Hirschsprung Disease Panel

Last modified: Jun 12, 2018

Summary

  • Is a 15 gene panel that includes assessment of non-coding variants
  • Is ideal for patients with a clinical suspicion of Hirschsprung disease (HSCR).

Analysis methods

  • PLUS
  • SEQ
  • DEL/DUP

Availability

3-4 weeks

Number of genes

15

Test code

MA1801

CPT codes

SEQ 81404
SEQ 81405
SEQ 81406
DEL/DUP 81479

Summary

The Blueprint Genetics Hirschsprung Disease Panel (test code MA1801):

  • Is a 15 gene panel that includes assessment of selected non-coding disease-causing variants
  • Some patients heterozygous for a de novo polyalanine repeat expansion mutations (PARMs) in the PHOX2B gene have isolated or more commonly syndromic Hirschsprung disease in association with with congenital central hypoventilation syndrome (CCHS, PMID 16888290). Repeat expansions are generally difficult to detect via NGS assays and their clinical validation at large scale is impossible due to lack of publicly available control samples with abnormal repeat expansions. So far, we have been able to detect and confirm stretches of 28 alanines instead of normal amount of 20 (genotype “20/28”). However, we do not know exact sensitivity or detection limit of our assay for these alanine repeats.

  • Is available as PLUS analysis (sequencing analysis and deletion/duplication analysis), sequencing analysis only or deletion/duplication analysis only

Test Specific Strength

Some patients heterozygous for a de novo polyalanine repeat expansion mutations (PARMs) in the PHOX2B gene have isolated or more commonly syndromic Hirschsprung disease in association with with congenital central hypoventilation syndrome (CCHS, PMID 16888290). Repeat expansions are generally difficult to detect via NGS assays and their clinical validation at large scale is impossible due to lack of publicly available control samples with abnormal repeat expansions. So far, we have been able to detect and confirm stretches of 28 alanines instead of normal amount of 20 (genotype “20/28”). However, we do not know exact sensitivity or detection limit of our assay for these alanine repeats.

ICD codes

Commonly used ICD-10 code(s) when ordering the Hirschsprung Disease Panel

ICD-10 Disease
Q43.1 Hirschsprung disease (HSCR)

Sample Requirements

  • EDTA blood, min. 1 ml
  • Purified DNA, min. 3μg
  • Saliva (Oragene DNA OG-500 kit)

Label the sample tube with your patient’s name, date of birth and the date of sample collection.

Note that we do not accept DNA samples isolated from formalin-fixed paraffin-embedded (FFPE) tissue.

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.

Genes in the Hirschsprung Disease Panel and their clinical significance

Gene Associated phenotypes Inheritance ClinVar HGMD
BDNF Central hypoventilation syndrome, congenital AD 1 22
CELSR3 Hirschsprung disease AD 16
EDN3 Hirschsprung disease, Central hypoventilation syndrome, congenital, Waardenburg syndrome AD/AR 6 21
EDNRB Hirschsprung disease, ABCD syndrome, Waardenburg syndrome AD/AR 8 66
KIF1BP Goldberg-Shprintzen megacolon syndrome AR 7 10
L1CAM Mental retardation, aphasia, shuffling gait, and adducted thumbs (MASA) syndrome, Hydrocephalus due to congenital stenosis of aqueduct of Sylvius, Spastic, CRASH syndrome, Corpus callosum, partial agenesis XL 71 287
MITF Tietz albinism-deafness syndrome, Waardenburg syndrome AD/AR 28 55
NRG1 Nonsyndromic Hirschsprung disease AD/AR 1 10
NRTN Hirschsprung disease AD 2
PAX3 Craniofacial-deafness-hand syndrome, Waardenburg syndrome AD/AR 33 138
PHOX2B Central hypoventilation syndrome, congenital, Neuroblastoma, susceptiblity to, Neuroblastoma with Hirschsprung disease AD 10 80
RET Hirschsprung disease, Central hypoventilation syndrome, congenital, Pheochromocytoma, Medullary thyroid carcinoma, Multiple endocrine neoplasia AD/AR 84 402
RMRP Cartilage-hair hypoplasia, Metaphyseal dysplasia without hypotrichosis, Anauxetic dysplasia AR 34 123
SOX10 Peripheral demyelinating neuropathy, central dysmyelination, Waardenburg syndrome, and Hirschsprung disease AD 41 136
ZEB2* Mowat-Wilson syndrome AD 135 280

* Some, or all, of the gene is duplicated in the genome. Read more.

