DISEASE INFORMATION
Hirschsprung disease (HSCR) is multigenic congenital disease of the large intestine characterized by absence of neuronal ganglia, colon enlargement, and constipation; it is typically suspected in newborns who have not passed meconium 48 hours after birth.¹ Hirschsprung disease has an estimated incidence of one out of 5,000 births, varying almost two-fold depending upon ethnicity, with males being four times more likely affected than females.^2,3 HSRC has been associated with two phenotypes: short segment (S-HSCR or Type I HSCR) and long segment (L-HSCR or Type II HSCR). S-HSCR accounts for 60%-85% of patients and affects the rectum along with a small portion of the colon (the sigmoid). The remaining 15%-25% of patients have L-HSCR which also affects the rectum and extends beyond the upper sigmoid.^1,4 HSCR is associated with multiple congenital abnormalities in approximately 30% of patients.^4. X-ray, barium enema studies, and rectal manometry can be helpful in establishing diagnosis of HSCR, but confirmation requires a rectal biopsy.^1,5 

HSCR is associated with mutations in eight partially-interdependent genes, with mutations occurring primarily in the RET gene. Dominant loss-of-function mutations in RET have been identified in 50% of familial and 35% of sporadic HSCR cases.⁶ RET has also been shown to act as a modifier gene in other syndromic forms of HSCR.⁷

TESTING BENEFITS & INDICATIONS
Diagnostic testing is helpful for individuals known or suspected to have Hirschsprung disease especially those with L-HSCR. Testing is available for relatives of HSCR patients in whom the mutation is known and for pregnancies at risk.

TEST DESCRIPTION
RET exons 2, 3, 5, 6, 9, 10, 12, 13, and 17 plus at least 20 bases into the 5’ and 3’ ends of all the introns are analyzed. If a disease causing mutation is not identified, the remainder of the 20 exons of RET plus at least 20 bases into the 5’ and 3’ ends of all the introns will be analyzed. Specific mutation analysis for individual RET mutations known to be in the family is also available.

MUTATION DETECTION RATE
Mutations in the RET gene account for 50% of familial cases of HSCR and up to 35% of sporadic cases.⁸ The Ambry Test: RET-Related Hirschsprung Disease is capable of detecting >99% of described mutations in RET.

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: Call for availability.
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. 

REFERENCES
¹Amiel J, Lyonnet S. J Med Genet. 2001 Nov;38(11):729-39.
²Badner JA et al. Am J Hum Genet. 1990 Mar;46(3):568-80.
³ Chin TW et al. J Chin Med Assoc. 2008 Aug;71(8):406-10.
⁴ Passarge E. Nat Genet. 2002 May;31(1):11-2.
⁵ Neville H. (2008) Hirschsprung Disease. eMedicine.
   http://emedicine.medscape.com/article/929733-diagnosis. Accessed October 12, 2009.
⁶ Bolk S et al. Proc Natl Acad Sci U S A. 2000 Jan 4;97(1):268-73.
⁷ Pontual L et al. J Med Genet. 2006;43:419-423.
⁸ Tam et al. Pediatr Surg Int. 2009;25:543-558.

DISCLAIMER
This test was developed and its performance characteristics were determined by Ambry Genetics Corporation.  The laboratory is regulated under the Clinical Laboratory Improvement Amendments 2003 as qualified to perform non-waived testing.  The Ambry Test: RET-Related Hirschsprung Disease (HSCR) analyzes the following types of mutations: nucleotide substitutions, small deletions, small insertions (including small repeat expansion), and small indels.  It is not intended to analyze the following types of mutations: gross deletions/duplications, gross rearrangements, deep intronic variations, and other unknown abnormalities.  The pattern of mutation types varies with the gene tested and the Ambry Test detects a high but variable percentage of known and unknown mutants of the classes stated.  A negative result from the analysis cannot rule out the possibility that the tested individual carries a rare unexamined mutation or mutation in the undetectable group.  The Ambry Test: RET-Related Hirschsprung Disease (HSCR) is designed and validated to be capable of detecting >99% of described mutations in RET (considering less than 1% to be the other types of mutations).   HSCR is a complex clinical disorder, which in most cases is due to alterations in RET generally detected by the Ambry Test: RET-Related Hirschsprung Disease (HSCR) except as noted above.  Mutations in other genes or the regions not tested by the Ambry Test: RET-Related Hirschsprung Disease (HSCR) can also give rise to clinical conditions similar to Hirschsprung disease (HSCR). Although molecular tests are highly accurate, rare diagnostic errors may occur.  Possible diagnostic errors include sample mix-up, erroneous paternity identification, technical errors, clerical errors, and genotyping errors. Genotyping errors can result from trace contamination of PCR reactions, from maternal cell contamination in fetal samples, from rare genetic variants, which interfere with analysis, or from other sources. This report does not represent medical advice.  Any questions, suggestions, or concerns regarding interpretation of results should be forwarded to a genetic counselor, medical geneticist, or physician skilled in interpretation of the relevant medical literature.