with hypertension as qualitative trait. The second consortium (CHARGE) studied 2.5 million genotyped or imputed SNPs in 29,136 subjects and found significant (P<4×10 -7 ) associations with systolic blood pressure for 13 SNPs, with diastolic blood pressure for 20 SNPs and with hypertension for 10 SNPs [START_REF] Levy | Genome-wide association study of blood pressure and hypertension[END_REF]. The data were than meta-analysed with those of the Global BPGen consortium [89] and revealed genome-wide significance (P<5×10 -8 ) for ATP2B1 Despite the impressive samples sizes and even more impressive P-values it should be noted that the effects of these SNPs on blood pressure are rather modest. For example, in Global BPgen the SNP showing the strongest association with systolic blood pressure (rs11191548, P=7×10 -24 ) increases systolic blood pressure by 1.16 mmHg per major allele. Generally an effect of about 1 mmHg per

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identified SNP can be assumed which may have major implications for hypertension related cardiovascular disease in the population but minor implications for an individual. Nevertheless, these projects have clearly demonstrated the existence of blood pressure susceptibility genes with small but measurable effects. Most of the SNPs are located in genes that have not immediately been thought to play a major role in the pathogenesis of hypertension, and it is hoped that these findings will improve our understanding of the underlying pathomechanisms.

Monogenic forms of hypertension

Since publication of our review in 2000 [START_REF] Lee | Genetics of hypertension: from experimental models to clinical applications[END_REF] there has been considerable progress in the area of rare Mendelian forms of hypertension. These syndromes account for less than 1% of human hypertension and because a clear and often treatable reason has been elucidated, do not immediately explain the genetics of essential hypertension. However, the Mendelian forms of hypertension are important because they constitute the archetype of hereditable hypertension; demonstrate the importance of genes expressed in the kidney and responsible for salt and water handling in the pathogenesis of hypertension; and may help to unravel mechanisms that also apply to the pathogenesis of essential hypertension. Mendelian forms of hypertension have been reviewed

recently [START_REF] Padmanabhan | Genetic factors in hypertension[END_REF][START_REF] Binder | A review of the genetics of essential hypertension[END_REF][START_REF] Lifton | Molecular mechanisms of human hypertension[END_REF] and are summarised in table 1. It should be noted that there are additional syndromes that would go beyond the scope of this review. For instance, loss-of-function mutations in the aldosterone synthase gene (CYP11B2) can lead to reduced aldosterone levels, hypovolaemia, hypotension and shock [START_REF] Mitsuuchi | Congenitally defective aldosterone biosynthesis in humans: inactivation of the P-450C18 gene (CYP11B2) due to nucleotide deletion in CMO I deficient patients[END_REF][START_REF] Mitsuuchi | Congenitally defective aldosterone biosynthesis in humans: the involvement of point mutations of the P-450C18 gene (CYP11B2) in CMO II deficient patients[END_REF][START_REF] Pascoe | Mutations in the human CYP11B2 (aldosterone synthase) gene causing corticosterone methyloxidase II deficiency[END_REF]. Other examples include familiar hyperaldosteronism type II, congenital adrenal hyperplasia, mutations in the PPARG gene and hypertension and bradydactyly [START_REF] Padmanabhan | Genetic factors in hypertension[END_REF][START_REF] Binder | A review of the genetics of essential hypertension[END_REF][START_REF] Lifton | Molecular mechanisms of human hypertension[END_REF]. Some of these syndromes can be caused by mutations in several genes, some have not yet been localised to a gene, or have been localised but still lack understanding of the mechanisms that lead to hypertension.

