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Introduction
Center for Medical Genetics
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Introduction
Center for Medical Genetics
Diagnosis of genetic disorders
Clinical assesment
Laboratory investigations (three labs)
Counseling
Cursus
Human Molecular Genetics
Les 1 : From human cytogenetics to
molecular cytogenetics
Les 2 : Monogenic disorders
Les 3 : Familial cancer
Les 4 : Multifactorial genetic disorders
Les 5 : Diagnosis and Research in Human Genetics
From human cytogenetics to
molecular cytogenetics
• introduction
• historical overview (the birth of human
cytogenetics)
• progress in (molecular) cytogenetics
• general aspects of (molecular) cytogenetics
• molecular mechanisms for constitutional
chromosomal rearrangements in humans
The birth of human cytogenetics
• 1956: Tjio and Levan count the full complement
of 46 human chromosomes
The birth of human cytogenetics
• 1956: Tjio and Levan count the full complement
of 46 human chromosomes
• serendipitous addition of water to a suspension
of fixed cells
• 3 years after description of DNA structure
• 30 years after count of 48 chromosomes by
Thomas Painter
The birth of human cytogenetics
• human chromosomes have a morphology which
allows classification
The birth of human cytogenetics
rapidly associations were found between
human diseases (syndromes) and specific
chromosome abnormalities
1959
Lejeune et al : +21 in Down syndrome
Ford et al.
: 45,X in Turner syndrome
Jacobs et al : 47,XXY in Klinefelter syndrome
1960 Nowel and Hungerford
Philadelphia chromosome in CML
1973 Rowley: t(9;22)(q34;q11) in CML
The birth of human cytogenetics
rapidly associations were found between
human diseases (syndromes) and specific
chromosome abnormalities
1963 chromosome 5 short arm partial deletion
in Cri du Chat syndrome
1963 D-chromosome deletion in patient
with bilateral retinoblastoma
Prenatal diagnosis
cordocentesis
amniocentesis
Preimplantation
genetic diagnosis
Chorion villi
sampling
www.visembryo.com/baby/hp.html
Further progress in human cytogenetics
is fueled by technical innovations (I)
• 1968 Caspersson et al
differential staining of chromosomes produces
a recognizable banding pattern (chromosomal
barcode) along the length of the chromosomes
• chromosome bands are related to differences in
base pair composition, gene density, repetitive
elements, chromatin packaging but molecular
basis is not understood
• greatly facilitates classification and recognition
of structural aberations
general aspects of (molecular) cytogenetics
ISCN 1995
International System for Human Cytogenetic Nomenclature
groep A (1-3)
groep B (4-5)
groep C (6-12, X)
groep D (13-15)
groep E (16-18)
groep F (19-20)
general aspects of (molecular) cytogenetics
general aspects of (molecular) cytogenetics
chromosomal rearrangements
• numerical chromosome changes/aneuploidy
result from errors occurring during
meiotic or mitotic segregation
• structural chromosome changes
translocations
inversions
insertions
deletions
duplications
CYTOGENETICA EN MOLECULAIRE CYTOGENETICA:
CONSTITUTIONELE EN VERWORVEN
CHROMOSOMALE DEFECTEN
general aspects of (molecular) cytogenetics
general aspects of (molecular) cytogenetics
general aspects of (molecular) cytogenetics
general aspects of (molecular) cytogenetics
reciprocal translocation
ISCN 1995
International System for Human Cytogenetic Nomenclature
http://www.waisman.wisc.edu/cytogenetics/abnormalities/abnor
malities.html
Robertsonian translocation
45,XX,der(13;14)(q10;q10)
Reciprocal translocation
46,XY,t(6;9)(q24;p23)
ISCN 1995
International System for Human Cytogenetic Nomenclature
http://www.waisman.wisc.edu/cytogenetics/abnormalities/abnor
malities.html
Reciprocal translocation (unbalanced)
46,XY,t(6;9)(q24;p23)
46,XY,der(6)t(6;9)(q24;p23)
ISCN 1995
International System for Human Cytogenetic Nomenclature
