Hum Genet (2014) 133:1359–1367 DOI 10.1007/s00439-014-1469-6
ORIGINAL INVESTIGATION
De novo MECP2 duplications in two females with intellectual disability and unfavorable complete skewed X-inactivation Nathalie Fieremans · Marijke Bauters · Stefanie Belet · Jelle Verbeeck · Anna C. Jansen · Sara Seneca · Filip Roelens · Elfride De Baere · Peter Marynen · Guy Froyen
Received: 21 April 2014 / Accepted: 9 July 2014 / Published online: 19 July 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract Xq28 microduplications of MECP2 are a prominent cause of a severe syndromic form of intellectual disability (ID) in males. Females are usually unaffected through near to complete X-inactivation of the aberrant X chromosome (skewing). In rare cases, affected females have been described due to random X-inactivation. Here, we report on two female patients carrying de novo MECP2 microduplications on their fully active X chromosomes. Both patients present with ID and additional clinical features. Mono-allelic expression confirmed complete skewing
of X-inactivation. Consequently, significantly enhanced MECP2 mRNA levels were observed. We hypothesize that the cause for the complete skewing is due to a more harmful mutation on the other X chromosome, thereby forcing the MECP2 duplication to become active. However, we could not unequivocally identify such a second mutation by array-CGH or exome sequencing. Our data underline that, like in males, increased MECP2 dosage in females can contribute to ID too, which should be taken into account in diagnostics.
Electronic supplementary material The online version of this article (doi:10.1007/s00439-014-1469-6) contains supplementary material, which is available to authorized users.
Introduction
N. Fieremans · M. Bauters · S. Belet · J. Verbeeck · G. Froyen Human Genome Laboratory, VIB Center for the Biology of Disease, Leuven, Belgium N. Fieremans · M. Bauters · S. Belet · J. Verbeeck · P. Marynen · G. Froyen (*) Human Genome Laboratory, Department of Human Genetics, KU Leuven, 3000 Leuven, Belgium e-mail:
[email protected] A. C. Jansen Department of Pediatric Neurology, Vrije Universiteit Brussel (VUB), UZ Brussel, Brussels, Belgium S. Seneca Center for Medical Genetics, Vrije Universiteit Brussel (VUB), UZ Brussel, Brussels, Belgium F. Roelens Department of Pediatric Neurology, AZ Delta, Roeselare, Belgium E. De Baere Center for Medical Genetics, Ghent University, Ghent University Hospital, Ghent, Belgium
Methyl-CpG binding protein 2 (MECP2; MIM 300005) encodes for an essential epigenetic regulator in postnatal brain development causing transcriptional activation as well as repression of many genes (Adkins and Georgel 2011; Gonzales and LaSalle 2010). Mutations and rearrangements in MECP2 are a major cause of neurodevelopmental disorders. Loss-of-function mutations in males are mostly lethal while in females, they are the cause of Rett syndrome (RTT; MIM #312750). In classical RTT, a period of apparently normal development is followed by regression—with loss of social, motor and communication skills—and the development of stereotypic hand movements (Hagberg et al. 1983). However, a wide degree of variability has been reported including the atypical preserved speech variant, congenital variant, early seizure variant, and forme fruste (Hagberg and Skjeldal 1994). Microduplications including the entire MECP2 gene result in a severe ID syndrome with progressive neurological symptoms in males (Bauters et al. 2008; del Gaudio et al. 2006; Van Esch et al. 2005). This is known as the Lubs X-linked mental retardation syndrome (MRXSL) (MIM #300260), or the MECP2 duplication
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syndrome. Clinical features include infantile hypotonia, recurrent respiratory infection, severe ID, absence of speech development, seizures and spasticity. Females, on the other hand, are usually unaffected because the MECP2 gene, which is located at Xq28, is subject to X chromosome inactivation (XCI) in female somatic cells. XCI is a random process with respect to the parental origin of the X undergoing inactivation. However, on rare occasions, ‘skewing’ of X-inactivation may occur. This is when one X chromosome becomes preferentially inactivated, i.e., when ≥80 % of cells inactivate the same X chromosome. Female carriers of a MECP2 duplication show near to complete skewing against the aberrant X chromosome, thereby sparing them from any clinical signs although in some of these carriers, neuropsychiatric symptoms have been described (Ramocki et al. 2010; Van Esch 2012). Recently, however, MECP2 duplications have been reported to cause disease phenotypes in female patients as well, which could be explained by the fact that skewing against the mutant X chromosome does not always take place (Bijlsma et al. 2012; Grasshoff et al. 2011; Kirk et al. 2009; Mayo et al. 2011; Reardon et al. 2010; Shimada et al. 2013b). Skewing of X-inactivation can happen by chance, but can also be caused by primary or secondary stochastic or genetic processes (Morey and Avner 2011). In primary skewing, the inactive X chromosome is chosen before silencing is initiated (Morey and Avner 2011). The best known example is a mutation in XIST or its antisense regulator TSIX, which affects its expression (Nesterova et al. 2003). The most common cause of secondary skewing is post-inactivation cell selection, occurring due to an X chromosome mutation that affects cell proliferation (Morey and Avner 2011). Therefore, a skewed X-inactivation ratio may point to a carrier status for an X-linked mutation. Here, we report on two female patients with de novo tandem microduplications at Xq28 resulting in significantly increased MECP2 expression. Interestingly, both female patients completely inactivated the apparently normal X chromosome. The clinical features of both girls, however, are less severe than those observed in the MECP2 duplication syndrome in male patients.
