CYSTIC FIBROSIS & AMBRY GENETICS
Cystic Fibrosis (CF) affects approximately 30,000 children and adults in the United States.¹ Common symptoms include chronic cough, recurrent lung infections leading to diminished pulmonary function, pancreatic insufficiency, elevated sweat chlorides, poor growth, pancreatitis, and infertility in males. At least eight million Americans are asymptomatic carriers of CF.² 

Ambry Genetics is committed to the CF community and has unprecedented knowledge and experience in analysis of the causative gene, CFTR. The Ambry lab has analyzed the complete CF gene for more than 20,000 patient samples, providing the largest single-laboratory cystic fibrosis test database in the world from which to draw result interpretations. Our test is capable of detecting over 99% of the 1500+ known mutations, as well as novel variants. Ambry Genetics offers various test options to best suit the clinical situation.

TEST OPTIONS
508 FIRST is the ideal test for patients who have had no previous DNA testing.  It provides a quick and inexpensive screen for ΔF508, the most common CF mutation.  Positive results are reported in 3-5 days.  Testing automatically continues with CF AMPLIFIED for diagnostic patients who do not have homozygous ΔF508 and carrier test patients who are negative for ΔF508.   508 FIRST combines rapid, cost-effective detection of the major CF mutation with the convenience and reassurance of high-sensitivity testing by CF AMPLIFIED.

CF AMPLIFIED is the most comprehensive CF test available, detecting approximately 99% of mutations, including gross deletions and duplications, in patients of all ethnicities. The test begins with full gene sequence analysis which detects 97-98% of mutations. If two mutations are detected in a diagnostic test, or one mutation in a carrier test, the testing is informative and the analysis is complete. If not, analysis automatically continues for gross deletions and duplications. Testing is done in steps to control costs; if deletion/duplication testing is not necessary, a discount is applied and the average turnaround time is reduced from 15-35 days to 14-28 days.  Ambry Genetics was the first to introduce clinical testing for gross deletions and duplications, which account for 1-2% of CF mutations overall.  In CF patients with one mutation detected by full gene sequence analysis, deletion/duplication testing can solve diagnostic dilemmas by finding the “missing mutation” in up to 20% of cases.^3-5

CF Full Gene Analysis without deletion/duplication testing remains available to detect 97-98% of mutations.  By client request within one month after results are reported, deletion/duplication analysis can be added for an additional fee as long as there is sufficient DNA available. Otherwise deletion/duplication testing can be done on a new sample.

As described in our Gene Report, we compared the yields of testing by the Ambry Test and the ACMG 23-mutation panel in a series of 877 samples. Approximately one-third of mutations were missed by the panel. Forty-one percent of the affected CF patients had homozygous ΔF508 mutations that could have been diagnosed inexpensively in less than one week using 508 FIRST. Therefore, 508 FIRST is indicated for patients with no prior DNA testing and CF AMPLIFIED is appropriate for any patient needing diagnostic testing to:

• confirm a suspected diagnosis of CF
• follow-up to abnormal newborn screen
• determine etiology of congenital bilateral absence of the vas deferens
• determine etiology of apparently isolated chronic or recurrent pancreatitis (see our Pancreatitis testing information)

CARRIER TESTING BENEFITS            
High-risk carrier test situations require higher standards of detection. Detection rates of mutation panels vary by ethnicity and for the basic 23-mutation panel, they range from 88% down to 49% in the major American ethnic groups.  Laboratories may test for additional mutations to increase the detection rates, but mutation panels do not achieve the Ambry Test: CF AMPLIFIED’s rate of approximately 99% in all ethnicities.  For general screening purposes, a small to moderate amount of uncertainty is acceptable to many patients. However, there are circumstances where a higher degree of confidence is desired including:

• one partner is known to be a CF carrier
• one partner is non-Caucasian
• fetal ultrasound shows echogenic bowel
• family history of CF
• gamete donors

The table below shows background carrier rates, detection rates,⁶ and revised carrier risks for various ethnicities. The revised carrier risk is the chance, which exists after any CF genetic test, that a mutation went undetected. The CF AMPLIFIED test provides a significant reduction to the revised carrier risk.

Ethnicity	23-Mutation Panel Detection Rate	CF AMPLIFIED Detection Rate	Background Carrier Risk before Any Test	Revised Risk After 23-Mutation Panel	Revised Risk after CF AMPLIFIED
Non-Hispanic Caucasian	88%	99%	1/25	1/206	1/2401
Hispanic American	72%	99%	1/58	1/203	1/5701
African American	64%	99%	1/61	1/171	1/6001
Asian American	49%	99%	1/94	1/183	1/9301
Ashkenazi Jewish	94%	99%	1/24	1/384	1/2301

Calculations assume a negative test result and no family history of CF.

Comprehensive testing by the Ambry Test provides the greatest chance of mutation detection possible for the most informed diagnosis, patient care, and genetic counseling.

