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DEVELOPMENTAL MEDICINE & CHILD NEUROLOGY ORIGINAL ARTICLE

Evaluation of non-coding variation in GLUT1 deficiency

YU-CHI LIU1,2,* JIA WEI AUDREY LEE1,* SUSANNAH T BELLOWS1 JOHN A DAMIANO1

SAUL A MULLEN1,3 SAMUEL F BERKOVIC1 MELANIE BAHLO2 INGRID E SCHEFFER1,3,4

MICHAEL S HILDEBRAND1 CLINICAL GROUP†1

1 Department of Medicine, Epilepsy Research Centre, Austin Health, University of Melbourne, Heidelberg, Vic.; 2 Population Health and Immunity Division, The Walter

and Eliza Hall Institute, Parkville, Vic.; 3 Florey Institute, Heidelberg, Vic.; 4 Department of Paediatrics, University of Melbourne, Royal Children’s Hospital, Parkville,

Vic., Australia.

Correspondence to Michael S Hildebrand, Epilepsy Research Centre, 245 Burgundy St Heidelberg, Vic. 3084, Australia. E-mail: [email protected]

*These authors contributed equally to this work.†Members of the group are listed in Appendix A.

PUBLICATION DATA

Accepted for publication 14th April 2016.

Published online

ABBREVIATIONS

CSF Cerebrospinal fluid

GLUT-1 Glucose transporter-1

AIM Loss-of-function mutations in SLC2A1, encoding glucose transporter-1 (GLUT-1), lead to

dysfunction of glucose transport across the blood – brain barrier. Ten percent of cases with

hypoglycorrhachia (fasting cerebrospinal fluid [CSF] glucose

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deficiency, making it difficult to identify genotype – pheno-type correlations, as emphasized by a recent French study of 265 patients.12 Despite this molecular diagnostic suc-cess, SLC2A1 mutations are not detected in 10% of patients with 16y)

3.3 – 4.4 (age 50% of pairedserum glucose levela

5 2.8 – 4.5

6 3.3 – 4.4

7 3.0 – 4.5

8 2.5 – 5.0

9 2.2 – 5.5

10 2.8 – 4.0

11 3.3 – 4.5

12 2.5 – 5.6

13 2.5 – 5.5

14 2.8 – 4.4

15 2.8 – 5.0

aOnly paired CSF and serum glucose levels were interpretable for

this laboratory.

What this paper adds• Deep intronic SLC2A1 mutations may cause GLUT1 deficiency.

• Awareness of age-related cerebrospinal fluid (CSF) glucose levels critical in

GLUT1 deficiency evaluation.

• Sequencing of SLC2A1 is worthwhile when GLUT1 deficiency is suspected.

2 Developmental Medicine & Child Neurology 2016

http://www.novocraft.com/http://www.novocraft.com/

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UCSC Genome Browser). PCR duplicates were removedusing MarkDuplicates from Picard version 1.117 (http://broadinstitute.github.io/picard/; Broad Institute, Cambridge,

MA, USA). Variant calling was performed with GATK HaplotypeCaller (version v3.2 – 2) and variant annotation

with ANNOVAR (http://annovar.openbioinformatics.org/en/latest/; University of Pennsylvania, Philadelphia, PA,USA). Variants were filtered according to the following cri-teria: location in exonic or splicing regions; mutation types(missense, stopgain/loss, coding indels, or potential splicesite mutations); a minor allele frequency ≤0.01 in the 1000Genomes dataset, Exome Aggregation Consortium database,or the 6500 NHLBI-ESP exomes dataset; and appearancein C; Fig. 1) and verifiedby Sanger sequencing. This variant was not found in pub-licly available databases including dbSNP, ExAC, the 1000Genomes and the 6500 NHLBI-ESP exomes dataset. Con-sidering its location within a splice site, the potential effect of the mutation on the splicing process was first evaluatedcomputationally. Splice-Site Analyzer Tool software indi-cated that the mutation reduced the similarity score of thesplice site to the consensus splice site sequence from 81.77to 69.29 (http://ibis.tau.ac.il/ssat/SpliceSiteFrame.htm; Tel

Aviv University, Tel Aviv, Israel), predicting the splicingmachinery may recognize an alternate splice site instead of the mutated site.

