A naturally occurring canine model of syndromic congenital microphthalmia.

In humans, the prevalence of congenital microphthalmia is estimated to be 0.2-3.0 for every 10,000 individuals, with nonocular involvement reported in ∼80% of cases. Inherited eye diseases have been widely and descriptively characterized in dogs, and canine models of ocular diseases have played an essential role in unraveling the pathophysiology and development of new therapies. A naturally occurring canine model of a syndromic disorder characterized by microphthalmia was discovered in the Portuguese water dog. As nonocular findings included tooth enamel malformations, stunted growth, anemia, and thrombocytopenia, we hence termed this disorder Canine Congenital Microphthalmos with Hematopoietic Defects. Genome-wide association study and homozygosity mapping detected a 2 Mb candidate region on canine chromosome 4. Whole-genome sequencing and mapping against the Canfam4 reference revealed a Short interspersed element insertion in exon 2 of the DNAJC1 gene (g.74,274,883ins[T70]TGCTGCTTGGATT). Subsequent real-time PCR-based mass genotyping of a larger Portuguese water dog population found that the homozygous mutant genotype was perfectly associated with the Canine Congenital Microphthalmos with Hematopoietic Defects phenotype. Biallelic variants in DNAJC21 are mostly found to be associated with bone marrow failure syndrome type 3, with a phenotype that has a certain degree of overlap with Fanconi anemia, dyskeratosis congenita, Shwachman-Diamond syndrome, Diamond-Blackfan anemia, and reports of individuals showing thrombocytopenia, microdontia, and microphthalmia. We, therefore, propose Canine Congenital Microphthalmos with Hematopoietic Defects as a naturally occurring model for DNAJC21-associated syndromes.

Frameshift Variant in AMPD2 in Cirneco dell'Etna Dogs with Retinopathy and Tremors.

While the manifestations of many inherited retinal disorders are limited to loss of vision, others are part of a syndrome that affects multiple tissues, particularly the nervous system. Most syndromic retinal disorders are thought to be recessively inherited. Two dogs out of a litter of Cirneco dell' Etna dogs, both males, showed signs of retinal degeneration, along with tremors and signs described as either atypical seizures or paroxysmal dyskinesias, while the other two male littermates were normal. We named this oculo-neurological syndrome CONS (Cirneco oculo-neurological syndrome), and undertook homozygosity mapping and whole-genome sequencing to determine its potential genetic etiology. Notably, we detected a 1-bp deletion in chromosome 6 that was predicted to cause a frameshift and premature stop codon within the canine AMPD2 gene, which encodes adenosine monophosphate deaminase, an enzyme that converts adenosine 5'-monophosphate (AMP) to inosine 5'-monophosphate (IMP). Genotyping of the available Cirneco population suggested perfect segregation between cases and controls for the variant. Moreover, this variant was absent in canine genomic databases comprised of thousands of unaffected dogs. The AMPD2 genetic variant we identified in dogs presents with retinal manifestations, adding to the spectrum of neurological manifestations associated with AMPD2 variants in humans.

Natural disease history of a canine model of oligogenic RPGRIP1-cone-rod dystrophy establishes variable effects of previously and newly mapped modifier loci.

Canine RPGRIP1-cone-rod dystrophy (CRD), a model for human inherited retinal diseases (IRDs), was originally identified as autosomal recessive early-onset blindness. However, later studies revealed extensive phenotypic variability among RPGRIP1 mutants. This led to the identification of a homozygous MAP9 variant as a modifier associated with early-onset disease. Based on further phenotypic variation affecting cone photoreceptor function, we report mapping of L3 as an additional modifier locus, within a 4.1-Mb locus on canine chromosome 30. We establish the natural disease history of RPGRIP1-CRD based on up to 9-year long-term functional and structural retinal data from 58 dogs including 44 RPGRIP1 mutants grouped according to the modifier status. RPGRIP1 mutants affected by both MAP9 and L3 modifiers exhibited the most severe phenotypes with rapid disease progression. MAP9 alone was found to act as an overall accelerator of rod and cone diseases, while L3 had a cone-specific effect. Ultrastructural analysis of photoreceptors revealed varying degrees of rod and cone damage, while the connecting cilia appeared structurally preserved in all groups. We conclude that RPGRIP1-CRD is an oligogenic disease with at least three loci contributing to the pathogenesis. While the RPGRIP1 variant is required for developing the disease, MAP9 and L3 modifiers exacerbate the phenotype, individually and cumulatively. Oligogenic canine RPGRIP1-CRD illustrates the impact of multiple genetic modifiers on disease phenotype and thus has the potential to reveal new targets for broad-spectrum therapies for oligogenic or polygenic forms of human IRDs.

