Lecture 28-Clinical Molecular Genetics 1 and 2 Flashcards
What is the difference between a genome mutation and a chromosome mutation?
- genome: whole chromosomes absent or present in excess (ex: downs)
- chromosome: chromosomes translocated
What is the difference between a mutation, polymorphism and a rare variant?
- mutation: permanent change in the nucleotide sequence that causes a disease
- polymorphism: a change in the DNA that does not cause disease and is in >1% of the population
- Rare variant: a change in the DNA sequence that does not cause diseases and is in <1%
When a change in DNA sequence is found it can be hard to tell whether this is a _____ or _____.
- mutation
- rare variant
compound heterozygote
a person with 2 mutant alleles on the same gene (ex: CF)
What is advanced maternal age and paternal age? Why is this age set for women?
- 35 for both
- this is the age in women because this is the age at which the risk for a chromosome abnormality equals the risk of miscarrying the child
Haploinsufficiency
Youre making the functional, normal protein just not enough of it
Dominant negative
when the mutant protein disrupts the function of the normal protein
Of dominant negative, haploinsufficiency, gain of function and loss of function which of these are dominant disorders?
- gain of function
- haploinsufficiency
- dominant negative
Achondroplasia inheritance
AD, always from father
What is the rate at which individuals develop new mutations?
- 80%
Achondroplasia results from a mutation in what gene? What kind of mutation is it and what does this cause?
- FGFR3
- its a gain of function mutation that keeps the receptor constitutively active limits skeletal growth
How can you test for achondroplasia?
- 99% of people with achondroplasia have a G–>A transition that alters restriction enzyme sites. The other 1% has a G –> C transversion that also alters restriction sites
- The G–>A digests with SfcI to give 109 and 55 bp segments
- The G–>C digests with MspI to give bands of 107 and 57
Are most people with dominant achondroplasia heterozygous or homozygous?
- heterozygous because both of the parents would’ve had to have had the condition
Is MR associated with achondroplasia?
- no
semidominant trait
can distinguish between hetero and homozygous trait (homozygous is lethal)
What is the most common fatal autosomal recessive disease in Caucasians?
- CF
(T/F) Different CF mutations are associated with different ethnic groups.
true
What chromosome is the CF gene on?
- 7
How many mutations for CF have been described?
1800
∆F508
most common CF mutation–in frame
p.W1282X
most common CF mutation in Ashkenazi Jews
- nonsense mutation so RNA is either degraded or you make a truncated protein
3120+1G–>A
most common CF mutation in AAs
- splicing variant
If you find one of these mutations does that automatically tell you that the patient has CF?
- no, you would have to find RNA or the protein
What facts do you need to make a DIAGNOSIS of CF?
- clinical feature, family history or positive neonatal screening test
AND - positive sweat Cl, 2 CFTR mutation or positive nasal transmembrane potential
What type of mutation is R117H? Does having this mean the person will have CF? What do you have to look for?
- missense
- no, you have to look at intron 8 for T repeats of 5, 7, or 9. If the T repeat is 5, then your child will have CF, if its 7 or 9 your child will not have CF
EXAM QUESTION: If you find a child with ∆F508 and R117H mutations, and also find that they have both 5T and 9T repeats, what does this tell you? What if there were 2 9T alleles?
- the ∆F508 allele ALWAYS has a 9T associated with it, therefore the R117H allele has the 5T associated with it and therefore the child will have classic CF.
- if there were 2, 9T alleles then this would not lead to disease because exon 9 wouldn’t be spliced out
Why do the 5, 7 and 9T repeats affect the protein?
- as the “tract” decreases, splicing efficiency decreases and results in skipping of exon 9. The 5T allele is in 90% of the transcripts that don’t have exon 9
∆F508, I148T and 3199del6 mutations are in linkage disequilibrium with ______ (i.e., they will not recombine without this, they are linked)
9T
Describe oligonucleotide ligation assay (OLA)
- used to detect CF mutations
- primers specific for mutant and normal alleles and a common primer or probe are mixed with test DNA.
- This process is like PCR but the 2 pieces need to be ligated before the PCR reaction can occur and the ligation won’t occur if theres a mismatch between them. So either the mutant or the WT probe will bind to the test DNA, be ligated and will undergo a PCR reaction
- fluorescent markers send a signal depending on which probe has bound and if there is a heterozygote then you get 2 peaks half the height of the homozygous peaks
What is the advantage to OLA?
- its very quick
If someone has OLA done and they come back as a carrier for a mutation does this mean that they won’t have the disease?
- no, they could still have a mutation in a CF gene that was not on the panel tested
Aside from the obvious reasons (prenatal diagnosis, routine prenatal testing) why would you do a genetic test for CF?
