Genetic Analysis of Hereditary Transthyretin Ala97Ser Related Amyloidosis

Summary

Here, we present a protocol to confirm the presence of point mutation for the diagnosis of hereditary transthyretin amyloidosis, using Ala97Ser, the most common endemic mutation in Taiwan, as an example.

Abstract

Genetic testing is the most reliable test for hereditary transthyretin related amyloidosis and should be performed in most cases of transthyretin amyloidosis (ATTR). ATTR is a rare but fatal disease with heterogeneous phenotypes; therefore, the diagnosis is sometimes delayed. With increasing attention and broader recognition on early manifestations of ATTR as well as emerging treatments, appropriate diagnostic studies, including the transthyretin (TTR) genetic test, to confirm the types and variants of ATTR are therefore fundamental to improve the prognosis. Genetic analyses with polymerase chain reaction (PCR) methods confirm the presence of TTR point mutations much more quickly and safer than conventional methods such as southern blot. Herein, we demonstrate genetic confirmation of the ATTR Ala97Ser mutation, the most common endemic mutation in Taiwan. The protocol comprises four main steps: collecting whole blood specimen, DNA extraction, genetic analysis of all four TTR exons with PCR, and DNA sequencing.

Introduction

Transthyretin (TTR) amyloidosis (ATTR) is the most common form of hereditary systemic amyloidosis¹, and can be caused by an autosomal dominantly inherited mutation in the transthyretin (TTR) gene². TTR mutations destabilize the tetrameric protein structure and lead to its dissociation into monomers that reassembles into amyloid fibrils². More than 100 amyloidogenic TTR mutations have been reported worldwide¹. Genetic analyses with polymerase chain reaction (PCR) methods confirm the presence of TTR point mutation and have advantages including avoiding the handling of radioac....

Protocol

The testing performed in the laboratory was carried out in accordance with the requirements of the Clinical Laboratory Improvement Amendments (CLIA) of 1988, the regulations approved by the Institutional Review Board of Chang Gung Memorial Hospital and University (License no. 100-4470A3 and 104-2462A3). Informed consent was obtained from all patients.

1. Blood Specimen Collection

1. Collect whole blood into commercially available EDTA-treated tubes. Mix gently and store blood sample at 4 °C until processing.

2. DNA Extraction from Peripheral Blood

Use a DNA Extractio....

Results

Agarose gel electrophoresis of two patients and one healthy individual revealed bands of the expected sizes, including a 454 bp PCR product for exon 4 of the TTR gene (Figure 1).

Discussion

There are two critical steps within the protocol. First, in order to have sufficient number of white blood cells, a hemodiluted specimen should be avoided¹¹. Second, the use of appropriate PCR primers is fundamental to obtain reliable results¹². We used the Primer-BLAST web tool to design the primers^4,13; a minimum of 40 base pairs on each side of the four TTR exons should be covered. We also run BLAST on NCBI to ch.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
EDTA-treated tubes	BD
DNA Extraction Kit	Stratagen	200600
NanoDrop ND2000 spectrophotometer	Thermo Fisher Scientific	NanoDrop 2000
Delicate Task Wipers	Kimberly-Clark	Kimtech Science Kimwipes
AmpliTaq Gold 360 DNA Polymerase kit	Applied Biosystems	4398823
TTR gene intronic primers	Exon1	F: 5’-TCAGATTGGCAGGGATAAGC-3’
	Exon1	R: 5’-GCAAAGCTGGAAGGAGTCAC-3’
	Exon2	F: 5’-TCTTGTTTCGCTCCAGATTTC-3’
	Exon2	R: 5’-TCTACCAAGTGAGGGGCAAA-3’
	Exon3	F: 5’-GTGTTAGTTGGTGGGGGTGT-3’
	Exon3	R: 5’-TGAGTAAAACTGTGCATTTCCTG-3’
	Exon4	F: 5’-GACTTCCGGTGGTCAGTCAT-3’
	Exon4	R: 5’-GCGTTCTGCCCAGATACTTT-3’
thermocycler	Applied Biosystems	GeneAmp PCR System 9700
electrophoresis cell	ADVANCE	Mupid-2plus
DNA ladder	Protech	PT-M1-100
dye	BioLabs	B7021
AlphaImager EC	Alpha Innotech	AlphaImager EC
automatic sequencer	Applied Biosystems	3730xl DNA Analyzer