Differential Analysis of Gly211Val and Gly286Val Mutations Affecting Sarco(endo)plasmic Reticulum Ca2+-ATPase (SERCA1) in Congenital Pseudomyotonia Romagnola Cattle.

Congenital pseudomyotonia in cattle (PMT) is a rare skeletal muscle disorder, clinically characterized by stiffness and by delayed muscle relaxation after exercise. Muscle relaxation impairment is due to defective content of the Sarco(endo)plasmic Reticulum Ca2+ ATPase isoform 1 (SERCA1) protein, caused by missense mutations in the ATP2A1 gene. PMT represents the only mammalian model of human Brody myopathy. In the Romagnola breed, two missense variants occurring in the same allele were described, leading to Gly211Val and Gly286Val (G211V/G286V) substitutions. In this study, we analyzed the consequences of G211V and G286V mutations. Results support that the reduced amount of SERCA1 is a consequence of the G211V mutation, the G286V mutation almost being benign and the ubiquitin-proteasome system (UPS) being involved. After blocking the proteasome using a proteasome inhibitor, we found that the G211V mutant accumulates in cells at levels comparable to those of WT SERCA1. Our conclusion is that G211/286V mutations presumably originate in a folding-defective SERCA1 protein, recognized and diverted to degradation by UPS, although still catalytically functional, and that the main role is played by G211V mutation. Rescue of mutated SERCA1 to the sarcoplasmic reticulum membrane can re-establish resting cytosolic Ca2+ concentration and prevent the appearance of pathological signs, paving the way for a possible therapeutic approach against Brody disease.

Short prolactin isoforms are expressed in photoreceptors of canine retinas undergoing retinal degeneration.

Prolactin (PRL) hormone functions as a pleiotropic cytokine with a protective role in the retina. We recently identified by transcriptome profiling that PRL is one of the most highly upregulated mRNAs in the retinas of mutant rcd1 (PDE6B) and xlpra2 (RPGR) dogs at advanced stages of photoreceptor disease. In the present study, we have identified the expression of a short PRL isoform that lacks exon 1 in canine retinas and analyzed the time-course of expression and localization of this isoform in the retinas of these two models. Using laser capture microdissection to isolate RNA from each of the retinal cellular layers, we found by qPCR that this short PRL isoform is expressed in photoreceptors of degenerating retinas. We confirmed by in situ hybridization that its expression is localized to the outer nuclear layer and begins shortly after the onset of disease at the time of peak photoreceptor cell death in both models. PRL protein was also detected only in mutant dog retinas. Our results call for further investigations into the role of this novel PRL isoform in retinal degeneration.

CCDC66 frameshift variant associated with a new form of early-onset progressive retinal atrophy in Portuguese Water Dogs.

