Clinical Molecular Genetics Flashcards
Mutation
Any intrinsic change in DNA
Genome mutation
Whole chromosomes are absent or present in excess (Down Syndrome)
Chromosome Mutation
Chromosome is rearranged (i.e. Translocation)
Gene Mutation
Mutation involving a single gene
Allele
Alternative variants of genetic information at a specific locus that control the same trait. Different alleles are caused by small polymorphisms and rare variants, which are non-disease causing changes.
Polymorphism
Non-disease causing change in allele sequence that is present in 1% of the population. The frequency of the less common base change must be >1% in order to be considered a polymorphism.
Rare Variant
Non-disease causing change in allele sequence that occurs in <1% in order for the change in allele sequence to be considered a rare variant.
Compound Heterozygote
Individual with 2 different mutations in the same gene.
Haploinsufficiency
One mechanism of a disease that is cause by a mutation that results in a normal functional protein but there is not enough protein for it to do its job properly.
Achondroplasia
Autosomal Dominant single gene gain of function mutation in FGFR3 receptor that leads to constant repression of cartilage formation. Majority of the individuals (~80%) do not have affected parents and their condition is the result of a new mutation on the PATERNAL chromosome. 99% of affected individuals have p.Gly380Arg transition, 1% have p.Gly380Cis transversion
Transition Mutation
Substitution of a purine for a purine/pyrimidine for a pyrimidine. Missense mutation.
Translation Mutation
Substitution of a pyrimidine for a purine or a purine for a pyrimidine. Missense mutation.
FGFR3 Mutation
Mutation causing Achondroplasia; diagnosed using restriction enzymes
- p.G 380A (99%) results in 55bp and 109bp fragments when cut with restriction enzyme SfcI
- p.G389C results in 57bp and 107bp fragments when cut with MspI restriction enzyme.
MspI
Restriction enzyme used in Achondroplasia diagnosis. Identifies minority (1%) of FGFR3 mutations. Cuts at G to C mutation producing 57bp and 107 bp fragments.
SfcI
Restriction enzyme used in Achondroplasia diagnosis. Identifies majority (99%) of FGFR3 mutations. Cuts at G to A mutation producing 55bp and 109 bp fragments.
Homozygous Mutant Achondroplasia
Lethal. In this case, the baby is AA. Homozygous mutant achondroplasia causes a restricted rib cage so the baby cannot breathe and ultimately they die.
What major factor contributes to the initial risk for Cystic Fibrosis?
Ethnicity. Carrier frequency for Cystic Fibrosis differs by ethnic group.
What are the different carrier frequencies for Cystic Fibrosis according to ethnicity?
1/25 Caucasian 1/29 Ashkenazi Jews 1/45 Hispanics 1/62 African Amer 1/150 Asians
CFTR
Gene with 27 exons located at 7q31 that encodes for a 1480 residue transmembrane protein responsible for transporting sodium across membrane in lungs. There are over 1800 mutations described (example of allelic heterogenetiy because different mutations on alleles of the same gene still produce cystic fibrosis). Most common mutations: deltaF508, pW1283X (Ashkenazi Jews), 3120+1G>A (African Amer.)
deltaF508
Mutation in CFTR gene that causes an inframe deletion of Phe at position 508. Most Cystic Fibrosis common mutation among all ethnicities. Is always accompanied (linkage disequilibrium) by poly 9T tract in intron 8 of CFTR.
p.W1282X
Nonsense mutation in CFTR gene that replaces Phe codon at position 1282 with a Stop codon causing Cystic Fibrosis. Most common mutation found in Ashkenazi Jewish pops.
What is the most common Cystic Fibrosis mutation?
deltaF508 (inframe deletion)
What is the most common Cystic Fibrosis mutation in Ashkenazi Jews?
p.W1282X (nonsense mutation)
3120+1G>A
Splicing mutation in CFTR gene that causes a loss of protein function resulting in Cyctic Fibrosis. Most common in African American populations.
What is the most common mutation in African Americans with Cystic Fibrosis?
3120+1G>A (splicing mutation)
What are the requirements for making a diagnosis of Cystic Fibrosis?
Patient MUST have one from each of the two groups listed.
