Genetic analysis of the dystroglycan gene in bronchopulmonary dysplasia affected premature newborns

Clinica Chimica Acta 378 (2007) 164 – 167 www.elsevier.com/locate/clinchim

Genetic analysis of the dystroglycan gene in bronchopulmonary dysplasia affected premature newborns Paola Concolino a , Ettore Capoluongo a , Concetta Santonocito a , Giovanni Vento c , Milena Tana c , Costantino Romagnoli c , Cecilia Zuppi a , Franco Ameglio a , Andrea Brancaccio b , Francesca Sciandra b,⁎ b

a Laboratorio di Biologia Molecolare, Università Cattolica del Sacro Cuore, Italy CNR, Istituto di Chimica del Riconoscimento Molecolare c/o Istituto di Biochimica e Biochimica Clinica, Università Cattolica del Sacro Cuore, Italy c Dipartimento di Pediatria, Divisione di Neonatalogia Università Cattolica del Sacro Cuore, Largo Francesco Vito 1, 00168 Rome, Italy

Received 24 October 2006; accepted 17 November 2006 Available online 25 November 2006

Abstract Background: Dystroglycan (DG) is an extracellular matrix receptor that serves as an adhesion molecule and is essential for the stability of the plasma membrane. DG is highly expressed within the epithelial cell layer where it supports morphogenesis, adhesion and wound repair. Mechanically ventilated newborns often develop bronchopulmonary dysplasia (BPD), characterized by a progressive impairment of wound repair capacity in their lung. Methods: To verify if the susceptibility to BPD might be linked to genetic abnormalities in the DG gene (DAG1), we searched for possible mutations in 33 premature newborns with gestational age b 34 weeks with risk of developing BPD. DAG1 genotype was determined in 11 premature newborns with BPD as compared to 22 premature infants without lung complications. Results: Eight polymorphisms were found, four of them being new DAG1 single nucleotide polymorphisms (SNPs). Only one significant association was found with BPD positive infants: the N494H homozygous genotype (p = 0.033). The same polymorphism was found significantly associated with BPD when allelic frequencies were considered (p = 0.0015). Conclusions: Our data enrich the list of DAG1 SNPs and could be useful to trigger further genetic studies about the involvement of DG in human diseases. © 2006 Elsevier B.V. All rights reserved. Keywords: Dystroglycan; Extracellular matrix; Single nucleotide polymorphisms; Bronchopulmonary dysplasia; DNA sequencing

1. Introduction Dystroglycan (DG) is a widely expressed extracellular matrix adhesion complex that anchors the cells to the surrounding extracellular matrix [1]. Originally, DG was

Abbreviations: DG, dystroglycan; DGC, dystrophin glycoprotein complex; BPD, bronchopulmonary dysplasia; SNPs, single nuclear polymorphisms. ⁎ Corresponding author. Istituto di Chimica del Riconoscimento Molecolare, Università Cattolica del Sacro Cuore, Largo F. Vito 1, 00168, Rome, Italy. Tel.: +39 0630155135; fax: +39 0630156706. E-mail address: [email protected] (F. Sciandra). 0009-8981/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2006.11.016

isolated from skeletal muscle, as part of a large multimeric dystrophin–glycoprotein complex (DGC) composed of dystrophin, sarcoglycans, syntrophins, dystrobrevins and sarcospan [2,3]. The DG complex is expressed as a single precursor that is posttranslationally cleaved to generate two subunits, αand β-DG. α-DG is a highly glycosylated peripheral protein that binds the laminin–neurexin-sex hormone-binding globulin (LNS) domain containing proteins, such as laminin, perlecan and agrin, and interacts non-covalently with the β subunit [4]. β-DG is a transmembrane protein whose cytoplasmatic tail interacts with dystrophin and other cytoskeletal proteins [5]. Therefore DG represents a functional non-integrin link between the extracellular matrix and the

