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Knockout of Tmlhe in mice is not associated with autism spectrum disorder phenotypes or motor dysfunction despite low carnitine levels


Deletion of exon 2 of the trimethyllysine hydroxylase epsilon (TMLHE) gene was identified in probands with autism spectrum disorder (ASD). TMLHE encodes the first enzyme in carnitine biosynthesis, N6-trimethyllysine dioxygenase (TMLD). Researchers have suggested that carnitine depletion could be important for the development of ASD and cognitive, locomotor and social dysfunctions, but previous findings have been inconclusive regarding the specific role of endogenous carnitine. We developed a mouse knockout model with constitutive TMLD enzyme inactivation that exhibited a significant decrease in the carnitine by more than 90% compared to wild-type (WT) mice. However, we did not observe any significant social, cognitive, or repetitive-behavior changes associated with ASD in the knockout mice; muscle strength and coordination were also not affected. In addition, the life expectancy of knockout mice was similar to that of WT mice. In conclusion, knockout of Tmlh in mice does not induce an ASD phenotype or motor dysfunction despite extremely low carnitine and gamma-butyrobetaine concentrations. Moreover, inactivation of TMLD does not induce a phenotype similar to previously described primary carnitine deficiency; indeed, our results showed that low levels of carnitine sustained adequate energy production, muscle function and social behavior in mice.


A search for genetic variants throughout the genome in individuals with autism spectrum disorder (ASD) and their families identified a novel deletion of exon 2 of the X-chromosomal trimethyllysine hydroxylase epsilon (TMLHE) gene [1]. Further studies identified such deletions in 16 probands with ASD, and these deletions are common in autistic but also healthy males [2]. This deletion results in loss of N6-trimethyllysine dioxygenase (TMLD) activity and the absence of a TMLD protein as well as metabolic abnormalities in plasma and urine [2]. TMLHE encodes the first enzyme in carnitine biosynthesis, TMLD, and enzyme deficiency was suggested to be a risk factor for ASD [2, 3]. Furthermore, this finding prompted speculation that carnitine metabolism is a target for therapeutic intervention [2]. Since mitochondrial dysfunction is associated with ASD [3], it was hypothesized that lower carnitine levels and changes in acylcarnitine profiles might be related to mitochondrial dysfunctions and abnormal fatty acid metabolism induced by deficiencies of carnitine synthesizing enzymes [2]. It has been speculated that this mechanism explains the many links of ASD with disturbed mitochondrial functions [4].

Carnitine is important for the mitochondrial metabolism of long- and medium-chain fatty acids. Endogenous carnitine biosynthesis begins with the TMLD-driven oxidation of TML and is then completed in three subsequent steps of enzyme-catalyzed reactions (5). The carnitine level in tissues is regulated by organic cation transporter novel type 2 (OCTN2, SLC22A5), which maintains carnitine homeostasis and ensures high tissue levels [6]. The main function of carnitine is the shuttling of long-chain fatty acid moieties across the inner mitochondrial membrane into the matrix for β-oxidation [6]. Therefore, carnitine supplementation is suggested for the treatment of several diseases related to insufficient mitochondrial activity [6].

In patients, primary carnitine deficiency is associated with inborn errors of carnitine biosynthesis enzymes, e.g., TMLD or a carnitine transporter. OCTN2 knockout (KO) mice are characterized by a 50% reduction in carnitine concentration, steatosis, hypoglycemia, intestinal ulcer formation, and decreased spontaneous activity, resulting in a 10% survival rate by one month after birth [7, 8]. However, in human studies of both children and adults, primary carnitine deficiency is characterized by highly variable clinical symptoms. The severity of symptoms varies from severe cardio-, hepato- and encephalopathy pathologies [6] to an asymptomatic course of disease in some individuals despite extremely low carnitine levels (i.e., 1–2 µM) in their plasma [9, 10]. These discrepancies might indicate that confounders are more important to the clinical manifestations of disorders than low carnitine levels per se.

