Glycosaminoglycans and proteoglycans in the skin of aneuploid fetuses with increased nuchal translucency

C.S. von Kaisenberg1,2,3,4, F. Prols2, K.H. Nicolaides3, N. Maass1, I. Meinhold-Heerlein1 and B. Brand-Saberi2

1 Department of Obstetrics and Gynaecology, University of Schleswig-Holstein, Kiel, 2 Department of Anatomy, University of Freiburg, Freiburg, Germany and 3 Harris Birthright Research Centre For Fetal Medicine, King’s College School of Medicine and Dentistry, London, UK

4 To whom correspondence should be addressed at: University of Schleswig-Holstein, Campus Kiel, Department of Obstetrics and Gynaecology, Michaelisstr. 16, 24105 Kiel, Germany. e-mail: vkaisenberg{at}email.uni-kiel.de


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: First trimester increased fetal nuchal translucency is associated with fetal aneuploidies. One of the mechanisms of pathophysiology could be an abnormal extracellular matrix facilitating the formation of an interstitial edema. A previous study investigating interstitial edema in first trimester fetuses found large amounts of hyaluronan in the skin of fetuses with trisomy 21. The aim of this study was to establish distribution patterns for a number of other glycosaminoglycans—dermatan, heparan and keratan sulphate, chondroitin-6-sulphate and chondroitin-4-sulphate proteoglycan—in the nuchal skin of normal and chromosomally abnormal fetuses at 11–14 weeks. We also investigated whether biglycan (BGN), which is located on chromosome X, is underexpressed in fetuses with Turner syndrome. Decorin (DCN), a similar-sized proteoglycan located on chromosome 12, was taken as a control. METHODS: We studied the distribution and concentration of various extacellular matrix components using immunohistochemistry, a double staining technique, in-situ hybridization, Northern and Western blot analysis. RESULTS: Chondroitin-6-sulphate and chondroitin-4-sulphate proteoglycan were increased in Turner syndrome fetuses and BGN seemed to be underexpressed compared with normal controls, while DCN was not. Dermatan, heparan and keratan sulphate showed no significant abnormal distribution in trisomies 21, 18, 13, or in Turner syndrome, compared with normal. Western and immunohistochemical analysis revealed that absence of a second X chromosome, as is the case in Turner syndrome, affects BGN protein pattern. CONCLUSIONS: An abnormal amount of glycosaminoglycans and proteoglycans presumably contributes to increased nuchal translucency.

Key words: aneuploidy/extracellular matrix/glycosaminoglycans/nuchal translucency/proteoglycans


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In fetuses with Turner syndrome and other chromosomal abnormalities, there is a collection of fluid in the nuchal region that can be visualized by ultrasonography as nuchal translucency at 11–14 weeks of gestation (Nicolaides et al., 1992Go). Turner syndrome is characterized by monosomy of the X chromosome in females only (45,X) and occurs with an incidence of ~1:2500 in live-born females. The main clinical features of this disorder are short stature, gonadal dysgenesis, webbing of the neck, aortic isthmus stenosis and lymphedemas of the hands and feet. Possible mechanisms for increased nuchal translucency include abnormalities of the heart and great arteries, cardiac dysfunction, abnormal development of the lymphatic system and abnormal composition of the extracellular matrix of tissues (Hyett et al., 1996Go, 1997Go; von Kaisenberg et al., 1997Go, 1998aGo,b, 1999Go). A previous study investigating the formation of an interstitial edema found large amounts of hyaluronan in the skin of fetuses with trisomy 21, which can lead to excessive hydration of the extracellular matrix, whereas in trisomies 18 and 13 and Turner syndrome the amount was similar to that in chromosomally normal controls (Böhlandt et al., 2000Go).

This study investigates further the hypothesis that increased nuchal translucency in Turner syndrome or aneuploidies may be the consequence of altered composition of the skin, in particular of glycosaminoglycans or proteoglycans. The aim of this study was to establish distribution patterns for a number of other glycosaminoglycans—dermatan, heparan and keratan sulphate, chondroitin-6-sulphate and chondroitin-4-sulphate proteoglycan—in the nuchal skin of normal and chromosomally abnormal fetuses at 11–14 weeks. Fibroblast cultures from postnatal Turner patients contained only half the amount of biglycan (BGN) mRNA and BGN protein compared with age-matched control cultures (Geerkens et al., 1995Go). Therefore we also determined BGN mRNA and protein levels in the skin of Turner syndrome fetuses. BGN maps to the X chromosome and undergoes inactivation. Normal males and females have only one active copy of BGN, and fibroblast cultures in postnatal normal males and females did not show any difference in BGN expression, while individuals with additional X or Y chromosomes showed substantial overexpression (Table I). This suggests that BGN expression does not correlate with the number of BGN copies, but is rather correlated with the number of sex chromosomes. A regulator on the sex chromosomes (X and/or Y) may drive the transcription rate of BGN (Figure 1, Table I). If a gene copy of such a regulator on the X chromosome escapes inactivation or is pseudoautosomally regulated, different transcription rates of BGN in Turner patients and healthy females could be explained. The gene copy of the regulator gene on the Y chromosome should be a homologue of the X chromosomal copy, explaining identical transcription rates in females and males (Hibi et al., 1993Go). BGN belongs to the family of the small-sized leucine-rich proteoglycans (SLRP) and is post-translationally modified by the addition of two glycosaminoglycan side-chains. The core protein may be modified by the addition of one or two glycosaminoglycan chains.


