The Holstein Friesian Lethal Haplotype 5 (HH5) Results from a Complete Deletion of TBF1M and Cholesterol Deficiency (CDH) from an ERV-(LTR) Insertion into the Coding Region of APOB

BACKGROUND: With the availability of massive SNP data for several economically important cattle breeds, haplotype tests have been performed to identify unknown recessive disorders. A number of so-called lethal haplotypes, have been uncovered in Holstein Friesian cattle and, for at least seven of these, the causative mutations have been identified in candidate genes. However, several lethal haplotypes still remain elusive. Here we report the molecular genetic causes of lethal haplotype 5 (HH5) and cholesterol deficiency (CDH). A targeted enrichment for the known genomic regions, followed by massive parallel sequencing was used to interrogate for causative mutations in a case/control approach. METHODS: Targeted enrichment for the known genomic regions, followed by massive parallel sequencing was used in a case/control approach. PCRs for the causing mutations were developed and compared to routine imputing in 2,100 (HH5) and 3,100 (CDH) cattle. RESULTS: HH5 is caused by a deletion of 138kbp, spanning position 93,233kb to 93,371kb on chromosome 9 (BTA9), harboring only dimethyl-adenosine transferase 1 (TFB1M). The deletion breakpoints are flanked by bovine long interspersed nuclear elements Bov-B (upstream) and L1ME3 (downstream), suggesting a homologous recombination/deletion event. TFB1M di-methylates adenine residues in the hairpin loop at the 3’-end of mitochondrial 12S rRNA, being essential for synthesis and function of the small ribosomal subunit of mitochondria. Homozygous TFB1M(-/-) mice reportedly exhibit embryonal lethality with developmental defects. A 2.8% allelic frequency was determined for the German HF population. CDH results from a 1.3kbp insertion of an endogenous retrovirus (ERV2-1-LTR_BT) into exon 5 of the APOB gene at BTA11:77,959kb. The insertion is flanked by 6bp target site duplications as described for insertions mediated by retroviral integrases. A premature stop codon in the open reading frame of APOB is generated, resulting in a truncation of the protein to a length of only <140 amino acids. Such early truncations have been shown to cause an inability of chylomicron excretion from intestinal cells, resulting in malabsorption of cholesterol. The allelic frequency of this mutation in the German HF population was 6.7%, which is substantially higher than reported so far. Compared to PCR assays inferring the genetic variants directly, the routine imputing used so far showed a diagnostic sensitivity of as low as 91% (HH5) and 88% (CDH), with a high specificity for both (≥99.7%). CONCLUSION: With the availability of direct genetic tests it will now be possible to more effectively reduce the carrier frequency and ultimately eliminate the disorders from the HF populations. Beside this, the fact that repetitive genomic elements (RE) are involved in both diseases, underline the evolutionary importance of RE, which can be detrimental as here, but also advantageous over generations.