To determine the impact of TMLD enzyme activity on the development of primary carnitine deficiency, we inactivated (knocked out) the Tmlhe gene (Tmlhe) in mice to study constitutive TMLD enzyme deficiency-induced changes in the levels of carnitine and its biosynthetic intermediates TML and γ-butyrobetaine (GBB) in plasma and brain tissue. In addition, we assessed ASD-related behavioral patterns of Tmlhe-KO and wild-type (WT) mice in a range of social behavior, cognitive and motor function tests.

Materials and methods

A detailed description of methods is available in Additional file 1.

Development of the Tmlhe-KO mouse model

The Tmlhe-KO mouse model was created using the CRISPR/Cas9 gene-editing protocol to generate random mutations [11]. Adult 11 weeks old C57BL/6 N mice (WT: 9 males and 14 females; Tmlhe-KO: 10 males and 11 females) were housed under standard conditions and tested for behavioral changes up till week 18. All WT (+/+) mice used in experiments were littermates of the KO mice. The experimental procedures were performed in accordance with the guidelines of the European Community and local laws and policies (Directive 2010/63/EU), and all of the procedures were approved by Food and Veterinary Service, Riga, Latvia.

Levels of TML, GBB, and carnitine

TML, GBB and carnitine levels in plasma and brain tissue samples were measured by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC‒MS/MS) in positive ion electrospray mode [5, 12].

Behavioral tests to evaluate social behavior and motor function

We performed the commonly used tests to determine the mouse behavioral profile associated with ASD: three-chamber social, marble burying, and nest shredding tests [13]. Passive avoidance and Y-maze tests were used to determine cognitive function [14, 15]. Grip strength and rotarod tests were performed to assess muscle strength and coordination.

Results and discussion

TML, GBB, and carnitine levels were substantially changed in both the plasma and brain tissues of Tmlhe-KO compared to those in their WT counterparts (Fig. 1), which is in line with the predicted functionality of the TMLD enzyme. We found up to 3.5-fold higher TML concentrations in plasma (1.51 ± 0.02 KO vs. 0.62 ± 0.10 WT nmol/ml) and brain tissue (37.50 ± 0.65 KO vs. 9.86 ± 0.26 WT nmol/g) of Tmlhe-KO mice than those of WT mice. GBB is an important intermediate metabolite in the carnitine synthesis pathway [16]. Since GBB was not detectable in Tmlhe-KO mouse plasma and brain tissues, we concluded that the carnitine synthesis pathway was severely interrupted in these animals. As a result, carnitine concentrations in Tmlhe-KO mouse plasma (0.92 ± 0.07 KO vs. 26.50 ± 1.93 WT nmol/l) and brain tissue (10.91 ± 0.39 KO vs. 121.1 ± 4.44 WT nmol/g) were significantly lowered to 90% of those in WT mice. Alterations in TML, GBB, and carnitine concentrations were observed in both male and female Tmlhe-KO mice. The Tmlhe-KO mice fully mimicked the most severe manifestations of inherited TMLHE gene deficiency in humans according to the carnitine, GBB, and TML levels.

Fig. 1
figure 1

TML, GBB, and carnitine concentrations in WT and Tmlhe-KO mouse brains (A) and plasma (B). Concentrations of GBB and carnitine were significantly lower in KO mice than in WT mice. Each value is the mean (± SEM) of 5–8 mice. **p < 0.0001 compared to the WT group (two-way ANOVA followed by unpaired t-test). Coresponding F and p values are: plasma TML F3, 16 = 28.48, p < 0.0001; plasma GBB F3, 16 = 22.62, p < 0.0001; plasma carnitine F3, 16 = 168.6, p < 0.0001; brain TML F3, 25 = 741.5, p < 0.0001; brain GBB F3, 24 = 457.6; p < 0.0001, brain carnitine F3, 25 = 479.6; p < 0.0001

