Congenital syndactyly in cattle: four novel mutations in the low density lipoprotein receptor-related protein 4 gene (LRP4)
© Drögemüller et al; licensee BioMed Central Ltd. 2007
Received: 24 November 2006
Accepted: 23 February 2007
Published: 23 February 2007
Isolated syndactyly in cattle, also known as mulefoot, is inherited as an autosomal recessive trait with variable penetrance in different cattle breeds. Recently, two independent mutations in the bovine LRP4 gene have been reported as the primary cause of syndactyly in the Holstein and Angus cattle breeds.
We confirmed the previously described LRP4 exon 33 two nucleotide substitution in most of the affected Holstein calves and revealed additional evidence for allelic heterogeneity by the identification of four new LRP4 non-synonymous point mutations co-segregating in Holstein, German Simmental and Simmental-Charolais families.
We confirmed a significant role of LRP4 mutations in the pathogenesis of congenital syndactyly in cattle. The newly detected missense mutations in the LRP4 gene represent independent mutations affecting different conserved protein domains. However, the four newly described LRP4 mutations do still not explain all analyzed cases of syndactyly.
Many inherited malformations of domestic animals are analogous to human hereditary anomalies and have proven to be valuable animal models for the investigation of the pathogenesis of rare human phenotypes with identical molecular basis .
Isolated congenital syndactyly in cattle only affecting the digits, also known as mulefoot, refers to the fusion or non-division of the two developed digits of the bovine foot . The variable expressed syndactyly phenotype in cattle is most often seen in the front feet, but all four feet underlying a right-left and front-rear gradient may be involved . The bovine syndactyly consists mainly of pairs of horizontally synostotic phalanges and adaptive structural changes develop proximal to the fused digits . Bovine syndactyly has been shown to segregate as a monogenic recessive trait with incomplete penetrance in many breeds of cattle in many countries (OMIA: 000963). Genetic mapping located the syndactyly locus on cattle chromosome 15 . This bovine chromosome region is homologous to a segment of mouse chromosome 2 containing the low density lipoprotein receptor-related protein 4 gene (Lrp4), alternatively designated as multiple epidermal growth factor-like domains 7 gene (Megf7). Homozygous Lrp4-deficient mice are growth-retarded, with fully penetrant polysyndactyly in their fore and hind limbs . Positional cloning of two recessive mutations of the mouse that cause polysyndactyly (dan and mdig) showed that the Lrp4 gene plays an essential role in the process of digit differentiation in mammalian species . Other members of the low density lipoprotein receptor gene family have been shown to regulate intracellular signaling cascades . In humans, syndactyly represents the most common congenital malformation of the hand and is characterized by the apparent fusion of soft tissue of the fingers and toes with or without bony fusion . Syndactyly may occur as an isolated malformation or as part of a syndrome. Until now the two human genes HOXD13 and GJA1 were identified harboring causative dominant mutations for isolated syndactyly types II (OMIM: 186000) and III (OMIM: 186100), respectively. However, while clinical studies in these human defects revealed variable phenotypical expression, the establishment of precise genotype-phenotype correlations for limb malformations is difficult and the molecular genetic basis of numerous human cases of syndacytly is still unknown .
Recently, two independent causative mutations in the bovine LRP4 ortholog were identified in the predominantly affected cattle breeds of Holstein and Angus [9, 10]. A homozygous LRP4 exon 33 substitution of two consecutive nucleotides (c.4863_4864delCGinsAT) was observed in 36 affected Holstein individuals, which lead to amino acid changes at two LRP4 codons (p. [Asn1621Lys; Gly1622Cys]) affecting a conserved EGF-like protein domain . In the two reported affected Angus cattle a homozygous LRP4 single nucleotide substitution at the first base of intron 37 (c.5385+1G>A) disrupted the 5'splice site, which introduced aberrant splicing of intron 36 and lead to a truncated translation product lacking the normal N-terminal cytoplasmic domain . The finding that mulefoot is caused by mutations within the same gene in both breeds is in agreement with earlier findings, where affected calves had experimentally been produced by mating of heterozygous Holstein and Angus cattle .
The aim of this study was to screen the bovine LRP4 gene for co-segregating mutations in sixteen animals from different breeds affected by congenital syndactyly.
