Anemia Panel

Last modified: Jun 12, 2018

Summary

  • Is a 78 gene panel that includes assessment of non-coding variants
  • Is ideal for patients suspected to have hereditary anemia who have had HBA1 and HBA2 variants excluded as the cause of their anemia or patients suspected to have hereditary anemia who are not suspected to have HBA1 or HBA2 variants as the cause of their anemia. The genes on this panel are included in the Comprehensive Hematology Panel.

    Is not recommended for patients suspected to have anemia due to alpha-thalassemia (HBA1 or HBA2). These genes are highly homologous reducing mutation detection rate due to challenges in variant call and difficult to detect mutation profile (deletions and gene-fusions within the homologous genes tandem in the human genome).

Analysis methods

  • PLUS
  • SEQ
  • DEL/DUP

Availability

3-4 weeks

Number of genes

78

Test code

HE0401

CPT codes

SEQ 81406
SEQ 81407
SEQ 81408
DEL/DUP 81479

Summary

The Blueprint Genetics Anemia Panel (test code HE0401):

  • Is a 78 gene panel that includes assessment of selected non-coding disease-causing variants
  • Is available as PLUS analysis (sequencing analysis and deletion/duplication analysis), sequencing analysis only or deletion/duplication analysis only

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.

Anemia is defined as a decrease in the amount of red blood cells or hemoglobin in the blood. The symptoms of anemia include fatigue, weakness, pale skin, and shortness of breath. Other more serious symptoms may occur depending on the underlying cause. The causes of anemia may be classified as impaired red blood cell (RBC) production or increased RBC destruction (hemolytic anemias). Hereditary anemia may be clinically highly variable, including mild, moderate, or severe forms. Hb Bart syndrome is a severe form of anemia secondary to alpha thalassemia. It is characterized by hydrops fetalis leading to death almost always in utero or shortly after birth. The thalassemias, sickle cell disease, and other hemoglobinopathies represent a major group of inherited disorders of hemoglobin synthesis (HBA1HBA2HBB). The thalassemias are among the most common genetic disorders worldwide, occurring more frequently in the Mediterranean region, the Indian subcontinent, Southeast Asia, and West Africa. Hereditary spherocytosis and hereditary elliptocytosis are examples of inherited hemolytic anemias. Hereditary spherocytosis is the most common congenital hemolytic anemia among Caucasians with an estimated prevalence ranging from 1:2,000 to 1:5,000.

