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Title: Curation of GLA screened cases

Document type: SOP (english) IT support

Document ID: SOPeIT-79

Author: Digital Data Products & Curation

Owner: Digital Data Products & Curation

Approver(s): Ellen Kargesapproved at 2020-01-28 15:21 (UTC +0100)

Approval date: 2020-01-28

Effective date: 2020-01-28

SOP (english) IT supportCentogene AG SOPeIT-79Curation of GLA screened cases Version: 2.0

Property of Centogene AG. Unauthorized distribution or copying prohibitedGenerated by Digital Data Products & Curation at 2020-02-21 14:35:08 (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 ofgenetic and phenotypic data from 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 AbbreviationsGLA: alpha- galactosidase

FD: Fabry Disease

HGNC: HUGO Gene Nomenclature Committee

HGVS: Human Genome Variation Society

HPO: Human Phenotype Ontology

CentoMD: Centogene’s Mutation Database

CuRepo: Curation repository

MOI: Mode of inheritance

CI: Clinical informatin

G2P: Genotype – to – Phenotype

CentoLSD: Centogene’s Lysosomal Storage Disease database

4. Applicable DocumentsSOPeIT-77 GLA gene- Fabry disease association and curation

SOPeIT-82 Classification of GLA variants

SOPeIT-86 GLA variant curation

SOPeIT-84 Reclassification of GLA variants

SOPeIT-28 Curation Repository Data Submission

SOPeIT-32 Curation Repository Software Description

SOPeIT-48 Quality checks - CuRepo

SOPeIT-72 Curation Repository General Quality Checks

SOPeIT-56 Curation repository Biochemistry import

SOPeIT-62 Curation Repository Other Import

SOPeIT-55 Curation Repository MLPA/qPCR Import

SOPeIT-46 SeqPilot Import into Curation Repository

SOPeIT-64 Curation Repository Illumina HTS Import



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5. ResponsibilitiesThis SOP applies to all employees responsible for curating patients screened within Fabry disease clinical context.

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

OMIM:https://www.omim.org

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

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

7. ProcedureContents

7.1. Curation of GLA screened patients on status pending

7.2. Curation of GLA patients on marked status

7.3. Curation of cases by warnings (patients on status Public)

7.4. Track case history

Before proceedingScope and goal of the case curation process

Curators are responsible for collection, association, update and review of genetic and phenotypic data of casesanalyzed 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 in order tomaximize curation accuracy, efficiency and assurance of the highest level of data quality in CentoMD andCentoLSD database.

Data gathering and curation are procedures developed and implemented in the Curation Repository system, thatis 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-oriented modelwhere characteristics belonging to a particular individual (patient information, clinical data, methodology anddetected genetic variants) are stored and associated together. Data gathering process is influenced by alreadyexisting 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 standard proceduresestablished in house (see additional documents). At database level, the reviewed cases change their status from“pending”, “marked” into “approved” case.

In order to start curation by case, all variants detected in this case must be approved. It aims at assuring that the



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

http://www.centomd.com

https://www.omim.org

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

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

entries belonging to an individual follow the rules for final statement closely, and that all associated data is inagreement with the agreed guidelines. The following factors are considered as critical for the final statement:variant significance, patient genotype (number of clinically relevant changes, their zygosity and location -i.e. cisvs. trans), inheritance pattern of the disorder, the sex of the patient (for X-linked diseases), the phenotypicdescription, and if available- levels of biomarkers.

NOTE: during curation by case, a review of the available documents in Gepado (medical reports, request forms,email communications, reclassification reports etc.) is a must. Whenever the curation by case ends with adiscordant conclusion comparing the issued medical report, request form or other related document, informimmediately your supervisor!

Workflow description

The curation process of GLA- screened individuals within FD clinical context takes place in CuRepo software.Data are imported weekly as briefly described below:

All new patients screened for GLA and linked with the status “completed” / “finalized” in Gepado are transferred inCuRepo (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 are retrieved. Additionally,the biochemical analyses are transferred and stored in the structured format CuRepo provides. On the importedcases, the GLA genetic variants are transferred from UniDB, and if Sanger sequencing performed, SeqPilot isused as most reliable source. Once import complete, patients are getting the status “pending” in Curepo.

When patient previously imported and curated (i.e. on status Public in CuRepo), but new information becomeavailable (like: analysis performed for further clarifications (i.e. MLPA, qPCR); new details on clinical status orfamily history provided; variants previously detected and classified are reclassified), patient turns into status“marked”. The status is automatically reversed from Public into Marked whenever new information is transferred /applied at CuRepo level.

