Forms

Familial Amyloidosis via the APOA1 Gene

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

Sequencing

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
1403 APOA1$580.00 81479 Add to Order
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 18 days.

Clinical Sensitivity

Hereditary or familial amyloidosis occurs due to mutations in several genes. While mutations in the transthyretin (TTR) gene are the most common, accounting for ~90% of hereditary (familial) amyloidosis, about 20 mutations have been identified in the APOA1 gene in individuals with amyloidosis. Our full gene sequencing test is expected to detect >99% of APOA1 causative mutations.

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Deletion/Duplication Testing via aCGH

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
600 APOA1$690.00 81479 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 Features

Amyloidosis is characterized by abnormal deposition of insoluble beta-pleated sheet aggregates (amyloid) of specific plasma proteins, resulting in disruption of organ and tissue function. Deposition can be localized or systemic, be restricted to a single organ or involve multiple organs respectively. The kidney is frequently affected. Clinically, the presence of proteinuria, renal insufficiency, heart failure, orthostatic hypertension, peripheral neuropathy or unexplained kidney, heart or systemic disease are suspicious for amyloidosis (Picken 2010). Presentation includes a spectrum of symptoms varying from asymptomatic to fatigue, extremity edema, angina or syncope, which is associated with more advanced disease (Leung et al 2012).

Hereditary apolipoprotein A-I mediated amyloidosis is heterogeneous with some patients developing extensive visceral amyloid deposits (liver, spleen, and kidneys, with occasional involvement of the heart, nerves, larynx, and gastrointestinal tract) and end-stage renal failure as young adults, while others have only laryngeal and/or skin amyloid deposits, which may be of little clinical consequence (Rowczenio et al. 2011). Although the condition may lead to end-stage renal disease and other organ failure, the natural history of the disease is often slow. The substantial phenotypic heterogeneity among patients with identical APOAI variants implies that other genetic and environmental factors influence clinical manifestations and supports the need for molecular testing in patients with apparent localized amyloidosis, particularly involving the larynx or skin.

Genetics

The majority of amyloidosis is somatic in nature, involving deposition of immunoglobulin light chain (AL) as in myeloma or monoclonal gamopathies, or serum amyloid protein (AA) in chronic inflammation. However, a substantial minority of cases are due to inherited changes in amylogenic proteins. (Picken 2010).

Hereditary or familial amyloidosis is a rare autosomal dominant condition that occurs due to heterozygous mutations in several genes including APOA1. APOA1 encodes apolipoprotein A-I (apoA-I) which is a component of high-density lipoprotein (HDL). HDL transports cholesterol and certain fats called phospholipids through the bloodstream from the body tissues to the liver. HDL is often referred to as "good cholesterol" because high levels of this substance reduce the chances of developing heart and blood vessel (cardiovascular) disease. The kidney and liver are the major sites of apo A-I catabolism, which is why nephropathy is mostly observed in the presence of APOA1 variants.

About 45 APOA1 variants (missense, nonsense and inframe deletions) have been described to be pathogenic (Joy et al. 2003; Rowczenio et al. 2011). Variants in APOA1 are associated with high-density lipoprotein (HDL) deficiency and familial visceral amyloidosis (both neuropathic and non-neuropathic forms). These phenotypically distinct groups of mutations are uniquely localized in different regions of the apoprotein sequence (Sorci-Thomas and Thomas 2002). No homozygote for an amyloidogenic apo A-I variant has yet been described, suggesting possible lethality.

Testing Strategy

This test involves bidirectional Sanger sequencing using genomic DNA of all coding exons of the APOA1 gene plus ~20 bp of flanking non-coding DNA on each side. We will also sequence any single exon (Test #100) in family members of patients with a known mutation or to confirm research results.

Indications for Test

Molecular genetic testing for APOA1 mediated amyloidosis should be considered in individuals with any of the following findings: biopsied tissues confirm amyloid deposition by Congo red staining which demonstrates a characteristic apple-green birefringence under polarized light, no pathogenic variant identified in the transthyretin gene (accounts for 90% of all familial amyloidosis), or amyloid protein typing using either using immunohistochemical staining of an affected tissue biopsy or liquid chromatography and tandem mass spectrometry (LC/MS/MS) of tryptic digests of micro-dissected amyloid plaques (Rowczenio et al. 2011).

