Final Exam for Advanced Medical Genetics and Genomics

Steps for clinical molecular genetic testing

1. DNA/RNA isolation

2. positive or negative selection by primer/probe, restriction enzyme digestion, bisulfite treatment

3. amplification by PCR

4. separation by size

5. detection

6. sequencing

DNA/RNA isolation

1. cell lysis or protein removal

2. selective nucleic acid extraction

3. purification by ethanol precipitation or solid-state binding/elution

4. resuspension of nucleic acids in dilute salt buffer

What anneals to DNA and serves as replication start?

primers

What hybridizes to DNA to detect and label presence?

probes

What does PCR do?

amplify specific DNA sequences

PCR components

DNA template, primers, Taq polymerase, dNTPs, Mg2+

Steps of PCR

1. Denaturation

2. Annealing

3. Extension

Targeted PCR-RFLP

PCR followed by restriction enzyme digestion and gel separation

Limitations of PCR-RFLP

only for known variants, requires informative restriction site, can be affected by nearby variants, ambiguous results

Allele-specific PCR (AS-PCR)

targeted PCR method designed to selectively amplify one allele based on a variant specific primer, so amplification occurs preferentially when corresponding allele is present

Limitations of AS-PCR

limited to known variants, highly targeted, primer-dependent, nearby variants can interfere

Microarray Genotyping

many different synthetic oligos attached to discrete spots on the membrane, DNA from one patient added, sites with homology hybridize and are identified based on fluorescence

Limitations of microarray genotyping

detects primarily predefined small variants, cannot identify novel or rare variants, results depend on probe design, does not provide complete sequence info

Multiplex PCR detection of exon deletions

multiple PCR primers amplify exons, products analyzed via gel

Limitations of multiplex PCR detection of exon deletions

detects only targeted deletion of exons, not any other variants, exact breakpoints not determined, primer binding variants may cause allele dropout, confirmation by an independent CNV method may be required

Multiplex Ligation Probe Amplification (MLPA)

identifies copy number variants at exon level

Steps of MLPA

1. design probes

2. multiplex hybridization

3. ligation

4. PCR amplification

5. fragment analysis

Limitations of MLPA

primarily detects CNVs, detects only targeted regions, exact breakpoints not determined, variants can interfere, limited sensitivity for low-level mosaicism

Microsatellites (STRs) Analysis

STR region amplified by fluorescently labeled primers, PCR products separated by capillary electrophoresis, product size can be converted to number of repeats

Triplet primed PCR

detects triplet repeat expansions by using a pair of unique gene primers and a triplet primer targeting repeat region

Limitations of triplet primed PCR

cannot accurately determine the repeat size of a mutation, very large expansions can be difficult to characterize, additional testing to determine size

Southern Blotting

detect trinucleotide repeats, can decide the size of repeat sequences

qPCR

real time quantification with high sensitivity and wide dynamic range

Bisulfite treatment

methylation status testing by converting unmethylated C to U, but methylated C does not convert

Sanger Sequencing

DNA template with sequence specific primer and small proportion of chain terminating ddNTPs

Testing methods for fragment size and quantification analysis

triplet primed PCR, STR analysis, Southern Blot

Testing methods for direct detection of specific alleles

AS-PCR

Testing methods for Copy number analysis

MLPA, qPCR

What is next generation sequencing?

Massively parallel, deep sequencing, high throughput, short read sequencing

How does Illumina sequencing work?

sequencing by synthesis (bridge amplification and reversible terminator sequencing)

NGS analysis bioinformatics workflow

1. basecalling

2. alignment

3. variant calling

4. filtering/annotation

5. casual variant

What is a VCF file?

variant call file

What are the key parameters for variants calling in NGS?

phred-scaled quality (Q) scores, depth of coverage

What depth do you need for germline, somatic, and mosaic sequencing?

germline = 30X for genomes, 50X for exomes
somatic = 100X tumor, 30X for normal
mosaic = 900X

Panel based NGS

sequencing of preselected genes or regions of DNA

Limitations of WES

not all exonic regions sequenced, noncoding variants missed, low sensitivity for structural variants, interpretation

Limitations of WGS

data storage and analysis, VUS, not good for low level mosaicism

When to choose panel based NGS

clear and specific phenotype, well-defined set of disease genes, need for high depth coverage, faster turnaround time, interpretation burden should be minimized

When to choose WES or WGS

phenotype is genetically heterogenous, panel testing negative or no gene panel available, complex genetic disorders without clear cause, rapid diagnosis (no time to try different panel based testing)

Technical challenges of NGS

coverage gaps, difficulty detecting large indels, structural variants, repeat expansion, sequencing errors

Analytical challenges of NGS

data volume, variability in bioinformatic pipelines, false positives/negatives in variant calling

Interpretation Challenges of NGS

high VUS, limited knowledge of noncoding or regulatory regions, incomplete annotations, challenges interpreting incidental findings

Limitations of 3rd gen sequencing

higher error rates, lower throughput, higher cost, longer lib prep

What criteria is considered very strong in determining pathogenicity?

predicted null variant in a gene where LOF is a known mechanism of disease (PVS1)

What criteria is considered strong in determining pathogenicity?

prevalence in affecteds is stat increased over controls (PS4), same AA change as established variant (PS1), functional studies shows deleterious effect (PS3), de novo (PS2)

What criteria is considered moderate in determining pathogenicity?

absent in population databases (PM2), novel missense change at AA residue where a different pathogenic missense change has been seen before (PM5), protein length changing variant (PM4), mutational hotspot (PM1), de novo (without paternity and maternity confirmed) (PM6), for recessive disorders detected in trans with pathogenic variant (PM3)

What criteria is considered strong in determining benign-ness?

