hours only. Property of Centogene AG. Unauthorized ......Generated by Digital Data Products & Curation at 2020-02-21 14:13:40 (UTC +0100) - Uncontrolled copy. Valid for 24 hours only

Title: Curation of GLA variants detected in Fabry screened patients

Document type: SOP (english) IT support

Document ID: SOPeIT-86

Author: Digital Data Products & Curation

Owner: Digital Data Products & Curation

Approver(s): Ellen Kargesapproved at 2020-02-20 15:11 (UTC +0100)

Approval date: 2020-02-20

Effective date: 2020-02-20

SOP (english) IT supportCentogene AG SOPeIT-86Curation of GLA variants detected in Fabry screened patients Version: 3.0

Property of Centogene AG. Unauthorized distribution or copying prohibitedGenerated by Digital Data Products & Curation at 2020-02-21 14:13:40 (UTC +0100) - Uncontrolled copy. Valid for 24hours only.

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1. Purpose and ObjectiveThis Standard Operating Procedure (SOP) describes the process of collection, association, update and review ofGLA variants detected in patients screened within Fabry disease clinical context at CENTOGENE AG, into astructured and standardized format. It utilizes a combination of computer-based tools and manual review in orderto assure the accuracy, efficiency and quality of the curation process.

2. Area of ApplicationThis SOP applies to Curation departments at Centogene.

3. Terms and AbbreviationsAA: amino acid

CentoLSD: Centogene’s Lysosomal Storage Disease database

CentoMD: Centogene’s Mutation Database

CI: Clinical informatin

CRV: clinically relevant variants

CuRepo: Curation repository

FD: Fabry disease

G2P: Genotype – to – Phenotype

GLA: alpha-galactosidase A

HGNC: HUGO Gene Nomenclature Committee

HGVS: Human Genome Variation Society

HPO: Human Phenotype Ontology

MOI: Mode of inheritance

STD: Standard deviation

VUS: variant of uncertain significance

WES: whole exome sequencing

WGS: whole genome sequencing

4. Applicable DocumentsSOPeIT-28 Curation Repository Data Submission

SOPeIT-32 Curation Repository Software Description

SOPeIT-46 SeqPilot Import into Curation Repository

SOPeIT-48 Quality checks - CuRepo

SOPeIT-55 Curation Repository MLPA/qPCR Import

SOPeIT-56 Curation repository Biochemistry import

SOPeIT-62 Curation Repository Other Import

SOPeIT-64 Curation Repository Illumina HTS Import



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SOPeIT-72 Curation Repository General Quality Checks

SOPeIT-77 GLA gene- Fabry disease association and curation

SOPeIT-79 Curation of GLA screened cases

SOPeIT-82 Classification of GLA variants following the ACMG guidelines

SOPeIT-84 Reclassification of GLA variants following the ACMG guidelines

SOPeR-36 Variant classification following ACMG guidelines at Centogene

VALeIT-58 Automatic Variant classification based on ACMG guidelines

5. ResponsibilitiesThis SOP applies to all employees responsible for curating GLA variants screened within Fabry disease clinicalcontext.

6. Reagents, materials and devicesSoftware:

UniDB: http://ts0001.russ.CENTOGENE.internal/unidbweb/variantsearch

CentoMD®: www.centomd.com

Curation Repository: https://srv-centomd.centogene.internal/curation-repo-acmg/login

OMIM:https://www.omim.org

Gepado: https://gepado-prod.centogene.internal/Xpro/

CentoLSD: https://www.centogene.com/centolsd.html

7. ProcedureContents

7.1 Data organization and available options under Unique variant view

7.2 Curation of GLA variants

7.3 Curation of GLA variants by warning

7.4 Track case history

Before proceeding

Scope and goal of the case curation process

Curators are responsible for collection, association, update and review of genetic and phenotypic data ofcases analyzed at Centogene (or externally) into the structured format of the Curation repository database.

During curation process, curator utilizes a combination of computer-based tools and manual review inorder to maximize curation accuracy, efficiency and assurance of the highest level of data quality inCentoMD and CentoLSD database.



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http://ts0001.russ.centogene.internal/unidbweb/variantsearch

http://www.centomd.com

https://srv-centomd.centogene.internal/curation-repo-acmg/login

https://www.omim.org

https://gepado-prod.centogene.internal/Xpro/

https://www.centogene.com/centolsd.html

Data gathering and curation are procedures developed and implemented in the Curation Repositorysystem, that is complaint with the HGNC, HGVS and HPO nomenclatures

Curators undergo extensive training to ensure curation consistency and standardization. They confirm thatdatabase is error-free (items properly associated and interpreted, no inconsistencies, and / or discrepanciesagainst detected observations in house and external sources), and close the curation process by manualapproval that reviewed and curated data agree with standard procedures established in house.

The data is gathered by a combination of manual submission and data import following a case-orientedmodel where characteristics belonging to a particular individual (patient information, clinical data,methodology and detected genetic variants) are stored and associated together. Data gathering process isinfluenced by already existing and processed master data, where genes-diseases- MOI are pre-linked.

The process is closed by manual confirmation that reviewed and curated data agree with standardprocedures established in house (see additional documents). At database level, the reviewed item changetheir status from “pending”, “marked” into “approved” item.

In order to start curation by case, all variants detected in this case of interest must be approved. It aims atassuring that the entries belonging to an individual follow the rules for final statement closely, and that allassociated data is in agreement with the agreed guidelines. The following factors are considered as criticalfor the final statement: variant significance, patient genotype (number of clinically relevant changes, theirzygosity and location -i.e. cis vs. trans), inheritance pattern of the disorder, the sex of the patient (for X-linked diseases), the phenotypic description, and if available- levels of biomarkers.

NOTE: during variant curation, a deep understanding of ACMG guidelines is a must. Whenever the variantcuration ends with a discordant conclusion, inform immediately your supervisor!

A) Workflow description

Curation of variants starts with ascertaining reference sequences, standard ontology and nomenclature system. Tomaintain uniformity for variants naming, the curation scientists are following closely the HGVS nomenclature. Theuse of g. for genomic, c. for coding DNA seq, p. for protein and m. for mitochondrial is mandatory. The referencetranscript is represented by longest transcript at amino acid level. Variants described on historical nomenclatureare matched to current transcript/ nomenclature. At CENTOGENE, NCBI transcripts are used as standard referencetranscript (RefSeq Gene) and the hg19 genomic build is considered. To standardize the use of gene symbols, thecuration scientists follow the HGNC guidelines.

HGVS- compliant variants are then subjected to variant classification following the ACMG recommendations.Curation scientist evaluates all available criteria under the following categories: general population and sub-population allele frequencies and observations, computational and predictive tools, functional assays performed inhouse and / or obtained from literature, segregation and de novo observations, annotations in literature and / orexternal and available databases (including disease-specific, locus-specific and variant databases), family historyand allelic and genotyping data.

