ClinVar Genomic variation as it relates to human health
NM_145868.2(ANXA11):c.118_119delinsAT (p.Asp40Ile)
The aggregate germline classification for this variant, typically for a monogenic or Mendelian disorder as in the ACMG/AMP guidelines, or for response to a drug. This value is calculated by NCBI based on data from submitters. Read our rules for calculating the aggregate classification.
Stars represent the aggregate review status, or the level of review supporting the aggregate germline classification for this VCV record. This value is calculated by NCBI based on data from submitters. Read our rules for calculating the review status. The number of submissions which contribute to this review status is shown in parentheses.
No data submitted for somatic clinical impact
No data submitted for oncogenicity
Variant Details
- Identifiers
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NM_145868.2(ANXA11):c.118_119delinsAT (p.Asp40Ile)
Variation ID: 2444454 Accession: VCV002444454.2
- Type and length
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Indel, 2 bp
- Location
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Cytogenetic: 10q22.3 10: 80170852-80170853 (GRCh38) [ NCBI UCSC ] 10: 81930608-81930609 (GRCh37) [ NCBI UCSC ]
- Timeline in ClinVar
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First in ClinVar Help The date this variant first appeared in ClinVar with each type of classification.
Last submission Help The date of the most recent submission for each type of classification for this variant.
Last evaluated Help The most recent date that a submitter evaluated this variant for each type of classification.
Germline Mar 18, 2023 Mar 18, 2023 Feb 24, 2023 - HGVS
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Nucleotide Protein Molecular
consequenceNM_145868.2:c.118_119delGAinsAT MANE Select Help Transcripts from the Matched Annotation from the NCBI and EMBL-EBI (MANE) collaboration.
missense NM_145868.2:c.118_119delinsAT MANE Select Help Transcripts from the Matched Annotation from the NCBI and EMBL-EBI (MANE) collaboration.
NP_665875.1:p.Asp40Ile missense NM_001157.3:c.118_119delinsAT NP_001148.1:p.Asp40Ile missense NM_001278407.2:c.118_119delinsAT NP_001265336.1:p.Asp40Ile missense NM_001278408.2:c.118_119delinsAT NP_001265337.1:p.Asp40Ile missense NM_001278409.2:c.19_20delinsAT NP_001265338.1:p.Asp7Ile missense NM_145869.2:c.118_119delinsAT NP_665876.1:p.Asp40Ile missense NC_000010.11:g.80170852_80170853delinsAT NC_000010.10:g.81930608_81930609delinsAT - Protein change
- D40I, D7I
- Other names
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- Canonical SPDI
- NC_000010.11:80170851:TC:AT
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Functional
consequence HelpThe effect of the variant on RNA or protein function, based on experimental evidence from submitters.
- functionally_abnormal Sequence Ontology [SO:0002218]
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Global minor allele
frequency (GMAF) HelpThe global minor allele frequency calculated by the 1000 Genomes Project. The minor allele at this location is indicated in parentheses and may be different from the allele represented by this VCV record.
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Allele frequency
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The frequency of the allele represented by this VCV record.
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- Links
Genes
Gene | OMIM | ClinGen Gene Dosage Sensitivity Curation |
Variation Viewer
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Links to Variation Viewer, a genome browser to view variation data from NCBI databases. |
Related variants | ||
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HI score
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The haploinsufficiency score for the gene, curated by ClinGen’s Dosage Sensitivity Curation task team. |
TS score
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The triplosensitivity score for the gene, curated by ClinGen’s Dosage Sensitivity Curation task team. |
Within gene
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The number of variants in ClinVar that are contained within this gene, with a link to view the list of variants. |
All
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The number of variants in ClinVar for this gene, including smaller variants within the gene and larger CNVs that overlap or fully contain the gene. |
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ANXA11 | - | - |
GRCh38 GRCh37 |
276 | 361 |
Conditions - Germline
Condition
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The condition for this variant-condition (RCV) record in ClinVar. |
Classification
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The aggregate germline classification for this variant-condition (RCV) record in ClinVar. The number of submissions that contribute to this aggregate classification is shown in parentheses. (# of submissions) |
Review status
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The aggregate review status for this variant-condition (RCV) record in ClinVar. This value is calculated by NCBI based on data from submitters. Read our rules for calculating the review status. |
