Identifying cholesterol deficiency in Holstein cattle

Cholesterol deficiency is a genetic defect which causes the early death of young Holstein cattle. To prevent more animals being affected by this disease, there was a need to detect which animals carried the genetic mutation responsible for it, in order to avoid using them for reproduction. Carole Charlier, project head at the Animal Genomics Research Unit at the ULg GIGA, and her team, identified this mutation and have developed a test to detect animals carrying it. The culprit is the APOB gene, which gives rise to an ‘abnormal’ protein.

Carole Charlier’s team has been working non-stop since the start of the academic year. In September, the Liègois team was approached by Holstein cattle breeders wanting to develop a test to detect the mutation responsible for a lethal disease in Holstein calves. Since then, the Liège team has achieved this objective and much more. ‘CDH, which stands for ‘cholesterol deficiency haplotype’, is a disease which leads to calves dying between three weeks and six months after birth’, explains Carole Charlier, project leader at the Animal Genomics Research Unit at the ULg GIGA. ‘Affected animals present severe diarrhoea, severe weight loss and almost no fat. Blood analyses shows an hypolipidaemic effect, particularly very low levels of cholesterol. There is currently no treatment for the illness’, she specifies. Given that the Holstein breed is the most widespread dairy breed in the world, you can imagine farmers’ distress faced with an illness such as this, which affects livestock production. This is particularly the case because CDH is not the only illness to affect the reproduction of Holstein cows (read the article Mutations that affect bovine fertility).

A unique database in Europe

To prevent Holstein cows giving birth to diseased calves, the breeders needed to find the mutation responsible for this illness in order to avoid using animals who carry that mutation for reproduction. ‘It was a German team who described the illness and the same team presented the location of the mutation responsible for this illness, at a seminar in July’, continues Carole Charlier. ‘They had localised this mutation at chromosome 11 but had not yet identified it. Based on this, they had developed an indirect haplotype test, in other words, a test which made it possible to identify the part of the chromosome which should carry the mutation. The test is not 100% reliable but it is useful to identify animals who may potentially carry the mutation’. Once they had this information, Carole Charlier’s team sought to identify the mutation responsible for CDH. To do so, they had one key tool: a database, generated be Wouter Coppieters and his team within GIGA’s genomic platform, covering the sequencing of 750 complete genomes from Holstein cows. ‘This database had been developed over the last two years as part of the DAMONA project led by Professor Michel Georges and funded by an ERC Advanced Grant’, clarifies Charlier. Based on a list of reproductive bulls likely to carry the mutation responsible for CDH, the researchers were able to identify four bulls who formed part of the DAMONA database. They then analysed the part of chromosome 11 where the German team had localised the mutation in question. ‘We didn’t find anything at that location’, says Charlier. ‘But one million base pairs from this location is the apolipoprotein B gene (APOB). In humans, we know that ‘loss of function’ type mutations of this gene provoke similar symptoms to those observed in calves with CDH’, explains Charlier. Could there be a mutation in the APOB gene which could explain the appearance of the cholesterol deficiency in Holstein cattle? This is what the researchers aimed to find out.

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.

Analysing the genome of small family structures

Initially, the DAMONA database was designed to identify de novo mutations. If a de novo mutation takes place within cells in the germline - the cells which are susceptible to forming the gametes (ovules or sperm cells) - of a given individual (father or mother) they will not be detectable in them but will be transmitted to the descendant conceived by the mutated gamete, who will then transmit it to half of their own descendants. During his doctoral studies, Chad Harland aimed to identify the classic de novo mutations for the entire DAMONA database. Classic mutations are those which involve the modification of one or a few nucleoties within a given sequence. Of the 750 individuals listed in the DAMONA database, there were 115 small family structures consisting of a ‘father-mother’ couple, one of their descendants and four or five individuals descended from this descendant. Charlier and her colleagues used this range of data to find ‘new retrotransposition’ type de novo mutations within these family structures. ‘We wanted to see the mobilisation of de novo elements similar to those found in the APOB gene within the germline’, she explains. ‘We were able to highlight five such de novo mutations among the 115 family structures available to us’. These mutations were therefore produced within a sperm cell or ovocyte of a father or a mother. Of these five mutations, four came from a male germline and one from a female germline. ‘This clearly shows that the this type of event can be triggered both in males and in females’  indicates Carole Charlier. ‘But we also realised, unexpectedly, that of the four de novo mutations which appeared in the males, three of them were produced in the germine of the same bull. Even more surprisingly, two of these three mutations had been transmitted by the very same sperm cell’ reveals the researcher. These significant results suggest that, at a specific point in time, an endogenous retroviral element was mobilised in the germline of this bull. The next stage for Carole Charlier’s team will be to find out whether there is, for this bull in particular (or others presenting the same type of events) an alteration of the genome defence mechanisms which are supposed to suppress the mobilisation of transposable elements.

In the context of another European project, the sequencing of the transcriptome of Holstein cattle is also underway. Carole Charlier and her colleagues already have the intention of using this new range of data to try to establish a correlation between the presence of transposable elements from one location on the genome of these animals and the level of gene transcription in or near to which the elements are inserted. ‘Some elements that we have found in the genome of Holstein or Belgian Blue cattle will very probably have a significant impact, given the significant genes in which they are inserted’, she says. At the very least, that the farmers’ call for a CDH detection test will have a domino effect on the Carole Charlier’s research and that of her team, which shows no signs of slowing down!