- At least one char. clinical feature
- Family history of CF
- Positive neonatal screening
- +sweat Cl- on 2 separate tests
- Presence of 2 CFTR mutations
- +nasal transmembrane potential
R117H
Missense mutation in in CFTR that may be accompanied by poly T tract in intron 8. If there is an R117H mutation present, you must determine if it is cis (on same chromosome) with intron 8 poly T sequence (5T, 7T, 9T). R117H cis with 5T results in ~90% of transcripts w/o exon 9 and a nonfunctional chloride channel causing Cyctic Fibrosis.
What do deltaF508, I148T and 3199del6 have in common?
They are all mutations in CFTR gene in linkage disequilibrium with poly 9T tract.
Which CFTR mutations are always in linkage disequilibrium with poly 9T tract?
deltaF508, I148T and 3199del6
What is the probability that a male with CF will pass on his mutated allele?
Without assisted reproduction: 0% because males with CF are infertile.
What is commonly referred to as being the largest gene in the human genome?
Dystrophin gene: 2.4Mb, 79 exons.
Mutations in this gene cause muscular dystrophy.
Becker Muscular Dystrophy
Milder form of Duchene Muscular Dystrophy that is caused by deletions or duplications in Dystrophin gene that code for the middle portion of the Dystrophin protein, leaving the N and C termini still in tact. Because the N and C termini are still in tact, there is still partially functional protein and the condition is not as bad.
Gonado Mosaicism
Mutations present only in the germ cells that cannot be seen in the blood.
Duchene Muscular Dystrophy
X-Linked with mutations in dystrophin gene. There are different mutations possible: deletions (~60%), premature stop (~15%), dupliation (~5%).
*The size of the deletion does not correlate with the severity of the condition. Large in frame deletions can result in a truncated protein with in tact N and C termini rendering it partially functional (Becker Muscular Dystrophy).
Friedreich’s Ataxia
Unique nucleotide repeat disorder. The only autosomal recessive disorder that has been found to be due to nucleotide repeats (all others are either X-linked or autosomal recessive.
If there is a nucleotide repeat >90 bp, what type of analysis must be done and why?
At repeats >90bp, a Southern Blot must be used for analysis because repeats of this length simply cannot be synthesized by Taq polymerase in PCR.
What can be determined by an amino acid (i.e polyglutamine or polyalanine) repeat seen in a protein?
There must be a repeated nucleotide sequence in the EXON of the gene because we see amino acid repeats.
This is the case in Huntington’s disease.
Huntington’s Disease
Progressive disorder of motor, cognitive & psychiatric disturbances.
- Autosomal dominant
- CAG repeat in 1st EXON of HTT gene located at 4p16.
- When there are 40 or more repeats, that is when the disease manifests itself.
- Symptoms do not usually appear until mid-adulthood (40-60y.o.)
HTT
Gene located at 4p16 (chromosome 4) that when mutated (CAG - Glutamine repeats) can cause Huntington’s disease.
CAG repeats in HTT gene - what are the thresholds?
Normal: 10 - 26 repeats with 18 being the median.
Affected: >40 repeats.
- CAG repeated 27-35 times the individual is not as risk themselves but their children have an increased risk
- CAG repeated 36-39 times, there is decreased penetrance for symptomatic Huntington’s and the individual may or may not develop symptoms.
Anticipation
Either:
- Earlier age of onset as mutation passes from generation to generation
- More severe phenotype in subsequent generations
DMPK
Myotin protein kinase gene. Located 19q13. In Myotonic Dystrophy, there is a CTG repeat in the 3’ UNTRANSLATED REGION (NONCODING region).
What is the difference between coding and non-coding region repeats?
Coding region repeats can only be a couple hundred repeats while repeats in the non-coding regions can be in the thousands.
CTG repeats in DMPK gene - what are the thresholds?
Normal repeat number is 5 - 35. Individuals affected with Myotonic dystrophy have >50 repeats.
