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Amyotrophic Lateral Sclerosis and Frontotemporal Dementia Sequencing Panel

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

Sequencing

Test Code TestCPT Code Copy CPT Codes
1965 ANG 81403 Add to Order
ARHGEF28 81479
C9orf72 81479
CDH13 81479
CHMP2B 81479
FUS 81406
GRN 81406
HNRNPA1 81479
HNRNPA2B1 81479
MAPT 81406
OPTN 81406
PFN1 81479
PSEN1 81405
PSEN2 81406
SOD1 81404
SQSTM1 81479
TARDBP 81405
TREM2 81479
UBQLN2 81479
VAPB 81479
VCP 81479
Full Panel Price* $1540.00
Test Code Test Total Price CPT Codes Copy CPT Codes
1965 Genes x (21) $1540.00 81403, 81404, 81405(x2), 81406(x5), 81479(x12) Add to Order
Pricing Comment

$1540 for full panel.

$250 for C9orf72 only. 

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

This NGS panel will detect pathogenic variants in at least 68% of patients with familial ALS and 11% of apparently sporadic cases of ALS (Renton et al. 2014). This panel will also detect pathogenic variants in up to 65% of patients with familial FTD (see Table, below). The following Table indicates sensitivity by gene and by phenotype.

Gene                 ALS            Reference         FTD                Reference

C9ORF72          40%           1                           25%                2

SOD1                 20%           1                           NA                  NA

TARDBP            1-5%          1                          One case       3

FUS                    1-5%          1                          One case      4

ANG                  <1%            1                           NA                 NA

OPTN               <1%            1                           NA                 NA

CHMP2B          4 cases     HGMD                  5 cases         HGMD

VCP                  <1%           1                            <1%              1

VAPB                <1%           1                            NA                 NA

UBQLN2          1.2%          5                            2.2%                5

PFN1                1 - 2 %       6                           NA                 NA

SQSTM1           3%             7                           3%                7

ARHGEF28       3 cases     8, 9                       NA                NA

CDH13              2 cases*   10, 11                  NA                NA

GRN                  One Case   12                     >20%            13

HNRNPA1       2 Families  14                       NA                 NA

HNRNPA2B      One case * 10                     NA                 NA

MAPT                 NA           NA                        Up to 20%     15

PSEN1               2 cases *  15                       10 cases       HGMD

PSEN2               NA            NA                        One case *   16

TREM2               NA            NA                        2 cases *      17, 18

 

Sensitivity corresponds to the percentage of all genotyped patients with a clinical diagnosis and a positive family history of either ALS or FTD.  For genes with rare pathogenic variants, the numbers of reported simplex cases are listed.  Lack of conclusive evidence for pathogenicity is indicated by an asterisk.

REFERENCES

1: Robberecht and Philips, 2013

2: Majounie, 2012

3: Borroni, 2010

4: Langenhove T van, 2012

5: Synofzik, 2012

6: Wu, 2012

7: Rubino, 2012

8: Droppelmann, 2013

9: Ma Y, 2014

10: Couthouis, 2014

11: Daoud, 2011

12: Sleegers, 2008

13: Baker, 2006

14: Kim, 2013

15: Rademakers, 2012

16: Ferrari, 2012

17. Thelen, 2014

18. Borroni, 2014

HGMD: Human Gene Mutation Database

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Deletion/Duplication Testing via aCGH

Test Code TestIndividual Gene PriceCPT Code Copy CPT Codes
600 ANG$690.00 81479 Add to Order
FUS$690.00 81479
OPTN$690.00 81479
PSEN1$690.00 81479
PSEN2$690.00 81479
SOD1$690.00 81479
SQSTM1$690.00 81479
TARDBP$690.00 81479
UBQLN2$690.00 81479
VAPB$690.00 81479
VCP$690.00 81479
Full Panel Price* $1290.00
Test Code Test Total Price CPT Codes Copy CPT Codes
600 Genes x (11) $1290.00 81479(x11) 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 pathogenic deletions appear to be rare. They were reported only in four genes: OPTN, GRN, MAPT, PSEN1 (Iida et al. 2012; Maruyama et al. 2010; Pickering-Brown et al. 2006; Rohrer et al. 2013; Rovelet-Lecrux et al. 2009; Evin et al. 2002).

