# The Creatine Transporter Gene Paralogous at 16p11.2   Is Expressed in Human Brain

Authors: Nadia Bayou; Ridha M'rad; Ahlem Belhaj; Hussein Daoud; Ramzi Zemni; Sylvain Briault; M. Béchir Helayem; Lamia Ben Jemaa; Habiba Chaabouni
Journal: Comparative and Functional Genomics
 (2008)

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## Abstract

Autism is a complex neurodevelopmental disorder characterized by impairment of social interaction, language, communication, and stereotyped, repetitive behavior. Genetic predisposition to autism has been demonstrated in families and twin studies. About 5–10% of autism cases are associated with chromosomal abnormalities or monogenic disorders. The identification of genes involved in the origin of autism is expected to increase our understanding of the pathogenesis.
We report on the clinical, cytogenetic, and molecular findings in a boy with autism carrying a de novo translocation t(7;16)(p22.1;p11.2). The chromosome 16 breakpoint disrupts the paralogous SLC6A8 gene also called SLC6A10 or CT2. Predicted translation of exons and RT-PCR analysis reveal specific expression of the creatine transporter paralogous in testis and brain. Several studies reported on the role of X-linked creatine transporter mutations in individuals with mental retardation, with or without autism. The existence of disruption in SLC6A8 paralogous gene associated with idiopathic autism suggests that this gene may be involved in the autistic phenotype in our patient.


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## Body

## 1. Introduction

Autism  is defined as a pervasive  neurodevelopmental disorder with onset
before 36 months of age,  resulting in impairment in  social interaction,
communication, and in a manifestation of repetitive stereotypic behavior. Its
incidence is estimated at about 1/1000  to 1/2000 with a biased male-to-female
ratio of three or four to one (3-4:1)  [1]. The exact aetiology of autism
remains unknown, although it is likely to result from a complex combination of
genetic, neurological, immunological, and environmental factors [2]. Genetic
factors for autism have been well established in family and twin studies. The
concordance rate in monozygotic twins is estimated to be 90%. This rate is
between 0% and 24% in dizygotic twins [3].About 3–6% of autism
cases are associated with chromosomal abnormalities [4]. Positional cloning of
translocation or inversion breakpoints has been a successful way of identifying
candidate genes for many genetic disorders. The underlying assumption is that a
gene disrupted by the breakpoint is causative of the phenotype. Therefore, as
an alternative way to identify candidate genes for autism, we analyzed the
breakpoint regions of de novo translocation t(7;16)(p22.1; p11.2) in a patient with autism. To the best of our
knowledge, there are few published reports of patients with autism carrying
chromosomal translocation located on 16p11.2 [5].


## 2. Materials and Methods

### 2.1. Case Report

Our
patient is the younger of two children born at term of non consanguineous
healthy parents.  Patient’s psychomotor delays were noticed in the first months
of life. He was unable to maintain eye contact and presented many motor
stereotypes and ritualistic behaviors such as cutting papers into small pieces
and aligning objects. Pregnancy was uneventful. Delivery was by caesarean
section secondary to a narrow basin. Birth weight was 3350 g, and no recognized
malformations were noted. The patient could not sit alone before 12 months
neither could he walk alone before 26 months. He never achieved continence and
still needed help in eating, and complete assistance in dressing and self care.The
patient had severe language impairment being only able to pronounce
monosyllabic words, without any relational intent. A clinical evaluation
performed at the age of 4 years revealed a weight of 13 kg (−2SD), a height of 102 cm (normal), and an
occipitofrontal head circumference of 48 cm (−2,3SD). No evident dysmorphic features
were noted.The
clinical diagnosis of autism was made according to the Diagnostic and Statistical Manual of Mental Disorders—Revision 4 (DSM-IV) and the Autism Diagnostic Interview,
French version 6 1993 (ADI-R) criteria at the age of 4 years and 11 months. The Childhood
Autism Rating Scale, French version (CARS-T) scale indicated a score of 45
documenting a high level of autistic behavior. The IQ test was crucial in
confirming the mental retardation diagnosis. It was not carried out as the
parents declined this. The MRI of the brain revealed a cerebellar megacisterna
in the posterior fossa. The MRS showed a normal spectral pattern as well for
the creatine as for the remaining metabolites. Creatine and creatinine
concentrations in body fluids were not evaluated as the parents refused this. Southern
blot analysis revealed the absence of FRAXA mutation. The healthy sister was
not available for analysis.