Materials and methods Samples The screening protocols were approved by the appropriate Institutional Review Board of the respective University Hospitals (Belgium) and informed consent was obtained from the parents of the affected patients. Genomic DNA from patients, their parents, and control females was isolated from peripheral blood according to standard
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procedures and stored at 4 °C. All primer sequences are provided in online supplementary Table S1. Oligo‑X array‑CGH and qPCR analysis X chromosome-specific array-CGH was performed using custom 244 k oligo-arrays (Agilent Technologies, Palo Alto, CA, USA). Hybridization and feature extraction were performed as described elsewhere (Froyen et al. 2008) and data were visualized with the Genomics Workbench software (Agilent Technologies). Confirmation of copy number change was done by realtime quantitation (qPCR) using SYBRgreen on an LC480 apparatus (Roche, Basel, Switzerland) as described previously (Van Esch et al. 2005). Data were normalized against the PAK3 locus at Xq23. MLPA, FISH and X‑inactivation To screen for copy number changes in MECP2, multiplex ligation-dependent probe amplification (MLPA) was performed with the probe mix P015-MECP2 (MRC Holland, Amsterdam, the Netherlands) according to the manufacturer’s instructions. The location of the duplication was analyzed by standard FISH on metaphase chromosome spreads of patients and controls. The DOP-PCR product of the BAC clone RP11119A22 (includes the entire MECP2 gene) was labeled with SpectrumOrange (Abbott Molecular Inc., Des Plaines, IL, USA) using the direct labeling kit (Invitrogen, Paisley, UK) as described elsewhere (Menten et al. 2006). Signals were visualized by digital imaging microscopy with Cytovision capturing software (Applied Imaging, Santa Clara, CA, USA). Lymphocyte-derived genomic DNA was subjected to the standard androgen receptor (AR) or FMR1 methylation assay (Allen et al. 1992; Carrel and Willard 1996) as previously reported (Van Esch et al. 2005). Expression analysis Total RNA was extracted from white blood cells or lowpassage Epstein–Barr virus-transformed peripheral blood lymphocytes (EBV-PBLs). qPCR, as described above, was performed on cDNA as described previously (Van Esch et al. 2005). Normalization was done to different housekeeping genes. Highly polymorphic SNPs in genes with significant expression in lymphocytes were selected. Primers were designed using the VectorNTi software and PCR was performed on DNA of the patients and their parents, followed by Sanger sequencing. The same SNPs were sequenced on cDNA obtained from the patients to assess the allelic expression ratios.
