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Disorders of Sex Development and Infertility Sequencing Panel with CNV Detection

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  • Clinical Features and Genetics
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  • Methods
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TEST METHODS

Sequencing

Test Code Test Copy GenesCPT Code Copy CPT Codes
2669 AIRE 81406, 81479 Add to Order
AMH 81479, 81479
AMHR2 81479, 81479
ANOS1 81406, 81479
AR 81405, 81479
ARL6 81479, 81479
ARX 81404, 81403
ATRX 81479, 81479
AURKC 81479, 81479
BBS1 81406, 81479
BBS10 81404, 81479
BBS12 81479, 81479
BBS2 81406, 81479
BBS4 81479, 81479
BBS5 81479, 81479
BBS7 81479, 81479
BBS9 81479, 81479
BMP15 81479, 81479
CATSPER1 81479, 81479
CBX2 81479, 81479
CFTR 81223, 81222
CHD7 81407, 81479
CLPP 81479, 81479
CYP11A1 81479, 81479
CYP17A1 81405, 81479
CYP19A1 81479, 81479
DHH 81479, 81479
DMRT1 81479, 81479
DMRT2 81479, 81479
DNAH1 81479, 81479
EIF2B1 81479, 81479
EIF2B2 81405, 81479
EIF2B3 81406, 81479
EIF2B4 81406, 81479
EIF2B5 81406, 81479
FGF8 81479, 81479
FGFR1 81405, 81479
FGFR2 81479, 81479
FIGLA 81479, 81479
FOXL2 81479, 81479
FSHB 81479, 81479
FSHR 81479, 81479
GALNTL5 81479, 81479
GALT 81406, 81479
GATA4 81479, 81479
GNRH1 81479, 81479
GNRHR 81405, 81479
HESX1 81479, 81479
HFE 81479, 81479
HFM1 81479, 81479
HS6ST1 81479, 81479
HSD17B3 81479, 81479
KISS1 81479, 81479
KISS1R 81479, 81479
LEP 81479, 81479
LEPR 81406, 81479
LHCGR 81406, 81479
LHX3 81479, 81479
LHX4 81479, 81479
MAMLD1 81479, 81479
MAP3K1 81479, 81479
MCM8 81479, 81479
MKKS 81479, 81479
NOBOX 81479, 81479
NPAS2 81479, 81479
NR0B1 81404, 81479
NR5A1 81479, 81479
NSMF 81479, 81479
PCSK1 81479, 81479
PICK1 81479, 81479
POR 81479, 81479
PRLR 81479, 81479
PROK2 81479, 81479
PROKR2 81479, 81479
PROP1 81404, 81479
RSPO1 81479, 81479
SEMA3A 81479, 81479
SLC26A8 81479, 81479
SOHLH1 81479, 81479
SOX2 81479, 81479
SOX3 81479, 81479
SOX9 81479, 81479
SPATA16 81479, 81479
SRD5A2 81479, 81479
SRY 81400, 81479
STAR 81479, 81479
TAC3 81479, 81479
TACR3 81479, 81479
TEX11 81479, 81479
TRIM32 81479, 81479
TTC8 81479, 81479
WDR11 81479, 81479
WNT4 81479, 81479
WT1 81405, 81479
WWOX 81479, 81479
ZP1 81479, 81479
Full Panel Price* $2240.00
Test Code Test Copy Genes Total Price CPT Codes Copy CPT Codes
2669 Genes x (96) $2240.00 81222, 81223, 81400, 81403, 81404(x4), 81405(x6), 81406(x10), 81407, 81479(x167) 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

This multi-gene panel analyzes genes involved in both syndromic and non-syndromic DSDs, and male/female infertility. 64 genes in this panel account for approximately 35% of cases of DSD (Baxter et al. 2015). Pathogenic variants in 28 genes cause hypogonadotropic hypogonadism in about 40-50% of patients. Pathogenic variants in 14 genes cause gonadal failure in 15% of affected females with hypergonadotropic hypogonadism (Layman 2013).

