Hirschsprung Disease Panel

Hirschsprung disease (HSCR), or congenital intestinal aganglionosis, is a birth defect characterized by complete absence of neuronal ganglion cells from a portion of the intestinal tract. Nerve cells are critical to the functioning of the colon as they control the regular muscle contractions that keep food moving through the bowels. In HSCR the aganglionic segment includes the distal rectum and a variable length of contiguous proximal intestine. In 80% of individuals, aganglionosis is restricted to the rectosigmoid colon (short-segment disease), in 15%-20%, it extends proximal to the sigmoid colon (long-segment disease) and in about 5%, aganglionosis affects the entire large intestine (total colonic aganglionosis). Affected infants typically have 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 in the first two months of life. However, in the milder forms the initial diagnosis of HSCR may be delayed until late childhood or adulthood, and therefore HSCR should be considered in anyone with lifelong severe constipation. Individuals with HSCR are at risk for enterocolitis and/or potentially lethal intestinal perforation. HSCR is considered a neurocristopathy, a disorder of cells and tissues derived from the neural crest, and may occur as an isolated finding or as part of a multisystem disorder. Both syndromic and nonsyndromic causes of HSCR are recognized. Roughly a third of children who have Hirschsprung’s disease have other organ system involvement. Examples of monogenic syndromic forms of HSCR (covered by this panel) are Waardenburg syndrome type 4 (autosomal recessive disease due to EDNRB, EDN3 mutations in which HSCR is common and autosomal dominant form with SOX10 mutations in which HSCR is present in almost 100% of cases), Mowat-Wilson syndrome (mutations in ZEB2, HSCR present in 41-71% of cases) and multiple endocrine neoplasia type 2 (MEN 2A and 2B) (mutations in RET). Approximately 50% of familial cases of HSCR are heterozygous for mutations in RET, however the penetrance of these mutations is only 50 to 70%, is gender-dependent, and varies according to the extent of aganglionosis. The incidence of HSCR is approximately 1/ 5,000 live births, but it varies among different populations.

The target region for each gene includes coding exons and ±20 base pairs from the exon-intron boundary. In addition, the panel includes non-coding and regulatory variants if listed above (Non-coding variants covered by the panel). Some regions of the gene(s) may be removed from the panel if specifically mentioned in the ‘Test limitations” section above. If the test includes the mitochondrial genome the target region gene list contains the mitochondrial genes. The sequencing data generated in our laboratory is analyzed with our proprietary data analysis and annotation pipeline, integrating state-of-the art algorithms and industry-standard software solutions. Incorporation of rigorous quality control steps throughout the workflow of the pipeline ensures the consistency, validity and accuracy of results. Our pipeline is streamlined to maximize sensitivity without sacrificing specificity. We have incorporated a number of reference population databases and mutation databases including, but not limited, to 1000 Genomes Project, gnomAD, ClinVar and HGMD into our clinical interpretation software to make the process effective and efficient. For missense variants, in silico variant prediction tools such as  SIFT, PolyPhen,MutationTaster are used to assist with variant classification. Through our online ordering and statement reporting system, Nucleus, ordering providers have access to the details of the analysis, including patient specific sequencing metrics, a gene level coverage plot and a list of regions with suboptimal coverage (<20X for nuclear genes and <1000X for mtDNA) if applicable. This reflects our mission to build fully transparent diagnostics where ordering providers can easily visualize the crucial details of the analysis process.

We provide customers with the most comprehensive clinical report available on the market. Clinical interpretation requires a fundamental understanding of clinical genetics and genetic principles. At Blueprint Genetics, our PhD molecular geneticists, medical geneticists, and clinical consultants prepare the clinical statement together by evaluating the identified variants in the context of the phenotypic information provided in the requisition form. Our goal is to provide clinically meaningful statements that are understandable for all medical professionals regardless of whether they have formal training in genetics.

Variant classification is the cornerstone of clinical interpretation and resulting patient management decisions. Our classifications follow the ACMG guideline 2015.

The final step in the analysis is orthogonal confirmation. Sequence and copy number variants classified as pathogenic, likely pathogenic, and variants of uncertain significance (VUS) are confirmed using bi-directional Sanger sequencing or by orthogonal methods such as qPCR/ddPCR when they do not meet our stringent NGS quality metrics for a true positive call.

Our clinical statement includes tables for sequencing and copy number variants that include basic variant information (genomic coordinates, HGVS nomenclature, zygosity, allele frequencies, in silico predictions, OMIM phenotypes, and classification of the variant). In addition, the statement includes detailed descriptions of the variant, gene, and phenotype(s) including the role of the specific gene in human disease, the mutation profile, information about the gene’s variation in population cohorts, and detailed information about related phenotypes. We also provide links to the references, abstracts, and variant databases used to help ordering providers further evaluate the reported findings if desired. The conclusion summarizes all of the existing information and provides our rationale for the classification of the variant.

Identification of pathogenic or likely pathogenic variants in dominant disorders or their combinations in different alleles in recessive disorders are considered molecular confirmation of the clinical diagnosis. In these cases, family member testing can be used for risk stratification. We do not recommend using variants of uncertain significance (VUS) for family member risk stratification or patient management. Genetic counseling is recommended.

Our interpretation team analyzes millions of variants from thousands of individuals with rare diseases. Our internal database and our understanding of variants and related phenotypes increases with every case analyzed. Our laboratory is therefore well-positioned to re-classify previously reported variants as new information becomes available. If a variant previously reported by Blueprint Genetics is re-classified, our laboratory will issue a follow-up statement to the original ordering healthcare provider at no additional cost, according to our latest follow-up reporting policy.