Aberrant photoreceptor function or morphogenesis leads to blinding retinal degenerative diseases, the majority of which have a genetic aetiology. A variant in PRCD previously identified in Portuguese Water Dogs (PWDs) underlies prcd (progressive rod-cone degeneration), an autosomal recessive progressive retinal atrophy (PRA) with a late onset at 3-6 years of age or older. Herein, we have identified a new form of early-onset PRA (EOPRA) in the same breed. Pedigree analysis suggested an autosomal recessive inheritance. Four PWD full-siblings affected with EOPRA diagnosed at 2-3 years of age were genotyped (173,661 SNPs) along with 2 unaffected siblings, 2 unaffected parents, and 15 unrelated control PWDs. GWAS, linkage analysis and homozygosity mapping defined a 26-Mb candidate region in canine chromosome 20. Whole-genome sequencing in one affected dog and its obligatory carrier parents identified a 1 bp insertion (CFA20:g.33,717,704_33,717,705insT (CanFam3.1); c.2262_c.2263insA) in CCDC66 predicted to cause a frameshift and truncation (p.Val747SerfsTer8). Screening of an extended PWD population confirmed perfect co-segregation of this genetic variant with the disease. Western blot analysis of COS-1 cells transfected with recombinant mutant CCDC66 expression constructs showed the mutant transcript translated into a truncated protein. Furthermore, in vitro studies suggest that the mutant CCDC66 is mislocalized to the nucleus relative to wild type CCDC66. CCDC66 variants have been associated with inherited retinal degenerations (RDs) including canine and murine ciliopathies. As genetic variants affecting the primary cilium can cause ciliopathies in which RD may be either the sole clinical manifestation or part of a syndrome, our findings further support a role for CCDC66 in retinal function and viability, potentially through its ciliary function.

Candidate Genetic Modifiers for RPGR Retinal Degeneration.

Purpose: To define genetic variants associated with variable severity of X-linked progressive retinal atrophy 1 (XLPRA1) caused by a five-nucleotide deletion in canine RPGR exon ORF15. Methods: A genome-wide association study (GWAS) was performed in XLPRA1 phenotype informative pedigree. Whole genome sequencing (WGS) was used for mutational analysis of genes within the candidate genomic region. Retinas of normal and mutant dogs were used for gene expression, gene structure, and RNA duplex analyses. Results: GWAS followed by haplotype phasing identified an approximately 4.6 Mb candidate genomic interval on CFA31 containing seven protein-coding genes expressed in retina (ROBO1, ROBO2, RBM11, NRIP1, HSPA13, SAMSN1, and USP25). Furthermore, we identified and characterized two novel lncRNAs, ROBO1-AS and ROBO2-AS, that display overlapping gene organization with axon guidance pathway genes ROBO1 and ROBO2, respectively, producing sense-antisense gene pairs. Notably, ROBO1-AS and ROBO2-AS act in cis to form lncRNA/mRNA duplexes with ROBO1 and ROBO2, respectively, suggesting important roles for these lncRNAs in the ROBO regulatory network. A subsequent WGS identified candidate genes within the genomic region on CFA31 that might be implicated in modifying severity of XLPRA1. This approach led to discovery of genetic variants in ROBO1, ROBO1-AS, ROBO2-AS, and USP25 that are strongly associated with the XLPRA1 moderate phenotype. Conclusions: The study provides new insights into the genetic basis of phenotypic variation in severity of RPGRorf15-associated retinal degeneration. Our findings suggest an important role for ROBO pathways in disease progression further expanding on our previously reported changes of ROBO1 expression in XLPRA1 retinas.

X-Linked Duchenne-Type Muscular Dystrophy in Jack Russell Terrier Associated with a Partial Deletion of the Canine DMD Gene.

A 9-month old male Jack Russell Terrier started showing paraparesis of the hindlimbs after a walk. Hospitalized, the dog went into cardiac arrest, and later died. Necroscopic examination revealed a severe thickness of the diaphragm, esophagus, and base of the tongue, leading to the diagnosis of muscular dystrophy. The histology confirmed the marked size variation, regeneration, and fibrosis replacement of the skeletal muscle fibers. Immunohistochemistry demonstrated the absence of dystrophin confirming the diagnosis. Transmission electron microscopy showed disarrangement of skeletal muscle fibers. Finally, whole-genome sequencing identified a ~368kb deletion spanning 19 exons of the canine dystrophin (DMD) gene. This pathogenic loss-of-function variant most likely explains the observed disease phenotype. The X-chromosomal variant was absent in seven controls of the same breed. Most likely, this partial deletion of the DMD gene was either transmitted on the maternal path within the family of the affected dog or arose de novo. This study revealed a spontaneous partial deletion in DMD gene in a Jack Russell Terrier showing a Duchenne-type muscular dystrophy due to non-functional dystrophin.

KDM2B-associated paunch calf syndrome in Marchigiana cattle.

