A Novel TaqI Polymorphism in the Coding Regionof the Ovine TNXB Gene in the MHC Class III Region:Morphostructural and Physiological Influences

Oyeyemi O. Ajayi • Mufliat A. Adefenwa • Brilliant O. Agaviezor •

Christian O. N. Ikeobi • Matthew Wheto • Moses Okpeku •

Samuel A. Amusan • Abdulmojeed Yakubu • Marcos De Donato •

Sunday O. Peters • Ikhide G. Imumorin

Received: 13 August 2012 / Accepted: 14 March 2013

� Springer Science+Business Media New York 2013

Abstract The tenascin-XB (TNXB) gene has antiadhesive effects, functions in

matrix maturation in connective tissues, and localizes to the major histocompatibility

complex class III region. We hypothesized that it may influence adaptive physio-

logical response through an effect on blood vessel function. We identified a novel

g.1324 A?G polymorphism at a TaqI recognition site in a 454 bp fragment of ovine

TNXB and genotyped it in 150 Nigerian sheep using PCR-RFLP. The missense

Electronic supplementary material The online version of this article (doi:10.1007/s10528-013-9622-9)

contains supplementary material, which is available to authorized users.

O. O. Ajayi � B. O. Agaviezor � C. O. N. Ikeobi � M. Wheto � S. A. Amusan � S. O. Peters

Department of Animal Breeding and Genetics, University of Agriculture, Abeokuta, Nigeria

O. O. Ajayi � M. A. Adefenwa � B. O. Agaviezor � A. Yakubu � M. De Donato �S. O. Peters � I. G. Imumorin (&)

Department of Animal Science, Cornell University, 267 Morrison Hall, Ithaca, NY 14853, USA

e-mail: igi2@cornell.edu

M. A. Adefenwa

Department of Cell Biology and Genetics, University of Lagos, Lagos, Nigeria

B. O. Agaviezor

Department of Animal Science and Fisheries, University of Port Harcourt, Port-Harcourt, Nigeria

M. Okpeku

Department of Livestock Production, Niger Delta University, Amassoma, Nigeria

A. Yakubu

Department of Animal Science, Nasarawa State University, Lafia, Nigeria

M. De Donato

Department of Biomedicine, Universidad de Oriente, Cumana, Venezuela

S. O. Peters

Department of Animal Science, Berry College, Mount Berry, GA 30149, USA

123

Biochem Genet

DOI 10.1007/s10528-013-9622-9

mutation changes glutamic acid (GAA) to glycine (GGA). Among SNP genotypes,

significant differences (P \ 0.05) were observed in body weight and fore cannon bone

length. Interaction effects of breed, SNP genotype, and geographic location had a

significant effect (P \ 0.05) on chest girth. The SNP genotype was significantly

(P \ 0.05) associated with physiological traits of pulse rate and skin temperature. The

observed effect of this novel polymorphism may be mediated through its role in

connective tissue biology, requiring further association and functional studies.

Keywords Tenascin-XB gene � Sheep � TaqI � Nigeria � Morphology �Physiological status

Introduction

Tenascin-XB, encoded by the TNXB gene in the major histocompatibility complex

(MHC) class III region in sheep, is a large extracellular matrix protein that plays a

key role in the deposition of collagen by dermal fibroblasts, with its expression

observed in the dermis of the skin and in the connective tissues of heart and skeletal

muscles (Burch et al. 1997; Matsumoto et al. 1994). Similarly, extracellular matrix

molecules like tenascin-X (TNX) and fibrilin encoded by TNXB and FBN1 genes are

known to be expressed in connective tissues of muscles, tendons, and joint capsules

(Bosman and Stamenkovic 2003). Given these observations, mutations in genes

encoding these extracellular matrix molecules may alter structure and function of

muscle and tendon connective tissues and thus contribute to both muscle weakness

and hypermobility or contractures (Voermans et al. 2009).

