Learning Objectives 1) Identify main goals of chromosome analysis!2) Basics of ISCN nomenclature for chromosome bands and abnormalities!3) Primary clinical indications for cytogenetic testing!4) Primary clinical findings assoc. with the common constitutional (individ. born with) chromosomal abnormalities!5) How nondisjuction and translocation can lead to trisomy 21!6) Definitions of balanced vs. unbalanced karyotype, mosaicism!!1) Chromosome analysis!- Conventional chromosome analysis= G-banded!- Only during mitosis are chromosomes visible under a light microscope--must have a dividing cell for conventional cytogenetic study!- Chromosomes are harvested during mitosis, spread onto glass slides and treated with protein and a stain (ex. Giemsa) to reveal "G"-band pattern!- Each chromosome has 2 sister chromatids, they + centromere= difficult to see, but bands are much easier to see!- Each chromosome has a unique G-banding pattern that is constant between individuals (ex. chr. 2 from one person bands like chrom. 2 from another)!- karyogram: when all the chroms are stained and lined up!- FISH: Fluorescence in situ hybridization!- Genomic microarrays, DNA based!!2) ISCN Nomenclature - Provides the published guidelines and standards for the designation of chromossomes, individual bands on chromosomes, and chromosome abnormalities!-p= short arm, q= long arm!- Each band is numbered:!- Centromeres p10 or q10!- Bands are numbered independently on the p and q arms!- Band #s increase as they move away from the centromere!- Each band usually encompasses >4 Mb of DNA!- Limits the resolution of G banding studies!- Most patients with abnormalities involving at least one G band have multiple other clinical issues!- EX. ISCN: 47,XY,+21 (47= number of chroms in the karyotype, the sex chrom. complement, then+ gain of whole chromosome 21)!!3) Primary clinical indications for cytogenetic testing!- Presence of multiple, congenital abnormalities!- May be detected prenatally in a fetus via ultrasound or post-natally after birth!- Baby born with clinical features of Down Syndrome!- Peripheral blood sample, takes about 48-72 hours to get a result!- Tissue most commonly studied in a newborn suspected of having a chrom. abnormality= peripheral blood!-1-3cc obtained by venipuncture and collected into a sodium heparin tube (won't clot blood)!- Peripheral blood T-lymphocytes need to be stimulated to divide in vitro (add mitogen to kick it into cycle). This is why, for G banded analysis it takes 48-72 hours for the physician to have a result!!4) Primary clinical findings assoc. with the common constitutional (individ. born with) chromosomal abnormalities - If you find a Robertsonian translocation in a baby with Down syndrome, it is clinically indicated toperform a chromosome study on the parents to see if one of them carries a balanced translocation!- More autosomal trisomies are lethal!- Autosome trisomies that can lead to a live baby (w/o mosaicism) are Trisomy 13, 18, and 21!- Mosaicism for other chroms (eg. 8, 9, 16) are viable if in assoc. with a normal cell line!- Trisomy 13!- Holoprosencephaly with incomplete development of the forebrain, olfactory, and optic nerves, severe cognitive defects!- Cleft lip/palata!- Polydactyly of hands/feet!- Cardiac anomalies!- Genitalia abnormalities!- Median survival= 2.5 days 5) How nondisjuction and translocation can lead to trisomy 21 - Trisomy: having 3 copies of a specific chrom.!- 95% due to meiosis nondisjuc. erros!- 4% due to Robertsonian translocation. Most common translocation we see in humans.!- Involves the joining of 2 acrocentric (13, 14, 15, 21, 22) chromosomes at their centromeres!- The p arms of these are composed on repetitive DNA sequences. Loss/gain of p arm of these is fine.!- Problem with RT, you end up with three copies of ex. chromosome 21--2 normal and 1 chromosome hooked up to the #14.!-1% due to mosaicism!- Most of these errors occur during maternal meiosis 1, risk increases with age. 35= advanced maternal age (1/350) from 1/1600 before! 6) Definitions of balanced vs. unbalanced karyotype, mosaicism - mosaicism: having two or more cell lines, each with a diff. karyotype. Can be any mix of abnormal/normal.!- Nondisjuction during meiosis can generate this (most common), also nondis. during mitosis!- Ex. 47, XY, +21[20]/46, XY[30]!- In gen. phenotype is less severe than pure trisomy, but can't tell what the different distribution of cell lines is in diff. tissues!- Balanced translocation: no net gain or loss of critical genetic material (p arms don't matter of the acrocentric chroms)!- Unbalanced translocation: the net gain or loss of critical genetic material. Ex. the baby has an extra
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