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Hypoparathyroidism Sequencing Panel

  • Summary and Pricing
  • Clinical Features and Genetics
  • Citations
  • Methods
  • Ordering/Specimens
Order Kits
TEST METHODS

NGS Sequencing

Test Code Test Copy GenesCPT Code Copy CPT Codes
2653 AIRE 81406 Add to Order
AP2S1 81479
CASR 81405
FAM111A 81479
GATA3 81479
GCM2 81479
GNA11 81479
GNAS 81479
HADHA 81406
HADHB 81406
PTH 81479
PTH1R 81479
SOX3 81479
STX16 81479
TBCE 81479
Full Panel Price* $1690.00
Test Code Test Copy Genes Total Price CPT Codes Copy CPT Codes
2653 Genes x (15) $1690.00 81405, 81406(x3), 81479(x11) Add to Order
Pricing Comment

If you would like to order a subset of these genes contact us to discuss pricing.

Targeted Testing

For ordering targeted known variants, please proceed to our Targeted Variants landing page.

Turnaround Time

The great majority of tests are completed within 28 days.

Clinical Sensitivity

In a cohort study of 37 pediatric hypoparathyroidism patients in Korea (Kim et al. 2015), 22 cases (59.5%) had 22q11.2 microdeletion syndrome. Other genetic defects were found in GATA3 (5/37, 13.5%), AIRE (1, 2.7%), FAM111A (1, 2.7%) and mitochondrial genome (1, 2.7%). The remaining 7 (18.9%) patients were classified as idiopathic hypoparathyroidism cases.

The cause of nonsurgical isolated hypoparathyroidism remains unknown in about 90% of sporadic cases and 30% of familial cases (Lambert et al. 2014).

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Deletion/Duplication Testing via aCGH

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
600 AIRE$690.00 81479 Add to Order
AP2S1$690.00 81479
CASR$690.00 81479
FAM111A$690.00 81479
GATA3$690.00 81479
GCM2$690.00 81479
GNA11$690.00 81479
GNAS$690.00 81479
HADHA$690.00 81479
HADHB$690.00 81479
PTH$690.00 81479
PTH1R$690.00 81479
SOX3$690.00 81479
STX16$690.00 81479
TBCE$690.00 81479
Full Panel Price* $1290.00
Test Code Test Copy Genes Total Price CPT Codes Copy CPT Codes
600 Genes x (15) $1290.00 81479(x15) Add to Order
Pricing Comment

# of Genes Ordered

Total Price

1

$690

2

$730

3

$770

4-10

$840

11-30

$1,290

31-100

$1,670

Over 100

Call for quote

Turnaround Time

The great majority of tests are completed within 28 days.

Clinical Sensitivity

No large deletions or duplications have been reported yet in these genes: FAM111A, AP2S1, GNA11, PTH, PTH1R, and TBCE. Deletions or duplications have been reported in these genes, but are uncommon: AIRE, CASR, GATA3, GCM2, HADHA, HADHB, and STX16. Deletions or duplications have been commonly reported in SOX3 and GNAS.

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Clinical Features

Hypoparathyroidism is an abnormality of calcium metabolism characterized by hypocalcemia and hyperphosphatemia due to inadequate (absent or markedly reduced) secretion of parathyroid hormone (Garfield et al. 2001; De Sanctis et al. 2012; Al-Azem et al. 2012; Kim et al. 2015). Hypoparathyroidism can be inherited (commonly seen in children) or acquired (usually in adults). Clinical manifestations of pediatric hypoparathyroidism include bronchospasm, laryngospasm, tetany and seizures in the neonatal period; and confusion, fatigue, muscle cramping, paraesthesia, twitching and arrhythmia in older children. Congenital hypoparathyroidism caused by genetic aberration can be syndromic or isolated.

