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. 2014 Jan 29;9(4):587–599. doi: 10.4161/epi.27806

Epigenomic and transcriptomic signatures of a Klinefelter syndrome (47,XXY) karyotype in the brain

Joana Viana 1,†, Ruth Pidsley 2,3,†, Claire Troakes 2, Helen Spiers 2, Chloe CY Wong 2, Safa Al-Sarraj 2, Ian Craig 2, Leonard Schalkwyk 2, Jonathan Mill 1,2,*
PMCID: PMC4121369  PMID: 24476718

Abstract

Klinefelter syndrome (KS) is the most common sex-chromosome aneuploidy in humans. Most affected individuals carry one extra X-chromosome (47,XXY karyotype) and the condition presents with a heterogeneous mix of reproductive, physical and psychiatric phenotypes. Although the mechanism(s) by which the supernumerary X-chromosome determines these features of KS are poorly understood, skewed X-chromosome inactivation (XCI), gene-dosage dysregulation, and the parental origin of the extra X-chromosome have all been implicated, suggesting an important role for epigenetic processes. We assessed genomic, methylomic and transcriptomic variation in matched prefrontal cortex and cerebellum samples identifying an individual with a 47,XXY karyotype who was comorbid for schizophrenia and had a notably reduced cerebellum mass compared with other individuals in the study (n = 49). We examined methylomic and transcriptomic differences in this individual relative to female and male samples with 46,XX or 46,XY karyotypes, respectively, and identified numerous locus-specific differences in DNA methylation and gene expression, with many differences being autosomal and tissue-specific. Furthermore, global DNA methylation, assessed via the interrogation of LINE-1 and Alu repetitive elements, was significantly altered in the 47,XXY patient in a tissue-specific manner with extreme hypomethylation detected in the prefrontal cortex and extreme hypermethylation in the cerebellum. This study provides the first detailed molecular characterization of the prefrontal cortex and cerebellum from an individual with a 47,XXY karyotype, identifying widespread tissue-specific epigenomic and transcriptomic alterations in the brain.

Keywords: Klinefelter syndrome; DNA methylation; gene expression; 47,XXY; prefrontal cortex; cerebellum

Introduction

Klinefelter syndrome (KS) is the most common sex-chromosome aneuploidy in humans, affecting approximately 1 in every 600 newborn males.1 Most individuals with KS carry one extra X-chromosome (karyotype 47,XXY), although other reported variants include 48,XXXY, 48,XXYY and 49,XXXXY.2 The condition presents with a broad range of phenotypes that often vary in severity.2 In addition to the well-characterized physical and physiological features—tall stature, gynecomastia, hypogonadism, and absent spermatogenesis3—KS is also often associated with psychiatric and neurodevelopmental phenotypes including language-based learning disabilities, decreased verbal intelligence and difficulties with task planning and inhibitory control.4 Of note, individuals with KS frequently exhibit symptoms related to schizophrenia including schizotypal traits,5 auditory hallucinations6 and verbal cognition impairment.7 Furthermore, several structural brain abnormalities are associated with the disorder including abnormal cerebral asymmetry8,9 and reductions in the size of specific brain regions,6,10,11 total brain volume,10,12,13 and white matter volume.9,10

The mechanism(s) by which the supernumerary X-chromosome determines the phenotypes evident in KS are poorly understood, although skewed X-chromosome inactivation (XCI), gene-dosage dysregulation, and the parental origin of the extra X-chromosome have all been implicated,14-16 suggesting that epigenetic processes play an important role. However, little is known about the specific epigenetic changes associated with KS, especially in tissues relevant to the KS phenotype. A study comparing global long interspersed nucleotide element-1 (LINE-1) DNA methylation in whole blood from individuals with Turner’s syndrome (45,XO), healthy males (46,XY), healthy females (46,XX) and KS patients (47,XXY) reported that increased chromosomal number was associated with hypomethylation across the genome.17 Studies of genome-wide patterns of DNA methylation in patients with trisomy 2118 and trisomy 819 also reveal large changes not limited to the supernumerary chromosome, indicating that widespread epigenetic changes may be a common feature of chromosomal aneuploidy. Moreover, differences in brain morphology have been associated with imprinting of the X-chromosome in Turner Syndrome, indicating that the parental origin of the X-chromosome may be important in mediating the psychiatric symptoms present in sex abnormalities.20

This study is the first to examine genome-wide patterns of DNA methylation and gene expression in two regions of the brain obtained post-mortem from a patient with a 47,XXY karyotype. We identify widespread tissue-specific epigenomic and transcriptomic alterations, providing potential clues about the molecular causes and consequences of KS.

Results and Discussion

As part of an integrated “-omics” study of schizophrenia (Pidsley et al., submitted), we examined genome-wide patterns of DNA methylation, gene expression, and genetic variation in post-mortem cerebellum and prefrontal cortex brain tissue samples from schizophrenia patients and controls. During the standard quality control steps of these data we identified a discrepancy between reported and measured sex for one schizophrenia patient who displayed the genomic characteristics expected of both male and female samples simultaneously. As expected, across the entire set of samples, males and females showed distinct levels of DNA methylation across probes on the X-chromosome, with the exception of one sample recorded as male, who clustered with the female samples (Fig. S1A). This individual also clustered with females for XIST gene expression (Fig. S2A) but with males when DNA methylation and gene expression across the Y-chromosome were assessed (Figs. S1B and S2B). A 47,XXY karyotype was confirmed via the high-resolution SNP genotyping array data (Figs. S3 and S4) and the presence of the Y-chromosome was confirmed via a PCR-based sex-typing assay (Fig. S5). A number of other genomic alterations were identified in this individual (Table S1), although there was no obvious excess burden of autosomal copy number variations (CNVs), except for one region with four copies of a region spanning the NKAIN2 locus. This is notable since copy number changes in this gene have previously been reported in neuropsychiatric phenotypes including neurodevelopmental disorders and schizophrenia.21-24 The patient’s autopsy report did not record KS, suggesting that the 47,XXY karyotype was undetected during the patient’s lifetime, although we did not have access to detailed pre-mortem medical records. In addition to schizophrenia, the autopsy report highlighted that the 47,XXY patient had a poor nutritional state, hepatomegaly, vascular spiders, nystagmus, dysdyadocokinesia, some degree of ataxia at the time of death, a known history of alcohol abuse and were prescribed the medications parentrocite and sulpiride.

