male control
0.51 ±0.05
0.49 ±0.09
0.46 ±0.09
female control
0.99 ±0.07
1.01 ±0.08
1.02 ±0.09
R1 (male)
0.53
0.63
-
R21 (male)
0.51
0.53
-
R37 (male)
0.53
0.53
-
R5
0.50
0.54
0.50
R23
0.46
1.04
0.51
R30
0.95
0.63
1.18
R7
0.98
1.06
0.95
R10
0.88
1.09
1.10
R11
1.00
1.14
0.80
R12
0.87
1.05
1.04
R26
1.02
1.02
1.05
R32
0.91
1.02
0.95
R35
1.10
1.13
0.81
R40
0.77
0.76
0.96
R41
0.87
0.95
0.93
R43
0.85
0.96
1.08
R44
0.85
0.8
0.92
R45
1.00
0.95
0.88
R48
1.00
0.94
1.00
R50
0.83
0.83
1.00
R14
1.44
1.35
1.40
R19
1.85
1.62
1.74
R20
1.41
1.38
1.40
R33
1.45
1.33
0.98
82
Figure 5.6. The plots of quantitative Real Time PCR and QF-PCR analyses results.
83
(a)
(b)
(c)
Figure 5.7. A representative QF-PCR analysis for a healthy female (a), R5 with exon 3
deletion (b), and R19 with exon 3 duplication (c), respectively.
84
Figure 5.8. QF-PCR analysis of patient R33.
5.1.4. X Chromosome Inactivation Status
X chromosome inactivation analysis was performed for 44 female patients; however,
the status of six patients (13.6 per cent) could not be identified because of homozygousity
for both AR and ZNF261 loci. X-inactivation was random in 23 (61.5 per cent) and skewed
in 15 (39.5 per cent) of 38 informative patients (Table 5.1). The paternal X chromosome
was active in one patient and eight patients showed preferential activation of the maternal
X chromosome. The origin of the active allele could not be determined in six patients
because of unavailability of the maternal sample. A representative XCI analysis of patients
with skewed, random, and non-informative XCI status is shown in Figure 5.9.
(+) (+)
(a)
(+) (+)
(b)
(+) (+)
(c)
Figure 5.9. X chromosome inactivation analysis of patients with skewed (a), random (b),
and non-informative (c) XCI pattern, respectively. (+) represents the PCR products from
HhaI digested DNA.
85
5.1.5. Genotype–Phenotype Correlations
The clinical severity scores of the patients were given in Table 5.1. High scores
(maximum score 9) indicate more severe disease phenotype. A statistically significant
correlation could not be identified between the mean severity score of patients and the
presence, type and location of mutation, and the XCI pattern (Table 5.3). However, the
patients with exon deletions were found to have higher clinical severity scores than all
other mutation-positive patients (8.33±0.58 vs 6.70±1.57, p=0.066). When site of mutation
is considered alone, we observed that the patients with affected TRD domain had more
severe phenotype than the patients with affected MBD domain (7.80±1.23 vs 6.88±1.64,
respectively). Mutation negative patients and patients with skewed XCI patterns had
slightly milder phenotypes when compared to mutation positive patients and patients with
random XCI, respectively.
When we performed statistical analysis of severity scores for specific clinical
features, we obtained a significant difference in the fields of ‘‘gait function’’ and “eye
contact”. None of the patients with MECP2 exon deletions have ever walked (p=0.019).
Eye contact is very difficult to obtain in patients with exon duplications when compared to
that of patients with missense mutation (p=0.016). Although the differences were not
significant, mutation positive patients had severe problems in their ability in purposeful
hand movement and walking skills when compared to mutation negative patients.
5.1.6. Prenatal Diagnosis
Upon request, prenatal diagnosis was performed in the families of the patient R29
with p.T158M, patient R42 with p.L386Hdel12, and patient R69 with p.R255X mutations.
Chorionic biopsy specimens were tested and found to be negative for index patient’s
mutation (Figure 5.10).
86
(a)
(b)
(c)
Figure 5.10. Agarose gel electrophoresis showing the prenatal diagnosis performed in the
families of the patient R29 with p.T158M (a), patient R42 with p.L386Hdel12 (b), and
patient R69 with p.R255X mutations (c).
