Whole Genome Sequencing Report

Patient: Primary Referrer:

Jean DOE Dr Make Youwell

1 Doe St The Best Hospital Darlinghurst NSW 2010 1 Hospital St Australia Darlinghurst NSW 2010 Australia Sex: Male Provider No. 123456AB DOB: 01-Jan-2000

Family ID: 17F12345 Request date: 10-Jan-2017 DNA Tube ID: FD01234567 Collection date: 15-Jan-2017 Unique Lab ID: 12345678 Received date: 20-Jan-2017 Your reference(s): ABC54321 Submitted specimen: EDTA blood

Test requested: Whole Genome Sequencing Primary Analysis: Bioinformatics analysis of all known protein coding genes Secondary Analysis: None requested Incidental Findings: Not reported as per consent form Samples analysed: Trio

Indications for testing: Seizures, severe intellectual disability, facial dysmorphism.

Family members tested: Name Relationship Status DOB Sex DNA Tube ID

Jean DOE Proband Affected 01-Jan-2000 M FR01234567 Ja… DO… Mother Unaffected 02-Feb-1980 F FR01234568 Bo… DO… Father Unaffected 03-Mar-1978 M FR01234569

Summary of results:

A de novo MOSAIC HEMIZYGOUS pathogenic variant has been identified in the WDR45 gene.

Result / Genotype: Gene Variant Location Zygosity Disorder(s) ACMG Classification

WDR45 ChrX(GRCh37):g.[48932540_ Exon Hemizygous Neurodegeneration Pathogenic 48932541=/del];[0] 13 of 13 (mosaic) with brain iron (Class 5) NM_007075.3:c.[1007_1008=/ accumulation 3 del];[0] (OMIM #123456) p.[(Tyr337=/Tyr337Cysfs*5)]; [0] This variant was present in the Proband, and confirmed by Sanger sequencing. The variant was NOT detected in the Mother or Father.

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Genome.One Name: Jean DOE DOB: 01-Jan-2000

Interpretation: Whole genome sequencing has identified a de novo hemizygous truncating variant in WDR45. Both whole genome sequencing (WGS) and Sanger sequencing data are consistent with mosaicism for this variant, with a minority of reads on WGS being non-deleted and small peaks consistent with the presence of the normal allele observed on Sanger sequencing.

De novo variants in WDR45 have been reported in patients with neurodegeneration with brain iron accumulation (NBIA), a condition characterized by relatively static intellectual disability in childhood, followed by a neurodegenerative course in adolescence or adulthood. The majority of affected individuals are female, and evidence for mosaicism has been seen in at least one other affected male. However, a more severe phenotype with epileptic encephalopathy presenting in infancy has been reported in a small number of males with apparently non-mosaic WDR45 variants. The c.1007_1008del variant is absent from population databases and has been reported previously, in two affected women.

This is classified as a PATHOGENIC variant, by ACMG/AMP criteria for the classification of sequence variants. The classification of this variant may change over time, with additions to the literature or new clinical information.

We recommend the following: • Correlation of this result with the clinical phenotype • Genetic counselling as appropriate

Comments: This family should be referred to a clinical genetics service for genetic counselling regarding this result.

Report Issued by Genome.One Pty Ltd

Authorised: 12:00 on 01-Jan-2017

Dr Genetic Pathologist Report Status: MBBS PhD FRCPA FHGSA Final

Genetic Pathologist

370 Victoria Street Darlinghurst Sydney NSW 2010 Australia E: [email protected] T: +61 2 9359 8002 W: www.genome.one

Report copied to: Sally Smith; Associate Genetic Counsellor; Best Hospital; Neurology Department; Sydney; Australia

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Genome.One Name: Jean DOE DOB: 01-Jan-2000