Identifying structural brain abnormalities associated with a 47,XXY karyotype is of relevance given the established link between KS and several neuropsychiatric disorders including schizophrenia.5,25 Detailed records taken by the neuropathologist at autopsy show that although the 47,XXY patient had a similar total brain mass to other patients (47,XXY = 1417 g, all other samples = 1410 ± 182 g, other males = 1454 ± 201 g, females = 1325 ± 92 g) they had a markedly lower cerebellum mass (47,XXY = 111 g, all other samples = 170 ± 24 g, other males = 175 ± 27 g, females = 160 ± 15 g), equating to more than two standard deviations below the mean of the other samples (Fig. 1; Fig. S6). The reduced cerebellum mass is consistent with the patient’s autopsy report of movement disorders, and previous studies demonstrate an association between cerebellar ataxia and reduced cerebellar size.26 Reductions in cerebral volume and decreased cortical thickness in the left inferior frontal, temporal, and superior motor regions have been previously reported in KS,13 and a recent imaging study showed significant reductions in the volume of several brain regions, including the cerebellum, in KS patients compared with controls.10 Other studies have also described brain abnormalities in KS patients, including reductions in total brain volume,12 specific brain regions6,11 and white matter volume,9 as well as abnormal cerebral asymmetry.8,9 These latter findings are consistent with the theory that brain asymmetry and cerebral dominance in humans is determined by a XY homologous gene pair.27 Structural brain abnormalities have also been identified in patients with other types of X-chromosome aneuploidy28; a recent study reported increased cerebellum volume in Turner syndrome (45,XO) patients, contrasting with the decreased cerebellum volume in our patient (47,XXY).20 However, because reduced cerebellum mass has been associated with alcoholism29 it is possible that the reduction observed in the 47,XXY patient is related to the their alcohol abuse history.

graphic file with name epi-9-587-g1.jpg

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Figure 1. Reduced cerebellar mass in a 47,XXY patient comorbid for schizophrenia. Shown is the average cerebellar mass (in grams) across all samples compared with the cerebellar mass of the 47,XXY patient. Shown are the minimum, maximum, median and interquartile range of the cerebellar mass.

Global DNA methylation levels were estimated in both brain regions across all individuals using bisulfite-pyrosequencing assays targeting LINE-1 and Alu repeat elements across the genome, as described previously.30,31 Relative to other samples, the 47,XXY patient was a striking hypomethylated outlier across both LINE-1 (47,XXY = 67.7%, other samples = 73.0 ± 2.3%) and Alu (47,XXY = 25.5%, other samples = 28.1 ± 2.6%) repetitive elements in the prefrontal cortex (Fig. 2A and B; Fig. S7A and B). Conversely in the cerebellum, the 47,XXY individual showed notable hypermethylation compared with the other samples at LINE-1 repetitive elements (47,XXY = 78.4%, other samples = 71.9 ± 2.1%), although no significant difference was observed at Alu repetitive elements (47,XXY = 24.7%, other samples = 24.8 ± 0.8%) (Fig. 2C and D; Fig. S7C and D).

graphic file with name epi-9-587-g2.jpg

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Figure 2. Tissue-specific differences in global DNA methylation in a 47,XXY patient comorbid for schizophrenia. The 47,XXY patient is significantly hypomethylated in the prefrontal cortex across both LINE-1 (A) and Alu repetitive elements (B) but hypermethylated in the cerebellum at LINE-1 elements (C), compared with the other samples. No significant difference was observed for DNA methylation across Alu repeat elements in the cerebellum (D). Shown are the minimum, maximum, median and interquartile range of the DNA methylation values.

Using the Infinium HumanMethylation450 BeadChip (Illumina Inc.) we identified numerous autosomal regions showing consistent differential DNA methylation in the 47,XXY patient compared with other samples (Tables 1 and 2). Of note, a region within the sperm-associated antigen 1 (SPAG1) gene is significantly hypermethylated in both the prefrontal cortex and the cerebellum, an interesting observation in the context of the infertility associated with KS given the role of this gene in spermatogenesis, fertilization, and infertility.32 Other differentially methylated regions (DMRs) were found to be tissue-specific, and/or only detectable relative to either female or male controls. Of potential relevance to KS, for example, are cerebellar DMRs in the vicinity of Piwi-like protein 1 (PIWIL1), which plays a role in the self-renewal of germline stem cells.33 Also of interest, given the cerebellar abnormalities seen in this individual, is evidence for cerebellum-specific hypermethylation of the LIM/homeobox 4 (LHX4) gene; mutations in this gene have been associated with altered brain development and cerebellar structure.34 Given the comorbid diagnosis of schizophrenia in this patient, it is interesting that several of the prefrontal cortex DMRs are located in close proximity to other neurobiologically-relevant genes, including NOTCH4, EPHB3 and KCNN1, that have been previously implicated in schizophrenia,35 brain development36-38 and regulation of microglia and neurons during neuroinflammation.38 The large differences in DNA methylation reported here are specific to the XXY individual; none of these regions are significantly differentially methylated in our analysis of schizophrenia and matched controls (Pidlsey et al., submitted).

Table 1. 47,XXY-associated differently methylated regions in the prefrontal cortex.

