De novo deletion in MECP2 in a monozygotic twin pair: a case report
© Mittal et al; licensee BioMed Central Ltd. 2011
Received: 7 January 2011
Accepted: 27 August 2011
Published: 27 August 2011
Rett syndrome (RTT) is a severe, progressive, neurodevelopmental disorder predominantly observed in females that leads to intellectual disability. Mutations and gross rearrangements in MECP2 account for a large proportion of cases with RTT. A limited number of twin pairs with RTT have also been reported in literature.
We investigated 13 year old, monozygotic twin females with RTT and some noticeable differences in development using a combinatorial approach of sequencing and Taqman assay. Monozygosity status of the twins was confirmed by informative microsatellite markers.
The twins shared a de novo deletion in exon 3 in the MBD domain of MECP2. To the best of our knowledge, this is only the second report of genetic analysis of a monozygotic twin pair.
Rett Syndrome (RTT; MIM # 312750) is a severe progressive neurodevelopmental disorder that predominantly affects females. It has an estimated global prevalence of approximately one in 10,000-15,000 female births [1, 2] and one in 100,000 male births [3, 4]. Typical/classical RTT is characterized by normal development up to the age of 7-18 months; then a period of developmental stagnation followed by rapid regression, deceleration of head growth, stereotypic hand movements, loss of speech and acquired motor skills. In contrast, atypical RTT refers to a subset of patients who do not meet all the criteria but manifest a variant form of the disease which exhibits heterogeneity in terms of age of onset, severity and clinical course [5, 6]. Point mutations and insertion/deletion variations in MECP2 (Xq28) account for approximately 70-80% of cases with classic RTT [7, 8] and a lower percentage of atypical cases [9–11]. Gross rearrangements in MECP2, which cannot be detected by sequencing or dHPLC, can be identified by a range of alternate methods such as Southern blot analysis [12, 13], gene dosage assays with quantitative fluorescent PCR [14, 15], and multiplex ligation-dependent probe amplification (MLPA) [16, 17]. These methods contribute to unequivocal diagnosis of an additional ~10% of mutation negative cases . Exons 3 and 4 in the MECP2 have been identified to be hotspots for rearrangements [1, 2].
A limited number of twin pairs with RTT, all clinically well characterized, have also been reported in literature. Some studies have described twins showing almost concordant clinical features suggesting a genetic basis for RTT syndrome [19, 20]. To the contrary, others described clinical discordance in monozygotic twins with RTT, with early developmental differences [21, 22] and also with regard to seizures, scoliosis and stereotypic hand movements during adolescence in a twin pair . However, genetic analysis has been reported for only in a single monozygotic twin pair . This study is a second report describing the genetic basis of RTT in a 13 year old monozygotic female twin pair.
The monozygotic twin females were born in an uneventful pregnancy by caesarean section with each having birth weight of 2.4 kg. The twins had normal motor milestones till about two years of age. Regression of milestones was observed following seizures, the younger of the twins at two years of age and the elder six months later. They also show minor phenotypic variation between them. The older twin had short stature (height -130 cm- < 5th Centile NCHS); and a head circumference of 51.5 cm; was non-verbal with poor response to commands; has a Vineland Social Maturity Scale (VSMS) score of 19 indicating profound mental retardation. The gait was wide based with no contractures and she had an attention span of 10-15 minutes. The younger twin also had short stature (height - 122 cm < 5th centile) and a head circumference of 48.5 cm; had a vocabulary of few single words; responded promptly to commands; as severely mentally retarded with a VSMS score of 23. She also had a wide based gait with mild knee contractures; and was on the move all the time. Both the twins had stereotypic behavior (hand biting and wringing movements); had thin and wasted limbs; had no organomegaly or evidence of head trauma or birth asphyxia; no difficulties in eating, chewing or swallowing; had normal vision; no sleep disturbances to date; no scoliosis; both were toilet trained and could indicate their needs well.
The study was approved by the institutional ethical committee. Informed consent was obtained from the parents and blood samples (and also photographs) were collected for genetic analysis from the twin pair and parents. Patients were diagnosed as per criteria previously described . Ten age and sex matched female healthy controls with no history of mental illness were also recruited from the participating hospitals. These healthy controls were used for normalization of the relative quantification assay. Fourteen highly polymorphic microsatellite repeat markers were genotyped in the patient family to confirm monozygosity.
a) Mutation Analysis
In order to detect point mutations and small homo-/heterozygous interstitial deletions/insertions all four exons of MECP2 were sequenced in both the twins. PCR amplification of complete exons including exon-intron boundaries were carried out using published primers  and the amplification products were sequenced using ABI3700 genetic analyzer.