At this time, the clinical sensitivity of deletion/duplication testing is difficult to estimate due to the lack of large cohort studies. So far, gross deletions or duplications have been reported in SOX3, LHCGR, SRY, NR0B1, DMRT1, NR5A1, GATA4, WT1, WNT4, and FGFR2 genes.

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Clinical Features

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). Disruption of either determination or differentiation can lead to disorders of sex development (DSDs) which are congenital conditions with atypical development of chromosomal, gonadal, or anatomic sex (Hughes et al. 2006). DSDs, ranging in severity from genital abnormalities to complete sex reversal, include 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 DSDs result from incomplete intrauterine virilization and are characterized by ambiguous or ‘female’ external genitalia, variable gonadal dysgenesis, hypospadias, reduced to no sperm production, and müllerian structures that range from absent to presence of a uterus and fallopian tubes (Ostrer et al. 2009). 46,XX DSDs often relate to excess androgen and are characterized by ambiguous or ‘male’ external genitalia, müllerian aplasia, hyperandrogenism and primary amenorrhea (Knarston et al. 2016).

Infertility is a disorder of the reproductive system defined by the failure to achieve a clinical pregnancy after 12 months or more of regular unprotected sexual intercourse. It affects 10-20% of couples worldwide and is generally attributed to males and females equally. In humans, sexual development and reproductive function occur by the actions of the hypothalamin-pituitary-gonadal axis induced by gonadotropin releasing hormone (GnRH). Aberrations in this axis can lead to pubertal and reproductive deficiencies. Diagnoses of infertility include hypognadotrophic hypogonadism, hypergonadotrophic hypogonadism, and obstructive disorders (Layman 2002). Patients with hypognadotrophic hypogonadism present with absent or deficient puberty and poorly defined secondary sexual characteristics owing to low serum gonadotrophin, follicle stimulating hormone (FSH), and luteinizing hormone (LH); while infertility in patients with hypergonadotrophic hypogonadism is usually caused by gonadal defect (primary amenorrhea and premature ovarian failure in female; oligospermia, azoospermia , or other abnormalities of sperm morphology or motility in male).

Genetics

DSDs 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 DSDs (Baxter et al. 2015). These genes are implicated in sex determination, sex differentiation and cause of hypogonadism. The most commonly involved genes in DSDs include SRY, NR5A1, MAP3K1, DHH, NR0B1 (DAX1), WNT4, CYP21A2, and SOX9.

It is estimated that genetic abnormalities including both chromosomal and single-gene alterations can account for up to 30% of cases of infertility (Hotaling 2014). Apart from chromosomal abnormalities, alterations in reproductive function can be caused by gene defects at various levels of the hypothalamin-pituitary-gonadal axis. At least 20 genes have been shown to cause hypogonadotropic hypogonadism; 14 genes are involved in gonadal failure in females with hypergonadotropic hypogonadism (Layman 2013). Moreover, single or multiple gene defects in other clinical conditions, such as cryptorchidism, premature ovarian failure and spermatogenic failure, have also been recently described.

Sex chromosome aneuploidy, structural abnormality and copy number variation are common genetic causes of DSDs and infertility. For this reason, we recommend chromosomal microarray analysis as the first genetic testing in the case of a patient with ambiguous genitalia, other suspected disorder of sex development, or infertility. See Test description #2000 for information on Whole-Genome Chromosomal Microarray (CMA-ISCA).

Testing Strategy

For this NextGen test, we sequence all coding exons of the genes listed below, plus ~20 nucleotides of flanking intronic DNA. 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 pathogenic, undocumented and questionable variant calls 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 >99% 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 include individuals with symptoms consistent with disorders of sex development including both non-syndromic DSD with ambiguous or abnormal genitalia and syndromic conditions that involve additional congenital anomalies as well as individuals with genetic infertility.

