Fragile X Syndrome

Genetics, Epigenetics & the Role of Unprogrammed Events in the expression of a Phenotype

A loss of function of the FMR-1 gene results in severe learning problems, intellectual disability (retardation) and, for some, autism or autism- like behaviors

Fragile X syndrome: variable expressivity and incomplete penetrance

1 DNA methylation-induced silencing of expanded mutant alleles results in reduced FMR-1 transcription  loss of gene function  severe effects on nervous system [FMR1 = Fragile X Mental Retardation 1] http://ghr.nlm.nih.gov/gene/FMR1

Two unprogrammed events must occur for the abnormal phenotype to be expressed: • expansion of the number of CGG repeats via a DNA replication error (meiosis or mitosis) or a crossing-over error during meiosis • methylation of cytosines in the expanded repeats -- chance of methylation increases with the number of CGG repeats

First a very short Primer on Epigenetics

Epigenetic effects or changes– heritable changes in gene expression that do not involve changes in the DNA sequence

What do we mean by gene expression?

4 Epigenetic mechanisms are important in many cell processes

• Progressive restriction of cell fate during embryonic development involving changes in chromatin structure associated with loss of pluripotency, lineage restriction and cell differentiation • X inactivation during embryogenesis in mammalian females – one X chromosome in each cell becomes highly condensed and transcriptionally inactive • Imprinted gene function in mammals -- the level of gene expression is dependent on whether the gene copy came from mom or dad • Adult neuronal plasticity and neurogenesis --changes in chromatin marks and transcriptional networks associated with sustained neuronal activity, mood disorders and addiction • Environmental effects (such as diet or stress) on phenotype may involve epigenetic changes in gene function • Abnormal gene expression (such as in Fragile –X syndrome or cancer cells): epigenetic drift

5 Mechanisms involved in chromatin modifications

Nature. 2010 June 10; 465(7299): 728–735. Five broad and interrelated mechanisms are known to affect chromatin structure. All five have been shown to be essential contributors to the development and cell fate determination of tissues 1. DNA methylation, 2. histone modification 3. insertion of histone variants 4. remodeling complexes 5. non-coding RNAs

DNMT

DNMT = DNA methyltransferase SAM= S=adenosyl methionine

7 DNA methylation status is heritable via this mechanism:

Dnmt1 and associated proteins scan newly replicated DNA for hemi- methylated sites and methylate the CpG’s on the newly synthesized strands

8 mechanisms of gene inactivation via DNA methylation: http://fire.biol.wwu.edu/trent/trent/DNAsilencing.pdf

Understanding Fragile X

• In the Fragile –X family we are considering, mom is heterozygous for a normal allele with 29 repeats and a “premutation” allele with 65 CGG repeats • A premutation allele is at higher risk for a further increase in CGG repeats than a normal allele (we’ll talk about why this is the case in a later lecture)

Alleles with expanded CGG repeats are at higher risk for DNA methylation- induced silencing of FMR-1 transcription than alleles with fewer CGG repeats

http://ghr.nlm.nih.gov/gene/FMR1

I-2 has a normal allele with ~ 29 CGG repeats and an allele with an 29/65 expanded repeat in the pre-mutation range (~ 65 repeats).

~66 & 300 ~130-200 ~100-270 ~170-200 ~300 ~ 29 ~130-670 ~170 ~170 100% ~200 & 530 45% 3% 3% 0% 45%

IQ 72 120 92 122 58 62 CmepG (methylated) alleles shown in bold and larger font; unmethylated copies in smaller font Overall methylation (at a specific site in the repeated area) indicated by degree of shading and by % of total gene copies examined NOTE: all offspring inheriting the permutation (fragile X allele with ~65 repeats) are somatic mosaics – different cells have alleles with different numbers of repeats and levels of DNA methylation.

12 Somatic mosaicism is the presence of genetically distinct populations of somatic cells in a given organism. It is frequently masked, but it can also result in major phenotypic changes

Mosaicism can be caused by • mutations • epigenetic alterations of DNA • chromosomal abnormalities • spontaneous reversion of inherited mutations

Nature Reviews Genetics 3, 748-758 (October 2002) | doi:10.1038/nrg906

13 Genes as a Mirror of your Life rather than your Life as a Mirror of your Genes

Epigenetic mechanisms are affected by several factors and processes including development in utero and in childhood, environmental chemicals, drugs and pharmaceuticals, aging, and diet

https://commonfund.nih.gov/EPIGENOMICS/figure.aspx

• Restriction of pluripotency • X inactivation

. SOLID BLACK ARROW Many epigenetic modifications become biologically stabilized at a particular stage of development, and are maintained subsequently throughout the lifetime of the organism DASHED BLACK ARROW However, at certain genomic loci, epigenetic marks can readily change over time. This ‘epigenetic drift’ is thought to depend both on environmental and intrinsic factors. This diagram shows an example of how epigenetic drift can lead to the loss of DNA methylation, with consequences for gene expression and cellular and organismal phenotypes. Nature Reviews Genetics Feb 2012