Download Diagnostic schedule of rare genetic disorders in the

Debrecen
DIAGNOSTIC SCHEDULE OF RARE
GENETIC DISORDERS IN THE UNIVERSITY
OF DEBRECEN, HUNGARY
Éva Oláh
CLINICAL GENETIC CENTER,
INSTITUTE OF PEDIATRICS,
MEDICAL AND HEALTH SCIENCE CENTER,
UNIVERSITY OF DEBRECEN,
HUNGARY
Bridge 2010, Bristol, May 13-14, 2010
Protestant College
1538
Main building of
University of Debrecen
Bridge 2010, Bristol, May 13-14, 2010
CLINICAL GENETIC CENTER
Diagnostic work focuses on two main fields:
– Rare genetic disorders
• Congenital malformations
• Mental disability of unknown origin
• Infertility
• Disturbance of sexual differentiation
• Short stature
– Acquired genetic changes of malignant diseases:
acute and chronic leukemias, MDS, HL, NHL,
solid tumors
Bridge 2010, Bristol, May 13-14, 2010
Why „rare” genetic disorders ?
• Incidence: <1:2000
• Significance:
– Although each of them is rare, altogether they
represent a high proportion of morbidity and mortality
– They cause about ¼ of infant mortality and
1/3 of hospitalization
– Nearly all of them are associated with mental
disability and/or behaviour disturbances.
– The majority of them are not treatable
– Modern molecular genetic methods offer good
possibilities to identify exact genetic diagnosis
Exact genetic diagnosis is of paramount importance
for early development and prevention
Bridge 2010, Bristol, May 13-14, 2010
Prevalence at birth of various etiological subgroups
of congenital genetic disorders
• Estimated prevalence at birth: 78‰
• Distribution by etiology:
Chromosome abnomalities: 6 ‰
Mendelian inheritance: 16.8‰
Non-mendelian inherited diseases
(uniparental disomy, genomic
imprinting)
Mitochondrial inherited diseases
Multifactorial disorders
Genetic syndromes with unknown etiology
56.2 ‰
Ensenauer RE, Reinke SS, Ackerman MJ, Tester DJ, Whiteman DA, Tefferi A: Primer on medical genomics. Part VIII: Essentials
of medical genetics for the practicing physician. Mayo Clin Proc 2003, 78: 846-857.
Primary Health Care Approaches for Prevention and Control of Congenital and Genetic Disorders – A Report on WHO meeting,
1999.
Bridge 2010, Bristol, May 13-14, 2010
Genetic methods applied in diagnostic
procedure
– Traditional cytogenetics: G-banding
– Fluorescence In Situ Hybridization: FISH, mFISH
– Molecular genetic methods: Southern blot, PCR,
RT-PCR, RFLP, sequencing
– Array CGH
– Syndrome „searching” / identification
– Complementary examinations: imaging
techniques, biochemical, metabolic tests
Bridge 2010, Bristol, May 13-14, 2010
Genetic diagnostic schedule
Known chromosomal syndromes, syndromes of unknown origin,
mental disability, dysmorphic symptoms
Chromosome analysis
1.
Numerical, structural
Uncertain
Normal karyotype
aberrations
Further steps delineated by the phenotype
Chromosome aberration
in accordance with the Fine chormosome alterations: Syndromes of
given clinical features
markers’ identification, sSMC, unknown origin
microdeletion syndromes,
subtelomeric rearrangements
Known monogenic
syndromes: Fra-X,
chondrodysplasia,
craniosynostoses
etc.
Syndrome search
FISH, MFISH
Subtelomeric FISH
Molecular genetic tests
Bridge 2010, Bristol, May 13-14, 2010
Cytogenetic studies (2004-2009)
normal karyotype: 813 (83.47%)
Number of chromosome
analyses: 974
autosomal: 126 (76.26%)
chromosome
gonosomal: 31 (19.25%)
aberrations: 161 (16.53%) both:
4 (2.49%)
250
200
200
150
150
ÖSSZES
Total No
KÓROS
Abnormal
100
100
50
50
0
2003 2004
2005 2006
2004
2005 2006
2007 2007
2008 2008
2009
Down syndrome: 68/126 (42.24%)
With parental consent
Bridge 2010, Bristol, May 13-14, 2010
Genetic diagnostic schedule
Known chromosomal syndrome, syndromes of unknown origin,
mental disability, dysmorphic symptoms
Chromosome analysis
Numerical, structural
aberrations
Uncertain
Normal karyotype
Further steps delineated by the phenotype
2.
Chromosome aberration Fine chromosome alterations: Syndromes of
is compatible with the
markers’ identification, sSMC, unknown origin
microdeletion syndromes,
clinical picture
subtelomeric rearrangements
Syndrome search
Known monogenic
syndromes: Fra-X,
FISH, MFISH, CGH
chondrodysplasia,
craniosynostoses
Subtelomeric FISH
etc.
