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Title: Classification of GLA Variants following ACMG guidelines at CENTOGENE

Document type: SOP (english) IT support

Document ID: SOPeIT-82

Author: Digital Data Products & Curation

Owner: Digital Data Products & Curation

Approver(s): Ellen Kargesapproved at 2020-02-20 15:46 (UTC +0100)

Approval date: 2020-02-20

Effective date: 2020-02-20

SOP (english) IT supportCentogene AG SOPeIT-82Classification of GLA 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 14:12:16 (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 GLA atCENTOGENE; thus, optimizing, improving and streamlining the accuracy and reproducibility of the variantinterpretation process.

2. Area of ApplicationThis SOP is mandatory for all employees responsible for classification, reclassification and curation of variants inGLA.

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

CRV: clinically relevant variants

CIV: clinically irrelevant variants

DBS: Dried blood spot

FD: Fabry Disease

GLA: alpha-galactosidase A

LOF: Loss of function

MLPA: multiplex ligation-dependent probe amplification

NGS: next generation sequencing

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-77 GLA Gene- Fabry Disease Association and Curation

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

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

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



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

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. Eachevidence 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 usesupdated 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



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

http://www.centomd.com

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

known proteins 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 TOOLSSIFT 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, DANN: a deep learning approach for annotating the pathogenicity of genetic 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, dbNSFP: a lightweight database of human nonsynonymous SNPs and their functional2011)

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

rf_score (Liu X, dbNSFP: a lightweight database of human nonsynonymous SNPs and their functional 2011)



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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 Clinically Relevant Variants

General considerationsThis variant classification document follows the guidelines and interpretation criteria established by ACMGand outlined in SOPeIT-36.

This classification is applicable to variants in GLA gene

This classification scheme is influenced by CENTOGENE’s knowledge in the diagnosis of FD (internalobserved frequencies, segregation data, genotype- phenotype correlation, co-occurrence, enzymatic andbiomarker levels)

FD is X-linked, meaning affected males are hemizygous and affected females may be heterozygous orhomozygous. Neither males nor females are considered “carriers” since a single copy in males and themultiple mechanisms that can result in the expression of X-linked traits in females.

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

As standard, during variant classification interpretation all CentoFiT included sources (internal and external)are mandatory to be used for CentoFiT –analyzed cases (see MAN2-20_ User manual CentoFiT).

The following additional documents must be considered:

Appendix 1: Classification scheme

Evidence for classification should 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)

Supporting (Benign supporting – BP)



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7.1 ACMG Guidelines Adaptation

PVS1Definition

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 GLA (Kobayashi M 2019, Moiseev S 2019). For thetruncating GLA variants classified as pathogenic, likely pathogenic, likely benign or benign, all are classified aspathogenic/likely pathogenic (n=175). The following coding effects are considered: frameshift, nonsense, newtranslation termination codon, splicing mutation, start loss.

Use caution with splice variants that are predicted to lead to exon skipping but leave the remainder of the proteinintact. Multiple GLA isoforms are known, use caution to determine the variant coding effect is truncating for alltranscripts. The GLA transcript reference at Centogene is NM_000169.2.

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



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Inconsistent ObservationsExclude common variants (BA1 is applied)

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



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PVS2

DefinitionRare GLA variant confirming at CENTOGENE a deleterious effect via in vivo measurements of Lyso-Gb3 biomarker(≥ 2.14 ng/ml) and clinical suspicion of Fabry disease.

Considerations for ApplicationLyso-Gb3 (sphingolipid globotriaosylceramide) is a reliable marker in FD, reflecting the severity and progress of thedisease (Aerts JM 2008, Sakuraba H 2018). PVS2 can be applied when pathological biomarker levels, lyso-Gb3 ≥ 2.14ng/ml, are identified in informative individuals, hemizygous males suspected of having FD. Use caution when co-occurring pathogenic/likely pathogenic GLA variants are detected.

To assign PVS2 to a variant, find the mean and standard deviation of Lyso-Gb3 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 results

Exclude common variants (BA1 or BS1 applied)



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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 ApplicationApply to variants with coding effect: Missense variants where the amino acid change is a perfect match for anothermissense variant. Confirm that the previously published variant is causative for FD by examining HGMD, ClinVar,CuRepo, and relevant publications from Pubmed.

