Sex Bias and the Genetics of AutismTychele N. Turner, Ph.D.
Assistant Professor
Washington University School of Medicine
Department of Genetics
March 25, 2020
@tycheleturner
Outline
✧ Background of autism epidemiology and genetics
✧ Sex bias in autism and the Diagnostic and Statistical Manual of Mental Disorders, 5th Edition (DSM-5)
✧ Potential biological reasons for a sex bias
✧ Evidence for a female-protective effect in autism
✧ Study of de novo variants by sex
✧ Gene regulatory networks in autism
Autism
² Childhood neurodevelopmental disorder
² Heritability: ~80% Sandin et al. 2017, Steffenburg et al. 1989, Bailey et al. 1995
² Prevalence: 1/59 Centers for Disease Control and Prevention
² Sex ratio of 4 males : 1 female Palmer et al. 2017, Jorde et al. 1991
Photo credit: Dr. John Constantino, MD Washington University School of Medicine
Genetic features of autism
²Single gene syndromic causes✧ ADSL, CDKL5, FMR1, MECP2, PTEN, SHANK3, TSC1
✧ Fragile X syndrome✧ Expansion of repeat sequence on the X chromosome and
affecting the FMR1 gene✧ Primarily affects boys
²Common variation: genome-wide association studies and polygenic risk scores
² Overall contribution up to 50%
² Five genome-wide significant loci
²Rare variation: copy number variants and de novogene-disrupting mutations
² 15q11-13 maternal duplications, 16p11.2, many others, most are recurrent
² 124 genes (e.g., CHD8, DYRK1A, FOXP1, GRIN2B, PTEN, SCN2A, TBR1)
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
ID or GDD Speech delay Motordifficulties
Microcephaly Feedingdifficulties
Visionabnormalities
5 or moresymptoms
DYRK1A Total (n = 61) DYRK1A ascertained for ASD (n = 18)Idiopathic ASD (n = 1981) Idiopathic ASD with IQ < 70 (n = 487)
Earl et al. 2017, Molecular Autism
Outline
✧ Background of autism epidemiology and genetics
✧ Sex bias in autism and the Diagnostic and Statistical Manual of Mental Disorders, 5th Edition (DSM-5)
✧ Potential biological reasons for a sex bias
✧ Evidence for a female-protective effect in autism
✧ Study of de novo variants by sex
✧ Gene regulatory networks in autism
Disorders in the DSM-5 with sex biases
0.09
0.250.33 0.33 0.33 0.33
0.57
0.67 0.67 0.67
0.80 0.80
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
anorexianervosa
kleptomania briefpsychoticdisorder
panicdisorder
agoraphobia generalizedanxietydisorder
intellectualdisability
attentiondeficit
hyperactivitydisorder
specificlearningdisorder
motordisorder
autismspectrumdisorders
tic disorder
fract
ion
of c
ases
that
are
mal
e
✧ Sex bias is seen in many neuropsychiatric and neurodevelopmental disorders✧ It is an important characteristic to consider when studying these phenotypes
Aggregated from “Understanding Mental Disorders, Your Guide to DSM-5” by the American Psychiatric AssociationA comprehensive review on sex bias in human disease is Ober et al. 2008, Nature Reviews Genetics
Outline
✧ Background of autism epidemiology and genetics
✧ Sex bias in autism and the Diagnostic and Statistical Manual of Mental Disorders, 5th Edition (DSM-5)
✧ Potential biological reasons for a sex bias
✧ Evidence for a female-protective effect in autism
✧ Study of de novo variants by sex
✧ Gene regulatory networks in autism
t = threshold
affected
tm tf
male (m), female (f)specific thresholds
liability
Liability distribution in multifactorial disorder
liability = genetic and environmental factors
unaffectedaffected malesunaffected males
affected femalesunaffected females
Reasons for sex bias² Sex chromosomes (females have two X chromosomes and males have one X, males have a Y chromosome
