Isolated Nonsyndromic Congenital Heart Defects via the NKX2-5 Gene

  • Summary and Pricing
  • Clinical Features and Genetics
  • Citations
  • Methods
  • Ordering/Specimens
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Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
944 NKX2-5$490.00 81479 Add to Order
Targeted Testing

For ordering targeted known variants, please proceed to our Targeted Variants landing page.

Turnaround Time

The great majority of tests are completed within 18 days.

Clinical Sensitivity

Mutations in the NKX2-5 gene have been reported in up to 4% of sporadic cases of CHDs (Goldmuntz et al. Circulation 104:2565-2568, 2001; McElhinney et al. J Am Coll Cardiol 42:1650-1655, 2003). One study found 7 mutations in 26 probands with AV block and cardiac septal defects with and without conotruncal defects (Benson et al. J Clin Invest 104:1567-1573, 1999).

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Deletion/Duplication Testing via aCGH

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
600 NKX2-5$990.00 81479 Add to Order
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Turnaround Time

The great majority of tests are completed within 20 days.

Clinical Features

Congenital heart defects (CHDs) are the most common birth defect, occurring in 6 to 10:1,000 live births and are a major cause of infant morbidity and mortality (Hoffman et al. J Am Coll Cardiol 39:1890-1900, 2002; Oyen et al. Circulation 120:295-301, 2009). Congenital heart diseases arise due to defects in cardiac morphogenesis during embryonic development, which leads to structural malformations in the heart and great vessels. Cardiac septal defects, which include atrial septal defects (ASD), ventricular septal defects (VSD), and atrioventricular septal defects (AVSD), are common, with an estimated incidence of 5:1,000 live births (Wessels et al. Clin Genet 78:103-123, 2010). Roughly 10% of patients with CHDs have conotruncal heart defects, which include tetralogy of Fallot (TOF), double outlet right ventricle (DORV), truncus arteriosus (TA), interrupted aortic arch (IAA), and transposition of the great arteries (TGA) (Oyen et al. Circulation 120:295-301, 2009).


CHDs have genetic and non-genetic causes. The majority of patients with CHDs are thought to have a complex, multifactorial etiology. CHDs can be caused by single gene or chromosomal abnormalities, exposure to teratogens, and other unknown mechanisms. Non-cardiac malformations are found in roughly 20% of patients with CHDs and chromosomal abnormalities account for ~7% of patients with CHDs (Eskedal et al. Cardiol Young 14:600-607, 2004; Oyen et al. Circulation 120:295-301, 2009). Monogenic non-syndromic CHD are caused by mutations in regulators of heart development (reviewed by Bruneau Nature 451:943-948, 2008). Autosomal dominant nonsyndromic CHDs can occur due to mutations in the homeobox transcription factor NKX2-5 (OMIM 600584). Causative mutations in NKX2-5 have been reported in patients with CHDs such as septal defects, AV conduction defects, conotrunctal defects, pulmonary stenosis and hypoplastic left heart (McElhinney et al. J Am Coll Cardiol 42:1650-1655, 2003; Stallmeyer et al. Clin Genet 78:533-540, 2010). The majority of documented causative variants in NKX2-5 are missense mutations; however, nonsense, small insertions, and small deletions have also been reported. In addition to CHDs, missense mutations in NKX2-5 have also been found in patients with congenital nongoitrous hypothyroidism (Dentice et al. J Clin Endocr Metab 91:1428-1433, 2006).

Testing Strategy

This test involves bidirectional DNA sequencing of both coding exons of the NKX2-5 gene plus ~10 bp of flanking non-coding DNA on either side of each exon. We will also sequence any single exon (Test #100) in family members of patients with known mutations or to confirm research results.

Indications for Test

Patients with non-syndromic CHD, including cardiac septal defects (ASD, VSD, AVSD), conotruncal defects (TOF, DORV, IAA, TGA), AV conduction defects, and pulmonary valve stenosis are candidates for this test.


