1. Lessons from DUF1220 Protein Domains, Cognitive Disease and Human Brain Evolution II James M. Sikela, Ph.D. Department of Biochemistry & Molecular Genetics Human Medical Genetics and Neuroscience Programs University of Colorado School of Medicine Advanced Genome Analysis Course University of Colorado School of Medicine April 23, 2015

2. Fortna, et al, PLoS Biol. 2004 Human & Great Ape Genes Showing Lineage-Specific Copy Number Gain/Loss

3. Fortna, et al, PLoS Biol. 2004 Human & Great Ape Genes Showing Lineage-Specific Copy Number Gain/Loss Genes showing human lineage- specific increases in copy number

4. DUF1220 Repeat Unit Popesco, et al, Science 2006

5. GenomePDE4DIP Total DUF1220 NBPF Genes Human227223 Chimp312519 Gorilla39915 Orangutan49211 Gibbon35310 Macaque13510 Marmoset13111 Mouse Lemur121 Bushbaby132 Tarsier110 Rabbit183 Pika110 Mouse110 Rat110 Guinea Pig111 Squirrel111 Tree Shrew143 Cow173 Dolphin141 Pig131 Horse183 Dog131 Panda121 Cat132 Megabat110 Microbat110 Hedgehog110 Shrew110 O’Bleness et al.. G3 Sept (2012). DUF1220 Copy Number in Mammalian Genomes -Copy amplification restricted to anthropoid species: monkeys, apes and humans

6. Sequences Encoding DUF1220 Domains Show the largest human lineage-specific increase in copy number of any protein coding region in the genome (160 HLS; >270 total in haploid genome) Show the largest human lineage-specific increase in copy number of any protein coding region in the genome (160 HLS; >270 total in haploid genome) Show signs of positive selection especially in primates Show signs of positive selection especially in primates In brain, are expressed only in neurons In brain, are expressed only in neurons Are highly amplified in human, reduced in great apes, further reduced in monkeys, single-or-low copy in prosimians and non-primate mammals, and absent in non-mammals Are highly amplified in human, reduced in great apes, further reduced in monkeys, single-or-low copy in prosimians and non-primate mammals, and absent in non-mammals Have increased in human primarily by domain hyper- amplification involving DUF1220 triplet Have increased in human primarily by domain hyper- amplification involving DUF1220 triplet

7. Key Human-Specific Evolutionary Features of 1q21.1 Region O’Bleness, et al, Nat Rev Genet, 2012 ‡*

8. 1q21.1 Deletions linked to Microcephaly* 1q21.1 Duplications linked to Macrocephaly* Recurrent Reciprocal 1q21.1 Deletions and Duplications Associated with Microcephaly or Macrocephaly and Developmental and Behavioral Disorders Brunetti-Pierri, et al, Nature Genetics 2008 Recurrent Rearrangements of Chromosome 1q21.1 and Variable Pediatric Phenotypes Mefford, et al, N. Engl. J. Med. 2008 *Implies the copy number (dosage) of one or more genes in this region is influencing brain size in a dose-dependent manner These CNVs encompass or are immediately flanked by DUF1220 sequences (Dumas & Sikela, Cold Spring Harbor Symposium Quant. Biol., 2009)

9. DUF1220/NBPF Sequences & Recurrent Disease-associated 1q21.1 CNVs

10. Dumas, et al, Amer. J. Hum. Gen., 2012

12. Correlation of DUF1220 Copy Number with Multiple Primate Brain MRI Phenotypes

13. Is DUF1220 copy number associated with brain size in humans? Custom, high-density 1q21 DNA microarrays to test copy number variation via arrayCGH 42 individuals with 1q21.1 CNVs Microcephaly (Type I and Type II Deletions) Macrocephaly (Duplications) Test for correlation of copy number with head circumference (FOC Z-score) 6 DUF1220 clades & 53 1q21 genes tested qPCR validation

14. Microcephaly –Class I Deletions –Class II Deletions Larger interval More severe Macrocephaly –Duplications 1q21 ArrayCGH Profiles for Disease Groups Dumas, et al, Amer. J. Hum. Gen., 2012

16. BCL9

17. GRP89A

18. DUF1220

19. Copy Number of Genes in the 1q21.1 Region vs. Brain Size in 5 Primate Species HumanChimpOrangutanMacaqueMarmoset HumanChimpOrangutanMacaqueMarmoset Brain (g)1350380390887Itga1011111 DUF1220*268125923530Nudt1711111 PPIAL451100Rnf11511111 Sec22b11111Cd16011111 Notch2nl11111PDZK131111 HFE211111GPR8931111 Txnip11111Hydin10000 Polr3gl11111Prkab211111 Polr311111Pdia3p11111 Ankrd3411111Fmo511111 Ankrd3511111Chd1l11111 Lix1l11111Bcl911111 Rbm8a11111Acp611111 Gnrhr211111GJA511111 Pex11b11111GJA811111 *DUF1220 protein domains: NBPF gene family Dumas, et al, Amer. J. Hum. Gen., 2012

