Sample
Cytosine methylation
Rad4
*CpC at -165/-164,
*CpA at +15/+16
Rad6
*CpA at -171/-170,
*CpC at -2/-1
Rad15
*CpC at +80/+81
Rad17
*CpG at -116/-115
Rad21
Hypermethylation between -249/-27
Rad27T
Hypermethylation between -226/+117
Rad28T
*CpG at -191/-190
Rad29T
*CpT at -133/-132,
*CpA at +11/+12
Rad29N
*CpC at -114/-113
Rad31T
Hypermethylation between -249/+117
Rad31N
Hypermethylation between +6/+117
Rad32T
Hypermethylation between -121/+117
Rad33T
*CpCCC at -305/-302,
*CpC at -190/-189
Rad48
*CpC +84/+85
*CpN indicates methylated cytosine.
100
Figure 5.20. Methylation analysis of 5’ flanking region of the hHR23A gene. (a) The
distribution of CpG di-nucleotides in 5’ flanking region (-310 to +140) of hHR23A gene
are depicted schematically. Methylation analysis was performed on a region covering the
putative promoter sequence and 59 CpG di-nucleotides. (b) The results of methylation
analysis of samples. Open circles indicate unmethylated CpGs, and filled circles
methylated CpGs.
,
, and
represent the methylated CpA, CpT, and CpC di-
nucleotides, respectively.
101
Figure 5.21. Bisulfite sequencing of 5’flanking region of hHR23A gene in tumor adjacent
tissue of patient Rad31.
102
Figure 5.22. Bisulfite sequencing of 5’flanking region of hHR23A gene in tumor tissue of
patient Rad31.
103
5.2.2.2. hHR23B. Cytosine methylation (CpG and non-CpG) in the upstream of hHR23B
gene was observed in 15 tumor tissues. A representative result for bisulfite sequencing
analysis of hHR23B gene is given in Figure 5.23. Detailed results are given in Table 5.6
and Figure 5.24. CpG methylation was observed in six patients (Rad9, 13, 16, 20, 22, and
27T) and non-CpG methylation was also present in three of them (Rad9, 20, and 22). Nine
patients have only non-CpG methylation. The methylated cytosine residues were within the
Sp1 binding sites in patients Rad9, 17, 22, 27T, and 32T. C*CpWGG motif was present in
four samples (Rad2, 9, 25T, and 30T).
Bisulfite sequencing revealed that three tumor samples (Rad17, 27T, and 32T) have
cytosine methylations in both hHR23A and hHR23B genes. The sample Rad27T showed
hypermethylated region (between nts -226/+117) in hHR23A and methylated cytosine
residue within the Sp1 binding site (-270/-261) in hHR23B gene. Similarly, upstream
region (between -121/+117 nts) of hHR23A gene was hypermethylated and cytosine
residue within the Sp1 binding site (-135/-126) in hHR23B gene in patient sample Rad32T.
Sample Rad17 has CpC methylation at position -116/-115 and methylated cytosine residue
within the Sp1 binding site (-135/-126) in hHR23A and hHR23B genes, respectively. The
summary of the methylation status and the histopathology of the samples analyzed in this
study are listed Table 5.7.
104
Figure 5.23. Chromatogram showing the bisulfite sequencing of 5’flanking region of
hHR23B gene in the tumor tissue of patient Rad22.
105
Table 5.6. Results of methylation analysis for 5’ flanking region of hHR23B.
Sample
Cytosine methylation
Rad2
C*CpAGG at -212/-208
Rad9
C*CpAGG at -151/-147,
GC*CpCCGCCCC (Sp1) at -135/-126,
*CpG at -84/-83
Rad13
*CpG at -172/-171
Rad14
G*CpCC at -205/-202
Rad16
*CpG at -172/-171
Rad17
GC*CpCCGCCCC (Sp1) at -135/-126
Rad20
*CpG at -144/-143,
C*CpCCA at -78/-73
Rad22
G*CAC at -78/-75,
*CpG at -185/-184,
GCCCCGCCC*CpA (Sp1) at -135/-126,
GC*CpC at -58/-55,
T*CpCCC at -43/-39,
*CpG at -28/-27
Rad25T
C*CpAGG at -151/-147
Rad27T
GCCC*CpGCCCC (Sp1) at -270/-261
Rad30T
C*CpAGG at -212/-208
Rad32T
GCCCCGCC*CpC (Sp1) at -135/-126
Rad46
A*CpCCC at -78/-73
Rad50
GCCCCGCC*CpC (Sp1) at -135/-126
Rad51
G*CpAA at -235/-232
*CpN indicates methylated cytosine.