- to do a genotype-phenotype correlation: some mutations are associated with pancreatic sufficiency
- helps you make a diagnosis when you can’t get sweat from a neonate or its indefinitive
- male infertility workup: men with CF are sterile
Duchenne Muscular Dystrophy (DMS) inheritance
- X linked
describe the DMS gene
HUGE: 2.4Mb, 79 exons
What accounts for the mutations that give rise to DMS? (3)
- large deletions: ~60% of mutations (most detectable with PCR)
- ~15% have premature stops (nonsense)
- 5% are duplications
(T/F) the larger the deletion, the worse the phenotype with DMS.
False
What is associated with a less severe prognosis in DMS?
- if the N and C termini are there and only part of the middle of the protein are missing because it will still maintain function. This is actually called Becker dystrophy.
in DMD what is the most common chromosomal alteration that causes this?
deletions
Hemizygote
a male iwth a single allele on an XL locus
What is the chance of a mother giving DMD (an XL disorder) when she has already had a child with it, and when you do NOT know if the mother is a carrier?
- 2/3 chance she’s a carrier (because there’s a 1/3 chance the condition could’ve arisen by a new mutation since it’s XL) X 1/2 chance she passes on the gene X 1/2 chance she has a boy = 1/6
What is the chance of a mother giving DMD (an XL disorder) when she has already had a child with it, and when you DO know the mother is a carrier?
1/2 chance of passing the gene X 1/2 chance she has a boy = 1/4
What is MLPA?
- A newer way to test for DMD (as opposed to PCR and densitometry) that is very similar to OLA
- 2 probes (mutant vs. normal) with a “stuffer sequence” that does not bind the DNA can lay down on the test strand next to each other and only if theres a perfect match can ligation occur between them.
- ligation, PCR (only if ligated)
- the probes that annealed are amplified by PCR and the length of the strands (that are given by the stuffer sequences) are used to distinguish which one bound the parent strand and amplified during PCR
How would you explain a mother who is not a carrier for DMD according to MLPA that has 2 children with DMD?
- she could have germline mosaicism which is fairly common with DMD and in this case we wouldn’t see the mutations in the blood sample tested
Where on genes to repeats occur?
ANYWHERE
Freidrick’s Ataxia
only AR disease caused by repeats
Polyglutamine/Polyalanine repeats
- repeats only in exons
Where are the Fragile X repeats located on a gene?
5’ UTR
How do polyglutamine repeats compare in size to other types of repeats?
- they’re smaller because they’re in an exon
All known polyglutamine disorders are characterized by ______ that begins when? What will eventually happen?
- neuronal dysfunction
- midlife
- results in severe neurodegeneration
polyglutamine disorders are caused by ______.
toxic gain of function mechanism
What kind of mutation are polyglutamine repeats? (i.e., loss of function, gain of function, haploinsufficient, or dominant negative)
- dominant negative because they interfere with the WT protein function
PolyQ repeats can be detected what 2 ways?
- nucleic acid: trinucleotide repeat disorders, if you have 90 repeats you do SB
2/3 of HD patients present with _____ while the other 1/3 present with _____
- neurological disease
- psychiatric changes
HD is from what repeat in what exon of what gene on what chromosome?
- CAG
- 1st exon
- HTT gene
- Ch4
How many repeats do you need to be considered normal in HD? Affected?
- normal: 10-26
- affected: 40+
How could you differentiate each phase of HD? Early, Intermediate and Later?
- Early: sublt changes in movements/coordination, often depressed/irritable mood)
- Intermediate: chorea more noticeable, difficulty with voluntary activity and trouble swallowing and speaking
- Later: total dependence, mute, incontinent
What makes it possible for us to test presymptomatically with HD and why is it so important that we confirm our diagnoses?
- we know the gene that’s affected
- we need to know we’re not dealing with something that looks like it
HD inheritance?
- AD, late onset usually after childbearing years
Do we use SB or PCR for HD?
PCR
Where do we get the prenatal child’s DNA for testing? Why do we have to be more sensitive about testing with this condition?
- DNA from CVS and amniocytes
- you may have to test in a way that doesn’t reveal the genotype of an at-risk parent
How do you test a child for HD without revealing the parent’s genotype?
- linkage approach: essentially telling the parents whether or not the potential carrier passed on the allele from the non affected parent or the affected parent (which may or may not be the allele with the mutation)
What are the 3 types of myotonic dystrophy?
- mild
- classical
- congenital
Myotonic dystrophy is suspected in adults with what symptoms?
- muscle weakness
- myotonia (sustained muscle contraction)
- cataracts
MD is suspected in neonates with what symptoms? (5)
- hypotonia
- facial muscle weakness
- generalized weakness
- club foot
- respiratory insufficiency or failure
What is the mutation in MD? (what gene on what chromosome, which repeat and in what region?)
- DMPK
- chromosome 19
- CTG repeats
- 3’ UTR
What is the normal number of repeats in someone with MD?