          Gene           DMR position (hg19)           Probes in DMR           47,XXY vs. all other           47,XXY vs. other males           47,XXY vs. females
          β difference           P value           β difference           P value           β difference           P value
          VWA1           chr1:1374601–1374669           2               -0.05           6.28E-05    
            chr1:182669244–182669315           2                   0.13           3.14E-06
            chr1:247681102–247681931           8               0.16           1.35E-06    
          OR2L13           chr1:248100585–248100614           4                   0.3           7.88E-06
            chr2:4931004–4931074           2           -0.23           2.07E-08           -0.23           3.66E-08           -0.23           3.93E-09
          MAP4K3           chr2:39665105–39665186           2                   -0.01           4.03E-05
          PCBP1           chr2:70313772–70313833           2                   0.01           2.57E-05
          LOC388965           chr2:84517321–84517950           9               -0.03           3.14E-05    
          CXCR1           chr2:219031640–219031719           2                   -0.05           2.61E-05
          IHH           chr2:219922729–219922998           2                   0.02           6.33E-07
            chr2:240530497–240530569           2                   -0.16           2.46E-05
          WNT5A           chr3:55515168–55515541           3                   0.03           2.18E-05
          NEK11           chr3:130745442–130745959           13           0.04           7.82E-05               0.04           < 2.00E-16
          DVL3           chr3:183887905–183888477           4                   -0.05           4.49E-05
          EPHB3           chr3:184297380–184297522           3               -0.07           1.33E-05    
            chr3:195578011–195578280           6                   0.22           2.18E-05
            chr4:53588360–53588850           6                   0.03           1.68E-06
          SNHG8           chr4:119199621–119200372           11           0.04           2.30E-07           0.04           3.82E-11           0.04           2.16E-06
            chr5:784832–784915           3           0.5           < 2.00E-16           0.5           < 2.00E-16           0.5           < 2.00E-16
          RIPK1           chr6:3077011–3077041           2                   -0.16           9.23E-05
          NOTCH4           chr6:32179862–32179971           2                   -0.02           1.43E-05
          TAPBP           chr6:33269769–33269832           2                   -0.2           1.29E-08
            chr6:168120556–168120635           2               -0.06           3.07E-06           -0.05           1.94E-06
          MAD1L1           chr7:2144559–2144767           3               -0.09           8.00E-07    
            chr7:157075207–157075303           2               -0.1           2.74E-05    
          PTPRN2           chr7:157744316–157744347           2           -0.06           < 2.00E-16           -0.06           < 2.00E-16           -0.06           < 2.00E-16
            chr8:58055876–58056026           2           0.39           1.20E-05           0.4           2.33E-06           0.38           3.12E-05
          C8orf71           chr8:58192753–58192883           2               -0.22           9.11E-05    
          SPAG1           chr8:101224915–101225361           5           0.13           < 2.00E-16           0.13           < 2.00E-16           0.14           < 2.00E-16
          SPAG1           chr8:101225800–101225902           2           0.22           < 2.00E-16           0.22           6.54E-12           0.23           < 2.00E-16
            chr10:2978126–2978687           5                   -0.1           7.97E-09
            chr10:8090846–8090924           2                   0.04           7.87E-05
          STK32C           chr10:134062522–134062614           2                   -0.21           1.95E-05
          TMBIM4           chr12:66563381–66563928           10           0.03           3.12E-06           0.03           2.10E-07           0.03           4.13E-08
          GALNT9           chr12:132904540–132904689           2               -0.11           3.24E-06    
          NDRG2           chr14:21493406–21493410           2                   0.03           2.15E-06
            chr14:95837801–95837929           3           -0.05           1.77E-06           -0.06           2.54E-06           -0.05           3.52E-07
          LBXCOR1           chr15:68125566–68125599           2                   -0.2           7.84E-06
            chr15:68126065–68126178           2                   -0.04           8.16E-05
            chr19:17504632–17504972           3                   0.15           2.61E-07
          KCNN1           chr19:18077727–18077834           3                   0.02           4.85E-08
          AXL           chr19:41731934–41732589           5                   0.03           3.79E-06
          KDELR1           chr19:48894694–48895030           9           0.03           4.30E-05               0.03           3.79E-11
            chr21:44573854–44574022           3           -0.09           1.02E-07           -0.09           9.20E-08           -0.08           9.79E-10
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Light gray boxes indicate non-significant results. Dark gray boxes indicate that no comparison was made (X-chromosome-linked genes were compared with females only whereas Y-chromosome-linked genes were compared with males only).

Table 2. 47,XXY-associated differently methylated regions in the cerebellum.

          Gene           Position (hg19)           Probes in DMR           47,XXY vs. all other           47,XXY vs. males           47,XXY vs. females
          β difference           P value           β difference           P value           β difference           P value
          LHX4           chr1:180201891–180201893           2                   0.26           5.42E-05
          LHX4           chr1:180202256–180202784           2                   0.09           2.92E-09
          LHX4           chr1:180203135–180203143           8                   0.05           1.49E-06
          LAMB3           chr1:209799191–209799353           4           -0.14           7.94E-05               -0.14           3.66E-07
          TTC15           chr2:3469154–3469529           2               -0.19           2.47E-07    
          TET3           chr2:74328923–74329082           2               -0.07           5.17E-05    
            chr3:87137933–87138700           2           0.11           < 2.00E-16           0.11           < 2.00E-16           0.1           2.66E-12
          CLDN18           chr3:137728810–137729296           9                   -0.13           2.25E-05
          NHEDC1           chr4:103940711–103941300           2               0.08           1.96E-07    
            chr5:784832–784915           2           0.43           < 2.00E-16           0.43           < 2.00E-16           0.43           < 2.00E-16
            chr5:68628240–68628738           2               0.1           3.90E-06    
          RIPK1           chr6:3077011–3077041           3                   -0.17           6.98E-05
          HIST1H3C           chr6:26045532–26045663           13           0.1           3.02E-12           0.1           4.68E-09           0.1           < 2.00E-16
          PTPRN2           chr7:157744316–157744347           4               -0.05           7.10E-10    
            chr8:58055876–58056175           3           0.43           8.50E-05           0.44           1.55E-06    
          C8orf71           chr8:58191386–58192065           6           -0.25           4.89E-08           -0.26           3.82E-09           -0.25           1.38E-07
          SPAG1           chr8:101224915–101225361           6           0.13           < 2.00E-16           0.13           < 2.00E-16           0.13           < 2.00E-16
          SPAG1           chr8:101225800–101225902           11           0.21           < 2.00E-16           0.21           < 2.00E-16           0.21           < 2.00E-16
          C9orf64           chr9:86571409–86572014           3           0.1           6.98E-05           0.1           6.25E-06    
          KIAA1274           chr10:72254314–72254335           2                   -0.11           1.70E-06
          CALHM1           chr10:105218160–105218286           2                   -0.1           6.26E-05
          CALCB           chr11:15093613–15093769           2           -0.16           2.67E-06           -0.16           1.66E-06           -0.18           3.66E-07
          TP53AIP1           chr11:128812804–128813442           2                   -0.19           1.24E-05
          TP53AIP1           chr11:128812846–128813008           3               -0.19           7.61E-05    
          PIWIL1           chr12:130822286–130822818           2               -0.25           4.26E-05    
          NBEA           chr13:36044860–36045352           2           0.05           3.26E-05           0.05           2.21E-06           0.05           8.17E-05
            chr15:26489846–26490045           2               -0.05           8.82E-05    
          KRTAP17–1           chr17:39472114–39472340           2                   -0.15           9.52E-05
            chr17:55213563–55213600           5                   -0.15           9.68E-05
            chr17:77680078–77680232*           2           -0.19           6.73E-05               -0.19           3.31E-07
          LRP5L*           chr22:25758621–25758749           5               -0.1           8.18E-05    
          MXRA5           chrX:3264517–3265089           2                   0.04           1.92E-09
          TTTY14           chrY:21238886–21239607           2               0.09           1.18E-11    
          * CNV with copy number of 3 overlapping with the region (see Table S1)
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Light gray boxes indicate non-significant results. Dark gray boxes indicate that no comparison was made (X-chromosome-linked genes were compared with females only whereas Y-chromosome-linked genes were compared with males only).