106
Figure 5.24. Methylation analysis of 5’ flanking region of the hHR23B gene. (a)
Schematic representation of the hHR23B gene upstream region comprising 40 CpG di-
nucleotides. (b) The samples with cytosine methylation. Open circles indicate
unmethylated CpGs, and filled circles methylated CpGs.
and
represent the
methylated CpT and CpC di-nucleotides, respectively.
107
Table 5.7. The histopathological and epigenetic findings of the tumor tissue analyzed in this study.
#
Age
Tissue type
Histopathological
type
Stage
LNM*
(+/ -)
Histological
Grade
ER
C-erbB2
Cytosine methylation
Rad23A Rad23B
Rad 1
73
Primary tumor
IDC
pT4bN1
+
3
-
?
-
-
Rad 2
55
Primary tumor
IDC
pT1N0
-
2
90 per cent
-
ND
+
Rad 3
50
Primary tumor
IDC
pT2N2
+
2
-
-
ND
ND
Rad 4
59
Primary tumor
IDC
pT3Nx
?
3
-
-
+
-
Rad 5
65
Primary tumor
IDC
pT2N0
-
2
60 per cent
-
-
-
Rad 6
42
Primary tumor
IDC
pT1N1
+
1
<5 per cent
-
+
-
Rad 7
82
Primary tumor
IDC
pT1N0
-
3
60 per cent
-
ND
ND
Rad 9
65
Primary tumor
IDC
pT2N1
+
3
70 per cent
-
ND
+
Rad 10
49
Primary tumor
Invasive apocrin
carcinoma
pT1N0
-
2
-
+
ND
-
Rad 11
64
Primary tumor
IDC
pT1N1
+
1
80 per cent
-
-
-
Rad 12
46
Primary tumor
IDC
pT2N2
+
2
-
+
ND
ND
Rad 13
58
Primary tumor
IDC
pT2N2
+
3
60 per cent
-
ND
+
Rad 14
52
Primary tumor
IDC
pT2N1
+
3
80 per cent
-
ND
+
Rad 15
78
Primary tumor
IDC
pT1Nx
?
2
80 per cent
-
+
-
Rad 16
39
Primary tumor
ductal carcinoma in
situ
10 per cent
-
ND
+
Rad 17
81
Primary tumor
IDC
pT2N0
-
2
70 per cent
-
+
+
Rad 18
71
Primary tumor
Mixed IDC-ILC
pT1N0
-
3
90 per cent
-
ND
-
Rad 19
79
Primary tumor
IDC
pT4bN1
+
3
-
+
ND
ND
Rad 20
66
Primary tumor
IDC
pT2N1
+
3
90 per cent
-
ND
+
Rad 21
43
Primary tumor
IDC
pT2N2
+
3
90 per cent
+
Hyperm.
-
* Lymph Node Metastases; IDC: Invasive (or Infiltrating) Ductal Carcinoma; NA: not available; ND: not determined.
108
Table 5.7. The histopathological and epigenetic findings of the tumor tissue analyzed in this study (continued).
#
Age
Tissue type
Histopathological
type
Stage
LNM*
(+/ -)
Histological
Grade
ER
C-
erbB2
Cytosine methylation
Rad23A Rad23B
Rad 22
60
Primary tumor
IDC
pT3N0
-
3
-
+
ND
+
Rad 23
63
Primary tumor
IDC
pT2N3
+
2
90 per cent
-
-
-
Rad 25N
45
Normal
-
-
-
-
ND
-
Rad 25T
Primary tumor
IDC
pT2N1
+
3
60 per cent
-
ND
+
Rad 26N
40
Normal
-
-
-
-
ND
-
Rad 26T
Primary tumor
IDC
pT1N0
-
1
90 per cent
-
-
-
Rad 27N
37
Normal
-
-
-
-
ND
ND
Rad 27T
Primary tumor
IDC
pT2N1
+
3
20 per cent
-
Hyperm.
