Modeling rare gene variation to gain insight into the oldest biomarker in autism: construction of the serotonin transporter Gly56Ala knock-in mouse
© Springer Science+Business Media, LLC 2009
Received: 19 March 2009
Accepted: 25 May 2009
Published: 9 June 2009
Alterations in peripheral and central indices of serotonin (5-hydroxytryptamine, 5-HT) production, storage and signaling have long been associated with autism. The 5-HT transporter gene (HTT, SERT, SLC6A4) has received considerable attention as a potential risk locus for autism-spectrum disorders, as well as disorders with overlapping symptoms, including obsessive-compulsive disorder (OCD). Here, we review our efforts to characterize rare, nonsynonymous polymorphisms in SERT derived from multiplex pedigrees carrying diagnoses of autism and OCD and present the initial stages of our effort to model one of these variants, Gly56Ala, in vivo. We generated a targeting vector to produce the Gly56Ala substitution in the Slc6a4 locus by homologous recombination. Following removal of a neomycin resistance selection cassette, animals exhibiting germline transmission of the Ala56 variant were bred to establish a breeding colony on a 129S6 background, suitable for initial evaluation of biochemical, physiological and behavioral alterations relative to SERT Gly56 (wildtype) animals. SERT Ala56 mice were achieved and exhibit a normal pattern of transmission. The initial growth and gross morphology of these animals is comparable to wildtype littermate controls. The SERT Ala56 variant can be propagated in 129S6 mice without apparent disruption of fertility and growth. We discuss both the opportunities and challenges that await the physiological/behavioral analysis of Gly56Ala transgenic mice, with particular reference to modeling autism-associated traits.
The serotonin system is implicated in autism
Elevated whole blood serotonin (5-hydroxytryptamine, 5-HT) was first reported in autism by the lab of Daniel X. Freedman in 1961 , well before 5-HT was fully accepted as a neurotransmitter . Serotonin was discovered as a serum factor leading to vasoconstriction in 1948 by Maurice Rapport and colleagues . This work converged with that of Vittorio Erspaner, who identified in 1952 that 5-HT was the agent secreted by enterochromaffin cells previously designated as ‘enteramine’ . Subsequent work demonstrated that 5-HT synthesized in enterochromaffin cells is released into the enteric circulation and taken up by platelets, which contain more than 99% of the 5-HT in peripheral blood . Due to the vastly greater amount of 5-HT contained in platelets, it is challenging to measure free 5-HT in plasma, and it is unclear if plasma 5-HT levels are changed in autism [6, 7]. Thus, hyperserotonemia appears to arise from elevated platelet stores of 5-HT.
Nearly fifty years have passed since the initial description of whole blood 5-HT as a biomarker in autism, described sixteen years earlier by Leo Kanner . Subsequent studies have repeatedly demonstrated that more than one-quarter of autistic children are found to have elevated whole blood 5-HT levels , and, indeed, hyperserotonemia is arguably the most highly reproduced biomarker linked to a neurobehavioral disorder. A recent study in a more homogeneous Dutch population suggests that whole blood 5-HT may actually show a bimodal distribution in autism . Despite the long-standing observation of hyperserotonemia in autism, our understanding of its origin and relationship to the etiology of autism remains incomplete.
Functional studies of the platelet serotonin system in autism have evaluated both the serotonin transporter and serotonin receptors. The 5-HT transporter (5HTT, SERT) is essential for the uptake of the biogenic amine into the platelet. Some but not all studies indicate alterations in platelet 5-HT uptake as well as SERT binding levels in autism [11–19]. Some studies have also shown decreased platelet serotonin receptor 5-HT2 binding [20–22], which could possibly influence platelet 5-HT levels by modulation of platelet activation and release. Of note, recent work from our laboratory indicates that the 5-HT2A receptor can modulate SERT associations with the platelet membrane skeleton . Inconsistency in transporter and receptor studies in autism likely reflects the complex regulatory systems that control platelet 5-HT utilization as well as platelet activation. Certainly, an important consideration is the regulation of gut production of 5-HT and its regulation by intrinsic signaling and transport mechanisms [24–28].
