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Page 100 Khajuria et al. J Transl Genet Genom 2020;4:91-103 I http://dx.doi.org/10.20517/jtgg.2020.06

In the present study, we were able to find MECP2 sequence variations in overall 90.3% of RTT patients
using DNA sequencing. Using MLPA analysis, we were able to detect large putative deletions of MECP2
in all the classical RTT patients, which were negative on DNA sequencing. MLPA increased the detection
rate of MECP2 sequence variants identified in RTT patients from 90.3% to 98.6%. We propose that MLPA
analysis of MECP2 is crucial and needs to be performed in classical RTT patients. Large deletions can be
missed using DNA sequencing and reaffirms the view that large MECP2 deletions are an important cause
of classical RTT [6,13] . In this study, the majority of the RTT patients were carrying the C>T transitions,
[12]
supporting the reported literature .

Data from different western studies have shown MECP2 sequence variation frequency between 70% and
97% in classical RTT [1,3,7,12,16,18] . A literature search revealed many studies on RTT from Asia that reported
MECP2 sequence variation frequency of 50%-92.5% in classical RTT patients [19-23] . In the two Indian studies
[8,9]
published thus far, the detection rates of MECP2 variations was lower as compared to our study . The
[9]
study by Lallar et al. , which included 19 RTT patients (14 classical and 5 atypical), reported a detection
rate of 93% in classical RTT girls using a combination of Sanger sequencing followed by MLPA analysis,
[8]
supporting our findings. In the other Indian study by Das et al. , investigating 90 individuals with
suspected RTT phenotype, 19 different MECP2 mutations and polymorphisms were identified in 27/90
(30%) patients while the rest remained uncharacterized.

The high rate of MECP2 sequence variation detected in the present study compared to the data from other
Asian, Indian, and western studies can be explained by the fact that a strict inclusion/exclusion of classical
RTT patients was adopted based on revised clinical diagnostic criteria of RTT and involvement of a
[4]
multidisciplinary team for clinical evaluation of the patients. Our study supports the previous findings that
clinical stringency based on diagnostic criteria can increase the mutation detection rate in RTT patients
[24]
and emphasizes the importance of diagnostic criteria in the assessment of RTT patients .
The worldwide reported eight hotspot MECP2 sequence variants p.R106W, p.R133C, p.T158M, p.R168X,
p.R255X, p.R270X, p.R294X, and p.R306C were identified with a frequency of 57% in our study, which
[3,7]
is similar to previously reported western and Asian studies . The hotspot variant p.R294X identified
recurrently in western population was found in only one patient in the present study, whereas other
hotspot variants, namely p.P152R, p.G269fs, and p.L386fs, were identified in more than one patient. In
another Indian study by Das et al. , which included 90 patients (suspected RTT patients), showed MECP2
[8]
sequence variations in only 30% of patients (27/90 patients), of which seven hotspot variants, namely
p.R106W, p.R133C, p.T158M, p.R168X, p.R255X, p.R270X, and p.R306C (sequence variant p.R294X was
[9]
not reported in this study), were identified with frequency of 51.8%. The other Indian study by Lallar et al.
reported four common hotspot variants (p.R168X, p.T158M, p.R306C, and p.R255X). The present study
indicates that the variant p.R294X may not be a hotspot sequence variation in Indian RTT patients and
instead p.G269fs, p.P152R, and p.L386fs, may be the hotspot sequence variations along with the seven other
recurrent variants identified. Multiple recurrences of these three variant (p.G269fs, p.P152R, and p.L386fs)
in the present study indicate that these variants could be the hotspot variants specific to Indian population.
Larger studies from India are needed to confirm and support these findings.

The dentification of 13 novel variants, including four large deletions, was another highlight of our study,
which emphasizes the genetic heterogeneity of MECP2 variants and underlines the need for generating
population specific data. In view of identification of other recurrent variants in our study along with
reported hot spot mutations, sequencing the MECP2 gene (beginning with exons 3 and 4) followed by
MLPA testing if sequencing results are negative is recommended rather than targeted testing.

The majority of the variants were distributed in the functional domain of MECP2 with most missense
variants clustered in MBD and truncating variants in ID and TRD of MECP2 [Figure 2], which support