Genomic Inform™ Genetic Test Results SUMMARY

Genomic Inform™ Genetic Test Results

Patient Ordering physician Specimen Name: ##### ##### ##### ##### Type: Saliva DOB: Jan 01, 1963 Collected: ### ##, #### Test: ######### Report date: ### ##, #### Received: ### ##, ####

Test performed: Singleton WGS

SUMMARY

RISK STATUS:

The following are pathogenic/likely pathogenic variants identified in this individual that have been reported to increase the risk to develop the associated diseases:

ABCA4 A heterozygous, likely pathogenic variant was identified in the ABCA4 gene. ABCA4-related disease: Heterozygous, pathogenic variants in the ABCA4 gene have been reported to cause Age-related macular degeneration. It should be noted that this is a susceptibility gene and may not confer disease and the visual disturbance may precede ophthalmoscopic findings. Individuals with a heterozygous pathogenic variant in this gene are also at a reproductive risk for the conditions listed under the carrier risk section of this report.

UNCERTAIN RISK STATUS:

A heterozygous variant of uncertain significance was identified in a gene where the clinical symptoms associated with the disease

may overlap with this individual's symptoms of elevated cholesterol levels. Variants of uncertain significance are neither pathogenic nor benign, but are likely to be reclassified over time as more evidence becomes available. Therefore, it is recommended that results containing uncertain variants be reinterpreted periodically to determine if they may be related to disease.

LDLR A heterozygous variant of uncertain significance was identified in the LDLR gene. LDLR-related disease Heterozygous (and rarely homozygous) pathogenic variants in the LDLR gene have been reported to cause an inherited form of

high cholesterol called familial hypercholesterolemia. As the excess cholesterol circulates through the bloodstream, it is deposited abnormally in tissues such as the skin, tendons, and coronary arteries, which increases risk for cardiovascular disease.

CARRIER STATUS:

Carriers are typically unaffected themselves but have a risk of passing the variant to their offspring. The reproductive risk for a particular gene or disease is based on whether both parents are carriers for pathogenic changes in the same gene. The following variants indicate this individual is a carrier for the following genes and related diseases:

ABCA4 In addition to a personal Age-related macular degeneration risk, this individual is at reproductive risk for the following diseases:

Cone-rod dystrophy, a condition associated with a loss of visual sharpness (acuity), an increased sensitivity to light (photophobia), and impaired color vision; Stargardt macular degeneration, characterized by vision loss that worsens over time, particularly affecting central and night vision; and Retinitis pigmentosa.

Variantyx Inc 1671 Worcester Rd, Suite 300 +1 617-209-2090 Lab Director: Christine M Stanley, PhD, FACMG ABMGG #2005163 Framingham, MA 01701 [email protected] CLIA #22D2140582, MA License #5438 Page 1/8 Genomic Inform™ Genetic Test Results

Patient: ##### ##### DOB: Jan 01, 1963 Test: #########

LAMB2

A likely pathogenic variant was identified in the LAMB2 gene.

LAMB2-related disease

Pathogenic variants in the LAMB2 gene cause Pierson syndrome (PIERSS) an autosomal recessive disorder characterized by nephrotic syndrome with neonatal onset, diffuse mesangial sclerosis and eye abnormalities with microcoria as the leading clinical features [MIM:609049].

SEPN1 A likely pathogenic variant was identified in the SEPN1 gene. SEPN1-related disease Homozygous and compound heterozygous pathogenic variants in the SEPN1 gene have been reported to cause several disorders: Multiminicore disease, Rigid spine muscular dystrophy, Congenital fiber-type disproportion; and desmin-related myopathy with Mallory body-like inclusions. Together, muscle diseases caused by SELENON gene mutations are known as

SELENON-related (or SEPN1-related) myopathies. The occurrence of Multiminicore disease (MMD) in two generations in a few families has been reported, suggestive of autosomal dominant inheritance.

No other pathogenic or likely pathogenic structural, mitochondrial, short tandem repeat, or small sequence variants were identified that have been associated with the family history or clinical symptoms provided. No variants were identified in the American College of Medical Genetics and Genomics (ACMG) minimum list of 59 genes to be reported as incidental or secondary findings.

There were no pathogenic variants in hereditary cancer genes identified.