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Duplication junction sequencing Mapping of the breakpoint junctions was performed as described previously (Bauters et al. 2008). The unique PCR product obtained in patient 1 was sequenced and aligned to Xq28 sequences with the ContigExpress tool of VectorNTi (Lifetechnologies). XIST and exome sequencing PCR followed by Sanger sequencing was performed on the XIST minimal promoter region and the beginning of exon 1. The sequenced region was 3.2 kb in size and is located at ChrX: 73,069,528–73,072,728 (UCSC Hg19). Five overlapping PCR products were generated. For exome sequencing, exome capture was performed with the SeqCap EZ Human Exome Library v3.0 (NimbleGen) and subsequently sequenced in a single end 50 bp run on a HiSeq 2000 instrument (Illumina, San Diego, CA, USA) with a mean coverage of at least 50X per sample (Genomics Core, Leuven, Belgium). Sequence reads were mapped to the reference genome using BWA 0.5.9rc1(Li and Durbin 2010). After variant calling with GATK 1.0.4974 (DePristo et al. 2011) and Casava 1.8.2 (Illumina), any base call that deviated consistently from the reference genome was regarded as a possible variation. Variant annotation was performed by Annovar 20-11-2011 and file conversion via SAMtools 0.1.12a. Variant confirmation was done by PCR and Sanger sequencing. SNP and microsatellite analysis To assess the parental origin of the X chromosome on which the duplication arose, we explored the exome sequencing data for heterozygous SNPs within the respective Xq28 duplications. PCR was performed on genomic DNA of the patients and their parents followed by sequencing. Informative SNPs were then sequenced on cDNA from the patients to confirm the parental origin of the active X chromosome. Alternatively, we analyzed heterozygous microsatellites within the duplicated regions via fragment analysis as described above. The relative areas under the curves indicated the allele that was duplicated in the patient, which was then checked in the parents.
Results Patients Patient 1 presented at age 13 with mild ID. She was the only child of healthy third degree consanguineous parents and was born after a normal pregnancy and delivery.
Fig. 1 Pictures of patient 2. The girl was diagnosed with a Rett-like phenotype. She carries a de novo 1.4 Mb MECP2 microduplication on her active X chromosome
Early developmental milestones were normal. She attended a regular kindergarten, but was transferred to special education at 5.5 years old. She was last examined at the age of 13 years. Her weight was at the 50th percentile, height at the 90th percentile, and head circumference at the 25th percentile. She had an elongated face, narrow eyes, and a straight nose. Her hands were always moist. She was well oriented and had normal speech. Gait, balance, coordination, and deep tendon reflexes were normal. She had occasional hand-washing movements and a mild tremor of the hands, which was influenced by stress and posture. Her Full Scale IQ score was 63. Brain MRI and work-up for inherited disorders of metabolism were normal. Karyotype, subtelomere FISH and screening for fragile X syndrome were normal. Deletion 22q11 was absent. Patient 2 presented with moderate to severe developmental delay (Fig. 1). Since early life, she suffers from persistent gastro-esophageal reflux with regurgitation. She learned to walk at 26 months of age. She always attended special education. The Stutsman developmental quotient at age 6 was 32. At the current age of 12, she only speaks a few words, and demonstrates an autistic behavior with stereotypic activities, as well as sleep disturbances. She is learning to communicate with a speech generating device. Breathing abnormalities, wringing and other repetitive hand movements, and epileptic seizures were never noticed. Her head circumference is normal (30th percentile), and weight and stature are on the 50th percentile. Although her feet are somewhat narrow with mild antepes adductus and hallux valgus, and knee extension
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Fig. 2 FISH on metaphase chromosomes of both female patients. Representative images to localize the Xq28 duplication (a) in patient 1 and (b) in patient 2. The BAC clone RP11119A22, containing MECP2 and labeled with SpectrumOrange, is present only at the distal part of the X chromosome. The centromeric X probe (Xcen) is labeled in green