# The gene has suboptimal coverage (means <90% of the gene’s target nucleotides are covered at >20x with mapping quality score (MQ>20) reads).

The sensitivity to detect variants may be limited in genes marked with an asterisk (*) or number sign (#)

Gene refers to the HGNC approved gene symbol; Inheritance refers to inheritance patterns such as autosomal dominant (AD), autosomal recessive (AR), X-linked (XL), X-linked dominant (XLD) and X-linked recessive (XLR); ClinVar refers to the number of variants in the gene classified as pathogenic or likely pathogenic in this database (ClinVar); HGMD refers to the number of variants with possible disease association in the gene listed in Human Gene Mutation Database (HGMD). The list of associated, gene specific phenotypes are generated from CGD or Orphanet databases.

Non-coding variants covered by the panel

Gene Genomic location HG19 HGVS RefSeq RS-number
EDN3 Chr20:57875743 c.-125G>A NM_000114.2
EDN3 Chr20:57875849 c.-19C>A NM_000114.2 rs375594972
L1CAM ChrX:153133652 c.1704-75G>T NM_000425.4
L1CAM ChrX:153128846 c.3531-12G>A NM_000425.4
L1CAM ChrX:153136500 c.523+12C>T NM_000425.4
PAX3 Chr2:223085913 c.958+28A>T NM_181459.3
RET Chr10:43613947 c.2392+19T>C NM_020975.4 rs778745375
RMRP Chr9:35657745 NR_003051.3 rs377349293
RMRP Chr9:35657746 NR_003051.3 rs551655682
SOX10 Chr22:38412215 c.-31954C>T NM_006941.3 rs606231342
SOX10 Chr22:38379877 c.-84-2A>T NM_006941.3
ZEB2 Chr2:145274987 c.-69-1G>A NM_014795.3

Added and removed genes from the panel

Genes added Genes removed
BDNF
CELSR3
L1CAM
NRTN
PHOX2B
RMRP

Test strength

Some patients heterozygous for a de novo polyalanine repeat expansion mutations (PARMs) in the PHOX2B gene have isolated or more commonly syndromic Hirschsprung disease in association with with congenital central hypoventilation syndrome (CCHS, PMID 16888290). Repeat expansions are generally difficult to detect via NGS assays and their clinical validation at large scale is impossible due to lack of publicly available control samples with abnormal repeat expansions. So far, we have been able to detect and confirm stretches of 28 alanines instead of normal amount of 20 (genotype “20/28”). However, we do not know exact sensitivity or detection limit of our assay for these alanine repeats.

The strengths of this test include:
  • CAP and ISO-15189 accreditations covering all operations at Blueprint Genetics including all Whole Exome Sequencing, NGS panels and confirmatory testing
  • CLIA-certified personnel performing clinical testing in a CLIA-certified laboratory
  • Powerful sequencing technologies, advanced target enrichment methods and precision bioinformatics pipelines ensure superior analytical performance
  • Careful construction of clinically effective and scientifically justified gene panels
  • Our Nucleus online portal providing transparent and easy access to quality and performance data at the patient level
  • Our publically available analytic validation demonstrating complete details of test performance
  • ~1,500 non-coding disease causing variants in Blueprint WES assay (please see below ‘Non-coding disease causing variants covered by this panel’)
  • Our rigorous variant classification based on modified ACMG variant classification scheme
  • Our systematic clinical interpretation workflow using proprietary software enabling accurate and traceable processing of NGS data
  • Our comprehensive clinical statements

Test limitations

Genes with partial, or whole gene, segmental duplications in the human genome are marked with an asterisk (*) if they overlap with the UCSC pseudogene regions. The technology may have limited sensitivity to detect variants in genes marked with these symbols (please see the Panel content table above).

This test does not detect the following:
  • Complex inversions
  • Gene conversions
  • Balanced translocations
  • Mitochondrial DNA variants
  • Repeat expansion disorders unless specifically mentioned
  • Non-coding variants deeper than ±20 base pairs from exon-intron boundary unless otherwise indicated (please see above Panel Content / non-coding variants covered by the panel).

