RESEARCH ARTICLE
Neurochemical Evaluation of Brain Function With 1 H Magnetic Resonance Spectroscopy in Patients With Fragile X Syndrome ¨.S¸. Kiper,1,2 B. Volkan-Salancı,1,2 Y. Alanay,1,2 D. Aktas¸,1,2 G.E. Utine,1,2 B. Akpınar,3 U. Arslan,4 P.O G. Halilog˘lu,1,5 K.K. Og˘uz,3 K. Bodurog˘lu,1,2 and M. Alikas¸ifog˘lu1,2 1
Department of Pediatrics, Faculty of Medicine, Hacettepe University, Ankara, Turkey Department of Pediatric Genetics, Faculty of Medicine, Hacettepe University, Ankara, Turkey
2 3
Department of Radiology, Faculty of Medicine, Hacettepe University, Ankara, Turkey
4
Department of Biostatistics, Faculty of Medicine, Hacettepe University, Ankara, Turkey Department of Pediatric Neurology, Faculty of Medicine, Hacettepe University, Ankara, Turkey
5
Manuscript Received: 6 November 2012; Manuscript Accepted: 12 August 2013
Fragile X syndrome (FXS) is the most common hereditary disorder of intellectual disability. Cognitive deficits involve executive function, attention, learning and memory. Advanced neuroimaging techniques are available, and 1H magnetic resonance spectroscopy (MRS) can be used as a complementary method to MR imaging to understand disease processes in brain, by in vivo demonstration of brain metabolites. MRS was performed in 13 male patients with FXS full mutation, and 13 age- and sex-matched healthy controls. FXS diagnosis was based on clinical evaluation, followed by detection of FMR1 full mutation. Axial T2 TSE, sagittal T1 SE and coronal 3D MPRAGE images were obtained for both morphological imaging and voxel localization. Following evaluation of conventional images, multivoxel MRS (CSI) through supraventricular white matter and single voxel MRS (svs) with an intermediate echo time (TE:135 ms) from the cerebellar vermis were performed. Choline/Creatine (Cho/Cr), N-acetyl aspartate/Creatine (NAA/Cr), and Choline/N-acetyl aspartate (Cho/ NAA) ratios were examined at right frontal (RF), left frontal (LF), right parietal (RP), left parietal (LP), and cerebellar vermian (C) white matter. Statistical analyses were done using t-test and Mann–Whitney U tests. A statistically significant difference was observed in RP Cho/NAA ratio (cell membrane marker/neuroaxonal marker), FXS patients having lower levels than controls (P ¼ 0.016). The results should be evaluated cautiously in parallel to consequences in brain metabolism leading to alterations in neurotransmitter levels, osmoregulation, energy metabolism and oxidative stress response described in animal models. MRS may serve to define a metabolic signature and biomarkers associated with FXS. Ó 2013 Wiley Periodicals, Inc.
Key words: fragile X syndrome; magnetic resonance spectroscopy; choline; N-acetyl aspartate; brain
Ó 2013 Wiley Periodicals, Inc.
How to Cite this Article:
¨ S¸, Utine GE, Akpınar B, Arslan U, Kiper PO Volkan-Salancı B, Alanay Y, Aktas¸ D, Halilog˘lu G, Og˘uz KK, Bodurog˘lu K, Alikas¸ifog˘lu M. 2014. Neurochemical evaluation of brain function with 1H magnetic resonance spectroscopy in patients with fragile X syndrome. Am J Med Genet Part A 164A:99–105.