is slightly restricted, there is no clear spasticity, and she is able to walk long distances. Brain MRI only showed a cavum septi pellucidi et vergae. Karyotyping and screening for fragile X syndrome were normal, as well as metabolic work-up. Identification of duplications at Xq28 In both female patients, MLPA revealed a 1.5-fold increased copy number of the four MECP2 exons (153.28– 153.36 Mb; UCSC Hg19) but not for VAMP7/SYBL1 (155.14 Mb) indicating that these are interstitial duplications (online supplementary Fig. S1). Both copy number gains occurred de novo. In patient 1, oligo-X arrayCGH mapped the location of the microduplication from 153.24 Mb to 153.68 Mb revealing a size of 0.44 Mb. This region harbors about 20 genes including the ID genes MECP2, FLNA and GDI1 (online supplementary Fig. S2A). In patient 2, the duplication was mapped from 152.23 Mb to 153.69 Mb by oligo-X array-CGH, and is 1.46 Mb in size. This region contains about 50 genes including the ID genes SLC6A8, L1CAM, MECP2, FLNA and GDI1 (online supplementary Fig. S2B). Both distal breakpoints seem to be located in the PLXNA3 gene. The Xq28 duplications were confirmed by qPCR. In patient 2, array-CGH revealed an additional very subtle 1 kb duplication at Xq28 partially including MTCP1 and BRCC3. However, qPCR also detected this copy number gain in her father and it is, therefore, regarded as non-pathogenic. Mapping of the MECP2 duplications For both patients, FISH analysis revealed signals for the MECP2-containing probe RP11-119A22 only at Xq28, demonstrating that the duplicated sequence had not integrated elsewhere in the genome. Importantly, we often detected three to four closely spaced signals on one X chromosome (Fig. 2a, b) strongly suggestive for a tandem
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duplication event. Further fine mapping of the copy number gains allowed to sequence over the junction in patient 1, proving the direct tandem duplication. The proximal breakpoint was located at ChrX: 153,247,624 and the distal one at ChrX: 153,688,854 in exonic sequences of the TMEM187 and PLXNA3 genes, respectively (online supplementary Fig. S3). No microhomology was found at the junction. However, an insertion of 25 bp was noticed, which points to non-homologous end joining (NHEJ) as this repair mechanism is prone to small insertions and deletions. In patient 2, the junction could not be defined despite the small breakpoint regions. At the proximal side, the breakpoint maps within a 0.8 kb region (ChrX: 152,228,318–152,229,112) while the distal one is located within a 1.1 kb interval (ChrX: 153,687,280–153,688,404) in intron 1 of PLXNA3. X‑inactivation study X-inactivation analysis revealed that patient 1 completely inactivates (100/0) her maternally inherited X chromosome (429 allele), which was also completely inactivated in her mother (Fig. 3a, online supplementary Fig. S4). Microsatellite and expression analyses further revealed that the de novo MECP2 duplication is located on the paternally inherited active X chromosome (see below). Patient 2 also presented with completely skewed X inactivation (100/0), with the paternally inherited X chromosome inactive. In this patient, the de novo MECP2 duplication is located on the active X chromosome inherited from the mother. This was confirmed by mono-allelic expression of the maternal SNP (see below). The mother of patient 2 showed random X inactivation (65/35) (Fig. 3b). To validate these X inactivation data, we investigated mono-allelic expression via SNP analysis of rs6638360 at Xp11.22 and rs2286977 at Xq24. Both SNPs were heterozygous (T/C and G/A, respectively) in each patient and homozygous in the parents (except for rs2286977 in the mother of patient
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Fig. 3 Pedigrees of the families showing the X-inactivation ratios and the de novo MECP2 duplication. a Patient 1 is homozygous for the AR locus. X chromosome inactivation (XCI) at the FMR1 locus revealed 100 % inactivation of the maternally inherited X chromosome (black) not carrying the MECP2 duplication (indicated by the
star). Moreover, the mother of patient 1 has the same X chromosome fully inactive. b XCI for patient 2, performed at the AR locus, showed 100 % skewing with the paternally inherited X chromosome (gray) inactivated. Again, this X chromosome does not carry the duplication. The mother of patient 2 shows a random (65/35) XCI
Table 1 SNP typing to determine the inheritance of the active X chromosomes
when compared to EBV-PBLs from two female controls (mean 3.5- and 3.8-fold, respectively) (online supplementary Fig. 5A). Similarly, we quantified the expression levels in RNA from blood lymphocytes of patient 2 and two female control lymphocyte samples. Again, mRNA levels of MECP2 and GDI1 were significantly elevated (mean 2.8- and 2.2-fold, respectively) (online supplementary Fig. S5B). As a control, we used the GUSB gene, which did not give altered expression. These significantly increased levels of genes within the Xq28 duplication clearly demonstrated that the X chromosome carrying the duplication is always active in the patients’ blood cells. The situation in blood can be used as a representative for the situation in the brain and other tissues (Bittel et al. 2008).