The robustness of hyperserotonemia as an autism biomarker has helped focus research on the genetic control of platelet 5-HT levels, with the belief that common regulatory mechanisms in the platelet and the central nervous system could reveal genes important to autism susceptibility. Indeed, one small study found higher whole blood 5-HT levels in children with ASD who had an affected sibling than in children with ASD but no affected siblings . In the Hutterites, a large founder population not ascertained for brain disorders, whole blood 5-HT levels were found to be under nearly complete genetic control, with a heritability of 0.99 . In a small twin study, the maximum rate of 5-HT uptake was also found to be highly heritable . The Cook lab has used linkage and association to map quantitative trait loci for whole blood 5-HT levels in the Hutterites . The initial study found significant association at a functional polymorphism in the integrin β3 subunit gene (ITGB3) as well as suggestive association at the Vitamin D receptor gene . Follow-up analyses revealed that ITGB3, as well as the 5-HT transporter gene (SLC6A4), were primarily associated with whole blood 5-HT levels in males . Carneiro and colleagues subsequently demonstrated physical and functional interactions between the ITGB3 and SERT proteins that regulates both platelet 5-HT uptake and aggregation . Consistent with these findings, three studies have now reported a gene-gene interaction between ITGB3 and SLC6A4 in association with autism [34–36], supporting the idea that whole blood 5-HT represents a genetically-determined, intermediate phenotype in autism that can be mined for elucidation of disease susceptibility genes.
Recent data support the idea that the 5-HT system is also altered in the brain in autism. If the peripheral findings translate into the brain, increased 5-HT uptake or storage in the presynaptic terminal would be expected to diminish extracellular 5-HT availability. Unfortunately, a direct assessment of synaptic 5-HT in patients with autism is not possible with present technologies. A surrogate measure is the assessment of brain 5-HT synthesis, and indeed this measure demonstrates an altered developmental pattern in autism . Other studies have evaluated 5-HT receptor binding. Two neuroimaging studies have found decreased 5-HT2 receptor binding, one a SPECT study in adults with Asperger’s syndrome  and the second a PET study in parents of children with autism . Blatt’s group has also presented data demonstrating decreased 5-HT2A binding in post-mortem samples from autism subjects . The overall consistent findings of decreased 5-HT2 receptor binding in platelet, neuroimaging, and post-mortem studies supports the hypothesis that peripheral alterations in 5-HT systems contribute to the behavioral abnormalities associated with autism.
Finally, limited pharmacology studies underscore a role for the 5-HT system in autism. Dietary depletion of the 5-HT precursor tryptophan, which is expected to cause decreased synaptic 5-HT, leads to worsened repetitive behaviors and irritability in autism . Consistent with these findings, several studies have reported that 5-HT reuptake inhibitors can relieve symptoms of irritability and rigid-compulsive behavior in autism [42, 43]; more recent data from clinical trials are less supportive, but the wide spectrum of autism traits complicates selection of appropriate target symptoms in these trials. More consistent data supports the use of risperidone [44, 45], an atypical antipsychotic with antagonism at multiple monoamine receptors, including the 5-HT2A receptor. It is striking that this agent has clinical utility in autism despite findings of reductions in 5-HT2A receptor levels. This discrepancy may be an important clue that a primary effect impacting 5-HT2A receptor sensitivity in these subjects may exist which may then trigger compensatory receptor down-regulation.
Rare variants offer one way to approach complexity at the SERT gene
Multiple lines of evidence favor involvement of the 5-HT transporter gene (SLC6A4) in autism; although the cumulative data are not straightforward or uniform in their support, which is perhaps not surprising in a complex behavioral disorder with a polygenic origin. Most of this evidence has emerged from candidate gene studies oriented around the platelet 5-HT system. Importantly, linkage studies have also implicated the chromosome 17q11-q21 region containing SLC6A4 [46, 47]. Follow-up linkage studies [48–50] have detected more significant evidence for linkage in this region when considering families with two or more affected male children and no affected females. These findings suggest that SERT might harbor sex-specific risk alleles for autism and possibly contribute to, or be responsive to, the gender bias of autism. Such an idea is in line with the findings that variation in SLC6A4 is primarily associated with whole blood 5-HT levels in males . It also matches accumulating evidence in the 5-HT transporter knockout mouse that altered expression of SLC6A4 has sexually dimorphic effects on brain 5-HT systems and resulting behavior (reviewed in ).
