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)