Summary of LRP4 mutations described within this study.
LRP 4 exon
Genomic DNA sequence change
LRP4 protein sequence change
Affected LRP4 protein domain
p. [Asn1621Lys; Gly1622Cys]
LDL receptor class A 2
LDL receptor class B 13
LDL receptor class B 8
Holstein family I
Holstein family II
This study indicates that congenital syndactyly may occur in different breeds of cattle due to mutations in LRP4. All reported family histories are compatible with autosomal recessive inheritance of bovine syndactyly. Also the known variability in the phenotypic expression characterized by the variable number of affected feet and the gradually different fusion of bones per foot could be confirmed by the reported sixteen cases. Most interesting, four novel mutations in the bovine LRP4 gene were detected which possibly lead to syndactyly in cattle as none of these sequence variants was observed in chromosomes from unaffected control animals.
The analysis of the syndactyly cases from the Holstein breed confirmed the presence of the recessively inherited c.4863_4864delCGinsAT mutation in eight cases, which were related to the recently identified founder cow named Raven Burke Elsie, born 1947 . In contrast, four out of twelve syndactyly affected Holstein calves from our Holstein family I shared only a single, paternally inherited copy of this mutation. Therefore we assume the existence of further disease causing mutations within the LRP4 gene within the international Holstein population. Unfortunately, we did not find evidence for such a mutation in these four cases within in the 37 analyzed LRP4 exons. The identified exon 7, 29, and 32 SNP could be probably excluded as causative mutations for syndactyly since they did not alter the amino acid sequence and due to the observed high frequencies for the mutated alleles in the controls. As no genomic sequence data for the regulatory 5'region of LRP4 including exon 1 is publicly available, we were not able to analyze this gene region for possible mutations. There might be even another gene involved in the pathogenesis, e.g. modifying genes that would impact LRP4 expression. The bovine ALX4 gene, located within the mulefoot linked region on cattle chromosome 15, previously has been excluded in our affected animals as candidate gene .
The detected c.4940C>T SNP in Holstein family II co-segregates perfectly with the disease according to recessive inheritance and provides evidence that independent non-synonymous LRP4 mutations occur within the Holstein population, The two missense mutations in LRP4 which co-segregate with syndactyly in the Simmental family underline the identified allelic heterogeneity at the bovine LRP4 gene. The observed perfect co-segregation of each of these mutations within the analyzed families provides evidence that these mutations are potentially causative for syndactyly. All available parents carried a single mutated allele and the affected animals showed two copies of mutated alleles, respectively. Finally, the LRP4 genotypes of the affected animals from the crossbred family illustrate the existence of compound heterozygotes carrying two different deleterious LRP4 alleles. Due to records from the breeder, we assume that the dam of the affected sire II-1 (Figure 4A), a Holstein cow, had a well known syndactyly carrier sire (Marathon) in her ancestry. This could explain the occurrence of the c.4863_4864delCGinsAT mutation within this family.
The p.Gly81Ser mutation affects an invariably conserved glycine in a ligand binding LDL receptor class A domain (Figures 5 and 6), characterized by successive cysteine-rich repeats of about 40 amino acids, which occur in low density lipoproteins and related receptors . Furthermore, it is highly likely that this mutation disrupts LRP4 function, as for example a frameshift mutation affecting residue 98 of human LDLR class A domain causes hypercholesterolemia in a Japanese family .
Two mutations (p.Gly907Arg, p.Gly1199Ser) affect low density lipoprotein receptor class B, or alternatively termed YWTD, domains which are found as multiple tandem repeats building the characteristic beta-propeller structure in low density lipoprotein receptors . The p.Gly907Arg mutation affects a conserved glycine in LDL receptor class B domain 8 and the p.Gly1199Ser affects an invariably conserved glycine in LDL receptor class B domain 13 (Figures 5 and 6). A substitution of a valine for a glycine at residue 544 of human LDLR causes hypercholesterolemia and functional analysis of this mutation gives rise to an LDL receptor that is not transported to the cell surface and is rapidly degraded . Several mutations of the human LDLR gene causing hypercholesterolemia affecting the LDL receptor class B domain have been reported . Therefore it is likely that these two bovine mutations impair LRP4 function.
