European Journal of Human Genetics (2009) 17, 344 – 351 & 2009 Macmillan Publishers Limited All rights reserved 1018-4813/09 $32.00 www.nature.com/ejhg ARTICLE Mutations in mammalian tolloid-like 1 gene detected in adult patients with ASD Paweł Stan´ czak1, Joanna Witecka2, Anna Szydło2, Ewa Gutmajster2, Małgorzata Lisik2, Aleksandra Augus´ciak-Duma2, Maciej Tarnowski2, Tomasz Czekaj2, Hanna Czekaj2 and Aleksander L Sieron´ *,2 1Swietokrzyskie Center for Cardiology, Regional Hospital, Kielce, Poland; 2Department of General and Molecular Biology and Genetics, CoE for Research and Teaching of Molecular Biology of Matrix and Nanotechnology, CoE Network, BioMedTech ‘Silesia’, Medical University of Silesia, Katowice, Poland Atrial septal defect (ASD) is an incomplete septation of atria in human heart causing circulatory problems. Its frequency is estimated at one per 10 000. Actions of numerous genes have been linked to heart development. However, no single gene defect causing ASD has yet been identified. Incomplete heart septation similar to ASD was reported in transgenic mice with both inactive alleles of gene encoding mammalian zinc metalloprotease a mammalian tolloid-like 1 (tll1). Here, we have screened 19 ASD patients and 15 healthy age-matched individuals for mutations in TLL1 gene. All 22 exons were analyzed exon by exon for heteroduplex formation. Subsequently, DNA fragments forming heteroduplexes were sequenced. In four nonrelated patients, three missense mutations in coding sequence, and one single base change in the 50UTR have been detected. Two mutations (Met182Leu, and Ala238Val) were detected in ASD patients with the same clinical phenotype. As the second mutation locates immediately upstream of the catalytic zinc-binding signature, it might change the enzyme substrate specificity. The third change, Leu627Val in the CUB3 domain, has been found in an ASD patient with interatrial septum aneurysm in addition to ASD. The CUB3 domain is important for substrate-specific recognition. In the remaining 15 patients as well as in 15 reference samples numerous base substitutions, deletions, and insertions have been detected, but no mutations changing the coding sequence have been found. Lack of mutations in relation to ASD of these patients could possibly be because of genetic heterogeneity of the syndrome. European Journal of Human Genetics (2009) 17, 344–351; doi:10.1038/ejhg.2008.175; published online 1 October 2008 Keywords: atrial septal defect; DNA sequencing; gene mutations; heart defects; heteroduplex analysis; mammalian tolloid-like 1 Introduction defects might result either from factors damaging the Congenital heart defects in general are related to aberrant fetus during development or they can be of genetic development of the circulation system in a fetus. The background. Some environmental factors, drugs, alcohol abuse, and infections contracted by the mother before, or during pregnancy (eg, rubella) may alter heart development.1–6 However, environmental and maternal *Correspondence: Dr AL Sieron, Department of General and Molecular Biology and Genetics, Medical University of Silesia in Katowice, factors have been linked to approximately 2% of con- ul. Medyko´w 18, Bldg. C-1, 40-752 Katowice, Poland. genital heart defects.7 In a significant number of heart Tel: þ 48 32 208 8394; Fax: þ 48 32 208 8382; abnormalities coexistence of a few anomalies has been E-mail: [email protected] Received 5 May 2008; revised 16 July 2008; accepted 29 August 2008; shown in different parts of the circulation system indicat- 1,8 published online 1 October 2008 ing genetic factors. Mutations in TLL1 causing ASD P Stan´czak et al 345 Chromosomal anomalies and single gene defects, how- gene for mutations by heteroduplex detection followed by ever, could be attributed to the background of 8% of DNA sequencing. congenital heart defects.7,9 – 11 Thus, the background of most of the congenital heart defects is still poorly understood. Materials and methods The heart itself and septation of the large vessels begin at Patients the fourth week of fetal development, but the process is not Hospital records of more than 100 cardiological patients completed until shortly after birth. Septation anomalies are have been reviewed for ASD cases. Twenty individuals have linked to abnormal development of endocardial cushions. been identified who fulfill the requirements of this project. They are necessary for completeness of interatrial as well as All of them were informed about procedures related to the interventrical septa. Incomplete interatrial septum is a study. The procedures were conducted in accordance with common feature of such genetic disorders as Noonan, the Institutional Guidelines and Regulations and with the Turner, and Holt-Oram syndromes.10 – 12 Guidelines for the Conduct of Research Involving Human The condition studied here is called atrial septal defect Subjects approved by the appropriate institutional review (ASD) and has some features common with the other just board (IRB) Bioethical Committee of the Silesian Medical listed heart-related syndromes. Currently, the defects