Molecular genetic tests
Bridge 2010, Bristol, May 13-14, 2010
Molecular cytogenetics: Fluorescens in situ
hybridisation (FISH)
Detection of presence or absence of specific DNA sequences using
various fluorescence-labelled probes on interphase or metaphase cells
Advantages:
•
•
•
•
No dividing cells (prior to cell culture) are needed
Better resolution (120 Kb-3Mb) (subtelomeric rearrangements, microdeletions, sSMC)
Great number of cells can be studied (Mosaicism, ratio of abnormal cells)
Rapid, sensitive
Disadvantage: Number of probes applied simultaneously is limited
Bridge 2010, Bristol, May 13-14, 2010
FISH probes
Centromere-specific:
(alpha-satellite)
numerical aberrations
Arm-specific
probes: inversion,
isochromosome
Locus-specific:
gene deletion,
microdeletion
mBand (550 bands)
(deletion,
duplication)
Whole chromosome
painting probes:
rearrangements
between
chromosomes
multicolor-FISH:
(spectral
karyotyping:
SKY)
Bridge 2010, Bristol, May 13-14, 2010
Multicolor-FISH (M-FISH)
Simultaneous visualization of all chromosomes labelling each
pair by different colours using 5 fluorochromes with different
spectrum in various combination (DAPI, FITC, Cy5, Texas red,
Spectrum Orange - Metasystems, 24 Xcyte kit )
INDICATIONS
• Interchromosomal rearrengements, translocations
• Marker chromosomes, insertions
• Hidden aberrations
• Complex rearrangements
SHORTCOMINGS
• Resolution is limited (<5Mb)
• Intrachromosomal rearrengements,
small deletions, duplications, exact
chromosomal breakpoints cannot be
identified
Bridge 2010, Bristol, May 13-14, 2010
To identify/clarify
uncertain cytogenetic changes
microdeletions
sSMC
subtelomeric rearrangements
Identification of uncertain
cytogenetic alterations – Case 1.
Down syndrome patient
FISH
21q22
Down-region
specific probe
21;21 tandem
translocation?
Bridge 2010, Bristol, May 13-14, 2010
To identify/clarify
uncertain cytogenetic changes
microdeletions
sSMC
subtelomeric rearrangements
With parental consent
Identification of uncertain
cytogenetic alterations – Case 2.
• Facial dysmorphy, low set ears
hypertelorism
• clinodactily, club feet
• hallux valgus, micropenis, hypospadiasis
• inquinal hernia l.d.
46,XY,der(22),t(7;22)
46,XY,der(22)
Bridge 2010, Bristol, May 13-14, 2010
To identify/clarify
uncertain cytogenetic changes
microdeletions
sSMC
subtelomeric rearrangements
Microdeletion syndromes
Deletions and duplications of chromosomal regions
smaller than 300 Kb involve several genes which are
physically close to but functionally independent from
each other
Locus-specific probes
Bridge 2010, Bristol, May 13-14, 2010
To identify/clarify
uncertain cytogenetic changes
microdeletions
sSMC
subtelomeric rearrangements
Prader-Willi syndrome
Chr 15
- prevalence: 1:10 000, 1:20 000
- 15q11.2 region: paternal allele deletion
- Responsible gene/enzyme: SNRPN
Clinical symptoms:
- congenital hypotonia
- severe feeding difficulties in the
neonatal period,
- marked developmental delay,
mental disability
- hyperphagia and consequent
obesity from early childhood on
- short stature
- hypogonadism
With parental consent
Bridge 2010, Bristol, May 13-14, 2010
To identify/clarify
uncertain cytogenetic changes
microdeletions
sSMC
subtelomeric rearrangements
Angelman syndrome
Chr 15
- prevalence: 1:10 000, 1:20 000
- 15q11.2 region, maternal allele deletion
- gene/enzyme: UBE3A
Clinical symptoms:
- severe cognitive problems
- microcephalia
- seizures
- sleep disturbances
- prognathia
- widely spaced teeth
- „happy puppet” laughing spells
With parental consent
Bridge 2010, Bristol, May 13-14, 2010
To identify/clarify
uncertain cytogenetic changes
microdeletions
sSMC
subtelomeric rearrangements
Chr 22
Di George /
Velocardiofacial syndrome
- Most common microdeletion syndrome
- prevalence: 1:5000
- 22q11.21 region: 30 genes deletion
Clinical symptoms:
- thymus aplasia
- cleft palate
- hypocalcaemia
- large vessel anomalies (aorta, truncus,
Fallot tetralogia, VSD)
- facial dysmorphy
- swallow and speech difficulties
- moderate mental disability
- hypotonia
- in adults: psychiatric diseases
Bridge 2010, Bristol, May 13-14, 2010
To identify/clarify
uncertain cytogenetic changes
microdeletions
sSMC
subtelomeric rearrangements
Williams syndrome - Elfin face
Chr 7
- prevalence: 1:20 000
- 7q11.23 region deletion
- protein: elastin (ELN), LIM
kinase (LIMK1)
Clinical symptoms
- somatic retadation
- mental disability
- hypercalcaemia
- wide, open mouth, full everted
lower lip, periorbital edema
- hoarse voice, loose skin,
- hyperacusis
- friendly, ongoing behaviour
- supravalvular aortic stenosis
- arteriopathies
With parental consent
Bridge 2010, Bristol, May 13-14, 2010