Background

Methyl-CpG-binding protein 2 (MECP2) located at Xq28 acts as a transcriptional repressor or activator regulating the genes associated with nerve system development [1]. Loss-of-function mutations of MECP2 gene are the cause of most cases of Rett syndrome (RTT, OMIM: 312750). RTT is a neurodevelopmental disorder which affects 1/10,000 girls. Recent studies found that duplications involving MECP2 gene and increased MECP2 protein dosage can cause a severe syndromic developmental delay (DD), mental retardation (MR), and recurrent infections. It was found in 1 % of patients with idiopathic X-linked MR (XLMR) and in 2 % of male patients with severe encephalopathy [2]. It was named MECP2 duplication syndrome (MECP2 DS), a newly described genetic condition, and gain-of-function mutations of MECP2.

The cardinal features of the syndrome include early onset hypotonia, moderate to profound mental retardation, delayed psychomotor development, speech delay or absent to very limited speech, seizures, progressive spasticity in male patients. Dysmorphism is common and some features changed with age. Midface hypoplasia, depressed nasal bridge and a hypotonic face such as open mouth with tented upper lip, excessive drooling present in most patients at young age. When time goes by, some additional facial features may appear: a narrow nose, prominent chin, deep-set eyes, large ears, and widely spaced teeth [3]. Genital anomalies, including cryptorchidism and small penis, were reported in some cases. Patients with congenital central hypoventilation syndrome (CCHS), hyperkinesis were also reported [4, 5]. Half of these patients have a short lifespan less than 25-year-old [6]. The animal model with over expression both the mouse and human MECP2 gene showed similar neurological symptoms including epilepsy, spasticity, hypoactivity and short lifespan. The severe phenotype is correlated with the higher Mecp2 protein level [7, 8].

Here we report a Chinese boy with MECP2 duplication syndrome, who inherited the duplicated gene from his unaffected mother, while his mother inherited the duplicated gene from his unaffected grandmother. The genetic characters and the clinical features are presented.

Case presentation

Case report

The patient is a 2-year-4-month-old boy, who is the first child of his nonconsanguineous Chinese parents, a 26-year-old mother and a 27-year-old father, both of them are healthy. Following an uneventful pregnancy, he was born at 38 weeks of gestation with a birth weight of 3.15 kg. Feeding difficulties and poor suck had been noticed since birth. Hypotonia was apparent at age of 4 months. He showed severe developmental delay. He could not raise his head until 4 months old, sit unsupported till 11 months. At 2-year-4-month-old, he could not walk and speak any meaningful words. Eye contact and social smiling were enabling. He had recurrent respiratory tract infections, and hospitalized for severe pneumonia for several times. At 12 months old, his head circumference was 47 cm, weight was 11 kg. The boy had mild dysmorphism with flat nasal bridge, anteverted nares, small mouth, high-arched palate, low-set ears and cryptorchidism of the right side. Hand stereotypes including hand wringing and hand shaking presented at 8 months. He had no chronic constipation, no seizures. Magnetic resonance imaging (MRI) at age 1 year was unremarkable. Karyotype analysis was normal. Serum amino acid and urine organic acid screen were unremarkable.

Family history: The boy’s mother had two younger brothers. Both of them had severe developmental delay. The elder one could not walk or speak before he died at age of 5. The best motor function was sitting unaided. His brain computed tomography (CT) scan showed generalized cerebral atrophy. The younger one had a history of recurrent infections, died of a sever pneumonia at the age of 1y. He could not sit or walk alone during his life, speak no meaningful words yet. Both the mother and the grandmother were asymptomatic, they did not have any symptoms of depression, anxiety and compulsions. Figure 1 showed the pedigrees of the family, the proband was showed by an arrow.