Official Gene Symbol OMIM ID
NKX2-5 600584
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT


Genetic Counselors
  • Benson DW, Silberbach GM, Kavanaugh-McHugh A, Cottrill C, Zhang Y, Riggs S, Smalls O, Johnson MC, Watson MS, Seidman JG, Seidman CE, Plowden J, Kugler JD. (1999) Mutations in the cardiac transcription factor NKX2.5 affect diverse cardiac developmental pathways. J Clin Invest 104(11):1567-73. PubMed ID: 10587520
  • Bruneau. (2008) The developmental genetics of congenital heart disease. Nature 451(7181):943-948. PubMed ID: 18288184
  • Dentice M, Cordeddu V, Rosica A, Ferrara AM, Santarpia L, Salvatore D, Chiovato L, Perri A, Moschini L, Fazzini C, Olivieri A, Costa P, Stoppioni V, Baserga M, De Felice M, Sorcini M, Fenzi G, Di Lauro R, Tartaglia M, Macchia PE. (2006) Missense mutation in the transcription factor NKX2-5: a novel molecular event in the pathogenesis of thyroid dysgenesis. J Clin Endocr Metab 91(4):1428-1433. PubMed ID: 16418214
  • Eskedal L, Hagemo P, Eskild A, Aamodt G, Seiler KS, Thaulow E. (2004) A population-based study of extra-cardiac anomalies in children with congenital cardiac malformations. Cardiol Young 14(6):600-607. PubMed ID: 15679995
  • Goldmuntz E, Geiger E, Benson DW. (2001) NKX2.5 mutations in patients with tetralogy of fallot. Circulation 104(21):2565-8. PubMed ID: 11714651
  • Hoffman JI, Kaplan S. (2002) The incidence of congenital heart disease. J Am Coll Cardiol 39(12):1890-900. PubMed ID: 12084585
  • McElhinney DB, Geiger E, Blinder J, Benson DW, Goldmuntz E. (2003) NKX2.5 mutations in patients with congenital heart disease. J Am Coll Cardiol 42(9):1650-1655. PubMed ID: 14607454
  • Stallmeyer B, Fenge H, Nowak-Göttl U, Schulze-Bahr E. (2010) Mutational spectrum in the cardiac transcription factor gene NKX2.5 (CSX) associated with congenital heart disease. Clin Genet 78(6):533-540. PubMed ID: 20456451
  • Wessels MW, Willems PJ. (2010) Genetic factors in non-syndromic congenital heart malformations. Clin Genet 78(2):103-23. PubMed ID: 20497191
  • Øyen N, Poulsen G, Boyd HA, Wohlfahrt J, Jensen PK, Melbye M. (2009) Recurrence of congenital heart defects in families. Circulation 120(4):295-301. PubMed ID: 19597048
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Bi-Directional Sanger Sequencing

Test Procedure

Nomenclature for sequence variants was from the Human Genome Variation Society (  As required, DNA is extracted from the patient specimen.  PCR is used to amplify the indicated exons plus additional flanking non-coding sequence.  After cleaning of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit.  Products are resolved by electrophoresis on an ABI 3730xl capillary sequencer.  In most cases, sequencing is performed in both forward and reverse directions; in some cases, sequencing is performed twice in either the forward or reverse directions.  In nearly all cases, the full coding region of each exon as well as 10 bases of non-coding DNA flanking the exon are sequenced.

Analytical Validity

As of February 2018, we compared 26.8 Mb of Sanger DNA sequence generated at PreventionGenetics to NextGen sequence generated in other labs. We detected only 4 errors in our Sanger sequences, and these were all due to allele dropout during PCR. For Proficiency Testing, both external and internal, in the 14 years of our lab operation we have Sanger sequenced roughly 14,300 PCR amplicons. Only one error has been identified, and this was an error in analysis of sequence data.

Our Sanger sequencing is capable of detecting virtually all nucleotide substitutions within the PCR amplicons. Similarly, we detect essentially all heterozygous or homozygous deletions within the amplicons. Homozygous deletions which overlap one or more PCR primer annealing sites are detectable as PCR failure. Heterozygous deletions which overlap one or more PCR primer annealing sites are usually not detected (see Analytical Limitations). All heterozygous insertions within the amplicons up to about 100 nucleotides in length appear to be detectable. Larger heterozygous insertions may not be detected. All homozygous insertions within the amplicons up to about 300 nucleotides in length appear to be detectable. Larger homozygous insertions may masquerade as homozygous deletions (PCR failure).

Analytical Limitations

In exons where our sequencing did not reveal any variation between the two alleles, we cannot be certain that we were able to PCR amplify both of the patient’s alleles. Occasionally, a patient may carry an allele which does not amplify, due for example to a deletion or a large insertion. In these cases, the report contains no information about the second allele.

Similarly, our sequencing tests have almost no power to detect duplications, triplications, etc. of the gene sequences.

In most cases, only the indicated exons and roughly 10 bp of flanking non-coding sequence on each side are analyzed. Test reports contain little or no information about other portions of the gene, including many regulatory regions.

In nearly all cases, we are unable to determine the phase of sequence variants. In particular, when we find two likely causative mutations for recessive disorders, we cannot be certain that the mutations are on different alleles.