With the accumulated evidence of alterations in the platelet 5-HT system in autism, it is not surprising that some of the earliest candidate gene association studies in autism focused on the SERT gene. Unfortunately, studies of SLC6A4 in autism have been complicated by the presence of multiple common functional variants in gene regulatory regions. The best-studied of these variants, the promoter length polymorphic repeat (5-HTTLPR) , was initially reported as associated with autism in haplotype analyses by the Cook lab . Subsequent studies have yielded inconsistent findings. Indeed, although most studies have reported association with the short allele, which leads to less gene transcription, others have reported association with the long allele (reviewed in a recent meta-analysis ). Genotyping errors could possibly account for some of this inconsistency given deviation from Hardy-Weinberg equilibrium in some samples and discrepancies in genotypes of the same samples across different laboratories .
Other studies have explored the possible involvement of SLC6A4 in autism by expanding the range of polymorphisms studied. Indeed, multiple, additional functional polymorphisms have been discovered within the SLC6A4 gene regulatory regions [56–58], including a single nucleotide polymorphism (SNP) that reduces the functional impact of the 5-HTTLPR long allele (La, Lg) . To date, these polymorphisms have not been systematically studied in large autism samples. Other SLC6A4 polymorphisms and haplotypes that have been reported to associate with autism have not been fully characterized for either their own functional significance or linkage disequilibrium with other possible functional variants [59, 60]. Indeed, one haplotype in linkage disequilibrium with the 5-HTTLPR short allele, which leads to decreased SERT expression, has actually been associated with increased platelet 5-HT uptake . These findings signify a still incomplete understanding of SERT genetic regulation. A comprehensive analysis focusing on the impact of multiple alleles on predicted SERT expression or function should greatly enhance our understanding of the SERT-serotonin-autism connection.
Another fruitful approach to understanding the possible involvement of SLC6A4 in autism susceptibility is to focus on either specific symptom clusters or on intermediate phenotypes. Four studies have reported association at various SLC6A4 polymorphisms when performing sub-analyses of repetitive behavior in autism [49, 60, 62, 63]. One of these studies also reported association of the 5-HTTLPR short allele with poor non-verbal communication . Two studies have reported association of various SLC6A4 polymorphisms with either whole blood 5-HT  or with 5-HT uptake in autism . Finally, one study reported association of the 5-HTTLPR short allele with increased cortical gray matter in two- to four-year-old children with autism . These collected studies of subphenotypes or intermediate phenotypes add strength to the hypothesis that SLC6A4 represents a susceptibility gene for subgroups of children with autism.
One intriguing possibility to emerge from the data on common SLC6A4 variants is that optimal SERT activity is likely to be highly constrained, with both high and low SERT activity impacting autism susceptibility, though likely with different patterns of symptoms and intermediate phenotypes. Such an idea parallels ideas emerging from recent findings at the MECP2 gene implicated in Rett syndrome . Additionally, this idea fits with the observation that genetic variation can lead to increased SERT function in association with repetitive behavior in autism [32, 46], as well as with OCD [56, 57, 67]; whereas genetic variation that supports decreased SERT function is associated with impaired social behavior  and increased cortical size in young children with autism , as well as in a mouse model of PTEN-associated autism .
Association of human SERT gene variants with autism traits
Despite intriguing findings of both common and rare functional SLC6A4 variants, the collective evidence does not come close to explaining the strong linkage findings in male sibling pairs in this chromosomal region [48–50]. Indeed, the collected evidence suggests that no single, common functional polymorphism at this locus underlies autism susceptibility, at least when considered individually. Recently, the Blakely and Sutcliffe labs explored the alternative hypothesis that multiple, rare functional variants account for some of the findings of linkage in this region. In a comprehensive analysis of SERT noncoding and coding exons from 120 families that contributed most to linkage at 17q11.2, our groups found 5 coding substitutions and 15 variants in 5´ noncoding and intronic regions transmitted in families . In the aggregate, these variants showed significant association to autism and tracked specifically with rigid-compulsive traits.