### 2.2. Cytogenetic and FISH Studies

Chromosome
analysis was performed on the proband’s and parent’s blood, using standard
high-resolution techniques. FISH was performed on the proband’s metaphase with BACs
that were selected according to the UCSC Genome Browser, and provided by BACPAC
Resource Centre (BPRC) and Pr. Mariano Rocchi (University of Bari,
Italy). BAC clones were biotinylated with biotin-11-dUTP (Sigma, Mo, USA)
by nick translation  using the BioNick labeling system (Invitrogen Life Technologies).


### 2.3. Bioinformatic Research

Bioinformatic
search was carried out using the UCSC Genome Browser to identify potential
autism candidate genes. The Genomatix Software was used for the promoter and the gene
structure predictions.


### 2.4. Expression Studies

Total RNA
was isolated and purified using the RNAgents Total RNA Isolation System
(Promega) from peripheral white blood cells and other human tissues (testis,
adult and foetal brain, fibroblasts, and kidney).1μg of total RNA was converted
to cDNA was used in 25μL reactions with AMV PCR Master Mix (Promega).A 4
nucleotides insertion in the 1st exon of the SLC6A8 paralogous gene let us to
design specific primers with Web Primer DNA program (insertion not found in
the SLC6A8 gene).RT-PCR
assays specific for the chromosome 16 paralogous SLC6A8 transcript and direct sequencing were done using these specific primers (5′-TACCGCTTCTTCTCGCGGCTCTTG-3′, and 5′-ATCGCGCGCGGCGGCACGGC-3′).


## 2.1. Case Report

Our
patient is the younger of two children born at term of non consanguineous
healthy parents.  Patient’s psychomotor delays were noticed in the first months
of life. He was unable to maintain eye contact and presented many motor
stereotypes and ritualistic behaviors such as cutting papers into small pieces
and aligning objects. Pregnancy was uneventful. Delivery was by caesarean
section secondary to a narrow basin. Birth weight was 3350 g, and no recognized
malformations were noted. The patient could not sit alone before 12 months
neither could he walk alone before 26 months. He never achieved continence and
still needed help in eating, and complete assistance in dressing and self care.The
patient had severe language impairment being only able to pronounce
monosyllabic words, without any relational intent. A clinical evaluation
performed at the age of 4 years revealed a weight of 13 kg (−2SD), a height of 102 cm (normal), and an
occipitofrontal head circumference of 48 cm (−2,3SD). No evident dysmorphic features
were noted.The
clinical diagnosis of autism was made according to the Diagnostic and Statistical Manual of Mental Disorders—Revision 4 (DSM-IV) and the Autism Diagnostic Interview,
French version 6 1993 (ADI-R) criteria at the age of 4 years and 11 months. The Childhood
Autism Rating Scale, French version (CARS-T) scale indicated a score of 45
documenting a high level of autistic behavior. The IQ test was crucial in
confirming the mental retardation diagnosis. It was not carried out as the
parents declined this. The MRI of the brain revealed a cerebellar megacisterna
in the posterior fossa. The MRS showed a normal spectral pattern as well for
the creatine as for the remaining metabolites. Creatine and creatinine
concentrations in body fluids were not evaluated as the parents refused this. Southern
blot analysis revealed the absence of FRAXA mutation. The healthy sister was
not available for analysis.


## 2.2. Cytogenetic and FISH Studies

Chromosome
analysis was performed on the proband’s and parent’s blood, using standard
high-resolution techniques. FISH was performed on the proband’s metaphase with BACs
that were selected according to the UCSC Genome Browser, and provided by BACPAC
Resource Centre (BPRC) and Pr. Mariano Rocchi (University of Bari,
Italy). BAC clones were biotinylated with biotin-11-dUTP (Sigma, Mo, USA)
by nick translation  using the BioNick labeling system (Invitrogen Life Technologies).


## 2.3. Bioinformatic Research

Bioinformatic
search was carried out using the UCSC Genome Browser to identify potential
autism candidate genes. The Genomatix Software was used for the promoter and the gene
structure predictions.