Diseases

Name Inheritance OMIM ID
3-Oxo-5 Alpha-Steroid Delta 4-Dehydrogenase Deficiency AD 264600
46,XX Sex Reversal, Type 1 AR 400045
46,XY Sex Reversal, Type 5 AD 613080
46,XY Sex Reversal, Type 6 AD 613762
46,XY Sex Reversal, Type 7 AR 233420
Adrenal Insufficiency, Congenital, With 46,XY Sex Reversal, Partial Or Complete AR 613743
Androgen Resistance Syndrome XL 300068
Antley-Bixler Syndrome AR 207410
Antley-Bixler Syndrome With Genital Anomalies And Disordered Steroidogenesis AR 201750
ATR-X Syndrome XL 301040
Bardet-Biedl Syndrome 1 AR 209900
Bardet-Biedl Syndrome 10 AR 615987
Bardet-Biedl Syndrome 11 AR 615988
Bardet-Biedl Syndrome 12 AR 615989
Bardet-Biedl Syndrome 2 AR 615981
Bardet-Biedl Syndrome 3 AR 600151
Bardet-Biedl Syndrome 4 AR 615982
Bardet-Biedl Syndrome 5 AR 615983
Bardet-Biedl Syndrome 6 AR 605231
Bardet-Biedl Syndrome 7 AR 615984
Bardet-Biedl Syndrome 8 AR 615985
Bardet-Biedl Syndrome 9 AR 615986
Blepharophimosis, Ptosis, And Epicanthus Inversus AR 110100
Camptomelic Dysplasia AD 114290
CATSPER-Related Male Infertility AR 612997
Cholesterol Monooxygenase (Side-Chain Cleaving) Deficiency AR 201710
Congenital Bilateral Absence Of The Vas Deferens AR 277180
Cystic Fibrosis AR 219700
Deficiency Of Steroid 17-Alpha-Monooxygenase AD,AR 202110
Familial Gynecomastia, Due To Increased Aromatase Activity AR 139300
Follicle-Stimulating Hormone Deficiency, Isolated XL 229070
Galactosemia AR 230400
Gonadotropin-Independent Familial Sexual Precocity AR 176410
Hemochromatosis Type 1 AR 235200
Hyperprolactinemia 615555
Hypogonadotropic Hypogonadism 10 with or without Anosmia AR 614839
Hypogonadotropic Hypogonadism 11 with or without Anosmia AR 614840
Hypogonadotropic Hypogonadism 12 with or without Anosmia AR 614841
Hypogonadotropic Hypogonadism 13 with or without Anosmia AR 614842
Hypogonadotropic Hypogonadism 14 with or without Anosmia AD 614858
Hypogonadotropic Hypogonadism 15 with or without Anosmia AD 614880
Hypogonadotropic Hypogonadism 16 with or without Anosmia AD 614897
Hypogonadotropic Hypogonadism 7 with or without Anosmia AR 146110
Hypogonadotropic Hypogonadism 8 with or without Anosmia AR 614837
Hypogonadotropic Hypogonadism 9 with or without Anosmia AD 614838
Hypospadias 2, X-Linked XL 300758
Infertility Associated With Multi-Tailed Spermatozoa And Excessive DNA AR 243060
Isolated X-Linked Adrenal Hypoplasia Congenita AR 300200
Kallmann Syndrome 1 XL 308700
Kallmann Syndrome 2 AD 147950
Kallmann Syndrome 3 AD 244200
Kallmann Syndrome 4 AD 610628
Kallmann Syndrome 5 AD 612370
Kallmann Syndrome 6 AD 612702
Leukoencephalopathy With Vanishing White Matter AR 603896
Mental Retardation-Hypotonic Facies Syndrome X-Linked, 1 XL 309580
Microphthalmia Syndromic 3 AR 206900
Mullerian Aplasia And Hyperandrogenism AD 158330
Obesity, Morbid, Due to Leptin Deficiency AR 614962
Obesity, Morbid, Due to Leptin Receptor Deficiency AR 614963
Oocyte Maturation Defect AR 615774
Ovarian Dysgenesis 1 AR 233300
Ovarian Dysgenesis 2 AD 300510
Ovarian Hyperstimulation Syndrome AR 608115
Palmoplantar Hyperkeratosis With Squamous Cell Carcinoma Of Skin And 46,XX Sex Reversal AR 610644
Panhypopituitarism X-Linked AD 312000
Perrault Syndrome 3 AR 614129
Persistent Mullerian Duct Syndrome AR 261550
Pituitary Hormone Deficiency, Combined 2 AR 262600
Pituitary Hormone Deficiency, Combined 3 AR 221750
Pituitary Hormone Deficiency, Combined 4 AD,AR 262700
Polyglandular Autoimmune Syndrome, Type 1 AD 240300
Premature Ovarian Failure 10 AR 612885
Premature Ovarian Failure 3 AD 608996
Premature Ovarian Failure 5 AD 611548
Premature Ovarian Failure 6 AR 612310
Premature Ovarian Failure 9 AR 615724
Proprotein Convertase 1/3 Deficiency AR 600955
Septooptic Dysplasia AR 182230
Spermatogenic Failure 2 AR 309120
Spermatogenic Failure 3 XL 606766
Spermatogenic Failure 6 AD 102530
Spermatogenic Failure 8 XL 613957
Testicular Anomalies with or without Congenital Heart Disease AD 615542
Testosterone 17-Beta-Dehydrogenase Deficiency AR 264300
Wilms' Tumor AD 194070
X-Linked Lissencephaly 2 AR 300215

