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Premature Ovarian Failure (POF) Sequencing Panel with CNV Detection

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

Sequencing

Test Code Test Copy GenesCPT Code Copy CPT Codes
5285 AIRE 81406, 81479 Add to Order
BMP15 81479, 81479
CLPP 81479, 81479
CYP17A1 81405, 81479
CYP19A1 81479, 81479
EIF2B2 81405, 81479
EIF2B4 81406, 81479
EIF2B5 81406, 81479
FIGLA 81479, 81479
FOXL2 81479, 81479
FSHR 81479, 81479
GALT 81406, 81479
HFM1 81479, 81479
LMNA 81406, 81479
MCM8 81479, 81479
MCM9 81479, 81479
NOBOX 81479, 81479
NR5A1 81479, 81479
PSMC3IP 81479, 81479
SOHLH1 81479, 81479
STAG3 81479, 81479
Full Panel Price* $1640.00
Test Code Test Copy Genes Total Price CPT Codes Copy CPT Codes
5285 Genes x (21) $1640.00 81405(x2), 81406(x5), 81479(x35) Add to Order
Pricing Comment

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

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

This multi-gene panel analyzes genes associated with premature ovarian failure (POF). The detection rate of this NGS panel in a large cohort of female patients is unavailable in the literature, but pathogenic variants in 6 of the genes within this panel have been discovered in 13/100 (13%) of patients with POF (Bouilly et al. 2016. PubMed ID: 27603904).

Gross deletions or duplications have been reported in AIRE, CLPP, CYP17A1, CYP19A1, EIF2B2, FOXL2, GALT, LMNA and NR5A1 (Human Gene Mutation Database). Overall clinical sensitivity is difficult to predict due to the paucity of documented cases, but is expected to be low.

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Clinical Features

Premature Ovarian Failure (POF), also known as primary ovarian insufficiency, is a genetically and phenotypically heterogeneous disorder characterized by primary amenorrhea or loss of menstrual function before the age of 40. Patients with POF often have elevated levels of follicle-stimulating hormone (FSH) and decreased levels of estrogen (Nelson. 2009. PubMed ID: 19196677). POF, in the most severe form, is a result of ovarian dysgenesis in which pubertal development is also severely affected. POF is a major cause of infertility and affects 1% of women before the age of 40. Approximately 50% of patients have varying and unpredictable ovarian function, and 5 to 10% of patients with POF may conceive without treatment (Nelson. 2009. PubMed ID: 19196677). There are several causes of premature ovarian failure, including chromosomal abnormalities, pathogenic sequence variants, autoimmune disorders, and environmental factors. POF can be isolated or part of a genetic syndrome (Kovanci and Schutt. 2015. PubMed ID: 25681846).

Genetics

Approximately 10-30% of POF cases have familial inheritance (Vegetti et al. 1998. PubMed ID: 9740426; van Kasteren et al. 1999. PubMed ID: 10527968). The inheritance can be autosomal recessive, dominant or X-linked. To date, defects in at least 21 genes have been documented to cause POF (Layman. 2013. PubMed ID: 23499866; Bouilly et al. 2016. PubMed ID: 27603904). Pathogenic variants in genes involved in meiosis (HFM1, STAG3), in DNA repair (MCM8, MCM9), in genes encoding transcription factors (NR5A1, NOBOX, FIGLA, FOXL2 and SOHLH1), and in genes encoding TGF-ß signaling pathway (BPM15) have all been reported in POF (Bouilly et al. 2016. PubMed ID: 27603904; Patiño et al. 2017. PubMed ID: 28505269). Moreover, this panel contains a set of other genes associated with syndromes that may also present with POF, such as aromatase deficiency, blepharophimosis, ptosis and epicanthus inversus syndrome (BPES), Malouf syndrome, ovarian hyperstimulation syndrome, and Perrault syndrome. See individual gene test descriptions for information on molecular biology of gene products and mutation spectra.

Testing Strategy

For this Next Generation Sequencing (NGS) test, sequencing is accomplished by capturing specific regions with an optimized solution-based hybridization kit, followed by massively parallel sequencing of the captured DNA fragments. Additional Sanger sequencing is performed for regions not captured or with insufficient number of sequence reads. All reported pathogenic, likely pathogenic, and 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.

Copy number variants (CNVs) are also detected from NGS data. We utilize a CNV calling algorithm that compares mean read depth and distribution for each target in the test sample against multiple matched controls. Neighboring target read depth and distribution and zygosity of any variants within each target region are used to reinforce CNV calls. All CNVs are confirmed using another technology such as aCGH, MLPA, or PCR before they are reported.

This panel typically provides at least 99.2% coverage of all coding exons of the genes listed, plus ~10 bases of flanking noncoding DNA. We define coverage as >20X NGS reads or Sanger sequencing.

