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Chronic Granulomatous Disease via the CYBB Gene

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

Sequencing

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
1651 CYBB$870.00 81479 Add to Order
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 18 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

This test involves bidirectional Sanger sequencing using genomic DNA of all coding exons (1-13) of the CYBB gene plus ~20 bp of flanking non-coding DNA on each side. We will also sequence any single exon (Test #100) or pair of exons (Test #200) in patients and relatives of patients or to confirm research results.

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

Bi-Directional Sanger Sequencing

Test Procedure

Nomenclature for sequence variants was from the Human Genome Variation Society (http://www.hgvs.org).  As required, DNA is extracted from the patient specimen.  PCR is used to amplify the indicated exons plus additional flanking non-coding sequence.  After cleaning of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit.  Products are resolved by electrophoresis on an ABI 3730xl capillary sequencer.  In most cases, sequencing is performed in both forward and reverse directions; in some cases, sequencing is performed twice in either the forward or reverse directions.  In nearly all cases, the full coding region of each exon as well as 20 bases of non-coding DNA flanking the exon are sequenced.

Analytical Validity

As of March 2016, we compared 17.37 Mb of Sanger DNA sequence generated at PreventionGenetics to NextGen sequence generated in other labs. We detected only 4 errors in our Sanger sequences, and these were all due to allele dropout during PCR. For Proficiency Testing, both external and internal, in the 12 years of our lab operation we have Sanger sequenced roughly 8,800 PCR amplicons. Only one error has been identified, and this was due to sequence analysis error.

Our Sanger sequencing is capable of detecting virtually all nucleotide substitutions within the PCR amplicons. Similarly, we detect essentially all heterozygous or homozygous deletions within the amplicons. Homozygous deletions which overlap one or more PCR primer annealing sites are detectable as PCR failure. Heterozygous deletions which overlap one or more PCR primer annealing sites are usually not detected (see Analytical Limitations). All heterozygous insertions within the amplicons up to about 100 nucleotides in length appear to be detectable. Larger heterozygous insertions may not be detected. All homozygous insertions within the amplicons up to about 300 nucleotides in length appear to be detectable. Larger homozygous insertions may masquerade as homozygous deletions (PCR failure).

Analytical Limitations

In exons where our sequencing did not reveal any variation between the two alleles, we cannot be certain that we were able to PCR amplify both of the patient’s alleles. Occasionally, a patient may carry an allele which does not amplify, due for example to a deletion or a large insertion. In these cases, the report contains no information about the second allele.

Similarly, our sequencing tests have almost no power to detect duplications, triplications, etc. of the gene sequences.

In most cases, only the indicated exons and roughly 20 bp of flanking non-coding sequence on each side are analyzed. Test reports contain little or no information about other portions of the gene, including many regulatory regions.

In nearly all 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 for example 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 and cycle sequencing.

Unless otherwise indicated, the sequence data that we report are based on DNA isolated from a specific tissue (usually leukocytes). Test reports contain no information about gene sequences in other tissues.

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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