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Identification of a large intronic transposal insertion in SLC17A5 causing sialic acid storage disease Tarailo-Graovac, Maja; Drögemöller, Britt I; Wasserman, Wyeth W; Ross, Colin J D; van den Ouweland, Ans M W; Darin, Niklas; Kollberg, Gittan; van Karnebeek, Clara D M; Blomqvist, Maria Feb 10, 2017

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RESEARCH Open AccessIdentification of a large intronic transposalinsertion in SLC17A5 causing sialic acidstorage diseaseMaja Tarailo-Graovac1,2,3†, Britt I. Drögemöller1,2,4†, Wyeth W. Wasserman1,2,3, Colin J. D. Ross1,2,4,Ans M. W. van den Ouweland5, Niklas Darin6, Gittan Kollberg7, Clara D. M. van Karnebeek1,3,8,9*and Maria Blomqvist7*AbstractBackground: Sialic acid storage diseases are neurodegenerative disorders characterized by accumulation of sialic acidin the lysosome. These disorders are caused by mutations in SLC17A5, the gene encoding sialin, a sialic acid transporterlocated in the lysosomal membrane. The most common form of sialic acid storage disease is the slowly progressiveSalla disease, presenting with hypotonia, ataxia, epilepsy, nystagmus and findings of cerebral and cerebellar atrophy.Hypomyelination and corpus callosum hypoplasia are typical as well. We report a 16 year-old boy with an atypicallymild clinical phenotype of sialic acid storage disease characterized by psychomotor retardation and a mixture ofspasticity and rigidity but no ataxia, and only weak features of hypomyelination and thinning of corpus callosum onMRI of the brain.Results: The thiobarbituric acid method showed elevated levels of free sialic acid in urine and fibroblasts,indicating sialic acid storage disease. Initial Sanger sequencing of SLC17A5 coding regions did not show anypathogenic variants, although exon 9 could not be sequenced. Whole exome sequencing followed by RNAand genomic DNA analysis identified a homozygous 6040 bp insertion in intron 9 of SLC17A5 correspondingto a long interspersed element-1 retrotransposon (KF425758.1). This insertion adds two splice sites, bothresulting in a frameshift which in turn creates a premature stop codon 4 bp into intron 9.Conclusions: This study describes a novel pathogenic variant in SLC17A5, namely an intronic transposalinsertion, in a patient with mild biochemical and clinical phenotypes. The presence of a small fraction ofnormal transcript may explain the mild phenotype. This case illustrates the importance of including lysosomalsialic acid storage disease in the differential diagnosis of developmental delay with postnatal onset andhypomyelination, as well as intronic regions in the genetic investigation of inborn errors of metabolism.Keywords: Sialic acid storage disease, Salla disease, SLC17A5, Whole exome sequencing, Transposon insertion* Correspondence: cvankarnebeek@cw.bc.ca; maria.k.blomqvist@vgregion.se†Equal contributors1BC Children’s Hospital Research Institute, University of British Columbia,Vancouver, BC, Canada7Department of Clinical Chemistry and Transfusion Medicine, Institute ofBiomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg,SwedenFull list of author information is available at the end of the article© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Tarailo-Graovac et al. Orphanet Journal of Rare Diseases  (2017) 12:28 DOI 10.1186/s13023-017-0584-6BackgroundThe primary catabolic pathway of sialo-glycoconjugates islysosomal degradation. In the lysosome, sialic acid residuesare sequentially removed from the carbohydrate chain bysialidases (neuraminidases), and then transported out of thelysosome by the transporter protein sialin [1, 2]. Sialin is amember of the SLC17 solute carrier family, a group ofstructurally related membrane proteins that is a part of themajor facilitator superfamily of transporters [3, 4]. TheSLC17 family proteins have diverse crucial biological func-tions and several members are associated with inheritedneurological or metabolic diseases [2].Sialic acid storage diseases (SASDs) are autosomal re-cessive neurodegenerative disorders that are characterizedby an excessive storage of sialic acid in the lysosomescaused by defective transport by the sialin protein. Twomain disorders represent this group i.e. infantile sialic acidstorage disease (ISSD, OMIM #269920) and the slowlyprogressive