Split hand-split foot malformation [Elektronische Ressource] : determining the frequency of genomic aberrations with molecular-genetic methods / von Charlotte W. Ockeloen

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Informations

• Publié par: charite_-_universitatsmedizin_berlin
• Publié le: 01 janvier 2010
• Nombre de lectures: 46
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Aus dem Institut für Medizinische Genetik
der Medizinischen Fakultät Charité – Universitätsmedizin Berlin




DISSERTATION


Split hand/split foot malformation: determining the frequency of
genomic aberrations with molecular-genetic methods



zur Erlangung des akademischen Grades
Doctor medicinae (Dr. med.)






vorgelegt der Medizinischen Fakultät
Charité – Universitätsmedizin Berlin





von


Charlotte W. Ockeloen

aus Nijmegen, Niederlande








































Gutachter: 1. Prof. dr. med. S. Mundlos
2. Prof. dr. med. A. Rauch
3. Prof. dr. med. G. Gillessen-Kaesbach





Datum der Promotion: 19.11.2010
TABLE OF CONTENTS

PREFACE ....................................................................................................................... 1
1. INTRODUCTION ......... 2
1.1 PATHOGENESIS OF LIMB DEVELOPMENT ................................. 3
1.2 SHFM1 (MIM 183600) ....................... 5
1.3 SHFM2 (MIM 313350) ................................................................ 6
1.4 SHFM3 (MIM 600095) 7
1.5 SHFM4 (MIM 605289) - MUTATIONS IN THE TP63-GENE ...................................... 9
1.6 SHFM 5 (MIM 606708) .................... 10
1.7 EVIDENCE FOR TWO NEW SHFM LOCI ................................. 11
1.8 SPLIT HAND/FOOT MALFORMATION AND LONG BONE DEFICIENCY ............................ 12
1.9 GENOTYPE/PHENOTYPE CORRELATION ................................ 12
2. HYPOTHESES .......................................................................... 14
3. MATERIALS AND METHODS .................................................. 15
3.1 PATIENTS ............... 15
3.2 MULTIPLEX LIGATION-DEPENDENT PROBE AMPLIFICATION (MLPA) ............................. 18
3.3 REAL-TIME QUANTITATIVE PCR SHFM 3 LOCUS (10Q24) .......................................... 22
3.4 ARRAY CGH ................................................................ 25
4. RESULTS .................. 29
4.1 ARRAY CGH RESULTS ............................................................................................ 39
4.2 FAMILIAL CASES ...................................... 41
5. DISCUSSION ............................................................................ 44
FUTURE PROSPECTS ..... 48
ZUSAMMENFASSUNG ................................................................................................ 49
REFERENCES .............. 51
LEBENSLAUF ................................................................................................ 57
ERKLÄERUNG ............. 58 Preface
PREFACE

First of all, I would like to thank Dr. rer. nat. Eva Klopocki, for her excellent supervision
and guidance throughout the project. She helped me accomplish my goals and was
always stimulating and helpful. Second, I would like to thank my laboratory colleagues,
Randy Koll and Fabienne Trotier, for their assistance with my experiments and being
such nice colleagues.
I owe much gratitude to Prof. Dr. Mundlos, who gave me the opportunity to work on this
project. Also, I would like to thank all other colleagues from the genetics department of
the Charité who helped or assisted me during my time in the laboratory.






















