Sex Bias and the Genetics of Autism · 2020-03-25 · Outline Background of autism epidemiology and genetics Sex bias in autism and the Diagnostic and Statistical Manual of Mental

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







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







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







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







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

http://denovo-db.gs.washington.edu/

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


✧de novo variants

✧denovo-db v.1.5

✧sex ratio = 2.2

replication cohort✧18,778 sequenced parent-child


✧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


Gene discovery is greater in females

0

10

20

30

40

50

Chromosome

-log 1

0(p)

1 3 5 7 9 11 13 15 17 19 212 4 6 8 10 12 14 16 18

●●●●●

●

●

●

●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●

●●

●●●●●●●●

●

●●●●●●●●●●

●

●●

●

●●●●●

●

●●●●●●●●●●●●

●

●●●

●

●●●●●●●●●●●●●●●●●

●

●

●

●●●●●●

●

●●●●●●●●●●●●●

●●●●●●●●●

●●

●●●●●

●

●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●

●

●●●●●

●

●●

●

●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●

●

●●●●●

●

●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●

●

●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●

●

●●●●●

●

●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●

●

●●●●●●●●●●

●

●●●●●●●●●●●●●●●●

●

●●●●●

●●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●

●

●●●●●●●●●

●

●●●

●

●

●

●●●●●●●●●●●

●

●●●●●●●●●●●●●

●

●●●●●●

●●●●●●●●●●●●●●●

●

●●●●●

●

●

●

●●

●

●●

●

●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●

●

●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●

●

●●●●●●

●●

●●●●●●●●●●●●●●

●

●●●●●●●●●●●●

●

●●●●●●●●●

●

●●●●●●●

●

●●●●●●●●●

●

●●

●

●●●●

●

●●●●●●●

●

●●●●●●

●

●●●●

●

●

●

●●●●●●●●●●●●●●●●●●●●●

●●

●●●●●●●●●●●●●●●●

●

●●

●

●

●

●●●●●●

●

●●●●●

●

●

●

●●

●

●●●●●●●●

●

●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●

●●

●●●●●●●●●

●

●●●●●●●●●●●●●●●●

●

●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●

●

●

●●●●●●●●

●●

●●●

●

●●●●●

●

●●●●●●●●●●●

●

●

●

●● ●●●●●●●●●●●●●●●●

●●

●●●●●●●●●

●●

●●●

●

●●●●●●●●●●●●●●●●●●●●

●

●●●●

●

●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●

●●●●●●

●

●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●

●

●●

●

●

●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●

●●

●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●

●●●

●

●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●

●

●●●●●●●●●●

●

●●●●●●●●●●●●●●●

●

●●●●

●

●●●●

●

●●●●●●●

●

●●●●●●●●●●

●

●●●●●●●●●●●●●

●

●●●●●●

●

●●●

●

●●●●●●●●●●●●

●

●●●●

●

●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●

●

●●●●●●●●●●

●

●●●●●●●

●

●

●●●●●●●●●●●●●●●●●

●●

●

●●●●●●●●●●●●●●●●●

●

●

●

●●●●●●●●●●●●●●●●

●

●

●●●

●

●●●●●●●●

●●●

●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●

●

●

●

●●●●●●●●●●

●

●●●●●●●●●●●●●●●●

●

●●●●●●●●●

●

●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●

●

●

●

●●●●●●●

●

●●●

●

●●●●●

●

●●

●

●●●●●●●●●●

●

●●●●●●●

●

●●●

●

●●●

●●

●●●●●●●●

●

●●●●

●●

●●●●●●

●

●

●

●●●●●●

●

●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●

●

●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●

●

●●

●●●●●●●●●●●●●●

●

●●●●

●

●●●●●●●●●●●●●

●

●●●●●●●●●

●

●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●

●●

●●●●●●●●●●●●

●

●●●

●

●●●●●●

●

●●●●

●

●●●●●●●

●

●●

●

●●●●

●

●

●

●●●●●●

●

●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●

●

●

●

●●●●●●●●●●

●

●●●●●●●●

●●●●●

●

●●●●

●

●●●●●

●

●●●●●

●

●●●●●●●●

●

●

●

●●●●●●●●●●●●●●

●

●●●●●●

●

●●●●●●●●●

●

●●●●●

●

●●

●

●●●●●●●●

●

●●●●

●

●

●●●●●●●●

●

●

●

●●●●●●●●●●●●●●●

●

●●

●

●●●●●

●

●●●

●

●●●●●●●●●●●●

●

●●●●●●●●●●●●

●

●●●●●●●

●

●●

●

●●●●●●

●

●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●●

●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●

●

●

●●●●●●●●●●●

●●

●●●●●●

●

●●●●●●●●●●●

●

●●●●

●

●●●●●●●●●●●●●●●●●●●●●●

●

●●●

●

●●●●●●●●●●●

●

●

●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●

●●●●

●

●●●●●●●●●●

●

●

●

●

●

●●

●

●●●●●●●●●●●●●●

●

●

●●●●●●●●●●●●●●●

●

●

●

●●●●●

●

●●●

●

●●●●●●

●

●●

●

●●

●

●●●

●

●●●●●●●●●

●

●●●●●●●●●●●

●

●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●

●

●●

●

●●

●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●

●

●●●●●

●

●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●

●

●●●●●●●●●●●●

●

●

●

●

●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●

●

●●

●

●

●

●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●

●

●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●

●

●●●●●●●●●

●●

●●●●●●●●●●●

●

●●●●●●●●

●

●●●●●●●●●●●●●●●

●

●●●●●

●●

●●

●

●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●

●●●●●●●●●●●●●●●●●●●

●

●●●

●

●●●●●●●

●

●●●

●

●●●●●

●

●●●●●●●●●●●●●

●

●●●●

●

●

●

●●●●

●

●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●

●

●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●

●

●●●

●●●●

●

●●

●

●

●

●●●●●●●●●●●

●

●●●●●●●●●●●●●●

●

●●●

●

●

●●●●●●●●

●

●●●●●●●●

●

●●●●

●

●●●●●●●●●●

●

●●●●●●●●●●●●●●

●●●●●●●●●●●●●●●●●●

●

●

●●●●●●●●

●

●●●●●●

●

●●●●●●●

●

●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●

●

●

●●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●

●

●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●

●●

●●●

●

●●●●●●●

●

●●●●●●

●●

●

●●

●●●●●●●

●

●●

●

●

●

●●●●

●

●●●●●●●●●●●●●●●●

●

●●●●●●

●

●●●●●●●●●●●

●

●●

●

●●●●●

●

●●●●●●

●

●●●●●●●●●●●●●●●

●

●●

●

●●

●

●●

●

●●●

●

●●●●

●

●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●●

●●●●●●

●●

●●

●

●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●

●

●●

●

●

●

●

●●

●

●●●●

●

●●●●●●●●●●●●

●

●●

●

●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●

●

●●●●●●●●●●●●●●

●

●●●●●●●●●●●●

●●

●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●

●

●●●

●●●●●●●●●

●

●●

●

●●●

●

●●●●●

●

●●●●●●●●●●●

●

●●●●●●●●

●●

●●●●●●●●●●●●●●●

●

●●●●

●

●●●●●●●●●●

●

●

●●●●●●●●●●●

●

●●●●●●●●●●

●

●

●●

●

●●●●●

●

●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●

●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●

●●●

●

●●●●●●●●

●

●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●

●

●●●●●●●●

●

●●●●●●●

●

●●

●

●●●●

●

●●●●●

●

●●●●●●●●●●●

●

●●

●

●●●●●●●●

●

●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●

●●●●●●

●

●●

●

●

●

●●

●

●●

●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●

●

●●

●

●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●

●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●

●

●●●●●

●●●●●●●●●●●●●●●●

●

●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●

●

●●●●●

●

●●●●●●●●●

●

●●●●●●●

●

●●

●

●●●

●

●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●

●

●●●

●

●●●●●●●●●●●●

●●

●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●

●

●●●

●

●●●●●●●●●●●●●●

●

●●●●●

●

●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●

●

●●●●●●

●

●●●●●●●●

●●

●●●●●●●●●●●●●

●

●●

●

●●●

●

●

●●●●●●●●●●●●●

●

●

●

●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●

●

●●●●●●●●●●

●

●●●●

●

