METHODS Subjects A large Japanese family composed of a total of 29 members across four generations contained 11 members with ASD (I-1, II-2, II-6, II-7, III-1, III-2, III-4, IV-1, IV-2, IV-5, and IV-6; fig 1A). ASD in five patients (II-7, III-1, III-2, IV-1, and IV-2) was surgically repaired, and two patients (II-2 and IV-1) also had pulmonary stenosis (PS). The heart defects in eight subjects (II-2, II-7, III-1, III-2, IV-1, and IV-2) including two diseased subjects (I-1 and II-6) had been clinically diagnosed by one of co-authors (BK) on the basis of their past histories, operation records, 12-lead electrocardiograms, and echocardiograms with colour Doppler apparatus (fig 1A), while those in three other subjects (III-4, IV-5, and IV-6) were retrospectively found using information provided by the mutation analysis described below. None of the 11 affected members had any other abnormalities in the cardiac conduction system or other organs. After informed consent was obtained, DNA was extracted from peripheral blood leukocytes of 22 family members.

Linkage and mutation analyses Linkage analysis was performed in 22 family members at 11 microsatellite marker loci around GATA4 at 8p23 (D8S561, D8S503, D8S1721, D8S550, D8S265, D8S552, D8S1790, D8S549, D8S254, D8S258, and D8S1771), and two-point linkage analysis was carried out using the computer program MLINK (FASTLINK software, version 4.1 P), as described previously. 11 III-4, IV-5, and IV-6 were regarded as unaffected at this initial linkage analysis.