and females do not, some X chromosome genes can escape X inactivation, …)² Hormones like estrogen and androgen² Imprinting ² Protective effect (greater load of mutation in severity or number)
for a disorder affecting more males
Falconer 1965, Ann. Hum. Genet. ; Strachan and Read, Human Molecular Genetics; Chakravarti and Turner 2016, Bioessays; Lady Washington, Vancouver Family Magazine
Outline
✧ Background of autism epidemiology and genetics
✧ Sex bias in autism and the Diagnostic and Statistical Manual of Mental Disorders, 5th Edition (DSM-5)
✧ Potential biological reasons for a sex bias
✧ Evidence for a female-protective effect in autism
✧ Study of de novo variants by sex
✧ Gene regulatory networks in autism
2011
2012
2013
2014
2015
2019
Higher frequency of de novo CNVs in females with autism (Levy et al., Neuron)
Increased rate of de novo variants in females with autism (Neale et al., Nature)
More autistic impairments in siblings of females with autism than males with autism (Robinson et al., PNAS)
Excess of deleterious autosomal CNVs in females with NDDs (Jacquemont et al., AJHG)
Higher frequency of de novo CNVs in females with autism (Sanders et al., Neuron)
Excess of females with recessive gene disruptions (Doan et al., Nature Genetics)
2015Gene discovery in female-enriched multiplex families (Turner et al., Nature)
Consistent with more severe mutations (rare variants of large effect) in females to develop autism
We (and others) have hypothesized that gene discovery would be greatest in the rarer class (females in NDDs)
Evidence for the female-protective effect in neurodevelopmental disorders (NDDs)
Outline
✧ Background of autism epidemiology and genetics
✧ Sex bias in autism and the Diagnostic and Statistical Manual of Mental Disorders, 5th Edition (DSM-5)
✧ Potential biological reasons for a sex bias
✧ Evidence for a female-protective effect in autism
✧ Study of de novo variants by sex
✧ Gene regulatory networks in autism
Three main questions
1. Are there any genes significant in only one sex and not the other?
2. Is there a differential gene discovery rate in males and females?(i.e., is gene discovery greater in females as the female-protective effect would suggest?)
3. Are there genes with differential counts of de novo variants in a direct comparison of males and females?
de novo mutations from whole-exome and whole-genome sequencing in NDDs
discovery cohort✧8,825 sequenced parent-child
trios (n = 26,475 individuals)
✧de novo variants
✧denovo-db v.1.5
✧sex ratio = 2.2http://denovo-db.gs.washington.edu/
Turner et al. 2016, Nucleic Acids Research
Twice as many males are needed to reach similar numbers of significant genes in the discovery cohort
X chromosome
autosomes
17 19 18
CASK, DCX, DDX3X, HDAC8,MECP2, NAA10,SMC1A, USP9X, WDR45, ZC4H2
CDK13, GATAD2B, ITPR1,
LEO1, NAA15,PPM1D, WAC
ADNP, ARID1B, ASXL3,CTNNB1, DYRK1A, EP300,FOXP1, HNRNPU, KANSL1,
KCNQ2, MED13L, POGZ,PPP2R5D, SCN2A, SETD5,
SLC6A1, STXBP1, TBL1XR1,TCF4
ANP32A, BRAF,CHD2, CHD8,
CSNK2A1, CTCF,DLX3, ENO3,
MBD5, MEF2C,MEIS2, NSD1, PTEN, SATB2,
SMARCD1, SPAST, SYNGAP1, WDR26
males (n=6,046)females (n=2,779)
Exome-wide significance at p < 4.3 × 10-7✧ 36 genes in females✧ 37 genes in males✧ 54 in the union
Gene discovery is greater in females
Greater significant gene discovery in females
✧ Downsampling analysis to the same number as the original NDD females(n = 2,779 females, 36 significant genes)
✧ 10,000 sets of only NDD malesSignificantly more genes discovered in females than male-only (p < 1x10-4)