20. Association of ArrayCGH-predicted DUF1220 Copy Number with Brain Size (FOC Z score) in 1q21 Disease Population Dumas, et al, Amer. J. Hum. Gen., 2012

22. Association of ArrayCGH-predicted DUF1220 Copy Number & Brain Size (FOC Z score) in Combined Deletion Groups Dumas, et al, Amer. J. Hum. Gen., 2012

24. Investigation of DUF1220 Copy Number in Non-disease Population >300 non-disease individuals previously analyzed by brain sMRI 59 DNA samples selected from extremes in gray matter volume (large [n=29] and small [n=30]) Analyzed by arrayCGH using custom 1q21 arrays

25. ArrayCGH Data for Non-Disease Group DUF1220 CON1 & CON2 Clades Dumas, et al, Amer. J. Hum. Gen., 2012

28. Factors that must be reconciled for model linking 1q21.1 instability, evolutionary adaptation & recurrent disease Evolutionary rapid DUF1220 copy number increase Estimate, on average, 28 DUF1220 copies added to human genome every 1 million years since the Homo/Pan split Underlying mechanism must account for continued, recurrent DUF1220 increases Underlying mechanism must account for excess of 1q21.1 disease-associated CNVs

29. Increase in DUF1220 Copy Number 1q21 Deletions: Microcephaly Schizophrenia 1q21.1 Duplications: Macrocephaly Autism Proposed Mechanism Linking DUF1220, Brain Evolution, and Disease * * Autism, Congenital Heart Disease, Congenital Anom. of Kidney/Urinary Tract, Epilepsy, Intellectual Disability, Intermittent Explosive Disorder, Macrocephaly, Mayer-Rokitansky-Kuster-Hauser Syn., Microcephaly, Neuroblastoma, Schizophrenia, Thrombocytopenia-absent-radius (TAR) Syn. Evolutionary Advantage of Increase in Brain Size Increase/Retained 1q21.1 Instability

30. DUF1220 Model DUF1220 model proposes that: 1) Increasing DUF1220 copy number (dosage) increases human brain size, and 2) the evolutionary advantage of rapidly increasing DUF1220 copy number in the human lineage has resulted in retention of the high genomic instability of the 1q21.1 region which, in turn, has precipitated a spectrum of recurrent human brain and developmental disorders DUF1220 model proposes that: 1) Increasing DUF1220 copy number (dosage) increases human brain size, and 2) the evolutionary advantage of rapidly increasing DUF1220 copy number in the human lineage has resulted in retention of the high genomic instability of the 1q21.1 region which, in turn, has precipitated a spectrum of recurrent human brain and developmental disorders Dumas & Sikela, Cold Spring Harbor Symposium Quant. Biol., 2009

31. Relative frequencies of copy-number deletions and duplications associated with autism or schizophrenia CNV locusCondition Deletion Duplication P-value cases cases(Fisher’s exact) 1q21.1 Autism 2 10 0.001 Schizophrenia 15 4 16p11.2Autism 14 5 0.00013 Schizophrenia 5 24 22q11.21Autism 1 80.000049 Schizophrenia 16 1 22q13.3Autism 5 0 0.0079 Schizophrenia0 4 Boldface italic font indicates that the CNV is documented as a risk factor for the condition specified. Relative frequencies of copy-number deletions and duplications associated with autism or schizophrenia CNV locusCondition Deletion Duplication P-value cases cases(Fisher’s exact) 1q21.1 Autism 2 10 0.001 Schizophrenia 15 4 16p11.2Autism 14 5 0.00013 Schizophrenia 5 24 22q11.21Autism 1 80.000049 Schizophrenia 16 1 22q13.3Autism 5 0 0.0079 Schizophrenia0 4 Boldface italic font indicates that the CNV is documented as a risk factor for the condition specified. Adapted from Crespi, Stead & Elliot, PNAS, 2009 Autism & schizophrenia as genomic sister disorders exhibiting opposite phenotypes

32. Gaussian distribution of DUF1220 copy number in human populations: Rich source of overlooked functional allelic variation 55-90 copies 20-55 copies 140-250 copies

33. DUF1220 and Autism High rates of brain growth in years 0-2 Greater brain growth -> greater severity Strong genetic component to etiology; no major-effect genes identified. 1q21 CNV linked to autism encompasses DUF1220 copies DUF1220 copy number not directly examined in autism/ASD Does DUF1220 copy number affect phenotype?