Genetics

Isolated hypoparathyroidism can be inherited in autosomal dominant (AP2S1, CASR, GNA11, PTH and GCM2), autosomal recessive (PTH and GCM2) or X-linked manner (SOX3) (Kim et al. 2015; De Sanctis et al. 2012; Mannstadt et al. 2013). Syndromic hypoparathyroidism is commonly seen in chromosome 22q11.2 microdeletion syndrome, but can be also caused by defects in single genes such as AIRE (polyglandular autoimmune syndrome, autosomal recessive), TBCE (hypoparathyroidism-retardation-dysmorphism syndrome, autosomal recessive), FAM111A (Kenny-Caffey syndrome, autosomal dominant), GATA3 (hypoparathyroidism-deafness-renal dysplasia syndrome, autosomal dominant), PTH1R (Blomstrand chondrodysplasia, autosomal recessive), GNAS and STX16 (pseudohypoparathyroidism, autosomal dominant) or HADHA and HADHB (trifunctional protein deficiency, autosomal recessive). Mitochondrial genome defects have been also associated with syndromic hypoparathyroidism such as Kearns-Sayre syndrome. The mutation spectrum throughout these genes includes all types of variants.

See individual gene test descriptions for information on molecular biology of gene products.

Testing Strategy

For this NGS panel, the full coding regions, plus ~20 bp of non-coding DNA flanking each exon, are sequenced for each of the genes listed below. Sequencing is accomplished by capturing specific regions with an optimized solution-based hybridization method, followed by massively parallel sequencing of the captured DNA fragments. Additional Sanger sequencing is performed for any regions not captured or with insufficient number of sequence reads. All pathogenic, undocumented and questionable variant calls are confirmed by Sanger sequencing.

Indications for Test

Candidates for this test are patients with hypoparathyroidism. This test especially aids in a differential diagnosis of similar phenotypes by analyzing multiple genes simultaneously.

Genes

Official Gene Symbol OMIM ID
AIRE 607358
AP2S1 602242
CASR 601199
FAM111A 615292
GATA3 131320
GCM2 603716
GNA11 139313
GNAS 139320
HADHA 600890
HADHB 143450
PTH 168450
PTH1R 168468
SOX3 313430
STX16 603666
TBCE 604934
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

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CONTACTS

Genetic Counselors
Geneticist
Citations
  • Al-Azem H., Khan A.A. 2012. Best Practice & Research. Clinical Endocrinology & Metabolism. 26: 517-22. PubMed ID: 22863393
  • De Sanctis V. et al. 2012. Current Opinion in Endocrinology, Diabetes, and Obesity. 19: 435-42. PubMed ID: 23128574
  • Garfield N., Karaplis A.C. 2001. Trends in Endocrinology and Metabolism: Tem. 12: 288-94. PubMed ID: 11504667
  • Kim J.H. et al. 2015. Clinical Endocrinology. 83: 790-6. PubMed ID: 26384470
  • Lambert A.S. et al. 2014. The Journal of Clinical Endocrinology and Metabolism. 99: E469-73. PubMed ID: 24423332
  • Mannstadt M. et al. 2013. The New England Journal of Medicine. 368: 2532-4. PubMed ID: 23802536
Order Kits
TEST METHODS

NextGen Sequencing using PG-Select Capture Probes

Test Procedure

We use a combination of Next Generation Sequencing (NGS) and Sanger sequencing technologies to cover the full coding regions of the listed genes plus ~20 bases of non-coding DNA flanking each exon.  As required, genomic DNA is extracted from the patient specimen.  For NGS, patient DNA corresponding to these regions is captured using an optimized set of DNA hybridization probes.  Captured DNA is sequenced using Illumina’s Reversible Dye Terminator (RDT) platform (Illumina, San Diego, CA, USA).  Regions with insufficient coverage by NGS are covered by Sanger sequencing.  All pathogenic, likely pathogenic, or variants of uncertain significance are confirmed by Sanger sequencing.

For Sanger sequencing, Polymerase Chain Reaction (PCR) is used to amplify targeted regions.  After purification of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit.  PCR products are resolved by electrophoresis on an ABI 3730xl capillary sequencer.  In nearly all cases, cycle sequencing is performed separately in both the forward and reverse directions.