Numerous genes were found to be differentially expressed (DE) in the 47,XXY prefrontal cortex and cerebellum compared with other samples (Tables 3 and 4) with a Bonferroni-corrected z-score P value < 0.05. In both brain regions the vast majority of DE genes were characterized by increased expression in the 47,XXY patient (prefrontal cortex: 12 loci significantly upregulated, 1 locus significantly downregulated; cerebellum: 18 loci significantly upregulated, 0 loci significantly downregulated), suggesting that the supernumerary X-chromosome may be upregulating transcription at multiple autosomal loci across the genome. Although some DE genes were observed only in comparisons with both 46,XY males and 46,XX females, most were sex-comparison-specific. Strikingly, across both brain regions, many more 47,XXY DE genes were observed in comparison with females than males in both tissues (prefrontal cortex: 47,XXY vs females = 59, 47,XXY vs males = 18; cerebellum: 47,XXY vs females = 50, 47,XXY vs males = 21). Furthermore, although some changes were consistently observed in both the prefrontal cortex and cerebellum (e.g., CAMP, EPCAM and LOC441208), the majority were tissue-specific to a single brain region. The large differences in gene expression reported here are specific to the 47,XXY individual; none of these transcripts are differentially expressed in our analysis of schizophrenia and matched controls (Pidlsey et al., submitted).

Table 3. 47,XXY-associated differentially expressed genes in the prefrontal cortex.

