Creatine Deficiency Syndrome via the SLC6A8 Gene
- Summary and Pricing
- Clinical Features and Genetics
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In cohorts of patients with X-linked intellectual disability (XLID), pathogenic variants in SLC6A8 have been reported to account for ~0.6 - 3.3% of cases. In cohorts of patients with generalized intellectual disability (syndromic or non-syndromic ID), pathogenic variants in SLC6A8 have been reported to account for ~1.4% of cases (Mercimek-Mahmutoglu and Salomons 2015).
In one study of patients suspected of a cerebral creatine deficiency syndrome (CCDS) based on clinical features, 26 pathogenic variants were identified in 195 patients, for a sensitivity of ~13.3% (Comeaux et al. 2013). In a retrospective study of 101 male patients with clinical, biochemical, enzymatic and neuroimaging test results consistent with creatine transporter deficiency, all patients carried a pathogenic variant in SLC6A8. In ~96% of these patients, the variant was a small variant detectable via direct sequencing, while the remaining ~4% were found to carry gross deletions (van de Kamp et al. 2013).
Creatine transporter (CrT) deficiency is an inborn error of creatine biosynthesis causing cerebral creatine deficiency syndrome (CCDS), which typically has an onset in childhood. The creatine transporter protein is required to move creatine out of the blood and into cells, as well as between different cells following endogenous creatine synthesis (Cheillan and Sedel 2016). Characteristic biochemical findings of CrT deficiency include normal levels of creatine and guanidinoacetate (GAA) in the plasma and urine and a normal GAA/creatinine ratio in the plasma. A finding considered pathognomic for CrT deficiency is a high creatine/creatinine ratio in the urine, although it should be noted that this can be normal in affected females (Mercimek-Mahmutoglu and Salomons 2015; Ardon et al. 2016; Cheillan and Sedel 2016). Direct measurement of total creatine levels in the brain is possible by in vivo proton magnetic resonance spectroscopy (1H-MRS) (Stromberger et al. 2003). In CrT deficient patients, 1H-MRS analysis reveals a complete absence or dramatic decrease in the brain creatine peak (Heussinger et al. 2017).
All patients with CrT deficiency present with non-progressive intellectual disability (ID) that can range from mild to severe and is associated with language delays (Cheillan and Sedel 2016). In addition to these patients with non-syndromic ID, other patients present with syndromic ID. Additional findings in the syndromic patients can include seizures, movement disorders, behavior disorders, hypotonia and spasticity, dysmorphic features, gastrointestinal findings, and rarely, cardiac findings (Mercimek-Mahmutoglu and Salomons 2015; Cheillan and Sedel 2016). Treatment of CrT deficient patient with dietary methods has thus far had limited success (Fons et al. 2008). However, clinical trials for pharmaceutical therapy are underway (https://clinicaltrials.gov).
It should be noted that secondary creatine deficiency can be caused by defects in the urea and remethylation cycles as well as by ornithine aminotransferase deficiency (Cheillan and Sedel 2016). If creatine deficiency is suspected based on biochemical tests, but primary cerebral creatine deficiency syndromes are ruled out via genetic and enzyme testing, consideration should be given to these disorders.
CrT deficiency is inherited in an X-linked recessive fashion. The majority of reported patients are male, though affected females have also been reported. Females carrying pathogenic SLC6A8 variants range from asymptomatic to severely affected (Mercimek-Mahmutoglu and Salomons 2015). Approximately 30% of pathogenic variants have been reported to occur de novo, while the rest are maternally inherited (Mercimek-Mahmutoglu and Salomons 2015). To date, over 100 pathogenic variants have been reported in the SLC6A8 gene. The most common types of variants reported are missense and small deletions, though nonsense, splicing, and small insertions and indels have been reported, as well as gross deletions and duplications (Human Gene Mutation Database). No obvious genotype-phenotype correlations have yet been made (Mercimek-Mahmutoglu and Salomons 2015). The most commonly reported pathogenic variants are p.Phe107del, p.Pro544Leu and p.Pro554Leu (van de Kamp et al. 2013; Mercimek-Mahmutoglu and Salomons 2015; SLC6A8 Variant Database).
