Chapter 5 - Principles of Clinical Cytogenesis and Genome Analysis

clinical cytogenesis

study of chromosomes, their structure, and their inheritance, as applied to the practice of medicine

• not focused on single mutations, bigger picture stuff

cytogenetic abnormalities

the results of chromosome disorders, developmental issues

• spontaneous pregnancy loss, congenital malformations, intellectual diability, cancer

clinical indications of chromosome and genome analysis

• failure to thrive

• stillbirth/neonatal death

• fertility issues

• structural characterization of genomic imbalances and fmily follow-up studies

• neoplasia

• pregnancy

Giemsa banding (G-banding)

• not often used

• gold standard fro consititutional and aquired diagnosis

• precise and unambiguous - can be used on indiv chromosomes

• 400-500 band resolution

high-resolution banding (prometaphase banding)

• less condensed = more detail

• detects rearrangements in 2-3Mb size range

metacentric chromosome

central centromere and arms of approx. equal length

• present in normal human karyotype

submetacentric chromosome

off-center centromere and arms of clearly different lengths

• present in normal human karyotype

acrocentric chromosome

centromere near one end

• present in normal human karyotype

telocentric chromosome

centromere at one end and only one arm

• only present with chromosome rearrangement

chromosome numbering system

• chromosome number = 1-22, X, Y

• short arm (p) or long arm (q)

• region, band, and subband

• ex - 5p51.2

Florescence In Situ Hybrididation (FISH)

• a method for detecting the presence or absence of a particular DNA sequence

• done under microscope - can see indiv. chromosomes

• “have to know what you are fishing for”

locus-specific probes

single copy DNA probes specific for sequences within given bonds

• in metaphase binds to each sister chromatid

Satellite DNA probes

repetitive alpha-satellite DNA probes specific for the centromeres of certain chromosomes used to count the number

• finds centromeres

Chromosome microarrays (CMA)

frontline diagnostic test for most clinical applications

• queries whole genome on a glass slide containing regularly spaced DNA probes that represent loci across the entire genome

• replaced g-banding

• show duplications and deletions

CMA advantages

don’t need to know what you are looking for, high through put, very sensitive, quick

CMA disadvantages

can’t tell is DNA translocated or rearranged or inverted - only deletions and duplications, can display variants in small differences that may not be significant (variants of unknown significance VUS)

WGS advantages

whole genome involves, efficient and becoming less expensive, don’t need to sequence whole genome to understand chroosome, identify specific genes linked to cancers and birth defects

WGS disadvantages

still more expensive than CMA or WES, limited ability to resolve complex structural variation, repetitive regions and genes with homologs in other regions

Whole Exome Sequencing

use of high-though put methods to sequence all the exons of protein-coding genes (the exome)

• comprises ~1.5% of the genome

• accurate detection of exonic sequence-level variants

• less accurate than WGS because less consistant and large unsequences regions between exons

gene dosage

the number of copies of a particular gene in the genome

• balance and imbalance is central to chomosomes and genomic disorders

heteroploidy

any chromosome number other than the normal 46

• triploidy, tetraploidy or aneuploidy

aneuploidy

an abnormal chromosome number due to an extra or missing chromosome

• most common chromosome disorder

• physical and/or neurodevelopmental abnormalities

polyploidy

an exact multiple of the haploid chromosome number (n)

• ex - 3n, 4n - you will die

• 2n is normal

triploidy

typically result from dspermy (1 egg + 2 sperm) or nondisjunction in diploid egg or sperm

• phenotype depends on source of extra chromosome set

• extra paternal set - molar pregnancy, terminates around 4th month

• extra maternal set - early pregnancy spontaneous abortion, terminates very early

tetraploidy

4n, always 92,XXXX or 92,XXYY

• results from failure of completion of an early cleavage division of the zygote

Autosomal aneuploidy

typically 13, 18, or 21

• lethal if trisomy of larger chromosomes

sex-linked aneuploidy

• monosomy x = turner syndrome

• trisomy x = triple x syndrome

• extra x = klinefelter (XXY)

• extra y = XYY syndrome

results of NDJ in meiosis 1

2 gametes with double the number of chromosomes and 2 gametes with no chromosomes

results of NDJ in meiosis 2

2 gametes with normal chromosome, 1 with double, 1 with none

Chromosomal deletions

partial monosomy - unbalanced

• haploinsufficiency - 1 copy cannot carry out the functions properly

• depends on location and size of deletion for clinical consequences

• spontaneously occur ot unequal crossing over

chromosomal duplications

partial trisomy - unbalanced

• spontaneous or unequal crossing over

• generally less harmful than deletion

marker/ring chromosomes

very small chromosome seen in addition to mornal chromosome

• no specific definition just extra uncertain DNA

• abnormality depends on the origin of the marker

• if marker lacks telomeres - it will form a ring

isochromosomes

unbalanced, 1 entire are missing, the other arm duplicated

• formation is not understood

dicentric chromosomes

2 chromosome pieces with centromeres fuse end-to-end

reciprocal translocation

exchange of chromosomal segments between two nonhomologous chromosomes

• typically without any medical significance in carriers but in offspring often unviable

• balanced

robertsonian translocation

2 acrocentric chromosomes fuse near centromere and 2 short arms are lost

• carriers have 45 chromosomes but normal phenotype

• higher risk of unbalanced offspring

insertion translocations

nonreciprocal, rare, requires 3 chromosome breaks

inversion

balanced, typically no medical significance but offspring often unviable

Unbalanced chromosomal rearrangements

abnormal phenotype leads to disruption to normal gene dosage (rarer)

• deletion, duplication, marker + ring, isochromosomes, dicentric chromosomes

balanced chromosomal rearrangements

more common, no pheonotypic abnormalities, all genomic material present just rearranged

• translocations and inversions

Mosaicism

some cells are normal, some cells are polyploid or aneuploid

• 2+ different chromosome complements are present among the cells in an individual

• difficult to assess significance because tissues analyzed may not reflect other tissues