Follow up recommendations Genetic counseling is recommended to review the personal and reproductive implications of these results. Variants of uncertain significance are neither pathogenic nor benign, but are likely to be reclassified over time as more evidence becomes available. Therefore, it is recommended that results containing uncertain variants be reinterpreted periodically to determine if they may be related to disease. Treatments and agents to avoid for SEPN1-related disease have been reported in Gene Reviews (PMID: 20301467).

Patient: ##### ##### DOB: Jan 01, 1963 Test: #########

Variants in genes linked to increased disease risk

Location Variant Disease / Inheritance Pathogenicity

ABCA4 c.5882G>A Age-related macular degeneration Likely pathogenic NM_000350.2 p.Gly1961Glu MOI: Autosomal dominant Evidence: PP3, PS1,

Heterozygous PP5 rs1800553

LDLR c.2541G>A Familial hypercholesterolaemia Uncertain significance NM_000527.4 p.Pro847Pro MOI: Autosomal dominant Evidence: PP3 Heterozygous rs137853964

ABCA4 NM_000350.2 c.5882G>A p.Gly1961Glu

This variant is a Nonsynonymous SNV mutation in the ABCA4 gene. According to published ACMG guidelines the pathogenicity of this variant is classified as Likely Pathogenic based on the following evidence: PP3, PS1, PP5. Zygosity state is Heterozygous. It is a variant with a 0.0113 maximal allele frequency in the population databases available for review. In peer reviewed clinical literature this variant has been reported to be associated with phenotype(s) listed in the Disease/Inheritance field in the table above. Calculated severity score is 0.79 on a scale of 0.0 – 1.0. The gene is considered non-essential and its tolerance score (RVIS) is in the top 86.31%. Additional comments and references: The variant, c.5882G>A identified in the ABCA4 gene in this patient satisfies the following lines of evidence: multiple lines of computational evidence support a deleterious effect on the gene or gene product (PP3), same amino acid change as a previously established pathogenic variant regardless of nucleotide change (PS1), reputable source recently reports variant as pathogenic but the evidence is not available to the laboratory to perform an independent evaluation (PP5). Based on the ACMG guidelines, the evidence is considered sufficient for classification of this variant as likely pathogenic. 1. Allikmets et al. (1997) Science 277:1805 2. Alapati et al. (2014) Molecular Diagnostic Testing by eyeGENE: Analysis of Patients With Hereditary Retinal Dystrophy Phenotypes Involving Central Vision Loss. IOVS 55:5510 3. Birtel et al. (2018) Clinical and genetic characteristics of 251 consecutive patients with macular and cone/cone-rod dystrophy. SCI REP 8:4824 4. Lee et al. (2017) Genotypic spectrum and phenotype correlations of ABCA4-associated disease in patients of south Asian descent. EJHG 25:735 5. Thiadens et al. (2012) Clinical course, genetic etiology, and visual outcome in cone and cone-rod dystrophy. OPHTHALMOL 119:819

LDLR NM_000527.4 c.2479G>A p.Pro847Pro This variant is a Nonsynonymous SNV mutation in the LDLR gene. According to published ACMG guidelines the pathogenicity of this variant is classified as Uncertain Significance based on the following evidence: PP3. Zygosity state is Heterozygous. It is a variant with a 0.0032 maximal allele frequency in the population databases available for review. In peer reviewed clinical literature this variant has been reported to be associated with phenotype(s) listed in the Disease/Inheritance field in the table above. Calculated severity score is 0.92 on a scale of 0.0 – 1.0. The gene is considered non-essential and its tolerance score (RVIS) is in the top 2.03%. Additional comments and references: The variant c.2479G>A, p.Val827Ile identified in the LDLR gene in this patient satisfies the following lines of evidence: multiple lines of computational evidence support a deleterious effect on the gene or gene product (PP3). Based on the ACMG guidelines, the evidence

Patient: ##### ##### DOB: Jan 01, 1963 Test: ######### is considered insufficient for classification as either benign or pathogenic and is, therefore, considered a variant of uncertain significance. 1. Hobbs et al. (1992) Hum Mutat 1:445 2. Bertolini et al. (2013) Spectrum of mutations and phenotypic expression in patients with autosomal dominant hypercholesterolemia identified in Italy. ATHEROS 227:342 3. Dorschner et al. (2013) Actionable, Pathogenic Incidental Findings in 1,000 Participants Exomes AJHG 93:631 4. Durst et al. (2017) Molecular genetics of familial hypercholesterolemia in Israel-revisited. ATHEROS 257:55

