USING e-ANNOTATION TOOLS FOR ELECTRONIC PROOF CORRECTION

Once you have Acrobat Reader open on your computer, click on the Comment tab at the right of the toolbar:

This will open up a panel down the right side of the document. The majority of

tools you will use for annotating your proof will be in the Annotations section,

pictured opposite. We’ve picked out some of these tools below:

1. Replace (Ins) Tool – for replacing text.

Strikes a line through text and opens up a text

box where replacement text can be entered.

How to use it

‚ Highlight a word or sentence.

‚ Click on the Replace (Ins) icon in the Annotations

section.

‚ Type the replacement text into the blue box that

appears.

2. Strikethrough (Del) Tool – for deleting text.

Strikes a red line through text that is to be

deleted.

How to use it

‚ Highlight a word or sentence.

‚ Click on the Strikethrough (Del) icon in the

Annotations section.

3. Add note to text Tool – for highlighting a section

to be changed to bold or italic.

Highlights text in yellow and opens up a text

box where comments can be entered.

How to use it

‚ Highlight the relevant section of text.

‚ Click on the Add note to text icon in the

Annotations section.

‚ Type instruction on what should be changed

regarding the text into the yellow box that

appears.

4. Add sticky note Tool – for making notes at

specific points in the text.

Marks a point in the proof where a comment

needs to be highlighted.

How to use it

‚ Click on the Add sticky note icon in the

Annotations section.

‚ Click at the point in the proof where the comment

should be inserted.

‚ Type the comment into the yellow box that

appears.

USING e-ANNOTATION TOOLS FOR ELECTRONIC PROOF CORRECTION

5. Attach File Tool – for inserting large amounts of

text or replacement figures.

Inserts an icon linking to the attached file in the

appropriate place in the text.

How to use it

‚ Click on the Attach File icon in the Annotations

section.

‚ Click on the proof to where you’d like the attached

file to be linked.

‚ Select the file to be attached from your computer

or network.

‚ Select the colour and type of icon that will appear

in the proof. Click OK.

6. Drawing Markups Tools – for drawing

shapes, lines and freeform annotations on

proofs and commenting on these marks.

Allows shapes, lines and freeform annotations to be

drawn on proofs and for comment to be made on

these marks.

How to use it

" Click on one of the shapes in the Drawing Markups

section.

" Click on the proof at the relevant point and draw the

selected shape with the cursor.

" To add a comment to the drawn shape, move the

cursor over the shape until an arrowhead appears.

" Double click on the shape and type any text in the

red box that appears.

In silico identification and three-dimensional modellingof the missense mutation in ADAMTS21 in a sheep flockwith dermatosparaxis

Luis V. Monteagudo*, Luis M. Ferrer†, Elena Catalan-Insa*†, Demetris Savva‡, Liam J. McGuffin‡ and

Maria T. Tejedor*

Department of *Anatomy, Embryology and Genetics, and †Animal Pathology, School of Veterinary Science, Miguel Servet 177, 50013, Zaragoza,

Spain

‡School of Biological Sciences, University of Reading, Whiteknights, Reading, Berkshire, RG6 6AH, UK2

Correspondence: Luis V. Monteagudo, Department of Anatomy, Embryology and Genetics, School of Veterinary Science, Miguel Servet 177,

50013 Zaragoza, Spain. E-mail: monteagu@unizar.es3

Background – Dermatosparaxis (Ehlers–Danlos syndrome in humans) is characterized by extreme fragility of

the skin. It is due to the lack of mature collagen caused by a failure in the enzymatic processing of procollagen I.

We investigated the condition in a commercial sheep flock.

Hypothesis/Objectives – Mutations in the ADAM metalloproteinase gene, specifically at the thrombospondin

type 1 motif 2 (ADAMTS2) locus, are involved in the development of dermatosparaxis in humans, cattle and the

dorper sheep breed; consequently, this locus was investigated in the flock.

Animals – A single affected lamb, its dam, the dam of a second affected lamb and the rams in the flock were

studied.

