Molecular Genetics Laboratory Report

July 30, 1999

 

1. Candidate Genes

The candidate genes were chosen with input from all representative FHA centers. The choice of candidates was based on the physiologic rationale for involvement of gene products in specific phenotypes. The candidate gene set of DNA samples includes 1404 DNA from whites, 161 DNA from blacks and 163 DNA from randoms, for a total of 1728 samples. For the earliest markers (LPL, D8S282, UT5187 and VLDLR) only 1601 samples were genotyped. Eighteen candidate genes and 20 markers were run, for an total of 34,052 genotypes attempted. As an average for all candidate gene markers, over 93.5% of genotypes were scored. The variation in percent scored among markers is the result of the intrinsic robustness of the different marker systems; certain PCR markers perform better than others. We have attempted to fill in, i.e. redo, genotypes for marker systems that during analysis yielded interesting results in order to maximize the informational content (APOAI/CIII/A4 locus - D11S4127) (FCHL - D1S104 and D1S1677).

The specific markers chosen to test candidate genes or regions are listed in the methodology sections that follow. Whenever possible, intragenic markers were utilized.

 

Hormone Sensitive Lipase

LIPE

Chr. 19q13.1-13.2

Genotyping Methods:

Genotyping for the dinucleotide marker LIPE-95 is based on PCR amplification of

genomic DNA. Forward and reverse primer sequences for LIPE-95 are found in Levitt, et al. Cytogenetics Cell Genetics 69: 211-214 and are A: AGG CTG TGT TTC CCC AGA CT, B: AAA CTG CAC CTA ATC TTC CC. PCR reactions were carried out in 96-well trays in a MJ Research PTC-225 thermal cycler instrument (Watertown, MA). The final volume for each reaction was 25m l. Each reaction contained 100ng of template DNA, 0.5 Unit of Taq DNA polymerase (Boehringer Mannheim, Indianapolis, IN), 10 picomoles of both forward and reverse primers, 1 picomole of 32P labeled primer B end labeled with T4 polynucleotide kinase (Molecular Biology Resources, Inc. Milwaukee, WI), 200m m of dNTPs, 5% DMSO, and PCR buffer (final concentration of 10mM Tris pH 8.4, 40mM NaCl, 1.5mM MgCl2, 0.25mM spermidine). The PCR temperature conditions for the LIPE-95 primers were as follows: after an initial 5 min denaturation at 94˚C, 30 PCR cycles were carried out with a denaturing phase of 94˚C for 20 sec., an annealing phase of 64˚C for 20 sec., and an extension phase at 72˚C for 40 sec. This was followed by a final cycle of 7 min. at 72˚C. PCR products were then loaded on a 7% acrylamide gel containing 6.7M urea and 32% formamide. Electrophoresis of the gel was carried out at a constant 80 Watts for 4-6 hours. The gels were exposed to X-ray film (Hyperfilm, Amersham Life Science, Buckinghamshire, England) for 12-16 hours. Genotypes were scored relative to an M13mp18 sequence ladder according to their size in base pairs.

Marker Location: Marker LIPE-95 is in intron 205 base pairs upstream of exon 8.

Reference: Levitt, et al. (1995) Mapping of the gene for hormone sensitive lipase (LIPE) to chromosome 19q13.1-q13.2, Cytogenetics Cell Genetics 69: 211-214.

 

Hepatic Lipase

LIPC

Chr. 15q21

Genotyping Methods:

Genotyping for the dinucleotide marker D15S117 is based on PCR amplification of

genomic DNA. Forward and reverse primer sequences for D15S117 are found in the Genethon database (http://www.genethon.fr) (Dib, et al. 1996, Nature 380:152-154) and are A: GCA CCA ACA ACT TAT CCC AA, B: CCC TAA GGG GTC TCT GAA GA. PCR reactions were carried out in 96-well trays in a MJ Research PTC-225 thermal cycler instrument (Watertown, MA). The final volume for each reaction was 25m l. Each reaction contained 100ng of template DNA, 0.5 Unit of Taq DNA polymerase (Boehringer Mannheim, Indianapolis, IN), 10 picomoles of both forward and reverse primers, 0.25 picomole of 32P labeled primer A end labeled with T4 polynucleotide kinase (Molecular Biology Resources, Inc. Milwaukee, WI), 200m m of dNTPs, 5% DMSO, and PCR buffer (final concentration of 10mM Tris pH 8.4, 40mM NaCl, 1.5mM MgCl2, 0.25mM spermidine). The PCR temperature conditions for the D15S117 primers were as follows: after an initial 5 min denaturation at 94˚C, 35 PCR cycles were carried out with a denaturing phase of 94˚C for 30 sec., an annealing phase of 58˚C for 30 sec., and an extension phase at 72 ˚ C for 1 minute. PCR products were then loaded on a 7% acrylamide gel containing 6.7M urea and 32% formamide. Electrophoresis of the gel was carried out at a constant 80 Watts for 4-6 hours. The gels were exposed to X-ray film (Hyperfilm, Amersham Life Science, Buckinghamshire, England) for 12-16 hours. Genotypes were scored relative to a M13mp18 sequence ladder according to their size in base pairs.

Marker Location: Proxy marker (D15S117)- LOD 11.74, theta 0.0 from intragenic LIPC marker according to Guerra reference.

Reference: Guerra, et al. (1997) A hepatic lipase (LIPC) allele associated with high plasma concentrations of high density lipoprotein cholesterol, Proceedings of the National Academy of Sciences 94:4532-4537.

LOD score obtained from CEPH database (http://www.cephb.fr.ogi-bin).

