The document summarizes research on identifying genetic risk factors for alopecia areata. It describes analyzing genetic data from over 700 patients to identify 64 candidate genes and 701 variants potentially involved in monogenic causes. Pathway analysis found these genes enriched for extracellular matrix functions. Further studies will validate co-segregation of variants in families and test for genetic burden using exome data from 10,000 controls. The research aims to elucidate pathogenesis by studying extracellular matrix integrity and signaling in patient samples.

1. Lynn Petukhova, Ph.D.
lynn.petukhova@columbia.edu
Assistant Professor
Columbia University
Alopecia Areata Research Summit
December 5, 2018
Vagelos College of
Physicians and Surgeons

2. the set of genetic variants that contribute to a disease in the population
Human genetics studies
genetic architecture
polygenic risk monogenic causes
genetic architecture of chronic disease
§ pathway overview, cell type
§ population relevance of target
§ genetic risk scores
§ interpretable biology
§ drug target identification
§ familial risk
genome-wide association studies (GWAS) linkage, exome sequencing
(SNPs) (mutations)

3. Genetic architecture of Chronic Diseases
Breast Cancer BRCA1, BRCA2 TP53, CHEK2, PALB2, ATM, CDH1, RECQL, FANCM 122,977 cases +200
[BRCA1]
IBD NOD2 ADAM17, AICDA, BTK, CYBA, CYBB, DCLRE1C, DOCK8, G6PC3, GUCY2C,
HPS1, HPS4, HPS6, ICOS, IKBKG, IL10, IL10RA, IL10RB, IL21, IPEX, ITGB2,
LRBA, MEFV, MVK, NCF1, NCF2, NCF4, PIK3R1, PLCG2, SLC37A4, STXBP2,
TTC37, TTC7A, WAS, XIAP, PRDM1, NDP52
25,000 cases +200
[NOD2]
Atopic Dermatitis FLG ADA, ADGRE2, ARPC1B, CARD11, CARMIL2, CDSN, CHD7, DCLRE1C, DOCK8,
DSG1, DSP, ERBB2IP, FOXP3, IFNGR1, IL2RA, IL2RG, IL4RA, IL7RA, JAK1, KIT,
LIG4, MALT1, PGM3, PLCG2, RAG1, RAG2, SPINK5, STAT1, STAT3, STAT5,
STAT5B, TGFBR1, TGFBR2, TPSAB1, WAS, WIPF1, ZAP70
18,900 cases 31
[FLG]
Alzheimer’s APP, PSEN1,
PSEN2
APOE, AKAP9, CD33, CR1, EPHA1, INPP5D, NME8, SORL1, TREM2, THBS2,
PLEKHG5
42,000 cases 21
[PSEN1]
LDL PCSK9, LDLR APOB, LDLRAP1, ABCG5, ABCG8 8,000 total 38
[ LDLR, PCSK9]
Disease Linkage Additional monogenic genes Cohort Loci
Monogenic (mutations)
linkage analysis, exome sequencing
Polygenic (SNPs)
GWAS
AA 3,000 cases 14
Type 2 diabetes HNF4A, GCK INS, BLK, ABCC8, KCNJ11, HNF1A, PDX1, HNF1B, NEUROD1, KLF11, CEL,
PAX4, APPL1
47,979 cases 76
[HNF4A, GCK]