          nuID           Gene           Probe position (hg19)           Strand           47,XXY vs. all other           47,XXY vs. males           47,XXY vs. females
          fold change           P value           fold change           P value           fold change           P value
          fVedl_tOxJHgKDgkUk           ANGPTL7           chr1:11255703–11255752           +           0.17           1.11E-16           0.17           5.11E-15           0.17           < 2.00E-16
          Zl4IEsHlNCOIgeii9c           VCAM1           chr1:101204019–101204068           +                   0.12           1.78E-08
          cqSF81At6xB7XyHjiQ           PRG4           chr1:186282872–186282921           +                   0.2           < 2.00E-16
          lojns5Hg3o6bW_X07 g           CFH           chr1:196648872–196648921           +                   0.23           1.43E-07
          Qo0T0_REI6OtlJLFOU           CFH           chr1:196658692–196658741           +                   0.38           1.11E-16
          NIDSs_kv.U.0.u7g.8           H3F3A           chr1:226259333–226259382           +               0.33           7.90E-07    
          NfqgAIRfuk8Lfn_vAk           EXOC8           chr1:231468728–231468777           -               0.21           1.68E-07    
          ZoXhLA6oEHo3o160B8           EPCAM           chr2:47606943–47606992           +                   0.69           1.11E-16
          BnrvPnvPuvuuMkvvno           EPCAM           chr2:47607046–47607095           +                   1.04           2.26E-08
          cpAs4gp4jAojq4jkyo           EPCAM chr2:47612328–47612349:
          47613711–47613738		           +           0.2           1.65E-09           0.2           2.98E-07           0.21           < 2.00E-16
          xd6Z60LRKEWCAU0bEY           EFEMP1           chr2:56149495–56149544           -               0.39           6.29E-10    
          9l31SOkN7XN3uegjnI           AOX1           chr2:201535434–201535483           +                   0.08           < 2.00E-16
          Zl7s26IBCIUH.09XlU           COL6A3           chr2:238232942–238232991           -                   0.39           6.62E-14
          QXNDUD1d6L9O85e_Sk           COL6A3           chr2:238233095–238233144           -                   0.13           6.77E-10
          rItcu7lcV6dKfep3iA           CAMP           chr3:48266905–48266954           +           0.2           < 2.00E-16           0.2           < 2.00E-16           0.2           < 2.00E-16
          69KffSiAkLEesX6HuQ           SLC25A20           chr3:48900042–48900091           -               0.36           5.69E-11    
          lnxCeHgiilejtJhR.U           DNASE1L3 chr3:58179070–58179093:
          58178505–58178530		           -                   0.13           < 2.00E-16
          6h.tJeHqxD3.vD_S7o           PHLDB2           chr3:111694722–111694770           +                   0.27           3.50E-09
          ommprt5mtriFZmriGc           FAIM           chr3:138327937–138327986           +                   0.11           3.54E-07
          ln0vn7fesf3vOp.6ok           TP63           chr3:189614936–189614985           +                   0.21           < 2.00E-16
          KpJwqU0.HokiEUlMrU           NULL           chr4:144496759–144496808           -                   0.09           5.31E-07
          35QRHIUd.o9CJHuzh4           GPX8           chr5:54460446–54460495           +                   0.15           < 2.00E-16
          riWe5en0.0i4hP7Huk           BTNL9           chr5:180488062–180488111           +                   0.2           3.57E-12
          QfyhEl_TCJbggfqkvk           LOC401233           chr6:3019504–3019553           -               0.15           7.79E-07    
          fajeo6Sh6PMkUgoxnk           BMP5 chr6:55625255–55625272:
          55623882–55623913		           -           0.19           2.30E-14           0.18           7.69E-12           0.19           < 2.00E-16
          K6oCu8IOLU3SUMufrs           TBX18           chr6:85444495–85444544           -                   0.12           6.56E-07
          07q5ey9ScdKKeKSiDg           IKBIP           chr6:168224491–168224540           -               -0.69           2.87E-07    
          0uHF8y.yziXFfoICwo           TMEM196           chr7:19759195–19759244           -                   0.21           4.12E-09
          Zn50ZUiIgAvCeeFeCk           INMT           chr7:30797037–30797086           +                   0.12           3.31E-12
          cyxSgtKKIa_cDsvc3o           LOC441208           chr7:32768986–32769035           +           0.32           < 2.00E-16           0.33           < 2.00E-16           0.32           7.88E-15
          iX3qB7qexxL0p66.jk           FGL2           chr7:76825768–76825817           -                   0.43           2.49E-11
          KmAXs1qVBeAE1SUIHo           COL1A2           chr7:94057677–94057726           +                   0.35           < 2.00E-16
          9jUT7yueFyEiOB4rX4           COL1A2           chr7:94060111–94060160           +                   0.45           < 2.00E-16
          QyCF6hIoee3ld3rRXk           DEFA1           chr8:6856660–6856709           -           0.12           1.38E-13           0.12           1.57E-12           0.12           1.11E-15
          xKBJBHXp4QcJQzpXK0           SCARA5           chr8:27727946–27727995           -                   0.26           9.82E-07
          0LiHngASd5JSA633Ro           SDCBP           chr8:59492276–59492306           +                   0.15           6.15E-07
          K7con83.RdfSKOc6rU           BNC2           chr9:16416697–16416746           -                   0.17           1.83E-13
          6oXSUI4JlkkvBc4W5I           CCL19 chr9:34689927–34689940:
          34689801–34689836		           -                   0.32           < 2.00E-16
          WrlIsC.cAwT13R_xVE           OGN           chr9:95146774–95146823           -                   0.77           4.11E-15
          BDhFLxNahZPgiMZeio           OGN           chr9:95147978–95148027           -                   0.88           < 2.00E-16
          ZjdFPOE1NVcnRRMkTI           OMD           chr9:95176881–95176930           -                   0.45           3.73E-07
          Kc4qiig6iKqdKpQwHc           SLC27A4           chr9:131123437–131123486           +                   0.09           3.26E-08
          oKAoV4glIpSToLgqkg           ITIH2           chr10:7788602–7788651           +                   0.2           1.24E-09
          lqMskrfH4OAt4z_eOk           OTUD1           chr10:23730535–23730584           +                   0.16           9.87E-11
          xqKT0lF6D5OJ2dL_o8           PDE6H           chr12:15134355–15134404           +                   0.16           2.54E-08
          ZfKOiSBOiKCP0.wnXU           SLCO1C1           chr12:20905935–20905984           +               0.13           2.83E-10    
          fX9eh..pR5XocuAg6E           PKP2           chr12:32944450–32944499           -                   0.21           2.92E-07
          cjif7cfQC.v58VfSXU           GJB2           chr13:20761910–20761959           -                   1           < 2.00E-16
          ciAGgiIoDiuq_igFTo           EDNRB           chr13:78470881–78470930           -           0.18           1.49E-07               0.18           1.11E-16
          xuigGSeQeCaIdwFSfk           PTGDR           chr14:52742862–52742911           +                   0.15           < 2.00E-16
          rl55P5uN0IXUlILV9Q           PTGDR           chr14:52743020–52743069           +                   0.32           < 2.00E-16
          0knRyVFXXc.6sIg.HE           TMEM30B           chr14:61744888–61744937           -                   0.45           < 2.00E-16
          TP6d2kdUp6iejF5XpI           LOC388152           chr15:84871644–84871693           -                   0.12           5.26E-09
          3eh0.Qkv5.70DbjiAU           NUDT21           chr16:56463489–56463538           -           0.15           1.04E-07           0.15           6.69E-08           0.15           5.92E-07
          KsuX1EryiLsDi3rL_0           TMEM220           chr17:10617182–10617231           -           0.09           2.07E-08           0.09           2.60E-09           0.08           3.79E-08
          x.Sd_F7Vd6eXeLeDdU           TOP2A           chr17:38545067–38545116           -           0.11           1.49E-07               0.12           < 2.00E-16
          EnpItS.SAMZeiUiS2E           FAM20A           chr17:66533566–66533615           -           0.12           2.70E-08           0.12           4.20E-09    
          9k3mzbqMPhOKn4iB1I           CD177           chr19:43867372–43867421           +           0.14           6.18E-14           0.13           8.74E-12           0.14           < 2.00E-16
          3CBVEhgxeipOOJilWo           MYL9           chr20:35176437–35176486           +                   0.11           1.34E-07
          No174RVAVBCigl6guU           SLPI chr20:43882216–43882241:
          43881769–43881792		           -                   0.27           < 2.00E-16
          ojLV_BETnlid6ABVEk           UBE2C chr20:44444504–44444552:
          44445348–44445348		           +                   0.1           < 2.00E-16
          r37yu690k8Sk.uedqI           LOC401397           chr20:57523176–57523225           -                   0.11           2.69E-08
          TdSCif1KMn_KMJ5o4k           LOC96610           chr22:22664198–22664247           +                   0.09           6.12E-08
          luuljtLxeOlOiCJCmE           ADRBK2           chr22:25817204–25817253           +                   0.1           1.11E-16
          TADKWp66dGXsNUkf6Q           GGA1           chr22:38013841–38013890           +           -0.83           9.84E-07           -0.86           2.61E-09    
          Z5K0omXVEuL9VRHadc           RRP7A           chr22:42907975–42908024           -                   0.08           < 2.00E-16
          EqZLp0Zez1SR.qKUKQ           RRP7B           chr22:42969512–42969561           -                   -0.55           6.55E-10
          0szegLje1eiuskL9Ro           NPM1           chrX:123414788–123414837           +               0.2           8.35E-08    
          lLh40p.7RHpRI4TceU           GYG2P1 chrY:14518992–14518993:
          14518689–14518736		           -                   0.11           1.21E-07
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Light gray boxes indicate non-significant results. Dark gray boxes indicate that no comparison was made (X-chromosome-linked genes were compared with females only whereas Y-chromosome-linked genes were compared with males only). Annotation data for each probe obtained using the Bioconductor package illuminaHumanv4.db.39

Table 4. 47,XXY-associated differentially expressed genes in the cerebellum.

