Origins of pseudogenes and their involvement in human disease

1. Origins of pseudogenes and their involvement in human disease Cat Yearwood St. George’s, London

2. Keywords • Pseudogene • Non-processed (duplicated) • Processed (retrotransposed) • Unequal crossing over • Reverse transcription • Gene conversion • Gene regulation through RNA

3. Pseudogene = defective copy of all or part of a gene or its coding sequence • High homology to original gene, but with some sequence changes/ missing parts of gene that make it essentially non-functional • Common in human genome

4. Two main types • Non-processed pseudogenes or duplicated pseudogenes • Copy of all or part of genomic DNA of gene including introns, promoter etc. • Formed by duplication of original gene by mechanisms such as unequal crossing over of homologous chromosomes or unequal sister chromosome exchange, or due to errors during repair of double-stranded DNA breaks Tandem gene duplication due to unequal crossing over – misalignment of repeat units

5. If whole gene duplicated then may be functional initially, however is an extra copy and so not subject to same natural selection pressures and so evolves more rapidly, accumulating mutations that make it non-functional and so becoming a pseudogene • Alternatively, may acquire a related function and become a different, but related functional gene

6. Non-processed pseudogenes often located next to or near eachother due to mechanism of duplication, e.g. CYP21A2 and CYP21A2P (congenital adrenal hyperplasia) and SMN1 and SMN2 (SMA) • However, can be located on different chromosomes e.g. NF1 Many partial copies of duplicated gene located near centromere of many chromosomes

7. 2.Processed or retrotransposed pseudogenes • Formed from RNA • Spontaneous reverse transcription of a RNA transcript of a gene by a reverse transcriptase enzyme results in a DNA copy of RNA transcript which is then inserted into the genome • This DNA copy of an RNA transcript has no introns or promoter, but does have UTRs and poly A tail. • Since no promoter, cannot be expressed – often termed “dead on arrival” • Exceptions if happen to integrate adjacent to an existing promoter – can be expressed (PTENP1 pseudogene thought to be an example of this)

8. Role in human disease:1. Congenital adrenal hyperplasia (CAH) • Recessive disease mostly caused by 21 hydroxylase deficiency • CYP21A2 is functional gene • CYP21A2P is non-processed pseudogene in adjacent tandemly repeated gene cluster with ~97% sequence identity • Almost all pathogenic mutations to functional gene brought about through interaction with pseudogene • Due to gene conversion (non-reciprocal exchange between similar sequences resulting in the donor sequence “correcting” the acceptor sequence, resulting in a gene with part or all of its sequence derived from another similar gene or another copy of the same gene) • Due to unequal crossing-over of the repeat unit resulting in loss of functional gene • Von-Willebrand disease type 3 (recessive) –also get gene conversion causing a proportion of mutations (often see several pathogenic mutations in cis due to gene conversion – need to distinguish true compound heterozygotes)

9. Theory of gene conversion

10. Mechanisms of mutation in CAH

11. 2. Spinal Muscular Atrophy (SMA) • Non-processed pseudogene SMN2 that is both transcribed and translated. 99% homology with SMN1, the crucial gene for SMA pathogenesis. • Knockout of ESE leads to skipping of exon 7 in majority of SMN2 transcripts, but some full length protein still produced • Majority of mutations in SMA (a recessive disorder caused by loss of function of SMN1 protein) are gene deletions of SMN1 or gene conversion of SMN1 into SMN2. These can both be tested for using an MLPA kit which detects a loss of the SMN1 exon 7 probe binding site

12. Crucial role for SMN2 in SMA disease severity • Number of copies of SMN2 variable in general population • Since SMN2 produces low levels of normal protein it can partially compensate for lack of SMN1 • Range of severity and age of onset seen in SMA patients • Mainly attributable to number of copies of SMN2, those with severe disease typically having 1 or 2 SMN2 copies (loss of SMN1 likely through true deletion) and those with milder disease 3 or more SMN2 copies (loss of SMN1 likely to be due to gene conversion of SMN1 into another copy of SMN2)

13. 3. PTEN – tumour suppressor gene • Processed but expressed pseudogene PTENP1 • Not translated due to abolition of start codon • However mRNA of pseudogene has role in regulating PTEN expression levels through competing for miRNAs • PTENP1 protects PTEN from binding of miRNAs as have many of the same miRNA binding sites in their 3’ UTRs

14. Without PTENP1 mRNA, more miRNAs bind to PTEN mRNA, so reducing levels of PTEN protein, cells very sensitive to variation in PTEN protein levels • Human cancers often show heterozygous loss of PTENP1 pseudogene • Despite not producing a protein of its own, PTENP1 is effectively a tumour suppressor itself as it regulates PTEN protein production • Implications for other pseudogenes that produce mRNA – may have a function through their RNA transcript despite producing no functional protein

15. Further reading • Strachan and Read, 1999. Human Molecular Genetics 2. Chapters 7 and 9 • www.geneclinics.org – SMA, CAH and VWD • www.mrc-holland.com – information on SMA MLPA kit P021 • Poliseno et al., 2010. A coding-independent function of gene and pseudogene mRNAs regulates tumour biology. Nature465: 1033-1040.