87
Table 5.3. Mean Phenotypic Severity Scores of female patients of first group.
* (P=0.016); **(p=0.019); *** (p=0.066)
88
5.1.7. Multiplexed ARMS-PCR Approach for the Detection of Common MECP2
Mutations
In the present study, we have established a multiplex multiplex amplification
refractory mutation system (ARMS) - PCR assay to detect the seven common MECP2
mutations p.R106W, p.R133C, p.T158M, p.R168X, p.R255X, p.R270X, p.R294X, and
p.R306C. Each primer set was tested and optimized using mutation positive DNA samples,
and then multiplexed (Figure 5.11). A representative multiplex ARMS-PCR assay analysis
is shown in Figure 5.12.
5.1.7.1. Assay optimization. Several factors, including the concentration of primers,
MgCl
2
and Taq polymerase, and PCR cycling conditions that can affect PCR specificity
and efficiency were optimized. The concentration of Taq polymerase and MgCl
2
both had
pronounced effects on the specificity and relative yield of the PCR products. Although
high MgCl
2
concentration increased the intensity of our desired bands, it resulted in non-
specific backgrounds. The optimum amount of MgCl
2
was found to be 2.5 mM to ensure
specificity of the multiplex PCR assay. A range of Taq polymerase concentration was also
tested and PCR specificity was found to be highest with 1.5 U of Taq Polymerase. To
improve the specificity and the sensitivity of the amplification, we have used a touchdown
PCR strategy with stepwise decrease of the annealing temperature from 63 to 59 C.
Different concentrations for each set of primers were tested until maximum sensitivity and
specificity were obtained (Table 4.3).
5.1.7.2. Validation of the assay. To evaluate the assay, we tested 14 patients with RTT for
whom we had previously determined the genotypes by PCR followed by restriction
enzyme digestion or DNA sequencing. We observed complete concordance between the
traditional and Multiplex-ARMS methods (Figure 5.13).
89
Figure 5.11. Agarose gel electrophoresis of the multiplex ARMS PCR assay products.
Each mutant ARMS primer was evaluated with corresponding mutation positive DNA
samples. Lane 1-8: Allele specific amplification of mutations p.R106W, p.R133C,
p.T158M, p.R168X, p.R255X, p.R270X, p.R294X, and p.R306C, respectively. Lane 9:
100 bp DNA ladder (MBI Fermentas). An aliquot of 10 µl of each PCR product was
loaded onto the gel.
Figure 5.12. A representative multiplex ARMS-PCR assay analysis. Lane 1: PCR with
wild-type primers of Panel 1; Lane 2-5: PCR with mutant primers of Panel 1 using samples
with p.R294X, p.R255X, p.R168X, and p.R133C mutations, respectively; Lane 6: PCR
with wild-type primers of Panel 2; Lane 7-9: PCR with mutant primers of Panel 2 using
samples with mutations p.R306C, p.R270X, and p.T158M, respectively. An aliquot of 15
µl of each PCR product was loaded on the gel.
90
Figure 5.13. Evaluation of the multiplexed ARMS-PCR assay using RTT patient samples
with known mutations. (a) Panel 1. Lane 1: PCR with wild type primers; Lane 2: patient
with p.R294X; Lane 3-7: patients with p.R255X; Lane 8-9: patients with p.R168X; Lane
10: patient with p.R133C. (b) Panel 2. Lane 1: PCR with wild type primers; Lane 2: patient
with p.R306C; Lane 3: patient with p.R270X; Lane 4-6: patients with p.T158M; Lane 7-
10: Analysis of patients with p.R106W using ARMS assay. An aliquot of 15 µl of each
PCR product was loaded onto the gel.
5.1.8. The Effect of DNA Concentration on Reliability and Reproducibility of SYBR
Green Dye-based Real Time PCR Analysis to Detect the Exon Rearrangements
In this part of the study, we have investigated the effect of DNA concentration on
reliability and reproducibility of Real Time PCR to detect the exon rearrangements. SYBR
(a)
(b)
91
Green dye-based Real Time PCR analysis was performed to detect the MECP2 exon 3
rearrangements in five samples with previously determined genotypes.