●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●

●

●●●●●

●

●●●●●

●

●●●●●

●

●●●●

●

●●●●●

●

●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●

●

●●●●●●●●●●●

●

●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●

●

●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●

●

●●●●●●●●

●

●

●

●●●●●●●●●●●●●●●●●●●●

●

●●

●

●●●●●●●●●●●●●●●●●●●●●●●

●

●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●

●

●●●●●●●●●●●●●

●

●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●

●

●●●●●●●●●●●●●●

●

●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●

●

●●

●

●●●●●●●●●●●●●●

●

●●●●●●●●●●●

●

●●●●●●

●

●●●●●●●

●

●●●●●●●●●●●●

●

●●●●●●●

●

●●●

●

●

●

●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●

●●

●●●●●●●

●

●

●

●

●●●●●●●●●●●●●

●

●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●

●

●●●

●●

●●●●

●

●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●

●

●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●

●

●●●

●

●●

●

●●

●

●●●●●●

●

●●●●●●●●

●

●●●●●

●

●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●

●

●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●

●

●

●

●●●●●●●●●●●●●●●●●●

●

●●●●

●

●●●●●●

●

●●●●●●●●●●●●●●

●

●●●●

●

●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●

●

●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●

●

●●●●●●●●●●

●

●●●●●●●●●●

●

●●●

●

●●●●●●●●●●●●●●●●

●

●●

●

●●●●●●●●●●●

●

●●

●

●●

●

●●

●

●●●●●●●●●●●

●

●●●●●●

●

●●●●●●●●●●●●

●●●●

●●●

●

●●●●●●●●●●●●●●●

●

●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●

●

●

●

●●●●●●●●●●●

●

●●

●

●●●●●

●

●●●

●

●●●●●●●●●

●

●●●●

●

●●●●●●●

●

●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●

●

●●●

●

●●●●●●●●●●●

●

●●●●●●

●

●●

●

●

●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●

●

●●●●

●

●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●

●

●●●●●●●

●

●●●●●●●

●

●●●●

●

●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●

●

●●●●

●

●●●●●●●●●●●●●●

●

●●

●

●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●

●

●●●●●●●●●●●●

●

●●●●●●●●●

●

●●●

●

●●●●●●

●

●●

●

●

●

●●●●●●●●●●●●●●●●●

●

●●●●●●●

●

●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●

●

●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●

●

●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●

●

●

●

●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●

●

●

●●

●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●

●●●●●●●●●●

●

●●●●

●

●●●●●●●

●●

●

●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●

●

●●●●●

●●

●●●●●

●●

●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●

●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●

●

●●●

●●

●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●

●

●●●●●●●●

●

●●●●●●

●

●●

●

●●

●

●●●●●

●

●●●●●●●●

●

●

●

●●

●

●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●

●

●●●●●

●●

●●●●●●●●●

●

●●●●●●●●●●●●●●●●●

●

●●

●

●●

●

●

●

●●●●●●●●●●●●●●●●●●●●●●

●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●

●

●●●●●●●●●●●●●●●●●●●●●●●

●

●●●

●

●●●

●

●●●●●●

●

●●●●●●●●●●

●

●●●●●●

●

●●

●

●●●●

●

●●●●●●

●

●●●●●●●

●●●●●●

●

●●●●●●●●●●●

●

●

●●●●

●

●●●●

●

●●

●

●

●●●●●

●

●●●●●●●●●●●●●●●●●●●●

●

●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●

●●●

●

●●●●●●●●●●●●●●●●●

●

●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●

●

●●

●

●●●●●●●●●●●●●●●●●●

●

●●●●

●

●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●

●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●

●

●●●●●●●●●●●●●●●

●

●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●

●

●●●●●●●●

●

●

●

●●●

●●

●●●●●●●●●●●●●●●●●●●●●●●●

●●

●●●●●●●●●

●

●●●

●

●●●●●●●●●●

●

●●●●●●●●●●●●●

●

●●●●●●●●

●

●●●●●●●●●●●●●●

●

●●●●●

●

●●

●

●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●

●

●●●●●●●●

●

●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●

●

●●●●

●●

●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●

●

●●●

●

●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●

●

●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●●

●●

●

●●●●●●●●

●●

●●●●●

●

●●

●

●●●●●●●●●●●●●●

●

●●●

●

●

●

●

●

●●●●●

●

●●

●

●●

●

●●●●●●●●

●●

●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●

●

●

●

●●●●●●●●●●

●

●●●●●●●●●●●●●

●

●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●

●

●●●●●

●

●●

●

●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●

●

●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●

●

●●

●

●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●

●

●

●

●●●●●●●●●●●●●●●●

●

●●●●●●●●●

●

●●●●●●●●●

●

●●

●

●

●

●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●

●

●

●

●●●●●●●●●●●●

●

●●●●

●

●●

●

●●●●●●●●●●●●●

●

●●●●●●

●

●●●

●

●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●

●

●●

●

●●

●

●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●

●

●●●

●

●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●

●

●●●

●

●●●●●●●●●●●●●●●

●

●●●●●●●

●

●●●●●

●

●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●

●

●●●●●●●●●●●●

●●

●●●●●●●●●●●●●●

●

●●●●●●●

●

●

●●●●

●

●●●

●

●●●●●●●●

●

●●●●●

●

●●●

●

●●●●●●●●●●●●●●●●●●

●

●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●

●

●

●●

●

●●

●●

●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●

●

●●●●●●●●●●●●

●

●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●

●

●●●●●

●

●●●●●●●●●●●●●●

●

●●●●●●●

●

●●●

●

●

●

●●●●●

●

●●●

●●

●

●

●●●●●●●●●●●●●●●●●

●

●●●●●●●●

●

●

●

●

●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●

●

●●●●●●●●●●●●●

●

●●●

●

●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●

●

●

●

●●●●●●●●●●●●●●

●

●●

●

●●●●●●●●●●●●●●●●●●●

●

●●●●●●●

●

●●●●●●●

●

●●●●

●

●●●

●

●●●●●●●

●

●●●●●●●●●●●●●●●●

●

●●

●

●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●

●

●●

●

●●●●●●●●●●

●

●●●●●●●●

●

●●●

●

●

●

●●●●●●

●

●●●●

●

●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●

●

●

●

●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●

●

●●●●●●

●

●●●●●●●●●●●●●

●

●●●●●●●●●●●●

●

●●●●●●●●

●

●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●

●

●●●●●●●●

●

●

●

●●●●

●

●●●●●●●●●●●●●●●●

●

●●●●●●●

●

●●●

●

●●●●●●●●●●●●●●●

●●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●

●●

●

●

●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●

●●

●●●●●●●●●●●●●

●

●●

●

●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●

●

●●●●●●●●●●●●●●●

●

●●●●●●●●●●●

●

●●●●●●●●●●

●

●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●

●

●●●●●

●

●

●●●

●●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●

●

●●●●●●●●●●●

●

●●●●●●●●●

●

●●●

●

●●●●●●●●●

●

●●

●

●●●●●●●●●●●●

●

●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●

●

●●●●●

●

●●●●

●

●●●●●●

●

●●●●●●●●●●●●●●●

●

●●●●●●●●●●

●

●

●

●

●

●●

●

●●●●●●

●

●●●●●●

●

●●●●●●●●●●●●●●●●

●●

●●

●

●●●●●●●●●●

●

●●●●●●●

●

●●●●

●

●

●

●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●

●

●●

●

●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●

●

●●●●●

●

●

●

●●●

●

●●●●●●●

●

●●●●●

●

●●●●●●●●

●

●●

●

●●●●●●●●●●●●

●

●●●●●●●●●

●

●●●●●

●

●●●●●●●●●●●●●●●●●●●

●

●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●

●

●●●●

●

●●●●●●●●

●

●●●●●●●●●●●●●●●●●

