Mutation identification in a canine model of X-linked ectodermal dysplasia

X-linked hypohidrotic ectodermal dysplasia (XHED), an inherited disease recognized in humans, mice, and cattle, is characterized by hypotrichosis, a reduced number or absence of sweat glands, and missing or malformed teeth. In a subset of affected individuals and animals, mutations in the EDA gene (formerly EDI), coding for ectodysplasin, have been found to cause this phenotype. Ectodysplasin is a homotrimeric transmembrane protein with an extracellular TNF-like domain, which has been shown to be involved in the morphogenesis of hair follicles and tooth buds during fetal development. Some human XHED patients also have concurrent immunodeficiency, due to mutations in the NF-κB essential modulator protein (IKBKG; formerly NEMO), which is also encoded on the X chromosome. In a breeding colony of dogs with XHED, immune system defects had been suspected because of frequent pulmonary infections and unexpected deaths resulting from pneumonia. To determine if defects in EDA or IKBKG cause XHED in the dogs, linkage analysis and sequencing experiments were performed. A polymorphic marker near the canine EDA gene showed significant linkage to XHED. The canine EDA gene was sequenced and a nucleotide substitution (G to A) in the splice acceptor site of intron 8 was detected in affected dogs. In the presence of the A residue, a cryptic acceptor site within exon 9 is used, leading to a frame shift and use of a premature stop codon that truncates the translation of both isoforms, EDA-A1 and EDA-A2, resulting in the absence of the TNF-like homology domain, the receptor-binding site of ectodysplasin.The sequence data described in this article have been submitted to GenBank under accession numbers AY924407–AY924414.     

Generation and Characterization of Function-blocking Anti-ectodysplasin A (EDA) Monoclonal Antibodies That Induce Ectodermal Dysplasia

Development of ectodermal appendages, such as hair, teeth, sweat glands, sebaceous glands, and mammary glands, requires the action of the TNF family ligand ectodysplasin A (EDA). Mutations of the X-linked EDA gene cause reduction or absence of many ectodermal appendages and have been identified as a cause of ectodermal dysplasia in humans, mice, dogs, and cattle. We have generated blocking antibodies, raised in Eda-deficient mice, against the conserved, receptor-binding domain of EDA. These antibodies recognize epitopes overlapping the receptor-binding site and prevent EDA from binding and activating EDAR at close to stoichiometric ratios in in vitro binding and activity assays. The antibodies block EDA1 and EDA2 of both mammalian and avian origin and, in vivo, suppress the ability of recombinant Fc-EDA1 to rescue ectodermal dysplasia in Eda-deficient Tabby mice. Moreover, administration of EDA blocking antibodies to pregnant wild type mice induced in developing wild type fetuses a marked and permanent ectodermal dysplasia. These function-blocking anti-EDA antibodies with wide cross-species reactivity will enable study of the developmental and postdevelopmental roles of EDA in a variety of organisms and open the route to therapeutic intervention in conditions in which EDA may be implicated.     

Bovine renal lipofuscinosis: Prevalence, genetics and impact on milk production and weight at slaughter in Danish cattle

Background

Bovine renal lipofuscinosis (BRL) is an incidental finding in cattle at slaughter. Condemnation of the kidneys as unfit for human consumption was until recently considered the only implication of BRL. Recent studies have indicated a negative influence on the health of affected animals. The present study investigated the prevalence, genetics and effect of BRL on milk yield and weight at slaughter.

Methods

BRL status of slaughter cattle was recorded at four abattoirs during a 2-year-period. Data regarding breed, age, genetic descent, milk yield and weight at slaughter were extracted from the Danish Cattle Database. The prevalence of BRL was estimated stratified by breed and age-group. Furthermore, total milk yield, milk yield in last full lactation and weight at slaughter were compared for BRL-affected and non-affected Danish Holsteins and Danish Red cattle.

Results

433,759 bovines were slaughtered and 787 of these had BRL. BRL was mainly diagnosed in Danish Red, Danish Holstein and crossbreds. The age of BRL affected animals varied from 11 months to 13 years, but BRL was rarely diagnosed in cattle less than 2 years of age. The total lifelong energy corrected milk (ECM) yields were 3,136 and 4,083 kg higher for BRL affected Danish Red and Danish Holsteins, respectively. However, the median life span of affected animals was 4.9 months longer, and age-corrected total milk yield was 1,284 kg lower for BRL affected Danish Red cows. These cows produced 318 kg ECM less in their last full lactation. Weight at slaughter was not affected by BRL status. The cases occurred in patterns consistent with autosomal recessive inheritance and several family clusters of BRL were found. Analysis of segregation ratios demonstrated the expected ratio for Danish Red cattle, but not for Danish Holsteins.

