4.1.10. The Effect of DNA Concentration on Reliability and Reproducibility of SYBR Green Dye-based Real Time PCR Analysis to Detect the Exon Rearrangements 184.108.40.206. Preparation of DNA Samples.Photometric measurement of the stock DNA and
the dilutions of 200 ng/µl were performed on an ND-1000 spectrophotometer (NanoDrop
Technologies). DNA was diluted first to 200 ng/µl, stored at 37 °C overnight, and then
diluted to final concentration of 0.025 ng/µl. Six dilutions at concentrations of 0.025, 0.25,
1.25, 12.5, 25, and 50 ng/µl were prepared from the patient R23 with MECP2 exon 3
deletion, patient R19 with MECP2 exon 3 duplication, and two healthy females and one
male. Standards (1.25, 2.5, and 5 ng/µl) were prepared by dilution of a healthy female
genomic DNA. All dilutions were prepared in a volume of 200 µl.
220.127.116.11. Quantitative Real Time PCR Conditions.The real time PCR was performed in a
total volume of 20 µl, containing 10 µl 2X SYBR Green PCR Master Mix (TaKaRa,
Japan), 1 µl of each primer per reaction, 4 µl of the genomic DNA dilution and distilled
water. The PCR protocol on Light Cycler (LC) (Roche Diagnostics, Mannheim, Germany)
was as follows: an initial denaturation step (95°C for 2 min) followed by amplification and
quantification steps repeated for 40-50 cycles (95°C for 5 sec, 59°C for 10 sec, 72°C for
20 sec, with a single fluorescence measurement at the end of the elongation step at 72°C), a
melting curve program (65–98°C with a heating rate of 0.2°C per second and a continuous
fluorescence measurement) and terminated by cooling to 40°C. Each sample was amplified
with the MeCP2 and reference gene primer pairs. The coding exon 3 of the MeCP2 was
amplified using the following primers designed by Bienvenu et al. (2000); Rett_exon3F:
gtgatacttacatacttgtt; Rett_exon3R: ggctcagcagagtggtgggc. The reference NDRG1 gene was
amplified using primers NDRG1_exon7F: aggctcccgtcactctg; NDRG1_exon7R:
gtcttccttcatcttaaaatg (Kalaydjieva et al., 2000).
Each target and reference gene assay included: 1) a standard curve of three dilution
points of healthy female DNA (20, 10, and 5 ng), 2) 20 ng of calibrator healthy female
DNA, and 3) test sample DNAs. Melting point analysis was conducted on all PCR
products to check for any nonspecific amplicons.
18.104.22.168. Quantification.Quantification was performed using both the standard curve
method and the comparative Ct method. Within each PCR batch three aliquots of wild type
control DNA in decreasing concentrations (20, 10, and 5 ng) from a healthy female were
included to construct a standard curve and the copy numbers of the exons in each sample
were interpolated. Since two different standard curves were constructed for the target and
reference genes, the copy numbers of MECP2 exon 3 were normalized against a calibrator
DNA sample. After normalization to the calibrator, the copy number of MECP2 exon was
calculated by dividing these normalized values by the copy number of the reference gene.
Instead of interpolating unknown samples from a standard curve, it is also possible to
calculate the copy number based on the observed Ct values as follows:
2 - CT = (1+E)
C T targetgene
C T referencegene
Where E is the efficiency of the PCR reaction (set at default value 0.95), C
difference in threshold cycle value between test sample and calibrator sample for the gene
under investigation (test gene), and C
is the difference in threshold cycle value
between test sample and calibrator sample for reference gene.
22.214.171.124. Statistical Analysis.The results were evaluated by a paired sample t-test and
Pearson correlation coefficient using SPSS v 15.0 software (SPSS Inc., Chicago, IL, USA).