INTRODUCTION Fragile X syndrome (FXS) is the most common hereditary disorder of intellectual disability and autism. Affected individuals display a combination of nonspecific cognitive and behavioral abnormalities, as well as clinically recognizable physical characteristics. Cognitive deficits of the affected patients mostly involve executive function, attention, learning and memory. Behavioral characteristics in the affected people reflect communicative problems owing to autistic features and hyper arousal, which leads to loss of communicative behavior. 1 H magnetic resonance spectroscopy (MRS) is a powerful clinical tool for interrogating the neurochemical environment and Conflicts of interest: None. Correspondence to: Gu¨len Eda Utine, M.D., Ph.D., Department of Pediatrics, Faculty of Medicine, Hacettepe University, 06100 Sihhiye, Ankara, Turkey. E-mail: [email protected] Article first published online in Wiley Online Library (wileyonlinelibrary.com): 20 November 2013 DOI 10.1002/ajmg.a.36207
99
100 characterizing the metabolic features of neurologic disease, and thus can be used as a complementary method to MR imaging (MRI) to understand disease process in the brain, particularly by in vivo demonstration of brain metabolites. MRS was previously performed in patients with various disorders of central nervous system, including mainly brain tumors, infectious processes, white matter disorders, inborn errors of metabolism, and neonatal injury, as a diagnostic, research and treatment follow-up tool [Panigrahy et al., 2010; Prust et al., 2011]. Executive dysfunction in male patients with FXS was investigated in one recent comparative study with MRS [Kesler et al., 2009]. This study analyzed cholinergic dysfunction in FXS, which is presumed to be involved in cognitive disabilities, as suggested by previous studies in humans, mouse and drosophila models. Results showed reduced choline to creatine ratio at dorsolateral prefrontal cortex in patients with FXS. Investigating such aspects of FXS could lead to improvements in treatment, guiding drug trials to specific cognitive deficits of patients with FXS. We performed 1H MRS in a group of 13 pediatric male patients with FXS to search for further clues on neurometabolite profile in pediatric patients with FXS. Imaging studies included the most commonly sampled right and left hemispheric frontal and parietal regions, as well as the cerebellum, which was reported to be affected by FXS [Huber, 2006].
PATIENTS AND METHODS Local institutional review board approved this prospective study and participants and/or relatives gave written informed consent. 1 H MRS was performed in 13 male patients with FXS full mutation, and 13 age- and sex-matched healthy controls. FXS diagnosis was based on clinical evaluation, followed by detection of FMR1 full mutation by polymerase chain reaction and standard Southern blot testing [Oostra and Willemsen, 2001]. Intellectual quotients (IQ) were measured using the revised Wechsler intelligence scale [Wechsler, 1999], except in two patients who had severe behavioral problems. The control group was recruited from healthy children tested to exclude presence of a FXS mutation. These children were functioning normally as stated by their families and were considered “healthy” based on detailed developmental history and physical and neurological examination. A formal IQ test was not performed in this group, as these children were assumed to have normal IQ based on their neurological examination and school performances. The mean age was 8.8 3.7 years in the FXS group and 8.9 3.1 years in the control group (P ¼ 0.606). IQ in the patient group was 54 19.7 (range: 25–90). Participants were not taking antiepileptic medication or did not experience recent seizures. No other medications known to alter spectral pattern of metabolites were taken by the participants. One patient formerly had simple febrile seizures and another had generalized seizures, both controlled with antiepileptic treatment. Two others had previously been on short-term valproic acid treatment because of electroencephalography (EEG) abnormalities, in the absence of clinical seizures. Control EEG recordings were normal in these patients. EEGs were obtained in 12 FXS patients and were abnormal in four (all without seizure histories). The EEG abnormalities included background abnormalities, and addition-