Variant-related information is predominantly extracted automatically, and when the case extracted / managedmanually.

CENTOGENE has been optimized the variant classification, implementing a new standalone criterion, namely PVS2.This very strong pathogenic criterion is assigned to variants that are confirming a deleterious effect via in vivomeasurements of biomarker levels.

At CENTOGENE variants are classified using five-tiered scheme: pathogenic, likely pathogenic, variant of uncertain



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significance (VUS), likely benign and benign. An additional clinical class, namely risk factor, represents a raresituation.

Variants are in general quarterly reviewed; however, if the system notifies curation scientists on new evidences tobe available, the re-evaluation based on the new knowledge is prioritized and performed immediately.

The curation process of GLA variants within FD clinical context takes place in CuRepo software. Data are importedweekly as briefly described below:

All new patients screened for GLA and linked with the status “completed” / “finalized” in Gepado aretransferred in CuRepo (see documents related to data import in CuRepo: SOPeIT- 56, SOPeIT- 62; SOPeIT-55; SOPeIT- 46; SOPeIT- 64). During this transfer, patient details, clinical information and family data areretrieved. Additionally, the biochemical analyses are transferred and stored in the structured formatCuRepo provides. On the imported cases, the GLA genetic variants are transferred from UniDB, and ifSanger sequencing performed, SeqPilot is used as most reliable source. Once import complete, patients aregetting the status “pending” in CuRepo.

GLA variants are subjected for independent review in CuRepo

During variant curation process, ALWAYS check the issued medical report. When inconsistencies / errorsdetected, inform your supervisor!

A DETAILED DESCRIPTION OF DATA ORGANIZATION IN CUREPO IS PROVIDED IN SOPeIT-32 CURATIONREPOSITORY SOFTWARE DESCRIPTION

7.1 Data organization and available options under Unique variant view

Below a detailed overview of the available data during variant curation is provided.

Curation of GLA variants is performed as described in 7.2 subchapter under the module Unique variants;https://srv-centomd.centogene.internal/curation-repo/curation/variant-unique

By default, under Unique variants view, the result table is by default empty. The following items areincluded:

a. Reserve: option to reserve the variants

b. Details/ ID: the unique identifier for each CuRepo variant; this number can be selected, and curator



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https://srv-centomd.centogene.internal/curation-repo/curation/variant-unique

jumps on each variant for more details and to perform curation (see 7.2)

c. Is Pub?: information if variant already described in external databases (i.e. HGMD, ClinVar)

d. Gene: the corresponding gene, in this situation GLA

e. Chrom: the chromosome where gene located, in this situation chromosome X

f. gDNA: the genomic coordinates, according to hg19 build

g. cDNA: the cDNA change/ coordinates on transcript of interest, for GLA the transcript NM_000169.2 isused

h. Location: location within exon/ intron, according to transcript of interest, for GLA the NM_000169.2 isused

i. Protein: the protein change according to transcript of interest, for GLA the transcript of interest isNM_000169.2 is used

j. Reference: publication linked to variant of interest

k. dbSNP ID: the rs ID when variant identified in NCBI

l. HGMD Accession: HGMD Accession number, if variant detected in HGMD database

m. Type on DNA Level: the change at DNA level

n. Clinical significance: the manual curated clinical significance

o. ACMG class: the variants class according to ACMG related SOPs

p. QC class: the Quality Check class, where manual curated clinical class (i.e. clinical significance) iscompared against ACMG class

q. PathoScore: the highest pathogenicity score of Lyso-Gb3 biomarker level (see APPX2 and SOPeIT-28Curation Repository Data Submission)

r. Coding effect: the effect on protein level

s. Submitter: user submitting for the first time variant into CuRepo (via manual submission orautomated import)

t. Comment available: Indicates if variant is linked or not with a comment (See 7.2 below)

u. Status: indicates the quality of variant (pending, marked or public)

v. Approve option: curator has here the option to mark or approve a variant

w. Delete Option: curator has here the option to remove a variant from database

x. History: curator can see here all applied changes to a variant

Below a screenshot of result page once GLA gene is used for searching; note that on the top of the resulttable total number of GLA variants is indicated (i.e. 1026 elements).

Left side screenshot: a)-to-m) points



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Right side of screenshot: n)- to- x) points

By selecting any variant ID, curator jumps on Variant view, and a new tab opens



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The variant view is divided into the following categories:

a) Variant information: by default all details shown

b) External information: by default all details shown

c) Curated ACMG Rationale: by default all details hidden

d) Patients: by default all details hidden

Screen shot of the GLA variant c.1286T>C, p.L429P by default Variant View:

On variant view, the variant of interest follows the Transcript (Gene): cDNA change rule. For the exampleabove (i.e. GLA c.1286T>C), Curation of variant NM_000169.2(GLA):c.1286T>C is indicated.

Under this information, two search boxes (rectangles with green lines) can be used to initiate anothersearch: Gene and cDNA change. To search here for another GLA variant, go under cDNA change search box(green line) and add cDNA change following HGVS nomenclature.

c.1196G>A variant search initiated from Variant View of c.1286T>C GLA variant:



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Press Search

The Variant view is updated, and c.1196G>A details are indicated:

NOTES:

The search box of cDNA change follows strictly the HGVS nomenclature. Therefore, system notifies the user whenthe change does not follow the HGVS guidelines. Below two examples:

Example 1: cDNA change is not complete; c.1196G> used instead of c.1196G>A

Example 2: cDNA change is not correct: c.1196G>T instead of c.1196G>A



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Each category of the Variant View is below in details characterized:

a) Category Variant Information:

Once the variant is selected/ searched, the following details are seen under Variant information category:

cDNA change: editable, following HGVS nomenclature

gDNA change: editable, following HGVS nomenclature

Protein change: editable, following HGVS nomenclature

Location: editable, automatically indicated by the system based on Gene- Transcript- cDNA change asstored under master data

Pathogenicity score

Type on DNA level: editable, drop-down list

Coding effect: editable, drop-down list

Clinical significance: editable, drop-down list

ACMG class: automatically calculated based on assigned rules under Curated ACMG Rationale (seeCategory Curated ACMG Rationale)

QC class: automatically calculated based on clinical significance and Curated ACMG class

Gene

Transcript: automatically based on Gene: Transcript association under Master data

Chromosome: automatically based on Gene: Chromosome location under Master data (chromosome Xfor GLA gene)

Publication: automatically indicated based on metadata (see External information category)

Published status: automatically indicated based on metadata (Published when variant previouslyreported in the literature; Unpublished when variant not reported in literature)

Last edited: last user adding corrections/ updates and automatically tracked based on log in credentials

Last justification: user adding rationale and automatically tracked based on log in credentials

No of patients: number of patients carrying the variant of interest; automatically calculated based oncase- variant associations

All mandatory fields are highlighted with the red asterisk:

Example of Variant Information category for GLA c.1196G>A variant. Data organization and mandatory fields areindicated:



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Under Variant Information, curator has the following options:

Review on one page all positive patients by clicking on the [cDNA change] option from the grey bar.Selection of the [cDNA change] opens a new tab (see below the screenshots- left and right).

