A sibship with duplication of Xq28 inherited from the mother; genomic characterization and clinical outcomes

X-linked intellectual disability (XLID) is found in approximately 5–10% of the intellectually disabled men [1]. XLID is divided into syndromic forms in which intellectual disability is one of many symptoms, and non-syndromic forms, in which intellectual disability is the only symptom. In a previous study, 102 genes were shown to be associated with 81 out of 160 cases of XLID syndromes in over 50 families with non-syndromic XLID [1]. Most of the genetic defects leading to XLID are loss-of-function mutations, including point mutations (frame shift, nonsense, and missense), microdeletions, and translocations. XLID caused by gain-of-function is via microduplication where causal genes are abnormally copied. A well-known XLID microduplication region is Xq28, in which there is duplication of X-linked genes such as methyl-CpG-binding protein 2 (MECP2; MIM *300005) a key gene involved in Rett syndrome, a neurodevelopmental disorder that affects mostly girls Xq28 duplication syndrome is an important aspect of XLID. Mutations in the MECP2 gene in Xq28 were first reported in patients with Rett syndrome in 1999 [2] and other X-linked severe neurodevelopmental disorders. Particularly, large deletions, single base mutations, or small frame shift mutations in MECP2 are mostly commonly associated with Rett syndrome [3, 4]. Classical Rett syndrome is typically characterized by a period of normal development until 6 to 18 months of age. After this age, motor and speak skills regress, followed by loss of useful hand skills; rather afflicted children exhibit repetitive hand motions, such as hand wringing. Additional symptoms of Rett syndrome include acquired microcephaly, profound intellectual disability, epilepsy, ataxia, and autistic behaviors. There is a wide spectrum of disease severity for Rett syndrome as determined by molecular diagnostics [5].

While loss-of-function mutations in MECP2 result in Rett syndrome gain-of-function mutations are associated with MECP2 duplication syndrome. MECP2 duplications were initially described in a female patient presenting with a speech variant symptom and was diagnosed molecularly with atypical Rett syndrome [6]. MECP2 duplication syndrome and Rett syndrome share overlapping clinical phenotypes include intellectual disability, motor deficits, epilepsy, hypotonia, and progressive spasticity [7, 8]. Males with Xq28 duplication, including duplications at the MECP2 locus spanning 0.3–4 Mb, have subsequently been reported by employing the multiplex ligation-dependent probe amplification (MLPA) and array-based comparative genomic hybridization (array-CGH) [7, 9‐11]. Common phenotypes in males with MECP2 duplication are severe to profound X-linked intellectual disability, Rett syndromic features, progressive spasticity, neonatal or infantile hypotonia, poor speech development, recurrent respiratory infections, epilepsy, and dysmorphic facial features such as large ears, mid-face hypoplasia, brachycephaly, and depressed nasal bridge [8].

In this study we identified one family with two brothers with Xq28 duplication syndrome and a mother who was a carrier with the same loci duplication. We characterized the duplicated region of the brothers and the mother using various molecular and cytogenetic techniques and to present clinical features.

High-resolution cytogenetic analysis showed apparently normal karyotypes in the proband and family members. However the MLPA analysis used the P245 microdeletion syndromes-1 probemix detected a duplication of the MECP2 region in the proband, his older brother and mother. These results were re-evaluated using the P015 MECP2 probemix and in addition to MECP2 gene, duplication signals for IRAK1, L1CAM and IDH3G were also identified (Fig. 1b and c).

In the HUMARA analysis the mother was heterozygous (302/314) and the proband was hemizygous (302) for HUMARA locus. After HpaII digestion (only unmethylated allele digested), only one allele (302) was amplified in the mother and no amplification product was obtained in the proband. These results indicated that XCI in the mother was completely skewed, only the X chromosome with duplication of the Xq28 region were inactivated in almost all cells (Fig. 3).

Array CGH analysis confirmed the earlier MLPA results and revealed the exact breakpoints and the size of the duplicated region. A gain signal at Xq28 region spanning about 411kb was identified in both patients and their mother (chrX:153,0027,303-153,438,781). Array CGH data are summarized in table 2.

A duplication of 411.478 kb in Xq28 including the MECP2 and IRAK1 genes, was identified in our study, while varying sizes of 200–4000 kb have been reported in other studies [10, 11, 17].

