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Bridge 2010, Bristol, May 13-14, 2010
Identification of sSMC – case 1.
47,XY, + mar(30%)/46,XY(70%) 47,XY,+der(8)
Clinical symptoms
- behaviour disturbances - mild mental disability (IQ:67) - long philtrum - V. finger clinodactyly - dysgenesis of the corpus callosum
With parental consent
To identify/clarify mosaicism uncertain cytogenetic changes microdeletions sSMC subtelomeric rearrangements
Bridge 2010, Bristol, May 13-14, 2010
• Subtelomeric regions are rich in genes severe dysmorphic symptoms• 50% of cases proved to be inherited (balanced carrier parent)• 5-10% of cases with idiopathic severe mental disability (deletion, duplication, translocation)• Some of them are associated with specific phenotype (1p, 4p, 5p, 9q, 17p, 22q• Probes: Vysis ToTelVysion Probe Panel: mixture of 15 individual „ready-to-use” direct labelled probes specific for telomeric regions of the p and q arms of all chromosomes (excepted the p arms of acrocentric chromosomes)
To identify/clarify mosaicism uncertain cytogenetic changes microdeletions sSMC subtelomeric rearrangements
Detection of subtelomeric rearrangements.
FISH: ToTelVysion Multi-color DNA probe Mixture
Bridge 2010, Bristol, May 13-14, 2010With parental consent.
Clinical symptoms- Hypotonia- Eye: Retinitis pigmentosa, decoloration of papilla, nystagmus, div. strabism- Polydactyly- Facial dysmorphy: depressed premaxillary region, hypertelorism, ptosis, small, low set ears, downward slanting mouth - Severe mental disability- Delayed psychomotoric development
Detection of subtelomeric rearrangement – cases 1.-2.
8p subtelomeric deletion; 12p subtelomericregion trisomy in unbalanced translocation
Bridge 2010, Bristol, May 13-14, 2010
Array Comparative genomic hybridisation (CGH)
Advantages • to identify the exact breakpoints, localization of
affected genes • to detect microdeletions, microduplications,
polymorphisms of pathological importance • to study genotype – phenotype correlations
Bridge 2010, Bristol, May 13-14, 2010
Specific dystrophin gene array analysis
Sample 3: 114/1 affected boy at contiguous gene deletion syndrome (Score
dystrophin gene: -0,536; Score neighbouring genes: -0,742)
Complex glycerol kinase deficiency (CGKD): microdeletion of Xp21
Bridge 2010, Bristol, May 13-14, 2010
Complex glycerol kinase deficiency (CGKD): Xp21 microdeletion
• dystrophin gene deletion by MLPA
Neighbouring genes: • GK gene: Xp21.1 – glycerol kinase deficiency • NR0B1 gene: Xp21.3-21.2 – adrenal hypoplasia • Il1RAP gene: Xp22.1-21.3 – mental disability• RPGR gene:
Accurate breakpoints Genotype-phenotype correlation
Bridge 2010, Bristol, May 13-14, 2010
Genetic diagnostic approaches
Chromosome aberrationis compatible with the clinical features
Further steps delineated by the phenotype
Fine chromosome alterations: markers’ identification, sSMC, microdeletion syndromes, subtelomeric rearrangements
FISH, MFISH, CGHMolecular genetic tetsts(mutation analysis: PCR, sequencing, RFLP etc.)
Syndromes ofunknown origin
Syndromeidentification:„search” >5600
Chromosome analysisChromosome analysis
Numerical, structuralaberrations
Uncertain Normal karyotype
Known monogenic syndromes: Fra-X, chondrodysplasia, syndromic craniosynostoses etc.
Known chromosomal syndromes, syndromes of unknown origin, mental disability, dysmorphic symptoms
3.
