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Title: Classification of GBA Variants following ACMG guidelines at CENTOGENE

Document type: SOP (english) IT support

Document ID: SOPeIT-81

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-81Classification of GBA Variants following ACMG guidelines at CENTOGENE Version: 3.0

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

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1. Purpose and ObjectiveThis SOP aims to standardize the application of ACMG guidelines for classification of variants detected in GBA atCENTOGENE in Gaucher patients; thus, optimizing, improving and streamlining the accuracy and reproducibility ofthe variant interpretation process.

2. Area of ApplicationThis SOP is mandatory for all employees responsible for classification, selection, reclassification and curation ofvariants in GBA gene.

3. Terms and AbbreviationsAVCA: Automatic Variant Classification based on ACMG guidelines

CRV: clinically relevant variants

CIV: clinically irrelevant variants

GD: Gaucher Disease

GBA: b-glucocerebrosidase

LOF: Loss of FunctionMLPA: multiplex ligation-dependent probe amplification

NGS: next generation sequencing

PSAP: Prosaposin

qPCR: quantitative polymerase chain reaction

VUS: variant of uncertain significance

4. Applicable DocumentsACMG Guidelines (Richards S, 2015)

SOPeR-36 Variant Classification following ACMG guidelines at Centogene

SOPeR-1 Validation and reporting of genetic results

SOPeIT-76 GBA Gene- Gaucher disease Association and Curation

MANe-20 User Manual CentoFiT Variant Filtering Software v1.3.6

VALeIT-58 Automatic Variant Classification based on ACMG guidelines (AVCA)

MANe7- Centogene Gepado Handbook

SOPeBS-24 Determination of biomarkers for the diagnosis of lysosomal storage diseases (Fabry disease, Gaucher´s disease, Niemann-Pick disease A/B/C) in plasma samples_ ABSciex 5500

SOPeBS-22 Determination of the biomarkers for the diagnosis of lysosomal storage diseases (Fabry disease,Gaucher's disease, Niemann-Pick disease A/B/C) in dry blood samples on TripleQuad 5500

SOPeCR-1 Report Sending

VALeBS-5 Concentration of biomarkers for Gaucher, Fabry and Niemann Pick in DBS by MRM-MS on ABSciex 5500

VALeBS-6 Concentration of biomarkers for Gaucher, Fabry and Niemann Pick in plasma by MRM-MS on ABSciex5500



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https://app.qualio.com/reference/MANe-20

https://app.qualio.com/reference/VALeIT-58

5. ResponsibilitiesThis SOP applies to all employees responsible for classifying genetic variants at CENTOGENE AG.

6. Reagents, materials and devices

INTERNAL DATABASES AND TOOLSUniDB: http://ts0001.russ.CENTOGENE.internal/unidbweb/variantsearch

CentoMD®: www.centomd.com

CuRepo: https://srv-centomd.CENTOGENE.internal/curation-repo/login

AVCA

A calculator tool used to generate supporting evidences to be evaluated for variant classification.Each evidence must be either accepted, rejected or marked as inconsistent/unknown/not applied. Adescription is necessary when selecting or rejecting an evidence. Please use full sentences and providecitations.

Instructions for using the AVCA calculator are described in MANe-20

CentoFiT

An interface for the filtering and evaluation of variants

Instruction for using CentoFiT are described in MANe-20

The latest version of AVCA is automatically used by CentoFiT.

EXTERNAL DATABASESThe Genome Aggregation Database (gnomAD) (Karczewski K, 2019)

a resource developed by an international consortium of investigators, with the goal of aggregating andharmonizing both exome and genome sequencing data from a wide variety of large-scale sequencingprojects, and making summary data available for the wider scientific community

The data set provided on this website spans 125,748 exome sequences and 15,708 whole-genomesequences from unrelated individuals sequenced as part of various disease-specific and populationgenetic studies.

HGMD (Stenson, 2003)

The Human Gene Mutation Database represents an attempt to collate all known (published) genelesions responsible for human inherited disease

The database contains 256,070 variants in 10,914 genes

ClinVar (Landrum MJ, 2018)

A public archive of reports of the relationships among human variations and phenotypes, withsupporting evidence

The database contains more than 500k unique variants representing over 30k genes

UCSC RepeatMasker (Kent WJ, 2002)

RepeatMasker is a program that identifies DNA sequences for interspersed repeats and low complexityDNA sequences.

UCSC uses the latest version of RepeatMasker (data last updated on 2009-04-24) and occasionally uses



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

http://www.centomd.com

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

updated versions of the software and libraries that are not yet available on the RepeatMasker website

InterPro (Mitchell AL, 2019)

o A database of protein families, domains and functional sites in which identifiable features found in knownproteins can be applied to new protein sequences in order to functionally characterize them

NCBI RefSeq (O'Leary NA, 2016)

o A comprehensive, integrated, non-redundant, annotated set of refernces eqeunces including genomic,transcript and protein.

PubMed

o A repository of biomedical literature (https://www.ncbi.nlm.nih.gov/pubmed/)

IN SILICO TOOLS

SIFT 4G (Vaser R, 2016)

A program that predicts whether an amino acid substitution affects protein function based onsequence

PolyPhen-2_HDIV and Polyphen2_HVAR (Adzhubei IA, 2010)

A tool that predicts the impact of amino acid substitutions on the stability and function of proteinsusing structural and comparative evolutionary data

The HVAR and HDIV versions provide scores after training on different datasets.

