Heterotaxy and Situs Inversus Panel

32 gene panel that includes assessment of non-coding variants

Ideal for patients with a diagnosis of laterality disorder.

Analysis methods Availability Number of genes Test code CPT codes
PLUS
SEQ
DEL/DUP
4 weeks 32 GHC0127 DEL/DUP 81479
SEQ 81479

Summary

ICD codes
Commonly used ICD-10 code(s) when ordering the Heterotaxy and Situs Inversus Panel

ICD-10 Disease
Q61.5 Nephronophthisis

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.

About

Heterotaxy is a disorder in which the internal organs are abnormally arranged in the chest and abdomen and there are often associated complex cardiovascular malformations. Right isomerism sequence (asplenia syndrome, Ivemark sequence, right atrial isomerism) causes a complex congenital heart disease, two morphologically right atria and often a single ventricle and AVSD, TGA and anomalous pulmonary venous drainage. The spleen can be absent and there may be abnormal folding of the intestines. In left isomerism sequence (polysplenia syndrome) there are two morphologically left atria and absence of the sinoatrial node that may cause complete heart block. The associated heart defects are not usually as severe as in right isomerism. Multiple small spleens may be found and there may be abnormal folding of the intestines. Isomerism sequence occurs with an incidence of 1/24,000 and accounts for about 1 % of congenital heart defects. Situs inversus is a condition with a complete mirror image arrangement of the internal organs. It affects approximately 1/10,000 individuals and is associated with an increased risk of congenital heart disease. Situs inversus can also occur in association with primary ciliary dyskinesia. A proportion of the laterality disorders are explained by mutations in known genes and can be inherited in an autosomal dominant, autosomal recessive or X-linked manner.

Panel Content

Genes in the Heterotaxy and Situs Inversus Panel and their clinical significance

Gene Associated phenotypes Inheritance ClinVar HGMD
ACVR2BHeterotaxy, visceral, 4, autosomalAD12
ANKS6NephronophthisisAR612
ARMC4Ciliary dyskinesiaAR1416
C21ORF59Ciliary dyskinesiaAR34
CCDC39Ciliary dyskinesiaAR2538
CCDC40Ciliary dyskinesiaAR2433
CCDC103Ciliary dyskinesiaAR44
CCDC114Ciliary dyskinesiaAR67
CCDC151Ciliary dyskinesia, primary, 30AR52
DNAAF1Ciliary dyskinesiaAR1431
DNAAF2Ciliary dyskinesiaAR114
DNAAF3Primary ciliary dyskinesiaAD/AR83
DNAAF5Ciliary dyskinesiaAR82
DNAH5Ciliary dyskinesiaAR95160
DNAH11Ciliary dyskinesiaAR51101
DNAI1Ciliary dyskinesiaAR1429
DNAI2Ciliary dyskinesiaAR146
DNAL1Ciliary dyskinesiaAR31
DYX1C1Ciliary dyskinesiaAR1111
FOXH1Congenital heart malformations, HoloprosencephalyAD32
GDF1Transposition of the great arteries, dextro-looped 3, Double-outlet right ventricleAD1115
INVSNephronophthisisAR1233
LEFTY2Left-right axis malformationsAD33
LRRC6Ciliary dyskinesiaAR717
MMP21Heterotaxy, visceral, 7AR418
NODALHeterotaxy, visceralAD413
PIH1D3Ciliary dyskinesia, primary, 36XL211
PKD1L1Heterotaxy, visceral, 8, autosomalAR25
SPAG1Primary ciliary dyskinesiaAD/AR1610
TTC25Ciliary dyskinesia, primary, 35AR22
ZIC3Heterotaxy, visceral, VACTERL association, Congenital heart defects, nonsyndromicXL1441
ZMYND10Ciliary dyskinesiaAR716

Non-coding variants covered by the panel

Gene Genomic location HG19 HGVS RefSeq RS-number
CCDC39Chr3:180367941c.1167+1248A>GNM_181426.1

Panel Update

Genes added

Genes removed

Test strength and Limitations

The strengths of this test include:

  • CAP and ISO-15189 accreditations covering all operations at GHC 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 GHC 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 The following exons are not included in the panel as they are not sufficiently covered with high quality sequence reads: *PPA2* (11, 12). 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 GHC Genetics.