BACKGROUND: Chianina, Romagnola, and Marchigiana are the 3 most important Italian breeds of cattle raised in the Apennine Mountains. Inherited disorders have been reported in the Chianina and Romagnola breeds but not in the Marchigiana breed. Recently, a case resembling recessively inherited KDM2B-associated paunch calf syndrome (PCS) in Romagnola cattle was identified in Marchigiana cattle. HYPOTHESIS/OBJECTIVES: To characterize the features of the observed congenital anomaly, evaluate its possible genetic etiology, and determine the prevalence of the deleterious allele in the Marchigiana population. ANIMALS: A single stillborn Marchigiana calf was referred for clinicopathological examination because of the presence of PCS-like morphological lesions. METHODS: The animal was necropsied and the calf and its parents were genotyped. A PCR-based direct gene test was applied to determine the KDM2B genotype and 114 Marchigiana bulls were genotyped. RESULTS: The pathological phenotype included facial deformities, enlarged fluid-filled abdomen, and hepatic fibrosis. The affected animal was the offspring of consanguineous mating and homozygous presence of the KDM2B missense variant was confirmed. Both parents were heterozygous for KDM2B and the prevalence of carriers in a selected population of Marchigiana bulls was <2%. CONCLUSIONS AND CLINICAL IMPORTANCE: The characteristic malformations and genetic findings were consistent with the diagnosis of PCS and provide evidence that the deleterious KDM2B variant initially detected in Romagnola cattle also occurs in the Marchigiana breed.

Complex Structural PPT1 Variant Associated with Non-syndromic Canine Retinal Degeneration.

Rod and cone photoreceptors are specialized retinal neurons that have a fundamental role in visual perception, capturing light and transducing it into a neuronal signal. Aberrant functioning of rod and/or cone photoreceptors can ultimately lead to progressive degeneration and eventually blindness. In man, many rod and rod-cone degenerative diseases are classified as forms of retinitis pigmentosa (RP). Dogs also have a comparable disease grouping termed progressive retinal atrophy (PRA). These diseases are generally due to single gene defects and follow Mendelian inheritance.We collected 51 DNA samples from Miniature Schnauzers affected by PRA (average age of diagnosis ∼3.9 ±1 years), as well as from 56 clinically normal controls of the same breed (average age ∼6.6 ±2.8 years). Pedigree analysis suggested monogenic autosomal recessive inheritance of PRA. GWAS and homozygosity mapping defined a critical interval in the first 4,796,806 bp of CFA15. Whole genome sequencing of two affected cases, a carrier and a control identified two candidate variants within the critical interval. One was an intronic SNV in HIVEP3, and the other was a complex structural variant consisting of the duplication of exon 5 of the PPT1 gene along with a conversion and insertion (named PPT1dci ). PPT1dci was confirmed homozygous in a cohort of 22 cases, and 12 more cases were homozygous for the CFA15 haplotype. Additionally, the variant was found homozygous in 6 non-affected dogs of age higher than the average age of onset. The HIVEP3 variant was found heterozygous (n = 4) and homozygous wild-type (n = 1) in cases either homozygous for PPT1dci or for the mapped CFA15 haplotype. We detected the wildtype and three aberrant PPT1 transcripts in isolated white blood cell mRNA extracted from a PRA case homozygous for PPT1dci , and the aberrant transcripts involved inclusion of the duplicated exon 5 and novel exons following the activation of cryptic splice sites. No neurological signs were detected among the dogs homozygous for the PPT1dci variant. Therefore, we propose PPT1dci as causative for a non-syndromic form of PRA (PRA PPT1 ) that shows incomplete penetrance in Miniature Schnauzers, potentially related to the presence of the wild-type transcript. To our knowledge, this is the first case of isolated retinal degeneration associated with a PPT1 variant.

A frameshift mutation in MOCOS is associated with familial renal syndrome (xanthinuria) in Tyrolean Grey cattle.

