Sitosterolemia via the ABCG8 Gene
- Summary and Pricing
- Clinical Features and Genetics
|Test Code||Test Copy Genes||Individual Gene Price||CPT Code Copy CPT Codes|
Our most cost-effective testing approach is NextGen sequencing with Sanger sequencing supplemented as needed to ensure sufficient coverage and to confirm NextGen calls that are pathogenic, likely pathogenic or of uncertain significance. If, however, full gene Sanger sequencing only is desired (for purposes of insurance billing or STAT turnaround time for example), please see link below for Test Code, pricing, and turnaround time information.
For ordering targeted known variants, please proceed to our Targeted Variants landing page.
The great majority of tests are completed within 28 days.
To date, only about 100 cases of sitosterolemia have been reported (Escolá-Gil et al. 2014). In a series of 25 unrelated families with a diagnosis of sitosterolemia based on elevated plasma sitosterol levels, pathogenic variants in the ABCG5 and ABCG8 genes were found in 9 of 25 and 16 of 25 families respectively (Lu et al. 2001). Analytical sensitivity for detection of pathogenic variants in the ABCG5 and ABCG8 genes is >95%. Two cases of a deletion of exon 3 in the ABCG5 gene have been reported and are not predicted to be detected by this sequencing method (Lu et al. 2001).
Sitosterolemia is a disorder hallmarked by a 30-100 fold increase in plasma plant sterols. Heightened levels are due to defects in the adenosine triphosphate-binding cassette transporter protein which effluxes free sterols from hepatocytes and enterocytes into the gut lumen. The primary clinical features of sitosterolemia patients are tendon or tuberous xanthomas and accelerated atherosclerosis which have been report in individuals as young as 4 years of age (Mymin et al. 2003). Hemolytic anemia, stomatocyte formation, and macrothrombocytopenia are common hematologic findings and may be the only clinically observed symptoms in patients (Wang et al. 2014; Escolá-Gil et al. 2014). Clinical colormetric enzyme assays to quantify sterols cannot discriminate between cholesterol and plant sterols making diagnosis difficult (Kidambi and Patel 2008). Genetic testing is helpful in the differential diagnosis of sitosterolemia from cerebrotendinous xanthomatosis, cardiovascular disease, and familial hypercholesterolemia. Ezetimibe, an inhibitor of intestinal cholesterol absorption, has been shown to be effective in reducing plasma sterol levels (Othman et al. 2015).
Sitosterolemia is inherited in an autosomal recessive manner with mutations in the ABCG5 and ABCG8 genes being responsible for 1/3 and 2/3 of cases respectively (Lee et al 2001; Escolá-Gil et al. 2014; Hubacek et al. 2001). The ABCG5 and ABCG8 genes encode the heterodimer adenosine triphosphate-binding cassette transporter protein present on enterocytes and hepatocytes. In enterocytes, the transporter is located on the apical membrane and promotes efflux of plant sterols back into the intestinal lumen. In the liver, the transporter is responsible for excretion of plant sterols into the bile (Lee et al. 2001). Pathogenic variants in the ABCG5 and ABCG8 genes impair transporter function or surface expression and result in elevated plasma plant sterol levels.
The c.1083G>A (previously reported as c.1173G>A) variant resulting in a nonsense change (p.Trp361*) accounts for half of all pathogenic alleles in the ABCG8 gene (Lu et al. 2001). Loss of function variants represent over two-thirds of the documented mutations and include nonsense, frameshift, and splice site alterations found throughout the gene. Missense variants represent about a third of cases, and no gross deletions have been reported (Lu et al. 2001; Escolá-Gil et al. 2014). Pathogenic variants in the in the ABCG8 gene are more commonly found in Caucasian populations (Kidambi and Patel 2008).
For this Next Generation Sequencing (NGS) test, sequencing is accomplished by capturing specific regions with an optimized solution-based hybridization kit, followed by massively parallel sequencing of the captured DNA fragments. Additional Sanger sequencing is performed for regions not captured or with insufficient number of sequence reads. All reported pathogenic, likely pathogenic, and variants of uncertain significance are confirmed by Sanger sequencing.
For Sanger sequencing, polymerase chain reaction (PCR) is used to amplify targeted regions. After purification of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit. PCR products are resolved by electrophoresis on an ABI 3730xl capillary sequencer. In nearly all cases, cycle sequencing is performed separately in both the forward and reverse directions.