+
Rad 28N
63
Normal
-
-
-
-
-
-
Rad 28T
Primary tumor
IDC
pT3N2
+
2
-
-
+
-
Rad 29N
62
Normal
-
-
-
-
+
-
Rad 29T
Primary tumor
IDC
pT2N(Mi)
+
3
100 per cent
-
+
-
Rad 30N
75
Normal
-
-
-
-
-
-
Rad 30T
Primary tumor
IDC
pT1N1
+
2
per cent80
-
-
+
Rad 31N
77
Normal
-
-
-
-
Hyperm.
-
Rad 31T
Primary tumor
IDC
pT2N(Mi)
+
3
100 per cent
-
Hyperm.
-
Rad 32N
81
Normal
-
-
-
-
ND
ND
Rad 32T
Primary tumor
IDC
pT2N1
+
3
90 per cent
-
Hyperm.
+
Rad 33N
48
Normal
-
-
-
-
-
-
Rad 33T
Primary tumor
IDC
pTxNx
+
3
NA
NA
+
-
Rad 43
47
Primary tumor
Invasive Lobular
pT2N1
+
2
80 per cent
-
-
-
Rad 44
52
Primary tumor
Atypical medullar
pT1N1
+
3
-
-
-
-
* Lymph Node Metastases; IDC: Invasive (or Infiltrating) Ductal Carcinoma; NA: not available; ND: not determined.
109
Table 5.7. The histopathological and epigenetic findings of the tumor tissue analyzed in this study (continued).
#
Age
Tissue type
Histopathological
type
Stage
LNM*
(+/ -)
Histological
Grade
ER
C-
erbB2
Cytosine methylation
Rad23A Rad23B
Rad 45
61
Primary tumor
IDC
pT2N0
-
1
30 per cent
-
-
-
Rad 46
55
Primary tumor
Invasive Lobular
pT2N0
-
2
90 per cent
-
-
+
Rad 47
45
Primary tumor
IDC
pT2N2
+
3
-
+
ND
ND
Rad 48
47
Primary tumor
ductal carcinoma in
situ
+
ND
Rad 49
41
Primary tumor
IDC
pT1N3
+
3
-
+
-
-
Rad 50
54
Primary tumor
IDC
pT2N1
+
2
80 per cent
-
ND
+
Rad 51
53
Primary tumor
IDC
pT1N1
+
3
30 per cent
-
ND
+
Rad 52
51
Primary tumor
IDC
pT1N0
-
3
30 per cent
+
-
-
Rad 53
48
Primary tumor
IDC
pT1N0
-
1
60 per cent
-
ND
-
Rad 54
57
Primary tumor
IDC
pT1N0
-
2
70 per cent
-
-
-
Rad 55
79
Primary tumor
IDC
pT2N1Mx
+
3
70 per cent
-
-
-
Rad 56
63
Primary tumor
IDC
pT1N0
-
2
90 per cent
-
ND
-
Rad 58
49
Primary tumor
Invasive Lobular
pT2N2
+
2
80 per cent
-
ND
-
Rad 59
77
Primary tumor
IDC
pT1N0
-
2
NA
NA
-
-
Rad 62T 76
Primary tumor
IDC
pT2Nx
?
2
60 per cent
-
-
-
Rad 62N
Normal
-
-
-
-
-
-
Rad 60
Normal
-
-
-
-
-
-
Rad 61
41
Normal
-
-
-
-
-
-
Rad24
Normal
-
-
-
-
-
-
* Lymph Node Metastases; IDC: Invasive (or Infiltrating) Ductal Carcinoma; NA: not available; ND: not determined.