MutationTaster (Schwarz JM, 2014)

A Bayes classifier to predict the disease potential of a variant, calculating probabilities for the variant tobe either a disease mutation or a harmless polymorphism based on a large training set

Phylop100way_vertebrate (Pollard KS, 2010)

A score measuring evolutionary conservation; Positive scores - sites that are predicted to be conservedand Negative scores - sites that are predicted to be fast-evolving

GERP++ (Davydov EV, 2010)

A tool for the identification of constrained elements in multiple alignments, by quantifying substitutiondeficits i.e., sites that show fewer substitutions than would be expected to occur during neutralevolution

CADD (Rentzsch P, 2019)

A tool for scoring the deleteriousness of single nucleotide variants as well as insertion/deletionsvariants by integrating multiple annotations into one metric by contrasting variants that survivednatural selection with simulated mutations

DANN (Quang D, 2015)

A tool to recognize pathogenic variants by annotating genetic variants, and especially noncoding variantusing the same feature set and training data as CADD

ada_score (Liu X, 2011)

A method for predicting the splice altering effect of a single nucleotide variant located within the splicingconsensus region

An ensemble prediction score based on ada-boost and scaled from 0 and 1, and higher values indicatea greater probability that the variant will alter the splicing of the gene



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rf_score (Liu X, 2011)

A method for predicting the splice altering effect of a single nucleotide variant located within the splicingconsensus region

An ensemble prediction score based on random forests and scaled from 0 and 1, and higher valuesindicate a greater probability that the variant will alter the splicing of the gene

7. Procedure

Contents7.1 ACMG Guidelines Adaptation

7.2 Uncertain Variants

7.3 Reporting Variants

General considerationsThis variant classification document follows the guidelines and interpretation criteria established by ACMGand outlined in SOPeR-36. Below only GBA specific ACMG adaptations are in detailed described.

This classification is applicable to variants in GBA within GD context making gene and disease specificclassification modifications to the way genetic findings are evaluated, interpreted, validated and reported inSOPeR-1.

This classification scheme is informed by CENTOGENE’s knowledge in the diagnosis of GD (internal observedfrequencies, segregation data, genotype- phenotype correlation, co-occurrence, enzymatic and biomarkerlevels)

Variants undergoing classification have already undergone all quality filters (MANe-20)

An ‘informative case’ is defined as an individual which carries:

Either a homozygous GBA CRV (i.e. pathogenic or likely pathogenic variant), or

Either two GBA CRV (i.e. pathogenic or likely pathogenic variant) in trans

Additionally patients homozygotes or compound heterozygotes for GBA VUS variants are continuouslymonitored until the VUS variants are reclassified (part of GBA variant curation process or GBAreclassification process)

The following additional documents must be considered:

Appendix 1: Classification scheme

Evidence must not be counted twice so be aware of overlapping rules!

Criteria for pathogenic

Very strong (Pathogenic Very Strong - PVS)

Strong (Pathogenic Strong – PS)

Moderate (Pathogenic Moderate – PM)

Supporting (Pathogenic supporting – PP)

Criteria for benign

Stand-alone (Benign Alone – BA)

Strong (Benign Strong – BS)



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Supporting (Benign supporting – BP)

7.1 ACMG Guidelines Adaptation

PVS1

Definition

Rare null variant (nonsense, frameshift, canonical +/-1 or 2; initiation codon, single or multi exon deletion) onmultiple NCBI transcripts (RefSeq), located outside the extreme 3’ end. The variant is located within a gene whereLOF is a known mechanism of the disease [min. 80% of the truncating variants described in HGMD (DM), ClinVar(consistently annotated pathogenic and likely pathogenic) and CentoMD® are pathogenic and / or likelypathogenic].

Considerations for ApplicationLoss of function is a known mechanism of disease for variants in GBA (Cormand B, 1998; Park HW, 2012). Forpathogenic, likely pathogenic, likely benign or benign truncating GBA variants, 38/39 (97%) of all loss of functionvariants are classified as pathogenic or likely pathogenic according to CentoMD 5.5. The following coding effects areconsidered leading to loss-of-function: frameshift, nonsense, new translation termination codon, splicing variant,stop loss, gross rearrangement, gene deletion or conversion.

Use caution with splice variants that are predicted to lead to exon skipping but leave the remainder of the proteinintact. Multiple GBA isoforms are known, therefore use caution when determining if the variant effect meets thedefinition of truncating on all transcripts. The GBA transcript reference at Centogene is NM_000157.3. Alltranscripts refer to NCBI transcripts and the hg19 genomic build.

For truncating variants, PVS1 is automatically assigned by the AVCA calculator:



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AVCA cannot assign PVS1 for GBA gene conversion with GBAP pseudogenes. Therefore, you must assign manuallyPVS1 for those mutations detected by other method than NGS (gel electrophoresis, MLPA, qPCR).

To assign PVS1, add the variant of interest in the HGVSC search box and the Gepado order ID of the patient inwhich the variant was detected in the OrderID search box:



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Open the ‘Computation and predictive data’ tab and in the PVS1 section, select Apply to add description and pressSave:



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Now AVCA indicates that PVS1 has been assigned to the variant of interest:



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Inconsistent ObservationsExclude variants that are common, meeting BA1 or BS1.