This test provides full coverage of all coding exons of the ABCG8 gene, plus ~10 bases of flanking noncoding DNA. We define full coverage as >20X NGS reads or Sanger sequencing.
Indications for Test
Candidates for testing include patients presenting with xanthomas, premature atherosclerosis, hemolytic anemia, and macrothrombocytopenia. Presence of stomatocytes in bloods smears and elevated plant sterol levels (>20-30mg/dl) are common laboratory findings. Gas chromatography or high performance liquid chromatography are necessary to specifically measure plant sterol levels as colormetric assays to measure sterols do not differentiate between cholesterol and plant sterols (Kidambi and Patel 2008; Lu et al. 2001).
|Official Gene Symbol||OMIM ID|
- Genetic Counselor Team - firstname.lastname@example.org
- Luke Drury, PhD - email@example.com
- EscolÃ -Gil J.C. et al. 2014. Current Atherosclerosis Reports. 16: 424. PubMed ID: 24821603
- Hubacek J.A. et al. 2001. Human Mutation. 18: 359-60. PubMed ID: 11668628
- Kidambi S., Patel S.B. 2008. Journal of Clinical Pathology. 61: 588-94. PubMed ID: 18441155
- Lee M.H. et al. 2001. Nature Genetics. 27: 79-83. PubMed ID: 11138003
- Lu K. et al. 2001. American Journal of Human Genetics. 69: 278-90. PubMed ID: 11452359
- Mymin D. et al. 2003. Circulation. 107: 791. PubMed ID: 12578886
- Othman R.A. et al. 2015. The Journal of Pediatrics. 166: 125-31. PubMed ID: 25444527
- Wang Z. et al. 2014. American Journal of Hematology. 89: 320-4.Â PubMed ID: 24166850
NextGen Sequencing using PG-Select Capture Probes
We use a combination of Next Generation Sequencing (NGS) and Sanger sequencing technologies to cover the full coding regions of the listed genes plus ~20 bases of non-coding DNA flanking each exon. As required, genomic DNA is extracted from the patient specimen. For NGS, patient DNA corresponding to these regions is captured using an optimized set of DNA hybridization probes. Captured DNA is sequenced using Illumina’s Reversible Dye Terminator (RDT) platform (Illumina, San Diego, CA, USA). Regions with insufficient coverage by NGS are covered by Sanger sequencing. All pathogenic, likely pathogenic, or variants of uncertain significance are confirmed by Sanger sequencing.
For Sanger sequencing, Polymerase Chain Reaction (PCR) is used to amplify targeted regions. After purification of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit. PCR products are resolved by electrophoresis on an ABI 3730xl capillary sequencer. In nearly all cases, cycle sequencing is performed separately in both the forward and reverse directions.
Patient DNA sequence is aligned to the genomic reference sequence for the indicated gene region(s). All differences from the reference sequences (sequence variants) are assigned to one of five interpretation categories, listed below, per ACMG Guidelines (Richards et al. 2015).
(1) Pathogenic Variants
(2) Likely Pathogenic Variants
(3) Variants of Uncertain Significance
(4) Likely Benign Variants
(5) Benign, Common Variants
Human Genome Variation Society (HGVS) recommendations are used to describe sequence variants (http://www.hgvs.org). Rare variants and undocumented variants are nearly always classified as likely benign if there is no indication that they alter protein sequence or disrupt splicing.
As of March 2016, 6.36 Mb of sequence (83 genes, 1557 exons) generated in our lab was compared between Sanger and NextGen methodologies. We detected no differences between the two methods. The comparison involved 6400 total sequence variants (differences from the reference sequences). Of these, 6144 were nucleotide substitutions and 256 were insertions or deletions. About 65% of the variants were heterozygous and 35% homozygous. The insertions and deletions ranged in length from 1 to over 100 nucleotides.
In silico validation of insertions and deletions in 20 replicates of 5 genes was also performed. The validation included insertions and deletions of lengths between 1 and 100 nucleotides. Insertions tested in silico: 2200 between 1 and 5 nucleotides, 625 between 6 and 10 nucleotides, 29 between 11 and 20 nucleotides, 25 between 21 and 49 nucleotides, and 23 at or greater than 50 nucleotides, with the largest at 98 nucleotides. All insertions were detected. Deletions tested in silico: 1813 between 1 and 5 nucleotides, 97 between 6 and 10 nucleotides, 32 between 11 and 20 nucleotides, 20 between 21 and 49 nucleotides, and 39 at or greater than 50 nucleotides, with the largest at 96 nucleotides. All deletions less than 50 nucleotides in length were detected, 13 greater than 50 nucleotides in length were missed. Our standard NextGen sequence variant calling algorithms are generally not capable of detecting insertions (duplications) or heterozygous deletions greater than 100 nucleotides. Large homozygous deletions appear to be detectable.