Left screen: Patients view for variant GLA, c.1196G>A. The variant annotations are indicated and number of co-concurrent CRV (same gene and other genes are indicated, if the case). The result table contains one positiveindividual per one row.

The Patients details can be here reviewed to understand case- related evidences (left side):



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Right side_ Patients view for variant GLA, c.1196G>A:



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Retrieve coordinates on transcript of interest (on cDNA or gDNA level; see 7.2 below); green symbol:

Editing options: all editable fields can be here processed (see below and chapter 7.2.1)

View History: curators sees here all applied changes (if any)

Check for duplicates: under this option, system checks if variant may be twice imported / submitted

Delete variant: curator can remove the variant, if variant is confirmed to be an artefact.

View and add comment: curator can, if necessary, add comments, which will be visible to other curators. Ifat least one comment available, system notifies via a red message, on the top of Variant view

Comment notification:



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To see comment, select the View and add comment option: a new window opens where user ca read the availablecomment(s).

View and Add comment:

Mark/ Approve: curator can mark an already approved variant, or can approve an variant, warning freevariant on status pending or marked (see 7.2)

If any change applied, Update option is ready for approval. Note that changes without pressing Updatebutton will not be saved!

b) Category External information

Under this category are indicated the metadata used to review during ACMG assignment or rejections.

Under this category, data is divided into sub-category:

All transcript annotations: the cDNA changes of the variant of interest are displayed on all knownNCBI transcripts, using hg19 assembly. Curator must quickly see if the variant type / coding effect staysunchanged on all transcripts, as at Centogene the “uniform model” is applied automatically.

Example screen shot below: the GLA variant c.1196G>A is annotated against the one known GLA NCBI transcriptsusing hg19 build.



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Frequencies: the following tools are represented: ESP, 1000 Genomes, gnomAD genex (genomes andexomes), gnomad genex Subpop (subpopulations), CentoMD. Versions of each tool are indicated. Whenvariants not detected, the system provides No Record Present message.

Predictions:

For missense variants the following in silico predictions tools are represented: SIFT, Polyphen,Mutationtaster

For splicing variants: ada and rf_Score

For all variant types: the conservation score using PhyloP100Way_ vertebrate to understand if thechange is conserved or not at nucleotide level

rsID indicates if the variant is known in NCBI database

External databases

HGMD is automatically retrieved showing the variant class, variant HGMD accession number and thephenotype for which variants was described. If variant not detected the HGMD, No Record Present isindicated



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ClinVar is automatically retrieved showing the link (if the case) and the clinical class(es). When variantnot detected in ClinVAr, No record Present is indicated.

User cannot here edit any item; this category is only for visualization of the metadata.

User has the option to jump to original source, when information available (rsID, HGMD and ClinVar) andindicated as links

c) Category Curated ACMG Rationale

By default, this category is hidden.

Click on Curated ACMG Rationale, and the data is organized as following (for visualization see the screenshot below) on https://srv-centomd.centogene.internal/curation-repo-acmg/

Category bar indicating the pre calculated ACMG class (indicated by class and color code; red forpathogenic/ likely pathogenic; dark orange for Uncertain; green for benign and likely benign)

Assigned rules (following the ACMG ID and the color code),

Rejected rules (following the ACMG ID and no color code; all black letters),

Supportive pathogenic evidences (including all ACMG rules supportive for pathogenic effect; red colorcode)

Supportive benign evidences (Including all ACMG supportive for tolerated effect; green color code)

Report summarizing all assigned rules (as ACMG rule ID, rule name, Strengths, Definitions andDescriptions).



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https://srv-centomd.centogene.internal/curation-repo-acmg/login

Curated ACMG Rationale bar . Indicates the pre-calculated ACMG class. This class automatically updateswith the classification scheme (see APPX1) via assignments and rejections actions (see 7.2.2 and 7.2.3).

Supporting pathogenic evidences: this part is in red and refers to the assigned pathogenic rules , if the



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case. Under this part there is a text box (see 7.2) where user can add description during rule assignment.

Each pathogenicity supporting ACMG rule has its own place indicated by a rectangle with red line andcorresponding ACMG ID and name (for example: PVS1= Null/ truncating). A not yet assigned rule, isindicated by a gray fill. An assigned rule is indicated by a red fill. See screen shot below and compare PVS1=Null/ Truncating versus PVS2= Internal biomarker.

Supporting benign evidences, this part is in green and refers to the assigned benign rules , if the case.Under this part there is a text box (see 7.2) where user can add description during rule assignment.

Each benign supporting ACMG rule has its own place indicated by a rectangle with green line andcorresponding ACMG ID and name (for example: BA1= Allele frequency >5%). A not yet assigned rule, isindicated by a gray fill. An assigned rule is indicated by a green fill. See screen shot below and compareBA1= Allele frequency >5% versus BP6= reputable source benign.



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Report of the assigned rules where ACMG rule ID, Rule name, Strength, Definition and Description aresummarized.

At this category, user has here the following options (described in detail under 7.2.2. and 7.2.3 subchapters):

Assign ACMG rules

Add / Edit descriptions for assigned rules

Reject assigned rules

Perform quick QC via report review

Update ACMG class

Correct QC class via assignment and rejection

Review applied changes via History option



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d) Category Patients

All patients carrying the variant of interest are indicated under Patients. The number of total patients isindicated between the squared brackets, and by default no extra details are indicated.

Click on patients, and the patient ID (following Gepado Patient ID terminology) are indicated

User has the option to click here on each Patient ID. Once Patient ID is clicked, a new tab opens, with UniqueIndividuals view, where patients details can be reviewed.



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7.2 Curation of GLA variants

Scope: assure that all entries belonging to a GLA variant are according to ACMG guidelines and internal establishedSOPs (following the GLA- FD specifics).

Procedure:

Go on https://srv-centomd.centogene.internal/curation-repo/ and log in with your credentials

Under Unique variants module search for GLA variants (as described above under 7.1)

To prioritize variants for curation, review the QC class (see screen shot below). The QC class calculatesautomatically the clinical significance of the variants against the ACMG pre-calculated class (see SOPeR- 36and SOPeIT-82 GLA variant classification).