Related Tests

Name
46,XX Disorder of Sex Development (DSD) via the WNT4 Gene
46,XY Disorder of Sex Development (DSD) via the HSD17B3 Gene
DHH-related Disorders of Sex Development via the DHH Gene
FGFR1-Related Disorders via the FGFR1 Gene
FGFR2-Related Disorders via the FGFR2 Gene
HESX1-Related Disorders via the HESX1 Gene
LHCGR-related Disorders via the LHCGR Gene
NR0B1-Related Disorders via the NR0B1 Gene
NR5A1-Related Disorders via the NR5A1 Gene
SOX2-Related Ocular Disorders via the SOX2 Gene
SRY-related Disorders of Sex Development via the SRY Gene
Alpha-Thalassemia X-linked Intellectual Disability Syndrome via the ATRX Gene
Androgen Insensitivity Syndrome (AIS) via the Androgen Receptor (AR) Gene
Anophthalmia / Microphthalmia Sequencing Panel
Autism Spectrum Disorders and Intellectual Disability (ASD-ID) Comprehensive Sequencing Panel with CNV Detection
Autoimmune Polyendocrinopathy Syndrome Type 1 via the AIRE Gene
Autosomal Recessive Limb Girdle Muscular Dystrophy (LGMD) Sequencing Panel
Autosomal Recessive Retinitis Pigmentosa Sequencing Panel with CNV Detection
Bardet-Biedl Syndrome Sequencing Panel
Bardet-Biedl Syndrome via the ARL6/BBS3 Gene
Bardet-Biedl Syndrome via the BBS1 Gene
Bardet-Biedl Syndrome via the BBS10 Gene
Bardet-Biedl Syndrome via the BBS12 Gene
Bardet-Biedl Syndrome via the BBS2 Gene
Bardet-Biedl Syndrome via the BBS4 Gene
Bardet-Biedl Syndrome via the BBS5 Gene
Bardet-Biedl Syndrome via the BBS7 Gene
Bardet-Biedl Syndrome via the BBS9 Gene
Bardet-Biedl Syndrome via the MKKS/BBS6 Gene
Bardet-Biedl Syndrome via the TRIM32/BBS11 Gene
Bardet-Biedl Syndrome via the TTC8/BBS8 Gene
Blepharophimosis-Ptosis-Epicanthus Inversus syndrome (BPES) via the FOXL2 Gene
Cancer Sequencing and Deletion/Duplication Panel
CHARGE and Kallmann Syndromes Sequencing Panel
CHARGE and Kallmann Syndromes via the CHD7 Gene
Chronic Pancreatitis Sequencing Panel
Ciliopathy Sequencing Panel
Combined Pituitary Hormone Deficiency (CPHD) Sequencing Panel with CNV Detection
Combined Pituitary Hormone Deficiency-2 (CPHD2) via the PROP1 Gene
Combined Pituitary Hormone Deficiency-4 (CPHD-4) via the LHX4 Gene
Comprehensive Cardiology Sequencing Panel with CNV Detection
Comprehensive Inherited Retinal Dystrophies (includes RPGR ORF15) Sequencing Panel with CNV Detection
Comprehensive Neuromuscular Sequencing Panel
Congenital Abnormalities of the Kidney and Urinary Tract (CAKUT) Sequencing Panel with CNV Detection
Congenital Hypothyroidism and Thyroid Hormone Resistance Sequencing Panel
Cornelia de Lange Syndrome and Cornelia de Lange Syndrome-Related Disorders Sequencing Panel
Craniosynostosis and Related Disorders Sequencing Panel
Cystic Fibrosis and CF-Related Disorders via the CFTR Gene
Disorders of Sex Development Sequencing Panel with CNV Detection
Early Infantile Epileptic Encephalopathy Sequencing Panel
Early Infantile Epileptic Encephalopathy, Recessive Sequencing Panel
Early Infantile Epileptic Encephalopathy:
Dominant and X-linked Sequencing Panel
Epilepsy: Ohtahara Syndrome Sequencing Panel
Facial Dysostosis Related Disorders Sequencing Panel
Female Infertility Sequencing Panel with CNV Detection
Galactosemia Type I via the GALT Gene
Galactosemia Type I via the GALT Gene, 5.5 kb Common Deletion
Globozoospermia via the SPATA16 Gene
Hereditary Hemochromatosis Sequencing Panel
Hereditary Hemochromatosis via the HFE gene
Hereditary Ovarian Cancer Sequencing and Deletion/Duplication Panel
Holoprosencephaly, Autosomal Dominant, Nonsyndromic, Sequencing Panel
Hypogonadotropic Hypogonadism/Kallmann Syndrome Sequencing Panel with CNV Detection
