Skeletal Dysplasia with Abnormal Mineralization Panel

Last modified: May 04, 2018

Summary

  • Is a 32 gene panel that includes assessment of non-coding variants
  • Is ideal for patients with a clinical suspicion of hypophosphatasia or hypophosphatemic rickets. The genes on this panel are included in the Comprehensive Growth Disorders / Skeletal Dysplasias and Disorders Panel.

Analysis methods

  • PLUS
  • SEQ
  • DEL/DUP

Availability

3-4 weeks

Number of genes

32

Test code

MA1301

CPT codes

SEQ 81404
SEQ 81405
SEQ 81408
DEL/DUP 81479

Summary

The Blueprint Genetics Skeletal Dysplasia with Abnormal Mineralization Panel (test code MA1301):

  • Is a 32 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

ICD codes

Commonly used ICD-10 code(s) when ordering the Skeletal Dysplasia with Abnormal Mineralization Panel

ICD-10 Disease
E83.31 Hypophosphatasia
E83.31 Hypophosphatemic rickets

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.

Hypophosphatasia is a rare inherited skeletal dysplasia due to loss of function mutations in the ALPL gene. It is characterized by defective mineralization of bone and/or teeth in the presence of low activity of serum and bone alkaline phosphatase. Clinical features range from stillbirth without mineralized bone at the severe end to pathologic fractures of the lower extremities in later adulthood at the mild end. At least six clinical forms are currently recognized based on age at diagnosis and severity of features. The differential diagnosis of hypophosphatasia depends on the age at which the diagnosis is considered. In utero, osteogenesis imperfecta (OI) type II and campomelic dysplasia are the most common differential diagnoses. Rare conditions such as Stuve–Wiedemann syndrome may also be involved. At birth, OI type II, campomelic dysplasia, and chondrodysplasias with bone mineralization defect are very similar diseases and are challenging to differentiate radiographically. In infancy and childhood, different OI types are the most common differential diagnosis, but rarer disorders such as cleidocranial dysostosis, Cole-Carpenter syndrome, idiopathic juvenile osteoporosis, and renal osteodystrophy should be considered. In adulthood, osteopenia/osteoporosis and more rarely osteoarthritis and pseudogout may be caused by hypophosphatasia. Serum alkaline phosphatase activity can suggest the diagnosis pending confirmation with genetic testing. Hypophosphatemic rickets (HR) is a genetic disorder which prevents sufficient reabsorption of phosphate in the proximal renal tubule, with increased phosphate excretion, resulting in rickets. Rickets is a metabolic disorder of the growing bone which occurs in children before fusion of the epiphysis and is characterized by impaired mineralization of the osteoid matrix during growth. The most common form of HR is inherited in an X-linked manner, but the remaining 20% of familial HR patients belong to the autosomal dominant HR and to the hereditary HR with calciuria types.

Genes in the Skeletal Dysplasia with Abnormal Mineralization Panel and their clinical significance

