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Epilepsy and Intellectual Disability in Females via the PCDH19 Gene

  • Summary and Pricing
  • Clinical Features and Genetics
  • Citations
  • Methods
  • Ordering/Specimens
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TEST METHODS

NGS Sequencing

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
4207 PCDH19$690.00 81405 Add to Order
Pricing Comment

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 Sanger Sequencing click here.
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 28 days.

Clinical Sensitivity

PCDH19 mutations have been identified in approximately 10% of females that present with clustered febrile seizures during infancy and that tested negative for SCN1A variants (Marini et al. 2010; Depienne et al. 2011) .

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Deletion/Duplication Testing via aCGH

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
600 PCDH19$690.00 81479 Add to Order
Pricing Comment

# of Genes Ordered

Total Price

1

$690

2

$730

3

$770

4-10

$840

11-30

$1,290

31-100

$1,670

Over 100

Call for quote

Turnaround Time

The great majority of tests are completed within 28 days.

Clinical Features

Female restricted epilepsy with mental retardation (EFMR; OMIM:300088) is a neurocognitive disorder that primarily affects females. Onset of epilepsy occurs during infancy or early childhood and patients present with clusters of focal febrile seizures. Seizures can manifest as periods of staring with motor arrest or bilateral clonic jerking and are often accompanied by fearful screaming (Marini et al. 2012). The seizures themselves are short in duration, but seizure clusters can be prolonged, lasting for weeks (Higurashi et al. 2013). Interictal EEG recordings reveal focal poly-spike and wave discharges on a slow background. Seizure frequency decreases with age and often seizures remit during adolescence (Depienne et al. 2011). Most patients have normal motor skills and can walk without assistance (Higurashi et al. 2013). EFMR patients display a range of intellectual disability from mild to severe. Psychiatric features commonly seen in EFMR include aggression, depression and obsessive behavior. Approximately 25% of EFMR patients fit the criteria for having an autism spectrum disorder (Camacho et al. 2012).

Genetics

EFMR is inherited in a female-centric X-linked dominant manner and is caused by mutations in the PCDH19 gene. EFMR predominantly affects females due to a process termed 'cellular interference' where populations of cells expressing wild type PCDH19 and cells expressing mutant PCDH19 are required to observe a disease phenotype (Wieland et al. 2004; Depienne et al. 2009). Since females carry two X chromosomes and X-inactivation is random, females are mosaic for PCDH19 expression. A rare male that was mosaic for a PCDH19 mutation was identified to have an epilepsy phenotype (Depienne et al. 2009). PCDH19 mutations can be inherited from unaffected males, inherited from mildly affected females or arise de novo. Penetrance of PCDH19 mutations is nearly 100% in females (Dimova et al. 2012). Missense, nonsense, frameshift and splice site mutations as well as large deletions have been reported in the PCDH19 gene (Marini et al. 2012; Vincent et al. 2012). Many of the identified PCDH19 mutations are clustered in exon 1, which encodes the large cytoplasmic domain of the protocadherin protein (Depienne et al. 2009). PCDH19 encodes a protocadherin protein. Protocadherins belong to the cadherin family of proteins. Cadherins have well-defined roles in cell adhesion. The exact role of protocadherins is unclear, but studies suggest that the large extracellular domain of protocahderins form homophillic interactions with other cadherin proteins to mediate cell adhesion (Emond et al. 2011). Studies in a zebrafish model demonstrate that PCDH19 is required for proper cell migration during neural development (Biswas et al. 2010). PCDH19 mRNA levels have also been shown to decrease in the rat hippocampus in response to electroconvulsive shock and it has been suggested PCDH19 plays a role in responding to stimuli and shaping neural circuitry (Kim et al. 2010).

Testing Strategy

For this Next Generation Sequencing (NGS) test, 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 regions not captured or with insufficient number of sequence reads. All reported pathogenic, likely pathogenic, and 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.

This test provides full coverage of all coding exons of the PCDH19 gene, plus ~10 bases of flanking noncoding DNA. We define full coverage as >20X NGS reads or Sanger sequencing.

Indications for Test

PCDH19 testing should be considered in females with epilepsy and intellectual disability of unknown cause. In addition, PCDH19 sequencing should be considered in females with a diagnosis for Dravet syndrome but for which no SCN1A mutation was identified.