Genes without complete coverage: MCM8, NR5A1, and STAG3. A full list of regions not covered by NGS or Sanger sequencing is available upon request.

Since this test is performed using exome capture probes, a reflex to any of our exome based tests is available (PGxome, PGxome Custom Panels).

Indications for Test

This test is for patients with premature ovarian failure and/or infertility.

Genes

Official Gene Symbol OMIM ID
AIRE 607358
BMP15 300247
CLPP 601119
CYP17A1 609300
CYP19A1 107910
EIF2B2 606454
EIF2B4 606687
EIF2B5 603945
FIGLA 608697
FOXL2 605597
FSHR 136435
GALT 606999
HFM1 615684
LMNA 150330
MCM8 608187
MCM9 610098
NOBOX 610934
NR5A1 184757
PSMC3IP 608665
SOHLH1 610224
STAG3 608489
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

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Perrault Syndrome Type 3 and Deafness, Autosomal Recessive 8 (DFNB8) via the CLPP Gene
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Sudden Cardiac Arrest Sequencing Panel with CNV Detection

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Bouilly et al. 2016. PubMed ID: 27603904
  • Human Gene Mutation Database (Bio-base).
  • Kovanci and Schutt. 2015. PubMed ID: 25681846
  • Layman. 2013. PubMed ID: 23499866
  • Nelson. 2009. PubMed ID: 19196677
  • Patiño et al. 2017. PubMed ID: 28505269
  • van Kasteren et al. 1999. PubMed ID: 10527968
  • Vegetti et al. 1998. PubMed ID: 9740426
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TEST METHODS

Exome Sequencing with CNV Detection

Test Procedure

For the PGxome we use Next Generation Sequencing (NGS) technologies to cover the coding regions of targeted genes plus ~10 bases of non-coding DNA flanking each exon. As required, genomic DNA is extracted from patient specimens. Patient DNA corresponding to these regions is captured using Agilent Clinical Research Exome hybridization probes. Captured DNA is sequenced on the NovaSeq 6000 using 2x150 bp paired-end reads (Illumina, San Diego, CA, USA). The following quality control metrics are generally achieved: >97% of target bases are covered at >20x, and mean coverage of target bases >120x. Data analysis and interpretation is performed by the internally developed software Titanium-Exome. In brief, the output data from the NovaSeq 6000 is converted to fastqs by Illumina Bcl2Fastq, and mapped by BWA. Variant calls are made by the GATK Haplotype caller and annotated using in house software and SnpEff. Variants are filtered and annotated using VarSeq (www.goldenhelix.com). Common benign, likely benign, and low quality variants are filtered from analysis. All reported pathogenic, likely pathogenic, and 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.

Copy number variants (CNVs) are also detected from NGS data. We utilize a CNV calling algorithm that compares mean read depth and distribution for each target in the test sample against multiple matched controls. Neighboring target read depth and distribution and zygosity of any variants within each target region are used to reinforce CNV calls. All CNVs are confirmed using another technology such as aCGH, MLPA, or PCR before they are reported.

Analytical Validity

Copy Number Variant Analysis: The PGxome test detects most larger deletions and duplications including intragenic CNVs and large cytogenetic events; however aberrations in a small percentage of regions may not be accurately detected due to sequence paralogy (e.g., pseudogenes, segmental duplications), sequence properties, deletion/duplication size (e.g., 1-3 exons vs. 4 or more exons), and inadequate coverage. In general, sensitivity for single, double, or triple exon CNVs is ~70% and for CNVs of four exon size or larger is close to 100%, but may vary from gene-to-gene based on exon size, depth of coverage, and characteristics of the region.

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 sequencing does not reveal any heterozygous differences from the reference sequence, we cannot be certain that we were able to detect both patient alleles.

For technical reasons, the PGxome test is not 100% sensitive. Some exons cannot be efficiently captured, and some genes cannot be accurately sequenced because of the presence of multiple copies in the genome. Therefore, a small fraction of sequence variants will not be detected.

We sequence coding exons for most given transcripts, plus ~10 bp of flanking non-coding DNA for each exon. Unless specifically indicated, test reports contain no information about other portions of the gene, such as regulatory domains, deep intronic regions, uncharacterized alternative exons, chromosomal rearrangements, repeat expansions, epigenetic effects, and mitochondrial genome variants.

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 amplification.

Unless otherwise indicated, DNA sequence data is obtained from a specific cell-type (usually leukocytes if taken 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.

Balanced translocations or inversions are only rarely detected.

Certain types of sex chromosome aneuploidy may not be detected.  

In nearly all cases, our ability to determine the exact copy number change within a targeted region is limited.

Our ability to detect CNVs due to somatic mosaicism is limited.

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.

A negative finding does not rule out a genetic diagnosis.

Genetic counseling to help to explain test results to the patients and to discuss reproductive options is recommended.

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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