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Autoimmune Lymphoproliferative Syndrome/ALPS Sequencing Panel

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

Sequencing

Test Code Test Copy GenesCPT Code Copy CPT Codes
4921 CASP10 81479 Add to Order
CASP8 81479
CTLA4 81479
FADD 81479
FAS 81479
FASLG 81479
ITK 81479
KRAS 81405
LRBA 81479
MAGT1 81479
NRAS 81479
PIK3CD 81479
PRKCD 81479
SH2D1A 81404
STAT3 81479
XIAP 81479
Full Panel Price* $1690.00
Test Code Test Copy Genes Total Price CPT Codes Copy CPT Codes
4921 Genes x (16) $1690.00 81404, 81405, 81479(x14) 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

Germline pathogenic variants in the FAS, FASLG and CASP10 genes account for about 75%, <5% and <5% of Autoimmune Lymphoproliferative Syndrome (ALPS) cases respectively. Somatic FAS variants account for about 15-20% of cases. Currently about 20% of cases of ALPS have an undefined genetic cause (Shah et al., 2014. PubMed ID: 25086580; Bleesing et al., 2017. PubMed ID: 20301287). Clinical sensitivity for the CASP8, FADD, ITK, KRAS, MAGT1, and NRAS genes for ALPS is currently unknown due to limited number of cases reported to date. Around 60% of X-linked Lymphoproliferative syndrome cases are attributed to pathogenic variants in the SH2D1A gene. Large deletions account for approximately 25% of all reported SH2D1A pathogenic variants (Arico et al., 2001. PubMed ID: 11159547).

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Deletion/Duplication Testing via aCGH

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
600 FAS$690.00 81479 Add to Order
KRAS$690.00 81479
NRAS$690.00 81479
SH2D1A$690.00 81403
STAT3$690.00 81479
XIAP$690.00 81479
Full Panel Price* $840.00
Test Code Test Copy Genes Total Price CPT Codes Copy CPT Codes
600 Genes x (6) $840.00 81403, 81479(x5) 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

Gross deletions in the FAS gene have only been reported in a few cases (Magerus-Chatinet et al., 2011. PubMed ID: 21183795; Pensati et al., 1997. PubMed ID: 9322534; van der Werff ten Bosch et al., 1998. PubMed ID: 9695976). Gain of function pathogenic variants in the STAT3, KRAS and NRAS genes have been reported to be causative for ALPS, and deletion/duplication testing has minimal clinical utility. Large deletions account for approximately 25% of all reported SH2D1A pathogenic variants (Arico et al., 2001. PubMed ID: 11159547). Large deletions in XIAP have been reported in Familial Hemophagocytic Lymphohistiocytosis patients, but make up < 10% of all reported XIAP pathogenic variants (Marsh et al., 2009. PubMed ID: 19398375).

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Clinical Features

Autoimmune Lymphoproliferative Syndrome (ALPS) is a disorder characterized by dysregulation of lymphocyte hemostasis resulting in elevation of non-malignant lymphocyte numbers. Affected individuals present with lymphadenopathy, hepatomegaly, and splenomegaly primarily in the first years of life. In the second decade of life, patients often experience autoimmune attacks on red blood cells (hemolytic anemia), neutrophils (neutropenia), or platelets (thrombocytopenia) due to production of autoantibodies. Other symptoms may include arthritis, vasculitis, oral ulcers, skin rash, and heightened risk for development of lymphoma (Bleesing et al., 2017. PubMed ID: 20301287). Adult forms of ALPS have recently been described with some individuals developing life threatening end organ lymphoproliferative disease (Niemela et al., 2011. PubMed ID: 21079152). Patients with ALPS may be treated with immunosuppressive therapies such as corticosteroids to maintain lymphocyte homeostasis (Rao and Oliveira, 2011. PubMed ID: 21885601). There are 5 different subtypes of ALPS distinguished by their underlying genetic cause: type 1A (FAS), type2B (FASLG), type 2A (CASP10), type 2B (CASP8), type 3 (PRKCD), type 4 (NRAS, KRAS), and type 5 (CTLA4). Genetic testing is helpful in identifying the subtype of ALPS as well as in differential diagnosis from other immune disorders. Other related disorders include common variable immunodeficiency disease (CVID), Hyper IgM syndrome, X-linked lymphoproliferative diseases, Wiskott-Aldrich syndrome and FADD deficiency, autoimmune disease multisystem infantile onset type 1/ADMIO1, and activated PI3K-δ syndrome type 1/APDS1 (Bleesing et al., 2017. PubMed ID: 20301287; Sumegi et al., 2000. PubMed ID: 11049992; Li et al., 2014. PubMed ID: 24550228; Linka et al., 2012. PubMed ID: 22289921; Stepensky et al., 2011. PubMed ID: 21109689; Bolze et al., 2010. PubMed ID: 21109225; Chun et al., 2002. PubMed ID: 12353035).