Qin et al. (2008) reported that the class III region of the sheep and human MHC

are very similar with respect to length, location, and orientation between the class I

and class II regions and their gene composition. Loci within the class III region are

often highly conserved; in general terms they evolved prior to the class I and II loci

(Kumanovics et al. 2003). TNXB, an important gene in the gene-rich subregion of

the MHC class III of mammals, was discovered because of its 30 overlap with the

human CYP21A2 gene (Morel et al. 1989). Tenascin-X deficiency, in recessive or

dominant form, has been proposed as a cause of hypermobility type and tenascin-X

deficiency type Ehlers-Danlos Syndrome (EDS) in humans (Schalkwijk et al. 2001;

Zweers et al. 2004). Symptoms range from musculoskeletal pain to easy bruising

and velvety skin (Beighton et al. 1998) in hypermobility type and muscle weakness,

skin hyperextensibility, velvety skin, and easy bruising (Voermans et al. 2007) in

tenascin-X deficiency type EDS.

Although tenascin-X protein is abundantly expressed in almost all the connective

tissues (Zweers et al. 2003), it is not expressed in lymphoid organs. Therefore, the

apparently incongruous location of TNXB within the MHC class III region prompted us

to hypothesize its association with morphostructural and physiological indices because

any of the MHC class III genes could contain sequences that affect productivity either

alone or in linkage disequilibrium with other disadvantageous alleles in other genes

(Walsh et al. 2003). In this study we identified a polymorphism defined by a TaqI

restriction site in the coding region represented by a 454 bp fragment of the ovine TNXB

Biochem Genet

123

gene in Nigerian sheep. This was genotyped in 150 animals and analyzed for association

of genotypes at this locus with morphostructural and physiological indices.

Materials and Methods

Animals and DNA Samples

Samples were collected from 150 randomly selected, sexually mature sheep, of

approximately the same age ([15.5 months), representing the four major breeds of

sheep in Nigeria: 40 Uda sheep, 40 Yankasa, 40 Balami, and 30 West African

Dwarf. The sheep were sampled from farms and markets across Nigeria, in the

states of Bauchi and Borno in the northeast, Kano and Sokoto in the northwest,

Plateau in the north central, and Ogun in the southwest zones of Nigeria (Fig. 1),

according to the geographic distribution of the breeds published by Blench (1999).

The animals were reared under the semi-intensive system of management.

Blood (5–7 mL) was collected from each sheep by jugular venipuncture into

heparinized tubes and stored in a battery-powered refrigerator before being

transferred to the laboratory for DNA isolation. Genomic DNA was isolated using

Fig. 1 Geographic zones of Nigeria. Sheep were sampled for this study from the states of Bauchi andBorno in the northeast, Kano and Sokoto in the northwest, Plateau in the north central, and Ogun in thesouthwest zones. Inset Location of Nigeria in relation to sub-Saharan Africa

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123

the ZymoBead Genomic DNA kit (Zymo Research Corp., Irvine, CA, USA). DNA

yield was quantified and its quality assessed using a Nanodrop ND-100 UV/Vis

Spectrophotometer (Nanodrop Technologies, DE, USA).

PCR Amplification, Sequencing, and Multiple Sequence Alignment

The ovine TNXB gene was amplified using forward 50-TCACCACAACAAAGAT-30

and reverse 50-GGACCTTGAAGGAGTCAAAT-30 primers (Qin et al. 2008) to

generate a fragment of 454 bp. PCR amplifications were carried out in a Techne

thermal cycler in a total reaction volume of 20 lL, containing an average of 20 ng

DNA and 10 pmol each primer, in AccuPower PCR Premix (Bioneer Corp., Alameda,

CA, USA). The PCR cycling conditions were denaturation at 95�C for 4 min; followed

by 35 amplification cycles of denaturation at 94�C for 30 s, annealing at 60�C for

1 min, and extension at 72�C for 3 min; and a final extension at 72�C for 10 min. PCR

products were detected on 1.5% agarose gel including ethidium bromide and

photographed under UV light. The amplified fragments were sequenced using the

same PCR primers with the Applied Biosystems Automated 3730 DNA Analyzer, Big

Dye Terminator chemistry, and AmpliTaq-FS DNA polymerase.

