Ultra-Rare Mutations in SRCAP segregate in Caribbean ... · 8/24/2017 · CH Case vs CH...
Transcript of Ultra-Rare Mutations in SRCAP segregate in Caribbean ... · 8/24/2017 · CH Case vs CH...
Ultra-Rare Mutations in SRCAP segregate in Caribbean Hispanic families with Alzheimer’s
disease
Supplementary Information
Supplementary Methods
Linkage analysis: Using common polymorphisms (MAF>0.05) detected by GWAS in the discovery families, we performed joint
linkage and association analysis with PSEUDOMARKER1 using all sequenced samples. Single nucleotide variants (SNVs) within 20
MB of a linkage peak with LOD>1.7 were prioritized for follow-up genotyping.
Haplotype analysis: To investigate the unusual pattern of segregation in one large Caribbean Hispanic family, we conducted
haplotype phasing of the 1MB region flanking the variant using genome-wide SNP data in the family members. We used R-based
haplo.stats to construct haplotypes2.
Blood collection and RNA extraction. Whole blood (20 mL) from affected mutation carriers, affected related and unrelated
individuals without mutations and healthy elderly unrelated controls, was obtained and mRNA was extracted using the PAXgene
Blood RNA kit (Qiagen) according to the manufacturer’s instruction. mRNA quality and integrity for all samples were assessed by
agarose (1.5%) gel electrophoresis.
RT-qPCR. cDNA of total collected blood RNA was produced with the First Strand cDNA Synthesis Kit (Origene) according to the
manufacturer’s instructions. A total of 100 ng of total RNA was used per reaction for consistency between experiments. qPCR
reactions were carried out using the Power SYBR® Green PCR master mix (ThermoFisher Scientific). Samples were assayed on a
real-time qPCR thermocycler (Eppendorf Mastercycler® Realplex2) in 96-well optical plates, with an initial melting step of 95°C (10
minutes), followed by 40 cycles of 95°C (1 minute), 59°C (30 seconds), 60°C (1 minute). The delta-delta cycle threshold (ddCt)
method was employed to determine relative gene expression differences between groups (controls, affected carriers, affected non-
carriers). Samples were normalized against the housekeeping gene, Glycerladehyde-3-phosphate dehydrogenase (GAPDH).
Supplementary Tables
Supplementary Table e-1a: Demographic information of the samples used in WES and follow up genotyping
Status Number
of Samples
Mean Age at Onset or last
examination: years ± SD
Women, % APOE e4, %
FAMILIAL SAMPLES IN WES (31 Discovery Families)
Affected 98 74.8 ± 8.3 63.1 2.9*
Unaffected 12 80.3 ± 3.6 62.3 14.2
FAMILIAL SAMPLES (31 families -Genotyping)
Affected 265 73.2 ± 9.9 60.64 25.1
Unaffected 61 60.3 ± 9.6 61.5 24.4
FAMILIAL SAMPLES (47 families -Genotyping)
Affected 234 74.36 ± 9.12 64.53 34.2
Unaffected 69 63 ± 8.99 65.22 31.4
UNRELATED CASES (genotyping) Affected 1949 76.2 ± 8.7 66.03 24.84
UNRLEATED CONTROLS (genotyping)
Unaffected 318 75.9 ±6.05 69.2 10.52
* Families were chosen for lack of APOE ε4 allele clustering in the affected members
We analyzed rare coding variants modifying risk of LOAD in the discovery whole exome sequencing cohort comprising of 31
Caribbean Hispanic families. In the follow-up genotyping step, we used all the familial samples from the 31 families including the
discovery cohort, all members from 47 additional families and an additional cohort of unrelated, ethnicity matched case-control set.
