Lysosomal Disorders and Mucopolysaccharidosis Panel

Last modified: Jun 12, 2018

Summary

  • Is a 102 gene panel that includes assessment of non-coding variants
  • Is ideal for patients with a clinical suspicion of lysosomal storage diseases (LSDs), mucolipidoses, mucopolysaccharidoses, glycoprotein storage disorders or lipid storage disorders. The genes on this panel are included in the Comprehensive Metabolism Panel.

Analysis methods

  • PLUS
  • SEQ
  • DEL/DUP

Availability

3-4 weeks

Number of genes

102

Test code

ME1501

CPT codes

SEQ 81405
SEQ 81406
SEQ 81407
DEL/DUP 81479

Summary

The Blueprint Genetics Lysosomal Disorders and Mucopolysaccharidosis Panel (test code ME1501):

  • Is a 102 gene panel that includes assessment of selected non-coding disease-causing variants
  • All exons of the GBA gene have segmentally duplicated pseudogenes that reduce sensitivity of NGS diagnostics in general. However, Blueprint Genetics custom assay has good coverage (>20x) with high mapping rates (mapping quality >40) for 100.0% of the target regions in GBA gene. Our validation showed high mean coverage of 184X for the GBA gene. Thus, our NGS Panel is not expected to have major limitations in detecting variants in GBA gene although clinical validation has not been performed at large scale for Gaucher disease.

  • Is available as PLUS analysis (sequencing analysis and deletion/duplication analysis), sequencing analysis only or deletion/duplication analysis only

Test Specific Strength

All exons of the GBA gene have segmentally duplicated pseudogenes that reduce sensitivity of NGS diagnostics in general. However, Blueprint Genetics custom assay has good coverage (>20x) with high mapping rates (mapping quality >40) for 100.0% of the target regions in GBA gene. Our validation showed high mean coverage of 184X for the GBA gene. Thus, our NGS Panel is not expected to have major limitations in detecting variants in GBA gene although clinical validation has not been performed at large scale for Gaucher disease.

ICD codes

Commonly used ICD-10 code(s) when ordering the Lysosomal Disorders and Mucopolysaccharidosis Panel

ICD-10 Disease
E75.00 Lipid storage disorders
E76.0 Mucopolysaccharidoses
E77.9 Glycoprotein storage disorders
E76.3 Mucolipidoses

Sample Requirements

  • EDTA blood, min. 1 ml
  • Purified DNA, min. 3μg
  • Saliva (Oragene DNA OG-500 kit)

Label the sample tube with your patient’s name, date of birth and the date of sample collection.

Note that we do not accept DNA samples isolated from formalin-fixed paraffin-embedded (FFPE) tissue.

About fifty different lysosomal storage diseases (LSDs) have been identified. These disorders are caused by mutations that result in the deficiency or reduced activity of intracellular enzymes that catabolize biological macromolecules. LSDs are caused by lysosomal dysfunction as the result of a single enzyme deficiency required for the metabolism of lipids, glycoproteins or mucopolysaccharides. These absence or impaired effectiveness of these enzymes results in accumulation of specific macromolecular compounds within lysosomes in various tissues and organs, causing progressive damage that can become life-threatening in some diseases. Although each LSD is individually rare, as a group they have an incidence of about 1/7,000-8,000 live births, varying between different populations. LSDs may be variably expressed as infantile, juvenile, or adult forms. In adult-onset diseases, the pathogenesis is usually slower than in the infantile or juvenile forms, and may include peripheral and CNS symptoms, whereas infantile and juvenile forms often involve progressive central nervous system involvement in addition to peripheral symptoms. LSDs exhibit clinical heterogeneity. Symptomatic pathology may be a function of mutation type and residual enzyme levels and specific mutations or types of mutations may be connected to discrete disease effects even if genotype-phenotype correlations are not strong. Most of LSDs are autosomal recessively inherited, however a few are X-linked recessively inherited, such as Fabry disease and Hunter syndrome (MPS2). Other examples of LSDs covered by this panel are Gaucher’s disease (the most common LSD), Tay-Sachs disease, Type II Pompe Disease, Salla disease, Krabbe disease and Hurler disease. Enzyme-replacement therapy (ERT) is now commercially available for six LSDs, typically used lifelong with specific management practices for each.