Gene
SNP
Genomic DNA
cDNA
Father
Mother
Patient
Patient
Patient 1 HUWE1
rs6638360
C
T
C/T
C
DOCK11 Patient 2
rs2286977
A
G
A/G
A
HUWE1
rs6638360
C
T
C/T
T
DOCK11
rs2286977
G
A/G
G/A
A
ARHGAP4
rs2070097
A
G/A
A/G
G
HCFC1
rs3027875
A
G/A
A/G
G
SNP typing of rs6638360 within the coding region of HUWE1 at Xp11, and rs2286977 within that of DOCK11 at Xq24, was performed on genomic DNA of the two female patients and their parents. Analysis of expressed SNPs was performed on cDNA obtained from the patients showing mono-allelic expression. The paternal X chromosome is the active one in patient 1, while the maternal X chromosome is fully active in patient 2. In addition, two heterozygous SNPs located within the duplicated region in patient 2, rs2070097 in ARHGAP4 and rs3027875 in HCFC1, confirmed these data
2) (Table 1). Next, expression analysis by RT-PCR on cDNA from the patients followed by sequencing revealed a single nucleotide at each SNP position proving monoallelic expression. For patient 1, the paternal alleles were exclusively used while for patient 2, these were the maternal alleles. MECP2 mRNA expression We previously demonstrated highly stable MECP2 mRNA levels in male and female controls (Van Esch et al. 2005). qPCR expression analysis in RNA from EBV-PBLs from patient 1 demonstrated increased mRNA levels for MECP2 and GDI1, both present within the duplication,
SNP and microsatellite analysis To determine the parental origin of the X chromosome on which the duplication arose de novo, we searched for polymorphic SNPs within the duplicated regions in our exome sequencing data. In patient 1, no heterozygous exonic SNPs were identified. We then tested five polymorphic intergenic SNPs and five microsatellite repeats spread across the duplicated region. From these only the (TG)n dinucleotide tandem repeat at ChrX: 153,381,403–153,381,470 was heterozygous. Comparison of the fragment analysis data of this patient with that of her parents demonstrated that the MECP2 duplication had arisen on the paternal X chromosome (online supplementary Fig. S4A). In patient 2, two heterozygous SNPs, rs2070097 (ChrX: 153,176,254) located in ARHGAP4 and rs3027875 (ChrX: 153,215,839) in HCFC1, detected by exome sequencing within the 1.4 Mb duplication, were confirmed by Sanger sequencing. These SNPs were hemizygous in the father (A) and heterozygous in the mother and patient (G/A). Therefore, the A allele was inherited from the father and the G allele from the mother. In the electropherograms, the higher
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heights of the G peaks compared to the A peaks for both SNPs strongly suggested that the duplication is located on the maternally inherited X chromosome (online supplementary Fig. S6B). Similarly, the number of reads obtained by exome sequencing showed that for rs2070097, 17 reads had the maternal G allele while only 6 reads showed the paternal A allele. Similarly for rs3027875, 54 reads carried the maternal G allele and only 34 the paternal A allele strongly pointing to a higher copy number of the maternally inherited alleles. Sanger sequencing of the patient’s cDNA only detected the G alleles for both SNPs, which is consistent with expression from the maternally inherited X chromosome only (Table 1, online supplementary Fig. S6B).
functional effect, if any, of this amino acid change needs further investigation. A potential effect on splicing could not be tested because this allele is completely skewed precluding its expression. We also detected the reciprocal fusion transcript between TMEM187 and PLXNA3 confirming the junction of the tandem duplication in this patient. In patient 2, exome sequencing revealed an unknown synonymous variant in SMC1A that could have an effect on splicing. However, since this gene is reported to escape X inactivation and this variant was also found in her healthy father, it is unlikely to induce skewing. No other potential X-linked mutations were found.
XIST sequencing
Discussion
To identify variants that may cause primary skewing in our female patients, Sanger sequencing was performed on part of the promoter region and the first exon of XIST (ChrX: 73,069,528–73,072,728). In patient 1, a single nucleotide A/T variant was detected at position ChrX: 73,072,402 in exon 1 of XIST. This variant was inherited from her mother. XIST cDNA analysis revealed that indeed this variant is located on the inactive X chromosome in both patient 1 and her mother. It has not been reported in SNP databases and was not detected in 30 random Belgian XCI females that we screened. In patient 2, besides SNP rs36033063, we did not find any sequence variants in the sequenced XIST region.