BACKGROUND: Renal syndromes are occasionally reported in domestic animals. Two identical twin Tyrolean Grey calves exhibited weight loss, skeletal abnormalities and delayed development associated with kidney abnormalities and formation of uroliths. These signs resembled inherited renal tubular dysplasia found in Japanese Black cattle which is associated with mutations in the claudin 16 gene. Despite demonstrating striking phenotypic similarities, no obvious presence of pathogenic variants of this candidate gene were found. Therefore further analysis was required to decipher the genetic etiology of the condition. RESULTS: The family history of the cases suggested the possibility of an autosomal recessive inheritance. Homozygosity mapping combined with sequencing of the whole genome of one case detected two associated non-synonymous private coding variants: A homozygous missense variant in the uncharacterized KIAA2026 gene (g.39038055C > G; c.926C > G), located in a 15 Mb sized region of homozygosity on BTA 8; and a homozygous 1 bp deletion in the molybdenum cofactor sulfurase (MOCOS) gene (g.21222030delC; c.1881delG and c.1782delG), located in an 11 Mb region of homozygosity on BTA 24. Pathogenic variants in MOCOS have previously been associated with inherited metabolic syndromes and xanthinuria in different species including Japanese Black cattle. Genotyping of two additional clinically suspicious cases confirmed the association with the MOCOS variant, as both animals had a homozygous mutant genotype and did not show the variant KIAA2026 allele. The identified genomic deletion is predicted to be highly disruptive, creating a frameshift and premature termination of translation, resulting in severely truncated MOCOS proteins that lack two functionally essential domains. The variant MOCOS allele was absent from cattle of other breeds and approximately 4% carriers were detected among more than 1200 genotyped Tyrolean Grey cattle. Biochemical urolith analysis of one case revealed the presence of approximately 95% xanthine. CONCLUSIONS: The identified MOCOS loss of function variant is highly likely to cause the renal syndrome in the affected animals. The results suggest that the phenotypic features of the renal syndrome were related to an early onset form of xanthinuria, which is highly likely to lead to the progressive defects. The identification of the candidate causative mutation thus enables selection against this pathogenic variant in Tyrolean Grey cattle.

An Intronic MBTPS2 Variant Results in a Splicing Defect in Horses with Brindle Coat Texture.

We investigated a family of horses exhibiting irregular vertical stripes in their hair coat texture along the neck, back, hindquarters, and upper legs. This phenotype is termed "brindle" by horse breeders. We propose the term "brindle 1 (BR1)" for this specific form of brindle. In some BR1 horses, the stripes were also differentially pigmented. Pedigree analyses were suggestive of a monogenic X-chromosomal semidominant mode of inheritance. Haplotype analyses identified a 5 Mb candidate region on chromosome X. Whole genome sequencing of four BR1 and 60 nonbrindle horses identified 61 private variants in the critical interval, none of them located in an exon of an annotated gene. However, one of the private variants was close to an exon/intron boundary in intron 10 of the MBTPS2 gene encoding the membrane bound transcription factor peptidase, site 2 (c.1437+4T>C). Different coding variants in this gene lead to three related genodermatoses in human patients. We therefore analyzed MBTPS2 transcripts in skin, and identified an aberrant transcript in a BR1 horse, which lacked the entire exon 10 and parts of exon 11. The MBTPS2:c1437+4T>C variant showed perfect cosegregation with the brindle phenotype in the investigated family, and was absent from 457 control horses of diverse breeds. Altogether, our genetic data, and previous knowledge on MBTPS2 function in the skin, suggest that the identified MBTPS2 intronic variant leads to partial exon skipping, and causes the BR1 phenotype in horses.

Correction: Hairless Streaks in Cattle Implicate TSR2 in Early Hair Follicle Formation.

[This corrects the article DOI: 10.1371/journal.pgen.1005427.].

Fast-twitch skeletal muscle fiber adaptation to SERCA1 deficiency in a Dutch Improved Red and White calf pseudomyotonia case.