- > 50
In MD, how does the length of the repeat correlate to the age of onset?
inverse correlation
What is unique about the transference of MD between generations?
- anticipation: when we either see an earlier age of onset as the mutation passes between generations or more severe phenotype because repeats expand as their passed on
Infants with MD almost always inherit the expanded allele from ______
their mother
If a woman has >300 repeats for MD what is the chance her child will be affected congenitally?
60%
If the woman with >300 repeats for MD wanted to test her child, would you use PCR or SB?
- SB: there would be 1000s of repeats!
Why are there generally smears on SBs for MD patient samples?
- the DNA is mitotically unstable so the amount of DNA in each cell varies slightly between cells
Where do you get the DNA from the neonate for testing for MD?
- CVS or amniocytes
Given that the amount of repeats is inversely related to the time of onset, can we use the amount of repeats to predict when the onset of the disease will be?
- no, but if there are >1000 repeats the child is more likely to have congenital MD
What is the prevalence of PWS and AS?
- PWS: 1/10-20,000
- AS: 1/20,000
Can imprinting patterns vary between tissues?
- yes, imprinting patters can change in the brain
What are 2 big clues that imprinting is going on in a disease?
- UPD is associated with abnormal development
- Consistent parental origin of the mutant allele in inherited disorder
The “imprinted allele” refers to which allele?
- the allele that’s silenced
Explain how gene expression changes between imprinted genes, non-imprinted genes in deletions of chromosome parts (ex: PWS).
- when you have one strand of DNA of a certain region, the imprinted genes will not be expressed so you don’t have enough of these
- the non imprinted version of genes that are imprinted will have enough product because usually their partner on the homologous chromosome is silenced anyways
- genes that don’t have imprinting probably have haploinsufficiency and this is why PWS patients usually have light hair and skin compared to their families, a gene for pigmentation is usually lost in these deletions
What is a good tool to check for deletions particularly in PWS and AS?
- FISH probes
heterodisomy
- what does it result in?
- M1 nondisjunction: results in 1 maternal and 1 paternal copy of the same chromosome in 1 gamete
isodisomy
- what does it result in?
- M2 nondisjunction: results in 2 paternal or 2 maternal copies of a chromosome in one gamete
trisomy rescue
- the fetus is initially trisomic but one chromosome is lost. Usually the trisomy is originally caused by the mother because nondisjunction events are more common in women
monosomy rescue
fetus is initially monosomic but the chromosome is duplicated resulting in complete isodisomy
Gamete complementation
- one game is disomic and the other is nullisomic resulting in a zygote that has the right number of those chromosomes
- this is more rare because there would have to be a nondisjunction event in both the mom and dad for this to occur
How does UPD differ from a deletion in one of the 2 homologous chromosomes that are imprinted? How does this differ from a methylation defect?
- you still have none of the genes that are normally imprinted
- you have double the genes that are the non-imprinted form of imprinted genes although this doesn’t really affect the person
- correct amount of non-imprinted genes
- this does NOT differ from a methylation defect other than that in a methylation defect the genes are turned off because they look like a male/female chromosome (ex: receiving our mother’s paternal allele from our her)
In both PWS and AS most of the time the disease is caused by what chromosomal change?
- deletion
Considering the distribution of causes between PWS and AS, they generally break down to the same causes in similar proportions except for what 2 things?
- in AS 11% are from mutations in UBE3A
- in AS 10% have normal molecular and cyto
NOTE: in the rest the next biggest cause of disease after deletions is UPD, then methylation defects, then translocations
How can you check the cause of PWS or AS?
- methylation analysis: 96% of the C’s in the critical region for these disorders are methylated in the maternal allele and none in the paternal
UBE3As functions are normally seen where?
in the brain
What are the 2 ways to do methylation analysis?
- SB with HPAII that recognizes (and will not cut) at methylated GC sites
- PCR after bisulfite treatment: changes unmethylated Cs –> Us. 2 primers are made (1 with C, 1 with U) and one common primer, run on gel
What is the advantage to using PCR and bisulfite for methylation analysis?
requires less DNA and is quicker
Of all the potential causes for PWS and AS, what causes should you think of if you see cousins with PWS and AS?
- methylation defects
- chromosomal rearrangements
What is OI inheritance?
AD
Collagen is composed from what end to what end? What does this mean in terms of OI patients?
- C terminus to N terminus
- mutations in the C terminus will be more detrimental
Type I OI
- null allele, less severe because you still have half functional collagen
How do mutations in alpha1 collagen present (molecularly)?
- mutations in the Gly
- mutant peptide messes up the normal collagen formation
Type II OI
- lethal
How do mutations in alpha2 collagen present (molecularly)?
- still get some normal and some abnormal collagen but this results in the more severe forms of Type II-IV (not type 1 since type 1 is the alpha 1 null)