Patient DNA sequence is aligned to the genomic reference sequence for the indicated gene region(s). All differences from the reference sequences (sequence variants) are assigned to one of five interpretation categories, listed below, per ACMG Guidelines (Richards et al. 2015).

(1) Pathogenic Variants
(2) Likely Pathogenic Variants
(3) Variants of Uncertain Significance
(4) Likely Benign Variants
(5) Benign, Common Variants

Human Genome Variation Society (HGVS) recommendations are used to describe sequence variants (http://www.hgvs.org).  Rare variants and undocumented variants are nearly always classified as likely benign if there is no indication that they alter protein sequence or disrupt splicing.

Analytical Validity

As of March 2016, 6.36 Mb of sequence (83 genes, 1557 exons) generated in our lab was compared between Sanger and NextGen methodologies. We detected no differences between the two methods. The comparison involved 6400 total sequence variants (differences from the reference sequences). Of these, 6144 were nucleotide substitutions and 256 were insertions or deletions. About 65% of the variants were heterozygous and 35% homozygous. The insertions and deletions ranged in length from 1 to over 100 nucleotides.

In silico validation of insertions and deletions in 20 replicates of 5 genes was also performed. The validation included insertions and deletions of lengths between 1 and 100 nucleotides. Insertions tested in silico: 2200 between 1 and 5 nucleotides, 625 between 6 and 10 nucleotides, 29 between 11 and 20 nucleotides, 25 between 21 and 49 nucleotides, and 23 at or greater than 50 nucleotides, with the largest at 98 nucleotides. All insertions were detected. Deletions tested in silico: 1813 between 1 and 5 nucleotides, 97 between 6 and 10 nucleotides, 32 between 11 and 20 nucleotides, 20 between 21 and 49 nucleotides, and 39 at or greater than 50 nucleotides, with the largest at 96 nucleotides. All deletions less than 50 nucleotides in length were detected, 13 greater than 50 nucleotides in length were missed. Our standard NextGen sequence variant calling algorithms are generally not capable of detecting insertions (duplications) or heterozygous deletions greater than 100 nucleotides. Large homozygous deletions appear to be detectable.   

Analytical Limitations

Interpretation of the test results is limited by the information that is currently available.  Better interpretation should be possible in the future as more data and knowledge about human genetics and this specific disorder are accumulated.

When Sanger sequencing does not reveal any difference from the reference sequence, or when a sequence variant is homozygous, we cannot be certain that we were able to detect both patient alleles.  Occasionally, a patient may carry an allele which does not amplify, due to a large deletion or insertion.   In these cases, the report will contain no information about the second allele.  Our Sanger and NGS Sequencing tests are generally not capable of detecting Copy Number Variants (CNVs).

We sequence all coding exons for each given transcript, plus ~20 bp of flanking non-coding DNA for each exon.  Test reports contain no information about other portions of the gene, such as regulatory domains, deep intronic regions or any currently uncharacterized alternative exons.

In most cases, we are unable to determine the phase of sequence variants.  In particular, when we find two likely causative mutations for recessive disorders, we cannot be certain that the mutations are on different alleles.

Our ability to detect minor sequence variants due to somatic mosaicism is limited.  Sequence variants that are present in less than 50% of the patient’s nucleated cells may not be detected.

Runs of mononucleotide repeats (eg (A)n or (T)n) with n >8 in the reference sequence are generally not analyzed because of strand slippage during PCR.

Unless otherwise indicated, DNA sequence data is obtained from a specific cell-type (usually leukocytes from whole blood).   Test reports contain no information about the DNA sequence in other cell-types.

We cannot be certain that the reference sequences are correct.

Rare, low probability interpretations of sequencing results, such as for example the occurrence of de novo mutations in recessive disorders, are generally not included in the reports.

We have confidence in our ability to track a specimen once it has been received by PreventionGenetics.  However, we take no responsibility for any specimen labeling errors that occur before the sample arrives at PreventionGenetics.