adult form that is prevalent in Finland calledSalla disease (OMIM #604369) [5]. ISSD is a more severeform resulting in an early-lethal multisystemic disease.Common symptoms are intellectual developmentaldisorder, muscular hypotonia, failure to thrive as well ascoarse features, seizures, bone malformations, hepatosple-nomegaly, and cardiomegaly in some patients. Further-more, this condition is associated with non-immunehydrops fetalis. Clinical symptoms of Salla disease includenystagmus, muscular hypotonia, ataxia, early psychomotorretardation and speech impairment. Epilepsy is a commonfeature and cerebral and cerebellar atrophy, hypomyelina-tion and corpus callosum hypoplasia are typical findingson these patients. The clinical progression is slow, andpatients can live throughout adulthood.In 1999 Verheijen et al identified SCL17A5 as the genecoding the sialin protein and found mutations in this genein SASD patients [6]. Since then, a wide spectrum ofdisease-causing variants have been reported in SLC17A5,including missense and nonsense mutations, splice site mu-tations and deletions. Of particular relevance to individualsfrom Finland and Sweden is the relatively high frequency ofthe c.115C >T transition (NM_012434.4; rs80338794). Thismissense mutation changes a highly conserved arginine toa cysteine (p.Arg39Cys) and, in homozygous form, causesthe classical slowly progressing Salla phenotype [7, 8]. Theestimated carrier frequency for this variant in Finland isreported to be as high as 1/200 [7]. Other SLC17A5 muta-tions that have more damaging effects on the sialin proteinfunction cause ISSD. Individuals diagnosed with intermedi-ate Salla disease usually have one allele of p.Arg39Cys incompound heterozygosity with a more severe mutation [7].As a result of the excessive storage of sialic acid, SASDpatients excrete large amounts of free sialic acid in theirurine. Thus this biomarker is used for biochemical diag-nosis of these patients. Classical Salla patients excreteabout 10 times more free sialic acid in urine comparedto healthy controls and in ISSD patients this elevation iseven higher (about 100 times the normal levels). Fur-thermore, the storage of sialic acid can also be detectedin tissue samples and cultured fibroblasts.This report describes a new intronic transposal insertionin SLC17A5 in a patient showing elevated free sialic acidin urine and fibroblasts, giving rise to a milder phenotypeof SASD than ISSD and Salla disease. The results furtherhighlight the importance of expanding molecular analysesto non-coding regions when biochemical signs pointtowards a certain diagnosis.MethodsClinical reportThis boy is the first child to healthy unrelated parents ofKurdish origin. Apart from transiently decreased fetalmovements reported by the mother (during 7th monthsof pregnancy), the pregnancy was uneventful. The boywas born after 37 weeks of gestation with a birth weightof 2630 g, length of 49 cm and head circumference of32 cm. The Apgar score was 10-10-10 and the perinatalperiod was uncomplicated. The boy was first admitted at9 months of age because of bilateral esotropia. He wasthen also found to have hyperopia (+7-8). Regularophthalmological investigation revealed no otherchanges. He was again admitted at 13 months of agebecause of delayed psychomotor development. He couldsit, grasp with his whole hands and move between themand babbled with two syllables, corresponding to adevelopmental age of around 6–7 months. Muscle toneand tendon reflexes were normal. He learned to say afew words and to walk unsupported around 3 years ofage. His communicative skills peaked at 5 years of agewhen he could combine 2–3 words, while his best motorfunction was at 9 years of age when he could walkunsupported both uphill and downhill. He has sincethen slowly lost developmental skills and has becomeincreasingly stiff. A trial of L-Dopa treatment had noeffect. A permanent gastrostomy was placed at 11 yearsof age because of swallowing difficulties although he stillmanaged to eat small pieces of food by himself. At thelast assessment at 16 years of age, he was a very happyand easy-going young man with the ability to communi-cate with gestures, sounds, pointing and about 30 signsand a few words. He was able to walk around 100 mwith support and had a comparably good fine motorfunction with