Exclude variants at the extreme 3′ end of the gene, i.e. occurring in the last exon or the last 50 bp of thepenultimate exon



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PVS2

DefinitionRare GBA variant with confirmed pathological functional effect via in vivo measurements of Lyso-Gb1 biomarker(≥10 ng/ml). Only informative cases are used for PVS2 assignment. Do not classify GBA variants detected inindividuals carrying only one GBA CRV or VUS variant, or two GBA variants in cis. These cases have Lyso-Gb1 levels<10 ng/ml.

Considerations for ApplicationLyso-Gb1 (glucosylsphingosine) is a fully validated and highly reliable marker in GD, reflecting the severity andprogress of the disease (Hurvitz N, 2019; Rolfs A, 2013). The sensitivity of the biomarker for GD for a concentrationof ≥10 ng/ml is >99.9%. PVS2 is only assigned to informative individuals, those suspected of having GD withhomozygous or compound heterozygous variants detected. Use caution when co-occurring pathogenic/likelypathogenic GBA variants are detected.

To assign PVS2 to a variant, find the mean and standard deviation of Lyso-Gb1 levels of all informative individuals.PVS2 can be applied by selecting ‘A’ in the ‘PVS2’ section of the ‘Functional Data’ tab, entering a description and click‘Save’:



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Now AVCA indicates that PVS2 has been assigned to the variant of interest:



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Inconsistent Observations

Exclude individuals with inconclusive or failed biomarker assay

Exclude non-informative individuals or patients which are labelled as healthy / asymptomatic (until further



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clarifications with physicians).

PS1Definition

Rare missense variant where the same amino acid has been previously established pathogenic variant regardlessof nucleotide change in HGMD (DM), ClinVar (consistently annotated pathogenic and likely pathogenic) andCentoMD® (pathogenic and likely pathogenic).

Considerations for ApplicationApplicable to missense variants for which the amino acid change is a perfect match another missense variant thatis classified by CentoMD® as pathogenic or likely pathogenic. If the variant has not been classified internally,publications including HGMD and ClinVar can be considered. Be aware of changes that impact splicing ratherthan/in addition to changes to the amino acid.

Variants that appear in publications are automatically identified in the ‘Databases’ section of CentoFiT:

Clicking on this link will take the user to the publications in HGMD containing the variant of interest:http://adm0010.blau.centogene.internal/hgmd/variantsearch?query=CM870010

Clicking on the ‘Pubmed’ ID link will take the user to the publication in PubMed:

https://www.ncbi.nlm.nih.gov/pubmed/2880291?dopt=Abstract



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http://adm0010.blau.centogene.internal/hgmd/variantsearch?query=CM870010


Following the check of the publication, store the information in the ‘Computational and predictive data’ section byentering a ‘Description’ and pressing save:

Now AVCA indicates that PS1 has been assigned to the variant of interest:



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Inconsistent ObservationsExclude variants with low quality or contradictory publications

PS2

Definition



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De novo, both maternity and paternity confirmed, in a patient with the disease and no family history.

Considerations for ApplicationBecause GD is autosomal recessive, de novo inheritance is usually not expected, however, there are patientsdescribed in the literature carrying one inherited GBA variant and on de novo GBA variant. So far, at Centogenethere is no such case identified. Nevertheless, there is a theoretical possibility, and therefore you must be aware ofsuch uncommon situations. Confirmation of both maternity and paternity is required in addition to targetedconfirmation that the variant was not inherited.

When applicable, the information can be stored the decision in the ‘Segregation data and de-novo’ tab of AVCA inthe PS2 section by selecting ‘Status A’, entering a description and click ‘Save’:



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Inconsistent ObservationsExclude individuals where only the genotypes of the questioned variant are available (e.g. WES in index,carrier testing in parents)



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Exclude individuals with a family history inconsistent with de novo inheritance i.e. family history of GDreported.

PS3Definition

Rare GBA variants confirming at CENTOGENE a deleterious effect via in vivo measurements of beta-glucocerebrosidase enzymatic activity (<4.1 μmol/l/h).

Or/ And

Well-established in vitro or in vivo functional studies in literature supporting a damaging effect on the gene or geneproduct.

Considerations for ApplicationFunctional studies that have been validated and shown to be reproducible and robust in a clinical diagnosticlaboratory setting are considered. This includes internal enzyme activities, tests reported by the physician andpublished functional assays. Variants in PSAP can result in a GD-like phenotype despite normal glucocerebrosidaseactivity. For all patients suspected of having GD, the biochemistry results much be checked in Gepado.

Variants that appear in publications are automatically identified in the ‘Databases’ section of CentoFiT using assource HGMD database:

Clicking on this link will take the user to the publications in HGMD containing the variant of interest:

The publication must then be examined in PubMed for any functional assay which may clarify the deleterious effectin vitro or in vivo:



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After examining the publications and determining that in vivo studies indicate the variant is pathogenic, PS3 can beapplied. Store the decision in the ‘PS3’ section of the ‘Functional Data’ tab and click on save:



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Now AVCA indicates that PS3 has been assigned to the variant of interest:



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Inconsistent ObservationsExclude variants with low quality or contradictory publications



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PS4Definition

The prevalence of the variant in affected individuals is significantly increased compared to prevalence in controls(OR > 5.0 in GWASdb).