Methods – DNA was purified from blood, PCR primers were used to detect parts of the ADAMS2 gene and

nucleotide sequencing was performed using Sanger’s procedure. Skin samples were examined using standard

histology procedures.

Results – A missense mutation was identified in the catalytic domain of ADAMTS2. The mutation is predicted to

cause the substitution in the mature ADAMTS2 of a valine molecule by a methionine molecule (V15M) affecting

the catalytic domain of the enzyme. Both the ‘sorting intolerant from tolerant’ (SIFT) and the PolyPhen-2 method-

ologies predicted a damaging effect for the mutation. Three-dimensional modelling suggested that this mutation

may alter the stability of the protein folding or distort the structure, causing the protein to malfunction.

Conclusions and clinical importance – Detection of the mutation responsible for the pathology allowed us to

remove the heterozygote ram, thus preventing additional cases in the flock.

Introduction

Dermatosparaxis (Ehlers–Danlos syndrome type VIIC in

humans) is characterized by extreme fragility of the con-

nective tissue, resulting in severe skin tearing during nor-

mal life. It is an autosomal recessive inherited disease,

associated either with deletions or with single nucleotide

substitutions resulting in the appearance of premature

stop codons in the ‘a disintegrin6 and metalloproteinase

with thrombospondin motifs 2’ (ADAMTS2) gene in

humans, cattle and dorper sheep.1,2 Such mutations

impair the normal processing of procollagen I by ADAM-

TS2, resulting in accumulation of precursor molecules

which fail to provide the normal strength to fibrils.1 Con-

sequently, the skin tears following minimal tension during

normal management practices.

A case of dermatosparaxis in a crossbreed ovine flock

was referred to us. We had access to the affected lamb,

its mother, the rams in the flock and a second ewe which

had previously given birth to a lamb (not available) with

identical clinical signs. Affected lambs suffered extensive

wounds and presented with large skin flaps unrelated to

accidental causes. Attempts to suture the flaps were

unsuccessful because silk suture produced cuts and

failed to join the flaps to the rest of the skin. Severe

wound infections quickly led to a critical situation, and the

lambs were euthanized for humanitarian reasons. Post-

mortem examination showed extreme fragility of the skin

(Figure 1 and Video S1). Parents appeared normal, sup-

porting recessive inheritance.

Predictions of the nucleotide sequence coding for

ovine ADAMTS2 are available in GenBank (accession

NW_004080168).3 In cattle (accession NW_003103998),4

exon 2 corresponds to the ADAMTS2 pro-domain, while

the mature enzyme is encoded by subsequent exons,

starting with the catalytic domain (exons 3–8).2 BLAST

alignment shows that the DNA for this domain is

highly conserved in cattle and sheep.5 Given that

premature stop codons in ADAMST2 are responsible for

Accepted 9 August 2014

Sources of Funding: This study was self-funded.

Conflict of Interest: No conflicts of interest have been declared.

Luis V. Monteagudo and Luis M. Ferrer are joint first authors.

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

61

62

© 2014 ESVD and ACVD, Veterinary Dermatology 1

Vet Dermatol 2014 DOI: 10.1111/vde.12178

VD

E12178

Dispa

tch:

6.9.14

CE:Ash

okra

jD.

JournalCode

ManuscriptNo.

No.

ofpa

ges:

4PE:Sha

rmila

dermatosparaxis in dorper sheep and cattle, this was cho-

sen as the candidate gene to investigate this flock.1,2

Materials and methods

Animals

A flock of 200 crossbred sheep was initially formed by acquiring

ewes and rams from different farms; breeding animals were then

replaced by animals bred on the same farm.

DNA studies

DNA was isolated from peripheral blood. Briefly, 15 lL of blood was

lysed in 180 lL of TE (10 mM Tris–Cl, 0.1 mM EDTA, pH 8). After

centrifugation for 2 min (12,000g), pellets were resuspended in

180 lL of 5% w/v Chelex resin in TE, incubated for 20 min at 100°C

and recentrifuged; the supernatant was stored at �20°C until DNA

amplification (25–50% of the reaction volume).