●

●●●●●●●

●

●●●●●●●●●●●●●

●

●●●●●●●●●●●●

●

●●

●

●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●

●

●●●●●●

●

●

●

●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●

●

●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●●

●●●●●●●●●●●

●

●●●●●

●

●●●●●●●●●●●●●●●●●●●●●

●

●

●

●●●●●●

●

●●●●●●●●●●●●●●●

●

●●

●●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●

●

●●●●

●

●●●●●●

●

●●●●●●

●

●●

●

●●●●●●●●●●●●●

●

●●

●

●●●●●●

●

●●

●

●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●

●

●

●

●●●●●

●

●●●●●●●●

●

●●

●

●●●●●●

●

●●●●●●●●

●

●●●●●●●●●●

●

●●●●●●●●●●●●●●

●

●●●●●●●

●

●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●

●

●●●●●●●●

●

●

●

●●●●●●●

●

●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●

●●

●●●●●

●

●●●●●●

●

●

●

●●●●

●

●●●●●●●●●●

●

●●

●

●●●●●●●●●●●●●●●●

●

●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●

●

●●●●●●●●●●●●

●

●●●●●●●

●●

●●●●●●●●●●●●●●●●●

●

●

●

●●

●

●●●●

●

●

●

●

●

●●●●●●●●●●●

●

●●●●●●●●

●

●

●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●

●

●●●

●

●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●

●

●●●●●●

●

●●●●●●●●●●●●●

●

●●

●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●

●●●●●●●

●

●

●

●●●●●●●●

●

●●●●●●●●●●●●●

●

●●●●●●●

●

●

●●●●●●●

●

●●●●●

●

●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●

●

●●●

●

●●●●●

●●

●●●●●

●

●●●●●●●

●

●●●●●●●●●●●●

●

●●●●●●●●●

●

●

●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●

●

●●●●●●●●●

●

●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●

●

●●●●

●

●●

●

●

●

●●●●●●

●

●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●

●●●●●●●

●

●●●●●●●●●●●●●●●●●●

●

●●

●

●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●

●

●●●●●

●

●

●

●●

●

●●●●●●

●

●●●●●

●

●●●●●●●●●●●●

●

●

●

●●●

●

●●●●

●

●●●●●●●●●●●●●●●●●

●

●

●

●●

●

●

●

●

●●

●

●●●●●●

●●

●

●

●●●●●●●●●●

●●●

●●●●●●●●●●

●

●●●●●●

●

●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●

●

●

●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●

●

●●

●

●●●●●●●●●●●●●●●●

●●

●●●●

●

●●●●●●●●●●●●●●●●

●●●●●●●●●●●●

●

●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●

●

●●

●

●●●

●

●●●●●●●●●●●●●●●

●

●●

●

●●●●●●●●●●●●●●●●●●●●

●

●●

●

●●●●●●●●

●

●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●

●

●●●

●

●

●

●●●●●●●●●

●

●

●

●

●●●●●●●●●●●

●

●●●●●●

●

●●●●●●●●●●●●●

●

●●●

●

●

●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●

●

●●●●●

●

●●●●●●●●●●

●●●

●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●

●

●●●●●●●

●

●●●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●

●

●●●●●●

●

●●●●●●●●●●●●●●

●

●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●

●●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●

●

●●●●●●●●

●

●●●●●●

●●

●

●

●●●●●●●●●●

●

●

●●●

●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●

●

●●●●●●●●●●●●●●●●●●●●●

●

●●

●

●●

●

●●●●●●●●●●●●●●●●●

●

●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●

●

●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●

●

●●●●●●

●

●●●●●●

●

●●

●

●●

●

●●●●●●●●●●●

●

●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●

●

●●●●●●●●

●

●

●

●●●●●●●●●●

●

●●●●●

●

●

●●●

●

●●●●●●●●

●

●●●

●

●●

●●

●●●●●●●●●

●

●●●

●

●●

●

●●●●●●●●●●●●●●●

●

●

●

●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●

●

●

●

●●●●

●

●

●

●●●●●●●●●●●

●

●●●●●

●

●●●●●●●●

●

●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●

●

●●●●●

●

●●●●

●

●●●●●●●

●

●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●

●

●●●●

●

●●●●●●●●

●

●●●

●

●●●●●●●●●●●●

●

●●●●●●

●

●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●

●

●●●●●●●

●

●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●

●

●●●

●

●●●●

●

●●

●

●

●

●●●

●

●●●●●●●●●●

●

●●●●●●●●●●

●

●●●●●●●

●

●●

●

●●●●●●●●●●●●●●●●●●●●●●●●

●●

●●●●

●

●●●●●●●

●

●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●

●●

●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●

●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●●

●

●

●●●●●●●

●

●●●●●●●●●●

●

●●●●●●●

●

●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●

●

●●●

●

●●●●●●

●

●●●●●●●●●●●●●

●

●●

●

●●●●●●●●

●

●●

●

●●●●●●●

●

●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●

●

●●

●

●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●

●

●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●

●

●●●●●●●●●

●

●●●●●●●●●●●●●●●●

●

●●●

●

●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●

●

●●●●●●●●●●●●

●

●●●●●

●

●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●

●

●●●●●●

●

●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●

●

●●●●●●●●●●●●

●

●●●●●●●●●●●●

●

●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●

●

●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●

●

●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●

●●

●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●

●

●

●

●●●●

●

●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●

●

●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●

●

●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●

●

●

●

●●●●●

●

●●

●

●●●●●●●

●

●●●●●●●●●●●●●●●

●

●●●●●●

●

●●●●●●●●●●●●●

●

●

●

●●●●

●

●●

●

●●

●

●●●●●●●●●●●

●

●●●●

●

●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●

●●

●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●

●

●●●●●●●●●●●●●●●●●●●

●

●●●●

●

●●●●●●●●●●●●●

●

●●●●●●●●

●

●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●

●

●●●

●

●●●●●●●●●●●●●●●●●●●●●●●

●

●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●

●●

●●●●●●●

●

●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●

●

●●●

●●

●●●●●

●

●●●●

●

●●●●●●●●●●●

●

●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●

●

●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●

●

●●

●

●

●●●●●●●●●●●●●●●●●

●

●

●

●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●

●

●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●

●

●

●

●●●

●

●

●

●●●●●●●●●●●●●●●●

●

●●●

●

●

●●

●●●●●●●●●●●●●

●

●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●

●

●●

●

●●●●●●●●●●●●

●

●●

●

●●●●●●●●●●●

●

●●

●

●●●

●

●●●●●●

●

●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●

●

●●●●●●●●●●●●●

●

●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●

●●●●

●

●●●●●●●●●●●

●

●●

●

●●●●●●●●●

●

●●●●●●●●

●

●●●●●●

●

●●●

●

●●●●●

●

●

●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●

●

●●●●

●●

●●

●

●

●

●

●●●●●●●●

●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●

●

●

●

●

●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●

●

●●●

●

●

●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●

●

●

●●●●●●

●●●

●

●●

●

●●●●●●●●●

●

●●●●●●●●●●●●●●●●

●

●●●●●

●

●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●

●

●●●

●●

●

●

●●●●●●●●●

●

●●●●●●●●

●●●●●●●●●●

●

●

●

●

●●

●

●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●

●

●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●

●●

●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●

●

●

●

●●●

●

●●●●●●●

●

●●●

●

●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

female-only

ITPR1

CDK13

DDX3X

WDR45

HDAC8

NAA10

USP9X

-log1

0(p-

valu

e)

