hours only. Property of Centogene AG. Unauthorized ... · CURATION REPOSITORY SOFTWARE DESCRIPTION 7.1 Curation of GBA screened patients on status pending Scope: assure that all entries

Title: Curation of GBA screened cases

Document type: SOP (english) IT support

Document ID: SOPeIT-78

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-78Curation of GBA screened cases Version: 2.0

Property of Centogene AG. Unauthorized distribution or copying prohibitedGenerated by Digital Data Products & Curation at 2020-02-21 14:34:11 (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 Gaucher disease clinical context at CENTOGENE AG,into a structured and standardized format. It utilizes a combination of computer-based tools and manual review inorder to assure the accuracy, efficiency and quality of the curation process.

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

3. Terms and AbbreviationsGBA: b-glucocerebrosidase

GD: Gaucher 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- 76 GBA gene- Gaucher disease association and curation

SOPeIT- 81 Classification of GBA variants

SOPeIT- 85 GBA variant curation

SOPeIT- 83 Reclassification of GBA 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 Gaucher 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

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 GBA screened patients on status pending

7.2. Curation of patients on marked status

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

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 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 procedures



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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

established 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 theentries 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 mandatory. Whenever the curation by case ends with adiscordant conclusion comparing the issued medical report, request form or other related document, informimmediately your supervisor!

A. Workflow description

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

All new patients screened for GBA 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 GBA 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 GBA screened case implies a review of the following types of evidences:

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

Internally generated results: GBA genetic variants, beta-glucocerebrosidase and Lyso-Gb1 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 GBA screened case curation is summarized



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Figure: Schematical representation of the curation process at CENTOGENE

Curation process is divided into the following three categories:

1. gene- disease association curation (see step 1; described in SOPeIT-76 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-85 GBA 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 outside CuRepo

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 GBA screened patients on status pending

Scope: assure that all entries belonging to a new individuals screened for GBA, follow closely the rules of



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Final statement, and associated data is in agreement with all issued documents for curators and availableguidelines.

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 GBA and under status Pending as indicated in the screenshot below:

Press enter

All patients screened for GBA 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:



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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 GBA 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

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 SOPeIT-85 GBA 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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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 (3rd option left; from top to bottom). 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 GBA. During typing matches are indicating accordingly (see screen shot below)

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



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

All available and pre-associated phenotypes under Master data (see figure Process Workflow above, beforeProcedure chapter) are displayed for selection. Chose the right GD form as indicated in SOPeIT-76 GBA Gene-Gaucher disease association and curation.



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

Select the appropriate genotype following the Appendix Genotype decision trees:



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Select Statement according to Clinical statement decision trees

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;compare the Mark Review bar, red above and grey below)



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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 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 GBA and under Statusfor Marked. Press Enter

Page indicates all screened GBA 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 GBA variants and actaccording to the decision trees:



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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 GBA variants, and their zygosity.Below, note that under Sanger, one pathogenic, heterozygous GBA variant is displayed, and variant is onPublic (i.e. approved).

Then open the MLPA /qPCR analysis tab, and note that there is a heterozygous GBA gross deletionidentified and classified as pathogenic.



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

According to genotype and statement decision trees apply corrections in the next step (if the case). Open thebiochemical analysis to check the Lyso-Gb1 levels



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In the example used here, genotype must be corrected from heterozygote into compound heterozygote andstatement from carrier into affected.

Add note: according to Lyso-Gb1 validation documents, presence of two heterozygotes GBA variants andLyso-Gb1 levels higher than 10 ng/ml (reference <=4.8 ng/ml) support a trans location of the two GBA geneticvariants.

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.

Undo under Reserved by you, press OK under the message and proceed to select another patient on marked



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(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 GBA and Gaucher related SOPs.

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

GBA genotype vs CI

GBA genotype vs Lyso-Gb1 levels and interpretation

GBA genotype vs Statement

Statement vs CI

CI vs Statement vs Lyso-Gb1 levels and interpretation

Lyso-Gb1 levels and interpretation vs Statement

Number & Class of GBA relevant variants- Zygosity vs Genotype

CI vs GBA genotype vs statement

GBA genotype vs CI vs genotype vs enzyme vs biomarker

GD type versus age of the patient

GD 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



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All changes applied are highlighted in yellow.

Left side screenshot:

Right side screenshot:



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8. ReferencesNone

9. Appendices1. SOPeIT-78 APPX3_Training module GBA Case curation_v0.1 pba.xlsx

2. SOPeIT-78 GBA Cases Curation APPX 2_ Statement decision trees_oge.docx

3. SOPeIT-78 GBA Cases Curation APPX 1_ Genotype decision trees_v2.0.docx



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https://app.qualio.com/attachments/427542?download

