Molecular Diagnosis

Question 1

polymorphisms

Answer 1

multiple forms of a gene that can exist in a population (i.e. sickle cell anemia is a balanced polymorphism)

not a mutation since it is common

functional change that still allows the expression of the protein

Question 2

single nucleotide changes

Answer 2

base changes
substitutions
point mutations
indels (insertions-deletions)
SNPs (single nucleotide polymorphisms) 
- variation of a single nucleotide in the DNA sequence among individuals

detection = easy to detect by traditional molecular genetic tech….like sequencing or PCR

Question 3

changes in 2bp up to 1,000bps

Answer 3

large nucleotide repeats

inversions

insertion-deletions

variable number tandem repeats (VNTRs)

detection = easy to detect by traditional molecular genetic tech….like sequencing or PCR

Question 4

changes in whole chromosome up to whole genome

Answer 4

detection = harder detect by traditional molecular genetic tech….like sequencing or PCR

instead use cytogenic methods = analyze karyotype under microscope, FISH

Question 5

what size genomic changes were hard to detect until recentyl

Answer 5

several kb to 1-5 mega-base

Question 6

germline mutation

Answer 6

originates in parental gametes or in the zygote at the single cell stage….affects every cell in an organism and is passed onto offspring

Question 7

somatic mutation

Answer 7

acquired in just one cell type….not detectable in other tissues

Question 8

types of point mutations

Answer 8

silent = same amino acid is still encoded

neutral = different but functionally equal amino acid

missense = functionally different amino acid

nonsense = stop codon

frame-shift = deletion or insertion = alters whole sequence

Question 9

methylation errors

Answer 9

doesn’t change the nucleotide sequence…but the modification of the nucleotides

commonly, methylation of a maternal or paternal allele and only one copy is expressed

in errors = genes can be inappropriately silenced (imprinting error)
–> example = uniparental disomy

Question 10

loss of heterozygosity (LOH)

Answer 10

can happen by a few ways

for example….initially there are 2 different copies of a gene, but a copy is lost, and the remaining copy is duplicated –> leads to homogeneity

Question 11

duplications, insertions, or deletions

Answer 11

can disrupt reading frame

insertion of a transposable element such as Alu or LINE repeat

detection = southern hybridization, fluorescent in-situ hydrbiization (FISH), pulsed field gel analysis, or PCR amplification

Question 12

ARMS =

Answer 12

amplification refractory mutation system

Question 13

MS-PCR or MS-southern blot =

Answer 13

methylation specific

Question 14

STR

Answer 14

short tandem repeat

Question 15

CGH =

Answer 15

comparative genomic hybridization

Question 16

detection of point mutations

Answer 16

PCR/RFLP

ARMS

sequencing

Question 17

detection of trinucleotide expansion

Answer 17

PCR

southern

Question 18

detection of methylation error

Answer 18

MS-PCR

MS-southern blot

Question 19

detection of copy number alterations (>1kb)

Answer 19

CGH and/or SNP array

Question 20

detection of LOH

Answer 20

STR typing

SNP array

Question 21

chromosomal rearrangements

Answer 21

array CGH

karyotyping

Question 22

PCR/RFLP

Answer 22

for small, known mutations

based on the sizes produced - can determine if the mutation is present

Question 23

PCR/ASO

Answer 23

allele-specific oligonucleotides

known, small mutations

2 probes are used, 1 WT and 1 mutant…differing by 1 nucleotide

WT probe will hybridize with the normal sequence (from WT DNA)

mutant probe will hybridize with the mutated sequence

Question 24

sequencing

Answer 24

find small nucleotide changes, deletions, insertions

each base has a specific corresponding color

compare sequences to see if something is different

Question 25

methylation specific southern blot

Answer 25

digest genomic DNA with enzymes that are sensitive to the methylation state of DNA

if DNA is methylated = restriction sites cannot be recognized –> longer fragments

Question 26

Prader-Willi Syndrome

Answer 26

chromosome 15

deletion in paternal allele

won’t be cut by Not1 when doing MS-southern blot analysis

15% of these patients have uniparental disomy for 2 maternal alleles

2-5% due to methylation of paternal allele

Question 27

Angelman syndrome

Answer 27

chromosome 15

deletion in maternal allele

Question 28

methylation specific PCR

Answer 28

can be used to diagnose prader-willi and angelman syndromes

faster than southern blot

if maternal allele is present and paternal lost –> PWS

vice versa –> AS

Question 29

fragile X syndrome

Answer 29

most common x-linked mental retardation

CGG expansion mutation at exon 1 of the FMR-1 gene

highly expanded CGG repeats are always methylated and methylation on the CpG island of the FMR-1 gene will inhibit FMR-1 expression

normal = 6-45 rpts
grey zone = 45-54
pre-mutation = 55-200
full mutation = more than 230

PCR can be used to detect up through pre-mutation

MS-southern is used in addition in case above pre-mutation levels

Nru-1 = methylation sensitive restriction enzzyme….will only cut a restriction site if unmethylated = smaller fragment

if methylated = cannot cut = longer fragments

Question 30

CGH (comparative genomic hybridization) assay

Answer 30

mix and hybridize control and pt DNA, label and analyze

use for unknown cause of

• dysmorphism
• congenital multiple abnormalities
• mental retardation
• developmental delay
• autism

powerful whole genome sequencing

• advantage = great detection sensitivity for deletion, duplication, insertion, non balanced rearragement, aneuploid, and triploid
• –> can also detect uniparental disomy and LOH

disadvantage = not good detection of balanced rearrangement and low % mosaicism

can detect DiGeorge’s syndrome

Question 31

SNP+CGH assay combination

Answer 31

use for detecting copt number changes; SNPs, and LOH

Question 32

how can LOH happend

Answer 32

1, copy neutral loss = copy number does not change but heterozygosity is lost (loss of chromosome and then duplication of the other chromosome)

2. mitotic recombination in the region of the mutation
3. gene conversion in the region of the mutation

Question 33

NGS - next generation sequencing (NGS)

Answer 33

whole genomic or whole exome sequencing

advantages = millions of DNA fragments can be sequenced simultaneously (high throughput)
- can detect homozygous deletion, hemizygous deletion, gain, point mutation, indel, pathogenic gene info if whole genome

disadvantages = high cost…can be cheaper if whole exome is done instead of whole genome…which can still be effective because mutations that cause diseae are more likely to happen in coding regions
- cannot detect epigenetic changes

whole genome= more diagnositic yield and incidental findings than whole exome

Question 34

light microscope –> G-banded karyotype –> microarrary –> whole exome sequencing –> whole genome

Answer 34

increasing…

resolution

number of nucleotides probed gets greater

more variants can be detected

diagnostic yield and incidental findings increase

Question 35

NGS application to rare pediatric diseases

Answer 35

tri-based whole genome = 42% can be detected

tri-based whole exome = 40%

proband only whole exome = 28%

**using gene panel NGS…detect much less than 28% (current use of NGS)

single gene sequencing = very few can be detected

Question 36

application of molecular diagnosis

with genetic disorders

Answer 36

direct analysis of genetic mutations

genetic linkage analysis

population screening

preimplantation diagnosis for IVF

Question 37

application of molecular diagnosis

with analysis of cancer genome

Answer 37

prognostic indicators

markers for therapy protocol selection

markers for personalized medicine

Question 38

application of molecular diagnosis

detection of infectious pathogens

Answer 38

quantitative analysis

strain genotyping

species identification

Question 39

application of molecular diagnosis

other

Answer 39

pharmacogenetic testing

human identification testing