De novo mosaic MECP2 mutation in a female with Rett syndrome

Key Clinical Message We describe a female with Rett syndrome carrying a rare de novo mosaic nonsense mutation on MECP2 gene, with random X‐chromosome inactivation. Rett syndrome severity in females depends on mosaicism level and tissue specificity, X‐chromosome inactivation, epigenetics and environment. Rett syndrome should be considered in both males and females.


| INTRODUCTION
This study is the first report of a female patient carrying a rare MECP2 point mutation in a mosaic form with typical RS phenotype. Copy number and methylation analysis excluded Prader-Willi and Angelman syndromes. Sanger sequencing of MECP2 gene revealed a de novo mosaic C>T nonsense mutation at position 139 of exon 3. In order to estimate the level of mosaicism, Next-Generation Sequencing was performed, showing approximately 25% abnormal cells. X-chromosome inactivation analysis of the patient showed a random pattern.
This is the first known female mosaic MECP2 mutation carrier, with a classical RS phenotype. The specific mutation in its full non-mosaic form was previously detected in only one RS patient.
Detailed investigation of rare mosaic patients is of significant value for the understanding of molecular mechanisms involved in tissue-specific gene expression and development of RS clinical features.
Rett syndrome (RTT) was first described by Andreas Rett in 1966 as a progressive neurodevelopmental disorder. 1 It currently accounts for a large portion of intellectual disability (ID) in females, with its prevalence estimated at 1:10 000 live female births. 2 The main clinical features of RS include developmental regression affecting motor and language skills, stereotypical hand movements, microcephaly, and ataxia. The criteria necessary for RS diagnosis are | 367 ALEXANDROU Et AL. normal prenatal and perinatal period; normal psychomotor development through the first 6 months of life; normal head circumference at birth, with subsequent deceleration of head growth; loss of purposeful hand skills; severely impaired expressive and receptive language; apparent severe intellectual disability; and gait apraxia and truncal apraxia/ataxia. 3 The phenotypic variation observed in RS patients over the years allowed for a definition of "classical" and "atypical" forms, with their diagnostic boundaries more clearly set in 2010. 4  Rett syndrome is an X-linked dominant disorder, with the vast majority of cases being caused by mutations or deletions in the MECP2 gene located on Xq28, coding for the methyl-CPG-binding protein 2. 5 However, it has recently been shown that approximately 5% of patients with RS features may carry mutations within other genes such as cyclin-dependent kinaselike 5 (CDLK5) and forkhead box G1 (FOXG1), as well as other genes related to epilepsy or intellectual disability. [6][7][8][9] MECP2 mutations can also be found in males, although at a lower frequency, since most full MECP2 mutations and deletions are lethal at the foetal stage. Other types of full mutations within MECP2 gene in males can result in a variety of phenotypes from mild ID to severe neonatal encephalopathy. 10 In a more recent report, a male with classic RS was described to carry a 5 bp duplication in the open reading frame of exonh1 of MECP2, thus highlighting the importance of considering RS in males, as well as in females. 11 In case of somatic MECP2 mosaicism, male patients can survive to birth and exhibit clinical features similar to the female full mutation 12 or an atypical RS phenotype. 13,14 The frequency of MECP2 mosaicism is currently unknown, with only male mosaic patient being reported so far and to our knowledge, no reports have been published to date describing MECP2 mutation mosaicism in females.
In this study, we present a unique case of a rare mosaic MECP2 point mutation found in a female patient with typical Rett syndrome. This new finding will add to the understanding of pathogenic mechanisms leading to phenotype manifestation in somatic mosaicism and may assist in the predictions concerning RS severity.

| Patients and samples
The patient was a five-year-old girl at the time of referral, with a phenotype highly indicative of typical Rett syndrome, including autistic features, psychomotor delay, severe intellectual disability, normal MRI, speech regression, and stereotypic hand movements. The patient's biological parents are unaffected. The patient was referred for investigation of Prader Willi-Angelman (PWA) syndromes as part of consented diagnostic services.
DNA samples of the patient and both biological parents were obtained by isolation from peripheral blood using the QIAamp DNA Midi kit (Qiagen, Hidden, Germany) according to the supplier's protocol.
Mutation analysis of all four coding exons and intron/ exon boundaries of the MECP2 gene were carried out using PCR amplification and direct sequencing, as described in Appendix S1.
Parental testing was performed using primer sequences for Exon 3 and confirmed by restriction enzyme analysis using the Bbv1 enzyme (New England BioLabs Inc., Ipswich, MA, USA).
Next-Generation Sequencing (NGS) was performed targeting, among others, all coding regions of MECP2 and FOXG1, using the Nextera XT DNA Library preparation kit (Illumina, San Diego, CA, USA) with the obtained PCR amplicons on NextSeq 500 system according to the manufacturer's protocol (Illumina Inc).
The X-chromosome inactivation (XCI) pattern was determined by methylation analysis for the region flanking the polymorphic CAG repeat in exon 1 of the AR gene. 15

