Fine Mapping of Lrp11 Trans-eQTL on Mouse Chromosome 5 Using SNP Markers

ABSTRACT MATERIALS AND METHODS Here we report the preliminary results of the effort to fine-map a previously identified 3.5 Mbp quantitative trait locus (QTL) associated with variation in Lrp11 expression levels using 110 MF1 outbred mice. The Undergraduate Genomics Research Initiative (UGRI) at UCLA is using genotype data to identify the causal variation for Lrp11. Based on the C57BL/J6 standard inbred mouse strain, we designed primer pairs and dye-labeled custom LI-COR primers for nine equidistant SNP sites selected to be polymorphic across eight inbred strains (C57BL/J6, BALB/c, AKR, DBA/2, A/J, C3H, RIIIS, I/LnJ) in the identified QTL region. We have sequenced PCR-amplified SNP regions for the eight standard inbred strains. We specifically sequenced regions surrounding SNPs rs29732770 and rs31562818, of which rs31562818 successfully identified the polymorphism in the eight strains. We plan to test our designed primers on the remaining seven SNP regions for all inbred strains to confirm polymorphisms across all eight strains. This will enable us to genotype the 110 MF1 mice using the designed primers to fine map and identify the candidate gene(s) for the Lrp11 trans-eQTL on Chr5 locus. These results should prove informative for understanding interactions among the human LRP11 transgene, LRP11, and LDL levels.

DNA ISOLATION AND QTL MAPPING• DNA isolated from 10 mg mouse spleen using standard Qiagen DNeasy® kit protocol• Focusing on previously identified Lrp11 trans-eQTL candidate region on mouse chromosome 5, between 78 and

81.5 Mbp, for fine mapping (Fig. 3)

FINE MAPPING STRATEGY USING SNP MARKERSIsolating polymorphic SNPs in QTL region

• Confirm polymorphism across 8 inbred strains,A/J, AKR, C57BL/J6, BALB/c, C3H/He, DBA/2J,RIIIS, I/LnJ using the following public resources:

Mouse Genome Informatics MGI Database5

• Obtained base pair identities at SNP sites• SNPs based on NCBI build 37 assembly

Mouse Phenome Database6

• Obtained flanking sequencesBLAT (BLAST-like Alignment Tool)7

• Ensured sequence rarity in genome• Used whole-genome BLAT

Primer DesignPrimer38

• Derived primers, obtained meltingtemperatures and lengths (Tables 1,2)

BLAT search of primers7

• Ensured uniqueness of primer sequences in genome• Ensured that primer sequences were conserved across initial 6 strains

SEQUENCING SNP REGION FROM GENOMIC AND AMPLIFIED DNA• Sequenced samples directly from genomic DNA, as well as PCR amplified SNP region DNA (Table 2)• Sequenced PCR amplified SNP regions using LI-COR® 4200 and 4300 DNA Analyzers (Fig. 5)

• PCR reaction performed using Bio-Rad MyCycler™ thermocycler: 92.0°C for 2 minutes; 30X: 92.0°Cfor 0:30, 54.0°C for 0:30, 70.0°C for 1:00; 70°C for 1:00; 4.0°C hold

• Sequencing reaction used labeled custom IRDye700 primers• 5.5% acrylamide gel using KB Plus Gel Matrix, run for 8 hours• Sequence exported via e-Seq V3.0 in FASTA format and confirmed using NCBI-BLAST

• Analyzed to locate SNP sites

INTRODUCTION Elevated levels of low-density lipoprotein (LDL) in humans have been implicated in atherosclerosis and coronary heart disease1. Low-density lipoprotein-related receptor protein 11 (Lrp11) on chromosome 10 regulates LDL levels in mice through clathrin-mediated endocytosis2,3. A recent study has identified a trans-eQTL locus for Lrp11 expression on mouse Chr5. The candidate region identified spans a 3.5 Mbp region (from 78Mbp to 81.5 Mbp on Chromosome 5). Furthermore, Lrp11 exhibits homology to human and chimpanzee LRP11 on chromosome 63. We are interested in fine mapping the location of the trans-eQTL by genotypping denser SNP markers within the Chromosome 5 locus.

To achieve high-resolution mapping, 110 mice of a commercially available outbred stock, MF1 (Harlan, Indianapolis, Indiana, USA), were used since they can be treated as an ultrafine mosaic of standard inbred strains as described in Yalcin et al4. The genome of MF1 mice resembles the hetergenous stock mice which were established from 8 standard inbred strains (C57BL/6, BALB/c, RIIIS, AKR, DBA/2, I/LnJ, A/J and C3H) in the 1970s (Fig. 1). Genome-wide association studies of 1813 SNP markers, conducted by UCLA collaborative researcher Dr. Anatole Ghazalpour, identified a 3.5 Mbp quantitative trait locus (QTL) influencing Lrp11 expression levels (Fig. 2).

SUMMARYRESULTS• Only PCR amplified DNA using unlabeled primer pairs (Table 1) yielded sequencing signal.• PCR primers for rs31562818 showed reliable signals after SNP region amplification (Fig. 4).

• DBA/2J showed questionable amplification. Remaining seven strains showed adequate amplification. • Polymorphisms for rs31562818 were successfully identified using LI-COR® sequencing (Fig. 5).

• rs31562818 marker found in sequences from AKR, RIII, A/J, C3H, BALB/c, and C57BL/J6.• rs29732770 marker PCR amplification failed to give a product for any of the 8 inbred strains.• Further improvements of primer design should enable successful PCR amplification and sequencing.