Considerations for ApplicationThere are no existing GBA or GD GWAS. This rule is not evaluated for GBA variants in the context of GD.

PM1Definition

Rare missense variant on multiple NCBI transcripts (RefSeq), located in a critical domain in InterPro / dbNSFP wheremin. 80% of the described variants (minimum 10) are pathogenic or likely pathogenic (in HGMD as DM, in ClinVarconsistently annotated as pathogenic or likely pathogenic, in CentoMD® as pathogenic or likely pathogenic).

Considerations for ApplicationThe functional domains of GBA, as defined by InterPro, include a Glycosyl hydrolase family 30, TIM-barrel domain atresidues 117-466 and a beta sandwich domain at residues 469-531. More than 80% of variants in the Glycosylhydrolase family 30, TIM-barrel domain are pathogenic or likely pathogenic (151 / 178 = 84.8%). Additionally, theactive site of the beta-glucocerebrosidase enzyme is within the TIM barrel domain and includes residues 235 and340. Less than 80% of the variants in the beta sandwich domain are pathogenic (13 / 21 = 61.9%). There for allmissense variants in the TIM barrel domain (c.49_1398) are included.

Assign PM1 to a missense in the functional domain (c.49_1398) and store the results in the ‘Functional data’ sectionof AVCA by selecting apply, entering a description and click on save:



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Now AVCA indicates that PM1 has been assigned to the variant of interest:



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Inconsistent ObservationsExclude truncating variants (type: exon_loss_variant, frameshift_variant, stop_gained, stop_lost, start_lost,splice_acceptor_variant, splice_donor_variant)

Exclude common variants (BA1 or BS1 are applied)



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PM2Definition

Extremely low variant frequency for the autosomal recessive GD in gnomAD and/ or CentoMD® (<=0.005).

Considerations for ApplicationThe estimated annual incidence of GD in the general population is 1 in 60,000 (0.001%) (Stirnemann J, 2017). Themajority of identified GBA variants are rare (97%), with an allele frequency less than 0.5%. Use care with largeinsertions/deletions as these variants can be underrepresented in public databases.

This information is automatically applied and is available in the ‘Population data’ section of the AVCA tool:



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Inconsistent ObservationsExclude common variants (BA1 or BS1 are applied)

PM3Definition

Rare variant is detected in trans with a pathogenic variant for an autosomal recessive disease and in a clinicallyaffected patient (parental testing included).

OR/ And

Rare variant can be assumed to be in trans with another pathogenic variant for an autosomal recessive disease ina clinically affected patient where PVS2 evidence is assigned.

Considerations for ApplicationFor application to a variant proven to be in trans orientation with a known pathogenic or likely pathogenic variantand detected in an affected individual. Testing of parents (or offspring) must confirm the trans orientation of thevariants. Use care to ensure the accurate pathogenic/likely pathogenic classification of the co-occurring variant.

The class of the other variants detected can be found in the ‘class_string’ column of CentoFiT. The detection in theparents/children for determining the orientation can be found in the ‘zygosities’ column of CentoFiT:

Save the applicable data in the ‘Allelic data’ tab of AVCA by selecting ‘Status A’ for PM3, entering a description andclicking ‘Save’:



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Inconsistent Observations:Exclude individuals where an alternative genetic cause is identified i.e. compound heterozygote orhomozygous pathogenic in GBA



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Exclude common variants (not meeting PM2)

PM4Definition

Rare variant is an in frame deletion or insertion on multiple NCBI transcripts (RefSeq), in a non- repeat region(according to Repeat Masker from UCSC) or a non-stop loss variant.

Considerations for ApplicationApplies to all in-frame insertions/deletions of variant type: disruptive in frame deletion, disruptive in frameinsertion, conservative in frame deletion, conservative in frame insertion. There are no repetitive, exonic regions ofGBA as defined by RepeatMasker to consider.

PM4 can be applied to any in-frame insertion/deletion by entering a description in the ‘PM4’ section of the‘Computation and predictive data’ tab of AVCA and click ‘save’:



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Inconsistent ObservationsExclude variants with a coding effect other than in frame insertion/deletion



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PM5Definition

Novel missense change at amino acid residue where a different missense change determined to be pathogenic inHGMD (DM), ClinVar (consistently annotated pathogenic and likely pathogenic), and CentoMD® (pathogenic andlikely pathogenic).

Considerations for ApplicationApplies to missense variant in a codon where a missense variant resulting in a different missense change has beenclassified by CentoMD® as pathogenic or likely pathogenic. If the variant has not been classified internally,publications including HGMD and ClinVar can be considered. Verify that the previously published variant iscausative, DM in HGMD, by evaluating relevant publications from PubMed. Be aware of changes that impactsplicing rather than/in addition to changing the amino acid.

Publications including the variant of interest can be found in the ‘Databases’ section of CentoFiT:

The applicable publications can be viewed in the Centogene HGMD web interfacehttp://adm0010.blau.centogene.internal/hgmd/variantsearch?query=CM993703 :

Using the links, the publications must be checked manually for quality (requirements in PS1) using PubMed:

When applying PM5, enter a description in the ‘PM5’ section of the ‘Computation and predictive data’ tab of AVCAand click ‘save’:



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Inconsistent ObservationsExclude variants with low quality or inconsistent publications



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PM6Definition

Assumed to be de novo but without confirmation of paternity or maternity

Considerations for ApplicationBecause GD is autosomal recessive, de novo inheritance is not expected however, there are patients described inthe literature carrying one inherited GBA variant and on de novo GBA variant. So far, at Centogene there is no suchcase identified. Nevertheless, there is a theoretical possibility, and therefore you must be aware of suchuncommon situations.