In the initial study, one of the autism-associated variants, Gly56Ala, showed constitutively elevated SERT activity that was not further up-regulated through the PKG and p38 MAPK pathways in transformed lymphoblasts from autism probands . This polymorphism, at an amino acid that is conserved across all mammalian SERTs, was the only SERT variant frequent enough to attempt independent statistical analyses, which revealed that the Ala56 allele: 1) was overtransmitted 3:1 to subjects with autism (P = 0.025), 2) displayed a gender-bias (males) when considering affected subjects within linked pedigrees (P = 0.017), and 3) was associated with Autism Diagnostic Interview component factors corresponding to rigid-compulsive traits (P = 0.0085) and sensory aversion (P = 0.0005). The SERT amino-terminus where Gly56 resides is an important regulatory area [69–71]. These considerations make the Gly56Ala allele an important allele to model for system level effects in an animal model (see below).
Two of the SERT coding variants found in autism subjects in our studies, Gly56Ala and Lys605Asn, were both shown to exhibit a gain of function despite normal surface expression . Our autism studies uncovered three additional SERT coding variants: Ile425Leu, Phe465Leu and Leu550Val. All three variants occur within transmembrane domains at amino acids that are highly conserved . Rigid-compulsive behaviors were associated with these three novel SERT variants when analyzed as a group (P = 0.0003) . When all linked families with individuals carrying a coding variant were considered together, the significance in elevated severity of rigid-compulsive behaviors increased (P = 0.0006).
Even these rare variants are unlikely to present a simple story given the presence in the SERT gene of multiple common functional variants that can affect gene expression. It is entirely possible that the functional effects of these rare variants, particularly the more subtle increased activity and dysregulation of Gly56Ala, could be partially offset in some individuals by common variants leading to decreased SERT expression or variation in other genes within 5-HT signaling networks. Indeed, one subsequent study did not find association of rare SERT variants with autism or rigid-compulsive behavior in families without evidence for linkage to this region . Another study did not find association of the Ala56 allele when considered alone in OCD ; although it is quite possible that these seemingly related disorders arise from distinct origins. Regardless, the discovery of functional amino acid variants in SERT offers a critical opportunity to translate altered SERT activity into an animal model for comprehensive assessment of genotype/phenotype assessments beyond what is feasible in humans.
In vitro analysis of autism-associated SERT coding variants
Of the five coding variants that we identified in probands from multiplex autism families, two of these, Gly56Ala and Lys605Asn, were actually first characterized, along with eight other rare SERT variants, independent of their potential roles in autism [72, 75]. In comparison to the reference hSERT cDNA (often termed “wild-type”) originally identified by Ramamoorthy and colleagues , both Ala56 and Asn605 demonstrated elevated 5-HT transport activity in transfected HeLa cells . This gain-of-function transport property was shared (and even exceeded) by several other SERT coding variants including Ile425Val, a variant reported by Ozaki and colleagues in association with two pedigrees bearing a complex neuropsychiatric phenotype including OCD, autism spectrum disorder, social phobia, and alcohol abuse . Since these studies, additional cases of Val425 in OCD have been reported . Clearly, the identification of two mutations at the same site (Val425 vs Leu425) in disorders with trait overlap increases the probability that both are functional and relevant to disease status.
Rare serotonin transporter coding variants identified in autism and autism spectrum (Jeremy)
Effect on Function
N-terminal intracellular tail
~70% increase in 5-HT uptake; increased cell surface expression 
~70% increase in 5-HT uptake; increased cell surface expression 
~70% increase in 5-HT uptake; increased cell surface expression 
C-terminal intracellular tail
~30% increase in 5-HT uptake 
Ideally, one would like to evaluate the impact of SERT coding variants on the brain of autism subjects. While we hope to pursue this need with transgenic mice, it seems unlikely that such analyses in humans will be available in the near future. It is well known, however, that SERT is expressed in transformed lymphoblasts , the very same models used to archive DNA from subjects recruited for genetic studies. We took advantage of this opportunity to examine basal transport activity and regulation in cells derived from autism subjects. Indeed, Ala56 , as well as the other autism-associated SERT variants, display elevated 5-HT transport activity (Fig. 1). As a control in the latter studies, L-glutamate transport was found to be equivalent to that in wildtype SERT, consistent with alterations derived from a specific influence on SERT expression and/or regulation. Interestingly, although transformed lymphoblasts do not express a vesicular storage mechanism for 5-HT (to our knowledge), SERT expression is low in these cells relative to platelet and raphe neurons, such that the growth perturbations noted above for stable CHO-Flp-In cells do not appear to reduce our ability to detect 5-HT transport enhancement. Studies with multiple lymphoblast lines expressing the Ala56 variant  also reveal that activators of PKG and p38 MAPK display the loss of kinase sensitivity seen with transfected HeLa cells. Together, these findings reinforce the properties of autism-associated SERT variants as gain-of-function mutants that also display complex regulatory deficits.
