Brain metabolic signatures in patients with genetic and nongenetic amyotrophic lateral sclerosis

Abstract Aims To study the brain metabolic signature in Chinese amyotrophic lateral sclerosis (ALS) patients and compare the difference in brain metabolic patterns between ALS with and without genetic variants. Methods We included 146 patients with ALS and 128 healthy controls (HCs). All patients with ALS underwent genetic testing to screen for ALS related genetic variants and were then divided into genetic (n = 22) and nongenetic ALS (n = 93) subgroups. All participants underwent brain 18F‐FDG‐PET scans. Group comparisons were performed using the two‐sample t‐test model of SPM12. Results We identified a large of hypometabolic clusters in ALS patients as compared with HCs, especially in the bilateral basal ganglia, midbrain, and cerebellum. Moreover, hypometabolism in the bilateral temporal lobe, precentral gyrus and hypermetabolism in the left anterior cingulate, occipital lobe, and bilateral frontal lobe were also found in ALS patients as compared with HCs. Compared with nongenetic ALS patients, genetic ALS patients showed hypometabolism in the right postcentral gyrus, precuneus, and middle occipital gyrus. The incidence of sensory disturbance in patients with genetic ALS was higher than that in patients with nongenetic ALS (5 of 22 [22.72%] vs. 7 of 93 [7.52%], p = 0.036). Conclusions Our investigation provided unprecedented evidence of relative hypometabolism in the midbrain and cerebellum in ALS patients. Genetic ALS patients showed a specific signature of brain metabolism and a higher incidence of sensory disturbance, indicating that genetic factors may be an underlying cause affecting the brain metabolism and increasing the risk of sensory disturbance in ALS.


| INTRODUC TI ON
Amyotrophic lateral sclerosis (ALS) is a rare neurodegenerative disorder characterized by weakness and atrophy due to loss of both upper and lower motor neurons, causing death due to respiratory paralysis within 3-5 years from the onset. 1 Although its etiology is still poorly understood, the interaction between genetic background, environmental, and lifestyle factors is a potential cause of ALS. 2 About 10% of cases are familial ALS (fALS), while the remaining 90% of cases are sporadic ALS (sALS). Since the SOD1 gene was reported in 1993, more than 40 genes have been reported to be linked with ALS. [1][2][3][4] ALS patients carrying specific mutations or variants may show distinct clinical phenotypes and prognosis. 5 Uncovering the relationship between genotype and phenotype has important implications for pathogenetic explanations in ALS. 18 F-fluorodeoxyglucose positron emission tomography ( 18 F-FDG-PET) is a powerful tool to display the brain metabolic signature in ALS. [6][7][8][9] Previous studies have shown that different ALS phenotypes displayed their specific brain metabolic changes. For example, ALS with cognitive impairment or frontotemporal dementia (FTD) demonstrated prefrontal, anterior cingulate, and insular hypometabolism when compared with ALS with normal cognition. [10][11][12] However, a large 18 F-FDG-PET study is still lacking in Chinese mainland.
Moreover, most 18 F-FDG-PET studies focused on the metabolic features in characterize patients carrying GGGGCC repeat expansion in C9orf72, [13][14][15] While the GGGGCC repeat expansion is rarely found in ALS patients in Asia, especially in China. 16 Moreover, genomewide pathogenic mutation metabolic pattern in ALS has not been systematically studied yet. Herein, the aim of this study was twofold: first, to elucidate the brain metabolic pattern in ALS patients in the mainland of China; second, to explore the brain metabolic changes characterizing genetic ALS as compared with nongenetic ALS.

| Genetic analysis
In this study, 115 patients with ALS in this study underwent genetic testing by whole exome sequencing (WES), standard polymerase chain reaction (PCR) and repeat primed polymerase chain reaction (RP-PCR) assay. The genomic data was stored in our inhouse data base (National Geriatric Clinical Medical Research Center, Xiangya Hospital, Bioinformatics Center). According to previous studies (Table S1) Conclusions: Our investigation provided unprecedented evidence of relative hypometabolism in the midbrain and cerebellum in ALS patients. Genetic ALS patients showed a specific signature of brain metabolism and a higher incidence of sensory disturbance, indicating that genetic factors may be an underlying cause affecting the brain metabolism and increasing the risk of sensory disturbance in ALS.

K E Y W O R D S
18 F-FDG-PET, amyotrophic lateral sclerosis, brain metabolism, genetic, whole exome sequencing a ReVe value more than 0.7. 20