When applicable, the information can be stored the decision in the ‘Segregation data and de-novo’ tab of AVCA inthe PS2 section by selecting ‘Status A’, entering a description and click ‘Save’:

Inconsistent ObservationsExclude variants that have been confirmed de novo (PS2 is applied)

Exclude individuals with a positive family history for GD

Exclude variants that are common (BA1 or BS2 are applied)

PP1



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Definition

Co-segregation with disease in multiple affected family members (at least five family members; or at least twoindependent trios) in a gene definitely known to cause the disease.

Considerations for ApplicationFor families with affected and unaffected individuals, the genotype-phenotype correlation must be consistent. Thephenotype of each affected individual must match the disease and for GD (SOPeIT-77). A minimum of 1 affectedand 3 unaffected OR 2 affected and 0 unaffected family members is required. The following genotype areacceptable: all affected are homozygotes and all unaffected are heterozygotes OR all affected are compoundheterozygotes and all unaffected are heterozygotes.

This information can be reported by the physician as found in the order in the ‘Documents’ tab of Gepado:

Additionally, apply to publications with a genotype-phenotype correlation in a family with minimum of 1 affectedand 3 unaffected OR 2 affected and 0 unaffected family members. This information can be found in thepublications section of CentoFiT:

The publications can be accessed following the link via HGMD(http://adm0010.blau.centogene.internal/hgmd/variantsearch?query=CM081636):

After reviewing the publication, enter the evidence in the ‘Segregation data and de novo’ section of AVCA byselecting ‘Status A’ for PP1, entering a description and clicking ‘Save’:



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Now AVCA indicates that PP1 has been assigned to the variant of interest:



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Inconsistent Observations:Exclude individuals with unknown phenotype



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PP2Definition

Missense variants on multiple NCBI transcripts (RefSeq), in a gene that has low rate of benign variants and in whichmissense are a common mechanism of the disease [min. 80% of the missense variants described in HGMD (DM),ClinVar (consistently annotated pathogenic and likely pathogenic) and CentoMD® are pathogenic and / or likelypathogenic].

Considerations for ApplicationGBA has a low rate of benign missense variation. For missense GBA variants classified as pathogenic, likelypathogenic, likely benign or benign, 8.5% (12/141) are classified as likely benign or benign. Additionally, missensevariation is a known cause of GD: HGMD reports 306 disease causing missense GBA variants. Therefore PP2 can beapplied for all missense variants in GBA.

Store the information in the ‘Functional Data’ tab of AVCA in the ‘PP2’ section and click ‘Save’:



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Inconsistent ObservationsExclude variants that are not missense in all transcripts

PP3



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Definition

Non- synonymous variants on multiple NCBI transcripts (RefSeq), where all in silico results (if any) must agree: SIFT(D), Polyphen2_HDIV (D), Polyphen2_HVAR (D, P), Mutation taster (A, D), phyloP100way_vertebrate (>=1.5), GERP++(>=2.0), CADD_raw (>=4.0), DANN score (>=0.5), RF_score >=0.6, Ada_ score >= 0.6.

Considerations for ApplicationBecause many in silico algorithms use the same or very similar input for their predictions, each tool cannot becounted as an independent criterion; PP3 can be used only once in any evaluation of a variant. A lack of predictiondoes not indicate a prediction of benign effect!

For Missense variants all in silico tools making a prediction must predict a pathogenic effect in the majority oftranscripts, ada and rf score are ignored:

Tool Deleterious threshold

SIFT D

Polyphen2_HDIV D or P

Polyphen2_HVAR D or P

Mutation taster A or D

PhyloP100way_vertebrate >=1.5

GERP++ >=2.0

CADD_raw >=4.0

DANN score >=0.5

The rf and ada score are used for splicing variants, together with phyloP and GERP conservation scores. Takespecial care for splice site variants as ada and rf scores are only available for substitutions but not for smallinsertions/deletions and only in the canonical splice region not for all other exonic/intronic regions.

Tool Deleterious threshold

rf_score >=0.6

ada_score >=0.6

PhyloP100way_vertebrate >=1.5

GERP++ >=2.0

The results for all tools are automatically generated and can be viewed in the ‘Computational and predictive data’section of the AVCA tool:



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Inconsistent ObservationsExclude Missense variants with any disagreement between prediction tools

Exclude truncating variants (PVS1 is applied)

PP4Definition

Patient phenotype of family history is highly specific for disease with a single gene etiology.

Considerations for ApplicationThe clinical manifestations of GD are highly variable, but the phenotype reported must be similar to symptomsknown for GD. The hallmark symptoms for the GD types 1, 2 and 3 can be found in SOPeIT-77. So far, atCentogene there is no such case identified. Nevertheless, there is a theoretical possibility, and therefore you mustbe aware of such uncommon situations.

