Forward vs. reverse genetics: a bovine perspective based on visible and hidden phenotypes of inherited disorders

In modern cattle production, we have seen a negative trend for decades in reproduction while productivity and performance have improved. Although considered genetically complex, part of these fecundity, fertility, and rearing success issues are caused by Mendelian monogenic disorders. Traditionally, such disorders are investigated opportunistically based on their sporadic occurrence and through subsequent targeted analysis of affected individuals. This approach is called the forward genetic approach (FGA). Modern genomic technologies, such as single nucleotide polymorphism (SNP) array genotyping and whole-genome sequencing (WGS), allow for straightforward locus mapping and the identification of candidate causal variants in affected individuals or families. Nevertheless, a major drawback is the arbitrary sampling and availability of well- phenotyped individuals for research, especially for mostly invisible defects affecting fecundity, early embryonic death, and abortions. Therefore, the reverse genetic approach (RGA) is applied to screen for underlying recessive lethal or sub-lethal variants. This approach requires the availability of massive population-wide genomic data. By applying a haplotype screen for a significant deviation of the Hardy-Weinberg equilibrium, genomic regions potentially harboring candidate causal variants are identified. The subsequent generation of WGS data of haplotype carriers allows for the mining for pathogenic variants potentially causing a reduction in homozygosity.

In the first part of my thesis, I present 18 successful, 1 inconclusive example, and 1 example addressing co-dominant effects of a known disorder. These FGA analyzes include heritable skin (n=7), bone (n=7), neuromuscular (n=1), eye (n=2), as well as syndromic disorders (n=3) in various European cattle breeds. Missense and frameshift variants in the IL17RA, DSP, and FA2H genes were described in three recessive genodermatoses: immunodeficiency with psoriasis-like skin alterations, syndromic ichthyosis, and ichthyosis congenita, respectively. Hypohidrotic ectodermal dysplasia was described as X-linked disorder that is associated with a gross deletion in the EDA gene. In dominant genodermatoses, a missense variant in COL5A2 was shown to lead to classical Ehlers-Danlos syndrome, an in-frame deletion in KRT5 was shown to cause epidermolysis bullosa simplex, and results of a study using an individual case of juvenile angiomatosis remained inconclusive. A recessive disorder described as hemifacial macrosomia was associated with a missense variant in LAMB1. Chondrodysplasia in a single family was shown to be caused by a de novo mutation in the bull leading to a stop- loss of the gene FGFR3. De novo mutations (missense and large deletions) in the COL2A1 and COL1A1 genes were associated with achondrogenesis type II (bulldog calf syndrome), and osteogenesis imperfecta type II, respectively. Another mutation that we found to affect bone morphology was a trisomy in chromosome 29 leading to proportional dwarfism with facial dysplasia. Congenital neuromuscular channelopathy was for the first time associated with a missense variant in KCNG1. Furthermore, a de novo missense variant in ADAMTSL4 and a recessive missense variant in CNGB3 were shown to cause congenital cataract and achromatopsia, respectively. Additionally, cases of pulmonary hypoplasia and anasarca syndrome were analyzed and shown to be caused by trisomy 20 in two unrelated calves and a recessively inherited missense variant in ADAMTS3. Moreover, the fatal syndromic disorder skeletal-cardo-enteric dysplasia was described to be caused by a de novo missense variant in MAP2K2. Finally, I investigated the effects on blood cholesterol and triglyceride levels of heterozygous carriers of the previously described APOB-related cholesterol deficiency.

In the second part of my thesis, I present the outcome of the RGA in four main Swiss populations, that was validated with the SWISScow custom array. In the Brown Swiss dairy population, 72 haplotype regions showed significant depletions in homozygosity. Four of these haplotypes (BH6, BH14, BH24, and BH34) were associated with missense and nonsense variants in different genes (MARS2, MRPL55, CPT1C, and ACSL5, respectively). In the Original Braunvieh population, eight haplotype regions were identified. Candidate causal variants included a missense variant in TUBGCP5 gene associated with haplotype OH2, and a splice site frameshift variant in LIG3 gene associated with haplotype OH4. In the Holstein population, 24 haplotype regions were identified with a significant reduction of homozygosity. Subsequently, four novel candidate variants were proposed: a nonsense variant in KIR2DS1 for haplotype HH13, in-frame deletion in the genes NOTCH3 for HH21 haplotype, and RIOX1 for HH25 haplotype, and finally, a missense variant in PCDH15 for HH35 haplotype. In the Simmental population, eleven haplotype regions were detected. The haplotype SH5 was associated with a frameshift variant in DIS3 gene and the haplotypes SH8 and SH9 with missense variants in the CYP2B6 and NUBPL genes, respectively. For the breeds Brown Swiss, Original Braunvieh, and Holstein, association studies were carried out including traits describing fertility, birth, growth, and survival. Thereby most of the described mentioned haplotypes show additive effects.

Regardless of the approach, all the described candidate causal variants can be used as a tool of precision diagnostics and represent a step forward towards personalized medicine in cattle. Furthermore, these variants can be easily genotyped and allow for targeted breeding to reduce the number of risk matings, which would lead to a reduction of affected animals and significant improvement in animal health and welfare.