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Human Molecular Genetics Advance Access originally published online on July 6, 2004
Human Molecular Genetics 2004 13(17):1849-1855; doi:10.1093/hmg/ddh203
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Human Molecular Genetics, Vol. 13, No. 17 © Oxford University Press 2004; all rights reserved

Effects on fear reactivity in XO mice are due to haploinsufficiency of a non-PAR X gene: implications for emotional function in Turner's syndrome

Anthony R. Isles1, William Davies1, Doreen Burrmann1, Paul S. Burgoyne2 and Lawrence S. Wilkinson1,*

1Neurobiology and Developmental Genetics Programmes, The Babraham Institute, Babraham, Cambridge, CB2 4AT, UK and 2MRC National Institute of Medical Research, Mill Hill, London NW7 1AA, UK

Received May 18, 2004; Accepted June 23, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Recent work has indicated altered emotional functioning in Turner's syndrome (TS) subjects (45,XO). We examined the role of X-chromosome deficiency on fear reactivity in X-monosomic mice (39,XO), and found that they exhibited anxiogenic behaviour relative to normal females (40,XX). A molecular candidate for this effect is Steroid sulfatase (Sts) as this is located in the pseudoautosomal region (PAR) of the X-chromosome and consequently is normally biallelically expressed. In addition, the steroid sulfatase enzyme (STS) is putatively linked to fear reactivity by an effect on GABAA receptors via the action of neurosteroids. Real-time PCR demonstrated that levels of Sts mRNA were reduced by half in the brains of 39,XO mice compared with 40,XX, and that expression levels of a number of GABAA subunits previously shown to be important components of fear processing (Gabra3, Gabra1 and Gabrg2) were also altered. However, 40,XY*X mice, in which the Y*X is a small chromosome comprising of a complete PAR and a small non-PAR segment of the X-chromosome, exhibited the same pattern of fear reactivity behaviour as 39,XO animals, but equivalent expression levels of Sts, Gabra1, Gabra3 and Gabrg2 to 40,XX females. This showed that although Sts may cause alterations in GABAA subunit expression, these changes do not result in increased fear reactivity. This suggests an alternative X-chromosome gene, that escapes inactivation, is responsible for the differences in fear reactivity between 39,XO and 40,XX mice. These findings inform the TS data, and point to novel genetic mechanisms that may be of general significance to the neurobiology of fear.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Differentiation of the X and Y chromosomes during mammalian evolution has created an X gene dosage imbalance between the sexes. In order to equalize X gene expression, one of the X-chromosomes of normal females (XX) is inactivated at random, ensuring equal expression of the majority of X-linked genes in male (XY) and female mammals (1). Some genes found in the so-called pseudoautosomal region (PAR), where meiotic recombination occurs between X and Y chromosomes, escape inactivation. In addition, in both mouse (2–4) and particularly humans (5,6), a number of X-specific genes have also been shown to escape inactivation. Interestingly, some of these genes do not have expressed Y-homologues (6), and the persistence of such a dosage imbalance may lead to sex-dimorphisms (1).

In humans, partial or complete loss of one of the sex chromosomes, either the second X or the Y, results in Turner's syndrome (TS, 45,XO) (7). TS subjects are phenotypically female and typically show a general failure of growth, infertility and a range of anatomic abnormalities. These features are assumed to be due to the reduced expression (haploinsufficiency) of bi-allelically expressed (non-dosage compensated) genes on the X (8). In addition, TS girls, despite having normal verbal intelligence, often have a number of subtle behavioural and cognitive deficits, including poor visuospatial skills and social adjustment problems (9). TS subjects have also been reported to show differences in emotional reactivity (10), especially in relation to heightened arousal under stressful situations (11), and difficulties in interpreting ‘fearful’ facial expressions (12,13). Taken together these data suggest the existence of gene-dosage sensitive mechanisms on the X-chromosome impacting on aspects of fear reactivity and anxiety.