Genes in the Lysosomal Disorders and Mucopolysaccharidosis Panel and their clinical significance

Gene Associated phenotypes Inheritance ClinVar HGMD
ABCC8 Hyperinsulinemic hypoglycemia, Diabetes, permanent neonatal, Hypoglycemia, leucine-induced, Diabetes mellitus, transient neonatal AD/AR 128 621
ACY1 Aminoacylase 1 deficiency AR 5 14
ADAMTSL2*,# Geleophysic dysplasia AR 8 28
ADSL Adenylosuccinase deficiency AR 24 56
AGA Aspartylglucosaminuria AR 41 37
ALDH5A1 Succinic semialdehyde dehydrogenase deficiency AR 14 69
ALDH7A1 Epilepsy, pyridoxine-dependent AR 50 113
AMT Glycine encephalopathy AR 25 95
ANTXR2 Hyalinosis, infantile systemic, Fibromatosis, juveline hyaline AR 16 45
ARG1 Hyperargininemia AR 17 54
ARSA Metachromatic leukodystrophy AR 96 223
ARSB Mucopolysaccharidosis (Maroteaux-Lamy) AR 27 199
ASAH1 Spinal muscular atrophy with progressive myoclonic epilepsy, Farber lipogranulomatosis AR 13 71
ASPA Aspartoacylase deficiency (Canavan disease) AR 39 102
ATP13A2 Parkinson disease (Kufor-Rakeb syndrome) AR 20 37
BTD Biotinidase deficiency AR 180 236
CLN3 Neuronal ceroid lipofuscinosis, type 3 AR 87 71
CLN5 Neuronal ceroid lipofuscinosis, type 5 AR 49 45
CLN6 Neuronal ceroid lipofuscinosis, type 6 AR 29 82
CLN8 Neuronal ceroid lipofuscinosis, type 8 AR 35 42
COL2A1 Avascular necrosis of femoral head, Rhegmatogenous retinal detachment, Epiphyseal dysplasia, with myopia and deafness, Czech dysplasia, Achondrogenesis type 2, Platyspondylic dysplasia Torrance type, Hypochondrogenesis, Spondyloepiphyseal dysplasia congenital (SEDC), Spondyloepimetaphyseal dysplasia (SEMD) Strudwick type, Kniest dysplasia, Spondyloperipheral dysplasia, Mild SED with premature onset arthrosis, SED with metatarsal shortening, Stickler syndrome type 1 AD 166 544
COL11A2 Weissenbacher-Zweymuller syndrome, Deafness, Otospondylomegaepiphyseal dysplasia, Fibrochondrogenesis, Stickler syndrome type 3 (non-ocular) AD/AR 28 55
CTNS Cystinosis AR 55 146
CTSA Galactosialidosis AR 17 36
CTSC Periodontitis, juvenile, Haim-Munk syndrome, Papillon-Lefevre syndrome AR 16 92
CTSD Ceroid lipofuscinosis, neuronal AR 12 18
CTSK Pycnodysostosis AR 25 54
DHCR7 Smith-Lemli-Opitz syndrome AR 67 216
DPYD 5-fluorouracil toxicity, Epilepsy, hearing loss, and mental retardation syndrome; EHLMRS AD/AR 54 85
DYM Dyggve-Melchior-Clausen dysplasia, Smith-McCort dysplasia AR 21 34
ETFA Glutaric aciduria, Multiple acyl-CoA dehydrogenase deficiency AR 9 29
ETFB Glutaric aciduria, Multiple acyl-CoA dehydrogenase deficiency AR 6 14
ETFDH Glutaric aciduria, Multiple acyl-CoA dehydrogenase deficiency AR 41 173
FH Hereditary leiomyomatosis and renal cell cancer AD/AR 156 205
FOLR1 Cerebral folate deficiency AR 10 27
FUCA1 Fucosidosis AR 19 32
GAA Glycogen storage disease AR 147 558
GALC Krabbe disease AR 74 234
GALNS Mucopolysaccharidosis (Morquio syndrome) AR 48 334
GAMT Guanidinoacetate methyltransferase deficiency AR 18 58
GBA* Gaucher disease AR 84 471
GCDH Glutaric aciduria AR 70 208
GLA Fabry disease XL 215 919
GLB1 GM1-gangliosidosis, Mucopolysaccharidosis (Morquio syndrome) AR 65 212
GLDC Glycine encephalopathy AR 105 424
GM2A GM2-gangliosidosis, AB variant AR 9 10
GNE Inclusion body myopathy, Nonaka myopathy, Sialuria AD/AR 58 201
GNPTAB Mucolipidosis AR 155 183
GNPTG Mucolipidosis AR 28 45