The sequences obtained were verified by comparing them to published sequences

of sheep and other species in Genbank. Multiple sequence alignment of deduced

protein sequences from humans, mice, and cows was accomplished using multiple

sequence comparison by log-expectation (Muscle) software (http://www.ebi.ac.uk/

Tools/msa/muscle/), which generally achieves better average accuracy and better

speed than ClustalW2 or T-Coffee, depending on the chosen options.

PCR-RFLP Genotyping of Sheep TNXB

To identify the SNP in the TNXB gene, we aligned the DNA sequences derived from

nine animals using the ClustalW program (http://www.ebi.ac.uk/Tools/clustalw/).

The nonsynonymous SNP in codon 106 was detected. This SNP was analyzed by PCR-

RFLP in 150 sheep. TaqI (Fermentas Life Sciences, Glen Burnie, MD, USA), which

recognizes the palindromic tetranucleotide sequence ;TCGA, was used for restriction

enzyme digestion according to NEB cutter version 2.0 (Vincze et al. 2003). About 10

lL of each PCR product, 2 lL 109 fast digest green buffer, 1 lL TaqI fast digest

enzyme, and 17 lL nuclease-free water were added in this order in a PCR tube and

incubated inside a heat block for 10 min. The restriction fragments were separated by

electrophoresis in 2.5% agarose gel dissolved in 19 TBE and stained with 0.5 lg/mL

ethidium bromide. Genotypes were scored from restriction fragments.

Morphostructural and Physiological Measurements

All measurements were made on each animal within a 2-week sampling period when

weather conditions were similar across the geographic regions (variation in environ-

mental conditions was ± 5%). Each animal was measured for body weight (kg) and

eight linear body measurements (cm), following standard procedures and anatomical

reference points (Yakubu et al. 2010). Height at withers was taken as the distance from

Biochem Genet

123

the surface of the ground to the withers; rump height was measured vertically from the

ground to the top of the pelvic girdle; body length was from the tip of the mouth to the

pin bone; ear length was from the base of the ear to its tip; fore cannon bone length

measured the lower part of the leg extending from the hock to the fetlock; chest girth

was the body circumference just behind the forelegs; chest depth was the dorsal–ventral

distance between the most dorsal point of the withers and the ventral surface of the

sternum; and rump width was the distance between the two tuber coxae. Rectal

temperature was taken on each animal using a digital thermometer inserted into the

rectum at the display of the lowest readable temperature (�C) and removed at the sound

of the alarm signal. Respiratory rate was determined by counting the number of

movements of the abdomen per minute using a stop watch. Pulse rate was determined

by placing a stethoscope just below the chest region and counting the number of pulses

per minute using a stop watch. The heat stress index was derived from the relationship

between pulse rate and respiratory rate together with their normal average values using

the formula of Oladimeji et al. (1993), H = (AR/AP) 9 (NP/NR), where H is the heat

stress index, AR is the average respiratory rate value, AP is average pulse rate value,

NP is normal pulse rate value, and NR is normal respiratory rate.

Statistical Analysis

Morphological and physiological trait data were analyzed using the generalized

linear model in the SAS (2009) software package. In the linear model,

Yijkl ¼ lþ Si þ Aj þ Bk þ Ll þ ðSAÞij þ ðSLÞil þ ðABÞjk þ ðALÞjl þ ðBLÞkl

þ ðSABÞijk þ ðALSÞjli þ ðBLSÞkli þ eijkl;

Yijkl is the trait of interest, l is the overall mean for the trait of interest, Si is SNP genotype

(i = 1–3), Aj is fixed effect of jth breed (j = 1–4), Bk is fixed effect of kth sex (k = 1–2),

Ll is fixed effect of lth geographic location (l = 1–4), (SA)ij is interaction effect of SNP

genotype and jth breed, (SL)il is interaction effect of SNP genotype and lth geographic

location, (AB)jk is interaction effect of jth breed and kth sex, (AL)jl is interaction effect of

jth breed and lth geographic location, (BL)kl is interaction effect of kth sex and lth

geographic location, (SAB)ijk is interaction effect of SNP genotype with breed and sex,