Variants in genes linked to carrier risk

Location Variant Disease / Inheritance Pathogenicity

ABCA4 c.5882G>A Age-related macular degeneration (AD), Cone- Likely pathogenic NM_000350.2 p.Gly1961Glu rod dystrophy (AR), Stargardt macular Evidence: PP3, PS1, Heterozygous degeneration (AR), Retinitis pigmentosa (AR) PP5 rs1800553 MOI: Autosomal recessive (AR), Autosomal dominant (AD) LAMB2 c.1477delT Pierson syndrome Likely pathogenic NM_002292.3 p.Cys493fs MOI: Autosomal recessive Evidence: PM2, PVS1 Heterozygous rs969481454

SEPN1 c.943G>A Multiminicore disease, Rigid spine muscular Likely pathogenic NM_020451.2 p.Gly315Ser dystrophy, Congenital fiber-type disproportion, PS4, PP3, PSV1, Heterozygous Desmin-related myopathy with Mallory body-like PM3, PP5 rs121908188 inclusions MOI: Autosomal recessive

ABCA4 NM_000350.2 c.5882G>A p.Gly1961Glu

Additional comments and references: The variant, c.5882G>A identified in the ABCA4 gene in this patient satisfies the following lines of evidence: multiple lines of computational evidence support a deleterious effect on the gene or gene product (PP3), same amino acid change as a previously established pathogenic variant regardless of nucleotide change (PS1), reputable source recently reports variant as pathogenic but the evidence is not available to the laboratory to perform an independent evaluation (PP5). Based on the ACMG guidelines, the evidence is considered sufficient for classification of this variant as likely pathogenic. 1. Allikmets et al. (1997) Science 277:1805 2. Alapati et al. (2014) Molecular Diagnostic Testing by eyeGENE: Analysis of Patients With Hereditary Retinal Dystrophy Phenotypes Involving Central Vision Loss. IOVS 55:5510 3. Birtel et al. (2018) Clinical and genetic characteristics of 251 consecutive patients with macular and cone/cone-rod dystrophy. SCI REP 8:4824

Patient: ##### ##### DOB: Jan 01, 1963 Test: #########

4. Lee et al. (2017) Genotypic spectrum and phenotype correlations of ABCA4-associated disease in patients of south Asian descent. EJHG 25:735 5. Thiadens et al. (2012) Clinical course, genetic etiology, and visual outcome in cone and cone-rod dystrophy. OPHTHALMOL 119:819

LAMB2 NM_002292.3 c.1477delT p.Cys493fs This variant is a Frameshift mutation in the LAMB2 gene. According to published ACMG guidelines the pathogenicity of this variant is classified as Likely Pathogenic based on the following evidence: PVS1, PM2. Zygosity state is Heterozygous. It is a variant with a 0.0001 maximal allele frequency in the population databases available for review. In peer reviewed clinical literature this variant has been reported to be associated with phenotype(s) listed in the Disease/Inheritance field in the table above. Calculated severity score is 1.0 on a scale of 0.0 – 1.0. The gene is considered essential and its tolerance score (RVIS) is in the top 3.76%. Additional comments and references: The variant, c.1477delT, p.Cys493fs, identified in the LAMB2 gene in this patient satisfies the following lines of evidence: frameshift/null variant in a gene where loss of function (LOF) is a known mechanism of disease (PVS1), absent from controls or at extremely low frequency if recessive (PM2). Based on the ACMG guidelines, the evidence is considered sufficient for classification of this variant as likely pathogenic. 1. Wuhl et al. (2007) Am J Med Genet A 143:311 2. Beaufils et al. (2018) Skeletal impairment in Pierson syndrome: Is there a role for laminin2 in bone physiology? BONE 106:187 3. Sen et al. (2017) Clinical genetic testing using a custom-designed steroid-resistant nephrotic syndrome gene panel: analysis and recommendations. JMG 54:795

