Confirming the Diagnosis: Blood Testing

There are two labs in the USA that can test for the PHOX2B gene mutation that causes about 90% of all CCHS cases. One lab is in Chicago, IL at Rush University Medical Center (RUMC). If you are submitting samples to RUMC, please download and complete the PDF file submission form. The other lab, Ambry Genetics, is located in Aliso Viejo, CA. Information about the two testing centers is as follows.

CHICAGO, IL USA

The Molecular Diagnostic Laboratory at Rush University Medical Center is pleased to offer a clinical DNA test for Congenital Central Hypoventilation Syndrome (CCHS) to physicians and medical institutions.

The test is a PCR assay which directly amplifies and sizes the second polyalanine-coding triplet repeat sequence in exon 3 of the PHOX2B gene. This triplet repeat is expanded in the majority (about 90%) of individuals with CCHS. The remaining individuals with CCHS (10%) will have mutations that can be identified by follow-up sequencing of the coding regions of the PHOX2B gene. It should be noted that 5-10% of CCHS patients inherited their PHOX2B mutation from a parent who has mosaicism or a ‘lesser dose’ of the mutation (which explains why the parents are not affected with the CCHS phenotype). Because mosaic parents can pass the same PHOX2B mutation on to other children, it is necessary to test all parents of CCHS probands for mosaicism. The PCR assay PHOX2B Screening Test (and not sequencing) is the best available assay for identifying and quantifying mosaicism. Therefore, children suspected to have CCHS should ideally be tested by the PCR assay PHOX2B Screening Test, with follow-up sequencing if no mutation is found. All parents of children with identified polyalanine expansion mutations should be screened by the PCR assay PHOX2B Screening Test to determine mosaicism. For further clarification of testing indications, please contact Dr. Elizabeth Berry-Kravis.

Three to 9cc of blood in an EDTA vacutainer is required. Call (312-942-6298), fax (312-942-2857) or email Nancy Becker or Dr. Paul Wong if less than 3cc of blood is available. Send the blood at room temperature by overnight delivery service like Federal Express. Because of transportation issues, do not obtain blood on Friday, Saturday or Sunday.

If kept overnight, the blood should be refrigerated. DO NOT FREEZE. No refrigeration is needed for same day deliveries. A turnaround time of 1 to 2 weeks is anticipated. Please include the requesting physician’s complete address, e-mail address and phone and fax numbers. For queries, please use e-mail contact for Nancy Becker, Dr. Weese-Mayer, or Dr. P. Wong.

The shipping address is:

Nancy Becker
1750 W. Harrison Street
Room 1501
Section of Genetics
Jelke Chicago, IL 60612
Rush University Medical Center

Phone: (312) 942-6298
Fax: (312) 942-2857

(Chicago, Rush Presbyterian Medical Center (continued)

Diagnosis, clinical information and billing information must accompany the blood sample. The patient can prepay with a cashier’s check or credit card or we can bill the referring institution. Pre-payment is required for all samples from outside the U.S. Rush University Medical Center will not bill third party payers (e.g. insurance, Medicare, Medicaid) for this testing. Payment is the responsibility of the submitting entity. The cost of the PHOX2B Screening Test is USD $399.00; this pricing is effective as of July 1, 2008 and is subject to change without notice.

CPT codes: 83891, 83894, 83898, 83912.

Please note: The cost of testing has been reduced to accommodate families with CCHS. More than half of all previous proceeds were applied directly to CCHS research. CCHS is a very rare disease, and like all rare diseases, it is difficult to obtain funding from public sources since it is not considered a major public health issue. The funds previously generated from PHOX2B testing have been invaluable to support much needed research into this gene and CCHS.

All test results will be reviewed by Drs. Weese-Mayer and Jennings (Children’s Memorial Hospital, Northwestern University) and Dr. Berry-Kravis (RUMC). Additional information and medical records may be requested.

ALISO VIEJO, CA USA

Ambry Genetics®

The Ambry Test®: Congenital Central Hypoventilation Syndrome

Disease Information Individuals with Congenital Central Hypoventilation Syndrome (CCHS) have adequate ventilation when awake and hypoventilation with normal respiratory rates and shallow breathing during sleep. In severe cases, hypoventilation can also occur while awake. Some children with CCHS also show symptoms of a generalized autonomic nervous system dysfunction including Hirschsprung Disease in 20% ¹. Neural crest tumors are found in 6%¹. Symptoms usually appear during the newborn period though CCHS is increasingly recognized in older children and adults.

In a few cases, CCHS is inherited in an autosomal dominant pattern, but the majority of cases are de novo. Approximately 92% of affected individuals have an in-frame expansion of a polyalanine repeat in exon 3 of the PHOX2B gene from the normal 20 repeats to 25-33 repeats. The remaining ~8% of patients have other mutations at the end of exon 2 or within exon 3¹, ², ³. Mutation type and repeat length generally correlate with disease severity. Non-repeat mutations are associated with increased frequencies of Hirschsprung Disease, neural crest tumors, and continuous ventilator dependence compared to patients with polyalanine repeat expansions¹, ³. Similarly, large repeat expansions correlate with a more severe respiratory phenotype and higher risk of Hirschsprung Disease and tumors than short expansions². Late-onset CCHS patients without Hirschsprung Disease are typically found to have small repeat expansions³.

Testing Benefits & Indications

Diagnostic confirmation in patients suspected to have congenital or late-onset CHS
Define level of suspicion for other autonomic dysfunction and tumors
Parental testing to rule out risk for late-onset symptoms

Test Description

The Ambry Test is a full gene sequence analysis. Double-stranded automated sequencing is performed in sense and antisense directions for all exons 1-4 of the PHOX2B gene, plus at least 20 bases into the 5’ and 3’ ends of all the introns. Alanine repeat numbers for the commonly-expanded region in exon 3 are determined and reported in all cases. Specific mutation analysis for individual PHOX2B mutations known to be in the family is also available.

Mutation Detection Rate

Approximately 99% of mutations are detectable by this test.

Turn-Around-Time

Full gene analysis: 10-21 days
Specific mutation analysis: 10-14 days

Specimen Requirements

Blood: Collect 3-5cc from adult or 2cc minimum from child into EDTA purple-top tube (first choice) or ACD yellow-top tube (second choice). Store at room temperature or refrigerate. Ship at room temperature.
Saliva: Collect 2ml into Oragene™ DNA Self Collection container. Store and ship at room temperature.
DNA: Send 20µg in TEat50-100 ng/µl. Store frozen and ship on ice or dry ice.
Prenatal: Prenatal testing is available. Please call an Ambry Genetic Counselor to discuss your case.
CPT Codes: Full gene analysis or specific mutation analysis 83891, 83894, 83898, 83904, 83909, 83912

Ambry Genetics®
100 Columbia #200
Aliso Viejo, CA 92656

Toll Free: 866.262.7943
Phone: 949.900.5500
Fax: 949.900.5501
www.ambrygen.com

References

¹ Berry-Kravis EM et al. Am J Respir Crit Care Med. 2006; 174:1139-1144.
² Trochet D et al. Hum Mol Genet. 2005; 14:3697-3708.
³ Trochet D et al. Am J Hum Genet. 2005; 76; 421-426.
© 2008 Ambry Genetics P0408-09-026-MKG-00