Test ID: TTRX
Amyloidosis, Transthyretin-Associated Familial, Reflex, Blood

Diagnosis of adult individuals suspected of having transthyretin-associated familial amyloidosis

Mass spectrometry to evaluate transthyretin (TTR) protein structure is performed first. In all cases demonstrating a structural change, the TTR gene will be further analyzed by DNA sequence analysis. If no alterations are detected, subsequent gene analysis will not be performed unless a request for AMYL/83667 Amyloidosis, Transthyretin-Associated Familial, DNA Sequence, Blood is submitted by the ordering physician or client.

If familial amyloidosis by liquid chromatography-mass spectrometry is abnormal, DNA sequence will be performed and charged separately.

See Amyloidosis (Familial) Test Algorithm in Special Instructions.

• Informed Consent for Genetic Testing
• Amyloidosis (Familial) Test Algorithm

Liquid Chromatography-Mass Spectrometry (LC-MS)

Specimen must arrive within 96 hours of draw.

Container/Tube:

Preferred: Lavender top (EDTA)

Acceptable: ACD

Specimen Volume: 3 mL

Collection Instructions:

1. Invert several times to mix blood. 

2. Send specimen in original tube.

Forms:

1.   1. New York Clients-Informed consent is required. Please document on the request form or electronic order that a copy is on file. An Informed Consent for Genetic Testing (Supply T576) is available in Special Instructions.

2.   2. If not ordering electronically, submit a Biochemical Genetics Request Form (Supply T439) with the specimen.

The systemic amyloidoses are a group of diseases that result from the abnormal deposition of amyloid in various tissues of the body. They have been classified into 3 major types: primary, secondary, and hereditary. The most common form of amyloidosis (AL) is a disease of the bone marrow called primary systemic AL (immunoglobulin light chain). Secondary AL usually occurs in tandem with chronic infectious or inflammatory diseases, such as rheumatoid arthritis, tuberculosis, or osteomyelitis. Familial or hereditary AL is the least common form. Determining the specific type of AL is imperative in order to provide both an accurate prognosis and appropriate therapies.

Familial or hereditary transthyretin AL is an autosomal dominant disorder caused by mutations in the transthyretin gene (TTR). The resulting amino acid substitutions lead to a relatively unstable, amyloidogenic transthyretin (TTR) protein. Most individuals begin to exhibit clinical symptoms between the third and seventh decades of life. TTR-associated AL is progressive over a course of 5 to 15 years and usually ends in death from cardiac or renal failure or malnutrition. Affected individuals may present with a variety of symptoms including peripheral neuropathy, blindness, cardiomyopathy, nephropathy, autonomic nervous dysfunction, and bowel dysfunction.

More than 90 mutations that cause TTR-associated familial AL have now been identified within the TTR gene. Most of the mutations described to date are single base pair changes that result in an amino acid substitution. Some of these mutations correlate with the clinical presentation of AL.

The Division of Laboratory Genetics recommends a testing strategy that includes both protein analysis by mass spectrometry (MS) and TTR gene analysis by DNA sequencing (AMYL/83667 Amyloidosis, Transthyretin-Associated Familial, DNA Sequence, Blood) for patients in whom TTR-associated familial AL is suspected. The structure of TTR protein in plasma is first determined by MS. Only the transthyretin (also known as prealbumin) is analyzed for amino acid substitutions. Other proteins known to be involved in other less common forms of familial amyloidosis are not examined. If no alterations are detected, gene analysis will not be performed unless requested by the provider (ie, when the diagnosis is still strongly suspected; to rule-out the possibility of a false-negative by MS). In all cases demonstrating a structural change by MS, the entire TTR gene will be analyzed by DNA sequence analysis to identify and characterize the observed alteration (gene mutation or benign polymorphism).

For predictive testing in cases where a familial mutation is known, testing for the specific mutation by DNA sequence analysis (AMYKM/83705 Amyloidosis, Transthyretin-Associated Familial, Known Mutation) is recommended. These assays do not detect mutations associated with non-TTR forms of familial AL. Therefore, it is important to first test an affected family member to determine if TTR is involved and to document a specific mutation in the family before testing at risk individuals.

An interpretive report will be provided.

