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Congenital Limb Malformation Sequencing Panel with CNV Detection

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

Sequencing

Test Code Test Copy GenesCPT Code Copy CPT Codes
5065 ARHGAP31 81479,81479 Add to Order
BMP2 81479,81479
BMPR1B 81479,81479
BRCA2 81479,81479
BRIP1 81479,81479
CC2D2A 81479,81479
CDH3 81479,81479
CEP290 81408,81479
CHSY1 81479,81479
COL2A1 81479,81479
DHODH 81479,81479
DOCK6 81479,81479
DYM 81479,81479
ERCC4 81479,81479
ESCO2 81479,81479
FANCA 81479,81479
FANCD2 81479,81479
FANCE 81479,81479
FANCF 81479,81479
FANCG 81479,81479
FANCI 81479,81479
FANCL 81479,81479
FANCM 81479,81479
FBLN1 81479,81479
FBXW4 81479,81479
FGF10 81479,81479
FGFR2 81479,81479
FGFR3 81479,81479
FIG4 81406,81479
FLNA 81479,81479
FMN1 81479,81479
GDF5 81479,81479
GLI3 81479,81479
GNAS 81479,81479
GREM1 81479,81479
HDAC4 81479,81479
HOXD13 81479,81479
IHH 81479,81479
KIF7 81479,81479
LMBR1 81479,81479
LRP4 81479,81479
MGP 81479,81479
MKS1 81479,81479
MYCN 81479,81479
NIPBL 81479,81479
NOG 81479,81479
NOTCH1 81408,81479
NSDHL 81479,81479
PALB2 81406,81479
PIGV 81479,81479
PITX1 81479,81479
RAD51C 81479,81479
RBM8A 81229,81479
RECQL4 81479,81479
ROR2 81479,81479
RPGRIP1L 81479,81479
SALL1 81479,81479
SALL4 81479,81479
SF3B4 81479,81479
SHH 81479,81479
SLX4 81479,81479
SOX9 81479,81479
TBX15 81479,81479
TBX3 81479,81479
TBX5 81405,81479
THPO 81479,81479
TP63 81479,81479
WNT3 81479,81479
WNT7A 81479,81479
Full Panel Price* $990.00
Test Code Test Copy Genes Total Price CPT Codes Copy CPT Codes
5065 Genes x (69) $990.00 81229, 81405, 81406(x2), 81408(x2), 81479(x132) Add to Order
Pricing Comment

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 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

In one study, pathogenic variants were found in 18% (36/199) of patients with a genetic etiology of Congenital Upper Limb Anomalies. Among them, 13/199 cases had a copy number variation on the chromosomal level, and 23/199 cases were found to have a pathogenic variant involving a single nucleotide substitution, or small deletion/insertion (Carli et al. 2013. PubMed ID: 24343878).

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Clinical Features

Congenital limb malformation refers to both gross reduction defects and more subtle alterations in the number, length, and anatomy of the legs, arms, and digits. The prevalence is ~ 1 in 500 to 1 in 1,000 live births (Wilkie. 2003). Congenital limb malformation includes many conditions such as: preaxial/postaxial polydactyly of the foot/hand (Burger et al. 2017. PubMed ID: 28946786), brachydactyly, and limb hypoplasia-reduction (Bonafe et al. 2015. PubMed ID: 26394607).

Genetics

This panel includes 69 genes associated with a variety of genetic congenital limb malformations (Wilkie. 2003; Bonafe et al. 2015. PubMed ID: 26394607). Genetic limb malformations are genetically heterogenous and can be inherited in an autosomal dominant (AD), autosomal recessive (AR), and X-linked (XL) manner.

This panel offers testing for the following and many other conditions: Brachydactyly, Ectrodactyly, Polydactyly, Syndactyly, Symphalangism, Townes-Brocks branchiootorenal-like syndrome, Duane-radial ray syndrome, Fanconi anemia, Pallister-Hall syndrome, Split-hand/foot malformation, some subtypes of Meckel syndrome, Holt-Oram syndrome, Robinow syndrome, Liebenberg syndrome, TP63-related conditions, Liebenberg syndrome, Keutel Syndrome, Smith McCort Dysplasia, Yunis-Varon Syndrome, Camptomelic Dysplasia, and Feingold syndrome 1.

