Orders
This page...
Forms
Order Kits
Order Kits
Order Kits
Loading...
×
Ambiguous Genitalia Sequencing Panel with CNV Detection
- Summary and Pricing
- Clinical Features and Genetics
- Citations
- Methods
- Ordering/Specimens
Order Kits
TEST METHODS
Exome with CNV
| Test Code | Test Copy Genes | CPT Code Copy CPT Codes | |
|---|---|---|---|
| 6903 | AKR1C4 | 81479,81479 | Add |
| ANOS1 | 81406,81479 | ||
| AR | 81405,81479 | ||
| ARX | 81403,81403 | ||
| ATRX | 81479,81479 | ||
| B3GLCT | 81479,81479 | ||
| BCOR | 81479,81479 | ||
| BMP4 | 81479,81479 | ||
| CCNQ | 81479,81479 | ||
| CDKN1C | 81479,81479 | ||
| CEP41 | 81479,81479 | ||
| CHD4 | 81479,81479 | ||
| CHD7 | 81407,81479 | ||
| CREBBP | 81407,81406 | ||
| CYB5A | 81479,81479 | ||
| CYP11A1 | 81479,81479 | ||
| CYP11B1 | 81405,81479 | ||
| CYP17A1 | 81405,81479 | ||
| CYP19A1 | 81479,81479 | ||
| DHCR24 | 81479,81479 | ||
| DHCR7 | 81405,81479 | ||
| DHH | 81479,81479 | ||
| DMRT1 | 81479,81479 | ||
| DNMT3B | 81479,81479 | ||
| DYNC2H1 | 81479,81479 | ||
| ESCO2 | 81479,81479 | ||
| FAT4 | 81479,81479 | ||
| FEZF1 | 81479,81479 | ||
| FGFR1 | 81405,81479 | ||
| FIG4 | 81406,81479 | ||
| FRAS1 | 81479,81479 | ||
| FREM2 | 81479,81479 | ||
| GATA4 | 81479,81479 | ||
| GNRHR | 81405,81479 | ||
| GRIP1 | 81479,81479 | ||
| HCCS | 81479,81479 | ||
| HOXA13 | 81479,81479 | ||
| HSD17B3 | 81479,81479 | ||
| HSD17B4 | 81479,81479 | ||
| HSD3B2 | 81479,81479 | ||
| ICK | 81479,81479 | ||
| IL17RD | 81479,81479 | ||
| IRF6 | 81479,81479 | ||
| KISS1R | 81479,81479 | ||
| LHCGR | 81406,81479 | ||
| LMNA | 81406,81479 | ||
| MAP3K1 | 81479,81479 | ||
| MKKS | 81479,81479 | ||
| MKS1 | 81479,81479 | ||
| NEK1 | 81479,81479 | ||
| NR0B1 | 81404,81479 | ||
| NR5A1 | 81479,81479 | ||
| NSMF | 81479,81479 | ||
| OPHN1 | 81479,81479 | ||
| POR | 81479,81479 | ||
| PROKR2 | 81479,81479 | ||
| PTPN11 | 81406,81479 | ||
| RIPK4 | 81479,81479 | ||
| ROR2 | 81479,81479 | ||
| RSPO1 | 81479,81479 | ||
| SALL1 | 81479,81479 | ||
| SEMA3A | 81479,81479 | ||
| SETBP1 | 81479,81479 | ||
| SOS1 | 81406,81479 | ||
| SOX10 | 81479,81479 | ||
| SOX2 | 81479,81479 | ||
| SOX3 | 81479,81479 | ||
| SOX9 | 81479,81479 | ||
| SPECC1L | 81479,81479 | ||
| SRD5A2 | 81479,81479 | ||
| SRY | 81400,81479 | ||
| STAR | 81479,81479 | ||
| TBX15 | 81479,81479 | ||
| TOE1 | 81479,81479 | ||
| TRAIP | 81479,81479 | ||
| TSPYL1 | 81479,81479 | ||
| TWIST2 | 81479,81479 | ||
| UBR1 | 81479,81479 | ||
| WDR60 | 81479,81479 | ||
| WNT4 | 81479,81479 | ||
| WNT5A | 81479,81479 | ||
| WNT7A | 81479,81479 | ||
| WT1 | 81405,81479 | ||
| WWOX | 81479,81479 | ||
| ZFPM2 | 81479,81479 | ||
| Full Panel Price* | $990 | ||
| Test Code | Test Copy Genes | Total Price | CPT Codes Copy CPT Codes | |
|---|---|---|---|---|
| 6903 | Genes x (85) |
$990 | 81400, 81403(x2), 81404, 81405(x7), 81406(x7), 81407(x2), 81479(x150) | Add |
Pricing Comments
We are happy to accommodate requests for testing single genes in this panel or a subset of these genes. The price will remain the list price. If desired, free reflex testing to remaining genes on panel is available. Alternatively, a single gene or subset of genes can also be ordered via our PGxome Custom Panel tool.
