Genotype Reporting

Methodology

NCRAD extracts DNA and generates a 96 SNP genetic fingerprint (StandardBiotools Juno 96.96 IFC/BioMarkHD, more information on our validation and SOPs available by request) for each participant banked in the repository. This SNP fingerprint is generated immediately following extraction and for identity confirmation when samples are distributed. As part of this fingerprint, APOE haplotypes are generated from the APOE SNPs, rs7412 and rs429358, which are validated by running the same primers in duplicate. LRRK2 G2019S genotype is generated using the rs34637584 SNP, also run in duplicate.

Apolipoprotein E

Apolipoprotein E (APOE) is the major genetic risk factor for late onset Alzheimer’s disease. It has three alleles typically observed, ε2, ε3, and ε4, as well as one extremely rare allele ε1, which are determined based on the genotypes of two SNPs, rs7412 and rs429358. The ε2 variant is considered protective against Alzheimer’s disease, while the ε4 variant is a risk factor; ε3 is considered risk neutral and is the most common. In the NCRAD reports, APOE output is two numbers in the following format: x/y, where x and y = 2, 3, or 4. For example, 2/3. This indicates the APOE genotype (ε2/ε3), calculated from the SNP data using the chart below.


APOE Genotype	rs429358 Alleles	rs7412 Alleles	Notes
ε1/ε1	CC	TT	Not observed
ε1/ε2	CT	TT	Not observed
ε1/ε3	CT	CT	Not observed
ε2/ε4	CT	CT	Very rare
ε1/ε4	CC	CT	Not observed
ε2/ε2	TT	TT	Rare
ε2/ε3	TT	CT
ε3/ε3	TT	CC	Most Common
ε3/ε4	CT	CC
ε4/ε4	CC	CC

Leucine-Rich Repeat Kinase 2

The LRRK2 G2019S variation originates from a guanine (G) > adenine (A) substitution at position 6055 of exon 41 of LRRK2 gene (SNP rs34637584) that results in the change of a glycine to serine at codon 2019 of LRRK2, which alters the protein kinase encoded by exon 41. This mutation is currently believed to be associated with between 1- 4% of all Parkinson’s disease cases worldwide, though it is significantly more frequent in certain North African and Jewish populationsi. In NCRAD reports, the “normal” nucleotide configuration is reported as GG. The heterozygous substitution variant is reported as GA, and the homozygous variant is reported as AA.

Additional Information

NCRAD genotype reports include several fields in addition to the APOE and LRRK2 G2019S genotypes.

CURRENT_STATUS is a summary field (PASS or FAIL). PASS indicates that the DNA was of good quality and generated the SNP fingerprint, and there are no outstanding quality control flags (fingerprint sex matches reported sex, any subject duplication has been confirmed with the site, etc.). NCRAD does not report genotype results for any sample without the PASS designation.
EXPECTED_BARCODE is the unique sample barcode for the DNA sample which was SNP fingerprinted. This is tracked so we can identify the sample from which the results were generated. When the fingerprint is generated, it is compared against the other SNP fingerprints in our database. If the SNP fingerprint is found to match a different sample in the database, NCRAD works with the study site(s) and laboratory team to investigate the possibility of duplicate enrollment, mislabeled/misidentified sample, processing error, etc
EXPECTED_SUBJECT is the participant ID (either NCRAD ID or ID provided by the study) to which the sample belongs. When the fingerprint is generated, it is compared against the other SNP fingerprints in our database. If the SNP fingerprint is found to match a different subject in the database, NCRAD works with the study site(s) and laboratory team to investigate the possibility of duplicate enrollment, mislabeled/misidentified sample, processing error, etc.
R_G is sex confirmation (Reported vs Genetic sex): The SNPs to generate this information are included in the fingerprint as a quality control measure to ensure a match between reported sex in the database and genetic sex. M/M (or F/F) indicates the participant was reported to be male and confirmed male (or female confirmed female), genetically. M/F or F/M indicates a mismatch between the reported and genetic data.

Data Dictionary

<sup>i</sup> Billingsley KJ, Bandres-Ciga S, Saez-Atienzar S, Singleton AB. Genetic risk factors in Parkinson's disease. Cell Tissue Res. 2018 Jul;373(1):9-20. doi: 10.1007/s00441-018-2817-y. PMID: 29536161; PMCID: PMC6201690.
Field	Description	Values
CURRENT_STATUS	Reportable status of the genotype data	PASS, FAIL
EXPECTED_BARCODE	Sample barcode of the fingerprinted DNA sample	Barcode
EXPECTED_SUBJECT	Patient ID of the fingerprinted DNA sample	Patient ID
R_G	Comparison of reported to genetic sex	M/M, F/F, M/F, F/M
FLD_LRRK2	LRRK2 G2019S genotype	GG, GA, AA
FLD_APOE	Combined APOE allelic configuration	3/3, 2/3, 2/4, 4/3, etc.
APOE_value_1	APOE allele 1 variant	E1, E2, E3, E4
APOE_value_2	APOE allele 2 variant	E1, E2, E3, E4

ⁱ Billingsley KJ, Bandres-Ciga S, Saez-Atienzar S, Singleton AB. Genetic risk factors in Parkinson's disease. Cell Tissue Res. 2018 Jul;373(1):9-20. doi: 10.1007/s00441-018-2817-y. PMID: 29536161; PMCID: PMC6201690.

Please Note: It is very important to note that these results are only to be used for research purposes. This test is not approved for clinical use and therefore the data cannot be returned to participants.

Cost

Dependent upon the type and number of samples received at NCRAD. Please inquire at 1-800-526-2839 or alzstudy@iu.edu.