Below are described three procedures: curation of patients on pending (i.e. new analyzed patients), curation ofpatients on marked (i.e. existing patients with new data / changed data) and, curation of patients on public (qualitychecks to detect possible errors/ inconsistencies; curation by warnings).

Curation of GLA screened case implies a review of the following types of evidences:

Patient and family details, provided by physician (external information): clinical status, family relationship,gender, family history, treatments, externally performed diagnostic tests, etc.

Internally generated results: GLA genetic variants, alpha- galactosidase and Lyso-Gb3 results

Patient genotype

Diagnosis statement (G2P correlation)

When running this process check all documented related to the patient to assure accuracy and completeness.During this process, opening the medical reports, request forms and any other related document is obligatory!

When inconsistencies / errors detected, inform your supervisor!

In the figure below, the workflow of GLA screened case curation is summarized

Figure: Schematical representation of the curation process at CENTOGENE



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Curation process is divided into the following three categories:

1. gene- disease association curation (see step 1; described in SOPeIT-77 Gene- disease association curation)

2. curation by variant (see steps 5), including curation by warnings at variant level via quality checks (see steps 7and 8) described in SOPeIT-86 GLA Variant curation

3. curation by case (see steps 2, 3 and 4), including curation by warnings at case level via quality checks (see steps7 and 8) described in this SOP, SOPeIT-48 and SOPeIT- 72.

A (plain grey background): processes within CuRepo; B and C (gray, vertical lines): processes outsideCuRepo

Below the following processes are described:

Curation of cases on pending (new screened patients)

Curation of cases on marked (patients set previously on public status, but with new information available)

Curation of cases by warning (patients on status public)

DURING THIS PROCEDURE, USE DECISION TREES FOR GENOTYPE AND STATEMENT AS DESCRIBEDIN APPENDICES

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

7.1. Curation of GLA screened patients on status pending

Scope: assure that all entries belonging to a new individual screened for GLA, follow closely the rules of Finalstatement, and associated data is in agreement with all issued documents for curators and availableguidelines.



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Procedure:

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

Select Curate symbol :

By default, Individuals module is active, and search is initiated form the by default window.

Add under Gene GLA and under status Pending as indicated in the screenshot below:

Press enter

All patients screened for GLA on pending are indicated:

Reserve the patient by selecting the Reserve? Option (see screenshot below)



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

Once you click, the patients is linked with your username, and no other curator can work on the same patient. Seescreen shot below to see how reservation message is seen by you:

Then click under Details on the blue arrow (see screen shot above)

Once patient is selected, a new tab open. In the screen below, note that by default all submodules are indicatedwith hidden details. To open a module, you need to select a module. All pending GLA cases have no statementsubmodule/ header (brown color). Analysis header is green, and biochemical header yellow. The patient-orientedview contains the following modules by default, after import: Clinical information, Family details, All orders. Theimported variants and biochemical data are stored under Analyses and Variants without phenotype



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Please note that patient cannot be approved without a statement generation or missing / incomplete mandatoryfiled.

All Variants must be on Public (see green header) in order to proceed with curation by case. If at least one varianton pending / marked, variant curation must occur first (See SOP GLA variant curation).

Select first Clinical information module and check if all mandatory fields are processed and accurate (againstrequest form in Gepado). Follow SOPeIT-28 CuRepo Data submission during curation of CI. Note that CI modulehas status Marked.



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Review fields according to request forms and when ready / complete press Approve

The CI information module is now linked to Public status



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Next step is to generate the genotype and the statement. Follow closely the genotype and final statement decisiontrees during this step!

To do so, select Add Phenotype option (see screen shot below). Note that after curator input on patient (i.e.approving CI module, Patient automatically changes the status into Marked)



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By selecting Add Phenotype option, a new tab opens (see screen shot below). The phenotype header isassociated with brown color.

Select Add New Phenotype option:



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New items are indicated: Disease and Gene. Remember (as described in SOPeIT-28 and SOPeIT-32) thatmandatory fields are marked with “*”. System denies proceeding with unprocessed mandatory fields!

Under Gene type GLA. During typing matches are indicating accordingly (see screen shot below)

When GLA typing is complete, system indicates all gene symbols containing “GLA”

Select the GLA gene. Once GLA selected, three more fields, mandatory for clinical statement are generated areready for processing: Inheritance, Genotype and Statement



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All available and pre-associated phenotypes under Master data (see figure SW and process workflow) aredisplayed for selection. Chose the right FD form as indicated in SOPeIT-77 Gene- Disease association.

