SAMPLE/TEST A J2446 Blood

Report Date 12/04/2020

Clinical Information

Joint pain, compressed spinal column, short stature, bowed legs.

Testing Performed

NGS493 - Hypophosphatasia and Hypophosphatemic Rickets Panel

Reports Sent To

Facility: MNG Laboratories Phone:678-225-0222 Fax:None; eMail:[email protected]

Result

_{Primary Findings:}

_NEGATIVE

Sequencing and Copy Number Summary:

No single nucleotide variants, small indels, or large deletions/duplications that are likely to be of medical relevance were identified in the genes assessed by this panel.

Genetic counseling is recommended.

Hereditary hypophosphatemic rickets is a group of genetic disorders with variable severity and age at onset. Signs and symptoms are typically present in early childhood and may include low levels of phosphate in the blood, and abnormalities of bone development, movement, and growth. The inheritance pattern varies depending on the causative gene. The most common form of the disorder, X-linked hypophosphatemia, is caused by pathogenic variants in the PHEX gene. X-linked hypophosphatemia accounts for approximately 80% of all cases of hereditary hyphosphatemic rickets. This test assists in the differential diagnosis of patients with unclear cases of hypophosphatemic rickets. [Ruppe, PMID: 22319799; Carpenter, PMID: 21538511; Haffner, PMID: 31068690] FINAL 630292 SAMPLE REPORT First Last Male 01/01/2020 Gender DOB NGS493-SAMPLE None Institution ID Dx Code

PATIENT

Reported by: Trey Langley, PhD 12/04/2020

S. Hussain Askree, MBBS PhD, Laboratory Director, Medical Neurogenetics, LLC

NUCLEAR GENE SINGLE NUCLEOTIDE POLYMORPHISM AND SMALL INDEL SEQUENCING ASSESSMENT:

Genomic regions of interest are selected using a custom capture reagent for target enrichment (Twist Bioscience) and sequenced via the Illumina® Novaseq 6000 next generation sequencing platform. Sequencing reads are aligned with the human genome reference GRCh37/hg19 build. Regions of interest include all exons and intron/exon junctions (+/-20 nucleotides) for each gene analyzed. A minimum of 99% of bases in targeted regions are covered at >15X. Analytical sensitivity is estimated to be >99% for single nucleotide variants, >97% for insertions/deletions less than six base pairs, and >95% for insertions/deletions between six and fifteen base pairs. Uncovered regions with known pathogenic variants are sequenced in a targeted manner (List based on ClinVar Database: June 2, 2020 release).

NUCLEAR GENE COPY NUMBER VARIANT ASSESSMENT: Next Generation Sequencing data used to call SNPs and

small indels are assessed with Illumina’s DRAGEN (Dynamic Read Analysis for GENomics) Bio-IT Platform. Genes listed in ClinVar with intragenic pathogenic deletions are padded with additional intronic probes to allow single exon resolution CNV detection (List based on ClinVar Deletion Database: January 2019 release; see bolded genes in appendix). For other genes, large deletions (>10 exons) can be detected. The resolution of this analysis can vary depending on region-specific features. Analytical sensitivity is estimated to be >95%.

RESULTS INTERPRETATION: Results should be used in the context of available clinical information and should not be

used as the sole basis for patient management or treatment. Genetic counseling is recommended. Variants are assessed according to ACMG criteria (Richards et al. 2015; PMID: 25741868). This report contains interpretation of PATHOGENIC and LIKELY PATHOGENIC variants (by ACMG Criteria) as well as VARIANTS OF UNCERTAIN SIGNIFICANCE (VUS) with pathogenic predictions related to the clinical information provided. Variants not reported: (1) Variants classified as BENIGN or LIKELY BENIGN by ACMG Criteria; (2) VARIANTS OF UNCERTAIN SIGNIFICANCE (VUS) with benign or likely benign predictions; (3) Variants related to carrier status; (4) Polymorphisms with implications in drug response (pharmacogenomics variants). We will reanalyze the data periodically at the clinician’s request to allow potential reinterpretation based on new research or evidence. GnomAD abbreviations for population frequency data: African/African American (AFR), Latino (AMR), Ashkenazi Jewish (ASJ), East Asian (EAS), Finnish (FIN), Non-Finnish European (NFE), South Asian (SAS), Other (OTH).