bilateral pincer grasp. On examination hehad generally increased muscle tone considered to be amixture of spasticity and rigidity with associated con-tractures in large joints and a right-sided scoliosis. Themuscle tendon reflexes were generally increased withleft-sided ankle clonus and a right-sided Babinski’s sign.There were no involuntary movements or signs of ataxia.Tarailo-Graovac et al. Orphanet Journal of Rare Diseases  (2017) 12:28 Page 2 of 10He had no seizures and EEG was normal. Head circumfer-ence and height has been normal and there were no signsof additional organ involvement. Routine laboratory inves-tigations including metabolic screening analyses have beennormal. A 3 T MRI of the brain was performed at 8 yearsof age showing mild features of hypomyelination and thin-ning of corpus callosum (Fig. 1). In view of the unusualphenotype, further investigation of the SLC17A5 gene wasdriven by the elevation of free sialic acid in urine andfibroblasts.MaterialsSkin fibroblasts were obtained and cultured according toroutine procedures in Eagle’s minimal essential mediumsupplemented with 10% foetal calf serum and 1% PEST.Confluent cells were harvested by trypsination andstored in -20 °C until analysis.Urine samples were collected as morning aliquots andstored at -20 °C until analysis.Analysis of sialic acidFibroblasts were suspended in water and homogenizedusing a glass/teflon homogenizer. After addition of sodiumchloride to a final concentration of 0.15 M, the cells werecentrifuged at 20 000xg for 30 min at +4 °C and the super-natant was collected for determination of free sialic acid(see below). Cell protein concentration was determined bythe BCA method (Pierce laboratories). Analysis of free sialicacid in urine was performed by thin-layer chromatography.The plates were developed in 1-butanol/acetic acid/water(2:1:1 by volume) and visualized with resorcinol reagent.Total (free and bound) sialic acid in urine was deter-mined with the resorcinol method [9] after hydrolysisby 0.05 M sulphuric acid and purification with ion-exchange chromatography. Free sialic acid in urine andfibroblasts were analyzed by the thiobarbituric acidmethod according to Aminoff [10] after purification byion-exchange chromatography.Sanger sequencing of SLC17A5Mutation analysis of all coding exon (including 30 nucle-otides before and after the exon) of SLC17A5 wasperformed by Sanger Sequencing (primers available onrequest) using an ABI 3730XL automated sequencer(Applied Biosystems, Foster City, CA, USA). Data wereFig. 1 Axial T2 sequences showed slightly increased T2 signal in supratentorial central white matter (a) while the cerebellar white matter lookednormal (b). Axial T1-weighted imaging showed normally signaling supratentorial white matter (c). Sagittal T2-weighted imaging revealed a somewhatthin corpus callosum and a small cyst (1.2 cm) of the corpus pineale (d)Tarailo-Graovac et al. Orphanet Journal of Rare Diseases  (2017) 12:28 Page 3 of 10analysed using SeqPilot software (version 4.2.1 build506; JSI medical systems GmbH, Ettenheim, Germany).Whole exome sequencingGiven the mild elevation of sialic acid and the results fromthe initial Sanger sequencing of the coding exons ofSLC17A5, a novel inborn error of metabolism wassuspected and thus whole exome sequencing (WES) wasperformed through the TIDEX gene discovery project (UBCIRB approval H12-00067). WES was performed for theindex and his unaffected parents using the Agilent SureSe-lect kit and Illumina HiSeq 2000 (Perkin-Elmer, USA). Thedata was analyzed using our semi-automated bioinformaticspipeline [11]. Briefly, the sequencing reads were aligned tothe human reference genome version hg19 and rare variantswere identified and assessed for their potential to disruptprotein function, and subsequently screened under a seriesof genetic models: homozygous, hemizygous, compoundheterozygous and de novo.SLC17A5 RNA/cDNA analysisTotal RNA was extracted from the cultured skin fibro-blasts using the AllPrep DNA/RNA Mini kit (Qiagen).RT-PCR was performed with the One-step RT-PCR kit(Qiagen) with forward primer 5’-TATTCCTGGTAGCTGCTGGC-3’ and reverse primer 5’- TCTGGCAACTAGTGATATTTCATGA-3’ predicted to amplify a prod-uct of 517 bp in length of NM_012434.4; SLC17A5cDNA (c.1130 – c.1646).PCR products were separated on agarose gels stainedwith GelStar® and