Bridge 2010, Bristol, May 13-14, 2010 DIAGNOSTIC SCHEDULE OF RARE GENETIC DISORDERS IN THE UNIVERSITY OF DEBRECEN, HUNGARY Éva Oláh Debrecen CLINICAL GENETIC

Bridge 2010, Bristol, May 13-14, 2010

DIAGNOSTIC SCHEDULE OF RARE GENETIC DISORDERS IN THE UNIVERSITY

OF DEBRECEN, HUNGARY

Éva Oláh

Debrecen

CLINICAL GENETIC CENTER, INSTITUTE OF PEDIATRICS,

MEDICAL AND HEALTH SCIENCE CENTER, UNIVERSITY OF DEBRECEN,

HUNGARY


Protestant College 1538

Main building ofUniversity of Debrecen


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

CLINICAL GENETIC CENTER


• 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

Why „rare” genetic disorders ?

Exact genetic diagnosis is of paramount importancefor early development and prevention


• 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.

Prevalence at birth of various etiological subgroups of congenital genetic disorders


– 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

Genetic methods applied in diagnostic procedure


Genetic diagnostic schedule

Chromosome aberrationin accordance with the

given clinical features

Further steps delineated by the phenotype

Fine chormosome alterations: markers’ identification, sSMC, microdeletion syndromes, subtelomeric rearrangements

FISH, MFISHSubtelomeric FISH Molecular genetic tests

Syndromes ofunknown origin

Syndrome search

Chromosome analysisChromosome analysis

Numerical, structuralaberrations

Uncertain Normal karyotype

Known monogenic syndromes: Fra-X, chondrodysplasia, craniosynostoses etc.

Known chromosomal syndromes, syndromes of unknown origin, mental disability, dysmorphic symptoms

1.


normal karyotype: 813 (83.47%)Number of chromosomeanalyses: 974 autosomal: 126 (76.26%)

chromosome gonosomal: 31 (19.25%) aberrations: 161 (16.53%) both: 4

(2.49%)

Cytogenetic studies (2004-2009)

Total NoAbnormal

50

100

150

200

2004 2005 2006 2007 2008 2009

Down syndrome: 68/126 (42.24%)

With parental consent


Genetic diagnostic schedule

Chromosome aberrationis compatible with the

clinical picture

Further steps delineated by the phenotype

Fine chromosome alterations: markers’ identification, sSMC, microdeletion syndromes, subtelomeric rearrangements

FISH, MFISH, CGHSubtelomeric FISHMolecular genetic tests

Syndromes ofunknown origin

Syndrome search

Chromosome analysisChromosome analysis

Numerical, structuralaberrations

Uncertain Normal karyotype

Known monogenic syndromes: Fra-X, chondrodysplasia, craniosynostoses etc.

Known chromosomal syndrome, syndromes of unknown origin, mental disability, dysmorphic symptoms

2.


Detection of presence or absence of specific DNA sequences usingvarious 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, sensitiveDisadvantage: Number of probes applied simultaneously is limited

Molecular cytogenetics: Fluorescens in situ hybridisation (FISH)


Arm-specific probes: inversion, isochromosome

FISH probes

Centromere-specific:(alpha-satellite)numerical aberrations

Locus-specific:gene deletion, microdeletion

Whole chromosomepainting probes:rearrangements between chromosomes

mBand (550 bands)(deletion,duplication)

multicolor-FISH: (spectral karyotyping:SKY)


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

Multicolor-FISH (M-FISH)


FISH

21q22

To identify/clarify uncertain cytogenetic changes microdeletions sSMC subtelomeric rearrangements

Identification of uncertain cytogenetic alterations – Case 1.

Down syndrome patient

21;21 tandem translocation?

Down-regionspecific probe



46,XY,der(22)

46,XY,der(22),t(7;22)

• Facial dysmorphy, low set ears hypertelorism• clinodactily, club feet• hallux valgus, micropenis, hypospadiasis• inquinal hernia l.d.

Identification of uncertain cytogenetic alterations – Case 2.



Locus-specific probes

To identify/clarify uncertain cytogenetic changes microdeletions sSMC subtelomeric rearrangements Deletions and duplications of chromosomal regions

smaller than 300 Kb involve several genes which are physically close to but functionally independent from each other

Microdeletion syndromes


Prader-Willi syndromePrader-Willi syndrome

- 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

Chr 15




Angelman syndromeAngelman syndrome

- 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

Chr 15




Di George / Di George / Velocardiofacial syndromeVelocardiofacial 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

Chr 22



Williams syndrome - Elfin faceWilliams syndrome - Elfin face

- 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

Chr 7



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Bridge 2010, Bristol, May 13-14, 2010 DIAGNOSTIC SCHEDULE OF RARE GENETIC DISORDERS IN THE UNIVERSITY OF DEBRECEN, HUNGARY Éva Oláh Debrecen CLINICAL GENETIC