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Table I. Transcription rates of biglycan (BGN) in fibroblasts of patients with numerical aberrations of the X or the Y chromosome (Vetter et al., 1993)
 


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Figure 1. In normal females, there are two X chromosomes of which one is inactivated and there are two regulator genes, acting on one BGN gene site (100% BGN expression). In normal males, there is one X chromosome and one Y chromosome with one BGN gene site, which is regulated by each one regulator gene on the X and the Y chromosomes (100% BGN expression). In Turner syndrome, there is one active X chromosome with one regulator site acting on one active BGN gene site (50% BGN expression). In triple X women, there are three X chromosomes, of which two are inactivated and there are three regulator genes, acting on one BGN gene site (>100% BGN expression); and supposedly in Klinefelter’s, there are two X chromosomes and one Y chromosome with one X chromosome inactivated and three regulator genes (two on the X and one on the Y chromosome) acting on one BGN site (>100% BGN expression). (Adapted from Hibi and Takano, 1993, with permission.)

 
Decorin (DCN), a similar-sized proteoglycan encoded on chromosome 12, does not show different protein levels in fibroblast cell lines from adult normal or Turner syndrome patients (Geerkens et al., 1995Go). Previous studies investigating the genetic regulation of extracellular matrix components found up-regulation for collagen type VI in first trimester fetuses with trisomy 21 (von Kaisenberg et al., 1998aGo,b). DCN can bind to collagen type I and inhibits collagen type I and II fibrillogenesis in vitro. We therefore investigated whether DCN was already co-localized with collagen fibrils at this early stage of development, because co-localization with collagen fibrils may also indicate interaction of DCN with other collagens.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Nuchal skin was obtained from seven fetuses with Turner syndrome, and one each with trisomies 21, 18 or 13 after termination of pregnancy at 11–14 weeks of gestation (Table II). Fetal skin was obtained from 10 age-matched normal fetuses ranging in total from 11 through 19 weeks (11 + 3, 11 + 4, 11 + 6, 12 + 1, 13 + 5, 14 + 4, 15 + 2, 16 + 4, 17 + 6 and 19 + 0 weeks) after termination of gestation for psychosocial reasons (Table II). Immediately after surgical termination of pregnancy, nuchal skin tissue from the posterior region of the fetal neck identical to the sonographically detected increased nuchal translucency was identified, dissected and processed as described below. The karyotype of the affected fetuses had been obtained by chorionic villus sampling at the request of the parents because of increased nuchal translucency. Control fetuses were considered normal with a nuchal translucency measurement of <2 mm. In the seven fetuses with Turner syndrome, there was no mosaicism and no structural abnormality of the X chromosome except one Turner fetus with a Robertsonian translocation 13/14. Written informed consent was obtained, the study was approved by the hospital ethics committee and tissue collection was made in accordance with the Polkinghorne (1989Go) guidelines on the research use of fetal tissues.


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Table II. Gestational age at termination, crown–rump length (CRL) and nuchal translucency (NT) of seven fetuses with Turner syndrome and one each with trisomies 21, 18 and 13 and the respective normal age-matched controls
 
Immunohistochemistry
Skin was embedded in Tissue Tek without prior fixation and frozen in liquid nitrogen. Cryosections of nuchal skin from chromosomally abnormal and normal fetuses were stained with monoclonal antibodies against dermatan sulphate, 1:100, monoclonal, clone 6-B-6, IgG1 kappa (Bioscience, Switzerland); heparan sulphate, 1:100, monoclonal, clone 7E12, IgG1 (Boehringer Mannheim, Germany); keratan sulphate, 1:100, monclonal, clone 5D4 IgG1 kappa (ICN, Germany); chondroitin-6-sulphate, 1:70, monoclonal, clone 3-B-3, IgG1 kappa; chondroitin-4-sulphate, 1:100, monoclonal, clone 2-B-6, mouse IgG1, kappa (ICN, Germany) and polyclonal antibodies against the core protein of BGN (LF-121), 1:100, and DCN (LF-122) 1:100 (Fisher et al., 1989Go).