Genetics

ALPS is inherited in an autosomal dominant manner through pathogenic variants in either the FAS, FASLG, or CASP10 genes. Pathogenic variants in the FAS gene primarily occur in the germline for individuals with ALPS (~70% of cases) but can also occur somatically in double negative (CD4-CD8-) a/b T cells (~15% of cases) leading to disease (Neven et al., 2011. PubMed ID: 21885602; Holzelova et al., 2004. PubMed ID: 15459302). Homozygous and compound heterozygous pathogenic variants in the FAS gene have been reported for ALPS with patients having severe disease. Pathogenic variants in the CASP10 or FASLG gene account for <5% and <1% of cases of ALPS respectively (Bleesing et al., 2017. PubMed ID: 20301287). A germline NRAS or somatic KRAS gain of function variant, designated p.Gly13Asp, has been reported in rare cases of ALPS (Oliveira et al., 2007. PubMed ID: 17517660; Niemela et al., 2011. PubMed ID: 21079152; Takagi et al., 2011. PubMed ID: 21063026). Gain of function pathogenic variants within the STAT3 and PIK3CD genes are responsible for ADMIO1 and APDS2 respectively, whereas loss of function pathogenic variants result in immunodeficiency syndromes (Angulo et al., 2013. PubMed ID: 24136356; Lucas et al., 2013. PubMed ID: 24165795; Haapaniemi et al., 2015. PubMed ID: 25349174). Disorders and inheritance patterns of other lymphoproliferative disorders are listed below.

Disorder

Gene

Inheritance

ALPS type 1A

FAS

AD, somatic

ALPS type 1B

FASLG

AD

ALPS type 2A

CASP10

AD

ALPS type 2B

CASP8

AR

ALPS type 3

PRKCD

AR

ALPS type 4

NRAS, KRAS

AD, somatic

ALPS type 5

CTLA4

AD

XLP type 1

SH2D1A

XR

XLP type 2

XIAP

XR

XMEN

MAGT1

XR

LPFS1

ITK

AR

FADD Deficiency

FADD

AR

APDS2

PIK3CD

AD

ADMIO1

STAT3

AD

CVID8

LRBA

AR

Apoptosis is an important process for maintaining lymphocyte homeostasis allowing responses to pathogens but avoiding autoimmune effects. Many of the genes involved in this panel facilitate apoptosis through a Fas-associated mechanism resulting in a cascade of caspase activation.

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.

This panel provides at least 99.5% coverage of all coding exons of the genes listed, plus ~10 bases of flanking noncoding DNA. We define coverage as >20X NGS reads for coding regions and 0-10 bases of flanking DNA, or Sanger sequencing. Only exons 5, 10, 11, 14 and 21-23 will be analyzed for the STAT3 as only gain of function variants in these regions have been reported for patients with ADMIO1.

Genes without complete coverage: STAT3. A 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

Diagnostic criteria for ALPS includes chronic, non-malignant, non-infectious lymphadenopathy, and elevated double negative CD3+ (CD4-CD8-) α/β T cells (>1.5% of total lymphocytes or 2.5% of CD3+ lymphocytes) in the setting of normal lymphocyte counts (Li et al., 2014. PubMed ID: 24550228). Elevated plasma IL-10, positive Coomb’s Test, defective lymphocyte apoptosis, elevated IgA, IgG, and IgM antibody levels are also indicative for ALPS. Complete diagnostic guidelines for ALPS were revised in 2009 (Oliveira et al., 2010. PubMed ID: 20538792). The strongest candidates have a family history for ALPS (Bleesing et al., 2017. PubMed ID: 20301287).