The presence of a structural change in transthyretin (TTR) is suggestive of a gene mutation that requires confirmation by DNA sequence analysis. A negative result by mass spectrometry does not rule-out a TTR mutation. Mass spectrometric (MS) results are falsely negative if the amino acid substitution does not produce a measurable mass shift for the mutation transthyretin. Approximately 90% of the TTR mutations are positive by MS (see Cautions).

After identification of the mutation at the DNA level, predictive testing for at-risk family members can be performed by molecular analysis (AMYKM/83705 Amyloidosis, Transthyretin-Associated Familial, Known Mutation).

There are 3 circumstances where testing by mass spectrometry will not identify amyloid-causing mutations:

-If the amino acid change results in a protein different by <10 atomic mass units (amu), the mutation will not be reliably detected.

-If an amino acid change results from a frequent nondisease-causing mutation (+30 amu). Since over 12% of the population has this innocuous polymorphism, it is an instance in which molecular testing must be done.

-Coinheritance of the polymorphism with a -30 amu mutation would also result in a transthyretin mass indistinguishable from normal.

Mass spectrometry was performed on 107 control specimens along with 18 specimens with known TTR mutations. Of the 18 mutants detected, 11 corresponded to a common benign polymorphism and 7 corresponded to a pathogenic mutation.

1. Benson MD: The hereditary amyloidoses. Best Pract Res Clin Rheumatol 2003;17:909-927

2. Shimizu A, Nakanishi T, Kishikawa M, et al: Detection and identification of protein variants and adducts in blood and tissues: an application of soft ionization mass spectrometry to clinical diagnosis. J Chromatogr B Analyt Technol Biomed Life Sci 2002 Aug 25;776(1):15-30

3. Lim A, Prokaeva T, McComb ME, et al: Characterization of transthyretin variants in familial transthyretin amyloidosis by mass spectrometric peptide mapping and DNA sequence analysis. Anal Chem 2002 Feb 15;74(4):741-751

4. Boston University School of Medicine, BUSM. Mass Spectrometry Resource. Available from URL: bumc.bu.edu/Dept/Home.aspx?DepartmentID=354

5. Eneqvist T, Sauer-Eriksson AE: Structural distribution of mutations associated with familial amyloidotic polyneuropathy in human transthyretin. Amyloid 2001;8:149-168

Familial Amyloidosis, Mass Spectrometry (MS):

Transthyretin (TTR) is purified from plasma using affinity chromatography. The chromatography is done using an antihuman-TTR antibody that has been coupled to POROS-aldehyde media. Plasma is reduced with 12.5 mM dithiothreitol (DTT) to simplify the mass spectra by removing Cys10 adducted species. The plasma:DTT solution is then injected onto the affinity column, which sequesters TTR using pH 7.4 phosphate buffer saline. TTR is then eluted from the affinity column with pH 2.5 100 mM glycine:2% acetic acid buffer and concentrated on a C4 column, which is then washed with acetic acid:methanol:water (1:2:97) to remove excess phosphate and other buffer components that suppress MS response. TTR is then eluted from the C4 column and introduced to the MS using methanol:water:glacial acetic acid:TFA (94.5:5:0.5:0.04). The acquired ion spectra are deconvoluted and reviewed for TTR variants. After deconvolution, normal patients present with a single peak corresponding to wild-type (wt) TTR, which serves as a reference. When positive, amyloid patients are typically heterozygous and are detected by the presence of 2 peaks (ie, wt TTR and mutant TTR) differing in mass.(Bergen HR 3rd, Zeldenrust SR, Butz ML, et al: Identification of transthyretin variants by sequential proteomic and genomic analysis. Clin Chem 2004 Sep;50[9]:1544-1552)

Familial Amyloidosis, DNA Sequence:

All 4 exons of the TTR gene are amplified by PCR and then subjected to direct DNA sequence analysis.(Bergen RH 3rd, Zeldenrust SR, Butz ML, et al: Identification of transthyretin variants by sequential proteomic and genomic analysis. Clin Chem 2004;50:1544-1552)

Monday; 8 a.m.

83788-Mass spectrometry-tandem mass spectrometry (MS-MS/MS)

81404-TTR (transthyretin) (eg, familial transthyretin amyolidosis), full gene sequence (if appropriate)