P. Concolino et al. / Clinica Chimica Acta 378 (2007) 164–167

cells playing an important role for the structural stability of the plasma membrane [6]. DG is expressed in many cellular types and tissues where it is involved in crucial cellular functions: it is essential for maintaining the correct structure and function of skeletal muscle and nervous system [7,8], it has been implicated in myelination and nodal architecture of peripheral nerves [9], synaptogenesis [10] and signalling [11]. DG is highly expressed also in epithelial cells where it is particularly prominent on the basal side facing the basement membranes [12]. DG is involved in epithelial morphogenesis [13] and it supports the maintenance of cell polarity [14]. Knockout DG mice undergo premature death early during development (6.5 days) mainly due to their inability to form and develop the Reichert's membrane, the first extraembryonic basement membrane [15]. Recent reports highlight a possible implication of DG also in the progression of some epithelial tumours, where a wrong processing of β-DG was observed [16]. In fact, in some cell lines and tumour biopsies, an abnormal proteolytic cleavage of the β-DG ectodomain that may lead to the loss of the α-DG subunit from the cell periphery was observed [16]. This phenomenon could generate a failure in establishing proper contacts between the DG complex and its extracellular matrix binding partners thus eventually chaperoning cell invasion and tumour progression. In epithelial cells, extracellular matrix receptors are also implicated in wound repair after injury [17]. The involvement of DG in wound repair was demonstrated in lung, where it is expressed both by smooth muscle and airway epithelial cells [12,18]. A decreasing capacity of wound repair in lung is often associated with chronic lung disease involving formation of scars that are replaced by fibrotic tissue. Pulmonary fibrosis is one of the main characteristics of bronchopulmonary dysplasia (BPD), which is considered as one of the most common complications in premature newborns [19]. The genetic factors that may influence the presence or severity of neonatal lung disorders are still largely unknown [20]. We analysed the coding region of the DG gene, DAG1, in a sample of 33 premature newborns, in order to investigate whether there is a correlation between a widely expressed adhesion complex such as DG and the susceptibility to BPD [12]. 2. Methods 2.1. Patients This study was carried out in the Neonatal Intensive Care Unit of the Catholic University of Rome between July 2005 and June 2006 and included 33 premature white Caucasian infants. Infants were enrolled in this study if they fulfilled the following inclusion criteria: gestational age b34 weeks, endotracheal intubations at birth and on-going intensive care required. Newborns with major congenital malformations, prenatal infections [positive blood or bronchoalveolar lavage fluid (BALF) cultures at birth], with severe asphyxia (Apgar score b 5 at 1′ and 5′), or treated with early (first week of life) dexamethasone therapy were all excluded from this study. Infants enrolled in this study but who died before the BPD diagnosis were also excluded from the final analysis. All newborns received surfactant therapy (200 mg/kg of Curosurf—Chiesi Farmaceutici, Italy) during the first 6 h of life. Mechanical ventilation was performed with BabyLog 8000 plus (Draeger, Lubeck, Germany) in high

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frequency oscillatory ventilation modality, as previously described [21]. BPD diagnosis was made when the infant needed more than 21% of oxygen and/or mechanical ventilation at 36 post-conception weeks, with characteristic radiographic features. The study was approved by the Ethical Committee of the Department of Pediatrics, following the indication of the Helsinki Declaration. Our analysis also included a control group of 20 healthy blood donors, 10 males and 10 females, all born at term. This group was used to compare premature subjects with at term individuals.

2.2. Genetic analysis Genomic DNA was extracted from blood samples containing EDTA, using a commercially available kit (High Pure PCR template preparation kit, Roche Diagnostic GmbH, Germany). The three DAG1 (Gene Bank NM_004393) exons and their intron/exon boundaries were PCR amplified from genomic DNA of premature newborns and adult donors. Primers utilized for amplification were previously described [22] and were synthesized by Pharmacia (Uppsala, Sweden). PCR reactions were performed in a volume of 50 μl with a 3 mM MgCl2 final concentration. After a hot start of 2 min at 95 °C, 40 cycles were performed, each consisting of a denaturation step (30 s) at 95 °C, an annealing step (45 s) at 60 °C and an extension (2 min) at 72 °C. The efficiency of the PCR reaction was checked in a 1% agarose gel electrophoresis. DNA sequence analysis was performed from PCR reactions using an automated DNA sequencer (ABI Prism 3100 Avant, Applied Byosistems) and the Big Dye terminator V.3.1 staining Kit (Applied Byosistems) according to the manufacturer's instructions. Results were analyzed using the Seqscape v2.1 software package (Applied Byosistems).