Confirmation of SLC2A1 intron retentionBecause SLC2A1 is expressed in lymphocytes, we examinedthe effect of the mutation by extracting RNA from venousblood of the proband and his unaffected mother. Oligonu-cleotides were designed to amplify sequence from exon 5to exon 8 (415 base pairs), and this size fragment wasamplified from the cDNA of the mother who was homozy-gous wild-type c.972+5G (Fig. 2). However, when the

Non-Coding Variation in GLUT1 Deficiency Yu-Chi Liu et al. 3

http://broadinstitute.github.io/picard/http://broadinstitute.github.io/picard/http://annovar.openbioinformatics.org/en/latest/http://annovar.openbioinformatics.org/en/latest/http://www.ncbi.nlm.nih.gov/nuccore/NM_006516http://www.ncbi.nlm.nih.gov/genehttp://www.mrc-holland.com/http://ibis.tau.ac.il/ssat/SpliceSiteFrame.htmhttp://ibis.tau.ac.il/ssat/SpliceSiteFrame.htmhttp://www.mrc-holland.com/http://www.ncbi.nlm.nih.gov/genehttp://www.ncbi.nlm.nih.gov/nuccore/NM_006516http://annovar.openbioinformatics.org/en/latest/http://annovar.openbioinformatics.org/en/latest/http://broadinstitute.github.io/picard/http://broadinstitute.github.io/picard/

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T a b l e

I I :

P h e n o t y p e a n d f a s t i n g c e r e b r o s p

i n a l f l u i d ( C S F ) g l u c o s e r e s u l t s o f p a t i e n t s w i

t h n o v e l v a r i a n t s i n S L C 2 A 1

P r o b a n d

V a r i a n t

E p i l e p s y

s y n d r o m

e

S e i z u r e t y p e

A g e o f

o n s e t

D e v e l o p m e n t /

i n t e l l e c t

O t h e r

c l i n i c a l

f e a t u r e s

F a s t i n g C

S F

g l u c o s e l e v e l

( m m o l / L )

[ a g e a t t e

s t ]

L a b o r a t o r y

r e f e r e n c e

r a n g e

( m m o l / L )

A g e - s p e c i fi c

r e f e r e n c e

r a n g e

( L e e n

e t a l . ,

2 0 1 0 )

( m m o l / L )

E E G fi n

d i n g s

H e a d

c i r c u m f e r e n c e

( c e n t i l e )

F a m i l y 1 ,

I I : 1

A ( c . 9 7 2 + 5 G > C )

G L U T 1

e n c e p h a l o p a t h y

A t y p i c a l

a b s e n c e ,

n o n - c o n v u l s i v e

s t a t u s

e p i l e p t i c u s

4 y e a r s

D e l a y e d ,

r e g r e s s i o n ,

m o d e r a t e

i n t e l l e c t u a l

d i s a b i l i t y

A t a x i a ,

d y s a r t h r i a ,

h y p e r -

v e n t i l a t i o n

i n d u c e d

t e t a n y

1 . 9

[ 5 y ]

2 . 8 – 4 . 0

2 . 5 – 4 . 0

G e n e r a

l i z e d

s p i k e w a v e ,

p o l y s p

i k e

w a v e ,

f r e q u e

n t

m u l t i f o c a l

d i s c h a

r g e s

2 0 t h – 3 0 t h

F a m i l y 2 ,

I I : 1

B ( c . 1

9 – 4 2 0 C > T )

E p i l e p s y

w i t h

m y o c l o

n i c -

a t o n i c s

e i z u r e s

T o n i c / c l o n i c ,

M y o c l o n i c ,

D r o p a t t a c k s

3 y e a r s

N o r m a l

2 . 7

[ 3 y ]

2 . 8 – 5 . 0

2 . 5 – 4 . 0

G e n e r a

l i z e d

s p i k e w a v e ,

p o l y s p

i k e w a v e

5 0 t h – 6 0 t h

F a m i l y 3 ,

I I : 1

C ( c . 1

9 – 2 0 7 T > C )

E a r l y - o n

s e t

a b s e n c e

e p i l e p s y

A b s e n c e

1 2 m o n t h s

D e l a y e d

2 . 6

[ 1 6 m o ]