Missense mutations in ATP2A1 gene, encoding SERCA1 protein, cause a muscle disorder designed as congenital pseudomyotonia (PMT) in Chianina and Romagnola cattle or congenital muscular dystonia1 (CMD1) in Belgian Blue cattle. Although PMT is not life-threatening, CMD1 affected calves usually die within a few weeks of age as a result of respiratory complication. We have recently described a muscular disorder in a double muscle Dutch Improved Red and White cross-breed calf. Mutation analysis revealed an ATP2A1 mutation identical to that described in CMD1, even though clinical phenotype was quite similar to that of PMT. Here, we provide evidence for a deficiency of mutated SERCA1 in PMT affected muscles of Dutch Improved Red and White calf, but not of its mRNA. The reduced expression of SERCA1 is selective and not compensated by the SERCA2 isoform. By contrast, pathological muscles are characterized by a broad distribution of mitochondrial markers in all fiber types, not related to intrinsic features of double muscle phenotype and by an increased expression of sarcolemmal calcium extrusion pump. Calcium removal mechanisms, operating in muscle fibers as compensatory response aimed at lowering excessive cytoplasmic calcium concentration caused by SERCA1 deficiency, could explain the difference in severity of clinical signs.

Hairless Streaks in Cattle Implicate TSR2 in Early Hair Follicle Formation.

Four related cows showed hairless streaks on various parts of the body with no correlation to the pigmentation pattern. The stripes occurred in a consistent pattern resembling the lines of Blaschko. The non-syndromic hairlessness phenotype observed occurred across three generations of a single family and was compatible with an X-linked mode of inheritance. Linkage analysis and subsequent whole genome sequencing of one affected female identified two perfectly associated non-synonymous sequence variants in the critical interval on bovine chromosome X. Both variants occurred in complete linkage disequilibrium and were absent in more than 3900 controls. An ERCC6L missense mutation was predicted to cause an amino acid substitution of a non-conserved residue. Analysis in mice showed no specific Ercc6l expression pattern related to hair follicle development and therefore ERCC6L was not considered as causative gene. A point mutation at the 5'-splice junction of exon 5 of the TSR2, 20S rRNA accumulation, homolog (S. cerevisiae), gene led to the production of two mutant transcripts, both of which contain a frameshift and generate a premature stop codon predicted to truncate approximately 25% of the protein. Interestingly, in addition to the presence of both physiological TSR2 transcripts, the two mutant transcripts were predominantly detected in the hairless skin of the affected cows. Immunohistochemistry, using an antibody against the N-terminal part of the bovine protein demonstrated the specific expression of the TSR2 protein in the skin and the hair of the affected and the control cows as well as in bovine fetal skin and hair. The RNA hybridization in situ showed that Tsr2 was expressed in pre- and post-natal phases of hair follicle development in mice. Mammalian TSR2 proteins are highly conserved and are known to be broadly expressed, but their precise in vivo functions are poorly understood. Thus, by dissecting a naturally occurring mutation in a domestic animal species, we identified TSR2 as a regulator of hair follicle development.

Epidermolysis bullosa in Danish Hereford calves is caused by a deletion in LAMC2 gene.

BACKGROUND: Heritable forms of epidermolysis bullosa (EB) constitute a heterogeneous group of skin disorders of genetic aetiology that are characterised by skin and mucous membrane blistering and ulceration in response to even minor trauma. Here we report the occurrence of EB in three Danish Hereford cattle from one herd. RESULTS: Two of the animals were necropsied and showed oral mucosal blistering, skin ulcerations and partly loss of horn on the claws. Lesions were histologically characterized by subepidermal blisters and ulcers. Analysis of the family tree indicated that inbreeding and the transmission of a single recessive mutation from a common ancestor could be causative. We performed whole genome sequencing of one affected calf and searched all coding DNA variants. Thereby, we detected a homozygous 2.4 kb deletion encompassing the first exon of the LAMC2 gene, encoding for laminin gamma 2 protein. This loss of function mutation completely removes the start codon of this gene and is therefore predicted to be completely disruptive. The deletion co-segregates with the EB phenotype in the family and absent in normal cattle of various breeds. Verifying the homozygous private variants present in candidate genes allowed us to quickly identify the causative mutation and contribute to the final diagnosis of junctional EB in Hereford cattle. CONCLUSIONS: Our investigation confirms the known role of laminin gamma 2 in EB aetiology and shows the importance of whole genome sequencing in the analysis of rare diseases in livestock.

Looking the cow in the eye: deletion in the NID1 gene is associated with recessive inherited cataract in Romagnola cattle.

