Faulty expression of the APOB gene

When looking for classic mutations, i.e. the modification of one or more nucleotides in the APOB gene sequence, Carole Charlier and her team didn’t see anything unusual. ‘However at the exon 5 of this gene, there was one particular sign: the insertion of a repeated sequence’, she reveals. ‘We characterised this sequence and found that this repeated element was part of a family of mobile elements known as ‘endogenous retroviral elements’ or ‘endogenous retroviruses’.’ Repeated sequences represent 50% of the mammalian genome. There are different categories and sub-categories which include transposable elements. ‘The repeated element found at the APOB gene was part of this category’, reveals Charlier. ‘This is the insertion of a complete element of 7,000 base pairs. This insertion should lead to a total stop of the transcription of the gene and should abolish its function’.

Thanks to collaborative work with the Faculty of Veterinary Medicine at the ULg (Drs Arnaud Sartelet and Emilie Knapp), Carole Charlier’s team was able to obtain biological material (DNA and tissue) from a calf suffering from CDH. ‘We sequenced its transcriptome based on its liver cells to check the impact of the mutation on the expression of the APOB gene’, explains Charlier. This is how they were able to confirm that this calf’s APOB gene had given rise to an ‘abnormal’ protein. Transcription of the gene was interrupted when only 3% of the protein had been formed.

This discovery enabled the Liège researchers to develop a test which directly investigated the mutation, in order to be able to validate or invalidate the results obtained from the existing, indirect, haplotype test. This test should now enable farmers to avoid using animals carrying the mutation for reproduction. ‘This mutation is fairly common within the Holstein population, affecting between 6 and 8% of animals. But, because of artificial insemination, if animals carrying the mutation are used for reproduction, the number of animals carrying the mutation and, thus, the number of calves who are affected can be multiplied considerably’, stresses Charlier.

Focus on the transposable elements in the cattle genome

Having achieved their goal of creating a 100% reliable test to detect cows and bulls who carry the mutation responsible for CDH, Carole Charlier and her team did not stop there. Normally, endogenous retroviruses are strongly suppressed by the organism’s defence mechanisms. If they are not suppressed in this way, they may transpose within the genome, be transcribed, or go on to integrate elsewhere in the genome and cause various consequences for the individuals carrying them. Hence, the genome’s defence system is usually highly effective in terms of working against these mutations. The ULg researchers wanted to understand the genomic distribution of these elements, their frequency, and their specificity in Holstein and Belgian Blue cattle.

Against this backdrop, Chad Harland, a doctoral student from New Zealand working in the Animal Genomics Research Unit, had developed a bioinformatic tool (LocaTER) to detect elements of the same type as that found in the APOB gene, within the genome. The results revealed 1,200 polymorphic events of this kind. ‘Some were specific to Belgian Blues, while others were specific to Holstein cattle. Finally, some were shared by the two breeds, which would seem to indicate that they therefore appeared in the cattle genome longer ago’, specifies Charlier. The scientists showed that, as had already been demonstrated particularly in mice, that the transposition elements of the cattle genome were subject to significant purifying selection pressure. ‘When such an element is mobilised, it will integrate randomly anywhere in the genome: in intergenic regions, in the introns or exons. The direction of its insertion has an impact upon the consequences of this insertion on the role of the affected gene’, explains Charlier. Indeed, if the element in question inserts itself in the direction of the transcription of the gene, it will disturb and/or stop the transcription of this gene. If the insertion takes place in the reverse direction of the transcription, its impact on the role of the gene will be more variable. ‘By looking both at the distribution and position of these 1,200 elements in the genome of Holstein and Belgian Blue cattle, we were able to show that there are fewer insertions at gene level than we would expect if we start from the principle that these insertions in the genome are random. Moreover, when these elements are inserted into a gene, it is twice as likely that they will be inserted in the opposite to the direction of transcription,’ explains Charlier. These observations confirm that a purifying selection procedure is at work: the elements inserted into the genes in the direction of the transcription were eliminated over time, due to their negative impact on the individuals carrying these mutations.