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Complex Hereditary Spastic Paraplegia 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
2677 AMPD2 81479, 81479 Add to Order
AP4B1 81479, 81479
AP4E1 81479, 81479
AP4M1 81479, 81479
AP4S1 81479, 81479
AP5Z1 81479, 81479
ARL6IP1 81479, 81479
ARSI 81479, 81479
ATL1 81406, 81479
B4GALNT1 81479, 81479
BICD2 81479, 81479
BSCL2 81406, 81479
C12orf65 81479, 81479
C19orf12 81479, 81479
CCT5 81479, 81479
CYP2U1 81479, 81479
CYP7B1 81479, 81479
DDHD1 81479, 81479
DDHD2 81479, 81479
ENTPD1 81479, 81479
ERLIN2 81479, 81479
EXOSC3 81479, 81479
FA2H 81479, 81479
FLRT1 81479, 81479
GAD1 81479, 81479
GBA2 81479, 81479
GJC2 81479, 81479
KIF1A 81479, 81479
KIF1C 81479, 81479
KIF5A 81479, 81479
L1CAM 81407, 81479
MAG 81479, 81479
MARS 81479, 81479
NIPA1 81404, 81479
NT5C2 81479, 81479
PGAP1 81479, 81479
PLP1 81405, 81404
PNPLA6 81479, 81479
RAB3GAP2 81479, 81479
REEP1 81405, 81479
SLC16A2 81405, 81404
SPAST 81406, 81405
SPG11 81407, 81479
SPG20 81479, 81479
SPG21 81479, 81479
SPG7 81406, 81405
TECPR2 81479, 81479
TFG 81479, 81479
USP8 81479, 81479
VPS37A 81479, 81479
WASHC5 81407, 81479
WDR48 81479, 81479
ZFYVE26 81479, 81479
Full Panel Price* $2340.00
Test Code Test Copy Genes Total Price CPT Codes Copy CPT Codes
2677 Genes x (53) $2340.00 81404(x3), 81405(x5), 81406(x4), 81407(x3), 81479(x91) Add to Order
Pricing Comment

If you would like to order a subset of these genes contact us to discuss pricing.

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

This multi-gene panel includes almost all the currently known causative genes for complex HSP. It is difficult to estimate the clinical sensitivity of this test due to the lack of data. Based on the frequencies of some causative genes in this panel (Lo Giudice et al. 2014), we predict that this test should detect causative variants in at least 55% of patients with complex HSP phenotype.

At this time, the clinical sensitivity of deletion/duplication testing is difficult to estimate due to the lack of large cohort studies. For PLP1, 14 large deletions and 37 large insertions/duplications have been reported. For each of the three genes (L1CAM, SLC16A2 and SPG7), about 15 deletions or insertions/duplications have been described so far. Copy Number Variants have been rarely found in the other genes of this test (Human Gene Mutation Database).

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Clinical Features

Hereditary spastic paraplegia (HSP) is a group of clinically and genetically diverse diseases, characterized by spasticity (rigid muscles) and lower extremity weakness (Fink 2014; Hensiek et al. 2015). Historically, some HSPs are classified as “complex” if the impairments in the lower limb are also accompanied by other systemic or neurologic abnormalities such as seizures, ataxia, intellectual disability, dementia, and vision and hearing impairment (Fink 2015).

Symptoms of the disease may begin at any age, from birth to late adulthood (Fink 2014). In addition to the age of onset variation, the severity and rate of progression are highly variable among different subtypes of complex HSPs, and even within a specific subtype (Tesson et al. 2015).

Due to the clinically heterogeneous features, complex HSP is sometimes misdiagnosed. Molecular genetic testing is particularly useful in a precise diagnosis of this type of disease. This NGS panel for complex HSP may confirm a clinical diagnosis and identify a causative sequence variant while avoiding costly and time-consuming sequential testing.

Genetics

To date, more than 80 genetic loci and over 60 genes have been shown to be involved in HSP (Tesson et al. 2015). Complex HSP is usually inherited in an autosomal recessive (AR) manner; autosomal dominant (AD) and X-linked (XL) forms are less common (Hensiek et al. 2015). For the inheritance mode of each subtype of complex HSP, see the table below. The genotype-phenotype correlation is poor, and some genes are associated with both complex and pure HSP (Fink 2013). This multi-gene panel contains causative genes solely for complex HSP as well as the ones that can cause both complex and pure HSP.