110
5.3. Molecular Basis of Congenital Hypothyroidism
In the present study, the genetic mechanisms leading to congenital hypothyroidism
(CH) and prolonged paralysis after mivacurium in a patient was investigated. The patient
was screened for the presence of mutations within the TTF2 and BChE genes responsible
of hypothyroidism and neuromuscular block after anesthetic administration, respectively.
5.3.1. Clinical Features of the Patient
The proband, a 3900-g female infant, was born to consanguineous parents by normal
vaginal delivery at 40 wk gestation after an uncomplicated pregnancy. Postnatal
examination revealed the patient to be hypotonic, hypoactive, hypothermic, and areflexic
with cleft palate, spiky hairs, and bilateral choanal atresia, subsequently confirmed by
paranasal sinus tomography. Meconium staining and perinatal respiratory distress
prompted admission to neonatal intensive care for ventilatory support. Immediately after
birth, the patient’s total serum T4 level was 0.758 µg/dl [normal range (NR), 6.1–14.9
µg/dl], and TSH was greater than 100 mIU/ml (NR, 1.7–9.1 mIU/ml). L-T4 replacement
therapy was started, and the baby was discharged at age 2 months. The proband’s parents
and older male sibling are biochemically euthyroid with no congenital anomalies.
Thyroid ultrasonography and gadolinium-enhanced computed tomography (CT)
examination of the proband indicated the thyroid tissue in a eutopic location (Figure 5.25).
5.3.2. Mutation Analysis of the TTF2 Gene
Direct sequencing of coding exon of TTF-2 gene from the patient revealed a C
T
transition at nucleotide position 304 (Figure 5.26). The mutation leads to the replacement
of amino acid arginine with cysteine at codon 102 (p.R102C) affecting the forkhead
domain of the protein. The arginine residue in this position is highly conserved in
H.Sapiens, M.Musculus, R.Norvegicus, C.Elegans
and among human forkhead proteins
(Figure 5.27).
111
Figure 5.25. Axial postcontrast CT image of the patient reveals a slightly enlarged thyroid
gland (arrows) in the paratracheal region with absent contrast enhancement (a).
Comparative imaging of the thyroid gland (arrows) in a healthy 9-yr-old child (b) (Barış et
al
., 2006).
304 C
T (R102C)
(a)
304 C
T (R102C)
(b)
Figure 5.26. DNA sequencing profile of the TTF-2 gene showing mutation in homozygous
condition in the patient (a). Her unaffected mother is heterozygous for the mutation (b).
The presence of the mutation was confirmed by restriction enzyme analysis. The
mutation creates a novel AlwNl site. Her unaffected consanguineous parents were found to
be heterozygote (Figure 5.28) and 100 control chromosomes tested negative for the same
mutation.
112
H.sapiens
M.musculus
R.norvegicus
C.elegans
(a)
(b)
Figure 5.27. Alignment of the TTF-2 forkhead DNA-binding domain with selected FOX
proteins between species (a) and within human proteins (b). At the bottom, ‘*’ indicates
conserved residues in all sequences. The arrow shows the Arg 102 residue (Barış et al.,
2006).
113
Figure 5.28. Two per cent agarose gel showing AlwNl digestion results for the patient and
her family members.
5.3.3. Functional Characterization
Functional analyses performed in University of Cambridge revealed that the
mutation is highly deleterious, with the p.R102C mutant protein exhibiting negligible DNA
binding and transcriptional activity (Barış et al., 2006).
5.3.4. Mutation Analysis of Butyrylcholinesterase (BChE) Gene
Patients with CH are candidates for multiple operations due to midline defects (cleft
palate and choanal atresia). When our CH patient was a 3-month-old baby, a gastrostomy
was required because of a severe nutritional problem secondary to cleft palate. After
operations, the patient showed prolonged neuromuscular block (paralysis) for four hours
after a single dose of mivacurium as a muscle relaxant. BChE activity was measured and
the proband was found to have a marked decrease in BChE activity compared with her
parents and brother. The enzyme activities were 408 IU/1 for patient, 4953 IU/l for mother,
671 bp
599 bp
114
4594 IU/1 for father and 6513 IU/1 for brother (normal 5400–13 200 IU/1) (Yıldız et al.,
2006).