2 . 8 – 4 . 5

2 . 4 – 4 . 2

G e n e r a

l i z e d

s p i k e w a v e

4 0 t h – 5 0 t h

F a m i l y 4 ,

I I I : 1

E p i l e p t i c

e n c e p h a l o p a t h y

F o c a l m o t o r

s e i z u r e s ,

g e n e r a l i z e d

t o n i c c l o n i c

s e i z u r e s ,

s t a t u s

e p i l e p t i c u s

6 w e e k s

D e l a y e d ,

r e g r e s s i o n ,

s e v e r e

i n t e l l e c t u a l

d i s a b i l i t y

N o n - v e r b a l ,

c o m p l e x

m o v e m e n t

d i s o r d e r ,

b e h a v i o u r a l

i s s u e s

2 . 9

[ 2 y ]

3 . 5

[ 1 3 y ]

4 . 4

[ 1 4 y ]

3 . 0 – 5 . 0

2 . 4 – 4 . 2

G e n e r a

l i z e d

s l o w i n

g ,

n o d e fi

n i t e

e p i l e p t i f o r m

d i s c h a

r g e s

1 0 t h


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same amplification was performed for the proband, the wild-type (415 base pairs) fragment and a larger fragment (~ 570 base pairs) were observed (Fig. 2). These resultsrevealed incorporation of intron 7 to 8 sequence in theRNA produced by the mutant allele. This intron inclusionis predicted to lead to a premature stop codon(p.S324+28*).

Screening for non-coding single nucleotide and copy

number variantsBased on the findings for Family 1, we searched for non-coding mutations in promoter and intronic regions of SLC2A1 in the 55 remaining probands by Sanger sequenc-ing and multiplex-ligation-dependent probe amplification.

Two novel single nucleotide variants in deep intronicregions were identified by sequencing in three families(one variant was found in two probands) (Fig. 1) but none

was found in the promoter regions. We also searched forcopy number variants at the SLC2A1 locus by multiplex-ligation-dependent probe amplification in the 55 remainingprobands, but none were found (data not shown).

Intronic variants reduce SLC2A1 mRNA transcriptRNA from fresh venous blood was obtained from two indi-

viduals from Family 4 with a deep intronic variant; bloodsamples were not available for transcript analysis fromFamilies 2 and 3. Real-time PCR studies using cDNA derived from patient RNA revealed markedly reducedSLC2A1 mRNA transcript in the proband (who carries

variant C) compared with her unaffected mother (without variant C) (Fig. 3). The proband had around half the

mRNA of her mother, consistent with near-failure of tran-scription from one allele of SLC2A1.

Phenotypic analyses of patients with intronic variants The three probands with intronic variants presented with arange of phenotypes (Table II): epilepsy with myoclonic-atonic seizures (Family 2), early-onset absence epilepsy

with developmental delay (Family 3), and GLUT1encephalopathy with seizures, developmental delay andregression, complex motor disorder (ataxia, dystonia, andepisodic events), and progressive microcephaly (Family 4).

The CSF glucose levels (2.6 – 2.9mmol/L) of all three

Controla b c

c.972+5 G>C c.19-420 C>T c.19-207 T>C

Control Control

Figure 1:5 4Novel SLC2A1 non-coding variants in families. Variant A is the splice site mutation identified by whole exome sequencing in Family 1. Variant

B was found in Family 2, and variant C in Families 3 and 4.

587

M

o t h er –RT

M

o t h er +RT

P r o b an d –RT

P r o

b an d +RT

p U C 1 8 DNA H

a el l l l a d d er

MUT splice product (~570 bp)

WT splice product (415 bp)

458434

298

Figure 2: Transcript analysis in Family 1 confirms intron retention. Reverse transcriptase PCR results showing inclusion of intron 7 to 8 sequence in the

c.972+5G>C mutant transcript. A pUC18 DNA HaeIII ladder was run. The wild-type fragments are 415 base pairs whereas the mutant fragment is largerdue to retention of intron 7 to 8 sequence in the transcript from one allele. No amplification is observed from the minus reverse transcriptase (RT) con-

trol samples.

L O W

R E S O L U T I O N C O L O R F I G

C O L O R


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probands were low according to laboratory-specific refer-ence ranges; however, when compared with age-specificreference ranges, the levels were within the normal range

(Table II).