Through optimization of reaction conditions, the optimal concentration of primers
was found to be 5 and 10 pmol for NDRG1 (reference gene) and MECP2, respectively. The
melting curves of the all PCR products showed a single peak with identical melting
temperature (Tm), indicating that there was no non-specific amplification. The specificity
of the real-time PCR was further confirmed by analysis of the PCR products on an agarose
gel which showed the expected amplification products of 172 and 175-bp for MECP2 exon
3 and NDRG1, respectively.
5.1.8.1. Comparison of Quantification Methods. Two methods for determination of the
MECP2
exon 3 copy numbers from the raw data of Real-Time PCR reaction are available.
The relative kinetic method is based on interpolated data from a standard curve, whereas
the comparative Ct method transforms a difference in Ct values (between the test sample
and the calibrator sample) into a copy number ratio. The relative kinetic method takes in
account the actual efficiency of the reaction. The comparative Ct method does not require
standard curves and 0.95 was used as default amplification efficiency in quantification. For
all samples the MECP2 exon 3 copy number was calculated using both methods. A paired
sample t-test showed that the obtained results by both methods were significantly similar
(P = 0.451>0.05). In addition, the Pearson correlation coefficient of 0.996 (P = 1E-6 <0.01)
demonstrated the equivalence of both methods.
5.1.8.2. Quantification. Using the control DNA samples (20 ng) for whom we had
previously determined the genotypes, the mean ratios were observed as 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 non-carriers (expected value: 1.0). The differences between
the three groups were highly significant (p<0.001) (ANOVA).
Triplicate measurements were performed on six DNA concentrations of 0.1, 1, 5, 50,
100, and 200 ng of the patient R23 with MECP2 exon 3 deletion, patient R19 with MECP2
exon 3 duplication, and two healthy females and one male (Table 5.4). The expected copy
number ratio was obtained in all cases when 1 ng, 5 ng, and 50 ng DNA used. However,
92
using 0.1 ng DNA, the ratio was out of expected range (± 2SD) in six of 15 measurements
resulting in misgenotyping for R5, R19 and female 2 samples. In case of 100 ng DNA,
expected ratio could not be obtained in four of 15 measurements leading to misgenotyping
of R19 and two female samples. We did not get any expected copy number while using
200 ng DNA (Figure 5.14).
The effect of the DNA concentration on the amplification efficiency and specifity
could be observed in amplification curve and melting curve analysis. Use of the high DNA
concentrations (100–200 ng) resulted in inhibition of the amplification and/or nonspecific
product formation in some cases (Figure 5.15 and 5.16).
Table 5.4. Ct values obtained from Real Time PCR analysis on different amounts of DNA.
Sample
DNA
conc.
Ct of
MeCP2
Ct of
NDRG1
ratio of
MeCP2/NDRG1 gene
35.00
32.64
1.20
37.00
37.00
1.15
0.1 ng
27.76
26.67
1.00
32.50
30.37
1.11
30.60
29.76
0.89
1 ng
31.82
31.58
0.97
29.61
27.84
1.06
28.16
27.50
1.17
5 ng
25.70
24.98
1.00
26.38
25.00
1.00
26.21
25.79
0.95
50 ng
26.21
25.93
0.95
24.88
23.26
0.80
26.05
23.98
0.27
100 ng
26.17
24.98
0.87
23.95
21.83
0.58
25.60
23.06
0.20
Female 1
200 ng
21.50
21.62
1.42
93
Table 5.4. Ct values obtained from Real Time PCR analysis on different amounts of DNA
(continued).
Sample
DNA
conc.