Fig. 1
figure 1

Pedigrees of the family. III-1 is the proband, I-1 and II-1 carry the MECP2 duplication, II-2 and II-3 died at the age of 5-year-old and the age of 1y, respectively

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MLPA tests

Genomic DNAs from the peripheral blood leukocytes of the patient, his parents and the maternal grandmother were extracted using standard methods. MLPA (SALSA MLPA kit P015 MECP2, MRC-Holland, Amsterdam, Holland) was performed to detect large deletions or duplications of MECP2 gene as previously described [9]. MLPA products were separated and analyzed using the ABI Prism 3100 Genetic Analyzer and Genescan software according to manufacturer’s recommendations.

aCGH analysis

In order to determine the precise size of the duplication, aCGH was performed on the mother’ DNA, using Affymetrix GeneChip System 3000Dx v.2 (by Tianjin kingmed center for clinical laboratory, Shanghai, China).

Fluorescence in situ hybridization (FISH)

FISH test was performed to interrogate the MECP2 copy number and to identify the location of the duplicated MECP2 gene in chromosome (done by Beijing Ahngook Pharm. Co., Ltd, Beijing, China).

X chromosome inactivation patterns (XCI)

The patterns of XCI of female carriers were analyzed according to the procedure described by Allen et al. [10]. Inactivation was considered to be non- random if the ratio was > 70:30.

Results

DNA fragment duplication was found in the patient, his mother and maternal grandmother by MLPA test. The fragment range from X-152,783,270 to X-153,239,085, including L1 cell adhesion molecule gene (L1CAM; OMIM #308840), interleukin-1 receptor-associated kinase 1 (IRAK1; OMIM #300283), MECP2, filamin A (FLNA; OMIM #300017) and other genes, that probes were not designed in the kit P015 (Fig. 2).

Fig. 2
figure 2

Genes duplication in the proband (a), his mother (b) and his grandmother (c), tested by MLPA

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aCGH was performed on the proband’s mother to test the precise size of the duplicated fragment. The result showed that the duplication fragment was 510, 270 bps (153,113,885–153,624,154) containing 17 genes (Table 1), including two subunits of Micro-RNA (MIR3202), starting from L1CAM and ending with emerin (EMD; OMIM #300384). There was no gene coming across the breakpoints.

Table 1 Duplicated genes and their positions
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FISH test was performed on the mother. The red fluorescence signals representing MECP2 gene were seen on each X chromosome, and only one fluorescence signal could be seen on each X chromosome (Fig. 3). That means the duplicated MECP2 gene was located at X chromosome next to the original MECP2 gene.

Fig. 3
figure 3

Two-color fluorescence in situ hybridization analysis of the mother with MECP2 gene duplication. The duplicated MECP2 gene was located at X chromosome, and only one fluorescence signal can be seen on each X chromosome. Green signal is a control single-copy probe, hybridized to alpha satellite DNA at the chromosome X centromere. Red signal on distal Xq is a probe for the MECP2 gene, which is present on each X chromosome

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The XCI pattern analysis showed the XCI ratio of the patient’s mother and maternal grandmother was 80:20 and 74:26, respectively. The X chromosome carrying the duplication was 80 % inactive in the mother and 74 % inactive in the grandmother. The proband inherited the X chromosome carrying the duplication variant from his mother.

Discussion

With the development of new techniques, some genomic disorders such as Williams-Beuren syndrome (WBS, MIM 194050), Potocki-Lupski syndrome (PTLS, MIM 610883), Miller-Dieker syndrome (MDS, MIM 247200), were found to be caused by the alteration of gene dosage, or copy number variation (CNV) [11]. MECP2 gene acting as a transcriptional regulator of gene expression should express very accurate level in cells. Loss-of-function of MECP2 leads to Rett syndrome while gain-of-function causes MECP2 duplication syndrome. In additional, patient with Xq28 rearrangement containing MECP2 triplication was considered to be more severe than MECP2 duplication [12, 13]. Animal models also demonstrated this phenomenon.

MECP2 duplication is most common among the Xq28 rearrangement. The reported duplications differed in size and location (except the carrier mom and inherited offspring), were defined as non-recurrent events. The flanking region around MECP2 gene is abundant of low-copy repeat (LCR) sequences and high GC content, which generated the genomic instability such as double-strand breaks (DSBs) and a collapsed fork. The molecular mechanisms of MECP2 copy number variation were proposed by mainly two ways: one is Fork Stalling and Template Switching (FoSTeS), another is microhomology mediated break-induced replication (MMBIR) [14–17].