GNS Mucopolysaccharidosis (Sanfilippo syndrome) AR 7 25
GPC3 Simpson-Golabi-Behmel syndrome XL 29 72
GUSB* Mucopolysaccharidosis AR 26 61
HEXA Tay-Sachs disease, GM2-gangliosidosis, Hexosaminidase A deficiency AR 112 184
HEXB Sandhoff disease AR 34 111
HGSNAT Mucopolysaccharidosis (Sanfilippo syndrome), Retinitis pigmentosa AR 24 70
HRAS Costello syndrome, Congenital myopathy with excess of muscle spindles AD 41 29
HYAL1 Mucopolysaccharidosis AR 2 3
IDS* Mucopolysaccharidosis XL 80 631
IDUA Mucopolysaccharidosis AR 57 274
L2HGDH L-2-hydroxyglutaric aciduria AR 13 76
LAMA2 Muscular dystrophy, congenital merosin-deficient AR 125 294
LAMP2 Danon disease XL 57 97
LDB3 Dilated cardiomyopathy (DCM), Myopathy, myofibrillar AD 9 14
LIPA Wolman disease, Cholesterol ester storage disease AR 13 84
MAN1B1 Mental retardation AR 8 25
MAN2B1 Mannosidosis, alpha B, lysosomal AR 37 144
MANBA Mannosidosis, lysosomal AR 12 18
MCOLN1 Mucolipidosis AR 46 35
MFSD8 Ceroid lipofuscinosis, neuronal AR 25 46
MOCS1* Molybdenum cofactor deficiency AR 7 35
MOCS2 Molybdenum cofactor deficiency AR 10 15
MYOT Myopathy, myofibrillar, Muscular dystrophy, limb-girdle, 1A, Myopathy, spheroid body AD 7 16
NAGA Kanzaki disease, Alpha-n-acetylgalactosaminidase deficiency, Schindler disease type I, Schindler disease type III AR 6 9
NAGLU Mucopolysaccharidosis (Sanfilippo syndrome), Charcot-Marie-Tooth disease, axonal, type 2V AR 35 168
NEU1 Sialidosis AR 22 60
NPC1 Niemann-Pick disease AR 120 453
NPC2 Niemann-pick disease AR 16 27
PEX1 Heimler syndrome, Peroxisome biogenesis factor disorder 1A, Peroxisome biogenesis factor disorder 1B AR 80 132
PEX3 Zellweger syndrome, Peroxisome biogenesis disorder AR 4 10
PEX5 Adrenoleukodystrophy, neonatal, Rhizomelic chondrodysplasia punctata, Zellweger syndrome, Peroxisome biogenesis disorder AR 8 14
PEX6 Heimler syndrome, Peroxisome biogenesis disorder 4A, Peroxisome biogenesis disorder 4B AR 32 106
PEX10 Adrenoleukodystrophy, neonatal, Zellweger syndrome, Peroxisome biogenesis disorder, Ataxia AR 19 29
PEX12 Zellweger syndrome, Peroxisome biogenesis disorder AR 22 37
PEX13 Adrenoleukodystrophy, neonatal, Zellweger syndrome, Peroxisome biogenesis disorder AR 5 10
PEX16 Zellweger syndrome, Peroxisome biogenesis disorder AR 8 12
PEX26 Adrenoleukodystrophy, neonatal, Zellweger syndrome, Peroxisome biogenesis disorder AR 13 27
PGK1 Phosphoglycerate kinase 1 deficiency XL 15 26
PHYH Refsum disease AR 11 36
PPT1* Ceroid lipofuscinosis, neuronal AR 86 77
PRODH* Hyperprolinemia AR 44 10
PSAP Krabbe disease, atypical, Metachromatic leukodystrophy due to saposin-b deficiency, Combined saposin deficiency, Gaucher disease, atypical, due to saposin C deficiency AR 18 26
QDPR Hyperphenylalaninemia, BH4-deficient AR 13 61
RAI1 Smith-Magenis syndrome AD 35 109
SGSH Mucopolysaccharidosis (Sanfilippo syndrome) AR 35 147
SLC17A5 Sialuria, Finnish (Salla disease), Infantile sialic acid storage disorder AR 41 52
SLC25A15* Hyperornithinemia-hyperammonemia-homocitrullinemia syndrome AR 22 36
SLC46A1 Folate malabsorption AR 17 20
SMPD1 Niemann-Pick disease AR 84 249
SUMF1 Multiple sulfatase deficiency AR 21 52
SUOX Sulfocysteinuria AR 6 28
TCF4 Corneal dystrophy, Fuchs endothelial, Pitt-Hopkins syndrome AD 86 141
TPP1 Spinocerebellar ataxia, Neuronal ceroid lipofuscinosis type 2 AR 56 110