Last evaluated
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The most recent date that a submitter evaluated this variant for the condition. |
Variation/condition record
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The RCV accession number, with most recent version number, for the variant-condition record, with a link to the RCV web page. |
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Pathogenic (1) |
criteria provided, single submitter
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Feb 24, 2023 | RCV003153237.1 |
Submissions - Germline
Classification
Help
The submitted germline classification for each SCV record. (Last evaluated) |
Review status
Help
Stars represent the review status, or the level of review supporting the submitted (SCV) record. This value is calculated by NCBI based on data from the submitter. Read our rules for calculating the review status. This column also includes a link to the submitter’s assertion criteria if provided, and the collection method. (Assertion criteria) |
Condition
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The condition for the classification, provided by the submitter for this submitted (SCV) record. This column also includes the affected status and allele origin of individuals observed with this variant. |
Submitter
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The submitting organization for this submitted (SCV) record. This column also includes the SCV accession and version number, the date this SCV first appeared in ClinVar, and the date that this SCV was last updated in ClinVar. |
More information
Help
This column includes more information supporting the classification, including citations, the comment on classification, and detailed evidence provided as observations of the variant by the submitter. |
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Pathogenic
(Feb 24, 2023)
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criteria provided, single submitter
Method: clinical testing, in vitro
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Oculopharyngeal muscular dystrophy 1
Affected status: not applicable, yes
Allele origin:
de novo,
not applicable
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Laboratory of Neurogenetics and Molecular Medicine, Institut de Recerca Sant Joan de Déu
Accession: SCV003804451.1
First in ClinVar: Mar 18, 2023 Last updated: Mar 18, 2023 |
Observation 1:
Clinical Features:
Ptosis (present) , Ophthalmoparesis (present) , Neck flexor weakness (present) , Weakness of facial musculature (present) , Scapular winging (present) , Dysphonia (present) , Dysphagia … (more)
Ptosis (present) , Ophthalmoparesis (present) , Neck flexor weakness (present) , Weakness of facial musculature (present) , Scapular winging (present) , Dysphonia (present) , Dysphagia (present) , Limb muscle weakness (present) , Childhood onset (present) (less)
Sex: male
Ethnicity/Population group: Caucasian
Geographic origin: Spain
Observation 2:
Sex: male
Tissue: Muscle
Result:
ANXA11 immunostaining of muscle biopsy revealed additional validation of the ANXA11 p.Asp40Ile pathogenicity. The most striking finding with respect to the histopathological descriptions of the individuals with adult-onset IBM was the detection of ANXA11 signal in the sarcoplasm and sarcolemma of the fibers and three different types of aggregates according to their size and location. Seventy-one out of 1887 muscle fibers had ANXA11+ aggregates representing 3.7%. They were distributed as follows: 30% were large aggregates related or not to vacuoles, 60% were small aggregates located in the sarcoplasm with or without relation to the sarcolemma, and 10% were located only in the sarcolemma. Super-resolution confocal images revealed ANXA11 aggregates shaped like pearl strips with varied sizes or large complex structures in the sarcoplasm and as layered subsarcolemmal chains. In addition, quantification of the aggregates area revealed significant differences between sarcoplasm and sarcolemma. The aggregate area ranged from <0.1 to 1000?µm2 Methodology Snap-frozen muscle biopsy specimens were sampled from right deltoids and processed according to standard muscle histopathological techniques. Immunofluorescence staining the muscle sample was cut into 10?µm thick sections, fixed (4% paraformaldehyde at room temperature (RT) for 7 min), rinsed with PBS-Tween (0.5%), and blocked with 1% Tween- 8% BSA- 10% donkey serum in PBS for 1 h at RT. Antibody incubation was performed overnight at 4°C using the following primary antibodies: rabbit polyclonal a-ANXA11 (1:1000), mouse monoclonal a-hnRNPA2B1 (1:50) and mouse monoclonal a-SQSTM1 (1:20). After primary antibody incubation, sections were washed in PBS-tween-triton and incubated for 3 h at RT with the secondary antibodies a-mouse Alexa Fluor 488 and a-rabbit Alexa Fluor 594. For counterstaining of nuclei, sections were incubated 5 min with DAPI (1:5000, Thermo Fisher Scientific, Inc.) and rinsed in PBS-Tween-triton before mounting onto Fluoromount G (Thermo Fisher Scientific, Inc.). Confocal and super-resolution imaging Confocal and super-resolution microscopy analysis was performed by Leica TCS SP8 equipped with a white light laser, HyVolution and Hybrid spectral detectors (Leica Microsystems GmbH, Mannheim, Germany). The confocal images of both muscle biopsies and fibroblast cultures were acquired using an HC?