Conclusion

The study confirmed that BRL is a common finding in Danish Holsteins and Danish Red cattle at slaughter. The disorder is associated with increased total milk yield due to a longer production life. However, a reduced milk yield was detected in the end of the production life in Danish Red. The study supports that BRL is inherited autosomal recessively in the Danish Red breed and Danish Holsteins, but with incomplete penetrance of the genotype in Danish Holsteins.     

Mutation p.Leu354Pro in EDA causes severe hypohidrotic ectodermal dysplasia in a Chinese family

X-linked recessive hypohidrotic ectodermal dysplasia (XLHED) is characterized by the defective morphogenesis of teeth, hair, and eccrine sweat glands. It is associated with mutations in the EDA gene. Up to now, more than 100 mutations in the EDA gene have been reported to cause XLHED. The product of EDA gene is a trimeric type II transmembrane protein that belongs to the tumor necrosis factor (TNF) family of ligands. In this study, we identified a Chinese family with XLHED. Direct DNA sequencing of the whole coding region of EDA revealed a novel missense mutation, p.Leu354Pro in a patient affected with XLHED. This mutation was not found in either unaffected male individuals of the family or 168 normal controls. The substitution of Leu354 with Pro was found to be located in the TNF-like domain of EDA and may influence the epithelial signaling pathway required for the normal ectodermal development through altering the topology of EDA. Our finding broadens the spectrum of EDA mutations and may help to understand the molecular basis of XLHED and aid genetic counseling.     

High level production and one-step purification of biologically active ectodysplasin A1 and A2 immunoadhesins using the baculovirus/insect cell expression system

Ectodysplasin A (EDA) is a ligand of the tumor necrosis factor (TNF) family that has been shown to play a crucial role in ectodermal differentiation. Mutations of the syntenic ectodysplasin A gene (Eda) are responsible for Tabby (Ta) phenotype in mice and human X-linked hypohidrotic ectodermal dysplasia (XLHED). EDA-A1 and EDA-A2 are the two main splice variants of Eda, which differ from each other in only two amino acid residues and engage the tumor necrosis factor (TNF) family receptors EDAR and XEDAR, respectively. We have used the baculovirus/insect cell system to express the recombinant EDA proteins fused to the Fc portion of a truncated human IgG1 immunoglobulin heavy chain. Immunoadhesins (4.5-4.7 mg/L) from crude supernatant could be purified to near homogeneity by using rProtein A affinity chromatography. The purified EDA immunoadhesins were endowed with ligand-binding activity as they could bind EDAR or XEDAR on the surface of 293T cells that had been transiently transfected with the corresponding plasmids. Functional activities of EDA immunoadhesins were demonstrated by their ability to activate the NF-kappaB pathway in cells expressing their cognate receptors. These results open up the possibility of obtaining large amounts of purified EDA proteins to investigate EDAR/XEDAR related signaling pathways and for the treatment of patients with X-linked hypohidrotic ectodermal dysplasia.     

Close linkage between X-linked ectodermal dysplasia and a cloned DNA sequence detecting a two allele restriction fragment length polymorphism in the region Xp11-q12

Summary EDA (ectodermal dysplasia, anhidrotic) is an X-linked recessive disorder characterized by hypohidrosis, hypoor anodontia, and hypotrichosis. A possible linkage between the gene for EDA and a number of restriction fragment length polymorphisms (RFLPs) spread over the X chromosome was investigated in two Danish families segregating EDA. No recombination between the gene for EDA and our probe pTAK8, which detects a two allele polymorphism in the region Xp11-q12, was found in nine informative meiotic events (seven of which are phase known), giving a maximal lod score of 2.41 at a recombination fraction of 0.00. This juxtacentromeric location of the gene for EDA agrees well with the linkage data obtained with the other markers used in this study.     