* Some, or all, of the gene is duplicated in the genome. Read more.

# The gene has suboptimal coverage (means <90% of the gene’s target nucleotides are 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 (#)

Gene refers to the HGNC approved gene symbol; Inheritance refers to inheritance patterns such as autosomal dominant (AD), autosomal recessive (AR), X-linked (XL), X-linked dominant (XLD) and X-linked recessive (XLR); ClinVar refers to the number of variants in the gene classified as pathogenic or likely pathogenic in this database (ClinVar); HGMD refers to the number of variants with possible disease association in the gene listed in Human Gene Mutation Database (HGMD). The list of associated, gene specific phenotypes are generated from CGD or Orphanet databases.

Non-coding variants covered by the panel

Gene Genomic location HG19 HGVS RefSeq RS-number
ABCC8 Chr11:17498513 c.-190C>G NM_000352.3
ABCC8 Chr11:17465872 c.1333-1013A>G NM_000352.3
ADSL Chr22:40742514 c.-49T>C NM_000026.2
ALDH7A1 Chr5:125907053 c.696-502G>C NM_001182.4
AMT Chr3:49459938 c.-55C>T NM_000481.3 rs386833677
ARG1 Chr6:131901748 c.306-611T>C NM_000045.3
ARSA Chr22:51064121 c.1108-12C>G NM_000487.5 rs757806374
BTD Chr3:15687154 c.*159G>A NM_000060.2 rs530872564
CLN3 Chr16:28497984 c.461-13G>C NM_000086.2 rs386833721
COL2A1 Chr12:48379984 c.1527+135G>A NM_001844.4
CTNS Chr17:3539712 c.-643G>T NM_004937.2
CTNS Chr17:3552117 c.141-24T>C NM_001031681.2
CTNS Chr17:3563518 c.971-12G>A NM_001031681.2 rs375952052
CTSC Chr11:88070895 c.-55C>A NM_001814.4 rs766114323
ETFDH Chr4:159593534 c.-75A>G NM_004453.2
GAA Chr17:78078369 c.-17C>T NM_000152.3
GAA Chr17:78078341 c.-32-13T>A NM_000152.3
GAA Chr17:78078341 c.-32-13T>G NM_000152.3 rs386834236
GAA Chr17:78078352 c.-32-2A>G NM_000152.3
GAA Chr17:78078351 c.-32-3C>A NM_000152.3
GAA Chr17:78078351 c.-32-3C>A/G NM_000152.3
GAA Chr17:78082266 c.1076-22T>G NM_000152.3 rs762260678
GAA Chr17:78092432 c.2647-20T>G NM_000152.3
GALC Chr14:88459917 c.-74T>A NM_001201402.1
GALNS Chr16:88908390 c.245-11C>G NM_000512.4
GAMT Chr19:1399508 c.391+15G>T NM_138924.2 rs367567416
GCDH Chr19:13010271 c.1244-11A>G NM_000159.3
GLA ChrX:100656225 c.547+395G>C NM_000169.2
GLA ChrX:100653945 c.640-11T>A NM_000169.2
GLA ChrX:100654735 c.640-801G>A NM_000169.2 rs199473684
GLA ChrX:100654793 c.640-859C>T NM_000169.2 rs869312374
GNPTG Chr16:1412562 c.610-16_609+28del NM_032520.4 rs193302853
HEXB Chr5:74014605 c.1243-17A>G NM_000521.3
HEXB Chr5:74016442 c.1509-26G>A NM_000521.3 rs201580118
HEXB Chr5:74016926 c.1614-16_1615dupTTCATGTTATCTACAGAC NM_000521.3 rs756912360