| RESULTS
Array-CGH analysis of the patient, as well as MS-MLPA for PWA resulted in no abnormal findings (data not shown). The phenotype of the patient was highly indicative of RS; therefore, targeted Sanger sequencing of 368 | ALEXANDROU Et AL.
MECP2 gene was performed and subsequently revealed a de novo mosaic C>T mutation at position 139 in exon 3, transcript NM_004992 ( Figure 1). The aberration is a nonsense mutation predicted to be p.Gln47X at the protein level. Restriction enzyme analysis gave normal results for both parents and also confirmed the mutation in the patient ( Figure 1). The level of mosaicism was estimated with NGS to be approximately 25% (Figure 2). X-chromosome inactivation (XCI) analysis of the patient showed a random XCI pattern.

| DISCUSSION
In this case report, we describe a female patient with a phenotype highly indicative of RS, carrying a rare mosaic MECP2 mutation. This c.139C=/>T de novo nonsense mutation on exon 3 creates a premature stop codon resulting in a truncated protein. The mutation is rare and has only been reported once in the literature 16 in a large study investigating RS genotypephenotype correlation. The presence of this mutation in its full non-mosaic form was detected in a female patient with classical RS phenotype, and further studies have shown an 83% XCI ratio. 16 Its localization within the coding region of the RS main causative gene in combination with its predicted truncating effect suggest that it is most probably causative of the phenotype described in our patient.
To the best of our knowledge, female MECP2 mosaic mutations have not been described in the literature. Male individuals carrying causative MECP2 mutations in a mosaic form have typical or mild/atypical RS, which is considerably milder than male carriers of the corresponding full mutations, who rarely survive to birth. 12 Extrapolating on female mosaics, one would theoretically expect a mild RS or a status of almost asymptomatic carrier. In our case, despite the relatively low level of mosaicism (~25%), the patient is exhibiting the full spectrum of Rett syndrome phenotype, including some key clinical features such as autistic traits, psychomotor delay, speech regression, and stereotypic hand movements.
This apparently unexpected severity may be partially explained by high penetrance of the new mutation due to its truncating nature. Truncating mutations within MECP2 have been associated with a more severe RS manifestation, as compared to missense mutations which may cause a milder or atypical RS. 17 On the same level, the patient's genetic background may also influence the severity of some symptoms attributed to RS. In contrast to male mosaic MECP2 mutation carriers, female mosaic phenotype is significantly influenced by Xchromosome inactivation (XCI) patterns. A skewed XCI could be an important factor, as it regulates the expression of the mutated MECP2 allele within the brain. 18 In the patient described in this study, XCI studies have shown a random pattern; therefore, the phenotypic outcome cannot be explained within this context.
Besides the percentage of mosaicism, the varying levels of mutated cells in different tissues may be also contributing to a more severe than expected phenotype in the patient. For example, higher levels of cells with MECP2 mutation may be present in the brain, as compared to blood. As brain is not an easily accessible tissue, a buccal swab would be a feasible alternative in order to get a closer estimation of the possible level of mosaicism in the brain. The utility of this approach has been demonstrated in testing for various psychiatric disorders using expression and XCI studies which have shown correlating results in buccal and brain tissue cells. 19,20 Despite the high potential value of buccal swab analysis, we were unable to obtain a sample, as the family was not available for further testing.
The introduction of Next-generation sequencing (NGS) into routine genetic diagnosis is expected to play a key role in the future investigation of mosaicism in general. Out of all the techniques applied in this study, NGS was the only one that offered the necessary sensitivity to allow an estimation of the exact percentage of the mosaicism level, even though the presence of mosaicism was also evident by Sanger sequencing. Moreover, recent studies apply NGS to screen various patient groups including patients with RS phenotypes, revealing new mutations within MECP2 thus proposing new pathogenic pathways. 21 Based on the above, NGS will not only add to the list of pathogenic mutations, but also provide more data concerning the frequency of somatic mosaicism in patients with various syndromes.
The detection and thorough investigation or somatic mosaicism in RS patients as well as in other syndromes is a valuable tool for uncovering unknown mechanisms of disease development and severity assessment based on tissue specificity.