Forms

Chronic Granulomatous Disease via the CYBB Gene

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

NGS Sequencing

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
6975 CYBB$690.00 81479 Add to Order
Pricing Comment

Our most cost-effective testing approach is NextGen sequencing with Sanger sequencing supplemented as needed to ensure sufficient coverage and to confirm NextGen calls that are pathogenic, likely pathogenic or of uncertain significance. If, however, full gene Sanger sequencing only is desired (for purposes of insurance billing or STAT turnaround time for example), please see link below for Test Code, pricing, and turnaround time information.

For Sanger Sequencing click here.
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

Mutations in the CYBB gene account for ~70% of cases of CGD (Leiding and Holand 2012; Roos et al. 2010). Analytical sensitivity is >95% for detection of causative mutations within the CYBB gene. Deletions of one or more exons are common and also detectable by Sanger sequencing in males. In females, analytical sensitivity is lower as we are unable to detect large heterozygous deletions by this method. Gross deletions of the CYBB gene have been reported in less than 5% of cases (Roos et al. 2010). Deletions have been reported to span several genes leading to associations of Kell phenotype/Mcleod syndrome (XK gene), Duchenne muscular dystrophy (DMD gene), and retinitis pigmentosa (RPGR gene) with X-linked CGD (Brown et al. 1996; Watkins et al. 2011).

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Deletion/Duplication Testing via aCGH

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
600 CYBB$690.00 81479 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 Features

Chronic granulomatous disease (CGD) an inherited immunodeficiency characterized by repeated infections with bacterial and fungal pathogens and formation of granulomas. CGD immunodeficiency is due to an impairment of the NADPH oxidase complex resulting in an inability to generate superoxide in phagocytic cells to lyse pathogens (Song et al. 2011). Common pathogens include Staphylococcus aureus, Pseudomonas species, Candida albicans, Aspergillus species, and Nocardia species. Pneumonia, granuloma formation within gastrointestinal and genitourinary tracts, and failure to thrive are hallmark symptoms of the disorder. In severe cases, granulomas can lead to abscess formation and organ failure. Treatments include long course antimicrobials to ward off infections (Leiding and Holland 2012). Simultaneous administration of antimicrobials and corticosteroids may be used to resolve colitis associated with heightened inflammatory responses to infection (Leiding et al. 2012). Patients with CGD should avoid areas where fungal spores are common such as mulch, gardens, and yard waste. Approximately one in 200,000 newborns in the US are affected with CGD (Winkelstein et al 2000). Genetic testing can aid in differential diagnosis of CGD from other disorders associated with granuloma formation and hyperinflammation such as cystic fibrosis, hyper IgE syndrome, Crohn’s disease, allergic bronchopulmonary aspergillosis, and glucose 6-phosphate dehydrogenase deficiency (Leiding and Holland 2012).

Genetics

CGD is inherited in an X-linked manner through mutations in the CYBB gene. Autosomal recessive forms of CGD also occur through mutations in the CYBA, NCF1, NCF2, and NCF4 genes (Roos and de Boer 2014). Disease onset with individuals with X-linked CGD is ~3 years with mortality occurring in 20% compared to autosomal recessive forms with onset ~7 years and mortality occurring in 8% of cases. The majority of CYBB mutations are truncating with nonsense (30%), deletions (22%) and splice site (19.5%) mutations being causative for CGD (Roos et al 2010; Piirilä et al. 2006). Missense mutations are found in 20% of cases with mutations residing in amino acids 1-309 minimally affecting superoxide production and associated with good prognosis compared to missense mutations in amino acid 310 affecting FAD and NAPDH binding domains and rendering NADPH complex nonfunctional leading to worse prognosis (Leiding and Holland 2012). Insertions, gross deletions, and promoter mutations are present in less than 5% of cases of X-linked CGD. Contiguous gene deletions have been reported to span several genes leading to associations of Kell phenotype/Mcleod syndrome (XK gene), Duchenne muscular dystrophy (DMD gene), and retinitis pigmentosa (RPGR gene) with X-linked CGD (Brown et al. 1996; Watkins et al. 2011). Adult-onset of CGD has been reported in females heterozygous for CYBB mutations and is thought to occur through skewed X-inactivation (Gono et al. 2008; Anderson-Cohen et al. 2003). The CYBB gene encodes the gp91phox subunit of the NADPH oxidase complex. This complex is responsible for transporting electrons from NAPDH to oxygen to generate superoxide within the phagolysosome to facilitate lysis of pathogens in phagocytic cells such as neutrophils and macrophages (Song et al. 2011).

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.