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

The applicable publications can be viewed in the Centogene HGMD web interface

http://adm0010.blau.centogene.internal/hgmd/variantsearch?query=CM051520:

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



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Save this information in the ‘Computation and predictive data’ section of AVCA by selecting ‘Status A’ for PS1,entering a description and clicking ‘Save’:

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



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Inconsistent ObservationsLow quality or contradictory publications



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PS2Definition

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

Considerations for ApplicationConfirmation of maternity is required in addition to targeted confirmation of that the variant was not inherited.Additionally, the phenotype of the index must match FD and a family history negative for FD. The hallmarkphenotypes for FD can be found in SOPeIT-76. The variant must be heterozygous in the index and absent from themother i.e. het:abs:abs or hem:abs:abs (excluding all other zygosities). So far, at Centogene there is no such caseidentified. Nevertheless, there is a theoretical possibility, and therefore you must be aware of such uncommonsituations.

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

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



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PS3Definition

Rare GLA variants confirming at CENTOGENE a deleterious effect via in vivo measurements alpha-galactosidase A inmales (< 15.3 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. Apply to variants with pathological results from CENTOGENE’s enzyme assay (> 15.3μmol/l/h) without biomarker level information.

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

The applicable publications can be viewed in the Centogene HGMD web interface:

Using the links, the publications must be checked manually for quality using PubMed:



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Store the information in the ‘Functional Data’ tab of AVCA in the PS3 section by selecting ‘Status A’, entering adescription and click ‘Save’:

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



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Inconsistent ObservationsLow quality or contradictory publications

PS4



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Definition

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 GLA or FD GWAS. This rule is not evaluated for GLA variants in the context of FD.

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 domain of GLA, an alpha galactosidase A, C-terminal beta sandwich domain as defined by InterPro,includes residues 325-411. More than 80% of variants in the functional domain are pathogenic or likely pathogenic(98 / 113 = 86.7%). PM1 can be applied to variants with coding effect missense in the alpha galactosidase A, C-terminal beta sandwich domain of GLA (c.973_1233).

Variants falling within this domain are identified by the ‘Functional Data’ section of the AVCA tool:



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



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Inconsistent ObservationsTruncating variants (Coding Effect: exon_loss_variant, frameshift_variant, stop_gained, stop_lost, start_lost,splice_acceptor_variant, splice_donor_variant)

Common variant (Application of BA1 or BS1)



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PM2Definition

Extremely low variant frequency for the X-linked Fabry disease in gnomAD and/ or CentoMD® (<=0.005).

Considerations for ApplicationThe estimated annual incidence of FD is 1 in 80,000 births but when late-onset variants are considered, aprevalence of 1 in 3,000 (0.03%) has been suggested (Hopkins PV 2015). The majority of identified GLA variants arerare (98%), with an allele frequency less than 0.5%. Apply to variants with a frequency <0.5% in gnomAD andinternal databases. Use care with large insertions/deletions as these variants can be underrepresented in publicdatabases.

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



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Inconsistent ObservationsCommon variant (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 ApplicationFD is inherited in an X-linked manner, multiple mechanisms can result in the expression of heterozygous X-linkedtraits in females. Therefore, the identification of a pathogenic variant co-occurring in trans would not be evidenceof pathogenicity for GLA variants.

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 (Variant type: disruptive in frame deletion, disruptive in frame insertion,conservative in frame deletion, conservative in frame insertion) GLA variants because there are no repetitive,exonic regions of GLA to consider.

Store the results in the ‘Computation and predictive data’ section of AVCA by selecting ‘Status A’, entering adescription and click ‘save’:



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Inconsistent ObservationsCommon Variants (Application of BA1, BS1)

PM5



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Definition

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 ApplicationApply to missense variants that where a different change to the same amino acid has been shown to bepathogenic. Determine if the previously published variant is causative; DM in HGMD, OR pathogenic / likelypathogenic in CuRepo AND HGMD, and relevant publications from PubMed should be evaluated. Beware ofchanges that impact splicing 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 interface:

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

Store the information in the ‘Computational and predictive data’ tab of AVCA in the PS3 section by selecting ‘StatusA’, entering a description and click ‘Save’:



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Exclude variants in low quality or inconsistent publications



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PM6Definition

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

Considerations for ApplicationConfirmation of maternity is required in addition to targeted confirmation of that the variant was not inherited.Additionally, the phenotype of the index must match FD and a family history negative for FD. The hallmarkphenotypes for FD can be found in SOPeIT-76. The variant must be hemizygous in the index and absent from themother i.e. hem:abs:abs (excluding all other zygosities).

