Jayne Duncan FRCPath Course December 2010

1. Describe the principles of autozygosity mapping and how it can be used to identify novel disease loci. What are the advantages of such studies? What types of diseases and families are most amenable to this type of gene discovery? Jayne Duncan FRCPath Course December 2010

2. Autozygosity • Autozygosity is a term used to describe homozygosity for markers identical by descent. • The two alleles are both copies of one specific allele that was present in a recent ancestor. • An individual who is homozygous for two such alleles is said to be autozygous at that particular locus. • Alternatively an individual may be homozygous for a particular allele because they have two alleles identical by state (a second independent example of the same allele has entered the family). • The rarer the alleles are in a population, the more likely the homozygosity represents autozygosity.

3. Consanguinity • In a consanguineous marriage both partners share some of their ancestors. • They may have inherited a copy of the same ancestral allele at a locus. • If they were second cousins (offspring of first cousins) they would be expected to share 1/32 of all their genes because of common ancestry. • Any children would be autozygous at 1/64 of all loci.

4. Autozygosity and Gene Discovery • Autozygosity can be utilized in gene discovery because: • For a very rare allele or haplotype, a single homozygous affected child born to second cousins generates a lod score log10(64) = 1.8. • If there are two affected sibs who are both also homozygous for the same allele, the lod score is log10 = (64x4x4) =3.0. • This is exceptionally high considering that the chance that a sib would have inherited the same pair of parental haplotypes, even if they are unrelated to the disease is 1/4.

5. Autozygosity and Gene Discovery (2) • Small consanguineous families can generate significant lod scores. • Autozygosity is a powerful tool for gene discovery especially if families can be found with multiple people affected by the same recessive condition in two or more siblings linked by consanguinity. • Such families are found in Middle Eastern countries and parts of the Indian subcontinent where first-cousin and other consanguineous marriages are common.

6. Advantages of Autozygosity Mapping • Can map genes for rare autosomal recessive conditions. • Useful for where there is locus heterogeneity. • High statistical power of studies as families analysed have high levels of consanguinity and large numbers of siblings per family.

7. Principles of Autozygosity Mapping • Find suitable families where the recessive condition of interest is presenting. • Identify regions of concordant homozygosity using highly polymorphic markers such as microsatellites or by performing a SNP microarray genome wide autozygosity scan. • It is very likely that affected individuals will be homozygous not only at the disease locus but at closely linked loci. • Only a small number of shared homozygous regions will be identified. • Identify candidate genes within these haplotype regions and screen for pathogenic mutations. • Perform studies such as protein expression to confirm gene is responsible for the disease of interest.

8. Case Study: Mapping a gene for benign recurrent intrahepatic cholestasis • Rare autosomal recessive condition. • Clinical manifestations include: -intermittant episiodes of cholestasis without extrahepatic bile duct obstruction. -Initial elevation of serum bile acids followed by cholestatic jaundice which resolves over a period of weeks or months.

9. Study Design • 3 patients distantly related through consanguineous marriage in the Netherlands were identified for the study. • DNA samples were collected from the patients and their parents (to enable construction of marker haplotypes) and the affected sib of one of the patients. • The affected sib was included to determine shared regions, as those not shared would be unlikely to contain the causative gene. • 256 microsatellite markers were tested spaced at 10-20cM across the autosomes.

10. Results • One area at located in 18q21-q22, covering approximately 19cM was shared by five out of 6 haplotypes. • This could be because the segments are inherited from a common ancestor or a high frequency of the alleles in the relatively isolated study population. • 60 additional markers were typed in each of the shared segments to extend the conserved haplotypes. • Two adjacent regions of homozygosity were identified on chromosome 18, including the one shared by the 5/6 haplotypes. • Probability calculations indicate that such segment sharing as shown here is unlikely to arise by chance.

11. Results (2) • Haplotypes of BRIC patients using 13 microsatellite markers over the putative candidate region on chromosome 18. • The markers from the original genome screen are shown in bold. • The disease haplotype is outlined with two areas possibly shared by all 6 chromosomes indicated. • The chromosomes of 10 may carry a mutation of different origin, or may provide further information to narrow down the BRIC candidate region

12. Example: Null mutations in FAM161A are a cause of Retinitis Pigmentosa • Retinitis Pigmentosa is the most common inherited retinal degeneration. • Worldwide prevalence of 1/4000. • Heterogeneous and can be inherited as an autosomal recessive (50-60%), autosomal dominant (30-40%) or X-linked (5-15%). • So far 32 loci have been associated with arRP. • In 5 of these (RP22, RP28, RP29, RP32 and RP54) the causative gene is unknown.

13. Study design • Rozenfeld et al used a combination of autozygosity mapping and homozygosity mapping to identify a 4Mb homozygous region on chromosome 2p15 in patients with arRP. • This region partially overlaps with the RP28, a previously identified arRP locus. • Whole genome SNP analysis was performed with the use of Affymetrix 250K microarrays. • Families studied were from Israel and the Palestinian territories.