This test provides full coverage of all coding exons of the CYBB gene, plus ~10 bases of flanking noncoding DNA. We define full coverage as >20X NGS reads or Sanger sequencing.

Indications for Test

Oxidative burst test (Nitroblue tetrazolium or dihydrorhodamine) indicating impaired superoxide production, and recurrent fungal and bacterial infections are characteristic of CGD. Protein expression analysis is not a reliable predictor of CGD as missense mutations may render CYBB protein nonfunctional (Kuhns et al. 2010).

Gene

Official Gene Symbol OMIM ID
CYBB 300481
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Disease

Name Inheritance OMIM ID
Granulomatous Disease, Chronic, X-Linked 306400

Related Test

Name
Chronic Granulomatous Disease Sequencing Panel

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Anderson-Cohen M, Holland SM, Kuhns DB, Fleisher TA, Ding L, Brenner S, Malech HL, Roesler J. 2003. Severe phenotype of chronic granulomatous disease presenting in a female with a de novo mutation in gp91-phox and a non familial, extremely skewed X chromosome inactivation. Clin. Immunol. 109: 308–317. PubMed ID: 14697745
  • Brown J, Dry KL, Edgar AJ, Pryde FE, Hardwick LJ, Aldred MA, Lester DH, Boyle S, Kaplan J, Dufier JL, Ho MF, Monaco AM, et al. 1996. Analysis of three deletion breakpoints in Xp21.1 and the further localization of RP3. Genomics 37: 200–210. PubMed ID: 8921393
  • Gono T, Yazaki M, Agematsu K, Matsuda M, Yasui K, Yamaura M, Hidaka F, Mizukami T, Nunoi H, Kubota T, Ikeda S-I. 2008. Adult onset X-linked chronic granulomatous disease in a woman patient caused by a de novo mutation in paternal-origin CYBB gene and skewed inactivation of normal maternal X chromosome. Intern. Med. 47: 1053–1056. PubMed ID: 18520120
  • Kuhns DB, Alvord WG, Heller T, Feld JJ, Pike KM, Marciano BE, Uzel G, DeRavin SS, Priel DAL, Soule BP, Zarember KA, Malech HL, et al. 2010. Residual NADPH oxidase and survival in chronic granulomatous disease. N. Engl. J. Med. 363: 2600–2610. PubMed ID: 21190454
  • Leiding JW, Freeman AF, Marciano BE, Anderson VL, Uzel G, Malech HL, DeRavin S, Wilks D, Venkatesan AM, Zerbe CS, Heller T, Holland SM. 2012. Corticosteroid therapy for liver abscess in chronic granulomatous disease. Clin. Infect. Dis. 54: 694–700. PubMed ID: 22157170
  • Leiding JW, Holland SM. 2012. Chronic Granulomatous Disease. 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: 22876374
  • Piirilä H, Väliaho J, Vihinen M. 2006. Immunodeficiency mutation databases (IDbases). Hum. Mutat. 27: 1200–1208. PubMed ID: 17004234
  • Roos D, Boer M de. 2014. Molecular diagnosis of chronic granulomatous disease. Clin. Exp. Immunol. 175: 139–149. PubMed ID: 24016250
  • Roos D, Kuhns DB, Maddalena A, Roesler J, Lopez JA, Ariga T, Avcin T, Boer M de, Bustamante J, Condino-Neto A, Matteo G Di, He J, Hill HR, Holland SM, Kannengiesser C, Köker MY, Kondratenko I, van Leeuwen K, Malech HL, Marodi L, Nunoi H, Stasia MJ, Ventura AM, Witwer CT, Wolach B, Gallin JI. 2010. Hematologically important mutations: X-linked chronic granulomatous disease (third update). Blood Cells Mol. Dis. 45: 246–265. PubMed ID: 20729109
  • Song E. et al. 2011. Clinical and molecular allergy : CMA. 9: 10. PubMed ID: 21624140
  • Watkins CE, Litchfield J, Song E, Jaishankar GB, Misra N, Holla N, Duffourc M, Krishnaswamy G. 2011. Chronic granulomatous disease, the McLeod phenotype and the contiguous gene deletion syndrome-a review. Clin Mol Allergy 9: 13. PubMed ID: 22111908
  • Winkelstein JA, Marino MC, Johnston RB Jr, Boyle J, Curnutte J, Gallin JI, Malech HL, Holland SM, Ochs H, Quie P, Buckley RH, Foster CB, Chanock SJ, Dickler H. 2000. Chronic granulomatous disease. Report on a national registry of 368 patients. Medicine (Baltimore) 79: 155–169. PubMed ID: 10844935
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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