✧ 10,000 sets of NDD males and femalesSignificantly more genes discovered in females than males and females combined (p = 0.02)
Potential NDD-related phenotypic differences in females
✧Correlation between fraction of individuals with developmental disorder (DD) and fraction that are female in downsampling sets
✧For individuals with autism where IQ data are available, IQ is lower in females than in males (p = 1.8 x 10-6)
✧ female mean = 75.0
✧male mean = 82.1
de novo mutations from whole-exome and whole-genome sequencing in NDDs
discovery cohort✧8,825 sequenced parent-child
trios (n = 26,475 individuals)
✧de novo variants
✧denovo-db v.1.5
✧sex ratio = 2.2
replication cohort✧18,778 sequenced parent-child
trios (n = 56,334 individuals)
✧de novo count data for genes significant in discovery cohort
✧GeneDx
✧sex ratio = 1.2
After replication, proportionally there are still more genes enriched in females
X chromosome
autosomes7 15 3
DDX3X,HDAC8,NAA10,USP9X, WDR45
CDK13,ITPR1
ADNP, ARID1B, CTNNB1,DYRK1A, FOXP1, HNRNPU,MED13L, POGZ, PPP2R5D,SCN2A, SETD5, SLC6A1,STXBP1, TBL1XR1, TCF4
CHD8,MBD5,
SYNGAP1
malesfemales
Exome-wide significance at p < 4.3 × 10-7✧ 22 genes in females✧ 18 genes in males✧ 25 in the union
Gene discovery is greater in females
0
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Chromosome
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1 3 5 7 9 11 13 15 17 19 212 4 6 8 10 12 14 16 18
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ITPR1
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 2122 X
male-only
SYNGAP1MBD5
CHD8-log1
0(p-
valu
e)
Chromosome
Discovery Replicationgene chromosome male minimum p-value female minimum p-value male minimum p-value female minimum p-valueCDK13 chr7 0.0623 6.05E-16* 7.00E-07 1.45E-07*DDX3X chrX NA 5.54E-50* 7.51E-02 4.41E-117*HDAC8 chrX 0.109 4.88E-13* NA 1.32E-16*ITPR1 chr3 0.0139 3.07E-07* 1.19E-05 1.03E-07*NAA10 chrX NA 7.47E-10* NA 1.45E-18*USP9X chrX 0.182 2.67E-09* 3.83E-01 6.54E-12*WDR45 chrX 0.0025 1.56E-08* 2.99E-04 8.83E-41*CHD8 chr14 2E-12* 5.64E-06 1.08E-12* 1.48E-04MBD5 chr2 3E-07* 2.35E-02 4.32E-08* 6.95E-02SYNGAP1 chr6 2E-09* 2.15E-04 3.39E-18* 2.09E-03
Replicated genes significant in only one sex
* multiple-test correction significant
♀
♂
Female-only enriched genes on the X chromosome✧ Three of the five genes are either constitutive (DDX3X, USP9X) or facultative (NAA10)
escapers of X inactivation
✧ Consistent with a model where females have buffered expression and develop NDD whereas male with same mutation is lethal
✧ Different than phenotypes like Fragile X Syndrome where females more often do not have the syndrome and males do
USP9XDDX3XWDR45
HDAC8
NAA10
Cerebellum Cortex Hippocampus Left Ventricleof the Heart
Liver
50
100
150
TPM
femalemale
Gene expression for DDX3X
Brain
https://gtexportal.org/home/gene/DDX3XGenerally, 13% of X chromosome genes constitutively escape inactivation (Tukiainen et al. 2017)
Female-only enriched genes on the X chromosome
USP9XDDX3XWDR45
HDAC8
NAA10
Gene Known phenotype
USP9X Intellectual disability in females (Reijnders et al. 2016, AJHG)
DDX3X Intellectual disability in females (Snijders Blok et al. 2015, AJHG)
WDR45 Neurodegeneration with brain iron accumulation mostly affecting females (Haack et al. 2012, AJHG)
HDAC8 Cornelia de Lange syndrome with more severe intellectual disability observed in males than females (Kaiser et al. 2014, HMG)
NAA10 Ogden syndrome which affects both males and females (Rope et al. 2011, AJHG)
