Cytogenetic and molecular cytogenetic analysis in clinical genetics 5th year Clinical genetics

Eduard Kočárek eduard.kocarek@lfmotol.cuni.cz Phone (office): 224 435 980 Phone (Plzenska): 257296151 Secretary of the Institute of biology and medical genetics phone 224 433 500 – 502

Basic cytogenetic examinations Interphase cells Barr body (sex chromatin) Metaphase cells – staining of chromosomes Solid staining G-banding R-banding C-banding Q-banding Ag-NOR

Molecular cytogenetic examinations Identification of chromosomal abnormalities by means of molecular biological methods Interphase cells could be used for analysis (with exception of whole chromosome painting probes and M-FISH) Examples of methods: in situ hybridization and its modifications (CGH, M-FISH, fiber FISH etc.) Gene chips, resp. array CGH etc. PRINS, PCR in situ quantitative fluorescent PCR, real time PCR methods based on amplification of probe attached to target sequence (MLPA, MAPH) hybridization PCR

FISH (fluorescent in situ hybridization) Locus specific probes Centromeric probe target DNA denaturation hybridization probe M-FISH Whole chromosome painting probes

Quantitative fluorescent PCR QF-PCR Primer 1 In case of informative polymorphism each peak represents one locus on one chromosome. denaturation, annealing Primer 2 PCR Capillary electrophoresis

QF-PCR – normal finding

QF-PCR – identification of trisomy

MLPA Multilocus ligase-dependent probe amplification P „stuffer“ sequence Hybridization sequence Target DNA P

MLPA

MLPA – computer evaluation

MLPA result – deletions in the dystrophine gene

DNA attached to the membrane MAPH Multilocus amplifiable probe hybridization DNA attached to the membrane Mixture of probes DNA sample hybridization Probe 1 Probe 2 primers primers Capillary electrophoresis amplification of attached probes

Indications of cytogenetic and molecular cytogenetic examinations

Basic cytogenetic chromosomal analysis Patient Basic cytogenetic chromosomal analysis Molecular cytogenetic analysis (mostly FISH) Molecular biological analysis

Indications for postnatal chromosomal analysis Suspicion to concrete chromosomal abnormality (concrete syndrome) Multiple congenital anomalies or developmental delay Mental retardation Gonadal dysgenesis Infertility Miscarriages Delivery of dead fetus or death of a newborn child Occurrence of certain malignancies

What to do before indication of postnatal chromosomal analysis? Exclude possibility of non-genetic affection Influence of teratogens or prenatal infections Complications during the birth (asphyxia, injuries of the newborn child) Postnatal non genetic influence (injuries, infections) Exclude monogenic or multifactorial disorder (= without abnormal chromosomal finding)

Tissue samples for postnatal chromosomal analysis Peripheral blood Fibroblasts from skin biopsy Epithelial cells from buccal smear (only in rare cases for Barr body identification or FISH) Bone marrow (hemoblastosis) Solid tumor Autopsy material (in case of the death of a patient)

How to take a blood sample for chromosomal analysis? Disinfect the skin with alcohol (96% ethanol) Use tube with heparin or lithium-heparin to prevent blood clotting. (The EDTA tube is used only for the DNA isolation.)

In which conditions we have to indicate FISH analysis? The material doesn't contain metaphase chromosomes Unsuccessful cultivation It isn't possible to cultivate the tissue from patient (preimplantation analysis, rapid prenatal examinations, examinations of solid tumors or autopsy material) Analysis of complicated chromosomal rearrangements Identification of marker chromosomes Analysis of low-frequency mosaic Diagnosis of submicroscopic (cryptic) chromosomal rearrangements Microdeletion syndromes Amplification of oncogenes and microdeletion of tumor-suppressor genes in malignancies

Indication Result INDICATION Various samples RESULT Metaphase cells Using molecular cytogenetic analysis the aberration was discovered in other family members in vitro cultivation is possible In vitro cultivation is impossible Metaphase cells (cultivated lymphocytes, amniocytes etc.) Interphase cells (e.g. tumor cells, tissues taken from autopsy material or histological sections) Various samples Chromosomal analysis Negative finding, detailed examination is necessary (e.g. microdeletions) Chromosomal abnormality remains unclear (e.g. marker chromosomes, rearrangements including 3 and more chromosomes, mosaics) Chromosomal aberration is found and described (e.g. aneuploidies, simple structural rearrangements) Molecular cytogenetic analysis RESULT Result

Basic cytogenetic chromosomal analysis Patient Basic cytogenetic chromosomal analysis Molecular cytogenetic analysis (mostly FISH) Molecular biological analysis

In which cases we have to indicate detailed molecular biological analysis? Neither chromosomal nor molecular cytogenetic analysis led to relevant conclusion Suspicion to monogenic disorder (e.g. fragile X syndrome) Some cases of microdeletion syndromes could be associated with very short deletions or spot mutations that can't be identified by means of common molecular cytogenetic methods. Identification of uniparental disomy, or other imprinting defects (especially in Prader-Willi/Angelman syndromes).

Patient 1

See the photo of the patient and note abnormal phenotypic features. 2-years old boy with mental retardation Inborn cardiac defect – supravalvular aortic stenosis. See the photo of the patient and note abnormal phenotypic features.

Patient 1 (boy, 2 years) irides stellatae hypertelorism low set ears abnormal teeth open mouth, thick lip „elfin face“

Patient 1 Phenotypic features and inborn defects are typical for Williams-Beuren syndrome This syndrome is caused by microdeletion of the long arm of the chromosome 7 (sub-band 7q11.23). In 95% of patients this microdeletion could be examined by the FISH method. Before the molecular cytogenetic analysis basic cytogenetic examination is recommended. Which type of probe you would use for FISH analysis of microdeletion of the chromosome 7?

Microdeletion should be confirmed by the FISH analysis Patient 1 - karyotype Normal finding: 46,XY Microdeletion should be confirmed by the FISH analysis

MOLECULAR CYTOGENETIC ANALYSIS OF 7q11.23 MICRODELETION ELN LIMK1 D7S613 CENTROMERE TELOMERE 180 kb LOCUS SPECIFIC PROBE FOR THE CRITICAL REGION ELN/LIMK/D7S613 (labeled with the Spectrum Orange, red signal) CONTROL PROBE D7S522 (labeled with the Spectrum Green, green signal)

Patient 1 – conclusion of the molecular cytogenetic examination Microdeletion of 7q11.23 chromosome confirmed. Diagnosis: Williams-Beuren syndrome

Prognosis of patients with the Williams-Beuren syndrome Neonatal hypercalcemia Mild to moderate mental retardation Supravalvular aortic stenosis could lead to a heart attack already in childhood (sudden death of the child).

Thank you for your attention and good bye!