Interpretation of the test results is limited by the information that is currently available. Better interpretation should be possible in the future as more data and knowledge about human genetics and this specific disorder are accumulated.
When Sanger sequencing does not reveal any difference from the reference sequence, or when a sequence variant is homozygous, we cannot be certain that we were able to detect both patient alleles. Occasionally, a patient may carry an allele which does not amplify, due to a large deletion or insertion. In these cases, the report will contain no information about the second allele. Our Sanger and NGS Sequencing tests are generally not capable of detecting Copy Number Variants (CNVs).
We sequence all coding exons for each given transcript, plus ~20 bp of flanking non-coding DNA for each exon. Test reports contain no information about other portions of the gene, such as regulatory domains, deep intronic regions or any currently uncharacterized alternative exons.
In most cases, we are unable to determine the phase of sequence variants. In particular, when we find two likely causative mutations for recessive disorders, we cannot be certain that the mutations are on different alleles.
Our ability to detect minor sequence variants due to somatic mosaicism is limited. Sequence variants that are present in less than 50% of the patient’s nucleated cells may not be detected.
Runs of mononucleotide repeats (eg (A)n or (T)n) with n >8 in the reference sequence are generally not analyzed because of strand slippage during PCR.
Unless otherwise indicated, DNA sequence data is obtained from a specific cell-type (usually leukocytes from whole blood). Test reports contain no information about the DNA sequence in other cell-types.
We cannot be certain that the reference sequences are correct.
Rare, low probability interpretations of sequencing results, such as for example the occurrence of de novo mutations in recessive disorders, are generally not included in the reports.
We have confidence in our ability to track a specimen once it has been received by PreventionGenetics. However, we take no responsibility for any specimen labeling errors that occur before the sample arrives at PreventionGenetics.
myPrevent - Online Ordering
- The test can be added to your online orders in the Summary and Pricing section.
- Once the test has been added log in to myPrevent to fill out an online requisition form.
- A completed requisition form must accompany all specimens.
- Billing information along with specimen and shipping instructions are within the requisition form.
- All testing must be ordered by a qualified healthcare provider.
(Delivery accepted Monday - Saturday)
- Collect 3 ml -5 ml (5 ml preferred) of whole blood in EDTA (purple top tube) or ACD (yellow top tube). For Test #500-DNA Banking only, collect 10 ml -20 ml of whole blood.
- For small babies, we require a minimum of 1 ml of blood.
- Only one blood tube is required for multiple tests.
- Ship blood tubes at room temperature in an insulated container. Do not freeze blood.
- During hot weather, include a frozen ice pack in the shipping container. Place a paper towel or other thin material between the ice pack and the blood tube.
- In cold weather, include an unfrozen ice pack in the shipping container as insulation.
- At room temperature, blood specimen is stable for up to 48 hours.
- If refrigerated, blood specimen is stable for up to one week.
- Label the tube with the patient name, date of birth and/or ID number.
(Delivery accepted Monday - Saturday)
- Send in screw cap tube at least 5 µg -10 µg of purified DNA at a concentration of at least 20 µg/ml for NGS and Sanger tests and at least 5 µg of purified DNA at a concentration of at least 100 µg/ml for gene-centric aCGH, MLPA, and CMA tests, minimum 2 µg for limited specimens.
- For requests requiring more than one test, send an additional 5 µg DNA per test ordered when possible.
- DNA may be shipped at room temperature.
- Label the tube with the composition of the solute, DNA concentration as well as the patient’s name, date of birth, and/or ID number.
- We only accept genomic DNA for testing. We do NOT accept products of whole genome amplification reactions or other amplification reactions.
(Delivery preferred Monday - Thursday)
- PreventionGenetics should be notified in advance of arrival of a cell culture.
- Culture and send at least two T25 flasks of confluent cells.
- Some panels may require additional flasks (dependent on size of genes, amount of Sanger sequencing required, etc.). Multiple test requests may also require additional flasks. Please contact us for details.
- Send specimens in insulated, shatterproof container overnight.
- Cell cultures may be shipped at room temperature or refrigerated.
- Label the flasks with the patient name, date of birth, and/or ID number.
- We strongly recommend maintaining a local back-up culture. We do not culture cells.