The QC class indicates one of the following items: Perfect match, Partial match, Medium match, No match

Perfect match: the clinical significance of the variant matches the ACMG class

Partial match: the clinical significance of the variant matches the super-class (i.e. Pathogenic or Benign;here are expected the following variations: likely pathogenic vs pathogenic; and likely benign vs benign,respectively)



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https://srv-centomd.centogene.internal/curation-repo/

Medium match: the clinical significance of the variants detects one class under a super- class and oneunder VUS class. Expected are: Likely pathogenic / pathogenic vs VUS; or Likely benign/ benign vs VUS

No match: the clinical significance of the variant follows under discordant super- classes. Expected areto be seen variants pathogenic/ likely pathogenic vs benign/ likely benign

Under Reserve? Press and note that system informs that variant is reserved by you, and all other curatorsare notified, thus avoiding to work on the same variant simultaneously

As next step, you need to open the variant view. To do so, select under Details / ID the variant ID (blue,marked with an blue up arrow)

Subjected for curation are Variant information details and Curated ACMG Rationale.

7.2.1 Curation under Variant information

Curator is responsible to bring all fields subjected for curations according to HGVS terminology, CuRepoterminology and closing this step of the process with a Public variant

CuRepo system is HGVS complaint and variants must follows throughout this submodule the qualityimplemented as described in CuRepo- software related documents. When terminology does not followthese quality standards, notifies the user by highlighting the deviating fields in red and by notifying the issueand which steps must be processed.

Below a such GLA variant where system detects empty fields:



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In order to retrieve the genomic coordinates and the integrated rules for variant annotation, press thegreen symbol

under cDNA Change.

System automatically retrieves the genomic coordinates and fills accordingly the protein change andlocation



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Process Type on DNA level and Coding effects according to SOPeIT- 28 Data submission from the availabledrop-down list

Note that warnings are deactivated, and all problematic fields processed. Once at least one change / actionby curator detected by the system, the option to Save the changes is active (note the Update blue option)

Press Update. The Update button changes from blue color into green color, and user is informed thatVariant is updated



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The clinical significance of the variant is initially the variant class used by reporters on the medical issuedreports (via import). When clinical significance is not retrieved from the original source (UniDB, Gepado orSeqPilot), it will be empty, and system notifies that Clinical significance is empty and it must be selected.

Therefore initially the variant is on pending and represents the reported clinical class. Curator must openand check the report in Gepado before approving the variant. At this step, curator ensures that Variantinformation in CuRepo is consistent with the reported details.

Once curator agrees with all fields under Variant information, press Approve variant. Variant changes thestatus from Pending/ Marked into Public.

NOTE: Curator cannot approve a variant with Warnings which are not yet resolved and will inform user accordingly.This implies all fields where system has the mandatory symbol and where terminology invalid or field is blank



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Once this step is complete, curator proceed with variant curation following the ACMG guidelines, andconsidering the specifics established and implemented at Centogene.

7.2.2 Curation under Curated ACMG Rationale

Subjected to this curation are variants with inconsistent Clinical significance and ACMG class. Systemcompares the clinical significance against the ACMG class (if any), and the aim of this step is to have a QCclass of Perfect match.

Remember that following ACMG rules are pre-calculated according to ACMG automation at Centogene:PVS1, PVS2, PS1, PM1, PM2, PM4, PM5, PP2, PP3, PP5, BA1, BS2, BP3, BP4, BP6 and BP7

Curator can manually reject or edit the automatically generated rules.

The following rules are subjected to manual assignment: PS2, PS3, PS4, PM3, PM6, PP1, PP4, BS1, BS3, BS4,BP1, BP2, BP5

During variant curation following ACMG guidelines, follow closely the appropriate related GLA SOP (seeapplicable documents)

Going through ACMG class evidences, curator reviews all most up to date available information and decidesif clinical significance class must be corrected or not.

Below specific curator actions and challenges using examples as available are described. If any uncertaintyencountered during this process, contact your supervisor.

One variant curation example is provided under 7.2.3 In this chapter are provided examples by ACMGevidence type.

In the situation that ACMG class after completing the steps below are not supporting the clinicalsignificance, and QC class is not on Perfect match, follow the SOPeIT- 84 Reclassification of GLA variants.

Review of PVS1 / Null or truncating for GLA gene:



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GLA variants assigned for PVS1 must follow under truncating effect definition as implemented (see VALeIT-58 Automatic Variant Classification based on ACMG guidelines (AVCA2.0).

Here is one example where PVS1 is automatically assigned (see screenshot below). Note that PVS1 isindicated under assigned rules, the PVS1 rectangle is red- filled by default.

You can select the rectangle corresponding to PVS1, and under Selected Pathogenic ACMG Rule (by defaultblank), is now indicated the available description (see below GLA variant c.128del; p.G43Afs*78)



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Limitation (I): this limitation refers to the following mutation types, which are not yet included in theautomatized PVS1 execution: gross and gene rearrangements, conversions and inversions. Such variant willnot have a PVS1 assigned, and under External information no data to display (the most gross / generearrangements do not follow HGVS guidelines in the original sources in order to be automaticallyretrieved). Therefore, curator must manually act here as described below for the GLA variant c.640_*18del,where deletion of exon 5 to 7 has been confirmed:

In order to assign PVS1 for this variant, click on PVS1 rectangle (by default no red fill). Type your descriptionand note that two options A (assign) or R (reject) are now indicated. Note that system denies A or R selectionwithout a description (Save option not active until description/ rationale entered)



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Add your rationale under Selected Pathogenic ACMG Rule: PVS1. Note that Save option is now active! Checkthe rationale against accuracy and against grammatical errors (if any; remember that grammar check isintegrated into the free text box). Once ready, and no other correction/ adjustment required, press A, andSave:

Once pressing Save option, system calculates automatically the ACMG class (note that from Not classified,ACMG class changes into Uncertain), and informs that data was saved successfully.



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Limitation (II): this limitation refers to those truncating variants and localized within the last exon or intron.

Therefore, GLA variants localized in exon 7 (GLA transcript NM_000169.2 contains seven exons), are notautomatically assigned for PVS1.

Curator must evaluate the impact of the truncating mutation type on protein by reviewing functionalevidences: this can refer to other already classified pathogenic GLA truncating variant at the same codon ordownstream from the last codon of interest; or levels of Lyso-Gb3 pathological biomarker in vivo.

For example, GLA variant c.1208del, p.L430X is a nonsense variant localized in exon 7. By default, PVS1 is notassigned (due to its location within last GLA exon).



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From the report of the assigned rules, PVS2 has been assigned based on clear Lyso-Gb3 pathological levelsobserved internally in vivo. Curator can check accuracy of this observation in Curepo and agree to assignPVS1 ACMG criterion based on functional impact observed Lyso-Gb3 levels.

This observation is important to understand the complete types of evidences for both the variant ofinterest, and for those variants having a truncating impact on this codon (i.e. 430) or downstream but notyet associated with functional evidences.



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Review of PVS2 / Internal biomarker for GLA gene:

The PVS2 rules has been developed and implemented at Centogene as pathogenic / very strong type ofevidence for variants which are BA1 free.

It belongs to variant based on evidences at case level (ONLY INFORMATIVE cases are considered) on Lyso-Gb3 biomarker

The PVS2 minimum cutoff of lyso-Gb3 is >=2.14 ng/ml with a reference of <=1.8 ng/ml. Note that 1.8 ng/mlrepresents the pathological diagnostic cutoff (not the PVS2 acceptable minimum cutoff).

