A 63-bp insertion in exon 2 of the porcine KIF21A gene is associated with arthrogryposis multiplex congenita.

A recessive form of arthrogryposis multiplex congenita (AMC) was detected 20 years ago in the Swiss Large White (SLW) pig population. A diagnostic marker test enabled the identification of carrier animals, but the underlying causal mutation remains unknown. To identify the mutation underlying AMC, we collected SNP chip genotyping data for 11 affected piglets and 23 healthy pigs. Association testing using 47 829 SNPs confirmed that AMC maps to SSC5 (P = 9.4 × 10-13 ). Subsequent autozygosity mapping revealed a common 6.06 Mb region (from 66 757 970 to 72 815 151 bp) of extended homozygosity in 11 piglets affected by AMC. Using WGS data, we detected a 63-bp insertion compatible with the recessive inheritance of AMC in the second exon of KIF21A gene encoding Kinesin Family Member 21A. The 63-bp insertion is predicted to introduce a premature stop codon in KIF21A gene (p.Val41_Phe42insTer) that truncates 1614 amino acids (~97%) from the protein. We found that this deleterious allele still segregates at a frequency of 0.1% in the SLW pig population. Carrier animals can now be detected unambiguously and excluded from breeding.

A CHRNB1 frameshift mutation is associated with familial arthrogryposis multiplex congenita in Red dairy cattle.

BACKGROUND: Bovine arthrogryposis multiplex congenita (AMC) is a syndromic term for a congenital condition characterized by multiple joint contractures. Rare inherited forms of bovine AMC have been reported in different breeds. For AMC in Angus cattle a causative genomic deletion encompassing the agrin (AGRN) gene, encoding an essential neural regulator that induces the aggregation of acetylcholine receptors (AChRs), is known. In 2015, three genetically related cases of generalized AMC affecting Red dairy calves were diagnosed in Denmark. RESULTS: The family history of three affected calves suggested an autosomal recessive inheritance. Single nucleotide polymorphism (SNP) genotyping showed a single genomic region of extended homozygosity of 21.5 Mb on chromosome 19. Linkage analysis revealed a maximal parametric LOD score of 1.8 at this region. By whole genome re-sequencing of the three cases, two private homozygous non-synonymous variants were detected in the critical interval. Both variants, located in the myosin phosphatase Rho interacting protein (MPRIP) and the cholinergic receptor nicotinic beta 1 subunit gene (CHRNB1), were perfectly associated with the AMC phenotype. Previously described CHRNB1 variants in humans lead to a congenital myasthenic syndrome with impaired neuromuscular transmission. The cattle variant represents a single base deletion in the first exon of CHRNB1 (c.55delG) introducing a premature stop codon (p.Ala19Profs47*) in the second exon, truncating 96 % of the protein. CONCLUSIONS: This study provides the first phenotypically and genetically characterized example of a bovine AMC phenotype that represents an inherited neuromuscular disorder corresponding to human congenital myasthenic syndrome. The identified CHRNB1 loss of function variant is predicted to have a deleterious effect on fetal AChR function, which could explain the lethal phenotype reported in this study. The identification of this candidate causative mutation thus widens the known phenotypic spectrum of CHRNB1 mutations and enables selection against this pathogenic variant in Red dairy cattle.

Genome-wide next-generation DNA and RNA sequencing reveals a mutation that perturbs splicing of the phosphatidylinositol glycan anchor biosynthesis class H gene (PIGH) and causes arthrogryposis in Belgian Blue cattle.