Monoclonal antibodies directed against chondroitin-4-sulphate proteoglycan and chondroitin-6-sulphate proteoglycan specifically recognize 4-sulphated and 6-sulphated chondroitin following chondroitinase ABC (ChABC) digestion of various proteoglycans. Self-association among the glycosaminoglycans (GAG) and interaction with other extracellular matrix components may be affected by the particular charge organizations of the GAG molecule endowed by the sulphate group. The two monoclonals react with the chondroitin sulphate ‘stubs’ which remain after extensive ChABC digestion. The antibody specificity is according to the sulphated position (4S, 6S) of these ‘stubs’ (ICN product description). Proteoglycans, for example one of the small-sized proteoglycans BGN or DCN, have one or more glycosaminoglycan (GAG) side-chains attached to a core protein. Cross-reactivity of monoclonal antibodies directed against chondroitin sulphate proteoglycans with small proteoglycans may exist, but seems unlikely, since the antibody specificity is according to the sulphated position. Therefore it is unlikely that the finding of intense fluorescence in the dermis of nuchal skin of Turner fetuses using chondroitin-6-sulphate antibodies is the result of cross-reactivity of this antibody with either BGN or DCN (Figures 2a, b, f, 8, 12 and 13). The BGN and DCN antibodies did not cross-react, neither with each other, nor with dermatan, heparan or keratan sulphate, because BGN and DCN antibodies are directed against a short peptide of the core protein. No detailed information was available for dermatan sulphate or heparan sulphate, but keratan sulphate did not cross-react with any of the other antigens (Boehringer Mannheim Biochemica and ICN product information).



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Figure 2. Immunohistochemistry in nuchal skin of a fetus with Turner syndrome at 12 weeks using antibodies against biglycan (BGN) (a), decorin (DCN) (b), keratan sulphate (c), heparan sulphate (d), dermatan sulphate (e) and chondroitin-6-sulphate (f). One of the most striking findings is the distribution of chondroitin-6-sulphate. Bar = 50 µm.

 


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Figure 8. Immunohistochemistry with antibody against chondroitin-6-sulphate in nuchal skin of fetuses with Turner syndrome (a), trisomy 21 (b), trisomy 18 (c), trisomy 13 (d) and a normal control (e). In Turner syndrome there was intense fluorescence in the dermis extending down to a chain of vessels at the dermis–subcutis junction. To some extent, this intense dermal staining was also observed in the nuchal skin of trisomic fetuses, but did not stretch downwards towards the dermis–subcutis junction. Bar = 50 µm.

 


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Figure 12. Antibody against biglycan (BGN) in nuchal skin of normal fetuses with increasing gestation: 12+1 weeks (a), 13 + 5 weeks (b), 14 + 4 weeks (c), 15 + 2 weeks (d), 16 + 4 weeks (e) and 17 + 6 weeks (f). Staining of the nuchal skin of normal fetuses with increasing gestation ranging from 12 to 17 weeks showed a more irregular staining pattern for BGN with substantial signal variation. There was no substantial variation with gestation for BGN.

 


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Figure 13. Antibody against decorin in nuchal skin of normal fetuses with increasing gestation: 12 + 1 weeks (a), 13 + 5 weeks (b), 14 + 4 weeks (c), 15 + 2 weeks (d), 16 + 4 weeks (e) and 17 + 6 weeks (f). Staining of the nuchal skin of normal fetuses with increasing gestation ranging from 12 to 17 weeks showed a more homogeneous staining pattern in decorin more correlated to the fibrillar components of the skin. There was no substantial variation with gestation for decorin.

 
Frozen sections (20 µm) were collected on chrome–alum–gelatin-coated slides. They were dried, blocked with 1% bovine serum albumin in 0.1 mol/l potassium phosphate buffer (pH 7.6) for 10 min and then incubated with primary monoclonal or polyclonal antibodies directed against different extracellular matrix components (60 min at room temperature). After being rinsed with phosphate buffer, the sections were incubated with affinity-purified goat anti-mouse IgG for the monoclonal antibodies and with goat anti-rabbit IgG for the polyclonal antibodies conjugated with Cy3 from Dianova (Germany) yielding a red signal. Anti-collagen type I antibodies were detected by a goat anti-rabbit antibody coupled with fluorescein (Dianova) yielding a green signal. Double-labelling was performed with both primary and secondary antibodies applied to the same sections in a sequence. Sections were covered with mowiol (Hoechst, Germany) and coverslips. Controls were carried out in two ways. Cryostat sections from trisomic and normal fetuses were processed in the same way, except for the omission of the primary antibody to assess the degree of non-specific staining. Alternatively, pre-immune mice serum was used instead of the first monoclonal antibody and pre-immune rabbit serum instead of the first polyclonal antibody for specific controls. The sections were viewed and micrographs were taken using an epifluorescence microscope (Carl Zeiss, Germany), and TMY-400 black-and-white films or colour films.

