Familial Dysautonomia via the IKBKAP Gene - Full Gene Sequencing
- Summary and Pricing
- Clinical Features and Genetics
|Test Code||Test Copy Genes||Individual Gene Price||CPT Code Copy CPT Codes|
Our most cost-effective testing approach is NextGen sequencing with Sanger sequencing supplemented as needed to ensure sufficient coverage and to confirm NextGen calls that are pathogenic, likely pathogenic or of uncertain significance. If, however, full gene Sanger sequencing only is desired (for purposes of insurance billing or STAT turnaround time for example), please see link below for Test Code, pricing, and turnaround time information.
For ordering targeted known variants, please proceed to our Targeted Variants landing page.
The great majority of tests are completed within 28 days.
This sequencing test will identify all previously reported pathogenic variants in the IKBKAP gene, and examine the remaining gene sequence to identify any other unreported pathogenic variants.
Familial dysautonomia (FD), also known as Riley–Day syndrome and hereditary sensory autonomic neuropathy type III, is characterized by hypotonia, gastrointestinal dysfunction, vomiting crises, recurrent pneumonia, altered sensitivity to pain, taste and temperature perception, absence of fungiform papillae on the tongue, decreased or absent deep tendon reflexes, absence of overflow tears, and cardiovascular instability (Shohat and Halpern 2010). These congenital symptoms result from the abnormal development and survival of the sensory and autonomic systems, which leads to progressive neurological abnormalities. FD almost exclusively occurs in individuals with Ashkenazi Jewish (AJ) ancestry. Penetrance is complete, although there is variable expressivity (Axelrod 2005). The incidence of FD among individuals with AJ heritage is 1:3600 births, and has an estimated carrier frequency of 1/30. Someone who is not of AJ ancestry has a carrier risk of less than 1:150 (Slaugenhaupt et al. 2001).
FD is inherited in an autosomal recessive manner. It is caused by pathogenic variants in the IKBKAP gene, which encodes a component of the Elongator complex. The complex allows for permissive chromatin structure for efficient mRNA elongation during transcription (Shohat and Halpern 2010). Two pathogenic variants account for more than 99% of affected individuals. (1) The c.2204+6T>C variant is a founder mutation that is responsible for disease in individuals with AJ heritage. It causes skipping of exon 20 resulting in a truncated IKAP protein (Dietrich et al. 2011). The abnormal transcript is tissue specific, as the abnormal transcript is found in the brain but not in lymphoblasts and fibroblasts (Slaugenhaupt et al. 2001). (2) The c.2087G>C (p.Arg696Pro) variant has been reported in the AJ population but is not as common as the intronic variant, and has not been reported in the homozygous state. Another variant c.2741C>T (p.Pro914Leu) has been reported in an individual with FD who inherited the variant from a non-AJ parent along with the common pathogenic variant from their AJ parent (Leyne et al. 2003). The missense variants appear to disrupt phosphorylation (Gold-von Simson and Axelrod 2006).
For this NextGen test, the full coding regions plus ~20 bp of non-coding DNA flanking each exon are sequenced for the gene listed below. Sequencing is accomplished by capturing specific regions with an optimized solution-based hybridization kit, followed by massively parallel sequencing of the captured DNA fragments. Additional Sanger sequencing is performed for any regions not captured or with insufficient number of sequence reads. All pathogenic, likely pathogenic, or variants of uncertain significance are confirmed by Sanger sequencing.
Indications for Test
Candidates for this test are patients clinically diagnosed with Familial Dysautonomia or carrier testing in family members. This test is specifically designed for heritable germline mutations.
|Official Gene Symbol||OMIM ID|
- Genetic Counselor Team - email@example.com
- Jiabin Zhang, PhD - firstname.lastname@example.org
- Axelrod FB. 2005. Familial dysautonomia: a review of the current pharmacological treatments. Expert Opin Pharmacother 6: 561–567. PubMed ID: 15934882
- Dietrich P, Yue J, E. S, Dragatsis I. 2011. Deletion of Exon 20 of the Familial Dysautonomia Gene Ikbkap in Mice Causes Developmental Delay, Cardiovascular Defects, and Early Embryonic Lethality. PLoS ONE 6: e27015. PubMed ID: 22046433
- Gold-von Simson G, Axelrod FB. 2006. Familial Dysautonomia: Update and Recent Advances. Current Problems in Pediatric and Adolescent Health Care 36: 218–237. PubMed ID: 16777588
- Leyne M, Mull J, Gill SP, Cuajungco MP, Oddoux C, Blumenfeld A, Maayan C, Gusella JF, Axelrod FB, Slaugenhaupt SA. 2003. Identification of the first non-Jewish mutation in familial Dysautonomia. American Journal of Medical Genetics 118A: 305–308. PubMed ID: 12687659
- Shohat M, Halpern GJ. 1993. Familial Dysautonomia. In: Pagon RA, Adam MP, Bird TD, Dolan CR, Fong C-T, and Stephens K, editors. GeneReviews™, Seattle (WA): University of Washington, Seattle,. PubMed ID: 20301359
- Slaugenhaupt SA, Blumenfeld A, Gill SP, Leyne M, Mull J, Cuajungco MP, Liebert CB, Chadwick B, Idelson M, Reznik L. 2001. Tissue-Specific Expression of a Splicing Mutation in the IKBKAP Gene Causes Familial Dysautonomia. The American Journal of Human Genetics 68: 598–605. PubMed ID: 11179008
NextGen Sequencing using PG-Select Capture Probes
We use a combination of Next Generation Sequencing (NGS) and Sanger sequencing technologies to cover the full coding regions of the listed genes plus ~20 bases of non-coding DNA flanking each exon. As required, genomic DNA is extracted from the patient specimen. For NGS, patient DNA corresponding to these regions is captured using an optimized set of DNA hybridization probes. Captured DNA is sequenced using Illumina’s Reversible Dye Terminator (RDT) platform (Illumina, San Diego, CA, USA). Regions with insufficient coverage by NGS are covered by Sanger sequencing. All pathogenic, likely pathogenic, or variants of uncertain significance are confirmed by Sanger sequencing.
