1.4.5. Thyroid Development and TTF-2
The mature mammalian thyroid gland evolves from two distinct embryologic
structures, the thyroid diverticulum, an endodermal component that gives rise to thyroid
follicular cells, and the neuroectodermal ultimobranchial bodies that differentiate into the
parafollicular calcitonin-producing C cells. Thyroid follicular cells originate by
invagination of pharyngeal endoderm beginning at E8–8.5 of mouse development (Ericson
and Frederiksson, 1990). The thyroid primordium migrates downward to reach its final
destination in front of the trachea at E13–14. It is only at E15, after completion of the
migration process, that thyroid follicular cells differentiate, as measured by expression of
several thyroid-specific genes (Tg, TPO and TSHr) (Lazzaro et al., 1991). Two
transcription factors, TTF-1 and Pax-8, have been proposed to be necessary for thyroid
differentiation (Damante and Di Lauro, 1994). These proteins are present in the thyroid at
late stage at E8.5, suggesting that an additional, essential event(s) must occur to trigger
differentiation of thyroid cells at E13–14 (Lazzaro et al., 1991). TTF-2 expression is turned
off exactly between E13 and E15 in the developing thyroid. The correlation between the
onset of thyroglobulin and thyroperoxidase gene expression and the disappearance of TTF-
2 mRNA suggests that in the embryo the role of TTF-2 is to block the activation of
thyroid-specific gene expression by TTF-1 and Pax-8. It has been proposed that thyroid
cell precursors enter into a determined state at E8.5, which is characterized, and possibly
induced, by the presence of TTF-1 and Pax-8. During the next 5 days, thyroid cell
precursors undergo a long migration, at the end of which they will express their full-
differentiated phenotype. The presence of TTF-2 in the migrating thyroid cell precursors
would prevent precocious expression of genes that might have an adverse effect on
migration, for example because of changes in the adhesive properties of the cells (Zannini
., 1997). In adult thyroid tissue TTF-2 expression is restored. It also shows a transient
expression pattern in the developing pituitary. When Rathke’s pouch is completed at E12-
12.5, TTF-2 is undetectable in pituitary cells (Hermesz et al., 1996).
TTF-2 is expressed in two glands, the thyroid and the pituitary, which are part of a
regulatory circuit responsible for homeostatic control of thyroid hormone production. The
hypothalamus is also part of this circuit, where TTF-1, another transcription factor of
relevance in thyroid cell function and differentiation, is expressed (Kimura et al., 1996). It
is inviting to think that recruitment of the same regulatory molecules in functionally related
organs in the thyroid–pituitary–hypothalamic axis is advantageous to coordinate
development and function.
TTF-2 has a dual function in the development of the thyroid gland (Zannini et al.,
1997). In the mouse, TTF-2 shows transient expression during the migration of thyroid
precursor cells from the invagination of the pharyngeal endoderm to the final destination in
front of the trachea. During this period, TTF-2 represses transcriptional activation of the
thyroglobulin and thyroperoxidase promoters by TTF-1 and PAX8, respectively.
Subsequently, TTF-2 expression is turned off; however, is restored in adult thyroid tissue.
In the adult thyroid, TTF-2 functions as a transcriptional activator of thyroglobulin
(Sinclair et al., 1990) and thyroperoxidase (Francis-Lang et al., 1992; Aza-Blanc et al.,
1.4.6. TTF-2 Gene Mutations
Two missense mutations (p.A65V and p.S57N) of human TTF-2 gene have been
reported in two families with CH. In a Welsh family, two male siblings with thyroid
agenesis, cleft palate, choanal atresia and bifid epiglottis together with spiky hair were
homozygous for a missense mutation (p.A65V) within the forkhead DNA binding domain.
Functional studies indicated that the p.A65V mutation is highly deleterious, with the
mutant protein exhibiting a complete lack of DNA binding and transcriptional activation.
In neither case thyroid tissue was detected by
I scanning and ultrasonography (Clifton-
Bligh et al., 1998). In the second family, a homozygous p.S57N missense mutation within
the forkhead domain was reported in two probands presented with CH. Both siblings
exhibited cleft palate, and the other cervical midline defects (choanal atresia, bifid
epiglottis) were absent. When compared directly in functional studies, the p.S57N mutation
is less deleterious than the p.A65V mutation, preserving some DNA binding and
transcriptional activity. Although p.S57N mutant protein retains 75 per cent of the maximal
transcriptional activity, thyroid tissue was absent in both siblings. Unlike the cases
described previously, these patients had an incomplete clinical phenotype, which may
indicate partial preservation of TTF-2 function in vivo (Castanet et al., 2002).
1.4.7. Plasma Cholinesterase Deficiency
Patients with CH are candidates for multiple operations due to midline defects (cleft
palate and choanal atresia) and their response to administration of muscle relaxants is
crucial. Several cases with different disease phenotype showing the prolonged
neuromuscular block (paralysis) have been reported following the administration of muscle
relaxants such as mivacurium (Kaiser et al., 1995; Chung et al., 2002). Mivacurium is a
short acting non-depolarising neuromuscular blocking agent (Kaiser et al., 1995).
