Amyotrophic Lateral Sclerosis / Motor Neuron Disease via the ANXA11 Gene
- Summary and Pricing
- Clinical Features and Genetics
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The great majority of tests are completed within 18 days.
Pathogenic variants in the ANXA11 gene were detected in about 1% of familial Amyotrophic Lateral Sclerosis cases, and 1.7% of sporadic cases (Smith et al. 2017).
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by a selective loss of motor neurons in the motor cortex, brain steam, and spinal cord (Tandan and Bradley 1985). The dysfunction and loss of these neurons results in rapid progressive muscle weakness, atrophy and ultimately paralysis of limb, bulbar and respiratory muscles. The mean age of onset of symptoms is about 55 years of age; most cases begin between 40 and 70 years of age. The annual incidence of ALS is 1-2 per 100,000 (Cleveland and Rothstein 2001).
The most common symptoms include twitching and cramping of muscles of the hands and feet, loss of motor control in the hands and arms, weakness and fatigue, tripping and falling. Symptoms usually begin with asymmetric involvement of the muscles. As the disease progresses, symptoms may include difficulty in talking, breathing and swallowing, shortness of breath, and paralysis.
Cognitive impairment was not been initially associated with ALS. However, frontotemporal dementia (FTD) has been reported in several cases. Dementia now has been documented in patients with ALS from different ethnic groups and affects both males and females (Wikström et al. 1982; Lipton et al. 2004; Mitsuyama and Inoue 2009).
About 10% of ALS cases are familial (Emery and Holloway 1982). In most of these families, ALS is inherited in an autosomal dominant manner (AD-ALS) and is age-dependent penetrance. In rare families, the disease is transmitted in an autosomal recessive or dominant X-linked pattern.
About 90% of patients with ALS are sporadic cases (SALS) with no known affected relatives. It is unclear how many of the apparently sporadic cases are inherited with low penetrance. The clinical presentations of familial ALS (FALS) and sporadic ALS (SALS) are similar. However, the onset of symptoms in FALS is usually earlier compared to that of SALS (Kinsley and Siddique 2015).
Autosomal Dominant ALS (AD-ALS) is a clinically and genetically heterogeneous disorder that affects all ethnic groups. Several genes have been implicated in the disease. They include C9orf72, SOD1, FUS, TARDBP, ANG, OPTN, VCP, VAPB, PFN1, MATR3, CHCHD10, TBK1 and ANG.
Recently, whole exome sequencing studies by an international consortium of researchers revealed heterozygous pathogenic variants in the ANXA11 gene in both familial and sporadic ALS patients. A total of six different variants, all missense, have been reported. One of these, p.Asp40Gly, was detected in four apparently unrelated families from the United Kingdom and Italy. All patients with the p.Asp40Gly variant shared a common founder haplotype.
Evidence for pathogenicity included co-segregation of the variants with ALS in families with a history of the disease. In-vitro expression studies indicated altered binding properties of the mutated ANXA11 proteins. In addition, pathological postmortem examinations showed aggregation of the mutated protein in the affected tissues of patients with the p.Asp40Gly variant (Smith et al. 2017).
Based on the available data, all patients with the p.Asp40Gly variant presented with a late-onset classic form of ALS, with no dementia. First symptoms included difficulty in speaking and swallowing, indicating a bulbar-onset disease.
The ANXA11 gene encodes Annexin A11, a calcium-dependent phospholipid-binding protein that is involved in intracellular vesicle trafficking (Lecona et al. 2003; Smith et al. 2017).
This test involves bidirectional DNA Sanger sequencing of all coding exons and splice sites of the ANXA11 gene. The full coding sequence of each exon plus ~10 bp of flanking DNA on either side are sequenced. We will also sequence any single exon (Test #100) in family members of patients with a known pathogenic variant or to confirm research results.
Indications for Test
All patients with symptoms suggestive of ALS or Motor Neuron Disease, and no pathogenic variants in the previously identified ALS genes: C9orf72, SOD1, FUS, TARDBP, ANG, OPTN, VCP, VAPB, PFN1, MATR3, CHCHD10, TBK1 and ANG.
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- Genetic Counselor Team - email@example.com
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- Cleveland D.W., Rothstein J.D. 2001. Nature Reviews. Neuroscience. 2: 806-19. PubMed ID: 11715057
- Emery A.E., Holloway S. 1982. Advances in Neurology. 36: 139-47. PubMed ID: 7180680
- Kinsley L, Siddique T. 2015 Amyotrophic Lateral Sclerosis Overview. In: Pagon RA, Adam MP, Bird TD, Dolan CR, Fong C-T, and Stephens K, editors. GeneReviews™, Seattle (WA): University of Washington, Seattle. PubMed ID: 20301623
- Lecona E. et al. 2003. The Biochemical Journal. 373: 437-49. PubMed ID: 12689336
- Lipton A.M. et al. 2004. Acta Neuropathologica. 108: 379-85. PubMed ID: 15351890
- Mitsuyama Y., Inoue T. 2009. Neuropathology. 29: 649-54. PubMed ID: 19780984
- Smith B.N. et al. 2017. Science Translational Medicine. 9: N/A. PubMed ID: 28469040
- Tandan R., Bradley W.G. 1985. Annals of Neurology. 18: 271-80. PubMed ID: 4051456
- Wikström J. et al. 1982. Archives of Neurology. 39: 681-3. PubMed ID: 7125994
Bi-Directional Sanger Sequencing
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 10 bases of non-coding DNA flanking the exon are sequenced.
As of February 2018, we compared 26.8 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 14 years of our lab operation we have Sanger sequenced roughly 14,300 PCR amplicons. Only one error has been identified, and this was an error in analysis of sequence data.
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).
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 10 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.
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- 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.
- 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.
(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.
(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.
(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.