3

The proband from Family 1 has trialled theketogenic diet, however they found it difficult to imple-ment because the patient refused food. Seizures in pro-bands of Families 3 and 4 have been well controlled onanti-epileptic medication. The proband from Family 4 hassevere intellectual disability and is severely restricted dueto autistic features and behavioural problems. A modified

Atkins or ketogenic diet is now being considered in thesepatients.

DISCUSSION A critical issue is the variation in the definition of normalCSF glucose levels in different laboratories (Table I).3,10

Yang et al.10 note that the normal range for CSF glucosehas never been properly defined, and this has led to con-siderable confusion about the lower limits of normal CSFglucose in patients whose presentation falls within theGLUT1 phenotypic spectrum.5,6,9 This is further compli-cated by the need for age-specific reference ranges asclearly delineated by Leen et al.3 When reviewing theseranges for the probands of Families 2 to 4 with intronic

variants, it is notable that all would be defined as havingnormal fasting CSF glucose for age in contradistinction totheir laboratory-specific ranges (Table II). The proband of Family 4 had three CSF glucose test results, which

interestingly show marked variability (2.9, 3.3, 4.4). Although testing should be performed in the fasting state,it is possible that adequate fasting is not always achievedand could result in pre-analytical error. The abnormalmRNA transcript levels found in one proband is highly suggestive of GLUT1 deficiency, implying that the morerecent parameters suggesting that CSF glucose lower than3.3mmol/L are more appropriate to consider for investiga-tion of this treatable disease. Even so, 52 of our 56 patientsdid not have mutations, and it remains unclear if theirCSF glucose level reflects normal physiological variationor GLUT1 deficiency. Although genetic heterogeneity may also underlie GLUT1 deficiency, our sequencing of related transporters GLUT3 and MCT1 – 4 did not identify mutations.14

Sequencing of SLC2A1 intronic and promoter sequencesrevealed a novel de novo single nucleotide splice site muta-tion in the proband of Family 1 by whole exome analysis,and novel deep intronic variants in the probands of Fami-lies 2, 3, and 4 (Fig. 1). SLC2A1 splice site mutations have

been reported in GLUT1 deficiency with abnormal splic-ing of the mRNA transcripts in some cases3,13,15,16

(Table SIII, online supporting information). These splicesite mutations have been found 1 to 3 base pairs from theintron – exon boundary, whereas our mutation was 5 basepairs from the boundary (Table SIII). Our transcript stud-ies showed that the de novo splice site mutation led to dis-ruption of the original donor splice site and utilization of acryptic splice site further into the intron, resulting inintron inclusion in the transcript (Fig. 2). This transcript islikely to lead to translation of aberrant protein due to apremature stop codon, and consequent GLUT1 trans-

porter deficiency.Here we show that deep intronic variants can also alterSLC2A1 transcription and are likely to lead to GLUT1deficiency (Fig. 3). To add further support for thepathogenicity of these variants, functional studies such aserythrocyte 3-O-methyl-d-glucose uptake assays could beconsidered. Recurrent, novel variant C (c.19 – 207 T>C),found in Families 3 and 4, is predicted by the Encode pro-

ject (http://www.genome.ucsc.edu/ENCODE/; StanfordUniversity, Stanford, CA, USA) to be in a region enrichedin H3K27Ac, H3K4me1, and H3K4me3 histone markingscharacteristic of active enhancer elements.17 Alteration of these epigenetic modifications has the potential to change

chromatin structure and de-regulate gene transcription.18

For example, binding of transcription factors to intronicenhancer elements has been found to regulate, through his-tone modifications, CFTR expression in cystic fibrosis.19

In silico predictions suggest that variant B (c.19 – 428C> T), found in Family 2, lies within a gene region

where DNA-dependent RNA polymerase II subunit A (POLR2A) – the largest subunit of RNA polymerase II – binds. Therefore, variant B could affect POLR2A bindingand enhancer interaction, leading to altered transcriptionof SLC2A1. This type of effect is not unprecedented inepilepsy as a deep intronic variant in a pseudoexon was

1.2

SLC2A1 mRNA

1

Mother

Proband

0.8

0.6

R e l a t i v e m R N A

0.4

0.2

0

Figure 3: Real-time PCR analysis reveals reduced transcript due to deep

intronic mutation. Real-time PCR results showing reduction of SLC2A1

mRNA transcript in blood due to the presence of variant C in the proband

from Family 4 compared with her unaffected mother who does not carry

the variant.