If applicable, save the evidence in the ‘Other databases and other data’ section of AVCA by selecting ‘Status A’ for



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PP4, entering a description and clicking ‘Save’:

Inconsistent ObservationsExclude individuals with unknown phenotype

PP5Definition

Reputable source reports variant as pathogenic, evidences is not available for laboratory to perform anindependent evaluation (HGMD- DM; ClinVar consistently pathogenic and likely pathogenic).

Considerations for ApplicationRequires ClinVar with at least 2 entries as pathogenic or including expert panel review, or disease- specificdatabase classifies the variants as pathogenic or likely pathogenic.

The internal and external classifications can be found in the ‘Other databases and other data’ section of the AVCAtool:



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Inconsistent ObservationsExclude variants with inconsistent ClinVar classifications: P/LP + L/B or B or VUS, or VUS + B or L/B



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BA1Definition

Allele frequency is >5% in any of the following databases: gnomAD and CentoMD®

Considerations for ApplicationThe annual incidence of GD is about 1/60,000 (0.0016%). A common variant is unlikely to cause GD howeverexceptions are known to exist in other rare diseases so care should be taken especially in internal databases whichare enriched for Gaucher patients.

The gnomAD (‘Gnomad Exomes’) and CentoMD® (‘healthy_cohort_af’) allele frequency can be found in CentoFiT inthe variant table:

Store the information in the ‘Population data’ section by entering a ‘Description’ and pressing ‘Save’:



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Now AVCA indicates that BA1 has been assigned to the variant of interest:



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Exclude rare variants (PM2 is applied)

Exclude common variants with allele frequency < 5% (BS1 is applied)

BS1



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Definition

Allele frequency is greater than expected for the disorder (Orphanet, Genetic HomeRef).

Considerations for ApplicationThe annual incidence of GD is about 1/60,000 (0.0016%). Variants with an allele frequency greater than 0.01 (1.0%)in either gnomAD or internal databases meet the requirements for BS1.

Application of BS1 AND BS2 overrides pathogenic supporting/suggestive evidences to maintain a benignclassification.

The gnomAD (‘Gnomad Exomes’) and CentoMD® (‘healthy_cohort_af’) allele frequency can be found in CentoFiT inthe variant table:

Store the information in the ‘Population data’ section by entering a ‘Description’ and pressing ‘Save’:



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Now AVCA indicates that BS1 has been assigned to the variant of interest:



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Inconsistent ObservationsExclude rare variants (PM2 is applied)

Exclude very common variants (BA1 is applied)

Exceptions:



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Variant gnomAD AF CentoMD® AF Class Notes

c.1226A>G 0.23% 1.38% Pathogenic Published as diseasecausing (Lei K, 2018; ReuterMS, 2018; Robak LA, 2017)

c.1448T>C 0.13% 1.06% Pathogenic Published as diseasecausing(Lei K, 2018; Russo R, 2018;Feng Y, 2018)

BS2

Definition

Observed in healthy adult for autosomal recessive GD in gnomAD and / or CentoMD®.

Considerations for ApplicationGD is inherited in an autosomal recessive manner, as such homozygous healthy adults are required for applyingBS2. Use caution as GD can be late onset; the internal age at diagnosis of is 19.8 ± 19.3 years. Application of BS1and BS2 overrides pathogenic supporting and suggestive evidences to remain benign.

The identification of healthy adults is automatically applied and can be viewed in the ‘Population data’ section ofthe AVCA tool:



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Exclude individuals with unknown health status



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BS3Definition

Variants not confirming at CENTOGENE a deleterious effect via in vivo measurements of enzymatic activity and / orbiomarker level.

Or/ And

Well-established in vitro or in vivo functional studies that show (in literature) no damaging effect on proteinfunction or splicing.

Considerations for ApplicationF

For all patients suspected of having GD, the biochemistry results must be checked in Gepado. Internal biomarkerlyso-Gb1 lower than 4.8 ng/ml and beta-glucocerebrosidase enzymatic activity greater than 4.1 μmol/l/h arerequired for application of BS3. Additionally, normal enzymatic activity reported by the physician can be used asevidence for BS3.

Results of biochemical test can be found in the ‘Analysis’ tab of Gepado:

Select any available L-Gb1 Analysis:

All results of biochemical tests are found in a table with the interpretation:

To apply BS3 to variants found in informative individuals with normal levels of biomarker and enzyme activity, addthe description in the ‘BS3’ section of the ‘Functional data’ tab and click save:



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Additionally, BS3 can be applied to variants where in vitro or in vivo functional studies that show (in literature) nodamaging effect on protein function or splicing. Variants that appear in publications are automatically identified inthe ‘Databases’ section of CentoFiT. Clicking on the link will take the user to the publications in HGMD containingthe variant of interest:



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http://adm0010.blau.centogene.internal/hgmd/variantsearch?query=CR110552

Clicking on the ‘Pubmed’ ID link will take the user to the publication in PubMedhttps://www.ncbi.nlm.nih.gov/pubmed/21087600?dopt=Abstract:

After reading the publication, if the variant has been shown by in vitro or in vivo assays not to be damaging toprotein function or splicing, store the information in the ‘Functional data’ section by entering a ‘Description’ andpressing save:



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http://adm0010.blau.centogene.internal/hgmd/variantsearch?query=CR110552





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Inconsistent ObservationsExclude individuals with inconclusive biomarker/enzyme levels

Exclude inconsistent and/or low quality published data



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Cannot be applied with PS3

BS4Definition

Lack of segregation in affected family members

Considerations for ApplicationTo be applied to a minimum of 2 GD affected family members and no genotype-phenotype correlation. Be awarethat families may have more than one pathogenic variant contributing to a disorder, further confounding anapparent lack of segregation. So far, at Centogene there is no such case identified. Nevertheless, there is atheoretical possibility, and therefore you must be aware of such uncommon situations.