We also compared the performance of the WT and Tmlhe-KO mice in a range of social behavior and motor function tests. In the three-chamber social test, Tmlhe-KO female and male mice displayed robust sociability by spending more time sniffing the novel mouse than the novel object (Table 1). In the marble-burying test, we observed that all mice were interested in marbles; there was no difference in marble burying between WT and Tmlhe-KO mice (Table 1). Nest-building behavior in the nest-building test also was unchanged in Tmlhe-KO mice (Table 1). Furthermore, Tmlhe-KO female mice demonstrated normal maternal behavior, including licking of pups, nest building, and crouching over grouped pups. We also tested mouse grip strength and performance on the accelerating rotarod test because both carnitine deficiency and ASD are associated with impairments in muscle strength and motor coordination [3]. The forelimb grip strength and rotarod performance of both male and female Tmlhe-KO mice was similar to WT mice, indicating that muscle strength and coordination in Tmlhe-KO mice were fully preserved despite very low carnitine concentrations (Table 1, Fig. 1). Patients with ASD might have variable degrees of intellectual disability [13]; therefore, we examined both working and contextual memory in mice using the Y-maze and passive-avoidance tests, respectively. We did not observe any differences in cognitive function between Tmlhe-KO mice and WT mice (Table 1). These results also confirmed that low levels of carnitine did not induce behavioral changes characteristic of ASD.

Table 1 WT and Tmlhe-KO mouse performance in social behavior, motor function and cognition tests

ASD has been associated with mitochondrial energy failure [3]. A retrospective study reported that 5% (vs. 0.01% in control) of children with ASD had a mitochondrial disease that was determined by abnormal concentrations of biomarkers such as increased plasma levels of lactate, alanine and acylcarnitines, which were suggested as markers of mitochondrial dysfunction [3]. The alanine plasma concentration was not increased in the Tmlhe-KO mice, but concentrations of lactate and acylcarnitines were significantly decreased, suggesting more efficient utilization of energy metabolism substrates in these mice [11]. Compared to WT mice, Tmlhe-KO mice exhibited improved mitochondrial functionality, particularly the OXPHOS-dependent respiration rate and OXPHOS coupling efficiency, in several tissues [11]. In a previous study, we reported that mitochondria in Tmlhe-KO mice are better protected against ischemia‒reperfusion-induced damage [11]. In contrast to OCTN2-deficient JVS mice [7, 8], signs of cardiac, muscle, and liver dysfunction were not observed over the 2-year lifespan of Tmlhe-KO mice, similar to WT mice (Table 1) [11]. This suggests that the transporter functions of carnitine or other organic cations might be more important for mitochondrial functionality than low levels of carnitine alone.


Despite the robust findings in the Tmlhe-KO mouse model, patients can be more sensitive to very low carnitine levels.


In conclusion, constitutive Tmlhe gene inactivation in mice did not induce an ASD phenotype or motor dysfunction despite extremely low carnitine and GBB concentrations. The typical manifestations of primary carnitine deficiency were not induced by TMLD dysfunction, and low levels of carnitine did not result in impaired motor functions or any obvious ASD-like behavior.

Availability of data and materials

All data generated or analyzed during this study are included in this published article and additional files (see Additional file 2). Any additional information related to the current study is available from the corresponding author on reasonable request.



Autism spectrum disorder


Trimethyllysine hydroxylase epsilon gene


N6-trimethyllysine dioxygenase




Trimethyllysine hydroxylase epsilon gene knockout mice


Organic cation transporter novel type 2




Ultra-performance liquid chromatography-tandem mass spectrometry


Oxidative phosphorylation


Juvenile visceral steatosis


  1. Celestino-Soper PB, et al. Use of array CGH to detect exonic copy number variants throughout the genome in autism families detects a novel deletion in TMLHE. Hum Mol Genet. 2011;20:4360–70.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Celestino-Soper PB, et al. A common X-linked inborn error of carnitine biosynthesis may be a risk factor for nondysmorphic autism. Proc Natl Acad Sci USA. 2012;109:7974–81.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Rossignol DA, Frye RE. Mitochondrial dysfunction in autism spectrum disorders: a systematic review and meta-analysis. Mol Psychiatry. 2012;17:290–314.

    Article  CAS  PubMed  Google Scholar 

  4. Weissman JR, et al. Mitochondrial disease in autism spectrum disorder patients: a cohort analysis. PLoS ONE. 2008;3: e3815.