4. Genetic architecture of Chronic Diseases
Breast Cancer BRCA1, BRCA2 TP53, CHEK2, PALB2, ATM, CDH1, RECQL, FANCM 122,977 cases +200
[BRCA1]
IBD NOD2 ADAM17, AICDA, BTK, CYBA, CYBB, DCLRE1C, DOCK8, G6PC3, GUCY2C,
HPS1, HPS4, HPS6, ICOS, IKBKG, IL10, IL10RA, IL10RB, IL21, IPEX, ITGB2,
LRBA, MEFV, MVK, NCF1, NCF2, NCF4, PIK3R1, PLCG2, SLC37A4, STXBP2,
TTC37, TTC7A, WAS, XIAP, PRDM1, NDP52
25,000 cases +200
[NOD2]
Atopic Dermatitis FLG ADA, ADGRE2, ARPC1B, CARD11, CARMIL2, CDSN, CHD7, DCLRE1C, DOCK8,
DSG1, DSP, ERBB2IP, FOXP3, IFNGR1, IL2RA, IL2RG, IL4RA, IL7RA, JAK1, KIT,
LIG4, MALT1, PGM3, PLCG2, RAG1, RAG2, SPINK5, STAT1, STAT3, STAT5,
STAT5B, TGFBR1, TGFBR2, TPSAB1, WAS, WIPF1, ZAP70
18,900 cases 31
[FLG]
Alzheimer’s APP, PSEN1,
PSEN2
APOE, AKAP9, CD33, CR1, EPHA1, INPP5D, NME8, SORL1, TREM2, THBS2,
PLEKHG5
42,000 cases 21
[PSEN1]
LDL PCSK9, LDLR APOB, LDLRAP1, ABCG5, ABCG8 8,000 total 38
[ LDLR, PCSK9]
Disease Linkage Additional monogenic genes Cohort Loci
Monogenic (mutations)
linkage analysis, exome sequencing
Polygenic (SNPs)
GWAS
AA 3,000 cases 14
Type 2 diabetes HNF4A, GCK INS, BLK, ABCC8, KCNJ11, HNF1A, PDX1, HNF1B, NEUROD1, KLF11, CEL,
PAX4, APPL1
47,979 cases 76
[HNF4A, GCK]
BRCA1 mutations among breast cancer patients

5. Preliminary Evidence for Alopecia Areata Mutations
Limitations
§ Small sample size (n=28 families)
§ Sparse markers (n=300)

6. Linkage Analysis Rationale
50,000 variants in a human exome
1,000 mutations affect protein structure
Parent-child transmission reduces the number of candidate mutations by half.
Disease co-segregation can further eliminate substantial genomic regions.
Exomes are noisy.

7. What How
Linkage Analysis Method
B C D A
mutation

8. Linkage Analysis Analytic Plan
1. Use tagSNPs to identify cosegregating haplotypes
2. Finemap regions by testing cosegregating
tagSNPs for association
3. Map associated tagSNPs to all genic SNPs to
identify candidate genes.
4. Use exome sequencing data to screen candidate
genes for mutations that alter protein sequence.

9. Cohorts constructed from the National Alopecia Areata Registry
Discovery Cohort
genotype 38 families, exome sequence for 28 probands
Replication Cohort
§ Exome sequence for 108 patients who
report family history.
§ Exome sequence for 620 patients who
report no family history

10. Identify Cosegregating Haplotypes
chr cytoband snp start start Hg19 snp stop stop Hg19 region size gene count max NPL LOD
1 1p36.1 rs10794531 26,694,245 rs9438620 26,716,136 21,891 1 1.08
1 1p13.2 rs155646 112,688,022 rs3128373 112,858,294 170,272 0 1.04
1 1q23.3 rs4657210 162,587,423 rs1704745 162,693,795 106,372 1 1.06
2 2q12.1 rs7571700 105,189,937 rs2679852 105,797,790 607,853 2 1.13
2 2q36.1-q37.3 rs2098533 225,182,419 rs12469535 243,044,147 17,861,728 158 4.17
4 4q34.3 rs1991983 182,454,723 rs6821430 182,499,174 44,451 0 1.31
5 5q13.1-q13.3 rs2047588 71,578,902 rs2114950 76,726,202 5,147,300 36 1.15
5 5q21.2 rs11955569 103,472,789 rs2034227 103,647,014 174,225 0 1.01
5 5q33.1-q34 rs4958486 151,058,334 rs10515827 160,754,957 9,696,623 49 1.71
6 6p12.2-q15 rs4715280 51,974,289 rs1504292 91,637,689 39,663,400 133 2.31
6 6q22.31-q24.1 rs9385268 123,041,722 rs7744152 139,663,885 16,622,163 84 2.18
8 8p23.1 rs7843504 8,439,068 rs7463440 12,652,105 4,213,037 37 1.9
9 9p24.3 rs10964134 204,201 rs755383 863,635 659,434 4 1.65
12 12q21.33-q23.1 rs2897852 90,768,440 rs11612901 96,221,318 5,452,878 30 1.71
15 15q22.2 rs11630244 60,021,637 rs7174483 60,123,836 102,199 0 1.02
17 17q25.3 rs1869932 77,318,565 rs2241886 78,113,832 795,267 9 1.13
18 18p11.21 rs1149360 13,540,713 rs3760534 13,874,975 334,262 4 1.09
18 18q21.2-q21.31 rs1105471 45,086,502 rs554192 55,784,763 10,698,261 42 1.27
20 20q13.11-q13.12 rs6065582 41,771,701 rs2179069 43,507,254 1,735,553 23 1.52
22 22q13.2 rs2072884 43,436,169 rs695537 43,769,871 333,702 6 1.05
Identified in Martinez-Mir et al.