14. Autozygosity Results

15. Results • Sequence analysis of 12 candidate genes from the 2p region overlapping RP28 revealed 3 null mutations in FAM161A in 20/172 families. • RT-PCR analysis in 21 human tissues showed high expression in the retina and low levels in the brain and testis. • Two alternatively spliced transcripts expressed in the retina, the major transcript lacks exon 4. • Expression studies on mice showed that during embryogenesis low levels of Fam161a transcripts were present in the optic cup and after birth expression was elevated and limited to the photoreceptor layer. • Despite these studies the function of FAM161A is unknown, however it is moderately conserved in mammals and loss of function mutations are likely to be responsible for the arRP phenotype in approximately 12% of families.

16. Autozygosity Mapping using the Affymetrix Genome-wide SNP Array DNA is cut with a restriction enzyme. Universal adptors (blue) are ligated to the fragments, allowing them to be amplified with a single pair of PCR primers. PCR products are fragmented, labeled and hybridised to 25-mer oligos on the microarray. Each oligo specifically hybridises to fragments containing one allele of one specific SNP. Strachan & Read 2010

17. Affymetrix Allele Detection The Affymetrix system uses 40 probes per nucleotide position, each about 25 nucleotides long. Probes are organized in sets of four, each having a different nucleotide at the central position. Five quartets query the forward strand and five query the reverse strand.. The five quartets are offset along the genomic sequence so that the variable nucleotide in the probe might be at position -2, -1, 0, +1 and +2 relative to the nucleotide being assayed. Base calling is based on algorithms that compare the hybridization intensities of all 40 probes.

18. Case Study: Identification of TUBA8 Gene as the cause of Polymicrogyria with Optic Nerve Hypoplasia • Polymicrogyria (PMG) is a malformation of the cerebral cortex. • Usual gyral pattern is replaced by numerous small infoldings. • The normally six layered cortex is replaced by a four layered or unlayered cortex. • It is a hallmark of Bilateral Frontoparietal Polymicrogyria (BFPP) that results from faulty N-glycosylation of GPR56 protein, which has a role in the maintenance of pial basement membrane integrity during development of the cortex.

19. Identification of the TUBA8 Gene • Performed Autozygosity mapping in Four children from two consanguineous families of Pakistani origin. • Autosomal recessive inheritance considered due to consanguinity in both families. • Children presented with severe developmental delay, hypotonia, seizures, profound neurological impairment and optic nerve hypoplasia. • Neurological imaging showed a pattern of extensive PMG with a dysplastic or absent corpus callosum anabnormality of the brain stem and hypoplastic optic nerves.

20. Identification of the TUBA8 Gene (2) • Regions of concordant homozygosity identified in affected individuals from both families by performing a SNP-microarray-genome-wide homozygosity scan • Used the Affymetrix Genome Wide Human SNP Array 6.0. • One 7.42Mb region of concordant homozygosity identified on chromosome 22q11.2 in affected individuals. • There were 929 SNPs and 230 annotated genes within the shared minimal autozygous region. • The presence of the α8-tubulin gene (TUBA8) which is expressed in the brain was thought significant and considered a candidate gene - dominant mutations in the α1-tubulin gene (TUBA1A) are known to cause lissencephaly associated with mild microcephaly and dysgenesis of the corpus callosum and brain stem hypoplasia. -More recently dominant mutations in TUBB2 resulting in asymmetric frontal predominant PMG with corpus collosum deysgenesis have been described

21. Results • Sanger sequencing in the affected individuals of the two families showed no coding region variants. • A homozygous 14bp deletion in intron 1 of TUBA8 was identified. • This segregated with the disease in both families and all obligate carriers were heterozygous for the mutation. • Mutation not found in 342 ethnically matched control chromosomes

22. Results continued • The 14bp deletion lies 11bp upstream of the exon 2 splice junction, eliminating a large portion of the acceptor site polypyrimidine tract. • Reverse transcriptase PCR analysis on lymphoblastoid RNA from one of the affected individuals showed the full length TUBA8 transcript was present at a greatly reduced level. • The major product present was a shortened transcript, confirmed by sequencing to lack exon 2.

23. Conclusions • Autozygosity mapping is applicable to rare autosomal recessive diseases. • Consanguineous families are studied. • Finding affected families is a challenge, due to location of families and prevalence of disease. • Haplotypes need to be identical by descent not state to yield results. • Method has been applied to identification of candidate genes for Bardet-Biedl syndrome, hearing loss and Fowler syndrome, to name a few.

24. Abdullahi et al Am J Hum Genet 85: 737-744: 2009 Strachan & Read. Human Molecular Genetics 4th Ed (2010) Houwen et al Nature Genetics 8: 380-386. Rozenfeld et al Am J Hum Genet 87: 382-391: 2010 References