Early Pharmacological Intervention in Autism: Wide

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Sep 2, 2013 - DLG. AP1. 18. 7.27. MVK. 12. 7.27. NALC. N. 13. 7.24. ISO. C1. 57.19. DST ..... No TV, ... Pharmacological. Intervention. Ion Channels. Prenatal.
Early Pharmacological Intervention in Autism: Wide-Locus GWAS Leading to Novel Treatment Options KM Wittkowski1, B Bigio2, V Sonakya2, MK Tonn3, F Shic4, M Ascano5, C Nasca6, G Gold-Von Simson7 1)RU:

4)Yale

The Rockefeller University, New York 2)CCTS, RU 3)Hochschule Koblenz, Germany University, New Haven 5)Tuschl Laboratory, RU 6)McEwen Laboratory, RU 7)NYU, New York

ABSTRACT

Childhood Absence Epilepsy

ASD: Significance / Replication

The prevalence of autism spectrum disorders (ASD) has increased dramatically over the past 25 years to >1% of U.S. children. Although twin studies attest to a high degree of heritability, the genetic risk factors are still poorly understood. Almost a decade after the completion of the Human Genome Project, the scientific and medical advances hoped for from genome-wide association studies (GWAS) have not yet been realized. Enlarging the sample size to tens of thousands of subjects greatly increases the duration and cost of data collection and, in nonrandomized studies, may not increase the signal/noise ratio. Here, we combine a recent wide-locus approach with novel decision strategies fine-tuned to GWAS. With these methodological advances, mechanistically related clusters of genes and novel treatment options, including prevention of more severe forms of ASD, can now be suggested from studies of a few hundred narrowly defined cases only.

µGWAS of data from only 185 children with CAE (Wittkowski 2013) compared against Illumina controls (Fig. 8) confirmed the involvement of ion channels and the Ras pathway (Fig. 9). Fig. 10 and 11 demonstrate the diagnostic output generated by µGWAS and the ability of µGWAS to hone in on epistasis between intragenic regions.

In two independent studies (AGP 1/II, Anney 2012) of 1071/576 subjects, 18/8 genes reached studyspecific GW significance (Fig. 12). The top results where highly enriched with Ras/Ca2+ associated genes (Fig. 13), in general, and PTPRs (Fig. 14), in particular. 7.50

SEC16B

Fig. 1: Assumed Genetic Structure. Neighboring SNPs (Y, Z) are assumed to be in LD with intermediate disease loci, unless separated by a recombination hotspot (X, Y).

SNP.A

SNP.Y

CREB5 ISOC1

CNTNAP2

DST

7.50

?

PARD3

RASSF8 ARHGAP32

RBFOX1 NALCN

DLGAP1

2013 Sep 02 21:06:44 7.12 6.55 6.58 6.61 6.66 6.69 6.73 6.76 6.77 6.79 6.87 6.92 6.95 6.95 7.02 7.02 7.10 7.15 7.18 7.19 7.24 7.27 7.27 7.29 7.29 7.30 7.32 7.38 7.40 7.50 7.61 7.61 7.73 7.82 7.84 8.03 8.18 8.21 8.31 8.35 8.53 9.41

● single SNP ◊ wide locus reliability ♦ high ♦ .. ♦ low

2 8 10 15 4 15 7 2 16 19 17 9 3 18 3 3 2 6 5 13 12 18 7 10 3 12 4 18 12 1 2 21 1 11 22 X 4 16 7 22 2

OPHN1/ARHGAP41

VPS5 4 OSR2 EXOC6 GABRB3 SETD 7 FAM8 1A SLC2 5A13 GRB14 CNTNAP4 TYK2 SCN4A LHX2 ITPR1 FAM5 9A ATP2 B2 COL8A1 BAZ2 B DST ISOC1 NALCN MVK DLGAP1 CNTNAP2 PARD3 TFDP2 MLEC PPP2 R2C ATP8 B1 RASSF8 NLRP3 BRE KCNJ15 SEC1 6B ARHGAP32 PITPNB OPHN1 FAT4 RBFOX1 CREB5 SYN3 EEF1 A1P12

SYN3

ATP8B1

Fig. 8: GWAS Results for CAE. Foreground: ssGWAS (the pseudogene EEF1A1P12 as the only significant finding). Background: µGWAS

Fig. 12: Enrichment of most significant genes with genes involved in Ras/Ca2+ bottom: study-specific cut-off (Fig. 3); top panels: µGWAS, bottom panels: ssGWAS.

