Genetic Testing- TNR Expansion Flashcards
What is trinucleotide repeat expansion (TNR)?
a unique mutations mechanisms where mutant alleles are unstable and actually change in size from one generation to the next
What is trinucleotide repeat expansion (TNR)?
a unique mutations mechanisms where mutant alleles are unstable and actually change in size from one generation to the next
T or F. allele size shows a direct correlation with disease severity
T
Name four important TNR disorders
- Fragile X syndrome
- Huntington disease
- Friedrich ataxia
- Myotonic dystrophy
What is the disease gene for Fragile X syndrome?
CGG repeat on the 5’ untranslated end of FMR1 gene on the X chromosome
What are the symptoms of Fragile X syndrome?
developmental delay and cognitive impairment
What is the number of CGG repeats for a normal FMR1 gene?
5-54 alleles
What is the number of CGG repeats for an intermediate FMR1 gene? What are the results of having this number of repeats?
In fragile X syndrome, there are intermediate alleles (gray, 55-200 repeats) that are not large enough to cause disease in a person who carries such an allele, but are large enough to be unstable during meiosis (aka the ‘premutation’ range). Thus, a full mutation and disease may arise in the next generation.
What is the number of CGG repeats for a fully mutated FMR1 gene?
> 200 alleles. symptomatic
What is the disease gene for Huntington disease?
CAG repeat within an exon of the HTT gene on chromosome 4
What are the symptoms of Huntington disease?
progressive neuropsychiatric disorder
What is the number of CAG repeats for a normal HTT gene?
10-35
What is the number of CAG repeats for a fully mutated HTT gene?
>
Note that there is a small intermediate range, ranging from 36-40
What is the disease gene for Friedrich ataxia?
GAA repeat within an intron of the FXN gene on chromosome 9
What are the symptoms of Friedrich ataxia?
progressive ataxia
What is the number of CAG repeats for a normal FXN gene?
What is the number of CAG repeats for a fully mutated FXN gene?
> ~70-200
What is the disease gene for Myotonic dystrophy?
CTG repeat in the 3’ untranslated region of the DMPK gene on chromosome 19
What are the symptoms of Myotonic dystrophy?
progressive muscle wasting
What is the number of CAG repeats for a normal DMPK gene?
What is the number of CAG repeats for an intermediate DMPK gene?
35-49 (premutation)
What is the number of CAG repeats for a fully mutated DMPK gene?
mild phenotype: ~50-150
classic phenotype: ~100-1000
congenital: >1000
TNR expansion disorders are typically associated with what kinds of symptoms?
neurologic and muscular
T or F. Within the normal size range, the repeats are perfectly stable, and there is no risk of disease
T. In general, if we examined multiple generations from a normal family, we would expect allele sizes to be perfectly maintained. However, at the same time, there is variation in allele sizes across the normal population. Using the HD gene in normal families as an example, if we looked at one family, we could track an allele with 6 repeats through multiple generations, and there would be no change in its size. A different normal family can have allele sizes anywhere within the normal range (up to ~35 repeats for HD), and these will segregate through that family in a stable manner.
What is the molecular mechanism for allele expansion?
In spite of much research, unanswered questions remain about the molecular mechanism for allele expansion. There is evidence of “looping” out of one strand of the repeat segment during DNA repair or replication that is thought to account for increased copy number.
In alleles that are large enough to be unstable, expansion occurs in germ cells, and an expanded mutant allele may be passed to the next generation.
Diseases caused by TNR expansion can be put in what two categories?
Those where the triplet repeat is in coding sequence, and those where the triplet repeat is in a noncoding portion of the gene.
In TNR related diseases where the triplet repeat is in coding sequence (exon), the trinucleotide repeating sequence is always?
CAG, coding for glutamine
What happens when runs of glutamine get too high?
When runs of glutamine get too big, the protein has a toxic effect on neurons. In affected individuals, fragments of the abnormal protein are visible as aggregates within neurons
The associated toxicity of too much glutamine on neuron is called?
a gain of function abnormality, since presence of the abnormal protein exerts a dominant deleterious effect. Although loss of function (absence) of huntington protein (for example) may have serious developmental consequences, it does NOT cause huntington disease. It’s the TNR expansion that accounts for the observed pathogenicity.
