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61
International Journal of Veterinary Science
www.ijvets.com P-ISSN: 2304-3075 E-ISSN: 2305-4360 editor@ijvets.com
SHORT COMMUNICATION
Detection of Silent Homozygous Polymorphism in Exon 4 of SLC35A3 Gene in a Holstein Cattle
Carrier for Complex Vertebral Malformation
Rosaiah Kotikalapudi, Rajesh K Patel*, Phani Sri S Sunkara and Arpita Roy
Sandor Proteomics Pvt. Ltd., Banjara Hills, Hyderabad-500 034, India
ARTICLE INFO
ABSTRACT
Received:
Revised:
Accepted:
May 29, 2013
June 04, 2013
June 10, 2013
Key words:
Complex vertebral malformation
Holstein bull
Mutation
Polymorphism
SLC35A3 gene
*Corresponding Author
Rajesh K Patel
rkpatel46@yahoo.com
The complex vertebral malformation (CVM) syndrome is a congenital
autosomal recessively inherited disorder first observed in Danish Holsteins. It is
caused by a point mutation (G→T) at nucleotide position 559 of the gene,
bovine solute carrier family 35 member 3 (SLC35A3). Bovine SLC35A3 plays
a vital role in the development of the axial skeleton. The aim of this study was
to detect carriers of CVM in Holstein population using Polymerase Chain
Reaction- Primer-introduced restriction analysis and Restriction Fragment
Length Polymorphism (PCR-PIRA and RFLP) methods. Our results show that
one out of 60 bulls tested exhibited polymorphism (G→T) at position 559 in
exon 4 of SLC35A3 gene. To confirm this polymorphism, the PCR product was
purified using ExoSAP-IT followed by sequencing by Applied Biosystems
3130XL Automated Sequencer using the ABI BigDye Ver 3.1. Gene sequences
from normal and carrier animals were compared using the software, codon code
Aligner 4.0.4. Surprisingly, the sequence analysis of PCR product also revealed
the presence of two previously unknown homozygous mutations (TG→CT) at
nucleotide positions 554 and 555 in addition to the previously reported
heterozygous mutation at position 559. The bull was immediately culled from
the breeding programme. To the our best of knowledge, this is the first study to
report the existence of homozygous and heterozygous mutations at positions
554, 555 and 559 in exon 4 of SLC35A3 gene in Indian Holstein cattle.
However, it is surprising that no phenotypic effects were observed in the carrier
bull, necessitating further studies to fully elucidate the effects of these novel
homozygous mutations.
Cite This Article as: Kotikalapudi R, RK Patel, PSS Sunkara and A Roy, 2013. Detection of silent homozygous
polymorphism in exon 4 of SLC35A3 gene in a Holstein cattle carrier for complex vertebral malformation. Inter J Vet
Sci, 2(2): 61-64. www.ijvets.com
INTRODUCTION
The complex vertebral malformation (CVM)
syndrome is a congenital autosomal recessively inherited
disorder in Holstein cattle (Agerholm et al., 2001).
Studies of Danish Holsteins with CVM have shown that
the extent of foetal mortality prior to gestation day 260 is
approximately 77% (Nielsen et al., 2003). Majority of the
calves that survive until the end of the gestation period are
stillborn and are phenotypically characterized by retarded
growth and mild bilateral flexion of the carpal and pastern
joints with rotation of the digits. Additionally, most of the
animals have vertebral malformation, malformed ribs, and
arthrogryposis of the tarsal and posterior pastern joints
(Agerhoim et al., 2004). Extensive malformation of the
cervical and thoracic vertebrae is observed in typical
cases, causing a shortening of the neck. Other
malformations have been reported as a part of this
syndrome, including cardiac interventricular septal
defects, malformation of the great vessels and myocardial
hypertrophy (Agerholm et al., 2001, Nielsen et al., 2003).
The syndrome was first discovered in the Danish Holstein
population in 1999 (Agerholm et al., 2001), but shortly
thereafter reported in the United States (Duncan et al.,
2001, Holstein Association, USA, 2006), the United
Kingdom (Revell, 2001), Netherlands (Wouda et al.,
2000), Japan (Nagahata et al., 2002), Germany
(Konersmann et al., 2003), Sweden (Berglund et al.,
2004), Denmark (Thomsen et al., 2006), and India
(Mahdipour et al., 2010). It is important to recognize that
CVM is not newly identified disease. It has been present
in the Holstein breed since many generations, and that
Inter J Vet Sci, 2013, 2(2): 61-64.
62
only the DNA test for its diagnosis is a new advancement.
Genealogical records traced the origin of the disease-
causing allele to a common ancestral bull, Carlin-M
Ivanhoe Bell, which has been extensively used in dairy
cattle breeding worldwide for over two decades due to the
superior lactation performance of his daughters
(Mahdipour et al., 2010). Coincidently, Carlin-M Ivanhoe
Bell was a carrier for two genetic disorders, CVM and
Bovine leukocyte adhesion deficiency (BLAD). The
BLAD and CVM genes are mapped to chromosomes 1
(Shuster et al., 1992) and 3 (Thomsen et al., 2006),
respectively. When the sire (father) of Carlin-M Ivanhoe
Bell, a bull named Pennstate Ivanhoe Star, was tested he
was found to be a carrier of both CVM and BLAD
(Thomsen et al., 2006). Carlin-M Ivanhoe Bell's grandsire
Osborndale Ivanhoe, however, carried only BLAD.