 

Apolipoprotein A1/CIII/A4

APOA1/CIII/A4

Chr. 11q23.3

Genotyping Methods:

Genotyping for the dinucleotide marker D11S4127 is based on PCR amplification of genomic DNA. Forward and reverse primer sequences for D11S4127 are found in the Genethon database (http://www.genethon.fr) (Dib, et al. 1996, Nature 380: 152-154) and are A: ATG AGA AGT GCC ATC CAG C, B: ACT ATG CCC AGT GTG TGT GC. PCR reactions were carried out in 96-well trays in a MJ Research PTC-225 thermal cycler instrument (Watertown, MA). The final volume for each reaction was 25m l. Each reaction contained 100ng of template DNA, 0.5 Unit of Taq DNA polymerase (Boehringer Mannheim, Indianapolis, IN), 10 picomoles of both forward and reverse primers, 0.25 picomoles of 32P labeled primer A end labeled with T4 polynucleotide kinase (Molecular Biology Resources, Inc. Milwaukee, WI), 200m m of dNTPs, 5% DMSO, and PCR buffer (final concentration of 10mM Tris pH 8.4, 40mM NaCl, 1.5mM MgCl2, 0.25mM spermidine). The PCR temperature conditions for the D11S4127 primers were as follows: after an initial 5 min denaturation at 94˚C, five PCR cycles were carried out with a denaturing phase of 94˚C for 20 sec., an annealing phase of 58˚C for 20 sec., and an extension phase at 72˚C for 40 sec. This was followed by an additional 30 cycles at similar conditions with the exception of the annealing phase which was done at 54˚C for 20 sec. PCR products were then loaded on a 7% acrylamide gel containing 6.7M urea and 32% formamide. Electrophoresis of the gel was carried out at a constant 80 Watts for 4-6 hours. The gels were exposed to X-ray film (Hyperfilm, Amersham Life Science, Buckinghamshire, England) for 12-16 hours. Genotypes were scored relative to an M13mp18 sequence ladder according to their size in base pairs.

Marker Location: Proxy marker (D11S4127)- LOD 13.0, theta 0.0 from gene cluster

Reference: Mapping information from CEPH database (http://www.cephb.fr.ogi-bin).

 

Cholesterol 7-Hydroxylase

CYP7A1

Chr. 8q11-q12

Genotyping Methods:

Genotyping for the dinucleotide marker D8S285 is based on PCR amplification of

genomic DNA. Forward and reverse primer sequences for D8S285 are found in the Genethon database (http://www.genethon.fr) (Dib, et al. 1996, Nature 380: 152-154) and are A: GCA TCA CAC AGA ATC TTT G, B: ATG GGT TTA TGG CCT TTA C. PCR reactions were carried out in 96-well trays in a MJ Research PTC-225 thermal cycler instrument (Watertown, MA). The final volume for each reaction was 25m l. Each reaction contained 100ng of template DNA, 0.5 Unit of Taq DNA polymerase (Boehringer Mannheim, Indianapolis, IN), 10 picomoles of both forward and reverse primers, 0.25 picomoles of 32P labeled primer A end labeled with T4 polynucleotide kinase (Molecular Biology Resources, Inc. Milwaukee, WI), 200m m of dNTPs, 5% DMSO, and PCR buffer (final concentration of 10mM Tris pH 8.4, 40mM NaCl, 1.5mM MgCl2, 0.25mM spermidine). The PCR temperature conditions for the D8S285 primers were as follows: after an initial 5 min denaturation at 94˚C, five PCR cycles were carried out with a denaturing phase of 94˚C for 20 sec., an annealing phase of 58˚C for 20 sec., and an extension phase at 72˚C for 40 sec. This was followed by an additional 30 cycles at similar conditions with the exception of the annealing phase which was done at 54˚C for 20 sec. PCR products were then loaded on a 7% acrylamide gel containing 6.7M urea and 32% formamide. Electrophoresis of the gel was carried out at a constant 80 Watts for 4-6 hours. The gels were exposed to X-ray film (Hyperfilm, Amersham Life Science, Buckinghamshire, England) for 12-16 hours. Genotypes were scored relative to an M13mp18 sequence ladder according to their size in base pairs.

Marker Location: Proxy marker used by Wang, et al. for CYP7A1

Reference: Wang, et al. (1998) Linkage Between Cholesterol 7a -Hydroxylase and High Plasma Low-density Lipoprotein Cholesterol Concentrations, Journal of Clinical Investigation 101(6): 1283-1291.

 

Plasminogen Activator Inhibitor-1

PAI-1

Chr. 7q21.3-q22

Genotyping Methods:

Genotyping for the dinucleotide marker in intron 3 of PAI1 is based on PCR amplification of genomic DNA. Forward and reverse primer sequences for PAI1 are found in Dawson, et al. (1991) Ateriosclerosis Thrombosis 11: 183-190 and are A: ATT TGA ACC GGA TTC GGA GGC TGC, B: CCC CTG TTC ACT GCT CCC CTA TTC. PCR reactions were carried out in 96-well trays in a MJ Research PTC-225 thermal cycler instrument (Watertown, MA). The final volume for each reaction was 25m l. Each reaction contained 100ng of template DNA, 0.5 Unit of Taq DNA polymerase (Boehringer Mannheim, Indianapolis, IN), 10 picomoles of both forward and reverse primers, 0.25 picomole of 32P labeled primer B end labeled with T4 polynucleotide kinase (Molecular Biology Resources, Inc. Milwaukee, WI), 200m m of dNTPs, 5% DMSO, and PCR buffer (final concentration of 10mM Tris pH 8.4, 40mM NaCl, 1.5mM MgCl2, 0.25mM spermidine). The PCR temperature conditions for the PAI1 primers were as follows: after an initial 5 min denaturation at 94˚C, five PCR cycles were carried out with a denaturing phase of 94˚C for 20 sec., an annealing phase of 58˚C for 20 sec., and an extension phase at 72˚C for 40 sec. This was followed by an additional 30 cycles at similar conditions with the exception of the annealing phase which was done at 54˚C for 20 sec. PCR products were then loaded on a 7% acrylamide gel containing 6.7M urea and 32% formamide. Electrophoresis of the gel was carried out at a constant 80 Watts for 4-6 hours. The gels were exposed to X-ray film (Hyperfilm, Amersham Life Science, Buckinghamshire, England) for 12-16 hours. Genotypes were scored relative to an M13mp18 sequence ladder according to their size in base pairs.

