Characterization of novel SLC6A8 variants with the use of splice-site analysis tools and implementation of a newly developed LOVD database

Abstract

The X-linked creatine transporter defect is caused by mutations in the SLC6A8 gene. Until now, 66 synonymous and intronic variants in SLC6A8 were detected in our laboratory. To gain more insight in the effect of the detected variants, we applied five free web-based splice-site analysis tools to 25 published variants that were stratified as (non-)disease causing. All were correctly predicted to have no effect (n¼18) or to cause erroneous splicing (n¼7), with the exception of a pathogenic de novo 24 bp intronic deletion. Second, 41 unclassified variants, including 28 novel, were subjected to analysis by these tools. At least four splice-site analysis tools predicted that three of the variants would affect splicing as the mutations disrupted the canonical splice site. Urinary creatine/creatinine and brain MRS confirmed creatine transporter deficiency in five patients (four families), including one female. Another variant was predicted to moderately affect splicing by all five tools. However, transient transfection of a minigene containing the variant in a partial SLC6A8 segment showed no splicing errors, and thus was finally classified as non-disease causing. This study shows that splice tools are useful for the characterization of the majority of variants, but also illustrates that the actual effect can be misclassified in rare occasions. Therefore, further laboratory studies should be considered before final conclusions on the disease-causing nature are drawn. To provide an accessible database, the 109 currently known SLC6A8 variants, including 35 novel ones, are included in a newly developed LOVD DNA variation database.

Phenotype and genotype in 101 males with X-linked creatine transporter deficiency

“…Missense variants can be classified by creatine uptake studies in CRTR deficient fibroblasts after in vitro overexpression of the mutant allele 40 41. Neutral or intronic variants should be studied by bioinformatic analysis and mRNA analysis 39. The recommended diagnostic workup is shown in figure 5.…”

“…d If missense variant, test for restoration of creatine uptake in CRTR deficient fibroblasts after transfection with SLC6A8 cDNA in which the variant has been introduced by site-directed mutagenesis 40 41. If neutral variant or intron variance sequence (IVS), perform mRNA analysis if bioinformatic analysis points to a splice site effect 39 . e If elevated urinary Cr to Crn, repeat urine analysis.…”

“…All 13 exons of the SLC6A8 gene (NM_005629.3) were sequenced as previously described39 at the VUMC in 87 of the 103 patients in this cohort. DNA analysis in the other 16 patients was performed in various other laboratories.…”

Screening for primary creatine deficiencies in French patients with unexplained neurological symptoms

“…Thus, for the first time, our screening study provides strong practice-based evidence confirming this estimated prevalence for PCD. The girl diagnosed with SLC6A8 deficiency presented a severe phenotype similar to affected male patients, an observation that is not surprising in view of recent work [34,61,62] also describing this X-linked disorder in the female population with intellectual disability. In this respect, screening for SLC6A8 deficiency in female patients should be included in the diagnostic workup since this disorder still remains under-diagnosed.…”

Diagnostic methods and recommendations for the cerebral creatine deficiency syndromes

“…In the CTD gene, for example, there have been over 150 different mutations found with no clear "hot spots" identified (54). With new mutations being identified regularly, it is important to sequence the gene to identify mutations as well as polymorphisms.…”