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Stickler Syndrome Sequencing Panel with CNV Detection

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

Sequencing with CNV

Test Code Test Copy GenesCPT Code Copy CPT Codes
10271 COL11A1 81479,81479 Add to Order
COL11A2 81479,81479
COL2A1 81479,81479
COL9A1 81479,81479
COL9A2 81479,81479
COL9A3 81479,81479
LOXL3 81479,81479
LRP2 81479,81479
VCAN 81479,81479
Full Panel Price* $960
Test Code Test Copy Genes Total Price CPT Codes Copy CPT Codes
10271 Genes x (9) $960 81479(x18) Add to Order

New York State Approved Test

Pricing Comments

Our favored testing approach is exome based NextGen sequencing with CNV analysis. This will allow cost effective reflexing to PGxome or other exome based tests. However, if a lower cost sequencing option without CNV detection is desired, please see this link for Test Code, pricing, and turnaround time information. If the alternative option is selected, CNV detection may be ordered through Test #600.

We are happy to accommodate requests for testing single genes in this panel or a subset of these genes. The price will remain the list price. If desired, free reflex testing to remaining genes on panel is available. Alternatively, a single gene or subset of genes can also be ordered via our PGxome Custom Panel tool.

Targeted Testing

For ordering sequencing of targeted known variants, please proceed to our Targeted Variants landing page.

Turnaround Time

The great majority of tests are completed within 26 days.

Clinical Sensitivity

Causative variants in COL2A1 and COL11A1 account for 80-90% and 10-20% of variants identified in autosomal dominant STL syndrome, respectively; causative variants in COL11A2 account for rare dominant cases. Causative variants in COL9A1, COL9A2, COL9A3 and LOXL3 and LRP2 have been found only in a few families affected with autosomal recessive inheritance of STL syndrome (Robin et al. 2011; Schrauwen et al. 2014; Alzahrani et al. 2015). Causative variants in the VCAN gene were identified in ten out of twelve families diagnosed with VCAN-related vitreoretinopathy (Kloeckener-Gruissem and Amstutz 2012).

The sensitivity for large deletions and duplications in the COL2A1, COL11A2, COL9A1, COL9A2, and COL9A3 genes is probably low, because only a few cases with large deletions and insertions involving the these five genes have been reported (van der Hout et al. 2002; Human Mutation Database). However, large deletions in the COL11A1 gene were detected in six unrelated Stickler syndrome patients by MLPA methods (Vijzelaar et al. 2013).

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Clinical Features

Stickler syndrome (STL) is a multisystem disorder characterized by ocular, skeletal, orofacial and auditory defects. Ocular defects include myopia, cataract, and retinal detachment. Hearing loss can be both conductive and sensorineural. Other features include midfacial underdevelopment and cleft palate and mild spondyloepiphyseal dysplasia and/or precocious arthritis (Robin et al. 2011). Pathogenic variants in COL2A1, COL11A1, COL11A2, COL9A1, COL9A2 and COL9A3 are responsible for Type I, Type II, Type III, Type IV, Type V and Type VI Stickler syndrome, respectively (Robin et al. 2011; Van Camp et al. 2006; Baker et al. 2011; Nikopoulos et al. 2011; Vijzelaar et al. 2013).

Stickler syndrome Type I (STL1) is characterized by “membranous” congenital vitreous anomaly; Stickler syndrome Type II (STL2) is characterized by “beaded” congenital vitreous anomaly, Stickler syndrome (STL3) has craniofacial and joint abnormalities and hearing loss, but no ocular findings. Stickler syndrome Type IV and Type V have moderate-to-severe sensorineural hearing loss, moderate-to-high myopia with vitreoretinopathy (Baker et al. 2011). Stickler syndrome Type VI features moderate-to-severe sensorineural hearing loss, moderate-to-high myopia, and midface retrusion (Faletra et al. 2014). One study suggested that hearing loss (mostly mild to moderate) was found in ~60% of Stickler patients. Hearing loss was seen in patients with COL11A1 (~80%), COL11A2 (~94%) and COL2A1 (~52%) pathogenic variants (Acke et al. 2012). Stickler syndrome was also found in 39 out of 141 newborns who were diagnosed with Pierre-Robin sequence (Thouvenin et al. 2012).

Only one undocumented homozygous missense LOXL3 variant was reported in two sibs affected with Stickler syndrome, who had micro/retrognathia, a U-shaped cleft palate, and high non-progressive myopia, with or without mild conductive hearing loss (Alzahrani et al. 2015). Pathogenic variants in LRP2 gene mainly cause Donnai–Barrow syndrome (DBS), a malformation disorder characterized by craniofacial features, agenesis of the corpus callosum, developmental delay, intellectual disability, ocular findings, low molecular weight proteinuria, and sensorineural hearing loss. Some of the DBS clinical features overlap with Stickler syndrome. Recently, an undocumented homozygous LRP2 variant was found in two sibs, from one Saudi consanguineous family, affected with a predominant eye phenotype similar to the Stickler syndrome (Schrauwen et al.2014). To date, only a few autosomal recessive Stickler syndrome cases have been reported.