Marker Location: PAI1 marker found in intron 3 of PAI1 gene

Reference: Dawson, et al. (1991) Genetic Variation at the Plasminogen Activator Inhibitor-1 Locus Is Associated With Altered Levels of Plasma Plasminogen Activator Inhibitor-1 Activity, Arterio. Thrombosis 11: 183-190.

 

Lipoprotein Lipase

LPL

Chr. 8p22

Genotyping Methods:

Genotyping for the dinucleotide markers D8S282 and LPL1GTR2, is based on PCR amplification of genomic DNA. Forward and reverse primer sequences for D8S282 are found in the Genethon database (http://www.genethon.fr) (Dib, et al. 1996, Nature 380: 152-154) and are A: GGG CAC AGG CAT GTG T, B: GGC TGC ATT CTG AAA GGT TA. Forward and reverse primer sequences for LPL1GTR2, located near the end of the 5' region of the LPL gene, are from J.-M. Lalouel (personal communication) and Odelberg and White (Odelberg and White 1993, A Method for Accurate Amplification of Polymorphic CA-repeat Sequences, PCR Methods and Applications 3(7): 12) and are A: AAT GGA TGG ATA GTT GGG AGA GGG, B: TGG TTC CCA CAT GCC CCA TTT C. PCR reactions were carried out in 96-well trays in a MJ Research PTC-225 thermal cycler instrument (Watertown, MA). The final volume for each reaction was 25m l. Each reaction contained 100ng of template DNA, 0.5 Unit of Taq DNA polymerase (Boehringer Mannheim, Indianapolis, IN), 10 picomoles of both forward and reverse primers, 0.25 picomole of 32P labeled primer A end labeled with T4 polynucleotide kinase (Molecular BiologyResources, Inc. Milwaukee, WI), 200m m of dNTPs, 5% DMSO, and PCR buffer (final concentration of 10mM Tris pH 8.4, 40mM NaCl, 1.5mM MgCl2, 0.25mM spermidine). Cycling times and temperatures for D8S282 and LPL1GTR2 marker systems were identical. After an initial 5 min denaturation at 94˚C, five PCR cycles were carried out with a denaturing phase of 94˚C for 20 sec., an annealing phase of 58˚C for 20 sec., and an extension phase at 72˚C for 40 sec. This was followed by an additional 30 cycles at similar conditions with the exception of the annealing phase which was done at 54˚C for 20 sec. PCR products were then loaded on a 7% acrylamide gel containing 6.7M urea and 32% formamide. Electrophoresis of the gel was carried out at a constant 80 Watts for 4-6 hours. The gels were exposed to X-ray film (Hyperfilm, Amersham Life Science, Buckinghamshire, England) for 12-16 hours. Genotypes were scored relative to an M13mp18 sequence ladder according to their size in base pairs.

Marker Locations: Marker LPL1GTR2 placed in the 5 region of the LPL gene by Odelberg and White.

D8S282 marker is 3cM from LPL gene and was used as proxy marker by Wu et al.

References: Wu, et al. (1996) Quantitative Trait Locus Mapping of Human Blood Pressure to a Genetic Region at or near the Lipoprotein Lipase Gene Locus on Chromosome 8p22, Journal of Clin. Invest. 97: 2111-2118.

Odelberg and White (1993) A Method for Accurate Amplification of Polymorphic CA-repeat Sequences, PCR Methods and Applications 3(7):12.

 

Microsomal Triglyceride Transfer Protein

MTP

Chr. 4q22-24

Genotyping Methods:

Genotyping for the dinucleotide marker in intron 10 of MTP is based on PCR amplification of genomic DNA. Forward and reverse primer sequences for MTP are found in Heath, et al. Prenatal Diagnosis 17: 1181-1186 and are A: TCC ACA GGA TTC ATA ACC, B: TTC TCC ACT CTT CCC CAT. PCR reactions were carried out in 96-well trays in a MJ Research PTC-225 thermal cycler instrument (Watertown, MA). The final volume for each reaction was 25m l. Each reaction contained 100ng of template DNA, 0.5 Unit of Taq DNA polymerase (Boehringer Mannheim, Indianapolis, IN), 10 picomoles of both forward and reverse primers, 0.25 picomole of 32P labeled primer A end labeled with T4 polynucleotide kinase (Molecular Biology Resources, Inc. Milwaukee, WI), 200m m of dNTPs, 5% DMSO, and PCR buffer (final concentration of 10mM Tris pH 8.4, 40mM NaCl, 1.5mM MgCl2, 0.25mM spermidine). The PCR temperature conditions for the MTP primers were as follows: after an initial 5 min denaturation at 94˚C, 30 PCR cycles were carried out with a denaturing phase of 94˚C for 1 min., an annealing phase of 57˚C for 1 min., and an extension phase at 72˚C for 1 min. This was followed by a final cycle of 5 min. at 72˚C. PCR products were then loaded on a 7% acrylamide gel containing 6.7M urea and 32% formamide. Electrophoresis of the gel was carried out at a constant 80 Watts for 4-6 hours. The gels were exposed to X-ray film (Hyperfilm, Amersham Life Science, Buckinghamshire, England) for 12-16 hours. Genotypes were scored relative to an M13mp18 sequence ladder according to their size in base pairs.