So far, at Centogene there is no such case identified. Nevertheless, there is a theoretical possibility, and thereforeyou must be aware of such uncommon situations.

If applicable, store the decision in the ‘Segregation data and de-novo’ tab of AVCA in the PM6 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

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



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PP1Definition

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 ApplicationA minimum of 1 affected and 3 unaffected OR 2 affected and 0 unaffected family members is required. For familieswith affected AND unaffected individuals, the genotype- phenotype correlation must be consistent. The phenotypeof each affected individual must match FD (SOPeIT-76).

So far, at Centogene there is no such case identified. Nevertheless, there is a theoretical possibility, and thereforeyou must be aware of such uncommon situations.

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:

Review the publications in PubMed:



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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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Variant is confirmed de novo (PS2 is applied)

Exclude individuals with unknown phenotype

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 ApplicationGLA has a low rate of benign missense variation; 4.1% of missense variants (13/320) are classified as likely benignor benign. Additionally, missense variation is a known cause of FD (Kobayashi M 2019); HGMD reports 628 diseasecausing missense GLA variants. Applies to all missense variants in GLA.

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



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Now AVCA indicates that PP2 has been assigned to the variant of interest:



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Inconsistent ObservationsExclude variants that are not missense in all transcripts



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PP3Definition

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. The rf and adascore are used for splicing variants, together with phyloP and GERP conservation scores. Take special care forsplice site variants as ada and rf scores are only available for substitutions but not for small insertions/deletionsand only in the canonical splice region not for all other exonic/intronic regions. For variants that are missense on alltranscripts, ada and rf score are ignored.

Use caution with intronic variants as these are known to cause FD (Vaz-Drago R 2017).

A lack of pathogenic prediction does not indicate a benign effect!

For Missense variants all in silico tools making a prediction must predict a pathogenic effect in a majority oftranscripts:

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

For splicing variants rf and ada predictions as well as conservation:

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 truncating variants (PVS1 is applied)

Exclude variants with an established detrimental effect (PS3 is applied)

Exclude variants with disagreement between prediction tools as automatically determined in the‘Population Data’ section of the AVCA tool:

PP4Definition

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



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Considerations for ApplicationApplicable to informative individuals suspected of having FD and a family history of FD. Be aware that there issignificant phenotypic variability in individuals carrying the same genetic variant, even within the same family(SOPeIT-76).

When applicable, in the ‘Other databases and other data’ section of AVCA select ‘Status A’ for PP4, write adescription and click ‘Save’:

Inconsistent ObservationsExclude variants that are de novo (PS2 or PM6 are applied)

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 ApplicationInclude variants for which ClinVar with at least 2 entries as pathogenic or including expert panel review, or disease-specific database classifies the variants as pathogenic or likely pathogenic.

The classifications can be found in the ‘other databases and other data’ section of the AVCA tool:



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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

BA1Definition

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

Considerations for ApplicationThe annual incidence of FD is up to 1 in 3,000 (0.03%). A common variant is unlikely to cause FD however



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exceptions are known to exist in other rare diseases so care should be taken especially in internal databases whichare enriched for Fabry patients. Applies to variants with a frequency > 5% in gnomAD or internal database.

Add the information to the ‘Population data’ section of AVCA by selecting ‘Status A’ for BA1, entering a descriptionand clicking ‘Save’:



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

Inconsistent ObservationsExclude rare variants (PM2 is applied)



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BS1Definition

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

Considerations for ApplicationThe annual incidence of FD is up to 1 in 3,000 (0.03%). Only 14 variants (1.4%) have an allele frequency greater than1% in either database, all of which are benign/likely benign. Apply to variants with an allele frequency greater than0.01 (1.0%) in either gnomAD or internal databases. Application of BS1 and BS2 overrides pathogenic supportingand suggestive evidences to remain Benign.