In-situ hybridization
Digoxigenin-labelled cRNA antisense riboprobes were taken as probes for in-situ hybridization according to the published protocol (Schaeren-Wiemers et al., 1993Go). The BGN and DCN probes were obtained from Dr Larry Fisher, NIH, USA (Fisher et al., 1989Go). The BGN probe P16 was a 1685 bp insert in pBluescript containing the complete coding sequence of human bone proteoglycan I PGI (BGN). The antisense strand was linearized with KpnI and transcribed from the T3 promoter. The sense strand linearized with XbaI and transcribed with T7 polymerase. The DCN probe P2 was a 1600 bp insert into pBluescript containing the complete coding sequence of human bone proteoglycan II PGII (DCN) (Fisher et al., 1989Go). The antisense strand was linearized with BamHI and transcribed from the T7 promoter, the sense strand linearized with KpnI and transcribed from the T3 promoter.

Isolation of RNA and protein
Skin tissues, dissected from control and Turner syndrome fetuses, were frozen immediately in RNase-free polypropylene tubes in liquid nitrogen and stored at –70°C. Total RNA and protein were isolated from identical probes by using the Trizol-based isolation procedure according to the protocol provided by the manufacturer (Life Technologies, Germany).

Northern blot analysis
We loaded 10 µg of total RNA per lane in the absence of ethidium bromide. RNA was blotted, UV-cross-linked, stained in methylene blue, and hybridized (Prols et al., 2001Go). Human BGN and DCN probes were labelled with [32P]dCTP using a random primer labelling kit (Life Technologies). For evaluating the quality of the individual RNA and to confirm equal loading per lane, the methylene blue-stained 28S and 18S rRNA are given as references.

Western blot analysis
Before electrophoresis the pelleted protein probes were rinsed three times in guanidium chloride (0.3 mol/l) and dissolved in 1% sodium dodecylsulphate overnight at 4°C. Dissolved protein samples were centrifuged at 10 000 g for 10 min at 4°C. The supernatants were transferred into new tubes and protein concentration was determined as described before (Prols et al., 1998Go). We loaded 25 µg protein per lane onto 12% polyacrylamide gels in the presence of 0.1% 2-mercaptoethanol. After electrophoresis, proteins were transferred onto nitrocellulose membranes in a semi-dry blotting chamber. To evaluate equal loading of the lanes, the membranes were stained with Ponceau S reagent (Sigma Aldrich Chemical Co., Germany).

Immunostaining was performed as previously described (Prols et al., 1998Go). Membranes were blocked in 5% non-fat dry milk in the cold overnight and stained with the polyclonal BGN antibody (1:2000). As second antibody we used horseradish-coupled goat-anti-rabbit antibody (1: 20 000). Staining was visualized using the ECL detection system (Pierce Reagent; KMF-Laboratories, St Augustin, Germany).


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 Abstract
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 Materials and methods
 Results
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Immunohistochemistry
The proteoglycans BGN and DCN and the glycosaminoglycans dermatan, heparan and keratan sulphate were expressed in the skin of both chromosomally normal fetuses and fetuses with trisomies 21, 18, 13 and Turner syndrome and there was no obvious difference in expression between the two groups (Figures 2345678 and 11). Chondroitin-6-sulphate proteoglycan was also expressed in the skin of both groups but in Turner syndrome there was intense fluorescence in the dermis extending down to a chain of vessels at the dermis–subcutis junction (Figures 2f, 8a and 14). The area below this junction was practically devoid of staining. To some extent, this intense dermal staining was also observed in the nuchal skin of trisomic fetuses, but did not stretch downwards towards the dermis–subcutis junction (Figure 8b, c, d). Chondroitin-4-sulphate proteoglycan showed a similar reactivity pattern in the skin of fetuses with Turner syndrome with intense staining in the lower dermis and absence of staining below a chain of vessels at the dermis–subcutis junction, which could not be observed in normal fetuses (data not shown). Staining of the nuchal skin of normal fetuses with increasing gestation ranging from 12 to 17 weeks showed a more irregular staining pattern for BGN with substantial signal variation (Figure 12), a more homogenous staining pattern in DCN correlated with the fibrillar components in the skin (Figure 13). There was no substantial variation with gestation for any of the antibodies tested (Figures 1214).