AMERICAN JOURNAL OF MEDICAL GENETICS PART A ally left posterior hemispheric paroxysmal epileptiform abnormality activated with sleep in one patient, and multifocal epileptiform abnormality in another. All imaging studies were performed on a 1.5 T MR system (Symphony, Siemens, Germany). Axial T2 TSE (TR/TE: 3,800/ 90 ms) and sagittal T1 SE (TR/TE: 500/20 ms) with a slice thickness of 5 mm and no gap between slices, and coronal 3D MPRAGE (TR/ TE: 2,130/3.9 ms; flip angle: 15; matrix: 192 256) images were obtained for morphological imaging and voxel localization in MRS. Following on-site evaluation of these conventional images, a dedicated radiologist (B.A.) performed 1H MRS. Multivoxel (CSI)(TR/ TE: 1,500/135 ms) through white matter at the level of corona radiata and single voxel 1H MRS (svs) (PRESS; TR/TE: 1,500/ 135 ms) from the cerebellar vermis at the level of 4th ventricle and middle cerebellar peduncles were performed. Two radiologists (B.A. and K.K.O) carried-out 1H MRS analyses in consensus, by using automated post processing software provided by the manufacturer on an off-line workstation (Leonardo Systems, Siemens AG, Munich, Germany). Resonance peaks of N-acetyl aspartate (NAA), choline (Cho), creatine (Cr) were identified at 2.0, 3.2, and 3.03 ppm, respectively, and ratios of NAA/Cr, Cho/Cr, and Cho/NAA were computed from right frontal (RF), left frontal (LF), right parietal (RP), left parietal (LP) white matter and cerebellar vermis (C). Figures 1–4 represent examples of voxels of interest and their resulting spectra. Gray matter and cerebrospinal fluid were left out of voxels from supratentorial imaging and measurements were made only over areas of white matter. The ROI size was 80 mm 100 mm 100 mm and the size of a single voxel was 12 mm 12 mm 12 mm (mediolateral anteroposterior thickness). Data were analyzed using SPSS 15.0 for Windows (SPSS, Inc., Chicago, IL). For normal distribution of quantitative data, independent samples t-test was used for comparison of two independent groups. For abnormal distribution of quantitative data, Mann– Whitney U test was used. Arithmetic mean, standard deviation,
FIG. 1. Multivoxel MRS through supraventricular white matter. White rectangle shows the multivoxel positioned at the upper part of the lateral ventricles. Blue square shows the region of interest, which is right parietal white matter in (a) and right frontal white matter in (b).
UTINE ET AL.
101
FIG. 2. Multivoxel MRS (CSI, TR/TE; 1,500/135 ms). An example of the spectrum in the right parietal white matter (a) and right frontal white matter (b) in a control subject. Blue square shows the region of interest. (Cho, choline; Cr, creatine; NAA, N-acetyl aspartate).
median, and range were used as descriptive statistics. Results was accepted as statistically significance when P was
Neurochemical Evaluation of Brain Function With 1 H Magnetic Resonance Spectroscopy in Patients With Fragile X Syndrome ¨.S¸. Kiper,1,2 B. Volkan-Salancı,1,2 Y. Alanay,1,2 D. Aktas¸,1,2 G.E. Utine,1,2 B. Akpınar,3 U. Arslan,4 P.O G. Halilog˘lu,1,5 K.K. Og˘uz,3 K. Bodurog˘lu,1,2 and M. Alikas¸ifog˘lu1,2 1
Department of Pediatrics, Faculty of Medicine, Hacettepe University, Ankara, Turkey Department of Pediatric Genetics, Faculty of Medicine, Hacettepe University, Ankara, Turkey
2 3
Department of Radiology, Faculty of Medicine, Hacettepe University, Ankara, Turkey
4
Department of Biostatistics, Faculty of Medicine, Hacettepe University, Ankara, Turkey Department of Pediatric Neurology, Faculty of Medicine, Hacettepe University, Ankara, Turkey
5
Manuscript Received: 6 November 2012; Manuscript Accepted: 12 August 2013
Fragile X syndrome (FXS) is the most common hereditary disorder of intellectual disability. Cognitive deficits involve executive function, attention, learning and memory. Advanced neuroimaging techniques are available, and 1H magnetic resonance spectroscopy (MRS) can be used as a complementary method to MR imaging to understand disease processes in brain, by in vivo demonstration of brain metabolites. MRS was performed in 13 male patients with FXS full mutation, and 13 age- and sex-matched healthy controls. FXS diagnosis was based on clinical evaluation, followed by detection of FMR1 full mutation. Axial T2 TSE, sagittal T1 SE and coronal 3D MPRAGE images were obtained for both morphological imaging and voxel localization. Following evaluation of conventional images, multivoxel MRS (CSI) through supraventricular white matter and single voxel MRS (svs) with an intermediate echo time (TE:135 ms) from the cerebellar vermis were performed. Choline/Creatine (Cho/Cr), N-acetyl aspartate/Creatine (NAA/Cr), and Choline/N-acetyl aspartate (Cho/ NAA) ratios were examined at right frontal (RF), left frontal (LF), right parietal (RP), left parietal (LP), and cerebellar vermian (C) white matter. Statistical analyses were done using t-test and Mann–Whitney U tests. A statistically significant difference was observed in RP Cho/NAA ratio (cell membrane marker/neuroaxonal marker), FXS patients having lower levels than controls (P ¼ 0.016). The results should be evaluated cautiously in parallel to consequences in brain metabolism leading to alterations in neurotransmitter levels, osmoregulation, energy metabolism and oxidative stress response described in animal models. MRS may serve to define a metabolic signature and biomarkers associated with FXS. Ó 2013 Wiley Periodicals, Inc.