See individual gene test descriptions for information on molecular biology of gene products, and spectra of pathogenic variants.

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.

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 typically provides >99.7% coverage of all coding exons of the genes listed, plus ~10 bases of flanking noncoding DNA. We define coverage as ≥20X NGS reads or Sanger sequencing.

Some genes have multiple copies in the haploid genome. In these cases, we may only analyze part of these genes. For example, exons 32 to 44 of the TNXB gene are highly homologous to the pseudogene TNXA, therefore, this region cannot be accurately analyzed in this NGS panel.

Other genes without full coverage include, but may not be limited to: CEP290, CHSY1, DOCK6, FMN1, LRP4, NIPBL, NOTCH1, FANCD2, SALL1, SALL4, SHH, SF3B4, FBXW4, SHH, SOX9, RPGRIP1L, and RBM8A. A full list of regions not covered by NGS or Sanger sequencing is available upon request.

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

Patients with limb defects should be considered.

Diseases

Name Inheritance OMIM ID
Acrocallosal Syndrome, Schinzel Type AR 200990
Acrofacial Dysostosis 1, Nager Type AD 154400
Adams-Oliver Syndrome 1 AD 100300
Adams-Oliver Syndrome 2 AR 614219
Adams-Oliver Syndrome 5 AD 616028
Apert Syndrome AD 101200
Aplasia Of Lacrimal And Salivary Glands AD 180920
Brachydactyly Type A1 AD 112500
Brachydactyly Type A2 AD 112600
Brachydactyly Type C AD 113100
Brachydactyly, Type B2 AD 611377
Camptomelic Dysplasia AD 114290
Child Syndrome XL 308050
Cornelia de Lange syndrome 1 AD 122470
Cousin Syndrome AR 260660
Crouzon Syndrome AD 123500
Duane-Radial Ray Syndrome AD 607323
Ectodermal Dysplasia, Ectrodactyly, and Macular Dystrophy AR 225280
Ectrodactyly, Ectodermal Dysplasia, And Cleft Lip/Palate Syndrome 3 AD 604292
Essential Thrombocythemia AD 187950
Fanconi Anemia, Complementation Group A AR 227650
Fanconi Anemia, Complementation Group D1 AR 605724
Fanconi Anemia, Complementation Group D2 AR 227646
Fanconi Anemia, Complementation Group E AR 600901
Fanconi Anemia, Complementation Group F AR 603467
Fanconi Anemia, Complementation Group G AR 614082
Fanconi Anemia, Complementation Group I AR 609053
Fanconi Anemia, Complementation Group J AR 609054
Fanconi Anemia, Complementation Group L AR 614083
Fanconi Anemia, Complementation Group N AR 610832
Fanconi Anemia, Complementation Group O AR 613390
Fanconi Anemia, Complementation Group P AR 613951
Fanconi anemia, Complementation Group Q AR 615272
Feingold Syndrome 1 AD 164280
Holt-Oram Syndrome AD 142900
Hyperphosphatasia With Mental Retardation AR 239300
Joubert Syndrome 7 AR 611560
Joubert Syndrome 9 AR 612285
Keutel Syndrome AR 245150
Kniest Dysplasia AD 156550
Lacrimoauriculodentodigital Syndrome AD 149730
Liebenberg Syndrome AD 186550
Meckel Syndrome 1 AR 249000
Meckel Syndrome 4 AR 611134
Miller Syndrome AR 263750
Oto-Palato-Digital Syndrome Type 1 XL 311300
Pallister-Hall Syndrome AD 146510
Polydactyly, Preaxial II AD 174500
Progressive Osseous Heteroplasia AD 166350
Roberts Syndrome AR 268300
Robinow Syndrome, Autosomal Recessive AR 268310
Rothmund-Thomson Syndrome AR 268400
Saethre-Chotzen Syndrome AD 101400
Single Upper Central Incisor AD 147250
Smith McCort Dysplasia AR 607326
Syndactyly Cenani Lenz Type AR 212780
Synpolydactyly 1 AD 186000
Synpolydactyly 2 AD 608180
Temtamy Preaxial Brachydactyly Syndrome AR 605282
Tetraamelia, Autosomal Recessive AR 273395
Thrombocytopenia-Absent Radius Syndrome AR 274000
Townes-Brocks Syndrome AD 107480
Ulna And Fibula Absence Of With Severe Limb Deficiency AR 276820
Ulnar-Mammary Syndrome AD 181450
Yunis-Varon Syndrome AR 216340

