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Ehlers-Danlos Syndrome 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
1391 ADAMTS2 81479 Add to Order
ATP7A 81479
B3GALT6 81479
B3GAT3 81479
B4GALT7 81479
CHST14 81479
CHST3 81479
COL1A1 81408
COL1A2 81408
COL3A1 81479
COL5A1 81479
COL5A2 81479
FKBP14 81479
FLNA 81479
FLNB 81479
PLOD1 81479
SLC39A13 81479
ZNF469 81479
Full Panel Price* $1890.00
Test Code Test Copy Genes Total Price CPT Codes Copy CPT Codes
1391 Genes x (18) $1890.00 81408(x2), 81479(x16) 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

ATP7A pathogenic variants were identified in about 80% of males affected with Menkes disease (Kaler, 2010).

In one study, ADAMTS2 pathogenic variants were identified in 2 out of 5 studied patients with Ehlers–Danlos Syndrome (EDS) Type VIIC (Colige et al. 2004).

COL1A1 and COL1A2 pathogenic variants were identified in 56% (14/35) and 44% (11/35) of the Osteogenesis Imperfecta cases, respectively (Stephen et al. 2014). Sequencing analysis can detect more than 95% of pathogenic variants in the COL1A1 and COL1A2 genes, while deletion and duplication studies can detect 1% -2% of COL1A1 and COL1A2 pathogenic variants (van Dijk et al. 2012, Steiner et al. 2013).

COL3A1 pathogenic variants have been identified in approximately 95% of individuals with EDS IV (Pepin and Byers 2011). COL5A1 or COL5A2 pathogenic variants have been identified in at least 50% of affected individuals with classic EDS (Malfait et al. 2011).  

In one study, B3GALT6 pathogenic variants were identified in 12 individuals from 10 studied families (Nakajima et al. 2013).

Only a few pathogenic variants were reported in B3GAT3, B4GALT7 and SLC39A13 (Human Gene Mutation Database; Baasanjav et al. 2011; Okajima et al. 1999; Fukada et al. 2008). Analytical sensitivity for these 3 genes should be high, because nearly all pathogenic variants found in these genes are point variants that are expected to be detected by direct sequencing analysis.

 By linkage and then sequencing analysis, CHST14 pathogenic variants were identified in all 4 studied consanguineous families with EDS, musculocontractural type 1 (also called Adducted Thumb-Clubfoot Syndrome) (Dundar et al. 2009).

Because only two reports have described three FKBP14 pathogenic sequence variants in a total of seven patients with progressive kyphoscoliosis, myopathy, and hearing loss (Baumann et al. 2012; Aldeeri et al. 2014), it is difficult to estimate the clinical sensitivity of this test.

FLNA pathogenic variants were identified in 26 out of 41 patients with FLNA-related disorders (otopalatodigital syndrome, frontometaphyseal dysplasia, Melnick-Needles syndrome) (Robertson et al. 2003).

~90 FLNB pathogenic variants have been reported. FLNB pathogenic variants are point variants and small deletions, which are expected to be detected by direct sequencing analysis. Clinical sensitivity may be high in patients diagnosed by radiology (Daniel et al. 2012).

PLOD1 pathogenic variants were identified in 9 out of 12 unrelated patients with EDS type VIA (Giunta et al. 2005). The common intragenic duplication of exons 10-16 in the PLOD1 gene cannot be detected by sequencing.

ZNF469 pathogenic variants were found in 12.5% of patients with Brittle cornea syndrome (Lechner et al. 2014).

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Deletion/Duplication Testing via aCGH

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
600 ATP7A$690.00 81479 Add to Order
CHST3$690.00 81479
COL3A1$690.00 81479
COL5A1$690.00 81479
COL5A2$690.00 81479
FLNA$690.00 81479
FLNB$690.00 81479
PLOD1$690.00 81479
Full Panel Price* $840.00
Test Code Test Copy Genes Total Price CPT Codes Copy CPT Codes
600 Genes x (8) $840.00 81479(x8) 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 Sensitivity

Large deletions/duplications in ATP7A account for ~27% of reported pathogenic variants (HGMD).

So far, less than 10 large deletions have been reported in COL3A1 (Meienberg et al. 2010; HGMD), and a few in COL5A1 (Symoens et al. 2012; HGMD). No large deletions/duplications have been reported in COL5A2 (HGMD).

No large deletions/insertions have been reported in CHST3 and FLNB (HGMD).