In this work, we examined the evidence for gene dosage effects on the behavioural response to a fear inducing stimulus using a 39,XO mouse model that would also be haploinsufficient for non-inactivated X-linked genes. The 39,XO mice were generated by the fertilization of a normal gamete by a sex chromosome null gamete, and not, as in most cases of TS, by chromosome loss in early embryogenesis (7). As a result, the mouse model was completely free from the major problem of mosaicism (where the extent of chromosomal loss differs between different populations of cell lineages) that can potentially confound the interpretation of TS data (14). We further utilized the experimental tractability of the mouse model in examining possible molecular substrates of any behavioural effects. In particular, we tested the extent to which the gene Sts was important in the behavioural phenotype. Sts was an attractive a priori candidate for gene dosage effects on fear reactivity given its location in the PAR region of the X and its possible role in mediating fear responses by its actions in modulating neurosteroid function and hence the control of GABAA receptor function (15). GABAA receptors are key elements in the neural mechanisms underlying fear and anxiety (16). Moreover, in the case of Sts we could further exploit the mouse model by making use of 40,XY*X mice resulting from the production of the 39,XO animals (17). Y*X is in effect an X chromosome with a deletion from just proximal to Amel, to within the DXHXF34 repeat adjacent to the X centromere (18). Consequently it provides a complete PAR including the gene Sts, together with the small cluster of X-specific genes from the PAR boundary up to Amel (Fig. 1). Hence, by comparing 39,XO with 40,XY*X mice we could directly test whether a double dose of PAR (and therefore Sts) would rescue any X-monosomy effects.



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  		Figure 1. Schematic showing the genes that are known to be located on the Y*X chromosome.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Behaviour
Behavioural data were collected for 11 39,XO mice with a maternal X (XMO), 13 39,XO mice with a paternal X (XPO) generated in two separate crosses (1 and 3, see Materials and Methods), and for seven 40,XY*X mice and 13 40,XX mice, each deriving from two crosses (2 and 3). Preliminary analysis of the data revealed no significant differences between the XMO and XPO groups (data not shown) nor any effect of the cross of origin of the 40,XY*X mice and 40,XX littermate control mice. The data for mice originating from different crosses were therefore pooled into three groups; 39,XO, 40,XY*X and 40,XX.

39,XO and 40,XY*X mice showed an increased level of fear reactivity relative to 40,XX mice, as indexed by reduced time spent on the open arm of the elevated-plus maze (Fig. 2A; main effect of KARYOTYPE, F2,43=7.46, P<0.005). Post hoc tests (Tukey–Kramer) revealed that 40,XX were significantly different from both 39,XO and 40,XY*X (q=4.98, P<0.01; and q=4.35, P<0.05, respectively) but that 39,XO and 40,XY*X were not significantly different from one another (q=0.76, NS). Further evidence for increased fear reactivity in 39,XO and 40,XY*X mice on the elevated-plus maze was provided by the number of entries into the open arm (Fig. 2B; main effect of KARYOTYPE, F2,43=8.42, P<0.001). Again, post hoc tests revealed that 40,XX is significantly different from 39,XO and 40,XY*X (q=5.60, P<0.001; and q=4.01, P<0.05, respectively), but that 39,XO and 40,XY*X were not significantly different from one another (q=0.11). The elevated-plus maze data were not confounded by differences in oestrus cycle as there was no difference between the groups with regards to cycle length (grand mean of 6.4 days, H2=0.66, NS), and this was reflected in the fact that on the test day there was no difference in the distribution of the groups between the different stages of the oestrus cycle (proestrus, oestrus, dioestrus, {chi}2=1.64, NS). The fear reactivity data were also not confounded by any locomotor activity differences, either as measured directly on the elevated-plus maze (Fig. 2B; total arm entries, main effect of KARYOTYPE, F2,43=1.56, NS), or on the independent measure of activity in the locomotor activity cages (Fig. 2C; total beam breaks, main effect of KARYOTYPE, F1,66=0.07, NS). Furthermore, the 39,XO, 40,XY*X and 40,XX mice showed no difference in their basic reactivity to novelty as indexed the rate of habituation to the activity boxes over three consecutive days of testing (Fig. 2C; main effect of DAY, F2,131=7.01, P<0.001, but no interaction with KARYOTYPE, F4,131=0.28, NS).



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  		Figure 2. Behavioural measures of fear reactivity, locomotor activity and reactivity to novelty in 39,XO, 40,XY*X and 40,XX mice. (A) Mean±SEM time spent on the open arm of the elevated-plus maze. (B) Mean±SEM number of entries into the open arm and total number of arm entries. (C) Mean±SEM total beam breaks in a session as measured in the locomotor activity cages on day 1, day 2 and day 3 of testing.