5.3.4.1. PCR-RFLP. The patient and family members were analyzed for the presence of
two most common BChE variants; p.Asp70Gly (A-variant) and Ala539Thr. Restriction
analysis revealed that the patient, her consanguineous parents and unaffected brother were
negative for these variants (Figure 5.29).
Figure 5.29. Sau3AI (upper panel) and Alul (lower panel) digestion analyses for the patient
and her parents (C: normal individual) (Yıldız et al., 2006).
115
6. DISCUSSION
Epidemiological studies promise to provide correlative data to permit researchers
understand the etiology of human diseases and develop efficient genetic testing assays.
Additionally, the accumulated data of genotyping, expression profiling and proteomics
allows disease diagnosis, to understand the molecular mechanisms leading to the disease
pathogenesis, and to develop efficient therapeutic approaches. In the framework of this
thesis, we have investigated genetic and epigenetic changes and provided genotype-
phenotype correlations to unravel the molecular mechanisms that lead to three different
diseases, Rett Syndrome, breast cancer, and congenital hypothyroidism. To our knowledge,
it is the first study on genetic basis of Rett Syndrome in our population. The results of this
analysis revealed that gene dosage can be a mechanism that leads to this devastating
genetic/epigenetic disease. Investigation of epigenetic changes in two human repair genes
has shown CpG and non CpG methylation in tumor samples of breast cancer patients that
was important with respect to the available literature. Identification of the causative
mutation in the CH patient and its functional study with a collaborative work also helped to
understand the genetic mechanisms and provided original evidence that implicated
differential effects of TTF-2 mutations on downstream target genes required for normal
human thyroid organogenesis. These and other findings are discussed below in respective
sections for each disease.
6.1. Molecular Basis of Rett Syndrome
In the framework of this study, the genetic basis of Rett Syndrome (RTT) was
investigated in a total of 71 patients. A heterogeneous spectrum of disease-causing
mutations was identified in 68.2 per cent of a clinically well defined group of cases. Our
results showed that exon duplication/deletions that could not be detected by standard
techniques contribute to 19.3 per cent of these MECP2 mutations. Only 12.5 per cent of the
patients, referred for differential diagnosis, were positive for MECP2 gene mutations
suggesting that this gene does not represent a major cause of the disease among patients
with Rett-like features. Comparison of the clinical severity scores of patients with respect
116
to the presence, type and location of mutation in the gene MECP2, in addition to the
pattern of XCI did not reveal a statistically significant correlation.
The molecular analysis revealed 18 different mutations in 30 of 44 (68.2 per cent)
female patients in the first group of classical/atypical RTT cases with detailed clinical data.
Of the 30 patients with mutations, 10 had a missense, eight had a nonsense mutation, six
had small nucleotide deletion/insertions. MECP2 exon rearrangements were identified in
six female patients; three patients with exon 2-4 duplications (R14, R19, R20) and three
patients with exon 3 and/or exon 4 deletions (R5, R23, R30). In patient R33, exon
duplication identified by Real Time analysis could not be verified by QF-PCR.
The deletions, we have identified in three classical RTT female patients, affect the
MBD and TRD domains of the MECP2 protein. For these patients, it is highly unlikely that
the protein produced from the mutant allele (if any) would have residual function thus they
can be expected to be associated with severe phenotypes. Although Archer et al. (2006)
noted that their deletion group was clinically indistinguishable from other mutation-
positive RTT patients, the patients in our study presented higher clinical severity scores
than all other mutation-positive patients. The differences in severity were not significant
(8.33±0.58 vs 6.70±1.57, p=0.066), but the findings were in accordance with the data of
Hardwick et al. (2007) and Scala et al. (2007). When we performed statistical analysis of
severity scores for specific clinical features, we obtained a significant difference in the
field of ‘‘gait function’’ (p=0.016), since none of patients with MECP2 exon deletions
have ever walked.
