® Genomic Unity Intellectual Disability Analysis Results

Patient Ordering physician Specimen Name: ###### ###### ############ Type: Saliva DOB: ### ##, 2017 ############ Collected: Jan 12, 2020 Sample barcode: ######## ############ Received: Jan 15, 2020

Test performed: Intellectual Disability Analysis

Positive result

A heterozygous, pathogenic variant was identified in the MECP2 in this individual. MECP2

A heterozygous ~10 kb deletion encompassing exons 3 and 4 of the MECP2 gene was identified. Conditions associated with alterations in the MECP2 gene include Rett Syndrome, Atypical Rett Syndrome and X-linked intellectual disability. This variant is classified as Pathogenic.

No other pathogenic or likely pathogenic structural, mitochondrial, short tandem repeat, or small sequence clinically relevant variants were identified based on the clinical symptoms provided.

Follow up recommendations Genetic Counseling is strongly recommended to review the personal and reproductive implications of this result.

Indication for testing Phenotype: Strabismus, Seizures, Relative macrocephaly, Lennox-Gastaut Syndrome, Ineffective airway clearance, Hypermetropia, Global developmental delay, Generalized hypotonia, Failure to thrive, Esotropia, Eczema, Dysphagia, Developmental regression, Bruxism, Brain atrophy, Asthma

Variants with genetic etiology linked to a disease that is consistent with the indication for testing

Location Variant Disease / Inheritance Pathogenicity chrXq28q28x1154,027,483- 9675 bp Deletion [HGMD]: Rett syndrome; Atypical Pathogenic 154,037,158) Rett syndrome, X-linked Heterozygous mental retardation syndromic 13 MOI: X-linked

chrXq28q28x1(154,027,483-154,037,158)

This variant is a Deletion mutation in X. It includes one or more exons of the following gene: MECP2. Zygosity state is Heterozygous. It is a variant with a 0.00676 maximal allele frequency in the population databases for review. This variant overlaps with large structural variant(s) reported in peer reviewed clinical literature to be associated with Rett syndrome; Atypical Rett syndrome.

Additional This is heterozygous Xq28 deletion encompassing the following coordinates chrX:154,026,500-154,037,000 [Hg38]. This copy number variant been not been reported in the Database of Genomic Variants which includes a curated collection of structural

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 1/4 Genomic Unity® Intellectual Disability Analysis Results

Patient: ###### ###### DOB: ### ##, 2017 Sample barcode: ######## variants in healthy controls. This and overlapping CNVs have been reported in the scientific literature. It has been associated with clinical phenotypes Rett Syndrome, Atypical Rett Syndrome and X-linked Mental Retardation. The gene encompassed by this deletion includes exons 3 & 4 of the MECP2 gene. Overlapping deletions have been reported in multiple publications including Kobayashi Y et al 2012 PMID: 22001500. Based on the guidelines published by the ACMG for the interpretation of copy number variants (2011), this deletion is considered a Pathogenic variant.

Electronically signed by

General information The Genomic Unity® Intellectual Disability Analysis is a clinical diagnostic genetic test designed to identify pathogenic variants in the panel of listed in this test associated with genetic forms of intellectual disability. By using a whole genome sequencing platform methodology in conjunction with the Variantyx Genomic Intelligence® interpretation software, many more variants can be evaluated than is possible with standard panel or exome tests. The Genomic Unity® Intellectual Disability Analysis includes small sequence changes (single nucleotide variants), small insertions and deletions (delins) and deletions/duplications (del/dups).

Statistics The minimum acceptable average sequencing coverage is 25X, which provides highly confident variant identification because this test uses PCR-free genome sequencing and does not require exon capture. The sensitivity and specificity for SNVs in this assay are 0.99905+/-0.00015 and >0.99999, respectively, and the positive predicted value is 0.99896.

Reporting standards Single Nucleotide Variants (SNVs) Single nucleotide variants and small delins (<50 bp) are reported if there is clinical correlation to the patient's clinical symptoms.

Structural Variants (SVs) Structural variants are reported if there is clinical correlation with the genes contained within the structural region.

Short Tandem Repeats (STRs) Short tandem repeats in pathogenic ranges, if identified and reported, are orthogonally confirmed (e.g. PCR, Southern blot or other appropriate technique) by a third party CLIA laboratory. Genes with STRs related to early-onset intellectual disability: AFF2, AFF3, DIP2B, FMR1.

The false negative rate for repeat expansions has not been determined for the following genes: AFF2, AFF3, DIP2B

ACMG Secondary Findings Secondary findings are not reported for gene panel tests.

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 2/4 Genomic Unity® Intellectual Disability Analysis Results

Patient: ###### ###### DOB: ### ##, 2017 Sample barcode: ########

Parental Analysis When parental samples are submitted they are used in the evaluation of the patient (proband) only. No specific parental results are issued under the family member’s name.

Societal Recommendations Variants are reported using 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.

Clinical Correlation, Reanalysis and Reclassification of Variants Variants are reported if they meet certain frequency thresholds and are correlated with the patient’s clinical symptoms as reported to the lab at the time of testing. Therefore, variants in genes associated with clinical symptoms unrelated to the patient's clinical symptoms may not be reported. Furthermore, if clinical symptoms develop in the patient or if new clinical symptoms are associated with variants in genes identified by sequencing, they may not have been reported and a new analysis updating the clinical correlation may be needed to identify causal 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. Therefore, it is recommended that results be reinterpreted periodically to determine if the variants identified by sequencing may be related to disease.