Fig. 9: Ras/Ca2+ Signaling in CAE. Bold/double/light blue borders indicate genes among the top 20/50/100.

MAF LDobs pHWE,1 NA

< > < >

MAF LDobs LDobs LDexp pHWE,1 NA ∆NA

high high

data quality

RHS diplotype structure

µStat Discrimination

+/− Sequential Interaction terms

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Fig. 3 : Study-Specific GW Significance. GWS (solid bar) is estimated as the median projection from chr’ s with lowest deviation from the projection (dashed curves/bars).

Curtailing Polarities

.02 .98 10−3 20%

high high low low

Polarities +/0/− PC µIC:µICsuperset

Fig. 10: µGWAS diagnostics. From left: position, counts by phenotype, data quality indiators, significance by diplotype width, observed and expected LD.

Fig. 13: Replication of the Ras/Ca2+ Signaling in autism (see Fig. 5 for legend) by independent stage population (AGP I/II). Pink circles: growth factor regulation, green circles: ion channels AGP 22 AGP

AGP 2 ?

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Extended Manhattan plot

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Fig. 2 : Analysis Workflow. Steps involving µscores based on different structures (QoD, Fig, 6, polarity conflicts, Fig. 2) are highlighted as green.

EEF1A1P12

FAT4 PPP2R2C

PITPNB

Recent increases in memory and massive parallel computing have spurred the methodological advances making wide-locus GWAS based on a nonparametric approach (u-statistics for multivariate data, µGWAS) feasible (Fig. 2). µGWAS increases power by comprehensively analyzing information from several neighboring SNPs, drawing on the spatial structure of SNPs within an LD block without introducing biases through unrealistic assumptions (independence and additivity) (Fig. 1). Shifting the focus from individual SNPs to wide loci, which are less likely to be selected against, further reduces the risk of false positives without requiring larger sample sizes. To account for GWAS being non-randomized and the null distribution of p-values not being uniform due to MAF-significance association, we propose to replace a fixed “genome-wide significance” with study-specific cut-offs (Fig. 3).

BRE

BAZ2B

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PPFIBP1 PPFIBP1 PPFIBP1 PPFIBP1 6.29

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Recombination rate (cM/Mb)

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N221 H0: chr 6 T hu Aug 01 22:27:07 EDT 2013 5.81

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Wei 2011

Christian 2008

Recombination rate (cM/Mb)

14

Fig. 11: Epistatic regions in ARHGAP32. (A) LD map (B) coding regions (C) µGWAS test results by diplotype length and contributing polarities followed by SNP pattern (orange/yellow/green) for controls and cases sorted by µ(E,P) = µ((E1, E2, E4), (P1, P3, P4)) (near right stub). Diplotypes ranked high (red) and low (green) by µ-scores for each region (left stub: µE = µ(E1, E2, E4), µP = µ(P1, P3, P4)) are shown more saturated. Insert: Manhattan plot. (E) LD between each of the 10 SNPs included in the two µGWAS regions (blue) and the intermediate region (purple) with members of the same tag set (gray).

−log (p) Observed 10 (-logP)

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Fig. 14: Replication of intragenic regions. Several receptor protein-tyrosine phosphatases (PTPRs) having the same wide-loci (see nearby rare variants) associated in both independent studies.

ASD: Epistatic Effects

ASD: Drug Development

DISCUSSION

The higher power of µGWAS over ssGWAS is largely based on its ability to account for (shortrange) epistasis between neighboring SNPs (Fig. 1). In turn, it allows researchers to design studies exploring epistasis between genes. We compared AGP males with 1047 controls from a melanoma study genotyped on the same chip platform (Fig. 15). Strict definition (SDA) and higher functioning (HFA) cases scored higher and lower than controls, respectively, so that no difference could have been detected by comparing all cases against controls. PTPRs regulate GF signaling through reversible protein tyrosine dephosphorylation. Hence, PTPR variations, in general, may merely affect body size (and, thus, are not selected against), but in the presence of other genetic risk factors contribute significantly to deciding the fate of an ASD case towards either HFA or SDA. Fig. 13 suggests Cl−/K+ channels as epistatic risk factors being more suitable drug targets for small children than growth factor receptor (Gleevac), further supported by the results of Fig. 16.

