Week 4 - Chromosome Microarray Analysis

Question 1

DNA Copy Number Variants and Human Genetic Disease

Answer 1

Deletions and duplications are an important cause of human genetic diseases
Loss or gain of:
- whole chromosome (aneuploidy)
- several adjacent genes in a contiguous gene syndrome (microdeletion/microduplication syndrome)
- single gene
- exons (part of a gene)

Question 2

Molecular Mechanisms by which the Phenotype for Genetic Disorders can Arise

Answer 2

1. Gene dosage - altering the copy number of a dosage sensitive gene
   - Haploinsufficiency
   - Triplosensitivity
2. Disruption of coding sequences e.g. exon deletion
3. Gene fusion event at the breakpoint generating a gain of function variant
4. Perturbing long-range gene regulation i.e position effect
5. Deletion unmasking a recessive variant on the other allele

Question 3

Methods of Detecting CNVs

Answer 3

Ordered from what it can measure from biggest to smallest

1. Karyotype
2. FISH (metaphase & interphase)
3. QF-PCR (Quantitative Fluorescent PCR)
4. DNA microarray/ Array CGH
5. MLPA
6. NGS

Question 4

Genomic DNA Microarrays

Answer 4

Whole genome analysis
Microarray -> Molecular Karyotype
1000x greater resolution
Higher diagnostic yield - 15-20%

Question 5

Types of Chromosome Microarrays

Answer 5

Array CGH
- genomic gains and losses
SNP array
- genomic gains and losses
- copy neutral aberrations
      - regions of homozygosity (ROH)
      - regions of loss of heterozygosity (LOH)

Question 6

Array-CGH vs SNP-Arrays

Answer 6

Array-CGH
- patient DNA compared to control DNA
- fragment DNA, fluorescently labelled, then competitively hybridised on to the array and read with a fluorescence scanner
SNP-Arrays
- do not use a control DNA
- fragmented patient DNA is hybridised to the array
- SNP allele frequency and absolute fluorescence levels are compared to a standard consisting of averaged results for multiple normal samples

Question 7

Detection of CNVs - LogR Ratio and BAF

Answer 7

Log R Ratio (LRR)
- measure of the total signal intensity for the SNP
- any deviations from zero for LRR is evidence for copy number change
- LRR shifts to left (LRR decreases) then there is a deletion, shifts right (LRR increases) means a duplication
- if LRR decreases, BAF will show only two tracks (one at 0 and one at 1), if LRR increases, BAF will show four tracks (one at 0, 0.33, 0.67, 1)
B Allele Frequency (BAF)
- measure of the allelic intensity ratio of two alleles
- deviation in expected BAF pattern indicates copy number change or uniparental disomy
- if there is a region of homozygosity (ROH), LRR is 0 and there is two tracks on the BAF (one at 0 and one at 1 due to their being no heterozygosity (no AB only A and B))

Question 8

CNV Classifications

Answer 8

Pathogenic
- variant contributes to the development of disease
Likely Pathogenic
- high likelihood that this variant is disease-causing
Uncertain
- not enough information at this time to support a more definitive classification of this variant
Likely Benign
- not expected to have a major effect on disease; however, the scientific evidence is currently insufficient to prove this conclusively
Benign
- variant does not cause disease

Question 9

Region of Homozygosity

Answer 9

This term is specific to copy number neutral homozygosity
Does not apply to hemizygous deletions
- LCSH: long contiguous stretch of homozygosity: this term is interchangeable with ROH
- LOH: loss of heterozygosity: describes an event where heterozygosity (once present) is now absent (copy number neutral or deletion)
- AOH: absence of heterozygosity: describes an observation where no heterozygosity is present (copy number neutral or deletion)

Question 10

ROH Diagnostic Implications - Identity by Descent

Answer 10

In isolated population there will be more ROH becaue of the close ancestry
Clinically, ROH of any size can aid in diagnosis of autosomal recessive disease through homozygosity mapping and selection of a candidate gene for sequence analysis
In consanguineous families, the risk for autosomal recessive disease is directly proportional to the degree of parental relationship
ROH looks like:
- LRR 0
- no middle track for BAF

Question 11

ROH Diagnostic Implications - Uniparental Disomy (UPD)

Answer 11

Single large ROH (or a couple of large ROH) on same chromosome
Inheritance of two homologous chromosomes from one parent
Two types:
1. Heterodisomy
- only inherit one pair of parents chromosome (for a specific chromosome)
2. Isodisomy
- only inherit one half of parents chromosomes twice

Question 12

Mosaicism Detected by Microarray

Answer 12

The presence of two or more cell lineages with different genotypes arising from a single zygote in a single individual
- somatic
- germline
In somatic mosaicism the genome is altered at meiosis or mitosis to give cells with two or more genotypes
Tissues affected depends on the amount and proportion of the cells affected

Question 13

Detection of Mosaicism by CMA

Answer 13

Mosaicism > 5-10% can be detected by SNP microarrays
Identified by changes in the B allele frequency (BAF) profile
Scattered genotype with wider BAF tracks caused by introduction of another cell line with the same haplotype
Extra BAF tracks representing the introduction of another cell line with a NEW haplotype
LogR ratio (dosage)
- may shift if high level mosaic deletion or duplication