BACKGROUND: Cattle populations are characterized by regular outburst of genetic defects as a result of the extensive use of elite sires. The causative genes and mutations can nowadays be rapidly identified by means of genome-wide association studies combined with next generation DNA sequencing, provided that the causative mutations are conventional loss-of-function variants. We show in this work how the combined use of next generation DNA and RNA sequencing allows for the rapid identification of otherwise difficult to identify splice-site variants. RESULTS: We report the use of haplotype-based association mapping to identify a locus on bovine chromosome 10 that underlies autosomal recessive arthrogryposis in Belgian Blue Cattle. We identify 31 candidate mutations by resequencing the genome of four cases and 15 controls at ~10-fold depth. By analyzing RNA-Seq data from a carrier fetus, we observe skipping of the second exon of the PIGH gene, which we confirm by RT-PCR to be fully penetrant in tissues from affected calves. We identify - amongst the 31 candidate variants - a C-to-G transversion in the first intron of the PIGH gene (c211-10C > G) that is predicted to affect its acceptor splice-site. The resulting PIGH protein is likely to be non-functional as it lacks essential domains, and hence to cause arthrogryposis. CONCLUSIONS: This work illustrates how the growing arsenal of genome exploration tools continues to accelerate the identification of an even broader range of disease causing mutations, therefore improving the management and control of genetic defects in livestock.

Porcine arthrogryposis multiplex congenita (AMC): new diagnostic test and narrowed candidate region.

In the Swiss Large White pig population a genetically caused arthrogryposis multiplex congenita (AMC) variant was identified. The disease is autosomal recessively inherited and is a fatal defect. Affected piglets are of normal size, but show malformed and permanently contracted joints in their legs. Often the spinal cord is curved and the lower jaw is shortened. Originally, AMC was mapped to a 5 Mb region on pig chromosome 5 (SSC5) between microsatellite markers SW152 and SW904. In order to detect unaffected carriers a diagnostic test using markers within the candidate region was developed. However, two independent recombination events occurred in a diseased and in a healthy piglet. Therefore, we selected 24 consecutive markers (3 microsatellites, 19 SNPs and 2 indels) in the candidate region, and determined the haplotypes in the two pedigrees with the recombinations. The parents and five offspring were investigated. In consequence, we were able to narrow down the candidate region and map AMC between SNPs ALGA0032767 and DRGA0006010 on SSC5 which span around 2.32 Mb. The candidate region shares homology to human chromosome 12. However, we are still lacking good candidate genes. A PCR-RFLP was developed and is used as an improved genetic test for AMC.

Occurrence of malformed calves in April-May 2021 indicates an unnoticed 2020 emergence of Schmallenberg virus in Denmark.

During the European emergence of Schmallenberg virus (SBV) in 2011, examination of Culicoides spp. showed that SBV-infected midges were present across Denmark. However, SBV-associated malformations in ruminant species have not been reported in Denmark. In April 2021, seven calves with severe congenital generalized arthrogryposis and reduced body weight originating from a narrow region of the Jutlandic peninsula were submitted for examination. Analysis of fetal brain tissue for SBV viral RNA and pleural effusion for fetal anti-SBV antibodies identified SBV as the cause of the congenital syndrome. Backwards calculation from the calving dates indicated the occurrence of an unnoticed emergence of SBV in Denmark from early August 2020 and during the late summer and autumn. As SBV-associated malformations may lead to severe dystocia requiring fetotomy or caesarean section, veterinarians performing obstetric intervention are first-line personnel in recognition of SBV emergence in domestic ruminants.

SNP-based association mapping of Arachnomelia in Fleckvieh cattle.

Bovine arachnomelia is an inherited congenital disorder with malformation mainly of the limbs, the vertebral column and the skull, following a monogenic autosomal recessive heredity. Despite almost identical pathological findings, arachnomelia has previously been mapped to bovine chromosome 23 and 5 in Fleckvieh and Braunvieh respectively. Therefore, this disorder may be an example of locus heterogeneity in cattle. This study aimed to refine the candidate region to allow positional cloning and sequence analyses of candidate genes in Fleckvieh cattle. For that purpose, a case-control association mapping design was set up with a case group of 16 pre-selected affected individuals and a control group of 50 unrelated animals. The subset of affected animals was selected from a total of 129 pathologically confirmed cases due to the occurrence of recombination(s) within a 14.5 cM candidate interval previously mapped to chromosome 23. Six linked microsatellites currently used for indirect gene testing in Fleckvieh were analysed for this purpose. In all selected cases, a genome-wide scan using 44 473 informative SNPs revealed shared segments of homozygosity at 15 adjacent SNPs on chromosome 23. Additional haplotype analysis of 37 carrier bulls confirmed the localization of the arachnomelia locus to a region of 927 kb (13.622-14.549 Mb) containing molybdenum cofactor biosynthesis protein 1 gene, the most likely candidate gene for arachnomelia in Fleckvieh. The number of recombinant haplotypes observed in cases was more than doubled compared with the number of expected recombinations. This remarkably increased mapping resolution and thus illustrates the benefit of pre-selection in association studies.