This study describes two female ID patients with de novo MECP2 duplications that are located on their fully active X chromosomes. Comparative analysis of the copy number gains as well as the clinical features of our patients with those of the 16 reported female patients with duplications including MECP2 is illustrated in Fig. 4 and online supplementary Table S2, respectively (Auber et al. 2010; Bijlsma et al. 2012; Grasshoff et al. 2011; Kirk et al. 2009; Makrythanasis et al. 2010; Mayo et al. 2011; Reardon et al. 2010; Scott et al. 2014; Shimada et al. 2013a, b). The clinical characteristics of all of these patients are highly variable (online supplementary Table S2) likely due to the kind of aberration, its size and gene content, the X-inactivation pattern and other genetic and environmental factors. Regarding the reported females of this study, the clinical features of patient 1 are quite different and significantly less severe than those of patient 2. The smaller duplication size and subsequent smaller gene content in patient 1 (0.4 Mb) versus patient 2 (1.4 Mb) did not likely contribute to this variability since in male patients the duplication size does not define disease severity either (Bauters et al. 2008; Ramocki et al. 2010). The main driver for clinical severity is simply thought to be the level of MECP2 protein. It is now well appreciated that even modest overexpression of Mecp2 in mice results in a behavioral phenotype in male as well as female mice (Collins et al. 2004). Therefore, in analogy with male patients, females with MECP2 duplications or with gain-of-function mutations could result in a clinical phenotype as well. The severity likely depends predominantly on the X-inactivation status of the CNV-carrying X chromosome. Furthermore, the MECP2 levels are likely associated with the MECP2 copy number. Triplication of MECP2 has been reported in males (del Gaudio et al. 2006; Tang et al. 2012) and females (Mayo et al. 2011) with more severe clinical features when compared to duplication patients. On the other hand, Ramocki and co-workers did not find
Exome sequencing Since the MECP2 duplications in both patients were located on their active X chromosomes, we hypothesized the presence of an additional mutation on the other X chromosome, responsible for skewing. As no other likely harmful CNVs were detected by high-resolution X-oligo arrayCGH, we performed exome sequencing on DNA from both patients. In patient 1, a missense change c.2673G>C (p.Q891D) located at ChrX: 76,937,961 was identified in ATRX (NM_138270.2). This variant was inherited from her mother and is, therefore, located on her completely inactive X chromosome as well. Although this variant has not been reported in dbSNP, the 1000 genomes project or our private clinical database, it is located in an apparently neutral region of ATRX. Interestingly, this codon is polymorphic at its first base (rs3088074; c.2671C>G) resulting in a CAG to GAG change (p.Q891E). Both the patient and her mother carry this SNP but since they also harbor the G>C variant at the third nucleotide of this codon, it changes from GAG to GAC (p.E891D). The wild-type glutamine (Q) and the polymorphic glutamic acid (E), but not the identified aspartic acid (D), are highly conserved in vertebrates. The
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Fig. 4 Schematic representation of MECP2 microduplications in female patients. The duplications can be the result of insertion into an autosome (green), an unbalanced translocation (yellow), or an intrachromosomal duplication (orange). A triplication event is indicated in red. The minimal critical region containing MECP2 is boxed.
Gene content of the region for a selected number of genes, including the known XLID genes indicated in bold, is shown as black arrows. The patient from Shimada et al. (2013a) has a duplication starting at 150 Mb, the first part of the duplication is not shown in the figure. Positions are based on UCSC Hg19
a correlation between the MECP2 mRNA levels measured in patients’ lymphocyte-derived cell lines and their clinical severity (Ramocki et al. 2009), questioning whether this cell type accurately reflects the situation in brain. Similarly in patient 1, the MECP2 expression levels were higher than in patient 2 who actually presents with a more severe phenotype. However, since mRNA levels were measured in an EBV-PBL cell line from patient 1 and in blood lymphocytes from patient 2, the expression data may not be compared. Another important parameter that can affect the clinical outcome is the integration site in the genome. If the duplicated region is inserted into an autosome (Bijlsma et al. 2012; Makrythanasis et al. 2010; Shimada et al. 2013a) or generated by inherited interchromosomal translocations (Auber et al. 2010; Bijlsma et al. 2012), the subsequent functional disomy always results in an increased MECP2 expression, thereby disturbing normal development. The clinical outcome might further be affected by the consequent disruption of genes. Therefore, it is important to map the breakpoint sites and define the junction segments. In patient 1, the duplicated copy is inserted in a direct tandem orientation generating reciprocal fusion genes between TMEM187 and PLXNA3 for which potential new functions remain unknown. However, both genes remain intact on that X chromosome precluding their disruption. Patient 2 seems to have a more complex duplication. Besides the potential interruption of PNMA3 or PLXNA3 located at the proximal and distal breakpoints respectively, we cannot exclude the disturbance of other genes. Mutations in neither PNMA3 nor PLXNA3 have been associated with any disease. A last factor to take into account is the possibility