Pathogenic variants in VCAN cause Wagner syndrome (WS), which is characterized by optically empty vitreous cavity with avascular strands, membranes and/or veils, which are the pathological hallmark features of WS (Kloeckener-Gruissem and Amstutz 2016). Advanced stages of WS symptoms feature retinal traction and retinal detachment, which eventually leads to vision loss. The overlapping clinical features in WS and Stickler syndrome are myopia, presenile cataract, vitreous degeneration, radial perivascular retinal degeneration, and tractional retinal detachments (Kloeckener-Gruissem and Amstutz 2016).

Genetics

Causative variants in COL2A1 and COL11A1 account for 80-90% and 10-20% of variants identified in autosomal dominant STL syndrome, respectively; causative variants in COL11A2 account for rare dominant cases. Causative variants in COL9A1, COL9A2, COL9A3, LOXL3 and LRP2 have been found only in a few families affected with autosomal recessive inheritance of STL syndrome (Robin et al. 2011; Schrauwen et al. 2014; Alzahrani et al. 2015). Causative variants in the VCAN gene were identified in ten out of twelve families diagnosed with autosomal dominant VCAN-related vitreoretinopathy (Kloeckener-Gruissem and Amstutz 2016).

In addition to STL, causative variants in these genes also cause other skeletal dysplasia disorders. These skeletal disorders have overlapping clinical features with Stickler syndrome, 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 Stickler syndrome genes is highly recommended. See individual gene test descriptions for information on molecular biology of gene products.

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.

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.

Copy number variants (CNVs) are also detected from NGS data. We utilize a CNV calling algorithm that compares mean read depth and distribution for each target in the test sample against multiple matched controls. Neighboring target read depth and distribution and zygosity of any variants within each target region are used to reinforce CNV calls. All CNVs are confirmed using another technology such as aCGH, MLPA, or PCR before they are reported.

This panel provides 100% coverage of all coding exons of the genes plus 10 bases of flanking noncoding DNA in all available transcripts along with other non-coding regions in which pathogenic variants have been identified at PreventionGenetics or reported elsewhere. We define coverage as ≥20X NGS reads or Sanger sequencing.

Since this test is performed using exome capture probes, a reflex to any of our exome based tests is available (PGxome, PGxome Custom Panels).

Indications for Test

Candidates for this test are patients with clinical and radiologic features consistent with Stickler syndrome 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 candidates.

Genes

Official Gene Symbol OMIM ID
COL11A1 120280
COL11A2 120290
COL2A1 120140
COL9A1 120210
COL9A2 120260
COL9A3 120270
LOXL3 607163
LRP2 600073
VCAN 118661
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Diseases

Name Inheritance OMIM ID
Achondrogenesis Type 2 AD 200610
Avascular Necrosis Of Femoral Head, Primary AD 608805
Czech Dysplasia Metatarsal Type AD 609162
Deafness, Autosomal Dominant 13 AD 601868
Deafness, Autosomal Recessive 53 AR 609706
Epiphyseal Dysplasia, Multiple, With Myopia And Conductive Deafness AD 132450
Fibrochondrogenesis AR 228520
Fibrochondrogenesis 2 AR, AD 614524
Kniest Dysplasia AD 156550
Legg-Calve-Perthes Disease AD 150600
Marshall Syndrome AD 154780
Multiple Epiphyseal Dysplasia 2 AD 600204
Multiple Epiphyseal Dysplasia 3 AD 600969
Multiple Epiphyseal Dysplasia 6 AD 614135
Osteoarthritis With Mild Chondrodysplasia AD 604864
Otospondylomegaepiphyseal Dysplasia AR 215150
Platyspondylic Lethal Skeletal Dysplasia Torrance Type AD 151210
Spondyloepimetaphyseal Dysplasia Strudwick Type AD 184250
Spondyloepiphyseal Dysplasia Congenita AD 183900
Spondyloperipheral Dysplasia AD 271700
Stickler Syndrome Type 1 AD 108300
Stickler Syndrome, Type 2 AD 604841
Stickler Syndrome, Type 3 AD 184840
Stickler Syndrome, Type 4 AD 614134
Stickler Syndrome, Type 5 AR 614284
Stickler Syndrome, Type I, Nonsyndromic Ocular AD 609508
Wagner Syndrome AD 143200
Weissenbacher-Zweymuller Syndrome 277610

