Anticipation, the evidence for it & possible mechanisms

1. Anticipation, the evidence for it & possible mechanisms MRCPath course 4th September 2009 Yogen Patel

2. Anticipation • A phenomenon in which the age of onset of a disorder is reduced and/or the severity of the phenotype is increased in successive generations • ?Clinical phenomenon of anticipation truly existed or was it simply an artefact of clinical study • Lack of any plausible mechanism => ascertainment bias • Discovery of unstable expanding trinucleotide repeats in Fragile-X syndrome, Myotonic dystrophy and Huntington • Molecular developments showed it to be a characteristic of “dynamic mutations” • Arises from the tendency of disease repeats to expand and the inverse correlation between repeat length and age of disease onset

3. Disorders that show Anticipation

4. Huntington Disease (HD) • Anticipation occurs more commonly in paternal transmission of the mutated allele • Arises from instability of the CAG repeat during spermatogenesis (Duyao et al 1993, Telenius et al 1995) • Large expansions (i.e. an increase in allele size >7 CAG repeats) occur almost exclusively through paternal transmission • Most often children with juvenile-onset disease inherit the expanded allele from their fathers • Although on occasion they inherit it from their mothers (Nahhas et al 2005)

5. Paternal transmission (HD) Transmission of a HD intermediate allele to the male fetus is shown. Expansions in the sperm could be transmitted, resulting in the onset (in adulthood) of HD in offspring of a intermediate carrier father HD expansions had occurred before meiosis, as they were already present in mitotic diploid germ cells and might have arisen at any time from primordial germ cell segregation in utero through to the life-long, post-pubertal, spermatagonal stem cell Divisions (Pearson 2003, Yoon et al 2003) increase in expansions, with increasing paternal age probably through errors of repli. and/or repair during the pre –meiotic proliferative stages of spermatogonial divisions (Mangiarini et al 1997 )

6. Spinocerabellar Ataxia (SCA) • SCA3 (CAG)n • Typical inverse correlation between repeat number and age of onset • Paternal expansion bias is characteristic • Documented by repeat sizing of sperm. • SCA7 (CAG)n • One of the most unstable of all polyglutamine disease genes • Sometimes expanding by as much as 250 repeats in a single generation • Pronounced paternal expansion bias, with large expansions occurring in male germ cells • Frequently causing embryonic lethality that results in reduced transmission.

7. Myotonic Dystrophy type 1 (DM1) • Child with congenital DM1 is often born to a mildly affected or asymptomatic mother • Inheritance from father is possible but anticipation typically occurs in maternal transmission (paternal transmission is normally small rpt expansions) • Possible selection in sperm against full mutation • Both pre-meiotic (in the grand maternal uterus) and post-zygotic events probably contribute to the large DM1 CTG expansions (>1,000 repeats)

8. Fragile X Syndrome (FRAX) • May demonstrate anticipation in some families • Anticipation found in families with members affected with fragile X syndrome is not classic (e.g. in DM) as premutation may be transmitted for many generations with little or no presentation of clinical symptoms until a full mutation is produced • Occurs when a un/affected premutation or mosaic mutation carriers transmit unstable FMR1 alleles to their offspring (e.g., transmission from a grandfather who carries a premutation to his daughter, whose premutation expands into a full mutation when she transmits it to her son)

9. Fragile X Syndrome (FRAX) • Fully expanded mutations of greater than 200 copies are not found in sperm, so males transmit only premutations • Selection against FM in sperm • Normal alleles contain 6–45 triplets punctuated by one or more AGGs (@rpt 9,10,19,20,30), which are considered to have a stabilizing influence on the repeat tract • AGGs appear to anchor the segment against repeat expansion probably by the disruption of DNA secondary structures that may from during replication • Disease alleles contain expansions beyond 200 and up to 2000 repeats, with no AGG interruptions • Pathogenic expansions result exclusively from maternal transmission