Xq28 duplication syndrome involving MECP2 gene is inherited as an X-linked recessive trait associated with severe to profound intellectual disability. Clinical manifestations are male-specific with symptoms such as recurrent infections that may lead to premature death, infantile hypotonia, mild dysmorphic features, progressive spasticity of the lower limbs observed after 3 years of age, autistic features, and generalized epilepsy [8]. These clinical features were first described as Lubs-type X-linked mental retardation syndrome (MIM #300260) [18], and current understanding of the genetic basis was recognized as chromosomal duplication of the MECP2 region [19]. The function of MeCP2 as repressor or activator of transcription, chromatin architecture, and regulation of RNA splicing contributes to essential brain development as a regulator of synaptic and neuronal plasticity [20, 21]. MECP2 overexpression in mice also present as a neurological phenotype. The mice exhibited hallmark symptoms such as abnormal forepaw movements, hypoactivity, and premature death [22]. Additionally, mice that overexpressed wild-type human MECP2 from its own promoter developed progressive seizures [22]. Interestingly enough, this study and additional scans of the literature indicate that epilepsy associated with MECP2 duplication syndrome cannot be considered as a useful marker for early diagnosis. However, epilepsy is present in >90% of adolescent patients and shows a peculiar electro-clinical pattern. In a recent review, Ramocki et al. reported in >50% incidence of epilepsy in 110 patients with MECP2 duplication, resulting in severe seizures of multiple types [8]. More than half of the patients were resistant to seizure drug medicine, and there was also a case in which a patient was on a combination of multiple seizure drugs, and a ketogenic diet [23]. The common interictal EEG pattern in patients with MECP2 duplication from this study revealed asynchronous waves and generalized discharge of spikes from the fronto-temporal area with abnormal EEG background frequency and rhythm, such as the abnormal K complex and sleep spindle [23]. These EEG patterns were similar to those found in other genetic syndromes, such as 4p- syndrome or Angelman syndrome [24], thus perhaps making diagnosis difficult. With regards to possible therapies, a previous study reported that deep brain stimulation reduced convulsions up to 65% in case of failed multiple anti-epileptic drugs [25]. Epilepsy is a significant sign of MECP2 duplication syndrome, and an EEG follow-up of these patients from early childhood should be encouraged. Moreover, the definition of a more specific epileptic phenotype could be useful in order to suspect MECP2 duplication syndrome in older, undiagnosed patients. In our study, Patient 1 was treated with oxcarbazepine and Patient 2 with valproic acid, and serial follow-up interictal EEGs are needed to confirm the clinical improvement.

MECP2 severity is linked to copy number. Del Gaudio et al. found a MECP2 triplication in one boy resulting in a more severe phenotype than MECP2 duplication [9]. This implies that copy number is associated with enhanced disease. Thus, the MECP2 gene is a dose-sensitive and a critical gene in neurological development and disorders. Additionally in our study, we note that among the dysmorphic and neurological XLID phenotypes observed in Patient 1 and 2, the influence of MECP2 was most critical in the manifestation of these features. Undiagnosed patients presenting with MECP2 duplication phenotypes and seizure should be tested for MECP2 duplications via the array-CGH or MLPA, diagnostics that were crucial in our study.

X-linked genetic etiologies associated with infantile central hypotonia are Pelizaeus-Merzbacher Allan-Herndon-Dudley, Coffin-Lowry, Lowe, alpha thalassemia, and MECP2 spectrum disorders such as Rett syndrome and MECP2 duplication at Xq28 [26]. Treatment for infantile central hypotonia is massive supportive and symptomatic care. In our study, Patient 1 was treated in the neonatal intensive care unit due to infantile central hypotonia with poor sucking. If a genetic study was carried out at that time of his birth, he could have been diagnosed. We suggest that infantile central hypotonia patients must be considered for testing for Xq28 duplication syndrome, particularly duplication at MECP2.

Several children with duplicated MECP2 and IRAK1 were reported to have suffered severe recurrent respiratory infections with decreased IgA levels and poor antibody responses due to polysaccharide antigens exhibited in some children [26]. Even though Patient 2 had severe recurrent respiratory infections requiring hospitalization thrice a year, there was no evidence of immunodeficiency as per our assessment of complement and Ig antibodies. In a previous study, patients with overexpressed MECP2 were partially immunodeficient possibly due to the overexpression of MECP2 suppressing interferon (IFN)-γ secretion from T helper cells [27]. The intermediate signaling molecule, IRAK1, is known to have a critical roles in the Toll-like receptor (TLR) signaling pathway and activation and regulation of innate and adaptive immunity [28, 29]. Thus, cellular immune dysfunction, due in part to the suppression of IFN-γ production from T helper cells, is potentially caused by overexpression of MECP2 and IRAK1 in Xq28 duplication syndrome. However, additional studies for humoral immune dysfunction in Xq28 duplication are required.

Females rarely display the canonical neurological phenotypes including intellectual disability, associated with MECP2 duplication is due to extreme (>80%) or complete (100%) skewed XCI [30, 31]. The Mother of Patients 1 and 2 had the same duplication profile as her son. However, she had completely skewed (100%) XCI, including the duplicated MECP2 region, and she significantly little neurological manifestations as compared to her sons. Female patients carrying MECP2 mutations in Rett syndrome display X-linked dominant inheritance and not skewed XCI. However, female carriers with MECP2 duplication exhibit X-linked recessive inheritance and skewed XCI. This implies that overexpression of the neighboring genes in the duplicated Xq28 region rather than MECP2 itself causes negative selection, leading to XCI during the early embryonic period [32]. Mayo et al. specifically noted that the arrest-defective protein 1 homolog A (ARD1A or NAA10; MIM *300013) and host cell factor C1 (HCFC1; MIM *300019) as genes responsible for induced preferential XCI in females [33]. In our study the Mother had duplication in parts of NAA10 and HCFC1, and thus the possibility of negative selection was considered. In prior studies, obvious clinical phenotypes of MECP2 duplication syndrome was reported in females with less than 70% skewed XCI [31, 34‐36]. Additional research on the mechanism of XCI is needed, especially since negative selection may not occur despite the duplication of NAA1 and HCFC1. Further studies dissecting the genotype-phenotype correlation against the degree of MECP2 expression are also required.

MECP2/IRAK1 duplication at Xq28 is inherited as an X-linked recessive trait and male-specific disorder associated with severe intellectual disability. MECP2/IRAK1 was associated with primary dose-sensitive and dose-critical gene for the neurological phenotypes. Carrier mother had mild neuropsychiatric symptoms despite of markedly skewed XCI. Additional research is needed to know the relationship between neuropsychiatric phenotype and degree of MECP2 expression.