3. Results and discussion 11 out of 33 premature infants developed BPD while the remaining 22 did not develop lung complications. The main characteristics of the three groups are reported in Table 1. No significant differences were observed for gender, gestational age and birth weight between BPD positive and BPD negative patients. Obviously, the group of healthy blood donors was selected between at term born individuals, but their birth weight was not available. Table 2 lists the 8 polymorphisms found in our analysis, the numbers referring to the amino acids (Fig. 1B). Four of these SNPs were already identified [22,23], namely D163E, P248A, H752H and V871A. In this report we present the identification of four new DAG1 genetic variants mapping in the exon 3 and located within the α subunit (Fig. 1A): A375G and E412D, in the mucin-like region, and S485R and N494H, in the C-terminal domain of α-DG (Fig. 1B). Only in one case the two SNPs, A375G and S485R, were concomitantly present in the same Table 1 Characteristics of patients and controls (medians and ranges or frequencies) Variables

CLD positive premature newborns (n = 11)

CLD negative premature newborns (n = 22)

Adult healthy blood donors (n = 20)

Gestational age (weeks) Birth weight (g) Males Females

27 (24.0–31.0)

28 (25.0–33.0)

At term

860 (510–1250)

1010 (460–2230)

Not available

11 11

6 5

10 10

Comparison were performed only considering CLD positive versus CLD negative groups. No significances were observed.

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Table 2 Distribution of DAG-1 genetic variants among 53 subjects analyzed

Table 3 Distribution of allelic frequencies of DAG-1 variants in 53 subjects analyzed

Gene variants a

Allelic variants⁎

D163E P248A A375G a E412D a S485R a N494H a H752H V871A

CLD positive

CLD negative

Adult healthy blood donors

WT

Ht

Homo

WT

Ht

Homo

WT

Ht

Homo

0 2 10 11 11 7 5 2

2 0 0 0 0 0 4 1

9 9 1 0 0 4 2 8

2 4 21 21 21 21 15 6

7 3 1 1 1 0 5 4

13 15 0 0 0 1 2 12

nd nd 20 20 20 16 nd nd

nd nd 0 0 0 1 nd nd

nd nd 0 0 0 3 nd nd

Comparison between CLD positive or CLD negative only gave a significant difference (p = 0.033) (Yates' corrected Chisquare) for N464H variant (bold). No significant differences were found between healthy blood donors and CLD positive or negative groups, respectively. WT, wild type DAG1 gene. Ht, heterozygotes DAG1 gene. Homo, homozygotes DAG1 gene. nd = not determined. a Newly identified DAG1 SNPs.

subject. E412D was identified only in a BPD negative affected heterozygous patient. The novel DAG1 SNP N494H was identified in four BPD affected patients as well as in one infant not affected by BPD (p = 0.033). Three N494H homozygous subjects and one heterozygous were observed in the healthy blood donors while the A375G, E412D and S485R DG SNPs were not found. Table 3 shows the distribution of the allelic frequencies of DAG variants among the 53 subjects analysed: only the N494H SNP showed significant differences between BPD positive and BPD negative newborns. Prematurely born infants who require mechanical ventilation frequently develop BPD. While in the past BPD was attributed only to environmental effects, recent advances suggest that also