In vivo manipulations of the mouse SERT gene
A number of methods are available to manipulate genes in the mouse, including gene knockout, regional or inducible gene knockout, and transgenic overexpression, including inducible and reversible systems. The first knockout of the mouse SERT gene was performed by deletion of exon 2 in the labs of Dennis Murphy and Klaus-Peter Lesch . Over two hundred papers have now been published describing this mouse’s phenotype, which includes increased anxiety-like behavior, increased stress reactivity, decreased aggression, obesity, and disruption of brain architecture in the sensory cortex and amygdala (reviewed in ). Despite seeing decreased platelet 5-HT levels, the opposite of what is observed in the hyperserotonemia of autism, these mice also show decreased sociability in the Crawley test . Interestingly, when bred with mice haploinsufficient for Pten, a putative autism susceptibility gene, SERT +/− knockout mice show an exacerbation of the macrocephaly and decreased sociability characteristic of the Pten +/− mice . These findings may be consistent with the idea that SERT activity may be constrained within a certain range, with elevated or diminished SERT function leading to vulnerability to autism-like behavior.
Another genetic manipulation was performed in a mouse line expressing a human SERT transgene generated in the lab of Trevor Sharp . These mice show a 2- to 3-fold increase in SERT binding sites and a resulting decrease in extracellular 5-HT. Their platelet 5-HT levels have not yet been assessed. Providing a contrast to the SERT knockout mice, these hSERT transgenic overexpressing mice show a decrease in anxiety-like behavior that reverses with acute treatment with a 5-HT reuptake inhibitor. Furthermore, they show increased sensitivity to 5-HT2 receptor stimulation , again in contrast to the SERT KO mice, which show decreased sensitivity . This mouse could potentially serve as a surrogate model of the increased SERT function hypothesized in autism and OCD, but the use of a human SERT locus introduced using a yeast artificial chromosome construct may lead to alterations in gene regulation and expression patterns, as well as altered protein regulation and function, when introduced into the mouse.
Based upon our belief that manipulation of a native gene was likely to yield a meaningful brain and behavioral read-out of increased SERT function, we chose to introduce a knock-in of the Ala56 allele into the native mouse SERT gene. We chose the Ala56 allele for three reasons: 1) it shows association with rigid-compulsive behavior and sensory aversion in autism; 2) it is the most common amino acid variant of human SERT; 3) it offers an opportunity to probe the importance of altered SERT regulation in vivo.
Production of mSERT Gly56Ala knock-in mouse
Genotyping and initial growth analysis of SERT Ala56 knock-in mice
At two weeks of age, a 3–5 mm section of tail was removed for DNA extraction using the Sigma REDExtract-N-Amp Tissue PCR Kit (Sigma-Aldrich, St. Louis, MO, http://www.sigmaaldrich.com). Each PCR reaction contained 2 μL of tail DNA extract, 5 μL of Sigma REDExtract-N-Amp PCR Reaction Mix, 0.5 μL of each of the two 25-μM primers (Sense 5′-cctggccctcttagcaggtt-3′; Antisense 5′- aggaggagaagccagcaagg-3′), and 2 μL of water, for a 10-μL total reaction volume. All of the PCR reactions were performed on an MJ Research DNA Engine PTC-200 thermocycler (Bio-Rad, Hercules, CA, http://www.bio-rad.com) under the following conditions: initial denaturation step of 94 ° C for 2 min; followed by 5 cycles of three alternating steps (94 ° C for 30 sec, 63 ° C for 45 sec, and 72 ° C for 60 sec); followed by 35 cycles of three alternating steps (94 ° C for 30 sec, 61 ° C for 45 sec, and 72 ° C for 60 sec); followed by one final extension step of 72 ° C for 8 min. Final PCR products were analyzed by horizontal gel electrophoresis in 1% agarose (GenePure LE, ISC BioExpress, Kaysville, UT, http://www.bioexpress.com). The PCR product from the wildtype 129S6 SERT gene allele is 642 bp long, and the PCR product from the mutant 129S6 SERT Ala56 allele is 752 bp long. The χ2 test was used to evaluate deviation from expected Hardy-Weinberg equilibrium allele frequencies.