Marker Location: MTP marker found in intron 10 of MTP gene

Reference: Heath, et al. (1997) The Use of a Highly Informative CA Repeat Polymorphism Within the Abetalipoproteinaemia Locus (4q22-24), Prenatal Diagnosis 17: 1181-1186.

 

Manganese Superoxide Dismutase

SOD2

Chr. 6q25.3

Genotyping Methods:

Genotyping for the dinucleotide marker D6S437 is based on PCR amplification of

genomic DNA. Forward and reverse primer sequences for D6S437 are found in the Genethon database (http://www.genethon.fr) (Dib, et al. 1996, Nature 380: 152-154) and are A: TGT CCT GGT GGA GGC A, B: GGT ACA GTG TTT GAC CCT AAG A. PCR reactions were carried out in 96-well trays in a MJ Research PTC-225 thermal cycler instrument (Watertown, MA). The final volume for each reaction was 25m l. Each reaction contained 100ng of template DNA, 0.5 Unit of Taq DNA polymerase (Boehringer Mannheim, Indianapolis, IN), 10 picomoles of both forward and reverse primers, 0.25 picomole of 32P labeled primer A end labeled with T4 polynucleotide kinase (Molecular Biology Resources, Inc. Milwaukee, WI), 200m m of dNTPs, 5% DMSO, and PCR buffer (final concentration of 10mM Tris pH 8.4, 40mM NaCl, 1.5mM MgCl2, 0.25mM spermidine). The PCR temperature conditions for the D6S437 primers were as follows: after an initial 5 min denaturation at 94˚C, five PCR cycles were carried out with a denaturing phase of 94˚C for 20 sec., an annealing phase of 58˚C for 20 sec., and an extension phase at 72˚C for 40 sec. This was followed by an additional 30 cycles at similar conditions with the exception of the annealing phase which was done at 54˚C for 20 sec. PCR products were then loaded on a 7% acrylamide gel containing 6.7M urea and 32% formamide. Electrophoresis of the gel was carried out at a constant 80 Watts for 4-6 hours. The gels were exposed to X-ray film (Hyperfilm, Amersham Life Science, Buckinghamshire, England) for 12-16 hours. Genotypes were scored relative to an M13mp18 sequence ladder according to their size in base pairs.

Marker Location: Marker D6S437, used as a proxy marker, maps close to RFLP in gene MnSOD4- LOD 3.01, theta 0.0

Reference: Church, et al. (1992) Sublocalization of the Gene Encoding Manganese Superoxide Dismutase (MnSOD/SOD2) to 6q25 by Fluorescence in Situ Hybridization and Somatic Cell Hybrid Mapping, Genomics 17: 823-825.

Mapping information from CEPH database (http://www.cephb.fr.ogi-bin).

 

Adducin (Alpha)

ADD1

Chr. 4p16.3

Genotyping Methods:

Genotyping for the dinucleotide marker D4S2936 is based on PCR amplification of

genomic DNA. Forward and reverse primer sequences for D4S2936 are found in the Genethon database (http://www.genethon.fr) (Dib, et al. 1996, Nature 380: 152-154) and are A: CAC TCA AGC CTG GGG G, B: TGG CAC ATC ACC AAC AAC. PCR reactions were carried out in 96-well trays in a MJ Research PTC-225 thermal cycler instrument (Watertown, MA). The final volume for each reaction was 25m l. Each reaction contained 100ng of template DNA, 0.5 Unit of Taq DNA polymerase (Boehringer Mannheim, Indianapolis, IN), 10 picomoles of both forward and reverse primers, 0.25 picomoles of 32P labeled primer A end labeled with T4 polynucleotide kinase (Molecular Biology Resources, Inc. Milwaukee, WI), 200m m of dNTPs, 5% DMSO, and PCR buffer (final concentration of 10mM Tris pH 8.4, 40mM NaCl, 1.5mM MgCl2, 0.25mM spermidine). The PCR temperature conditions for the D4S2936 primers were as follows: after an initial 5 min denaturation at 94˚C, five PCR cycles were carried out with a denaturing phase of 94˚C for 20 sec., an annealing phase of 58˚C for 20 sec., and an extension phase at 72˚C for 40 sec. This was followed by an additional 30 cycles at similar conditions with the exception of the annealing phase which was done at 54˚C for 20 sec. PCR products were then loaded on a 7% acrylamide gel containing 6.7M urea and 32% formamide. Electrophoresis of the gel was carried out at a constant 80 Watts for 4-6 hours. The gels were exposed to X-ray film (Hyperfilm, Amersham Life Science, Buckinghamshire, England) for 12-16 hours. Genotypes were scored relative to an M13mp18 sequence ladder according to their size in base pairs.

Marker Location: Proxy marker (D4S2936)- LOD 17.0, theta 0.03 from intragenic marker D4S95.

Reference: Goldberg, et al. (1992) Cloning and mapping of the -adducin gene close to D4S95 and assessment of its relationship to Huntington disease, Human Molecular Genetics 1(9): 669-675.

Mapping information from CEPH database (http://www.cephb.fr.ogi-bin).