Ct of
MeCP2
Ct of
NDRG1
ratio of
MeCP2/NDRG1 gene
35.00
35.00
4.74
38.00
38.00
1.09
0.1 ng
31.68
31.21
0.83
32.55
30.13
0.89
30.79
29.93
0.97
1 ng
32.66
32.27
0.83
30.25
27.98
0.98
29.33
28.85
0.82
5 ng
28.33
28.90
1.21
27.57
25.32
1.00
26.88
26.55
0.86
50 ng
26.94
25.53
1.20
28.11
24.26
0.33
26.30
24.65
0.35
100 ng
26.14
25.06
0.93
28.42
23.53
0.16
Female 2
200 ng
28.74
23.14
0.10
32.28
30.90
0.45
32.21
30.08
0.29
0.1 ng
35.00
35.00
0.27
31.01
28.92
0.50
30.96
29.24
0.62
1 ng
28.35
26.93
0.66
29.54
27.69
0.46
29.89
27.65
0.34
5 ng
30.15
29.49
0.43
27.71
26.52
0.49
28.66
25.98
0.50
50 ng
29.12
26.09
0.39
27.33
25.18
0.49
27.1
24.69
0.42
100 ng
28.13
25.64
0.22
Male
200 ng
28.49
24.25
0.08
94
Table 5.4. Ct values obtained from Real Time PCR analysis on different amounts of DNA
(continued).
Sample
DNA
conc.
Ct of
MeCP2
Ct of
NDRG1
ratio of
MeCP2/NDRG1 gene
37.00
37.00
1.98
37.00
35.27
0.51
0.1 ng
37.00
37.00
1.98
31.50
29.24
0.47
31.10
29.24
0.44
1 ng
29.92
27.83
0.37
30.27
27.92
0.30
29.78
27.49
0.32
5 ng
28.14
26.81
0.53
27.71
26.52
0.49
28.66
25.98
0.50
50 ng
29.12
26.09
0.39
26.59
24.82
0.48
27.66
25.98
0.51
100 ng
27.98
26.65
0.55
Patient R5
with exon 3
deletion
200 ng
30.57
22.28
0.03
35.00
35.00
1.06
29.21
29.48
1.82
0.1 ng
30.95
30.79
1.10
31.87
32.55
1.70
30.61
30.51
1.46
1 ng
30.20
30.19
1.68
27.90
27.55
1.39
5 ng
29.11
29.62
1.52
27.17
27.38
1.51
50 ng
27.53
27.98
1.31
28.22
25.56
0.17
26.17
25.85
1.42
100 ng
26.22
25.84
1.38
Patient R19
with exon 3
duplication
200 ng
28.47
24.87
0.09
95
Figure 5.14. The summary of the Real Time analysis of MECP2 exon 3 rearrangements
using 0.1 – 200 ng DNA. Gray shaded boxes indicate the results in out of range.
Figure 5.15. The profile of the amplification products of sample R5 with different
concentrations of template DNA. Arrows show the unexpected amplification curves.
Figure 5.16. Melting curve analysis for PCR products of sample R5 using 0.1, 10, 50, 200
ng DNA. The line shows the expected Tm value for MECP2 exon 3 products.
96
5.2. Methylation Analyses of the Putative Promoter Region of Rad23 Genes in Breast
Tumor Tissues
In this study, the methylation status of 5’ flanking regions (including the CpG islands
and putative promoter sequence) of hHR23A and hHR23B genes was investigated in
primary breast tumor, tumor adjacent tissues, and normal breast tissues.
5.2.1. Characterization of the 5' flanking region of the hHR23 Genes
5.2.1.1. hHR23A Gene. A part of 5’ flanking region (1450-bp upstream and 180-bp
downstream sequence, relative to the translation start site (+1 ATG)) of hHR23A gene
(GenBank accession NT_011295.10) was analyzed to predict the putative eukaryotic Pol II
promoter. Web-based MethPrimer software revealed two CpG islands in the upstream
region (Figure 5.17). The first CpG island was 127 bp long and between nucleotide (nt)
positions -580 and -454.. A 287 bp long second island is located between nucleotides -341
and -55. PROSCAN program predicted a putative eukaryotic Pol II promoter region within
the second CpG island (position -48 to -298 nts) with a score of 98.79 (Promoter Cutoff
score = 53.000000). The promoter region lacks the CCAAT and TATA-like elements, a
common feature of the house-keeping genes. The analysis revealed potential binding sites
for the transcription factor Sp1 (-243/-231 and -233/-241 nts) as inverted overlapping sites,
overlapping ATF and CREB sites (-303/-294 nts), and an Elk-1 site (-113/-99 nts). The
primers were designed to investigate the methylation status of CpG di-nucleotides within
the -310/+140 nts, a region covering the putative promoter sequence and 59 CpG di-
nucleotides.