The c.856delA and c.826-829delGTGG deletions identified in patients R3 and R49,
respectively, present novel small deletions in exon 4. They cause a frame-shift introducing
a premature stop codon at position 288 in TRD domain of the protein and probably alter
the ability of the protein to recruit co-repressor complexes and affect its function in the
process of transcription repression. The third novel frame-shift mutation due to one-bp
deletion (c.744delG), in patient R46, creates a stop codon at the beginning of TRD domain
(p.Ser194fsX208) and causes lack of both TRD and NLS domains. It is known that MBD-
containing mutant proteins without TRD might accomplish some degree of silencing,
either by recruiting the silencing complex by a TRD-independent mechanism or by directly
117
interfering with binding of transcription factors (Ballestar et al., 2000). However, loss of
NLS and TRD domains in the case of p.Ser194fsX208 mutation might interfere with
proper localization to nucleus and its functioning. The novel deletions in the other patients
(R2, R8, and R42) hypothetically affect the C-terminal domain of the protein and may give
rise to nonfunctional proteins. The c. 1156-1192del36 (p.Leu386Hisdel12) mutation in
patients R2 and R42 are small deletions, however, the mutation in patient R8 causes in a
frame-shifted protein starting from Lys345 residue (p.K345fs). Patient R8 is more severely
affected in all respects compared to patient R2 and R42, with shorter period of normal
development, earlier appearance of epileptic seizures, abnormal gait function with a
severity score of 9 versus 7 and 5, in accordance with previous findings for C-terminal
deletion mutations (Smeets et al., 2005).
In this study, exon duplications were identified in two patients with early seizure and
in one with congenital variant of RTT. Although the extent of the duplications and whether
they are tandem repeats could not be investigated, we have shown the duplication of exons
that are known to be the expressed. Thus, it is the first report implicating gene duplications
as causative mutations in female atypical RTT cases. Previously, one female patient with
PSV variant of RTT.has been reported to carry three copies of exon four of the MECP2
gene (Ariani et al., 2004). In addition to this finding, Meins et al. (2005) has shown that
duplication in Xq28 causes increased expression of the MECP2 gene in a boy with features
of Rett Syndrome. Collins et al. (2004) has developed a mouse model that transgenically
over-expressed MECP2 under the endogenous human promoter. These mice developed
seizures, hypoactivity and spasticity with several other progressive neurological
abnormalities. These results support the possibility that duplication of MECP2 may lead to
increased expression and underlie some cases of X-linked delayed-onset neurobehavioral
disorders including Rett Syndrome. It can be suggested that gene duplications might cause
a gain of function rather than a loss of function via (i) repressing its target genes strongly,
(ii) preventing their derepression, and/or (iii) repress novel genes that are not the targets in
normal cellular conditions. The latter mechanism may be more likely considering the fact
that all of our patients with exon duplications present additional neurodevelopmental
symptoms leading to atypical phenotypes. Interestingly, eye contact is very difficult to
obtain in these patients when compared to that of patients with missense mutation
118
(p=0.016). Further analysis should be performed to unravel the pathogenic mechanism
caused by these duplications.
Previous studies suggest that patients with missense mutations tend to have
significantly milder disease than patients with truncating mutations (Cheadle et al., 2000;
Colvin et al., 2004). On the basis of statistical analysis, no significant correlation could be
inferred between the overall disease severity and the type of the mutation in our cohort of
patients. However, the patients with exon deletions or nonsense mutations affecting the
TRD domain of the protein had higher clinical severity scores. Mutation negative patients
and patients with skewed XCI patterns had slightly milder phenotypes whereas mutation
positive patients had severe problems in their ability in purposeful hand movement and
walking skills.
It has been shown that there is a tendency for skewing of XCI in lymphocytes in
RTT patients when compared with age-matched controls. Our data is also suggestive for
skewed XCI pattern to confer a protective effect on the phenotype especially for severe
mutations that lead to production of truncated MECP2 protein. Among our 12 patients
presenting skewed XCI pattern along with MECP2 mutations, 10 of them had
nonsense/deletion/insertions, and only two of them had missense (p.R306C and p.T158M)
mutations. Although we could not show that there is a statistically significant relationship
between severity and XCI pattern by mutation, patients with the same mutation and
different XCI status had clinical variability. In this situation, each individual might be
expected to have differences in inter-tissue and intra-tissue XCI status, as is sometimes
observed in mouse models (Young et al., 2004; Gibson et al., 2005).
