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CHARGE and Kallmann Syndromes Sequencing Panel

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

NGS Sequencing

Test Code Test Copy GenesCPT Code Copy CPT Codes
3267 ANOS1 81406 Add to Order
CHD7 81407
SEMA3E 81479
Full Panel Price* $1440.00
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

The sensitivity of this test varies based on the criteria used for diagnosis. Pathogenic variants in CHD7 are detected in over 95% of patients with a clinical diagnosis based on Blake or Verloes criteria (Blake et al. 1998; Verloes et al. 2005). CHD7 pathogenic variants are found in 60-70% of patients who are suspected to have CHARGE syndrome (Blake et al. 2011). About 11% of patients with a clinical diagnosis of Kallmann syndrome have pathogenic variants in CHD7 (Marcos et al. 2014).

Pathogenic variants in the ANOS1 gene account for ~ 8% of all KS cases (Dode et al. 2009). Pathogenic variants in the SEMA3E gene appear to be a rare cause of CHARGE or Kallmann syndromes.

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Deletion/Duplication Testing via aCGH

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
600 ANOS1$690.00 81479 Add to Order
CHD7$690.00 81479
Full Panel Price* $730.00
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

Large pathogenic deletions in CHD7 have been reported in less than 5% of patients with a clinical diagnosis of CHARGE syndrome (Bergman et al. 2008; Wincent et al. 2009; Blake et al. 2011).

Large deletions in the ANOS1 gene have been reported in up to 25% of X-linked Kallmann syndrome patients analyzed (Ahmadzadeh et al. 2015).

Large pathogenic deletions in the SEMA3E gene have not been reported.

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Clinical Features

CHARGE syndrome is a severe developmental disorder characterized by multiple congenital defects involving sensory and mediastinal organs. It is a clinically heterogeneous disorder in regards to symptoms and severity. Hallmark features include ocular coloboma; choanal atresia; cranial nerve abnormalities leading to facial palsy, loss of sense of smell, feeding, swallowing and breathing difficulties; and external and inner ear malformations resulting in hearing loss and reduced sense of balance. Additional features include hypogonadotropic hypogonadism, which manifests as incomplete or absent puberty and infertility; genital hypoplasia; distinctive facial features growth and developmental delay; a wide variety of heart defects; cleft lip and/or palate; and olfactory dysfunction in the form of aplasia or hypoplasia (Blake et al. 1998; Pinto et al. 2005). CHARGE syndrome is usually diagnosed during childhood. Diagnosis is made based on the presence of a combination of major and minor clinical features (Blake et al. 1998; Verloes et al. 2005). Magnetic resonance imaging (MRI) of the temporal bones reveals abnormalities in the semicircular canal (Amiel et al 2001). In rare cases, CHARGE syndrome has been detected in adults only after the birth of a child with the major characteristic features of the disease (Hughes et al. 2014). It has also been diagnosed antenatally (Legendre et al. 2012). CHARGE syndrome affects individuals worldwide with an incidence of approximately 1 case in 12,500 live births (Källén et al. 1999). Higher incidences have been reported in the Atlantic provinces of Newfoundland and Labrador, and the Maritime Provinces (Issekutz et al. 2005). See also (Lalani et al. 2012) and the CHARGE Syndrome Foundation (http://www.chargesyndrome.org/foundation.asp).

Kallmann syndrome (KS) is characterized by hypogonadotropic hypogonadism and impaired sense of smell as the result of deficient hypothalamic gonadotropin-releasing hormone and agenesis of the olfactory lobes. Additional features include unilateral failure of kidney development; abnormalities in tooth development; cleft lip and/or palate; and bimanual synkinesis, which is manifested by involuntary movements of one hand that mimic the other hand (Kaplan et al. 2010).

CHARGE syndrome has phenotypic overlap with Kallmann syndrome and hypogonadotropic hypogonadism (Kim et al. 2008; Jongmans et al. 2009). It has been argued that some patients presenting with a clinical diagnosis of Kallmann syndrome may represent unrecognized mild cases of CHARGE syndrome (Ogata et al. 2006).