Select the appropriate FD form. The tool will automatically fill the MOI according to Master data:



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Select the appropriate genotype following the Appendix Genotype decision trees:

Select Statement according to Clinical statement decision trees



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Press Store Phenotype Data

The Case view updated automatically. Status of the patient is marked. Clinical information (gray) Statement(brown) and Analysis (green) headers are on Public.Under Analysis and Variants without Phenotype (dark blue)there is no other analysis and variants to be associated with a statement. Therefore, system automaticallyactivates the option “Mark Reviewed” (option is now red; i.e. “ready to be clicked”).



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Press Mark Reviewed; Status of the Patient changes into Public and Mark Reviewed is again inactive (grey; seescreen shot below)

Now you can remove the reservation by clicking the Undo symbol:

Once is clicked, a message appears to ask for confirmation:



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Press OK, and Reservation is now ended, and if the case other curator can pick and select the case (containingthe last editing)

The Public status of the patient completes the curation of patients on pending, and another patient can be nowselected for curation

Patients linked to status Public are subjected to Curation by warning (see 7.3 Curation by warning; this SOP)

7.2. Curation of GLA patients on marked status

A patient on Marked status implies addition of new information, or a change under mandatory fields whichare key points for clinical statement (see Appendix clinical statement trees).

Procedure:

In order to identify the patients subjected for curation of marked cases, go under https://srv-centomd.centogene.internal/curation-repo/curation/individual and search under Gene for GLA and under Statusfor Marked. Press Enter

Page indicates all screened GLA patients o status Marked

Reserve the patient of interest:



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

Reserve and open one marked individual. A new window opens:

Check if genotype and statement are still according to the decisions trees.

To do so, open the available analysis tabs, review the zygosity and clinical class of the GLA variants and act



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according to the decision trees:

Description of the example patient below: the patient reserved is liked with two analyses (a Sanger and anMLPA/qPCR). Therefore, to understand the impact of the change on the patient, open each analysis andsee the clinical class of the GLA variants, and their zygosity.Below, note that under Sanger Analysis there is no pathogenic variant (0 variants).From MLPA/ qPCR one variant is detected. Therefore, the wildtype genotype and unaffected statementmay be impacted by the MLPA.Then open the MLPA /qPCR analysis tab, and note that there is a hemizygous GLA gross deletionidentified and classified as pathogenic.

Before applying any change to genotype and statement according to GLA- FD decision trees, review thebiochemical results. Note that Lyso-Gb3 levels are within pathological range.



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Therefore, according to decision trees, the new test (i.e. MLPA) brings relevant information affecting thecurrent genotype (i.e. hemizygous) and statement (i.e. affected).

Apply the corrections according to decision trees. Once a field is changed / processed, the Update option is active(below blue option)

Press Update. This option turns into green. Note that Statement header is still on Marked!

Now press Approve option. The Statement header (brown) is linked to Public status.



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With all headers on Public, Patient is now linked to an active Mark Reviewed option (see above the red header)

Press Mark Reviewed and note that Patient status is now Public.



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Undo under Reserved by you, press OK under the message and proceed to select another patient on marked(see 5.2 for more details).

7.3. Curation of cases by warnings (patients on status Public)

All individuals set on Public, are subjected to a quality control (QC) step, in order to identify any deviation fromthe established decision trees and to lower the risk of human errors.

Therefore, a number of scripts have been developed, implemented and regularly run by bioinformaticians (seeCuration Repository General Quality Checks SOPeIT-72) at CuRepo level. Bioinformaticians deliver excel fileswhere warnings and the identified deviations from SOP are indicated. Curators assign themselves for eachwarning and manually resolve it in CuRepo tool according to all GLA and Fabry related SOPs.

Once all warnings resolved, scripts run again until no warning received / no deviation from SOP identified.Below are summarized only GLA and Fabry specific QC at case level (see all performed QCs in SOPeIT-72):

GLA genotype vs CI vs gender

GLA genotype vs gender vs Lyso-Gb3 levels and interpretation

GLA genotype vs Statement

Statement vs CI vs gender



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CI vs Statement vs gender vs Lyso- Gb3 levels and interpretation

Number & Class of GLA relevant variants- Zygosity vs Genotype vs gender

CI vs GLA genotype vs statement

GLA genotype vs CI vs genotype vs enzyme vs biomarker

FD type versus age of the patient

FD type vs CI

7.4. Track case history

Every change applied in CuRepo during case curation process, are stored and visible under Show History option.

In order to review the changes applied to the case, select from Edit Data for/ Show History:

All changes applied are highlighted in yellow.



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Left side screenshot:

Right side screenshot:

8. ReferencesNone

9. Appendices1. SOPeIT-79 GLA Cases Curation APPX 1_ Genotype decision trees_oge.docx

2. SOPeIT-79 GLA Cases Curation APPX 2_ Statement decision trees_oge.docx

3. SOPeIT-79 APPX3_Training module GLA Case curation_v0.1 pba.xlsx



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