MAF is too high for disorder (BA1/BS1) or observation in controls is inconsistent with disease penetrance (BS2), well established functional studies show no deleterious effect (BS3), nonsegregation with disease (BS4)

CADD score 20 vs 30

variant is amongst the top 1% versus top 0.1% of deleterious variants

What are the clinical indications for cytogenetic analysis?

problems in early growth/development, stillborn, fertility problems, family history of a known or suspected chromosome abnormality, pregnancy in women of advanced age, neoplasia

Conventional chromosome analysis methods

G-banding, other banding, NORs silver staining

Molecular cytogenetic techniques

FISH, CMA

Newer technologies for structural chromosomal abnormalities

optical genome mapping, long read sequencing

How to get chromosomes for analysis

blood, add phythemagglutinin to stimulate T cells and add culture medium, culture, add colchicine and saline to stop in M phase, fix cells, spread by dropping, digest with trypsin and Giemsa stain, analyze spread

G negative (light) bands

GC rich, SINE/Alu rich, housekeeping genes, rich in transcribed sequences

G positive (dark) bands

High AT, LINE, genes are tissue specific, sparse genes

Limitations of FISH

targeted, cannot detect chromosomal rearrangement in less than 1-2% of total cells, cannot detect SNVs, indels, or intragenic tandem duplications, cannot detect chromosomal gain/loss <100kb

Difference between Array CGH and SNP array

array requires two labeled DNAs, SNP only needs patient DNA

array resolution is exons, SNP is by distribution

array cannot detect UPD or consanguinity, SNP can

Limitations of CMA

cannot detect balanced rearrangements, cannot detect low level mosaicism, cannot detect SNVs, expensive

Pharmacogenetics

study of how a single gene affects response to meds

Pharmacogenomics

study of how multiple genes affect response to meds

Pharmacokinetics

what the body does to the drug (metabolism and transport)P

Pharmacodynamics

what the drug does to the body (receptors, targets)

Prodrug

enters body as inactive drug, must be activated by enzyme

haplotype

a combination of a single or multuple spelling changes within a genes

diplotype

a specific combination of two haplotypes

*1

wildtype

Limitations of PGx

data utility, lack of confidence, non-genetic factors can impact effectiveness, insurance reimbursement, EMR alerts, inappropriate use of race

What are lysosomes

subcellular organelles from Golgi apparatus that catabolizes intra- and extracellular macromolecules

What are the final step of autophagy to degrade cellular macromolecules?

lysosomes

Impaired clearance of macromolecules underlies late-onset ________ conditions

neurodegenerative

Where are lysosomal enzymes synthesized?

rough ER and Golgi

How are lysosomal enzymes targeted?

via mannose-6-phosphate receptors

Gaucher disease predominately affects?

Ashkenazi Jews

General clinical features of LSDs?

course facies, skeletal abnormalities, macular cherry red spot, organomegaly, CNS deterioration, normal early development then regression

Which late onset LSDs have hgih incidence/

Pompe, Fabry, Niemann-Pick C

Mucopolysaccharidoses

disorders of GAG catabolism

What are GAGs?

proteoglycans, sulfated and unbranched polysaccharides, components of cells and extracellular matrix

Chondroitin sulfate (CS)

GlcUA-GalNac

cartilage, bone, heart valves

Heparan sulfate (HS)

GlcUA-IdUA-GlcNAc
basement membrane, outer cell surfaces

Dermatan sulfate (DS)

GlcUA-IdUA-GalNAc

skin, blood vessels, heart valves

Keratan sulfate (KS)

Gal-GlcNAc

cornea, bone, cartilage

Hurler Syndrome

Iduronidase deficiency (dermatan sulfate), dysmorphic syndrome, developmental delay, organomegaly, corneal clouding

Hunter Syndrome

Iduronate sulfatase (dermatan sulfate; X-linked), dysmorphic syndrome, developmental delay, organomegaly

San Fillipo A-D

4 heparan sulfate catabolic enzyme deficiency, learning difficulties, behavioral disturbances, dementia

Morquio A and B

2 keratan sulfate catabolic enzyme deficiency, severe bone dysplasia

Marateaux Lamy

galactosamine sulfatase deficiency (dermatan sulfate), dysmorphic syndrome, developmental delay

MPS VII

glucuronidase deficiency, hydrops, short stature, cognitive impairment, rare

MPS IX

hyaluronidase deficiency, extremely rare

Hurler syndrome inheritance

autosomal recessive

Hurler syndrome GAG elevations

DS, HS

Hunter syndrome GAG elevations

DS, HS

San Fillipo GAG elevations

HS

Morquio A and B GAG elevations

A: KS,CS

B: KS

Marateaux Lamy GAG elevations

DS

MPS VII GAG elevations

DS, HS, CS

Mucopolysaccharidosis Management

supportive care (PT, CPAP, hearing aids, surgery)

hematopoietic stem cell transplantation (standard of care for Hurler Syndrome)

enzyme replacement therapy

Most common sphingolipidoses

Gaucher, Fabry

Gaucher disease

glucocerebrosidase deficiency, hepatosplenomegaly, bone abnormalities, anemia

Fabry disease

a-galactosidase deficiency, acroparesthesia, angiokeratomas, hypohidrosis, corneal opacities, kidney dysfunction

What are sphingolipids?

complex lipids with membrane function including signal transduction and cell recognition

have fatty acid group, sphingomyelin, monosaccharide, and gangliosides