The MECP2 mutation detection rate was higher in the first group subjects (68.2 vs.
12.5 per cent). Furthermore, 26 patients of this group were diagnosed using a stringent
criteria and the mutations were identified in 79 per cent. Our results show that a strict
adherence to the RTT criteria and careful evaluation of the patients improve the rate of
MECP2 mutation detection. The diagnosis of RTT is mainly based on clinical criteria and
this is a critical step to decide or not to offer genetic testing. From a socio-economic point
of view, the use of efficient and well defined clinical criteria is very important since the
119
cost of MECP2 testing is high. Currently, the cost varies from $300 to $600 and is higher
than the official minimum wage in Turkey.
Mutation analyses revealed a total of 31 pathogenic variations of which 15 of 31 (48
.4 per cent) detected by PCR-RFLP, 10 of 31 (32.3 per cent) by SSCP-DNA sequencing
and 6 of 31 (19.3 per cent) by quantitative PCR assay. Thus, the PCR-RFLP method can be
used as a preliminary step to detect the most common mutations observed in MECP2 gene
in Turkish RTT patients. Our results showed that exon duplication/deletions contribute to
19.3 per cent of MECP2 mutations, and these rearrangements escape the PCR-based
screening strategy. Quantitative analysis of this gene should also be considered in RTT
patients, in order to determine the actual significance of the MECP2 gene in the etiology of
RTT.
With excluding 6 exon duplication/deletions, nearly 80 per cent (20 of 25) of the
point or small deletion/insertion mutations were detected in the exon 4 of the MECP2
gene. The rest of the mutations (20 per cent) were located within exon 3. This finding
suggests that the mutation in MECP2 exon 1 and 2 appears to be rare in Turkish RTT
patients and an initial analysis of exon 4 would provide the most efficient approach in a
mutation detection protocol.
Huppke et al. (2003) recommend using a cutoff point of 8 for genetic testing to
exclude girls with MECP2-negative results from the test.
However, the two patients with
exon duplications (patients R19 and R20) and the patient R22 with p.R106W mutation had
Huppke scores of 5-7, implicating that genetic diagnosis should be performed even when
the scores are lower than 8.
In three prenatal diagnostic tests performed, both chorionic biopsy specimens and
parents were tested and found to be negative for the index patient’s mutation. Germ line
mosaicism of MECP2 mutations is an important problem in genetic counseling for both
familial and sporadic RTT cases (Yaron et al, 2002). In the recent literature, Caselli et al.
(2004) reported nine cases that were evaluated for prenatal diagnosis. Only one fetus
carried the same mutation with affected sister (1/9, 11 per cent) and the pregnancy was
terminated. Although the majority of the patients have de novo mutations, the prenatal
120
diagnosis might be offered for the family with RTT daughter and the parents should be
informed about the possibility of germ line mosaicism.
Pathogenic sequence variations could not be identified in 14 of 44 (31.8 per cent)
sporadic female patients in the first group. These patients may have MECP2 mutations in
the promoter region or introns introducing novel splice sites that could not be detected by
PCR-SSCP analysis. RTT can be a genetically heterogeneous disorder, and other causative
genes might exist. Several recent studies identified mutations in the CDKL5 gene (OMIM
300203) encoding cyclin-dependent kinase like 5 in patients with an atypical, early seizure
variant of RTT (Weaving et al., 2005; Evans et al., 2005; Scala et al., 2005). Thus,
mutation screening of CDKL5 should be performed in MECP2 gene mutation-negative
patients with early-seizure variant of RTT. Finally, we suggest that quantitative analysis of
MECP2
has to be considered in especially RTT variants in order to determine the actual
significance of the gene in the etiology of RTT.
5> Dostları ilə paylaş: |