For GLA gene, a patient is informative with clinical picture suggestive for FD, or no info/ unknown, with ahem, het or hom (very rare) genotype, and Lyso-Gb3 of min 2.14 ng/ml, when reference for pathogeniccutoff is <=1.8

One informative case is considered sufficient to assign PVS2

Treat with caution the “asymptomatic” label, since it might me “on treatment” or “atypical/ mild” (i.e. notinformative case)

Do not consider Lyso-Gb3 biomarker results from follow up patients during PVS2 assignment

Do not consider patients with other genotype as specified under “informative case” definition

Exception: you might apply PVS2 based on Lyso-Gb3 levels of min 2.14 ng/ml for a PVS1 variant inpresence of a missense (but not for the missense variant until informative case available to furtherclarify the impact on the biomarker levels)

Do not consider patients with no FD suggestive picture (note that GLA belongs to incidental findings genescategory)

The Lyso-Gb3 >= 2.14 ng/ml PVS2 cutoff (ref <=1.8 ng/ml) applies to GLA variants which are rare (<0.01MAF) and not already classified as B/LB

A rare GLA variant with Lyso-Gb3 >= 2.14 ng/ml but <2.8 ng/ml and no further supporting -ACMG evidence(except for PM2, i.e. rare) must be initially classified and curated with precaution and consultation withmedical director /CGO is mandatory



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PVS2 is compatible with PS3 (see PS3 below), but not with BS3

To curate PVS2, repeat steps as described under PVS1: select the PVS2 rectangle, edit description (note thatPVS2 has by default the description template available), assign and save.

Below the GLA variant c.1090_1103del; p.Y365Cfs*5

Before editing (note that under Selected Pathogenic ACMG Rule: PVS2, the description indicates the standardtemplate for PVS2 rationale

Since PVS2 is already assigned, you need only to edit the description (replace the ADD MAX VALUE accordingly). TheSave button is thus activated, and press Save! In this situation, no ACMG class change expected to occur.

NOTES:

Variants which are linked with Lyso-Gb3 levels between the minimum pathological diagnostic cutoff (i.e. >1.8 ng/ml;reference <=1.8 ng/ml) and not reaching a maximum of PVS2 cutoff (i.e.< 2.14 ng/ml; reference <=1.8 ng/ml) areevaluated as PS3 (see below the PS3 criterion).

In presence of PVS2, no need to assign PS3 based on internal observations only.

A GLA variant pre-linked to PS3 due to no informative case reaching the minimum PVS2 cutoff of 2.14 ng/ml(reference <=1.8 ng/ml), must be assigned to PVS2 and the PS3 rationale adjusted accordingly (see PS3 below).Thus, the new evidences are tracked and visible to each curator, and the reason for reclassification (for thosevariants where PVS2 assignment changes the variant class) are easy to identify.

Review of PS1 / Same Amino acid for GLA gene:



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This rule is automatically assigned, and curator should check accuracy of the variant evaluation in theoriginal source(s), when other than CentoMD.

Remember that the same amino acid change must be previously established as pathogenic regardless ofnucleotide change. Beware of changes that impact splicing rather than the predicted amino acid change.

Based on the manual evaluation, curator can approve or reject this pre-assignment. In both circumstances,curator must add a summary of the findings from literature.

Below such example for the GLA variant c.1160T>C; p.L387P. The PS1 rule has been automatically assigned,based on the information that HGMD classifies the same amino acid as DM (for the cDNA changec.1160_1161delTCinsCT).

In order to check accuracy of this statement, click on HGMD link under External information (note thatClinVar does not contain this variant, and no annotation retrieved):



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The link leads to a new tab, where the internal HGMD interface opens and details are ready for evaluation:

Open the two publications, identify the key findings, summarize under the PS1 text field via editing, andsave. Remember that important is to summarize the evidences, which support a pathogenic impact. Wheninconsistent observations, not enough evidences or no statements towards pathogenicity identified in theoriginal publication(s), document accordingly and reject the PS1 criterion.

Accessing the PMID 28497441 (Oyanguren et al., 2017), curator cannot establish the pathogenicity of thevariant due to the following findings:

Only abstract avaible



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The GLA mutation identified in the case presentation is not named (and no match with the variant ofinterest; i.e. p.L387P possible)

From the second PMID (28509189; Sakamaki et al., 2014) curator identifies the following supportive findings:

The p.L387P was identified in a four-generation Japanese family with positive genotype- phenotypecorrelation

Six members of this family were confirmed to suffer from Fabry disease (based on deficient alpha-galactosidase activity.

The index patient and another two family members have been diagnosed with proteinuria; mother andbrother with cardiomyopathy.

Add these findings under PS1, and save.

Review of PS2 / Confirmed de novo for GLA gene

FD is an X-linked disease, and de novo inheritance is not expected to occur in FD patients typically

There are few FD patients described in the literature carrying de novo GLA variants. Usually the malepatients will present negative Fabry family history, and the genetic testing of the mother negative for theGLA variant of interest (for review see Pisani et al., 2015 PMID 26602202; Lee at al., 2018 PMID 31620600)

So far, at Centogene there is no such case identified. Nevertheless, there is a theoretical possibility, and



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therefore curator must be aware of such uncommon situations.

Review of PS3 / Functional assay for GLA gene

This ACMG rule referring to functional studies supportive of a damaging effect implies observations ofenzymatic activity in vitro and/ or in vivo. GLA variants associated already with PVS2 rule go through PS3evaluation in a manual manner whenever a PMID is linked to the variant of interest (meaning that varianthas been at least one time reported in the literature). The observations from literature are processedaccording to the SOPeIT-82 GLA classification SOP and are restricted to those characterized in FD- patients.

There are different situations (i-vi) expected to happen:

i) Internal observations (+PVS2) are concordant with published statements, and no conflict identified

ii) Internal observations (+PVS2) are concordant with published statements, and partial conflict identified

iii) Internal observations (+PVS2) are discordant with published statements (including full conflict)

iv) No internal observation available (-PVS2), and concordant published statements

v) No internal observation available (-PVS2), and discordant published statements

vi) Internal observations (-PVS2) supporting PS3 (enzyme activity, pathological Lyso-Gb3 within pathologicalrange but below PVS2 cutoff)

Subjected to PS3 review are all GLA variants linked with an external publication. Curators receive exportsbased on CuRepo content with the pre-associated PMID (s). At database level, curator sees these variantswith entries under References (note the screenshot below; two GLA variants are linked to correspondingpublication indicated as PMID)

Below, indications how to proceed for each situation are summarized; in case of unclear or not yet describedsituations, contact your supervisor.