(ALS)jli is interaction effect of breed with geographic location and SNP genotype,

(BLS)kli is interaction effect of sex with geographic location and SNP genotype, and eijkl

is the random error associated with each record (normally, independently, and identically

distributed with zero mean and constant variance). Significant means were separated

using Duncan’s multiple range test procedure from the same statistical software. Inter-

actions that were not significant were removed from the model. Correlations were also

computed to ascertain relationships among measurable traits.

Results

The TNXB gene fragment was successfully amplified with primers designed by Qin

et al. (2008) to produce a DNA fragment of 456 bp after amplicon sequencing. The

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123

sequences have been submitted to Genbank (awaiting accession numbers). The

alignment of sequences led to the discovery of the g.1324 A?G mutation (Genbank

accession no. EF 197845.1) in the coding region. The mutation was a transition at a

TaqI restriction enzyme site. Blastn analysis of the TNXB sequence in Nigerian

sheep revealed 99% nucleotide identity with Ovis aries (EF 197845.1). Prediction of

the amino acid sequence from the TNXB sequence in Nigerian sheep showed 90%

similarity with that of Bos taurus (acc. no. NP_777128.1), and this led to the

discovery of a p.E2000G missense mutation resulting in a change from glutamic

acid (GAA) to glycine (GGA). Enzymatic digestion of PCR products at the TaqI

polymorphic site showed AA homozygotes with two digested fragments of 316 and

140 bp, GG homozygotes with an undigested fragment of 456 bp, and AG

heterozygotes with all three bands.

The observed polymorphic site corresponded to lysine from the complete

alignment by Muscle of the human, mouse, and bovine protein sequences. Tracing

this position, it was observed that in humans and sheep, glutamic acid (E) was

present, and that position corresponded to amino acids K in cows and W in mice,

proving that the amino acid position is not conserved.

Effects of SNP Genotypes on Morphology

The highest frequency of the AA genotype (0.5) was found in Uda sheep, West

African Dwarf sheep had the highest AG frequency (0.6), and Uda sheep also had

the highest GG frequency (0.15). The highest frequency of the A allele was found in

Uda sheep, and the lowest was found in West African Dwarf sheep. The frequencies

of the A allele in the Yankasa and Balami breeds were intermediate between these

two extremes. AG was the most frequent genotype in the breeds studied, except for

the Uda breed. Chi-square values observed to be significant among the four breeds

were suggestive of Hardy–Weinberg equilibrium (Table 1).

The SNP genotype had significant effects (P \ 0.05) on body weight and fore

cannon bone length, with the highest values observed in the AG genotype

(36.14 ± 1.72 kg and 14.57 ± 0.54 cm, respectively). No significant differences

(P [ 0.05) were observed among the SNP genotypes for the remaining morpho-

logical traits examined (Table 2). For all the morphological indices, the highest

values were observed in the AG genotype. The lowest values were observed in the

GG genotype, except for rump width and ear length. The differences observed

among the AA, AG, and GG genotypes were not significant, however, except for

body weight and fore cannon.

The interaction effects of breed and SNP genotype (Table 3) did not significantly

affect morphological indices, except for chest girth. The highest value for chest

girth (81.52 ± 3.00 cm) was observed in Uda with the genotype AG, and the lowest

value (56.94 ± 2.46 cm) was seen in West African sheep homozygous for A. A

significant effect (P \ 0.01) on chest girth was also observed in the interaction

between location and SNP genotype (Supplementary Table 1), with the highest

value (81.80 ± 2.75 cm) observed in sheep from the north central region with the

AG genotype; the lowest value (60.15 ± 2.99 cm) was observed in sheep from the

southwest with the GG genotype.