Targeted Testing
For ordering sequencing of targeted known variants, please proceed to our Targeted Variants landing page.
Turnaround Time
The great majority of tests are completed within 26 days.
Clinical Sensitivity
This multi-gene panel analyzes genes involved in both syndromic and non-syndromic Disorders of Sex Development (DSD). 64 genes in this panel account for approximately 35% of cases of 46,XY DSD (Baxter et al. 2015. PubMed ID: 25383892). In one cohort, clinically significant CNVs were detected in 25% of patients with ambiguous genitalia (Tannour-Louet et al. 2010. PubMed ID: 21048976). So far, gross deletions or duplications have been reported in SOX3, LHCGR, SRY, NR0B1, DMRT1, NR5A1, GATA4, WT1, and WNT4.
Clinical Features
Ambiguous genitalia is a rare condition in which external genitalia do not appear to have the typical appearance of either male or female. Ambiguous genitalia is a subset of disorder of sex development (DSD). Sex development is a complex process under genetic control directing the initially bi-potential gonad to develop into either a testis or an ovary (sex determination), and the consequent differentiation of internal ducts and external genitalia (sex differentiation) (Laino et al. 2014. PubMed ID: 25248670). Disruption of either determination or differentiation can lead to DSD which are congenital conditions with atypical development of chromosomal, gonadal, or anatomic sex (Hughes et al. 2006. PubMed ID: 18947601). DSD, ranging in severity from genital abnormalities to complete sex reversal, includes congenital development of ambiguous genitalia, disjunction between the internal and external sex anatomy, incomplete development of sex anatomy, sex chromosome anomalies (Turner Syndrome; Klinefelter Syndrome) and disorders of gonadal development (Park et al. 2006).
Three subtypes of DSD are generally recognized: Sex Chromosome DSD, 46,XX DSD and 46,XY DSD. 46,XY DSD result from incomplete intrauterine virilization and are characterized by ambiguous or ‘female’ external genitalia, variable gonadal dysgenesis, hypospadias, oligospermia, azoospermia, and müllerian structures that range from absence to presence of a uterus and fallopian tubes (Mohnach et al. 2016. PubMed ID: 20301714). 46,XX DSD relate to excess androgen and are characterized by ambiguous or ‘male’ external genitalia, müllerian aplasia, hyperandrogenism and primary amenorrhea (Knarston et al. 2016. PubMed ID: 26846580).
Genetics
DSD are complex conditions caused by a wide range of genetic anomalies. They can be inherited in an autosomal dominant, autosomal recessive, X-linked, or Y-linked manner depending on the gene involved. To date, more than 60 genes have been showed to be involved in DSD (Baxter et al. 2015. PubMed ID: 25383892). These genes are implicated in sex determination, sex differentiation and causes of hypogonadism. The most commonly involved genes in DSD include SRY, NR5A1, MAP3K1, DHH, NR0B1 (DAX1), WNT4, CYP21A2, and SOX9. See individual gene test descriptions for information on molecular biology of gene products and mutation spectra.
Copy number variants (CNVs) are also a common genetic cause of ambiguous genitalia. Clinically significant CNVs were detected in 25% of patients with ambiguous genitalia (Tannour-Louet et al. 2010. PubMed ID: 21048976). For this reason, genetic testing to detect large cytogenetic events and CNVs is recommended for patients with ambiguous genitalia. Our CNV analysis enables these large cytogenetic abnormalities as well as some exon level CNVs to be identified from NGS data.
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.
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 ≥98% 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.
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
Candidates for this test are individuals with symptoms consistent with disorders of sex development especially ambiguous or abnormal genitalia.
Genes
| Inheritance | Abbreviation |
|---|---|
| Autosomal Dominant | AD |
| Autosomal Recessive | AR |
| X-Linked | XL |
| Mitochondrial | MT |
Diseases
CONTACTS
Genetic Counselors
- Genetic Counselor Team - clinicaldnatesting@preventiongenetics.com
Geneticist
- Fang Xu, PhD, FACMG - fang.xu@preventiongenetics.com
Citations
- Baxter et al. 2015. PubMed ID: 25383892
- Hughes et al. 2006. PubMed ID: 18947601
- Knarston et al. 2016. PubMed ID: 26846580
- Laino et al. 2014. PubMed ID: 25248670
- Mohnach et al. 2016. PubMed ID: 20301714
- Park et al. 2006. Consortium on the Management of Disorders of Sex Development.
- Tannour-Louet et al. 2010. PubMed ID: 21048976
Order Kits
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).
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
NextGen Sequencing: 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.
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 >95%, 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.
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.
Loading...
×
Copy Text to Clipboard
×