Hypogonadotropic Hypogonadism/Kallmann Syndrome via the PROK2 Gene
Hypogonadotropic Hypogonadism/Kallmann Syndrome via the PROKR2 Gene
Hypoparathyroidism Sequencing Panel
Idiopathic Hypogonadotropic Hypogonadism (IHH) via the GNRHR Gene
Idiopathic Hypogonadotropic Hypogonadism (IHH) via the KISS1 Gene
Idiopathic Hypogonadotropic Hypogonadism (IHH) via the KISS1R Gene
Idiopathic Hypogonadotropic Hypogonadism (IHH) via the GNRH1 Gene
Idiopathic Hypogonadotropic Hypogonadism (IHH) via the TAC3 Gene
Idiopathic Hypogonadotropic Hypogonadism (IHH) via the TACR3 Gene
Interstitial Lung Disease Sequencing Panel with CNV Detection
Isolated Nonsyndromic Congenital Heart Defects via the GATA4 Gene
Kallmann Syndrome (KS) Sequencing Panel
Kallmann Syndrome via the KAL1(ANOS1) Gene
Leber Congenital Amaurosis 10 (LCA10) via the CEP290 Gene
Leukoencephalopathy with Vanishing White Matter and Ovarioleukodystrophy Sequencing Panel
Leukoencephalopathy with Vanishing White Matter and Ovarioleukodystrophy via the EIF2B1 Gene
Leukoencephalopathy with Vanishing White Matter and Ovarioleukodystrophy via the EIF2B5 Gene
Leukoencephalopathy with Vanishing White Matter and Ovarioleukodystrophy via the EIF2B2 Gene
Leukoencephalopathy with Vanishing White Matter and Ovarioleukodystrophy via the EIF2B3 Gene
Leukoencephalopathy with Vanishing White Matter and Ovarioleukodystrophy via the EIF2B4 Gene
Limb-Girdle Muscular Dystrophy (LGMD) Sequencing Panel
Male and Female Infertility via the FSHB Gene
Male Infertility Sequencing Panel with CNV Detection
Male Infertility via the CATSPER1 Gene
Male Infertility with Large-headed Spermatozoa via the AURKC Gene
Metabolic Hypoglycemia Sequencing Panel
Monogenic Obesity via the MC4R Gene
Nephrotic Syndrome (NS)/Focal Segmental Glomerulosclerosis (FSGS) Sequencing Panel
Non-syndromic Intellectual Disability (NS-ID) Sequencing Panel with CNV Detection
Non-Syndromic Monogenic Obesity Sequencing Panel
Non-Syndromic Monogenic Obesity via the LEP Gene
Non-Syndromic Monogenic Obesity via the LEPR Gene
Non-Syndromic Monogenic Obesity via the PCSK1 Gene
Non-Syndromic Monogenic Obesity via the POMC Gene
Oocyte Maturation Defect (OOMD) via the ZP1 Gene
Ovarian Dysgenesis via the BMP15 Gene
Ovarian Dysgenesis via the FSHR Gene
Ovarian Hyperstimulation Syndrome via the FSHR Gene
P450 Oxidoreductase Deficiency via the POR Gene
Perrault Syndrome Type 3 and Deafness, Autosomal Recessive 8 (DFNB8) via the CLPP Gene
Premature Ovarian Failure (POF) Sequencing Panel with CNV Detection
Premature Ovarian Failure via HFM1 Gene Sequencing with CNV Detection
Premature Ovarian Failure via the FIGLA Gene
Premature Ovarian Failure via the MCM8 Gene
Premature Ovarian Failure/Ovarian Dysgenesis via the SOHLH1 Gene
Primary Ciliary Dyskinesia (PCD) via the DNAH1 Gene
Primary Ciliary Dyskinesia (PCD)/Immotile Cilia Syndrome and Cystic Fibrosis Sequencing Panel
Primary Ciliary Dyskinesia (PCD)/Immotile Cilia Syndrome Sequencing Panel
Renal Cancer Sequencing Panel
Retinitis Pigmentosa (includes RPGR ORF15) Sequencing Panel with CNV Detection
Rett Syndrome, Angelman Syndrome and Variant Syndromes Sequencing Panel with CNV Detection
Septo-optic Dysplasia Spectrum Sequencing Panel
Short Rib Skeletal Dysplasia Sequencing Panel
Skeletal Disorders and Joint Problems Sequencing Panel with CNV Detection
Spinocerebellar Ataxia-12 via the WWOX Gene
Steroid-Resistant Nephrotic syndrome via the WT1 Gene
Wilms Tumor via the WT1 Gene
X-Linked Intellectual Disability Sequencing Panel with CNV Detection
X-linked Lissencephaly-2 via the ARX Gene