 

Familial Combined Hyperlipidemia

FCHL

Chr. 1q21-23

Genotyping Methods:

Genotyping for the STR markers D1S104 and D1S1677 is based on PCR amplification of genomic DNA. Forward and reverse primer sequences for D1S104 are found in the Genethon database (http://www.genethon.fr) (Dib, et al. 1996, Nature 380: 152-154) and are A: ATC CTG CCC TTA TGG AGT GC, B: CCC ACT CCT CTG TCA TTG TA . Forward and reverse primer sequences for D1S1677 are found in CHLC database (http://www.chlc.org) (Murray, et al. 1994, Science 265: 2049-2054) and are A: AGT CAG CTT GAT TGA CCC AG, B: CTT AGT GTG ACA GGA AGG ACG . PCR reactions were carried out in 96-well trays in a MJ Research PTC-225 thermal cycler instrument (Watertown, MA). The final volume for each reaction was 25m l. Each reaction contained 100ng of template DNA, 0.5 Unit of Taq DNA polymerase (Boehringer Mannheim, Indianapolis, IN), 10 picomoles of both forward and reverse primers, 200m m of dNTPs, 5% DMSO, and PCR buffer (final concentration of 10mM Tris pH 8.4, 40mM NaCl, 1.5mM MgCl2, 0.25mM spermidine). Each reaction with D1S1677 contained 0.25 picomoles of 32P labeled primer A end labeled with T4 polynucleotide kinase (Molecular Biology Resources, Inc. Milwaukee, WI), while each reaction with D1S104 contained 1 picomole of 32P labeled primer B. Cycling times and temperatures for D1S104 and D1S1677 marker systems were identical. After an initial 5 min denaturation at 94˚C, five PCR cycles were carried out with a denaturing phase of 94˚C for 20 sec., an annealing phase of 58˚C for 20 sec., and an extension phase at 72˚C for 40 sec. This was followed by an additional 30 cycles at similar conditions with the exception of the annealing phase which was done at 54˚C for 20 sec. PCR products were then loaded on a 7% acrylamide gel containing 6.7M urea and 32% formamide. Electrophoresis of the gel was carried out at a constant 80 Watts for 4-6 hours. The gels were exposed to X-ray film (Hyperfilm, Amersham Life Science, Buckinghamshire, England) for 12-16 hours. Genotypes were scored relative to an M13mp18 sequence ladder according to their size in base pairs.

Marker Location: D1S104 maps adjacent to APOA2: LOD 3.50, theta 0.02

D1S1677 maps 0.4 cM from D1S104: LOD 0.91, theta 0.10

combined LOD 5.93. D1S104 and D1S1677 are the markers used in the FCHL study of Pajukanta.

Reference: Pajukanta, et al. (1998) Linkage of familial combined hyperlipidaemia to chromosome 1q21-q23, Nature Genetics 18: 369-373.

 

Interleukin 1-alpha

IL1A

Chr. 2q12-13

Genotyping Methods:

Genotyping for the dinucleotide marker IL1A/222-223 is based on PCR amplification of genomic DNA. Forward and reverse primer sequences for IL1A/222-223 are found in Todd, et al. Nucleic Acids Research 19(13): 3756 and are A: ATG TAT AGA ATT CCA TTC CTG, B: TAA AAT CAA GTG TTG ATG TAG . PCR reactions were carried out in 96-well trays in a MJ Research PTC-225 thermal cycler instrument (Watertown, MA). The final volume for each reaction was 25m l. Each reaction contained 100ng of template DNA, 0.5 Unit of Taq DNA polymerase (Boehringer Mannheim, Indianapolis, IN), 10 picomoles of both forward and reverse primers, 0.25 picomoles of 32P labeled primer A end labeled with T4 polynucleotide kinase (Molecular Biology Resources, Inc. Milwaukee, WI), 200m m of dNTPs, 5% DMSO, and PCR buffer (final concentration of 10mM Tris pH 8.4, 40mM NaCl, 1.5mM MgCl2, 0.25mM spermidine). The PCR temperature conditions for the IL1A/222-223 primers were as follows: after an initial 5 min denaturation at 94˚C, five PCR cycles were carried out with a denaturing phase of 94˚C for 20 sec., an annealing phase of 58˚C for 20 sec., and an extension phase at 72˚C for 40 sec. This was followed by an additional 30 cycles at similar conditions with the exception of the annealing phase which was done at 54˚C for 20 sec. PCR products were then loaded on a 7% acrylamide gel containing 6.7M urea and 32% formamide. Electrophoresis of the gel was carried out at a constant 80 Watts for 4-6 hours. The gels were exposed to X-ray film (Hyperfilm, Amersham Life Science, Buckinghamshire, England) for 12-16 hours. Genotypes were scored relative to an M13mp18 sequence ladder according to their size in base pairs.

Marker Location: IL1A/222-223 is an intragenic marker in the IL1A gene at nucleotide 7695.

Reference: Todd, et al. Dinucleotide repeat polymorphism in the human interleukin 1, alpha gene (IL1A), Nucleic Acids Research 19(13): 3756

 

Low Density Lipoprotein Receptor

LDLR

Chr. 19p13

Genotyping methods:

Genotyping for the tetranucleotide marker UT5187 is based on PCR amplification of genomic DNA. Forward and reverse primer sequences for UT5187 are from the Utah Marker Development Group (1995) Amer. Journ. Genet. 57: 619-628 and are A: TCG AGA CTA CAG TGA GCT G, B: ATT ATG GGT GTC TTC CTG AC. PCR reactions were carried out in a MJ Research PTC-225 thermal cycler instrument (Watertown, M.A.). The final volume for each reaction was 25m l. Each reaction contained 100ng of template DNA, 0.5 Units of Taq DNA polymerase (Boehringer Mannheim, Indianapolis, IN) 10 picomoles of both forward and reverse primers, 0.25 picomoles of 32P labeled primers A end labeledwith T4 polynucleotide kinase (Molecular Biology Resources Inc, Milwaukee, WI), 200m m of dNTPs, 5% DMSO, and PCR buffer (final concentration of 10mM Tris pH8.4, 40 mM NaCl, 1.5mM MgCl2, 0.25mM spermidine). Cycling times and temperatures for UT5187 were as follows: after an initial 5 min denaturation at 94˚C, seven PCR cycles were carried out with a denaturing phase of 94˚C for 20 seconds, an annealing phase beginning at 60˚C for 20 seconds and decreasing 1˚C for each of the next six cycles and an extension phase of 72˚ for 40 seconds. This was followed by an additional 24 cycles at similar conditions with the exception of the annealing phase which was done at 54˚ for 20 seconds. PCR products were then loaded on a 7% acrylamide gel containing 6.7M urea and 32% formamide. Electrophoresis of the gel was carried out at a constant 80 Watts for 4-6 hours. The gels were exposed to X-ray film (Hyperfilm, Amersham Life Sciences, Buckinghamshire, England) for 12-16 hours. Genotypes were scored relative to an M13mp18 sequence ladder according to their size in base pairs.