Bridge 2010, Bristol, May 13-14, 2010
Molecular diagnosis of monogenic disorders
Diseases studied:• Fragile–X: FMR1 gene – Southern blot • Craniosynostoses: Apert, Crouzon, Pfeiffer
FGFR2 gene – PCR, sequencing, RFLP
• Achondroplasia: FGFR 3 gene - RFLP Hypochondroplasia: FGFR 2, 3 genes –
sequencing • Lissencephaly: LIS1 gene – FISH, sequencing
Bridge 2010, Bristol, May 13-14, 2010
L TM TK1 TK2AIgI IgIIIIgII
Ser252Trp Pro253Arg: 99%
Apert syndrome: AD – FGFR2 mutation
Clinical symptomsCraniofacial dysmorphy: clover shaped cranium, high forehead,flat occiput, hypertelorism, deep nasal root, small, beaked nose, cleft palateSyndactyly, small muscular VSD
With parental consent
Bridge 2010, Bristol, May 13-14, 2010
Patient
Healthy
172153118
54
300250
200150
100
50
E B M
c.C755G ( Ser252Trp) mutation
Genetic study
DNA isolationAmplification of exon 8 in FGFR2 gene using PCR techniqueSequencing, RFLP analysis (MboI)
Bridge 2010, Bristol, May 13-14, 2010
Apert syndrome 2.
FGFR2c.C758G (p.Pro253Arg) Gain of function:
Increased ligand-binding capacityLoss of ligand-binding specificity
Genotype-phenotype correlation: Ser252Trp: cleft palate more common
better recovery after operationPro253Arg: more severe syndactyly
With parental consent
Bridge 2010, Bristol, May 13-14, 2010
achondroplasia Apert sy DMP Rett sy FGFR2 FGFR3 dystrophin MECP2
PKU, CF, CAH, Werdnig-Hoffmann, Smith-Lemli-Opitz, glycosilation disturbances, haemophilia, Russel-Silver, Lysosomal storage diseases (Gaucher, Fabry, MPS I. Niemann-Pick, Pompe) , etc.
ORPHANET
Significance: • to avoid unncessary investigations• prognosis,• praenatal diagnosis • praesymptomatic diagnosis• prevent recurrence
Further genetic tests to detect gene mutationsperformed in collaboration inside Hungary
Bridge 2010, Bristol, May 13-14, 2010
Genetic diagnostic approaches
Chromosome aberrationis compatible with the
clinical picture
Further steps delineated by the phenotype
Fine chromosome alterations: markers’ identification, sSMC, microdeletion syndromes, subtelomeric rearrengements
FISH, MFISH, CGHMolecular genetic tetsts(mutation analysis: PCR, sequencing etc.)
Syndromes ofunknown origin
Syndromeidentification:„search” >5600
Chromosome analysisChromosome analysis
Numerical, structuralaberrations
Uncertain Normal karyotype
Known monogenic syndromes: Fra-X, achondroplasia, Apert sy. etc.
Known chromosomal syndrome, syndromes of unknown origin, mental disability, dysmorphic symptoms
4.
Bridge 2010, Bristol, May 13-14, 2010
• blepharophimosis• ptosis• external acustic meatus stenosis +/- deafness• hypotonia• mental disability
Diagnostic criteria:
Photos are shown with parental consent
Diagnosis: Ohdo syndrome
Bridge 2010, Bristol, May 13-14, 2010
B=0 FA TRACE
Standard MRI vs. Neuroimaging:
T1 and T2 weighed MRI revealed no organic alteration in the brain, while fractional anisotropy and fiber trajectory imaging diclosed a demyelinated area close to the anterior horn of the ventricle causing a complete „communication” blockade
Magnetic resonance Imaging
Bridge 2010, Bristol, May 13-14, 2010
CONCLUSION
• To identify the genetic background of congenital genetic disorders the whole spectrum of currently available genetic tests have to be applied according to a proper algorythm
• The genetic diagnosis (etiology) determines the syndrome-specific additional symptoms as well as pattern and severity of intellectual disability.
• The genetic diagnosis has to be considered when developing the early intervention, rehabilitation and preventive measurements.
Bridge 2010, Bristol, May 13-14, 2010
Director of working group: Éva Oláh, MD, D.Sc.
Members:
Cytogenetics: Erzsébet Balogh, biol. PhD.
FISH, PCR: Anikó Ujfalusi, MD. PhD Beáta Bessenyei, mol. biol.
Syndrome diagnosis: Katalin Szakszon, MD. Gabriella P. Szabo, MD.
Technical assistants: Mrs. Gábor Orvos
Mrs. Ferenc BodnárMs. Éva Nagy
PhD students: Attila Mohánszky, mol. biol.
Gabriella P. Szabó, MD. Katalin Szakszon, MD. Ivett Körhegyi, MD.
: Student Scientific Circle: students
Clinical Genetic Center, University of Debrecen