A pedigree-analysis approach to the descriptive epidemiology of autosomal-recessive disorders.

We describe a pedigree-analysis approach to estimating descriptive epidemiological parameters for autosomal-recessive disorders when the ancestral source of the disorder is known. We show that the expected frequency of carriers in a cohort equals the gene contribution of the ancestral source to that cohort, which is equivalent to the direct (additive) genetic relationship of that ancestor to the cohort. Also, the expected incidence of affected foetuses ranges from (1/2)F* to F*, where F* is the mean partial inbreeding coefficient (due to the ancestor) of the cohort. We applied this approach to complex vertebral malformation (CVM) in Holstein-Friesians in Australia, for which the ancestral source is a USA-born bull, Carlin-M Ivanhoe Bell. The estimated frequency of carriers was 2.47% for the 1992-born and 4.44% for the 1997-born cohort of Holstein-Friesian cows in Australia. The estimated incidence of affected foetuses/calves was considerably less than one per thousand, ranging from 0.0024 to 0.0048% for the 1992-born cohort, and from 0.0288 to 0.0576% for the 1997-born cohort. These incidences correspond to expected numbers of affected female foetuses/calves ranging from 2 to 4 for the 1992-born cohort and from 28 to 56 for the 1997-born cohort. This approach is easy to implement using software that is readily available.

Partial tarsal arthrodesis and botulinum toxin A injection for correction of tarsal arthrogryposis in a cat.

An 11-week-old, domestic shorthair cat was presented for evaluation of a congenital right tarsal deformity. The cat was non-weightbearing lame on the right hindlimb. There was severe tarsal hyperextension and concurrent spasticity/contracture/shortening of the gastrocnemius muscle-tendon. The cat was otherwise healthy. Radiographically there was luxation of the proximal intertarsal joint and talocrural hyperextension. Treatment with 10 mu/kg botulinum toxin A (Botox; Allergan) intramuscular injections directly into the gastrocnemius muscle and passive physiotherapy marginally improved the range of motion in the talocrural joint. Definitive surgical treatment with partial tarsal arthodesis produced an excellent clinical outcome. This case report demonstrates the successful treatment of a severe congenital tarsal abnormality and also documents the first clinical use of botulinum toxin in a feline patient.

Reproductive losses caused by the ingestion of Poincianella pyramidalis in sheep.

Malformations have been observed in sheep and goats in the Brazilian semiarid region in areas where Poincianella pyramidalis is dominant. The objective of this trial was to determine whether Poincianella pyramidalis causes reproductive changes in pregnant sheep. Sixteen non-pregnant sheep were mated with two rams. After confirmation of the pregnancy by ultrasonography on the 18th day after mating, pregnant sheep were randomly divided into four groups (#1, 2, 3, and 4), with four animals each. Sheep received roughage in an amount equivalent to 2% of their body weight, mixed with 0%, 10%, 20% and 40% of dried leaves of P. pyramidalis for groups 1, 2, 3 and 4, respectively. In Groups 1 and 2, all animals lambed normally. In Group 3 (20% P. pyramidalis in the roughage), one lamb was born with arthrogryposis and three sheep gave birth to premature weak lambs within 128, 132, and 133 days of gestation. In Group 4 (40% P. pyramidalis in the roughage), one sheep lambed a normal lamb; another ewe had embryonic mortality after seven days of plant consumption, and two aborted on days 103 and 144 of pregnancy. One of the aborted fetuses was normal and the other showed arthrogryposis and prognathism. These results suggest that P. pyramidalis causes embryonic deaths, abortions, and malformations in sheep. Grazing pregnant sheep in areas where this plant is dominant should be avoided, and roughage for confined pregnant females should not contain more than 10% P. pyramidalis.