Genetics

CHARGE syndrome is an autosomal dominant condition. About 95% of patients with a clinical diagnosis of CHARGE syndrome based on the Blake or Verloes criteria have heterozygous pathogenic variants in the CHD7 gene (Vissers et al. 2004; Verloes et al. 2005; Blake et al. 2011). Over 680 different causative variants, located throughout the length of the gene, are listed in public databases (Human Gene Mutation Database; CHD7 Mutation Database). The great majority result in premature termination of protein synthesis, and include nonsense, splicing, small deletions and insertions. Large pathogenic deletions have been reported in less than 5% of patients with a clinical diagnosis of CHARGE syndrome (Bergman et al. 2008; Wincent et al. 2009; Blake et al. 2011). Chromosomal abnormalities as the result of balanced translocations, rearrangements, or interstitial deletions have also been reported in rare cases (Hurst et al. 1991; Johnson 2006; Arrington et al. 2005). Although most disease-causing variants are de novo, familial cases have been reported (Jongmans et al. 2008; Hughes et al. 2014). In these families, clinical features are usually variable among affected individuals and may be very mild. Parental mosaicism, both somatic and germline, has been detected (Jongmans et al 2006; Pauli et al. 2009).

CHD7 encodes the chromodomain helicase DNA-binding protein 7 that is required for normal mammalian development.

Kallmann syndrome is genetically heterogeneous with various inheritance patterns. Several genes have been associated with the disorder, including ANOS1, CHD7, and SEMA3E.

About 30 CHD7 pathogenic variants have been reported in patients with Kallmann syndrome; they account for ~ 11% of patients with a clinical diagnosis (Marcos et al. 2014). Unlike CHARGE-causative variants, the majority of Kallmann syndrome causative variants are missense. To date, no large deletions, duplications, or complex rearrangements were reported. Most cases are sporadic.

Pathogenic variants in the SEMA3E gene appear to be a rare cause of CHARGE or Kallmann syndromes. To date, only one patient with CHARGE syndrome was reported to have a pathogenic missense variant in SEMA3E (Lalani et al. 2004). A different missense variant was reported in one patient with Kallmann syndrome (Cariboni et al. 2015).

The semaphorin 3E protein is involved in the control of vascular patterning, which is critical for normal organogenesis (Gu et al. 2005).

Over 150 pathogenic variants in the ANOS1 gene have been reported in patients with Kallmann syndrome. The majority of these variants are truncating (HGMD). They account for ~ 8% of all KS cases (Dode et al. 2009). Large deletions have been reported in up to 25% of X-linked KS patients analyzed (Ahmadzadeh et al. 2015). Female carriers of ANOS1 pathogenic variants are usually not affected (Oliveira et al. 2011).

The ANOS1 gene is located on the X chromosome. It encodes anosmin-1, which is involved in the growth and migration of olfactory neurons.

Testing Strategy

For this NGS test, 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

Patients presenting with the major clinical criteria or a combination of minor and major criteria for CHARGE syndrome as described (Blake et al. 1998; Verloes et al. 2005). Patients with hypogonadotropic hypogonadism with or without impaired sense of smell are also candidates for this test (Ogata et al. 2006).

Genes

Official Gene Symbol OMIM ID
ANOS1 300836
CHD7 608892
SEMA3E 608166
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Diseases

Name Inheritance OMIM ID
CHARGE Association AD 214800
Kallmann Syndrome 1 XL 308700
Kallmann Syndrome 5 AD 612370

Related Tests

Name
Autism Spectrum Disorders and Intellectual Disability (ASD-ID) Comprehensive Sequencing Panel with CNV Detection
CHARGE and Kallmann Syndromes via the CHD7 Gene
CHARGE Syndrome via the SEMA3E Gene
Congenital Abnormalities of the Kidney and Urinary Tract (CAKUT) Sequencing Panel with CNV Detection
Cornelia de Lange Syndrome and Cornelia de Lange Syndrome-Related Disorders Sequencing Panel
Disorders of Sex Development and Infertility Sequencing Panel with CNV Detection
Disorders of Sex Development Sequencing Panel with CNV Detection
Female Infertility Sequencing Panel with CNV Detection
Hypogonadotropic Hypogonadism/Kallmann Syndrome Sequencing Panel with CNV Detection
Kallmann Syndrome (KS) Sequencing Panel
Kallmann Syndrome via the KAL1(ANOS1) Gene
Male Infertility Sequencing Panel with CNV Detection

