Lecture 4: Mutations and Disease Models

1. Genome 351, May 19, 2014 Lecture 4: Mutations and Disease Models Objectives: • Spectrum of human genetic variation • Sporadic vs. inherited disease • Triplet repeat mutations and genetic anticipation • Microdeletions and microduplications • Spontaneous point mutations and autism

2. What is the model of inheritance? • Is it inherited or did it occur as a result of a new mutation in germline of one of the parents? • Is the mutation recessive or dominant? • Is there one gene or many mutations in different genes that are required? • Are there different genes that can have the same effect? • What is the impact of the environment on whether you will develop the disease?

3. What type of mutation might be responsible for the disease? Sequence • Single base pair changes – single base pair substitutions • Small insertions/deletions – frameshift, microsatellite, minisatellite • Large-scale genomic variation (>1 kb) • Large-scale deletions, inversions, and insertions • Changes in copy (gains and losses) called CNVs (copy number variants) • Chromosomal mutations – gain or loss of entire chromosome Cytogenetics

4. I. Fragile X Syndrome and Triplet Repeats • Most common form of mental retardation: 1/2000 male births • Mild to severe mental retardation • 70% have other features: large head, joint elasticity and large testes • X-linked disorder not really dominant or recessive • Female carriers: mildly affected, female premature ovarian failure • When chromosomes from patients were grown under specific conditions showed an unusual constriction called a fragile site

5. Unusual Inheritance Pattern for an X-linked Trait • T = normal transmitting male • Risk of mental retardation depended on position within the pedigree • Daughters of normal transmitting males = 0% risk • Grandsons’ risk = 40-50% • Later generations had a higher risk of developing disease then preceding generations • Genetic anticipation

6. Targeted the constriction and identified a gene at that location called FMR1(fragile X mental retardation-1) Found an unusual repeat in the untranslated region of the gene transcript CGG triplet repeat was very variable between people Hypothesis: Variation in triplet repeat led to disease Cloning of FMR1 Gene Promoter Exon 1 primer (CGGCGGCGGCGG)n

7. Families with Fragile X • Among the general population and families without fragile X, the number of CGG repeats ranges from 5 to 55 repeat units. • Among families with fragile X, the number of CGG repeats among carriers was much larger (55-200 copies). • Among males with fragile X syndrome, the number of repeat units frequently exceeded 1000!

8. Molecular Explanation • CGG repeats are generally unstable and can increase and decrease in size but usually take many generations to change in copy • Once they reach 55 repeat units the chance that they will increase in the next generation increases very rapidly • Once they reach 200 repeats there is a 100% chance that they will jump to 1000 repeat units or more but this jump requires that it be transmitted from a mother • Once the repeat is >200 repeats it interferes with replication of the DNA (fragile site upon culture) and transcription—no transcription = no protein • The FMR1 protein is important in regulating other genes near the synapse—absence of protein = disease

9. Genetic Anticipation Explained A fragile X family • Progressive increase in size of CGG repeat • Requires a female transmission to go to full mutation

10. C G G G C C Errors during Replication or Repair falls off & re-lands on copied repeat nick & repair expanded repeat

11. Examples of disorders caused by repeat expansions Disorder Gene Unit Location

12. Summary • Triplet repeats are unstable and change length over short periods of time. • Threshold effect—once repeats get beyond a certain length, there is a higher chance that they will increase as opposed to decrease—once get too big then disrupt transcription (FMR1) or function of protein (Huntington). • Genetic anticipation—increasing chance of developing disease, increased severity or earlier age of onset occurs. • Originally considered non-Mendelian but now explained by unusual transmission characteristics of triplet repeats.