visualized on a Dark Reader Blue lighttransilluminator. Sequencing analysis was performedusing an ABI PRISM® 3100 Genetic Analyzer and theBigDye Terminator v.1.1 Cycle Sequencing Kit (AppliedBiosystems).PCR and Sanger sequencing of the genomic DNAPrimers were designed using the SLC17A5 DNA refer-ence sequence (Ensembl Gene ID ENST00000355773).An initial long-range PCR was performed usingprimers spanning the suspected location of the inser-tion (forward primer: 5’ - CTT CTG GAT TTA GCATCA ACC A - 3’ and reverse primer: 5’ - AGT ATTCCT GGT AGC TGC TG – 3’). The resultant PCRproducts were used as templates for the followingnested PCRs:i) A long range PCR (forward primer: 5’ - CTT CTGGAT TTA GCA TCA ACC A - 3’ and reverseprimer: 5’ - CAA CTT CCT GCT TTA ATT ATTGTG – 3’) to determine the location and sequenceof the identified insertion using Sanger sequencingii) A PCR-based assay to confirm the presence/absenceof the insertion using two primers outside of theinsertion (forward primer: 5’ - CTT CTG GAT TTAGCA TCA ACC A - 3’ and reverse primer: 5’ - CAACTT CCT GCT TTA ATT ATT GTG – 3’) and athird primer located inside of the insertion (forward pri-mer: 5’ - AAT ATT CGG GTG GGA GTG AC – 3’).Sanger sequencing was performed using BigDye®Terminator v3.1 Cycle Sequencing chemistry (Life Tech-nologies) and subsequent capillary electrophoresis wasperformed by the CMMT/CFRI DNA Sequencing CoreFacility using a Prism 3130xl 16-capillary automatedgenetic analyzer (Applied Biosystems). In silico analysesto determine the class of transposon and the effect onsplicing were performed using (i) RepeatMasker [12] and(ii) NetGene2 [13] and Alternative Splice Site Predictor(ASSP) [14].ResultsSialic acidSialic acid (total and free) was analyzed twice when thepatient was 11 years old (Table 1). While total sialic acidin urine was borderline normal, thin-layer chromatog-raphy showed an abnormal pattern with large amountsof free sialic acid (data not shown). Subsequent quantita-tive analysis revealed elevated amounts of free sialic acidin urine and fibroblasts (Table 1).Sanger sequencing of the coding regions of SLC17A5In the first attempt to find pathogenic variants in theSLC17A5 gene, Sanger sequencing of the coding regionswas performed not showing any pathogenic variants, al-though exon 9 could not be sequenced (data not shown).Whole exome sequencingUsing our semi-automated bioinformatics approach [11],20 candidate genes affected by rare variants predicted toaffect protein function and segregating according toMendelian inheritance models were identified. Based oninheritance patterns, these could be grouped into: homo-zygous (SPTY2D1, LRP2 and ERCC5), hemizygous(FLNA, ZNF275, GRIPAP1, AMER1, PLXNB3, TAS2R43,ARSH, MAGEA11 and NLGN4X), compound heterozy-gous (PLXND1, NHSL1, COBL, PIEZO1, NUP153 andMYH7B) and de novo (TCTE1 and PUSL1). However,none of these candidate genes could reasonably beTable 1 Sialic acid in urine and fibroblastsAnalyte Sampling 1 Sampling 2 Normal rangeSialic acid (total)—urinenmol/mol creatinine68 69 31–69Sialic acid (free) urinenmol/mol creatinine60 57 7–21Sialic acid (free) fibroblastsnmol/mg protein16 - <1,3Tarailo-Graovac et al. Orphanet Journal of Rare Diseases  (2017) 12:28 Page 4 of 10assumed to cause the biochemical and clinical phenotypeof the index. Interestingly, in addition to these, the WESanalysis revealed a presence of clustered mismatches indi-cative of a homozygous insertion of an unknown size andorigin in intron 9 of SLC17A5, with one of the breakpointslocated 24 bp from the intron/exon boundary. Given thepreviously described spectrum of Salla disease-causingvariants in SLC17A5 and the fact that exon 9 could not beamplified by Sanger sequencing, a putative insertion wasconsidered the best candidate and was further analyzed.SLC17A5 RNA/cDNA analysisRT–PCR of SLC17A5 cDNA from cultured skin fibro-blasts, spanning cDNA c.1130 – c.1646, followed by gelelectrophoresis, revealed three products instead of theexpected single transcript of 517 bp in the patient but notin the control subject (Fig. 2a). One of the additional frag-ments was approximately 620 bp and the other about1000 bp. All three bands were cut out and sequenced bySanger sequencing. Sequencing analysis of the 620 bpfragment revealed an insertion 106 bp in length,where the first 24 bp corresponded