Supplementary Table e-1b: Sample Sets used for single marker and gene-based tests
Experiment
Sample Sets
Used (From
Supplementary
Table 1a)
Variants
Tested Results
Segregation and annotation analyses in
exome-sequencing data 1
Exome-
Wide Prioritized 78 variants for follow-up genotyping
Single Marker Association with LOAD 2+5 78 22 out of 78 variants were significant at a Bonferroni correct
p=6.4e-03, prioritized SRCAP for gene-based testing
Gene-based GEE analyses for SRCAP
missense variants 1+2 10 OR=5.94 95% CI 1.59-22.27, p=8.2e-03
Gene-based GEE analyses for SRCAP
missense variants 1+2+3 10 OR=2.54 95% CI: 1.03-6.21, p=0.04
Gene-based GEE analyses for SRCAP
missense variants 1+2+3+4+5 10 OR=1.82; 95% CI 1.04-3.19, p=0.036
Fisher test comparing SRCAP variants
with ExAC data
1+2+3+4+5+
ExAC datasets 10 1.19E-16
Supplementary Table e1-c: Characteristics of the 31 discovery LOAD families
Total individuals ascertained for LOAD 326
Affected 265
Unaffected 61
Average family size 10.19 ± 5.60
Average affected individuals per family 8.28±3.80
Average unaffected individuals per family 2.90±2.64
SNP Gene Amino Acid Change SIFT POLY PHEN
Ref Obs WHOLE EXOME SEQUENCING
FOLLOW-UP GENOTYPING
FAMILIES
WITH MUT
AFF WITH MUT
CTRLS WITH MUT
BETA SE Z P
11_62343562 TUT1 NM_022830:c.C1743G:p.D581E D D G C 2 7 0 1.6E+00 1.4E-01 1.3E+02 0
12_49164644 ADCY6 NM_015270:c.C3161G:p.S1054C D D G C 1 4 0 4.0E+01 1.0E+00 1.6E+03 0
12_58020552 B4GALNT1 NM_001478:c.G1577A:p.R526Q T D C T 1 5 0 1.1E+00 1.0E-01 1.0E+02 0
12_62954811 MON2 NM_015026:c.C3950T:p.P1317L D D C T 1 5 0 4.0E+01 1.0E+00 1.6E+03 0
16_30732600 SRCAP NM_006662:c.G3344A:p.R1115H D NA G A 1 7 0 8.9E-01 1.0E-01 7.4E+01 0
19_39220007 ACTN4 NM_004924:c.G2671A:p.A891T D B G A 1 4 0 4.0E+01 1.0E+00 1.6E+03 0
19_7690771 XAB2 NM_020196:c.G817A:p.E273K D D C T 1 4 0 4.0E+01 1.0E+00 1.6E+03 0
20_33584889 MYH7B NM_020884:c.G3396C:p.K1132N D NA G C 1 6 0 4.0E+01 1.0E+00 1.5E+03 0
20_44313506 WFDC10B NM_172131:c.C233T:p.S78L D D G A 1 5 0 1.7E+00 1.2E-01 2.1E+02 0
7_138347831 SVOPL NM_174959:c.T2C:p.M1T D NA A G 7 10 0 3.9E+01 2.7E-01 2.0E+04 0
12_29908802 TMTC1 NM_001193451:c.T571C:p.C191R D B A G 1 4 0 4.0E+01 1.0E+00 1.6E+03 0
17_2290703 MNT NM_020310:c.C1241T:p.P414L D NA G A 2 9 0 1.2E+00 2.1E-01 3.4E+01 4.98E-09
12_120260630 CIT NM_001206999:c.C1105A:p.R369S T D G T 5 13 3 1.2E+00 2.3E-01 2.9E+01 8.23E-08
11_128840894 ARHGAP32 NM_014715:c.G3125A:p.R1042Q D D C T 2 10 1 1.4E+00 3.0E-01 2.1E+01 3.70E-06
14_55629787 DLGAP5 NM_001146015:c.G1555A:p.D519N D D C T 8 17 2 1.4E+00 3.1E-01 2.1E+01 5.44E-06
19_2936698 ZNF77 NM_021217:c.T135G:p.C45W T D A C 5 13 1 1.2E+00 2.7E-01 1.9E+01 1.05E-05
1_18023606 ARHGEF10L NM_001011722:c.C3454T:p.R1152W D D C T 6 13 4 1.2E+00 2.7E-01 1.9E+01 1.49E-05
19_1457179 APC2 NM_005883:c.G1144A:p.E382K D D G A 5 21 3 1.6E+00 4.0E-01 1.6E+01 7.35E-05
4_941532 TMEM175 NM_032326:c.C5G:p.S2C D P C G 8 15 2 1.9E+00 5.1E-01 1.4E+01 1.89E-04
21_47722064 C21orf58 NM_058180:c.C818T:p.P273L D NA G A 3 11 2 8.8E-01 2.4E-01 1.4E+01 1.90E-04
5_74892955 POLK NM_016218:c.G2437A:p.D813N D B G A 4 10 0 9.4E-01 2.6E-01 1.3E+01 3.15E-04
12_44915791 NELL2 NM_001145108:c.C2167T:p.R723C D D G A 3 10 1 2.1E+00 6.0E-01 1.3E+01 3.77E-04
Supplementary Table e-2: Top missense mutations significant at a Bonferroni correct p-value<6.4e-03 identified by WES and
validated by genotyping 31 families and an additional case control cohort
Supplementary Table e-3