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Figure 3. Immunohistochemistry with antibody against biglycan in nuchal skin of fetuses with Turner syndrome (a), trisomy 21 (b), trisomy 18 (c), trisomy 13 (d) and a normal control (e). There was no obvious difference between the various aneuploidies, compared with normal. Bar = 50 µm.

 


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Figure 4. Immunohistochemistry with antibody against decorin in nuchal skin of fetuses with Turner syndrome (a), trisomy 21 (b), trisomy 18 (c), trisomy 13 (d) and a normal control (e). There was no obvious difference between the various aneuploidies, compared with normal. Bar = 50 µm.

 


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Figure 5. Immunohistochemistry with antibody against keratan sulphate in nuchal skin of fetuses with Turner syndrome (a), trisomy 21 (b), trisomy 18 (c), trisomy 13 (d) and a normal control (e). There was no obvious difference between the various aneuploidies, compared with normal. Bar = 50 µm.

 


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Figure 6. Immunohistochemistry with antibody against heparan sulphate in nuchal skin of fetuses with Turner syndrome (a), trisomy 21 (b), trisomy 18 (c), trisomy 13 (d) and a normal control (e). There was no obvious difference between the various aneuploidies, compared with normal. Bar = 50 µm.

 


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Figure 7. Immunohistochemistry with antibody against dermatan sulphate in nuchal skin of fetuses with Turner syndrome (a), trisomy 21 (b), trisomy 18 (c), trisomy 13 (d) and a normal control (e). There was no obvious difference between the various aneuploidies, compared with normal. Bar = 50 µm.

 


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Figure 11. Double staining technique of nuchal skin from fetuses with Turner syndrome (ac) and normal age-matched controls (df) at 12 weeks: polyclonal antibody against decorin (red in the colour figure available online) (a, d), monoclonal antibody against collagen type I (green in the colour figure available online; b, e) and both polyclonal antibody against decorin and monoclonal antibody against collagen type I (c, f). Double staining reveals that decorin co-localizes with collagen type I especially in the upper dermis in fetuses with Turner syndrome and normal controls. Bar = 50 µm. This figure can be viewed in colour as Supplementary data at Human Reproduction Online.

 


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Figure 14. Antibody against chondroitin-6-sulphate in nuchal skin of normal fetuses with increasing gestation: 12 + 1 weeks (a), 13 + 5 weeks (b), 14 + 4 weeks (c), 15 + 2 weeks (d), 16 + 4 weeks (e) and 17 + 6 weeks (f). Staining of the nuchal skin of normal fetuses with increasing gestation ranging from 12 to 17 weeks for chondroitin-6-sulphate proteoglycan showed a pattern that extended down to a chain of vessels at the dermis–subcutis junction. There was no substantial variation with gestation for chondroitin-6-sulphate.

 
Double staining technique
DCN staining revealed that it was evenly distributed both in the Turner fetus and normal controls, decreasing towards the subcutis (Figure 11a, d). Collagen type I staining gave a similar pattern, but highlighted a more fibrillar structure of the matrix (Figure 11b, e). Using both DCN and collagen type I in the same slide revealed that DCN co-localizes with collagen type I especially in the upper dermis in both groups of fetuses (Figure 11c, f).

In-situ hybridization
Staining with BGN showed weak fluorescence in the area underlying the epidermis with homogeneous distribution. BGN seemed to be underexpressed in the nuchal skin of fetuses with Turner syndrome compared with an age-matched control (Figure 9), whereas DCN staining showed a comparable staining pattern in the nuchal skin tissue of the Turner and normal fetuses (Figure 10).



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Figure 9. In-situ hybridization using a riboprobe for BGN in the nuchal skin of a fetus with Turner syndrome (a) and a normal age-matched control (b). Staining with BGN showed pale fluorescence in the area underlying the epidermis with homogeneous intensity. BGN seemed to be underexpressed in the nuchal skin of fetuses with Turner syndrome compared with an age-matched control. Bar = 50 µm. This figure can be viewed in colour as Supplementary data at Human Reproduction Online.

 


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Figure 10. In-situ hybridization using a riboprobe for decorin in the nuchal skin of one fetus with Turner syndrome (a) and a normal age-matched control (b). Bar = 50 µm. This figure can be viewed in colour as Supplementary data at Human Reproduction Online.

 
Northern blot analysis
Out of three Turner syndrome fetuses, the RNA of one fetus was degraded and could not be evaluated further. The probes under investigation proved to be intact, as shown by the methylene staining of the 28S and 18S rRNA (Figure 15A, B). The RNA isolated from the second fetus (Figure 15A, probe 7) showed BGN mRNA concentrations similar to those of controls, whereas the third fetus (Figure 15A, probe 8) exhibited decreased BGN mRNA levels. Both tissues derived from Turner syndrome fetuses showed smaller BGN transcripts than control tissue, pointing towards truncation or increased instability of the BGN RNA. DCN mRNA levels were comparable in concentration and size in tissue derived from normal and Turner syndrome fetuses (Figure 15B).