Genes in the Anemia Panel and their clinical significance

Gene Associated phenotypes Inheritance ClinVar HGMD
ABCB7 Anemia, sideroblastic, and spinocerebellar ataxia XL 8 9
ADAMTS13 Schulman-Upshaw syndrome, Thrombotic thrombocytopenic purpura, familial AR 27 167
ALAS2 Anemia, sideroblastic, Protoporphyria, erythropoietic XL 27 98
AMN Megaloblastic anemia-1, Norwegian AR 26 33
ANK1 Spherocytosis AD/AR 14 88
ATM Breast cancer, Ataxia-Telangiectasia AD/AR 860 1026
ATR Cutaneous telangiectasia and cancer syndrome, Seckel syndrome AD/AR 8 18
ATRX Carpenter-Waziri syndrome, Alpha-thalassemia/mental retardation syndrome, Holmes-Gang syndrome, Juberg-Marsidi syndrome, Smith-Fineman-Myers syndrome, Mental retardation-hypotonic facies syndrome XL 59 163
BLM Bloom syndrome AR 91 107
BRCA2 Fanconi anemia, Medulloblastoma, Glioma susceptibility, Pancreatic cancer, Wilms tumor, Breast-ovarian cancer, familial AD/AR 2959 2364
BRIP1 Fanconi anemia, Breast cancer AD/AR 182 166
C15ORF41 Congenital dyserythropoietic anemia AR 3 2
CDAN1 Anemia, dyserythropoietic congenital AR 12 49
CLCN7 Osteopetrosis AD/AR 13 95
CUBN* Megaloblastic anemia-1, Finnish AR 37 52
CYB5R3 Methemoglobinemia due to methemoglobin reductase deficiency AR 21 66
DHFR* Megaloblastic anemia due to dihydrofolate reductase deficiency AR 2 5
DNAJC21 Bone marrow failure syndrome 3 AR 5 8
EPB42 Spherocytosis AR 8 14
ERCC4 Fanconi anemia, Xeroderma pigmentosum, XFE progeroid syndrome AR 11 59
FANCA Fanconi anemia AR 76 541
FANCB Fanconi anemia XL 10 20
FANCC Fanconi anemia AR 70 58
FANCD2* Fanconi anemia AR 14 57
FANCE Fanconi anemia AR 5 16
FANCF Fanconia anemia AR 7 16
FANCG Fanconi anemia AR 13 89
FANCI Fanconi anemia AR 12 43
FANCL Fanconi anemia AR 7 22
FANCM Fanconi anemia AR 2 49
G6PD Glucose-6-phosphate dehydrogenase deficiency XL 42 222
GATA1 Anemia, without thrombocytopenia, Thrombocytopenia with beta-thalessemia,, Dyserythropoietic anemia with thrombocytopenia XL 19 15
GPI Hemolytic anemia, nonspherocytic due to glucose phosphate isomerase deficiency AD 11 37
GSS Glutathione synthetase deficiency AR 8 34
HBA1* Alpha-thalassemia (Hemoglobin Bart syndrome), Alpha-thalassemia (Hemoglobin H disease) AR/Digenic 16 205
HBA2*,# Alpha-thalassemia (Hemoglobin Bart syndrome), Alpha-thalassemia (Hemoglobin H disease) AR/Digenic 41 286
HBB Sickle cell disease, Thalassemia-beta, dominant inclusion body, Other Thalassemias/Hemoglobinopathies, Beta-thalassemia, Hereditary persistence of fetal hemogoblin AD/AR/Digenic 200 854
HFE Hemochromatosis AR/Digenic 10 56
KLF1 Anemia, dyserythropoietic congenital, Blood group, Lutheran inhibitor, Hereditary persistence of fetal hemoglobin AD/BG 16 46
LPIN2 Majeed syndrome AR 9 12
MTR Methylmalonic acidemia AR 13 40
NBN Breast cancer, Nijmegen breakage syndrome AD/AR 141 87
NT5C3A Uridine 5-prime monophosphate hydrolase deficiency, hemolytic anemia due to AR 10 28
PALB2 Fanconi anemia, Pancreatic cancer, Breast cancer AD/AR 422 358
PC Pyruvate carboxylase deficiency AR 31 40
PDHA1 Leigh syndrome, Pyruvate dehydrogenase E1-alpha deficiency XL 62 191
PDHX Pyruvate dehydrogenase E3-binding protein deficiency AR 14 22
PIEZO1 Dehydrated hereditary stomatocytosis, Lympehedema, hereditary III AD 22 44
PKLR Pyruvate kinase deficiency, Elevation of red blood cell ATP levels, familial AR 17 263
PUS1 Mitochondrial myopathy and sideroblastic anemia AR 7 8
RAD51C Fanconi anemia, Breast-ovarian cancer, familial AD/AR 92 112
REN Hyperuricemic nephropathy, Hyperproreninemia, familial, Renal tubular dysgenesis AD 8 18
RHAG Overhydrated hereditary stomatocytosis, Anemia, hemolytic, Rh-null, regulator type, Anemia, hemolytic,Rh-Mod type, RHAG blood group AD/AR/BG 13 27
RPL5 Diamond-Blackfan anemia AD 14 72
RPL11 Diamond-Blackfan anemia AD 9 41
RPL15* Diamond-Blackfan anemia AD 2 2
RPL35A Diamond-Blackfan anemia AD 5 14
RPS7 Diamond-Blackfan anemia AD 2 9
RPS10 Diamond-Blackfan anemia AD 3 5
RPS19 Diamond-Blackfan anemia AD 22 168
RPS24 Diamond-Blackfan anemia AD 5 9
RPS26 Diamond-Blackfan anemia AD 10 30
RPS29 Diamond-Blackfan anemia AD 4 3
SBDS* Aplastic anemia, Shwachman-Diamond syndrome, Severe spondylometaphyseal dysplasia AD/AR 21 90
SEC23B Anemia, dyserythropoietic congenital AR 15 120
SLC4A1 Spherocytosis, Ovalcytosis, Renal tubular acidosis, distal, with hemolytic anemia, Cryohydrocytosis, Acanthocytosis, Band 3 Memphis AD/AR/BG 33 114
SLC19A2 Thiamine-responsive megaloblastic anemia syndrome AR 13 49
SLC25A38 Anemia, sideroblastic 2, pyridoxine-refractory AR 6 25
SLX4 Fanconi anemia AR 14 54
SPTA1 Spherocytosis, Ellipsocytosis, Pyropoikilocytosis AD/AR 24 42
SPTB Spherocytosis, Anemia, neonatal hemolytic, Ellipsocytosis AD/AR 18 74
TCN2 Transcobalamin II deficiency AR 9 33
TF Atransferrinemia AR 8 16
THBD Thrombophilia due to thrombomodulin defect, Hemolytic uremic syndrome, atypical AD 5 22
TMPRSS6 Iron-refractory iron deficiency anemia AR 13 76
TPI1 Triosephosphate isomerase deficiency AR 8 19
XRCC2 Hereditary breast cancer AD/AR 10 20
YARS2 Myopathy, lactic acidosis, and sideroblastic anemia AR 26 11

