Congenital Creatine Transporter Deficiency

T. J. deGrauw1, 4 G. S. Salomons5 K. M. Cecil2, 4 G. Chuck1 A. Newmeyer3

Congenital Creatine Transporter Deficiency Rapid Article 232

M. B. Schapiro1, 3, 4 C. Jakobs5

Abstract

Introduction

Background: Two inborn errors of metabolism of creatine synthesis as well as the X-linked creatine transporter (SLC6 A8) deficiency have been recognized. This report describes the features of five identified male patients and their female relatives who are carriers of the X-linked creatine transporter deficiency syndrome. Methods: Proton MR spectroscopy was used to recognize creatine deficiency in the patients. Molecular analysis of the SLC6A8 gene was performed, confirming the diagnosis of homozygous males and heterozygous females. Results: We describe four families from a metropolitan area in the U. S. with X-linked creatine transporter deficiency. All affected males present with developmental delay and severe developmental language impairment. Proton MR spectroscopy shows significantly depressed to essentially absent creatine and phosphocreatine in the male patients. Nonsense mutations and amino acid deletions were found in the SLC6A8 gene in the affected families. Conclusion: Creatine transporter deficiency may be a more common X-linked genetic disorder than originally presumed. The affected males exhibit mental retardation with severe expressive language impairment.

Inborn errors of creatine metabolism and of creatine transport were not recognized until magnetic resonance spectroscopy (MRS) became widely available to academic hospitals and specialty imaging centers. The creatine and phosphocreatine composite signal is one of three major metabolites observed with proton MRS. Deficiency syndromes that significantly minimize the brain creatine levels can be readily recognized with this technique.

Key words Creatine Transporter Deficiency ´ Developmental Language Impairment

Creatine and phosphocreatine are recognized as essential components for energy storage and transfer. High creatine concentrations are present in skeletal muscle, heart, retina and spermatozoa; brain has intermediate levels and low levels are found in lungs, liver and kidney [18]. Creatine biosynthesis involves two enzymes: L-arginine:glycine amidinotransferase (AGAT; EC 2.1.4.1) and guanidinoacetate methyltransferase (GAMT, EC 2.1.1.2), which both occur mainly in liver, pancreas and kidney. Creatine is transported via the blood and is taken up via the creatine transporter (SLC6A8/CT1/CRTR1, MIM 300036), which is predominantly expressed in tissue void of creatine synthesis. The first inborn error of creatine metabolism to be reported was GAMT deficiency [12] (MIM 601240) and recently two related patients with AGAT (MIM 602360) were described [7]. GAMT deficient patients (N > 15 known) present with central nervous system (CNS) symptoms and signs such as developmental delay, seizures and movement disorders [11,12,14]. Last year, a defect of the X-linked creatine transporter (MIM 300352) in a boy pre-

Affiliation 1 Division of Neurology, Cincinnati Childrens Hospital Medical Center, Cincinnati, Ohio, USA 2 Division of Radiology, Cincinnati Childrens Hospital Medical Center, Cincinnati, Ohio, USA 3 Division of Developmental Disabilities, Cincinnati Childrens Hospital Medical Center, Cincinnati, Ohio, USA 4 College of Medicine, University of Cincinnati, Cincinnati, Ohio, USA 5 Department of Clinical Chemistry, VU University Medical Center, Amsterdam, The Netherlands Correspondence Ton J. deGrauw, M. D., Ph. D. ´ Cincinnati Childrens Hospital ´ Division of Neurology ´ 3333 Burnet Avenue ´ Cincinnati, OH 45229 ± 3039 ´ USA ´ E-mail: [email protected] Received: August 16, 2002 ´ Accepted after Revision: August 22, 2002 Bibliography Neuropediatrics 2002; 33: 232 ± 238  Georg Thieme Verlag Stuttgart ´ New York ´ ISSN 0174-304X

Table 1 Biochemical findings of SP (index patient; family 1) Labs

Normal

Pre creatine

7.5 GMS/day

15 GMS/day

22.5 GMS/day

Creatine ± Plasma

17 ± 109

71

1836

1066

2282

± Urine

17 ± 721

2694

13958

n.d.

n.d.