Chromosome1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 2122 X

0

10

20

30

40

50

Chromosome

-log 1

0(p)

1 3 5 7 9 11 13 15 17 19 212 4 6 8 10 12 14 16 18

●●●●●●

●

●●●●●●●●●●●

●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●

●

●●●●

●●

●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●

●●●●●●

●●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●●

●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●

●●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●

●

●●●●●

●

●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●

●

●●●●●●●●●●●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●

●

●●

●

●

●

●●●●

●

●●●●●●●●●

●

●●●

●

●●●●●●●●●●●●●

●

●●●●●●

●●●●●●

●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●

●

●●●●●●

●

●●

●

●●●●●●●●

●

●

●

●●●

●

●●●●●●●●●●●●●

●

●

●

●

●

●

●

●●●

●

●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●

●

●●●●●●●●●

●

●●●

●

●●●●●●●●

●

●●●●●

●

●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●

●

●

●

●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●

●

●

●●●●●●●●●●●●●●●●

●

●

●

●●●●●

●

●●●●

●

●●●●

●●

●●●●●

●

●●●●

●

●

●

●

●●

●●●●

●

●●●●●●

●●●●●●●●●●●●●●●●●●

●

●●●●●

●

●●●

●

●●●●●●●●●●●●●●●●●●●●●●●

●●●●●●●●

●

●●

●

●●●●

●●

●

●●●●●●●

●

●●●●●●●

●

●●●●●●●●●●

●

●●●●●●●●●

●

●

●

●●●

●

●

●

●●●●●●

●

●●●●●●●●●●●●●●●

●●●●

●

●●●●●●●●●

●

●●●●●●●●●●

●

●●●●●●●●

●●●●

●

●●

●●●●●●●●●●●●●●

●

●●●●●

●

●● ●●●●●●●

●

●●●●●●●

●

●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●

●

●

●

●●●●●●●●●●●●●●●●●●●●●●

●●●

●●●●●●●

●

●●●●

●

●●●

●●

●

●●●●●●●●●●●●●●●●●●●●●●

●●●●●●●●●●●●●●●●●●

●

●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●

●

●

●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●

●

●●

●

●

●●

●●●

●●●

●

●●●●●●●●●●●●●●●●●●●

●●

●●●●●●●●●●●●●●●●●●●●●●●●●●●

●●

●●●●●●●

●

●

●

●●●●●

●

●

●

●●●●

●

●●

●

●●

●

●●●●●●●●●●●●●●●

●

●●●●●●●●●●

●●●●●

●

●●

●

●●●●

●●●●●●●●●●●●●●

●

●●●●●●●●●

●●

●●●

●

●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●

●●●●●●●●●●●●

●

●

●

●●●●

●●

●

●●●●●●●●●●

●

●●●●●●●●

●

●●●●●●●●●●●●●●

●

●●●●

●

●●

●

●●●●●●●●

●

●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●●●●●●●●●

●

●

●

●●●

●

●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●

●

●●●●

●

●●●●●●●●●●●●

●

●●●●

●

●●●●●●●

●

●●●●●●●

●

●●●●●

●

●●●●

●

●●●●●●●●●●

●

●●●●●●●●

●

●●

●●●●●●●●●●●●●●●

●

●●

●

●●●●

●

●●●●

●

●●●●

●

●

●●

●●●●●●●●●●●●●

●

●

●●●●●●●●●●●

●

●●

●

●●

●●

●●●●●●

●

●●●●●

●

●●●●●

●

●●●●●●●

●

●●●

●

●●●●●●●●●●●●●●●●●●●

●

●●●●●●

●

●

●

●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●●●●

●

●●●●●●●

●

●●

●

●

●

●

●●

●

●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●

●

●●

●

●●●●●●●●●●

●

●

●

●●●●

●

●

●●●●●●●●●

●

●●●●●

●

●●●●●

●

●●

●

●●●

●

●

●

●●●●●●●●●●●●●

●●●

●●●●●●●

●

●●●●●

●

●●●●●

●

●●●●●●●

●●

●

●●●●●●●

●

●●●●●

●

●●●●

●

●

●

●

●

●

●

●●●

●

●●

●

●

●

●●

●

●●●●●

●

●●●●●●●●●●●●●●●

●

●●●●●●●●●

●

●●●●●●●

●

●

●●●

●

●

●●●●●●●

●

●

●

●●●

●

●●●

●

●●●●●●

●●●●●●●●●●●●●

●

●●

●

●●

●

●●●●●

●

●●●●●●●●●●●

●

●●●●

●

●

●●●●●●●●

●

●●●●●●

●

●●●●●●

●●

●●

●

●●●●

●●

●●

●

●

●●

●●●●●

●●

●●●●●●

●●

●

●

●●●

●

●●●●

●

●●●●●●●●●●●●●●●●

●

●

●

●●●●

●●●●●●●●

●

●●

●

●

●

●●●●●●

●

●●●●●●●●●●

●

●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●●

●●

●●●●●

●

●●●●●●●●●●●●●●

●●●●●

●

●●●●●●●●●

●

●●●●

●

●

●●●●●●

●

●●●●●●●●●●

●

●●●●●●●

●

●●●●●●●●

●

●

●●●●●●●●

●●

●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●

●●

●

●●

●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●

●

●●●●

●

●●

●

●●●●●

●

●●

●

●●●●●●●●●●●●●●●●●●●

●

●●●●●●

●

●●●●●●●●

●

●●●

●

●●●●●●

●

●●

●●

●

●

●

●

●

●●●●●●●●●●●●●●●●●

●

●

●●●●●

●

●●

●

●●●

●

●●●●●●

●

●

●

●

●

●

●●●●●

●

●●

●

●

●

●

●

●

●

●●

●●

●●

●●

●●●

●

●

●

●

●

●●●●●

●

●●●●

●●

●●●●●●●●●●●

●

●●●●●

●●●●●

●

●●●●●●●●●●

●

●●●●●●

●

●●●●●●

●

●●●●●●●●●●●●●

●

●●●●●●●

●

●●●●

●

●●●●●●●●●●

●

●●●●●●●●●●

●●

●

●●●●

●

●

●

●●

●

●●●●

●

●

●

●

●●●●●●●●●●●●●●●●

●●●●●●●●●

●

●●●

●

●

●●●

●

●

●

●●

●●

●

●

●●●●●●●●●●●●●●●

●

●

●

●

●

●●●●●

●●

●●●●

●●

●

●

●

●

●

●

●●●●●●

●

●●●●

●

●●●●●●●●

●●●●●●●●●

●

●●●●●●●●●●●●●●

●

●●

●

●●●●●●●●

●

●●●●●●

●

●●●●●

●

●●●●●●●●●●●●●●●

●

●●

●

●●●●●

●

●●●●

●●●●

●

●●●

●

●●●●●●●●●●

●

●●●●●●●

●

●●●●●

●●●●●●●●

●

●●●

●●●

●

●●

●

●●●●●●●●●●

●

●

●

●●

●

●●●●●●●●●●●

●

●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●

●●

●●

●

●●●●

●●●

●

●●●

●

●●●●●

●

●●●●

●

●●

●

●

●

●●●●●●●●

●

●●●●●●

●

●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●●●●●

●

●●

●

●●●●

●●

●●●

●●●●

●

●

●

●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●

●

●●●●●●●●●

●●●

●

●●●

●

●●●●●●●●

●

●●

●●●●●●●●●●●

●

●

●

●●●●●●

●

●

●

●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●

●●●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●

●●●●●●●●●●●●●

●

●●●●●

●

●●●●●●●●●●●●

●

●●●●

●

●

●

●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●●●

●

●●●●●●●●●●●●●●●●●●●●●

●

●●●●

●

●●●

●

●●●●●●

●

●●●

●

●●●●●●●

●●

●●●●●●

●

●●●●●●●