Large deletions/duplications account for ~10% and 13% of reported pathogenic mutations in FLNA and PLOD1, respectively (HGMD).

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Clinical Features

Ehlers-Danlos syndrome (EDS) is a clinically and genetically heterogeneous group of heritable connective tissue disorders that mainly affect the skin, joints, ligaments, blood vessels, and internal organs (Byers and Murray 2012). This EDS panel focuses on, but is not limited to the following conditions: all types of EDS, frontometaphyseal dysplasia, Larsen syndrome, Brittle cornea syndrome (also called Ehlers-Danlos syndrome TYPE VIB, or keratoconus) and Occipital horn syndrome.

Genetics

This test analyzes genes involved in EDS as well as EDS-related conditions.

X-Linked Recessive Conditions: ATP7A-related occipital horn syndrome and FLNA-related Frontometaphyseal dysplasia are X-linked recessive conditions.

Autosomal Dominant Conditions:  COL1A1, COL3A1, COL5A1, COL5A2-related Ehlers-Danlos syndrome and FLNB-related Larsen syndrome are inherited in autosomal dominant manner.

Autosomal Recessive Conditions: ADAMTS2, B3GALT6, B3GAT3, B4GALT7, CHST14, CHST3, FKBP14, FLNA, PLOD1, and SLC39A13-related Ehlers-Danlos syndrome, CHST3-related Spondyloepiphyseal dysplasia with congenital joint dislocations and ZNF469-related Brittle cornea syndrome are inherited in an autosomal recessive manner.

Both Autosomal dominant/Autosomal Recessive Conditions: Pathogenic variants in COL1A2 cause both autosomal dominant as well autosomal recessive Ehlers-Danlos syndrome.

NOTE: Pathogenic variants in FLNA also cause X-linked dominant conditions, such as Heterotopia, periventricular/ Otopalatodigital syndrome and other conditions. Pathogenic variants in FLNB also cause autosomal recessive Spondylocarpotarsal synostosis syndrome. COL1A1 and COL1A2 are the major genes for osteogenesis imperfects. ATP7A is the major gene responsible for autosomal recessive Menkes disease.

See individual test descriptions for information on molecular biology of gene products and mutation spectra.

Testing Strategy

For this NextGen panel, 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.

Of note, TNXB is not included in this panel. However, TNXB sequencing is available.

Indications for Test

Candidates for this test are patients with clinical features of Ehlers-Danlos syndrome or Ehlers-Danlos syndrome related conditions, such as frontometaphyseal dysplasia, Larsen syndrome, Brittle cornea syndrome (also called Ehlers-Danlos syndrome TYPE VIB, or keratoconus) and Occipital horn syndrome.

Genes

Official Gene Symbol OMIM ID
ADAMTS2 604539
ATP7A 300011
B3GALT6 615291
B3GAT3 606374
B4GALT7 604327
CHST14 608429
CHST3 603799
COL1A1 120150
COL1A2 120160
COL3A1 120180
COL5A1 120215
COL5A2 120190
FKBP14 614505
FLNA 300017
FLNB 603381
PLOD1 153454
SLC39A13 608735
ZNF469 612078
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Diseases

Name Inheritance OMIM ID
Brittle Cornea Syndrome 1 AR 229200
Ehlers-Danlos Syndrome with Progressive Kyphoscoliosis, Myopathy, and Hearing Loss AR 614557
Ehlers-Danlos Syndrome with Short Stature and Limb Anomalies AR 130070
Ehlers-Danlos Syndrome, Autosomal Recessive, Cardiac Valvular Form AR 225320
Ehlers-Danlos Syndrome, Hydroxylysine-Deficient AR 225400
Ehlers-Danlos Syndrome, Musculocontractural Type AR 601776
Ehlers-Danlos Syndrome, Progeroid Type, 2 AR 615349
Ehlers-Danlos Syndrome, Type 1 AD 130000
Ehlers-Danlos Syndrome, Type 4 AD 130050
Ehlers-Danlos Syndrome, Type VIIA and VIIB AD 130060
Ehlers-Danlos Syndrome, Type VIIC AR 225410
Frontometaphyseal Dysplasia XL 305620
Larsen Syndrome, Dominant Type AD 150250
Multiple Joint Dislocations, Short Stature, Craniofacial Dysmorphism, and Congenital Heart Defects AR 245600
Occipital Horn Syndrome XL 304150
Spondylocheirodysplasia, Ehlers-Danlos Syndrome-Like AR 612350
Spondyloepiphyseal Dysplasia with Congenital Joint Dislocations AR 143095