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i) Internal observations (PVS2) are concordant with published statements, and no conflict identified

This situation refers to GLA variants pre-assigned with PVS2 (i.e. internal pathogenic Lyso-Gb3 levels observed invivo) and supportive damaging effects in the literature. It is important that all publications evaluated, are linked tothe FD phenotype and all experiments performed in vitro or in vivo are conflict- free.

Although, the PS3 ACMG rule will not be considered for calculating ACMG class (PVS2 is internally established andvalidated as pathogenic very strong criterion), it is important to gathered as much knowledge as possible, toanalyze and understand how Centogene’s classification scheme performs compared with other laboratories.

Example: GLA variant c.1160T>C; p.L387P. For this variant PVS2 is assigned. Under External databases, HGMDlabels the variant DM in the context of Fabry disease (see hgmd_ phenotype).

Open the HGMD and summarize the functional findings accordingly. For this variant there is no conflict andsources support a damaging effect of this variant in FD context. Additionally, we have internal PS3 evidences (fourmale patients with alpha- galactosidase performed).

Therefore, select the PS3, add your description and save. Variant is updated, and ACMG class unchanged:



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ii) Internal observations (+PVs2) are concordant with published statements, and partial conflict identified

This situation refers to conflicting observations in the literature, where Centogene’s internal data (i.e. PVS2) confirmthe damaging effect described by some authors. The right action here is adding description as identified in theliterature (i.e. discordant results and summarizing the main findings), and reject the PS3 (based on the internalSOPs, we do not score inconsistent or conflicting information). The rejection of PS3 will not impact the ACMG class(PVS2 is pathogenic very strong) but offer a complete analysis and understanding of the up-to-date knowledgegathered internally and in the literature.

No example at this moment available.

iii) Internal observations (+PVS2) are discordant with published statements (including full conflict)

This situation refers to the theoretical case that GLA variants are linked to PVS2 based on internal evidences, butthe most/ all of the available observations in the literature are against damaging effect (i.e. BS3). In this situation,BS3 (see below a detailed description of the BS3 evaluation ) is rejected; the stored information under BS3 is importantfor further analysis and validation of PVS2 superiority compared with external findings.

No example at this moment available.



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iv) No internal observation available (-PVS2), and concordant published statements

Some GLA variants identified in FD context cannot be yet linked to FD- informative cases. This refers to screening ofeither female patients with normal Lyso-Gb3 measurements (i.e. carriers), or to males where two GLA variantsidentified, and the pathological Lyso-Gb3 levels cannot (yet) be assigned to any of the mutation (further analysisrequired to clarify if the 2 GLA variants function as haplotype or not).

Therefore, PS3 evaluation is mandatory and review of the existing literature is initiated. PS3 is assigned when onlydamaging effects are described in vitro or in vivo (no conflicting or tolerated effect reported). In this circumstance,PS3 counts for the calculation of ACMG class.

Example: GLA variant c.1067G>A; p.R356Q. This variant is free of PVS2 and HGMD database annotates as DM,ClinVar as likely pathogenic.

Internal data is based on five individuals (four males and one female). Four males are hemizygotes for this variant,with alpha-galactosidase reduced activity (confirmed in two males; two other males are associated with pre-analytical failure). The Lyso-Gb3 biomarker levels are within normal range, except for one male patient, linked topathological Lyso-Gb3 levels. But note that this male carries another GLA variant (genotype is on Other/ complex)in cis (X-linked disease; males carry one X chromosome). Since the other three males carrying only the c. 1067G>Avariant have Lyso-Gb3 levels within normal range, the pathological Lyso-Gb3 result from the Other/ complex malecannot be assigned to this mutation (see PVS2 criterion and informative case definition). The male withother/complex genotype cannot be considered informative for this mutation and cannot be used as evidence toassign PVS2 criterion (bayed on Lyso-Gb3 levels). Further clarification is required to understand the clinical impactof this GLA missense variant on FD phenotype.



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To further clarify, review the publications and summarize the functional characterization of this variant. At the endsummarize all identified findings, both from external databases as well as from internal enzymatic assays. Once thesupportive findings are complete, press A (assign) and save.

NOTE: remember that individuals with pre- analytical failure under enzymatic analysis results are excluded fromevaluation!

v) No internal observation available (-PVS2), and discordant published statements

As described under iv, in absence of internal observations and literature findings are not consistent, concluded orenough, PS3 is not assigned, but summary of the literature evaluation is documented under description, and PS3 isrejected.

At this moment, no example available.



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vi) Internal observations (-PVS2) supporting PS3 (enzyme activity, pathological Lyso-Gb3 within pathological rangebut below PVS2 cutoff)

S3 assigment in this situation refer to:

1. Enzyme activity observations

2. Lyso-Gb3 levels between diagnostic and PVS2 cutoff

1. Enzyme activity

The internal observations based on enyzme activity must originate from in vivo measurements in male Fabrypatients (i.e. with clinical picture suggestive for Fabry).

The internal observations should be completed, if the case, with external observations and documented when fullconsensus identified, or partial conflict identified.

Do not assign for borderline, slightly decreased and analytical failure. At least one informative case (definition iskept) must be identified to fulfill all requirements for PS3 in this situation.

2. Lyso-Gb3 levels between diagnostic and PVS2 cutoff

Variants linked at case level with Lyso-Gb3 biomarker values between pathological diagnostic value and PVS2 cutoffare assigned for PS3: Lyso-Gb3 levels: >1.82 ng/ml but <2.14 ng/ml (when reference is <=1.8 ng/ml)

In the absence of PVS2 criterion,all types of evidences supporting PS3 evidence must be summarized. Thus,enzymatic results from Fabry male patients , Lyso-Gb3 levels with max levels of >=1.82 to <2.14ng/ml originatingfrom both Fabry males and females must be documented.

When internal observations are originating only from Fabry females (i.e. Lyso- Gb3 between 1.82 and 2.13 ng/mland no enzymatic activity at hand), you need to complete the PS3 assignment with external observations (when thecase; look for enzymatic activity in vivo or in vitro as recommended by ACMG). When no external observationsavailable, remember that assignment of PS3 based on Lyso-Gb3 with maximum of 1.82 to 2.13 ng/ml must behandled with precaution and consultation with medical director is mandatory.

When variant is linked already with a publication, perform literature review as described above. Document thefindings as described above under this type of evidence.

Review of PS4/ Affected > controls for GLA gene

This ACMG rule is not evaluated for GLA gene in the context of FD.

Review of PM1/ Hot spot- critical domain for GLA gene

This rule runs automatically, and does not require manual curator interaction.

Review of PM2/ Absent in controls for GLA gene



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This rule runs automatically, and does not require manual curator interaction

Review of PM3= AR in trans for GLA gene

FD is X-linked disease

Review of PM4/ Protein length for GLA gene


Review of PM5/ Different AA for GLA gene

This rule must be reviewed as described under PS1 ACMG rule. The system automatically retrievesinformation if a different amino acid at the same codon has been previously reported as DM in HGMD,pathogenic/ likely pathogenic in ClinVar and CentoMD.