Mild: 50 to ~150 repeats
Classical: ~100 to ~1000 repeats
Congenital: >2000 repeats
Myotonic Dystrophy
Multisystem disorder that affects skeletal muscle, smooth muscle, eye, heat, endocrine sys and CNS. Caused by >50 CTG repeats in 3’ UNTRANSLATED REGION of DMPK gene on 19q13. There is an inverse correlation between repeat length and age at onset (the more repeats present, the earlier the age of onset.
What diagnostic test must be used for Myotonic Dystrophy and why?
Must use a southern blot because the number of repeats is >90 and in some cases in the 1000’s making PCR impossible
If a mother has >300 CTG repeats in DMPK gene what is the risk for her having a child with congenital myotonic dystrophy?
60%
Imprinting
Differential modification and expression of alleles of a gene depending on the sex of the parent of origin (i.e Prader-Willi and Angelman Syndromes)
- About 31 human genes are reported to show imprinted expression.
Uniparental Disomy
Both copies of chromosomes come from one parent. Can lead to an autosomal recessive condition in a child with only one parent as a carrier (i.e. Cystic Fibrosis)
Imprinted allele
Refers to the silent, inactivated allele
Paternally imprinted
The paternal allele is silent/inactivated so the maternal allele is the one that is expressed
Maternally imprinted
The maternal allele is silent/inactivated so the paternal allele is expressed.
In which parent are nondisjunction events more common?
Moms (thus they are more likely to be involved in trisomy rescue because they are more likely to pass on two chromosomes instead of one).
List the underlying molecular mechanisms of Prader Willi Sundrome
- 70% have deletions in 15q11q13 (paternal chromosome 15-paternal imprinting)
- 25% have maternal uniparental disomy
- <1% have translocations involving the 15q11q13 region
List the underlying molecular mechanisms of Angelman Syndrome
- 68% have deletions in 15q11q13 (maternal chromosome 15-maternal imprinting)
- 7% have paternal uniparental disomy
- <1% have translocations involving 15q11q13 region
- 11% have UBE3A mutations
- 10% have normal molecular and cyto
Why is paternal uniparental disomy less common in Angelman Syndrome than maternal uniparental disomy is in Prader Willi Syndrome?
Because maternal nondisjuction is less common than maternal nondisjunction.
UBE3A
Gene that can have a point mutation and result in the phenotype of Angelman’s Syndrome. Comes into play in the brain.
What features of the genome allow for the possibility of methylation analysis?
> 96% of cytosines in the Prader Willi/Angelman’s critical region are methylated on the MATERNAL allele while NONE are methylated on the PATERNAL allele. This characteristic can be used to differentiate maternal and paternal alleles.
What is the first test that should be used to diagnose Prader Willi Syndrome?
Methylation analysis (detects 99% of PWS cases)
Which end of the collagen triple helix is formed first and what are the implications of this for osteogenesis imperfecta?
Collagen triple helix is first formed on the carboxy terminal end. This means that mutation in the carboxy terminus will result in a more severe phenotype than mutations in the amino terminus.
What are the two ways that methylation analysis for PWS and AS can be done?
- Southern blot with HpaII
2. PCR with bisulfite
HpaII
Restriction enzyme used in Southern blot analysis of PWS/AS. It recognizes a specific sequence in the PWS/AS critical region but does NOT recognize the sequence if methylation is present. Paternal allele will give a 0.9kb band on Southern Blot if present and maternal allele if methylated will give a 4.3kb band.
In southern blot, wild type will have 4.3kb and 0.9kb bands. AS will only have 0.9b band (paternal expression) and PWS will have 4.3kb band (maternal expresson).
HpaII
Restriction enzyme used in Southern blot analysis of PWS/AS. It recognizes a specific sequence in the PWS/AS critical region but does NOT recognize the sequence if methylation is present. Paternal allele will give a 0.9kb band on Southern Blot if present and maternal allele if methylated will give a 4.3kb band.
In southern blot, wild type will have 4.3kb and 0.9kb bands. AS will only have 0.9b band (paternal expression) and PWS will have 4.3kb band (maternal expresson).
Bisulfite and PCR
Bisulfite converts unmethylated cytosines to uracil. This can then be used with PCR by making probes with uracil and cytosine sequences. Maternal alleles in PWS/AS critical region will have more cytosines because there will be more methylated Cs.