13. Spectrum of Genetic Variation Sequence • Single base pair changes – point mutations • Small insertions/deletions – frameshift, microsatellite, minisatellite • Structural variation (>10 kb) • Large-scale deletions, inversions, and insertions • Changes in copy (gains and losses) called CNVs (copy number variants) • Chromosomal variation – translocations, inversions, fusions Cytogenetics

14. II. Structural Variation Deletion Duplication Inversion Copy Number Variants (CNVs) Balanced Events

15. Autism A group of disorders Impaired social interaction Delayed or impaired language development Repetitive and stereotypical patterns of behavior, interest and activities Strict “Autism Disorder” shows impairment in all 3 areas Leo Kanner Autistic Disturbances of Affective Contact, 1943

16. Autism Society of America

17. Autism • 4:1 bias males to females • ~1/100 births • Parents of autism patients sometimes show “aloofness, shyness, pragmatic language impairment” • Environmental factors are minor risk factors: e.g., maternal thalidomide, valproic acid use, maternal alcohol abuse, congenital rubella but NO evidence for vaccination • ~2-3% of autism patients show chromosomal abnormalities

18. Autism Genetics • Studies for common genetic variants (variants found in more than 1%) found few consistent findings • Characterization of X-linked candidate genes failed to identify genes • Most common “genetic” cause of autism is a recurrent 2 million base pair duplication on chromosome 15 • Accounts for ~1% of all autism

19. Autism and Copy Number Variation • Hypothesis: Autism is caused by spontaneous gains and losses of large segments of DNA • Copy Number Variation—gains and losses of larger segments of DNA (>1000 bp) • Screen families where there was one child affected with autism but parents were normal • Compare to DNA from “normal” individuals and families where autism is inherited for multiple generations • How?

20. Autism and Copy Number Variation • Compared genomes of autism and controls • Focus on sporadic cases • Autism patients are 10 times more likely to carry a large deletion or duplication (>200,000 bp) that is not seen in parents when compared to their parents • Certain regions of the genome are particularly prone to recurrently delete or duplicate

21. Genetic Basis of Neurocognitive Disease • 8-15% of severe neurocognitive and neurobehavioral disease is caused by spontaneous large changes in the copy of large segments of DNA • The events have a strong effect—if they are transmitted within pedigrees • Most of the events are very rare, but many 100s of sites have been identified • ~1/3 of the lesions are recurrent and can manifest as intellectual disability, schizophrenia, autism, epilepsy • Collectively common but individually rare, genetic events

22. “Normal” Human Genomes • Sequencing of multiple human genomes indicates that every human has ~250 copy number differences greater than 10,000 bp in size • Most of these are inherited • 8% of “normal” humans are missing or have gained large chunks of least 400,000 bp of DNA corresponding to 10-15 genes compared to 25% of children with developmental delay • Most of the large events are seen once or twice • 60% of the larger events >1 Mbp are not seen in parents

23. No Perfect Human Genome • Each individual has their own constellation of copy number variants, a small fraction that are not shared with a parent • In addition, all humans carry 100s of deleterious mutations that contribute to risk • Furthermore, there are many new single base pair mutations per individual • All individuals are genetically unique and “special” • Humans seem to be particularly prone to large events

24. Why are copy number changes so abundant? • Clue: Large repeat sequences (also called segmental duplications) are located at the breakpoints of the duplications or deletions • 25-fold enrichment for breakpoints to occur at these large repeat sequences • Human genome is enriched for these sequences

25. Human Genome has Large Repeatsthat are spread throughout! chr1 chr2 chr3 chr4 chr5 chr6 chr7 chr8 chr9 chr10 chr11 chr12 chr13 chr14 • ~4% duplication • >20 kb, >95% • ~4 average # duplicates • 59.5% interspersed (>1 Mbp) chr15 chr16 chr17 chr18 chr19 chr20 chr21 chr22 chrX chrY She, X et al., (2004) Nature 431:927-30

26. Mouse Genome does not! • ~3% duplication • >20 kb, >95% • Feb. 2006; mm8 • 87% are tandem pairwise

27. 16p 16p 16p 16m 16m 16m Repeated sequence copies in meiosis Replication Figure 12.7

28. 16p 16p 16p 16m 16m 16m Normal alignment Recombination Correct pairing of homologs Figure 12.7

29. 16p 16p 16m 16m Recombination Misalignment 16p One copy and deletion of a region 16m Three copies with duplication Similar sequences may lead to mispairing Figure 12.7

30. A B C TEL A B C TEL NAHR GAMETES A B C A B C TEL TEL Intellectual Disability/Autism/Epilepsy As a result of dosage sensitivity of genes Interspersed Duplications Create Lots of Copy Number Variation in Humans