* 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
ALAS2 ChrX:55054634 c.-15-2186C>G NM_000032.4
ALAS2 ChrX:55054635 c.-15-2187T>C NM_000032.4
ALAS2 ChrX:55054636 c.-15-2188A>G NM_000032.4
ALAS2 ChrX:55057617 c.-258C>G NM_000032.4 rs140772352
AMN Chr14:103396458 c.1007-29_1006+36delTCGCCCCGCCGCGGG NM_030943.3 rs386834162
AMN Chr14:103396444 c.1007-31_1006+34delCCTCGCCCCGCCGCG NM_030943.3 rs386834161
AMN Chr14:103395424 c.514-34G>A NM_030943.3 rs144077391
ANK1 Chr8:41655127 c.-73_-72delTG NM_020476.2 rs786205242
ANK1 Chr8:41655209 c.127-39554G>A NM_001142446.1 rs183894680
ANK1 Chr8:41566510 c.1900-17G>A NM_001142446.1 rs786205243
ANK1 Chr8:41566511 c.1900-18C>A NM_001142446.1
ATM Chr11:108093770 c.-174A>G NM_000051.3
ATM Chr11:108098321 c.-30-1G>T NM_000051.3 rs869312754
ATM Chr11:108094508 c.-31+595G>A NM_000051.3
ATM Chr11:108121024 c.1236-404C>T NM_000051.3
ATM Chr11:108138753 c.2639-384A>G NM_000051.3
ATM Chr11:108141209 c.2839-579_2839-576delAAGT NM_000051.3
ATM Chr11:108151710 c.3403-12T>A NM_000051.3 rs201370733
ATM Chr11:108158168 c.3994-159A>G NM_000051.3 rs864622543
ATM Chr11:108179837 c.5763-1050A>G NM_000051.3 rs774925473
BRCA2 Chr13:32889805 c.-40+1G>A NM_000059.3
BRCA2 Chr13:32953872 c.8954-15T>G NM_000059.3
BRCA2 Chr13:32971007 c.9502-28A>G NM_000059.3 rs397508059
BRIP1 Chr17:59858864 c.1629-498A>T NM_032043.2
CLCN7 Chr16:1506057 c.916+57A>T NM_001287.5
CUBN Chr10:17088532 c.3330-439C>G NM_001081.3 rs386833782
FANCA Chr16:89849346 c.1567-20A>G NM_000135.2 rs775154397
FANCA Chr16:89836805 c.2223-138A>G NM_000135.2
FANCA Chr16:89836111 c.2504+134A>G NM_000135.2
FANCA Chr16:89831215 c.2778+83C>G NM_000135.2 rs750997715
FANCA Chr16:89818822 c.2982-192A>G NM_000135.2
FANCA Chr16:89816056 c.3239+82T>G NM_000135.2
FANCA Chr16:89864654 c.893+920C>A NM_000135.2
FANCC Chr9:98011653 c.-78-2A>G NM_000136.2 rs587779898
FANCD2 Chr3:10083186 c.696-121C>G NM_033084.3
FANCI Chr15:89825208 c.1583+142C>T NM_001113378.1
GATA1 ChrX:48649496 c.-19-2A>G NM_002049.3
GSS Chr20:33543525 c.-9+5G>A NM_000178.2
HBA2 Chr16:223691 c.*92A>G NM_000517.4 rs63750067
HBB Chr11:5246720 c.*108A>C/G NM_000518.4
HBB Chr11:5246718 c.*110T>A/C NM_000518.4 rs33978907
HBB Chr11:5246718 c.*110T>G NM_000518.4
HBB Chr11:5246713 c.*110_*114delTAAAA NM_000518.4 rs35949130
HBB Chr11:5246713 c.*110_*114delTAAAA NM_000518.4 rs606231219
HBB Chr11:5246713 c.*110_*114delTAAAA NM_000518.4 rs606231219,rs35949130
HBB Chr11:5246717 c.*111A>G NM_000518.4 rs63751128
HBB Chr11:5246716 c.*112A>G/T NM_000518.4 rs63750954
HBB Chr11:5246715 c.*113A>G NM_000518.4 rs33985472
HBB Chr11:5246699 c.*129T>C NM_000518.4
HBB Chr11:5246696 c.*132C>A/T NM_000518.4
HBB Chr11:5246796 c.*32A>C NM_000518.4
HBB Chr11:5246781 c.*47C>G NM_000518.4
HBB Chr11:5246754 c.*74A>G NM_000518.4 rs369101035
HBB Chr11:5248351 c.-100G>A NM_000518.4 rs281864524
HBB Chr11:5248357 c.-106G>C NM_000518.4 rs63750681
HBB Chr11:5248372 c.-121C>T NM_000518.4 rs281864518
HBB Chr11:5248374 c.-123A>T NM_000518.4
HBB Chr11:5248377 c.-126C>A NM_000518.4
HBB Chr11:5248378 c.-127G>C NM_000518.4
HBB Chr11:5248263 c.-12C>T NM_000518.4 rs113115948
HBB Chr11:5248387 c.-136C>A/G/T NM_000518.4 rs33994806
HBB Chr11:5248388 c.-137C>A/G/T NM_000518.4 rs33941377