●●●●●

●

●●

●

●●●●●●

●

●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●

●

●●●●●●●●●

●

●●●●●

●

●●●●

●

●

●

●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●

●

●

●●

●●

●

●●●●●●●●●

●

●●●●●

●

●●●●●●●●●●●●

●

●●●●

●

●●●

●

●●●

●

●●●●●●●●●●●●●●●

●

●●●●●●●●●

●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●

●

●

●

●●●●●●●●●●

●

●●●●●●●●

●

●●●●●●

●●

●

●

●●●●

●

●●●●●●●●●

●

●●●●●●●

●●●

●●

●

●●●●●●●

●●

●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●

●

●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●

●

●●●●●●●

●

●

●

●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●

●

●●●●

●

●●●●●●

●

●

●

●

●

●

●

●●●●

●

●●●●●●●●●●

●

●●●

●

●

●

●●

●

●●●●●●●●●●●●●●●●●

●●

●●●●●●

●

●

●

●●

●

●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●

●

●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●

●

●●●●●●

●

●●●●●●●●●●●●●●●●●●●

●

●●

●

●●●●●●●●

●

●●●●●●●●●●●●●

●

●●●

●●

●●

●

●●●●●●●●●●●

●

●

●

●●●●●●●●●●●●●●●●●●●●●

●

●●

●

●●●●●●●●●●●

●

●●●●●

●

●●●●

●

●●

●

●●●●●●●●●●●●●●●●●●●●●●

●●●●●●●●●●●●

●

●

●

●●●●●●●●

●

●●●●●●●●●

●

●●●●

●

●

●●●

●

●

●

●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●

●

●●●●

●●

●

●

●●

●

●●●●

●

●●●●●●●

●●

●●●

●

●●●●●●●●●●●●●●●●●●●

●

●●

●

●●●●●

●

●●●●●

●

●●

●

●●

●

●●●●●●●●●●●●●●●●●●

●●●●●●●●●●●

●

●

●●●●●

●

●●●●●●●●●●

●

●

●

●

●

●●

●

●●●●●●●●●●●●●●

●

●●

●

●●●

●

●●●

●

●

●

●●●●●●●●●●●●●●●●●

●●●●●

●●●●●●●●●●●●●●●●●

●

●●●

●

●●●●●●

●

●●●●●●●●●●●●●●●●

●●●●●

●

●●●●●●

●

●

●

●●●●●●●

●

●●

●

●

●●●●●●●

●●●●●

●

●●●●

●

●●●●●●●●●

●

●●●●

●

●

●

●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●

●●●●●●●●●●●●●

●

●●●

●

●●●●

●

●●

●●

●●●●●●●●●●●●●●

●

●●●●

●

●●●●

●

●●●●

●

●●●

●●●●●●●●●●●●●●●

●

●●●

●

●●

●●●

●

●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●

●

●●●●●●

●

●

●●

●●●●●●●●

●

●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●

●●

●●●●●●●●●●

●

●●●●●●

●

●●●

●

●●●

●

●●●●●●●●●

●

●●●●●

●

●

●●●●

●

●●●●●●●

●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●

●

●●●●●●

●●●●●●

●

●●●●●●●●●●●●●●

●

●●●●●●

●

●●●

●●

●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●

●

●●●

●

●●●●●●●

●

●●●●●

●

●●●●●●●●●●

●

●

●●●●●●●●●●●●●

●●●●●●●●●●

●

●●●●

●

●●●●●

●

●●●●

●●●●●●●●●●●●●●

●

●●

●

●●●●●●

●

●●●●●●●●●●●●

●

●

●

●●●●●●

●

●●●●

●

●

●

●●●●●●

●●

●●●●●●●●●●●●●●●●●●●●●●●●●

●●

●●●●●●●●●●●

●

●

●

●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●

●

●●●●●●●●

●

●●●●●●●●●●●●●●●●

●

●●●

●

●●●

●

●●●●●

●

●●●●

●

●●

●

●●●●●●●●●●●●●●●●●

●

●●●●

●

●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●

●

●●●●●●●●●●●●

●

●●●●●

●

●●●●

●

●●●●●●●●●

●

●●●●

●

●●●●●●●●●●●●●●●●●●●●●●

●●●●●●●●●●

●

●●●●●●

●

●●●

●●

●●●●●●●●●●●●●●●●

●

●●●

●●

●

●

●

●●

●

●●●●

●●●●●●●●●●

●●

●●

●●

●●●●●●●●●●

●

●●●●●●●●

●

●●●●●●●●●●●●●●●

●

●●●●●

●

●●●●●

●

●●●●●●●●●●●●●●●●

●

●●●●

●

●●●●●●●●●●

●

●●●●●●

●

●

●

●●●●●●●●●●●●●●●

●

●●●●●●

●

●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●

●

●●●●●●●●●

●

●

●●●●

●

●

●

●

●●●

●

●●●●

●

●

●●●●●●●

●

●●

●

●

●

●●●●●●●●●●●●●●

●

●●●●●●●●●

●●

●●●

●

●●●●

●

●

●

●

●

●●

●

●

●

●●●●●

●

●

●

●●●

●

●●

●

●●●●●●●

●

●●●

●

●●●●●

●●

●●●●●●●●●●

●●

●●●●●●●●●●●

●

●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●

●

●●●●●●●●●●●●●●●●

●

●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●

●

●●●●●●

●

●●●●●

●

●●●●●●●●●●●●●●●●

●

●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●

●

●●

●

●●●●●

●

●

●

●●●●●

●

●●●●

●

●●●●

●

●●●●●●●●●●

●

●●●●●

●

●●●●●●●

●●

●●●●●●●●●●

●

●●●

●

●●●●●●●

●

●

●

●

●

●●

●

●●●●●●●

●

●●●

●●

●●●●●●●●●●●

●

●

●●

●●●●●

●

●●●●●●●●●

●●●●●●●●●●●●●●●●●●

●

●●

●●

●●●

●

●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●

●●●●●●●●●●●●●

●

●●●●●●●●●●●

●

●●●●●●●

●

●●

●

●●

●●

●●●●●●●●●●●●●●

●●

●●●

●

●●●●●●●●●●

●

●●

●●

●

●

●●●●

●

●

●

●●●●●●●●●●●

●●●●●●●

●●

●●●●

●

●●●●●●●●●

●●

●●

●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●

●

●●●●●●●●●

●

●●●●●●●●●●●●●

●●

●●●●●●●●●●●

●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●

●

●

●●

●

●

●

●

●●●●●●●

●

●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●

●

●●

●

●●●

●

●●●

●

●●●●

●

●●●●●●

●

●●

●●●●●●●

●

●●●

●

●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●

●

●●●●

●

●●●●

●

●

●

●●●●●

●

●●●●

●

●●●●●●●●●●●●

●

●●●●●●●●●●●

●

●●●

●

●

●

●●●

●

●●●●

●

●

●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●

●

●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●

●

●●●●●●●

●

●●●

●

●●●

●

●●●

●

●

●●

●

●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●

●

●

●

●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●

●

●

●

●●●●●●●●●●●●

●

●●●

●

●●

●●●●●●

●

●●●

●

●●●●●

●

●

●

●●●●

●

●●●●●●

●

●●

●

●●●●●●●●●●●●●●●●●

●

●●●●●●

●

●●●●●●

●

●●

●

●●●●●●●●●

●

●●●

●

●●

●

●●●●●●●●●

●●●●●●●●●●●●●●●●●●●●

●

●●●

●

●●

●●

●

●●●●

●

●●●

●

●

●

●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●●

●

●●●●

●

●●●●

●

●●●●●

●●

●

●●●

●

●

●●●●●●●●●

●

●●●●●●●

●

●●●●

●●●●●●●●●●●●●●●●●

●

●

●