          nuID           Gene           Probe position (hg19)           Strand           47,XXY vs. all other           47,XXY vs. other males           47,XXY vs. other females
          fold change           P value           fold change           P value           fold change           P value
          uO7tSXg9R5ohX55GF4           TNFRSF18           chr1:1140794–1140843           -                   0.15           1.01E-14
          TQ5MLOicLl.6KP36CI           FAM76A           chr1:28087875–28087924           +                   0.07           2.70E-07
          3VHqE.9440_ek4J75o           PCNXL2           chr1:233275460–233275463: 233270891–233270936           -                   0.14           6.48E-12
          ZoXhLA6oEHo3o160B8           EPCAM           chr2:47606943–47606992           +                   0.54           5.47E-12
          BnrvPnvPuvuuMkvvno           EPCAM           chr2:47607046–47607095           +                   0.9           3.44E-13
          6KDbq30yk7941631Kg           KCNH7           chr2:163228344–163228393           -                   0.12           7.42E-07
          6R96hdG6D63d4rLojk           LOC401052           chr3:10048205–10048254           -                   0.22           1.16E-10
          TQeEon6idqJ_oip878           ARPP21           chr3:35722564–35722613           +                   0.1           1.46E-12
          rItcu7lcV6dKfep3iA           CAMP           chr3:48266905–48266954           +           0.09           3.46E-12           0.09           1.86E-09           0.1           < 2.00E-16
          QEKvDrJSV50hXQ4Rno           ANKRD17           chr4:74005263–74005312           -                   0.22           1.98E-07
          0q44on0oRTyiSnoQTc           SH3TC2           chr5:148384360–148384409           -                   0.1           5.29E-11
          WVElhxxViAlxN9F9ec           RASGEF1C           chr5:179564876–179564899: 179564687–179564712           -               0.13           4.15E-07    
          01Xd110F6gx9ITdQHQ           RNF39           chr6_mcf_hap5:1420068–1420117           -                   0.16           6.29E-07
          0cVOsh0SeXuuB.p1eU           PRRC2A           chr6_dbb_hap3:2890088–2890137           +                   0.35           2.22E-07
          Tqg7v6gLQi6yj6_nqY           C6orf132           chr6:42070437–42070486           -                   0.23           3.15E-10
          HqI33tUcoB5g_xRIqI           C6orf176           chr6:166338009–166338058           -                   0.11           1.15E-08
          cyxSgtKKIa_cDsvc3o           LOC441208           chr7:32768986–32769035           +           0.14           3.61E-07        
          cyxSgtKKIa_cDsvc3o           LOC441208           chr7:32768986–32769035           +                   0.15           < 2.00E-16
          3WfZR.WSd6.5x7nndc           C7orf52           chr7:100813863–100813912           -           0.15           4.28E-09           0.15           1.03E-08           0.14           1.43E-09
          K_N4laRVOKE5IkXkig           CNTFR           chr9:34552159–34552166: 34552028–34552069           -           0.23           < 2.00E-16           0.23           3.99E-14           0.23           < 2.00E-16
          uJdPSKuSnCZOhiSaug           GPSM1           chr9:139252547–139252596           +                   0.25           2.95E-08
          90NSdfgVdSVn0l6k0c           ENTPD2           chr9:139942751–139942800           -                   0.09           6.35E-08
          3Z2Vqi1Jrte6J5ZTpU           IL15RA           chr10:6019449–6019498           -           0.08           8.68E-07               0.08           6.27E-08
          ZIRQ6g1Q6oLY56590U           IGF2           chr11:2159459–2159460: 2156712–2156759           -           0.1           9.90E-14           0.09           1.45E-12           0.1           < 2.00E-16
          Wl36k3qhE6w57l_Egs           CALCA           chr11:14988290–14988339           -           0.23           8.30E-09           0.23           5.73E-09           0.22           5.94E-09
          x9f4JKzfk6VFquC1eU           TMEM223           chr11:62558209–62558258           -                   0.28           6.22E-07
          9ooIoDrhULuqvuvSgI           SLC22A6           chr11:62744143–62744192           -           0.18           3.65E-07           0.18           9.29E-07           0.18           9.39E-08
          Z9IldVfociaepInc.BI           CNIH2           chr11:66051236–66051285           +           0.35           9.53E-07               0.36           4.88E-15
          HFwXl7tTpgV3E1wXlo           SMARCC2           chr12:56558210–56558259           -           0.18           8.81E-08           0.19           4.36E-08           0.18           6.14E-07
          QO_wesnjwsy9XkVfeo           LOC220115           chr13:53161055–53161104           +               0.12           8.32E-09    
          c70LXLcyj6S.A5.HVU           OLFM4           chr13:53626107–53626156           +           0.09           4.37E-07           0.09           1.59E-07    
          36JWb571RKl2v.XB_c           SPSB3           chr16:1826791–1826840           -                   -0.89           9.17E-07
          BloWN0NHoOuep7agcE           TNRC6A           chr16:24834917–24834966           +           0.13           9.96E-09           0.13           1.69E-08           0.13           9.43E-09
          QLR0VHu.euUKd_KlUc           FAM64A           chr17:6354072–6354121           +                   0.1           1.33E-07
          EdOgRNRCeCkhClZIJ0           RPL19           chr17:37360385–37360427           +                   0.7           6.11E-08
          x6gXnXotNXeQEiJUi4           RBFOX3           chr17:77303806–77303842: 77231875–77231887           -                   0.57           2.85E-09
          iIoD3lFP9UreSVdSeo           RBFOX3           chr17:77303839–77303888           -                   0.3           3.77E-07
          f4oYbqlTz8YVp9fc6U           FN3K           chr17:80708373–80708422           +           0.22           1.29E-10           0.21           7.08E-10           0.23           1.34E-14
          x3S.SNd5j.Pi6At_Z4           MGC70870 chr17_gl000205_random:
          119141–119190		           +                   0.31           3.30E-07
          oCVdUCZaWHZ7dnqh38           NFIC           chr19:3463515–3463564           +                   0.1           1.67E-08
          QudxDtFe.RtNJF3qhU           OLFM2           chr19:9964866–9964915           -           0.54           9.61E-07           0.57           1.20E-08    
          9RSiJQISr7l8VSiUVc           NACC1           chr19:13251492–13251541           +           0.49           7.50E-11           0.49           1.49E-08           0.5           < 2.00E-16
          uCKpSOEVwJKeNqQ6is           RAB3A           chr19:18307891–18307940           -                   0.93           1.71E-12
          l3roFeVfFJfjpJqDes           POU2F2           chr19:42595686–42595735           -                   0.09           6.52E-08
          T3DouuhUIkzS5yQDpI           UBE2V1           chr20:48700666–48700677: 48699413–48699451           -                   0.19           8.02E-09
          uXOOCKm1HogpVesKUk           KIAA1647           chr22:18958144–18958171           +                   0.12           6.51E-07
          i1_RF4d7R0Jf3UUpV0           SERPIND1           chr22:21141675–21141724           +                   0.1           4.00E-07
          Z5K0omXVEuL9VRHadc           RRP7A           chr22:42907975–42908024           -                   0.16           4.44E-15
          EU_ve.pMFc8DrmSJ4M           LOC389834           chrUn_gl000218:51064–51113           -               0.14           1.87E-07    
          lt1Hf71dcOcPygIhR4           CHIC1           chrX:72903733–72903782           +                   0.08           4.14E-06
          WnUxEZ5faxUURBNQuk           L1CAM           chrX:153127445–153127494           -                   0.77           1.28E-05
          HLEukIKIKKaSqkOEnQ           L1CAM           chrX:153128160–153128209           -                   0.08           2.23E-05
          fKg7fXeNt9dKDIdCzk           RPS4Y1           chrY:2712151–2712200           +               1.48           < 2.00E-16    
          Q8jADkAqS017VJIV90           NLGN4Y           chrY:16953254–16953303           +               0.06           1.27E-08    
          6tUwTEFxS.3kFYCVKk           AL833666           chrY:21724080–21724129           -               0.18           3.20E-10    
Open in a new tab

