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Multiple Epiphyseal Dysplasia Sequencing Panel

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

NGS Sequencing

Test Code Test Copy GenesCPT Code Copy CPT Codes
1317 COL2A1 81479 Add to Order
COL9A1 81479
COL9A2 81479
COL9A3 81479
COMP 81479
MATN3 81479
SLC26A2 81479
Full Panel Price* $1890.00
Test Code Test Copy Genes Total Price CPT Codes Copy CPT Codes
1317 Genes x (7) $1890.00 81479(x7) 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

Causative mutations in COMP, MATN3 and type IX collagen genes (COL9A1, COL9A2 and COL9A3) account for 70% , ~20% and ~10% of dominant MED, respectively (Jackson et al. 2012; Briggs et al. 2013). To date, only two missense mutations in COL2A1 have been reported in MED patients (Jackson et al. 2012). Mutations in the in SLC26A2 gene are responsible for ~70% of recessive form of MED (Bonafé et al. 2010).

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Deletion/Duplication Testing via aCGH

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
600 COL2A1$690.00 81479 Add to Order
COL9A1$690.00 81479
COL9A2$690.00 81479
COL9A3$690.00 81479
COMP$690.00 81479
MATN3$690.00 81479
SLC26A2$690.00 81479
Full Panel Price* $840.00
Test Code Test Copy Genes Total Price CPT Codes Copy CPT Codes
600 Genes x (7) $840.00 81479(x7) 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

The sensitivity for large deletions  and duplications may be low, because only a few cases with large deletions and insertions involving the COL9A3COMP and COL2A1 genes have been reported ( van der Hout et al. 2002; Human Mutation Database).

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Clinical Features

Multiple epiphyseal dysplasia (MED) is a clinically and genetically heterogeneous chondrodysplasia with either autosomal dominant or recessive inheritance. Dominant MED (ADMED) occurs early in childhood and usually involves pain in the hips and/or knees after exercise. Some patients may have a waddling gait. Adult height can be in the lower range of normal or mildly shortened. The limbs are relatively short in comparison to the trunk. Other features may include progressive pain and joint deformity which can lead to early-onset osteoarthritis. ADMED is caused by mutations in the COMP, MATN3, COL9A1, COL9A2, COL9A3 and COL2A1 genes (Briggs et al. 2013). Recessive MED (ARMED) is characterized by joint pain (usually in the hips or knees) occurring in late childhood; malformations of hands, feet, and knees; and scoliosis. Height is usually within the normal range prior to puberty; Adult height can range from 150 to 180 cm. ARMED is caused by mutations in the SCL26A2 gene (Bonafé et al. 2010).

Genetics

Causative mutations in COMP, MATN3 and type IX collagen genes (COL9A1, COL9A2 and COL9A3) account for 70% , ~20% and ~10% of dominant MED, respectively (Jackson et al. 2012; Briggs et al. 2013). To date, only two missense mutations in COL2A1 have been reported in MED patients (Jackson et al. 2012). Mutations in the in SLC26A2 gene are responsible for ~70% of recessive form of MED (Bonafé et al. 2010).

In addition to MED, mutations in these genes also cause other skeletal dysplasia disorders (see related diseases). These skeletal disorders have overlapping clinical features with MED, which cause difficulties in reaching a correct clinical diagnosis. Molecular diagnosis of the skeletal dysplasia subtypes is also complex because extensive genetic heterogeneity exists for each disorder (Warman et al. 2011). Considering the clinical and genetic heterogeneity, a molecular testing approach that interrogates all known MED genes is highly recommended. See individual gene test descriptions for information on molecular biology of gene products.

Testing Strategy

For this Next Generation (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.

Indications for Test

Candidates for this test are patients with clinical and radiologic features consistent with MED and related disorders. This test especially aids in a differential diagnosis of similar phenotypes, rules out particular syndromes, and provides the analysis of multiple genes simultaneously. Individuals who are suspected of any of these disorders, especially if clinical diagnosis is unclear, and individuals who have been found to be negative by mutation analysis for single gene tests are also candidates.