Gene

Official Gene Symbol OMIM ID
PCDH19 300460
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Disease

Name Inheritance OMIM ID
Epileptic Encephalopathy, Early Infantile, 9 300088

Related Tests

Name
Autism Spectrum Disorders and Intellectual Disability (ASD-ID) Comprehensive Panel
Early Infantile Epileptic Encephalopathy Sequencing Panel
Early Infantile Epileptic Encephalopathy:
Dominant and X-linked Sequencing Panel
Epilepsy: Dravet Syndrome Sequencing Panel
X-Linked Intellectual Disability Sequencing Panel

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Biswas S, Emond MR, Jontes JD. 2010. Protocadherin-19 and N-cadherin interact to control cell movements during anterior neurulation. The Journal of Cell Biology 191: 1029–1041. PubMed ID: 21115806
  • Camacho A, Simón R, Sanz R, Viñuela A, Martínez-Salio A, Mateos F. 2012. Cognitive and behavioral profile in females with epilepsy with PDCH19 mutation: Two novel mutations and review of the literature. Epilepsy & Behavior 24: 134–137. PubMed ID: 22504056
  • Depienne C, Trouillard O, Bouteiller D, Gourfinkel-An I, Poirier K, Rivier F, Berquin P, Nabbout R, Chaigne D, Steschenko D, Gautier A, Hoffman-Zacharska D, et al. 2011. Mutations and deletions in PCDH19 account for various familial or isolated epilepsies in females. Human Mutation 32: E1959–E1975. PubMed ID: 21053371
  • Depienne C. et al. 2009. Plos Genetics. 5: e1000381. PubMed ID: 19214208
  • Dimova PS, Kirov A, Todorova A, Todorov T, Mitev V. 2012. A Novel PCDH19 Mutation Inherited From an Unaffected Mother. Pediatric Neurology 46: 397–400. PubMed ID: 22633638
  • Emond MR, Biswas S, Blevins CJ, Jontes JD. 2011. A complex of Protocadherin-19 and N-cadherin mediates a novel mechanism of cell adhesion. The Journal of Cell Biology 195: 1115–1121. PubMed ID: 22184198
  • Higurashi N, Nakamura M, Sugai M, Ohfu M, Sakauchi M, Sugawara Y, Nakamura K, Kato M, Usui D, Mogami Y, Fujiwara Y, Ito T, et al. 2013. PCDH19-related female-limited epilepsy: Further details regarding early clinical features and therapeutic efficacy. Epilepsy Research 106: 191–199. PubMed ID: 23712037
  • Kim SY, Mo JW, Han S, Choi SY, Han SB, Moon BH, Rhyu IJ, Sun W, Kim H. 2010. The expression of non-clustered protocadherins in adult rat hippocampal formation and the connecting brain regions. Neuroscience 170: 189–199. PubMed ID: 20541594
  • Marini C, Darra F, Specchio N, Mei D, Terracciano A, Parmeggiani L, Ferrari A, Sicca F, Mastrangelo M, Spaccini L, Canopoli ML, Cesaroni E, et al. 2012. Focal seizures with affective symptoms are a major feature of PCDH19 gene-related epilepsy: PCDH19-Related Epilepsy. Epilepsia 53: 2111–2119. PubMed ID: 22946748
  • Marini C, Mei D, Parmeggiani L, Norci V, Calado E, Ferrari A, Moreira A, Pisano T, Specchio N, Vigevano F, Battaglia D, Guerrini R. 2010. Protocadherin 19 mutations in girls with infantile-onset epilepsy. Neurology 75: 646–653. PubMed ID: 20713952
  • Vincent A, Noor A, Janson A, Minassian B, Ayub M, Vincent J, Morel C. 2012. Identification of genomic deletions spanning the PCDH19 gene in two unrelated girls with intellectual disability and seizures. Clinical Genetics 82: 540–545. PubMed ID: 22091964
  • Wieland I, Jakubiczka S, Muschke P, Cohen M, Thiele H, Gerlach KL, Adams RH, Wieacker P. 2004. Mutations of the ephrin-B1 gene cause craniofrontonasal syndrome. Am. J. Hum. Genet. 74: 1209–1215. PubMed ID: 15124102
Order Kits
TEST METHODS

NextGen Sequencing using PG-Select Capture Probes

Test Procedure

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.

Analytical Validity

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.   

Analytical Limitations

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.

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.

Order Kits

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.
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.

SPECIMEN TYPES
WHOLE BLOOD

(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.

DNA

(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.

CELL CULTURE

(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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