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Figure 15. RNA and protein were extracted from the skin of five normal controls (week 12, fetal probes 1 to 4) and week 19 (fetal probe 5), one Turner syndrome fetus at week 16 (fetal probe 6), and two Turner syndrome fetuses at week 14 (fetal probes 7 and 8). The skin was isolated from the nuchal region (fetal probes 2, 4, 5, 7, 8), from the back (fetal probe 1) and the scalp (fetal probe 3). (A and B) Northern blot analysis for evaluating biglycan (BGN) and decorin (DCN) transcript sizes and amounts in control versus Turner syndrome fetuses. Equal loading of the lanes was confirmed by methylene blue staining of the blots. To evaluate the integrity of the RNA, both rRNA bands (28S rRNA and 18S rRNA) are given as reference. (A) Hybridization of the blot with the BGN probe revealed BGN transcripts of various sizes (4.7, 2.6, 1.8 and 1 kb). (B) Decorin hybridization revealed a double band of ~2 kb in all fetal probes under investigation. (C) Western blot of proteins extracted from the normal fetal probes 2, 3, 4 (week 12) and 5 (week 19) compared with three fetuses with Turner syndrome (fetal probes 6, 7 and 8). It becomes apparent that the 42 kDa BGN band as well as a 65 kDa and small-sized bands (16–19 kDa) that hybridize with the BGN antibody are largely depleted in fetuses with Turner syndrome. (D) PonceauS staining of the Western blot in (C) is given as a reference for comparison of the amount of total protein loaded per lane. The PonceauS staining reveals that the amount of protein loaded per lane was within comparable ranges (see large band of ~80 kDa) whereas smaller-sized proteins already revealed substantial differences in Turner syndrome fetuses when compared with normal controls.

 
Western blot analysis
Western blot analysis using the BGN antibody revealed bands of various sizes: 64, 37 and 42 kDa bands and smaller bands in the range of 16–19 kDa (Figure 15). According to the literature, the protein bands at 37 and 42 kDa correspond to the core BGN protein, where the 42 kDa form is the intracellular form and the 37 kDa band the secreted form (Fisher et al. 1989Go). Provided that the 64 kDa as well as the 16–19 kDa BGN hybridizing bands are not due to cross-hybridization, the 64 kDa protein could originate from post-translational modifications that had not been cleaved by the protein isolation procedure, whereas the small bands (16–19 kDa) could originate from BGN cleavage. All Turner syndrome fetuses exhibited largely reduced amounts of the 64 kDa as well as of the 16–19 kDa protein bands when compared with the 12 week old normal fetuses (Figure 15C, probe 2–4) and the 19 week normal fetus (probe 5). However, since we had not isolated and identified these bands as BGN protein products, we can only address the 37/42 kDa band as BGN protein.

A consistent finding is that the 37/42 kDa BGN protein band is largely reduced in age-matched Turner fetuses (fetal probes 7 and 8) when compared with normal fetuses (fetal probes 2 to 5). However, the fetal probe 6, which originated from a 16 week old Turner fetus, showed more of the 37/42 kDa BGN protein, the amount being similar to the one in the normal 19 week fetus (Figure 15).


    Discussion
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 Abstract
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 Materials and methods
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 Discussion
 References
 
The simplified picture of connective tissues as frameworks of insoluble fibrils and soluble polymers highlights the importance of collagen fibres in resisting tensile stress and of proteoglycans in binding large amounts of water, thus swelling and resisting compressive forces (Tables III and IV). The key feature of many of the glycoproteins is their ability to interact not only with cells, but also with other matrix proteins or growth factors. Adhesive glycoproteins thus have the ability to influence cell behaviour by allowing attachment and migration of cells (Ayad et al., 1994Go).


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Table III. Properties of the small proteoglycans biglycan (BGN) and decorin (DCN) characterized by a core protein to which is attached one or more glycosaminoglycan (GAG) side-chains
 

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Table IV. The glycosaminoglycan group of complex carbohydrates includes dermatan sulphate (DS), heparan sulphate (HS), keratan sulphate (KS), chondroitin sulphate (CS), hyaluronan (HA) and the aggrecan macromolecule
 
The findings of this study suggest that in the nuchal skin of fetuses with Turner syndrome, proteoglycan expression is substantially different from normal in the first trimester of pregnancy. In particular, chondroitin-6-sulphate is increased and stains a much wider area in the skin (Figures 2f, 8 and 14) while BGN is underexpressed (Figures 9 and 15). Distribution of the proteoglycan DCN and the glycosaminoglycans dermatan, heparan and keratan sulphates is not affected in fetuses with Turner syndrome (Figures 2, 57 and 10). In fetuses with trisomies, substantial changes from normal were not observed (Figures 38). In addition, in normal fetuses BGN, DCN and chondroitin-6-sulphate stainings did not show increased protein levels with increasing gestation ranging from 12 to 17 weeks (Figures 1214).