×?PL APO 20×/0.75 dry and an HC?×?PL APO 63×/1.4 oil immersion objectives. The super-resolution images were acquired using an HC?×?PL APO 100×/1.4 oil immersion objective, the HyD detector, and HyVolution. DAPI was excited with a blue diode laser (405?nm) and detected in the 425–480?nm. Antibodies bond to Alexa Fluor 488 were excited with an argon laser (488?nm) and detected in the 505–550?nm. Antibodies bond to Alexa Fluor 594 were excited with a white light laser (594?nm) and detected in the 610–795?nm. In fibroblasts and muscular biopsy, Z stacks of 10–15 sections were acquired every 0.5–0.8 µm along with the sample thickness. Appropriate negative controls were used to adjust confocal settings to avoid non-specific fluorescence artifacts. Image pixel was 8 bits depth and diameter pinhole diameter was 1 AU. To compare the confocal data, identical confocal settings were used for image acquisition of different experiments. Maximum intensity projections were performed using LAS X software and fluorescence quantification by the ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Observation 3:
Result:
Aggrescan predicts an increase in aggregation propensity, relative to ANXA11WT, of the region of sequence close to the mutation site in the case of ANXA11Asp40Gly, which was higher in ANXA11Asp40Tyr and even higher in ANXA11Asp40Ile. We investigated whether AnxA11p.Asp40Ile affects liquid droplet formation similarly to ANXA11Asp40Gly.6 LLPS assays were performed using purified ANXA11WT, ANXA11Asp40Gly, and ANXA11Asp40Ile full-length recombinant proteins. As previously described, we found that ANXA11WT undergoes LLPS at high temperature while ANXA11Asp40Gly undergoes LLPS at the same temperature although to a lesser extent (the maximum absorbance reached is half that of ANXA11WT, and there are fewer and smaller droplets by DIC microscopy). ANXA11WT droplets formation could be reversed by lowering the temperature (4°C for 30?min), whereas ANXA11Asp40Gly droplets remained abnormally aggregated under this condition.6 In the case of the patient's variant, our results showed that ANXA11Asp40Ile does not undergo LLPS under the same conditions but instead forms aggregates over the entire temperature range and those are not temperature reversible. Taken together, all these data argue that the p.Asp40Ile variant has a greater deleterious impact on ANXA11 solubility and ability to undergo LLPS than other pathogenic substitutions of ANXA11 at the Asp40 position. Methodology Plasmid constructions for recombinant ANXA11 An insert containing the SUMO-ANXA11 (1-505) protein sequence was designed and ordered using the GeneArt software in Thermo Fisher Scientific. The insert was cloned in a pDONR211 vector and then subcloned into the expression vector pDEST17, resulting in the 6His-SUMO-ANXA11 construct. p.Asp40Ile and p.Asp40Gly variants were obtained using the Q5 site-directed mutagenesis kit (New England Biolabs, Ipswich, MA, USA). Recombinant proteins were expressed in transformed Escherichia coli B834 cells. The cells were grown in LB medium at 37°C until an OD = 0.6, and induced with 0.1?mM IPTG overnight at 25°C. The cultures were then centrifuged for 30?min at 4000?rpm and the cells were resuspended in lysis buffer (PBS, 1?mM DTT, 0.05% NaN3, PIC, and PMSF, at pH 7.4). The cells were lysed by sonication and centrifuged for 30?min at 20,000?rpm. The pellet was washed twice with wash buffer (PBS, 1?mM DTT, 500?mM NaCl, 1% TritonX-100, PIC, PMSF, DNAse, and RNAse, at pH 7.4). The pellet was resuspended in resuspension buffer (20?mM Tris–HCl, 8 M urea, 50?mM NaCl, 1?mM DTT, pH 8.0) and centrifuged for 30?min at 20,000?rpm. The sample was injected in a Ni2+ affinity column and eluted with a gradient from 0 to 500?mM imidazole (at 4°C and without urea for the cases in which the protein was present in the wash buffer supernatants, and at RT and with 8 M urea for the cases in which the protein was present in the resuspension buffer supernatant). The samples in urea were then stepwise dialyzed from 8 to 0?M urea. The 6His-SUMO tag was cleaved with SUMO protease through dialysis for 2 h at 4°C in cleavage buffer (20?mM Tris–HCl, 50?mM NaCl, 1?mM DTT, pH 8.0). 8?M urea was added to the sample and injected into a nickel column to remove the cleaved tags. The reverse Ni2+ column was run at RT with the 8 M urea buffers containing 0 and 500?mM imidazole. The flow-through was injected in a size exclusion superdex200 (GE Healthcare), running in 20?mM HEPES, 200?mM NaCl, 1?mM TCEP, 0.05% NaN3, pH 7.4 buffer. The fractions with protein were pooled and concentrated to 25–50?µM, fast-frozen in liquid nitrogen and stored at -80°C. Sample preparation for in vitro LLPS experiments All samples were prepared on ice as follows. First, a buffer stock solution consisting of 20?mM HEPES, 1?mM TCEP and 0.05% NaN3 was pH adjusted to 7.4 and filtered using 0.22?µm sterile filters (Buffer Stock). A 1 M NaCl solution in the same buffer was also pH adjusted to 7.4 and filtered (Salt Stock). Then, the protein samples were thawed from -80°C on ice, pH adjusted to 7.4, and centrifuged for 5 min at 15,000?rpm. The supernatant (Protein Stock) was transferred to a new Eppendorf tube and the protein concentrations were determined by their absorbance at 280?nm using 42,750?M-1 cm-1 as the extinction coefficient value using a Cary100 ultraviolet–visible spectrophotometer. The indicated samples were prepared by mixing the right amounts of Buffer Stock, Protein Stock, and Salt Stock to reach the desired final protein and NaCl concentrations