## 2.4. Expression Studies

Total RNA
was isolated and purified using the RNAgents Total RNA Isolation System
(Promega) from peripheral white blood cells and other human tissues (testis,
adult and foetal brain, fibroblasts, and kidney).1μg of total RNA was converted
to cDNA was used in 25μL reactions with AMV PCR Master Mix (Promega).A 4
nucleotides insertion in the 1st exon of the SLC6A8 paralogous gene let us to
design specific primers with Web Primer DNA program (insertion not found in
the SLC6A8 gene).RT-PCR
assays specific for the chromosome 16 paralogous SLC6A8 transcript and direct sequencing were done using these specific primers (5′-TACCGCTTCTTCTCGCGGCTCTTG-3′, and 5′-ATCGCGCGCGGCGGCACGGC-3′).


## 3. Results

The
G-band pattern established a 46, XY, t(7;16)(p22; p11)  karyotype (Figure1(a)). The mapping of the chromosome 7p22.1
breakpoint demonstrates that no gene was disrupted in the interval  (data not
shown).Figure 1
Physical mapping of the breakpoint on chromosome 16. (a) The patient derivative chromosomes 7 and 16 are shown. By comparison to the both respective ideogrammed chromosomes, the breakpoints were located in 7p22 and 16p11.2. (b) FISH analysis with the BAC RP11-264M14B22 (green) located in 16p11.2 and a centromeric probe of chromosome 7 (red) shows that this BAC is spanning the breakpoint on chromosome 7. (c) Physical map of genomic region 16p11.2 derived from the May 2006 version of the UCSC Genome Browser (http://www.genome.ucsc.edu/). The genomic clones covering the region flanking the translocation breakpoint are indicated by simple black lines. The arrow indicates the position of the translocation breakpoint with regard to the genomic clones. The red line represents the location of the predicted gene.As shown
in Figure1(c),  the RP11-264M14 BAC hy bridized both to derivative 7 and  derivative 16 and to the normal chromosome 16 but not to the normal chromosome 7 (Figure 1(b)), indicating that this clone
spans the translocation breakpoint on chromosome 16p11.2 (Figure 1(c)).The clone
RP11-264M14, on 16p11.2, was found to be devoid of known genes but it included
an entire mRNA predicted to be paralogous to the human creatine transporter
gene (SLC6A8) located on Xq28. The chromosome 16 paralogous gene is spanning
486 952 bp of chromosome 16 (33,205,876-33,692,827).Our bioinformatic
research ruled out with the Genomatix Software predicted for the paralogous gene a
structure of 13 exons with intron-exon junctions conforming to splice-site
consensus sequences (Table1).Table 1
Exon/intron
organization of the human SLC6A8 paralogous gene.
Exon no.Exon sizeSequence at exon/intron boundaries(bp)5′ donor3′ acceptor1266NACGGAGgtgag2132cccagGTGTGCAAAGgtgag3250cccagGCCTGTGGGAgtgag4133cctagGAACAGAAAGgtacc5135cccagATCGTCTCAGgtgag6103tctagGTATGTACAAgtaag7125cccagCAGCCGTCAGgtagg8113cacagGGCGGGCTTGgtctc9138cacagTTTGTCTGATgtgag10103cccagGGTGGGTATGgtagg11101cacagGAGCTGCATGgtaag12171tgtagGGCATCTGAGgtaag131984tgcagTGCTGNA* Intron nucleotides are in lowercase letters, exon
nucleotides are in capital letters.**NA: not applicable.The
predicted mRNA for this paralogous gene (XR_ 017514) spans 3300 bp with a coding
sequence of 1326 bp and an ORF of 441 amino acids.  A predicted 1414 bp promoter
is located between nucleotides 33, 684, 872, and 33, 686, 285 of the genomic
DNA sequence contig NT_010393. The cDNA sequence contained 1846 bp of5′-UTR
and 1230 bp of 3′-UTR.NCBI
BLAST homology search analysis revealed that the nucleotide sequence of this
paralogous gene is 97.1% similar to the SLC6A8 gene.RT-PCR
assays specific for the chromosome 16 paralogous SLC6A8 transcript and direct
sequencing by primers identified a specific expression in foetal and adult
brain and in testis. The results indicated no expression of this gene control
blood cells and fibroblasts (Figure2). Although we did not identify the actual
sequence of the breakpoint, we mapped it between 33.28 Mb and 33.31 Mb
corresponding to the promoter region.Figure 2
Expression pattern of the SLC6A8
paralogous gene. (RT-PCR was done on RNA extracted from various human tissues. L: ladder; T: testis; B: brain; fB: foetal brain; Bl: blood; F: fibroblast; K: kidney).


