Diane Cairns Liverpool

1. ‘For many disorders with a genetic basis, phenotype variability is dependent upon which parent the mutant sequence was inherited from’. Discuss this statement in relation to trinucleotide repeat diseases using as many examples as possible. Diane Cairns Liverpool

2. Key Words Trinucleotide Paternal transmission Maternal transmission Intergenerational expansion (contraction) Phenotypic variability Anticipation

3. Trinucleotide repeat diseases Trinucleotide repeat diseases are associated with symptoms in a wide range of tissues, nearly all involve neurodegeneration or some other aspect of cognitive dysfunction. Almost all display wide symptomatic variability in terms of both severity and age of onset, ranging from mild late onset forms to severely debilitating, often fatal, congenital or childhood forms. The repeats can be translated (exonic) or untranslated (intronic / UTR). Expanded trinucleotide repeats which are pathogenic can be dynamic (unstable) or fairly stable.

4. They can be grouped according to size: Normal - polymorphic range not associated with disease. Intermediate/pre-mutation - no clinical features of disease but repeat chance of increasing to pathogenic range meiotically. Pathogenic/Full mutation - usually fully penetrant showing disease, tend to be ‘pure’ repeats. Coding Unstable at ~ 29-35 repeats, Expansions tend to be < 100 repeats. The changes in tract size are modest ≤ 10 per generation. Non–coding Expansions can be 1,000’s of repeats. Can increase by 100’s of repeats per generation. Anticipation The disease becomes more severe and has an earlier age of onset in successive generations. There is usually an inverse correlation between pathogenic repeat size and age of onset / disease severity.

5. The parental origin of the mutation is important factor in anticipation. Paternal origin mutation Expansions in coding repeats occur intergenerationally when inherited from the father. These expansions occur during cell replication, small changes occur per division – 1-3 repeats. Large repeat tracts in sperm are shortened during fetal development (genetic selection among sperm for cells with smaller repeats can’t be ruled out). Maternal origin mutation Expansions in non-coding repeats occur intergenerationally when inherited from the mother. Large expansions can occur in non-dividing cells (quiescent oocytes) via a repair-dependant mechanism.

6. Paternal Origin Majority are due to CAG expansions, the expanded repeat is translated into a polyglutamine tract. E.g. HD, SCA2, SCA7. Huntington disease (HD) Huntington disease is a progressive and fatal disorder characterised by motor, cognitive and psychiatric disturbances. • Gene: HTT. • Repeat: CAG repeat in exon 1. • Repeat sizes: normal 26-29, intermediate 29-37, affected 38-180. • Reduced penetrance 36-40 repeats - individuals carrying these mutations may not develop the disease. • Inheritance: Autosomal dominant.

7. Huntington Disease Large expansions (>7 CAGs) occur almost exclusively through paternal transmission due to the instability of the CAG repeat during spermatogenesis. Children with juvenile-onset disease inherit the expanded allele from their father. Offspring of affected mothers are more likely to show no change or contractions – even if the maternal allele is of a similar size to one that expands through a paternal transmission Genotype/phenotype CAG pathogenic repeat range correlates inversely with age of onset of symptoms. Juvenile onset very large repeat >60. Reduced penetrance "grey area" repeats - 36-40 repeats – in individuals carrying these mutations see very late onset or non penetrance. HD is a true dominant as individuals homozygous for two repeat expansions show a similar disease phenotype to heterozygous individuals.

8. Spinocerebellar ataxia’s (SCA) Patients generally present with progressive cerebellar ataxia & may have other neurological signs. Average age of onset 3rd - 4th decade however can vary widely (juvenile & very late onset cases). Death usually within 10 - 15years of onset – variable (i.e.: juvenile cases = shorter time between onset and death).

9. SCA2 Gene: ATXN2. Repeat: CAG repeat in coding region. Repeat sizes: normal <31, intermediate 31-3, affected 32-200. Inheritance: Autosomal dominant. SCA2 normal repeats usually interrupted with CAA repeats - interruptions thought to stabilise alleles. Paternal transmission of alleles shows meiotic instability and results in anticipation. E.g. - report of a man - 43 repeats, onset of symptoms at 22 – had infant with apnea, hypotonia, dysphagia - 202 CAG repeats. Genotype/phenotype Inverse correlation between size of repeat and age of onset. Increasing severity of the disease with larger pathogenic repeat. Variability in age of onset in individuals with fewer than 40 repeats. Homozygosity for an expanded allele does not appear to influence age of onset.

10. SCA 7 Blindness is a common symptom in affected adults. SCA7 • Gene: ATXN7. • Repeat: CAG repeat in coding region. • Repeat sizes: normal 4-17, intermediate 28-33, affected >36- >460. • Inheritance: Autosomal dominant. SCA7 - most unstable of the disorders with CAG repeats. SCA7 expanded paternal repeats - very unstable meiotically leading to large increase in repeats in offspring and juvenile onset. A child may be diagnosed with a sporadic neurodegenerative disease years before a parent or grandparent with the gene expansion becomes symptomatic. Genotype/phenotype Generally inverse correlation between size of repeat and age of onset. Increasing severity of the disease with larger pathogenic repeat.