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. 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 provides full coverage of all coding exons of the genes listed, plus ~10 bases of flanking noncoding DNA. We define full 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

Individuals with symptoms consistent with complex spastic paraplegia are candidates for this test. Because this complex HSP NGS panel covers genes with dominant, recessive, and X-linked inheritance patterns, this test may be particularly useful if the inheritance pattern is unclear based on the patient’s family history.

Diseases

Name Inheritance OMIM ID
Allan-Herndon-Dudley Syndrome XL 300523
Cerebral Palsy, Spastic Quadriplegic, 1 AR 603513
Mast Syndrome AR 248900
Neuropathy, Hereditary Sensory, With Spastic Paraplegia AR 256840
Spastic Paraplegia 1 XL 303350
Spastic Paraplegia 10 AD 604187
Spastic Paraplegia 11 AR 604360
Spastic Paraplegia 15 AR 270700
Spastic Paraplegia 17 AD 270685
Spastic Paraplegia 18 AR 611225
Spastic Paraplegia 2 XL 312920
Spastic Paraplegia 26 AR 609195
Spastic Paraplegia 28 AR 609340
Spastic Paraplegia 3 AD 182600
Spastic Paraplegia 30 AR 610357
Spastic Paraplegia 31 AD 610250
Spastic paraplegia 35 AR 612319
Spastic Paraplegia 39 AR 612020
Spastic Paraplegia 4 AD 182601
Spastic Paraplegia 43 AR 615043
Spastic Paraplegia 44 AR 613206
Spastic Paraplegia 45 AR 613162
Spastic Paraplegia 46 AR 614409
Spastic Paraplegia 47 AR 614066
Spastic Paraplegia 48 AR 613647
Spastic Paraplegia 49 AR 615031
Spastic Paraplegia 50 AR 612936
Spastic Paraplegia 51 AR 613744
Spastic Paraplegia 52 AR 614067
Spastic Paraplegia 53 AR 614898
Spastic Paraplegia 54 AR 615033
Spastic Paraplegia 55 AR 615035
Spastic Paraplegia 56 AR 615030
Spastic Paraplegia 57 AR 615658
Spastic Paraplegia 5A AR 270800
Spastic Paraplegia 6 AD 600363
Spastic Paraplegia 61 AR 615685
Spastic Paraplegia 63 AR 615686
Spastic Paraplegia 64 AR 615683
Spastic Paraplegia 7 AD,AR 607259
Spastic Paraplegia 75 AR 616680
Spastic Paraplegia 8 AD 603563
Troyer Syndrome AR 275900