 
Gene expression analysis
We assessed the expression level of the pseudoautosomal X-chromosome gene Sts in the brains of 40,XX and 39,XO mice using quantitative real-time PCR (Qt-PCR). In addition, given the central role played by Sts in regulating the switch between sulphated neurosteroids [antagonistic modulators of GABAA receptors (19,20)] and non-sulphated neurosteroids [agonistic effects on GABAA receptors (21)], we also assessed the relative expression levels of a number of key GABAA subunits; Gabra1, Gabra2, Gabra3 and Gabrg2. Using glyceraldehyde-3-phosphate dehydrogenase (Gapdh) to generate {Delta}CT values, expression of Sts (Fig. 3), was found to be significantly lower in brain tissue taken from 39,XO mice than in 40,XX (t=4.08, P<0.01). Moreover, the difference in {Delta}CT values between 40,XX and 39,XO was c. +1, consistent with the predicted halving of expression levels in 39,XO mice were Sts to be biallelically expressed. 39,XO mice also showed a relative reduction in expression in the brain of Gabra1 (t=4.517, P<0.01) and Gabrg2 (t=5.402, P<0.01) and an increase in Gabra3 (t=2.28, P<0.05) relative to 40,XX mice (Fig. 3). In contrast, expression levels of Gabra2 were unaltered between the two groups (t=0.04, NS). However, Qt-PCR analysis of gene expression in 40,XY*X relative to 40,XX mice, revealed that none of the genes that showed altered levels of expression in the brains of 39,XO mice were significantly changed in 40,XY*X mice (Fig. 4). The PAR gene Sts (t=0.49, NS), and the GABAA subunits, Gabra1 (t=0.15, NS), Gabra3 (t=0.43, NS) and Gabrg2 (t=0.90, NS), were all expressed at equivalent relative levels in the brains of 40,XY*X and 40,XX mice. The same patterns of gene expression levels in all groups were also seen when using hypoxanthine phosphoribosyltransferase (Hprt) to generate {Delta}CT levels (data not shown), indicating that the observed differences were real changes and not due to artefacts in the normalization process.



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  		Figure 3. Mean fold change in expression of Sts, Gabra1, Gabra2, Gabra3 and Gabrg2 in the brains of 39,XO mice (relative to 40,XX). The mean fold change is generated by transforming {Delta}{Delta}CT (see Materials and Methods) in the following manner; 2–{Delta}{Delta}CT (48). Consequently, the error bars (±SEM) are asymmetric. There was a reduction by half in the expression of Sts in the brains of 39,XO mice. There was also a significant reduction in expression of Gabra1 (c. –20%), a significant increase in the expression of Gabra3 (c. +30%) and a significant reduction (c. –40%) of Gabrg2.

 


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  		Figure 4. Mean fold change in expression of Sts, Gabra1, Gabra3 and Gabrg2 in the brains of 40,XY*X mice (relative to 40,XX). As described previously, the mean fold change is generated by transforming {Delta}{Delta}CT (see Materials and Methods) in the following manner; 2–{Delta}{Delta}CT (48) and consequently, the error bars (±SEM) are asymmetric. No significant differences in expression levels between the groups were detected for any gene analyzed.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
A main finding was that 39,XO mice showed an increased fear response in comparison with 40,XX control animals, as indexed by behaviour on the elevated-plus maze. Importantly, in light of human data examining other behaviours (22), this monosomy-X effect was indifferent to the parental origin of the X-chromosome (data not shown). The behavioural data were also not confounded by any pre-existing group differences in locomotor activity (23) or reactivity to novelty. These data indicate the existence in mice, of a gene (or genes) on the X-chromosome that mediates fear reactivity, and is biallelically expressed in 40,XX females (i.e. escapes X-inactivation). The 39,XO mice are haploinsufficient for this gene(s) and the effect of the ensuing reduced gene dosage is increased fearfulness.

We hypothesized that the PAR gene Sts may provide a valid candidate for the effects on fear reactivity. First, there is strong evidence that Sts escapes X-inactivation in the mouse (and probably also in humans) (24), a conclusion verified by our own expression analysis data. In addition, Sts is likely to be linked to fear reactivity via interactions with neurosteroids. Specifically, the product encoded by the Sts gene, STS, is an enzyme that is responsible in brain for the switch between sulphated and non-sulphated forms of neurosteroids such as the 3{alpha}-hydroxysteroids (allopregnalone, pregnalone) (15). These neurosteroids in their free form are known to be agonistic modulators of GABAA receptors (21), and have anxiolytic (25), anticonvulsant (26,27) and sedative properties (28). Conversely, neurosteroid sulphate esters appear to have antagonistic effects on GABAA receptors (19,20). In terms of the current findings, a reduction in Sts expression, as seen in our 39,XO animals, would be expected to lead to lower levels of free form neurosteroids and increased sulphate forms (15), which would, in turn, predict anxiogenesis. It was interesting, therefore, that we found that changes in expression of Sts were accompanied by alterations in the expression levels of the GABAA subunit genes Gabra3 (increased), and Gabra1 and Gabrg2 (both decreased) in the brains of 39,XO mice. Furthermore, two of these GABAA subunits, Gabra3 and Gabrg2, are important components of those GABAA receptor sub-types that mediate fear reactivity behaviour (29,30).