Related Tests

Name
FLNB-Related Disorders via the FLNB Gene
Autism Spectrum Disorders and Intellectual Disability (ASD-ID) Comprehensive Panel
Bleeding Disorders Sequencing Panel
Brittle Cornea Syndrome Sequencing Panel
Comprehensive Neuropathy Sequencing Panel
Distal Arthrogryposis Sequencing Panel
Ehlers-Danlos Syndrome with Progressive Kyphosis, Myopathy, and Hearing Loss (EDSKMH) via the FKBP14 Gene
Ehlers-Danlos Syndrome, Classic Type via the COL5A1 Gene
Ehlers-Danlos Syndrome, Classic Type via the COL5A2 Gene
Ehlers-Danlos Syndrome, Kyphoscoliotic Form via the PLOD1 Gene
Ehlers-Danlos Syndrome, Type IV via the COL3A1 Gene
Menkes Disease and Hereditary Motor Neuropathy via the ATP7A Gene
Osteogenesis Imperfecta and Hypophosphatasia (HPP) Sequencing Panel
Osteogenesis Imperfecta and Hypophosphatasia (HPP), and Inherited Hypophosphatemic Rickets Sequencing Panel
Osteogenesis Imperfecta via the COL1A1 Gene
Osteogenesis Imperfecta via the COL1A2 Gene
Otopalatodigital Spectrum Disorders, Periventricular Nodular Heterotopia and Cardiac Valvular Dystrophy via the FLNA Gene
Thrombocytopenia Sequencing Panel - Expanded
X-Linked Intellectual Disability Sequencing Panel

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Aldeeri A.A. et al. 2014. Clinical Genetics. 86: 469-72. PubMed ID: 24773188
  • Baasanjav S. et al. 2011. American Journal of Human Genetics. 89: 15-27. PubMed ID: 21763480
  • Baumann M. et al. 2012. American Journal of Human Genetics. 90: 201-16. PubMed ID: 22265013
  • Byers P.H, Murray M.L. 2012. The Journal of Investigative Dermatology. 132: E6-11. PubMed ID: 23154631
  • Colige A. et al. 2004. The Journal of Investigative Dermatology. 123: 656-63. PubMed ID: 15373769
  • Daniel P.B. et al. 2012. Human Mutation. 33: 665-73. PubMed ID: 22190451
  • Dündar M. et al. 2009. American Journal of Human Genetics. 85: 873-82. PubMed ID: 20004762
  • Fukada T. et al. 2008. Plos One. 3: e3642. PubMed ID: 18985159
  • Giunta C.et al. 2005. American Journal of Medical Genetics. 133A: 158-64. PubMed ID: 15666309
  • Human Gene Mutation Database (Bio-base).
  • Kaler, S.G. 2010. ATP7A-Related Copper Transport Disorders. 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: 20301586
  • Lechner J. et al. 2014. Human Molecular Genetics. 23: 5527-35. PubMed ID: 24895405
  • Malfait F. et al. 2011. Ehlers-Danlos Syndrome, Classic Type. 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: 20301422
  • Meienberg J. et al. 2010. European Journal of Human Genetics. 18: 1315-21. PubMed ID: 20648054
  • Nakajima M. et al. 2013. American Journal of Human Genetics. 92: 927-34. PubMed ID: 23664117
  • Okajima T. et al. 1999. The Journal of Biological Chemistry. 274: 28841-4. PubMed ID: 10506123
  • Pepin M.G. and Byers P.H. 2011. Ehlers-Danlos Syndrome Type IV. 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: 20301667
  • Robertson S.P. et al. 2003. Nature Genetics. 33: 487-91. PubMed ID: 12612583
  • Steiner R.D et al. 2013. COL1A1/2-Related Osteogenesis Imperfecta. 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: 20301472
  • Stephen J. et al. 2014. American Journal of Medical Genetics. Part A. 164A: 1482-9. PubMed ID: 24668929
  • Symoens S. et al. 2012. Human Mutation. 33: 1485-93. PubMed ID: 22696272
  • van Dijk FS. et al. 2012. European Journal of Human Genetics : Ejhg. 20: 11-9. PubMed ID: 21829228
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TEST METHODS

NextGen Sequencing using PG-Designed 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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