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Khajuria et al. J Transl Genet Genom 2020;4:91-103 I http://dx.doi.org/10.20517/jtgg.2020.06 Page 93

Sciences New Delhi, India. Patients were defined as classical when they showed a period of regression and
fulfilled all the main inclusion criteria (partial or complete loss of acquired purposeful hand skills; partial
or complete loss of acquired spoken language; gait abnormalities; impaired or absence of ability to walk;
stereotypic hand movements) as per the revised diagnostic criteria for classical RTT . The patients not
[4]
[4]
fulfilling the major criteria and having the clinical signs mentioned in exclusion criteria were excluded
from the study. Exclusion criteria were: evidence of brain injury secondary to perinatal or postnatal events,
neuro-metabolic disorders or infection causing neurological problems. Additionally, children having
grossly abnormal psychomotor development in first six months of life were also excluded. Ethical approval
for the present study was taken from the Ethics Committee of the Institute. Proper information about
the study was given to all families and written informed consent was obtained from the parent/guardian.
Enrolled patients represented all regions of India, with the majority from northern India. Patients were
evaluated by a team comprising of a clinical geneticist, a pediatric neurologist, and a child psychologist
before inclusion in the study. All clinical details were recorded in a predesigned proforma.

Five-milliliter blood samples were collected from all patients in EDTA vacutainer and DNA was
extracted by standard phenol-chloroform method. DNA samples were analyzed using bidirectional
Sanger sequencing for sequence variations followed by quantitative analysis using Multiplex Ligation-
dependent Probe Amplification (MLPA) for deletion/duplication analysis of MECP2 gene. The gene
nomenclature used was according to guidelines of HGNC (Hugo Genome Nomenclature Committee) and
[10]
the recommended sequence variant nomenclature of HGVS (Human Genome Variation Society) . Any
change found in DNA sequence of the RTT patients was also analyzed in their family members (except
three cases) to confirm its origin. The sequence variant profile was compared with the clinical presentation
to generate genotype-phenotype correlations. All novel variants identified in this study were submitted to
National Centre for Biotechnology Information (NCBI) GenBank (http://www.ncbi.nlm.nih.gov/GenBank)
[11]
and RettBASE: IRSF MECP2 Variation Database (http://mecp2.chw.edu.au) .
MECP2 screening
The coding region of exons 2-4 of MECP2 gene (transcript 1, MeCP2_e2) including flanking exon/intron
boundaries was amplified by PCR using seven overlapping primer sets (2.1, 2.2, 3.1, 3.2, 3.3, 4.1, and 4.2)
of MECP2 gene published elsewhere [1,12] . PCR amplification was performed in a final volume of 25 µL
containing 10× PCR buffer with 1.5 mM MgCl , 0.25 mM dNTPs, 0.625 U Taq polymerase, 1 pM/µL each
2
of forward and reverse primer, and 50 ng of DNA. All samples were analyzed by direct bidirectional Sanger
sequencing. The data were interpreted and compared with reference sequence of MECP2 (NM_004992.3)
gene. MLPA technique was used to screen the RTT patients who were negative for MECP2 sequence
variations on DNA sequencing to check for gross rearrangements [6,13,14] . SALSA MLPA kits P015-D2 and
P015-E2 (MRC-Holland, Amsterdam, The Netherlands) were used. All cases with positive or aberrant
results were rerun in a second MLPA reaction for confirmation.

Bioinformatic analysis
In the present study, various bioinformatic software packages were used for prediction of pathogenicity
of MECP2 sequence variations. Prediction tools such as PolyPhen (Polymorphism Phenotyping) (http://
genetics.bwh.harvard.edu/pph/), SIFT (Sorting Tolerant from Intolerant) (http://sift.jcvi.org/), and SNPs3D
(http://www.snps3d.org/) were used to predict if the non-synonymous particular variants are likely to
be deleterious. Prediction of the effect of synonymous variants was done using Codon Usage Database
(www.kazusa.or.jp/codon/). In-silico splice site analysis was done using Alamut (http://www.interactive-
biosoftware.com/alamut/) for prediction of splice-site variants. We used MeCP2_e2 transcript of MeCP2 for
pair wise alignment and the sequences were derived from NCBI HomoloGene (http://www.ncbi.nlm.nih.
gov/homologene). The alignments were produced using ClustalW (http://www.ebi.ac.uk/Tools/clustalw/).