## 4. Discussion

Substantial
evidence suggests that chromosomal abnormalities contribute to autism risk but
the exact prevalence is unclear because literature surveys span different
diagnostic and cytogenetic approaches and sample sizes. Recent studies show rates
of detected chromosomal abnormalities in 5%–10% of affected
individuals [6].A genome-wide
analysis of structural variations ruled by Marshall et al. [7] identified an
autism susceptibility locus on 16p11.2 involving  deletions and duplications.
They reported that this copy number variation (CNV) region was found  at near to
1% in the studied autistic cohort and not in controls.A region
on chromosome 16p11.2 (from genomic coordinates 29.5 Mb to 30.1 Mb) was found
deleted de novo in autistic patients [8]. Kumar et al.  [9] suggested that the
16p11.2 microdeletion is one of the most common recurrent genomic disorders
associated with autism. The question to rise is whether these deletions removed
the coding regions of some genes. This
region is nearby of the translocation breakpoint characterized in our autistic
patient (from genomic coordinates 33.28 Mb to 33.31 Mb) corresponding to the
promoter region of the human creatine transporter gene (SLC6A8) paralogous.This  gene
has been localized to 16p11.2 by two studies [10, 11]. It is also known as
SLC6A10 or CT2  [12]. The mRNA sequence BC012355 reported by Strausberg et al.
[13] is 2168 bp with a coding sequence of 1908 bp and 636 amino acids
predicted.Eichler et al. [11] and
Höglund et al. [14] have identified the SLC6A10 gene as a pseudogene
with an early stop codon. However, this gene has been considered to be a
functional gene both in UniGene
at NCBI as well as in a review article by Chen et al. [15].This gene
was found specifically expressed in testis by Iyer et al. [10]. They suggested
that this gene function is critical for creatine transport into the cell to
ensure normal sperm mobility.Our data
extend this finding; this is the first time this phenotype has been reported
for the chromosome 16 SLC6A8 paralogous, and the first time expression of this
gene has been reported in brain as well as testis.The
SLC6A8 genes are members of a superfamily of pro teins that includes the family
of Na+ and Cl− dependent transporters responsible for the
uptake of certain neurotransmitters (e.g., dopamine, GABA, serotonin, and
noradrenaline) and amino acids (e.g., glycine, proline, and taurine) [16].Szatmari
et al. [6] reported that some members of the solute carrier family fall in
positive linkage regions for autism or close to linkage peaks. Even if they
have not glutamatergic synaptic function, some members affect brain development
and constitute excellent candidate genes for autism.There is
a high prevalence of SLC6A8 mutations in X-linked mental retardation. Common
features of X-linked mental retardation are neurological disturbances including
seizures, behavioral problems, speech delay, and inability to engage in structured
play [17]. Our patient was diagnosed with a high level of autistic behavior and
severe delay both in speech and in expressive language function. We
hypothesized that this phenotype is the result of the creatine transporter
paralogous disruption by the translocation breakpoint on 16p11.2.The
disruption of this gene may cause a decreased dosage responsible for specific
tissue and developmental effects leading to autistic traits.This data
is in agreement with the deletions on 16p11.2 reported by Marshall et al. and
carried only by autistic patients [7]. The authors suggested that the loss of
some genes in the interval is critical for the development of speech, language,
and communication.As gene
expression is the basis of many crucial functions in the cell, the hypothesis
is that perturbation in the network of genes by the translocation or the CNVs
in this specific region cause
the related pathology by dosage differences and underlie the autism phenotype. The
absence of expression of the predicted human creatine transporter paralogous
gene (SLC6A10) in accessible tissues makes it currently impossible to
investigate this further.Confirmation
of whether a defective chromosome 16 paralogous SLC6A8 gene causes autism
should come from observing rearrangements of this gene in other autistic
patients. Coding and intronic regions have to be screened to establish if
mutations in the SLC6A8 paralogous gene are the cause of autism in a fraction
of nontranslocation subjects. Validation of the candidate gene identified here
will rely on association studies in patients and families with autism.



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*Source: 609684-2008-05-19.xml*