Marker Location: The cDNA clone (pHH1) of the LDL receptor contains a 2 allele RFLP that maps at the same location as UT5187 (D19S581) theta 0.0, LOD 18.1.

References: Utah Marker Development Group (1995) Amer. Journ. Genet. 57: 619-628.

Leppert, et al. (1986) A DNA Probe for the LDL Receptor Gene Is Tightly Linked to Hypercholesterolemia in a Pedigree with Early Coronary Disease, Am J Hum Genet 39:300-306.

Mapping information from CEPH database (http://www.cephb.fr.ogi-bin).

 

Leptin Receptor

LEPR

Chr. 1p31

Genotyping Methods:

Genotyping for the dinucleotide marker D1S2852 is based on PCR amplification of

genomic DNA. Forward and reverse primer sequences for D1S2852 are found in the Genethon database (http://www.genethon.fr) (Dib, et al. 1996, Nature 380: 152-154) and are A: GGG CAA CAC AGC GAG G, B: AAC TAC TGT CAC TGT TCA CAC TCA G. PCR reactions were carried out in 96-well trays in a MJ Research PTC-225 thermal cycler instrument (Watertown, MA). The final volume for each reaction was 25m l. Each reaction contained 100ng of template DNA, 0.5 Unit of Taq DNA polymerase (Boehringer Mannheim, Indianapolis, IN), 10 picomoles of both forward and reverse primers, 0.25 picomoles of 32P labeled primer A end labeled with T4 polynucleotide kinase (Molecular Biology Resources, Inc. Milwaukee, WI), 200m m of dNTPs, 5% DMSO, and PCR buffer (final concentration of 10mM Tris pH 8.4, 40mM NaCl, 1.5mM MgCl2, 0.25mM spermidine). The PCR temperature conditions for the D1S2852 primers were as follows: after an initial 5 min denaturation at 94˚C, five PCR cycles were carried out with a denaturing phase of 94˚C for 20 sec., an annealing phase of 58˚C for 20 sec., and an extension phase at 72˚C for 40 sec. This was followed by an additional 30 cycles at similar conditions with the exception of the annealing phase which was done at 54˚C for 20 sec. PCR products were then loaded on a 7% acrylamide gel containing 6.7M urea and 32% formamide. Electrophoresis of the gel was carried out at a constant 80 Watts for 4-6 hours. The gels were exposed to X-ray film (Hyperfilm, Amersham Life Science, Buckinghamshire, England) for 12-16 hours. Genotypes were scored relative to an M13mp18 sequence ladder according to their size in base pairs.

Marker Location: D1S2852 maps to the same contig and between primer sets from the 5' to 3' untranslated region of the LEPR gene.

Reference: Winick, et al. (1996) Identification of Microsatellite Markers Linked to the Human Leptin Receptor Gene on Chromosome 1, Genomics 36: 221-222.

 

Peroxisome Proliferative-Activated Receptor Gamma

PPAR

Chr. 3p25

Genotyping Methods:

Genotyping for the dinucleotide marker D3S3608 is based on PCR amplification of

genomic DNA. Forward and reverse primer sequences for D3S3608 are found in the Genethon database (http://www.genethon.fr) (Dib, et al. 1996, Nature 380: 152-154) and are A: ACG CCA TCT NCA GGC T, B: TGG GTG CAG CAC ACC A . PCR reactions were carried out in 96-well trays in a MJ Research PTC-225 thermal cycler instrument (Watertown, MA). The final volume for each reaction was 25m l. Each reaction contained 100ng of template DNA, 0.5 Unit of Taq DNA polymerase (Boehringer Mannheim, Indianapolis, IN), 10 picomoles of both forward and reverse primers, 0.25 picomole of 32P labeled primer A end labeled with T4 polynucleotide kinase (Molecular Biology Resources, Inc. Milwaukee, WI), 200m m of dNTPs, 5% DMSO, and PCR buffer (final concentration of 10mM Tris pH 8.4, 40mM NaCl, 1.5mM MgCl2, 0.25mM spermidine). The PCR temperature conditions for the D3S3608 primers were as follows: after an initial 5 min denaturation at 94˚C, five PCR cycles were carried out with a denaturing phase of 94˚C for 20 sec., an annealing phase of 58˚C for 20 sec., and an extension phase at 72˚C for 40 sec. This was followed by an additional 30 cycles at similar conditions with the exception of the annealing phase which was done at 54˚C for 20 sec. PCR products were then loaded on a 7% acrylamide gel containing 6.7M urea and 32% formamide. Electrophoresis of the gel was carried out at a constant 80 Watts for 4-6 hours. The gels were exposed to X-ray film (Hyperfilm, Amersham Life Science, Buckinghamshire, England) for 12-16 hours. Genotypes were scored relative to an M13mp18 sequence ladder according to their size in base pairs.