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.

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

Limitations of analysis Technical Limitations The Genomic Unity® Intellectual Disability Analysis uses a whole genome sequencing methodology to provide superior coverage of the listed genes as compared with other sequencing technologies. However, 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 this 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 technologies, including genome-based analyses, may generate false positive results. Results are applicable to the tissue type used and may not reflect the variation in other tissue types. While the minimum average read depth is at least 25X, whole genome sequencing 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), and 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. In addition, this test is not intended to detect aberrations not caused by variants in DNA sequence,

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 3/4 Genomic Unity® Intellectual Disability Analysis Results

Patient: ###### ###### DOB: ### ##, 2017 Sample barcode: ######## 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, Inc. 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.

Use of Test 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.

Method of analysis Methods For this test, the patient’s DNA sequence was generated by PCR-free whole genome sequencing performed using the Illumina TruSeq DNA PCR-Free kit followed by next generation sequencing at Psomagen Inc. located at 1330 Piccard Drive, Suite 103, Rockville, MD 20850, CLIA ID Number 21D2062464, Lab Director: Carlos Ruiz, MD, FACP, and the resultant FASTQ data file was securely transferred to Variantyx, Inc., where selective, discreet, in-silico analyses were used to detect, analyze and report clinically relevant variants using the Variantyx Genomic Intelligence® platform version 2.7.4.0.

Gene List ABCD1, ACSL4, ADNP, AFF2, AFF3, AGA, AHDC1, AIFM1, ALG13, AMMECR1, ANKRD11, AP1S2, AP4B1, ARHGEF6, ARHGEF9, ARID1A, ARID1B, ARSE, ARX, ASXL1, ASXL3, ATP6AP2, ATP7A, ATRX, AUTS2, BCAP31, BCOR, BCORL1, BDNF, BRWD3, CA8, CACNA1A, CASK, CC2D1A, CCDC22, CDKL5, CHD2, CHD7, CHD8, CLCN4, CNKSR2, CNTNAP2, CREBBP, CSNK2A1, CTCF, CTNNB1, CTNND2, CUL4B, DCX, DDX3X, DHCR7, DIP2B, DKC1, DLG3, DMD, DNM1, DNMT3A, DYNC1H1, DYRK1A, EBP, EFTUD2, EHMT1, EIF2S3, EP300, EZH2, FANCB, FGD1, FLNA, FMR1, FOLR1, FOXG1, FOXP1, FRMPD4, FTCD, FTSJ1, GAMT, GATAD2B, GATM, GDI1, GK, GNAO1, GPC3, GRIA3, GRIN1, GRIN2A, GRIN2B, HAL, HCCS, HCFC1, HCN1, HDAC6, HDAC8, HIVEP2, HMGB3, HNRNPH2, HNRNPU, HOXA1, HPRT1, HSD17B10, HUWE1, IDS, IGBP1, IKBKG, IL1RAPL1, IQSEC2, ITPR1, KANSL1, KAT6A, KCNB1, KCNJ10, KDM5C, KDM6A, KIF1A, KIF4A, KLF8, KLHL15, KMT2A, KMT2D, L1CAM, LAMP2, LAS1L, LINS1, MAGT1, MAN1B1, MAOA, MAP2K1, MBD5, MBTPS2, MECP2, MED12, MED13L, MED23, MEF2C, MID1, MID2, MTM1, MTOR, MYT1L, NAA10, NALCN, NDP, NDUFA1, NEXMIF, NF1, NHS, NIPBL, NLGN3, NLGN4X, NONO, NR2F1, NRXN1, NSD1, NSDHL, NSF5, NSUN2, OCRL, OFD1, OGT, OPHN1, OTC, PACS1, PAK3, PAX6, PCDH19, PDHA1, PGAP2, PGK1, PHF6, PHF8, PIGA, PIGO, PIGV, PLA2G6, PLP1, PNKP, POGZ, POLA1, PORCN, PPP2R5D, PPT1, PQBP1, PRPS1, PTCHD1, PTEN, PTPN11, PURA, RAB39B, RAD21, RAI1, RBM10, RIT1, RLIM, RNF113A, RPL10, RPS6KA3, SATB2, SCN1A, SCN2A, SCN8A, SETBP1, SETD5, SHANK3, SHROOM4, SLC16A2, SLC35A2, SLC6A1, SLC6A8, SLC9A6, SMARCA2, SMARCA4, SMARCB1, SMC1A, SMC3, SMS, SOS1, SOX3, SRPX2, SSR4, ST3GAL3, STAG2, STXBP1, SYN1, SYNGAP1, SYP, TAF1, TBC1D24, TBL1XR1, TBR1, TCF4, THOC2, TIMM8A, TMLHE, TRAPPC9, TRIO, TSC1, TSC2, TSPAN7, TUSC3, UBE2A, UBE3A, UPF3B, USP27X, USP9X, VPS13B, WAC, WDR45, WT1, ZC4H2, ZDHHC15, ZDHHC9, ZEB2, ZMYM3, ZNF41, ZNF711, ZNF81

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.

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 4/4