Detailed Mapping of a Congenital Heart Disease Gene in Chromosome
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J Med Genet 2000;37:581–587 581 Detailed mapping of a congenital heart disease J Med Genet: first published as 10.1136/jmg.37.8.581 on 1 August 2000. Downloaded from gene in chromosome 3p25 Elaine K Green, Matthew D Priestley, Jonathan Waters, Chris Maliszewska, Farida Latif, Eamonn R Maher Abstract analysis of large AVSD families have excluded Distal deletion of chromosome 3p25-pter linkage to the Down syndrome critical region (3p− syndrome) produces a distinct clinical on chromosome 21, so establishing locus syndrome characterised by low birth heterogeneity in the genetic pathogenesis of weight, mental retardation, telecanthus, AVSD.23 In view of the complexity of cardiac ptosis, and micrognathia. Congenital heart development, such heterogeneity is expected. disease (CHD), typically atrioventricular Linkage analysis in a large extended family septal defect (AVSD), occurs in about a mapped an atrioventricular septal defect sus- third of patients. In total, approximately 25 ceptibility locus to chromosome 1p31-p21.4 In cases of 3p− syndrome have been reported this kindred inheritance was observed to be world wide. We previously analysed five autosomal dominant with incomplete pen- cases and showed that (1) the 3p25-pter etrance. However, large AVSD families are deletions were variable and (2) the pres- rare, and so the potential for identifying AVSD ence of CHD correlated with the proximal genes using linkage is limited. extent of the deletion, mapping a CHD Cytogenetic deletion syndromes are rare, but gene centromeric to D3S18. To define the can provide important clues to the localisation of molecular pathology of the 3p− syndrome developmental genes. Classical examples of this further, we have now proceeded to analyse approach include the association of cardiac and the deletion region in a total of 10 patients other developmental defects with chromosome (five with CHD), using a combination of 22q11 deletions, and the occurrence of Hirsch- FISH analysis and polymorphic markers, sprung disease (HSCR) with chromosome 10 for up to 21 loci from 3p25-p26. These addi- deletions, leading to the identification of as tional investigations further supported the RET location of an AVSD locus within 3p25 and a HSCR susceptibility gene. Cytogenetic dele- tion syndromes associated with AVSD include refined its localisation. Thus, the critical 56 7 region was reduced to an interval between del(8)(p23) and del(3)(p25). Analysis of D3S1263 and D3S3594. Candidate 3p25 del(8)(p23) cases mapped the critical deletion region for heart defects and microcephaly to a 6 http://jmg.bmj.com/ CHD genes, such as PMCA2 (ATP2B2), 8 fibulin 2, TIMP4, and Sec13R, were shown Mb region of 8p23.1. Distal deletion of the to map outside the target interval. Addi- short arm of chromosome 3 (3p− syndrome) is ∼ tionally, the critical region for the pheno- a rare disorder ( 25 reported cases) associated typic features of the 3p− phenotype was with developmental and growth retardation as mapped to D3S1317 to D3S17 (19-21 cM). well as a characteristic dysmorphology (includ- Section of Medical and These findings will accelerate the identifi- ing ptosis, telecanthus, and micrognathia). Molecular Genetics, cation of the 3p25 CHD susceptibility locus Additional, but variable, developmental defects Department of and facilitate investigations of the role of include postaxial polydactyly, renal anomalies, on September 27, 2021 by guest. Protected copyright. Paediatrics and Child this locus in non-syndromic AVSDs, which cleft palate, and gastrointestinal anomalies. Health, University of are a common form of familial and isolated Congenital heart defects occur in about a third Birmingham, 79 Edgbaston, CHD. of patients and are typically AVSDs. To date, Birmingham B15 2TG, (J Med Genet 2000;37:581–587) all patients with 3p− syndrome have had large UK cytogenetically visible deletions, although there E K Green Keywords: congenital heart disease; chromosome 3p25 are also reports of patients with large deletions M D Priestley and a normal phenotype10 (unpublished obser- F Latif E R Maher Congenital heart defects (CHD) are an impor- vations). tant cause of morbidity and mortality aVecting We and others have reported that the extent West Midlands ∼ of distal 3p deletions in 3p− syndrome patients 8 in every 1000 children. Although most cases 79 Regional Genetics are sporadic, the role of genetic factors in CHD is variable. Furthermore, we found that of Service, Birmingham five patients with 3p− syndrome studied, the Women’s Hospital, has received increasing attention because of the major advances in paediatric cardiac surgery, three with CHD had the most proximal break- Birmingham B15 2TG, 11 UK such that now more than 