10. Maternal transmission (FRAX) Transmission of a FRAXA premutation to full mutation might occur during either oogenesis or early post-zygotic events. FRAXA males have fully methylated expanded CGG in all tissues except sperm, where only unmethylated premutation lengths exist. Oogenic meiosis begins in utero, arrests for years, resumes only at ovulation and is not completed until after fertilization, possible role of highly extended time for oogenic meiosis

11. Mechanisms of dynamic mutations • Repeat instability shows complex patterns between and within tissues that vary with developmental, epigenetic, proliferative and possibly environmental cues • So maybe processes occur individually or in combination, depending on the tissue, proliferative status and developmental stage of the cell • Concentrate on those during DNA replication, repair and recombination • All trinucleotide repeat (TNR) diseases involve mutations during parent-to-offspring transmission, implicating germline mutations in TNR instability • Paternal and maternal expansion biases are evident and might be driven by processes that are specific to sperm or oocyte development • Both pre-meiotic and post-zygotic events probably contribute to the large DM1 CTG expansions

12. Mechanisms of dynamic mutations • Investigated in sperm, transgenic mice and E.coli • Replication slippage - caused by misalignment during DNA replication (slipped strand mispairing) • The formation of unusual DNA secondary structures (e.g. hairpin, Quadruplex or H-DNA) leading to replication fork blockage and DNA slippage during lagging-strand synthesis might facilitate instability • Mis-processing of Okazaki fragments in replication • Unequal crossing-over, in which alleles mispair during meiotic crossing-over, resulting in one expanded and one contracted tract

13. Replication (origin-switch) Replication across the hairpin might result in expansions or deletions for nascent or template hairpins, respectively

14. Replication (fork-shift) Alterations in the Okazaki initiation zone (OIZ) relative to repeat and Okazaki initiations might influence the formation of hairpins Shifting the location of the origin, but not the direction of replication, might also affect instability by altering the location of the repeat relative to the OIZ

15. Repair During genome duplication. Following replication fork stalling, the induction of a double-strand break (DSB) (first column) OR fork reversal might result in length alterations being maintained during proceeding rounds of replication (second column) During genome maintenance, the presence of a DSB or a nick within the repeat tract might lead to strand fraying and TNR-specific structures. Failure of repair to correct the alteration will result in length differences

16. Recombination Homologous recombination between allelic repeats might occur with or without the exchange of flanks

17. Polyalanine Diseases • Rare AD developmental malformations • Characterized by small expansions • Repeats encoding alanine tracts • In contrast to polyglutamine expansions these are small and stable, not exceeding 30 triplets • Most are composed of ‘‘imperfect’’ alanine tracts, inc (GCG, GCA, GCC, and GCT codons) • This argues against an expansion mechanism involving single-strand, hairpin-like secondary structures as in other repeat disorders • More plausible mechanism for polyalanine expansion is unequal crossing-over, in which alleles mispair during meiotic crossing-over, resulting in one expanded and one contracted tract.

18. References • Encyclopedia of Molecular Cell Biology and Molecular Medicine, 2nd Edition. Volume 15, Edited by Robert A. Meyers 2005 • Introduction to risk calculation in genetic counseling By Ian D. Young 2007 • MacDonald et al 1993 Journal of Medical Genetics 1993;30:982-986 • Duyao et al 1993 Nat Genet. Aug;4(4):329-30 • Telenius et al 1995 Hum Mol Genet May;4(5):974 • Nahhas et al 2005 Am J Med Genet Sep 1;137A(3):328-31 • Cleary & Pearson 2005 Trends Genet. May;21(5):272-80. Review • Pearson 2003 Trends Mol. Med. 9,490–495 • Yoon et al 2003 Proc. Natl Acad. Sci. USA 100, 8834–8838 • Kaytor et al 1997 Hum. Mol. Genet. 6, 2135–2139 • Mangiarini et al 1997 Nature Genet. 15, 197–200 • Cleary & Pearson 2005b Nat Rev Genet. Oct;6(10):729-42. Review