4.2. Methylation Analyses of the Putative Promoter Region of hHR23 Genes in Breast Tumor Tissues
4.2.1. Non-heating DNA Extraction Protocol
Genomic DNA was extracted from archival formalin-fixed, paraffin-embedded
human primary breast tumors and normal breast tissues by using a modified version of
non-heating DNA extraction protocol described by Shi et al. (2002). Two sections (10 µm
thick) were obtained from each archival tissue. Sections were deparaffinized by adding 1
ml of xylene to the eppendorf tube for 30 min for two changes, followed by 100 per cent
and 75 per cent ethanol for 15 min with three changes. After a washing step with PBS for
15 min in two changes, 500 µl of tissue lysis buffer was added and incubated at 50 ºC for
48-72 hours until the whole tissue were dissolved completely. The mixture was centrifuged
at 13,000 rpm for 5 min and the supernatant fluid was transferred to a clean eppendorf
tube. Five hundred µl of phenol:chloroform:isopropanol solution (25:24:1) was added,
vortexed and centrifuged at 13,000 rpm for 10 min. The upper aqueous layer was carefully
transferred to a clean tube, 0.1 volume of 3M sodium acetate and 1 volume of isopropanol
were added, mixed by vortexing, and incubated at – 20 ºC for 10 min. The DNA was
precipitated by centrifugation at 13,000 rpm at 4 ºC. The supernatant fluid was discarded
and the precipitate washed once with 75 per cent ethanol. The DNA pellet was dissolved in
50 µl of distilled H
O. The concentration of the isolated DNA was calculated after
measuring the optical density at 260 nm on ND-1000 spectrophotometer (NanoDrop
4.2.2. Promoter Region Analyses and Primer Design
The web-based PROSCAN program (Prestridge, 1995) was used to predict the
putative eukaryotic Pol II promoter sequences in primary sequence data of hHR23A and
hHR 23B genes. MATCH and PROSCAN programs were used to identify high scoring
transcription factor (TF) binding sites. MethPrimer program (Li and Dahiya, 2002) was
used to identify the CpG islands and design the primers using the standard criteria, i.e.,
sequence was considered as a CpG island if there was a minimum G+C content of 50 per
cent with a minimum CpG (obs)/CpG (exp) of 0.6 in a 200-bp window length, and the
sequence length was at least 500-bp.
4.2.3. Bisulfite Modification
Genomic DNA was modified by Methylamp
DNA Modification Kit (Epigentek,
NY, USA) according to the manufacturer's protocol. Briefly, 0.25-1 µg of DNA in a
volume of 24 µl was denatured by adding 1 µl of denaturing buffer for 10 min at 37°C.
Bisulfite-Conversion buffer (125 µl) was added and mixed, and samples were incubated at
65 °C for 2 h. Modified DNA samples were applied to columns, washed, and then eluted
with 20 µl of elution buffer.
4.2.4. Amplification and Sequencing of the Bisulfite Modified DNA
Semi-nested PCR strategy was used to investigate the methylation status of the
putative promoter region of hHR23 genes (Figure 4.2). Several primer combinations were
used to get successful amplification product. The primer combinations were shown in
Figure 4.2. In general, 2.5 µl of the modified DNA was used in subsequent PCR reactions.
First-round PCR reactions were performed in a total volume of 25 µl containing 2.5 µl of
modified DNA, 2.5 µl of 10X polymerase buffer, 2.5 mM MgCl
, 2.5 µl of dimethyl
sulfoxide (DMSO), 200 µM dNTPs, 0.4 µM of each primer, and 2 U of Taq polymerase
(Fermentas). The following PCR program was used: 94ºC for 4 min, followed by 40 cycles
of 45 sec at 94 ºC, 45 sec at annealing temperature, 1 min at 72ºC, and a final extension
step of 8 min at 72ºC. One micro liter of the first-round PCR product was then used as a
template in the second round of PCR with a mixture that contains 2.5 µl of 10X
polymerase buffer, 2.0 mM MgCl2, 200 µM dNTPs, 0.4 µM of each primer, and 1 U of
Taq polymerase (Fermentas). The cycling condition was as follow: denaturation at 94°C
for 5 min was followed by 35 cycles of amplification: 94°C for 30 sec, 30 sec at annealing
temperature, and extension at 72°C for 30 sec. After the last cycle, an 8-min extension at
72°C was performed.
Five µl from the PCR product was run on two per cent agarose gel to check for the
quality of amplification. PCR products were purified using QIAQuick PCR purification kit
(QIAGEN) and sequenced with automated sequencer ABI 3130 PRISM (Applied
Biosystems) in Burç Laboratory (Istanbul, Turkey).
Figure 4.2. Semi-nested PCR strategy showing the primers and PCR cycling conditions
used to investigate the methylation status of hHR23 genes.
4.3. Molecular Basis of Congenital Hypothyroidism (CH)
4.3.1. Mutation Analysis of the TTF2 Gene
Genomic DNA was isolated from peripheral blood sample of the patient with CH,
her consanguineous parents and unaffected brother using salting out method as described
in section 4.1.1.
126.96.36.199. Direct DNA Sequencing Analysis.The entire coding region of TTF2 gene
(accession no. NM_004473) was amplified in two overlapping fragments using the primers
designed by Castanet et al (2002). PCR reaction was performed in a total volume of 25 µl
containing approximately 100 ng DNA, 2.5 µl of 10X polymerase buffer, 2.0 mM MgCl2,
10 per cent dimethyl sulfoxide (DMSO), 200 µM dNTPs, 0.4 µM of each primer, and 1 U
of Taq polymerase (Fermentas,). The following PCR program was used: 94ºC for 4 min,
followed by 35 cycles of 45 sec at 94 ºC, 30 sec at annealing temperature (58ºC for
TTF2A-D, 53ºC for TTF2C-E), 1 min at 72ºC, and a final extension step of 8 min at 72ºC
(Table 1.1.1). The PCR products were purified and bi-directionally sequenced using the
primers TTF2A and TTF2C as described in section 4.1.4.