Our ability to detect minor sequence variants, due for example to somatic mosaicism is limited. Sequence variants that are present in less than 50% of the patient’s nucleated cells may not be detected.

Runs of mononucleotide repeats (eg (A)n or (T)n) with n >8 in the reference sequence are generally not analyzed because of strand slippage during PCR and cycle sequencing.

Unless otherwise indicated, the sequence data that we report are based on DNA isolated from a specific tissue (usually leukocytes). Test reports contain no information about gene sequences in other tissues.

Deletion/Duplication Testing via Array Comparative Genomic Hybridization

Test Procedure

Equal amounts of genomic DNA from the patient and a gender matched reference sample are amplified and labeled with Cy3 and Cy5 dyes, respectively. To prevent any sample cross contamination, a unique sample tracking control is added into each patient sample. Each labeled patient product is then purified, quantified, and combined with the same amount of reference product. The combined sample is loaded onto the designed array and hybridized for at least 22-42 hours at 65°C. Arrays are then washed and scanned immediately with 2.5 µM resolution. Only data for the gene(s) of interest for each patient are extracted and analyzed.

Analytical Validity

PreventionGenetics' high density gene-centric custom designed aCGH enables the detection of relatively small deletions and duplications within a single exon of a given gene or deletions and duplications encompassing the entire gene. PreventionGenetics has established and verified this test's accuracy and precision.

Analytical Limitations

Our dense probe coverage may allow detection of deletions/duplications down to 100 bp; however due to limitations and probe spacing this cannot be guaranteed across all exons of all genes. Therefore, some copy number changes smaller than 100-300 bp within a targeted large exon may not be detected by our array.

This array may not detect deletions and duplications present at low levels of mosaicism or those present in genes that have pseudogene copies or repeats elsewhere in the genome.

aCGH will not detect balanced translocations, inversions, or point mutations that may be responsible for the clinical phenotype.

Breakpoints, if occurring outside the targeted gene, may be hard to define.

The sensitivity of this assay may be reduced when DNA is extracted by an outside laboratory.

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Ordering Options

myPrevent - Online Ordering
  • The test can be added to your online orders in the Summary and Pricing section.
  • Once the test has been added log in to myPrevent to fill out an online requisition form.
  • A completed requisition form must accompany all specimens.
  • Billing information along with specimen and shipping instructions are within the requisition form.
  • All testing must be ordered by a qualified healthcare provider.


(Delivery accepted Monday - Saturday)

  • Collect 3 ml -5 ml (5 ml preferred) of whole blood in EDTA (purple top tube) or ACD (yellow top tube). For Test #500-DNA Banking only, collect 10 ml -20 ml of whole blood.
  • For small babies, we require a minimum of 1 ml of blood.
  • Only one blood tube is required for multiple tests.
  • Ship blood tubes at room temperature in an insulated container. Do not freeze blood.
  • During hot weather, include a frozen ice pack in the shipping container. Place a paper towel or other thin material between the ice pack and the blood tube.
  • In cold weather, include an unfrozen ice pack in the shipping container as insulation.
  • At room temperature, blood specimen is stable for up to 48 hours.
  • If refrigerated, blood specimen is stable for up to one week.
  • Label the tube with the patient name, date of birth and/or ID number.


(Delivery accepted Monday - Saturday)

  • Send in screw cap tube at least 5 µg -10 µg of purified DNA at a concentration of at least 20 µg/ml for NGS and Sanger tests and at least 5 µg of purified DNA at a concentration of at least 100 µg/ml for gene-centric aCGH, MLPA, and CMA tests, minimum 2 µg for limited specimens.
  • For requests requiring more than one test, send an additional 5 µg DNA per test ordered when possible.
  • DNA may be shipped at room temperature.
  • Label the tube with the composition of the solute, DNA concentration as well as the patient’s name, date of birth, and/or ID number.
  • We only accept genomic DNA for testing. We do NOT accept products of whole genome amplification reactions or other amplification reactions.


(Delivery preferred Monday - Thursday)

  • PreventionGenetics should be notified in advance of arrival of a cell culture.
  • Culture and send at least two T25 flasks of confluent cells.
  • Some panels may require additional flasks (dependent on size of genes, amount of Sanger sequencing required, etc.). Multiple test requests may also require additional flasks. Please contact us for details.
  • Send specimens in insulated, shatterproof container overnight.
  • Cell cultures may be shipped at room temperature or refrigerated.
  • Label the flasks with the patient name, date of birth, and/or ID number.
  • We strongly recommend maintaining a local back-up culture. We do not culture cells.
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