●●●●●●●●●●●●●●●●●●●

●●

●●●●●●

●

●●

●

●●●●

●

●

●

●●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●

●

●●●●●●●

●

●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●

●

●●●●●●

●

●●●●●●●●●●●●●●●

●

●●●●●●●●●●●

●

●

●

●●●●●●●●

●

●●●●●●●●●●●

●

●●●●●●●●●●

●

●●●●

●

●

●

●●●●●●●●

●

●●●●●

●

●●●●●●●●●

●

●●●●●●

●

●●●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●

●●●●●●●●●●●●●●●●●

●

●●●●●

●

●●●●●

●

●●●●●

●

●●●●●●

●

●●●●●●●●

●●●●●●●●●●●●●●●●

●

●●●●●●

●

●●●●

●

●●●●●

●

●

●

●

●

●●●●

●●

●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●

●

●●●●●●●●●●●●●

●●●●●●●

●

●●●●●●●●●●●

●

●●●

●

●●●●●

●

●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●

●

●

●

●

●●●

●

●●●●●●●●

●

●●●●●

●

●●●●●●

●●

●●●●●●●●

●

●

●

●●●●●●●

●

●●●●●●●●●●●●●●

●

●●

●

●●●●●

●

●●●●●

●

●●●●●●

●

●●●●●●●●●

●

●●●●

●

●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●

●

●

●

●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●

●

●●●●●●●●

●

●●

●●●●●●●

●

●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●

●●

●

●●●●●●●●●●●●●

●

●●●●

●

●●●

●

●

●

●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●

●

●●●●●●

●

●●●

●●●●●●●●●●●●

●

●●●●●●●●

●

●●●●●●●●●●●●

●

●●●●

●

●

●

●●

●

●●●●

●

●●●●●●●●●●●

●

●●●

●

●●

●

●●

●

●●●●●●

●●●●●●●●●●

●

●

●

●●

●●●●●

●

●●

●

●●●●●

●

●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●

●●

●●●●●●●●

●

●●●●●●

●

●●

●

●●●●●●●●●●●

●●

●

●

●

●

●●●●●●●

●●●

●

●●●●●●●●●●●●●●●●

●

●●●●●●●●

●

●●

●

●●●●

●

●●●●●

●

●●●●●●●● ●●●●●●●●

●

●●●●●●●●●●●●●●●●

●

●

●

●●

●●

●●●●●●●●●●●●●

●

●

●●●●●●●●●

●

●●

●

●●

●

●●●●●●●●

●

●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●

●

●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●

●

●●●

●

●●●●

●

●●●●●●

●

●●●

●

●●●●●●●●●●●●●●●●●●●●

●

●●●

●

●●●●●●●●●●●●

●

●●

●

●●

●

●●●●●●●●●

●

●

●●●●●●●●●

●

●●●●●●●

●

●

●

●●●●●●

●

●●●●

●

●●●●●●●●

●

●●●●●●●●●

●

●●

●

●●●

●

●●●●●●●

●

●●●●●●●

●

●●●●●

●

●●

●

●●●●●●

●

●

●●

●●●●●

●●

●

●

●●

●

●●●●●●●●●

●

●●●●●●●●●

●

●●●●●●●●●●●

●

●●●●●●

●

●●

●

●●●●●●●

●

●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●

●●●●●●●●●●●●●●●●●●●●●●●●

●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●

●

●

●

●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●

●

●

●

●●●

●

●

●

●●●●

●

●●●●●●●●●●●

●●

●●●●●●●●●●●●●

●

●●●●●●

●

●●●

●

●●●

●

●●●●●●●●●●●●●●●●

●

●●

●●

●

●●●●

●

●●●●

●

●●●●●●●●●●●●●●

●

●

●●●●●●●●

●

●●●●●●●

●

●●●

●

●●●●●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●

●●

●●●●●●●●●●●●●●

●

●●

●

●●●

●

●●●●●●●●●

●

●●●

●●●

●●

●

●

●

●●●●●

●

●●●●

●

●●●●●

●●

●●●●

●

●●●

●●

●

●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●

●●

●●

●

●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●

●

●●

●●

●

●

●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●

●

●●●

●●●●●●●●●●

●

●●●●●●

●●●●●●●●

●●

●

●●●●●

●

●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●

●

●●●●●●●●●●●

●

●●●●●●●●

●

●●●●●●●●●

●

●●●●●●●●●●●●●●●●

●

●●●●●●

●

●●●●

●

●●●●●●

●

●●

●

●●●

●

●●●

●

●●●●●●

●

●

●●

●●●●●●●●●

●

●●●

●

●●●

●

●●●●●●

●

●●●●●

●

●●●●●

●

●●●

●

●●●●●

●

●●

●

●●●●●●●

●

●●●●●●●●●●

●

●●●●●

●

●●●

●

●●●●●●●●

●

●

●

●●●●●●●●●●●●●●●●●●●

●

●

●●●●●●●●●●●●●●●●●●●

●

●●●●●

●

●●●●

●

●●●●●●●●●●●●●●●

●

●

●

●●●●●●●●

●

●●

●

●●●

●

●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●

●

●●●●

●

●●●●

●

●●●●●●●●●●●●●●●

●

●

●

●

●●

●

●●

●

●●●●

●

●●

●

●●●●●●●●●●●●●

●

●●●●●

●

●●●●●

●

●●●

●

●●●

●

●●●●●●●●●●●●●

●

●●●●●●●●●●●

●●

●●●●

●●

●●●●●●

●●

●

●

●●●

●●●●●●●●●●●●●●●●●●●

●

●

●●

●●●●●●

●

●●●●●●●●●●●●●●

●

●●●●●

●

●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●

●

●

●

●●●●●●●●●●●

●

●●●

●

●

●●

●

●●●●●●●●●●●●●●●●●●●

●

●

●

●●●●●●●●●●●●

●

●

●

●●●●●●●●●●●●●●●●●

●

●●●●

●

●

●

●●●●●

●

●●●●●●●●●

●

●

●

●●●●●●●●●●●

●

●●

●

●

●●●●●●●●●●●●●

●

●●●●●

●●●

●●●●●●●●

●

●

●

●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●

●

●

●●

●●

●●

●●●●●●●●●

●

●●●●●●●●●●●●●●●●●

●

●●

●

●●●●●●●●●●●●●●●●●●●●●●●

●

●

●

●

●

●●

●●

●

●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●

●●●●●●

●

●

●

●●●●●

●●

●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●

●●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●

●●●●●●●●●●●●

●

●●●

●

●

●

●●●●●

●

●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●

●

●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●

●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●

●●●●●●●●●

●

●●●●●●●●

●

●●●●●●

●

●●●●●●

●

●●●●

●

●●●●●

●

●●

●

●●●●●●●●●●●

●

●●●●●●●●

●

●●●●

●●

●●●

●

●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●

●

●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●

●

●

●

●●●

●●●

●●

●

●●

●

●●●●●●●●●●●●●●●

●

●●●●●

●

●●●●●●●●●●

●●

●●●

●●

●●

●

●●

●●●●●

●

●●●●

●

●

●

●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●

●

●●●●●

●●

●●

●

●●●●●

●●●●●●●●●

●

●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●

●

●

●●●●●●●●●●●●●●●●●●

●

●●●

●

●●●●●●

●

●

●●●●●●●●●●

●

●●●●●●

●

●●●

●

●●●●●●●

●

●●●●●●●●●

●

●●●●●●●●●●●●●

●

●●

●

●●●●●●●