Add the information to the ‘Population data’ section of AVCA by selecting ‘Status A’ for BS1, entering a descriptionand clicking ‘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)



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BS2Definition

Observed in healthy adult for X-linked Fabry disease in gnomAD and / or CentoMD®.

Considerations for ApplicationFD is X-linked, as such heterozygous/homozygous female or hemizygous male healthy adults are required forapplying BS2. Apply to any hemizygous, heterozygous or homozygous adult, healthy individual in internal orexternal databases. Application of BS1 AND BS2 overrides pathogenic supporting and suggestive evidences toresult in a benign classification.

The identification of healthy adults can be viewed in the ‘Population data’ section of the AVCA tool:




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Exclude individuals with no clinical information in internal databases.

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 ApplicationFor all patients suspected of having FD, the biochemistry results much be checked in Gepado. Internal biomarkerlyso-Gb3 lower than 1.8 ng/ml and alpha-galactosidase A enzymatic activity greater than 15.3 μmol/l/h in males arerequired for application of BS3. Additionally, normal enzymatic activity reported by the physician or reported in theliterature can be used as evidence for BS3.

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

Select any available L-Gb3 Analysis:

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

Store the information in the ‘Functional data’ tab of AVCA by entering a description and clicking on ‘Save’:



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Additionally, variants that appear in publications are automatically identified in the ‘Databases’ section of CentoFiT:



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The applicable publications can be viewed in the Centogene HGMD web interfacehttp://adm0010.blau.centogene.internal/hgmd/variantsearch?query=CR952427 :

Using the links, the publications must be checked manually for quality using PubMed:

Following the check of the publication, store the information in the ‘Functional’ section by entering a ‘Description’and pressing save:




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http://adm0010.blau.centogene.internal/hgmd/variantsearch?query=CR952427


Exclude individuals with inconclusive functional assay

Exclude variants with inconsistent or low quality published data



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Application of PS3

BS4Definition

Lack of segregation in affected family members

Considerations for ApplicationMinimum 2 affected family members with no genotype- phenotype correlation. Be aware that families may havemore than one pathogenic variant contributing to a disorder, further confounding an apparent lack of segregation.The family genotypes and phenotypes must be examined in Gepado.

This information can be reported by the physician as found in the order 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 ObservationsApplication of PP1



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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 GLA as truncating variants are not the primary cause of disease; 34.1% of pathogenic/likelypathogenic variants are truncating (nonsense, splice variant 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 ApplicationFD is x-linked inherited, therefore, BP2 can be applied for any variant detected in cis with a pathogenic variant. Theaccurate classification of the co-occurring variant must be verified. The orientation of variants as confirmed byparental testing must be determined manually.

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 variants that are common (BA1 or BS1 are 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 ApplicationNot applicable because UCSC RepeatMasker identifies no repetitive elements in the exons of GLA.

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

CADD_raw < 4.0

DANN score < 0.5

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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Exclude variants with inconsistent predictions

Exclude variants with missing predictions

BP5Definition

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

Considerations for Application



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Limited to patients with a phenotype consistent with FD. Be aware that a dual diagnosis is possible. The accuracy ofthe classification of the potentially causal variant should be verified.

All detected GLA variants are visible in the variant table of CentoFiT.

If applicable, save the evidence in the ‘Other databases and other data’ section of AVCA by selecting ‘Status A’ forBP6, entering a description and clicking ‘Save’:



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Now AVCA indicates that BP5 has been assigned to the variant of interest:



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

BP6



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Definition

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, two high confidence reports in ClinVar or disease-specificdatabase classifying the variant as benign can be used as evidence that a variant is not disease causing.

The classifications can be found in the ‘other databases and other data’ section of the AVCA tool:




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

BP7Definition

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 ApplicationApply to a variant that synonymous on all transcripts where there is no predicted effect on splicing (ada_score < 0.6and rf_score < 0.6) and the position is not highly conserved (GERP++ <2). Take additional care for variants outsideof the canonical splice region or insertions/deletions that may impact splicing.

