Dynamic mutations

1. Dynamic mutations are an important mutation class. Describe the full range of methods and strategies that are currently being used to diagnose 3 inherited diseases of your choice. Choose examples which maximise the number of methods and strategies you describe.Sarah Waller Key words: Trinucleotide repeat; Huntington disease; polyglutamine; myotonic dystrophy; Fragile X syndrome; Southern blot; triplet-primed PCR; exclusion testing. Useful reference: La Spada & Taylor, Nat Rev Genet 11:247 (2010)

2. Dynamic mutations • A dynamic mutation is an unstable heritable element, where the initial change to the DNA sequence alters the chance of further changes to it. • These mutations typically occur in areas of tandem repeats, and are exemplified in the trinucleotide repeat disorders. • Triplet expansion is caused by slippage during DNA replication. ‘Loop out’ structures may form during DNA replication due to the repetitive nature of these DNA sequences; if this occurs on the daughter strand the number of repeats will be increased. • If the loop out structure occurs on the parent strand the number of repeats can decrease and the mutation can revert to a normal or premutation carrier state.

3. Trinucleotide repeat disorders • The length of the repeat determines the probability and extent of its pathogenicity. • Anticipation – the tendency of the age of onset to decrease and the severity of symptoms to increase through successive generations due to continued repeat expansion. More common in paternal transmission due to instability of the CAG repeat in spermatogenesis. • Most of these diseases have neurological symptoms. • Methods & strategies currently in use to diagnose: • Huntington disease • Myotonic dystrophy • Fragile X syndrome

4. Huntington disease (HD) • HD is a progressive neuronal degenerative disease caused by expansion of a CAG repeat in exon 1 of the HTT gene, resulting in production of a toxic protein containing a polyglutamine repeat. • Pathogenicity of HD allele is determined by its size: • 26 or fewer repeats – normal • 27-35 – intermediate; due to repeat instability the allele may expand to a HD-causing allele in subsequent generations • 36-39 – reduced penetrance HD-causing allele; patient is at risk but may not develop HD • 40 or more – full penetrance HD-causing allele • There is a significant correlation between CAG repeat size and the age of onset and rate of deterioration of motor & cognitive function • CAG tracts interrupted with CGG are more stable  Accurate sizing of HD allele crucial for proper diagnosis & genetic counselling

5. PCR of HD alleles • HD alleles can be accurately sized by PCR followed by capillary gel electrophoresis: • Sequence analysis can be performed on gel purified products of intermediate or reduced penetrance alleles to determine if there are any stabilising CGG interruptions.

6. Triplet-primed PCR (TP-PCR) TP-PCR can also be used to detect the presence of CAG repeats: • 3 primers used: forward (different to that used in genomic PCR), triplet repeat primer, consisting of GTC repeats linked to annealing site for 3rd primer. • Triplet repeat primer anneals randomly along CAG tract, generating ladder of products.

7. Exclusion testing • Exclusion testing can be offered to couples who wish to have prenatal testing but who do not want to know their own HD status. Analysis of polymorphic markers is carried out to determine if the fetus has inherited the HD locus from the affected grandparent:

8. Diagnosis of HD using PCR & TP-PCR • Both of these methods are: • cheap • quick and straightforward to perform • have very high specificity and sensitivity

9. Myotonic dystrophy • DM1 caused by CTG repeat expansion in 3’ UTR of DMPK gene, which encodes a myosin kinase. The predominant symptom in classic DM1 is distal muscle weakness. • Expanded RNA sequesters muscleblind 1 (MBNL1) protein, which leads to down-regulation of mRNAs which normally interact with MBLN1, eg genes involved in Ca signalling. Alternative splicing of certain genes is also disrupted. • As with HD, pathogenicity is determined by allele size: • - 37 or fewer repeats: normal • - 38-49 repeats: premutation, patients asymptomatic but children at risk of inheriting larger allele with pathologically expanded repeat • - 50-4000 repeats: full penetrance

10. Myotonic dystrophy • Larger repeat expansions correlate with earlier age of onset and more severe disease phenotype, particularly if less than 400 repeats. Alleles with greater than 1000 repeats give rise to congenital DM1, a severe disease often presenting before birth. • Anticipation usually occurs through maternal inheritance – very large alleles believed to be toxic to sperm. However smaller alleles inherited from father can also expand to full mutation. • Sizing of alleles performed by PCR; if only one allele detected then Southern blotting is performed since large expansions are beyond detection limit of PCR & capillary electrophoresis. This will distinguish patients with homozygous normal alleles and those with one normal and one expanded allele.

