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Ectodermal Dysplasia Sequencing Panel

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

NGS Sequencing

Test Code Test Copy GenesCPT Code Copy CPT Codes
3433 EDA 81479 Add to Order
EDAR 81479
EDARADD 81479
KRT85 81479
NECTIN1 81479
WNT10A 81479
Full Panel Price* $1440.00
Test Code Test Copy Genes Total Price CPT Codes Copy CPT Codes
3433 Genes x (6) $1440.00 81479(x6) 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. If you would like to order a subset of these genes contact us to discuss pricing.

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

Mutations in EDAEDARWNT10A and EDARADD together are responsible for ~90% of clinically diagnosed HED patients (Cluzeau et al. 2011). Due to limited publications, clinical sensitivity for the KRT85 and NECTIN1 genes is currently unknown. Analytical sensitivity may be high as the only reported pathogenic variants are detectable by sequencing.

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Deletion/Duplication Testing via aCGH

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
600 EDA$690.00 81479 Add to Order
EDAR$690.00 81479
EDARADD$690.00 81479
WNT10A$690.00 81479
Full Panel Price* $840.00
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 Sensitivity

Gross deletions account for ~7% of pathogenic variants found in the EDA gene (Cluzeau et al. 2011; Orstavik et al. 2007; Human Gene Mutation Database). Only one large deletion was reported in each of the EDAR  and WNT10A genes (Griggs et al. 2009; Iglesias et al. 2014), and no large deletions or duplications involving KRT85, NECTIN1. and EDARADD have been reported to date (Human Gene Mutation Database).

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Clinical Features

Ectodermal dysplasia (ED) is a clinically and genetically heterogeneous disorder characterized by abnormal development of hair, teeth, nail or sweat glands (Visinoni et al. 2009). ED can be clinically divided into more than 150 subtypes. Hypohidrotic ectodermal dysplasia (HED) is characterized by hypodontia, hypohidrosis and hypotrichosis. The major clinical signs are thin and dry scalp hair, eyelashes and eyebrows, reduced ability to sweat, and congenital missing or abnormal formed teeth. Some patients may have abnormal craniofacial features such as prominent forehead, saddle-back nose, and protruding lips (Visinoni et al. 2009; Wright et al. 2014). HED is currently known to be caused by pathogenic variants in the EDA, EDAR, EDARADD, WNT10A, KRT85 and NECTIN1 genes (Cluzeau et al. 2011; Shimomura et al. 2010; Sözen et al. 2001).

Genetics

Pathogenic variants in EDA cause X-linked HED, while pathogenic variants in EDAR, WNT10A, and EDARADD cause autosomal dominant and recessive forms of ectodermal dysplasia and tooth agenesis. In general, female carriers of EDA pathogenic variants and males and females with autosomal dominant HED have mild clinical features. A variety of pathogenic variants in EDAEDAR, WNT10A, and EDARADD account for 55-60%, 16%, 15-20% and 1-2% of pathogenic variants identified in HED patients, respectively (Cluzeau et al. 2011). Truncating EDAR pathogenic variants exhibit loss of function, while heterozygous missense pathogenic variants in the functional domain of the EDAR gene may have dominant negative effect (van Der Hout et al. 2008).

The EDA, EDAR, and EDARADD proteins are the key proteins in the EDA-EDAR-EDARADD and the NF-kappa-B pathways, which play a prominent role in the development of ectodermal structures such as hair follicles, sweat glands, and teeth (Cluzeau et al. 2011; Wright et al. 2014). The WNT10A protein is a secreted signaling protein and serves as a ligand for members of the frizzled family of seven transmembrane receptors which are involved in regulation of cell fate and patterning during embryogenesis.

Pathogenic variants in the KRT85 gene cause autosomal recessive ectodermal dysplasia 4, hair/nail type, which is characterized by congenital abnormal development of the hair and nails (Naeem et al. 2006; Shimomura et al. 2010). KRT85 protein coded by KRT85 belongs to the type II keratin gene family, a basic protein which heterodimerizes with type I keratins to form hair and nails. To date, only one small homozygous truncating pathogenic variant (c.1448_1449delCT, p.Pro483Argfs*18) was identified in two affected consanguineous Pakistani families (Shimomura et al. 2010 and Human Gene Mutation Database). 

