A reverse genetic approach identifies an ancestral frameshift mutation in RP1 causing recessive progressive retinal degeneration in European cattle breeds

Background[br/] Domestication and artificial selection have resulted in strong genetic drift, relaxation of purifying selection and accumulation of deleterious mutations. As a consequence, bovine breeds experience regular outbreaks of recessive genetic defects which might represent only the tip of the iceberg since their detection depends on the observation of affected animals with distinctive symptoms. Thus, recessive mutations resulting in embryonic mortality or in non-specific symptoms are likely to be missed. The increasing availability of whole-genome sequences has opened new research avenues such as reverse genetics for their investigation. Our aim was to characterize the genetic load of 15 European breeds using data from the 1000 bull genomes consortium and prove that widespread harmful mutations remain to be detected.[br/] Results[br/] We listed 2489 putative deleterious variants (in 1923 genes) segregating at a minimal frequency of 5 % in at least one of the breeds studied. Gene enrichment analysis showed major enrichment for genes related to nervous, visual and auditory systems, and moderate enrichment for genes related to cardiovascular and musculoskeletal systems. For verification purposes, we investigated the phenotypic consequences of a frameshift variant in the retinitis pigmentosa-1 gene segregating in several breeds and at a high frequency (27 %) in Normande cattle. As described in certain human patients, clinical and histological examination revealed that this mutation causes progressive degeneration of photoreceptors leading to complete blindness in homozygotes. We established that the deleterious allele was even more frequent in the Normande breed before 1975 (>40 %) and has been progressively counter-selected likely because of its associated negative effect on udder morphology. Finally, using identity-by-descent analysis we demonstrated that this mutation resulted from a unique ancestral event that dates back to ~2800 to 4000 years.[br/] Conclusions[br/] We provide a list of mutations that likely represent a substantial part of the genetic load of domestication in European cattle. We demonstrate that they accumulated non-randomly and that genes related to cognition and sensory functions are particularly affected. Finally, we describe an ancestral deleterious variant segregating in different breeds causing progressive retinal degeneration and irreversible blindness in adult animals

MOESM3 of A reverse genetic approach identifies an ancestral frameshift mutation in RP1 causing recessive progressive retinal degeneration in European cattle breeds

Additional file 3: Tables S3, S4, S5, S6 and S7. Additional information on the results of the gene enrichment analysis performed with Ingenuity Pathway Analysis.  This file contains five tables providing additional details on the results of the gene enrichment analysis: a summary of the significant diseases and disorders annotations (Table S3), a summary of significant physiological system development and function annotations (Table S4), a summary of significant molecular and cellular functions annotations (Table S5), the key-word attribution to each significant functional annotation conserved for the analysis (Table S6) and the significant canonical pathways (Table S7)

Real-time PCR expression analyzes of the deleted genes in different affected tissues at 90 dpc.

<p><i>ZEB2</i>, <i>GTDC1</i>, <i>ARHGAP15</i> (exon13), and <i>KYNU</i> (absent from the deleted fragment) expression is measured in various tissues from wild type (+/+; black histograms) or mutant (+/−; white histograms) fetuses.</p

Histological analyses of wild-type (+/+) and PMS (+/−) horn bud and forehead skin.

<p>(A) and (B) Head of PMS (+/−) and wt (+/+) fetuses. Horn bud of wt fetus is indicated by an arrow. (C) and (D) Histological sections of the “horn bud” of PMS and wt fetuses respectively. (E) Magnification (X10) of (C) showing one hair follicle primordium. (F) and (G) Magnifications (X10 and X3 respectively) of (D) showing respectively keratinizing epidermal cells and clusters of dermal cells displaying glandular/ductal differentiation. (H) and (I) Histological sections of the forehead skin of PMS and wt fetuses respectively. (J) and (K) Magnifications (X10) of (H) and (I) showing hair follicles primordial; note the slight difference between PMS and wt genotypes; statistical analysis also showed a significant difference in epidermis thickness: 15.8±3.0 µm in PMS vs 22.1±3.7 µm in wt; p-value = 2.3e-28 (Welch’s t-test). Scale bars in (C), (D), (H) and (I) represent 1 mm whereas scale bars in (E), (F), (G), (J) and (K) represent 100 µm.</p

Clinical features of Polled and Multisystemic Syndrome.

<p>(A) Two-and-half-year old wild-type heifer that was mechanically dehorned when approximately one-year old. (B) Two-and-half-year old affected heifer. Note the slender build, the shaggy hair coat demonstrating the bad health condition, and the hypotonia of hind limbs. (C) Upper part of the skull of the same affected heifer. Note the absence of corneous growth, the ridge-shaped extra bone deposition along the frontal suture and the narrowness of the muzzle insertion. (D) On-farm performance testing statistics of affected (PMS) and wild-type half-sibs. Values expressed as: means ± standard deviation (number of observations). *p<0.05, **p<0.01 and ***p<0.001 versus wild-type half-sisters (Welch’s t-test). Weaning corresponds to 210 days of age. (E and F) Ovaries of the affected (+/−) heifer displayed in (B) and (C). (I) Ovary of a wild-type (+/+) matched control. (G and H, and J and K) Histological analyses of the ovaries displayed in (E) and (I) respectively. (H) and (K) are higher magnifications (X5.5) of (G) and (J). Note the numerous large lacunae surrounded by connective tissue and the absence of follicles in the ovary from the affected heifer. Follicles are surrounded with a green dotted line in the photography of the wild-type ovary. Scale bars represent 1 cm in (F), (E) and (I); 500 µm in (G) and (J); and 50 µm in (H) and (K).</p

Mapping and characterization of the causative mutation for PMS syndrome.

<p>(A) and (B) Results of Mendelian error mapping using the Illumina 50 K and 777 K SNP beadchips, respectively. Markers displaying Mendelian errors between at least one PMS heifer and her sire are represented in purple whereas markers for which at least one of the three PMS animals is heterozygous are represented in blue. Other markers are not represented. (C) Plot of the whole-genome sequencing read depth coverage on the same region. *: artifact due to a local error in genome assembly. (D) Gene content of the region. (E) FISH-mapping with BAC clones located in the deleted region (labeled in red) and in the juxtacentromeric region of BTA2 (labeled in green) on fibroblasts of a PMS animal. (F) Magnification of (E) showing normal (above) and deleted (below) BTA2 chromosomes. (G) Genotyping of PMS using a three-primer PCR system (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049084#s3" target="_blank">methods</a>). Neg.: negative control.</p