SEPN1 NM_020451.2 c.943G>A p.Gly315Ser This variant is a Nonsynonymous SNV mutation in the SEPN1 gene. According to published ACMG guidelines the pathogenicity of this variant is classified as Likely Pathogenic based on the following evidence: PS4, PP3, PSV1, PM3, PP5. Zygosity state is Heterozygous. It is a variant with a 0.0002 maximal allele frequency in the population databases available for review. In peer reviewed clinical literature this variant has been reported to be associated with phenotype(s) listed in the Disease/Inheritance field in the table above. Calculated severity score is 1.0 on a scale of 0.0 – 1.0. The gene is considered non-essential and its tolerance score (RVIS) is in the top 83.42%. Additional comments and references: The variant c.943G>A, p.Gly315Ser identified in the SEPN1 gene in this patient satisfies the following lines of evidence: the prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls (PS4), for recessive disorders, detected in trans with a pathogenic variant (PM3), multiple lines of computational evidence support a deleterious effect on the gene or gene product (PP3), reputable source recently reports variant as pathogenic but the evidence is not available to the laboratory to perform an independent evaluation (PP5), cosegregation with disease in multiple affected family members, including multiple families from diverse ethnic backgrounds (PSV1). Based on the ACMG guidelines, the evidence is considered sufficient for classification of this variant as likely pathogenic. 1. Maggi et al. (2013) Congenital myopathies - Clinical features and frequency of individual subtypes diagnosed over a 5-year period in the United Kingdom. NEU DIS 23:195

Electronically signed by

Allan J. Fisher, MD, FACMG, FACOG Medical Director

Patient: ##### ##### DOB: Jan 01, 1963 Test: #########

Variantyx, Inc. Framingham, MA, USA

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General information This Genomic Inform™ genetic test is a clinical diagnostic test designed to identify variants that correlate with increased disease risk and carrier status. By using whole genome sequencing methodology and the Variantyx Genomic Intelligence® platform, many more variants can be evaluated than is possible with whole exome sequencing, including more small sequence changes (single nucleotide variants) and small insertions and deletions (indels), as well as variants not typically evaluated by exomes, including deep intronic variants, structural/copy number variants, deletion/duplications, short tandem repeats and mitochondrial sequence variants.

Method of analysis For this test, whole genome sequencing was performed using the Illumina TruSeq DNA PCR-Free kit followed by next generation sequencing (NGS) by Macrogen Inc., 1330 Piccard Drive, STE 103, Rockville, MD 20850, CLIA# 21D2062464. The resultant FASTQ data file was securely transferred to Variantyx and detected, analyzed and reported using the Variantyx Genomic Intelligence® platform version 2.7.3.0.

Statistics The minimum acceptable average coverage of sequence is 25X. Because the test uses PCR-free techniques and because genome sequencing does not require exon capture, these coverages provide highly confident variant identification. The sensitivity and specificity of the assay is 0.99735 and 1.00000, and the positive predicted value is 0.99715 for SNVs and small insertions and deletions less than 35 base pairs.

Report standards Variants are reported using Human Genome Variation Society (HGVS) recommendations, when available. Variants are classified using one of five interpretation categories recommended by the American College of Medical Genetics and Genomics (ACMG): pathogenic, likely pathogenic, uncertain, likely benign, and benign (PMID: 25741868). Benign and likely benign variants are typically not reported. Variants of uncertain clinical significance are reported in select cases where there is a strong clinical correlation to the patient’s clinical symptoms.

Annotations To maintain the most up-to-date annotations, the Variantyx database is updated quarterly and, as a result, variant classification and/or interpretation may change over time as more information becomes available. The following databases and tools are included in Variantyx Genomic Intelligence® platform:

1. Disease association: HGMD Professional (http://www.hgmd.cf.ac.uk/), Genome Trax (http://www.biobase- international.com/product/genome-trax), ClinVar (http://www.ncbi.nlm.nih.gov/clinvar/), OMIM (http://www.omim.org/), Orphanet (www.orpha.net/ ), GeneTests (https://www.genetests.org/ ). 2. Population frequencies: gnomAD (http://gnomad.broadinstitute.org/), dbSNP (http://www.ncbi.nlm.nih.gov/SNP/), ensembl (www.ensembl.org/), 1000 Genomes Project (www.1000genomes.org/), ExAC (http://exac.broadinstitute.org/), NHLBI Exome Sequencing Project (http://evs.gs.washington.edu/EVS/) and the Variantyx allele frequency database (http://variantyx.com/). 3. Severity prediction: SIFT, MutationAssessor, MutationTaster, GWAVA, PolyPhen2, FATHMM, LRT. 4. Conservation prediction: SiPhy, GERP++, PhyloP and PhastCons. 5. Gene Essentiality: According to published work 10.1371/journal.pgen.1003484 6. Gene tolerance: RVIS score, according to published work 10.1371/journal.pgen.1003709

A glossary of terms can be found at https://www.variantyx.com/glossary/.