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

Test Performance

The GHC Genetics panel covers classical genes associated with Brugada syndrome, catecholaminergic polymorphic ventricular tachycardia (CPVT), cardiac arrest underlying cardiac condition, cardiac arrest cause unspecified, syncope and collapse, abnormal ECG, Long QT syndrome, arrhythmogenic right ventricular cardiomyopathy (ARVC) and Short QT syndrome. 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. GHC 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 GHC 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%

Our mission is to improve the quality of the sequencing process and each modification is followed by our standardized validation process. Detection of Del/Dup of several genes is by MLPA analysis (MS Holland). All genes are performed by CNV analysis through the genome depending on exon size, sequencing coverage and sequence content. We have validated the assays for different starting materials including isolated DNA from EDTA blood that provide high-quality results.

Bioinformatics & clinical interpretation

The sequencing data generated in our laboratory is analysed by our bioinformatic pipeline, integrating state-of-the art algorithms and industry-standard software solutions. We use also JSI medical systems software for sequencing data analysis. JSI medical systems is a certified system offering sophisticated bioinformatic software solutions covering a wide field of sequencing techniques.

Incorporation of rigorous quality control steps throughout the workflow of the pipeline ensures the consistency, validity and accuracy of results.

Every pathogenic or probably pathogenic variant is confirmed by the Sanger sequencing method. Sanger sequencing is also used occasionally with other variants reported in the statement. In the case of variant of uncertain significance (VUS) we do not recommend risk stratification based on the genetic finding. The analysis of detected variants was performed on the basis of the reference database of polymorphisms and international mutation databases: UMD, LOVD and ClinVar.

The consequence of variants in coding and splice regions are estimated using Alamut software. The Alamut database contains more than 28000 coding genes, non-protein coding genes and pseudogenes. This database (shared with the high throughput annotation engine for NGS data, Alamut Batch) is frequently updated. Information comes from different public databases such as NCBI, EBI, and UCSC, as well as other sources including gnomAD, ESP, Cosmic, ClinVar, or HGMD and CentoMD (for those a separate subscription from Qiagen/Biobase and Centogene respectively is required). Alamut Visual finds information about nucleotide conservation data through many vertebrates’ species, with the phastCons and phyloP scores, amino acid conservation data through orthologue alignments and information on protein domains.

Moreover, we integrate several missense variant pathogenicity prediction tools and algorithms such as SIFT, PolyPhen, AlignGVGD or MutationTaster. It also offers a window dedicated to the in silico study of variants’ effect on RNA splicing, allowing the assessment of their potential impact on splice junctions and visualization of cryptic or de novo splice sites. Impact on splicing regulation is also assessed.


Clinical interpretation

At GHC Genetics our geneticists and clinicians, who together evaluate the results from the sequence analysis pipeline in the context of phenotype information provided in the requisition form, prepare the clinical report. We recommend an interpretation of the findings of this molecular genetic analysis, including subsequent oncological consultation for the patient in the context of genetic counselling for the patient.

We strive to continuously monitor current genetic literature identifying new relevant information and findings and adapting them to our diagnostics. This enables relevant novel discoveries to be rapidly translated and adopted into our ongoing diagnostics development without delay. The undertaking of such comprehensive due diligence ensures that our diagnostic panels and clinical statements are the most up-to-date on the market.

Variant classification is the corner stone of clinical interpretation and resulting patient management decisions. 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 analysed at our laboratories enables 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 three times at GHC Genetics. Reported variants of uncertain significance (VUS) 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 GHC 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, 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 counselling.

Our Clinical interpretation team analyses 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 laboratories are therefore well positioned to re-classify previously reported variants as new information becomes available. If a variant previously reported by GHC Genetics is re-classified, our laboratories will issue a follow-up statement to the original ordering health care provider at no additional cost.