When other variant is reported pathogenic/ likely pathogenic according to CentoMD, no evaluation isexpected.

When variant is detected in the external sources only (HGMD and/ or ClinVar), no information about qualityof annotation is provided. To avoid an annotation in these sources which is not according to the initialpublication, curator needs to check the publication and at the end to decide if agrees or not with PM5 pre-assignment. In case curator does not agree with pathogenic/ likely pathogenic statements, rejects PM5 andadjusts the description (summarizing the identified evidences).

Curator needs to adjust the rationale according to the identified findings if agrees with PM5 assignmentsbased on supportive pathogenic/ likely pathogenic statements authors provides in their work.

The aim is to adjust if the case the accuracy of PM5 assignment/ rejection and bring the rationality up-to-date to be used then in downstream processes.

Example: GLA variant c.1229C>T/ p.T410I. This variant has been pre-associated with PM5 during pre- assignmentprocess. Two other AA at this codon, have been described according to HGMD as disease causing mutations: T410Pand T410K.



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Search on http://adm0010.blau.centogene.internal/hgmd/genesearch under GLA gene for T410P and T410Kmutations.

Open and evaluate the available publications, summarizing the findings and deciding if DM annotation fits to thisvariant class.

Based on the supportive evidence, both variants are indeed correctly annotated in HGMD as DM. Adjust rationaleaccording to the findings and save (compare the automatically generated rationale against the manually adjusteddescription).



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http://adm0010.blau.centogene.internal/hgmd/genesearch

Review of PM6/ Assumed de novo for GLA gene

This rule does not apply to GLA- FD association. Be aware of the rare situation described under PS2=Confirmed de novo.

Review of PP1/ Segregation for GLA gene

This rule is manually to be adjusted by curator if the case. Most likely this rule is supposed to be processedfor patients screened initially against WES or WGS (not Fabry screening).

To assign this rule you need either at least two independent trios (parents and index) or at least one familycomprising minimum five- family members.

A perfect genotype- phenotype correlation must be detected in order to assign PP1. Family members whereCI are missing or are not informative, are excluded. Be careful with “asymptomatic”status (i.e. healthy or ontreatment)

No example available at this moment

Review of PP2/ Low rate benign missense for GLA gene




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Review of PP3/ In silico or computational for GLA gene


Review of PP4/ Phenotype – family history for GLA gene

In some instances, when enzymatic analysis and Lyso-Gb3 biomarker not possible to measure, other typesof evidences shall be consider. This ACMG rule is such criterion, when detailed information about positiveFD family history received, variant not described elsewhere and only clarification about carrier statusrequested.

Therefore, at Centogene the PP4 criterion is applied in the absence of FD- informative cases in house, and /or in absence of any external databases to further clarify the variant clinical class. Most likely, PP4 isevaluated for carriers with positive FD- specific phenotype in the family.

No example to be here described available at the moment

Review of PP5/ Reputable source for GLA gene

This rule is automatically generated but requires some additional evaluation by curator. As reputable sourcethree databases are called: CentoMD, HGMD and Clinvar. Some circumstances can be encountered bycurator:

The only reputable source is CentoMD (including when CentoMD is discordant to HGMD/ ClinVar). It isexpected that assignment of PP5 based on CentoMD is associated with other types of evidences(biochemical, phenotype related, segregation etc.). Therefore, GLA variants linked with PP5 based onlyon CentoMD but no other evidence (to easily understand why CentoMD calls pathogenic/ likelypathogenic) implies searching for additional criteria (before agreeing with PP5 assignment). If curatordoes not find that piece(s) of evidence(s) based on which CentoMD says pathogenic/ likely pathogenic,its rejection is expected.

Only one external database (HGMD or ClinVar) contributes to PP5. In this situation, curator mustanalyzed the evidences for pathogenicity statement (to avoid any confusion or miss-classification).When those evidences are available (for example PS3 assigned and functional evidences summarized)and consistent with PP5, no additional evaluation is required. As in situation above, there is a need tounderstand the underlying evidence for “that reputable source” to conclude variant pathogenicity.

Example: GLA variant c.326A>G. Automatically, PS1, PM2, PP2, PP3 and PP5 are pre-assigned. Under PP5 isindicated that CentoMD and HGMD classifies the variant as pathogenic. No other evidence is activated, andtherefore the reason for classifying this variant as pathogenic in CentoMD is not yet transparent. The variant, isbased on this pre-assigned rules, predicted to be likely pathogenic. The reported class is pathogenic, and QC classon Partial match.



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In order to understand why CentoMD classifies as pathogenic this GLA variant, review the positive patients inhouse. One male patient is found: 1101667. This patient, despite being male is not informative for FD: no CIprovided, the enzyme not reliable (the result is linked to pre-analytical failure) and the biomarker is in the normalrange.

Left view:



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Right view:

Since in house no FD informative case to clarify the clinical class of the variant, a literature check must be initiated.The D109G variant was reported in an 80-year-old male with low alpha-galactosidase A enzyme activity and kidneydysfunction requiring hemodialysis. This variant was also identified in several of this patient's relatives, includingtwo females on hemodialysis; however detailed clinical information was not provided (Herrera et al., 2014). Basedon these observations, curator can assign PS3 ACMG rule as argument for DM annotation in HGMD.



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Review of BA1/ Allele frequency >=5% for GLA gene


Review of BS1/ Allele frequency < disease frequency for GLA gene

This rule is not at the moment evaluated for GLA- FD associations

Review of BS2/ Observed in healthy for GLA gene

This rule runs automatically, and does not typically require manual curator interaction.

The only specific information refers to the observations in female patients which are homozygote (notheterozygote) and in adult healthy males (as hemizygote)

Review of BS3 / Functional assay against for GLA gene

This evidence type is subjected to manual evaluation.



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In order to assign BS3, the results must originate from FD- informative cases (i.e. hemizygote male patients).

As functional evidences are used enzymatic activity, Lyso-Gb3 measurements and if the case findings fromliterature.

Only consistent findings (i.e. benign/ tolerated) are used to assign BS3. Inconclusive or conflicting findingsare documented, but BS3 rejected.

Internal observation on alpha-galactosidase and / or Lyso-Gb3 are treated as most reliable functionalevidence.

In the presence of PVS2 assignment, but discordant evidences from literature (see PS3 ACMG rule, point iii),curator adds under BS3 the external findings and rejects the rule.

In presence of normal alpha- galactosidase activity and / or Lyso-Gb3 within normal range (i.e. <1.8 ng/ml)curator must ensure that individual is not on ERT/ or other FD- specific treatment, before closing the variantevaluation.

Review of BS4 / No segregation for GLA gene

This rule is reviewed manually by curators and refers to the patients screened via WES or WGS with aphenotype FD- related or not.