Gene

Official Gene Symbol OMIM ID
APOA1 107680
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Human Gene Mutation Database (Bio-base).
  • Joy T, Wang J, Hahn A, Hegele RA. 2003. Apoa1 related amyloidosis: a case report and literature review. Clinical Biochemistry 36: 641–645. PubMed ID: 14636880
  • Leung N, Nasr SH, Sethi S. 2012. How I treat amyloidosis: the importance of accurate diagnosis and amyloid typing. Blood 120: 3206–3213. PubMed ID: 22948045
  • Picken MM. 2010. Amyloidosis-where are we now and where are we heading? Archives of pathology & laboratory medicine 134: 545–551. PubMed ID: 20367306
  • Rowczenio D, Dogan A, Theis JD, Vrana JA, Lachmann HJ, Wechalekar AD, Gilbertson JA, Hunt T, Gibbs SDJ, Sattianayagam PT, Pinney JH, Hawkins PN, et al. 2011. Amyloidogenicity and Clinical Phenotype Associated with Five Novel Mutations in Apolipoprotein A-I. The American Journal of Pathology 179: 1978–1987. PubMed ID: 21820994
  • Sorci-Thomas MG, Thomas MJ. 2002. The effects of altered apolipoprotein AI structure on plasma HDL concentration. Trends in cardiovascular medicine 12: 121–128. PubMed ID: 12007737
Order Kits
TEST METHODS

Bi-Directional Sanger Sequencing

Test Procedure

Nomenclature for sequence variants was from the Human Genome Variation Society (http://www.hgvs.org).  As required, DNA is extracted from the patient specimen.  PCR is used to amplify the indicated exons plus additional flanking non-coding sequence.  After cleaning of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit.  Products are resolved by electrophoresis on an ABI 3730xl capillary sequencer.  In most cases, sequencing is performed in both forward and reverse directions; in some cases, sequencing is performed twice in either the forward or reverse directions.  In nearly all cases, the full coding region of each exon as well as 20 bases of non-coding DNA flanking the exon are sequenced.

Analytical Validity

As of March 2016, we compared 17.37 Mb of Sanger DNA sequence generated at PreventionGenetics to NextGen sequence generated in other labs. We detected only 4 errors in our Sanger sequences, and these were all due to allele dropout during PCR. For Proficiency Testing, both external and internal, in the 12 years of our lab operation we have Sanger sequenced roughly 8,800 PCR amplicons. Only one error has been identified, and this was due to sequence analysis error.

Our Sanger sequencing is capable of detecting virtually all nucleotide substitutions within the PCR amplicons. Similarly, we detect essentially all heterozygous or homozygous deletions within the amplicons. Homozygous deletions which overlap one or more PCR primer annealing sites are detectable as PCR failure. Heterozygous deletions which overlap one or more PCR primer annealing sites are usually not detected (see Analytical Limitations). All heterozygous insertions within the amplicons up to about 100 nucleotides in length appear to be detectable. Larger heterozygous insertions may not be detected. All homozygous insertions within the amplicons up to about 300 nucleotides in length appear to be detectable. Larger homozygous insertions may masquerade as homozygous deletions (PCR failure).

Analytical Limitations

In exons where our sequencing did not reveal any variation between the two alleles, we cannot be certain that we were able to PCR amplify both of the patient’s alleles. Occasionally, a patient may carry an allele which does not amplify, due for example to a deletion or a large insertion. In these cases, the report contains no information about the second allele.

Similarly, our sequencing tests have almost no power to detect duplications, triplications, etc. of the gene sequences.

In most cases, only the indicated exons and roughly 20 bp of flanking non-coding sequence on each side are analyzed. Test reports contain little or no information about other portions of the gene, including many regulatory regions.

In nearly all 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 for example 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 and cycle sequencing.

Unless otherwise indicated, the sequence data that we report are based on DNA isolated from a specific tissue (usually leukocytes). Test reports contain no information about gene sequences in other tissues.

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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