to an intronic se-quence immediately adjacent to the exon 9 splice site,followed by an 82 bp sequence corresponding to pos-ition 6033-5952 of a previously described transposableelement KF425758.1 [15] (Fig. 2b). The sequencingdata from the analysis of the > 1000 bp product wasnot readable due to too much noise and background.The 517 bp product corresponded to the referencesequence NM_012434.4 for both patient and control(not shown).PCR and Sanger sequencing of the genomic DNASubsequent Sanger sequencing of the region identifiedby the exome sequencing and RNA/cDNA analyses con-firmed the presence of a 6040 bp insertion, which waslocated in intron 9 of SLC17A5 (24 bp downstream ofexon 9) (Fig. 3a). RepeatMasker analyses [16] revealedthat this insertion was a long interspersed element-1(LINE-1, L1) retrotransposon [17]. As expected based onthe exome sequencing data and the RT-PCR analysis,the index was homozygous for this insertion, while theunaffected parents were heterozygous (Fig. 3b).In addition to the predicted splice site located at theexon-intron boundary, splice site analyses with Net-Gene2 revealed an additional predicted splice-site 82 bpinto the insertion, while ASSP analyses identified a thirdpredicted splice-site 679 bp into the insertion (Fig. 4a).The transcripts created by the three predicted splicesites would amplify a 517 bp fragment (wildtype), a623 bp fragment and a 1220 bp fragment, in agreementwith the cDNA analyses. The products from these twoalternate splice-sites result in a frameshift, which causesa premature stop codon 4 bp into intron 9. In case oftranslation of the aberrant transcripts, the prematurestop will be at amino acid 421 (Fig. 4b). The sequencesFig. 2 Genetic analyses. RT-PCR followed by gel electrophoresis of products spanning cDNA position c.1130 – c.1646 in the SLC17A5 gene showingtwo extra fragments of abnormal size in addition to the expected 517 bp fragment in the patient. The abnormal transcripts were absent in the controlsample (a). RNA was extracted from cultured skin fibroblasts. Direct sequencing of the 620 bp fragment revealed an apparently homozygous insertionof 106 bp in length, where the first 24 bp corresponded to an intronic sequence immediately adjacent to the exon 9 splice site, followed by an 82 bpsequence corresponding to position 6033-5952 of the transposable element KF425758.1 (b)Tarailo-Graovac et al. Orphanet Journal of Rare Diseases  (2017) 12:28 Page 5 of 10for the resulting cDNA products are provided in theAdditional file 1.Investigation of structural variation in the 1000 GenomeProject/Ensembl databases did not reveal any previousreports of this variant in healthy individuals.DiscussionThe lysosomal free sialic acid storage disorders presentwith a broad clinical spectrum. Salla disease representsthe mildest phenotype and occurs most frequently inFinland, and other Nordic countries such as Sweden andDenmark [7, 8, 18, 19]. The infantile form of sialic acidstorage disease shows a more severe clinical phenotypeand has no geographic predominance [7, 20, 21]. Therealso exists SASD forms that are intermediate in severitybetween Salla and ISSD [22–26]. Whereas Salla patientsusually present with hypotonia, ataxia and nystagmusthe first year of life, our patient showed delayed psycho-motor development together with hyperopia at age3 years. Ataxia usually remains a prominent feature asSalla disease progresses, however in the present caseataxia has not incurred. Instead, our patient showsincreased muscle tone, most likely due to a mixture ofspasticity and rigidity. The only clinical symptoms over-lapping with Salla disease are in fact early psychomotorretardation and speech problems, which by itself is notvery disease specific. MRI findings further support themilder clinical phenotype with weak features of hypo-myelination and thinning of corpus callosum in contrastto Salla patients where cerebral and cerebellar atrophy,hypomyelination and corpus callosum hypoplasia aretypical findings. Thus, the patient described in this papershows an even milder clinical phenotype of SASD thanSalla disease, which makes it difficult to pinpoint thecorrect diagnosis. Our findings suggest that analysis offree sialic acid needs to be considered in patients withencephalopathy and mild hypomyelination and thinningof corpus callosum.The