Cataract is a known condition leading to opacification of the eye lens causing partial or total blindness. Mutations are known to cause autosomal dominant or recessive inherited forms of cataracts in humans, mice, rats, guinea pigs and dogs. The use of large-sized animal models instead of those using mice for the study of this condition has been discussed due to the small size of rodent lenses. Four juvenile-onset cases of bilateral incomplete immature nuclear cataract were recently observed in Romagnola cattle. Pedigree analysis suggested a monogenic autosomal recessive inheritance. In addition to the cataract, one of the cases displayed abnormal head movements. Genome-wide association and homozygosity mapping and subsequent whole genome sequencing of a single case identified two perfectly associated sequence variants in a critical interval of 7.2 Mb on cattle chromosome 28: a missense point mutation located in an uncharacterized locus and an 855 bp deletion across the exon 19/intron 19 border of the bovine nidogen 1 (NID1) gene (c.3579_3604+829del). RT-PCR showed that NID1 is expressed in bovine lenses while the transcript of the second locus was absent. The NID1 deletion leads to the skipping of exon 19 during transcription and is therefore predicted to cause a frameshift and premature stop codon (p.1164fs27X). The truncated protein lacks a C-terminal domain essential for binding with matrix assembly complexes. Nidogen 1 deficient mice show neurological abnormalities and highly irregular crystal lens alterations. This study adds NID1 to the list of candidate genes for inherited cataract in humans and is the first report of a naturally occurring mutation leading to non-syndromic catarct in cattle provides a potential large animal model for human cataract.

Deletion in the EVC2 gene causes chondrodysplastic dwarfism in Tyrolean Grey cattle.

During the summer of 2013 seven Italian Tyrolean Grey calves were born with abnormally short limbs. Detailed clinical and pathological examination revealed similarities to chondrodysplastic dwarfism. Pedigree analysis showed a common founder, assuming autosomal monogenic recessive transmission of the defective allele. A positional cloning approach combining genome wide association and homozygosity mapping identified a single 1.6 Mb genomic region on BTA 6 that was associated with the disease. Whole genome re-sequencing of an affected calf revealed a single candidate causal mutation in the Ellis van Creveld syndrome 2 (EVC2) gene. This gene is known to be associated with chondrodysplastic dwarfism in Japanese Brown cattle, and dwarfism, abnormal nails and teeth, and dysostosis in humans with Ellis-van Creveld syndrome. Sanger sequencing confirmed the presence of a 2 bp deletion in exon 19 (c.2993_2994ACdel) that led to a premature stop codon in the coding sequence of bovine EVC2, and was concordant with the recessive pattern of inheritance in affected and carrier animals. This loss of function mutation confirms the important role of EVC2 in bone development. Genetic testing can now be used to eliminate this form of chondrodysplastic dwarfism from Tyrolean Grey cattle.

A nonsense mutation in the IKBKG gene in mares with incontinentia pigmenti.

Ectodermal dysplasias (EDs) are a large and heterogeneous group of hereditary disorders characterized by abnormalities in structures of ectodermal origin. Incontinentia pigmenti (IP) is an ED characterized by skin lesions evolving over time, as well as dental, nail, and ocular abnormalities. Due to X-linked dominant inheritance IP symptoms can only be seen in female individuals while affected males die during development in utero. We observed a family of horses, in which several mares developed signs of a skin disorder reminiscent of human IP. Cutaneous manifestations in affected horses included the development of pruritic, exudative lesions soon after birth. These developed into wart-like lesions and areas of alopecia with occasional wooly hair re-growth. Affected horses also had streaks of darker and lighter coat coloration from birth. The observation that only females were affected together with a high number of spontaneous abortions suggested an X-linked dominant mechanism of transmission. Using next generation sequencing we sequenced the whole genome of one affected mare. We analyzed the sequence data for non-synonymous variants in candidate genes and found a heterozygous nonsense variant in the X-chromosomal IKBKG gene (c.184C>T; p.Arg62*). Mutations in IKBKG were previously reported to cause IP in humans and the homologous p.Arg62* variant has already been observed in a human IP patient. The comparative data thus strongly suggest that this is also the causative variant for the observed IP in horses. To our knowledge this is the first large animal model for IP.