This information, as reported by the physician in the order, can be found in the ‘Documents’ tab of Gepado:

Enter the evidence in the ‘Segregation data and de novo’ section of AVCA by selecting ‘Status A’ for PP1, entering adescription and clicking ‘Save’:

Inconsistent ObservationsCannot be applied with PP1



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Exclude individuals with no clinical information

BP1Definition

Missense variants on multiple NCBI transcripts (RefSeq) in a gene for which primarily truncating variants (min 80%)are known to cause the disease (from the total of pathogenic and likely pathogenic variants described in HGMD,ClinVar and CentoMD®)

Considerations for ApplicationNot applicable to GBA variants as truncating variants are not the primary cause of disease; 18.5% ofpathogenic/likely pathogenic variants are truncating (nonsense or frameshift).

BP2Definition

Observed in trans with a pathogenic variants for a fully penetrant dominant gene/ disorder or observed in cis witha pathogenic variant in any inheritance pattern.

Considerations for ApplicationFor any heterozygous or homozygous variant detected in cis with a pathogenic variant. The orientation of thevariant must be confirmed by parental (or child) testing. The accurate classification of the co-occurring variantmust be verified.

The class of the other variants detected can be found in the ‘class_string’ column of CentoFiT. The detection in theparents/children for determining the orientation can be found in the ‘zygosities’ column of CentoFiT:

Save the applicable data in the ‘Allelic data’ tab of AVCA by selecting ‘Status A’ for PM3, entering a description andclicking ‘Save’:



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Now AVCA indicates that BP2 has been assigned to the variant of interest:



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Inconsistent ObservationsExclude common variants (BA1 applied)



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BP3Definition

Variant is an in frame deletion or insertion on multiple NCBI transcripts (RefSeq), in a repetitive region (according toRepeat Masker from UCSC).

Considerations for ApplicationThis evidence is not applicable to variants in GBA because the UCSC RepeatMasker identifies no repetitiveelements in the exons of GBA.

BP4Definition

Variants where all in silico results (if any) must agree: SIFT (T), Polyphen2_HDIV (B), Polyphen2_HVAR (B, T), Mutationtester (N, P), phyloP100way_vertebrate (<1.5), GERP++ (<2.0), CADD_raw (<4.0), DANN score (<0.5), RF_score <0.6,Ada_ score <0.6.

Considerations for ApplicationBecause many in silico algorithms use the same or very similar input for their predictions, each algorithm cannotbe counted as an independent criterion. BP4 can be used only once in any evaluation of a variant.

For Missense variants, all in silico tools making a prediction must predict a benign effect in a majority of transcripts:

Tool Benign Threshold

SIFT T

Polyphen2_HDIV B

Polyphen2_HVAR B

Mutation taster N or P

PhyloP100way_vertebrate < 1.5

GERP++ < 2.0

CADD_raw < 4.0

DANN score < 0.5

For splicing mutations, ada and rf scores, together with conservations are considered:

Tool Benign Threshold

rf_score <0.6

ada_score <0.6

GERP++ < 2.0

The results for all tools are automatically generated and can be viewed in the ‘Computational and predictive data’section of the AVCA tool:



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Inconsistent Observations:Cannot be applied with PP3

Exclude variants with inconsistent predictions

Exclude variants when any tool fails to make a prediction

BP5Definition

Variant found in a case with an alternative molecular basis for disease.



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Considerations for ApplicationLimited to patients with a phenotype consistent with GD (SOPeIT-76 ). Be aware that a dual diagnosis is possible.The accuracy of the classification of the potentially causal variant should be verified.

All detected GBA variants are visible in the variant table of CentoFiT:

When applicable, save the evidence in the ‘Other databases and other data’ section of AVCA by selecting ‘Status A’for BP6, entering a description and clicking ‘Save’:



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Now AVCA indicates that BP6 has been assigned to the variant of interest:



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Inconsistent ObservationsExclude individuals that are known to be from a consanguineous union.



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BP6Definition

Reputable source reports variant as benign, but evidence is not available to perform an independent evaluation(HGMD- DP, ClinVar consistently benign and likely benign)

Considerations for ApplicationFor variants where no functional data is published, 2 high confidence reports as benign or including expert panelreview in ClinVar can be used as evidence that a variant is not disease causing.

The internal and external classifications can be found in the ‘Other databases and other data’ section of the AVCAtool:



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Inconsistent ObservationsCannot be applied with PP5

Exclude variants with inconsistent ClinVar classifications; P/LP + L/B or B or VUS, or VUS + B or L/B

BP7

Definition

Not conserved (GERP++ < 2.0) silent variant on all NCBI transcripts (RefSeq) for which splicing prediction is notsupportive (RF_score <0.6; Ada_ score : <0.6).

Considerations for ApplicationFor variants that are synonymous on all transcripts and at a position that is not highly conserved (GERP++ <2).