Key words: fragile X syndrome; magnetic resonance spectroscopy; choline; N-acetyl aspartate; brain
Ó 2013 Wiley Periodicals, Inc.
How to Cite this Article:
¨ S¸, Utine GE, Akpınar B, Arslan U, Kiper PO Volkan-Salancı B, Alanay Y, Aktas¸ D, Halilog˘lu G, Og˘uz KK, Bodurog˘lu K, Alikas¸ifog˘lu M. 2014. Neurochemical evaluation of brain function with 1H magnetic resonance spectroscopy in patients with fragile X syndrome. Am J Med Genet Part A 164A:99–105.
INTRODUCTION Fragile X syndrome (FXS) is the most common hereditary disorder of intellectual disability and autism. Affected individuals display a combination of nonspecific cognitive and behavioral abnormalities, as well as clinically recognizable physical characteristics. Cognitive deficits of the affected patients mostly involve executive function, attention, learning and memory. Behavioral characteristics in the affected people reflect communicative problems owing to autistic features and hyper arousal, which leads to loss of communicative behavior. 1 H magnetic resonance spectroscopy (MRS) is a powerful clinical tool for interrogating the neurochemical environment and Conflicts of interest: None. Correspondence to: Gu¨len Eda Utine, M.D., Ph.D., Department of Pediatrics, Faculty of Medicine, Hacettepe University, 06100 Sihhiye, Ankara, Turkey. E-mail: [email protected] Article first published online in Wiley Online Library (wileyonlinelibrary.com): 20 November 2013 DOI 10.1002/ajmg.a.36207
99
100 characterizing the metabolic features of neurologic disease, and thus can be used as a complementary method to MR imaging (MRI) to understand disease process in the brain, particularly by in vivo demonstration of brain metabolites. MRS was previously performed in patients with various disorders of central nervous system, including mainly brain tumors, infectious processes, white matter disorders, inborn errors of metabolism, and neonatal injury, as a diagnostic, research and treatment follow-up tool [Panigrahy et al., 2010; Prust et al., 2011]. Executive dysfunction in male patients with FXS was investigated in one recent comparative study with MRS [Kesler et al., 2009]. This study analyzed cholinergic dysfunction in FXS, which is presumed to be involved in cognitive disabilities, as suggested by previous studies in humans, mouse and drosophila models. Results showed reduced choline to creatine ratio at dorsolateral prefrontal cortex in patients with FXS. Investigating such aspects of FXS could lead to improvements in treatment, guiding drug trials to specific cognitive deficits of patients with FXS. We performed 1H MRS in a group of 13 pediatric male patients with FXS to search for further clues on neurometabolite profile in pediatric patients with FXS. Imaging studies included the most commonly sampled right and left hemispheric frontal and parietal regions, as well as the cerebellum, which was reported to be affected by FXS [Huber, 2006].