Light gray boxes indicate non-significant results. Dark gray boxes indicate that no comparison was made (X-chromosome-linked genes were compares to females only whereas Y-chromosome-linked genes were compared with males only). Annotation data for each probe obtained using the Bioconductor package illuminaHumanv4.db.39

Given previous evidence that KS is associated with skewed XCI in peripheral blood,14 we next assessed allelic patterns of DNA methylation in the proximity of a polymorphic repeat (CAG)n in the androgen receptor (AR) gene. Although the prefrontal cortex was characterized by subtle allelic imbalance of XCI (7.5% skewing), this did not exceed the range of normal skewing observed in our previous analysis of healthy individuals,40 and there was no evidence of allelic skewing in the cerebellum (0.4% skewing). We were also interested in examining the expression and DNA methylation status of X-linked genes believed to escape XCI in females.41 Between 5 and 15% of genes on the X-chromosome are thought to escape XCI in healthy females,42,43 and it has been suggested that these loci may play an important role in the KS phenotype.44,45 Although genes escaping XCI might be expected to show consistently high expression levels in females compared with males, many loci actually show variable levels of expression in females46 with evidence of tissue-specificity.45,47

Although the 47,XXY patient is an outlier for the expression of many genes thought to escape XCI, we find no consistent pattern of altered transcription; some loci are upregulated and others downregulated, with many showing changes specific to either male or female comparison groups (Table S2). There is also considerable evidence for tissue-specific differences in the expression of these genes, concurring with data from a study of genes escaping XCI in 41,XXY mice compared with karyotypically normal male and female mice.45 One of the loci showing noticeably higher expression in the 47,XXY patient compared with both females and males across both brain regions is the gene encoding Eukaryotic Translation Initiation Factor 1AX (EIF1AX) (Fig. S8), which has been suggested as a possible candidate gene for Turner syndrome (45,XO).48 Browseable tracks for viewing within the Integrative Genomics Viewer (IGV) (http://www.broadinstitute.org/igv/home) showing 47,XXY-associated changes in DNA methylation and gene expression for other loci are downloadable from our laboratory website (http://epigenetics.iop.kcl.ac.uk/XXY).

We also looked at the expression of genes located in the pseudoautosomal regions (PAR) 1 and 249 (http://www.genenames.org/genefamilies/PAR), which are represented by three copies in individuals with a 47,XXY karyotype. Again, although often an outlier for transcription at these loci (Table S3), the observed pattern in the 47,XXY individual is heterogeneous, differing across tissues and between male and female comparison groups. Some PAR genes (e.g., SLC25A6) are clearly upregulated in the cerebellum but not the prefrontal cortex, while others (e.g., DHRSX) are upregulated across both tissues. Other genes such as GTPBP6, for example, are consistent outliers for reduced expression in the 47,XXY patient suggesting that transcription is not always positively correlated with copy number in the PAR. Further work is needed to explore the regulatory mechanisms influencing expression of loci on the extra X-chromosome, and the processes involved in controlling dosage compensation. Another region of interest in KS is the X transposed region (XTR) on the Y chromosome,50 created by a 3.5 Mb duplication from Xq21.3 to Yp11.2 during hominin evolution.51-53 In Figure S4 the allele frequency plot for the 46,XY male (B) shows three bands across both chromosomal regions, whereas the 47,XXY patient (A) is characterized by four bands, most likely resulting from cross hybridization of microarray probes. Although epigenetic deregulation of the Protocadherin 11 X-linked (PCDH11X) and Y-linked (PCDH11Y) genes in these regions are of great interest in KS and neuropsychiatric disease,54-56 DNA methylation array probes in the vicinity of these genes were excluded during our stringent quality control steps due to cross-reactivity57 and could not be assessed in this study (see the “Materials and Methods” section). Because it is plausible that the XTR contains sequence and epigenetic differences that are important in KS and schizophrenia, future studies should utilize methods that can unambiguously profile variation in this region.

In summary, this study identifies widespread transcriptomic and epigenomic changes in the prefrontal cortex and cerebellum associated with a 47,XXY karyotype. Although our findings are based on data from only a single 47,XXY individual, and it will be important to confirm the observed patterns samples from additional patients, this study represents the first detailed molecular characterization of brain tissue from an individual with a 47,XXY karyotype. The patient, who was comorbid for schizophrenia, was found to have a notably reduced cerebellum mass and was characterized by considerable locus-specific changes in DNA methylation and gene expression, with many of these differences being autosomal and tissue-specific. Strikingly, global DNA methylation, assessed via the interrogation of LINE-1 and Alu repetitive elements, was significantly altered in the 47,XXY patient in a tissue-specific manner. Finally, we find evidence for alterations in gene expression at loci believed to normally escape XCI in females.

Materials and Methods

Samples and nucleic acid isolation

Post-mortem brain samples were obtained from the MRC London Neurodegenerative Diseases Brain Bank (http://www.kcl.ac.uk/iop/depts/cn/research/MRC-London-Neurodegenerative-Diseases-Brain-Bank/MRC-London-Neurodegenerative-Diseases-Brain-Bank.aspx). Patients (n = 49) were approached in life for written consent for brain banking. All samples were dissected by a trained neuropathologist, snap-frozen and stored at -80°C following legal and ethical guidelines. The time between death and removal of the brain was recorded as post-mortem interval (PMI). The samples used in this study comprised of prefrontal cortex (PFC, n = 49) and cerebellum samples (CER, n = 48), from 23 schizophrenia patients (including the 47,XXY patient) and 26 unaffected controls. All schizophrenia patients were diagnosed pre-mortem by psychiatrists in the UK using standardized diagnostic criteria. Demographic information about the samples is summarized in Table S4. Genomic DNA was extracted from each tissue sample using a standard phenol-chloroform extraction and tested for purity and degradation using spectrophotometry and gel electrophoresis, respectively. RNA was extracted using a standard Trizol extraction method and purified using an RNeasy Mini Kit with DNase I digestion (Qiagen), according to manufacturer’s instructions. RNA was tested for degradation and purity using an Agilent 2100 Bioanalyzer and RNA 6000 Nano kit (Agilent Technologies). All samples were randomized with respect to gender and disease status throughout all stages of the project to avoid potential batch effects.