PCR primers were designed for the conserved region of the first

segments of the ADAMTS2 catalytic domain (Table 1). Nucleotide

sequences of PCR products were determined using Sanger’s proce-

dure.

Histology

Tissues were fixed in formaldehyde, embedded in paraffin, cut in

sections (5 lm thick) and stained with haematoxylin and eosin after

paraffin removal.

In silico predictions and three-dimensional analysis

The ‘sorting intolerant from tolerant’ (SIFT) algorithm and the

PolyPhen-2 approach were applied to predict the effects of the

single nucleotide polymorphism (SNP) found in the ovine

ADAMTS2 gene.6–8 The three-dimensional (3D) structures of the

wild-type (WT) and mutant sequences were modelled on the

IntFOLD2 server, predicting disordered (natively unstructured)

regions, assigning putative domain boundaries and indicating

likely binding sites and ligand interactions;9–12 these models were

validated using the ModFOLD4 model quality assessment

method.13

Results

The epidermis was unchanged; a band of loose connec-

tive tissue rich in procollagen fibres showing different

sizes and orientations was visible in the deep dermis and

the hypodermis (Figure 2).

The sequencing of the ADAMST2 pro-domain (involved

in dermatosparaxis in dorper sheep) did not reveal any

premature stop codon in the lamb.2 Three sets of primers

were therefore designed to amplify the third, fourth and

fifth exon, according to precedent nomenclature

(Table 1).2

A missense SNP was detected in homozygosis in

the fourth exon in the affected lamb (g.1862883G>A

in ovine3 and g.2030792G>A in bovine4). This SNP

results in the substitution of valine (V) by methionine

(M) in the catalytic domain of the mature ADAMTS2

enzyme (V15M). Interestingly, both the ewe that gave

birth to this lamb and the ewe that gave birth to an

affected lamb previously were heterozygous (G/A) for

this SNP. A screening of the male reproducers in the

flock using microsatellite markers allowed identification

of the ram siring the affected lamb; ADAMST2

sequences showed that this was the only heterozy-

gous ram in the flock for this SNP. This provides

additional confirmation for recessive autosomal inheri-

tance; heterozygous G/A parents with normal pheno-

type produce homozygous A/A descendants affected

by dermatosparaxis.

The evaluation of the possible effect of the V15M

substitution using the SIFT algorithm and the PolyPhen-

2 approach produced consistent results. The SIFT algo-

rithm classified it as ‘damaging’, scoring it with 0.01 in

a 0–1 (damaging–tolerated) range. PolyPhen-2 indicated

that it was ‘probably damaging’, with scores of 1 and

0.98 on the 0–1 scales for the HumDiv and HumVar

method, respectively).6–8 Based on information available

at GenBank, the valine residue in the ADAMST2 cata-

lytic domain is conserved amongst mammals, marsupi-

als (Sarcophilus harrisii), birds (Gallus gallus) and fish

(Takifugu rubripes). However, this is the first time

methionine has been found at this position in any spe-

cies.

Further understanding on the effect of the mutation

was obtained from the 3D analysis of the protein. From

the initial run of the IntFOLD2 server,9 the ordered struc-

ture of the catalytic domain was shown to be flanked by

two natively unstructured regions (M1–R253 and R554–

F1205). As the mutation was in the catalytic domain, the

300 ordered residues (H254–K553) were selected for

higher quality modelling, and the models were validated

Figure 1. Skin wound and flap in an affected lamb. Injuries did not

appear to be linked to accidents or trauma because the lamb was

kept under normal management with other lambs. Lesions led to

infection, septicaemia and death 7.