Mutations leading to X-linked hypohidrotic ectodermal dysplasia affect three major functional domains in the tumor necrosis factor family member ectodysplasin-A

Mutations in the epithelial morphogen ectodysplasin-A (EDA), a member of the tumor necrosis factor (TNF) family, are responsible for the human disorder X-linked hypohidrotic ectodermal dysplasia (XLHED) characterized by impaired development of hair, eccrine sweat glands, and teeth. EDA-A1 and EDA-A2 are two splice variants of EDA, which bind distinct EDA-A1 and X-linked EDA-A2 receptors. We identified a series of novel EDA mutations in families with XLHED, allowing the identification of the following three functionally important regions in EDA: a C-terminal TNF homology domain, a collagen domain, and a furin protease recognition sequence. Mutations in the TNF homology domain impair binding of both splice variants to their receptors. Mutations in the collagen domain can inhibit multimerization of the TNF homology region, whereas those in the consensus furin recognition sequence prevent proteolytic cleavage of EDA. Finally, a mutation affecting an intron splice donor site is predicted to eliminate specifically the EDA-A1 but not the EDA-A2 splice variant. Thus a proteolytically processed, oligomeric form of EDA-A1 is required in vivo for proper morphogenesis.     

Screening of EDA1 gene in X-linked anhidrotic ectodermal dysplasia using DHPLC: identification of 14 novel mutations in Italian patients

Mutations within EDA1 gene, which encodes for the ectodysplasin, cause X-linked anhidrotic ectodermal dysplasia. In this study, 23 Italian patients with anhidrotic ectodermal dysplasia were analyzed for mutations in EDA1 gene. We set up a rapid protocol through denaturing high-performance liquid chromatography, followed by sequencing, that allowed the characterization of 18 mutations, 14 novel and 4 recurrent: 8 missense mutations (p.L51Q, p.H54R, p.R156H twice, p.C332F, p.D316H, p.T378M, and p.A349T), 3 in-frame deletions (p.G82_P84del, p.A179_P191del, and p.L354del), 1 gross deletion (p.G168_G265del, identified through direct sequencing and PCR), 4 altered splicing (c.949-13T > C, c.741 + 1G/T, c.793 + 4A > T, and c.924 + 1G/T), 1 nonsense (p.Y3X), and 1 synonymous mutation (c.741G > A). Moreover, structural analysis of three missense mutations shows that alteration of the electrostatic surface of the protein (p.D316N), the break of intermonomer interactions (p.A349T) and destabilization of the single monomer structure (p.T378M), may irreversibly invalidate the EDA-A1 binding properties. Our data confirm and extend the large spectrum of EDA1 mutations and provide a rapid and efficient molecular protocol for testing EDA1 mutations in EDA patients.     

Identification of complex vertebral malformation carriers in Holstein cattle in south China

Complex vertebral malformation (CVM) is a recently described monogenic autosomal recessive hereditary defect of Holstein dairy cattle that causes premature birth, aborted fetuses and stillborn calves. Guanine is substituted by thymine (G>T) in the solute carrier family 35 member A3 gene (SLC35A3). A valine is changed to a phenylalanine at position 180 of uridine 5'-diphosphate-N-acetyl-glucosamine transporter protein. CVM is expected to occur in many countries due to the widespread use of sire semen. We developed a created restriction site PCR (CRS-PCR) method to diagnose CVM in dairy cows. This was tested on 217 cows and 125 bulls selected randomly from a Holstein cattle population in south China. Five Holstein cows and five Holstein bulls were identified to be CVM carriers; the percentages of CVM carriers were estimated to be 2.3, 4.0 and 2.9% in the cows, bulls and entire Holstein cattle sample, respectively.     

A hereditary disposition for bovine peripheral nerve sheath tumors in Danish Holstein cattle

Background

Peripheral nerve sheath tumors (PNSTs) are frequently found in Danish cattle at slaughter. Bovine PNSTs share several gross and histopathological characteristics with the PNSTs in humans with heritable neurofibromatosis syndromes. The aim of the present study was to investigate a possible hereditary disposition to PNSTs in dairy cattle by statistical analysis performed on data from 567 cattle with PNSTs. Furthermore, a preliminary genome-wide association study (GWAS) was performed on DNA isolated from 28 affected and 28 non-affected Holstein cows to identify loci in the bovine genome involved in the development of PNSTs.

Results

PNSTs were significantly more common in the Danish Holstein breed than in other breeds with 0.49% of Danish Holsteins slaughtered during an eight-year-period having PNSTs. PNSTs also occurred significantly more frequently in the offspring of some specific Holstein sires. Examination of three generation pedigrees showed that these sires were genetically related through a widely used US Holstein sire. The PNSTs included in GWAS were histologically classified as neurofibroma-schwannoma (43%), schwannoma (36%) and neurofibroma (21%) and derived from Holstein cows with multiple PNSTs. A single SNP on chromosome 27 reached genome-wide significance.

Conclusions

Gross and histological characteristics of bovine PNSTs are comparable to PNSTs in humans (schwannomatosis). Danish Holsteins are genetically disposed to develop PNSTs but the examined materials are insufficient to allow determination of the mode of inheritance.