Observation 4:
Result:
Since ANXA11 and hnRNPA2B1 participate in the formation and dynamics of Stres Granules (SGs) we experimentally assessed the SGs dynamics. We used fibroblasts from the patient and healthy controls, which we treated with 0.5?mM sodium arsenite for 1 h. First, we quantified by immunostaining with a-ANXA11 and a-hnRNPA2B1 the signal of these proteins in fibroblasts. The patient showed a significantly lower ANXA11 signal in both conditions compared to the control (basal p: <0.0001, stress p: <0.0001, recovery p: <0.0001) whilst the hnRNPA2B1 signal was reduced exclusively under basal conditions (p: <0.0001 for basal, p: 0.5923 for stress). Although the levels of both proteins were decreased in the patient's fibroblasts, we observed the formation of SGs, where ANXA11 and hnRNPA2B1 colocalized. Quantification of the soluble fraction of fibroblast protein extracts by Western blot showed the tendency for soluble ANXA11 levels to decrease under basal conditions while soluble hnRNPA2B1 levels were significantly decrease under both basal and stress conditions. The study of ANXA11 and HNRNPA2B1 mRNAs by qRT-PCR revealed no changes (ANXA11, basal p: 0.400; stress p: 0.999; HNRNPA2B1, p: 0.700 for basal, p: 0.400 for stress). To further investigate SGs defects we immunostained G3BP Stress Granule Assembly Factor 1 (G3BP1), which is a bona fide component of cytoplasmic SGs. As in control fibroblasts, ANXA11 strongly colocalized with G3BP1 in the SGs of the patient's fibroblasts. We found no differences among samples in SGs assemble (p: 0.358). After 1.5 h of recovery condition, only 9% of SGs were retained in control fibroblasts, whilst more than 90% of SGs did not disassemble in the patient's fibroblasts (p: <0.0001). This defect in SG disassembly has been reported in other ANXA11-related ALS-linked variants. Our results demonstrate that the new ANXA11 p.Asp40Ile variant has an impact on ANXA11 and hnRNPA2B1 biology and causes defective dynamics of SGs by altering their disassembly and clearance process. Methodology Endogenous G3BP1-positive stress granules (SGs) in control and patient fibroblasts were quantified following stress induction (0.5?mM sodium arsenite for 1 h) for assembled SGs and after recovery (fresh medium for 1.5 h) for the remained SGs. 100 cells per condition were quantified blindly per independent experiment (n = 3).
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Germline Functional Evidence
Functional
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The functional consequence of the variant, based on experimental evidence and provided by the submitter. consequence |
Method
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A brief description of the method used to determine the functional consequence of the variant. A citation for the method is included, when provided by the submitter. |
Result
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A brief description of the result of this method for this variant. |
Submitter
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The submitting organization for this submitted (SCV) record. This column also includes the SCV accession and version number, the date this SCV first appeared in ClinVar, and the date that this SCV was last updated in ClinVar. |
More information
Help
This column includes more information supporting functional evidence for the germline classification, including citations, the comment on classification, and detailed evidence provided as observations of the variant by the submitter. |
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functionally_abnormal
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Laboratory of Neurogenetics and Molecular Medicine, Institut de Recerca Sant Joan de Déu
Accession: SCV003804451.1
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Citations for germline classification of this variant
HelpTitle | Author | Journal | Year | Link |
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Common pathophysiology for ANXA11 disorders caused by aspartate 40 variants. | Natera-de Benito D | Annals of clinical and translational neurology | 2023 | PMID: 36651622 |
Text-mined citations for this variant ...
HelpRecord last updated Feb 20, 2024
This date represents the last time this VCV record was updated. The update may be due to an update to one of the included submitted records (SCVs), or due to an update that ClinVar made to the variant such as adding HGVS expressions or a rs number. So this date may be different from the date of the “most recent submission” reported at the top of this page.