- Is a 119 gene panel that includes assessment of non-coding variants.
Is ideal for patients with a clinical suspicion of Bardet-Biedl syndrome, Joubert syndrome, Meckel syndrome, nephronophthisis with or without retinal dystrophy, or complex ciliopathy phenotype.
Isn’t ideal for a patient with primary ciliary dyskinesia or isomerism/heterotaxy. For patients with a suspicion of primary ciliary dyskinesia, Primary Ciliary Dyskinesia Panel is recommended. For patients with isomerism/heterotaxy, Heterotaxy and Situs Inversus Panel is recommended.
The Blueprint Genetics Ciliopathy Panel (test code KI0701):
Refer to the most current version of ICD-10-CM manual for a complete list of ICD-10 codes.
- Blood (min. 1ml) in an EDTA tube
- Extracted DNA, min. 2 μg in TE buffer or equivalent
- Saliva (Please see Sample Requirements for accepted saliva kits)
Label the sample tube with your patient's name, date of birth and the date of sample collection.
We do not accept DNA samples isolated from formalin-fixed paraffin-embedded (FFPE) tissue. In addition, if the patient is affected with a hematological malignancy, DNA extracted from a non-hematological source (e.g. skin fibroblasts) is strongly recommended.
Please note that, in rare cases, mitochondrial genome (mtDNA) variants may not be detectable in blood or saliva in which case DNA extracted from post-mitotic tissue such as skeletal muscle may be a better option.
Read more about our sample requirements here.
Ciliopathies are a group of disorders resulting from either abnormal formation or function of cilia. Mutations in ciliary gene are known to cause single organ phenotypes, as well as complex syndromes. Ciliopathies have a broad range of phenotypes encompassing a number of different autosomal recessive, dominant and X-linked syndromes. As cilia are a component of almost all cells, ciliary dysfunction can manifest as a collection of features that include retinal degeneration, renal disease and brain malformations. Additional features may include congenital fibrocystic diseases of the liver and pancreas, diabetes, obesity and skeletal dysplasias. Ciliopathies can result from a mutation at a single locus in one patient while mutations affecting a number of different loci can, at the same time, can result in a similar phenotype in other patients. Ciliopathies can be classified according to whether there is aberrant function in an intact cilium or complete absence/loss of the mature cilium. The latter is the case with severe multi-organ phenotypes.
Genes in the Ciliopathy Panel and their clinical significance
|ACVR2B||Heterotaxy, visceral, 4, autosomal||AD||1||2|
|ARL3||Retinitis pigmentosa, Joubert syndrome||AD/AR||1|
|ARL6||Bardet-Biedl syndrome, Retinitis pigmentosa||AR||14||21|
|ARMC9||Joubert syndrome 30||AR||12||11|
|BBIP1||Bardet-Biedl syndrome 18||AR||1||1|
|BBS2||Bardet-Biedl syndrome, Retinitis pigmentosa||AR||58||91|
|C21ORF2||Retinal dystrophy with or without macular staphyloma (RDMS), Spondylometaphyseal dysplasia, axial (SMDAX)||AR||13||22|
|C2CD3||Orofaciodigital syndrome XIV||AR||9||10|
|C5ORF42||Orofaciodigital syndrome, Joubert syndrome||AR||97||103|
|C8ORF37||Retinitis pigmentosa, Cone rod dystrophy, Bardet-Biedl syndrome 21||AR||8||17|
|CC2D2A#||COACH syndrome, Joubert syndrome, Meckel syndrome||AR||76||91|
|CENPF||Ciliary dyskinesia -Lethal Ciliopathy||AR||13||8|
|CEP120||Short-rib thoracic dysplasia 13 with or without polydactyly||AR||9||9|