IDS ChrX:148564764 c.1181-15C>A NM_000202.5
IDS ChrX:148578704 c.709-657G>A NM_000202.5
L2HGDH Chr14:50735527 c.906+354G>A NM_024884.2
LAMA2 Chr6:129636608 c.3556-13T>A NM_000426.3 rs775278003
LAMA2 Chr6:129714172 c.5235-18G>A NM_000426.3 rs188365084
LAMP2 ChrX:119604078 c.-1054A>C NM_001122606.1
MOCS1 Chr6:39874534 c.*365_*366delAG NM_005943.5 rs397518419
NPC1 Chr18:21132700 c.1554-1009G>A NM_000271.4
NPC1 Chr18:21137182 c.882-28A>G/T NM_000271.4
PEX6 Chr6:42933952 c.2300+28G>A NM_000287.3 rs267608237
PEX6 Chr6:42933858 c.2301-15C>G NM_000287.3 rs267608236
PPT1 Chr1:40539203 c.*526_*529delATCA NM_000310.3 rs386833624
PPT1 Chr1:40558194 c.125-15T>G NM_000310.3 rs386833629
PSAP Chr10:73583679 c.778-26C>A NM_001042465.1
QDPR Chr4:17500790 c.436+2552A>G NM_000320.2
TPP1 Chr11:6637752 c.887-18A>G NM_000391.3

Added and removed genes from the panel

Genes added Genes removed
GM2A
LAMP2
MAN2B1
NAGA
HPD

Test strength

All exons of the GBA gene have segmentally duplicated pseudogenes that reduce sensitivity of NGS diagnostics in general. However, Blueprint Genetics custom assay has good coverage (>20x) with high mapping rates (mapping quality >40) for 100.0% of the target regions in GBA gene. Our validation showed high mean coverage of 184X for the GBA gene. Thus, our NGS Panel is not expected to have major limitations in detecting variants in GBA gene although clinical validation has not been performed at large scale for Gaucher disease.

The strengths of this test include:
  • CAP and ISO-15189 accreditations covering all operations at Blueprint Genetics including all Whole Exome Sequencing, NGS panels and confirmatory testing
  • CLIA-certified personnel performing clinical testing in a CLIA-certified laboratory
  • Powerful sequencing technologies, advanced target enrichment methods and precision bioinformatics pipelines ensure superior analytical performance
  • Careful construction of clinically effective and scientifically justified gene panels
  • Our Nucleus online portal providing transparent and easy access to quality and performance data at the patient level
  • Our publically available analytic validation demonstrating complete details of test performance
  • ~1,500 non-coding disease causing variants in Blueprint WES assay (please see below ‘Non-coding disease causing variants covered by this panel’)
  • Our rigorous variant classification based on modified ACMG variant classification scheme
  • Our systematic clinical interpretation workflow using proprietary software enabling accurate and traceable processing of NGS data
  • Our comprehensive clinical statements