●

●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●

●

●●●●

●

●●●●●●●●

●

●

●●●●

●●

●

●

●●●●●●●●●

●

●

●●●●●●●●●●●●

●

●●●●●●●●●●●●●

●●

●

●

●●●●●●●●

●

●

●●●

●●

●●

●

●●●●●●

●

●●●●●●●●●●

●

●●

●

●●●●●●●●●●●●●●●●●●●

●

●●●●●●

●

●●●●●●●●

●

●●

●

●

●

●

●●●

●

●

●

●

●●●●●

●

●

●

●●●●

●

●●●

●

●●

●●●

●

●●●●●●

●

●●●●

●

●●●●●●●●●●●●●●

●

●●●●●

●

●●●

●

●●●●●●●●●●●●●●●●●●

●

●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●

●

●

●

●●●●

●●

●

●

●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●

●

●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●

●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●

●

●

●

●

●

●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●

●

●●●●●

●

●●●●●●●

●

●●●●●●●●●●●●●●●●●●●

●

●●●●

●

●●

●

●●●●●

●●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●

●

●●●●●●●●●●●●●●●●●●●●

●

●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●

●

●●●

●●

●●●●●

●

●●●●●●

●●●●

●

●●

●●

●●●

●

●

●●●●●●●●●●●

●

●●

●

●●●●●●●●●●●●●●●●●●

●

●

●

●

●●●●●●●●●●●●●●●●

●

●●●●●●●●●

●

●●●

●

●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●

●

●●●●●●●●●●●●●●●●●

●●●

●●●●●●●●●●

●

●●●●●●

●

●

●

●●●●●●

●

●

●●●●●●●●●●●●●●●●●●

●

●●●●●●●

●

●

●

●●

●

●●

●

●

●

●●

●

●●●●●●●●●●●●

●

●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●

●

●

●

●

●●●●●●●●●●●●●●

●

●●●●●●

●

●●●●●●●●●●●●●●

●

●●●●●●

●

●●●●●●●●●●●●●●●●

●

●●●●●●●

●

●●

●●

●●●●

●

●●●●●

●

●●●●●●●●●●●●

●

●

●

●●●●●●●●●●●●●●●●

●

●●●●●●●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●

●

●●

●

●●●●●●●●●●●●●●●●●●●●●

●

●●

●●●

●●●●●●●●●●

●

●●●●●●●●●●●●●

●

●●●●●●●●●●●

●

●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●

●

●

●●●●●●●●●●

●

●●●●●

●

●●●

●

●●●●●●●●●

●

●●●●●●●●●●●●●

●

●●●●

●

●

●●

●

●●●●

●

●●

●●●

●

●

●

●●●●●

●

●●

●

●●●●●●●

●●

●●●●●●

●

●●●●●●●●●●●●●●●

●

●●●●●●●●●●

●

●●●●●●●●

●

●●

●●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●

●

●●●●●●

●

●●●●●●●●●

●

●●●●

●●

●●●●●●●

●

●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●

●●

●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●

●

●●

●

●●●●●●●●●●●●●●●●

●●●●●

●●

●●

●

●●

●

●●●●●●●●●

●

●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●

●

●

●

●●●●●●●●●

●

●●●●●●●●

●

●●●●●●●●●●●●●●●●

●

●●●●●●●

●

●●

●

●●●●●●●●●●●●●●●●●●●●

●

●

●

●●●●●●●●●●●

●

●●●

●

●●●

●

●●●●●●●●●

●

●●

●

●

●●●

●

●●●●

●

●●●

●

●●

●

●●●●●●●●●●●●●●●●●●●●●

●

●

●●●●●●●

●

●

●●●●●●●

●

●

●

●●●●●●●●

●

●●●●●●●●●●●●

●

●

●●

●●●●●●●

●

●●●●●●●●●●●●●●●

●●

●●

●●●●●●●

●

●●●●●●●●●●●●●

●

●●

●

●●●●●●●●●●●

●

●

●●●●●

●

●●●●

●

●●

●

●●

●●●●●●●●

●

●●●●

●●

●

●

●●●●●

●

●●●●●

●

●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●

●

●●●

●

●●●●●●●

●

●●●●

●●

●●●●

●

●

●

●

●

●●

●●

●●

●

●●●●●●●

●

●●●●●●●

●

●●●●●●●●●●●●

●

●●

●

●●●●●●●●●●

●

●●●

●

●●●●●●

●

●●●●●

●

●

●●●●●●●●●●●

●

●●

●

●●●●●●●

●

●●●●●●●●●●●●

●

●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●

●●●●●●●●●●●●●●

●

●●●●●●●●

●

●●●●●●●●●●●●

●

●●●●●●●●●

●●

●

●

●

●●●●

●

●●●●●●●

●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●

●

●●●●●●

●●

●●●

●

●●●●●●●●●●

●

●●●

●●

●●●●

●

●●●●●

●

●

●

●●●●●●●●●●●●●●●●●●●●●

●

●●●●●

●

●●●●

●

●

●

●

●●

●

●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●

●●●●●●●●●●●●●●●●●●●●●●●●

●

●●

●

●●

●●

●●●●●●●●●●●●●●

●

●●●●●●●●●●

●

●●●●

●

●●●●●●●●●●●●

●

●●●●●●●●

●●●●●●●●●●●

●

●●●

●

●●●●●●●●●●●

●

●●●●●●●

●●●●●●●●●●●●

●

●●

●

●●

●●

●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●

●

●●●●●●●●

●●

●●●●●●●●

●

●●●●●●●

●

●●●●●●●●

●

●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●

●●

●●

●

●●

●

●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●

●●●●

●●

●●

●

●

●●

●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●

●●●●●●●

●

●●●●●●●●●●

●●

●●●●

●●

●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●

●

●

●

●●●●●●●●●●●●●●●

●

●●●●●

●●●

●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●

●

●●●●

●

●●●●●●●●●●●●●

●

●●●●●●●●●

●

●●●●●●

●●●●

●

●

●

●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●

●

●

●

●●

●

●●●●●●●●●●●●●●●●●●●●●●●

●

●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●

●

●●●

●

●●●●●●●●●

●

●●●●●●●●●●●●

●

●●●●●●●●●●●

●

●●●

●●

●

●

●●●●●●●

●

●

●

●●●●

●

●●●●●●●●●●

●

●●

●

●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●

●●●●●●●

●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●

●

●●●●●●●●●●●

●

●●●●●●●●●●●

●

●●●●●●●

●

●●●●●

●

●●●●●

●

●●●●●●●●●●●●●●

●

●

●

●

●

●●

●

●●●

●

●●●●●●

●

●●●

●

●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●

●

●●●●●●●

●

●●●●

●

●●●●●●●

●

●

●

●

●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●

●

●●●

●

●

●

●●●●●●●●●●●●●●

●

●●●●●●●●●●●

●

●●●●●

●

●●●●●●●●●●

●

●●

●

●●●●

●

●●●●●●●●

●

●●●●●●

●

●

●

●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●

●

●●●●

●

●

●●

●●●●●●●●●●●●●●●

●

●●●

●

●●●●●●●●●

●

●●●●●●

●

●●●●●●●●●

●●●

●

●●●●●●

●

●●●●●●●●●●●●

●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●

●

●●●●●

●

●●

●●

●

●●●

●

●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●