Related Tests

Name
AMPD2-Related Disorders via AMPD2 Gene Sequencing with CNV Detection
C12orf65-Associated Optic Atrophy via the C12orf65 Gene
KIF1A-Related Disorders via the KIF1A Gene
PLP1-Related Disorders via the PLP1 Gene
REEP1-Related Disorders via the REEP1 Gene
TFG-Related Disorders via the TFG Gene
Allan-Herndon-Dudley Syndrome or Monocarboxylate Transporter 8 Deficiency via the SLC16A2 Gene
Autism Spectrum Disorders and Intellectual Disability (ASD-ID) Comprehensive Sequencing Panel with CNV Detection
Charcot Marie Tooth - Axonal Neuropathy Sequencing Panel
Charcot Marie Tooth - Comprehensive Sequencing Panel
Charcot Marie Tooth Type 2U via the MARS Gene
Comprehensive Inherited Retinal Dystrophies (includes RPGR ORF15) Sequencing Panel with CNV Detection
Comprehensive Neuromuscular Sequencing Panel
Comprehensive Neuropathy Sequencing Panel
Congenital Generalized Lipodystrophy (CGL) Sequencing Panel
Congenital Hypothyroidism and Thyroid Hormone Resistance Sequencing Panel
Congenital Myopathy Sequencing Panel
Distal Hereditary Motor Neuropathy Sequencing Panel
Hereditary Lymphedema via the GJC2 Gene
Hereditary Sensory and Autonomic Neuropathy Sequencing Panel
Hereditary Sensory Neuropathy with Spastic Paraplegia via the CCT5 Gene
Hereditary Spastic Paraplegia Comprehensive Sequencing Panel with CNV Detection
L1 Syndrome via the L1CAM Gene
Lymphedema Sequencing Panel
Mitochondrial Genome Maintenance/Integrity Nuclear Genes Sequencing Panel
Mitochondrial Membrane Protein-Associated Neurodegeneration via the C19orf12 Gene
Neurodegeneration with Brain Iron Accumulation and Infantile Neuroaxonal Dystrophy Sequencing Panel with CNV Detection
Non-syndromic Intellectual Disability (NS-ID) Sequencing Panel with CNV Detection
Optic Atrophy and Hereditary Spastic Paraplegia via the SPG7 Gene
Optic Atrophy Sequencing Panel
Pontocerebellar Hypoplasia Type 1B via the EXOSC3 Gene
Pure Hereditary Spastic Paraplegia Sequencing Panel with CNV Detection
Seipin-Related Disorders via the BSCL2 Gene
Spastic Paraplegia 10 via KIF5A Gene Sequencing with CNV Detection
Spastic Paraplegia 11 via SPG11 Gene Sequencing with CNV Detection
Spastic Paraplegia 15 via ZFYVE26 Gene Sequencing with CNV Detection
Spastic Paraplegia 18 via ERLIN2 Gene Sequencing with CNV Detection
Spastic Paraplegia 20 (Troyer Syndrome) via SPG20 Gene Sequencing with CNV Detection
Spastic Paraplegia 21 (Mast Syndrome) via the SPG21(ACP33) Gene
Spastic Paraplegia 26 via B4GALNT1 Gene Sequencing with CNV Detection
Spastic Paraplegia 28 via the DDHD1 Gene
Spastic Paraplegia 35 via the FA2H Gene
Spastic Paraplegia 3A via the ATL1 Gene
Spastic Paraplegia 4 via the SPAST Gene
Spastic Paraplegia 46 via GBA2 Gene Sequencing with CNV Detection
Spastic Paraplegia 47 via AP4B1 Gene Sequencing with CNV Detection
Spastic Paraplegia 48 via AP5Z1 Gene Sequencing with CNV Detection
Spastic Paraplegia 49 via TECPR2 Gene Sequencing with CNV Detection
Spastic Paraplegia 50 via AP4M1 Gene Sequencing with CNV Detection
Spastic Paraplegia 51 via AP4E1 Gene Sequencing with CNV Detection
Spastic Paraplegia 52 via the AP4S1 Gene
Spastic Paraplegia 53 via the VPS37A Gene
Spastic Paraplegia 54 via DDHD2 Gene Sequencing with CNV Detection
Spastic Paraplegia 56 via the CYP2U1 Gene
Spastic Paraplegia 58 via the KIF1C Gene
Spastic Paraplegia 59 via the USP8 Gene
Spastic Paraplegia 5A via the CYP7B1 Gene
Spastic Paraplegia 6 via the NIPA1 Gene
Spastic Paraplegia 61 via the ARL6IP1 Gene
Spastic Paraplegia 75 via the MAG Gene
Spastic Paraplegia 8 via the WASHC5/KIAA0196 Gene
Warburg Micro Syndrome Sequencing Panel
X-Linked Intellectual Disability Sequencing Panel with CNV Detection

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Fink J.K. 2014. Seminars in Neurology. 34: 293-305. PubMed ID: 25192507
  • Fink J.K. 2015. Hereditary Spastic Paraplegia Overview. In: Pagon RA, Adam MP, Bird TD, Dolan CR, Fong C-T, Smith RJ, and Stephens K, editors. GeneReviews™, Seattle (WA): University of Washington, Seattle. PubMed ID: 20301682
  • Hensiek A. et al. 2015. Journal of Neurology. 262: 1601-12. PubMed ID: 25480570
  • Human Gene Mutation Database (HGMD).
  • Lo Giudice T. et al. 2014. Experimental Neurology. 261: 518-39. PubMed ID: 24954637
  • Tesson C. et al. 2015. Human Genetics. 134: 511-38. PubMed ID: 25758904
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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 close to 100%, 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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