Scientists therefore believe that the mutation responsible
for CVM occurred either in Pennstate Ivanhoe Star (Sire)
or somewhere in his maternal family.
Biochemical aspect of the CVM reveals that this
disease results from an impaired protein molecule,
Uridine diphosphate N-acetylglucosamine (UDP-N-
acetylglucosamine) transporter or Golgi UDP-GlcNAc
transporter in the Golgi apparatus membrane (Patel, 2012)
. These transporter proteins transport a nucleotide sugar,
UDP-N-acetylglucosamine or UDP-GlcNAc and
coenzyme during metabolism from cytosol (site of
synthesis) into the Golgi lumen before these can be
substrates for the glycosylation of proteins, lipids, and
proteoglycans (Patel, 2012). The UDP-GlcNAc plays an
important role in the structure of the cytoskeleton. The
molecular basis of CVM is a substitution of guanine by
thymine (G→T) in a solute carrier family 35 member 3
gene (SLC35A3) which encodes a UDP-N-
acetylglucosamine transporter. The gene is located on
bovine chromosome BTA3 (Thomsen et al., 2006). This
mutation results in the substitution of Valine by
Phenylalanine at position 180 (V180F), impairing the
function of the transporter membrane protein. This single
point mutation in SLC35A3 gene with no restriction site
can be analysed using single-stranded conformation
polymorphism (PCR-SSCP) (Orita et al., 1989). An
alternate method is PCR-Primer Introduced Restriction
Analysis (PCR-PIRA) which creates a Pst I restriction site
in the wild-type gene during PCR (Kanae et al., 2005).
Once the restriction site is created, restriction fragment
length polymorphism (RFLP) analysis can be performed.
This paper describes a unique case of CVM carrier which
was diagnosed by creating a restriction site for RFLP and
further confirmed by gene sequencing.
MATERIALS AND METHODS
Blood samples were collected into EDTA blood
collecting vials, from 60 apparently healthy Holstein bulls
maintained at different frozen semen banks for routine
investigation of autosomal diseases including CVM. DNA
was extracted from blood cells by phenol-chloroform
method (Sambrook et al., 1989) which was followed by
qualitative and quantitative verification of DNA. For
detection of point mutation in SLC35A3 gene, polymerase
chain reaction (PCR) was performed to create a restriction
site for Pst I (PCR-PIRA). As described by Kanae et al.,
(2005), the 233 bp DNA fragment was amplified by PCR,
which was set by adding sense primer consisting of 23
bases each Forward- 5'- CAC AAT TTG TAG GTC TCA
CTG CA -3' and an antisense primer 5'- CGA TGA AAA
AGG AAC CAA AAG GG -3'. The forward primer
introduced a PstI site in the amplified product from the
wild-type allele. PCR mix contained 10X PCR buffer, 10
mM dNTPs, 5U/ µl of Taq DNA polymerase (Kapa Bio
systems), 5 pM each of sense and antisense primer
(MWG-Biotech AG), 1.5 mM MgCl2, 80 ng genomic
DNA and sterilized distilled water to make a final volume
of 20µl. The following PCR conditions were used:
predenaturation for 5 min at 95°C, denaturation for 60s at
95°C, annealing at 56°C for 60s, extension at 72°C for
90s followed by 35 cycles of: 60s at 95°C, 60s at 56°C,
90s at 72°C, and ending with 10 min at 72°C for final
extension. The PCR products were subjected to
electrophoresis on a 1.5 % agarose gel with ethidium
bromide and visualized under a UV transilluminator to
verify the amplified products. Amplified PCR product
was digested with Pst 1 (Fermentas life science, India)
restriction enzyme as per the standard protocol provided
by the supplier. The digested fragments were
electrophoresed on a 3% agarose gel stained with
ethidium bromide and observed under an UV
transilluminator. In order to confirm the polymorphism in
exon 4 of SLC35A3 gene, the PCR product was
subsequently sequenced. As described by Jonathan
(2008), a simple method to treat PCR products prior to
sequencing using ExoSAP-IT was performed. After the
purification step of the PCR product, it was sequenced in
an Applied Biosystems 3130XL Automated Sequencer
using the ABI BigDye Ver 3.1. Sequence analysis
comparison of the gene sequences was performed using
the Codon Code Aligner 4.0.4 Software.