One of the major discoveries in recent years was the description of the WNK (with no K [lysine])

kinases and their role in the pathogenesis of hypertension. The WNK1 and WNK4 serine-threonine

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kinases regulate the sodium, chloride and potassium transport mechanisms in the distal nephron [START_REF] Kahle | Molecular physiology of the WNK kinases[END_REF][START_REF] San Cristobal | WNK kinases, renal ion transport and hypertension[END_REF][START_REF] Flatman | Cotransporters, WNKs and hypertension: an update[END_REF]. WNK1 and WNK4 mutations have been identified to cause Gordon syndrome (pseudohypoaldosteronism type 2) [START_REF] Wilson | Human hypertension caused by mutations in WNK kinases[END_REF]. It has then be demonstrated that variants of the WNK1 gene not only cause this severe Mendelian form of hypertension but that more subtle variants are also associated with blood pressure variation in a severely hypertensive [START_REF] Newhouse | Haplotypes of the WNK1 gene associate with blood pressure variation in a severely hypertensive population from the British Genetics of Hypertension study[END_REF] 50 and 60 years, respectively, in carriers of the mutation compared to non-carriers. Thereby, these rare mutations in just three genes contributed equally or more to blood pressure variation in the general population than the common variants identified by the recent GWAS [89,[START_REF] Levy | Genome-wide association study of blood pressure and hypertension[END_REF]. Direct sequencing may be a promising tool to identify rare (i.e. minor allele frequency <1 %) mutations

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that are not usually covered in GWAS. The above example demonstrates that such rare variants in genes whose pathophysiological role in the regulation of blood pressure is beyond any doubt can help to explain blood pressure variability in the population.

Summary and open questions

As a result of enormous effort a number of plausible and validated genetic variants that are associated with blood pressure and/or hypertension have recently been found. It emerged that these variants are either common and in turn contribute little to blood pressure variation [89,[START_REF] Levy | Genome-wide association study of blood pressure and hypertension[END_REF] or are rare and have a relatively strong effect [102]. The future development of this area is difficult to predict but will most likely see a combination of GWAS and sequencing studies in large cohorts.

The past ten years have also demonstrated that accurate phenotyping is one of the keys to discover genetic determinants of disease. This may be less of an issue in relatively clearly defined diseases such as type 1 diabetes or Crohn's disease but is a major problem in hypertension where there is a somewhat arbitrary cut-off for definition of disease, marked sexual dimorphism, marked age effect, and a substantial influence of environmental factors. Unsurprisingly genetics of hypertension has been less of a success story compared to other common diseases, but the high impact of hypertension on cardiovascular morbidity and mortality more than justifies further efforts. The role of intermediate phenotypes including endothelial function and vascular stiffness but also associated phenotypes such as body weight and renal function [START_REF] Wallace | Linkage analysis using co-phenotypes in the BRIGHT study reveals novel potential susceptibility loci for hypertension[END_REF] in gene discovery remains to be elucidated.

It is also not clear whether an approach in the general population targeted towards blood pressure variation (quantitative trait) or an approach in selected extremes of cases and controls (quantitative trait) is superior. Most likely both approaches have their own merits.

Finally, we have witnessed substantial progress in both human genetics and animal models of hypertension, but have seen few truly translational studies. It appears that both approaches have been developed in parallel with little focus on informing each other. This will almost certainly change in the next few years, but probably not necessarily in the direction "animal to man" but more

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likely in the direction "man to animal". The highly sophisticated tools that are now available for human genomic studies have led to the identification of numerous causative genes and genetic loci that require detailed studies of function in experimental models.

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(

  chromosome 12q21; plasma membrane calcium ATPase 1), CYP17A1 (chromosome 10q24; steroid 17-alpha-monooxygenase), PLEKHA7 (chromosome 11p15; pleckstrin homology domain containing, family A member 7), SH2B3 (chromosome 12q24; SH2B adaptor protein 3), CACNB2 (chromosome 10p12; calcium channel, voltage-dependent, beta 2 subunit), CSK-ULK3 (chromosome 15q24; adjacent to c-src tyrosine kinase and unc-51-like kinase 3 loci), TBX3-TBX5, (chromosome 12q24; adjacent to T-box transcription factor TBX3 and T-box transcription factor TBX5 loci) and ULK4 (chromosome 3p22; unc-51-like kinase 4) for association with systolic or diastolic blood pressure or hypertension.