In order to assign this rule, at least two trios are required, or a family comprising at least 5 members.

All GLA variants not segregating within these families with FD- phenotype are assigned for BS4 andrationales generated; remember that inconsistent observations are not expected to be assigned; but can bedocumented and BS4 rejected

No example can be provided at this moment.

Review of BP1 / Missense in LOF for GLA gene


Review of BP2/ Co-occurrences cis/ trans (AD/AR) same gene for GLA gene

This rule requires manual curator interaction

Males screened for GLA gene indicates if the GLA variants identified are or not in cis. Therefore, a rare GLAvariant, which is identified in a male carrying another clear pathogenic GLA variant, can be assigned for BP2.

Example GLA variant c.625T>A/ p.W209R. This variant is pre-assigned with PM2, PP2 and BP6.



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Under BP6, is mentioned as source CentoMD. From the pre-assigned rules, it is not obvious based on which criteriaCentoMD annotates this GLA variant as likely benign.

Looking at the positive patients in house, observe that one male is identified at Centogene: Patient ID 1133870.

This patient is linked with a statement Affected and under Number of associated CRV, note that there is anindication of co-occurrences (and the co- occurring variant is a truncating GLA variant):



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Based on this observation, curator assigns BP2:

Review of BP3 / Repeat in frame for GLA gene


Review of BP4/ In silico or computational benign for GLA gene


Review of BP5/ Diagnosis confirmed by other variant or gene for GLA gene

This rule implies manual evaluation by curator



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Curator gathers information on two different aspects: i) genetic diagnosis of FD was clarified by another GLAvariant(s) or/ and ii) genetic diagnosis was confirmed by other gene (no FD confirmed). For the 2nd diagnosisprecaution must be taken when dual diagnosis possible.

The same variant (i.e. c.625T>A) as described under BP2 can be assigned for BP5. The co-occurring variantin a male patient with X-linked MOI where a clear GLA pathogenic variant identified (based on CI and clearLyso-Gb3 levels) supports a benign impact on FD phenotype.

Curator assigns and save the rationale in CuRepo.

Review of BP6/ Reputable source benign for GLA gene

The BP6 rule is automatically generated, however requires manual evaluation by curator. The BP6 rulesmust associate with other benign types of evidences to understand the underlying reasons for the reputablesource to classify it as benign/ likely benign.

As mentioned under its compatible rule, i.e. PP5, the reputable source (CentoMD, HGMD and ClinVar) issubjected to extract the supportive benign findings

As example you can use the GLA variant c.625T>A. This variant was initially assigned for BP6 based on theCentoMD source. To understand the evidences leading to the benign class by CentoMD, curator performedcase- specific evaluation for functional assays and co-occurrences. After check, curator assigned beside theBP6 the BP2 and BP5 evidences.

All GLA variants linked to BP6 but with no other / not enough evidences returns via QC checks back tocurators.

Review of BP7/ Synonymous for GLA gene


7.2.3. Example of GLA variant curation



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Below it is described the process of complete GLA variant curation using one example

The final outcome of variant curation is to obtained a variant on status Public, and Perfect match for QCclass

GLA variant c.155G>T; p.C52F

From Variant Information, note that this missense variant has been reported as Pathogenic (clinical significance),and the ACMG class is pre-calculated as Likely pathogenic. The QC class is on Partial match.

Review the assignment report: This GLA missense variant is predicted to be deleterious (PP3), rare (PM2), and otherAA changes have been described as disease causing in HGMD. The GLA gene has a low rate of benign missensevariations (PP2) and CentoMD described this variant as Pathogenic.



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From this report, two questions must be answered:

1. Based on which internal evidences is CentoMD database classifying this variant as pathogenic

2. Are the two AA changes already described in HGMD supporting the deleterious effect?

In order to evaluate the internal observations, open the six patients carrying this variant in Individuals view:

Left:



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Right:



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From these six patients, five are females and one is a male. This male is clinically suspected to suffer from Fabrydisease, and the alpha- galactosidase levels were found to be clear pathological (at LLOQ level; matching the PS3ACMG rule). Additionally, the Lyso-Gb3 levels were higher than PVS2 cutoff for Lyso-Gb3 in males (73.5 ng/ml; PVS2Lyso-Gb3 cutoff is >= 2.14 ng/ml). Thus, for this variant, the assignment of PVS2 evidence must be considered.

Evaluate now the publication for the two other AA changes described in HGMD as DM (i.e. p.C52G and p.C52W). todo so, go under http://adm0010.blau.centogene.internal/hgmd/genesearch , search under gene for GLA. Thenselect Get Missense / nonsense and search for the C52 protein change to evaluate all AA changes at this codon.Search in literature the supporting deleterious impact on GLA, summarize, edit the PM5 accordingly and save.



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http://adm0010.blau.centogene.internal/hgmd/genesearch

Note that after PVS2 assignment, the ACMG class turns from Likely pathogenic into Pathogenic and the QC classfrom Partial match into Perfect match.

Review once again the assignment report:



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When no change / edit required and variant associated with Public status and Perfect match, variant curation isconsidered complete.

7.3 Curation of GLA variants by warning

Bioinformaticians are regularly running scripts to detect any deviations from the current SOPs, process known asQC checks (see SOPeIT-72). The QC are run as following:

1. Quarterly, before CentoMD and CentoLSD content release

2. Monthly / quarterly according to the metadata version updates

Curators may expect to receive re-evaluation notifications from the bioinformaticians whenever one of thefollowing situations is identified:

The GLA variant does not anymore follow the rare definition



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The GLA variant is with the new metadata update discordantly annotated

The GLA variant is now linked to an informative case

The GLA variant does not correlate with phenotype (no segregation)

The CRV GLA variant is identified in healthy/ asymptomatic

The CRV GLA variant is now linked with normal Lyso-Gb3 levels in informative GD patient

ACMG pre-annotated rules change the status based on fulfill criteria (PS1, PM1, PM2, PM4, PM5, PP3, BS2,BP3, BP4, BP7)

All these warnings are manually reviewed by curators within CuRepo system and acts according to all related GLAprocedures.

7.4 Track case history

System track automatically all changes applied during variant curation.

In order to understand the applied changes, select the History option, under the assignment report

Once History selected, a new window opens. The changes are highlighted in yellow.

For example, GLA variant used as example, i.e. c.155C>T the assignment of PVS2 evidence is highlighted (seeleft side of the screenshot) and the ACMG class update as well (see right side of the screenshot):

8. Referenceshttps://www.acmg.net/docs/Standards_Guidelines_for_the_Interpretation_of_Sequence_Variants.pdf

9. Appendices



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1. SOPeIT-86 APPX1_Classification scheme v4 pba.docx

2. SOPeIT-86 APPX2 Lyso-Gb3 pathogenicity score pba.docx

3. SOPeIT-86 APPX3_Training module GLA variant curation pba.xlsx



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https://app.qualio.com/attachments/442378?download