Our test of the causal links between Sts haploinsufficiency and the changes in fear reactivity of the 39,XO mice was to examine behaviour and brain gene expression in 40,XY*X mice. 40,XY*X mice possess a normal X (maternally inherited) and Y*X chromosome. As described earlier (see Introduction), the Y*X chromosome provides a complete PAR (and therefore Sts) plus a limited number of other genes, and as a consequence is an excellent test of whether a double dose of PAR rescues any X-monosomy effects (31). Expression analysis confirmed that the level of Sts in the brains of 40,XY*X mice was equivalent to 40,XX. However, having a double dose of PAR failed to rescue the fear related behavioural phenotype as 40,XY*X subjects performed to an equivalent degree as 39,XO mice on the elevated-plus maze test. Furthermore, expression analysis revealed that this behavioural trait was present in XY*X mice despite normal expression levels of the GABAA subunits that were shown to be altered in the brains of 39,XO mice. Overall, these results suggest two things; first, there is a non-PAR gene that escapes X-inactivation and influences fear reactivity; and second, X-monosomy changes in GABAA subunit expression in the brain are caused by a lack of PAR, and probably due to a half dosage of Sts, but crucially these GABAA subunit expression changes are not responsible for the increased fear reactivity.

The earlier mentioned dissociations suggest a novel genetic mechanism of potential relevance to the molecular neurobiology of fear, but leave the precise mechanism(s) and identity of the causal gene(s) unknown. In terms of physiological mechanism, the X-related effects on fear reactivity may be related to underlying effects on hormone levels in the XO mice. This possibility arises from the observations of altered hormonal status in TS girls, most pertinently oestrogen, and evidence that sex steroids can affect fear responses (32). However, XO mice may be considered as a poor model for the hormonal aspects of TS in that they are universally fertile and, as demonstrated in the present work, do not show differences in the oestrus cycle when compared with XX females. Furthermore, any direct link between variability in oestrogen levels and behaviour on the elevated-plus maze with respect to the present data is further undermined by the absence of any systematic relationship between stage of oestrus and fear reactivity. It is possible therefore, that the monosomy-X effects did not involve effects that were secondary to changes in oestrogen, but were instead due to direct effects on the brain circuitry mediating fear.

There are also uncertainties as to the gene candidate(s) ultimately responsible for the fear phenotype in the XO mice. However, our experiments limit the characteristics of any such gene(s). First, it must be located outside the PAR; and second, it must escape inactivation. There are currently seven genes known to escape (or at least partially escape) X-inactivation in the mouse, although the more extensively analyzed human X is thought to contain many more (33). Two of these seven genes on the mouse X are also present on the Y*X chromosome (Sts and Mid1; Fig. 1), and so both can be excluded as candidates. The remaining five (Enox, Utx, Dbx, Smcx and Eif2s3x) are therefore all potential causal genes. Of particular interest is Utx as this gene is known to escape activation in both mouse and humans, and is found within a 4.96 Mb region of the human X (Xp11.3) that influences fear reactivity and development of the amygdala and orbitofrontal cortex in TS subjects (13). However, further work is required to establish whether Utx has a role in the neural circuitry of fear related behaviour.