Direct comparison of males and females reveals DDX3X as the one genome-wide significant gene
Discovery Replication
Gene Chromosome male counts
female counts
Fisher’s exact test p-value
Fisher’s exact test odds ratio
male counts
female counts
Fisher’s exact test p-value
Fisher’s exact test odds ratio
genome-wide significant in both
nominal significance in both
DDX3X X 0 26 4.73x10-9 Inf 2 65 1.76x10-11 20.15 yes yes
MECP2 X 1 12 1.26x10-3 13.08 8 54 1.77x10-5 4.18 no yes
NAA10 X 0 5 2.52x10-2 Inf 0 11 9.12x10-3 Inf no yes
HDAC8 X 1 7 3.23x10-2 7.62 0 11 9.12x10-3 Inf no yes
The other genes reaching nominal significance are also on the X chromosome
Power for the direct comparison of males and females
●●●●●●
●
●
●●
0 2000000 6000000 10000000
010
0020
0030
0040
00
power calcuations to 10,000,000 additional parent−child
sequenced trios
additional parent−child sequenced trios
num
ber o
f sig
nific
ant s
ex−b
iase
d ge
nes
A
●●●●●
●
●
●
●●
●●●●●
●
●●
● ●
Power80%90%100% ●●●●● ●
●
●
0 200000 400000 600000 800000 1000000
010
0020
0030
0040
00
subset of power calcuations to 1,000,000 additional parent−child
sequenced trios
additional parent−child sequenced trios
num
ber o
f sig
nific
ant s
ex−b
iase
d ge
nes
B
●●●●●
●
●
●
●●●●●
●
●●
Power80%90%100%
Supplemental Figure S2
Additional parent-child sequenced
trios
Genes at 80% power
Genes at 90% power
Genes at 100% power
10,000 1 1 1
20,000 4 4 1
30,000 7 4 1
40,000 17 8 2
50,000 19 18 4
100,000 148 126 18
500,000 4,208 1,918 1,886
1,000,000 4,253 4,245 4,208
5,000,000 4,660 4,623 4,615
10,000,000 4,660 4,660 4,657
Conclusions for “Study of de novo variants by sex”
✧ Sex bias is an important consideration when assessing a phenotype
✧Gene discovery, based on rare variants of large effect, is greatest in the rare class (females with NDDs) and can also reveal important genes on the X chromosome
✧While studies of NDDs have focused on a pooled (male and female) approach, other researchers studying new phenotypes may want to focus on the rarer class to detect genes with variants of large effect
✧Genes with rare variants of large effect provide key insight into the biology of the disorder (next part of talk)
Outline
✧ Background of autism epidemiology and genetics
✧ Sex bias in autism and the Diagnostic and Statistical Manual of Mental Disorders, 5th Edition (DSM-5)
✧ Potential biological reasons for a sex bias
✧ Evidence for a female-protective effect in autism
✧ Study of de novo variants by sex
✧ Gene regulatory networks in autism
Inputs / signalingGene Regulatory
Networks (GRNs)
Direct Molecular Output
Altered RNA and protein production
Terminal Output
Altered cell behavior and functions
Gene regulatory networks (GRNs) and their effect on biological activities
Feedback mechanisms
Gene regulatory networks are composed of a set of regulators, working together, that control output of RNA and protein levels
Davidson 2006, The Regulatory Genome: Gene Regulatory Networks in Development and Evolution
B
An example of a transcriptional GRN
✧ Input signals use cis-regulatory elements to regulate multiple genes
✧ Expression of these genes can regulate other genes and provide + or – feedback on the network
✧ Genes A and B have different effects because of their position in the network
✧ Deletion of A abrogates all downstream effects (is rate-limiting in the GRN)
✧ Deletion of B only has local effects
A
signal inputs
Individuals at the extreme for discovery of key rate-limiting genes
Chakravarti and Turner 2016, Bioessays
tm tf
male (m), female (f)specific thresholds
liability
