Polyglucosan Body Myopathy Type I, with or without Immunodeficiency, via the RBCK1 Gene
- Summary and Pricing
- Clinical Features and Genetics
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The great majority of tests are completed within 18 days.
Based on cumulative totals of patients reported in the literature, 29 small sequence variants were detected in a total of 16 patients, for an estimated sensitivity of ~91% (29 of 32 alleles). The remaining 3 alleles were multi-exonic deletions that would not be expected to be detected via direct sequencing (Boisson et al. 2012; Nilsson et al. 2013; Wang et al. 2013).
Polyglucosan body myopathy type I is caused by a genetic deficiency of the ubiquitin ligase RBCK1. The majority of patients that have been described to date have presented with progressive muscle weakness that typically begins with leg weakness, dilated cardiomyopathy, and polyglucosan accumulation in the skeletal muscle, heart, and/or liver. Some of these patients have also presented with ptosis, scoliosis, hepatomegaly, elevated liver transaminases, and growth retardation. Onset is typically during childhood or adolescence (Nilsson et al. 2013; Wang et al. 2013).
A small number of more severely affected patients have been reported. These patients have presented with severe immune dysfunction, including B-cell memory deficiency and hyper-IgA syndrome. As a result, they suffered frequent bacteriological infections from infancy and throughout childhood, chronic systemic inflammation, gastrointestinal symptoms (including vomiting, diarrhea, abdominal pain, and blood and mucous in stools), and dermatological abnormalities. RBCK1 deficiency was fatal in all three of the patients described with this more severe presentation (Boisson et al. 2012).
Polyglucosan body myopathy type I is an autosomal recessive disorder caused by pathogenic variants in the RBCK1 gene. Over 10 pathogenic variants have been described in this gene. The majority of reported variants are expected to lead to premature protein termination, and include nonsense variants, small frameshift deletions and insertions, and splice variants. A small number of missense variants have been reported, as well as one multi-exonic deletion (Human Gene Mutation Database). It has been proposed that the severity of the phenotype may be related to the type and location of sequence variant within RBCK1. For example, patients with a missense variant tend to have a mild form of disease with either no cardiomyopathy, or later-onset cardiomyopathy. The small number of patients with immunological symptoms have had loss-of-function variants that reside within the N-terminal region of RBCK1, whereas the patients without immunological symptoms have had at least one variant located in the middle or C-terminal region of RBCK1 (Nilsson et al. 2013).
The RBCK1 gene encodes an E3 ubiquitin ligase. It interacts with a variety of different proteins, including EYA1 (involved in myogenesis) and IRP2 (involved in control of iron homeostasis). It is also part of the linear ubiquitin assembly complex (LUBAC), which is important for regulation of the NFkB pathway that plays an important role in regulation of the immune system (Nilsson et al. 2013).
This test involves bidirectional Sanger sequencing using genomic DNA of all coding exons of the RBCK1 gene plus ~20 bp of flanking non-coding DNA on each side. We will also sequence any single exon (Test #100) or pair of exons (Test #200) in family members of patients with known pathogenic variants or to confirm research results.
Indications for Test
Patients with clinical features consistent with polyglucosan body myopathy type I, with or without immunodeficiency, are good candidates for this test. Family members of patients who have known RBCK1 pathogenic variants are also good candidates. We will also sequence the RBCK1 gene to determine carrier status.
|Official Gene Symbol||OMIM ID|
|Polyglucosan Body Myopathy 1 with or without Immunodeficiency||AR||615895|
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Bi-Directional Sanger Sequencing
Nomenclature for sequence variants was from the Human Genome Variation Society (http://www.hgvs.org). As required, DNA is extracted from the patient specimen. PCR is used to amplify the indicated exons plus additional flanking non-coding sequence. After cleaning of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit. Products are resolved by electrophoresis on an ABI 3730xl capillary sequencer. In most cases, sequencing is performed in both forward and reverse directions; in some cases, sequencing is performed twice in either the forward or reverse directions. In nearly all cases, the full coding region of each exon as well as 20 bases of non-coding DNA flanking the exon are sequenced.
As of March 2016, we compared 17.37 Mb of Sanger DNA sequence generated at PreventionGenetics to NextGen sequence generated in other labs. We detected only 4 errors in our Sanger sequences, and these were all due to allele dropout during PCR. For Proficiency Testing, both external and internal, in the 12 years of our lab operation we have Sanger sequenced roughly 8,800 PCR amplicons. Only one error has been identified, and this was due to sequence analysis error.
Our Sanger sequencing is capable of detecting virtually all nucleotide substitutions within the PCR amplicons. Similarly, we detect essentially all heterozygous or homozygous deletions within the amplicons. Homozygous deletions which overlap one or more PCR primer annealing sites are detectable as PCR failure. Heterozygous deletions which overlap one or more PCR primer annealing sites are usually not detected (see Analytical Limitations). All heterozygous insertions within the amplicons up to about 100 nucleotides in length appear to be detectable. Larger heterozygous insertions may not be detected. All homozygous insertions within the amplicons up to about 300 nucleotides in length appear to be detectable. Larger homozygous insertions may masquerade as homozygous deletions (PCR failure).
In exons where our sequencing did not reveal any variation between the two alleles, we cannot be certain that we were able to PCR amplify both of the patient’s alleles. Occasionally, a patient may carry an allele which does not amplify, due for example to a deletion or a large insertion. In these cases, the report contains no information about the second allele.
Similarly, our sequencing tests have almost no power to detect duplications, triplications, etc. of the gene sequences.
In most cases, only the indicated exons and roughly 20 bp of flanking non-coding sequence on each side are analyzed. Test reports contain little or no information about other portions of the gene, including many regulatory regions.
In nearly all 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 for example 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 and cycle sequencing.
Unless otherwise indicated, the sequence data that we report are based on DNA isolated from a specific tissue (usually leukocytes). Test reports contain no information about gene sequences in other tissues.
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.