Genes

Official Gene Symbol OMIM ID
CASP10 601762
CASP8 601763
CTLA4 123890
FADD 602457
FAS 134637
FASLG 134638
ITK 186973
KRAS 190070
LRBA 606453
MAGT1 300715
NRAS 164790
PIK3CD 602839
PRKCD 176977
SH2D1A 300490
STAT3 102582
XIAP 300079
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Diseases

Name Inheritance OMIM ID
Autoimmune Disease, Multisystem, Infantile-Onset, 1 AD 615952
Autoimmune Lymphoproliferative Syndrome AD 601859
Autoimmune Lymphoproliferative Syndrome, Type 2 AD 603909
Autoimmune Lymphoproliferative Syndrome, Type III AR 615559
Autoimmune Lymphoproliferative Syndrome, Type V AD 616100
Caspase-8 Deficiency AR 607271
Immunodeficiency 14 AD 615513
Immunodeficiency, Common Variable, 8, with Autoimmunity AR 614700
Immunodeficiency, X-Linked, With Magnesium Defect, Epstein-Barr Virus Infection, And Neoplasia XL 300853
Infections, Recurrent, With Encephalopathy, Hepatic Dysfunction, And Cardiovascular Malformations AR 613759
Lymphoproliferative Syndrome, Ebv-Associated, Autosomal, 1 AR 613011
Lymphoproliferative Syndrome, X-Linked, 1 XL 308240
Lymphoproliferative Syndrome, X-Linked, 2 XL 300635
RAS-Associated Autoimmune Leukoproliferative Disorder AD 614470

Related Tests

Name
KRAS-Related Disorders via the KRAS Gene
Autism Spectrum Disorders and Intellectual Disability (ASD-ID) Comprehensive Panel
Autoimmune Lymphoproliferative Syndrome via the FAS Gene
Autosomal Dominant Hyper IgE Syndrome via the STAT3 Gene
Comprehensive Cardiology Sequencing Panel
Comprehensive Fetal and Neonatal Loss Panel
Familial Hemophagocytic Lymphohistiocytosis (FHL) Sequencing Panel
Familial Hemophagocytic Lymphohistiocytosis, X-linked Lymphoproliferative Disease via the SH2D1A Gene
Familial Hemophagocytic Lymphohistiocytosis, X-linked Lymphoproliferative Disease via the XIAP/BIRC4 Gene
Fetal Concerns Sequencing Panel
Hyper IgE Syndrome Sequencing Panel
Hypertrophic Cardiomyopathy Sequencing Panel
Interstitial Lung Disease Sequencing Panel
Noonan Spectrum Disorders/Rasopathies Sequencing Panel
Noonan Syndrome via the NRAS Gene
Pan Cardiomyopathy Sequencing Panel
Primary Immunodeficiency via the PIK3CD gene
Sudden Cardiac Arrest Sequencing Panel

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Angulo et al., 2013. PubMed ID: 24136356
  • Arico et al., 2001. PubMed ID: 11159547
  • Bleesing et al., 2017. PubMed ID: 20301287
  • Bolze et al., 2010. PubMed ID: 21109225
  • Chun et al., 2002. PubMed ID: 12353035
  • Haapaniemi et al., 2015. PubMed ID: 25349174
  • Holzelova et. al., 2004. PubMed ID: 15459302
  • Li et al., 2014. PubMed ID: 24550228
  • Linka et al., 2012. PubMed ID: 22289921
  • Lucas et al., 2013. PubMed ID: 24165795
  • Magerus-Chatinet et al., 2011. PubMed ID: 21183795
  • Marsh et al., 2009. PubMed ID: 19398375
  • Neven et. al., 2011. PubMed ID: 21885602
  • Niemela et al., 2011. PubMed ID: 21079152
  • Oliveira et al., 2007. PubMed ID: 17517660
  • Oliveira et al., 2010. PubMed ID: 20538792
  • Pensati et al., 1997. PubMed ID: 9322534
  • Rao and Oliveira, 2011. PubMed ID: 21885601
  • Shah et al., 2014. PubMed ID: 25086580
  • Stepensky et al., 2011. PubMed ID: 21109689
  • Sumegi et al., 2000. PubMed ID: 11049992
  • Takagi et al., 2011. PubMed ID: 21063026
  • van der Werff ten Bosch et al., 1998. PubMed ID: 9695976
Order Kits
TEST METHODS

Exome Sequencing

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 using Illumina's Reversible Dye Terminator (RDT) platform NextSeq 500 using 150 by 100 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 NextSeq 500 is converted to fastqs by Illumina Bcl2Fastq 1.8.4, 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.

Analytical Validity
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. Occasionally, a patient may carry an allele which does not capture or 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 tests (including PGxome) are generally not capable of detecting Copy Number Variants (CNVs).

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 relevant to the patient's health 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.

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.

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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