11. Southern blotting procedure

12. Diagnosis of DM1 using Southern blots • Time consuming • Expensive • Requires large amounts of DNA • Requires skill and considerable training • Use of DIG probes has advantages over radioactively labelled probes (sensitivity, exposure time & safety) • Can detect mosaics

13. Fragile X syndrome (FRAX) • FRAX is the leading cause of mental retardation and autism, with an incidence of approx 1:2500. • Caused by large expansion to more than 200 repeats of a CGG repeat in the 5’ UTR of the FMR1 gene. Concomitant hypermethylation of these full mutations causes transcriptional silencing of the FMR1 gene. • Females with a full mutation are often unaffected but can have mild learning disability. • Premutation alleles (59-200 repeats) are not hypermethylated but can expand to a full mutation if transmitted from the mother. • 20% of females with a premutation develop POF; males with a premutation are likely to develop fragile X associated tremor/ataxia syndrome. Due to toxic gain of function of FMR1 RNA.

14. Diagnosis of FRAX • Accurate sizing of the FMR1 allele is important due to the possibility of premutation alleles expanding to full mutations in a single generation. Intermediate alleles (50-58 repeats) may also show instability but over several generations. • Fluorescent PCR and capillary electrophoresis can detect up to about 130 repeats in males and only about 100 repeats in females due to preferential amplification of the smaller allele. • Male samples in which no band is detected or female samples in which only one band is detected are assayed by Southern blot. • To determine the methylation status of the FMR1 CGG tract, patient DNA is digested with both EcoR1 and a methylation-sensitive enzyme such as Eag1.

15. FMR1 southern blot • FMR1 gene on the inactivated X chromosome in a female and fully expanded FMR1 mutations will be methylated & not digested by Eag1. • Unmethylated FMR1 in normal males (1,7,8 & 10) and on active X in females (2,5,9) gives small band (NL). • Methylated FMR1 on inactive X doesn’t cut with Eag1 and gives larger NL band (2, 5, 9) • Unmethylated premutation gives slightly larger band (4, PM) • Methylated full mutation gives much larger smeared band (FM, 2, 4, 6) EcoR1 Eag1 (CGG)n Probe EcoR1

16. PCR for large FMR1 expansions • The disadvantages of Southern blotting techniques for diagnosis of FRAX (expensive, time-consuming, requires large amount of DNA & technical difficulties) have lead to the recent development of long range PCR methodologies that can amplify very large GC-rich FMR1 expansions, eg the Asuragen Amplidex FMR1 kit. • Able to amplify FMR1 alleles with large GC-rich expansions that would not be detected by current PCR techniques. Males with no bands seen by PCR and females with single bands can therefore be resolved, and premutations can be sized. • Rapid results and easy to perform.

17. Asuragen AmplidexTM FMR1 PCR kit Example data from Amplidex TP-PCR assay. A & B show AmplidexTM TP-PCR results for two females who had a single band on LR-PCR. The main image shows the full view of the run & the inset highlights the TP-PCR products. Sample A shows both an expansion of >200 repeats (pink area) and a range of TP-PCR products that confirm an expansion is present. Sample B shows no evidence of expansion and is therefore homozygous for a 30 repeat allele. (Data from Nicola Ibberson, Cambridge) A B Use of such PCR kits will drastically reduce the number of samples that need Southern blots (males with 0 bands, females with single bands & intermediate/premutation patients), which are still required for determining methylation status of full expansions. However release of a methlyation-sensitive long-range FMR1 PCR kit from Asuragen is imminent and may eliminate need for Southern blotting for diagnosis of FRAX.

18. Conclusions • Instability of dynamic mutations renders intermediate sized alleles susceptible to expansion to pathogenic mutations in subsequent generations. Accurate sizing of these disease-associated genes is vital for diagnosis & genetic counselling. • Fluorescent PCR and TP-PCR are cheap, straightforward and accurate methods for detecting small expansions, particularly in diseases where the triplet expansion occurs in the coding region of the gene. • Southern blotting is the current method of choice for detecting large expansions and methylation status, however it is expensive, time-consuming and users often experience technical difficulties. • Recent developments of PCR methodologies that can amplify large GC-rich expansions will help to reduce the need for Southern blots in genetic diagnosis.