Pups from SERT 56Gly/Ala heterozygote matings were weighed weekly from one week of age until eight weeks of age. Repeated Measures Analysis of Variance was used to evaluate sources of variation between mice of different genotypes over time.
Results and discussion
Genotype distribution of Gly56Ala offspring from heterozygous breeding pairs
Pups from 56Gly/Ala breeding
With the successful generation of both homozygous Ala/Ala mice as well as heterozygous carriers, our thoughts now turn to analyses that are predicted by trait associations in children possessing the variant, as well as biochemical and behavioral traits seen in autism more generally. Modeling the SERT Ala56 variant in transfected cells and in transformed cells has identified an intriguing phenotype of increased baseline activity and failure to further upregulate via the PKG and p38-MAPK pathways. In vivo modeling of this variant in a knock-in mouse will allow us to answer a number of questions, ranging from basic to translational. We will need to verify that mRNA and protein expression patterns resulting from the introduction of this knock-in allele will parallel native mouse SERT. We will want to establish that the SERT Ala56 in vitro phenotype of increased transport is recapitulated in the mouse, initially using synaptosome preparations or platelets and progressing to in vivo assays. We will also evaluate whether the biomarker of elevated platelet 5-HT is present in the SERT Ala56 mouse.
After initial studies establish the characteristics of the mouse SERT Ala56 variant in vivo, a number of intriguing questions await. Some of these questions correspond to establishing face validity for behavioral traits that map onto autism symptoms (reviewed in ). Will the SERT Ala56 mice show alterations in social behavior ? Will they show repetitive behavior, perseverative exploration, or deficits in reversal learning, paralleling the association of SERT Ala56 with rigid-compulsive behavior ? Will they show increased sensory sensitivity, paralleling the association with sensory aversion ? Other questions relate to predicted behavioral changes due to altered SERT function. Will SERT Ala56 mice show altered response to pharmacological probes of the 5-HT system, including 5-HT reuptake inhibitors and 5-HT receptor agonists? Might these mice help resolve the conundrum of replicated reductions in 5HT2 receptor density but utility of the 5HT2a antagonist risperidone? Will they demonstrate changes in anxiety or stress reactivity that contrast with the SERT knockout mouse ?
Our efforts to establish a behavioral readout of altered SERT function will rest on an understanding of underlying changes in brain development, synaptic function and signaling. Indeed, we believe this can help address the impact of SERT variation on brain architecture. Our initial focus will be targeted to brain regions that are altered in the SERT knockout mouse [91, 92] and/or those regions that may act as substrates for the behavioral alterations found in individuals with the SERT Ala56 variant. We then hope to advance from any observed changes in brain architecture to elucidate the underlying alterations in neurodevelopment that lead to these changes. Ultimately, our hope would be not just to characterize a brain and behavioral phenotype in the SERT Ala56 knock-in mouse, but also to capitalize on the model to develop novel methods of reversing the phenotype, either genetically or pharmacologically.
We have entered a new era in autism research with the transfer to animal models of functional human mutations linked to autism symptom domains. Time will tell if our efforts help unravel the complexities of autism, including the well known male bias, the long-standing biomarker of hyperserotonemia, and the seemingly independent trait domains that have been suggested to arise from distinct genetic mechanisms . We have optimism that success in this venture can advance critical new models for the design of gene- or biomarker-based therapeutics.
We gratefully acknowledge the input and support of Ana Carneiro, Chong-Bin Zhu, David Airey and investigators/staff of the Vanderbilt Silvio O. Conte Center for Neuroscience Research. We also appreciate the expert laboratory oversight provided by Jane Wright, Qiao Han, Angela Steele, Sarah Whitaker, and Kathryn Lindler. Research efforts were supported by NIH Award K08MH081066, an American Academy of Child and Adolescent Psychiatry Pilot Research Award and an Autism Speaks Pilot Award to J.V., a Scientist-Educator Award to B.J.T, a NARSAD Young Investigator Award to H.C.P. and NIH Awards NS049261 to J.S.S., DA07390 and MH078028 to R.D.B., and HD15052 for support of the Vanderbilt Transgenic Mouse/ESC Shared Resource.
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