Summary of comparison of allele frequencies in Caribbean Hispanic (CH) LOAD cases with Caribbean Hispanic controls and controls
from different ExAC sub-populations
TEST AFF
ALLELES UNAFF
ALLELES AFF
TOTAL UNAFF TOTAL
ODDS RATIO P-VALUE
CH Case Vs CH Control* 151 15 38742 6616 1.721885 2.25E-02
CH Case vs CH Control+Exac LAT** 151 188 38742 122348 2.542491 1.19E-16
CH Case vs CH Control+Exac AFA*** 151 302 38742 110332 1.425587 2.99E-04
CH Case vs CH Control+Exac NFE**** 151 1038 38742 672480 2.531023 1.51E-21
Legend CH: Caribbean Hispanics
*CH Case Vs CH Control: Comparing independent Caribbean Hispanic Cases with controls from the same cohort
** CH Case vs CH Control+Exac LAT: Comparing independent Caribbean Hispanic Cases with controls from the same cohort and the ExAC Latino population
*** CH Case vs CH Control+Exac AFA: Comparing independent Caribbean Hispanic Cases with controls from the same cohort and the ExAC African American population
**** CH Case vs CH Control+Exac NFE: Comparing independent Caribbean Hispanic Cases with controls from the same cohort and the ExAC Caucasian (Non-Finnish European) population
AFF ALLELES: Number of SRCAP alleles in the ten mutations (Table 1) carried by affected individuals
AFF ALLELES: Number of SRCAP alleles in the ten mutations (Table 1) carried by unaffected individuals
AFF TOTAL: Total number of alleles for the ten mutations (Table 1) successfully genotyped in affected individuals
UNAFF TOTAL: Total number of alleles for the ten mutations (Table 1) successfully genotyped or sequenced (for ExAC population) in affected individuals
ODDS RATIO: Odds ratio in a Fisher’s Test
P-VALUE: P-value from a Fisher’s Test
Supplementary Table e-4A: Haplotype analysis of common polymorphisms in the 1MB region flanking R1115H in SRCAP in the
pedigree described in Figure 2.
ID HAPLOTYPE 1 HAPLOTYPE 2 724_41 1 1 GENERATION 1
724_99 4 5
GENERATION 2
724_5 4 5
724_3 4 5
724_4 2 4
724_8 2 4
724_6 2 4
724_9 2 4
724_12 4 4
CURRENT GENERATION
724_15 1 4
724_16 1 4
724_20 1 4
724_13 1 5
724_14 1 5
The R1115H variant in SRCAP tracks with haplotype 4, all samples that carry the variant also carry haplotype 4. The aunt (sample
41), who has AD but does not carry the variant has different ancestral haplotypes from the samples in the sibship that carry the
R1115H.
Supplementary Table e-4B: Haplotype definitions
HAPLOTYPE CODE rs7186852 rs885107 rs2919433 rs7187359 rs11642466 rs11642862 rs11645175 rs4889490
1 A A A G A G G A
2 A A A G A G G C
3 A G G A G A A A
4 G A A G A G G C
5 G G G A G A A C
The haplotype codes in Supplementary table 3a are defined by SNPs 1MB around the R1115H variant in SRCAP
Supplementary Table e-5: SRCAP expression is associated with 4 cognitive domains except visuospatial ability
Domains DF Estimate SE t Value Pr > |t|
Episodic Memory 1 -0.97652 0.24013 -4.07 <.0001
Semantic Memory 1 -1.24007 0.27262 -4.55 <.0001
Working Memory 1 -0.6643 0.18993 -3.5 0.0005
Perceptual Speed 1 -0.79383 0.23987 -3.31 0.001
Visuospatial Ability 1 -0.37095 0.22439 -1.65 0.099
References
1. Hiekkalinna, T. et al. PSEUDOMARKER: a powerful program for joint linkage and/or linkage disequilibrium analysis on mixtures of singletons and related individuals. Hum Hered 71, 256-66 (2011).
2. Schaid, D.J., Rowland, C.M., Tines, D.E., Jacobson, R.M. & Poland, G.A. Score tests for association between traits and haplotypes when linkage phase is ambiguous. Am J Hum Genet 70, 425-34 (2002).