This test may not reliably detect the following:

  • Low level mosaicism
  • Stretches of mononucleotide repeats
  • Indels larger than 50bp
  • Single exon deletions or duplications
  • Variants within pseudogene regions/duplicated segments

The sensitivity of this test may be reduced if DNA is extracted by a laboratory other than Blueprint Genetics.

For additional information, please refer to the Test performance section and see our Analytic Validation.

The Blueprint Genetics Hirschsprung disease panel covers classical genes associated with Hirschsprung disease (HSCR). The genes on the panel have been carefully selected based on scientific literature, mutation databases and our experience.

Our panels are sliced from our high-quality whole exome sequencing data. Please see our sequencing and detection performance table for different types of alterations at the whole exome level (Table).

Assays have been validated for different starting materials including EDTA-blood, isolated DNA (no FFPE), saliva and dry blood spots (filter card) and all provide high-quality results. The diagnostic yield varies substantially depending on the assay used, referring healthcare professional, hospital and country. Blueprint Genetics' Plus Analysis (Seq+Del/Dup) maximizes the chance to find a molecular genetic diagnosis for your patient although Sequence Analysis or Del/Dup Analysis may be a cost-effective first line test if your patient's phenotype is suggestive of a specific mutation type.

Performance of Blueprint Genetics Whole Exome Sequencing (WES) assay. All individual panels are sliced from WES data.

Sensitivity % (TP/(TP+FN) Specificity %
Single nucleotide variants 99.65% (412,456/413,893) >99.99%
Insertions, deletions and indels by sequence analysis
1-10 bps 96.94% (17,070/17,608) >99.99%
11-50 bps 99.07% (957/966) >99.99%
Copy number variants (exon level dels/dups)
Clinical samples (small CNVs, n=52)
1 exon level deletion 92.3% (24/26) NA
2 exons level deletion/duplication 100.0% (11/11) NA
3-7 exons level deletion/duplication 93.3% (14/15) NA
Microdeletion/-duplication sdrs (large CNVs, n=37))
Size range (0.1-47 Mb) 100% (37/37)
Simulated CNV detection
2 exons level deletion/duplication 90.98% (7,357/8,086) 99.96%
5 exons level deletion/duplication 98.63% (7,975/8,086) 99.98%
     
The performance presented above reached by WES with the following coverage metrics
     
Mean sequencing depth at exome level 174x
Nucleotides with >20x sequencing coverage (%) 99.4%

Bioinformatics

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 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. 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 such as, 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, the customer has an access to details of the analysis, including patient specific sequencing metrics, a gene level coverage plot and a list of regions with inadequate coverage if present. This reflects our mission to build fully transparent diagnostics where customers have easy access to crucial details of the analysis process.

Clinical interpretation

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 corner stone of clinical interpretation and resulting patient management decisions. Our classifications follow the Blueprint Genetics Variant Classification Schemes based on the ACMG guideline 2015. Minor modifications were made to increase reproducibility of the variant classification and improve the clinical validity of the report. Our experience with tens of thousands of clinical cases analyzed at our laboratory allowed us to further develop the industry standard.

The final step in the analysis of sequence variants is confirmation of variants classified as pathogenic or likely pathogenic using bi-directional Sanger sequencing. Variant(s) fulfilling all of the following criteria are not Sanger confirmed: 1) the variant quality score is above the internal threshold for a true positive call, 2) an unambiguous IGV in-line with the variant call and 3) previous Sanger confirmation of the same variant at least three times at Blueprint Genetics. Reported variants of uncertain significance are confirmed with bi-directional Sanger sequencing only if the quality score is below our internally defined quality score for true positive call. Reported copy number variations with a size <10 exons are confirmed by orthogonal methods such as qPCR if the specific CNV has been seen less than three times at Blueprint Genetics.

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 used, congress abstracts and mutation databases to help our customers 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 within the family. In the case of variants of uncertain significance (VUS), we do not recommend family member risk stratification based on the VUS result. Furthermore, in the case of VUS, we do not recommend the use of genetic information in patient management or genetic counseling. For eligible cases, Blueprint Genetics offers a no charge service to investigate the role of reported VUS (VUS Clarification Service).

Our interpretation team analyzes millions of variants from thousands of individuals with rare diseases. Thus, our database, and our understanding of variants and related phenotypes, is growing by leaps and bounds. 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 health care provider at no additional cost.