|CEP19||Morbid obesity and spermatogenic failure, Bardet-Biedl syndrome||AR||2||2|
|CEP290*||Bardet-Biedl syndrome, Leber congenital amaurosis, Joubert syndrome, Senior-Loken syndrome, Meckel syndrome||AR||130||289|
|CEP55||Multinucleated neurons, anhydramnios, renal dysplasia, cerebellar hypoplasia and hydranencephaly (MARCH)||4||2|
|CPE||Obesity, severe, and type II diabetes||AR||2|
|CRB2||Focal segmental glomerulosclerosis, Ventriculomegaly with cystic kidney disease||AR||12||22|
|CSPP1||Jeune asphyxiating thoracic dystrophy, Joubert syndrome||AR||32||27|
|DCDC2||Deafness, Nephronophthisis, Sclerosing cholangitis, neonatal||AR||13||9|
|DDX59||Orofaciodigital syndrome V||AR||2||6|
|DYNC2H1||Short -rib thoracic dysplasia with or without polydactyly type 1, Short -rib thoracic dysplasia with or without polydactyly type 3, Asphyxiating thoracic dysplasia (ATD; Jeune), SRPS type 2 (Majewski)||AR/Digenic||148||205|
|DYNC2LI1||Short-rib throacic dysplasia 15 with polydactyly||AR||19||14|
|EVC||Weyers acrofacial dysostosis, Ellis-van Creveld syndrome||AD/AR||58||83|
|EVC2||Ellis-van Creveld syndrome, Weyers acrodental dysostosis||AD/AR||78||75|
|FAM58A||Toe syndactyly, telecanthus, and anogenital and renal malformations (STAR syndrome)||XL||8||11|
|FAN1||Interstitial nephritis, karyomegalic||AR||12||21|
|GLI3||Acrocallosal syndrome, Pallister-Hall syndrome, Grieg cephalopolysndactyly syndrome, Postaxial polydactyly type A, Preaxial polydactyly type 3, Preaxial polydactyly type 4||AD||70||235|
|ICK||Endocrine-cerebroosteodysplasia, Epilepsy, juvenile myoclonic||AD/AR||1||3|
|IFT122*||Sensenbrenner syndrome, Cranioectodermal dysplasia (Levin-Sensenbrenner) type 1, Cranioectodermal dysplasia (Levin-Sensenbrenner) type 2||AR||13||23|
|IFT140||Short -rib thoracic dysplasia with or without polydactyly, Asphyxiating thoracic dysplasia (ATD; Jeune)||AR||38||63|
|IFT172||Retinitis pigmentosa, Short -rib thoracic dysplasia with or without polydactyly, Asphyxiating thoracic dysplasia (ATD; Jeune)||AR||22||25|
|IFT27||Bardet Biedl syndrome 19||AR||1||4|
|IFT43||Cranioectodermal dysplasia 3||AR||4||7|
|IFT52||Short-rib thoracic dysplasia 16 with or without polydactyly||AR||3||4|
|IFT74||Bardet Biedl syndrome 20||2||4|
|IFT80||Short -rib thoracic dysplasia with or without polydactyly, Asphyxiating thoracic dysplasia (ATD; Jeune)||AR||11||11|
|IFT81#||Short rib thoracic dysplasia with polydactyly, Cone-Rod dystrophy, autosomal recessive||AR||4||9|
|INPP5E||Joubert syndrome, Mental retardation, truncal obesity, retinal dystrophy, and micropenis (MORM syndrome)||AR||25||50|
|KIAA0556||Joubert syndrome 26||AR||2||2|
|KIAA0586#||Short rib thoracic dysplasia with polydactyly, Joubert syndrome||AR||29||31|
|KIAA0753||Orofaciodigital syndrome XV||AR||6||7|
|KIF14||Meckel syndrome 12||AR||9||16|
|KIF7||Acrocallosal syndrome, Hydrolethalus syndrome, Al-Gazali-Bakalinova syndrome, Joubert syndrome||AR/Digenic||24||44|
|LEFTY2*||Left-right axis malformations||AD||1||3|
|LZTFL1||Bardet-Biedl syndrome 17||AR||6||3|
|MKKS||Bardet-Biedl syndrome, McKusick-Kaufman syndrome||AR||21||59|
|MKS1||Bardet-Biedl syndrome, Meckel syndrome||AR||50||52|
|NEK1||Short -rib thoracic dysplasia with or without polydactyly, SRPS type 2 (Majewski)||AR/Digenic||22||23|
|NPHP1||Nephronophthisis, Joubert syndrome, Senior-Loken syndrome||AR||19||76|
|NPHP3||Nephronophthisis, Renal-hepatic-pancreatic dysplasia, Meckel syndrome||AR||38||75|
|NPHP4||Nephronophthisis, Senior-Loken syndrome||AR||20||113|