elevation of free urinary sialic acid has been con-sidered the biochemical hallmark of SASD and has beenobserved as early as 3 days of age [27]. Thus, the levelsof total urinary sialic acid (free and bound) is elevated inthese patients and colorimetric measurement of thisfraction is commonly used as the first screening bio-marker for SASD and other lysosomal diseases storingsialylated oligosaccharides. In our experience, the vastmajority of SASD patients show increased total sialicacid in urine. However, the patient described here showsborderline total sialic acid concentrations at two separatesampling occasions. As a complement to the quantitativeassay, oligosaccharide screening in urine samples is rou-tinely performed by thin-layer chromatography which inthis case was suggestive of increased levels of free sialicacid. This was confirmed by quantitative measurementsFig. 3 Sequencing of this ~6 kb fragment confirmed the presence of a 6040 bp LINE-1 retrotransposon, which was inserted in intron 9 (a). PCRanalyses confirmed that the unaffected parents were heterozygous for this insertion, while the index was homozygous (b)Tarailo-Graovac et al. Orphanet Journal of Rare Diseases  (2017) 12:28 Page 6 of 10of free sialic acid in urine and fibroblasts. The increaseof free sialic acid was somewhat lower than previouslydescribed Salla patients [27]. These results further high-light the importance of using qualitative analysis of urineoligosaccharides combined with quantitative analysis oftotal sialic acid. Another possibility to overcome theseproblems is to use mass spectrometry and simultan-eously measure total and free sialic acid [28, 29] whichmight be the golden standard in the near future. Itshould also be mentioned that SASD has been reportedin two siblings without sialuria, both homozygous forthe Lys136Glu mutation in SLC17A5 [30]. Increased freesialic acid was present in CSF, detected by H-NMR spec-troscopy, and this finding together with hypomyelinationwas suggestive of SASD.In view of the unusual phenotype in our patient, furtherinvestigation of SLC17A5 was driven by the elevation offree sialic acid in urine and fibroblasts. In the first attemptto find pathogenic variants, Sanger sequencing of the cod-ing regions of SLC17A5 was performed with negativeresults of all exons except for exon 9 which could not beamplified. Thus, an untargeted diagnostic approach wasused to find out the cause of the elevated SASD biomarkerby including the family in the TIDEX gene discoverystudy. WES analysis identified 20 candidate genes affectedby rare variants predicted to affect protein function,however none of these variants was deemed a goodexplanation for the observed phenotype in the patient.Interestingly, looking more closely into the SLC17A5 gene,the WES analysis revealed the presence of a homozygousinsertion of an unknown size or origin in intron 9. RT-PCR and Sanger re-sequencing further confirmed thepresence of a 6040 bp insertion (RefSeq KF425758.1),which was located in intron 9 of SLC17A5 (24 bp down-streamof exon 9) and showed this insertion to be a LINE-1 retrotransposon [17]. The presence of a large insertion inintron 9 might explain the problems of amplifying exon 9by the initial Sanger sequencing. This large transposon in-sertion has previously been described in SLC25A13 causingcitrin deficiency [15]. Furthermore, retrotranspositionalFig. 4 Splice site analyses using NetGene2 and ASSP, revealed that in the presence of the insertion, in addition to the correct splice-site occurring atthe exon- intron boundary, a further two splice sites are present in the insertion (a). These alternate splicing events result in a frameshift mutation,which results in a premature stop codon at amino acid 421, indicated by the arrow (b). Grey shading indicated the 12 transmembrane regions of thesialin proteinTarailo-Graovac et al. Orphanet Journal of Rare Diseases  (2017) 12:28 Page 7 of 10insertion of L1 elements resulting in genomic deletions hasbeen shown to cause pyruvate dehydrogenase complexdeficiency [31].Whole exome sequencing profiles only a small portionof the human genome (~1%) by capturing the protein-coding sequences, and one of the disadvantages of thisapproach is that a ‘pathogenic’ variant may be locatedoutside of the captured region, e.g. missed exonicregions, deep intronic