As well as the effects on fear, the 39,XO mice showed changes in the pattern of GABAA subunit gene expression in the brain, and these data may also be of relevance to the Turner's literature. As discussed previously, this molecular phenotype (in contrast to the fear phenotype) was rescued in the 40,XY*X mice, suggesting that it is dependent on a double dose of PAR, and most probably Sts, which is also thought to escape inactivation in humans (6,24). Although a link between GABAA and Sts has been established via the latter's action on neurosteroids (mentioned earlier), the mechanism by which GABAA subunit expression levels are altered remains to be established, although, there is evidence to suggest that one of the consequences of the actions of neurosteroids is to alter the pattern of GABAA subunit expression (34,35), which may, in turn, change the stoichiometry of GABAA receptor subunits. Previous work has shown the lability of GABAA receptor subunit composition in terms of compensatory changes following the selective knock-out of GABAA receptor subunits. For example, mice null for Gabra1 show a relative increase in expression of Gabra2 and, in particular, Gabra3 in the brain (36,37). This molecular lability can have functional consequences. In the case of the Gabra1 null mice, the alterations in subunit assembly gave rise to a shift in the pharmacological sensitivity of the benzodiazepine site that was related to an underlying change from type I BZ receptors (alpha1 containing) to type II BZ receptors (alpha2 or 3 containing) (38). Although we have shown in this work that the changes in GABAA subunit expression are unlikely to be responsible for the alteration in fear reactivity seen in 39,XO mice, they may cause other neural or behavioural changes. The possibility exists therefore, that similar changes in GABAA receptor functioning may contribute to the wide range of non-emotional behavioural changes found in TS girls, such as those indexing alterations in attentional and cognitive abilities (39).

In summary, we have for the first time demonstrated the existence of X-linked gene dosage effects on fear reactivity using the 39,XO mouse model. The 39,XO mice also showed highly specific alterations in GABAA subunit expression in the brain. A rescue experiment using 40,XY*X mice demonstrated that the fear reactivity is due to haploinsufficiency of a non-PAR gene, and is independent of the alterations in GABAA subunit expression, which are probably due to a haploinsufficiency for Sts. The data are consistent with work in TS girls and suggest novel neurobiological substrates of fear and anxiety. In addition, these data may have wider implications in that those X-linked genes that escape inactivation have the potential to generate sexually dimorphic traits owing to the fact that males have half the gene dosage of females (1,40). Even those genes that escape inactivation and have Y homologues, such as Utx, also have the potential to generate sexual dimorphisms, as often the Y-homologue in males does not fully compensate for the lack of a second X-homologue (41). Although the wider literature is far from conclusive, a recent heritability analysis suggests that rodents do display sex differences in fear reactivity, and as would be predicted by the data presented here, it is males who display increased fear reactivity (42). Clearly, the extent to which gene dosage mechanisms operating on the sex chromosomes contribute to sex differences in emotional behaviours both within patient and normal populations remains an important issue.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 REFERENCES
 
Animals
All mice were produced on a random bred MF1 albino (NIMR stock) background and derived from three crosses: (1) 40,XXx40,XPafY*. This cross generates a high frequency of 39,XMO (single X maternally inherited) together with 40,XXPaf females (17). (2) 40,XXx40,XY*. This cross generates a much lower frequency of 39,XMO females (43), but was included because it provides 40,XX litter mates that do not carry the Paf mutation and provides a greater number of XY*X females. (3) 40,In(X)1H/XPafx40,XY. This cross generates a high frequency of 39,XPO females owing to sex chromosomal loss following crossing over within the large X inversion In(X)1H (44,45). The Paf mutation was included on the normal X in order to provide Paf-carrying females (40,XPafX) for comparison with the Paf-carrying 40,XXPaf females from cross 1. Normal XX females are also produced following transfer of Paf from the X to the In(X) by recombination. All In(X)1H carrier females were excluded from the study following PCR identification as described by Ishikawa et al. (46). All the animals were generated at the MRC-National Institute of Medical Research (London, UK) and transported to The Babraham Institute (Cambridge, UK) as adults (~8 weeks old). Here, they were allowed to settle for 2 weeks and were then handled daily for 2 weeks before being assessed in the behavioural tasks at ~12 weeks of age. The animals were housed in groups of between two and five under temperature-controlled conditions and a 12 h light and 12 h dark cycle (lights on at 07.30 h). Standard laboratory chow and water was available ad libitum throughout testing. Oestrus cycle was analyzed by vaginal smear and staining using 10% toluidine blue (w/v). Smears were classified into three types indexing dioestrus, proestrus and oestrus stages of the oestrus cycle. The length of oestrus cycle was analyzed using the Kruskal–Wallis test. All experimenters were blind to the identity of the animals until the end of the experiment when the animals were culled, bone marrow removed from the femur, and chromosome spreads generated in order to allow karyotyping (47). All procedures were conducted in accordance with the requirements of the UK Animals Scientific Procedures Act (1986), under project license number PPL 80/1315.