PATIENTS AND METHODS Local institutional review board approved this prospective study and participants and/or relatives gave written informed consent. 1 H MRS was performed in 13 male patients with FXS full mutation, and 13 age- and sex-matched healthy controls. FXS diagnosis was based on clinical evaluation, followed by detection of FMR1 full mutation by polymerase chain reaction and standard Southern blot testing [Oostra and Willemsen, 2001]. Intellectual quotients (IQ) were measured using the revised Wechsler intelligence scale [Wechsler, 1999], except in two patients who had severe behavioral problems. The control group was recruited from healthy children tested to exclude presence of a FXS mutation. These children were functioning normally as stated by their families and were considered “healthy” based on detailed developmental history and physical and neurological examination. A formal IQ test was not performed in this group, as these children were assumed to have normal IQ based on their neurological examination and school performances. The mean age was 8.8 3.7 years in the FXS group and 8.9 3.1 years in the control group (P ¼ 0.606). IQ in the patient group was 54 19.7 (range: 25–90). Participants were not taking antiepileptic medication or did not experience recent seizures. No other medications known to alter spectral pattern of metabolites were taken by the participants. One patient formerly had simple febrile seizures and another had generalized seizures, both controlled with antiepileptic treatment. Two others had previously been on short-term valproic acid treatment because of electroencephalography (EEG) abnormalities, in the absence of clinical seizures. Control EEG recordings were normal in these patients. EEGs were obtained in 12 FXS patients and were abnormal in four (all without seizure histories). The EEG abnormalities included background abnormalities, and addition-
AMERICAN JOURNAL OF MEDICAL GENETICS PART A ally left posterior hemispheric paroxysmal epileptiform abnormality activated with sleep in one patient, and multifocal epileptiform abnormality in another. All imaging studies were performed on a 1.5 T MR system (Symphony, Siemens, Germany). Axial T2 TSE (TR/TE: 3,800/ 90 ms) and sagittal T1 SE (TR/TE: 500/20 ms) with a slice thickness of 5 mm and no gap between slices, and coronal 3D MPRAGE (TR/ TE: 2,130/3.9 ms; flip angle: 15; matrix: 192 256) images were obtained for morphological imaging and voxel localization in MRS. Following on-site evaluation of these conventional images, a dedicated radiologist (B.A.) performed 1H MRS. Multivoxel (CSI)(TR/ TE: 1,500/135 ms) through white matter at the level of corona radiata and single voxel 1H MRS (svs) (PRESS; TR/TE: 1,500/ 135 ms) from the cerebellar vermis at the level of 4th ventricle and middle cerebellar peduncles were performed. Two radiologists (B.A. and K.K.O) carried-out 1H MRS analyses in consensus, by using automated post processing software provided by the manufacturer on an off-line workstation (Leonardo Systems, Siemens AG, Munich, Germany). Resonance peaks of N-acetyl aspartate (NAA), choline (Cho), creatine (Cr) were identified at 2.0, 3.2, and 3.03 ppm, respectively, and ratios of NAA/Cr, Cho/Cr, and Cho/NAA were computed from right frontal (RF), left frontal (LF), right parietal (RP), left parietal (LP) white matter and cerebellar vermis (C). Figures 1–4 represent examples of voxels of interest and their resulting spectra. Gray matter and cerebrospinal fluid were left out of voxels from supratentorial imaging and measurements were made only over areas of white matter. The ROI size was 80 mm 100 mm 100 mm and the size of a single voxel was 12 mm 12 mm 12 mm (mediolateral anteroposterior thickness). Data were analyzed using SPSS 15.0 for Windows (SPSS, Inc., Chicago, IL). For normal distribution of quantitative data, independent samples t-test was used for comparison of two independent groups. For abnormal distribution of quantitative data, Mann– Whitney U test was used. Arithmetic mean, standard deviation,
FIG. 1. Multivoxel MRS through supraventricular white matter. White rectangle shows the multivoxel positioned at the upper part of the lateral ventricles. Blue square shows the region of interest, which is right parietal white matter in (a) and right frontal white matter in (b).
UTINE ET AL.
101
FIG. 2. Multivoxel MRS (CSI, TR/TE; 1,500/135 ms). An example of the spectrum in the right parietal white matter (a) and right frontal white matter (b) in a control subject. Blue square shows the region of interest. (Cho, choline; Cr, creatine; NAA, N-acetyl aspartate).
median, and range were used as descriptive statistics. Results was accepted as statistically significance when P was