Evidence of extensive renewed Schmallenberg virus circulation in Belgium during summer of 2016 - increase in arthrogryposis-hydranencephaly cases expected.

A seroprevalence study carried out between June and September 2016 in the Belgian sheep population showed a significant increase in overall (from 25% to 62%) and between-herd (from 60% to 96%) seroprevalence against Schmallenberg virus (SBV) during this period, indicating the most extensive recirculation of SBV since its original emergence in 2011. SBV recirculation was confirmed by the detection of SBV RNA-positive Culicoides obsoletus complex midges collected in the region of Antwerp in August 2016, reaching a minimum infection rate of 3%. The recirculation of SBV in the largely unprotected ruminant population during summer 2016 will likely cause an increase in the number of arthrogryposis-hydranencephaly cases in newborn ruminants during the coming months.

Congenital malformations and other reproductive losses in goats due to poisoning by Poincianella pyramidalis (Tul.) L.P. Queiroz (=Caesalpinia pyramidalis Tul.).

In the semiarid region of Brazil, in areas with vegetation composed mainly of Poincianella pyramidalis, several cases of congenital malformation and reproductive losses were observed in goats and sheep from 2012 to 2014. To determine the teratogenic effect of P. pyramidalis, two groups of eight goats each were used. Goats from Group 1 received fresh P. pyramidalis, harvested daily, as the only roughage during the whole breeding and pregnancy period. Goats in Group 2 (control) received Cynodon dactylon (tifton) hay free choice. Ultrasound examination for pregnancy diagnosis was performed every 28 days. Four goats from Group 1 were pregnant on day 28 but not on day 56, suggesting embryonic death or abortion. Another goat from Group 1 died at day 70 of pregnancy, and the fetuses exhibited micrognathia. The other three goats bore six kids, three of which showed bone malformations in the limbs, spine, ribs, sternum, and head, including arthrogryposis, scoliosis and micrognathia. One kid also showed hypoplasia of the left pulmonary lobes. In the control group, all goats bore a total of 13 kids and none of them exhibited malformations. These results demonstrated that P. pyramidalis causes congenital malformations and other reproductive losses in goats.

Arthrogryposis multiplex congenita in Aberdeen Angus cattle in Uruguay / Artrogripose múltipla congênita em bovinos Aberdeen Angus no Uruguai

Arthrogryposis multiplex congenita is reported for the first time in the Aberdeen Angus (AA) breed in Uruguay. In a commercial herd of 30 purebred Aberdeen Angus cows, two calves with severe musculoskeletal malformations died at birth. The cows had been inseminated using semen imported from Argentina from one elite AA sire only. At necropsy, one calf showed severe muscular atrophy, arthrogryposis affecting all four limbs and the spine, kyphoscoliosis and torticollis. Histopathology showed muscular atrophy with marked fiber size variation and abundant fibroadipose fibers. The central nervous system only showed congestion and edema due to dystocia, whereas the peripheral nerves and the number of motor neurons in the spinal appeared normal. DNA analysis confirmed arthrogryposis multiplex congenita. It is concluded that disease in Aberdeen Angus cattle is due to failure in the neuromuscular junction.(AU)

Artrogripose múltipla congênita é relatada pela primeira vez em bovinos Aberdeen Angus (AA) no Uruguai. Num rebanho comercial de 30 vacas a Aberdeen Angus, dois bezerros com graves malformações musculoesqueléticas morreram logo após o nascimento. As vacas foram inseminadas utilizando sêmen importado da Argentina, de apenas um touro de elite de AA. Na necropsia, um dos bezerros apresentava atrofia muscular grave, artrogripose afetando todos os quatro membros e a coluna vertebral, cifoscoliose e torcicolo. A histopatologia demonstrou atrofia muscular com acentuadas variações no tamanho das fibras e abundantes fibras fibroadiposas. O sistema nervoso central apresentava apenas congestão e edema devido à distocia, enquanto os nervos periféricos e o número de neurônios motores na medula espinhal pareciam normais. A análise de DNA confirmou artrogripose múltipla congênita. Concluiu-se que a doença em bovinos Aberdeen Angus se deve a falha na junção neuromuscular.(AU)

Ovine fetal malformations induced by in utero inoculation with Main Drain, San Angelo, and LaCrosse viruses.