b) Gene dosage analysis: detection of gene rearrangements
Details of primers and probes for the Taqman Assay
Forward Primer sequence
Reverse Primer sequence
5' AGCGGCGCTCCATCATC 3'
5' TTCCGTGTCCAGCCTTCAG 3'
5' CATGGGTCCCCGGTCAC 3'
5' GCTCCTTGTCAAGATGCCTTTTC 3'
5' CCATGACCTGGGTGGATGTG 3'
5' CCCTCAGCCTTGCCC 3'
5' GCGTCTGCAAAGAGGAGAAGAT 3'
5' GCGGGCTGAGTCTTAGCT 3'
5' CAGCCGTCGCTCTC 3'
Microsatellite markers for confirmation of monozygosity
We genotyped 14 microsatellite markers in the two parental samples, of these only nine markers were informative. These informative markers were genotyped in the twin pair. We observed identical alleles at all these nine markers confirming their monozygosity.
We screened the four exons and exon-intron boundaries among the twin pair by sequencing. No mutation was observed ruling out the involvement of any point mutations or small homo-/heterozygous interstitial deletions/insertions in the twin pair.
Gene dosage analysis
In the absence of any mutation in the four exons of MECP2, we carried out gene dosage analysis to identify heterozygous deletions/duplications, if any, in the rearrangement hotspot regions of the gene. Amplification efficiencies of the target and control (RNAseP) primers were observed to be similar (~96-100% range). This is a prerequisite for using the comparative Ct method for gene dosage analysis. To determine the range of RelQ values in normal population we analyzed 10 healthy controls (5 males and females each). There was no overlap in the RelQ between the male and female controls, thus validating the use of this assay for gene dosage. RelQ values in cases deviating significantly from the controls were considered as deletions or duplications. A RelQ ratio of ~1.5 represents three copies (duplication) whereas ~0.5 represents one copy (deletion) as compared to the two copies in the female sample.
RelQ values for Taqman probes in the RTT family under study
Location of Taqman Probe
Mutations/gene rearrangements in MECP2 resulting in RTT are mostly germ line events resulting in sporadic cases of RTT. A small proportion of affected sibs but with unshared mutations with mother implying germline mosaicism have also been observed [13, 27–29]. In addition, reports of rare cases of inherited mutations also exist [30, 31]. However, in such families, the mothers showed either very mild  or almost unaffected [32, 33] phenotype which has been explained on the basis of skewed/non-random X chromosome inactivation . In the case of proven monozygotic RTT twins, shared mutation may be expected. Probability of monozygosity is > 99.9% when more than five highly polymorphic markers have identical genotypes within a twin pair . Thus, 100% concordance with all the nine markers in our study confirms the monozygotic origin of the twin pair.
Mutation screening of the four exons of MECP2 in the twin pair did not show any point mutation. However, both monozygotic twins showed a heterozygous deletion in exon3 identified by Taqman assay (Table 2). The rearrangement was not shared with either of the parents, thus representing a de novo origin of the probable pathological variation in the family. As mentioned earlier, there were minor phenotypic differences in these twins. This may either be due to non-random (in)activation of the paternal/maternal X chromosomes  or may be due to yet poorly understood epigenetic signatures, if any, at MECP2 locus. The former assumption has partly been proved by another study where the twin sisters shared the mutation (R294X), but showed discordant clinical phenotype; they observed skewing in favour of the paternal allele in the twin with more severe phenotype . Preferential paternal X chromosome involvement has been shown in several other sporadic RTT cases [35, 36]. Twin studies may be useful to understand the genetic as well as the non-genetic contribution to the disease phenotype. Discordance witnessed among some twin pairs including the pair described in this study warrant further studies to unravel epigenetic mechanisms influencing RTT phenotype.
Written informed consent was obtained from the parents of the patients for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.
Acknowledgements and Funding
We gratefully thank RTT patients and their family for their willing support and participation in this study; Mrs. Jaya Krishnaswamy and Dr. Padmalochani of the Madhuram Narayan Centre for Exceptional children, Chennai, for immense support in recruitment and follow up of study samples; Prof. John Vincent, Centre of Addiction & Mental Health, University of Toronto, Canada, for greatly improving the quality of the manuscript with his critical inputs; Dr. Mainak Majumder, Labindia, Gurgaon, India for help with the Taqman assay. We also acknowledge the Central Instrumentation facility at University of Delhi South Campus for DNA sequencing. This work was supported by financial assistance from Department of Biotechnology, Govt. of India, New Delhi (#BT/PR4959/Med/14/C75/2004) and a research fellowship from University Grants Commission, New Delhi to KM.
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