Patient: ##### ##### DOB: Jan 01, 1963 Test: #########

Mitochondrial variants Mitochondrial variants are reported if the heteroplasmy level is above 5%. Large deletions are not reported.

Structural variants (SVs) Structural variants are reported if there is clinical correlation with the genes contained within the structural region. Structural variants are orthogonally confirmed by a third party CLIA laboratory, if identified and reported.

Short tandem repeats (STRs) Short tandem repeats in pathogenic ranges, when identified and reported, are orthogonally confirmed by a third party CLIA laboratory (e.g. PCR, Southern blot or other appropriate technique).

Genes for disorders involving early-onset intellectual disability: AFF2, AFF3, DIP2B, FMR1

Genes for disorders involving adult-onset movement with or without cognitive involvement: AR, ATN1, ATXN1, ATXN2, ATXN3, ATXN7, ATXN8OS, ATXN10, C9ORF72, CACNA1A, CNBP, CSTB, DMPK, FMR1, FXN, PP2R2B

Based on recommendations by the ACMG, the HTT and JPH3 genes are excluded from this analysis by default, but may be included if a specialized consent form has been signed by the patient/guardian and ordering clinician.

Limitations Technical limitations Although whole genome sequencing provides superior coverage of genes compared to other sequencing technologies, it should be noted that no sequencing platform detects all variants and, due to the vast amount of data produced using whole genome sequencing, not all variants are analyzed. A negative result from the analysis cannot rule out the possibility that the tested individual carries a rare, unexamined pathogenic variant or a pathogenic variant in an undetectable region. All next generation sequencing (NGS) technologies, including WGS analysis, may generate false positive results. Results are applicable to the tissue type used for this sequence test and may not reflect the variation in other tissue types. While the minimum average read depth is at least 25X, WGS cannot cover the entire genome. At the variant level, read depths fewer than 8X are not reported, which for any given test is approximately 0.5% of the reference genome (GRCh38). Each individual may have slightly different coverage yield distributions within the genome. While most variant types are detectable, some genetic aberrations, such as gross genomic rearrangements or variants in portions of genes with highly homologous pseudogenes, are identified with a lower efficiency. This test is not currently validated for large mitochondrial deletions. In addition, this test is not intended to detect aberrations not caused by variants in DNA sequence, such as gene expression, epigenetic modifications, fusion, chromosome conformational changes, X-linked recessive variants in females who manifest disease due to skewed X-inactivation and other unknown abnormalities. Variantyx is not responsible for specimen errors (e.g. labeling, extraction) of samples received that may have occurred prior to our receipt. Variants are not reported if they are not uniquely mappable, are of low coverage or are otherwise determined to be of low quality.

Reanalysis and reclassification of variants Variant classification and/or interpretation may change over time as more information becomes available on the clinical symptoms associated with the gene/variant. Not all variants of uncertain significance (VUS) identified by sequencing are reported. Typically, VUS are reported when they are rare and in genes associated with diseases with symptoms that partially or completely correlate with the patient’s disease spectrum and severity. VUS are neither pathogenic nor benign, but are likely to be reclassified over time as more evidence becomes available. New associations of symptoms to diseases and genes are likely to occur with time. In addition, if the clinical symptoms reported to Variantyx were incomplete or if there has been a change in symptoms, new correlations may be revealed upon reanalysis. Therefore, it is recommended that results be reinterpreted periodically to determine if they may be related to disease.

Use of test results by clinician Results should be interpreted by the ordering clinician in the context of the patient’s personal medical and family history.

Annotation limitations Sequence variation is compared to reference data using genome build GRCh38 and the lab cannot guarantee the accuracy of this data nor the accuracy of databases listed in the ‘Annotations’ section of this report.

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FDA notes: This laboratory is certified under the Clinical Laboratory Improvement Amendments (CLIA) of 1988 as qualified to perform high complexity laboratory testing. The US Food and Drug Administration (FDA) does not require this test to go through premarket clearance. This lab developed test (LDT) was developed and its performance characteristics determined by Variantyx, Inc. to be used for clinical purposes and not as investigational or as research. These results should be used in the context of the patient’s clinical findings and family history and not as the sole basis for diagnosis and/or treatment.