Figure 1 .Fig. 1

 11 Figure 1. Selected analytical tools in rat genetics. A) The construction of recombinant inbred

  This finding supports the hypothesis that common variants underlying the Mendelian forms of hypertension could affect blood pressure variation in the general population and thereby be involved in the pathogenesis of hypertension. In 2008,Tobin et al. [101] demonstrated that variants of the KCNJ1 gene causing Bartter syndrome type 2 (Table1) were strongly associated with mean 24-hour systolic or diastolic blood pressure. Associations were also found for CASR, NR3C2, SCNN1B and SCMM1G (Table1). Although this study was a milestone in understanding the role of common variants in genes causing monogenic hypertension for regulation of blood pressure in the general population it indeed dealt with common variants (minor allele frequency >1 %). The study byTobin et al. [101] is a candidate gene study and awaits replication in independent cohorts. In the same year, however, Ji et al. [102] opened a completely new avenue of research by also testing three genes which underlie Mendelian forms of hypertension (SLC12A3, SLC12A1 and KCNJ1) in a general population cohort derived from the Framingham Heart Study. In contrast to the work by

	and in the general
	population [100].
	Tobin et al. [101], these authors have screened all coding exons and flanking introns for rare DNA
	sequence variants and found a total of 138 different coding sequence variants in 2,492 subjects
	[102]. These mutations were associated with reduced prevalence of hypertension. Quantitatively,
	mean systolic and blood pressure was 5.7 mmHg, 6.4 mmHg and 9.0 mmHg lower at the age of 40,

Table 1 . Mendelian forms of hypotension and hypertension. SYNDROMES ASSOCIATED WITH LOW BLOOD PRESSURE

 1

	Disorder	Gene/Locus	Age of onset	Disease mechanism	Features	References
	Gitelman syndrome	SLC12A3	Adolescence or	Loss-of-function mutations in the gene encoding	Hypokalaemia with metabolic	[103,104]
		16q3	adulthood	the thiazide-sensitive Na-Cl cotransporter of the	alkalosis. Low serum Mg 2+ and low
				distal collecting duct causes salt wasting and	urinary Ca 2+ levels.
				activation of the renin-angiotensin-aldosterone
				system. Thereby maintenance of serum sodium but
				loss of potassium and H + by augmentation of the
				epithelial sodium channel.
	Bartter syndrome				Often associated with preterm
					delivery. In contrast to Gitelman
					syndrome, normal or only mildly
					reduced serum Mg 2+ and increased
					urinary Ca 2+ levels.
	Type 1	SLC12A	Neonatal	Loss of function of the apical Na-K-2Cl		[105]
		15q21		cotransporter.
	Type 2	KCNJ1	Neonatal	Mutations of the ATP-sensitive K + channel ROMK		[106]
		11q24		affect K + recycling and thereby inhibit Na +
				reabsorption in the thick ascending limb of Henle.
	Type 3	CLCNKB	School age	Loss-of-function mutations in the Cl -channel		[107]
		1p36		CLCNKB in the thick ascending limb of Henle.
				Some of these mutations have arisen from unequal
				crossing over between CLCNKB and the nearby
				related gene CLCNKA.
	Type 4	BSND	Neonatal	BSND encodes Barttin which functions as a beta-	Associated with sensorineural	[108]
		1p32		subunit for CLCNKA and CLCNKB chloride	deafness. inhibition of NaCl
				channels.	reabsorption in type IV Bartter
					syndrome is not restricted to the
					thick ascending limb of Henle.
	Type 5	CASR	Adulthood	Mutations activating the calcium-sensing receptor	Associated with autosomal dominant
		3q13		CaSR which then inhibits sodium transport in the	hypocalcaemia (ADH).
				thick ascending limb of the loop of Henle

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