Global DNA methylation assay

Bisulfite-PCR pyrosequencing was used to assess the methylation status of LINE-1 and Alu repeats as a proxy of global DNA methylation levels, as described previously.30,31 Samples were run on the Pyromark Q24 pyrosequencer (Qiagen) according to manufacturer’s instructions. DNA methylation levels for each sample were calculated as the average of the three interrogated CpG sites on each assay. Fully methylated and fully unmethylated control samples were included in all procedures to act as assay controls.

Genome-wide DNA methylation array processing

500ng of genomic DNA from each sample was treated with sodium bisulfite in duplicate, using the EZ-96 DNA methylation kit (Zymo Research) following the manufacturer’s standard protocol. Duplicates were pooled and the samples (PFC n = 46 and CER n = 46) were assessed using the Illumina Infinium HumanMethylation450 BeadChip (Illumina Inc.) run on the HiScan System (Illumina Inc.). All samples were randomized with respect to gender and disease status to avoid batch effects, and processed on eight BeadChips.

Methylomic data processing and analysis

Signal intensities were extracted using Illumina GenomeStudio software (Illumina Inc.) and imported into R58 using the methylumi and minfi packages.59,60 Multi-dimensional scaling plots of variable probes on the X- and Y-chromosome were used to check concordance between predicted and reported sex for each individual (see the “Results and Discussion” section). The comparison of non-CpG SNP probes on the array confirmed that the PFC and CER were sourced from the same individual where expected. Raw β values of CpG probes within brain region-specific differentially methylated regions (DMRs) (extracted from ref. 61) were used to confirm that the predicted and reported brain region corresponded for each sample. Probes containing a SNP with MAF > 5% within 10 bp of the CG target site based on the Illumina annotation data (n = 35 413) and non-CG probes (n = 65) were removed. Further stringent data quality control and processing steps were conducted using the dasen function in the wateRmelon package as previously described.62 The pfilter function was used to filter data by beadcount and detection P value to stringently control for poor quality probes (PFC n = 5623 probes and CER n = 10 417 probes removed across all samples). Prior to statistical analyses cross-reactive probes co-hybridizing to the sex-chromosomes, as previously identified,57 were removed. The pnorm function used to identify differentially methylated CpG sites in the 47,XXY patient and the comb-p package63 was used to identify 500bp regions of 2 or more adjacent differentially-methylated probes. The identified regions of differential DNA methylation were compared with copy number variation (CNV) data of the 47,XXY patient to screen for overlaps with any large genomic aberrations.

Genome-wide expression array processing

An amount of 100 ng RNA from each sample (PFC n = 47 and CER n = 48) was biotinylated and amplified using the Illumina TotalPrep RNA Amplification kit (Life Technologies) to produce cRNA. cRNA was quantitated using a NanoDrop NO-1000 (Thermo Fisher Scientific) and RediPlate 96 RiboGreen RNA Quantitation Kit (Life Technologies). Genome-wide expression was assessed using the Illumina HumanHT-12 v4 Expression BeadChip (Illumina Inc.) according to manufacturer’s instruction.

Expression data processing and analysis

Signal intensities for each probe were extracted using Illumina GenomeStudio software (Illumina Inc.) and imported into R using the lumi package within Bioconductor.64 Initial quality control checks using functions within lumi identified clear outlying samples, which were removed from subsequent analyses (PFC n = 5, CER n = 4). The sex of the samples was checked by comparing the sex predicted by the expression levels of the XIST gene with the reported sex for each individual (see the “Results and Discussion” section). Probes targeting transcripts of genes in the vicinity of brain region-specific DMRs61 were used to confirm that the predicted brain region corresponded with the reported region for each sample. Remaining samples were processed using the lumi64 and MBCB65 Bioconductor packages in R. During processing, probes with a detection P value > 0.01 across all samples were considered non-detectable and removed from subsequent analysis. The ComBat function within the sva package in R66 was used to adjust the data to remove batch effects. The pnorm function was used to identify differentially expressed transcripts in the 47,XXY sample. Genes identified as differentially expressed were compared with the CNV data of the 47,XXY patient to record overlaps with large genomic aberrations.

Genome-wide CNV detection

200ng of genomic DNA from each prefrontal cortex sample were genotyped using the Illumina HumanOmniExpress BeadChip (Illumina Inc.). All samples were randomized with respect to gender and disease status to avoid batch effects. Illumina GenomeStudio was used to call genotypes (using the HumanOmniExpress-12v1_C.egt cluster file) with the default GenCall cut-off of 0.15. To compare the sex predicted by the genetic data with the reported sex for each individual previously published recommendations were followed.67 PLINK was used to assess the heterozygosity rate of the probes on the X-chromosome68 (see the “Results and Discussion” section). Autosomal CNVs were called using PennCNV.69

PCR-based sex-typing assay

A PCR-based sex-typing assay was performed as described previously.70 In brief, the X and Y amelogenin (AMELX) sequences were amplified, with amplicons distinguished on the basis of size; the X-chromosome produces a 977bp amplicon, whereas the Y-chromosome produces a 788bp amplicon.

X-Chromosome Inactivation assay

The allelic X-Chromosome Inactivation (XCI) ratios of both tissues of the 47,XXY sample were determined by assessing DNA methylation in the proximity of a polymorphic repeat (CAG)n in the human androgen receptor (AR) gene, as described previously.71,72 In brief, 50 ng of genomic DNA was incubated with HpaII, MspI or water in triplicate. The digestion product was amplified using fluorescently labeled primers flanking the polymorphic repeat (CAG)n. An ABI3130 (Life Technologies) was used to separate the fluorescently labeled amplification products and quantify the peak heights of each allele. The XCI ratio was then calculated as previously described.40

Supplementary Material

Additional material
epi-9-587-s01.pdf (860.7KB, pdf)
Additional material
epi-9-587-s01.pdf (860.7KB, pdf)

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

This work was supported by a UK Medical Research Council (MRC) project grant to J.M. R.P. was funded by an MRC PhD studentship. We thank Annet Damhuis at the Royal Devon and Exeter NHS Trust for the technical assistance.

Glossary

Abbreviations:

KS

Klinefelter syndrome

XCI

X-Chromosome inactivation

LINE-1

long interspersed nucleotide element-1

PMI

post-mortem interval

CER

cerebellum

PFC

prefrontal cortex

DMR

differentially methylated regions

DE

differentially expressed

CNV

copy number variation

PAR

pseudoautosomal regions

10.4161/epi.27806

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