D163E P248A A375G E412D S485R N494H H752H V871A

D E P A A G E D S R N H H H V A

CLD positive

CLD negative

Adult healthy blood donors

N = 22

%

N = 44

%

N = 40

%

2 20 4 18 20 2 22 0 22 0 14 8 14 8 5 17

9.1 90.9 18.2 81.8 90.9 9.1 100 0 100 0 63.6 36.4 63.6 36.4 22.7 77.3

11 33 11 33 43 1 43 1 43 1 42 2 35 9 16 28

25.0 75.0 25.0 75.0 97.7 2.30 97.7 2.30 97.7 2.30 95.5 4.5 79.5 20.5 36.4 63.6

nd nd nd nd 40 0 40 0 40 0 33 7 nd nd nd nd

nd nd nd nd 100 0 100 0 100 0 82.5 17.5 nd nd nd nd

⁎All comparisons between CLD positive and CLD negative were not significant with the exception of N494H variant giving a p = 0.0015 (Yates' corrected Chisquare). All comparisons between premature newborns and adult healthy blood donors were not significant.

genetic factors may play an important role [20]. BPD is characterized by oxidative injury and mechanical disruption of the lung. Alterations in either the composition of the basement membrane or in the ability of the airway epithelium to bind the surrounding extracellular proteins may influence the repair process required during and after inflammatory injury to the mucosa [18]. The alveolar septation and the whole development of the immature lung are consequently deeply impaired. Indeed, the list of candidate genes potentially involved in BPD may also include genes responsible for lung morphogenesis and/or repair. The DG gene, DAG1, is located on

Fig. 1. A, Diagram of the structure of human DAG1 gene (not drawn to scale): DAG1 spans ≈ 65 kb of genomic DNA and it is composed by 3 exons: noncoding exon 1, exon 2 containing the ATG codon and part of the α subunit, exon 3 encoding for the remaining portion of α-DG, the whole β subunit ending with the stop TAA codon. B, Scheme of DG and its domain organization. The α subunit has a dumbbell-structure, with two globular N- and C-terminal domains separated by a highly glycosylated mucin-like region. β-DG is a transmembrane protein, binding α-DG through its N-terminal ectodomain while its cytodomain interacts with the cytoskeleton. The 4 newly identified SNPs, mapping within the central mucin-like region and the C-terminus domain of α-DG, are depicted (arrows); the numbers refer to their amino acidic position.

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chromosome 3p21, spans ≈ 65 kb of the human genome and consists of 3 exons (Fig. 1A) [24]. The first exon consists of a noncoding sequence; the exon 2 encodes a small N-terminal portion of the α subunit, whereas the third exon contains the remaining portion of the protein. The exons and the surrounding intronic regions of DAG1 were sequenced for each patient (Fig. 1A). DAG1 protein (Fig. 1B) is a receptor for extracellular matrix molecules that plays a crucial role for the structural stability of cells and tissues [4]. The DG complex is highly expressed in the lung and it is involved in the development of its epithelial tissue [13] where it is thought to be involved also in the wound repair of epithelial airway cells upon injury [18]. No natural mutations occur in human DAG1 and the DG knockout mouse was found to be embryonic lethal due to the disruption of the basement membrane at the peri-implantation stage, indicating that DG is a key player in development [15]. However, numerous human DAG1 SNPs have already been identified [22,23]. We have sequenced DAG1 of 11 premature infants affected with BPD, as compared to 22 premature babies not developing lung complications and to 20 adult healthy donors born at term, in order to address if DAG1 SNPs may be associated as risk factor to BPD susceptibility. We have found 4 new SNPs, namely A375G, E412D, S485R and N494H. A375G and S485R map within the mucin-like domain and in the C-terminal region of α-DG respectively. The SNP N494H hits the C-terminal domain of α-DG, and it was found to be increased (both as allelic frequency and homozygous genotype) in BPD positive patients that were homozygous. The putative functional role played by DAG1 SNPs and in particular by N494H is not yet known and it should be carefully assessed in the future by site-directed mutagenesis [25,26]. Given the great biomedical implications due to the involvement of DAG1 in several human pathologies, the search for novel SNPs, or mutations leading to even more severe genetic abnormalities (such as partial deletions or nonsense mutations), possibly correlating with novel neuromuscular disorders or other human diseases, should be constantly expanded and considered of primary importance [27,28].

[5]

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[22]

Acknowledgements This study was partially supported by MIUR COFIN 2003: “Functional basis and structural aspects of a natively unfolded protein, the extracellular domain of beta-dystroglycan”.

[23]

[24]

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