C O L O R


http://www.genome.ucsc.edu/ENCODE/http://www.genome.ucsc.edu/ENCODE/

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discovered that altered the expression of ALDH7A1 inpyridoxine-dependent epilepsy.20 However, we could not determine the effect of this variant, because blood samplesfrom suitable family members were unavailable for tran-script analysis.

A molecular diagnosis was not found for most of ourcohort with low CSF glucose levels according to current clinical laboratory reference ranges, despite examination of SLC2A1 coding and non-coding regions. Part of the expla-nation may be that we did not sequence introns 1 and 2comprehensively because of their large size (~ 15 and 12megabases, respectively), and there may be other distantly located non-coding sites important for SLC2A1 regulation.

Our findings suggest that, in a patient with clinical andlaboratory features of GLUT1 deficiency, and a normalresult from standard sequencing of SLC2A1, specific stud-ies looking for smaller intragenic, or contiguous copy number variants and structural variants, should be per-formed, followed by sequencing of intronic regions.

A C K N O W L E D G E M E N T S We thank the patients and their families for participation in this

study. Elena Aleksoska (Epilepsy Research Centre) is acknowl-

edged for performing genomic DNA extractions. This study was

supported by a National Health and Medical Research Council

(NHMRC) Program Grant (628952) to SFB and IES, a Practi-

tioner Fellowship (1006110) to IES and a Career Development

Fellowship (1063799) to MSH. MB was supported by an

NHMRC Senior Research Fellowship (1002098) and NHMRC

Program Grant (1054618). This work was also supported by Vic-

torian State Government Operational Infrastructure Support and

Australian Government NHMRC IRIISS funding to MB and YL.

Authors report grant funds that contributed to this project as out-

lined above. IES discloses payments from UCB Pharma, Eisai,

GSK, Athena Diagnostics, and Transgenomics for lectures and

educational presentations, and a patent for SCN1A testing held by

Bionomics Inc. licensed to various diagnostic companies. SFB dis-

closes payments from UCB Pharma, Novartis Pharmaceuticals,

Sanofi-Aventis, and Jansen Cilag for lectures and educational pre-

sentations, and a patent for SCN1A testing held by Bionomics

Inc. licensed to various diagnostic companies.

S U P P O R T I N G I N F O R M A T I O N

The following additional material may be found online:

Table SI: Oligonucleotides used in this study. Table SII: Exome coverage statistics.

Table SIII: Reported SLC2A1 splice site mutations.

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http://dx.doi.org/10.1111/epi.12007http://dx.doi.org/10.1111/epi.12007http://dx.doi.org/10.1111/epi.12007http://dx.doi.org/10.1111/epi.12007

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APPENDIX A:CLINICAL GROUP

The following authors are clinicians who provided patientsfor this study: Monique M. Ryan, Department of Neurol-ogy, Royal Children’s Hospital, and Department of Paedi-atrics, University of Melbourne, Royal Children’s Hospital,Parkville, Victoria; Richard J. Leventer, Department of

Neurology, Royal Children’s Hospital; Department of Pae-diatrics, University of Melbourne, Royal Children’s Hospi-tal; and Murdoch Children’s Research Institute, RoyalChildren’s Hospital, Parkville, Victoria; Jeremy L. Free-man, Department of Neurology, Royal Children’s Hospi-tal, Parkville, Victoria; Mark T. Mackay, Department of Neurology, Royal Children’s Hospital, and Department of

Paediatrics, University of Melbourne, Royal Children’sHospital, Parkville, Victoria; Michael Hayman, Depart-ment of Neurology, Royal Children’s Hospital, and Mur-doch Children’s Research Institute, Royal Children’sHospital, Parkville, Victoria; Victoria Rodriguez-Casero,Department of Neurology, Royal Children’s Hospital,Parkville, Victoria; Gopi Subramanian, Paediatric Neurol-ogy Unit, John Hunter Children’s Hospital, New LambtonHeights, NSW; Richard Webster, TY Nelson Department of Neurology and Neurosurgery, The Children’s Hospitalat Westmead, Sydney, NSW, Australia. Lynette G. Sadleir,Department of Paediatrics, School of Medicine and HealthSciences, University of Otago, Wellington, New Zealand.


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