variants, regulatory elements or inother non-coding regions of the genome. Moreover,WES data is not optimal for detection of larger variants,such as copy number variants and structural variants.Using whole genome sequencing (WGS) instead of WEScan overcome these problems since WGS refers to ana-lysis of the entire human genome [32] (and referencestherein). In our patient, the transposon insertion occurredrelatively close (within 24 bp) to the exon-intron boundaryand we were able to detect it using WES, highlighting theimportance of thorough analysis and interpretation ofNGS data [11] as well as further molecular analyses to val-idate the findings discovered using NGS data in patientswith suspected genetic disorders and persistent uniquebiochemical phenotypes.The possible consequences of this homozygous insertionin the index were investigated through in silico analyses.These analyses predicted that the insertion of this sequence24 bp into intron 9 created two splice sites, occurring 82 bpand 679 bp into the insertion sequence, in addition to thenormal splice site between exon 9 and intron 9. cDNA ana-lyses confirmed the presence of three SLC17A5 transcriptsin the index providing support for the presence of all threesplice sites in this region. In silico translation of the twotranscripts created by the alternate splice sites predicts apremature stop codon 4 bp into intron 9 and truncation atamino acid 421. The sialin protein consists of 12 trans-membrane regions and the variant found in our patientshould result in the absence of two transmembranedomains at the carboxyl end of the protein.The 32 mutations previously defined in SLC17A5 [33]show a wide spectrum (missense and nonsense mutations,splice-site mutations, insertions and deletions) and nodirect mutational hot-spots have been suggested. Thephenotypic variation observed in SASD seems to correl-ate, to some extent, with the presence of the Arg39Cysmutation [7, 8], where this variant seems to cause a morepreserved sialin function compared to other mutationsfound in compound heterozygous patients. Thus, otherSLC17A5 gene mutations that have more damaging effectson the sialin protein function are suggested to cause ISSD.However, other reports challenge this hypothesis. For ex-ample, homozygosity of the Lys136Glu variant has beenfound to cause both severe Salla disease [23] and a mildSASD phenotype [30]. Landau et al furthermore report aphenotypic variability of SASD in affected patients of asingle inbred kindred with the same homozygous missensemutation [34]. These findings suggest that polymorphismsin SLC17A5 or other genes involved in the metabolism offree sialic acid may account for the phenotypic variability.Mutations resulting in the deletion of exons 10 and 11 havepreviously been reported to have severe consequences [35],which contrasts the milder phenotype in our patient. Thecase discussed in here presents an interesting explanationfor the milder phenotype that is observed, even though itmay be presumed that exon 10 and 11 cannot be translated.We therefore propose the following explanation: In ourpatient, in addition to the two new splice sites resulting inaberrant transcription, we did identify normal transcriptwhich is the result of the retention of the normal splice site.The presence of low levels of wild type protein may providesome capacity to transport sialic acid, although not enoughto keep the patient healthy.ConclusionsWe report for the first time a patient with SASD caused bya large intronic transposon insertion in the SLC17A5 gene.Through careful analysis of whole exome, cDNA andgDNA sequencing results we were able to characterize theeffects of the complex variation identified in this patient.We provide an explanation for the mild clinical presenta-tion that was observed in this patient, which is somewhatdifferent to the phenotypes observed in classical Mb Sallapatients. In fact, SASD could be underdiagnosed because oftheir sometimes non-specific clinical findings. LysosomalSASD should be considered and included in the differentialdiagnosis of developmental delay with postnatal onset andsigns of white matter disease with hypomyelination.Additional fileAdditional file 1: Predicted sequences resulting from the LINE-1 retro-transposon insertion. (PDF 162 kb)AbbreviationsISSD: Infantile sialic acid storage