| Statistical analysis
Data are shown as the mean (standard deviation, SD) or percent- Neurological Institute (MNI) space, resliced to 2 × 2 × 2 mm. An 8-mm full-width half-maximum Gaussian kernel was used to improve between-participant spatial alignment and smooth data for statistical analysis. 22 Once the images were spatially normalized and smoothed, a general linear model was used to carry out the appropriate voxel-by-voxel univariate statistical tests. Image intensity was normalized between participants to prevent interparticipant variability in cerebral tracer uptake from masking regional changes. 22 This was done using proportional scaling, which scales each image proportionally to the mean global brain activity.
Comparisons between different groups were performed using the two-sample t-test model of SPM12. When each patient's group was compared with HCs, sex, age at PET, and years of education were used as covariates. In the comparison of subgroups of patients with ALS, sex, age at PET, disease duration, years of education, and ALSFRS-R score were used as covariates. The height threshold of metabolic changes was set at p ˂ 0.001 (p ˂ 0.05 Family-wise error [FEW]-corrected at cluster). If no cluster of significant difference was identified, a height threshold of p < 0.005 (p < 0.05 FWEcorrected at cluster) was set to perform further exploratory analyses. After data was preprocessed using SPM12, significant clusters were visualized, reported, and anatomically labeled using the xjView (http://www.alive learn.net/xjview) and BrainNet Viewer. 23

| Ethics approval
Written informed consent was obtained from all participants, and the study protocol was approved by the Ethics Committee and the Expert Committee of Xiangya Hospital, Central South University.

| Demographic and clinical features of subjects
The demographic and clinical features of all subjects are listed in  Compared with HCs, patients with ALS showed relative hypometabolism in the bilateral temporal lobe, precentral gyrus, basal ganglia, midbrain, and cerebellum as compared with HCs ( Figure 1; Table 2).

| Genetic features of the ALS patients
Some regions with relatively increased metabolism in ALS were found in the left anterior cingulate, occipital lobe, and bilateral prefrontal lobe as compared with HCs ( Figure 1; Patients with genetic ALS showed relative hypometabolism in the bilateral sublobar, parahippocampal gyrus, occipital lobe, and cerebellum, and right temporal lobe as compared with HCs ( Figure 3A; Table S3). A cluster of hypermetabolism was found in the bilateral frontal lobe and left sublobar in patients with genetic ALS as compared with HCs ( Figure 3A; Table S3).

| Patients with nongenetic ALS versus HCs
(height threshold at p < 0.001, p < 0.05 FWE-corrected at cluster level) Patients with nongenetic ALS showed relative hypometabolism in the left temporal lobe and precentral gyrus and bilateral sublobar ( Figure 3B; Table S4). We identified a cluster of relative hypermetabolism in patients with nongenetic ALS as compared to HCs, including the left cingulate gyrus and occipital lobe and bilateral inferior frontal gyrus ( Figure 3B; Table S4).

TA B L E 2
Clusters showing a statistically significant relative hypermetabolism or hypometabolism in ALS patients as compared to HCs.  Figure S3a; Table S8).

| ALS patients with sensory normal versus
HCs (height threshold at p < 0.001, p < 0.05 FWEcorrected at cluster level) ALS patients showed relative hypometabolism in sublobar and temporal lobe and relative hypermetabolism in frontal lobe and limbic system as compared with HCs. There were no differences of metabolism between ALS patients with sensory normal and HCs in postcentral gyrus. ( Figure S3b; Table S9).

| DISCUSS ION
In the present study, we specifically investigated brain metabolic disturbances in ALS patients and explored the relationship between brain metabolism and genotypes in a Chinese ALS cohort.
As compared to HCs, we found hypometabolism in the primary motor cortex, frontal lobe, and temporal lobe in ALS patients in agreement with the results of previous studies. 9,24 Interestingly, as compared with HCs, we found that ALS patients also showed hypometabolism in the midbrain and cerebellum, which was inconsistent with previous studies. 9,14 Moreover, we also found that patients with genetic ALS showed hypometabolism in the right parietal and occipital lobe as compared to patients with nongenetic