Chondroitin sulphate proteoglycans are known to have an influence on cell migration, in particular the migration of neural crest cells and outgrowing axons (Landolt et al., 1995Go). Potentially, the migration of blood and lymphatic vessel-forming cells could also be modified by an abnormal extracellular matrix (Miyabara et al., 1989Go). This might be an explanation for lymphatic vessel hypoplasia, in particular in the upper part of the skin in Turner syndrome fetuses (von Kaisenberg et al., 1999Go), where large skin edemas in the nuchal region at 11–14 weeks can frequently be observed.

The finding that in Turner syndrome fetuses there was underexpression for BGN is not surprising since this small proteoglycan is encoded by the X chromosome (Figure 9). The BGN gene was mapped to chromosome Xq13-qter (McBride et al., 1990Go), Xq27-ter (Fisher et al., 1991Go), Xq27-q28 (Traupe et al., 1992Go), and recently to the distal long arm of the X chromosome, band Xq28, near the second pseudo-autosomal region (Geerkens et al., 1995Go).

None of the seven fetuses with Turner syndrome showed mosaicism and there were no structural abnormalities of the X chromosome. However, one Turner fetus showed a Robertsonian translocation 13/14. This is of interest, because mosaicism was reported in 58/91 (66.7%) of Turner syndrome fetuses and 11/91 (12.6%) showed non-mosaic structural aberrations of the X chromosome, for example ring formation (Held et al., 1992Go). The existence of a normal additional sex chromosome in a mosaic could have substantially influenced gene expression.

BGN is expressed in a range of tissues and cell types, including connective tissue, human skin at the cell surface of differentiating keratinocytes and growing bone (Bianco et al., 1990Go; Traupe et al., 1992Go). A critical role particularly in long-bone growth was therefore postulated—Turner syndrome individuals (45X) are known to be small in stature and have a short femur. This phenotype has also been observed in BGN-deficient mice (Xu et al., 1998Go), while Klinefelter individuals (47XXY) and triple-X women (47XXX) are tall (Table I, Figure 1). BGN-deficient mice demonstrated both increased apoptosis of bone marrow stromal cells and decreased proliferation, leading to a deficiency in the generation of mature osteoblasts resulting in clinical osteopenia (Chen et al., 2002Go). BGN knockout mice are defective in the synthesis of type I collagen mRNA and protein and mimic Ehlers–Danlos-like changes in bone and other connective tissues (Chen et al., 2002Go; Corsi et al., 2002Go). BGN is also located in the aorta and fetuses with Turner syndrome often suffer from extreme narrowing of the aortic isthmus presenting with aortic isthmus stenosis at birth. Fibroblast cultures from postnatal Turner patients contained only 50% of BGN mRNA and protein as age-matched control cultures (Table I, Figure 1), whereas normal males, who also have only one X chromosome, show transcript levels of 100% (Geerkens et al., 1995Go). Abnormal distribution of BGN in Turner syndrome, found using in-situ hybridization, could not be confirmed by immunohistochemical studies and there was some degree of signal variation (Figures 2a, 3a, e and 12). Northern and Western blot analyses were performed in the skin of three fetuses with Turner syndrome and age-matched controls to quantify BGN mRNA and protein levels (Figure 15). BGN RNA seemed to be at similar levels compared with normal, but the stability of BGN RNA seemed to be reduced, which becomes apparent through the appearance of smaller BGN hybridizing bands. BGN protein patterns in Turner syndrome fetuses were largely different from normal. These data confirm the observation that BGN protein levels are decreased already in first trimester fetuses with Turner syndrome compared with normal controls (Figure 15). Further experiments could not be carried out, because Turner syndrome is one of the rarest numerical chromosomal abnormalities and further fetal samples were not available.