University in Katowice, Poland, No. L.dz.NN-013-339/03. related to ASD are corrected during puberty. However, in In addition, appropriate informed consent was obtained the past, when it was untreated, the patients often suffered from the human volunteers. from numerous severe cardiovascular and pulmonary Finally, 19 individuals agreed to participate in the study complications. and they all have been surveyed for medical and family In recent years, results of research with the use of health history. On the basis of the survey’s results and transgenic mice models revealed that a gene named medical records they were divided into three groups tolloid-like 1 (tll1) is critical for septa development in the (Table 1). mouse heart.13 The mice lacking active TLL1 died at mid- As a reference, blood samples from 15 age-matching gestation because of severe blood circulation failure. The volunteers (11 females and 4 males) were used to isolate tll1 gene product is a metalloprotease with domain DNA for genetic analyses. None of the volunteers have had structure identical to one of the alternatively spliced inherited developmental heart conditions. variants of the bmp-1 gene product, mammalian tolloid (mTLD). The mTLD was detected in mammals and some Samples collection and handling other invertebrates.14 – 18 The homologous protein BMP-1 From each patient two blood samples were drawn. One was originally reported by Wozney et al19 in a context of 3 ml sample was mixed with heparin and used for ectopic bone formation in rats when protein extracts from cytogenetic diagnosis to exclude possible chromosomal demineralized bovine bones were used.20 Subsequently, anomalies. The methods for lymphocytes isolation and based on protein domain structure it was classified in the preparation for karyotyping were the classical methods family of astacin, a group of metalloproteases involved described before.35,36 Slides were examined with a Zeiss in embryo development of different species.17,21 – 23 It is Axioskop microscope. Images were obtained using a Sony also commonly accepted that BMP-1 is important for ExwaveHAD digital camera and MultiScanBase software numerous physiological activities acting in seemingly (Computer Scanning System Ltd., PL) with the karyotype unrelated processes, such as extracellular matrix quality option, enabling manual karyotyping. For each patient, a control involving formation and activation of numerous total of 15 metaphases in each of five prepared karyograms factors that act in the regulation of different genetic were examined to determine the number of chromosomes. programs.17 – 21,23 – 32 The second sample of blood (5.0 ml) was mixed with An investigation of different mouse model with ventrical EDTA and used for the isolation of genomic DNA. spetum defect (VSD) but with active tll1 gene indicated that a different mechanism must be involved, possibly PCR conditions related to abnormal cell proliferation and differentia- The DNA for mutation detection was isolated with the use tion.33,34 Developmental defects of tll1À/À embryos seemed of commercial blood mini-kit (A&A Biotechnology, to be exclusively restricted to the heart.13 The comparison Gdansk, Poland) according to supplier instructions. The of the defects with expression of the tll1 revealed, in quality of purified genomic DNA was verified by spectro- normal mice embryo, its elevated expression levels during metry at A260/280 and agarose gel electrophoresis. heart septation. Thus, it is possible that the expression of Fragments of the TLL1 gene were amplified using pairs of TLL1 in humans could also be critical for correct heart primers designed to complement intronic sequences positioning and its proper septation. flanking coding sequences (Table 2). The conditions for Here, we have tested 19 adult individuals with clinically amplification PCR were as follows: step (1) denaturation confirmed ASD by karyotyping and screening of the TLL1 at 951C for 4 min, step (2) denaturation at 951C for 30 s, European Journal of Human Genetics Mutations in TLL1 causing ASD P Stan´czak et al 346 Table 1 Classification of ASD patients based on their individual clinical symptoms Patient number Sex (F/M) Patient’s age (years) Additional defects Group 1: ASD without additional defects 3 M 51 ND 5 M 55 ND 7 M 71 ND 10a M35ND 11 M 52 ND 15 F 57 ND 18 F 52 ND Group 2: ASD with diagnosed aneurysm of interatrial septum and other defects 1 M 70 Aneurysm, heart attack, cerebral stroke, atrial fibrillation, and cardiac pacemaker implantation 4 M 55 Heart attack 6 M 45 Hypertrophic cardiomyopathy 9 M 72 Aneurysm of interatrial septum sinus bradycardia 12 F 58 Asymptomatic aneurysm of interatrial septum 13 F 48 Aneurysm of interatrial septum 17 F 48 Asymptomatic aneurysm of interatrial septum 19 F 62 Artificial mitral valve implantation Group 3: ASD with irregular heart beating rhythm 2 M 52 Atrial fibrillation, cardiac pacemaker implantation 8 M 51 Atrial fibrillation, cardiac pacemaker implantation 14 F 56 Cardiac pacemaker implantation 16 F 67 Atrial fibrillation ASD, atrial septal defect; F, female; M, male; ND, Not determined.