|OFD1||Simpson-Golabi-Behmel syndrome, Retinitis pigmentosa, Orofaciodigital syndrome, Joubert syndrome||XL||153||160|
|PDE6D||Joubert syndrome 22||AR||3||1|
|PKD1*||Polycystic kidney disease||AD||237||1923|
|PKD2||Polycystic kidney disease||AD||55||333|
|PKHD1||Polycystic kidney disease||AR||249||557|
|PMM2||Congenital disorder of glycosylation||AR||76||128|
|PNPLA6||Laurence-Moon syndrome, Boucher-Neuhauser syndrome, Spastic paraplegia 39||AR||26||58|
|POC1B||Cone-rod dystrophy 20||AR||4||7|
|RPGRIP1L#||COACH syndrome, Joubert syndrome, Meckel syndrome, Retinal degeneration in ciliopathy, modifier||AR||39||49|
|SCAPER||Retinal dystrophy, Retinitis pigmentosa||AR||4||7|
|SCLT1#||Senior-Loken syndrome, Retinal dystrophy||AR||3|
|SDCCAG8||Bardet-Biedl syndrome, Senior-Loken syndrome||AR||14||18|
|SUFU||Medulloblastoma, Basal cell nevus syndrome||AD||22||44|
|TCTEX1D2||Short-rib thoracic dysplasia 17 with or without polydactyly, Jeune Asphyxiating Thoracic Dystrophy||AR||4||6|
|TCTN2||Joubert syndrome, Meckel syndrome||AR||20||15|
|TCTN3||Orofaciodigital syndrome (Mohr-Majewski syndrome), Joubert syndrome||AR||9||12|
|TMEM216||Joubert syndrome, Meckel syndrome||AR||17||8|
|TMEM231||Joubert syndrome, Meckel syndrome||AR||12||19|
|TMEM67||Nephronophthisis, COACH syndrome, Joubert syndrome, Meckel syndrome||AR||87||170|
|TRAF3IP1||Senior-Loken syndrome 9||AR||11||15|
|TRIM32||Bardet-Biedl syndrome, Muscular dystrophy, limb-girdle||AR||13||16|
|TTC21B||Short-rib thoracic dysplasia, Nephronophthisis, Asphyxiating thoracic dysplasia (ATD; Jeune)||AR||23||63|
|TTC8||Bardet-Biedl syndrome, Retinitis pigmentosa||AR||5||16|
|USP9X||Mental retardation, X-linked 99, Mental retardation, X-linked 99, syndromic, female restricted||XL||30||27|
|WDPCP||Meckel-Gruber syndrome, modifier, Bardet-Biedl syndrome, Congenital heart defects, hamartomas of tongue, and polysyndactyly||AR||6||8|
|WDR19||Retinitis pigmentosa, Nephronophthisis, Short -rib thoracic dysplasia with or without polydactyly, Senior-Loken syndrome, Cranioectodermal dysplasia (Levin-Sensenbrenner) type 1, Cranioectodermal dysplasia (Levin-Sensenbrenner) type 2, Asphyxiating thoracic dysplasia (ATD; Jeune)||AR||33||43|
|WDR34||Short -rib thoracic dysplasia with or without polydactyly, Asphyxiating thoracic dysplasia (ATD; Jeune)||AR||18||21|
|WDR35||Cranioectodermal dysplasia (Levin-Sensenbrenner) type 1, Cranioectodermal dysplasia (Levin-Sensenbrenner) type 2, Short rib-polydactyly syndrome type 5||AR||28||31|
|WDR60||Short-rib thoracic dysplasia 8 with or without polydactyly||AR||12||13|
|XPNPEP3||Nephronophthisis-like nephropathy 1||AR||3||2|
|ZIC3||Heterotaxy, visceral, VACTERL association, Congenital heart defects, nonsyndromic||XL||15||41|
|ZNF423||Nephronophthisis, Joubert syndrome||AD/AR||10||7|
* 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), and/or the gene has exons listed under Test limitations section that are not included in the panel as they are not sufficiently covered with high quality sequence reads.
The sensitivity to detect variants may be limited in genes marked with an asterisk (*) or number sign (#). Due to possible limitations these genes may not be available as single gene tests.
Gene refers to the HGNC approved gene symbol; Inheritance refers to inheritance patterns such as autosomal dominant (AD), autosomal recessive (AR), mitochondrial (mi), 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 Mitomap databases.
