Pitt-Hopkins Syndrome via the TCF4 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
1533 TCF4$1100.00 81406 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

Pathogenic variants in TCF4 were confirmed by molecular analysis in 13 of 36 patients (~36%) who were either clinically diagnosed with Pitt-Hopkins syndrome (25 patients), or with Rett/Angelman syndrome, but had negative findings in MECP2 and UBE3A (10 patients), or with Mowat-Wilson syndrome, but had negative findings in ZFHX1B (de Pontual et al 2009).

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Deletion/Duplication Testing via aCGH

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
600 TCF4$690.00 81405 Add to Order
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Turnaround Time

The great majority of tests are completed within 28 days.

Clinical Features

Pitt-Hopkins syndrome is a rare disorder characterized by extensive developmental delay, severe intellectual disability, intermittent hyperventilation followed by apnea, tendency to epilepsy, absence of speech, stereotypic hand movements, postnatal microcephaly, ocular anomalies and distinctive facial dysmorphism (cupid’s bow mouth, fleshy lips, broad and beaked nasal bridge). To date, no major congenital malformations have been described. Onset of this disease occurs in infancy and childhood (Zweier et al 2007; Peippo and Ignatius 2012; Sweatt 2013). It has been suggested that this disorder may be underdiagnosed. Pitt-Hopkins syndrome overlaps with features from other disorders such as Rett syndrome, Angelman syndrome, Joubert syndrome, Mowat-Wilson syndrome, and ARX, NRXN1 or CNTNAP2 related intellectual disability disorders.


Pitt-Hopkins syndrome is inherited in an autosomal dominant manner and is caused by defects in the TCF4 gene encoding the transcription factor-4, a member of class I basic helix-loop-helix family. TCF4 is the only gene in which pathogenic variants are known to cause Pitt-Hopkins syndrome. The human TCF4 gene consists of 20 exons (exons 2 through 19 are coding exons) and spans 360 kb. It is transcribed into isoforms with 18 different N-terminals, as well as several isoforms with alternative splicing of internal exons (Sweatt 2013). The transcription factor-4 (TCF4) shares homology with several other basic helix-loop-helix transcription factors (Zweier et al. 2007). It is ubiquitously expressed during embryo development, particularly in the brain and retina. It is continuously expressed in adult brain and other organs (Sepp et al 2011). TCF4 forms a homodimer or heterodimers with other proteins, in order to bind DNA and regulate gene expression. Depending on the binding partner, TCF4 may be either a transcription activator or suppressor. Upstream and downstream effectors of the TCF4 signaling pathway are largely unknown (Sweatt 2013). 

Haploinsufficiency of the TCF4 gene causes Pitt-Hopkins syndrome, while over-expression of the TCF4 gene in the brain is a risk factor to schizophrenia. Pathogenic variants in TCF4 gene include frameshift, nonsense, splice site, and missense, as well as large deletions. Most cases are caused by de novo pathogenic variants (Amiel et al. 2007; Zweier et al. 2007). The missense variants are generally within the basic helix-loop-helix domain, a hotspot for pathogenic variants (Sweatt 2013). DNA sequence analysis detects ~70% of TCF4 pathologic variants. However, ~30% of TCF4 pathologic variants are due to whole or partial gene deletions, which are generally not detectable by sequencing (Whalen et al. 2012).

Testing Strategy

Testing is accomplished by amplifying each coding exon of the TCF4 gene and ~20 bp of adjacent noncoding sequence, then determining the nucleotide sequence using standard Sanger dideoxy sequencing methods and a capillary electrophoresis instrument. 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

TCF4 gene sequencing test is recommended for patients who are suspected to have Pitt-Hopkins syndrome. Molecular genetic testing is the only confirmation for this disease. Candidates for TCF4 gene testing also include patients with suspected Rett syndrome, Angelman syndrome, Joubert syndrome, Mowat-Wilson syndrome, but have negative findings in genetic testing specifically for those syndromes. Prenatal diagnosis is possible if the genetic diagnosis has been established in an affected family member.


Official Gene Symbol OMIM ID
TCF4 602272
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT


Name Inheritance OMIM ID
Pitt-Hopkins Syndrome 610954


Genetic Counselors
  • Amiel J. et al. 2007. American journal of human genetics. 80: 988-93. PubMed ID: 17436254
  • de Pontual L. et al. 2009. Human mutation. 30: 669-76. PubMed ID: 19235238
  • Peippo M., Ignatius J. 2012. Molecular syndromology. 2: 171-180. PubMed ID: 22670138
  • Sepp M. et al. 2011. PloS one. 6: e22138. PubMed ID: 21789225
  • Sweatt J David. 2013. Experimental & Molecular Medicine. 45: e21 PubMed ID: 23640545
  • Whalen S. et al. 2012. Human mutation. 33: 64-72. PubMed ID: 22045651
  • Zweier C. et al. 2007. American journal of human genetics. 80: 994-1001. PubMed ID: 17436255
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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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