Table 1. PCR primers

Exon Forward 8 Reverse Annealing temperature Reference

2 CCCTTCCCTTCGCAGTGG GAGACAAGCGCGTACTCAC 60°C 2

3 CTCACACCTAGCTGGGGTTC GTCAGCAAGGGTGGTCTCAG 62.6°C Present study

4 TGATGATGGCAGGCTGATGG GAGCCTCCCTGTGACCATTT 62.6°C Present study

5 AATGCCTGAGCGTGAAGTGT TCTTACAGGAAACACGGGGC 62.6°C Present study

COLOR

© 2014 ESVD and ACVD, Veterinary Dermatology2

Monteagudo et al.

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

61

62

using the ModFOLD4 model quality assessment

method.13 The results of the 3D modelling of the WT AD-

AMTS2 and mutant are shown in Figure 3a–h. In Fig-

ure 3a–c, the models of WT and mutant proteins show

similar global structures and have similarly high global

quality scores (P � 0.001; much <1 in 1000 chance of

the model being incorrect).13 Closer examination of the

site around the mutation shows the implications of the

mutation (Figure 3d–f). In Figure 3f, it can be seen that

the L32 side-chain is in a different orientation in the

mutant than in the WT; perhaps this is due to lack of

space caused by the longer M15 side-chain of the

mutant. In Figure 3g,h, the side-chains are shown as

spheres to indicate atomic clashes. In Figure 3h, we can

see overlapping spheres, indicating clashes occurring

in the mutant protein between the side-chains on the

b-strand and a-helix. However, in the WT protein (Fig-

ure 3g) no side-chain clashes are observed between the

b-strand and a-helix residues. The clashes observed in

the mutant protein may alter the stability of the fold or dis-

tort the structure, causing the protein to malfunction.

Discussion

The histology observations in this case are consistent

with previous descriptions of accumulation of immature

procollagen fibres leading to loose connective tissue that

lacks normal strength and resistance,1 resulting in skin

fragility, severe wounds and infections that lead to early

death9 .

Dermatosparaxis has been repeatedly reported in

sheep as an autosomal recessive trait.14,15 Recessive

inheritance is well known to be linked to defects in enzy-

matic activity.16 Insertions, deletions, nonsense muta-

tions and other changes, such as chromosome

inversions, have traditionally been identified as the origins

of inherited diseases. Nowadays, the analysis of the pos-

sible phenotypic consequences of missense SNP takes

advantage of the vast amount of information available on

genome sequences in humans and animals. State-of-the-

art methodologies, such as SIFT and PolyPhen, were orig-

inally employed to analyse human genome sequences.6–8

However, as these procedures were developed (among

other reasons) for the analysis of conservation in

sequences from different species, their use is spreading

to animal genomics analysis. Recently, SIFT and Poly-

Phen-2 were applied to the identification of deleterious

haplotypes in three different genes involved in bovine

prenatal death.17 For instance, a substitution affecting a

putative manganese binding site in the glycinamide ribo-

nucleotide transformylase protein (N290T) is considered a

critical mutation, with the authors remarking that this

asparagine-290 is conserved among eukaryotes, pointing

to a key role of this amino acid. A similar situation occurs

in the case of the V15M substitution in ADAMTS2, as

shown in the present study; the conservation of valine-15

in the active domain of the proteinase points to a critical

function of this amino acid in procollagen maturation.

Figure 2. Histology of the skin of the affected lamb. Arrows indicate

a band of connective tissue consisting of accumulated procollagen

fibres showing different sizes and orientations in the deep dermis

and the hypodermis. Epidermal and adnexal structures appear normal

(haematoxylin and eosin stain).

(a) (b)

(d)

(f)

(g) (h)

(e)

(c)

Figure 3. Three-dimensional (3D) modelling of single nucleotide

polymorphism mutation in ADAMTS2. (a) IntFOLD2 3D model of the

wild-type (WT) structure showing the 300 ordered residues in a car-

toon view. Amino acid residue V15 is shown in stick form and high-

lighted in dark blue. (b) IntFOLD2 3D model of the mutant structure

showing 300 ordered residues in cartoon view. The M15 mutation is

shown in stick form and highlighted in red. (c) Superposition of the

WT (cyan) and mutant (green) models. (d) Zoomed view of WT struc-

ture with key amino acids labelled as sticks. (e) Zoomed view of

mutant structure with key amino acids labelled as sticks. (f) Superpo-

sition of key residues in WT and mutant. (g,h) The same as (d,e),

respectively, but here the side-chains are represented as spheres to

indicate the van der Waals volume of the atoms and reveal overlaps/

clashes.