Related Tests

Name
COL2A1-Related Disorders via the COL2A1 Gene
FGFR2-Related Disorders via the FGFR2 Gene
FGFR3-Related Disorders via the FGFR3 Gene
GLI3-Related Disorders via GLI3 Gene Sequencing with CNV Detection
GNAS-Related Disorders via GNAS Gene Sequencing with CNV Detection
NSDHL-Related Disorders via NSDHL Gene Sequencing with CNV Detection
TP63-Related Disorders via TP63 Gene Sequencing with CNV Detection
Achondroplasia via the FGFR3 Gene, Exon 10
Acrocallosal, Fetal Hydrolethalus, and Joubert Syndromes via the KIF7 Gene
Acrofacial Dysostosis 1, Nagar Type via SF3B4 Gene Sequencing with CNV Detection
Adams-Oliver Syndrome via the DOCK6 Gene
Autism Spectrum Disorders and Intellectual Disability (ASD-ID) Comprehensive Sequencing Panel with CNV Detection
Autosomal Recessive Retinitis Pigmentosa Sequencing Panel with CNV Detection
Brachydactyly via the CHSY1 Gene
Breast Cancer - Comprehensive Risk Sequencing Panel with CNV Detection
Breast Cancer - High / Moderate Risk Sequencing Panel with CNV Detection
Breast Cancer - High Risk Sequencing Panel with CNV Detection
Cancer Sequencing Panel with CNV Detection
Charcot Marie Tooth Type 4J via the FIG4 Gene
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Comprehensive Cardiology Sequencing Panel with CNV Detection
Comprehensive Epilepsy and Seizure Sequencing Panel with CNV Detection
Comprehensive Inherited Retinal Dystrophies (includes RPGR ORF15) Sequencing Panel with CNV Detection
Congenital Central Hypoventilation Syndrome (CCHS) via BMP2 Gene Sequencing with CNV Detection
Congenital Fibrosis of Extraocular Muscles (Ocular Motility Disorder) or Strabismus Syndromes Sequencing Panel with CNV Detection
Congenital Hypothyroidism and Thyroid Hormone Resistance Sequencing Panel with CNV Detection
Cornelia de Lange Syndrome via the NIPBL Gene
Disorders of Sex Development and Infertility Sequencing Panel with CNV Detection
Disorders of Sex Development Sequencing Panel with CNV Detection
Duane-Radial Ray Syndrome and Acro-Renal-Ocular Syndrome via SALL4 Gene Sequencing with CNV Detection
Fanconi Anemia via BRCA2/FANCD1 Gene Sequencing with CNV Detection
Fanconi Anemia via BRIP1/FANCJ Gene Sequencing with CNV Detection
Fanconi Anemia via PALB2/FANCN Gene Sequencing with CNV Detection
Fanconi Anemia via RAD51C/FANCO Gene Sequencing with CNV Detection
Fanconi Anemia via the FANCD2 Gene
Fanconi Anemia via the FANCE Gene
Fanconi Anemia via the FANCF Gene
Fanconi Anemia via the FANCI Gene
Fanconi Anemia via the FANCL Gene
Fanconi Anemia via the FANCM Gene
Fanconi Anemia via the SLX4/FANCP Gene
Female Infertility Sequencing Panel with CNV Detection
Focused Inherited Retinal Disorders Sequencing Panel with CNV Detection
GDF5-related Disorders via the GDF5 Gene
Hereditary Breast and Ovarian Cancer - Expanded and Lynch Syndrome Sequencing Panel with CNV Detection
Hereditary Breast and Ovarian Cancer - High Risk and Lynch Syndrome Sequencing Panel with CNV Detection
Hereditary Breast and Ovarian Cancer BRCA1/2 Sequencing Panel with CNV Detection
Hereditary Endometrial Cancer Sequencing Panel with CNV Detection
Hereditary Mixed Polyposis Syndrome via the GREM1 Gene
Hereditary Ovarian Cancer Sequencing Panel with CNV Detection
Hereditary Thrombocythemia via THPO Gene Sequencing with CNV Detection
Holoprosencephaly-3 (Autosomal Dominant Nonsyndromic) via SHH Gene Sequencing with CNV Detection
Holt-Oram Syndrome (HOS) via TBX5 Gene Sequencing with CNV Detection
