PITX2- Related Disorders via PITX2 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
1675 PITX2$580.00 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

Approximately 25%-60% of Axenfeld-Rieger syndrome cases are due to FOXC1 or PITX2 pathogenic variants (Tümer and Bach-Holm 2009; Reis et al. 2012; Alward 2000). In approximately half of ARS affected patients the genetic cause is unknown, which indicates that there are still more genes to be identified (Tümer and Bach-Holm 2009). Several gross deletions, duplications and complex rearrangements have been reported in PITX2 that are associated with ARS and related or overlapping phenotypes (Human Gene Mutation Database).

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Deletion/Duplication Testing via aCGH

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
600 PITX2$690.00 81479 Add to Order
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Turnaround Time

The great majority of tests are completed within 28 days.

Clinical Features

Axenfeld-Rieger syndrome (ARS) is a rare, highly penetrant autosomal dominant disorder characterized by varying degrees of eye anterior segment anomalies with systemic malformations such as dental hypoplasia and a protuberant umbilicus (Hjalt and Semina 2005; Berry et al. 2006; Waldron et al. 2010; Tümer and Bach-Holm 2009; Chang et al. 2012). Dental abnormalities in this syndrome helps in the diagnosis and also to distinguish ARS from other eye anterior segment abnormalities. Early diagnosis of ARS from its dento-facial and systemic features is essential in treating or preventing the most serious consequence of ARS (O’Dwyer and Jones 2005). The major clinical concern is high risk of developing open-angle glaucoma. Approximately 50% of the ARS affected patients develop glaucoma (Shields et al. 1985; Alward 2000; Hjalt and Semina 2005; Chang et al. 2012). ARS affected patients also need surveillance and management of sensorineural hearing loss, and cardiac, endocrinological, craniofacial and orthopaedic defects (Chang et al. 2012). Rieger syndrome (RIEG) and Axenfeld anomaly are both anterior chamber cleavage anomalies and considered to be variations of a single developmental disorder and belong to the ARS group (Reese and Ellsworth 1966). RIEG is characterized by malformations of the eyes, teeth, and umbilicus; whereas, Axenfeld anomaly displays only ocular features (Amendt et al. 2000).


Axenfeld-Rieger syndrome (ARS) is an autosomal dominant disorder caused by mutation in the PITX2 gene. Linkage studies mapped four chromosomal loci (4q25, 6p25, 13q14 and 16q24) that are involved with ARS and related or overlapping phenotypes. The genes that have been identified on chromosomes 4q25 and 6p25, PITX2 (the pituitary homeobox 2 gene) and FOXC1 (the forkhead box C1 gene, also known as FKHL7), respectively (Alward 2000; Lines et al. 2002; Tümer and Bach-Holm 2009), are known to be associated with ARS pathogenesis (Tümer and Bach-Holm 2009). PITX2 and FOXC1 are two transcription factor genes, which are expressed throughout eye ontogeny (Lines et al. 2002). PITX2 encodes a Paired-like homeodomain transcription factor, which plays a critical role in cell proliferation, differentiation, hematopoiesis and organogenesis (Huang et al. 2009). Also, PITX2 integrates retinoic acid and canonical Wnt signaling during eye anterior segment development (Gage et al. 2008; Gage and Zacharias 2009). Genotype-Phenotype correlation studies indicated that patients with PITX2 mutations have a more severe prognosis for glaucoma development as compared to FOXC1 mutations. Patients with FOXC1 duplications are at high risk for the development of glaucoma, which emphasizes the importance of genetic testing. Also, glaucoma in patients with PITX2 mutations is reported to be more difficult to treat than that in patients with FOXC1 mutations (Strungaru et al. 2007). It has been reported that PITX2 and FOXC1 interact with each other, which is essential for the regulation of common downstream target genes within specific cell lineages (Berry et al. 2006). Also, co-inheritance of PITX2 and FOXC1 mutations has been reported in a family, which segregated with the disease and showed variable phenotypic expression. The most severely affected individual had mutations in both genes, whereas the single heterozygous mutations caused mild ARS phenotypes (Kelberman et al. 2011). So far, about 80 mutations have been identified in PITX2 that are involved with ARS and related or overlapping phenotypes (Human Gene Mutation Database).

Testing Strategy

This test involves bidirectional DNA Sanger sequencing of all coding exons and ~ 20 bp of flanking noncoding sequence of PITX2 gene. We will also sequence any single exon (Test #100) in family members of patients with a known mutation or to confirm research results.

Indications for Test

All patients with symptoms suggestive of Axenfeld-Rieger syndrome and related phenotypes are candidates.


Official Gene Symbol OMIM ID
PITX2 601542
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT


Genetic Counselors
  • Alward WL. 2000. American Journal of Ophthalmology. 130: 107-15. PubMed ID: 11004268
  • Amendt BA. et al. 2000. Cellular and Molecular Life Sciences : Cmls. 57: 1652-66. PubMed ID: 11092457
  • Berry FB. et al. 2006. Human Molecular Genetics. 15: 905-19. PubMed ID: 16449236
  • Chang TC. et al. 2012. The British Journal of Ophthalmology. 96: 318-22. PubMed ID: 22199394
  • Gage PJ. et al. 2008. Developmental Biology. 317: 310-24. PubMed ID: 18367164
  • Gage PJ., Zacharias AL. 2009. Developmental Dynamics : an Official Publication of the American Association of Anatomists. 238: 2149-62. PubMed ID: 19623614
  • Hjalt TA., Semina EV. 2005. Expert Reviews in Molecular Medicine. 7: 1-17. PubMed ID: 16274491
  • Huang Y. et al. 2009. Febs Letters. 583: 638-42. PubMed ID: 19174163
  • Human Gene Mutation Database (Bio-base).
  • Kelberman D. et al. 2011. Human Mutatation. 32: 1144–52. PubMed ID: 21837767
  • Lines MA. et al. 2002. Human Molecular Genetics. 11: 1177–84. PubMed ID: 12015277
  • O'Dwyer E.M., Jones D.C. 2005. International Journal of Paediatric Dentistry. 15: 459-63. PubMed ID: 16238657
  • Reese AB., Ellsworth RM. 1966. Archives of Ophthalmology. 75: 307-18. PubMed ID: 5948260
  • Reis LM, Tyler RC, Volkmann Kloss BA, Schilter KF, Levin AV, Lowry RB, Zwijnenburg PJG, Stroh E, Broeckel U, Murray JC, Semina EV. 2012. PITX2 and FOXC1 spectrum of mutations in ocular syndromes. Eur. J. Hum. Genet. 20: 1224–1233. PubMed ID: 22569110
  • Shields MB. et al. 1985. Survey of Ophthalmology. 29: 387-409. PubMed ID: 3892740
  • Strungaru MH. et al. 2007. Investigative Ophthalmology & Visual Science. 48: 228-37. PubMed ID: 17197537
  • Tümer Z, Bach-Holm D. 2009. European Journal of Human Genetics : Ejhg. 17: 1527-39. PubMed ID: 19513095
  • Waldron JM. et al. 2010. Special Care in Dentistry : Official Publication of the American Association of Hospital Dentists, the Academy of Dentistry For the Handicapped, and the American Society For Geriatric Dentistry. 30: 218-22. PubMed ID: 20831741
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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 20 bases of non-coding DNA flanking the exon are sequenced.

Analytical Validity

As of March 2016, we compared 17.37 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 12 years of our lab operation we have Sanger sequenced roughly 8,800 PCR amplicons. Only one error has been identified, and this was due to sequence analysis error.

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 20 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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