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Ahmadzadeh A. et al. 2015. International Journal of Molecular and Cellular Medicine. 4: 152-9. PubMed ID: 26629483
  • Amiel J. et al. 2001. American Journal of Medical Genetics. 99: 124-7. PubMed ID: 11241470
  • Arrington C.B. et al. 2005. American Journal of Medical Genetics Part A. 133A: 326-30. PubMed ID: 15672384
  • Bergman J.E. et al. 2008. European Journal of Medical Genetics. 51: 417-25. PubMed ID: 18472328
  • Blake K. et al. 2011. European Journal of Human Genetics. 19: N/A. PubMed ID: 21407266
  • Blake K.D. et al. 1998. Clinical Pediatrics. 37: 159-73. PubMed ID: 9545604
  • Cariboni A. et al. 2015. The Journal of Clinical Investigation. 125: 2413-28. PubMed ID: 25985275
  • CHARGE Syndrome Foundation
  • CHD7 Mutation Database
  • Dodé C., Hardelin J.P. 2009. European Journal of Human Genetics. 17: 139-46. PubMed ID: 18985070
  • Gu C. et al. 2005. Science. 307: 265-8. PubMed ID: 15550623
  • Hughes S.S. et al. 2014. American Journal of Medical Genetics. Part A. 164A: 48-53. PubMed ID: 24214489
  • Human Gene Mutation Database (Bio-base).
  • Hurst J.A. et al. 1991. Journal of Medical Genetics. 28: 54-5. PubMed ID: 1999835
  • Issekutz K.A. et al. 2005. American Journal of Medical Genetics. Part A. 133A: 309-17. PubMed ID: 15637722
  • Johnson D. et al. 2006. Journal of Medical Genetics. 43: 280-4. PubMed ID: 16118347
  • Jongmans M.C et al. 2009. Clinical Genetics. 75: 65-71. PubMed ID: 19021638
  • Jongmans M.C. et al. 2006. Journal of Medical Genetics. 43: 306-14. PubMed ID: 16155193
  • Jongmans M.C. et al. 2008. American Journal of Medical Genetics. Part A. 146A: 43-50. PubMed ID: 18074359
  • Kaplan J.D. et al. 2010. American Journal of Medical Genetics. Part A. 152A: 2796-801. PubMed ID: 20949504
  • Källén K. et al. 1999. Teratology. 60: 334-43. PubMed ID: 10590394
  • Kim H.G. et al. 2008. American Journal of Human Genetics. 83: 511-9. PubMed ID: 18834967
  • Lalani S. et al. 2004. Journal of Medical Genetics. 41: e94. PubMed ID: 15235037
  • Lalani S.R. et al. 2012. CHARGE Syndrome. In: Pagon RA, Adam MP, Ardinger HH, Bird TD, Dolan CR, Fong C-T, Smith RJ, and Stephens K, editors. GeneReviews(®), Seattle (WA): University of Washington, Seattle. PubMed ID: 20301296
  • Legendre M. et al. 2012. Journal of Medical Genetics. 49: 698-707. PubMed ID: 23024289
  • Marcos S. et al. 2014. The Journal of Clinical Endocrinology and Metabolism. 99: E2138-43. PubMed ID: 25077900
  • Ogata T. et al. 2006. Endocrine Journal. 53: 741-3. PubMed ID: 16960397
  • Oliveira L.M. et al. 2001. The Journal of Clinical Endocrinology and Metabolism. 86: 1532-8. PubMed ID: 11297579
  • Pauli S. et al. 2009. Clinical Genetics. 75: 473-9. PubMed ID: 19475719
  • Pinto G. et al. 2005. The Journal of Clinical Endocrinology and Metabolism. 90: 5621-6. PubMed ID: 16030162
  • Verloes A. 2005. American Journal of Medical Genetics. Part A. 133A: 306-8. PubMed ID: 15666308
  • Vissers L.E. et al. 2004. Nature Genetics. 36: 955-7. PubMed ID: 15300250
  • Wincent J. et al. 2009. European Journal of Medical Genetics. 52: 271-2. PubMed ID: 19248844
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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