Deficiency or abnormality of plasma cholinesterase (also called pseudocholinesterase–
PChE, butyrylcholinesterase–BChE) may cause prolonged duration of action of
Barta et al., 2001
). Up to date, more than 20 genetic variants of BChE have
been described and p.Asp70Gly (A-variant) and Ala539Thr (K-variant) are the most
common ones responsible for reduced activity of Butyrylcholinesterase (La Du, 1993).
2. AIM OF THE STUDY
In the context of this study, the aim was to investigate the genetic mechanisms
responsible for Rett Syndrome (RTT), Breast Carcinogenesis, and Congenital
To provide further delineation of MECP2 mutations in RTT patients, we have
investigated the mutation profile of the entire coding region of the MECP2 gene and XCI
pattern in a cohort of 71 patients with classical or atypical RTT. The analyses in RTT
patients were extended:
• to establish quantitative Real Time PCR and quantitative fluorescent multiplex PCR
assays to detect the MECP2 exon rearrangements in mutation negative patients,
• to evaluate the impact of the use of stringent clinical criteria on MECP2 mutation
• to establish XCI analysis using two different reference genes,
• to evaluate the contribution of XCI to RTT clinical phenotype,
• to perform genotype/phenotype correlation based on comparison of severity score of
patients with the type and location of the mutation and the XCI pattern.
• to develop a
rapid and efficient MECP2 mutation screening strategy to be used as a
preliminary step for genetic diagnosis of RTT.
We aimed to design a simpler
multiplex ARMS-PCR strategy that allows identification
of seven mutations
accounting for up to two thirds of pathogenic MECP2 mutations.
• to analyse the effect of DNA concentration on reliability and reproducibility of Real
Time PCR analysis for identification of MECP2 exon rearrangments.
Aberrant methylation of CpG-rich sites (CpG islands) was identified as an epigenetic
mechanism for the transcriptional silencing of repair genes in different types of cancer. In
this study, the methylation status of 5’ flanking regions (including the CpG islands and
putative promoter sequence) of hHR23A and hHR23B genes were investigated in primary
breast tumor, tumor adjacent tissues, and normal breast tissues. Since the methylation
status of these genes was not investigated before, we aimed;
• to characterize the CpG islands and the putative promoter region in the 5' flanking
region of the hHR23 genes using web-based analysis,
• to design primer sequences to investigate the methylation status of CpG di-
• to determine the methylation status of the putative promoter region of hHR23 genes
in archival formalin-fixed, paraffin-embedded breast tumor and normal tissues.
The genetic mechanisms leading to congenital hypothyroidism and prolonged
paralysis after mivacurium in a patient with Bamforth Syndrome were investigated. For
• The patient was screened for the presence of mutations within the TTF2 gene
responsible of hypothyroidism. The effect of the identified mutation on DNA binding
ability of the TTF2 protein was tested based on a collaborative study.
• Since our CH patient was the first case with Bamforth Syndrome showing plasma
cholinesterase deficiency, the patient DNA sample was investigated for the presence
of BChE variants responsible for prolonged neuromuscular block (paralysis) after
administration of mivacurium as a muscle relaxant.
3.1. Subjects and Samples
Peripheral blood samples of patients with Rett Syndrome were provided with an
informed consent by Istanbul University (Department of Neurology, Division of Child
Neurology), Marmara University Hospital (Department of Pediatrics, Division of Child
Neurology), Health Ministry Tepecik Education Hospital (Child Health and Diseases
Clinics), and other centers.
The patient with Congenital Hypothyroidism was referred from Kocaeli University,
Faculty of Medicine, Department of Pediatrics.
Archival formalin-fixed, paraffin-embedded tissues were kindly provided by
Marmara University Hospital (Department of Pathology) and Nişantaşı Pathology
Laboratories (Istanbul, Turkey).
All solid and liquid chemicals used in this study were purchased from Merck
(Germany), Sigma (USA), Riedel de-Häen (Germany), and Carlo Erba (Germany), unless
stated otherwise in the text.
3.3. Fine Chemicals
DNA Polymerases were purchased from Fermentas (MBI Fermentas, Lithuania).
The restriction enzymes were purchased from Promega (USA), Fermentas (Lithuania), and
New England Biolabs (England).
3.3.2. Oligonucleotide Primers
The primers used in the framework of this thesis were synthesized by Integrated
DNA Technologies (USA), Alpha DNA (Canada), or Iontek (Istanbul). The sequence and
PCR conditions for the primers used throughout the thesis are given in Table 3.1 through
Table 3.1. Sequence of the primers used for exon amplification of the MECP2 gene.
Rett 2.1F: gagcccgtgcagccatcagc
Rett 3AF: tgtgtctttctgtttgtccc
Rett 3AR: gatttgggcttcttaggtgg
Rett 3BF: cctcccggcgagagcagaaa
Rett 3BR: tgacctgggtggatgtggtg
Rett 3CF: tgccttttcaaacttcgcca
Rett 3CR: tgaggaggcgctgctgctgc
Rett 3CF: tgccttttcaaacttcgcca
Rett 3MR: tggcctgagggtcggcctcagctttgc
Rett 3DF: gcagcagcagcgcctcctca
Rett 3DR: tggcaaccgcgggctgaggca
Rett 3EF: tgccccaaggagccagctaa
Rett 3ER: gctttgcaatccgctccgtg
Table 3.2. Primers used in X chromosome inactivation analysis.