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Variants that do not change the amino acid may still have an impact on splicing, therefore ada_score and rf_scoremust be < 0.6.

The coding effect and results of splicing prediction can be found in the ‘Computational and predictive data’ sectionof the AVCA tool:

Inconsistent ObservationsExclude variants where either splicing predictions are missing

7.2 Uncertain VariantsA variant is classified as Uncertain when not enough evidences to score for LP/P, B/LB.

Variants with contradictory evidence, that is to say when criteria for both pathogenic/likely pathogenic and



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benign/likely benign are met, will be classified as VUS.

Variants with no criteria met will be classified as VUS.

7.3 Reporting VariantsAll classifications and descriptions produced by and entered into AVCA are stored and can be accessed viaCentoFiT (MANe-20). Pathogenic variants, likely pathogenic variants and variants of uncertain clinical significanceare included in the report. When a clinically relevant variant is classified, it is reported according to SOPeR-1 andSOPeCR-1.

8. ReferencesAdzhubei IA, S. S. (2010). A method and server for predicting damaging missense mutations. Nat Methods., 248-249.

Cormand B, G. D. (1998). Molecular analysis and clinical findings in the Spanish Gaucher disease. Hum Mutat, 295-305.

Davydov EV, G. D. (2010). Identifying a high fraction of the human genome to be under selective constraint. PLoSComput Biol.

Feng Y, H. Y. (2018). Clinical and molecular characteristics of patients with Gaucher disease in Southern China. loodCells Mol Dis, 30-34.

Hurvitz N, D. T.-C.-V. (2019). Glucosylsphingosine (lyso-Gb1) as a Biomarker for Monitoring Treated and UntreatedChildren with Gaucher Disease. Int J Mol Sci.

Karczewski K, e. a. (2019). Variation across 141,456 human exomes and genomes reveals the spectrum of loss-of-function intolerance across human protein-coding genes. bioRxiv.

Kent WJ, S. C. (2002). The human genome browser at UCSC. Genome Res, 996-1006.

Konrad J. Karczewski, L. C. (2019). Variation across 141,456 human exomes and genomes reveals the spectrum ofloss-of-function intolerance across human protein-coding genes. bioRxiv.

Landrum MJ, L. J. (2018). ClinVar: improving access to variant interpretations and supporting evidence. Nucleic AcidsRes.

Lei K, Z. Y. (2018). A pilot screening of high-risk Gaucher disease children using dried blood spot methods inShandong province of China. Orphanet J Rare Dis .

Liu X, J. X. (2011). dbNSFP: a lightweight database of human nonsynonymous SNPs and their functional. Hum Mutat894-899.

Mitchell AL, e. a. (2019). InterPro in 2019: improving coverage, classification and access to protein sequenceannotations. Nucleic Acids Research .

O'Leary NA, W. M.-W.-A. (2016). Reference sequence (RefSeq) database at NCBI: current status, taxonomicexpansion, and functional annotation. Nucleic Acids Res, 733-745.

Park HW, L. Y. (2012). Novel frameshift mutation (Pro171fsX21) in neonatal type 2 Gaucher's disease. Gene, 170-173.

Pollard KS, H. M. (2010). Detection of nonneutral substitution rates on mammalian phylogenies. Genome Res, 110-121.

Quang D, C. Y. (2015). DANN: a deep learning approach for annotating the pathogenicity of genetic. Bioinformatics.,761-763.

Rentzsch P, W. D. (2019). CADD: predicting the deleteriousness of variants throughout the human genome. NucleicAcids Res, 886-894.



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Reuter MS, W. S. (2018). The Personal Genome Project Canada: findings from whole genome sequences of theinaugural 56 participants. CMAJ, 126-136.

Richards S, A. N.-F. (2015). Standards and guidelines for the interpretation of sequence variants: a joint consensusrecommendation of the American College of Medical Genetics and Genomics and the Association for MolecularPathology. Genet Med, 403-425.

Robak LA, J. I. (2017). Excessive burden of lysosomal storage disorder gene variants in Parkinson's disease. Brain,3191-3203.

Rolfs A, G. A. (2013). Glucosylsphingosine is a highly sensitive and specific biomarker for primary diagnostic andfollow-up monitoring in Gaucher disease in a non-Jewish, Caucasian cohort of Gaucher disease patients. PLoS One.

Russo R, A. I. (2018). Multi-gene panel testing improves diagnosis and management of patients with hereditaryanemias. Am J Hematol, 672-682.

Schwarz JM, C. D. (2014). MutationTaster2: mutation prediction for the deep-sequencing age. Nat Methods, 361-362.

Stenson, e. a. (2003). The Human Gene Mutation Database (HGMD®): 2003 Update. Hum Mutat, 577-581.

Stirnemann J, B. N. (2017). A Review of Gaucher Disease Pathophysiology, Clinical Presentation and Treatments. Int JMol Sci.

Vaser R, A. S. (2016). SIFT missense predictions for genomes. Nat Protoc.

9. Appendices1. SOPeIT-81 Classification of GBA Variants APPX1_Classification Scheme .docx

2. SOPeIT-81 APPX2 Training module GBA GD Classification pba.xlsx



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https://app.qualio.com/attachments/442494?download

https://app.qualio.com/attachments/442495?download