ALS.
Although previous studies have investigated the pattern of brain metabolism in ALS, an 18  and temporal lobe as described in previous studies. 9,24 Our findings further strengthen the evidence that 18 F-FDG-PET could be used as a biomarker to evaluate the degeneration of upper motor neurons and cognitive dysfunction in ALS.
Interestingly, we found brain hypometabolism in the region of midbrain and cerebellum in patients with ALS as compared with HCs ( Figure S1), which was inconsistent with previous 18 F-FDG-PET studies. 9,14 Most of previous studies revealed the relative hypermetabolism in midbrain and cerebellum in ALS patients. 8,9,25,26 Some researchers suggest that hypermetabolism in the midbrain and cerebellum may be resulted by the astrocytosis and activated microglia. 9,27 Nevertheless, theoretically, ALS is a neurodegenerative disease characterized by the progressive loss of motor neurons in the brain, brainstem, and spinal cord, 1,3 thus the expected effect of ALS is hypometabolism in the midbrain caused by neuronal loss.
Recently, an MRI study disclosed that patients with ALS exhibited focal cerebellar degeneration and cerebro-cerebellar connectivity alterations. 28 As with hypometabolism in the frontal and temporal in ALS, 10,12,27 the degeneration of the cerebellum and midbrain inevitably leads to a reduction in tissue metabolic rate. Hence, we proposed a hypothesis that the metabolic states of the midbrain and cerebellum in patients with ALS are determined by which of the two pathological states of inflammation and degeneration is dominant.
Further postmortem or specific PET tracer studies are required to validate the hypothesis.
In this study, 115 patients with ALS underwent genetic test.
The most common mutant gene was SOD1, followed by OPTN and CACNA1H. This result was in line with previous studies in China. 16,18 As we all know, SPG11 is a common AR-inherited ALS causative gene. 29 However, no mutations were detected in SPG11. There are two possible reasons listed below. First, in our cohort, none of the patients with ALS have autosomal recessive family history. Second, the AR-inherited mutations in SPG11 in Chinese ALS patients were rare. 16,18 As compared with HCs, the cluster of relative hypometabolism of patients with nongenetic ALS was major located in the temporal and frontal cortex, in line with the previous studies. 9,24 As compared with HCs, patients with genetic ALS showed relative hypometabolism in the occipital lobe, sublobar, and limbic lobe, which indicated that genetic ALS patients showed a specific brain metabolism signature.
Another interesting finding was hypermetabolism in the postcentral gyrus in patients with genetic ALS. The primary somatosensory cortex is located in the postcentral gyrus and widely interconnected with other brain regions, including the primary motor cortex. A previous study found that the number of neurons in the motor cortex and the somatosensory cortex were a positively correlated in ALS, suggesting that the somatosensory cortex is affected, once the degeneration of the motor cortex is initiated. 30 These findings were also supported by other clinical researches on ALS. An MRI study found that patients with ALS showed parietal lobe atrophy during disease progression. 31 Functional evaluation of the sensory cortex in patients with ALS using the high-frequency somatosensory evoked potentials (HF-SEP) disclosed significant somatosensory cortex dysfunction in patients with a disease duration of more than 2 years. 32 In familial ALS patients, a more frequent occurrence of sensory features at presentation was reported. 33 A previous study found that sensory disturbance was a more frequent feature in C9orf72associated ALS patients than nonC9orf72-associated ALS patients. 34 Numerous studies reported that patients with ALS carrying SOD1 causative mutations were more likely to have sensory abnormalities during the course of the disease. [35][36][37] Consistent with previous studies, we also found the incidence of sensory disturbance in genetic  38 A study about clusters of anatomical disease-burden patterns in ALS confirmed the imaging signatures of sensory cortex could be distinct disease subtypes. 39 Thus, these studies suggest that patients with ALS have somatosensory cortex involvement, although the molecular mechanism is unclear.
Combining with our results, we hypothesize that genetic factors may be an underlying cause of sensory disturbances in ALS.
Similar to the previous PET studies, 9,40 we also found hypometabolism in occipital lobes in genetic ALS patients as compared with nongenetic ALS patients. Several MRI studies reported reductions of cortical thickness, gray matter volume, and functional connectivity in occipital lobes in patients with ALS. [41][42][43] The above studies indicated the abnormal alterations in the occipital lobes of ALS patients, while the molecular mechanism is unknown. C9orf72-linked FTD-ALS patients were found to present parietal and occipital lobe atrophy using structural MRI scans. 44 A postmortem study found that two aberrant SOD1 mRNAs were detected from occipital cortex of ALS patients. 45 These findings suggested that mutations in known causative ALS genes may affect the metabolism of the occipital lobes of ALS patients. Neuroaxonal retinal abnormalities were detected in neurodegenerative diseases like Parkinson's Disease, progressive supranuclear palsy, multiple system atrophy by optical coherence tomography. 46,47 Moreover, a previous study reported that retinal nerve fiber layer thinning was associated with the atrophy of occipital lobe in Alzheimer's disease. 48 These studies indicate that pathological changes of visual pathway were solid evidence in neurodegenerative diseases. Recently, reported alteration of the retinal nerve in ALS was reported implied that the ALS visual pathway may be damaged. 49 The effects of genetic factors on pathological changes in the visual pathway in ALS require more investigation.
There are several limitations in our study. First, the relatively small sample size of the genetic group might influence the results of our study. Second, ALS patients were recruited from a single center in China and our findings regarding the correlations between genotype and brain metabolism in Chinese ALS patients cannot be generalized to European or American ALS populations. Third, we could not correct the effects of cortical atrophy, since not all patients underwent brain MRI scans in this study. Nevertheless, previous studies showed that the result of brain metabolism is relatively independent of cortical atrophy. 50 Fourth, since the specific functional scale of the occipital lobe in our study is lacking, we were unable to assess the difference in the function of the occipital lobe between patients with genetic ALS and nongenetic ALS. The correlation between clinical phenotype and hypometabolism in the occipital lobe in ALS needs further studies to discover. Moreover, cognitive screening is not available in part of ALS patients in our study. It does make some bias of the results in our study. Some patients overlapping ALS and other dementia diseases were not excluded in this study due to the lack of cognitive screening.
In conclusion, our observations strengthen the evidence that 18 F-FDG-PET is a reliable tool to assess the motor in ALS, and our investigation provided unprecedented evidence of relative hypometabolism in midbrain and cerebellum in ALS patients as compared with HCs. Genetic ALS patients showed a specific signature of brain metabolism and a higher incidence of sensory disturbance, indicating that genetic factors may be an underlying cause affecting the brain metabolism and increasing the risk of sensory disturbance in ALS.
Further studies may be required to confirm our preliminary findings.