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Introduction Center for Medical Genetics. Staff (81) Clinicians and psychologists Laboratory supervisors Researchers Laboratory technicians Secretary. Introduction Center for Medical Genetics. Diagnosis of genetic disorders Clinical assesment Laboratory investigations (three labs)

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slide1

Introduction

Center for Medical Genetics

Staff (81)

Clinicians and psychologists

Laboratory supervisors

Researchers

Laboratory technicians

Secretary

slide2

Introduction

Center for Medical Genetics

Diagnosis of genetic disorders

Clinical assesment

Laboratory investigations (three labs)

Counseling

slide3

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

slide4

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
slide5

The birth of human cytogenetics

  • 1956: Tjio and Levan count the full complement
  • of 46 human chromosomes
slide6

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
slide7

The birth of human cytogenetics

  • human chromosomes have a morphology which
  • allows classification
slide8

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
slide10

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

slide12

cordocentesis

amniocentesis

Chorion villi

sampling

Preimplantation

genetic diagnosis

Prenatal diagnosis

www.visembryo.com/baby/hp.html

slide13

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
slide16

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)

slide18

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
slide19

CYTOGENETICA EN MOLECULAIRE CYTOGENETICA:

CONSTITUTIONELE EN VERWORVEN

CHROMOSOMALE DEFECTEN

slide24

ISCN 1995

International System for Human Cytogenetic Nomenclature

http://www.waisman.wisc.edu/cytogenetics/abnormalities/abnormalities.html

Robertsonian translocation

45,XX,der(13;14)(q10;q10)

Reciprocal translocation

46,XY,t(6;9)(q24;p23)

slide25

ISCN 1995

International System for Human Cytogenetic Nomenclature

http://www.waisman.wisc.edu/cytogenetics/abnormalities/abnormalities.html

Reciprocal translocation (unbalanced)

46,XY,t(6;9)(q24;p23)

46,XY,der(6)t(6;9)(q24;p23)

slide26

ISCN 1995

International System for Human Cytogenetic Nomenclature

insertion

inversion

46,XY,ins(5;2)(p14;q22q32)

46,XX,inv(9)(p13q13)

slide27

ISCN 1995

International System for Human Cytogenetic Nomenclature

duplication

deletion

del(18)(pterp11.2)

del(18)(p11.2)

46,X,dup(X)(p11.2p22.1)

slide28

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
slide29

Further progress in human cytogenetics

is fueled by technical innovations (II)

Somatic cell hybrids

slide30

Further progress in human cytogenetics

is fueled by technical innovations (II)

slide31

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
slide32

Molecular mechanisms for constitutional

chromosomal rearrangements in humans

Positional cloning of t(1;17) breakpoints

slide34

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
slide36

Fluorescence in situ hybridisation

DNA

Commercial Cot1 DNA

Labeling: nick translation

Mix

10ml culture

Denaturation and incubation at 37°C

Wash, detection

and counterstain

Denaturation

Hybridisatie o/n

slide37

2 X chromosome 13

2 X chromosome 18

2 X chromosome 21

1 X chromosome Y

Control

lymphocytes

(FISH 952-35)

slide42

part I

disadvantages

limited resolution (~10 Mb del/dup)

laborious

only gains and losses / no balanced rearrangements

no information on the nature of the aberrations

CGH

advantages

whole genome in 1 experiment

no need to culture tumor cells

sensitive detection of gene amplification

retrospective analysis

slide44

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
slide45

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
slide46

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
slide47

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
slide50

Molecular mechanisms for constitutional

chromosomal rearrangements in humans

DiGeorge/velo-cardio-facial syndrome

recurrent reciprocal translocation t(11;22)

cat eye syndrome (CES)

slide51

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

slide52

Molecular mechanisms for constitutional

chromosomal rearrangements in humans

DiGeorge syndrome

velo-cardio-facial syndrome

slide53

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
slide54

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

slide55

Molecular mechanisms for constitutional

chromosomal rearrangements in humans

DiGeorge syndrome

velo-cardio-facial syndrome

slide56

Molecular mechanisms for constitutional

chromosomal rearrangements in humans

DiGeorge syndrome

velo-cardio-facial syndrome

slide57

Molecular mechanisms for constitutional

chromosomal rearrangements in humans

DiGeorge syndrome

velo-cardio-facial syndrome

slide58

Molecular mechanisms for constitutional

chromosomal rearrangements in humans

t(11;22)

cat eye syndrome

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

slide59

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
slide60

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).
slide61

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.
slide62

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
slide63

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
slide64

Molecular mechanisms for constitutional

chromosomal rearrangements in humans

Flat midface, epicanthal folds, long philtrum

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

slide65

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
slide66

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

slide67

Microbrachycephaly, prognathia, protruding tongue, macrostomia, widely spaced teeth,

severe MR, paroxysmal laughter, absent speech, ataxia with jerky arm movements,

seizures, hypopigmentation

slide68

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

slide69

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
slide70

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
slide71

Molecular mechanisms for constitutional

chromosomal rearrangements in humans

slide72

Molecular mechanisms for constitutional

chromosomal rearrangements in humans

Mechanistic models for rearrangements

slide73

Molecular mechanisms for constitutional

chromosomal rearrangements in humans

Mechanistic models for rearrangements

slide74

Molecular mechanisms for constitutional

chromosomal rearrangements in humans

Mechanistic models for rearrangements