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Interaction effects of breed, sex, and SNP genotype on morphological indices

(Supplementary Table 2) revealed significant effects (P \ 0.01) for rump height,

chest girth, rump width, and body weight. For body weight, the highest value

(54.83 ± 15.41 kg) was observed in female Balami with the AG genotype and the

lowest (15.42 ± 1.96 kg) in female West African Dwarf with the AA genotype. The

highest value (87.62 ± 12.21 cm) for rump height was observed in female Balami

with the AG genotype and the lowest (48.58 ± 2.58 cm) in female West African

Dwarf with the AA genotype. Chest girth was also highest (88.75 ± 4.21 cm) in

male Balami with the AA genotype; the lowest value (55.67 ± 3.16 cm) was

observed in female West African Dwarf homozygous for the AA genotype.

Interaction effects of breed, location, and SNP genotype were observed to be

significant (P \ 0.01) only for fore cannon bone length and chest girth (P \ 0.05)

(Table 4). North central Balami sheep with the AG genotype had the greatest chest

girth (90.60 ± 0.40 cm), and Yankasa from the northeast with the AG genotype had

the smallest (35.25 ± 26.75 cm). Finally, Yankasa sheep from the southwest with

the AG genotype had the longest fore cannon bone (18.57 ± 1.03 cm).

Table 1 Frequency of g1324 A?G SNP at the TNXB locus among four Nigerian sheep breeds

Breed Genotype Allele frequency v2

Number Frequency

AA AG GG AA AG GG A G

West African Dwarf 8 18 4 0.270 0.600 0.130 0.570 0.430 1.4748*

Uda 20 14 6 0.500 0.350 0.150 0.67 0.325 1.6367*

Yankasa 16 21 3 0.400 0.525 0.075 0.660 0.340 1.2112*

Balami 16 19 5 0.400 0.475 0.125 0.640 0.360 0.0307*

AA dominant, GG recessive

* Significant at P \ 0.05; indicates Hardy–Weinberg equilibrium

Table 2 Effect of SNP genotype on body weight and other morphological traits of Nigerian sheep breeds

Trait Genotype, mean ± SEM Level of

significanceAA (n = 60) AG (n = 72) GG (n = 18)

Body weight (kg) 32.56 ± 1.69ab 36.14 ± 1.72a 28.92 ± 2.5b P \ 0.05

Withers height (cm) 65.55 ± 1.43a 65.05 ± 1.65a 63.92 ± 2.71a

Rump height (cm) 67.34 ± 1.31a 67.78 ± 1.29a 65.81 ± 2.48a

Body length (cm) 77.46 ± 3.85a 83.27 ± 2.9a 76.76 ± 7.78a

Ear length (cm) 14.2 ± 0.42a 15.25 ± 0.54a 14.47 ± 0.89a

Fore cannon bone length (cm) 13 ± 0.56b 14.57 ± 0.5a 12.77 ± 1.06b P \ 0.05

Chest girth (cm) 72.41 ± 1.49a 73.32 ± 1.5a 72.92 ± 2.23a

Chest depth (cm) 26.39 ± 1.84a 28.36 ± 1.63a 25.37 ± 2.61a

Rump width (cm) 13.2 ± 0.32a 13.5 ± 0.23a 13.3 ± 0.5a

a, b Means in the same row bearing the same superscripts do not differ significantly (P [ 0.05)

