Patient: DoB: 1 / 11 Sample report as of June 9, 2020. Regional differences may apply. For complete and up-to-date test methodology description, please see your report in Nucleus online portal. Accreditation and certification information available at blueprintgenetics.com/certifications Congenital Structural Heart Disease Panel Plus REFERRING HEALTHCARE PROFESSIONAL NAME HOSPITAL PATIENT NAME DOB AGE GENDER ORDER ID Unknown PRIMARY SAMPLE TYPE SAMPLE COLLECTION DATE CUSTOMER SAMPLE ID DNA SUMMARY OF RESULTS PRIMARY FINDINGS The patient is heterozygous for KMT2D c.10342C>T, p.(Pro3448Ser), which is a variant of uncertain significance (VUS). Del/Dup (CNV) analysis Negative for explaining the patient’s phenotype. ADDITIONAL FINDINGS The patient is heterozygous for PKD1L1 c.5553-1G>A, which is likely pathogenic. Please see APPENDIX 2: Additional Findings for further details PRIMARY FINDINGS: SEQUENCE ALTERATIONS GENE TRANSCRIPT NOMENCLATURE GENOTYPE CONSEQUENCE INHERITANCE CLASSIFICATION KMT2D NM_003482.3 c.10342C>T, p.(Pro3448Ser) HET missense_variant AD Variant of uncertain significance ASSEMBLY POS REF/ALT ID GRCh37/hg19 12:49428608 G/A gnomAD AC/AN POLYPHEN SIFT MUTTASTER PHENOTYPE 1/249254 possibly damaging deleterious disease causing Kabuki syndrome SEQUENCING PERFORMANCE METRICS PANEL GENES EXONS / REGIONS BASES BASES > 20X MEDIAN PERCENT COVERAGE > 20X Congenital Structural Heart Disease Panel 114 1948 427191 426750 260 99.9 Blueprint Genetics Oy, Keilaranta 16 A-B, 02150 Espoo, Finland 1 / 11 VAT number: FI22307900, CLIA ID Number: 99D2092375, CAP Number: 9257331 Patient: DoB: 2 / 11 TARGET REGION AND GENE LIST The Blueprint Genetics Congenital Structural Heart Disease Panel (version 4, Oct 19, 2019) Plus Analysis includes sequence analysis and copy number variation analysis of the following genes: ABL1, ACTA2, ACTB*, ACTC1, ACTG1*, ACVR1, ACVR2B, ADAMTS10, ADAMTS17, AFF4, AMMECR1, ARHGAP31, ARID1A, ARID1B, B3GAT3*,#, BCOR, BMPR2, BRAF*, C12ORF57, CBL, CDK13, CDK9, CFAP53, CHD4, CHD7, CHRM2, CREBBP, CRELD1, CTC1, DHCR7, DLL4, DOCK6, EFTUD2, EHMT1, EIF2AK4, ELN, ENG, EOGT, EP300, EVC, EVC2, FLNA, FOXC1, FOXF1, FOXH1, FOXP1, GATA4*, GATA5, GATA6, GDF1, GJA1*, GJA5, GPC3, HAND1, HAND2, HDAC8, HNRNPK*, HOXA1, HRAS, JAG1, KDM6A, KMT2D, KRAS*, KYNU, LEFTY2*, MED12, MED13L, MEIS2, MMP21, MYCN, MYO18B, MYRF, NAA15, NF1*, NIPBL, NKX2-5, NKX2-6, NODAL, NONO, NOTCH1, NOTCH2*, NR2F2, NSD1, PITX2, PKD1L1, PPP1CB, PRDM6, PRKD1, PTPN11, PUF60, RAB23, RAF1, RBM10, RERE*, RIT1, SALL4, SMARCB1, SMC1A, SMC3, SOS1, SOS2, STAG2, STRA6, TAB2, TBX1, TBX20*, TBX5, TFAP2B, TGDS, TLL1, TMEM94, ZEB2*, ZFPM2 and ZIC3. The following exons are not included in the panel as they are not covered with sufficient high quality sequence reads: B3GAT3 (NM_001288722:5). This panel targets protein coding exons, exon-intron boundaries (± 20 bps) and selected non-coding, deep intronic variants (listed in Appendix 5). This panel should be used to detect single nucleotide variants and small insertions and deletions (INDELs) and copy number variations defined as single exon or larger deletions and duplications. This panel should not be used for the detection of repeat expansion disorders or diseases caused by mitochondrial DNA (mtDNA) mutations. The test does not recognize balanced translocations or complex inversions, and it may not detect low-level mosaicism. *Some, or all, of the gene is duplicated in the genome. Read more: https://blueprintgenetics.com/pseudogene/ #The gene has suboptimal coverage when >90% of the gene’s target nucleotides are not covered at >20x with mapping quality score (MQ>20) reads. The sensitivity to detect variants may be limited in genes marked with an asterisk (*) or number sign (#). Blueprint Genetics Oy, Keilaranta 16 A-B, 02150 Espoo, Finland 2 / 11 VAT number: FI22307900, CLIA ID Number: 99D2092375, CAP Number: 9257331 Patient: DoB: 3 / 11 STATEMENT CLINICAL HISTORY This tested prenatal sample is the DNA extracted from the amniocytes of a 35-year-old female. The fetus has a complex congenital heart defect: pulmonary artery and pulmonary valve stenosis; tricuspid atresia; ventricular septal defect; and ductal and aortic arch anomalies. Maternal cell contamination testing has been carried out by the customer. The results of the analysis show no signs of maternal contamination. CLINICAL REPORT Sequence analysis using the Blueprint Genetics (BpG) Congenital Structural Heart Disease Panel identified a heterozygous missense variant KMT2D c.10342C>T, p.(Pro3448Ser). KMT2D c.10342C>T, p.(Pro3448Ser) There is 1 individual heterozygous for this variant in the Genome Aggregation Database (gnomAD, n>120,000 exomes and >15,000 genomes). Database curators have made every effort to exclude individuals with severe pediatric diseases from these cohorts. Moreover, there is another missense variant, p.