Non-coding variants covered by Ciliopathy Panel
|Gene||Genomic location HG19||HGVS||RefSeq||RS-number|
Added and removed genes from the panel
|Genes added||Genes removed|
|ADAMTS9 ARL3 ATXN10 CEP55 FAN1 ICK IFT57 IFT74 INTU PIBF1 SUFU XPNPEP3|
- CAP accredited laboratory
- 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
- Some of the panels include the whole mitochondrial genome (please see the Panel Content section)
- Our Nucleus online portal providing transparent and easy access to quality and performance data at the patient level
- Our publicly available analytic validation demonstrating complete details of test performance
- ~2,000 non-coding disease causing variants in our clinical grade NGS assay for panels (please see ‘Non-coding disease causing variants covered by this panel’ in the Panel Content section)
- Our rigorous variant classification scheme
- Our systematic clinical interpretation workflow using proprietary software enabling accurate and traceable processing of NGS data
- Our comprehensive clinical statements
The following exons are not included in the panel as they are not sufficiently covered with high quality sequence reads: CC2D2A (NM_020785:7), IFT81 (NM_031473:12), KIAA0586 (NM_001244189:6, 33), RPGRIP1L (NM_015272:23), SCLT1 (NM_001300898:6), TCTN1 (NM_001173976:2;NM_024549:6). 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).
- Complex inversions
- Gene conversions
- Balanced translocations
- Some of the panels include the whole mitochondrial genome but not all (please see the Panel Content section)
- 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).
- Low level mosaicism in nuclear genes (variant with a minor allele fraction of 14.6% is detected with 90% probability)
- Stretches of mononucleotide repeats
- Low level heteroplasmy in mtDNA (>90% are detected at 5% level)
- Indels larger than 50bp
- Single exon deletions or duplications
- Variants within pseudogene regions/duplicated segments
- Some disease causing variants present in mtDNA are not detectable from blood, thus post-mitotic tissue such as skeletal muscle may be required for establishing molecular diagnosis.
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 genes on the panel have been carefully selected based on scientific literature, mutation databases and our experience.
Our panels are sectioned from our high-quality, clinical grade NGS assay. Please see our sequencing and detection performance table for details regarding our ability to detect different types of alterations (Table).
Assays have been validated for various sample types including EDTA-blood, isolated DNA (excluding from formalin fixed paraffin embedded tissue), saliva and dry blood spots (filter cards). These sample types were selected in order to maximize the likelihood for high-quality DNA yield. The diagnostic yield varies depending on the assay used, referring healthcare professional, hospital and country. Plus analysis increases the likelihood of finding a genetic diagnosis for your patient, as large deletions and duplications cannot be detected using sequence analysis alone. Blueprint Genetics’ Plus Analysis is a combination of both sequencing and deletion/duplication (copy number variant (CNV)) analysis.
The performance metrics listed below are from an initial validation performed at our main laboratory in Finland. The performance metrics of our laboratory in Seattle, WA, are equivalent.
Performance of Blueprint Genetics high-quality, clinical grade NGS sequencing assay for panels.
|Sensitivity % (TP/(TP+FN)||Specificity %|
|Single nucleotide variants||99.89% (99,153/99,266)||>99.9999%|
|Insertions, deletions and indels by sequence analysis|
|1-10 bps||99.2% (7,745/7,806)||>99.9999%|
|11-50 bps||99.13% (2,524/2,546)||>99.9999%|
|Copy number variants (exon level dels/dups)|
|1 exon level deletion (heterozygous)||100% (20/20)||NA|
|1 exon level deletion (homozygous)||100% (5/5)||NA|
|1 exon level deletion (het or homo)||100% (25/25)||NA|
|2-7 exon level deletion (het or homo)||100% (44/44)||NA|
|1-9 exon level duplication (het or homo)||75% (6/8)||NA|
|Simulated CNV detection|
|5 exons level deletion/duplication||98.7%||100.00%|
|Microdeletion/-duplication sdrs (large CNVs, n=37))|
|Size range (0.1-47 Mb)||100% (25/25)|
|The performance presented above reached by Blueprint Genetics high-quality, clinical grade NGS sequencing assay with the following coverage metrics|
|Mean sequencing depth||143X|
|Nucleotides with >20x sequencing coverage (%)||99.86%|
Performance of Blueprint Genetics Mitochondrial Sequencing Assay.