34. Experimental Design 183 Autism Cases 135 Controls ddPCR used to measure CON1 copy number CON1 selected as starting point due to association with brain size Examined differences in overall copy number between cases and controls Examined associations between DUF1220 and phenotype severity within cases Severity determined from Autism Diagnostic Interview- Revised (ADI-R) scores Three main measures of severity: Impaired social reciprocity Impaired communication Increased repetitive behaviors

35. CON1 copy number: ASD vs Controls CON1 ASD ASD copy number similar to controls What about severity? – DUF1220 has broad copy number range: has the potential to confer a broad range of phenotypic effect – CON1 also linked to brain growth (brain growth  severity) CON1 Controls

36. DUF1220 CON1 dosage is linearly associated with increasing severity of the primary symptoms of autism – Controlled for multiple potential confounders Results of Initial Study: CON1 Copy Number vs ASD Severity Davis, et al, PLoS Genetics, 2014

37. DUF1220 CON1 dosage is linearly associated with increasing severity of the primary symptoms of autism – Controlled for multiple potential confounders – Per single copy increase of CON1: 0.25 increase in Social Diagnostic Score (p=0.021) 0.18 increase in Communicative Diagnostic Score (p=0.030) 0.10 increase in Repetitive Behaviors Diagnostic Score (p=0.047) Results of Initial Study: CON1 Copy Number vs ASD Severity Davis, et al, PLoS Genetics, 2014

38. Results of Replication Study: CON1 Copy Number vs ASD Severity Repeated analyses in 166 additional cases Per single copy increase in CON1: – 0.24 increase in Social Diagnostic Score (p=0.036) – 0.16 increase in Communicative Diagnostic Score (p=0.07) – No association with Repetitive Behaviors Diagnostic Score Davis, et al, Human Genetics 2015

39. Autism and DUF1220: Conclusions DUF1220 CON1 dosage is linearly related to severity of symptoms in autism – more CON1 -> more severe social impairment – more CON1 -> more severe verbal communication impairment Could DUF1220 be involved is autism disease risk? Gene sequences important to autism may have been (and continue to be) missed by conventional genome-wide analyses – Highly duplicated, copy number polymorphic coding sequences Genes important to autism (and schizophrenia) may also be involved in human brain evolution – Double-edged sword model (Crow papers and Burns book)

40. What about DUF1220 and cognitive aptitude? Experimental Design: ArrayCGH and ddPCR analysis European population; brain MRI measurements and WISC IQ scores New Zealand population; multiple cognitive measurements including mathematical aptitude Looked for associations between CON2 copy number and cognitive function in each population

41. We identified a linear association between CON2 copy number and cognitive function in two independent populations of European descent. In North American males, an increase in CON2 copy number corresponded with an increase in WISC IQ (R2 = 0.13, p = 0.02), which may be driven by males aged 6–11 (R2 = 0.42, p = 0.003). We utilized ddPCR in a subset as a confirmatory measurement. This group had 26–33 copies of CON2 with a mean of 29, and each copy increase of CON2 was associated with a 3.3-point increase in WISC IQ (R2 = 0.22, p = 0.045). In individuals from New Zealand, an increase in CON2 copy number was associated with an increase in math aptitude ability (R2 = 0.10 p = 0.018). These were not confounded by brain size. To our knowledge, this is the first study to report a replicated association between copy number of a gene coding sequence and cognitive aptitude. Remarkably, dosage variations involving DUF1220 sequences have now been linked to human brain expansion, autism severity and cognitive aptitude, suggesting that such processes may be genetically and mechanistically inter-related. DUF1220 copy number is linearly associated with increased cognitive function as measured by total IQ and mathematical aptitude scores -Davis et al Human Genetics, 2015 (From the abstract):

42. Davis et al, Hum Genet 2015

43. Linear Association of DUF1220 CON2 Copy Number and Cognitive Aptitude Davis et al, Hum Genet 2015 Fig. 2 Linear association of CON2 with IQ in North American males. The triangles display array-based CON2 copy ratio versus WISC IQ in younger males (≤10.9). The gray dots display the same but in older males. The line is a least squares line of best fit in the younger male group (R2 = 0.42, p = 0.003, n = 19).

44. Summary DUF1220 domain copy number: Largest human-specific increase in copy number of any protein coding region (~270 human copies) Human increase accompanied by large disease burden Highly correlated with brain size (p<1.8x10 -6 ) & cortical neuron number (p<0.0011) across primate lineages Highly correlated with brain size in 1q21 disease population (microcephaly/macrocephaly): (p<1.56x10 -7 ) Dosage linked with increasing gray matter volume in non- disease population: (CON1:p<0.025); (CON2: p<0.033) CON2 dosage linked to cognitive aptitude (IQ and math) Increasing CON1 dosage linked with increasing severity of social impairment in autism Suggests DUF1220 dosage may be a key driver of human brain evolution & cognition, & modulator of autism severity

45. A Walk Through Our Genome --All regions of the genome are not created equal