The teratogenic potential of three bunyaviruses, two California serogroup bunyaviruses, LaCrosse virus and San Angelo virus, and a Bunyamwera serogroup member, Main Drain virus, in sheep was studied following in utero inoculation of ewes in early gestation. Although Main Drain virus appeared to be most teratogenic, all three viruses induced a range of lesions including arthrogryposis, hydrocephalus, fetal death, axial skeletal deviations, anasarca, and oligohydramnios. The teratogenic effects of these viruses are identical to those described in ovine infections by Cache Valley and Akabane viruses. Demonstration of a common bunyaviral tropism for fetal tissue infection that results in congenital brain and musculoskeletal malformations provides evidence that human in utero infection by bunyaviruses could result in similar malformations in human infants.

Evidence that Cache Valley virus induces congenital malformations in sheep.

An outbreak of congenital abnormalities occurred in sheep at San Angelo, Texas, between December 1986 and February 1987. Of 360 lambs born, 19.2% had arthrogryposis or other musculo-skeletal problems and hydranencephaly (AGH), and the total neonatal loss was 25.6%. In 1987, all ewes that were tested with AGH lambs had antibody to Cache Valley virus (CVV), whereas 62% of the ewes with normal lambs had CVV-specific antibody. Pre-colostral serum samples from AGH lambs had neutralizing antibody to CVV. An increase in prevalence of CVV-specific antibody, from 5% during the spring of 1986 to 63.4% during the winter of 1987, occurred during a time that included the gestation of these affected lambs, as well as a period of increased rainfall. The isolation of a CVV-related strain from a sentinel sheep in October 1987 confirmed the continued presence of this virus in the pasture where this outbreak occurred and provided a recent field strain for future studies.

Laboratory investigation of a naturally occurring outbreak of arthrogryposis-hydranencephaly in Texas sheep.

An epizootic of abortions, weak lambs, stillbirths, and congenital arthrogryposis-hydranencephaly occurred in a sheep flock in West Texas. The outbreak began during the first week of January 1987 and continued through the third week of February 1987. Lambs born after February 1987 were not affected. A high incidence of antibodies to Cache Valley virus (Texas 7856 isolate) was demonstrated in the ewes' serum and in precolostral serum from affected lambs. No virus was isolated from tissues and body fluids of the affected lambs. The clinical, pathological, and immunological features of the epizootic were similar to those reported in Akabane virus infection in sheep. Although serological findings strongly suggest Cache Valley virus as the etiological agent of this outbreak, transmission studies are needed.

Morphological variation of "complex vertebral malformation" in Holstein calves.

A study was performed to investigate the morphological expression of the inherited syndrome "complex vertebral malformation" (CVM) in Holstein calves. A total of 107 late-term aborted, premature, or neonatal calves suspected of having CVM were necropsied and retrospectively analyzed for the causal mutation in the gene SLC35A3. Sixty-two calves were homozygous affected, 16 were heterozygous, and 29 were homozygous normal. Calves affected by CVM were growth retarded. Vertebral lesions identified by radiography were present in 61 cases, of which 58 also had costal malformation. Malformation of the head, primarily in the form of dysplasia or palatoschisis, was present in 15 cases. Bilateral symmetric flexion of the carpal and metacarpophalangeal joints was present in all cases, whereas posterior arthrogryposis was found in 54 cases. Interventricular septal defects occurred in 33 calves, often in combination with other cardiac malformations. A wide spectrum of additional malformations was found. Other congenital syndromes were in most cases distinguishable from CVM on a morphological basis. However, a calf with a prenatal infection with bovine virus diarrhea virus constituted a phenocopy. The study demonstrated that the morphological expression of CVM is wide, but certain aspects, i.e., growth retardation, vertebral malformation, and symmetric arthrogryposis, are almost constant findings. However, cases without vertebral defects and phenocopies constitute a diagnostic problem. A presumptive diagnosis of CVM can in most cases be based on necropsy findings combined with information on descent and paternal CVM genotype, whereas a definitive diagnosis requires genotyping.

