Dynamic Mutation A Trinucleotide Repeat Disorder

DYNAMIC MUTATIONS …now the defect found in Arabidopsis thaliana

Sandep Yadav

DYNAMIC MUTATION ??  Unstable heritable element where probability of mutation is function of no. of copies of mutation.

Unique Properties:  Expanding, Unstable DNA repeats.  Repeats are usually polymorphic in copy number, with mitotic and meiotic instability.  Copy number related to the severity and/or age of onset  Imprinting effects  Reverse mutation - The mutation can revert to normal or to a premutation carrier state.  Founder effects are observed in association with dynamic mutation as a higher frequency of particular allele in the affected population.

TYPES OF DYNAMIC MUTATION

Genic location of disease-associated repeats

Expanded repeats that cause disease are found in many different genic locations

How do expanded repeats cause disease?  Gain of function: • Repeats in protein coding sequences - toxic proteins e.g. polyglutamine disease. • Repeats in RNA coding regions - altered RNA function e.g. splicing in Myotonic dystrophy.  Loss of function: • Repeats in non-coding regions - reduced transcription or translation e.g. fragile X syn. & IIL1 in A. thaliana

Processes involved in repeat expansion

Repeat expansion can take place in the germ line or in the somatic cell

Genetic Variation in A. thaliana’s Bur-0 strain     

Strains were grown at 27 C and short day condition All except Bur-0 strain showed normal growth. Bur-0 strain showed abnormal growth and no flowering. Phenotype was named as “irregularly impaired leaves”(iil). At 23 C this defect disappeared but worsen at 30 C.

Pf-0 at 27 C

Bur-0 at 27 C

Bur-0 at 23 C

Genetic basis of iil defect

Location of expanded repeats

RNA level of At4g13430

 Interval mapping by PCR revealed expansion of GAA repeats in Chrm. 4  At 23 C RNA level didn’t differ between genotypes while  RNA level decreased by 50% in Bur-0 strain at 27 C

Overexpression of mature Bur-0 transcript

Bur-0 plant at 27 C (5 week)

IIL expression w.r.t. Overexpression

Bur-0 plant harboring 35S:IIL1-Bur-0 transgene

GAA repeat and somatic variability  Phenotypic revertant showed the variability in the repeat no.  EMS promotes the contraction of triplet repeats.  After treatment 156 phenotypic revertants, 27 had reduced triplet copy no.

DNA blot analysis of IIL1 triplet repeat containing region

PCR analysis of IIL1 repeat.

CONCLUSION: • Bur-0 strain provided a genetically tractable model for TNR studies. • Phenotypic revertants highlight the potential of IIL1 repeats for further study. • The molecular mechanisms underlying the repeat disorders involve either loss of protein function or gain of function at the protein or RNA level. • Repeat instability involves the formation of unusual DNA structures during DNA replication, repair and recombination. • Simple repeats can be associated with phenotypic variability of evolutionary significance.

Suggested reading: •Kumar, S., Todesco, M., Schneeberger, K., Harilal, R., Balasubramanian, S., Weigel, D. (2009). A Genetic Defect Caused by a Triplet Repeat Expansion in Arabidopsis thaliana. Sciencexpress 1126: 110. •Al-Mahdawi, S. et al. (2008). The Friedreich ataxia GAA repeat expansion mutation induces comparable epigenetic changes in human and transgenic mouse brain and heart tissues. Hum. Mol. Genet. 17, 735-746. •Richards, R.I. (2001). Dynamic mutations: a decade of unstable expanded repeats in human genetic disease. Human Molecular Genetics 10: 21872194. •Pearson, C. E., Edamura, K. N., Cleary, J. D. (2005). Repeat instability: mechanisms of dynamic mutations. Nat. Rev. Gen. 6: 729-742.

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Expansion mechanisms Replication associated instability

Repair associated instability

Recombination associated