No interventions treat core / severe symptoms (Table 1). The two approved drugs, resperidone (Risperdal) and aripripazole (Ability), merely reduce irritability in children >5 yrs. Most behavioral therapies target milder forms of ASD. We propose a repurposed drug to prevent progression to more severe forms of autism by reducing hyperexcitation without interfering with not a psychoactive drug), neuro-transmitters (not drug in a unique formulation (not available for use anywhere in the world) for IP and improved safety. safety

Previous ssGWAS and CNV analyses have largely failed to elucidate the precise mechanism by which Ras and excitatory Ca2+ signaling interact and how to identify effective pharmacological interventions, With the novel computational biostatistics approach applied to the AGP data, the consistent results of from two independent populations are broadly consistent with previous isolated findings, confirm the Ras/Ca2+ hypothesis, and provide evidence-based insights into the etiology of ASD, a novel treatment paradigm, and approved drugs that might be repurposed for ASD. Our repeated findings in PTPRs complement previous findings in FMR1 / PTEN / MSNP1AS / SYNGAP1 in suggesting impaired neuronal growth regulation as a critical aspect in the etiology of SDA and, thus, a pharmacological intervention beginning at the of early symptoms, around 12 months of age. We posit a counterproductive maladaptive socioemotional response to exposure to unfamiliar people, caused by sensory overload involving disorganized perception of salient social features, potentially leading to experiences as intolerable as migraines, and, subsequently, pruning of structures for speech and ‘social intelligence’, consistent with aberrant brain growth pattern during early childhood. As a testable hypothesis, we suggest drugs that have been used to target ion channels in even younger children for decades (to treat pain in juvenile idiopathic arthritis) as a pharmacological intervention to decrease this hyperexcitation to a level where a child does not feel the need to withdraw from social interaction.

CTRL

SDA

AGP II

AGP I

HFA

6

Fig. 15: PTPRT allelotype profiles in SDA cases, melanoma controls, and HFA cases. Rows indicating individual subjects’ SNP profile (orange/green: homozygous; yellow: heterozygous) are sorted within each population by diplotype µ-score (dark green to dark read) computed from the three consensus SNPs (rs6102794, rs6072694, and rs6102795 out of the six-SNP PTPRT region of Figure 3), which are highlighted as more saturated. Dotted lines added for visual guidance.

4.65 4.67 4.67 4.67 4.68 4.69 4.69 4.71 4.72 4.75 4.76 4.76 4.76 4.76 4.81 4.83 4.94 4.94 4.95 4.96 4.96 4.98 5.00 5.02 5.03 5.03 5.04 5.12 5.22 5.22 5.23 5.24 5.36 5.39 5.56 5.70 5.77 5.79 5.82 6.19 6.21 C ACN A1 C PRPH2 R HOBTB1 KCNAB1 IL28R A ZFPM2 THOC 2 PFN2 ABCC4 H TR1 B MYO5 B ICOS KISS1 R PANK2 C UL1 ARHGAP48 ARHGAP24 C RAD D SEPT9 SL C25 A21 ETV6 PTENP1 SPG20 PPFIBP1 ATP1A3 /GR IK5 TAT..AP1G1 C DH 13 API5 KAL1 R APGEF3 APLN L IMK1 SSBP3 R BPJ PCDH 7 MAP2 D MD ACOT1 1 PMP2 ANO2 ANO4

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Fig. 16: Comparison of HFA cases against all parental controls. PTENP1 may act as a decoy miRNAs targeting PTEN. Cytogenic bands: ANO2: 12p13.3, ANO4: 12q23.3.