of genomic imprinting of the X chromosome. To date, there is increasing evidence that a parent-of-origin effect of the X chromosome could influence neurogenetic, psychiatric and even physical characteristics (Lepage et al. 2013; Skuse et al. 1997). Although no X-linked imprinted genes have been identified so far, we cannot exclude the presence of any such imprinted factors to be located in or near MECP2. Therefore, in combination with the X-inactivation pattern, imprinting could be an important regulator of phenotypic variation in female individuals. Knowing the underlying mechanisms of skewing is crucial for understanding X-linked disorders in female patients. Female carriers of mutations in many diseaseassociated genes on the X chromosome show preferential X-inactivation against the chromosome that harbors the mutation (Orstavik 2009; Plenge et al. 2002). Therefore, we hypothesize that in the two female patients reported in this study, a harmful mutation on the other X chromosome forced the X chromosome with the MECP2 duplication to become active in all cells. The mother of patient 1 also completely inactivates the same X chromosome as transmitted to her daughter. This finding supports the hypothesis that a highly pathogenic mutation on that X chromosome caused the inactivation in the mother and daughter. To search for the presumed second mutation, we sequenced the minimal promoter and exon 1 of the noncoding XIST RNA. We selected this 3.2 kb region because of the highest degree of conservation. Previously, an apparent mutation in the promoter (−43C>G) was proposed to predispose for skewed inactivation in two families but no direct proof was obtained (Plenge et al. 1997). In our study, patient 1 carried a maternally inherited novel A>T variant in exon 1
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of XIST. Exome sequencing further revealed a novel missense variant in ATRX which was also inherited from her mother. While many mutations in ATRX have been associated with severe skewing in carrier females (De La Fuente et al. 2011), this variant has not yet been reported even though this gene has extensively been screened in patients with alpha thalassemia and ID (MIM 301040). Unfortunately, DNA from additional family members was unavailable. The potential relation of both variants with skewing is yet unknown and requires further functional investigation. In patient 2, XIST and exome sequencing did not reveal any candidate variants that could have resulted in the skewing process. Recently, female patients with mutations in X-linked genes and predominant X-inactivation of the apparent normal X chromosome have been reported for Wiskott–Aldrich syndrome (MIM 614493) (Boonyawat et al. 2013; Daza-Cajigal et al. 2013), Duchenne muscular dystrophy (MIM 310200) (Juan-Mateu et al. 2012), Fabry disease (MIM 301500) (Bouwman et al. 2011) and mucopolysaccharidosis type II (MIM 309900) (Kloska et al. 2011; Pina-Aguilar et al. 2013). These findings strongly suggest that this ‘female X-linked two-hit model’ can result in several X-linked conditions. We propose exome sequencing in these cases to search for the proposed second mutation that caused skewing. Our finding of two female patients with ID and duplication of MECP2 resulting in significantly increased MECP2 expression underlines the importance of quantitative analysis of MECP2 in blood lymphocytes of female ID patients. Here, we also underline the importance of X-inactivation analysis in female patients to unveil X-linked mutations. Acknowledgments The authors thank the family members for their willingness to contribute to this research project. We are grateful to the excellent technical assistance of Karen Govaerts (KU Leuven, Belgium). This work was supported by grants from the VIB (Vlaams Instituut voor Biotechnologie), the Geconcerteerde Onderzoeks Acties (GOA) of the University of Leuven [grant number GOA/12/015 to GF], and from the Belgian Science Policy Office Interuniversity Attraction Poles (BELSPO-IAP) programme [grant number IAP P7/43-BeMGI to GF]. NF is a PhD fellow from the IWT (Agentschap voor Innovatie door Wetenschap en Technologie), Belgium. MB is a post-doctoral fellow and EDB is senior clinical investigator of the Fund for Scientific Research (FWO) Vlaanderen, Belgium. Conflict of interest The authors declare no conflict of interest.
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