Biochem Genet

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Biochem Genet

123

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113.5

0±

1.1

917.6

3±

0.8

0ab

c18.0

0±

1.2

9

AG

45.0

0±

3.6

794.4

3±

1.6

265.7

5±

4.8

070.1

8±

1.6

774.2

5±

1.7

2ab

cd

e30.8

9±

0.9

313.0

0±

0.4

318.5

7±

1.0

3a

17.2

1±

0.8

8

Bal

ami

NE

AA

32.8

0±

3.6

834.3

0±

3.3

865.9

2±

4.8

269.6

2±

2.5

373.7

0±

3.8

5ab

cd

e11.5

6±

0.9

013.9

8±

0.8

56.8

0±

0.3

7h

14.7

±0.5

8

AG

33.6

4±

2.9

956.3

5±

8.8

066.3

3±

3.9

172.5

4±

1.6

974.9

3±

2.4

7ab

cd

e31.0

6±

9.6

113.8

5±

0.4

811.7

3±

1.3

2cd

efg

h14.0

5±

0.9

0

GG

26.5

0±

6.5

037.7

5±

4.7

565.5

0±

2.5

067.2

0±

3.2

073.5

0±

2.5

0ab

cd

e11.7

5±

0.7

512.4

5±

1.7

57.2

5±

0.2

5g

h15.7

5±

0.7

5

NC

AA

29.6

7±

7.9

654.4

0±

11.3

670.9

0±

8.2

564.8

7±

5.3

969.0

0±

9.0

7b

cd

e16.2

3±

3.3

013.8

7±

1.8

39.5

3±

2.3

8efg

h14.3

3±

2.0

3

AG

40.5

0±

1.5

042.7

5±

0.2

576.6

0±

0.6

078.6

0±

3.6

090.6

0±

0.4

0a

14.3

0±

0.8

015.1

0±

1.7

07.3

0±

0.1

0g

h12.2

0±

0.2

0

NW

AA

36.4

3±

5.6

394.3

6±

12.3

272.0

0±

2.1

971.2

9±

3.8

376.0

7±

4.6

1ab

cd

e28.7

0±

3.1

914.2

0±

1.1

512.8

9±

1.2

7b

cd

efg

11.8

6±

0.5

5

AG

77.1

7±

28.9

117.8

3±

3.4

084.5

0±

5.8

8101.0

±24.1

778.2

5±

2.8

6ab

cd

e35.0

0±

1.1

815.5

0±

1.0

515.3

3±

0.7

1ab

cd

ef

12.6

7±

1.1

5

GG

47.5

±4.5

0113.5

±4.5

073.5

0±

1.5

076.5

0±

0.5

083.2

5±

0.2

5ab

c36.5

0±

3.5

015.0

0±

0.0

016.0

0±

1.0

0ab

cd

e11.5

±0.5

0

Lev

elof

signifi

cance

P\

0.0

5P

\0.0

1

Val

ues

are

leas

tsq

uar

em

eans

±st

andar

der

ror;

body

wei

ght

inkg;

all

oth

erm

easu

rem

ents

incm

a–

hM

eans

inth

esa

me

colu

mn

bea

ring

the

sam

esu

per

scri

pts

do

not

dif

fer

signifi

cantl

y(P

[0.0

5)

Biochem Genet

123

Effects of SNP Genotypes on Physiological Indices

SNP genotype had significant effects (P \ 0.05) on pulse rate and skin temperature

(Table 5). The pulse rate was highest (119.11 ± 6.67 beats/min) among GG

genotypes and lowest (104.96 ± 4.21 beats/min) for AG genotypes. Skin temper-

ature was highest (38.62 ± 0.15�C) for the AG and lowest (37.23 ± 1.43�C) for the

GG genotypes. The interaction effect for breed and SNP genotype (Table 6) was

significant (P \ 0.05) for skin temperature, with the highest value (39.03 ± 0.15�C)

occurring in Uda sheep with the AG genotype and the lowest in West African Dwarf

with the AA genotype.

Table 5 Heat tolerance traits as affected by SNP genotype in Nigerian sheep

Trait Genotype

AA (n = 60) AG (n = 72) GG (n = 18)

Pulse rate (beats/min) 110.80 ± 4.00ab 104.96 ± 4.21b 119.11 ± 6.67a

Respiratory rate (breaths/min) 51.00 ± 2.19 53.81 ± 3.11 57.56 ± 2.40

Rectal temperature (�C) 39.13 ± 0.16 39.26 ± 0.09 39.16 ± 0.14

Heat stress index 2.03 ± 0.03 2.00 ± 0.03 2.00 ± 0.05

Skin temperature (�C) 38.20 ± 0.48ab 38.62 ± 0.15a 37.23 ± 1.43b

Values are means ± standard errora, b Means in the same row bearing the same superscripts do not differ significantly (P [ 0.05)