Marker Location: Proxy marker (D3S3608) maps close to D3S1585 - LOD 16.6, theta 0.0

Reference: Vigouroux, et al. (1998) Human Peroxisome Proliferator-Activated Receptor-g 2, Diabetes 47: 490-492.

 

Low Density Lipoprotein Related Protein 1

LRP

Chr. 12q13.1-q13.3

Genotyping Methods:

Genotyping for the dinucleotide marker D12S1691 is based on PCR amplification of genomic DNA. Forward and reverse primer sequences for D12S1691 are found in the Genethon database (http://www.genethon.fr) (Dib, et al. 1996, Nature 380: 152-154) and are A: GAT GGG AAT CAA TCA TAC CCA AAA C, B: ATG AGC CAC CGT GCC TG. PCR reactions were carried out in 96-well trays in a MJ Research PTC-225 thermal cycler instrument (Watertown, MA). The final volume for each reaction was 25m l. Each reaction contained 100ng of template DNA, 0.5 Unit of Taq DNA polymerase (Boehringer Mannheim, Indianapolis, IN), 10 picomoles of both forward and reverse primers, 0.25 picomoles of 32P labeled primer B end labeled with T4 polynucleotide kinase (Molecular Biology Resources, Inc. Milwaukee, WI), 200m m of dNTPs, 5% DMSO, and PCR buffer (final concentration of 10mM Tris pH 8.4, 40mM NaCl, 1.5mM MgCl2, 0.25mM spermidine). The PCR temperature conditions for the D12S1691 primers were as follows: after an initial 5 min denaturation at 94˚C, five PCR cycles were carried out with a denaturing phase of 94˚C for 20 sec., an annealing phase of 58˚C for 20 sec., and an extension phase at 72˚C for 40 sec. This was followed by an additional 30 cycles at similar conditions with the exception of the annealing phase which was done at 54˚C for 20 sec. PCR products were then loaded on a 7% acrylamide gel containing 6.7M urea and 32% formamide. Electrophoresis of the gel was carried out at a constant 80 Watts for 4-6 hours. The gels were exposed to X-ray film (Hyperfilm, Amersham Life Science, Buckinghamshire, England) for 12-16 hours. Genotypes were scored relative to an M13mp18 sequence ladder according to their size in base pairs.

Marker Location: LRP maps physically very close (300kb) to GLI (glioma-associated oncogene homolog). The marker D12S1691 maps to within 1 cM of GLI.

Reference: Forus, et al. (1992) A Physical Map of a 1.3-Mb Region on the Long Arm of Chromosome 12, Spanning the GLI and LRP Loci, Genomics 35: 586-589.

GB4 map of GeneMap (http://www.ncbi.nlm.nih.gov/genemap/map.cgi).

 

Paraoxonase/arylesterase

PON1

Chr. 7q21-q22

Genotyping Methods:

Genotyping for the dinucleotide marker in intron 4 of PON1 is based on PCR amplification of genomic DNA. Forward and reverse primer sequences for PON1 are found in Clendenning, et al. (1996) Structural Organization of the Human PON1 Gene, Genomics 35: 586-589 and are A: CAT AAT GGT GAT GAA TGG GTG, B: CCT ATA GAC ACT TTA AAC TC. PCR reactions were carried out in 96-well trays in a MJ Research PTC-225 thermal cycler instrument (Watertown, MA). The final volume for each reaction was 25m l. Each reaction contained 100ng of template DNA, 0.5 Unit of Taq DNA polymerase (Boehringer Mannheim, Indianapolis, IN), 10 picomoles of both forward and reverse primers, 1 picomole of 32P labeled primer B end labeled with T4 polynucleotide kinase (Molecular Biology Resources, Inc. Milwaukee, WI), 200m m of dNTPs, 5% DMSO, and PCR buffer (final concentration of 10mM Tris pH 8.4, 40mM NaCl, 1.5mM MgCl2, 0.25mM spermidine). The PCR temperature conditions for the PON1 primers were as follows: after an initial 5 min denaturation at 94˚C, five PCR cycles were carried out with a denaturing phase of 94˚C for 20 sec., an annealing phase of 58˚C for 20 sec., and an extension phase at 72˚C for 40 sec. This was followed by an additional 30 cycles at similar conditions with the exception of the annealing phase which was done at 54˚C for 20 sec. PCR products were then loaded on a 7% acrylamide gel containing 6.7M urea and 32% formamide. Electrophoresis of the gel was carried out at a constant 80 Watts for 4-6 hours. The gels were exposed to X-ray film (Hyperfilm, Amersham Life Science, Buckinghamshire, England) for 12-16 hours. Genotypes were scored relative to an M13mp18 sequence ladder according to their size in base pairs.

Marker Location: PON1 marker found in intron 4 of PON1 gene

Reference: Clendenning, et al. (1996) Structural Organization of the Human PON1 Gene, Genomics 35: 586-589.

 

Tunicamycin Responsive Protein

RTP

Chr. 8q24.21

Genotyping Methods:

Genotyping for the tetranucleotide marker D8S1128 is based on PCR amplification of genomic DNA. Forward and reverse primer sequences for D8S1128 are found in the CHLC database (http://www.chlc.org) (Murray, J.C. et al, 1994, Science 265: 2049-2054) and are A: AAA CAA TCA AAG GCC CAG G, B: CCC ATT GGA AAC AGA GTT GA . PCR reactions were carried out in 96-well trays in a MJ Research PTC-225 thermal cycler instrument (Watertown, MA). The final volume for each reaction was 25m l. Each reaction contained 100ng of template DNA, 0.5 Unit of Taq DNA polymerase (Boehringer Mannheim, Indianapolis, IN), 10 picomoles of both forward and reverse primers, 0.25 picomole of 32P labeled primer A end labeled with T4 polynucleotide kinase (Molecular Biology Resources, Inc. Milwaukee, WI), 200m m of dNTPs, 5% DMSO, and PCR buffer (final concentration of 10mM Tris pH 8.4, 40mM NaCl, 1.5mM MgCl2, 0.25mM spermidine). The PCR temperature conditions for the D8S1128 primers were as follows: after an initial 5 min denaturation at 94˚C, five PCR cycles were carried out with a denaturing phase of 94˚C for 20 sec., an annealing phase of 58˚C for 20 sec., and an extension phase at 72˚C for 40 sec. This was followed by an additional 30 cycles at similar conditions with the exception of the annealing phase which was done at 54˚C for 20 sec. PCR products were then loaded on a 7% acrylamide gel containing 6.7M urea and 32% formamide. Electrophoresis of the gel was carried out at a constant 80 Watts for 4-6 hours. The gels were exposed to X-ray film (Hyperfilm, Amersham Life Science, Buckinghamshire, England) for 12-16 hours. Genotypes were scored relative to an M13mp18 sequence ladder according to their size in base pairs.

Marker Location and Reference: Atwood, et al. unpublished

 

Very Low Density Lipoprotein Receptor

VLDL-R

Chr. 9p24

Genotyping Methods:

Genotyping for the STR marker VLDL-R is based on PCR amplification of genomic DNA. Forward and reverse primer sequences for the trinucleotide repeat (CGG) marker located in the 5' untranslated region (-19) of the VLDL-R gene are from Jokinen, et al. (1994) Human Molecular Genetics 3(3):521 and are A: AAT GAA TTC ACC TTC TTC CTC CTT TCG GAA GGG C, B: AAT GAA TTC TCC TCA CTC ACC GGT TCC GGT GGC. PCR reactions were carried out in 96-well trays in a MJ Research PTC-225 thermal cycler instrument (Watertown, MA). The final volume for each reaction was 25m l. Each reaction contained 100ng of template DNA, 0.5 Unit of Taq DNA polymerase (Boehringer Mannheim, Indianapolis, IN), 10 picomoles of both forward and reverse primers, 0.25 picomoles of 32P labeled primer A end labeled with T4 polynucleotide kinase (Molecular Biology Resources, Inc. (Milwaukee, WI), 200m m of dNTPs, 10% DMSO, and PCR buffer (final concentration of 10mM Tris pH 8.4, 40mM NaCl, 1.5mM MgCl2, 0.25mM spermidine). The PCR temperature conditions for the VLDL-R primers were as follows: after an initial denaturation at 94 C for 5 min, 30 PCR cycles were carried out with a denaturing phase of 94˚C for 20 sec, an annealing phase of 68 C for 20 sec., and an extension phase of 72˚C for 40 sec. A final extension phase of 72 C for 5 min. followed these cycles. PCR products were then loaded on a 7% acrylamide gel containing 6.7M urea and 32% formamide. Electrophoresis of the gel was carried out at a constant 80 Watts for 4-6 hours. The gels were exposed to X-ray film (Hyperfilm, Amersham Life Science, Buckinghamshire, England) for 12-16 hours. Genotypes were scored relative to an M13mp18 sequence ladder according to their size in base pairs.

Marker Location: Trinucleotide repeat localized in 5 untranslated region (-19) of the VLDL-R gene

Reference: Sakai, et al. (1994) Structure, Chromosome Location, and Expression of the Human Very Low Density Lipoprotein Receptor Gene, Journal of Bio. Chem. 269: 2173-2182.

 

2. Coarse Map Genome Scan

The genome wide scan will utilize 240 highly polymorphic markers when completed. To date 180 markers, or 75% of the total, have been run, scored and the genotypes electronically shipped to the Coordinating Center. The last shipment of 40 markers, set #5, was sent on July 29,1999. The sample size for the coarse map scan is 1394 DNAs which includes "phantom" duplicates. The total number of genotypes attempted is 180 x 1394 = 250,920. Genotypes for the next set of 32 markers (set #6) is now being generated and we expect to complete this set of markers by October 1, 1999. The final set of 28 markers is scheduled to be completed by approximately December 1, 1999.

Genotypes from the genome wide scan are generated by probe hybridization instruments designed and built at the University of Utah Human Genome Center. Two instruments are currently dedicated to the FHS project. The genotyping is based on multiplexing of PCR based marker systems. Initially, this system was designed for large-scale DNA sequencing and the instrumentation and methodology is described in a 1994 Genomics article (Cherry, J. et al., 1994, Genomics 20, 68-74). For genotyping, PCR marker products are individually amplified, then pooled (up to 40 marker systems at once) and run on a polyacrylamide gel. The separated products are transferred onto a slowly moving, nylon membrane at the bottom of the gel. The membrane is placed onto a rotating drum in the probe hybridization chamber, and the membrane then automatically undergoes sequential rounds of probe hybridization and stripping for each marker system. The positions of the marker alleles are revealed by an enzyme-linked fluorescent detection system and the images captured by a CCD camera. The images are displayed on a screen and a computer system then automatically calls the size of the allele in base pairs relative to a M13 ladder sequence. For robust markers without imperfect repeats, the automated calling program can call alleles more than 90% of the time. The system is designed so that an operator can visually check all allele calls and manually call or change any of the automated calls. The allele sizes are automatically entered into a relational database and electronically transferred to the coordinating center where quality control issues are addressed.

The diagrams below indicate the marker systems chosen for the genome scan, their map order and distances, their heterozygosities, the number of alleles observed and the length of the short random repeat. Markers in bold are markers completed (n=180) and sent to the Coordinating Center. Markers in italics are markers not yet completed. The markers included in the next set, set 6, are labeled as belonging to set

6.