●●

●●●●●●

●

●●●

●

●●●●●●●●

●

●●●●●●●●

●

●●●●●●

●

●●●●●●●●●●

●

●

●●

●

●●●

●

●●

●

●●●●●●

●

●●●●●●●

●

●

●

●●●

●

●●●●●●●●

●

●●●●●●●●

●

●●●●

●

●

●

●

●●

●

●●

●●

●●●●●●

●

●●

●

●●●●●●●●●●●●●●●●●●●●●●●●

●

●

●

●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●

●

●●●

●

●●●●●●●●●

●

●

●

●●●●●●

●

●●●●

●

●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●

●

●

●●●

●●●●●●●●

●

●●●●

●

●

●

●●

●●●●●●●●●●●●

●

●

●

●●

●

●●●

●

●

●

●●●●●

●

●●●●●●●

●

●●

●

●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●

●

●

●

●●●

●●●●●●●●

●

●●●●

●

●●●●

●

●

●●●

●

●●

●

●●●●●●●●●●

●

●●●

●

●●●●●●●●●●●●

●

●●●●

●

●●●●●●●●●●●●●●●●●

●

●●

●

●●●

●

●

●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●

●

●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●

●●●●●●●●●

●

●

●

●●●●●●●

●

●●●●●●●●●●●●●●●●●

●●●●●●●●●●●●●●●

●

●●●●●●●●●●●

●

●●●

●

●●●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●●●●●●●●●

●

●●●●●●●●●●●

●

●●●●●●●●●●●●●●

●

●

●

●●●●

●

●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●

●●

●●●

●

●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●

●

●●●●

●

●●●●

●

●●

●

●●●●●●●●●●●●●●●

●

●●

●

●●●●●●

●

●

●

●●●●●●●●●●●●●●●●

●

●●●●●

●

●●●●●

●

●●●●●●

●

●●●●●●●●●●●●●●●●●

●●

●●●●

●

●●●●●

●

●●

●

●●●●●●●●●●●

●

●●●●●●●●●●●

●

●●

●

●●●●●●●●

●

●●●●●●●●●●●●

●

●●●●●●●●●●

●

●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●

●

●●

●●●●

●

●●●●●●●●●●●●

●

●●

●

●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●

●

●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●

●

●●●●

●

●●●

●

●●●●●

●

●●●●●●

●●

●●●●

●

●●●●●●●●●●●●●●●●●●●

●●●●●●●●●●●●

●

●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●

●●

●●●●●●●●●●●●

●

●

●

●●

●●●

●●

●●●●

●●

●

●●●●●●●●●●●●●●●●●●●●●●

●

●●●●

●

●●●●●●●●●●●●●●●●●●●●

●

●

●

●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●

●

●●●●●●

●●●●●

●●●

●

●●●●●

●●●●●●●●●

●

●

●

●●●●●

●●●●●●●

●

●●

●

●●●●●●●●●●●●●●●●

●

●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●

●

●

●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●

●●●

●●●●●●●●●●●●●

●

●●●●●●●

●

●●●●●●●●●●●●●●

●

●●●●

●

●●

●

●●●●

●

●●

●

●●●●●●●●

●

●●●●●●●●●●●●

●

●

●

●●●●●●●●

●

●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●

●

●

●

●●●●●●●●●●●

●

●●

●

●

●●●●●●●●●●

●

●●●

●

●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●

●

●●●●●

●

●●●●●●

●●●●●●●●

●

●●●●●●●●

●

●

●●●●●●●●●●●●●

●

●●●●

●

●●●●●●●●●●●●●●●●

●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●

●

●●●

●

●●●●●●●●●●●●●●●●●

●

●●●●●

●

●●●

●

●●●●

●

●●●●●

●

●●

●●●●●●●●●●

●●●●

●

●●●●●●●●

●●

●●●●

●

●●●●

●

●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●

●

●

●

●●●●●●●

●●●●●

●●●●

●

●

●●●

●

●●●●●●●●●●●●●●●

●

●●●●

●

●●

●

●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●

●●●●

●

●

●

●●●●●●●●

●●

●●

●

●●●

●

●●●

●

●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●

●●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●

●●

●●●●●●●●●●●●●●●

●

●

●

●●●●●●●●●●●●●

●●●●●●●●

●

●

●

●●●

●

●●●●●

●

●

●

●●●●●●●●●

●

●●●●●●●●●

●

●●●●●●

●●●●●

●●●●●●●

●

●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●

●●

●●●●●●●●●●

●

●●●●●●●●

●

●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●

●

●●

●●●●●●●●●●●●●●●●●

●

●●●●

●

●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●

●

●●●

●

●●●●●●●●●●●●●●●

●

●●●●●

●

●●●●●●●●●●●●●●●

●

●●●●●

●

●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●

●●

●●

●

●●●●●●●●●●

●●

●●●●●●●●●●●●●●●●●●●●

●●

●●●●●●●●

●●●●●

●

●●

●

●●●●●●●●●●●●●●●●●●●●●●●

●

●

●

●●●

●

●●●●●●●●

●

●

●●●

●

●●

●

●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●

●●

●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●

●

●●●●●●●●●●●●

●●

●●●

●

●●●●●●●●●●●●●●●●●●●

●

●●●

●

●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●

●

●●●

●

●●●●●●●

●

●●●

●

●

●

●

●●●●●●●●●●●●●●●

●

●●●

●

●●●●●

●

●●●●●●

●

●●●

●

●●●●●●●●

●

●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●

●

●●●●●

●

●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●●

●

●●●●●●●●●●●●●●●●●●●●

●

●●●●

●

●

●●●●●●●●●

●

●●●●●●●●●●●●●●●●

●

●●●

●

●●

●

●●●●

●

●●●

●

●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●

●

●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●

●

●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●

●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●

●●

●●●●●●●

●

●●●

●

●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●

●

●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●

●

●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

●

●●●●●●●●●●●●●

●

●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

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)

https://gtexportal.org/home/gene/DDX3X

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







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


✧ 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.

http://turnerlab.wustl.edu/

Sex Bias and the Genetics of Autism · 2020-03-25 · Outline Background of autism epidemiology and genetics Sex bias in autism and the Diagnostic and Statistical Manual of Mental

Documents