TEST METHODS
508 FIRST is performed by RFLP analysis for only the ΔF508 mutation.  CFTR full gene sequence analysis is performed by modified Temporal Temperature Gradient Electrophoresis (mTTGE) through 983 bases of 5’ untranslated region (UTR), exons 1 to 24 (a total of 27 exons), plus at least 20 bases into the 5’ and 3’ ends of all the introns. The exact nature of any sequence variation(s) detected by mTTGE are subsequently identified by PCR-based double-stranded automated sequencing in the sense and antisense directions. Intronic regions containing the two known mutations 3849+10kbC>T in intron 19 and intron 11’s 1811+1634A>G mutation (also known as 1811+1.6kbA>G) are also analyzed. CFTR poly T status, TG repeat, and exon 9 are analyzed by direct sequencing.  Gross deletion/duplication analysis determines gene copy number for all of the 27 exons using MLPA^®. All ACOG and ACMG recommended mutations are analyzed by the Ambry Test: CF AMPLIFIED and CF Full Gene Analysis.

RELATED TESTS                                                                                     

• TG Repeat Analysis can be ordered as a stand-alone test. (TG repeat numbers are automatically reported with CF AMPLIFIED diagnostic results when a 5T allele is detected.)
• CF Deletion/Duplication analysis can be ordered for any patient who has previously had full gene sequence analysis.
• Specific mutation analysis for individual CFTR mutations known to be in the family is also available.
• Aminoglycoside-Related Hearing Loss testing by sequencing the MT-RNR1 gene. This test can be ordered separately or as a reflex after positive CF AMPLIFIED diagnostic testing.

MUTATION DETECTION RATE
The CF AMPLIFIED test detects approximately 99% of CFTR mutations. Full gene analysis detects 97-98% of CFTR mutations.

SPECIMEN REQUIREMENTS
Blood: Collect 3-5 cc from adult or 2 cc 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.
Blood Spot: Minimum of one complete spot approximately 0.5 inch in diameter on S&S 903 collection paper or similar. Store in a clean plastic bag at room temperature. Ship at room temperature.
Saliva: Collect 2 ml into Oragene™ DNA Self-Collection container. Store and ship at room temperature.
DNA: Send 20 µg in TE at 50-100 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.

PATIENT BROCHURES
Diagnostic Genetic Testing for Cystic Fibrosis

Genetic Testing for Cystic Fibrosis Carrier Status

GENE REPORT
A Comparison of Ambry Genetics’ Full Gene Sequence Analysis to Common Mutation Testing in the Diagnosis of Cystic Fibrosis 

AMBRY PUBLICATIONS
A guanine to thymidine substitution, located 1643 nucleotides into intron 11 of the CFTR gene (1811+1643G>T), could represent a novel splicing mutation, found exclusively in CF patients of Hispanic descent.
2009 North American Cystic Fibrosis Conference

Impact of IVS8-(TG)m(T)n haplotype on IRT and sweat chloride levels in a cohort of hypertrypsinogenemic newborns with genotype ΔF508/5T identified by California CF Newborn Screening.
2009 North American Cystic Fibrosis Conference

The role of the F508C mutation in congenital bilateral absence of the vas deferens.
Genet Med. 2008:10:910–914.  Please call us for reprint.

Clinical features of three children with novel CFTR mutations.
2008 North American Cystic Fibrosis Conference

Comprehensive genetic analysis of the cystic fibrosis transmembrane conductance regulator from dried blood specimens – Implications for newborn screening.
Genet Med. 2006;8:557-562. Please call us for reprint.

CFTR genotypes in late diagnosed versus very early diagnosed CF patients.
2006 North American Cystic Fibrosis Conference

Homozygous CFTR mild mutations detected in CF patients diagnosed at an earlier age.
2006 Annual Meeting of the Association for Molecular Pathology

Identification of a novel combination of cystic fibrosis mutations in an asymptomatic woman following prenatal
risk assessment.
2006 Annual Meeting of the American College of Medical Genetics

Diagnostic testing by CFTR mutation analysis in a large group of Hispanics: Novel mutations and assessment of a population-specific mutation spectrum.
J Mol Diag. 2005;7:289-299

A case study illustrating the benefits of full sequence analysis for diagnosing CF.
2005 Annual Meeting of the American College of Medical Genetics

100% CFTR mutation detection in a group of patients affected with CF using The Ambry Test®: CF AMPLIFIED.
2005 North American Cystic Fibrosis Conference

Improved detection of cystic fibrosis mutations in infertility patients with DNA sequence analysis.
Hum Reprod. 2004;19:540-546

REFERENCES
¹ Cystic Fibrosis Foundation website. Available at www.cff.org. Accessed May 28, 2008.
²NIH Publication No. 97-4200, July 1997. Available at http://www.cureresearch.com/artic/cystic_fibrosis_
research_directions_niddk.htm.  Accessed May 29, 2008.
³Audrezet MP et al. Hum Mut. 2004;23:343-357.
⁴Niel F et al. J Med Genet. 2004;41:e118.
⁵Chevalier-Porst F et al. Hum Mut. 2005;25:504-510.
⁶ACMG Technical Standards and Guidelines for CFTR Mutation Testing, 2006 Edition.Available at http://www.acmg.net/Pages/ACMG_Activities/stds-2002/cf.htm.  Accessed May 29, 2008.