GLI3-Related Disorders via the GLI3 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
380 GLI3$1020.00 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
This test will detect causative mutations in about 70% of GCPS patients (Biesecker 2009), and  about 95% of PSH patients (Biesecker 2012).

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Deletion/Duplication Testing via aCGH

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
600 GLI3$690.00 81479 Add to Order
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Turnaround Time

The great majority of tests are completed within 28 days.

Clinical Features
Greig Cephalopolysyndactyly Syndrome (GCPS) is a clinically heterogeneous disorder that is characterized by craniofacial and digital malformations.  The clinical diagnosis of GCPS is based on the presence of preaxial polydactyly, syndactyly, macrocephaly, and ocular hypertelorism (Johnston et al. Hum Mutat 31:1142-54, 2010).  Additional features include prominent forehead, seizures,  hydrocephalus,  developmental delay, learning difficulties and postaxial polydactyly. For more information see Biesecker GeneReviews 2009 (

Pallister-Hall Syndrome (PHS) characterized by postaxial polydactyly and hypothalamic hamartoma (Johnston, 2010).  Additional features include gelastic seizures, small nails, imperforate anus, bifid epiglottis and laryngotracheal clefts.  PHS patients may have pituitary insufficiency which can result in death from adrenal insufficiency.  Symptoms are variable in appearance and severity.  Inter-familial variability has been reported to be much greater than intra-familial variability.  For more information see Biesecker GeneReviews 2012 (

Nonsyndromic polydactyly, including preaxial and postaxial, is characterized by abnormalities in the digits of the hands or feet with no other symptoms or abnormalities that are usually present in GCPS or PHS (Radhakrishna et al.  Nat Genet 17:269–71, 1997; Radhakrishna et al. Am J Hum Genet 65:645–55, 1999).
GCPS is an autosomal dominant disorder that is caused by loss of function mutations in the GLI3 gene (Vortkamp et al. Nature 352:539-40; 1991; Kalff-Suske et al. Hum Mol Genet 8:1769-77, 1999; Johnston et al. Am J Hum Genet 76: 609-22, 2005).  Mutations occur either de novo or are inherited dominantly.  The fraction of patients with de novo mutations is unknown.  GLI3 is the only gene that has been implicated in GCPS.  Causative mutations are located throughout the length of the gene, but especially within the first and last thirds of the gene (Johnston et al. 2005; Johnston, 2010). About 5-10% of GCPS patients have large deletions or translocations involving the GLI3 gene (Johnston et al. Am J Med Genet A 123:236-42, 2003; Biesecker 2009).

PHS is an autosomal dominant disorder that is caused by mutations in the GLI3 gene. These mutations result in a constitutive repressor protein leading to a loss of balance between the activator and repressor forms of the GLI3 protein, which is critical for Sonic Hedgehog (SHH) signaling (Kang et al. Nat Genet 15:266-8, 1997; Johnston, 2005).  Mutations either occur de novo or are inherited dominantly.  De novo mutations occur in ~ 25 % of patients and are usually associated in a more severe phenotype compared to that of familial cases (Biesecker, 2012).  PHS mutations are mostly (possibly even entirely) truncating (nonsense, frameshift, and obvious splicing defects) and are usually located within the middle third of the gene (Johnston, 2005).  No large deletions or complex rearrangements have been reported to date.  GLI3 is the only gene that has been implicated in PHS. 

In addition to GCPS and PHS, heterozygous mutations in the GLI3 gene cause nonsyndromic polydactyly (Radhakrishna 1997; Radhakrishna 1999).

GLI3 encodes a zinc finger protein of the GLI family, which has a double role.  The GLI3 protein acts as a DNA-binding transcription factor and as a mediator of SHH signaling, which is involved in the regulation of vertebrate organogenesis.
Testing Strategy
This test involves bidirectional Sanger sequencing of all 14 coding GLI3 exons plus ~20 bp of non-coding flanking DNA on each side. The large exon 14 (2311 bp coding region) is sequenced as five overlapping fragments. 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 symptoms consistent with GCPS, PHS or nonsyndromic polydactyly are candidates for this test. 


Official Gene Symbol OMIM ID
GLI3 165240
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT


Genetic Counselors
  • Johnston et al. (2003). "Clinical and molecular delineation of the Greig cephalopolysyndactyly contiguous gene deletion syndrome and its distinction from acrocallosal syndrome." Am J Med Genet A 123A(3): 236-42. PubMed ID: 14608643
  • Johnston et al. (2010). "Molecular analysis expands the spectrum of phenotypes associated with GLI3 mutations." Hum Mutat 31: 1142-54. PubMed ID: 20672375
  • Johnston (2005). "Molecular and clinical analyses of Greig cephalopolysyndactyly and Pallister-Hall syndromes: robust phenotype prediction from the type and position of GLI3 mutations." Am J Hum Genet 76(4): 609-22. PubMed ID: 15739154
  • Kalff-Suske (1999). "Point mutations throughout the GLI3 gene cause Greig cephalopolysyndactyly syndrome." Hum Mol Genet 8: 1769-77. PubMed ID: 10441342
  • Kang al. (1997). "GLI3 frameshift mutations cause autosomal dominant Pallister-Hall syndrome." Nat Genet 15(3): 266-8. PubMed ID: 9054938
  • Leslie G Biesecker. "Greig Cephalopolysyndactyly Syndrome." GeneReviews, 2009. PubMed ID: 20301619
  • Leslie G Biesecker. "Pallister-Hall Syndrome." GeneReviews, 2012. PubMed ID: 20301638
  • Radhakrishna et al. (1997). "Mutation in GLI3 in postaxial polydactyly type A. Nat Genet 17:269–71". PubMed ID: 9354785
  • Radhakrishna et al. (1999). "The phenotypic spectrum of GLI3 morphopathies includes autosomal dominant preaxial polydactyly type-IV and postaxial polydactyly type-A/B; no phenotype prediction from the position of GLI3 mutations". Am J Hum Genet 65:645–55. PubMed ID: 10441570
  • Vortkamp et al. (1991).  "GLI3 zinc-finger gene interrupted by translocations in Greig syndrome families." Nature 352:539-40. PubMed ID: 1650914
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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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