COLOR

COLOR

© 2014 ESVD and ACVD, Veterinary Dermatology 3

Inherited ovine dermatosparaxis

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

61

62

Here, 3D analysis provides additional evidence of the

importance of the V15M substitution, because it causes

critical structural changes in the active domain of the

enzyme.

As in humans, this report demonstrates that ovine

dermatosparaxis may be due to different mutations in

the locus ADAMST2.1 Dorper is a synthetic breed cre-

ated in South Africa in the 1930s by crossing Dorset

and blackheaded Persian sheep.18 Reproductive isola-

tion can be assumed amongst affected dorper and

the flock studied here. It explains genetic heterogene-

ity for this pathology instead of a common origin for

all cases.

In conclusion, in the light of in silico approach results,

the V15M substitution is likely to be responsible for criti-

cal modifications in the ADAMTS2 enzymatic activity that

lead to ovine autosomal recessive inherited dermatospar-

axis.

Acknowledgements

The authors would like to acknowledge the use of Servi-

cio General de Apoyo a la Investigaci�on-SAI, Universidad

de Zaragoza, Jos�e. A. Garc�ıa de Jal�on for histological

analysis and Mar�ıa Pe~na and Ane Rivas for help in labora-

tory tasks.

References

1. Colige A, Sieron AL, Li SW et al. Human Ehlers-Danlos syn-

drome type VII C and bovine dermatosparaxis are caused by

mutations in the procollagen I N-proteinase gene. Am J Hum

Genet 1999; 65: 308–317.

2. Zhou H, Hickford JGH, Fang Q. A premature stop codon in the

ADAMTS2 gene is likely to be responsible for dermatosparaxis

in Dorper sheep. Anim Genet 2012; 43: 471–473.

3. Archibald AL, Cockett NE, Dalrymple BP et al. The sheep gen-

ome reference sequence: a work in progress. Anim Genet 2010;

41: 449–453.

4. Zimin AV, Delcher AL, Florea L et al. A whole-genome assem-

bly of the domestic cow, Bos taurus. Genome Biol 2009; 10:

R42.

5. Altschul SF, Gish W, Miller W et al. Basic local alignment search

tool. J Mol Biol 1990; 215: 403–410.

6. Kumar P, Henikoff S, Ng PC. Predicting the effects of coding

nonsynonymous variants on protein function using the SIFT algo-

rithm. Nat Protoc 2009; 4: 1073–1081.

7. Ramensky V, Bork P, Sunyaev S. Human non-synonymous

SNPs: server and survey. Nucleic Acids Res 2002; 30: 3894–

3900.

8. Adzhubei IA, Schmidt S, Peshkin L et al. A method and server

for predicting damaging missense mutations. Nat Methods

2010; 7: 248–249.

9. Roche DB, Buenavista MT, Tetchner SJ et al. The IntFOLD ser-

ver: an integrated web resource for protein folds recognition, 3D

model quality assessment, intrinsic disorder prediction, domain

prediction and ligand binding site prediction. Nucleic Acids Res

2011; 39(Suppl. 2): W171–W176.

10. Buenavista MT, Roche DB, McGuffin LJ. Improvement of 3D

protein models using multiple templates guided by single-tem-

plate model quality assessment. Bioinformatics 2012; 28: 1851–

1857.

11. McGuffin LJ. Intrinsic disorder prediction from the analysis of

multiple protein fold recognition models. Bioinformatics 2008;

24: 1798–1804.