|Sensitivity %||Specificity %|
|ANALYTIC VALIDATION (NA samples; n=4)|
|Single nucleotide variants|
|Heteroplasmic (45-100%)||100.0% (50/50)||100.0%|
|Heteroplasmic (35-45%)||100.0% (87/87)||100.0%|
|Heteroplasmic (25-35%)||100.0% (73/73)||100.0%|
|Heteroplasmic (15-25%)||100.0% (77/77)||100.0%|
|Heteroplasmic (10-15%)||100.0% (74/74)||100.0%|
|Heteroplasmic (5-10%)||100.0% (3/3)||100.0%|
|Heteroplasmic (<5%)||50.0% (2/4)||100.0%|
|CLINICAL VALIDATION (n=76 samples)|
|Single nucleotide variants n=2026 SNVs|
|Heteroplasmic (45-100%)||100.0% (1940/1940)||100.0%|
|Heteroplasmic (35-45%)||100.0% (4/4)||100.0%|
|Heteroplasmic (25-35%)||100.0% (3/3)||100.0%|
|Heteroplasmic (15-25%)||100.0% (3/3)||100.0%|
|Heteroplasmic (10-15%)||100.0% (9/9)||100.0%|
|Heteroplasmic (5-10%)||92.3% (12/13)||99.98%|
|Heteroplasmic (<5%)||88.9% (48/54)||99.93%|
|Insertions and deletions by sequence analysis n=40 indels|
|Heteroplasmic (45-100%) 1-10bp||100.0% (32/32)||100.0%|
|Heteroplasmic (5-45%) 1-10bp||100.0% (3/3)||100.0%|
|Heteroplasmic (<5%) 1-10bp||100.0% (5/5)||99,997%|
|SIMULATION DATA /(mitomap mutations)|
|Insertions, and deletions 1-24 bps by sequence analysis; n=17|
|Homoplasmic (100%) 1-24bp||100.0% (17/17)||99.98%|
|Heteroplasmic (50%)||100.0% (17/17)||99.99%|
|Heteroplasmic (25%)||100.0% (17/17)||100.0%|
|Heteroplasmic (20%)||100.0% (17/17)||100.0%|
|Heteroplasmic (15%)||100.0% (17/17)||100.0%|
|Heteroplasmic (10%)||94.1% (16/17)||100.0%|
|Heteroplasmic (5%)||94.1% (16/17)||100.0%|
|Copy number variants (separate artifical mutations; n=1500)|
|Homoplasmic (100%) 500 bp, 1kb, 5 kb||100.0%||100.0%|
|Heteroplasmic (50%) 500 bp, 1kb, 5 kb||100.0%||100.0%|
|Heteroplasmic (30%) 500 bp, 1kb, 5 kb||100.0%||100.0%|
|Heteroplasmic (20%) 500 bp, 1kb, 5 kb||99.7%||100.0%|
|Heteroplasmic (10%) 500 bp, 1kb, 5 kb||99.0%||100.0%|
|The performance presented above reached by following coverage metrics at assay level (n=66)|
|Mean of medians||Median of medians|
|Mean sequencing depth MQ0 (clinical)||18224X||17366X|
|Nucleotides with >1000x MQ0 sequencing coverage (%) (clinical)||100%|
|rho zero cell line (=no mtDNA), mean sequencing depth||12X|
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 and regulatory 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. If the test includes the mitochondrial genome the target region gene list contains the mitochondrial genes. 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 including, 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, ordering providers have access to the details of the analysis, including patient specific sequencing metrics, a gene level coverage plot and a list of regions with suboptimal coverage (<20X for nuclear genes and <1000X for mtDNA) if applicable. This reflects our mission to build fully transparent diagnostics where ordering providers can easily visualize the crucial details of the analysis process.
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 ACMG guideline 2015.
The final step in the analysis is orthogonal confirmation. Sequence and copy number variants classified as pathogenic, likely pathogenic and variants of uncertain significance (VUS) are confirmed using bi-directional Sanger sequencing or by orthogonal methods such as qPCR/ddPCR when they do not meet our stringent NGS quality metrics for a true positive call.
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, abstracts and variant databases used to help ordering providers 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. We do not recommend using variants of uncertain significance (VUS) for family member risk stratification or patient management. Genetic counseling is recommended.
Our interpretation team analyzes millions of variants from thousands of individuals with rare diseases. Our internal database and our understanding of variants and related phenotypes increases with every case analyzed. 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.