Deletion/Duplication Testing Via Array Comparative Genomic Hybridization

Test Procedure

Equal amounts of genomic DNA from the patient and a gender matched reference sample are amplified and labeled with Cy3 and Cy5 dyes, respectively. To prevent any sample cross contamination, a unique sample tracking control is added into each patient sample. Each labeled patient product is then purified, quantified, and combined with the same amount of reference product. The combined sample is loaded onto the designed array and hybridized for at least 22-42 hours at 65°C. Arrays are then washed and scanned immediately with 2.5 µM resolution. Only data for the gene(s) of interest for each patient are extracted and analyzed.

Analytical Validity

PreventionGenetics' high density gene-centric custom designed aCGH enables the detection of relatively small deletions and duplications within a single exon of a given gene or deletions and duplications encompassing the entire gene. PreventionGenetics has established and verified this test's accuracy and precision.

Analytical Limitations

Our dense probe coverage may allow detection of deletions/duplications down to 100 bp; however due to limitations and probe spacing this cannot be guaranteed across all exons of all genes. Therefore, some copy number changes smaller than 100-300 bp within a targeted large exon may not be detected by our array.

This array may not detect deletions and duplications present at low levels of mosaicism or those present in genes that have pseudogene copies or repeats elsewhere in the genome.

aCGH will not detect balanced translocations, inversions, or point mutations that may be responsible for the clinical phenotype.

Breakpoints, if occurring outside the targeted gene, may be hard to define.

The sensitivity of this assay may be reduced when DNA is extracted by an outside laboratory.

Order Kits

Ordering Options


myPrevent - Online Ordering
  • The test can be added to your online orders in the Summary and Pricing section.
  • Once the test has been added log in to myPrevent to fill out an online requisition form.
REQUISITION FORM
  • A completed requisition form must accompany all specimens.
  • Billing information along with specimen and shipping instructions are within the requisition form.
  • All testing must be ordered by a qualified healthcare provider.

SPECIMEN TYPES
WHOLE BLOOD

(Delivery accepted Monday - Saturday)

  • Collect 3 ml -5 ml (5 ml preferred) of whole blood in EDTA (purple top tube) or ACD (yellow top tube). For Test #500-DNA Banking only, collect 10 ml -20 ml of whole blood.
  • For small babies, we require a minimum of 1 ml of blood.
  • Only one blood tube is required for multiple tests.
  • Ship blood tubes at room temperature in an insulated container. Do not freeze blood.
  • During hot weather, include a frozen ice pack in the shipping container. Place a paper towel or other thin material between the ice pack and the blood tube.
  • In cold weather, include an unfrozen ice pack in the shipping container as insulation.
  • At room temperature, blood specimen is stable for up to 48 hours.
  • If refrigerated, blood specimen is stable for up to one week.
  • Label the tube with the patient name, date of birth and/or ID number.

DNA

(Delivery accepted Monday - Saturday)

  • Send in screw cap tube at least 5 µg -10 µg of purified DNA at a concentration of at least 20 µg/ml for NGS and Sanger tests and at least 5 µg of purified DNA at a concentration of at least 100 µg/ml for gene-centric aCGH, MLPA, and CMA tests, minimum 2 µg for limited specimens.
  • For requests requiring more than one test, send an additional 5 µg DNA per test ordered when possible.
  • DNA may be shipped at room temperature.
  • Label the tube with the composition of the solute, DNA concentration as well as the patient’s name, date of birth, and/or ID number.
  • We only accept genomic DNA for testing. We do NOT accept products of whole genome amplification reactions or other amplification reactions.

CELL CULTURE

(Delivery preferred Monday - Thursday)

  • PreventionGenetics should be notified in advance of arrival of a cell culture.
  • Culture and send at least two T25 flasks of confluent cells.
  • Some panels may require additional flasks (dependent on size of genes, amount of Sanger sequencing required, etc.). Multiple test requests may also require additional flasks. Please contact us for details.
  • Send specimens in insulated, shatterproof container overnight.
  • Cell cultures may be shipped at room temperature or refrigerated.
  • Label the flasks with the patient name, date of birth, and/or ID number.
  • We strongly recommend maintaining a local back-up culture. We do not culture cells.
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