31. An Unstable Genome The duplication architecture predisposes specific regions of our genome to delete and duplicate Each site is individually rare (1/2,000 to 1/100,000 sperm/egg) but collectively may contribute significantly to disease Individuals are more or less the sum of the genetic information given from their parents Surveys of “normal” individuals suggest that as much as 8% may have large copy number differences

32. Summary • Large copy number changes explain a significant fraction of neurocognitive disease (8-15% of children with developmental delay, intellectual disability, and autism) • Many of the events are sporadic (e.g., not found in parents) but also can be transmitted for a few generations • Large-scale deletions and duplications are individually rare but collectively common in humans in part because of the architecture of our genome

33. Lessons Learned from CNVs • New mutations or very young mutations are important • Many different regions and genes when compromised can lead to the same disease (still one normal copy—i.e., heterozygous) • Individually rare but collectively common

34. III. New Mutations in Protein-Coding Regions • Sequence the protein-coding regions (termed exomes) of families with sporadic autism • Compare sporadic mutations in child when compared to mother, father, child and a subset of siblings • What does the pattern of new mutations look like in autistic children when compared to normal siblings? O’Roak et al., O’Neale et al., Sanders et al., Nature, 2012

35. New Mutations and Old Men • Every child has about 40-50 new mutations. • 80% of new mutations arise from mutations that occur in production of sperm as opposed to egg. • As fathers age they contribute more new mutations to their younger children.

37. Why? Male vs. Female Germ Cell Divisions • Paternal bias for new mutations explained partially by increased number of cell divisions of stem cell as father ages, while number of female divisions is set. • Sperm from a 50-year-old male (840 divisions) vs. 25-year-old (265 divisions).

38. Autism Sporadic SNP Mutations • New mutations in autism probands were more severe when compared to siblings • Significant 2- to 3-fold increase in number of disruptive or protein truncating—i.e., frameshift and stop codons—when compared to unaffected brother or sister • In an analysis of ~1000 autism family exomes only a handful of genes seen more than once (the few genes hit more than once are thought be very important in brain function, e.g., CHD8, GRIN2B, SCN2A) • Predict 500 different genes

39. An “Autism” Protein Interaction Network • 39% of the severe mutations are part of one highly interconnected pathway • More highly interconnected than expected by chance and unaffected siblings do not show enrichment in this pathway

40. Proving Specific Genes CHD8 (7/8) • Resequence candidate genes in thousands of patients and compare to controls • 5 genes show an excess of sporadic mutations in autism patients (~1% of autism) GRIN2B (3/4) DYRK1A (3/3) TBR1 (3/3)

41. Many genes need to be expressed at many different times and places for brain development Brain development is sensitive to gene expression differences Many different genes and pathways when compromised by a CNV or a point mutation can result in the autistic child Loss-of-function new mutations Genetic Model of Autism

42. Summary • Sporadic protein-disruptive mutations are much more common among individuals with autism than in their parents or unaffected siblings. • Specific genes and pathways are being identified based on this model of new mutation. • “Autisms”—not just one cause but >500 different genes—each family has a different genetic cause.

43. NY Times Article April 4, 2012Scientists link gene mutation to autism risk. • What type of mutations are being identified and what fraction of disease risk may be explained? • Why is it such a big deal and what is the hope? • Why will it be a “hard slog” forward? NY Times Article June 12, 2010 A decade later and no cures. • How many genetic causes have been identified for complex disease? Why the frustration? • What shortcut did geneticists use to find the “genes”?

44. References and Additional Reading • Required: • NY Times Articles • A decade later and no cures. June 12, 2010 • Scientists link gene mutation to autism risk. April 4, 2012 • Background: Richards & Hawley • Ch. 2 pp.71-77 • Ch. 5 pp. 167-180

45. Detecting large variants—a genome chip

46. Array Comparative Genomic Hybridization Array of Oligos Normal Human DNA Sample Disease Individual DNA Sample Hybridization 12 mm Merge • High-throughput detection of large-scale deletions and duplications