11. Maternal Origin The pathogenic mechanism of these triplet repeat diseases varies e.g. loss of function, aberrant mRNA processing, altered gene expression. E.g. FRAXA & DM1. Fragile X (FRAXA) Fragile X syndrome is characterised by mental retardation. Behavioural features include, autistic spectrum disorder, gaze avoidance, hyperactivity, hand-flapping and perseverative speech. Physical features include long face with large protruding ears, macro orchidism, hyperextendible finger joints. • Gene: FMR1 • Repeat: CGG repeat expansion in untranslated first exon. • 1% of cases due to other mutations. • Repeat sizes: Normal 6-50, intermediate 55-200, affected 200 – thousands. • Inheritance: X-linked dominant.

12. Fragile X (FRAXA) Expansion only seen on maternal transmissions. Males who inherit a full mutation allele from their mother harbour the expanded allele in their somatic cells BUT do not transmit the expanded allele to their progeny. Males only transmit premutations (normal transmitting males – NTMs – Sherman paradox’). NTM’s are unaffected and pass on the high risk allele to their unaffected daughters who then have affected children. The risk of expansion of the premutation upon maternal transmission is related to its size and has been estimated at <20% for small premutations (<70 CGG) and >80% for large premutations (>80 CGG). The loss of the two AAG interruptions in the CGG tract also leads to increased instability.

13. Fragile X (FRAXA) Genotype/phenotype Females display a much more varied phenotype than males, due to skewed X inactivation. The risk for instability of alleles with 41-49 repeats when transmitted from parent to child is minimal. Any changes in repeat number are typically very small (± 1 or 2 repeats). The frequency and magnitude of repeat instability increases with alleles containing more than 50 repeats. Instances of maternal transmission of 59 repeat alleles producing full mutations have been reported, this is the smallest repeat known to expand to full mutation in a single transmission.

14. Myotonic Dystrophy Type 1 Multisystem disorder that affects skeletal and smooth muscle as well as the eye, heart, endocrine system, and central nervous system. Mild - cataract and mild myotonia life span is normal. Classic - muscle weakness and wasting, myotonia, cataract, and often cardiac conduction abnormalities, may have a shortened life span. Congenital - hypotonia and severe generalized weakness at birth, often with respiratory insufficiency and early death. • Gene: DMPK. • Repeat: CTG repeat in 3’UTR. • Repeat sizes: normal 5-37, intermediate 37-50, affected >50. • Inheritance: Autosomal dominant.

15. Myotonic Dystrophy Type 1 Full mutations are almost exclusively transmitted maternally – length of the expansion correlates with maternal age. Repeats contract in germ cells of DM1 males if a disease-length repeat tract is large – plus the contraction frequency during male transmission increases with allele length. There is a rapid increase in CTG repeats through maternal line to congenital (though congenital cases where father affected). Alleles containing repeats longer than 35 CTG are unstable and may expand during meiosis. Genotype/phenotype Broad correlation repeat size and clinical severity (within families rather than between).

16. Conclusion Trinucleotide repeat diseases show intergenerational phenotypic variability. In the dynamic coding repeat diseases such as Huntington disease the expansion is inherited from the father and there is an inverse correlation between the size of the inherited repeat and the age of onset of and severity of symptoms. Sperm divide rapidly and this allows small changes to accumulate through the paternal germline. Long coding expansions tend to be selected against if the encoded gene product is too dysfunctional for viability. In non-coding repeat diseases such as Fragile X full mutations are transmitted maternally and there is not always a correlation with repeat size, age of onset and disease severity. Large expansions are more likely to be tolerated in untranslated or intronic regions, these large expansions occur in quiescent oocytes and are therefore not shortened like paternal expansions during replication and can expand to a far greater extent.

17. References Gene Reviews – http://www.ncbi.nlm.nih.gov/sites/GeneTests. Mechanisms of trinucleotiderepeat instability during human development., Cynthia T McMurray, Nature Reviews, 11: Nov 2010, 786-799. Fernandez-Lopez, L. et al.Induction of instability of normal length trinucleotide repeats within human disease genes. J. Med. Genet. 2004. McGlennen, R. C. Dynamic mutations pose unique challenges for the molecular diagnostics laboratory. Clinical Chemistry. 42(10): 1582-1588. Richards, R. I. Dynamic mutations: a decade of unstable expanded repeats in human genetic disease. Human Molecular Genetics. 10(20): 2187-2194. 2001. Sutherland, G. R. and Richards, R. I. Simple tandem DNA repeats and human genetic disease. Proc. Natl. Acad. Sci. 92: 3636-3641. 1995. Andrew, S. E. et al. Rethinking genotype and phenotype correlations in polyglutamine expansion disorders. Human Molecular Genetics. 6(12): 2005-2010. 1997. Molecular Features of the CAG Repeats of Spinocerebellar Ataxia 6 (SCA6), Matsuyama et al, Hum. Mol. Genet. (1997) 6 (8): 1283-1287.