    Article  PubMed  PubMed Central  Google Scholar 

  5. Kazaks A, et al. Expression and purification of active, stabilized trimethyllysine hydroxylase. Protein Expr Purif. 2014;104:1–6.

    Article  CAS  PubMed  Google Scholar 

  6. Longo N, Amat di San Filippo C, Pasquali M. Disorders of carnitine transport and the carnitine cycle. Am J Med Genet C Semin Med Genet. 2006;142C:77–85.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Knapp AC, Todesco L, Torok M, Beier K, Krahenbuhl S. Effect of carnitine deprivation on carnitine homeostasis and energy metabolism in mice with systemic carnitine deficiency. Ann Nutr Metab. 2008;52:136–44.

    Article  CAS  PubMed  Google Scholar 

  8. Kuwajima M, et al. Reduced carnitine level causes death from hypoglycemia: possible involvement of suppression of hypothalamic orexin expression during weaning period. Endocr J. 2007;54:911–25.

    Article  CAS  PubMed  Google Scholar 

  9. Spiekerkoetter U, et al. Silent and symptomatic primary carnitine deficiency within the same family due to identical mutations in the organic cation/carnitine transporter OCTN2. J Inherit Metab Dis. 2003;26:613–5.

    Article  CAS  PubMed  Google Scholar 

  10. Rasmussen J, Kober L, Lund AM, Nielsen OW. Primary Carnitine deficiency in the Faroe Islands: health and cardiac status in 76 adult patients diagnosed by screening. J Inherit Metab Dis. 2014;37:223–30.

    Article  CAS  PubMed  Google Scholar 

  11. Liepinsh E, et al. Low cardiac content of long-chain acylcarnitines in TMLHE knockout mice prevents ischaemia-reperfusion-induced mitochondrial and cardiac damage. Free Radic Biol Med. 2021;177:370–80.

    Article  CAS  PubMed  Google Scholar 

  12. Liepinsh E, et al. Inhibition of L-carnitine biosynthesis and transport by methyl-gamma-butyrobetaine decreases fatty acid oxidation and protects against myocardial infarction. Br J Pharmacol. 2015;172:1319–32.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Drapeau E, Riad M, Kajiwara Y, Buxbaum JD. Behavioral phenotyping of an improved mouse model of phelan-mcdermid syndrome with a complete deletion of the Shank3 gene. eNeuro. 2018;5.

  14. Zvejniece L, et al. The cognition-enhancing activity of E1R, a novel positive allosteric modulator of sigma-1 receptors. Br J Pharmacol. 2014;171:761–71.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Zvejniece L, et al. Investigation into stereoselective pharmacological activity of phenotropil. Basic Clin Pharmacol Toxicol. 2011;109:407–12.

    Article  CAS  PubMed  Google Scholar 

  16. Dambrova M, Liepinsh E. Risks and benefits of carnitine supplementation in diabetes. Exp Clin Endocrinol Diabetes. 2015;123:95–100.

    CAS  PubMed  Google Scholar 

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This research was supported by the Latvian Council of Science, project TRILYSOX (Grant No. LZP-2018/1-0082). The authors were supported by the European Union’s Horizon 2020 Research and Innovation Programme, Project FAT4BRAIN (Grant No. 857394).

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Authors and Affiliations



EL, MD, LZ, BS designed research; BS, GS, SG performed research; EL, LZ, BS, GS, SG analyzed data; and EL, MD, HS, LZ, BS, GS, SG wrote the paper.

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Correspondence to Edgars Liepinsh.

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The experimental procedures were performed in accordance with the guidelines of the European Community and local laws and policies (Directive 2010/63/EU), and all of the procedures were approved by Food and Veterinary Service, Riga, Latvia.

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The authors declare that they have no competing interests.

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Supplementary Information

Additional file 1:

A detailed description of methods and statistical analysis.

Additional file 2:

Raw data of carnitine, GBB and TML measurements.

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Liepinsh, E., Svalbe, B., Stelfa, G. et al. Knockout of Tmlhe in mice is not associated with autism spectrum disorder phenotypes or motor dysfunction despite low carnitine levels. Molecular Autism 14, 29 (2023).

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