188.8.131.52. AlwNI Digestion. The PCR products from the patient, her parents, unaffected
brother and 100 control chromosomes were subjected to AlwNI digestion. Genomic DNA
was amplified using the primers TTF2C/TTF2E as described in section 184.108.40.206. A total of
ten µl of the PCR product was digested with 3U of AlwNI restriction enzyme (Fermentas)
and 2 µl of 10X reaction buffer in a 20 µl reaction volume. The mixture was incubated at
37 ºC for 4 hours. The digested products were electrophoresed on two per cent agarose gel
at 100 V for 30 min. The c. 304 C>T (p.R102C) mutation creates a new restriction site
resulting in 72 bp and 599 bp fragments whereas wild type allele remains undigested.
4.3.2. Functional Characterization of p.R102C Mutant TTF2
Functional analyses were performed by Dr. Chatterjee’s laboratory in University of
Cambridge, UK. The DNA binding and transcriptional properties of the mutant and wild
type TTF2 have been investigated as described by Clifton-Bligh et al. (1998). Detailed
information was given in Appendix A.
4.3.3. Mutation Analysis of Butyrylcholinesterase (BChE) Gene
The DNA samples of the patient with CH and her family members were analyzed for
the presence of two most common BChE variants; p.Asp70Gly (A-variant) and
p.Ala539Thr (K-variant) according to Asanuma et al. (1999) and Maekawa et al. (1995),
respectively. PCR reaction was performed in a total volume of 25 µl containing
approximately 100 ng DNA, 2.5 µl of 10X polymerase buffer, 2.0 mmol/l MgCl
mmol/L dNTPs, 0.4 µmol/l of each primer, and 1 U of Taq polymerase (MBI Fermentas).
The PCR program on Icycler
thermal cycler was as follows: an initial denaturation step
at 94 ºC for 4 min, followed by 33 cycles of 30 sec at 94 ºC, 30 sec at annealing
temperature (52 ºC for M6/M115, 60 ºC for AP5/C539), 30 sec at 72 ºC, and a final
extension step of 8 min at 72 ºC.
The 373 bp region containing the p.Asp70Gly mutation site was amplified using the
primers M6/M115 and digested with the 3U of the Sau3AI restriction enzyme (MBI
Fermentas). The PCR product was digested to 214 and 135 bp fragments from the wild
type allele and remained undigested from the mutant allele. Similarly, 103 bp product
harboring the p.Ala539Thr mutation site was amplified using primers AP5/C539. The
mutation abolishes AluI restriction recognition site resulting in an undigested 103 bp
product for mutant allele whereas wild type allele digested into two fragments of 83 and 20
bp. The digested products were electrophoresed on three per cent agarose gels at 100 V for
30 min. The fragments were visualized by ethidium bromide staining under UV light.
5.1. Molecular Basis of Rett Syndrome
The molecular basis of Rett Syndrome (RTT) in our population was investigated in a
total of 71 isolated RTT cases (68 female and 3 male). A detailed clinical data were
available for 47 patients (44 females and 3 males). Huppke clinical scoring (Huppke et al.,
2003) was also available for 33 of these cases. The clinical features of these patients
analyzed in this study are summarized in Table 5.1. Twenty-four female patients had been
referred to our laboratory for differential diagnosis but the clinical data were not available.
All patients were screened for MECP2 gene mutations but quantitative PCR and XCI
analyses were performed only for the first group of 47 patients.
5.1.2. Mutation Analysis of MECP2 Gene
All patients were initially screened for the six recurrent MECP2 mutations
(p.R106W, p.T158M, p.R168X, p.R255X, p.R270X, and p.R306C) since these mutations
are known to account for up to two thirds of pathogenic mutations in RTT cases
(RettBASE). The PCR-RFLP based analyses detected mutation in 15 patients: four patients
(R6, R18, R22, and R28) with p.R106W, three patients (R25, R29, and R47) with
p.T158M, two patients (R15 and R17) with p.R168X, four patients (R9, R13, R24, and
R39) with p.R255X, patient R34 with p.R270X, and patient R16 with p.R306C mutation
(Figure 5.1). Patients tested negative for the recurrent mutations were further analyzed for
the four coding exons of the
MECP2 gene using SSCP analysis. Subsequent DNA
sequencing of patients showing altered SSCP pattern revealed ten different pathogenic
c.397C>T (p.Arg133Cys), c.1034_1042insGCGGATTGC (p.Lys345fs), c.455C>G
(p.Ser194fsX208), and c.826-829delGTGG (p.Val276fsX288) mutations in R2 and R42,
R3, R4, R8, R18, R31, R36, R46, and R49, respectively.