Teratogenicity of Aino virus in the chick embryo.

Aino virus (JaNAr 28 strain), a possible agent of a congenital anomaly of calves, was inoculated into the yolk sac of chick embryos at a dose of 10(2), 10(3), 10(4) or 10(5) TCID50 0.2 ml-1 at four, six or eight days of incubation. At 21 days of incubation all the unhatched embryos or hatched chickens in the experimental and control groups were sacrificed for pathological examination. The incidence of hydranencephaly, cerebellar hypoplasia or agenesis and arthrogryposis or scoliosis was highest (85 per cent in the chicks inoculated with 10(3) TCID50 0.2 ml-1 at eight days of incubation. The lesions were very similar to those found in congenital abnormalities in calves suspected of a natural infection with Aino virus.

Comparison of plasma disposition of alkaloids after lupine challenge in cattle that had given birth to calves with lupine-induced arthrogryposis or clinically normal calves.

OBJECTIVE: To compare plasma disposition of alkaloids after lupine challenge in cattle that had given birth to calves with lupine-induced arthrogryposis and cattle that had given birth to clinically normal calves and determine whether the difference in outcome was associated with differences in plasma disposition of anagyrine. ANIMALS: 6 cows that had given birth to calves with arthrogryposis and 6 cows that had given birth to clinically normal calves after being similarly exposed to lupine during pregnancy. PROCEDURES: Dried lupine (2 g/kg) was administered via gavage. Blood samples were collected before and at various time points for 48 hours after lupine administration. Anagyrine, 5,6-dehydrolupanine, and lupanine concentrations in plasma were measured by use of gas chromatography. Plasma alkaloid concentration versus time curves were generated for each alkaloid, and pharmacokinetic parameters were determined for each cow. RESULTS: No significant differences in area under the plasma concentration versus time curve, maximum plasma concentration, time to reach maximum plasma concentration, and mean residence time for the 3 alkaloids were found between groups. CONCLUSIONS AND CLINICAL RELEVANCE: Because no differences were found in plasma disposition of anagyrine following lupine challenge between cattle that had given birth to calves with arthrogryposis and those that had not, our findings do not support the hypothesis that between-cow differences in plasma disposition of anagyrine account for within-herd differences in risk for lupine-induced arthrogryposis.

Nicotiana glauca-induced congenital deformities in calves: clinical and pathologic aspects.

Seven calves born to 7 cows fed Nicotiana glauca during portions of the 1st trimester of gestation were deformed at birth. Deformities increased in severity as the calves aged. At birth, calves typically had arthrogryposis of the forelimbs or curvature of the spine; severity varied among calves. In 4 calves necropsied at about 15 months of age, there was general malpositioning and misalignment of the distal ends of the radius and ulna and the proximal ends of the metacarpal bones. Carpal joints were severely affected, fetlock joints were moderately affected, and pastern joints were slightly affected, with lateral rotation of forelimbs common; severity varied among calves. In 1 of the 4 calves, there was moderate torticollis and scoliosis resulting from wedging of some of the cervical and thoracic vertebrae and abnormal cranial curvature of the left thoracic ribs. Histologic changes were not noticed in muscle, brain, spinal cord, or endocrine organs of the 4 calves.

Congenital anomalies in a neonatal white-tailed deer in Alberta.

In September, 1973, a live male 2-3 day old white-tailed deer (Odocoileur virginianus) with multiple anomalies of the head and forelimbs was found near Lethbridge, Alberta. Notable gross findings inculded brachygnathia inferior, atypical incisor tooth alignment, medially folded ears, humero-radial arthrogryposis and bowing of the radius and ulna. Hematologic determinations indicated increased erythrocyte, hemoglobin and hematocrit levels. Histopathologic examination revealed mineralized spherites in the cerebellum and midbrain.