DCN, which is encoded on chromosome 12 (McBride et al., 1990Go), was as expected unchanged in the nuchal skin of Turner syndrome fetuses by immunohistochemistry (Figures 2b, 4a and e), in-situ hybridization (Figures 10 and 11) and Northern blotting (Figure 15). DCN is associated with fibrillar collagen I and II and its distribution is mutually exclusive to BGN (Bianco et al., 1990Go). Its distribution suggests that DCN is involved in regulating collagen fibril formation and organization. Moreover it has been shown that it can modulate the activity of growth factors by binding them (Yamaguchi et al., 1990Go). By using a double staining technique, we could demonstrate that DCN and collagen type I are already co-localized in the fetal skin (Figure 11). It was shown in vitro that DCN of bovine tendon demonstrated a unique ability to inhibit fibrillogenesis of both type I and type II collagen from bovine tendon and cartilage respectively (Vogel et al., 1984Go; Uldbjerg et al., 1988Go; Brown et al., 1989Go). Unlike BGN, DCN and collagen levels did not differ from control in Turner patients (Figure 11).

Keratan, heparan and dermatan sulphate did not show any obvious difference in distribution or expression in the nuchal skin of Turner syndrome fetuses, fetuses with trisomies or normal controls (Figures 2 and 57). The balance between proteases and inhibitors is critical for the steady state level of glycosaminoglycans, which involves the rate of protein synthesis, of post-translational modifications by glycosidases and sulphatases as well as proteases. The study of dermatan and heparan sulphate was performed because the enzyme iduronate-2-sulphatase (IDS), which metabolizes both molecules, is located on Xq28. We therefore assumed that the activity of this enzyme could be reduced in Turner syndrome resulting in an accumulation of its substrates. However, no changes have been observed at this early stage of pregnancy.

One of our most striking findings was the intense staining for chondroitin-6-sulphate in the dermis of nuchal skin of Turner fetuses, which extends down to a chain of vessels at the dermis–subcutis junction (Figures 2f, 8 and 14). The area below this junction was practically devoid of staining. This is compatible with previous findings in the nuchal skin of fetuses with Turner syndrome, where aberrant distribution of lymphatic vessels in the upper dermis was found using a PTN63 antibody against 5'-nucleotidase (von Kaisenberg et al., 1999Go). The scarcity of lymphatics in the upper dermis and the congestion of large diameter vessels at the dermis–subcutis junction was presumably the cause for the increase in nuchal translucency arising from failure of interstitial fluid drainage in this area.

Potential mechanisms for increased nuchal translucency are increased proteoglycans, which bind large amounts of water, thus creating swelling of the skin. This is true for chondroitin-6-sulphate in the dermis of nuchal skin of Turner fetuses found in this study and for hyaluronan in the dermis of nuchal skin of trisomy 21 fetuses (Böhlandt et al., 2000Go). Furthermore, adhesive glycoproteins influence cell behaviour by attachment and abnormal migration, potentially altering the migration of blood and lymphatic vessel-forming cells (Ayad et al., 1994Go), leading to lymphatic vessel hypoplasia (von Kaisenberg et al., 1999Go) or narrowing of the aortic isthmus (Hyett et al., 1997Go). BGN is located in the aorta. In Turner syndrome fetuses BGN is underexpressed in the nuchal skin as shown in our study and is likely to be reduced in the aortic arch. Turner syndrome fetuses frequently suffer from narrowing of the aortic isthmus, potentially as a result of decreased BGN, or of abnormal migration of cells from the neural crest, which form the aortic arch. In Turner fetuses with narrowing of the aortic arch, there is increased impedance to flow in the aorta and potentially overperfusion of the head and neck, leading to the highest degree of increase in nuchal translucency. In fetuses with trisomies 21, 18 and 13, unlike in Turner’s syndrome, there is normal BGN expression, and the mechanism of increased nuchal translucency is different. There is however, as the result of a gene dosage effect, overexpression of insoluble fibrils as part of the framework of connective tissues, in particular collagen type VI, laminin and collagen type IV respectively (von Kaisenberg et al., 1998aGo,b).

In conclusion, this study shows that fetuses with Turner syndrome show a distribution pattern for chondroitin-6-sulphate and BGN that is substantially different from normal. This indicates a genetic regulation of extracellular matrix components in first trimester fetuses and may help explain the pathophysiology of increased nuchal translucency.


    Acknowledgements
 
The study was supported by a grant from the Deutsche Forschungsgemeinschaft (Number Ka 1136/1-1, Ka 1136/1-2) and from the Fetal Medicine Foundation. Dr Larry Fisher from the NIH, USA, supplied the BGN plasmid P16 and antiserum LF121, and the DCN plasmid P2 and antiserum LF-122. We would like to thank Walter Just, MD, PhD, from the Department of Human Genetics, Ulm University Hospital, for very helpful comments in preparing the manuscript. Ellen Gimbel was of great technical assistance and Christa Micucci provided excellent technical and photographic assistance. Heidemarie Ihms was very supportive in reproducing the figure on the BGN gene expression.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on December 12, 2002; resubmitted on April 3, 2003; accepted on September 8, 2003.