Behaviour
The 39,XO and 40,XY*X mice were compared with 40,XX controls pooled from crosses 2 and 3. To account for possible order effects subjects were randomly assigned to two groups and assessed in two separate behavioural tests. The first group of animals was assessed in the following order; locomotor activity, elevated-plus maze. The second group was tested in the reverse order (elevated-plus maze, locomotor activity). Locomotor activity was measured using a battery of activity cages fitted with infrared beams. Activity data were collected in 5 min bins over a period of 1 h under red illumination. The subjects were assessed on three successive days. The elevated-plus maze consisted of a cross shaped maze (175 mmx78 mm) raised off the ground by 500 mm with two opposite arms open, and the other two arms enclosed by walls (150 mm high). The test was run in low level white and red light. The animals were placed on the maze and allowed to explore freely for 5 min. Each session was videotaped by a camera attached to the ceiling of the room and the videotapes analyzed afterwards. The main measures were the time spent in each location (closed and open arms, and the middle) and the entries into each arm. All data were analyzed by ANOVA, with factors KARYOTYPE (39,XO, 40,XX or 40,XY*X) and DAY (day of locomotor testing). All animals were smeared and tested for stage of oestrus cycle on each day of behavioural testing. The distribution of individuals from each group between the three stages of the oestrus cycle was analyzed by {chi}2 test.

Gene expression analysis
Gene expression analysis was performed by Qt-PCR on cDNA samples derived from total RNA extracted from sagitally dissected hemi-brains. The individual samples represented a subset of the behavioural cohort in each experiment. RNA was extracted from homogenized tissue (Ambion) according to the manufacturer's instructions. To remove residual DNA the RNA was then DNase treated (Ambion). RNA quality and concentration were then assessed by measurement of optical density at 260 and 280 nm. An amount of 1 µg of good quality RNA (260 : 280>1.85) was then reverse transcribed with 0.5 µg AMV reverse transcriptase (Roche) and poly-dT primers. Qt-PCR was performed using an ABI Prism 7700 sequence-detection system (PE Applied Biosystems, Warrington, UK). A 25 µl volume including diluted cDNA sample, 0.5 µmol/l primers (see Supplementary Material for primer sequences), 2 mmol/l magnesium chloride, nucleotides, Taq DNA polymerase and buffer included in the SYBR Green I Mastermix (PE Applied Biosystems). All samples were tested in triplicate in order to eliminate pipetting errors, and all primer pairs were designed to span two exons (i.e. the intron–exon boundary) thus eliminating possible signals from contaminating DNA. PCR cycling conditions were: 50°C for 2 min, 95°C for 10 min, 40 cycles of 94°C for 15 s and 60°C for 1 min. Qt-PCR data were obtained with the Sequence Detector Software (SDS version 1.6, PE Applied Biosystems). The amplification plots and predicted CT values from the exponential phase of the PCR were exported directly into Microsoft Excel (version 1998) worksheets for further analysis. The main measure taken to assess gene expression levels between groups was {Delta}CT. This is calculated by normalizing the CT value for a given sample to the CT value of a housekeeping gene, in this case Gapdh, for the same sample. Thus for each sample;

In order to check for possible between-group variations in Gapdh, we also calculated {Delta}CT using Hprt. Both {Delta}CTGapdh and {Delta}CTHprt values were transformed (2–{Delta}CT), then statistically analyzed using the Student's t-test to compare the two groups (39,XO and 40,XX or 40,XY*X and 40,XX) (48). The relationship between {Delta}CT and log input mRNA concentration was linear for all primer sets analyzed. The slope of the standard curve should be 0.1 for accurate mRNA transcript level determination. All standard curve and sample assays were performed in duplicate to improve the accuracy of mRNA transcript detection. No-template control assays were performed for each primer set used, which produced negligible signal detection, typically 38–40 CTs in value. All primer sets used for analyzing diluted cDNA samples typically produced 15–30 CTs on average, i.e. well within the accepted (0–40 CTs in value) detection window. In order to represent relative expression level graphically the numbers were expressed as a fold change relative to 40,XX expression as follows (48);


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
REFERENCES
 
Supplementary Material is available at HMG Online.


   ACKNOWLEDGEMENTS
 
We thank Gareth Hathway and David Skuse for helpful discussion, and Obah Ojarikre and Shantha Mahadevaiah for their help karyotyping the mice. This work was supported by the BBSRC, UK. W.D. was sponsored by a BBSRC Animal Science Committee studentship and the Oon Khye Beng Ch'Hia Tsio prize studentship for preventative medicine offered by Downing College, Cambridge.


   FOOTNOTES
 
* To whom correspondence should be addressed. Fax: +44 1223496022; Email: lawrence.wilkinson{at}bbsrc.ac.uk


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 

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