In the previous reports, the sizes of the duplications encompassing MECP2 locus vary between 79.7 kbs and 15.5 Mbs [18, 19]. The smallest duplication only contains MECP2 and IRAK1 genes. The duplication size in our patient was about 510 kbs, including 17 genes, starting from L1CAM and ending with EMD. L1CAM, IRAK1, MECP2 and FLNA were embraced in the duplication. These four genes play important roles in postnatal nervous system development. L1CAM and MECP2 were associated with X-linked mental retardation (XLMD). IRAK1, encoding an interleukin receptor–associated kinase was involved in the toll–like receptor transduction pathway, was a critical signaling mediator of innate immunity. Gottipati et al. found that over expression of this gene might lead to the susceptibility to infections [20]. However, Yang and colleagues identified immune defects among the patients with MECP2 DS had no association with IRAK1 duplication by transgenic mice that overexpressed MECP2 without IRAK1 duplication [21]. This conclusion was supported by Sawalha [22]. Chronic intestinal pseudo–obstruction (CIPO), thrombcytopaenia, patent ductus arteriosus (PDA), malrotation were found in two families with the duplications involved only the FLNA gene [23]. One case suffered CCHS also had chronic constipation without duplication of FLNA [24]. Some other genes in the duplicated area of our patient may be associated with mental retardation and development delay. Huang et al. reported 2 male-dizygotic-twins with a 17.9-kbs deletion of Xq28 including Arginine vasopressin receptor-2 gene (AVPR2; OMIM 300538) and extending into intron 7 of the RHO GTPase-activating protein 4 gene (ARHGAP4; OMIM 300023). They were diagnosed with nephrogenic diabetes insipidus (NDI) as well as psychomotor developmental delay [25]. Host cell factor C1 gene (HCFC1; OMIM 300019) was reported to be associated with X-linked mental retardation (XLMR) [26]. The clinical manifestation of our patient was characterized by developmental delay, early hypotonia, recurrent infection, and mild dysmorphism, without CIPO, PDA and NDI. Combined our study and previous reports, most of the symptoms of MECP2 DS should be attributed to gain of MeCP2 function.

Family study showed the mother and maternal grandmother also carried the MECP2 duplication. The patient inherited the mutation from them. FISH test on the mother showed the duplicated MECP2 gene was located at X chromosome. However, the mother and the maternal grandmother were asymptomatic. Further XCI study showed the XCI pattern was non-random in both of them. The ratio was 80:20 and 74:26 respectively. The X chromosome carrying the duplication was prioritized inactive. This molecular phenomenon may explain their asymptomatic phenotype. In the previous reports, almost all the asymptomatic carriers were with a skewed XCI except one with XCI ratio 43:57 [27]. However, the XCI ratio in peripheral blood DNA may not represent the ratio in Brain. It could be inconsistent in different tissues. A mother of a MECP2 DS patient had mild learning difficulties with a complete skewed XCI in the blood DNA while the ratio was 74:26 in her hair roots. The authors speculated that the inactivation of X chromosome with MECP2 duplication in her brain was not completely leading to her mild clinical features [28]. This is the only literature about XCI test performed on the other tissue rather than blood in patients with MECP2 DS. In this family, two maternal uncles of the boy had severe developmental delay, severe mental retardation and recurrent infections. Both of them died at young age. They were highly suspected to be patients with MECP2 DS.

Conclusions

In this study, we demonstrate one more Chinese family with MECP2 duplication syndrome. The main clinical features include early onset hypotonia, mental retardation, recurrent respiratory tract infections, limited speech and dysmorphism. Female carrying the MECP2 duplication could be asymptomatic due to the skewed XCI. In order to make an accurate diagnosis and provide a right genetic counseling, genomic structure rearrangement, especially MECP2 duplication should be tested in the patients with the above symptoms as well as other family members.

Consent

Gene mutational analysis on the family members and the study of the XCI patterns in female carriers began after signing an informed consent by the parents. Ethical approval was obtained from the hospital research ethic board. Written informed consent was obtained from the legal guardian of the patient for publication of this Case report and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.