Xinact F: gctgtgaaggttgctgttcctcat
Xinact R: tccagaatctgttccagagcgtgc
Xinact2 F: atgctaaggaccatccagga
Xinact2 R: ggagttttcctccctcacca
Table 3.3. Sequences and PCR conditions for the primers used in quantitative Real Time
Table 3.4. Sequence of the primers used in quantitative fluoresent multiplex PCR analysis.
Table 3.5. Sequence of the primers used in methylation analyses of the putative promoter
region of hHR23A and hHR23B genes.
Table 3.6. Sequence of the primers used for exon amplification of the TTF2 gene.
Table 3.7. Sequence of the primers used for exon amplification of the BChE gene.
3.3.3. DNA Size Marker
Size marker used in this study was 100-bp DNA ladder between 100 and 1000 bp
(MBI Fermentas, Lithuania).
QIAquick PCR Purification Kit was purchased from Qiagen (Germany). SYBR
Premix Ex Taq was purchased from TaKaRa (Japan).
Kit was purchased from Epigentek (USA). Deoxyribonucleoside triphosphates (dNTPs)
were purchased from Fermentas (MBI Fermentas, Lithuania).
3.5. Buffers and Solutions
3.5.1. DNA Extraction from Peripheral Blood
Cell Lysis Buffer
155 mM NH
10 mM KHCO
1 mM Na
EDTA (pH 7.4)
Nuclei Lysis Buffer
10 mM Tris-HCl (pH 8.0)
400 mM NaCl
2 mM Na
EDTA (pH 7.4)
10 per cent SDS (w/v) (pH 7.2)
20 mM Tris-HCl (pH 8.0)
0.1 mM Na
EDTA (pH 8.0)
5 M NaCl solution
292.2 g NaCl in 1 l dH
3.5.2. Polymerase Chain Reaction (PCR)
10 X MgCl
500 mM KCl
100 mM Tris-HCl (pH 9.0)
1 per cent Triton X-100 (Promega, USA)
10 X PCR Buffer
100 mM Tris-HCl (pH 8.8 at 25 °C)
500 mM KCl
0.8 per cent Nonidet P40 (Fermentas,
10 X PCR Buffer with (NH
750 mM Tris-HCl (pH 8.8 at 25 °C)
200 mM (NH
0.1 per cent Tween 20 (Fermentas, Lithuania)
25 mM MgCl
(Fermentas, Lithuania and
3.5.3. Agarose Gel Electrophoresis
10 X Tris-Borate-EDTA Buffer
0.89 M Tris-Base
0.89 M Boric Acid
20 mM Na
EDTA (pH 8.3)
1, 2 or 3 per cent Agarose Gel
1, 2 or 3 per cent (w/v) Agarose in 0.5 X
10 X Loading Buffer
2.5 mg/ml Bromophenol Blue
1 per cent SDS in 2 ml glycerol
3.5.4. Polyacrylamide Gel Electrophoresis
10 X TBE Buffer
0.89 M Tris-Base
0.89 M Boric Acid
20 mM Na
EDTA (pH 8.3)
30 per cent Acrylamide Stock :
29 per cent Acrylamide
1 per cent N, N'-methylenebisacrylamide
8 per cent Denaturing Gel
8 per cent Acrylamide Stock (19:1)
8.3 M Urea
1X TBE Buffer (pH 8.3)
10 per cent APS (w/v)
10X Denaturing Buffer
95 per cent Formamid
20 mM EDTA
0.05 per cent Xylene Cyanol
0.05 per cent Bromophenol Blue
3.5.5. Silver Staining
10 per cent Ethanol
0.5 per cent Glacial Acetic Acid
0.1 per cent AgNO
1.5 per cent NaOH
0.01 per cent NaBH
0.015 per cent Formaldehyde
0.75 per cent Na
Automated DNA Sequencing and Quantitative Fluorescent Multiplex PCR analyses
were perfomed using ABI 3100 and 3130 PRISM (Applied Biosystems) in Iontek and Burc
Laboratories (Istanbul, Turkey), respectively. Other experiments were performed using
facilities of the Department of Molecular Biology and Genetics at Boğaziçi University
(Istanbul, Turkey). The equipments used were as follows:
Model MAC-601 (Eyela, Japan)
Electronic Balance Model VA124 (Gec Avery, UK)
Electronic Balance Model CC081 (Gec Avery, UK)
Centrifuge 5415C (Eppendorf, Germany)
Universal 16R (Hettich, Germany)
-20°C (Bosch, Germany)
-70°C (GFL, Germany)
GelDoc Documentation System (Bio-Rad, USA)
Electrophoretic Equipments :
Horizon 58, Model 200 (BRL, USA)
Sequi-Gen Sequencing Cell (Bio-Rad,USA)
DGGE System Model # DGGE-200 (C.B.S.
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