For Sanger sequencing, Polymerase Chain Reaction (PCR) is used to amplify targeted regions. After purification of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit. PCR products are resolved by electrophoresis on an ABI 3730xl capillary sequencer. In nearly all cases, cycle sequencing is performed separately in both the forward and reverse directions.
Patient DNA sequence is aligned to the genomic reference sequence for the indicated gene region(s). All differences from the reference sequences (sequence variants) are assigned to one of five interpretation categories, listed below, per ACMG Guidelines (Richards et al. 2015).
(1) Pathogenic Variants
(2) Likely Pathogenic Variants
(3) Variants of Uncertain Significance
(4) Likely Benign Variants
(5) Benign, Common Variants
Human Genome Variation Society (HGVS) recommendations are used to describe sequence variants (http://www.hgvs.org). Rare variants and undocumented variants are nearly always classified as likely benign if there is no indication that they alter protein sequence or disrupt splicing.
As of March 2016, 6.36 Mb of sequence (83 genes, 1557 exons) generated in our lab was compared between Sanger and NextGen methodologies. We detected no differences between the two methods. The comparison involved 6400 total sequence variants (differences from the reference sequences). Of these, 6144 were nucleotide substitutions and 256 were insertions or deletions. About 65% of the variants were heterozygous and 35% homozygous. The insertions and deletions ranged in length from 1 to over 100 nucleotides.
In silico validation of insertions and deletions in 20 replicates of 5 genes was also performed. The validation included insertions and deletions of lengths between 1 and 100 nucleotides. Insertions tested in silico: 2200 between 1 and 5 nucleotides, 625 between 6 and 10 nucleotides, 29 between 11 and 20 nucleotides, 25 between 21 and 49 nucleotides, and 23 at or greater than 50 nucleotides, with the largest at 98 nucleotides. All insertions were detected. Deletions tested in silico: 1813 between 1 and 5 nucleotides, 97 between 6 and 10 nucleotides, 32 between 11 and 20 nucleotides, 20 between 21 and 49 nucleotides, and 39 at or greater than 50 nucleotides, with the largest at 96 nucleotides. All deletions less than 50 nucleotides in length were detected, 13 greater than 50 nucleotides in length were missed. Our standard NextGen sequence variant calling algorithms are generally not capable of detecting insertions (duplications) or heterozygous deletions greater than 100 nucleotides. Large homozygous deletions appear to be detectable.
Interpretation of the test results is limited by the information that is currently available. Better interpretation should be possible in the future as more data and knowledge about human genetics and this specific disorder are accumulated.
When Sanger sequencing does not reveal any difference from the reference sequence, or when a sequence variant is homozygous, we cannot be certain that we were able to detect both patient alleles. Occasionally, a patient may carry an allele which does not amplify, due to a large deletion or insertion. In these cases, the report will contain no information about the second allele. Our Sanger and NGS Sequencing tests are generally not capable of detecting Copy Number Variants (CNVs).
We sequence all coding exons for each given transcript, plus ~20 bp of flanking non-coding DNA for each exon. Test reports contain no information about other portions of the gene, such as regulatory domains, deep intronic regions or any currently uncharacterized alternative exons.
In most 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 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.
Unless otherwise indicated, DNA sequence data is obtained from a specific cell-type (usually leukocytes from whole blood). Test reports contain no information about the DNA sequence in other cell-types.
We cannot be certain that the reference sequences are correct.
Rare, low probability interpretations of sequencing results, such as for example the occurrence of de novo mutations in recessive disorders, are generally not included in the reports.
We have confidence in our ability to track a specimen once it has been received by PreventionGenetics. However, we take no responsibility for any specimen labeling errors that occur before the sample arrives at PreventionGenetics.
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.