Supplementary Figures
Supplementary Figure e-1a) Protocol for prioritizing SNVs from WES data (yield=67 SNPs)
High quality variants in 111 subjects
~200K Variants
Novel, coding, damaging (damaging in SIFT or possibly
or probably damaging in PolyPhen) variants
4525 Variants
SNPs
3370 Variants
Present in affecteds only
2648 variants
Present in 4 or more affecteds in multiple families or segregating in one family
92 SNPs
Present in 2 or more families
71 SNPs
Within 20 MB of a linkage peak with HLOD>1.7
67 SNPs
Supplementary Figure e-1b) Protocol for prioritizing Indels from WES data (Yield=11 indels)
High quality variants in 111 subjects
~200K Variants
Novel, coding, damaging or functional variants
4525 Variants
Indels
1104 Indels
Present in affecteds only
925 Indels
Present in 5 or more affectedsin multiple families
50 Indels
Functionally damaging Indels(Frameshift)
20 Indels
Frequency in public databases<2%
11 Indels
Carrier of p.R1115H in SRCAP
Wild Type
16:30732600, R1115HSupplementary Figure e-2: : R1115H in SRCAP is found in
all affecteds in a sibship in a large multiplex Caribbean
Hispanic family
Sample 41 who is affected and carries the SRCAP
wild type has different ancestral haplotypes in that
region (see Supplementary Table 3), suggesting that
she is likely a phenocopy. Sample 12 is 72 years
old, carries the SRCAP variant and is diagnosed
with mild cognitive impairment. The other carriers
(Samples 15, 16 and 20) in the most recent
generation are <65 years old (early to diagnose late-
onset Alzheimer’s Disease). These samples are
being continuously followed up to assess cognitive
performance and diagnosis of Alzheimer’s Disease.
Figure LegendImport ID: Subject IDAAO_Affected_Else_AgeLast_Seen: Age at onset of LOAD or age of last examination for healthy individuals
Supplementary Figure e-3A: Segregation patterns of missense SRCAP mutations
16:30732143, S1003P
Mutation Carrier
Wild Type
Figure LegendImport ID: Subject IDAAO_Affected_Else_AgeLast_Seen: Age at onset of LOAD or age of last examination for healthy individuals
Supplementary Figures e-3B: Segregation patterns of missense SRCAP mutations
16:30732558, p.T1101K
Mutation Carrier
Wild Type
Figure LegendImport ID: Subject IDAAO_Affected_Else_AgeLast_Seen: Age at onset of LOAD or age of last examination for healthy individuals
Supplementary Figures e-3C: Segregation patterns of five missense SRCAP mutations
16:30734934, p.I1397V
Mutation Carrier
Wild Type
Figure LegendImport ID: Subject IDAAO_Affected_Else_AgeLast_Seen: Age at onset of LOAD or age of last examination for healthy individuals
Supplementary Figures e-3D: Segregation patterns of five missense SRCAP mutations
16:30749583 , p.P2741R
Mutation Carrier
Wild Type
Figure LegendImport ID: Subject IDAAO_Affected_Else_AgeLast_Seen: Age at onset of LOAD or age of last examination for healthy individuals
Supplementary Figure e-4: Location of LOAD SRCAP variants relative to Floating Harbor Syndrome Variants
R1115H
T1101K
S1033P I1397V
P2741R
V2764G
L2919F
S2524C
R940QP1130SATPase
Domains I,Ia-IVATPase Domains V-VI
Exons with Floating Harbor Syndrome Mutations
Name ChrGRCh37Location
NM_006662.2(SRCAP):c.189C>G (p.Pro63=) 16 30715519
NM_006662.2(SRCAP):c.1559G>A (p.Ser520Asn) 16 30723222
NM_006662.2(SRCAP):c.3302C>A (p.Thr1101Lys) 16 30732558
NM_006662.2(SRCAP):c.3388C>T (p.Pro1130Ser) 16 30732644
NM_006662.2(SRCAP):c.3868A>G (p.Ser1290Gly) 16 30734045
NM_006662.2(SRCAP):c.4293C>G (p.Val1431=) 16 30735038
NM_006662.2(SRCAP):c.4355C>T (p.Ser1452Leu) 16 30735100
NM_006662.2(SRCAP):c.4563T>A (p.Pro1521=) 16 30735308
NM_006662.2(SRCAP):c.5705A>G (p.Glu1902Gly) 16 30740333
NM_006662.2(SRCAP):c.7263C>A (p.Arg2421=) 16 30748624
NM_006662.2(SRCAP):c.7303C>T (p.Arg2435Ter) 16 30748664
NM_006662.2(SRCAP):c.7330C>T (p.Arg2444Ter) 16 30748691
NM_006662.2(SRCAP):c.8755C>T (p.Leu2919Phe) 16 30750116
NM_006662.2(SRCAP):c.9444T>C (p.Ser3148=) 16 30750805
List of SRCAP variants implicated Floating Harbor Syndrome in ClinVar(evidence from multiple sources)