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Novel point mutation in the NKX2-5 gene in a Moroccan family with atrioventricular conduction disturbance and an atrial septal defect in the oval fossa

Published online by Cambridge University Press:  22 December 2006

Laïla Rifai ,
Wajih Maazouzi  and
Abdelaziz Sefiani
Laïla Rifai
Affiliation:
Department of Medical Genetics, National Institute of Health, Rabat, Morocco
Wajih Maazouzi
Affiliation:
Department of Cardiovascular Surgery, Ibn Sina Hospital, Rabat, Morocco
Abdelaziz Sefiani
Affiliation:
Department of Medical Genetics, National Institute of Health, Rabat, Morocco
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Abstract

Defects of the oval fossa usually occur as isolated malformations, but can show an autosomal dominant pedigree in familial cases. Several mutations have been described for the transcription factor NKX2-5, and co-segregate with varied cardiac anomalies. We have identified by sequence analysis a novel missense heterozygous mutation in the NKX2-5 gene, specifically a substitution of glutamine for proline at codon 160, in a Moroccan family, the affected members having a deficiency of the floor of the oval fossa and atrioventricular block.

Keywords

Congenital cardiopathymutation detectionmolecular biology

Information

Type
Brief Report
Information
Cardiology in the Young , Volume 17 , Issue 1 , February 2007 , pp. 107 - 109
Copyright
2007 Cambridge University Press

Deficiencies of the atrial septum are found in up to one-tenth of all people born with congenital cardiac disease, and can show an autosomal dominant pedigree in familial cases. Those seeking to understand the genetic bases of cardiac development have paid particular interest to some homeobox-containing genes. Recent advances have provided evidence that the protein products of three genes known to produce atrial septal defects within the oval fossa, specifically GATA4, TBX5, and NKX2-5, may complex with one another.1–2

Heterozygosity for mutations of the NKX2-5 gene in families are known to underscore a variety of congenital cardiac abnormalities.3–7 The most common phenotypes are atrial septal defects within the oval fossa, at a penetrance of 77%, and abnormalities of atrioventricular conduction, with 95% penetrance.8

We have now performed genetic testing in a Moroccan family with atrial septal defects in the oval fossa and disturbances of atrioventricular conduction, referred by a cardiovascular surgeon for genetic counselling to investigate whether the NKX2-5 gene was involved.

Case report

The proband (II2) was diagnosed at 15 years of age with an atrial septal defect within the oval fossa and prolongation of the PR interval. He underwent surgical closure of the atrial septal defect, and was found to have developed second degree atrioventricular block 4 years later. His son (III1) was diagnosed at the age of 6 months. He presented with rapid breathing, dyspnoea, tachycardia, difficulty of feeding, showed poor weight gain, and had a cardiac murmur audible at auscultation. The electrocardiogram showed an incomplete right bundle branch block. Echocardiography with colour flow Doppler showed a deficiency of 17millimetres in the floor of the oval fossa, with left-to-right flow across the defect. The rest of the clinical examination was normal. Analysis of the family pedigree revealed two other affected kindred, both with atrial septal defects in the oval fossa and first degree atrioventricular block, demonstrating autosomal dominant transmission of this trait.

In accordance with the recommendations of the ethics committee of the National Institute of Health, having obtained informed consent, blood samples were obtained from eight members of the family, four of whom are known to have atrial septal defects within the oval fossa.

The NKX2-5 gene, encoding the homeobox transcription factor, contains two exons that encode a 324-amino acid protein, including a homeodomain between amino acids 138 and 197. Polymerase chain reaction primers were designed to amplify both exons, using genomic deoxyribonucleic acid extracted from whole peripheral blood samples of the family members. The amplified products were directly sequenced using an ABI PRISM 310 Genetic Analyzer (Perkin-Elmer Biosystems), and the sequencing analysis showed, in the deoxyribonucleic acid of the proband, a transversion of A to C at codon 160 of the NKX2-5 gene (Fig. 1), recording to NCBI sequence NM_004387. This mutation is predicted to substitute a CCG codon, namely Proline, for a CAG codon of Glutamine, designated Gln160Pro, and occurred in the homeodomain, which is a highly conserved region. This new mutation was found in one allele of all clinically affected members, but not in the unaffected members of the family. Other previously reported mutations of NKX2-5 were not found.

Figure 1. Sequence analysis indicates a A to C transversion that leads to an exchange of glutamine to proline in codon 160.

Cleavage of amplified deoxyribonucleic acid from the family members was performed using BtgI restriction endonuclease. It demonstrated co-segregation of the mutation with the phenotype for the atrial septal defect (Fig. 2). Samples of deoxyribonucleic acid from control subjects of Morrocan origin did not show the presence of this site of restriction of BtgI.

Figure 2. The pedigree of the family, and allele specific polymerase chain reaction for Q160P mutation. A fragment of 340 pairs of bases corresponds to the wild allele present in all individuals. A fragment of 210 pairs of bases corresponds to the mutated allele, present only in the affected individuals. “M” is the molecular weight marker.

Discussion

We consider the identified mutation as pathogenic because of the implication of a highly conserved amino acid, its co-segregation with the disease, and the negative results from 100 normal chromosomes from a random control population. Analysis of missense mutations in the conserved homeodomain has shown that the principle determinant of the two most common phenotypes is the total dose of NKX2-5 capable of binding to deoxyribonucleic acid.9 This new mutation is also likely to have the same effects. It can be added to further functional studies to prove this hypothesis.

Progressive electrophysiologic abnormalities in individuals after spontaneous or surgical correction of atrial septal defects indicate that normal amounts of NKX2-5 are required for normal function of the atrioventricular node throughout life.3 Sometimes, disturbances of conduction, in particular atrioventricular block, are unique symptoms.5 Patients who are phenotypically normal, but mutated for the NKX2-5 gene, might have achieved spontaneous closure of their atrial septal defect, or simply not yet shown the clinical symptoms of the disturbance of conduction.10

None of the unaffected individuals in our family showed the mutation. They were subsequently reassured. Those who were found to have the mutation, and had undergone surgical closure of the atrial septal defect, were advised to undergo very strict cardiologic follow up, in order to detect and treat any disturbance of conduction that might appear later. They were also warned that they had a risk of 50% of transmitting the atrial septal defect, or other congenital cardiac anomalies, to their offspring.

Progress in finding new genes responsible for congenital cardiac disease, and exploring their role, leads to a better understanding of the pathogenesis of the malformations. It is essential to make accurate correlations between genotype and phenotype in families known to have mutations of the NKX2-5 gene, because afflicted individuals are at high risk of developing late onset problems of atrioventricular conduction, and therefore exposed to the risk of sudden death.

Acknowledgements

Our study was supported by the Moroccan Ministry of Health. We express our thanks to all the members of the family which participated in this study for donating their samples of blood. We thank in particular Laamri Aboubakr for his excellent and skillful assistance. His vast experience and constant support were key to the success of this study.

References

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Figure 0

Sequence analysis indicates a A to C transversion that leads to an exchange of glutamine to proline in codon 160.

Figure 1

The pedigree of the family, and allele specific polymerase chain reaction for Q160P mutation. A fragment of 340 pairs of bases corresponds to the wild allele present in all individuals. A fragment of 210 pairs of bases corresponds to the mutated allele, present only in the affected individuals. “M” is the molecular weight marker.