NEXT GENERATION SEQUENCING (NGS) TEST LIMITATIONS: This assay will not consistently detect germline

mosaicism below 50% or rule out the presence of large chromosomal aberrations, including rearrangements and inversions that do not change copy number of genomic regions. The assay does not detect repeat expansions. Possible intergenic variant interactions are not commented on. False positive or false negative results may occur for reasons that include: insufficient information available about rare genetic variants, sex chromosome abnormalities, pseudogene interference, homologous regions, blood transfusions, bone marrow transplantation, somatic or tissue-specific mosaicism, mislabeled samples, or erroneous representation of family relationships. Variants that do not alter an amino acid composition of a protein may be difficult to assess for pathogenicity since they may produce abnormalities in structures NOT assessed by conventional analysis paradigms (e.g. mRNA expression and processing; Science 2006:314 (5807):1930-1933).

Interpretation of the clinical significance of gene variations is limited by information about the variant that is available at the time of reporting, and by the quality and quantity of clinical information provided with the sample. As the understanding of human genetic diversity improves, the interpretation of the clinical significance of variants may change.

IN SILICO ASSESSMENT: In silico assessments do NOT definitively identify a variant as benign or pathogenic and should

NOT be relied upon as the sole criteria for ANY patient diagnosis. MutationTaster, PolyPhen-2 and SIFT are tools which predict the possible impact of an amino acid substitution on the structure and function of human proteins (PMID 19561590, 22261837, 20354512, 21520341, 24681721; http://www.mutationtaster.org). A cross comparison of in silico methods is

FALSE POSITIVES AND NEGATIVES: For specimens sent to ANY laboratory, a consideration for false positive or false

negative test results is the improper labeling of the patient’s sample during the acquisition, shipping, receipt and testing process. We take no responsibility for any specimen labeling errors that occur before the sample arrives at Medical

Neurogenetics. LLC. If test results are inconsistent with the clinical presentation, call Medical Neurogenetics, LLC to discuss the case or submit another sample for confirmatory testing.

REQUEST FOR ADDITIONAL DATA: For researchers or clinicians interested in receiving a list of all variants identified in

the analysis, we will provide this list without interpretation at your request and at no additional charge. We will also provide details of gene coverage for the analysis at your request. Please submit all requests online at our website:

https://mnglabs.com/support/.

CONSENT FOR TESTING: Obtaining and maintaining patient consent for genetic testing is the sole responsibility of the

managing physician and institution. A genetic testing consent form is available on our website.

FDA NOTES: This test was developed and its performance characteristics determined by Medical Neurogenetics, LLC. It

has not been cleared or approved by the US Food and Drug Administration. FDA does not require this test to go through premarket FDA review. This test is used for clinical purposes. It should not be regarded as investigational or for research. This laboratory is certified under the Clinical Laboratory Improvement Amendments (CLIA) as qualified to perform high complexity clinical laboratory testing. CLIA ID# 11D0703390; Georgia Lab Lic. Code 060-381.

Appendix:

GENES ASSESSED BY THIS TEST:ALPL (171760), CLCN5, CYP27B1, CYP2R1, DMP1, ENPP1 (173335), FGF23, PHEX

(300550), SLC34A3, VDR.

NGS493

630292

SAMPLE REPORT

First Name

Last Name _{HPP/Hypophosphatemia}

Panel NGS493-SAMPLE Institution ID A00070208 MNG ID 01/01/2020 DOB

Clinical Information

Joint pain, compressed spinal column, short stature, bowed legs.

Testing Performed

NGS493 - Hypophosphatasia and Hypophosphatemic Rickets Panel

Reports Sent To

Facility: MNG Laboratories Phone:678-225-0222 Fax:None; eMail:[email protected]

Result

_{Primary Findings:}

_POSITIVE

Sequencing and Copy Number Summary:

A PATHOGENIC variant was detected in the PHEX gene. Targeted testing for this variant in family members is available. Please see our website for ordering information.

No large deletions/duplications that are likely to be of medical relevance were identified in the genes assessed by this panel.

Clinical correlation and genetic counseling are recommended.