5.2.1.2. hHR23B Gene. Peng et al. (2005) has shown that the CpG di-nucleotides in the
upstream region (from -338 to -64 nts, relative to the transcription start site) of the
hHR23B gene (GenBank accession NW_924539) were methylated in Interleukin-6-
responsive Multiple Myeloma KAS-6/1 cell lines. This region was analyzed to characterize
the putative promoter sequence. PROSCAN software revealed a CCAAT and TATA- box
lacking putative Pol II promoter sequence in between nucleotide positions -14 to -264 with
a score of 217.35 (Promoter Cutoff score = 53.000000). Four Sp1 binding sites were
identified in positions -286/-279, -270/-261, -134/-127, and -118/-110. Using MethPrimer,
97
the primers were designed to cover the putative promoter region and 40 CpG di-
nucleotides between nucleotides -328 to -19 (Figure 5.18).
Figure 5.17. MethPrimer program output showing the CpG islands and the investigated
region at 5’ of the hHR23A gene.
Figure 5.18. MethPrimer program output showing the CpG island and the investigated
sequence in 5’ flanking region of hHR23B gene.
5.2.2. Bisulfite Sequencing of hHR23A and hHR23B in paraffin-embedded tissues
Sixty-one archival formalin-fixed, paraffin-embedded tissues consisting of 50
primary breast tumors (diagnosed as Invasive Ductal Carcinoma), nine tumor adjacent
tissues and four normal breast tissues were included in this study (Table 5.5). Genomic
DNA was extracted from tissues by using a modified version of non-heating DNA
extraction protocol described by Shi et al. (2002). Tissues were incubated with lysis buffer
at 50 ºC for 48-72 hours instead of overnight at 45 ºC recommended by Shi et al. (2002).
98
DNA isolation was successful in all samples with concentrations ranging from 3 to 470
ng/µl (Figure 5.19).
The methylation status of the CpG islands was investigated by bisulfite-sequencing
method. Semi-nested PCR strategy was used to amplify the bisulfite modified DNA
samples. PCR fragments could be produced in 38 samples for hHR23A gene and 58
samples for hHR23B gene out of 61 samples tested. The methylation status of hHR23A
and hHR23B genes could be determined in 35 of 38 and 51 of 58 samples, respectively.
Figure 5.19. Agarose gel electrophoresis showing the quality of the genomic DNAs
isolated from paraffin embedded tissues. Lane 1: 100-bp DNA ladder (MBI Fermentas);
Lane 2 and 3: DNA isolated from peripheral blood samples, Lane 4-6: DNA isolated from
paraffin embedded tissues, Lane 7 and 8: bisulfite treated DNA samples, Lane 9: 1-kb
DNA ladder (MBI Fermentas). Approximately 200-400 ng DNA was loaded onto the slots.
5.2.2.1. hHR23A. Methylation analysis of 5’ flanking region of hHR23A gene revealed
cytosine methylation in 12 tumor and 2 tumor adjacent tissues. The results are summarized
in Table 5.5 and Figure 5.20. Hypermethylation was observed in four tumors (from
patients Rad21, 27T, 31T, and 32T) and one tumor adjacent tissue sample (Rad31N). The
region between nucleotide positions +6/+117 was hypermethylated in tumor adjacent
tissue Rad31N whereas hypermethylation spread to the region between -249/+117 nts in
tumor tissue of the same sample (Rad31T) (Figure 5.21 and 5.22). Patient Rad29 has CpC
methylation at position -114/-113 in tumor adjacent tissue (Rad29N) whereas CpT and
CpA di-nucleotides were methylated at positions -133/-132 and +11/+12 in the tumor
tissue (Rad29T). Seven patients (Rad4, 6, 15, 29T, 29N, 33T, and 48) have only non-CpG
methylation. Bisulfite sequencing revealed no methylation in 21 tissue samples.
99
Table 5.5. Methylation analysis results for the 5’ flanking region of hHR23A.
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