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Clinical Features

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by a selective loss of motor neurons in the motor cortex, brain stem, and spinal cord (Tandan et al. 1985). The dysfunction and loss of these neurons results in rapid progressive muscle weakness, atrophy and ultimately paralysis of limb, bulbar and respiratory muscles. The mean age of onset of symptoms is about 55 years of age; most cases begin between 40 and 70 years of age. The annual incidence of ALS is 1-2 per 100,000 (Cleveland and Rothstein  2001).

The most common symptoms include twitching and cramping of muscles of the hands and feet, loss of motor control in the hands and arms, weakness and fatigue, tripping and falling. Symptoms usually begin with asymmetric involvement of the muscles. As the disease progresses, symptoms may include difficulty in talking, breathing and swallowing, shortness of breath, and paralysis.

Frontotemporal dementia (FTD), previously referred to as Pick’s disease, is a clinically heterogeneous syndrome due to the progressive degeneration and atrophy of various regions of the frontal and temporal lobes of the brain. Symptoms are insidious and begin usually during the fourth and sixth decades of life; although earlier and later onsets have been documented (Neary et al. 1998; Snowden et al. 2002; Bruni et al. 2007).  The annual incidence of FTD is 3-4 per 100,000 (Onyike and Diehl-Schmid 2013).

Two major forms, the behavioral-variant (FTD-bv) and the primary progressive aphasia (PPA), are recognized based on the site of onset of degeneration and the associated symptoms.

In FTD-bv the degenerative process begins in the frontal lobes and results in personality changes and deterioration of social conducts. Most common behavioral changes are: disinhibition, apathy, deterioration of executive function, obsessive thoughts, compulsive behavior, and neglect of personal hygiene (Rascovsky et al. 2011).

In PPA the degenerative process begins in the temporal lobes. PPA is a language disorder that is further divided into two sub-forms: progressive non-fluent aphasia (PNFA) and semantic dementia (SD). PNFA is characterized by difficulty in verbal communications, word retrieval, and speech distortion. Reading, writing and spelling are also affected; while memory is relatively preserved. SD is characterized by the progressive impairment of word comprehension, object and face recognition, and loss of semantic memory. Reading and writing skills are relatively preserved (Gustafson et al. 1993).

Cognitive impairment was not initially associated with ALS. However, frontotemporal dementia (FTD) has been reported in several cases. Dementia has been documented in patients with ALS from different ethnic groups and affects both males and females (Wikström et al. 1982; Lipton et al. 2004; Mitsuyama and Inoue, 2009).

A more recent prospective study showed that FTD occurred in up to 14% of patients with ALS.  Furthermore, cognitive impairment was detected in more than 40% of patients (Phukan et al. 2012).

Definite ALS has been reported in patients with a clinical diagnosis of FTD (Lomen-Hoerth et al. 2002).

In addition to pure ALS and pure FTD, a combination of ALS and FTD clinical features have been reported in both sporadic and familial cases (Morita et al. 2006; Ferrari et al. 2011).

Genetics

About 10% of ALS cases are familial (Emery and Holloway 1982). In most of these families, ALS is inherited in an autosomal dominant manner (AD-ALS) and is age-dependent with high penetrance. In rare families, the disease is transmitted in an autosomal recessive or dominant X-linked pattern.

About 90% of patients with ALS are sporadic cases (SALS) with no known affected relatives. It is unclear how many of the apparently sporadic cases are inherited with low penetrance. The clinical presentations of familial ALS (FALS) and sporadic ALS (SALS) are similar. However, the onset of symptoms in FALS is usually earlier compared to that of SALS (Kinsley and Siddique 2015).

Autosomal Dominant ALS (AD-ALS) is a clinically and genetically heterogeneous disorder that affects all ethnic groups. The following genes have been implicated in the disease: ANG, ARHGEF28, C9orf72, CDH3, CHMP2B, FUS, GRN, HNRNPA1, HNRNP2B1, OPTN, PFN1, SOD1, SQSTM1, VAPB, VCP, PSEN1,  TARDBP, and UBQLN2.

FTD is inherited in about 40% of cases (Rosso et al. 2003). In these families, the disease is inherited in an autosomal dominant manner. The remaining cases appear to be simplex with no known affected relatives. Similar to ALS, it is unclear how many of the apparently sporadic cases of FTD are inherited with low penetrance (Cruts et al. 2006; Le Ber et al. 2007).