SYNOPTIC REPORT

Gene Variant Location Zygosity Disorder(s) ACMG Classification

Mendelian disorders review: Neurodegeneration Population frequency: The variant is absent from with brain iron accumulation 5, also known as beta- the ExAC v0.3, 1000 genomes, and Genome.One propeller protein-associated neurodegeneration clinical databases. (BPAN), is an X-linked disorder characterized by global developmental delay in childhood, followed Protein functional domain: The variant results in by progressive dystonia, Parkinsonism, truncation of the protein, distal to the most C- extrapyramidal signs and dementia (Haack et al., terminal of the WD repeat domains. 2012; Hayflick et al., 2013; Saitsu et al., 2012). Biophysical changes: N/A Neurodegenerative features typically become apparent in adolescence or early adulthood, Conservation: The C-terminus of the WDR45 although later onset has been seen (Hayflick et al., protein, predicted to be truncated by this variant, is 2013). The majority of affected individuals are highly conserved in vertebrates. female, and it has been suggested that somatic In silico predictions: Not applicable mosaicism for a normal allele is required for viability in males, with evidence of mosaicism seen in at In silico splicing predictions: Splicing effect least one affected male (Haack et al., 2012). prediction software predicts no effect on splicing However, three males with West syndrome, a form ACMG/AMP evidence summary: of early-onset epileptic encephalopathy, were • PS (PVS1): Null variant in a gene where LOF is recently reported with apparently non-mosaic a known mechanism of disease. Note: This has variants in WDR45 (Nakashima et al., 2016). The been conservatively interpreted as "strong" majority of reported variants are predicted to result evidence for pathogenicity, rather than "very in loss of protein function. strong", because the variant is located in the Protein function: The WD Repeat Domain 45 last exon, distal to the most 3' known functional (WDR45) protein is characterized as a beta-propeller domain. There is evidence that, as would be scaffold which plays an important role in the expected given the variant's location in the budding of autophagosomes during autophagy, a final exon, the resulting mRNA escapes process by which cellular components are degraded nonsense-mediated decay (Haack et al., 2012). (Hayflick et al., 2013; Nakashima et al., 2016). The • PS2: De novo variant. Note: Use of this precise mechanism by which deficiency of WDR45 criterion assumes that the family history is not leads to neurological disease has not been relevant to this patient's phenotype. established. • PM (PS4): Variant reported in two affected individuals previously Published papers or reports: Two previous • PM2: Absent from controls. individuals have been reported with the c.1007_1008del variant (Haack et al., 2012). References: Variant database review: This variant is recorded • Haack, et al. (2012). Am J Hum Genet 91:1144- in ClinVar (RCV000034828.11, classified as 1149. pathogenic) and in OMIM (30056.0001) (incorrectly • Hayflick, et al. (2013). Brain 136:1708-1717. described as a 1bp deletion in OMIM). • Nakashima, et al. (2016). J Hum Genet 61:653- 661. • Saitsu, et al. (2013). Nat Genet 45(4):445-450.

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Genome.One Name: Jean DOE DOB: 01-Jan-2000