RESULTS AND DISCUSSION
The PCR-PIRA was performed with genomic DNA
samples extracted from the blood of 60 Holstein bulls
prior to RFLP analysis. The size of the PCR product was
233bp and it was subjected to further RFLP analysis using
Pst 1 restriction enzyme. In normal bulls, the PCR product
could be digested by the restriction enzyme yielding one
fragment of 212bp whereas in the carrier (heterozygous)
two fragments of 233 (uncut) and 212 bp were observed
(Fig. 1). One bull exhibited polymorphism (G→T) at
position 559 in exon 4 of SLC35A3 gene out of the 60
bulls tested (1.67%), a result that was far less than what
was reported earlier by Mahdipour et al., (2010) in Karan
Friesian cattle (23.1%) in India. The genotype and gene
frequency of heterozygous allele were estimated to be
0.0167 and 0.008, respectively. In order to confirm the
mutation, the PCR product was sequenced which
confirmed the polymorphism at position 559 (see the
chromatogram in Fig. 2). However, while comparing the
obtained sequence with NCBI sequence (Accession No.
NC_007301.5) and the documented sequence (Kanae et
al. 2005), two homozygous mutations (TG→CT) at 554
and 555 nucleotide positions were also found as indicated
in figure 2. To our surprise, these two mutations have no
obvious phenotypic effects on the bull as indicated by the
fact that the bull is apparently healthy at 18 months of
Inter J Vet Sci, 2013, 2(2): 61-64.
63
age. There are many reports of polymorphism at position
559 but none of them reported mutations at positions 554
and 555 as observed in our studies. In India, the studies on
Karan Fries, a breed that was developed by crossbreeding
between Holstein and Tharparkar (Bos indicus), also
exhibited polymorphism (T/G) at 559 in 12 out of 52 bulls
tested (Mahdipour et al., 2010). However, mutations at
positions 554 and 555, as observed by us, were not
reported by them.
Fig. 1: Analysis of CVM allele by the PCR-PIRA method.
Electrophoretogram of PstI digested PCR product generated by
amplification of genomic DNA using CVM specific primers.
Lane # 1: PCR product of 233bp, Lanes # 2, 4 & 5: 212bp band
for normal animals, Lane #3: 233bp and 212 bp bands for
heterozygous carrier animal. Lane # 6: O’Range Ruler™ 100bp
DNA ladder (Fermentas).
The defective allele for CVM had spread in Holstein
populations worldwide due to an extensive use of
breeding bulls that were later turned out to be carriers of
the defective gene. Konersmann et al., (2003) reported
that 13.2 % of 957 sires used for insemination in Germany
were diagnosed as carriers of CVM, while a prevalence of
31%, 32.5% and 23.07% was found in Denmark
(Thomsen et al., 2006), Japan (Nagahata et al., 2002) and
India (Mahdipour et al., 2010), respectively. Also, the
Holstein Association of the USA reported in 2006 that of
11868 bulls examined, 2108 were found to be carriers for
CVM i.e., 17.76 %, which was higher than our studies. In
their studies, no productive and reproductive differences
between carrier and normal animals were reported. The
only difference which was very obvious was the increase
in the rate of intra-uterine mortality. However, the risk of
return to service was also significantly higher in carrier
animals (Berglund et al., 2004). In this context, various
methods have been used for identification of single
nucleotide polymorphism in SLC35A3 gene. Agreholm et
al., (2001) performed genotyping of the CVM locus in a
template directed single-base extension assay and Kanae
et al., (2005) introduced PCR-primer introduced
restriction analysis (PCR-PIRA) for detecting a single
nucleotide mutation in any gene that lacks a restriction
site. Rusc and Kaminski (2007) used PCR-Single Strand
Confirmation Polymorphism (SSCP) method. Regardless
of the method used to detect CVM, the presence of CVM
carriers in population is of great concern to the breeders.
Our present study also revealed the prevalence of the
mutant gene in Indian Holsteins; it is therefore, advisable
to screen for all the possible autosomal recessive diseases,
especially in Holstein bulls before they are added to any
breeding programmes to avoid the risk of spread of any
such diseases.
In conclusion, the sequence comparison of PCR
products revealed two novel homozygous mutations
(TG→CT) at 554 and 555 nucleotide positions in addition
to the previously known heterozygous mutation at
position 559. To the best of our knowledge, this is the first
study to report the existence of both homozygous and
heterozygous mutations (TG→CT) at positions 554, 555,
and 559 in Indian Holstein cattle. Therefore, our study
reveals the presence of novel polymorphism in exon 4 of
SLC35A3 gene of Holstein which surprisingly has no
aberrant phenotypic effects. Hopefully, our novel findings
will alert animal scientists to look for new polymorphisms
despite a lack of effects on the phenotype of the animals.
Further studies are required to fully elucidate the impact
of these novel mutations on the productive and
reproductive performance of Holsteins.
Fig. 2: Comparing partial sequences of exon 4 of SLC35A3 gene obtained in our study (3rd line) with the sequences available on NCBI
site (1st line) and that documented by Kanae et al (2nd line), reveals the presence of two novel homozygous mutations (TG→CT) at
positions 554 & 555 together with a previously known heterozygous mutation (G→T) at position 559.
Inter J Vet Sci, 2013, 2(2): 61-64.
64
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