inversion
insertion
46,XY,ins(5;2)(p14;q22q32)
46,XX,inv(9)(p13q13)
ISCN 1995
International System for Human Cytogenetic Nomenclature
duplication
46,X,dup(X)(p11.2p22.1)
deletion
del(18)(pterp11.2)
del(18)(p11.2)
Further progress in human cytogenetics
is fueled by technical innovations (II)
methods for mapping (disease) genes based
upon chromosomal rearrangements
• Somatic cell hybrids
• flow sorted chromosomes
• FISH
Further progress in human cytogenetics
is fueled by technical innovations (II)
Somatic cell hybrids
Further progress in human cytogenetics
is fueled by technical innovations (II)
Molecular mechanisms for constitutional
chromosomal rearrangements in humans
Positional cloning of t(1;17) breakpoints
• constitutional (1;17)(p36.2;q11.2) in patient
with neuroblastoma
• 1p36 region is frequently lost in NB
• association of a translocation with a particular disease
phenotype may point at the chromosomal localisation
of the disease gene
• additional evidence from eg LOH, linkage,mouse,…
• positional cloning: cloning of disease gene based upon
the assumption of the chromosomal localisation
• physical mapping, identification of candidate genes
mutation analysis, expression studies, functional evidence
Molecular mechanisms for constitutional
chromosomal rearrangements in humans
Positional cloning of t(1;17) breakpoints
Further progress in human cytogenetics
is fueled by technical innovations
late ’80ies introduction of FISH
• significant increase of sensitivity
(10.000x)
• new possiblities eg interphase
• various applications eg gene mapping,
genetic diagnosis, research
• “the FISH have spawned”
CGH
M-FISH/SKY
FICTION
fibre FISH
FISH (fluorescentie in situ hybridisatie)
Fluorescence in situ hybridisation
DNA
Commercial Cot1 DNA
Labeling: nick translation
Mix
10ml culture
Denaturation and incubation at 37°C
Denaturation
Wash, detection
and counterstain
Hybridisatie o/n
2 X chromosome 13
2 X chromosome 18
2 X chromosome 21
1 X chromosome Y
Control
lymphocytes
(FISH 952-35)
CGH
part I
CGH
part I
CGH
part I
advantages
whole genome in 1 experiment
no need to culture tumor cells
sensitive detection of gene amplification
retrospective analysis
disadvantages
limited resolution (~10 Mb del/dup)
laborious
only gains and losses / no balanced rearrangements
no information on the nature of the aberrations
Molecular mechanisms for constitutional
chromosomal rearrangements in humans
• chromosomal rearrangements require the formation
of double strand breaks (DSBs) and subsequent
rejoining of the broken ends between two (or more)
breakpoints
• exogenous causes of structural aberrations
X-rays, -rays, -particles and other forms of
ionizing radiation
cause formation of oxidants which are powerful
clastogens
duration of exposure
Molecular mechanisms for constitutional
chromosomal rearrangements in humans
exogenous causes of structural aberrations
• chemicals: alkylating agents, purine and pyrimidine
analoges, alkyl epoxides, aromatic amines, nitroso
compounds and heavy metals
most often generation of breaks at G2
• viral infections
• lesions may undergo repair or misrepair by a wide range
of DNA repair systems
Molecular mechanisms for constitutional
chromosomal rearrangements in humans
endogenous causes of structural aberrations
• rare autosomal recessive chromosome breakage syndromes
caused by defective DNA repair enzymes
(AT, ATM; BS, BLM; NBS, NBS1)
• transposable elements
• short and long interspersed elements (LINE, SINE)
300 bp Alu (every 4 kb, gene rich), longer LINE (gene poor)
• segmental duplication, gene duplication
• fragile sites
Molecular mechanisms for constitutional
chromosomal rearrangements in humans
Segmental duplications: an ‘expanding’ role in genomic
instability and disease. Emanuel and Shaikh, Nature Reviews
Genetics, Volume 2, October 2001, 791-800.