disease; LINE-1 (L1): Long interspersedelement-1; NGS: Next generation sequencing; SASD: Sialic acid storagedisease; WES: Whole exome sequencing; WGS: Whole genome sequencingAcknowledgmentsWe gratefully acknowledge the family for their participation in this study;Mr B. Sayson and Ms. A. Ghani for consenting and coordination ofsample collection; Mrs. X. Han for Sanger sequencing; Mrs. M. Higginsonfor gDNA extraction, sample handling and technical data (University ofBritish Columbia, Vancouver, CA); Ms. E. Lomba for administrativesupport; and Dr. Jorge Asin Cayuela for revision of the manuscript.FundingThis work was supported by funding from the BC Children’s HospitalFoundation (1st Collaborative Area of Innovation, www.tidebc.org) andthe Canadian Institutes of Health Research (#301221 grant), Informaticsinfrastructure was supported by Genome BC and Genome Canada(ABC4DE Project). CvK is supported by a Michael Smith Foundation forHealth Research Scholar award. CJDR is supported by a CIHR NewInvestigator award.Tarailo-Graovac et al. Orphanet Journal of Rare Diseases  (2017) 12:28 Page 8 of 10Availability of data and materialsData is presented in the manuscript and its Additional file 1. The datasetanalyzed during the WES is available from the corresponding author onreasonable request.Authors’ contributionsMB contributed to interpretation of data and drafting of the paper well as finalapproval and submission of the manuscript. MTG performed bioinformaticsanalysis and interpretation of the WES data suggestive of an insertion, andcontributed to manuscript drafting and editing. GK performed the RNA/cDNAanalysis and identified the insertion, contributed to manuscript drafting andediting. BD identified the exact location and sequence of the insertion throughPCR and Sanger analyses, determined the functional consequences ofthe mutation through in silico analyses, interpreted these results,contributed to the manuscript drafting and editing. WW supervised thebio-informatics analysis. CR supervised the molecular (Sanger, RT-PCR/transposon) analysis. AvO contributed with the initial Sanger Sequencingand manuscript drafting. ND contributed with the clinical work-up of thepatient, and the manuscript drafting and editing. CvK designed thestudy, supervised the WES and molecular analysis, and contributed tothe manuscript draft. All authors read and approved the manuscript.Competing interestThe authors declare that they have no competing interest.Consent for publicationConsent to publish has been obtained from the guardians of the patient.Ethics approval and consent to participatePatient and family were enrolled into the TIDEX gene discovery study (UBC IRBapproval H12-00067) and provided informed consent for data and samplecollection, whole exome sequencing (WES), as well as the current case report.Author details1BC Children’s Hospital Research Institute, University of British Columbia,Vancouver, BC, Canada. 2Department of Medical Genetics, University ofBritish Columbia, Vancouver, Canada. 3Centre for Molecular Medicine andTherapeutics, Vancouver, Canada. 4Pharmaceutical Sciences, University ofBritish Columbia, Vancouver, BC, Canada. 5Department of Clinical Genetics,Erasmus Medical Center, Rotterdam, The Netherlands. 6Department ofPediatrics, Sahlgrenska Academy, Gothenburg University, Gothenburg,Sweden. 7Department of Clinical Chemistry and Transfusion Medicine,Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg,Gothenburg, Sweden. 8Department of Pediatrics, University of BritishColumbia, Vancouver, Canada. 9Department of Pediatrics, Academic MedicalCentre, Amsterdam, The Netherlands.Received: 18 October 2016 Accepted: 1 February 2017References1. 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J Med Genet. 2005;42:829–36.•  We accept pre-submission inquiries •  Our selector tool helps you to find the most relevant journal•  We provide round the clock customer support •  Convenient online submission•  Thorough peer review•  Inclusion in PubMed and all major indexing services •  Maximum visibility for your researchSubmit your manuscript atwww.biomedcentral.com/submitSubmit your next manuscript to BioMed Central and we will help you at every step:Tarailo-Graovac et al. Orphanet Journal of Rare Diseases  (2017) 12:28 Page 10 of 10


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