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Page 94 Khajuria et al. J Transl Genet Genom 2020;4:91-103 I http://dx.doi.org/10.20517/jtgg.2020.06

Statistical analysis
We used Analysis of Variance (ANOVA) to compare the group mean score on dependent variables,
2
covarying for age. Categorical variables were analyzed for significant associations using Pearson χ . STATA
version 9 was used for all statistical analysis.

RESULTS
Phenotypic features
All patients had apparently normal prenatal and perinatal history with normal early psychomotor
development. We did not have head circumference records of all patients at birth, but all had microcephaly
at the time of inclusion. The mean age of onset of symptoms in classical RTT patients was 16 ± 4.6 months
(range: 6-30) and the median age was 16 months. The mean age at diagnosis of classical RTT patients was
54 ± 34.9 months (range: 18-186 months) while the median age was 42 months. All patients had partial or
complete loss of acquired purposeful hand skills. Stereotypic hand movements were present in all patients
and the common hand stereotypes observed in our patients were hand wringing, washing, mouthing,
clenching, finger rolling, tapping, and clapping. All patients had partial or complete loss of acquired
language and most patients spoke only monosyllables or babbling. All patients had gait problems, of which
40% (29/72 patients) could not walk, 39% (28/72 patients) had an impaired gait, and 21% (15/72 patients)
could walk with support.

MECP2 sequence variants
Using DNA sequencing, we identified MECP2 sequence variants in coding region with a frequency of
88.9%. Using MLPA, large deletions of MECP2 gene were identified in 9.7% of patients. One patient was
identified with intronic variant and no other sequence variant was identified in this patient. In total, 38
different types of MECP2 sequence variations (25 reported and 13 novel) were identified in all 72 classical
RTT patients [Table 1].

Six recurrent sequence variants (p.P152R, p.T158M, p.R168X, p.R270X, p.G269fs, and p.R306C) were
identified in 36/72 RTT patients. All of these variants were identified in heterozygous form. The p.T158M
and p.R270X were the most recurrent sequence variants observed, followed by p.R168X, p.G269fs, p.R306C,
and p.P152R variants [Table 2]. Twenty-nine patients (40.3%) were found positive with other uncommon
heterozygous MECP2 sequence variants and one patient (1.38%) was identified with only one intronic
variant c.378-74C>T [Table 1].

Seven patients (six negative for MECP2 sequence variants on DNA sequencing and one patient who was
identified with only one intronic variant c.378-74C>T) were screened using MLPA analysis for ruling out
large deletion/duplications of MECP2 gene that cannot be identified using Sanger sequencing. In six out of
seven (8.33%) RTT patients, large deletions of one or more contiguous exons, especially of exon 3 and 4 of
MECP2, gene were identified. In one patient, an undefined deletion in 3’UTR of MECP2 gene was detected
[Table 1], and it was confirmed that there was no sequence change at the probe hybridization site, thus it
most likely was a real deletion.

Most of the mutations or sequence variants identified in patients were de novo and were not found in the
family members except two cases where different mutation or variant was identified in family members.
In one family, the asymptomatic mother of one RTT with p.D17fs mutation was found carrying missense
mutation p.H51Q (GenBank accession No. GU812286.1) of MECP2. In another family, the patient was
carrying p.R106W mutation, whereas her asymptomatic father was a carrier of intronic c.378-74C>T
variant of MECP2 gene. However, in three cases, the origin of novel large deletions could not be confirmed
as the parents were not available for testing.