1 1. Introduction
1. INTRODUCTION

Split hand/split foot malformation (SHFM), also known as ectrodactyly or cleft hand/foot,
is a complex congenital limb defect that is characterized by a deep median cleft with
absence of central ray(s). SHFM presents as a non-syndromic entity or as part of a
syndrome. It occurs either sporadically or in families. Reduced penetrance is frequently
4 observed, and has been documented in several pedigrees. One of the well-recognized
hallmarks of SHFM is the inter- and intra-familial phenotypic variability; limb defects
range from minor syndactyly of the digits to severe syndactylous hypoplasia of several
3digits or monodactyly.
Oligodactyly, presenting as three or more digits in association with syndactyly and a
4 deep median cleft, is by far the most common pattern. The other two core phenotypes
are monodactyly and bidactyly, formerly known as “lobster claw” malformation. Noncore
phenotypic manifestations include polydactyly, triphalangeal thumb, clinodactyly,
4,6camptodactyly, transverse phalanges, and ulnar deviation. Approximately 40% of
individuals presenting with SHFM have associated non-limb congenital anomalies, for
example mental retardation, cleft palate or ectodermal dysplasia. The overall
prevalence of SHFM is reported to range from approximately 0.6/10 000 newborns to
70.51/10 000 newborns.
SHFM can be categorized as typical or atypical. This differentiation was originally made
8 9,10 by Lange in 1937 and has been maintained by others . Atypical split hand is usually
unilateral, without associated foot involvement, and occurs sporadically. Regarding the
nomenclature and classification, there is significant confusion. It has been postulated
13that atypical cleft hand may be caused by vascular disruption. According to the
Committee of the International Federation of Societies for Surgery of the Hand, the term
11atypical split hand should be replaced by “symbrachydactyly”. However, many clinical
geneticists continue to refer to this entity as atypical split hand. In typical split hand,
bilateral involvement can occur as well as involvement of the feet. Patients may have a
positive family history. The split hand/split foot malformation is usually inherited in an
autosomal dominant manner, although autosomal recessive inheritance has also been
12described.

2 1. Introduction
So far 5 different genetic loci have been mapped for non-syndromic SHFM, and recently
17,19,21,22,28,27,39evidence for two new loci has been found.

1.1 PATHOGENESIS OF LIMB DEVELOPMENT

thThe formation of the upper limb occurs in the 4 week of embryonic development and is
completed approximately 8 weeks later. The initiation of the lower limb bud formation is
delayed by 2 days, but the factors that control limb development are the same for both
upper and lower limbs. Thus, it is not uncommon that limb abnormalities occur
3symmetrically.
The outgrowth and patterning of the limb occur in three dimensions: proximo-distal
(shoulder-finger direction), antero-posterior (thumb-little finger direction) and dorso-
ventral (back-palm direction). The apical ectodermal ridge (AER) is a thickened ridge of
ectoderm at the apex of the limb bud; it controls the outgrowth of the limb bud along the
proximo-distal axis. Directly underlying the AER is the progress zone (PZ), an area of
rapid cell division. Signals from the AER allow the underlying cells of the PZ to maintain
their proliferative activity. The zone of polarizing activity (ZPA), which is located in the
posterior region of the developing limb bud, controls the antero-posterior patterning of
the limb. Dorso-ventral patterning of the limb is controlled by the genes Wnt7a and
Lmx1. Surgical removal of the AER results in truncation of all skeletal elements of the
3,24,25limb (stylopod, zeugopod, and autopod).

A number of key players in the AER have recently been identified; these include
fibroblast growth factors (Fgfs), bone morphogenetic proteins (Bmps), Wnt signalling
molecules, and homeobox containing proteins, such as Msx1 and Msx2 (Fig.1). AER
formation is induced by mesodermal signalling to the overlying ectoderm, using Fgf10
and Bmps. Bmps control the ectodermal expression of Msx transcription factor genes.
The two major functions of Fgfs induce the proliferation of mesenchymal cells in the PZ,
and they are required by the ZPA to maintain Sonic Hedgehog (Shh) expression. Sonic
Hedgehog mediated Bmp signalling is essential to maintain the AER. This shows that a
co-dependence exists between the AER and the ZPA.
3 1. Introduction
Fgfs are crucial for limb development; Fgf4 and Fgf8 knockout mice develop a normal
AER, but mesenchymal gene expression is disturbed. This results in aplasia of the
24proximal and distal limb elements.
Several homeobox genes, such as HoxD and HoxA, are responsible for maintaining the
relationship between the AER and the PZ. In addition, Bmp signalling plays an
important role in this process. The homeobox genes are also essential for the formation
3, 24,25 of the individual digits of the fetal hand.


Figure 1. Signalling pathways in the developing limb bud. Failure to maintain the AER or defective
AER signalling underlies SHFM. Correct signalling in the anterior and posterior apical ectodermal ridge
(AER; light grey), but not in the median AER (yellow), may explain the relatively normal development of
t