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Recently, heterozygous mutations of human NKX2.5 were identified in patients with congenital heart disease. (1 2) The most common phenotypes were progressive atriaventricular conduction delays (AV block) and secundum atrial septal defect (ASD), but other anatomical abnormalities, such as ventricular septal defect, tetralogy of Fallot, or tricuspid valve abnormalities, including Ebstein's anomaly, and progressive left ventricular failure, were also reported. These findings strongly suggest that NKX2.5 is important in the later stages of heart development and maturation in addition to its role in cardiac progenitor commitment and patterning in the developing heart.
To characterise further the genotype-phenotype correlation of NKX2.5 mutations, we used NKX2.5 as a candidate gene in two kindreds where subjects in multiple generations had congenital heart disease and AV block. In this report, we describe phenotypes in two families with novel frameshift mutations in NKX2.5. The findings suggest an expanded population of subjects who could now be examined for mutations within this transcription factor and its upstream and downstream regulatory elements.
MATERIALS AND METHODS
All studies were carried out in accordance with the institutional guidelines for human research. Subjects were evaluated by history, review of medical records, physical examination, 12 lead electrocardiogram (EGG), and two dimensional transthoracic echocardiography with colour flow Doppler. Cardiac catheterisation, abdominal echography, and/or cardiac surgery have been performed in some subjects.
Following the methods of Benson et al, (1) the products, including all coding regions and intervening intron of NKX2.5, were amplified with the primers (1F and 4R) in a single polymerase chain reaction (PCR), using standard conditions and concentrations of reagents. Products were analysed on standard 1% agarose gels and stained with ethidium bromide. Sequencing primers used were previously designed for specific region sequencing (1) and carried out on the Applied Biosystems DNA Sequencing system (Model 373 or 377). For family 1, the deletion mutation was originally identified as two apparently different sequences overlapping at the deletion point in direct sequencing analysis. To clarify the actual sequence change on the mutant allele, the PCR products were amplified with 1F and 1R, cloned by a TA cloning kit, and sequenced. Four individual clones were sequenced with two for the wild type and two for the mutant allele identified. To confirm 215-221 de1, sequence length differences were determined using primer s oywA17 (5'-CTAAACCTGGAACAGCAGCA-3') and oywA18 (5'-TTTTCGGCTCTAGGGTCCTT-3'), designed at the flanking region to the deletion mutation in exon 1. A BssHII site was abolished by 223-224del allowing independent confirmation of this deletion mutation (not shown).
A 7 bp (AGCTGGG) deletion in exon 1 at nucleotide +215 from the translation starting point of the NKX2.5 gene …