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Baxter R.M. et al. 2015. The Journal of Clinical Endocrinology and Metabolism. 100: E333-44. PubMed ID: 25383892
  • Hotaling J.M. 2014. The Urologic Clinics of North America. 41: 1-17. PubMed ID: 24286764
  • Hughes I.A. et al. 2006. Journal of Pediatric Urology. 2: 148-62. PubMed ID: 18947601 PubMed ID: 18947601
  • Knarston I. et al. 2016. Clinical Science. 130: 421-32. PubMed ID: 26846580
  • Laino L. et al. 2014. Endocrine Connections. 3: 180-92. PubMed ID: 25248670
  • Layman L.C. 2002. Journal of Medical Genetics. 39: 153-61. PubMed ID: 11897813
  • Layman L.C. 2013. Molecular and Cellular Endocrinology. 370: 138-48. PubMed ID: 23499866
  • Ostrer H. 2009. Disorder of Sex Development and 46,XY Complete Gonadal Dysgenesis. In: Pagon RA, Adam MP, Bird TD, Dolan CR, Fong C-T, Smith RJ, and Stephens K, editors. GeneReviews™, Seattle (WA): University of Washington, Seattle. PubMed ID: 20301714
  • Park R. et al. 2006. Consortium on the Management of Disorders of Sex Development.
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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.

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Ordering Options


myPrevent - Online Ordering
  • The test can be added to your online orders in the Summary and Pricing section.
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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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