Table 6 Interaction effects of breed and SNP genotype on heat tolerance traits in Nigerian sheep

Breed Genotype Trait

Pulse rate

(beats/min)

Respiratory

rate

(breaths/min)

Rectal

temperature

(�C)

Heat stress

index

Skin

temperature

(�C)

West African

Dwarf

AA 101.00 ± 7.74 63.50 ± 10.70 39.51 ± 0.27 1.92 ± 0.06 35.48 ± 3.50b

AG 113.17 ± 7.11 75.11 ± 8.34 39.28 ± 0.19 1.91 ± 0.04 38.21 ± 0.48a

GG 109.00 ± 7.19 69.00 ± 3.79 39.28 ± 0.23 1.89 ± 0.08 38.85 ± 0.24a

Uda AA 116.60 ± 6.73 50.20 ± 1.63 39.15 ± 0.17 2.04 ± 0.05 38.39 ± 0.27a

AG 116.29 ± 8.80 51.43 ± 3.60 39.42 ± 0.18 2.04 ± 0.05 39.03 ± 0.15a

GG 104.00 ± 11.03 54.00 ± 4.10 38.87 ± 0.17 1.97 ± 0.09 37.92 ± 0.67a

Yankasa AA 98.50 ± 8.31 43.75 ± 4.37 39.35 ± 0.11 2.05 ± 0.06 38.74 ± 0.12a

AG 77.62 ± 6.76 39.24 ± 4.22 39.37 ± 0.13 2.00 ± 0.05 38.81 ± 0.13a

GG 128.75 ± 12.85 57.33 ± 5.81 39.50 ± 0.21 2.04 ± 0.09 38.77 ± 0.20a

Balami AA 120.75 ± 7.64 53.00 ± 3.44 39.79 ± 0.53 2.04 ± 0.05 38.79 ± 0.23a

AG 119.05 ± 7.53 51.47 ± 4.00 38.98 ± 0.22 2.06 ± 0.05 38.47 ± 0.24a

GG 140.00 ± 14.42 52.80 ± 2.94 39.24 ± 0.38 2.11 ± 0.07 34.18 ± 5.20b

Level of significance P \ 0.05

Values are least square means ± standard errora, b Means in the same column bearing the same superscripts do not differ significantly (P [ 0.05)

Biochem Genet

123

Discussion

Genetic variability within the MHC region has been discovered to influence

resistance or susceptibility to diseases and productivity of sheep (Larruskain et al.

2012; Hui et al. 2012; Hickford et al. 2011; Castillo et al. 2011; Keane et al. 2007;

Geldermann et al. 2006). In the present study, the g.1324G?A mutation resulted in

an amino acid change from hydrophilic glutamic acid to hydrophobic glycine,

which may plausibly alter the structure of the expressed tenascin-XB protein.

Qin et al. (2008) suggested that despite structural similarity between the human and

sheep MHC, it is likely that further studies would show species-specific differences

between the class III members of the sheep MHC and those of other species. Since this

is the first study of a mutation in this gene associated with morphostructural and

physiological indices, it is difficult to compare our results with similar studies. It is

interesting to note that significant differences in morphological traits for the AG

genotype indicated heterozygous superiority similar to the overdominant effect

inferred from studies involving MHC genes in mice coinfected with Salmonella and

Theiler’s murine encephalomyelitis virus (McClelland et al. 2003).

The prominent expression of tenascin-X protein as revealed by intense staining in

the muscle (Matsumoto et al. 1994) lends credence to its ubiquitous expression in

collagen, which is the most abundant protein in vertebrates. Muscle cells not only

play a great role in the body’s internal life, helping other systems to function, but

also may likely affect body weight, possibly through collagen deposits. Mutations in

the TNXB gene have been associated with reduced deposition of collagen (Egging

et al. 2006) and muscle weakness (Voermans et al. 2009). The mutant genotype

(GG) observed in Nigerian sheep may plausibly contribute to the inability of

individuals of that genotype to cover large distances in search of good-quality

forage, possibly due to musculoskeletal pain, a situation decidedly exacerbated by

the extensive or semi-intensive system of management practiced by most local

farmers in sub-Saharan Africa. This may be supported by observations that tenascin-