The p.Pro1647Lys mutation is located within a particular type of extracellular EGF-like motif, termed LDL-type EGF-like, which is characteristic for low density lipoprotein receptor related proteins . This domain is also affected by the previously reported Holstein c.4863_4864delCGinsAT mutation  and the majority (47%) of LDLR mutations causing hypercholesterolemia in man were found in the EGF-like domain . Additionally, the p.Pro1647Lys mutation may be dysfunctional due to the software predicted consequences (Table 1).
In summary, these comparisons provide support for the probable causality of the four newly identified bovine LRP4 mutations affecting codons 81, 907, 1199 and 1647, respectively. The three silent exonic mutations identified in the Holstein family I are very likely not causative, particularly as they occur at a rather high frequency in unaffected control animals. Finally, the published exon 33 substitution of two consecutive nucleotides could be confirmed in some of the affected Holstein calves and the recently reported LRP4 single nucleotide substitution at the first base of intron 37 (c.5385+1G>A) observed in two syndactyly affected Angus cattle was not observed in any of the examined cases within this study.
We confirmed a significant role of LRP4 mutations in the pathogenesis of congenital syndactyly in cattle. This represents the third LRP4 report of mutations in cattle while the first two LRP4 mutations were described recently in Holstein and Angus cattle, respectively [9, 10]. The data indicate that extensive allelic heterogeneity exists in cattle and within the Holstein breed. However, the four newly described LRP4 mutations do still not explain all analyzed cases of syndactyly. Therefore genetic testing for the carrier status of single individuals remains difficult, because at present obviously not all causal mutations have been detected. Thus, only presence of a known mutated allele can be recorded and unequivocal carriers are detected. Further studies of the regulatory 5'-region and exon1 of the bovine LRP4 gene will help to clarify if these regions are involved in the development of syndactyly.
We analyzed a total of sixteen affected animals and samples of sixteen available relatives from three cattle populations (Holstein, Simmental and Holstein × Simmental × Charolais crossbred) with the clinical diagnosis of congenital syndactyly. The clinical diagnosis was confirmed by radiography. In a previous linkage study we reported eight affected German Holstein calves belonging to a single eight generation family, named Holstein family I . This pedigree was extended by the inclusion of three cases of syndactyly in Italian Holstein and a single affected male German Holstein calf (VIII 1–4; Figure 1A). Each of these four cases could be maternally and paternally traced back to the single common male ancestor of the established Holstein family I. Additionally, a second German Holstein pedigree, named Holstein family II, with a single affected female calf without any relationship for five generations to family I was analyzed. Finally, a single affected purebred female German Simmental calf (Simmental family) and two affected crossbred bulls (father and son) belonging to a Holstein × Simmental × Charolais pedigree (crossbred family) were examined. All control samples used to check the distribution of sequence alterations were taken from an archive of unrelated artifical insemination sires matching to the German Holstein (n = 48), German Simmental (n = 48) and German Charolais populations (n = 16), respectively.
Genomic DNA was isolated from blood samples by standard methods. The protein coding LRP4 exons 2 to 38 with the intronic splice site junctions were PCR amplified with primers described before . The subsequent re-sequencing of the PCR products was performed after Shrimp Alkaline Phosphatase (Roche, Basel, Switzerland) and Exonuclease I (N.E.B., Axonlab, Baden, Switzerland) treatment using both PCR primers with the ABI BigDye Terminator Sequencing Kit 3.1 (Applied Biosystems, Rotkreuz, Switzerland) on an ABI 3730 capillary sequencer (Applied Biosystems). Sequence data were analyzed with Sequencher 4.6 (GeneCodes, Ann Arbor, MI, USA).
aristaless-like homeobox 4
epidermal growth factor
gap junction protein alpha 1
homeo box D13
low density lipoprotein
low density lipoprotein receptor
low density lipoprotein receptor-related protein 4
multiple epidermal growth factor-like domains 7
online mendelian inheritance in animals
online mendelian inheritance in man
sorting intolerant from tolerant
protein family and domain database
This study was supported by a grant of the German Research Council, DFG, Bonn to CD (DR 398/3-1). The authors thank all breeders and veterinarians providing affected animals.
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