• Segmental duplications = region or chromosome specific
low-copy repeats, new class of repetitive DNA elements
recently identified
• resulting genetic aberrations
deletions
interstitial duplications
translocations
inversions
marker chromosomes
Molecular mechanisms for constitutional
chromosomal rearrangements in humans
DiGeorge/velo-cardio-facial syndrome
recurrent reciprocal translocation t(11;22)
cat eye syndrome (CES)
Molecular mechanisms for constitutional
chromosomal rearrangements in humans
red blocks: low copy repeats
ADU: DGS patient with translocation
TDR: common 3 Mb typically deleted region
a-f: unusual deletions
Molecular mechanisms for constitutional
chromosomal rearrangements in humans
DiGeorge syndrome
velo-cardio-facial syndrome
Molecular mechanisms for constitutional
chromosomal rearrangements in humans
DiGeorge syndrome
velo-cardio-facial syndrome
•
•
•
•
•
1/4000 live births
gene haploinsufficiency syndrome
90% de novo, 10% inherited
deletion encompasses ~30 genes
clinical features are highly variable (table),
variable expressivity and incomplete penetrance
• affect pharyngeal and neurobehavioural development
• which genes are critically involved ????
mouse models: candidate TBX1, T-box family of genes
highly expressed in pharyngeal arches, TBX1 KO
Molecular mechanisms for constitutional
chromosomal rearrangements in humans
VCFS: CP, velopharyngeal insufficiency, small mouth, retrognathia, bulbous nasal tip,
microcephaly, concotruncal heart defects, MR, learning disabilities, short stature,
DGS: parathyroid hypoplasia, thymic hypoplasia and immune defect due to T cell deficit
Molecular mechanisms for constitutional
chromosomal rearrangements in humans
DiGeorge syndrome
velo-cardio-facial syndrome
Molecular mechanisms for constitutional
chromosomal rearrangements in humans
DiGeorge syndrome
velo-cardio-facial syndrome
Molecular mechanisms for constitutional
chromosomal rearrangements in humans
DiGeorge syndrome
velo-cardio-facial syndrome
Molecular mechanisms for constitutional
chromosomal rearrangements in humans
cat eye syndrome
t(11;22)
carriers have normal phenotype
are at risk for unbalanced progeny
1:3 segregation leading to
47,XX,+der(22)
MR, multipel malformation syndrome
including characteristic eye abnormalities
Molecular mechanisms for constitutional
chromosomal rearrangements in humans
CMT1A/HNPP
• Charcot-Marie-Tooth disease 1A
• duplication within 17p12
• peripheral myelin protein 22
• most common inherited peripheral neuropathy
• 70% of CMT1 inherited demyelating neuropathy
Molecular mechanisms for constitutional
chromosomal rearrangements in humans
CMT1A/HNPP
• most common inherited peripheral neuropathy
• 70% of CMT1 inherited demyelating neuropathy
First described in 1886 by Charcot and Marie in Paris, France and Tooth
in Cambridge, England. Most common inherited disorder of the peripheral
nerves affecting 1 in 2500 individuals in their 20s and 30s. Characterised
by distal muscle atrophy and weakness, first involving the legs and
particularly the peritoneal muscles. Sensory loss may be present but is
always less pronounced than muscle weakness, and tendon reflexes are
absent or diminished. High arched feet (pes cavus) are often present.
Also described as Hereditary and Motor Sensory Neuropathy (HMSN).
Molecular mechanisms for constitutional
chromosomal rearrangements in humans
CMT1A/HNPP
• hereditary neuropathy with liability
the pressure palsies
• idem inherited peripheral neuropathy
but with episodic and milder manifestations
Hereditary Neuropathy with liability to Pressure Palsies or HNPP is a slowly
progressive, hereditary, neuromuscular disorder which makes an individual very
susceptible to nerve injury from pressure, stretch or repetitive use. When injured,
the nerves demyelinate or lose their insulating covering. This causes episodes of
numbness and weakness in the injured area, which are referred to as the ‘pressure
palsies'. These episodes can be mild and more of a nuisance than anything, or so
severe almost all movement in the affected limb is impossible. They may last
several minutes to months. Because the symptoms can come and go, and most
neurologists have not yet heard of or seen a case of HNPP, it can be very difficult
and lengthy process to be diagnosed.