METHODOLOGY 1. TEST ACCREDITATION This analysis strategy will not identify all potential incidental This test was developed and its performance characteristics findings. A lack of such findings does not imply the absence of determined by Genome.One (ACN 608 029 732), a wholly owned clinically significant variation in any specific gene. Analysis of a subsidiary of the Garvan Institute for Medical Research (384 panel of genes, as opposed to genome-wide analysis, reduces Victoria Street Darlinghurst, Sydney, NSW 2010, Australia). The the likelihood of incidental findings. If analysis of a gene(s) Genome.One laboratory is located within the Kinghorn Centre for unrelated to the proband’s phenotype is indicated based on Clinical Genomics. family history or other clinical factors, targeted analysis of this gene(s) can be performed on request. This test has been accredited for clinical use by the National Association of Testing Authorities, Australia, under the Carrier status for recessive phenotypes are not reported, but such NATA/RCPA Medical Testing program in accordance with the analyses can again be facilitated for specific genes on request. requirements of the National Pathology Accreditation Advisory 3. VARIANT CLASSIFICATION Council of Australia. Variants are classified according to the joint consensus 2. ANALYTICAL METHOD recommendations of: the American College of Medical Genetics Whole Genome Sequencing was performed on Illumina HiSeq X and Genomics and the Association for Molecular Pathology for instruments on DNA fragment libraries generated using the the interpretation of sequence variants (Richards et al. Genet TruSeq Nano DNA Library Preparation kit with extracted DNA Med. 2015;17(5):405-424. PMID: 25741868), with modifications from the specimen(s) provided. Genomes are sequenced to an (Amendola et al. Am J Hum Genet. 2016;98:1067-1076. PMID: average coverage of 30X, with 96.6% of protein coding bases and 27181684); the Association for Clinical Genetic Science; the Dutch splice sites consistently covered (≥15X). Note that for an X-linked Society of Clinical Genetic Laboratory Specialists; and aspects of gene, only 3X coverage would be sufficient to call a hemizygous the scoring system reported by Karbassi et al. (Hum Mutat. variant with 95% confidence using our bioinformatics pipeline 2016;37(1):127–134. PMID: 26467025). Variants are reported (Meynert et al., BMC Bioinformatics, 2014;1–11. PMID: 25038816). according to HGVS nomenclature (v15.11). Paired-end reads are aligned to the NCBI reference sequence 4. TEST LIMITATIONS (GRCh37) using the Burrows-Wheeler Aligner (BWA-MEM), and This test does not sequence all bases in a human genome, and variant calls are made using the Genomic Analysis Tool Kit does not include regions such as centromeres, telomeres, and (GATK). Analytical validity was established using the variant some repetitive elements. Not all disease-associated genes have location relative to GiaB v2.19 NA12878 Certified Reference been identified. Not all variants have been identified or Material. interpreted. The clinical significance of variation in many genes is Variants are filtered by the in-house SEAVE-Gemini variant not well understood. prioritisation platform using: This report is limited to sequence variants of size <20 nucleotides. 1. The pattern of inheritance within each family; Synonymous variants, triplet repeat expansions, translocations, 2. A minor allele frequency consistent with the mode of GC-rich regions, some pseudogenes, mosaic variants, inheritance and disease frequency; generally, these are ≤2% methylation changes and large copy number changes are (AR) or ≤0.1% (AD) in the Exome Aggregation Consortium currently not included in our genome analysis. database (exac.broadinstitute.org); 3. Disease gene specific filtering derived from Orphanet and The clinical significance of variants identified and included in this OMIM, when phenotype-specific analyses are requested. report is determined by the clinical indications for testing specified in the genomic testing request and consent forms. The evidence for phenotype-causality is evaluated for each variant resulting from the filtering strategies above, and variants 5. DATA REINTERPRETATION are prioritised according to Genome.One criteria. Only validated This report is based on information that is current at the time of variants with evidence for causing or contributing to a disease reporting. Genomic medicine is a rapidly advancing field, and phenotype are reported (see Test Limitations). Each variant is new associations between genetic variants and disease are evaluated based on the available information from the following: continuously being discovered. As such, we recommend that databases (including ClinVar, LSDBs, NHLBI Exome Sequencing genomic sequencing data are periodically reinterpreted, Project, 1000 Genomes and ExAC), published literature, clinical particularly if new symptoms or clinical information become correlation, segregation analysis, functional studies, and available. Genome.One does not reinterpret or reissue reports predicted functional or splicing impact using evolutionary automatically; a request for reinterpretation in light of new conservation analysis and computational tools (including knowledge must be explicitly made in the form of a new referral PolyPhen-2, SIFT, CADD, Provean, RVIS and structural to Genome.One. information from PDB/Uniprot). Whole genome sequencing may potentially facilitate gene During the course of analysis, clinically significant variants may be discovery or new genotype-phenotype associations. A protocol identified in genes unrelated to the proband’s phenotype. These for release of raw data (FASTQ, BAM or VCF files) for approved are known as “incidental”, “secondary”, or “additional” findings. research purposes is available on request. In our laboratory, these variants will be identified if they have been previously classified as likely pathogenic or pathogenic in (End of Report) the ClinVar database, or are predicted to be protein-truncating. Variants identified in this way will only be included in the report if determined to be likely pathogenic or pathogenic by this laboratory, and only if the patient has consented to the reporting of such variants.

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