Autosomal Dominant Nocturnal Frontal Lobe Epilepsy via the CHRNA4 Gene
- Summary and Pricing
- Clinical Features and Genetics
|Test Code||Test||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.
Mutations in CHRNA4 were identified in 1 of 21 (~5%) of families with ADNFLE and 1 in 33 (~3%) of individuals with sporadic NFLE (Chen et al. 2009; Rozycka et al. 2003).
Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is a partial seizure disorder characterized by seizures that occur during non-REM sleep. Seizures in ADNFLE patients can present as repetitive limb movements, dystonic posturing, sleep walking, or elevation of the body or head with fear (Kurahashi and Hirose 2012). Seizures are brief, lasting from 5 seconds to 5 minutes, and often patients maintain consciousness throughout the attack. Frontal origin of ADNFLE seizures is revealed by ictal EEG recordings; interictal EEGs are normal. ADNFLE associated seizures are often managed with low doses of antiepileptic drugs (AEDs). ADNFLE is not generally associated with severe cognitive deficits or psychiatric problems, however recent evidence suggests that ADNFLE may have wider neurological phenotypes (Steinlein et al. 2012; Wood et al. 2010). It can be difficult to clinically distinguish ADNFLE from other non-epileptic paroxysmal sleep disorders, therefore a video-polysomnograph recording brain/eye/muscle activity during sleep is considered essential for diagnosis.
ADNFLE is inherited in an autosomal dominant manner and can be caused by missense mutations in the CHRNA4 or CHRNB2 genes. The S280F and S284L substitutions in CHRNA4 have been reported in ADNFLE patients of multiple ethnicities (Hwang et al. 2011). Mutations in either CHRNA4 or CHRNB2 produce indistinguishable ADNFLE phenotypes (McLellan et al. 2003).
CHRNA4 encodes a neuronally expressed subunit of the nicotinic acetylcholine receptor (nAChR). The nAChRs belong to a super-family of ligand-gated ion channels. Of the ADNFLE-associated CHRNA4 mutations reported, all are missense mutations and most are within the transmembrane domains of the receptor. Highly conserved residues within the transmembrane domain dictate the ligand-specificity and activity of the receptor. Reported mutations in CHRNA4 are gain of function, resulting in an increased sensitivity of neuronal nAChRs to the agonist acetylcholine (Phillips et al. 2001). It is hypothesized that increased nAchR activity underlies the epilepsy phenotype associated with CHRNA4 mutations.
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 CHRNA4 sequencing include patients with nocturnal seizures or individuals with a family history of ADNFLE.
|Official Gene Symbol||OMIM ID|
- Genetic Counselor Team - firstname.lastname@example.org
- Li Fan, MD, PhD, FCCMG, FACMG - email@example.com
- Chen Y, Wu L, Fang Y, He Z, Peng B, Shen Y, Xu Q. 2009. A novel mutation of the nicotinic acetylcholine receptor gene CHRNA4 in sporadic nocturnal frontal lobe epilepsy. Epilepsy Research 83: 152–156. PubMed ID: 19058950
- Hwang S-K, Makita Y, Kurahashi H, Cho Y-W, Hirose S. 2011. Autosomal dominant nocturnal frontal lobe epilepsy: a genotypic comparative study of Japanese and Korean families carrying the CHRNA4 Ser284Leu mutation. J. Hum. Genet. 56: 609–612. PubMed ID: 21753767
- Kurahashi H, Hirose S. 2012. Autosomal Dominant Nocturnal Frontal Lobe Epilepsy. In: Pagon RA, Adam MP, Ardinger HH, Bird TD, Dolan CR, Fong C-T, Smith RJ, and Stephens K, editors. GeneReviews(®), Seattle (WA): University of Washington, Seattle. PubMed ID: 20301348
- McLellan A, Phillips HA, Rittey C, Kirkpatrick M, Mulley JC, Goudie D, Stephenson JBP, Tolmie J, Scheffer IE, Berkovic SF, Zuberi SM. 2003. Phenotypic comparison of two Scottish families with mutations in different genes causing autosomal dominant nocturnal frontal lobe epilepsy. Epilepsia 44: 613–617. PubMed ID: 12681012
- Phillips HA, Favre I, Kirkpatrick M, Zuberi SM, Goudie D, Heron SE, Scheffer IE, Sutherland GR, Berkovic SF, Bertrand D. 2001. CHRNB2 Is the Second Acetylcholine Receptor Subunit Associated with Autosomal Dominant Nocturnal Frontal Lobe Epilepsy. The American Journal of Human Genetics 68: 225–231. PubMed ID: 11104662
- Rozycka A, Skorupska E, Kostyrko A, Trzeciak WH. 2003. Evidence for S284L mutation of the CHRNA4 in a white family with autosomal dominant nocturnal frontal lobe epilepsy. Epilepsia 44: 1113–1117. PubMed ID: 12887446
- Steinlein, O.K. et al. (2012). "Mutations in familial nocturnal frontal lobe epilepsy might be associated with distinct neurological phenotypes." Seizure 21(2):118:123. PubMed ID: 22036597
- Wood AG, Saling MM, Fedi M, Berkovic SF, Scheffer IE, Benjamin C, Reutens DC. 2010. Neuropsychological function in patients with a single gene mutation associated with autosomal dominant nocturnal frontal lobe epilepsy. Epilepsy & Behavior 17: 531–535. PubMed ID: 20189461
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.