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



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Inconsistent ObservationsExclude variants with missing splicing predictions

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

Variants with no criteria met will be classified as VUS.



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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.

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Karczewski K, et al. 2019. "Variation across 141,456 human exomes and genomes reveals the spectrum of loss-of-function intolerance across human protein-coding genes." bioRxiv.

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Kobayashi M, Ohashi T, Kaneshiro E, Higuchi T, Ida H. 2019. "Mutation spectrum of α-Galactosidase gene inJapanese patients with Fabry." J Hum Genet 695-699.

Landrum MJ, Lee JM, Benson M, Brown GR, Chao C, Chitipiralla S, Gu B, Hart J, Hoffman D, Jang W, Karapetyan K,Katz K, Liu C, Maddipatla Z, Malheiro A, McDaniel K, Ovetsky M, Riley G, Zhou G, Holmes JB, Kattman BL, MaglottDR. 2018. "ClinVar: improving access to variant interpretations and supporting evidence." Nucleic Acids Res.

Liu X, Jian X, Boerwinkle E. 2011. "dbNSFP: a lightweight database of human nonsynonymous SNPs and theirfunctional." Hum Mutat 894-899.

Liu X, Jian X, Boerwinkle E. 2011. "dbNSFP: a lightweight database of human nonsynonymous SNPs and theirfunctional predictions." Hum Mutat 894-899.

Mitchell AL, et al. 2019. "InterPro in 2019: improving coverage, classification and access to protein sequence annotations." Nucleic Acids Research.

Moiseev S, Fomin V, Savostyanov K, Pushkov A, Moiseev A, Svistunov A, Namazova-Baranova L. 2019. "ThePrevalence and Clinical Features of Fabry Disease in Hemodialysis Patients:Russian Nationwide Fabry DialysisScreening Program." Nephron. 249-255.

O'Leary NA, Wright MW, Brister JR, Ciufo S, Haddad D, McVeigh R, Rajput B, Robbertse B, Smith-White B, Ako-AdjeiD, Astashyn A, Badretdin A, Bao Y, Blinkova O, Brover V, Chetvernin V, Choi J, Cox E, Ermolaeva O, Farrell CM,Goldfarb T, Gupta T, Haft D, Ha. 2016. "Reference sequence (RefSeq) database at NCBI: current status, taxonomic expansion, and functional annotation." Nucleic Acids Res 733-745.

Pollard KS, Hubisz MJ, Rosenbloom KR, Siepel A. 2010. "Detection of nonneutral substitution rates on mammalian phylogenies." Genome Res 110-121.

Quang D, Chen Y, Xie X. 2015. "DANN: a deep learning approach for annotating the pathogenicity of genetic."Bioinformatics. 761-763.

Quang D, Chen Y, Xie X. 2015. "DANN: a deep learning approach for annotating the pathogenicity of genetic



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variants." Bioinformatics. 761-763.

Rentzsch P, Witten D, Cooper GM, Shendure J, Kircher M. 2019. "CADD: predicting the deleteriousness of variantsthroughout the human genome." Nucleic Acids Res 886-894.

Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, Grody WW, Hegde M, Lyon E, Spector E, Voelkerding K,Rehm HL. 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.

Sakuraba H, Togawa T, Tsukimura T, Kato H. 2018. "Plasma lyso-Gb3: a biomarker for monitoring fabry patientsduring enzyme replacement therapy." Clin Exp Nephrol 843-849.

Schwarz JM, Cooper DN, Schuelke M, Seelow D. 2014. "MutationTaster2: mutation prediction for the deep-sequencing age." Nat Methods 361-362.

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

Vaser R, Adusumalli S, Leng SN, Sikic M, Ng PC. 2016. "SIFT missense predictions for genomes." Nat Protoc.

Vaz-Drago R, Custódio N, Carmo-Fonseca M. 2017. "Deep intronic mutations and human disease." Hum Genet 1093-1111.

9. Appendices1. SOPeIT-82 Classification of GLA Variants APPX1_Classification Scheme pba.docx

2. SOPeIT-82 APPX2 Training module GLA FD Classification pba2.xlsx



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https://app.qualio.com/attachments/442596?download

https://app.qualio.com/attachments/442597?download