Figure 5.1. PCR-RFLP analysis for the detection of the common MECP2 mutations in
patients R17 (a), R24 (b), R6 (c), R29 (d), and R16 (e).
The molecular analysis revealed 18 different MECP2 mutations in 30 of 44 (68.2 per
cent) female patients in the first group of classical/atypical RTT cases with detailed clinical
data (Figure 5.2). Of the 30 patients with mutations, 10 had a missense, eight had a
nonsense mutation, six had small nucleotide deletion/insertions. The p.R255X and p.
R106W were the most common mutations with an equal frequency of 8.9 per cent in our
cohort of patients. Five mutations were novel to this study: p.Ser194fsX208 (c.744delG),
p.Val276fsX288 (c.826-829delGTGG), p.Leu286fsX288 (c.856delA), p.Leu386Hisdel12
(c.1156-1192del36) and p.Lys345fs (c.1034_1042insGCGGATTGC) (Figure 5.3 and 5.4).
Eighty percent (20 of 25) of the small deletion/insertion or point mutations were detected
in the exon 4 of the MECP2 gene. The rest of the mutations (20 per cent) were located
within exon 3. Exons 1 and 2 were free of sequence variations. All patients reported were
heterozygous for the identified mutations except patient R18 that was found to be a
compound heterozygote for p.R106W and p.P152R mutations. All available family
members were tested negative for the identified variations implicating de novo nature of
the mutations. MECP2 gene mutations could not be identified in any of the three male
MECP2 mutations could be detected in three of the 24 patients in the second group
that were referred for differential diagnosis. One of these mutations was a complex small
insertion in patient R60 and the others were p.R106W and p.R255X identified in patients
R68 and R69, respectively.
5.1.3. Quantitative PCR Analyses
We have developed a quantitative real time PCR strategy to screen for MECP2 exon
rearrangements in 23 samples (20 females and 3 males) that were negative for MECP2 point mutations. Each sample was amplified with the MECP2 and reference NDRG1 gene
primer pairs. The amplification products were analyzed using the Light Cycler analysis
software (version 4.0) (Figure 5.5). Using the Fit Points Method, the DNA was quantified
relative to the standard curve for each exon. Subsequently, the ratios between target
(MECP2) and reference (NDRG1) exon were calculated for each test person.
The observed mean ratios are 0.52±0.12 for deletion carriers (expected value: 0.5)
and 1.56±0.18 for duplication carriers (expected value: 1.5) vs. 1.022±0.17 for control
individual (expected value: 1.0). MECP2 exon rearrangements were identified in seven
female patients; four with exon 2-4 duplications (R14, R19, R20, and R33), one with exon
3 deletion (R23), one with exon 4 deletion (R30), and one with exon 3-4 deletions (R5)
(Table 5.2, Figure 5.6).
QF-PCR assay was used to verify the results obtained by quantitative real time PCR
analysis (Figure 5.7). The observed ratios of MECP2 exon 3/PRNP exon 2 were 1.02±0.09,
0.47±0.04, and 1.69±0.21 for the control individuals, exon 3 deletion carriers and exon 3
duplication carriers, respectively (Table 5.2, Figure 5.6). QF-PCR confirmed the presence
of exon rearrangements except the MECP2 gene duplication observed in patient R33
Figure 5.2. Schematic representation of the MeCP2 (a) and MECP2 gene (b) showing the
position of the mutations identified in this study. Numbers in brackets represent the
number of the patients with the same mutation.
Figure 5.4. Chromatograms showing sequencing profiles of sense (left panel) and
antisense (right panel) strands of MECP2 gene for the novel mutations identified in the
present study. (a, b) Patient R2 with c.1156-1192del36; (c, d) patient R3 with c.856delA;
(e, f) patient R8 with c.1034_1042insGCGGATTGC; (g, h) patient R46 with c.744delG;
and (i, j) patient R47 with c.826-829delGTGG. Arrows show the site of the mutations.
Table 5.1. The age, gender, and clinical and genetic features of the first group of 47 patients.
Table 5.1. The age, gender, and clinical and genetic features of the first group of 47 patients (continued).
*ND: no data; NI: not-informative; **The duplication identified in this patient by Real Time PCR could not be reproduced with QF-PCR
Figure 5.5. A representative Real Time analysis for a healthy female (a), R5 with exon 3
deletion (b), and R19 with exon 3 duplication (c), respectively.
Table 5.2. Quantitative Real Time PCR and QF-PCR analyses result.
Quantitative Real Time PCR
Samples Exon 3