± CSF

24 ± 66

n.d.

62

n.d.

n.d.

Guanidinoacetate ± Plasma ± Urine

0.036 ± 0.224

1.79 65 n.d.

1.42 63 0.07

1.37

1.02

n.d.

n.d.

n.d.

n.d.

Creatinine ± Plasma

17.7 ± 61.9

BUN (mg/dL)

26.5

88.4

70.7

14

18

16

256.4 19

NH3 Plasma

0 ± 34

37

11

8

16

NA (mMol/L)

134 ± 143

143

143

141

140

K (mMol/L)

3.3 ± 4.6

4.5

4.2

CL (mMol/L)

96 ± 109

103

108

AST (IU/L)

15 ± 60

31

29

ALT (IU/L)

10 ± 35

21

4

4.3 103 29 4 yrs (n = 39)

0.06 ± 5.0

NH2

1

TM1 TM2

3 TM3

4 TM4

5 TM5

50 ± 110

1.0 ± 3.5 6 ± 1208 17 ± 721

Y262X

2

0.35 ± 1.8

Del F408

6

7

TM6

TM7

8 TM8

R514X

9 TM9

10 11 TM10

four families within a period of 18 months in one childrens hospital using this technique. This technique requires sedation for subjects who are unable to remain motionless within the MR scanner. The cognitively impaired male patients needed conscious sedation in order to complete the MR examination. Unfortunately, sedation does expose the children to the risk of apnea. Creatine levels within the urine are not routinely included in basic screens of metabolic disorders, mental retardation, pervasive developmental disorders or autistic syndromes. Our biochemical data on these four families suggest that high normal to increased creatine levels in urine is suggestive for a SLC6A8 defect. This is in agreement with another recently reported family with this X-linked disorder [6]. Studies by proton MRS, creatine

12

TM11 TM12

13

COOH

10.3 ± 99 10.3 ± 99

Fig. 4 Overview of the mutations identified in the creatine transporter SLC6A8. The novel mutations in the creatine transporter gene SLC6A8 are shown. The scheme presents the SLC6A8 mRNA, including the boundary of the 13 exons. The conserved transmembrane domains (TM) and the mutations identified are indicated.

uptake assay in cultured fibroblasts of males, and/or DNA diagnostics are warranted in MR patients in whom increased creatine is found in blood and/or urine. Conversely, low concentrations of creatine and creatinine also need to be further explored with MR spectroscopy, because it can be a sign of a creatine synthesis defect [12]. The impaired creatine uptake in cultured fibroblasts of the male patients prompted DNA sequence analysis of the SLC6A8 gene. Indeed the disease causing mutations could be identified: a nonsense mutation R514 X (family 1), predicting a truncated protein and two independent amino acid deletions; delF107 (family 2) and delF408 (family 3). Both phenylalanines are located in highly conserved regions of the gene. No heterozygosity of the delF408 allele could be detected in somatic DNA of the mother in family 3, indicating that this allele was a de novo mutation which had either arisen in the patient or in part of the

deGrauw TJ et al. Congenital Creatine Transporter ¼ Neuropediatrics 2002; 33: 232 ± 238

Del F107

17.7 ± 61.9

Family 1

Family 2

Family 3

Family 4

1 delF107/wt

R514X/wt

2 3

SB

nd R514X/wt

SP

R514X/wt

wt

Y262X/wt

MB

delF107 delF107 delF107/wt

wt

wt

SBe

CH

delF408

Y262X

Fig. 5 This figure shows the pedigrees of the four families with creatine transporter deficiency. Affected individuals are indicated by completely blackened symbols, symptomatic carriers by a large dot within a circle, and asymptomatic carriers by a dot within a circle. The genotype is depicted in the figure as well as the initials of the male patients.