Hypochondroplasia via the FGFR3 Gene
Hypoparathyroidism Sequencing Panel with CNV Detection
Joubert and Meckel-Gruber Syndromes via the CC2D2A Gene
Joubert and Meckel-Gruber Syndromes via the CEP290 Gene
Joubert and Meckel-Gruber Syndromes via the RPGRIP1L Gene
Leber Congenital Amaurosis 10 (LCA10) via the CEP290 Gene
Leber Congenital Amaurosis Sequencing Panel with CNV Detection
Male Infertility Sequencing Panel with CNV Detection
Meckel-Gruber Syndrome via the MKS1 Gene
Melanoma Sequencing Panel with CNV Detection
Miller Syndrome via the DHODH Gene
Multiple Epiphyseal Dysplasia Sequencing Panel
Neonatal Crisis Sequencing Panel with CNV Detection
Otopalatodigital Spectrum Disorders, Periventricular Nodular Heterotopia and Cardiac Valvular Dystrophy via the FLNA Gene
Pancreatic Cancer Sequencing Panel with CNV Detection
Prostate Cancer Sequencing Panel with CNV Detection
Retinitis Pigmentosa (includes RPGR ORF15) Sequencing Panel with CNV Detection
Roberts Syndrome via the ESCO2 Gene
Robinow Syndrome and Autosomal Recessive Brachydactyly, Type B1 via ROR2 Gene Sequencing with CNV Detection
Rothmund Thomson Syndrome via the RECQL4 Gene
Saethre-Chotzen Syndrome Via the TWIST1 Gene
Skeletal Disorders and Joint Problems Sequencing Panel with CNV Detection
Stargardt Disease (STGD) and Macular Dystrophies Sequencing Panel with CNV Detection
Stickler Syndrome Sequencing Panel
Symphalangism, Proximal, Multiple Synostoses Syndrome, Stapes Ankylosis with Broad Thumb and Toes, Tarsal-Carpal Coalition Syndrome, and Brachydactyly, Type B2 via the NOG Gene
Thanatophoric Dysplasia (TD) via the FGFR3 Gene
Thrombocytopenia Absent Radius (TAR) Syndrome via the RBM8A 1q21.1 Deletion
Thrombocytopenia Absent Radius (TAR) Syndrome via the RBM8A Gene
Thrombocytopenia Absent Radius (TAR) Syndrome via the RBM8A Gene - Sequencing and 1q21.1 Deletion
Townes-Brocks Syndrome via SALL1 Gene Sequencing with CNV Detection
Ulnar-Mammary Syndrome via TBX3 Gene Sequencing with CNV Detection
X-Linked Intellectual Disability Sequencing Panel with CNV Detection
Xeroderma Pigmentosum via the ERCC4 Gene

CONTACTS

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Geneticist
Citations
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TEST METHODS

Exome Sequencing with CNV Detection

Test Procedure

For the PGxome we use Next Generation Sequencing (NGS) technologies to cover the coding regions of targeted genes plus ~10 bases of non-coding DNA flanking each exon. 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). Common benign, likely benign, and low quality variants are filtered from analysis. 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.

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

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 most given transcripts, plus ~10 bp of flanking non-coding DNA for each exon. Unless specifically indicated, test reports contain no information about other portions of the gene, such as regulatory domains, deep intronic regions, uncharacterized alternative exons, chromosomal rearrangements, repeat expansions, epigenetic effects, and mitochondrial genome variants.

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.  

In nearly all cases, our ability to determine the exact copy number change within a targeted region is limited.

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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