ACKNOWLEDGEMENTSSpecial thanks to Dr. Gaston Matthias-Udo Pfluegl and Karen Flummerfelt for their invaluable mentorship, instruction, guidance, and technical assistance, Drs. Anatole Ghazalpour and Aldons J. Lusis for all mouse DNA and QTL region data, and Kristin Toliver and Priya Thaker for their contributions to primer design and sequencing.

Aliyah Khan1, Karolina Khlebnikova2, Gracejeet Sroya2, Anjali D. Tapadia3

1Dept. of Physiological Sciences, 2Dept. of Chemistry and Biochemistry, 3Dept. of Molecular, Cell, and Developmental Biology, UCLA, Los Angeles, CA

Fig. 3: QTL mapping of SNPs in Lrp11-associated region of chromosome 5. Image courtesy of Dr. Anatole Ghazalpour, UCLA.

MF1

Fig. 1: The proposed eight ancestral inbred mouse strains for outbred MF1 stock. Image courtesy of Dr. Aldons J. Lusis, UCLA.

CONCLUSIONSDISCUSSIONBased on the results yielded thus far, we will continue to test the remaining SNP regions for the eight inbred strains. Thereafter, we will amplify, sequence, and genotype SNP markers for the 110 MF1 mice. We plan to use genotype data to identify the causal variation for Lrp11. Association analysis using marker regression will be used to determine SNPs most highly correlated with the Lrp11 trans-eQTL.

APPLICATIONS AND FUTURE RESEARCH• Confirmation studies using Lrp11 transgenic or knockout technology to validate the casual gene• Study the molecular interaction between the causal gene on Chr 5 and Lrp11• To study if the Chr5 in mouse affects LDL levels

REFERENCES1. Miller, M, Ginsberg, HN, and Schaefer, EJ. 2008. Relative atherogenicity and predictive value of non-high-density lipoprotein cholesterol for coronary heart disease. Am J Cardiol 101:1003-8.

2. Chung, NS and Wasan, KM. 2004. Potential role of the low-density lipoprotein receptor family as mediators of cellular drug uptake. Advanced Drug Delivery Reviews 56:1315-1334.

3. Retrieved from “MGI_4.01: Gene Detail”, April 2008 <http://www.informatics.jax.org/javawi2/servlet/WIFetch?page= markerDetail&key=84155>.

4. Yalcin et al. 2004. Genetic Dissection of a behavioral quantitative trait locus shows that Rgs2 modulates anxiety in mice. Nature Genetics 36:1197-1202.

5. Eppig JT, Blake JA, Bult CJ, Kadin JA, Richardson JE, et al. 2007. The Mouse Genome Database (MGD): new features facilitating a model system. Nucleic Acids Res 35(Database issue):D630-D637.6.

6. Bogue, MA, Grubb, SC, Maddatu, TP, Bult, CJ. 2007. Mouse Phenome Database (MPD). Nucleic Acids Res 35(Database issue):D643-9.

7. Kent, WJ. 2002. BLAT--the BLAST-like alignment tool. Genome Research 4:656-64.

8. Rozen, S and Skaletsky, HJ. 2000. Primer3 on the WWW for general users and for biologist programmers. Bioinformatics Methods and Protocols: Methods in Molecular Biology. 132:365-386.

110 MF1 mice

Fig. 2: Study design to correlate genotype with expression and physiological data from MF1 mice. Image courtesy of Dr. Anatole Ghazalpour, UCLA.

Gene Expression(24078 traits)

Physiological Traits(21 traits)

Genome Wide Association

Genotypes(1813 SNPs)

Fig. 5: LI-COR sequencing gel of SNP rs31562818 for strains AKR, RIIIS, A/J, C3H, BALB/C, and C57BL/J6 using LI-COR® IRDye700 labeled custom primer (Table 2). Lane order: ACTG.

The long term aim of this project is to fine map the Chr5 locus to find the causal gene in this region. To this end, we have designed primers for nine equidistant SNP sites (Fig. 3) using eight inbred strains and are PCR amplifying and sequencing the products.

The LI-COR® sequencer plays a monumental role in our research project in terms of verifying the presence of published SNPs in the different mouse strains (Fig. 4). Thus far, primers for nine markers have been designed for the eight inbred strains. Following successful preliminary sequencing of SNP regions in inbred mice, we will genotype SNP sites of the 110 MF1 mice of the original QTL study. It is estimated that approximately 62 runs on the LI-COR® sequencer would be required to complete our research project.

A. B.

Fig. 4: 1% agarose gel confirmation of PCR product of 1:1, 1:10, and 1:100 dilutions of template mouse genomic DNA using unlabeled primer pair for rs31562818 (Table 1). A) A/J, 1 kbp ladder (Promega), C3H, BALB/C, C57BL/J6. B) DBA/2J, I/LnJ, RIIIS, 1 kbp ladder (Promega), AKR. All reactions follow dilution pattern of 1:1, 1:10, 1:100.

Table 2: LI-COR labeled primer design using MGI sequences and Primer3. Primers for nine SNPs polymorphic across all eight inbred mouse strains are being used to sequence SNP regions for the eight inbred strains and the MF1 outbred stock. Primer sequences are conserved across the eight inbred mouse strains and checked for uniqueness using NCBI-BLAST.

Table 1: PCR primer pair design using MGI sequences and Primer3. Primer sequences are conserved across all eight inbred mouse strains and checked for uniqueness using NCBI-BLAST (data not shown). Amplified region includes LI-COR sequencing primer sequence. “any”, self-complimentarity score. “3’”, 3’ self-complementarity score.