12. Roche DB, Tetchner SJ, McGuffin LJ. FunFOLD: an improved

automated method for the prediction of ligand binding residues

using 3D models of proteins. BMC Bioinformatics 2011; 12: 160.

13. McGuffin LJ, Buenavista MT, Roche DB. The ModFOLD4 server

for the quality assessment of 3D protein models. Nucleic Acids

Res 2013; 41: W368–W372.

14. Fjolstad M, Helle O. A hereditary dysplasia of collagen tissues in

sheep. J Pathol 1974; 112: 183–188.

15. Wong S. Dermatosparaxis in Dorper sheep. Australian Society

for Veterinary Pathology. Vet Pathol Rep 2006; 72: 10–11. 10

16. Nicholas FW. Veterinary Genetics. Oxford, UK: Oxford Univer-

sity Press, 1987; 92–93.

17. Fritz S, Capitan A, Djari A et al. Detection of haplotypes associ-

ated with prenatal death in dairy cattle and identification of dele-

terious mutations in GART, SHBG and SLC37A2. PLoS ONE

2013; 8: e65550.

18. Database Breeds of Livestock, Oklahoma State University,

Department of Animal Science. Available at: http://www.ansi.

okstate.edu/breeds/. Accessed April 10, 2014.

Supporting Information

Additional Supporting Information may be found in the

online version of this article.

Video S1. Video recorded during the postmortem exami-

nation of an affected lamb immediately after euthanasia.

R�esum�e – Xxxxxxxx.

Resumen – Xxxxxxxx.

Zusammenfassung – Xxxxxxxx4 .

© 2014 ESVD and ACVD, Veterinary Dermatology4

Monteagudo et al.

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

61

62

Graphical Abstract

The contents of this page will be used as part of the graphical abstract

of html only. It will not be published as part of main.

Background – Dermatosparaxis (Ehlers–Danlos syndrome in humans) is characterized by extreme fragility of the skin. It is

due to the lack of mature collagen caused by a failure in the enzymatic processing of procollagen I. We investigated the

condition in a commercial sheep flock. Hypothesis/Objectives –Mutations in the ADAMmetalloproteinase gene, specifi-

cally at the thrombospondin type 1 motif 2 (ADAMTS2) locus, are involved in the development of dermatosparaxis in

humans, cattle and the dorper sheep breed; consequently, this locus was investigated in the flock. Conclusions and clin-

ical importance – Detection of the mutation responsible for the pathology allowed us to remove the heterozygote ram,

thus preventing additional cases in the flock.

© 2014 ESVD and ACVD, Veterinary Dermatology 5

Inherited ovine dermatosparaxis

Author Query Form

Journal: VDEArticle: 12178

Dear Author,

During the copy-editing of your paper, the following queries arose. Please respond to these by marking up

your proofs with the necessary changes/additions. Please write your answers on the query sheet if there is

insufficient space on the page proofs. Please write clearly and follow the conventions shown on the attached

corrections sheet. If returning the proof by fax do not write too close to the paper’s edge. Please remember

that illegible mark-ups may delay publication.

Many thanks for your assistance.

Query reference Query Remarks

1 AUTHOR: it is normal journal style for abbreviations of gene names to be

italic and proteins roman. Please confirm that this has been done correctly

and consistently throughout the manuscript or amend if necessary. Please

confirm that the gene is correct as ADAMTS2 or should it be Adamts2 for a

sheep gene?

2 AUTHOR: Please check that authors and their affiliations are correct.

3 AUTHOR: Please check the corresponding author’s details.

4 WILEY: Please provide French, Spanish and German abstract.

5 WILEY: Please confirm that the short abstract text is okay.

6 AUTHOR: is “a disintegrin” OK or does it need to be “A disintegrin” (as

opposed to “B disintegrin”, perhaps)?

7 AUTHOR: Please carefully check all the figures.

8 AUTHOR: do you need to specify 30–50 or vice versa?

9 AUTHOR: is “infections that lead to early death” OK now?

10 AUTHOR: Please check the abbreviated journal title for reference [15].