Molecular mechanisms for constitutional
chromosomal rearrangements in humans
CMT1A/HNPP
• hereditary neuropathy with liability
the pressure palsies
• idem inherited peripheral neuropathy
but with episodic and milder manifestations
SMS Smith-Magenis syndrome
• mental retardation/malformation syndrome
• ~5 Mb deletion
Molecular mechanisms for constitutional
chromosomal rearrangements in humans
Williams-Beuren syndrome
• deletion of the elastin gene, responsable
for supravalvular aortic stenosis
• ~1.6 Mb deletion at 7q11.23
• heart defects, facial dysmorphy,
mental retardation, behavioural abn
Molecular mechanisms for constitutional
Flat midface, epicanthal folds, long philtrum
chromosomal rearrangements
in humans
thik lips, depressed
nasal bridge, anteverted nares,
hypodontia, microdontia, harsh voice, MR
(average IQ 56), attention deficit disorder,
hypersensitivity to sound, coctail party personality,
short stature, hypoplastic nails, supravalvular stenosis
Molecular mechanisms for constitutional
chromosomal rearrangements in humans
PWS/AS
• ~4 Mb deletion of imprinted region on 15q12
• maternal deletion or paternal disomy
leads to AS (profound MR, no speach
development, uncontrolled laughter),
deletion of UBE3A (mouse KO)
• paternal deletion or maternal disomy
leads to PWS (MR, obesity, dysmorphic)
SNRP associated imprinting center
Failure to thrive in infancy, obesity, dolichocephaly,
narrow bitemporal diameter, almond-shaped eyes,
strabismus, thin upper lip, small appearing mouth,
down turned corners of the mouth, hypogonadism
small hands, hypopigmentation, learning disabilities,
behavioural problems
Microbrachycephaly, prognathia, protruding tongue, macrostomia, widely spaced teeth,
severe MR, paroxysmal laughter, absent speech, ataxia with jerky arm movements,
seizures, hypopigmentation
Molecular mechanisms for constitutional
chromosomal rearrangements in humans
X-linked ichtyosis
• deletion of the steroid sulphatase gene
haemophilia A
• inversion that disrupts factor VIII gene
int22h in intron 22 and two inverted
int22h at ~500 kb telomeric
Emery-Dreifuss muscular dystrophy
• inversion in the emerin gene
x
tel
tel
tel
Molecular mechanisms for constitutional
chromosomal rearrangements in humans
simple segmental duplication
• CMT1A-REP
two copies that flank the region
24 kb in size
98.7% identity
• S323 elements on Xp22, separated by 1.9 Mb
• two 11.3-kb inverted repeats that mediate the inversion
in the emerin gene, >99% identity
• int22h (intron homologous region) sequence which
mediates the inversion in the factor VIII gene
Molecular mechanisms for constitutional
chromosomal rearrangements in humans
complex segmental duplication
• 22q11 repeat
differences in size, content, organisation
truncated gene segments and pseudogenes
potentially recombinogenic sequences including
palindromic (A+T) rich repeats (PATRR) and VNTRs
• SMS at least four genes or pseudogenes
• PWS/AS duplications of HERC2
• BWS several (pseudo)genes in complex configurations
• evidence for presence recombinational hot spots
Molecular mechanisms for constitutional
chromosomal rearrangements in humans
Molecular mechanisms for constitutional
chromosomal rearrangements in humans
Mechanistic models for rearrangements
Molecular mechanisms for constitutional
chromosomal rearrangements in humans
Mechanistic models for rearrangements
Molecular mechanisms for constitutional
chromosomal rearrangements in humans
Mechanistic models for rearrangements