Hereditary hypophosphatemic rickets is a group of genetic disorders with variable severity and age at onset. Signs and symptoms are typically present in early childhood and may include low levels of phosphate in the blood, and abnormalities of bone development, movement, and growth. The inheritance pattern varies depending on the causative gene. The most common form of the disorder, X-linked hypophosphatemia, is caused by pathogenic variants in the PHEX gene. X-linked hypophosphatemia accounts for approximately 80% of all cases of hereditary hyphosphatemic rickets. This test assists in the differential diagnosis of patients with unclear cases of hypophosphatemic rickets. [Ruppe, PMID: 22319799; Carpenter, PMID: 21538511; Haffner, PMID: 31068690] 630292 SAMPLE REPORT First Last Gender DOB NGS493-SAMPLE None Institution ID Dx Code A00070208 J2446 Blood MNG ID Container ID Tissue NGS493 Version 10/26/2020 10/26/2020 10/26/2020 Accession Approval Date Collected

Reported by: Trey Langley, PhD 12/04/2020

S. Hussain Askree, MBBS PhD, Laboratory Director, Medical Neurogenetics, LLC

SINGLE NUCLEOTIDE VARIANT SUMMARY

DETAILED SINGLE NUCLEOTIDE VARIANT INFORMATION

PATHOGENIC VARIANT · PHEX · Exon 7/22 · c.830T>A · ENST00000379374.5 · p.Leu277Ter · ENSP00000368682.4 ·

Heterozygous · ChrX:22112198 · Reference GRCh37

Thousand Genomes: Not Available · GnomAD: Not Available · PolyPhen-2: Not Available · SIFT: Not Available · dbSNP ID: rs137853268 · ACMG Criteria: PVS1, PM2, PM6 · Locus Coverage: 500 · Gene Percent Covered: 100%

The PHEX gene has been associated with the following phenotypes: Hypophosphatemic rickets, X-linked dominant

A heterozygous variant was detected. This variant is a change of the leucine acid at amino acid position 277 to a termination or STOP codon, which is predicted to cause premature truncation of the protein. In PMID 9106524, this variant, also referred to as Leu274Ter, was detected in a female patient with a sporadic case of hypophosphatemia. This variant has been

submitted to ClinVar once as pathogenic (Submission Accession ID: SCV000031795.2). The available data justify the classification as PATHOGENIC.

PEER REVIEWED CLASSIFICATION (PMID) 9106524 PATHOGENIC/LIKELY PATHOGENIC VARIANTS

Gene

Nucleotide Position

Amino Acid

Position Zygosity Origin Associated Phenotypes

PHEX c.830T>A p.Leu277Ter Heterozygous Not

Determined

Hypophosphatemic rickets, X-linked dominant

NUCLEAR GENE SINGLE NUCLEOTIDE POLYMORPHISM AND SMALL INDEL SEQUENCING ASSESSMENT:

Genomic regions of interest are selected using a custom capture reagent for target enrichment (Twist Bioscience) and sequenced via the Illumina® Novaseq 6000 next generation sequencing platform. Sequencing reads are aligned with the human genome reference GRCh37/hg19 build. Regions of interest include all exons and intron/exon junctions (+/-20 nucleotides) for each gene analyzed. A minimum of 99% of bases in targeted regions are covered at >15X. Analytical sensitivity is estimated to be >99% for single nucleotide variants, >97% for insertions/deletions less than six base pairs, and >95% for insertions/deletions between six and fifteen base pairs. Uncovered regions with known pathogenic variants are sequenced in a targeted manner (List based on ClinVar Database: June 2, 2020 release).

NUCLEAR GENE COPY NUMBER VARIANT ASSESSMENT: Next Generation Sequencing data used to call SNPs and

small indels are assessed with Illumina’s DRAGEN (Dynamic Read Analysis for GENomics) Bio-IT Platform. Genes listed in ClinVar with intragenic pathogenic deletions are padded with additional intronic probes to allow single exon resolution CNV detection (List based on ClinVar Deletion Database: January 2019 release; see bolded genes in appendix). For other genes, large deletions (>10 exons) can be detected. The resolution of this analysis can vary depending on region-specific features. Analytical sensitivity is estimated to be >95%.