85% of children with points. Subsequently, Drumheller et al re- J Waters CHD survive into adulthood. Overall, the ported data on five patients (including one case C Maliszewska recurrence risk for most types of isolated studied previously by us7) that provided further E R Maher ∼ evidence for a chromosome 3p25 CHD gene. Correspondence to: congenital heart defect is 5% but atrioven- Professor Maher, tricular septal defects (AVSD) appear to have a To confirm and extend these findings we have [email protected] greater genetic contribution.1 Thus, follow up collected a total of 10 cases of 3p− syndrome of adults with AVSD suggests that at least 20% and performed molecular cytogenetic and Revised version received of apparently isolated cases result from a molecular genetic studies to define the extent 25 March 2000 Accepted for publication dominant gene. AVSD is classically associated of the 3p deletions and correlate this with the 14 April 2000 with Down syndrome, but genetic linkage presence or absence of CHD. www.jmedgenet.com 582 Green, Priestley, Waters, et al Table 1 Details of FISH probes and microsatellite mal recovery of PAC DNA were used. The P1 markers used in the study. The probes marked by * are PAC clones isolated are shown in table 1. J Med Genet: first published as 10.1136/jmg.37.8.581 on 1 August 2000. Downloaded from clones derived from RPCI1 library, and those probes starting with AC are cosmid probes isolated from the library LLO3NCO1, both obtained from the UK HGMP resource ISOLATION OF COSMIDS centre Cosmids were isolated for the markers Locus FISH probe Polymorphism D3S3088, D3S3714, and D3S3589 from the chromosome 3 library LLO3NO3 obtained D3S1304 — Microsatellite D3S18 cLIB 1 — from the UK HGMP resource centre. Hybridi- D3S1597 — Microsatellite sation probes were prepared from the amplified D3S1317 — Microsatellite PCR products from YAC 753F7 and 70D6 D3S601 cos 7 — D3S1038 cos 14 Microsatellite DNA, purified by Qiagen PCR product kit and D3S3589 61P20* / AC50K12 Microsatellite radiolabelled by random priming ∼25 ng using D3S587 cLIB 12-48 — the Rediprime kit (Amersham). Repetitive D3S3601 — Microsatellite D3S3594 226D20 Microsatellite sequences were blocked by preannealing with D3S1263 283J16* Microsatellite Cot-1 DNA (Gibco BRL) and hybridising NIB1677 201I11* Microsatellite under standard conditions12 at 65°C. The D3S3714 183M12* / AC12J24, AC19C22 Microsatellite D3S3680 183M12* Microsatellite identified clones (table 1) were obtained from D3S3088 225B20* / AC38A7 — the UK HGMP resource centre then main- D3S1259 248M7* / 275B20* Microsatellite tained in LB media supplemented with ampi- D3S3701 — Microsatellite D3S3602 — Microsatellite cillin. Cosmid DNA was isolated using the D3S3693 — Microsatellite Qiagen Midi kit. D3S3610 — Microsatellite D3S1585 289I19* Microsatellite FLUORESCENCE IN SITU HYBRIDISATION (FISH) Metaphase chromosome spreads were pre- Materials and methods pared from EBV transformed cell lines using PATIENTS AND SAMPLES standard methods. Before chromosome har- Lymphoblastoid cell lines were available for vest, cells were blocked in metaphase by the nine patients: P1, P2 (CUMG3.1), P3 (GM addition of colcemid to a concentration of 10 10922), P4, P5 (CUMG3.4), P6, P7, P8 (GM µg/ml. Probes (PACs, cosmids, and YACs) 10985), and P9, but not P10 (CUMG3.10). were biotinylated by nick translation with DNA was collected from the aVected child and biotin-11dUTP (Bionick labelling system, both parents in seven families (P1, P2, P5, P6, Gibco BRL). Chromosomal in situ suppression P7, P9, and P10), from the aVected child and (CISS) hybridisation was performed to im- mother in one case (P4), and only the aVected prove the specificity of the hybridisation, with child for P3 (GM 10922) and P8 (GM 10995). Cot-1 DNA (Gibco BRL) added to the biotin labelled probe at a ratio of 50:1. For all P1 ARTIFICIAL CHROMOSOME (PAC) ISOLATION hybridisations, a biotin labelled chromosome 3 AND MAINTENANCE alpha satellite centromere probe (Oncor) was http://jmg.bmj.com/ P1 clones were isolated by PCR based screen- used as a control to identify chromosome 3. ing using polymorphic marker primers (3p25- Hybridisation signals were visualised by a 26) from the UK HGMP resource centre three layer avidin-fluorescein isothiocyanate RPCI1 library.12 P1 clones were maintained in (FITC), biotinylate anti-avidin detection sys- LB media supplemented with 25 µg/ml Kana- tem. Analysis was carried out using a BX50 mycin and PAC DNA was isolated from the Olympus microscope and images captured host strains by column purification using the with an automated image analysis system Qiagen Maxi kit (Qiagen). The manufacturer’s (Cytovision, Applied Imaging). At least 20 on September 27, 2021 by guest. Protected copyright. recommended protocol adaptations for opti- metaphases were analysed for