Test limitations

The following exons are not included in the panel as they are not sufficiently covered with high quality sequence reads: ADAMTSL2 (11-19). Genes with suboptimal coverage in our assay are marked with number sign (#) and genes with partial, or whole gene, segmental duplications in the human genome are marked with an asterisk (*) if they overlap with the UCSC pseudogene regions. Gene is considered to have suboptimal coverage when >90% of the gene's target nucleotides are not covered at >20x with mapping quality score (MQ>20) reads. The technology may have limited sensitivity to detect variants in genes marked with these symbols (please see the Panel content table above).

This test does not detect the following:
  • Complex inversions
  • Gene conversions
  • Balanced translocations
  • Mitochondrial DNA variants
  • Repeat expansion disorders unless specifically mentioned
  • Non-coding variants deeper than ±20 base pairs from exon-intron boundary unless otherwise indicated (please see above Panel Content / non-coding variants covered by the panel).

This test may not reliably detect the following:

  • Low level mosaicism
  • Stretches of mononucleotide repeats
  • Indels larger than 50bp
  • Single exon deletions or duplications
  • Variants within pseudogene regions/duplicated segments

The sensitivity of this test may be reduced if DNA is extracted by a laboratory other than Blueprint Genetics.

For additional information, please refer to the Test performance section and see our Analytic Validation.

The Blueprint Genetics lysosomal disorders and mucopolysaccharidosis panel covers classical genes associated with lysosomal storage diseases (LSDs), lipid storage disorders, mucopolysaccharidoses, glycoprotein storage disorders and mucolipidoses. The genes on the panel have been carefully selected based on scientific literature, mutation databases and our experience.

Our panels are sliced from our high-quality whole exome sequencing data. Please see our sequencing and detection performance table for different types of alterations at the whole exome level (Table).

Assays have been validated for different starting materials including EDTA-blood, isolated DNA (no FFPE), saliva and dry blood spots (filter card) and all provide high-quality results. The diagnostic yield varies substantially depending on the assay used, referring healthcare professional, hospital and country. Blueprint Genetics' Plus Analysis (Seq+Del/Dup) maximizes the chance to find a molecular genetic diagnosis for your patient although Sequence Analysis or Del/Dup Analysis may be a cost-effective first line test if your patient's phenotype is suggestive of a specific mutation type.

Performance of Blueprint Genetics Whole Exome Sequencing (WES) assay. All individual panels are sliced from WES data.

Sensitivity % (TP/(TP+FN) Specificity %
Single nucleotide variants 99.65% (412,456/413,893) >99.99%
Insertions, deletions and indels by sequence analysis
1-10 bps 96.94% (17,070/17,608) >99.99%
11-50 bps 99.07% (957/966) >99.99%
Copy number variants (exon level dels/dups)
Clinical samples (small CNVs, n=52)
1 exon level deletion 92.3% (24/26) NA
2 exons level deletion/duplication 100.0% (11/11) NA
3-7 exons level deletion/duplication 93.3% (14/15) NA
Microdeletion/-duplication sdrs (large CNVs, n=37))
Size range (0.1-47 Mb) 100% (37/37)
Simulated CNV detection
2 exons level deletion/duplication 90.98% (7,357/8,086) 99.96%
5 exons level deletion/duplication 98.63% (7,975/8,086) 99.98%
     
The performance presented above reached by WES with the following coverage metrics
     
Mean sequencing depth at exome level 174x
Nucleotides with >20x sequencing coverage (%) 99.4%

