Techniques for Pseudogene Identification and Prediction of Gene

Conversion

University of IowaCenter for Bioinformatics and Computational Biology (CBCB)Coordinated Laboratory for

Computational Genomics (CLCG)http://genome.uiowa.edu

Jared Bischof, Todd Scheetz, Tom Casavant, Ed Stone, Val

Sheffield, Terry Braun

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Overview

• Definition of pseudogenes and gene conversion

• Describe methods for identification of pseudogenes in the human genome

• Identify characteristics of pseudogenes that may implicate gene conversion

• Validate through sequence disablements• Rank pseudogenes as gene conversion

candidates• Examine candidates for potential

disease-causing mutations• Data available via the web

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What are pseudogenes and how do they arise?

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Why do we care about pseudogenes?

• Human genome• Estimated 30,000 genes• ? pseudogenes

• Genes encode a functional product --> protein

• Mutations in genes• Alters proteins• Disease, deficiencies

• Pseudogenes may be a source of mutations in genes

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Gene Conversion

• The transformation of the sequence of one gene to that of another separate and distinct gene.

• The nonreciprocal transfer of genetic information between two similar but non-identical sequences.

• May result in gene mutations that affect and possibly delete the function of the gene.

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Gene Conversion

Non-processed pseudogenesare the best candidates for gene conversion

Heteroduplex

5' 3'5'3'

Gene conversion

No gene conversion

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meiosis I

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prophase I

-chromosomes more spread out (relative to mitosis)

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prophase I

-identical pairs matched

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prophase I

-homologous pairs match up (called a bivalent)-crossing over can now occur

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prophase I

-as chromatids shorten, and thicken, they are called “tetrads”-“chiasmata” – regions where crossover occurs-virtually all tetrads form at least one “chiasma”-thought to stabilize the tetrad

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metaphase I

-tetrads line up-microtubules attach to sister chromosomes

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anaphase I

–sister chromatids are pulled to the same pole (in mitosis, sister chromatids were pulled apart)

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Telophase I

•cell divides

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Metaphase Imaternal centromeres

migrate together (but independent of other chromosome pairs) paternal chromosomes

migrate together

crossing over

tetradaster

nuclear membrane breaks down

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Anaphase II, Telophase II

-sister pairs are pulled apart - cell constricts and divides

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Gene Conversion

Heteroduplex

Non-processed pseudogenesare best candidates for gene conversion

5' 3'5'3'

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Defining pseudogenes

1) Genomic DNA sequence that does not overlap known genes as defined by NCBI’s Reference Sequence gene set.* NCBI: National Center for

Biotechnology Information

AND

2) Genomic DNA sequence with sequence and gene structure similarity to genuine genes.

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Pseudogenes and Gene Conversion

Gene Co-localized Pseudogene

Processed/Non Pseudogene

Deleterious effects

5/14.1 YES Non-processed B cell deficiency

ABCC6 YES Non-processed Pseudoxanthoma elasticum (PXE)

CRYBB2 YES Non-processed Autosomal dominant cataract

CYP21 YES Non-processed Congenital adrenal hyperplasia

FR YES Non-processed Neural tube defects

GBA YES Non-processed Type 2 Gaucher disease

IDS YES Non-processed Hunter syndrome

p47-phox YES Non-processed Chronic granulomatous disease

PKD1 YES Non-processed Autosomal dominant polycystic kidney disease

SBDS YES Non-processed Shwachman-Diamond syndrome

VWF NO Non-processed Type 3 von Willebrand disease

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Pseudogene: Previous Work

1) Zhang and colleagues at Yale University• Identified processed and non-

processed• Non-processed are not available

2) Ohshima and colleagues in Japan• Identified processed pseudogenes

only

3) Torrents and colleagues at the European Molecular Biology Laboratory (EMBL)• Identified processed and non-

processed

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Pseudogene Study by EMBL

• Searched for pseudogenes in the human genome using protein sequences from the SpTrEMBL database to query the genome.o 830,525 entries (September 2, 2003)

o Including at least 10 species

• Gene location is not available• Exon/Intron structure is not available

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Pseudogene Search

UCSCHuman GenomeJuly 2003 (hg16)

UCSC annotationGenomic locationof refseq genes

Mask out RefSeq genes

Exons of refseq genes in FASTA

format

Human GenomeWith refseq genes

Masked out

BLAST exons vs. masked genome

BLAST results Candidate list of pseudogenes

UCSC human genome assembly masked for repeats

UCSC annotationGenomic alignments

of RefSeq genes

Exons of RefSeq genes in FASTA

format

Human genomewith RefSeq genes

masked out

BLAST results Identify pseudogenes

Pseudogenes, including

candidates for gene conversion

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ReportPaths Program

• RefSeq Gene A has 3 exons

• The exons (blue) and the BLAST alignments (red) of those exons against a RefSeq gene-masked genome are shown below with their genomic indices:

Gene A Exon 1 Exon 2 Exon 3

BLAST alignments (e < 0.0001)

chr110,400 11,000

2,000 X X X

910 11,080

10,20010,000chr1chr1

10,600 10,800

2,200 2,520 2,650 3,120 3,320

730

109,100

chr1 chr1 chr1

990 11,140 109,140

790

chrX chr2 chr10

chr9 chr12,680 2,720

22,330 22,370chr12

50% coverage

cutoff

X

X

X

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ReportPaths Program

• RefSeq Gene A has 3 exons

• The exons (blue) and the BLAST alignments (red) of those exons against a RefSeq gene-masked genome are shown below with their genomic indices:

2,000 X X X2,200 2,520 2,650 3,120 3,320chr1 chr1 chr1

chr12,680 2,720

BLAST alignments (e < 0.0001)

910 990chrX

X

Gene A Exon 1 Exon 2 Exon 3

chr110,400 11,00010,20010,000

chr1chr110,600 10,800

109,100 109,140chr10

11,080 11,140chr2X

730 790chr9

22,330 22,370chr12

X

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Pseudogenes Identified

6,898

7,103

Overlap

1,0711,77816,68819,537EMBL

3,8783,3697,08914,336UI

AmbiguousNonProcessedAllStudyType

0.01

0.0001

E-value cutoff

0.30 %9.3 Mbp476 bpEMBL

0.39 %11.9 Mbp830 bpUI

Coverage of genome

Total base pairs of pseudogenes

Average length of pseudogenes

Category

Study

• UI -- 50% of one exon from the progenitor gene must be

represented in a pseudogene sequence

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Classifying for Processing

Pseudogenes classified as processed ornon-processed

0

200

400

600

800

1000

1200

1400

-0.9 -0.6 -0.3 0 0.3 0.6 0.9 1.2 More

Percentage change of intronic sequence retained

Number of pseudogenes

7,098

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Pseudogene Disablements Identified

UI Pseudogenes EMBL Pseudogenes

Number of disablement

s

Pseudogenes w/disablemen

t

Number of disablement

s

Pseudogenes w/disablemen

t

Early stops 29,724 7,951

(55.5%)

12,568 6,972

(35.7%)

Frameshifts 17,546 7,335

(51.2%)

20,064 8,968

(45.9%)

Early stops or Frameshifts

47,270 9,169

(64.0%)

32,632 11,293

(57.8%)

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Bias of Co-localized Pseudogenes

• 1,945 (56.8%) of non-processed pseudogenes are on the same chromosome as the progenitor gene

• 375 (5.2%) of processed pseudogenes are on the same chromosome as the progenitor gene

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Co-localized Processed Pseudogenes

Histogram of experimental and simulated distances between co-localized, processed gene-pseudogene pairs

0

10

20

30

40

50

60

70

5000000150000002500000035000000450000005500000065000000750000008500000095000000

More

Distance between pseudogene and progenitor gene (bp)

Number of pseudogenes

Experimental Simulated

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Co-localized Non-processed Pgenes

Histogram of experimental and simulated distances between co-localized, non-processed gene-pseudogene pairs

0

200

400

600

800

1000

1200

1400

5000000150000002500000035000000450000005500000065000000750000008500000095000000

More

Distance between pseudogene and progenitor gene (bp)

Number of pseudogenes

Experimental Simulated

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Known Gene Conversion Cases

Gene Pgene ID

Co Chrom Dist Sim Len GC% GC3%

5/14.1 08894 YES 22 1.8M 96% 576 63% 77%

ABCC6 04734 YES 16 2.3M 99% 1198 61% 74%

CRYBB2 08798 YES 22 224k 95% 525 59% 78%

CYP21 11041 YES 6 29k 99% 2006 61% 81%

FR 02141 YES 11 30k 93% 714 55% 72%

GBA 00535 YES 1 7k 97% 2273 56% 65%

IDS 13690 YES X 20k 99% 237 50% 58%

p47-phox 11753 YES 7 369k 99% 1339 62% 82%

PKD1 04876-81 YES 16 5-10k 98% 6385-10155 67% 84%

SBDS 11923 YES 7 5.8M 97% 1578 42% 50%

VWF 02767 NO 12,22() NA 98% 2874 59% 73%

Note: All pseudogenes involved in known cases were identified as non-processed.