11. Results: Variant Filtering
10,000,000
373
6,688
SNPs Genes
11,272
300,000
Mutations
In the genome.
Genotyped TagSNPs
with evidence for linkage
and family-based association.
SNPs tagged by 373 SNPs.
Exome mutations (f<.05) in proband that alters protein sequence
and in a region with evidence for cosegregation with AA in proband’s family
~18,000
619
179
Genotype 38 families in discovery cohort
Exome sequence 28 probands in discovery cohort
223
148
108
87
Same mutation (f<.05) seen in replication cohort
Exome sequence replication cohort of 108 probands
New mutation (f<.001) in a same gene in replication cohort
5843
Exome sequence in a cohort of nonfamilial 620 patients
10349
16168union
49664New mutation (f<.001) in a same gene in replication cohort
701

12. Results: Functional connections among 64 genes
Term Count Fold Enrichment PValue
GO:0030574-collagen catabolic process 5 21.86 7.5E-05
GO:0005578-proteinaceous extracellular matrix 7 7.80 2.5E-04
GO:0030198-extracellular matrix organization 6 8.57 6.2E-04
GO:0005201-extracellular matrix structural constituent 4 17.08 0.002
GO:0050790-regulation of catalytic activity 4 16.96 0.002
GO:0030334-regulation of cell migration 4 15.13 0.002
GO:0005604-basement membrane 4 15.13 0.002
GO:0005581-collagen trimer 4 12.99 0.003
GO:0005788-endoplasmic reticulum lumen 5 7.78 0.004
GO:0007155-cell adhesion 7 4.27 0.005
GO:0043231-intracellular membrane-bounded organelle 7 3.75 0.010

13. Conclusion and Future Directions
§ We have prioritized 64 candidate genes and 701 variants as potential monogenic causes
of alopecia areata.
§ This gene set enriches pathways and gene ontology terms that suggests the ECM as a
potential mediator of alopecia areata pathogenesis and provides a rationale for future
studies of ECM integrity and signal transduction in patient samples.
§ We will be performing genic burden testing and variant association testing using exome
data in a cohort of 10,000 controls. We also will validate the cosegregation of a select set
of variants by genotyping family members.

14. Acknowledgements
National Alopecia Areata Registry
Angela Christiano
Vera Price
Maria Hordinsky
David Norris
Madeline Duvic
NIAMS 1P30 AR069632-01
NIAMS 1P50 AR070588-01
Christiano Lab
Stephanie Erjavec
Alexa Abdelaziz
Tarek Yammany
Angela M. Christiano
AACORT core services
Sahar Gelfman
Aris Floratos
The Feinstein Institute for Medical Research
Annette Lee
Peter Gregersen
Baylor College of Medicine
Wei V. Chen
Christopher Amos