Fine Mapping and Discovery of Recessive Mutations that Cause Abortions in Dairy

Cattle

P. M. VanRaden1, D. J. Null1*, T.S. Sonstegard2, H.A. Adams3, C.P. Van Tassell2, and K.M. Olson4

1Animal Improvement Programs Laboratory, ARS, USDA, Beltsville, MD 2Bovine Functional Genomics Laboratory, ARS, USDA, Beltsville, MD

3 Institute of Genomic Biology, University of Illinois, Urbana, IL 4National Association of Animal Breeders, Columbia, MO

Abstr. LB6

2012

Introduction Sequencing and Discovery of HH1

• 50K genotypes were used for haplotype discovery. Methods tested on

Brachyspina.

• Numbers of expected homozygous animals were calculated using real matings

and ranged from 7 to 90 for haplotypes examined further.

• Fertility records were used to confirm lethality, the number of carrier x carrier

matings ranged from 936 to 52,449.

• 5 haplotypes, named by breed and number within breed, (Table 1) were

confirmed to be lethal with affects on conception rate (CR) ranging from 3.0% to

3.7%.

• CR loss occurs <60 days HH3 and JH1, <100 days for HH2, and throughout

gestation for HH1 (Figure 1).

Conclusions

Fine Mapping the Haplotypes

Objectives

• Locate haplotypes with significant expected homozygous animals but none

observed.

• Narrow down suspect region using fine mapping.

• Use sequence data to locate the causative mutation within the fine mapped

region.

• Explore ways to use causative mutation test results in the future.

Chromo-some Location

Carrier Freq Founder

Name BTA Mbase % Birth yearHH1 5 62-68 4.5 1962HH2 1 93-98 4.6 1975HH3 8 92-97 4.7 1954JH1 15 11-16 23.4 1962BH1 7 42-47 14.0 1972

Table 1. Haplotypes affecting fertility.

60 100 140 2800

1

2

3

4

5

HH1 HH2 HH3 JH1 BH1

Gestation Length (days)Co

ncep

tion

Rat

e lo

ss

(%)

Figure 1. CR loss during gestation.

• Source haplotype is ~75 markers or ~ 5 Mb.

• Only used crossovers between a 50K animal and a 50K parent.

• Animals that have the source haplotype and a crossover of the source haplotype

were used for fine mapping.

• Interval that never becomes homozygous in live animals is the suspect region.

• Haplotypes narrowed down to a suspect region ranging from 4.5 Mb for BH1 to

0.8 Mb for JH1 (Figure 2).

Finding Haplotypes Affecting Fertility

• HH1 fine mapped to 3.2 Mb suspect region.

• Founder bull (Chief) and 3 sons (Mark, Ivanhoe Chief, and Valiant) were

sequenced by U. Illinois.

• Sequence analysis found 12 candidate SNPs but only 3 exonic. The suspect

allele for a SNP in gene APAF1 produces a stopgain mutation in its

homozygous recessive state.

• APAF1 knockout also causes embryonic loss in mice.

• 758 animals, including 486 HH1 carriers, were selected for APAF1 validation

genotyping.

• Concordance was 100% between HH1 carrier status and APAF1 mutation

carrier status.

Sequencing and Discovery of JH1

Source

Combined

With

Source

Suspect

Area

Carrier

Possible

Figure 2. Fine Mapping of JH1.

• 5 haplotypes affecting fertility have been reported to breeders since August

2011.

• Suspect regions narrowed using crossovers.

• Sequence data revealed the causative mutations for 2 of the 5, located in

APAF1 for HH1 and CWC15 for JH1.

• Imputation can combine information from both SNPs and QTLs to determine

status.

• Mating of heterozygotes should be avoided.

Tracking Known Recessives

• Located the source haplotype for 3 BS and 3 HO known recessives

(Table 2).

Recessive Brd BTATested

animalsConcord-ance (%)

New carriers

BLAD HO 1 11,782 99.9 314DUMPS HO 1 3,242 100.0 3Mulefoot HO 15 87 97.7 120Weaver BS 4 163 96.3 32SMA BS 24 568 98.1 111SDM BS 11 108 94.4 108

Table 2. Using SNP haplotypes to track known recessives.

Imputing Causative Mutations

• Carriers can be determined by haplotypes, gene tests, or combining and

imputing.

• For JH1 730 causative mutation test results were used to impute test

results for 6,784 50K animals.

• Causative mutation test results were also imputed for the HO known

recessives CVM, BLAD and DUMPS; 84,713, 83,694 and 92,234 tests were

imputed respectively.

• New carrier animals were discovered through imputation; 2,176 for CVM ,

332 for BLAD and 3 for DUMPS.

• Large numbers of genotyped animals and affordable DNA sequencing now allow

rapid discovery of new recessive defects.

• Breeders can use haplotype tests or gene tests in selection and mating programs.

• JH1 fine mapped to 0.8 Mb suspect region.

• Founder bull (Soldier) and 10 other carriers sequenced at 30X coverage by

BFGL with funding from American Jersey Cattle Association.

• 15 candidate SNPs found but only 1 exonic.

• Causative mutation (stopgain) was found in CWC15 gene.

• 758 animals, including 486 JH1 carriers, were selected for CWC15 validation

genotyping.

• Concordance was 99.3% between JH1 carrier status and CWC15 mutation

carrier status.

HH2, HH3, BH1 Sequencing

• These methods were automated to update the suspect region and report

haplotypes monthly.

• Breeders receive official haplotype status for all genotyped animals (3K are

unofficial).

• Sequenced exome (expressed DNA) for 8 carriers of each defect.

• Causative mutations are not obvious yet.

• BH1 includes 2 consecutive SNPs that never become homozygous. Some

genomic evaluations may exclude these because of Hardy-Weinberg edits, but

could indicate location of defect.

Acknowledgments

• Sequencing and analysis of HH1 conducted by H. Lewin, D. Larkin, A.

Beavers, M. McClure, and Geneseek.

• Fertility analyses conducted by J. Hutchison.