Bioinformatics

The target region for each gene includes coding exons and ±20 base pairs from the exon-intron boundary. In addition, the panel includes non-coding variants if listed above (Non-coding variants covered by the panel). Some regions of the gene(s) may be removed from the panel if specifically mentioned in the ‘Test limitations” section above. The sequencing data generated in our laboratory is analyzed with our proprietary data analysis and annotation pipeline, integrating state-of-the art algorithms and industry-standard software solutions. Incorporation of rigorous quality control steps throughout the workflow of the pipeline ensures the consistency, validity and accuracy of results. Our pipeline is streamlined to maximize sensitivity without sacrificing specificity. We have incorporated a number of reference population databases and mutation databases such as, but not limited, to 1000 Genomes Project, gnomAD, ClinVar and HGMD into our clinical interpretation software to make the process effective and efficient. For missense variants, in silico variant prediction tools such as SIFT, PolyPhen, MutationTaster are used to assist with variant classification. Through our online ordering and statement reporting system, Nucleus, the customer has an access to details of the analysis, including patient specific sequencing metrics, a gene level coverage plot and a list of regions with inadequate coverage if present. This reflects our mission to build fully transparent diagnostics where customers have easy access to crucial details of the analysis process.

Clinical interpretation

We provide customers with the most comprehensive clinical report available on the market. Clinical interpretation requires a fundamental understanding of clinical genetics and genetic principles. At Blueprint Genetics, our PhD molecular geneticists, medical geneticists and clinical consultants prepare the clinical statement together by evaluating the identified variants in the context of the phenotypic information provided in the requisition form. Our goal is to provide clinically meaningful statements that are understandable for all medical professionals regardless of whether they have formal training in genetics.

Variant classification is the corner stone of clinical interpretation and resulting patient management decisions. Our classifications follow the Blueprint Genetics Variant Classification Schemes based on the ACMG guideline 2015. Minor modifications were made to increase reproducibility of the variant classification and improve the clinical validity of the report. Our experience with tens of thousands of clinical cases analyzed at our laboratory allowed us to further develop the industry standard.

The final step in the analysis of sequence variants is confirmation of variants classified as pathogenic or likely pathogenic using bi-directional Sanger sequencing. Variant(s) fulfilling all of the following criteria are not Sanger confirmed: 1) the variant quality score is above the internal threshold for a true positive call, 2) an unambiguous IGV in-line with the variant call and 3) previous Sanger confirmation of the same variant at least three times at Blueprint Genetics. Reported variants of uncertain significance are confirmed with bi-directional Sanger sequencing only if the quality score is below our internally defined quality score for true positive call. Reported copy number variations with a size <10 exons are confirmed by orthogonal methods such as qPCR if the specific CNV has been seen less than three times at Blueprint Genetics.

Our clinical statement includes tables for sequencing and copy number variants that include basic variant information (genomic coordinates, HGVS nomenclature, zygosity, allele frequencies, in silico predictions, OMIM phenotypes and classification of the variant). In addition, the statement includes detailed descriptions of the variant, gene and phenotype(s) including the role of the specific gene in human disease, the mutation profile, information about the gene’s variation in population cohorts and detailed information about related phenotypes. We also provide links to the references used, congress abstracts and mutation databases to help our customers further evaluate the reported findings if desired. The conclusion summarizes all of the existing information and provides our rationale for the classification of the variant.

Identification of pathogenic or likely pathogenic variants in dominant disorders or their combinations in different alleles in recessive disorders are considered molecular confirmation of the clinical diagnosis. In these cases, family member testing can be used for risk stratification within the family. In the case of variants of uncertain significance (VUS), we do not recommend family member risk stratification based on the VUS result. Furthermore, in the case of VUS, we do not recommend the use of genetic information in patient management or genetic counseling. For eligible cases, Blueprint Genetics offers a no charge service to investigate the role of reported VUS (VUS Clarification Service).

Our interpretation team analyzes millions of variants from thousands of individuals with rare diseases. Thus, our database, and our understanding of variants and related phenotypes, is growing by leaps and bounds. Our laboratory is therefore well positioned to re-classify previously reported variants as new information becomes available. If a variant previously reported by Blueprint Genetics is re-classified, our laboratory will issue a follow-up statement to the original ordering health care provider at no additional cost.