affected malesunaffected males
affected femalesunaffected females
for a disorder affecting more males
B
A
signal inputs
Females with NDDs (rarer class) may have rarevariants of large effect in genes that are rate-limitingto GRN (e.g., they may regulate many other genes)
Replicated, significant genes in NDDs
X chromosome
autosomes7 15 3
DDX3X,HDAC8,NAA10,USP9X, WDR45
CDK13,ITPR1
ADNP, ARID1B, CTNNB1,DYRK1A, FOXP1, HNRNPU,MED13L, POGZ, PPP2R5D,SCN2A, SETD5, SLC6A1,STXBP1, TBL1XR1, TCF4
CHD8,MBD5,
SYNGAP1
malesfemales
Exome-wide significance at p < 4.3 × 10-7✧ 22 genes in females✧ 18 genes in males✧ 25 in the union
✧ Almost all genes are significant in females
✧ The 3 male-only are also nominally significant in females
Expression of significant gene sets
✧ Sex-biased genes show broad expression across adult human cortical cell types
✧ Shared risk genes ✧ are expressed in significantly more cell types than control genes✧ are enriched in significantly more neuronal cell types than non-neuronal cell types
0.00
0.25
0.50
0.75
1.00
0 10 20 30 40Number of clusters with expression
Pro
porti
on o
f gen
es
Gen set typeControl
ASD (sex-biased)
Gene setASD (Syn)
Control (DNM)
Females
Males
Males and Females
Inhibitory neurons
0.00
0.25
0.50
0.75
1.00
0 5 10 15 20 25Number of clusters with expression
Pro
porti
on o
f gen
es
set_typeControl
ASD_sex_biased
setASD_SYN
Control_DNM
Females
Males
Males_Females
Excitatory neurons
0.00
0.25
0.50
0.75
1.00
0 2 4 6Number of clusters with expression
Pro
porti
on o
f gen
es
Non-neuronal cellsA B C
Replicated significant genes are highly connected and are localized to the nucleus
✧ 22 of the 25 genes code for proteins within the
same PPI network
(p < 1.0 x 10-16)
✧ Associated with transcriptional processes (p =
5.4 x 10-4) and chromatin binding (4.7 x 10-3)
✧ 90.9% localize to the nucleus
(p = 9.3x10-4)
✧ Very consistent with one of the core modules
identified previously by many other groups
✧ These are key regulators of other genes
Conclusions for “Gene regulatory networks in autism”
✧GRNs are important for our understanding of translation of gene discovery
to molecular impact
✧Study of the rarer class (females in autism) can identify genes that are rate-
limiting to GRNs thereby revealing the pathways of focus
✧When looking at NDDs, significant genes are highly connected and related to
one another
✧These genes are responsible for regulating many other genes
Conclusions and Future Directions
✧Sex bias is an important consideration when assessing a phenotype
✧Gene discovery, based on rare variants of large effect, is greatest in the rare class (females with NDDs) and can also reveal important genes on the X chromosome✧ However, it is important to note that most significant genes are not exclusively
discovered in males or females
✧Work remains in understanding the sex difference in autism with regards to both genetic (e.g., inherited variation) and non-genetic factors
Acknowledgments
http://turnerlab.wustl.edu/
This work was supported by:² Autism Science Foundation postdoctoral fellowship ² National Institute of Health Pathway to Independence Award (K99/R00)² McDonnell Center for Cellular and Molecular Neurobiology
Turner LabJeffrey Ng, M.S.Evin Padhi Eleanor SamsStephanie Sarkar
University of WashingtonEvan E. Eichler, Ph.D.Amy B. Wilfert, Ph.D.Raphael A. Bernier, Ph.D.Micah Pepper, MPH
GeneDxKyle Retterer, M.S.Rebecca I. Torene, Ph.D.Zhancheng Zhang, Ph.D.
Allen InstituteTrygve E. Bakken, M.D., Ph.D.
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