HBB Chr11:5248389 c.-138C>A/T NM_000518.4 rs33944208
HBB Chr11:5248391 c.-140C>T NM_000518.4 rs34999973
HBB Chr11:5248393 c.-142C>T NM_000518.4 rs34883338
HBB Chr11:5248394 c.-143C>G NM_000518.4 rs63751043
HBB Chr11:5248402 c.-151C>T NM_000518.4 rs63751208
HBB Chr11:5248403 c.-152C>A NM_000518.4
HBB Chr11:5248269 c.-18C>G NM_000518.4 rs34135787
HBB Chr11:5248272 c.-21T>A NM_000518.4
HBB Chr11:5248491 c.-240G>A NM_000518.4 rs753344875
HBB Chr11:5248524 c.-273T>C NM_000518.4 rs139703273
HBB Chr11:5248280 c.-29G>A NM_000518.4 rs34704828
HBB Chr11:5248282 c.-31C>T NM_000518.4 rs63750628
HBB Chr11:5248294 c.-43C>T NM_000518.4
HBB Chr11:5248301 c.-50A>C NM_000518.4 rs34305195
HBB Chr11:5248301 c.-50A>G/T NM_000518.4
HBB Chr11:5248326 c.-75G>C NM_000518.4 rs63750400
HBB Chr11:5248326 c.-75G>T NM_000518.4
HBB Chr11:5248327 c.-76A>C NM_000518.4 rs281864525
HBB Chr11:5248328 c.-77A>G/T NM_000518.4
HBB Chr11:5248329 c.-78A>C/G NM_000518.4 rs33931746
HBB Chr11:5248330 c.-79A>G NM_000518.4 rs34598529
HBB Chr11:5248331 c.-80T>A/C NM_000518.4 rs33980857
HBB Chr11:5248332 c.-81A>C/G NM_000518.4 rs33981098
HBB Chr11:5248333 c.-82C>A/T NM_000518.4 rs34500389
HBB Chr11:5248342 c.-91A>C NM_000518.4
HBB Chr11:5248343 c.-92C>G NM_000518.4 rs397515291
HBB Chr11:5247062 c.316-106C>G NM_000518.4 rs34690599
HBB Chr11:5247081 c.316-125A>G NM_000518.4 rs63751175
HBB Chr11:5247102 c.316-146T>G NM_000518.4 rs35328027
HBB Chr11:5246970 c.316-14T>G NM_000518.4 rs35703285
HBB Chr11:5247153 c.316-197C>T NM_000518.4 rs34451549
HBB Chr11:5247216 c.316-260T>C NM_000518.4
HBB Chr11:5247046 c.316-90A>G NM_000518.4 rs63750433
HBB Chr11:5248044 c.93-15T>G NM_000518.4 rs35456885
HBB Chr11:5248050 c.93-21G>A NM_000518.4 rs35004220
HFE Chr6:26087649 c.-20G>A NM_000410.3 rs138378000
KLF1 Chr19:12998078 c.-124T>C NM_006563.3
KLF1 Chr19:12998108 c.-154C>T NM_006563.3 rs372651309
MTR Chr1:237057461 c.3205-196A>G NM_000254.2 rs544410324
MTR Chr1:236971838 c.340-166A>G NM_000254.2
MTR Chr1:236977232 c.609+1088G>A NM_000254.2 rs752526782
PALB2 Chr16:23649285 c.109-12T>A NM_024675.3 rs774949203
PC Chr11:66620883 c.1369-29A>G NM_000920.3
PDHA1 ChrX:19377861 c.*79_*90dupAGTCAATGAAAT NM_001173454.1
PDHA1 ChrX:19372579 c.625-30G>A NM_001173454.1
PDHA1 ChrX:19373648 c.873+26G>A NM_001173454.1
PDHX Chr11:34988372 c.816+11C>G NM_003477.2
PKLR Chr1:155271258 c.-72A>G NM_000298.5
PKLR Chr1:155271259 c.-73G>C NM_000298.5
PKLR Chr1:155271269 c.-83G>C NM_000298.5
PKLR Chr1:155263185 c.1269+44C>T NM_000298.5
PKLR Chr1:155265208 c.507+20C>A NM_000298.5
RPS7 Chr2:3622941 c.-19+1G>T NM_001011.3
RPS7 Chr2:3622942 c.-19+2T>C NM_001011.3
SEC23B Chr20:18488615 c.-16A>G NM_006363.4
SEC23B Chr20:18488060 c.-571A>G NM_006363.4 rs559854357
SEC23B Chr20:18526845 c.1743+168A>G NM_006363.4 rs111951711
SEC23B Chr20:18491863 c.221+163A>G NM_006363.4 rs573898514
SEC23B Chr20:18491731 c.221+31A>G NM_006363.4
SEC23B Chr20:18492791 c.222-78C>T NM_006363.4 rs150393520
SLC4A1 Chr17:42340296 c.-62G>A NM_000342.3 rs387906565
SPTA1 Chr1:158626459 c.2806-13T>G NM_003126.2
TCN2 Chr22:31011112 c.581-176A>T NM_000355.3
THBD Chr20:23030292 c.-151G>T NM_000361.2 rs16984852
THBD Chr20:23030443 c.-302C>A NM_000361.2