In TNR related diseases where the triplet repeat is in non-coding sequence (be it at either end of the gene transcript or in an intron), the trinucleotide repeating sequence is always?
variable, could be CGG, GAA, CTG, and locations vary within the genes
In TNR related diseases where the triplet repeat is in non-coding sequence (be it at either end of the gene transcript or in an intron), what is the result of TNR expansion?
no protein produced and RNA toxicity
In fragile X, an expanded CGG repeat in the 5’ UTR (in exon 1, upstream of the initiating AUG) leads to what?
methylation of the promoter region and silencing of the gene
In myotonic dystrophy, an expanded repeat in in the 3’ UTR causes what?
since it is downstream of the normal stop codon, it’s deleterious effects of repeat expansion are exerted at the RNA level
In Huntington disease, the CAG repeat is within the encoded protein. What results from expansion?
expanded polyglutamine causes toxic aggregates to form
What is genetic anticipation?
a condition where disease severity worsens in successive generations. Anticipation is explained by the fact that repeat copy number tends to expand each time it passes through meiosis (gametogenesis), and disease severity does correlate with repeat copy number.
leads to earlier progressive onset (not always) and increased severity in subsequent generations
What is the general approach to testing for TNR expansion?
Considered a targeted approach to testing since assays look directly at the TNR and provide an estimate of repeat number. Tests use patient DNA, and the result is reported as the number of repeats in each allele that the patient has. Main approach is PCR
T or F. Although the repeat copy number may vary, the flanking sequences are highly conserved
T. The goal of TNR testing is to generate a PCR product that spans the repeat segment and has defined end points in conserved flanking sequences. Thus, the size of the PCR product reflects the number of repeats present. The forward and reverse PCR primers would be specific for the relevant gene sequences on either side of the TNR repeat. Thus, for example, a PCR product from a mutant allele with 40 repeats would be 90bp larger than the product from a normal allele that had 10 repeats. Since a person has two copies of the HTT gene, both alleles would be sized. T
Notes on the accuracy of genetic allele testing
For all indications, testing has the capacity to provide an accurate estimate of allele size. While testing can determine allele size in a parent and, also in a fetus or child, it is not possible to make precise predictions about how much a parent’s unstable allele will expand in the next generation
Is Huntington disease the only TNR expansion disease related to coding segments?
NO, others include:
- Spinocerebellar ataxia
- Kennedy disease
- Machado-Joseph disease
Note that all these disorders are autosomal dominant, except for Kennedy disease, which is caused by expansion within a gene on the X chromosome
What is the inheritance mode of HD?
autosomal dominant
What is the average age of onset of HD and average life expectancy?
40 y/o; 15 years; progressive disease
What is the prevalence of HD?
~5;100,000
What causes HD?
TNR expansion leading to toxic aggregates of neurons involved in protein-protein interactions. The polyglutamine tract resulting from the expansion is near the N-terminus
Notes on the ranges of TNR repeats of normal, unstable, and mutated genes for HD.
normal- 10-35 (most common is 18)
unstable- 36-40 (reduced penetrance and may expand during meiosis)
mutated- >40 (symptomatic and will continue to expand in further generations)
T or F. People who are heterozygous for a repeat number in the low 40s will have the typical onset of symptoms in middle age.
T. However, while we think of HD as an adult onset disorder, those with very large expansions (100+) can express symptoms in the first or second decade of life.
What is the mode of inheritance of Friedrich ataxia?
autosomal recessive
What is the mode of inheritance of myotonic dystrophy type 1 (MDT1)?
autosomal dominant
What are some common symptoms of MDT1?
progressive muscle weakness and wasting, cardiac conduction defects, cataracts, prolonged muscle contraction, variable expressivity correlated to allele size
A common description of people with this disorder is that when they engage in a handshake or grab a doorknob, they often have difficulty letting go.
Doe MDT1 show anticipation?
Yes, there is earlier onset and increased severity with successive generations
In comparison, in HD, the primary evidence of anticipation is age of onset. Anticipation may vary in the specifics, but it is a general feature of TNR disorders.
How does TNR expansion cause disease in MDT1?
The pathogenic mechanism in DM1 is not well understood. The expanded TNR in the 3’ UTR of the mRNA causes the mRNA to accumulate in cells and to sequester certain RNA-binding proteins. This sequestration seems to exert a negative trans effect on the ability of other mRNAs to undergo normal translation (i.e. mutant alleles are transcribed but not translated). Thus, the mutation is thought to exert a dominant toxic effect on the DMPK gene and other genes
What is a normal DMPK gene typically used for?
the normal protein is a protein kinase involved in intercellular conduction and impulse transmission in heart and skeletal muscle
What is the prevalence of MDT1?
~1:20,000
What is friedrich ataxia? when is the typical onset?
slow progressive ataxia with onset at puberty. Cardiomyopathy is common
What does the normal FXN gene (that when mutated causes Friedrich ataxia) code for?
FXN encoded Frataxin, a nuclear-encoded gene involved in mitochondrial function