Related Test

Name
PGxome®

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Acke F.R. et al. 2012. Orphanet Journal of Rare Diseases. 7: 84. PubMed ID: 23110709
  • Alzahrani F. et al. 2015. Human Genetics. 134: 451-3. PubMed ID: 25663169
  • Baker S. et al. 2011. American Journal of Medical Genetics. Part A. 155A: 1668-72. PubMed ID: 21671392
  • Faletra F. et al. 2014. American Journal of Medical Genetics. Part A. 164A: 42-7. PubMed ID: 24273071
  • Human Gene Mutation Database (Bio-base).
  • Kloeckener-Gruissem B, Amstutz C. 2016. VCAN-Related Vitreoretinopathy. 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: 20301747
  • Nikopoulos K. et al. 2011. Investigative Ophthalmology & Visual Science. 52: 4774-9. PubMed ID: 21421862
  • Robin N.H. et al. 2011. Stickler Syndrome. 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: 20301479
  • Schrauwen I. et al. 2014. Clinical Genetics. 86: 282-6. PubMed ID: 23992033
  • Thouvenin B. et al. 2013. American Journal of Medical Genetics. Part A. 161A: 312-9. PubMed ID: 23303695
  • Van Camp G. et al. 2006. American Journal of Human Genetics. 79: 449-57. PubMed ID: 16909383
  • Van Der Hout A.H. et al. 2002. Human Mutation. 20: 236. PubMed ID: 12204008
  • Vijzelaar R. et al. 2013. Bmc Medical Genetics. 14: 48. PubMed ID: 23621912
  • Warman M.L. et al. 2011. American Journal of Medical Genetics. Part A. 155A: 943-68. PubMed ID: 21438135
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TEST METHODS

Exome Sequencing with CNV Detection

Test Procedure

For PGxome® we use Next Generation Sequencing (NGS) technologies to cover the coding regions of targeted genes plus 10 bases of flanking non-coding DNA in all available transcripts along with other non-coding regions in which pathogenic variants have been identified at PreventionGenetics or reported elsewhere. As required, genomic DNA is extracted from patient specimens. Patient DNA corresponding to these regions is captured using Agilent Clinical Research Exome hybridization probes. Captured DNA is sequenced on the NovaSeq 6000 using 2x150 bp paired-end reads (Illumina, San Diego, CA, USA). The following quality control metrics are generally achieved: >97% of target bases are covered at >20x, and mean coverage of target bases >120x. Data analysis and interpretation is performed by the internally developed software Titanium-Exome. In brief, the output data from the NovaSeq 6000 is converted to fastqs by Illumina Bcl2Fastq, and mapped by BWA. Variant calls are made by the GATK Haplotype caller and annotated using in house software and SnpEff. Variants are filtered and annotated using VarSeq (www.goldenhelix.com).

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.

Copy number variants (CNVs) are also detected from NGS data. We utilize a CNV calling algorithm that compares mean read depth and distribution for each target in the test sample against multiple matched controls. Neighboring target read depth and distribution and zygosity of any variants within each target region are used to reinforce CNV calls. All CNVs are confirmed using another technology such as aCGH, MLPA, or PCR before they are reported.

Analytical Validity

NextGen Sequencing: 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.

Copy Number Variant Analysis: The PGxome test detects most larger deletions and duplications including intragenic CNVs and large cytogenetic events; however aberrations in a small percentage of regions may not be accurately detected due to sequence paralogy (e.g., pseudogenes, segmental duplications), sequence properties, deletion/duplication size (e.g., 1-3 exons vs. 4 or more exons), and inadequate coverage. In general, sensitivity for single, double, or triple exon CNVs is ~70% and for CNVs of four exon size or larger is >95%, but may vary from gene-to-gene based on exon size, depth of coverage, and characteristics of the region.

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 sequencing does not reveal any heterozygous differences from the reference sequence, we cannot be certain that we were able to detect both patient alleles.

For technical reasons, the PGxome test is not 100% sensitive. Some exons cannot be efficiently captured, and some genes cannot be accurately sequenced because of the presence of multiple copies in the genome. Therefore, a small fraction of sequence variants will not be detected.

We sequence coding exons for all available transcripts plus 10 bp of flanking non-coding DNA for each exon. We also sequence other regions within or near genes in which pathogenic variants have been identified at PreventionGenetics or reported elsewhere.  Unless specifically indicated, test reports contain no information about other portions of genes.

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 amplification.

Unless otherwise indicated, DNA sequence data is obtained from a specific cell-type (usually leukocytes if taken 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.

Balanced translocations or inversions are only rarely detected.

Certain types of sex chromosome aneuploidy may not be detected.  

Our ability to detect CNVs due to somatic mosaicism is limited.

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.

A negative finding does not rule out a genetic diagnosis.

Genetic counseling to help to explain test results to the patients and to discuss reproductive options is recommended.

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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