     

A nonsense mutation in the optic atrophy 3 gene (OPA3) causes dilated cardiomyopathy in Red Holstein cattle

Cardiomyopathies are severe degenerative disorders of the myocardium that lead to heart failure. During the last three decades bovine dilated cardiomyopathy (BDCMP) was observed worldwide in cattle of Holstein-Friesian origin. In the Swiss cattle population BDCMP affects Fleckvieh and Red Holstein breeds. The heart of affected animals is enlarged due to dilation of both ventricles. Clinical signs are caused by systolic dysfunction and affected individuals die as a result of severe heart insufficiency. BDCMP follows an autosomal recessive pattern of inheritance and the disease-causing locus was mapped to bovine chromosome 18 (BTA18). In the present study we describe the successful identification of the causative mutation in the OPA3 gene located on BTA18 that was previously reported to cause 3-methylglutaconic aciduria type III in Iraqi-Jewish patients. We demonstrated conclusive genetic and functional evidence that the nonsense mutation c.343C>T in the bovine OPA3 gene causes the late-onset dilated cardiomyopathy in Red Holstein cattle.     

Female with hypohidrotic ectodermal dysplasia and de novo (X;9) translocation

Summary We present here a historical documentation of a female with X-linked hypohidrotic ectodermal dysplasia (XHED) and a de novo X/9 chromosome translocation. The patient was verbally reported by Dr. P.L. J. Cook to the HGM conference in 1973, but was subsequently lost to follow up. We have since traced her and confirmed the diagnosis of XHED with moderately severe mental retardation. According to Dr. P. L. J. Cook's records, fibroblast cell line AnLy GMO 705, was derived from this patient. Another female with a de novo X/12 chromosome translocation and hypohidrotic ectodermal dysplasia was recently reported. In both cases, the X chromosome breakpoint appears to be at Xq13.1     

X-linked anhidrotic ectodermal dysplasia and de novo t(X;1) in a female

Summary A de novo translocation (X;1)(q13.1;p36.33) was found in a 2-year-old girl with typical clinical features of X-linked anhidrotic ectodermal dysplasia (EDA). The breakpoint at Xq13.1 is approximately the same as has been described in 2 other EDA females with X;autosome translocations.     

Permanent correction of an inherited ectodermal dysplasia with recombinant EDA

X-linked hypohidrotic ectodermal dysplasia (XLHED; OMIM 305100) is a genetic disorder characterized by absence or deficient function of hair, teeth and sweat glands. Affected children may experience life-threatening high fever resulting from reduced ability to sweat. Mice with the Tabby phenotype share many symptoms with human XLHED patients because both phenotypes are caused by mutations of the syntenic ectodysplasin A gene (Eda) on the X chromosome. Two main splice variants of Eda, encoding EDA1 and EDA2, engage the tumor necrosis factor (TNF) family receptors EDAR and XEDAR, respectively. The EDA1 protein, acting through EDAR, is essential for proper formation of skin appendages; the functions of EDA2 and XEDAR are not known. EDA1 must be proteolytically processed to a soluble form to be active. Here, we show that treatment of pregnant Tabby mice with a recombinant form of EDA1, engineered to cross the placental barrier, permanently rescues the Tabby phenotype in the offspring. Notably, sweat glands can also be induced by EDA1 after birth. This is the first example of a developmental genetic defect that can be permanently corrected by short-term treatment with a recombinant protein.     

牛脊柱畸形综合征检测方法的建立与应用

牛脊柱畸形综合征(Complex vertebral malformation, CVM)是近年来新发现的致死性牛常染色体隐性遗传缺陷病。由于编码UDP-N-乙酰葡糖胺载体的SLC35A3基因发生G→T的突变而引起本病的发生, 可引起胎牛死胎、流产、早产。为了解我国正常的荷斯坦牛(黑白花奶牛)的CVM携带和发生情况, 建立、应用创造酶切位点PCR(Created restriction site PCR, CRS-PCR)、等位基因特异性PCR(Allele-specific polymerase chain reaction, AS-PCR)检测方法检测了表型正常的436头荷斯坦母牛和93头荷斯坦公牛, 检测到3头CVM携带者, 其中杂合母牛1头, 杂合公牛2头, 携带率分别为0.60%、2.20%。此方法简便、可靠, 为奶牛CVM有害基因的分型和筛选提供了新的方法和思路, 为我国奶牛的分子选育提供了可靠的理论依据。