Pathogenic variants in the NECTIN1 gene (also known as PVRL1) cause autosomal recessive cleft lip/palate-ectodermal dysplasia (CLPED1, also known as Zlotogora-Ogur syndrome and Margarita Island ectodermal dysplasia), autosomal recessive Orofacial cleft 7, as well as non-syndromic cleft of the lip/palate. NECTIN1 protein coded by NECTIN1 is a member of the immunoglobin (Ig) superfamily and plays a role in cell–cell adhesion. To date, more than 10 unique pathogenic variants have been reported. They include: missense (7), nonsense (2), small del/dup (3) and splicing (1). No large deletions/duplications have been reported (Sözen et al. 2001; Sözen et al. 2009; Avila et al. 2006, Huma Gene Mutation Database). The 554G>A, p.Trp185* variant is commonly found in non-syndromic sporadic clefting cases in Northern Venezuela, with a carrier frequency of 1 in 26 of the native population in that particular region (Sözen et al. 2001). In addition, heterozygous carriers of the 554G>A, p.Trp185* variant often have a broad and flat upper lip (Avila et al. 2006).

Testing Strategy

For this NextGen test, the full coding regions plus ~20 bp of non-coding DNA flanking each exon are sequenced for each of the genes 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 with symptoms consistent with HED and tooth agenesis, autosomal recessive form of cleft lip/palate-ectodermal dysplasia (CLPED1, also known as Zlotogora-Ogur syndrome and Margarita Island ectodermal dysplasia), autosomal recessive Orofacial cleft 7, as well as non-syndromic cleft of the lip/palate and the family members of patients who have known EDA, EDAR, WNT10A,  EDARADD, KRT85 and NECTIN1 gene mutations.

Genes

Official Gene Symbol OMIM ID
EDA 300451
EDAR 604095
EDARADD 606603
KRT85 602767
NECTIN1 600644
WNT10A 606268
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Related Tests

Name
Ectodermal Dysplasia via the EDA Gene
Ectodermal Dysplasia via the EDAR Gene
Ectodermal Dysplasia via the EDARADD Gene
Ectodermal Dysplasia via the KRT85 Gene
Ectodermal Dysplasia via the NECTIN1 Gene
Ectodermal Dysplasia via the WNT10A Gene

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Avila J.R. et al. 2006. American Journal of Medical Genetics. Part A. 140: 2562-70. PubMed ID: 17089422
  • Ørstavik K.H. et al. 2007. American Journal of Medical Genetics. Part A. 143A: 1510-3. PubMed ID: 17568423
  • Cluzeau C. et al. 2011. Human Mutation. 32: 70-2. PubMed ID: 20979233
  • Griggs B.L. et al. 2009. European Journal of Human Genetics. 17: 30-6. PubMed ID: 18854857
  • Human Gene Mutation Database (Bio-base).
  • Iglesias A. et al. 2014. Genetics in Medicine. 16: 922-31. PubMed ID: 24901346
  • Naeem M. et al. 2006. Journal of medical genetics. 43: 274-9. PubMed ID: 16525032
  • Sözen M.A. et al. 2001. Nature Genetics. 29: 141-2. PubMed ID: 11559849
  • Sözen M.A. et al. 2009. Genetic Testing and Molecular Biomarkers. 13: 617-21. PubMed ID: 19715471
  • Shimomura Y. et al. 2010. The Journal of Investigative Dermatology. 130: 892-5 PubMed ID: 19865094
  • van der Hout A.H. et al. 2008. European Journal of Human Genetics : Ejhg. 16: 673-9. PubMed ID: 18231121
  • Visinoni A.F. et al. 2009. American Journal of Medical Genetics. Part A. 149A: 1980-2002. PubMed ID: 19681154
  • Wright J.T. et al. 2014. Hypohidrotic Ectodermal Dysplasia. 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: 20301291
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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