(Pro3448Thr) at this position in the gnomAD. Both of the carriers of these variants existing in the reference population are heterozygous that suggest that these variants may be tolerated. However, all in silico tools utilized predict this variant to be damaging to protein structure and function. The affected amino acid is moderately conserved in mammals as well as in evolutionarily more distant species, which suggests that this position may not tolerate variation. There is a moderate physicochemical difference between proline and serine, therefore this is considered a non- conservative substitution (Grantham score = 74). To the best of our knowledge, this variant has not been described in the medical literature or reported in disease-related variation databases such as ClinVar or HGMD. KMT2D KMT2D (MIM *602113) gene encodes a histone methyltransferase that methylates the Lys-4 position of histone H3. The protein is part of a large protein complex called ASCOM, which has been shown to be a transcriptional regulator of the beta-globin and estrogen receptor genes. Heterozygous pathogenic variants in this gene have been shown to cause Kabuki syndrome (KS; MIM #147920). KS is a multiple congenital anomaly syndrome characterized by typical facial features, skeletal anomalies, mild to moderate intellectual disability and postnatal growth deficiency (ORPHA:2322). Individuals with KS often (70%) have congenital heart defects, coarctation of aorta being the most common, frequent ear infections, hearing loss and early puberty (GeneReviews NBK62111). The diagnosis of KS is established in a proband of any age with a history of infantile hypotonia, developmental delay, and/or intellectual disability AND one or both of the following 1) Typical dysmorphic features (long palpebral fissures with eversion of the lateral third of the lower eyelid, and ≥2 of the following: arched and broad eyebrows with the lateral third displaying notching or sparseness; short columella with depressed nasal tip; large, prominent, or cupped ears; persistent fingertip pads) or 2) a heterozygous pathogenic variant in KMT2D or a heterozygous or hemizygous pathogenic variant in KDM6A. KS was initially described in Japan, but has now been observed in all ethnic groups. Prevalence of KS is estimated to be 1:32,000. The inheritance pattern of KS is autosomal dominant. Majority of the KS cases are associated with mutations in the KMT2D gene. Deletions in the KDM6A (MIM *300128) gene have also been reported in few cases. In a study of 347 patients with clinical diagnosis of KS, 208 mutations were identified in the KMT2D gene (60%) and 12 in the KDM6A gene (3%) while 37% of the cases could not be molecularly confirmed (PMID: 27302555). The proportion of KS caused by de novo variants is unknown but it is likely to be high based on clinical experience. Human Gene Mutation Database (HGMD Professional 2019.4) reports >600 disease-causing variants in the KMT2D gene. These include small deletions, insertions and insertion-deletions (44%) most of those causing frameshift, nonsense variants (31%), missense variants (15%), splice site variants (9%) gross deletions (1%), gross insertions (0.7%) and more complex rearrangements (0.3%). Vast majority of Kabuki associated variants cause loss of gene function. Patients with whole-gene deletion of KMT2D or pathogenic Blueprint Genetics Oy, Keilaranta 16 A-B, 02150 Espoo, Finland 3 / 11 VAT number: FI22307900, CLIA ID Number: 99D2092375, CAP Number: 9257331 Patient: DoB: 4 / 11 truncating variants that occur in the first half of the gene may have more severe intellectual disability (PMID: 28295206). Mutation nomenclature is based on GenBank accession NM_003482.3 (KMT2D) with nucleotide one being the first nucleotide of the translation initiation codon ATG. CONCLUSION KMT2D c.10342C>T, p.(Pro3448Ser) is classified as a variant of uncertain significance (VUS), as there is insufficient evidence to evaluate its clinical relevance. This variant should not be used for clinical decision-making or risk evaluation in family members. Management of the patient and family should be based on clinical evaluation and judgment. Genetic counselling is recommended. The identified KMT2D c.10342C>T, p.(Pro3448Ser) variant is not eligible for the VUS Clarification Service at this time as family member testing is not sufficient to result in reclassification to likely pathogenic (please refer to our variant classification schemes on our website for additional information). The BpG VUS Clarification Service is offered when testing additional family members is likely to result in reclassification of the variant to likely pathogenic. Testing of the VUS in family members is available as part of our Familial Variant