FTD is genetically heterogeneous. Several genes have been implicated in the disorder: C9orf72, CHMP2B, FUS, GRN, SQSTM1, MAPT, PSEN1, PSEN2, TARDBP, TREM2 and UBQLN2.

The C9ORF72, GRN, and SQSTM1 genes have been implicated in patients with a combination of ALS and FTD  (ALS-FTD).

Overall, the vast majority of pathogenic variants are missense. Few truncating variants were reported in SOD1, FUS, OPTN, CHMP2B, SQSTM1, ARHGEF28, GRN, PSEN1, PSEN2 and TREM2. Large pathogenic deletions appear to be rare. They were reported only in four genes: OPTN, GRN, MAPT and PSEN1 (Iida et al. 2012; Maruyama et al. 2010; Pickering-Brown et al. 2006; Rohrer et al. 2013; Rovelet-Lecrux et al. 2009; Evin et al. 2002).

With few exceptions, pathogenic variants in the genes included in this panel are inherited with an autosomal dominant manner or occurred de novo. The exceptions are:

One single variant, p.Asp90Ala, in SOD1 was reported at the homozygous state in patients with ALS (Andersen et al. 1995).

Recessive variants in OPTN were reported in Japanese cases (Maruyama et al. 2010; Iida et al. 2012).

UBQLN2-Related ALS and FTD are inherited in an X-linked dominant manner with reduced penetrance in females. The age of onset appears to be earlier in males with no difference in the duration of the disease (Deng et al.  2011).

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

Testing Strategy

Because a pathogenic expansion of the GGGGCC hexanucleotide repeat in a non-coding region of C9orf72 has been reported as the most common genetic cause of ALS, FTD, and ALS-FTD (Renton et al. 2011; DeJesus-Hernandez et al. 2011; Byrne et al. 2012), we will first screen the patients’ DNA for the presence or absence of this expansion. When we find a pathogenic expansion, we stop testing. When there is no evidence for the pathogenic expansion, we sequence all coding exons of the 20 genes listed above using Next Generation Sequencing (NGS).

The repeat-primed PCR test is used as a screening method for the presence or absence of a pathogenic GGGGCC hexanucleotide repeat expansion located in the first intron of C9orf72. Of note, this test is not designed to determine the number of GGGGCC repeats in alleles carrying the pathogenic expansion (Warner et al. 1996; Renton et al. 2011).

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

Patients with symptoms suggestive of ALS, FTD and ALS-FTD.

Genes

Official Gene Symbol OMIM ID
ANG 105850
ARHGEF28 612790
C9orf72 614260
CDH13 601364
CHMP2B 609512
FUS 137070
GRN 138945
HNRNPA1 164017
HNRNPA2B1 600124
MAPT 157140
OPTN 602432
PFN1 176610
PSEN1 104311
PSEN2 600759
SOD1 147450
SQSTM1 601530
TARDBP 605078
TREM2 605086
UBQLN2 300264
VAPB 605704
VCP 601023
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Diseases

Name Inheritance OMIM ID
Amyotrophic Lateral Sclerosis Type 1 105400
Amyotrophic Lateral Sclerosis Type 10 612069
Amyotrophic Lateral Sclerosis Type 12 613435
Amyotrophic Lateral Sclerosis Type 14 613954
Amyotrophic Lateral Sclerosis Type 15 300857
Amyotrophic Lateral Sclerosis Type 17 614696
Amyotrophic Lateral Sclerosis Type 18 614808
Amyotrophic Lateral Sclerosis Type 20 615426
Amyotrophic Lateral Sclerosis Type 6 608030
Amyotrophic Lateral Sclerosis Type 8 608627
Amyotrophic Lateral Sclerosis Type 9 611895
CHMP2B-Related Frontotemporal Dementia 600795
Frontotemporal Dementia 600274
Frontotemporal Dementia And/Or Amyotrophic Lateral Sclerosis 105550
Frontotemporal Dementia and/or Amyotrophic Lateral Sclerosis 3 616437
Frontotemporal Dementia, Ubiquitin-Positive 607485
Inclusion Body Myopathy with Early-Onset Paget Disease with or without Frontotemporal Dementia 2 615422

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CONTACTS

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Citations
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Order Kits
TEST METHODS

NextGen Sequencing

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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