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Known Gene Conversion Cases

Gene involved in known gene conversion case

Pseudogene identified by EMBL

5/14.1 Identified as processed pseudogene

ABCC6 Not identified

CRYBB2 Not identified

FR Identified as non-processed pseudogene

GBA Identified as non-processed pseudogene

IDS Identified as processed pseudogene

p47-phox Not identified

PKD1 Identified as processed pseudogene

SBDS Not identified

VWF Identified as processed pseudogene

Note: CYP21p is not present in the July 2003 genome release

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Ranking Gene Conversion Candidates

• Non-processed, co-localized pseudogeneso 1,945 candidates

• Sequence characteristics (Score between 0 and 1)1. Similarity2. Pseudogene length/progenitor gene

length3. Pseudogene length/max pseudogene

length4. GC content5. GC3 content6. Distance from progenitor gene

1 - (distance/max distance)

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Ranking Gene Conversion Candidates

• Six variableso a,b,c,d,e,f

(0.00 to 1.00)o Six weights (w1,w2, … w6)

0.40, 0.02, 0.05, 0.00, 0.80, 1.00o Assign weights and rank gene conversion candidates (gene-pseudogene pairs)

o Try all combinations1.00a + 0.00b + 0.00c + 0.00d + 0.00e + 0.00f

0.98a + 0.02b + 0.00c + 0.00d + 0.00e + 0.00f

…0.00a + 0.00b + 0.00c + 0.00d + 0.00e +1.00f

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Ranking Gene Conversion Candidates

• Optimal weightso Best ranking of known cases (9 of 11) Sum of all rankings

2,228

2,228

SUM

5091055514546719218179126Rank

SBDSp47IDSGBAFRCYP21CRYBB2ABCC65/14.1Gene

39618688163Pubmed

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Ranking Gene Conversion Candidates

• Optimal weightso Best ranking of known cases (9 of 11) Sum of all rankings

Variable Similarity Length/ gene length

Length/ maxlength

GC GC3 Distance

Weight 0.26 0.02 0.00 0.08 0.02 0.62

2,228

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Publicly Available Data

• Web siteo http://genome.uiowa.edu/pseudogenes/

o Identified pseudogenes- July 2003- May 2004

o Gene conversion candidateso Statisticso Programs

- Reportpaths- Rank

- Looked closely at known retinal disease genes (155)- Novel gene conversion event

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Disease-causing Candidates

• Retinal Information Networko 155 human genes related to inherited retinal disorders

o 25 of these intersect the UI pseudogenes

o 13 have a co-localized pseudogeneo 6 have a non-processed pseudogeneo 4 have a non-processed, co-localized pseudogene-ABCC6, OPN1LW, RP9, ALMS1-(in training set)

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Disease-causing Candidates

• ABCC6 pseudogeneo Known case of pseudogene involvement in gene conversion has been linked with Pseudoxanthoma elasticum (PXE)

• OPN1LW pseudogeneo Involved in gene conversions with OPN1MW (24kb away) that can cause color blindness

• RP9 pseudogeneo Mutation (GAT-GGT) present in the Human Gene Mutation Database (HGMD) has been found to cause autosomal dominant retinitis pigmentosa

• ALMS1 pseudogeneo No literature to support gene conversion theory

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Disease-causing Candidates

• Highest-ranked validating gene conversion candidateo HBZ gene and a pseudogene 8.5kb awayo Gene conversion activates pseudogene

• Processed, co-localized gene-pseudogene pairso IMPDH1 pseudogene

- (GAC-AAC) is associated with the RP10 form of autosomal dominant retinitis pigmentosa

o PGK1 pseudogene- (ATT-ACT) has been associated with a PGK deficiency

- Final thought: gene conversion may be transparent to linkage disequilibrium (give example of LD)

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Repositories for mutations?• Pseudogenes may act as repositories for mutations -- where mutations may collect over time -- and then aberrantly be "gene converted" into the genuine gene.

• Conversely, perhaps the genuine gene actually helps maintain a copy of the p-gene, by continually "gene converting" a recognizable copy of the p-gene?

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An in silico discovery"Among the pseudogenes identified is a

retinitis pigmentosa 9 (RP9) pseudogene that carries a c.509A>G mutation which produces a p.Asp170Gly substitution that is associated with the RP9 form of autosomal dominant retinitis pigmentosa (adRP). The c.509A>G mutation in RP9 is a previously unrecognized example of gene conversion between the progenitor gene and its pseudogene."

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Questions?

Acknowledgements:

Jared BischofVal SheffieldEd StoneTodd ScheetzTom Casavant

CLCG

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End

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Gene Conversion

• The transformation of the sequence of one gene to that of another separate and distinct gene.

• The nonreciprocal transfer of genetic information between two similar but non-identical sequences.

• May result in gene mutations that affect and possibly delete the function of the gene.

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Simulation of Co-localized Pairs

• N1, N2, … represents the number of gene-pseudogene pairs co-localized to Chromosome 1, 2, …

• N1, N2, … number of genes and their pseudogenes were each randomly distributed on Chromosome 1, 2, …

• The distributions of these gene-pseudogene pairs were then analyzed in two groups (processed and non-processed)

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Final Thoughts

• 1,945 ranked candidates for gene conversion

• Similar analysis could be performedo OMIM - Online Mendelian Inheritance in Man

o HGMD - Human Gene Mutation Database

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Gene Conversion

Heteroduplex

Non-processed pseudogenesare best candidates for gene conversion