Gene Associated phenotypes Inheritance ClinVar HGMD
ALPL Odontohypophosphatasia, Hypophosphatasia perinatal lethal, infantile, juvenile and adult forms AD/AR 55 288
ANKH Calcium pyrophosphate deposition disease (familial chondrocalcinosis type 2), Craniometaphyseal dysplasia autosomal dominant type AD 12 20
B4GALT7 Ehlers-Danlos syndrome, progeroid form AR 9 8
CASR Hypocalcemia, Neonatal hyperparathyroidism, Familial Hypocalciuric hypercalcemia with transient Neonatal hyperparathyroidism AD/AR 95 392
CLCN5 Proteinuria, low molecular weight, with hypercalciuric nephrocalcinosis, Hypophosphatemic rickets,, Nephrolithiasis, I, Dent disease XL 40 263
COL1A1 Ehlers-Danlos syndrome, Caffey disease, Osteogenesis imperfecta type 1, Osteogenesis imperfecta type 2, Osteogenesis imperfecta type 3, Osteogenesis imperfecta type 4 AD 212 929
COL1A2 Ehlers-Danlos syndrome, cardiac valvular form, Osteogenesis imperfecta type 1, Osteogenesis imperfecta type 2, Osteogenesis imperfecta type 3, Osteogenesis imperfecta type 4 AD/AR 118 490
COL3A1 Ehlers-Danlos syndrome AD 476 620
COL5A1 Ehlers-Danlos syndrome AD 62 136
COL5A2 Ehlers-Danlos syndrome AD 15 23
CRTAP Osteogenesis imperfecta type 2, Osteogenesis imperfecta type 3, Osteogenesis imperfecta type 4 AR 11 27
CYP27B1 Vitamin D-dependent rickets AR 22 72
ENPP1 Arterial calcification, Hypophosphatemic rickets AR 20 67
FBN1 MASS syndrome, Marfan syndrome, Acromicric dysplasia, Geleophysic dysplasia, Weill-Marchesani syndrome AD 711 2070
FGF23 Tumoral calcinosis, hyperphosphatemic, Hypophosphatemic rickets AD/AR 10 16
FKBP10 Bruck syndrome type 2, Osteogenesis imperfecta type 3, Osteogenesis imperfecta type 4 AR 18 33
GALNT3 Tumoral calcinosis, hyperphosphatemic AR 15 34
MGP Keutel syndrome AR 5 7
P3H1 Osteogenesis imperfecta AR 13 55
PHEX Hypophosphatemic rickets XL 254 426
PLOD2 Bruck syndrome, Osteogenesis imperfecta type 3 AR 8 13
PLS3 Osteoporosis and osteoporotic fractures XL 12
PPIB Osteogenesis imperfecta type 2, Osteogenesis imperfecta type 3, Osteogenesis imperfecta type 4 AR 8 13
PTDSS1 Lenz-Majewski hyperostotic dwarfism AD 5 5
SERPINF1 Osteogenesis imperfecta type 3, Osteogenesis imperfecta type 4 AR 8 35
SLC34A3 Hypophosphatemic rickets with hypercalciuria AR 21 36
SLC39A13 Spondylodysplastic Ehlers-Danlos syndrome AR 2 8
SNX10 Osteopetrosis, autosomal recessive 8 AR 2 12
SOX9 Campomelic dysplasia, 46,XY sex reversal, Brachydactyly with anonychia (Cooks syndrome) AD 34 139
TNFRSF11A Familial expansile osteolysis, Paget disease of bone, Osteopetrosis, severe neonatal or infantile forms (OPTB1) AD/AR 8 23
TNFRSF11B Paget disease of bone, juvenile AR 8 18
VDR Vitamin D-dependent rickets AD/AR 18 65

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
ALPL Chr1:21835920 c.-195C>T NM_000478.4
ANKH Chr5:14871567 c.-11C>T NM_054027.4
CASR Chr3:121994640 c.1378-19A>C NM_001178065.1
COL1A1 Chr17:48272201 c.1354-12G>A NM_000088.3 rs72648337
COL1A1 Chr17:48268147 c.2343+31T>A NM_000088.3
COL1A1 Chr17:48267611 c.2451+77C>T NM_000088.3 rs72651665
COL1A1 Chr17:48267594 c.2451+94G>T NM_000088.3
COL1A1 Chr17:48273742 c.904-14G>A NM_000088.3
COL1A2 Chr7:94025130 c.70+717A>G NM_000089.3 rs72656354
COL3A1 Chr2:189872183 c.3256-43T>G NM_000090.3 rs587779667
COL5A1 Chr9:137680989 c.2647-12A>G NM_000093.4
COL5A1 Chr9:137686903 c.2701-25T>G NM_000093.4 rs765079080
COL5A1 Chr9:137726806 c.5137-11T>A NM_000093.4 rs183495554
CRTAP Chr3:33160815 c.472-1021C>G NM_006371.4 rs72659360
FBN1 Chr15:48739106 c.5672-87A>G NM_000138.4
FBN1 Chr15:48739107 c.5672-88A>G NM_000138.4
FBN1 Chr15:48720682 c.6872-14A>G NM_000138.4
FBN1 Chr15:48721629 c.6872-961A>G NM_000138.4
FBN1 Chr15:48707358 c.8051+375G>T NM_000138.4
FBN1 Chr15:48818478 c.863-26C>T NM_000138.4
IFITM5 Chr11:299504 c.-14C>T NM_001025295.2 rs587776916 Explain almost all cases of OI type V PMID 23240094
PHEX ChrX:22266301 c.*231A>G NM_000444.4
PHEX ChrX:22237137 c.1701-16T>A NM_000444.4
PHEX ChrX:22113485 c.849+1268G>T NM_000444.4
PLS3 ChrX:114856534 c.74-24T>A NM_005032.5
SERPINF1 Chr17:1665408 c.-9+2dupT NM_002615.5 rs398122519
SERPINF1 Chr17:1679209 c.787-617G>A NM_002615.5

Added and removed genes from the panel

Genes added Genes removed
GALNT3
MGP
PLS3
PTDSS1
SNX10

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
  • 1479 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

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 skeletal dysplasia with abnormal mineralization panel covers classical genes associated with hypophosphatasia, hypophosphatemic rickets and osteogenesis imperfecta. 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.