X interacts with types I, III, and V fibrillar collagen molecules when in native

conformation, suggesting that tenascin-X, via trimerization and multiple interac-

tions with components of collagenous fibrils, plays a crucial role in the organization

of extracellular matrices (Lethias et al. 2006). Although the exact mechanism of

how TNXB contributes to variations in body weight remains obscure, its effect in

regulating the structure and mechanical properties of connective tissues has been

well documented (Elefteriou et al. 1997, 1999; Lethias et al. 2006; Egging et al.

2007; Jollymore et al. 2009). For example, results from using single-molecule

atomic force microscopy to investigate the mechanical properties of bovine

tenascin-X indicated that it is an elastic protein, in that the fibronectin type III

domains can unfold under a stretching force and refold to regain their mechanical

stability when the force is removed. Mechanical stability, mechanical unfolding

kinetics, and contour length increment upon domain unfolding may be disrupted by

mutations yet uncharacterized (Jollymore et al. 2009). This is further buttressed by

the presence of distinct tenascin-X proteins composed of distinct molecular species

that retain functional activity (Egging et al. 2007), although none of these proteins

found in humans have been reported in sheep.

Biochem Genet

123

The SNP genotype had significant effects on pulse rate and skin temperature.

This could be attributed to a reduction in the collagen deposition, which greatly

affects the tensile strength of the blood vessels, thereby leading to stiffness and

hardening of the arteries, arterioles, capillaries, and veins (Lethias et al. 2006;

Egging et al. 2007; Jollymore et al. 2009). Reduction in the amount of blood

circulating around the body due to the stiffness of the blood vessels was

compensated for by an increase in the pulse rate. Our hypothesis is supported by the

ubiquitous and prominent expression of tenascin-X protein in blood vessels and

heart muscles, validated by intense staining in these regions (Matsumoto et al.

1994). Similarly, loss of tenascin-X from the vessel wall could affect collagen fibril

and elastic fiber properties, thereby causing weakness of the aortic wall (Zweers

et al. 2004), consistent with recent findings that a TNX-knockout mouse model

showed muscle weakness, mild myopathic features on histology, and functional

upregulation of genes encoding proteins involved in extracellular matrix degrada-

tion and synthesis (Voermans et al. 2011).

Tenascin-X expression in the heart during embryogenesis and adulthood suggests

its vital role in the development of the heart (Matsumoto et al. 1994, 2012; Burch

et al. 1995; Jing et al. 2011). Absence of TNXB in gene-targeted mice, however, did

not result in an apparent cardiac phenotype (Mao et al. 2002), but skin biopsies of

tenascin-X deficient humans showed severe abnormalities in elastic fibers and

collagen, which are also principal components of blood vessels (Zweers et al. 2004).

There is currently no published work on TNXB gene mutations on physiological

indices in ovine species. Therefore, its possible use as a candidate gene that affects

the rhythmic expansion of blood vessels in sheep awaits further investigation. The

reason for the obvious effect of SNP genotypes and interaction effect of breed and

SNP genotype on skin temperature is largely unknown. Significant results obtained

for interactions of breed with SNP genotype and location with SNP genotype on

chest girth suggest the sensitivity of the expressed forms of TNXB genotypes to

environmental components and breed differences. This may reflect a role in skeletal

muscle, heart muscle, esophagus, and gut, given the large amounts of deposited

collagen in these organs (Matsumoto et al. 1994; Burch et al. 1997). The observed

effect of this polymorphism may be mediated through its role in connective tissue

biology, and it may be a potential molecular genetic marker for morphological

growth and physiological indices when validated in a larger population supported by

functional studies. In addition, further studies are required to test the biochemical

effects of TNXB isoforms on quantitative traits.

Acknowledgments Financial support from the College of Agriculture and Life Sciences, Cornell

University, Ithaca, New York, is gratefully acknowledged.

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