Gene·gene·environment·behavior·development interaction at the core of autism: • (Nonsyndromic) autism starts with largely unknow prenatal events (♂: age, ♀: virus/stress ...) • Mutations in growth factor regulators (PTPRs) lead to neuronal overgrowth (brain sizes). • Mutations in K+/Cl− channels cause Ca2+ mediated overexcitation of neurons (“intense world”). • Stressful environments (urbanization) contribute to epistatic interaction (increasing prevalence). • This GGE interaction causes “migraine-like” experiences during the “stranger anxiety” period where children learn verbal/social skills, leading to behavioral maladaptation (“tune-out”). • The lack of verbal/social stimuli causes “patches of disorganization” (Stoner 2014, NEJM) as a form of developmental maladaptation when underutilized brain areas are permanently “pruned”. The PTPRs point to a short window of opportunity (WoO) for pharmacological intervention: • Treatment has to begin as early as possible, while neurons are still growing (12 mos of age. • “Patches Patches of disorganization” disorganization in >2 yr old brains. • Romanian orphans developed “quasi quasi--autism” autism when placed into foster care at >24 mos of age. • Hearing impairment leading to intellectual disability when diagnosed >24 mos of age. A rational drug target: treating either of two epistatic risk factors suffices: • Blocking growth factors (Gleevac, ...) is unacceptable in children merely at risk of ASD. • Ion channel modulators have been used in small children for arthritis and seizures. An orphan drug (mutual prodrug, pat. pending) • reduces risk of complications and overdose • guarantees market exclusivity (IP protection). 2 yrs to a $20B/yr market: After decades of use in related diseases, no phase 0/1/II trials are needed. We propose a RCT (n = 200) with minisentences within 12 months as clinical outcome. Strong IP protection • Drug not available for use anywhere in the world, • Higher safety prevents off-label prescriptions. • After “Makena”, FDA enforces compounding law. law • Orphan drug / pat. pending / few other indications • Data base driven companion test (US 7,664,616) Tab. 1: Drugs for core symptoms of ASD in phase III trials. Name, sample size, age group, begin, result

Fig. 17: AGP Manhattan Plot. Top: AGP I, bottom: AGP II. The top 20 genes in either stage are labeled in black, as are the top 11 genes by joint significance. Genes related to the Ras/Ca2+ pathway are highlighted in bold. Dots in regions primarily significant in male or female cases are shown as squares and circles, respectively. Results shown in red have low reliability (µIC)(Wittkowski 2013), names in gray indicate additional genes of potential interest, space permitting.

• Curemark CM-AT • buspirone • sapropterin • D-cycloserine • bumetanide • oxytocin • acamprosate • memantine

182: 6–16, 2009, no results 166: 2– 6, 2009, ongoing 41: 3– 6, 2009, no effect 102: 5–11, 2010, no results 60: 3– 6, 2010, “promising” 30: 12–18, 2013, ongoing 36: 5–18, 2013, recruiting 90: 12–18, 2014, not started

Childhood Environment Nutrition ...

Prenatal Environment Maternal Stress

Genetic Risk Growth Factors, Ras signaling Genetic Risk De novo variations Paternal age

Stress Unfamiliar Faces and Voices Protective Environment Few Caretakers, No TV, ...

Genotyping

Phenotyping

Special Education ...

Age ≈1 yr

Age >3 yr

Autism

Pharmacological Intervention ...

Pharmacological Intervention Ion Channels

Genetic Risk Calcium signaling

Fig. 18: Hypothesized interventions in children with ASD. During the critical period of developing cortical structures for social interactions the risk of stress-induced regression might be reduced through a combination of strategies including a protective environment with limited exposure to unfamiliar people and pharmacological interventions to reduce hyperexcitability by targeting ion channels.

CONCLUSION Genetic data from many more studies are already publicly available, so that the proposed computational biostatistics approach will advance personalized medicine and comparative effectiveness research. The genetic data collected over the last decade, could finally yield profound insights into the mechanistic bases of many common diseases and subgroup analyses of Phase III trials can now suggest risk factors for adverse events and novel directions for drug development.

REFERENCES Morales JF, Song T, Auerbach AD, Wittkowski KM (2008) Phenotyping genetic diseases using an extension of µ-scores for multivariate data. Stat Appl Genet Mol. 7:19 Wittkowski KM, Song T (2010) Nonparametric methods for molecular biology. Methods Mol Biol. 620:105 Wittkowski KM, et al. (2013) From single-SNP to wide-locus: genome-wide association studies identifying functionally related genes and intragenic regions in small sample studies. Pharmacogenomics. 14(4):391. Available from: http://www.ncbi.nlm.nih.gov/pubmed/23438886 Wittkowski KM, et al. (2014) A Novel Computational Biostatistics Approach Implies Impaired Dephosphorylation of Growth Factor Receptors As Associated With Severity of Autism. Translational Psychiatry. 4:e354. Available from: http://www.nature.com/tp/journal/v4/n1/full/tp2013124a.html.

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