Genetic Analysis of Hereditary Transthyretin Ala97Ser Related Amyloidosis

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 radioactively labeled probes in comparison with southern blot³. PCR is a fast, easy, cheap, and reliable technique that has been applied to numerous fields in modern sciences⁴.

The early diagnosis of this progressive and fatal disease is challenging given its phenotypic heterogeneity. With increasing attention and broader recognition on the early manifestations of ATTR as well as the emerging treatments⁵, appropriate diagnostic studies including TTR genetic test are therefore critically fundamental to improve prognosis. Furthermore, different mutations are associated with different penetrance of the trait, age of onset, patterns of progression, disease severity, median survival, efficacy of liver transplantation, or TTR stabilizers^2,6, and variable degrees of neurological and cardiological involvement, which have great implications for genetic counseling^7,8,9. Besides, a highly accurate genetic test is the only tool that differentiates the two distinct types of ATTR: hereditary (mutant) and wild type (non-mutant form, senile systemic amyloidosis, SSA)⁷. It is imperative to confirm the types of ATTR, because the therapies vary widely². Therefore, there is an increasing necessity to describe the stepwise protocol of the TTR genetic test.

The molecular approach to detect the mutation will be illustrated using Ala97Ser, the most common endemic mutation in Taiwan, as an example. Modifications in the DNA extraction step reduce the amount of the three solutions used and yields a sufficient amount of DNA. In this protocol, all four TTR exons were analyzed, while regions including 5' upstream, 3' downstream, promoters, introns, and untranslated regions (UTR) were not sequenced.

Protokół

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 Extraction Kit for the genomic DNA extraction (see Table of Materials).

1. Add 10 mL of Solution 1 to the 4.0 mL blood sample. Incubate the sample on ice for 10 min.
2. Centrifuge the sample at 2,000 x g for 5 min at 4 °C. Remove and discard supernatant carefully. Resuspend the pellet in 3 mL of Solution 1.
3. Centrifuge the sample at 2,000 x g for 5 min at 4 °C again and discard supernatant. Resuspend the pellet in 3 mL of Solution 2.
4. Add 10 µL of pronase stock solution (225 mg/mL) to get a final concentration of 100 µg/mL and mix well. Incubate at 37 °C overnight.
5. Chill the tube on ice for 10 min. Add 0.8 mL of Solution 3 to the sample and invert 3 - 5 times. Place sample on ice for 5 min.
6. Centrifuge the sample at 3,000 x g for 15 min at 4 °C. Carefully transfer the supernatant to a 15 mL sterile conical centrifuge tube.
7. Add 6 µL of RNase stock solution (10 mg/mL) to get a final concentration of 20 µg/mL. Incubate at 37 °C for 15 min.
8. Add 2.5 mL of isopropyl alcohol and mix thoroughly by gently inverting several times to precipitate DNA (strands of white, flocculent material will form). Using a large-bore pipet, transfer the DNA precipitant to a new 1.5 mL centrifuge tube.
9. Centrifuge at 12,000 x g for 3 min at 4 °C. Discard supernatant carefully. Wash DNA pellet by adding 500 µL of 70% ethanol.
10. Centrifuge at 12,000 x g for 1 min and discard supernatant. Air dry DNA pellet at room temperature. Add 100 µL of ddH₂O and incubate at 65 °C for 15 min to resuspend DNA.

3. Genomic DNA Quantification

Use a spectrophotometer (see Table of Materials) to detect the quantity and quality of the genomic DNA.

1. Log into the computer attached to the spectrophotometer machine. Open the software.
2. Initialize the spectrophotometer machine:
   1. Click "Nucleic Acid" on the spectrophotometer software.
   2. Clean the pedestal with a disposable paper wipe and purified water.
3. Blank the spectrophotometer:
   1. Load 2 µL of purified water on the pedestal.
   2. Lower the upper arm of the spectrophotometer and click "Blank" to calibrate it.
   3. When it is done, lift the upper arm and dry the pedestal with a wipe.
4. Measure the sample:
   1. Click "DNA-50" on Sample Type.
   2. Load 2 µL of DNA sample on the pedestal.
   3. Lower the upper arm and click "Measure" to measure the A260/A280 ratio and the concentration of the sample.
   4. Clean the pedestal in between each run with a wipe and purified water.
5. Click "Print Screen" to collect the data.