Added and removed genes from the panel

Genes added Genes removed
CLCN7
CYB5R3
DHFR
DNAJC21
NT5C3A
PIEZO1
REN
RHAG
SLC25A38
TCN2
TF
RPS17

Test strength

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

HBA1 and HBA2 genes have identical sequences at coding region and their mapping rely purely on differences at intronic/UTR regions. This reduces sensitivity for detecting variants in these region by using standard NGS diagnostics. However, Blueprint Genetics custom assay has good coverage (>20x) with improved mapping rates (mapping quality >40) within the target regions of these genes:  HBA1 80.7% and HBA2 59.4%. Our validation showed high mean coverage of 604x for HBA1 gene and 463x for HBA2. We have been able to detect sequence variants and some of the known disease causing deletions using our assay but some limitations in sensitivity is expected to exist at the moment. The following exons are not included in the panel as they are not sufficiently covered with high quality sequence reads: RPL15 (5). 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 anemia panel covers classical genes associated with beta-thalassemia, alpha-thalassemia, sickle cell anemia, x-linked sideroblastic anemia, Diamond-Blackfan anemia, Fanconi anemia, Grasbeck-Imerslund disease, hemoglobin H disease, hereditary elliptocytosis, congenital dyserythropoietic anemia, hemolytic anemia, Hb Bart's hydrops fetalis, congenital thrombotic thrombocytopenic purpura, Shwachman-Diamond syndrome, hereditary anemia and hereditary spherocytosis. 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.