Hirschsprung Disease (Non-syndromic) Panel

Hirschsprung disease (HSCR), aka congenital intestinal aganglionosis, is a birth defect characterized by complete absence of neuronal ganglion cells from a portion of the intestinal tract (Eng and Mulligan 1997). In 80% of individuals aganglionosis is restricted to the rectosigmoid colon (S-HSCR); in 15%-20% aganglionosis extends beyond the sigmoid colon (L-HSCR); and in about 5% aganglionosis is present in the entire large intestine (total colonic aganglionosis) (Amiel et al. 2008). HSCR is the main genetic cause of functional intestinal obstruction in infants and children.

Affected infants frequently present in the first two months of life with symptoms of impaired intestinal motility such as failure to pass meconium within the first 48 hours of life, constipation, emesis, abdominal pain or distention, and occasionally diarrhea. However, because the initial diagnosis of HSCR may be delayed until late childhood or adulthood, HSCR should be considered in anyone with lifelong severe constipation. HSCR can also be associated with cardiac defects and autonomic dysfunction. Individuals with HSCR are at risk for enterocolitis and/or potentially lethal intestinal perforation (Parisi 2011).

HSCR (non-syndromic) is caused by mutations in at least six genes: RET, EDN3, EDNRB, GDNF, NRTN and ECE1 (Parisi and Kapur 2000). Mutations in RET alone account for 7-41% of all HSCR cases (Sancandi et al. 2000), while EDNRB (Kusafuka et al. 1996) and EDN3 (Svensson et al. 1999) mutations account for approximately 10% of individuals with HSCR. Mutations in GDNF and NRTN have been identified in only a small minority of individuals with HSCR. Mutations in ECE1 are extremely rare and have been reported in only one individual, who also had craniofacial anomalies and a heart defect (Hofstra et al 1999).

RET: Causative for HSCR1 and occurs due to heterozygous missense and truncating mutations, suggesting an autosomal dominant mode of inheritance. RET protein is essential for the normal development of several kinds of nerve cells, including nerves in the intestine (enteric neurons). Loss of function of RET leads to improper development of enteric nerves, resulting in severe constipation or blockage of the intestine. L-HSCR and S-HSCR are regarded as the variable clinical expression of mutations at the RET locus. Mutations in RET are also involved in syndromic HSCR - Congenital Central Hypoventilation Syndrome (CCHS) as well as Multiple Endocrine Neoplasia II and Pheochromocytoma.

EDNRB: Causative for HSCR2, mostly due to missense and truncating mutations. Can be inherited as autosomal dominant or recessive. The protein encoded, endothelin receptor type B, is a cell surface protein which functions by interacting with endothelin 3. Together they play a very important role in neural crest cells. Disruption or loss of the EDNRB protein results in the loss of enteric nerve cells that are critical to intestinal development, resulting in Hirschsprung disease. Mutations in EDNRB are also associated with syndromic HSCR - Waardenburg-Shah syndrome.

GDNF: Causative for HSCR3 due to heterozygous missense mutations, suggesting an autosomal dominant mode of inheritance. GDNF is a highly conserved neurotropic factor that promotes the survival and differentiation of dopaminergic neurons. GDNF protein functions as a ligand for the RET proto-oncogene, and therefore loss of function mutations in the gene (missense or nonsense) impacts ligand binding resulting in disease. Mutations in GDNF are also associated with syndromic HSCR - Congenital Central Hypoventilation Syndrome (CCHS).

EDN3: Causative for HSCR4, due to missense and truncating mutations. Can be inherited as autosomal dominant or recessive. EDN3 protein (endothelin 3) functions by interacting with the endothelin receptor type B. Together they play an important role in neural crest cells and pigment-producing cells called melanocytes. Disruption or loss of endothelin 3 results in the loss of enteric nerve cells that are critical to intestinal development, resulting in Hirschsprung disease. Mutations in EDN3 are also associated with syndromic HSCR - Waardenburg-Shah syndrome.

ECE1: Causative for Hirschsprung Disease, Cardiac Defects and Autonomic Dysfunction. Heterozygous mutations in ECE1 though extremely rare, suggest an autosomal dominant mode of inheritance (Hofstra et al. 1999). ECE1 encoded protein, endothelin convertin enzyme-1, is involved in proteolytic processing of endothelin precursors to biologically active peptides. Disruption or loss of the ECE1 protein results in disease. Mutations in ECE1 are also associated with Essential Hypertension.

NRTN: Causative for Hirschsprung Disease. It encodes a neurotrophic factor, neurturin, belonging to the TGF-beta subfamily. Neurturin signals through the RET and GPI-linked coreceptors and promotes the survival and differentiation of neurons. A NRTN mutation that was previously observed in an individual with HSCR was not sufficient to cause disease, but when and only when a RET mutation was present did family members have HSCR (Doray et al. 1998). However, recently three other variants (two found on the same allele in the same patient) were found in two individuals with Hirschsprung disease (Ruiz-Ferrer et al. 2011). These individuals also did not have any pathogenic variants in the RET gene or other HSCR associated genes, nor were the NRTN variants found in controls. In addition, one of these variants was shown in vitro to reduce RET phosphorylation levels, which could lead to downstream defects in the proliferation, migration, and/or differentiation of neural crest cells leading to HSCR.

Mutations in RET alone account for 7-41% of all HSCR cases (Sancandi et al. 2000), while EDNRB (Kusafuka et al. 1996) and EDN3 (Svensson et al. 1999) mutations account for approximately 10% of individuals with HSCR. Mutations in GDNF and NRTN have been identified in only a small minority of individuals with HSCR. Mutations in ECE1 are extremely rare and have been reported in only one individual, who also had craniofacial anomalies and a heart defect (Hofstra et al 1999).

The clinical sensitivity of gross deletions in the RET gene in Hirschsprung disease is currently unknown. However, the whole RET gene has previously been observed to deleted and associated with HSCR (Yin et al. 1996).

This test is performed using Next-Gen sequencing with additional Sanger sequencing as necessary.

This panel provides 100% coverage of all coding exons of the genes plus 10 bases of flanking noncoding DNA in all available transcripts along with other non-coding regions in which pathogenic variants have been identified at PreventionGenetics or reported elsewhere. We define coverage as ≥20X NGS reads or Sanger sequencing. PGnome panels typically provide slightly increased coverage over the PGxome equivalent. PGnome sequencing panels have the added benefit of additional analysis and reporting of deep intronic regions (where applicable).

Dependent on the sequencing backbone selected for this testing, discounted reflex testing to any other similar backbone-based test is available (i.e., PGxome panel to whole PGxome; PGnome panel to whole PGnome).