NGS493

630292

SAMPLE REPORT

First Name

Last Name _{HPP/Hypophosphatemia}

Panel NGS493-SAMPLE Institution ID A00070208 MNG ID 01/01/2020 DOB

RESULTS INTERPRETATION: Results should be used in the context of available clinical information and should not be

used as the sole basis for patient management or treatment. Genetic counseling is recommended. Variants are assessed according to ACMG criteria (Richards et al. 2015; PMID: 25741868). This report contains interpretation of PATHOGENIC and LIKELY PATHOGENIC variants (by ACMG Criteria) as well as VARIANTS OF UNCERTAIN SIGNIFICANCE (VUS) with pathogenic predictions related to the clinical information provided. Variants not reported: (1) Variants classified as BENIGN or LIKELY BENIGN by ACMG Criteria; (2) VARIANTS OF UNCERTAIN SIGNIFICANCE (VUS) with benign or likely benign predictions; (3) Variants related to carrier status; (4) Polymorphisms with implications in drug response (pharmacogenomics variants). We will reanalyze the data periodically at the clinician’s request to allow potential reinterpretation based on new research or evidence. GnomAD abbreviations for population frequency data: African/African American (AFR), Latino (AMR), Ashkenazi Jewish (ASJ), East Asian (EAS), Finnish (FIN), Non-Finnish European (NFE), South Asian (SAS), Other (OTH).

NEXT GENERATION SEQUENCING (NGS) TEST LIMITATIONS: This assay will not consistently detect germline

mosaicism below 50% or rule out the presence of large chromosomal aberrations, including rearrangements and inversions that do not change copy number of genomic regions. The assay does not detect repeat expansions. Possible intergenic variant interactions are not commented on. False positive or false negative results may occur for reasons that include: insufficient information available about rare genetic variants, sex chromosome abnormalities, pseudogene interference, homologous regions, blood transfusions, bone marrow transplantation, somatic or tissue-specific mosaicism, mislabeled samples, or erroneous representation of family relationships. Variants that do not alter an amino acid composition of a protein may be difficult to assess for pathogenicity since they may produce abnormalities in structures NOT assessed by conventional analysis paradigms (e.g. mRNA expression and processing; Science 2006:314 (5807):1930-1933).

Interpretation of the clinical significance of gene variations is limited by information about the variant that is available at the time of reporting, and by the quality and quantity of clinical information provided with the sample. As the understanding of human genetic diversity improves, the interpretation of the clinical significance of variants may change.

IN SILICO ASSESSMENT: In silico assessments do NOT definitively identify a variant as benign or pathogenic and should

NOT be relied upon as the sole criteria for ANY patient diagnosis. MutationTaster, PolyPhen-2 and SIFT are tools which predict the possible impact of an amino acid substitution on the structure and function of human proteins (PMID 19561590, 22261837, 20354512, 21520341, 24681721; http://www.mutationtaster.org). A cross comparison of in silico methods is reported to demonstrate the following sensitivities and specificities: [MutationTaster2 88.7%, 87.4%] [PolyPhen2-DIV 85.8%, 82.1%] [SIFT 82.7%, 85.8%] (See www.mutationtaster.org for details).

FALSE POSITIVES AND NEGATIVES: For specimens sent to ANY laboratory, a consideration for false positive or false

negative test results is the improper labeling of the patient’s sample during the acquisition, shipping, receipt and testing process. We take no responsibility for any specimen labeling errors that occur before the sample arrives at Medical

Neurogenetics. LLC. If test results are inconsistent with the clinical presentation, call Medical Neurogenetics, LLC to discuss the case or submit another sample for confirmatory testing.

REQUEST FOR ADDITIONAL DATA: For researchers or clinicians interested in receiving a list of all variants identified in

the analysis, we will provide this list without interpretation at your request and at no additional charge. We will also provide details of gene coverage for the analysis at your request. Please submit all requests online at our website:

https://mnglabs.com/support/.

CONSENT FOR TESTING: Obtaining and maintaining patient consent for genetic testing is the sole responsibility of the

managing physician and institution. A genetic testing consent form is available on our website.

FDA NOTES: This test was developed and its performance characteristics determined by Medical Neurogenetics, LLC. It

has not been cleared or approved by the US Food and Drug Administration. FDA does not require this test to go through premarket FDA review. This test is used for clinical purposes. It should not be regarded as investigational or for research. This laboratory is certified under the Clinical Laboratory Improvement Amendments (CLIA) as qualified to perform high complexity clinical laboratory testing. CLIA ID# 11D0703390; Georgia Lab Lic. Code 060-381.

Appendix:

GENES ASSESSED BY THIS TEST:ALPL (171760), CLCN5, CYP27B1, CYP2R1, DMP1, ENPP1 (173335), FGF23, PHEX