Creatine is required for the storage and transmission of high energy phosphates (such as ATP) in the muscle and brain (Cheillan and Sedel 2016). Endogenous creatine synthesis occurs via the action of the L-arginine:glycine amidinotransferase (AGAT) and N-guanidinoacetate methyltransferase (GAMT) enzymes. The SLC6A8 gene encodes the creatine transporter protein which is required for the uptake of creatine from the blood into cells. In addition, the creatine transporter is required for the transport of guanidinoacetate (GAA) between cells in organs that do not express both AGAT and GAMT in the same cells. This is particularly true in the brain, and explains why deficiencies in the AGAT, GAMT and SLC6A8 proteins result in a large cerebral creatine deficit (Cheillan and Sedel 2016).
The SLC6A8 gene has high sequence homology to two pseudogenes on chromosome 16, which makes analysis of this gene challenging. In order to ensure we are correctly sequencing the functional SLC6A8 gene, we employ a long-range PCR with Sanger sequencing to analyze the gene. This allows us to use primer sequences that target the nucleotide sequences unique to the region containing the SLC6A8 gene rather than either pseudogene. All coding exons of the SLC6A8 gene plus ~10 bp of flanking non-coding DNA on each side are sequenced using bidirectional Sanger sequencing. This test also includes coverage for the intronic variants c.1392+24_1393_30del and c.1016+21_1016+54del (Hathaway et al. 2010; Cervera-Acedo et al. 2015).
We will also sequence any single exon (Test #100) or pair of exons (Test #200) in family members of patients with known mutations or to confirm research results.
Indications for Test
Patients with biochemical test results (especially a high creatine/creatinine ratio in the urine), impaired creatine uptake in cultured fibroblasts, and/or an absent or greatly decreased brain creatine peak by 1H-MRS analysis are good candidates for this test, particularly if X-linked inheritance is suspected. In addition, young children with global developmental delay, hypotonia, seizures and a movement disorder or older children with intellectual disability with speech delay, epilepsy, a movement disorder and behavior problems are also good candidates for this test. Testing is also indicated for maternal family members of patients with known SLC6A8 pathogenic variants, and we will also sequence the SLC6A8 gene to determine carrier status.
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|Autism Spectrum Disorders and Intellectual Disability (ASD-ID) Comprehensive Sequencing Panel with CNV Detection|
- Genetic Counselor Team - email@example.com
- McKenna Kyriss, PhD - firstname.lastname@example.org
- Ardon O. et al. 2016. Molecular Genetics and Metabolism Reports. 8: 20-3. PubMed ID: 27408820
- Cervera-Acedo C. et al. 2015. Human Genome Variation. 2: 15037. PubMed ID: 27081545
- Cheillan D. and Sedel F. 2016. Disorders of Creatine Metabolism. In: Hollak C.E.M. and Lachmann R.H., editors. Inherited Metabolic Disease in Adults: A Clinical Guide. New York: Oxford University Press, p 541-551.
- ClinicalTrials.gov (https://clinicaltrials.gov)
- Comeaux M.S. et al. 2013. Molecular Genetics and Metabolism. 109: 260-8. PubMed ID: 23660394
- Fons C. et al. 2008. Journal of Inherited Metabolic Disease. 31: 724-8. PubMed ID: 18925426
- Hathaway S.C. et al. 2010. Journal of Child Neurology. 25: 1009-12. PubMed ID: 20501887
- Heussinger N. et al. 2017. Pediatric Neurology. 67: 45-52. PubMed ID: 28065824
- Human Gene Mutation Database (Bio-base).
- Mercimek-Mahmutoglu S. and Salomons G.S. 2015. Creatine Deficiency Syndromes. In: Pagon RA, Adam MP, Ardinger HH, Bird TD, Dolan CR, Fong C-T, Smith RJ, and Stephens K, editors. GeneReviews(®), Seattle (WA): University of Washington, Seattle. PubMed ID: 20301745
- SLC6A8 Variant Database: http://grenada.lumc.nl/LOVD2/vumc/home.php?select_db=SLC6A8
- Stromberger C. et al. 2003. Journal of Inherited Metabolic Disease. 26: 299-308. PubMed ID: 12889668
- van de Kamp J.M. et al. 2013. Journal of Medical Genetics. 50: 463-72. PubMed ID: 23644449
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 10 bases of non-coding DNA flanking the exon are sequenced.
As of February 2018, we compared 26.8 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 14 years of our lab operation we have Sanger sequenced roughly 14,300 PCR amplicons. Only one error has been identified, and this was an error in analysis of sequence data.
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 10 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.
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- 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.
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- Culture and send at least two T25 flasks of confluent cells.
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- Send specimens in insulated, shatterproof container overnight.
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- We strongly recommend maintaining a local back-up culture. We do not culture cells.