Genes

Official Gene Symbol OMIM ID
COL2A1 120140
COL9A1 120210
COL9A2 120260
COL9A3 120270
COMP 600310
MATN3 602109
SLC26A2 606718
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Diseases

Name Inheritance OMIM ID
Achondrogenesis Type 2 200610
Achondrogenesis, Type Ib 600972
Atelosteogenesis, Type II 256050
Avascular Necrosis Of Femoral Head, Primary 608805
Czech Dysplasia Metatarsal Type 609162
Diastrophic Dysplasia 222600
Kniest Dysplasia 156550
Legg-Calve-Perthes Disease 150600
Multiple Epiphyseal Dysplasia 1 132400
Multiple Epiphyseal Dysplasia 2 600204
Multiple Epiphyseal Dysplasia 3 600969
Multiple Epiphyseal Dysplasia 4 226900
Multiple Epiphyseal Dysplasia 5 607078
Multiple Epiphyseal Dysplasia 6 614135
Osteoarthritis With Mild Chondrodysplasia 604864
Otospondylomegaepiphyseal Dysplasia 215150
Pseudoachondroplastic Spondyloepiphyseal Dysplasia Syndrome 177170
Spondyloepimetaphyseal Dysplasia Matrilin-3 Related 608728
Spondyloepimetaphyseal Dysplasia Strudwick Type 184250
Spondyloepiphyseal Dysplasia Congenita 183900
Spondyloperipheral Dysplasia 271700
Stickler Syndrome Type 1 108300
Stickler Syndrome, Type 4 614134
Stickler Syndrome, Type 5 614284
Stickler Syndrome, Type I, Nonsyndromic Ocular 609508

Related Tests

Name
COMP-Related Disorders via the COMP Gene
Achondrogenesis Type 1B (ACG1B) via the SLC26A2 Gene
Atelosteogenesis type 2 (AO2) via the SLC26A2 Gene
Diastrophic dysplasia (DTD) via the SLC26A2 Gene
Multiple Epiphyseal Dysplasia and Stickler Syndrome, Autosomal Recessive via the COL9A1 Gene
Multiple Epiphyseal Dysplasia and Stickler Syndrome, Autosomal Recessive, via the COL9A2 Gene
Multiple Epiphyseal Dysplasia via the COL9A3 Gene
Multiple Epiphyseal Dysplasia via the MATN3 Gene
Multiple Epiphyseal Dysplasia, Recessive (EDM4/rMED) via the SLC26A2 Gene
Short Rib Skeletal Dysplasia Sequencing Panel
Stickler Syndrome Sequencing Panel
Vitreoretinopathy Sequencing Panel

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Bonafé L, Mittaz-Crettol L, Ballhausen D, Superti-Furga A. 2010. Multiple Epiphyseal Dysplasia, Recessive. 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: 20301483
  • Briggs MD, Wright MJ, Mortier GR. 2013. Multiple Epiphyseal Dysplasia, Dominant. 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: 20301302
  • Human Gene Mutation Database (Bio-base).
  • Jackson GC, Mittaz-Crettol L, Taylor JA, Mortier GR, Spranger J, Zabel B, Merrer M Le, Cormier-Daire V, Hall CM, Offiah A, Wright MJ, Savarirayan R, et al. 2012. Pseudoachondroplasia and multiple epiphyseal dysplasia: A 7-year comprehensive analysis of the known disease genes identify novel and recurrent mutations and provides an accurate assessment of their relative contribution. Human Mutation 33: 144–157.
    PubMed ID: 21922596
  • Van Der Hout A.H. et al. 2002. Human Mutation. 20: 236. PubMed ID: 12204008
  • Warman ML, Cormier-Daire V, Hall C, Krakow D, Lachman R, LeMerrer M, Mortier G, Mundlos S, Nishimura G, Rimoin DL. 2011. Nosology and classification of genetic skeletal disorders: 2010 revision. American journal of medical genetics Part A 155: 943–968. PubMed ID: 21438135
Order Kits
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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