4. Genetic analyses of mutations

Amplify the target DNA with the PCR⁴. Perform PCR in a 25 µL reaction mixture using a DNA Polymerase kit (see Table of Materials). Table 1 shows the TTR gene intronic primers.

gene	Primer sequence
Exon1	F: 5’-TCAGATTGGCAGGGATAAGC-3’ R: 5’-GCAAAGCTGGAAGGAGTCAC-3’
Exon2	F: 5’-TCTTGTTTCGCTCCAGATTTC-3’ R: 5’-TCTACCAAGTGAGGGGCAAA-3’
Exon3	F: 5’-GTGTTAGTTGGTGGGGGTGT-3’ R: 5’-TGAGTAAAACTGTGCATTTCCTG-3’
Exon4	F: 5’-GACTTCCGGTGGTCAGTCAT-3’ R: 5’-GCGTTCTGCCCAGATACTTT-3’

Table 1. TTR gene intronic primers. (NCBI Reference Sequence: NC_000018.10)

1. Add the reagents in Table 2 to a 0.2 mL PCR tube.

Components	Volume (µL)	Final concentration
10x Buffer	2.5	1x
MgCl2 (25 mM)	1.5	1.5 mM
360 GC Enhancer	1	-
360 DNA Polymerase	0.2	2 U/reaction
dNTP Mix (25 mM each)	2	2 mM each
Primer F (10 µM)	1	0.4 µM
Primer R (10 µM)	1	0.4 µM
DNA (100 ng)	2
PCR-grade water	13.8	-
Total Volume	25	-

Table 2. PCR reaction conditions.

2. Mix gently and briefly centrifuge on a benchtop centrifuge to collect all components to the bottom of the tube. Place tube in a thermocycler.
3. Perform PCR for 30 cycles. Each cycle consists of a denaturation step for 30 s at 94 °C, primer annealing for 30 s at 58 °C, and polymerase extension for 45 s at 72 °C (see Table 3).

Step	Temperature (°C)	Time	cycle
Initial denaturation	95	5 min	1
DNA denaturation step	94	30 s	30 cycles
Primer annealing step	58	30 s	30 cycles
Polymerase extension step	72	45 s	30 cycles
Post elongation step	72	10 min	1
End of PCR cycling	4	Indefinite

Table 3. Cycling conditions for amplifying PCR products.

4. Validate the PCR product by visualizing with gel electrophoresis:
   1. Prepare a 1.2% agarose gel:
      1. Mix 1.2 g of agarose powder with 100 mL of 0.5x TBE in a microwavable flask. Microwave for 2 min until the agarose is completely dissolved. Cool down the agarose mixture to 55 °C.
      2. Add 2 µL of a ethidium bromide solution (10 mg/mL) to the agarose mixture and mix gently. Pour the agarose mixture into a gel tray to about 5 - 7 mm, then add the comb(s) in place. Allow it to solidify (at least 30 min) and carefully remove comb(s).
   2. Load samples and run an agarose gel:
      1. Place gel and tray into the electrophoresis cell. Fill the electrophoresis cell with 0.5x TBE until the gel is covered.
      2. Carefully load 2 µL of a 100 bp DNA ladder (see Table of Materials) into the one lane of the gel as molecular size marker. Load dye/sample mixture (5 µL of PCR product mix with 1 µL 6x loading dye) into the additional lanes.
      3. Turn on the power supply. Run the gel for 25 min at 100 V.
      4. Remove the gel from the electrophoresis cell. Visualize the DNA fragments under UV light in an imaging system.

5. DNA sequencing

1. Purify PCR products using a commercial kit and sequence commercially or with an automatic sequencer (see Table of Materials).
2. Submit nucleotide sequences to the NCBI BLAST server to compare the nucleotide query to the nucleotide databases¹⁰. After the results are displayed, perform sequence alignments and identify the expected mutation.

Wyniki

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).

Figure 1. Gel electrophoresis depicting PCR amplified TTR gene. Normal: a healthy indiv...

Dyskusje

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...

Materiały

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