R514X

Our patients all have a diagnosis of mental retardation with specific developmental speech language impairment (SLI). Some children with SLI have abnormalities on MRI as seen in our patient MB of the second family [2]. It is not clear how these imaging abnormalities relate to the SLI. Also, MRS metabolite abnormalities have been found in patients with dyslexia, but these abnormalities did not involve the creatine signal [9]. Our report illustrates that creatine transporter deficiency is probably not a static encephalopathy, and at least on imaging studies in the members of the second family is associated with progressive atrophy of the brain. Our patients have not lost developmental milestones, although the older patients have developed increasing behavior problems. The patients exhibit mild epilepsy that can be controlled with anti-epileptic drugs. The cognitive dysfunction, epilepsy and speech disability seems to characterize all congenital disorders of abnormal creatine metabolism [7,12].

deGrauw TJ et al. Congenital Creatine Transporter ¼ Neuropediatrics 2002; 33: 232 ± 238

Muscle contains the highest concentration of creatine, and creatine depletion in animals causes severe muscle weakness [18]. Surprisingly, our patients do not have clinical evidence of myopathy. MRS of muscle in SP showed a normal appearing level of creatine upon proton MRS and diminished phosphocreatine upon phosphorus MRS [4]. However, some GAMT and AGAT deficient patients are associated with motor developmental delay, and muscle weakness. This suggests that the low creatine levels as a result of the absence of creatine biosynthesis do affect the muscle in humans. Indeed in muscle of a GAMT deficient patient, reduced creatine levels of 8.8 mmol/kg; normal ca. 19 ± 21 mmol/ kg) were found by proton and phosphorus MRS of the muscle [5]. The absence of severe muscle weakness in all our patients and the presence of near-normal creatine signal for patient SP may indicate that more creatine transporters or alternatively spliced isoforms exist than previously assumed. In addition, the presence of creatine in CSF in the two CRTR patients who underwent a lumbar puncture is a sign that creatine transport through the blood-CSF barrier in the choroid plexus takes place through a different mechanism than the creatine transporter SLC6A8. Unfortunately, the treatment with high doses of creatine in our patient SP was unsuccessful. The uptake studies in fibroblasts suggested that high levels of extracellular creatine in brain should result in some creatine uptake of brain cells via alternative mechanisms. Such extracellular levels may not be reached in brain due to the blood-brain barrier. Further experiments will

be necessary to elucidate the exact mechanism of creatine uptake at high concentrations. In the immature rat, the brain is capable of biosynthesis of creatine [1]. The absence of detectable creatine in the brain of our index patient SP suggests that human brain cells by the age of 6 years are void of robust creatine biosynthesis, particularly within the basal ganglia and the cerebral gray and white matter. Analysis of the cerebellar hemisphere of MB with proton MRS demonstrated a small creatine resonance comparable to that found in the cerebrum of patients SBE and CH. The presence of small creatine signals in the 2- and 4-yearold patients, respectively, may arise from in situ synthesis in the brain or from differences in phenotypes for the different mutations.

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germline cells of the mother. This mutation has also been identified and reported in an Italian family [2]. In addition, we identified a novel hemizygous nonsense mutation Y262 X (family 4).

The recent identification of the three disordes of creatine synthesis and transport indicate that creatine is very important for proper brain function, but it is not clear why creatine deficiency in the brain results in the specific symptoms of cognitive insufficiency and speech impairment. Although a previous report deemed screening of patients with mental retardation, developmental dysphasia and behavioral problems with or without mild epilepsy unrealistic [14], we believe a more widespread evaluation with proton MRS and/or creatine measurements in urine is recommended due to the syndromal differences in the transporter and synthesis defects. This may result in the prompt identification of additional patients with creatine deficiency syndromes.

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deGrauw TJ et al. Congenital Creatine Transporter ¼ Neuropediatrics 2002; 33: 232 ± 238