Eur Arch Psychiatry Clin Neurosci DOI 10.1007/s00406-014-0524-6
ORIGINAL PAPER
AMPD1 functional variants associated with autism in Han Chinese population Lusi Zhang • Jianjun Ou • Xiaojuan Xu • Yu Peng • Hui Guo • Yongcheng Pan Jingjing Chen • Tianyun Wang • Hao Peng • Qiong Liu • Di Tian • Qian Pan • Xiaobin Zou • Jingping Zhao • Zhengmao Hu • Kun Xia
•
Received: 24 January 2014 / Accepted: 9 August 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Autism is a childhood neurodevelopmental disorder with high heterogeneity. Following our genomewide associated loci with autism, we performed sequencing analysis of the coding regions, UTR and flanking splice junctions of AMPD1 in 830 Chinese autism individuals as well as 514 unrelated normal controls. Fourteen novel variants in the coding sequence were identified, including 11 missense variants and 3 synonymous mutations. Among these missense variants, 10 variants were absent in 514 control subjects, and conservative and functional prediction was carried out. Mitochondria activity and lactate dehydrogenase assay were performed in 5 patients’ lymphoblast cell lines; p.P572S and p.S626C showed decreased mitochondrial complex I activity, and p.S626C increased lactate dehydrogenase release in medium. Conclusively, our data suggested that mutational variants in AMPD1 contribute to autism risk in Han Chinese population, uncovering the Electronic supplementary material The online version of this article (doi:10.1007/s00406-014-0524-6) contains supplementary material, which is available to authorized users. L. Zhang X. Xu Y. Peng H. Guo Y. Pan J. Chen T. Wang H. Peng Q. Liu D. Tian Q. Pan J. Zhao Z. Hu (&) K. Xia (&) State Key Laboratory of Medical Genetics, Central South University, 110, Xiangya Road, Changsha Hunan, China e-mail: [email protected] K. Xia e-mail: [email protected] L. Zhang Y. Peng H. Guo K. Xia School of Life Sciences, Central South University, Changsha, Hunan, China
contribution of mutant protein to disease development that operates via mitochondria dysfunction and cell necrosis. Keywords
Autism AMPD1 Mutation Mitochondria
Introduction Autism is a broadly defined childhood neurodevelopmental disorder with onset prior to 3 years of age. The core symptoms were characterized by impairment in language and social communication, and repetitive and restrictive behaviors [1]. Autism has emerged as a major public health concern with an estimated prevalence of 1:50 in USA [2] and is considered to be the most inheritable neuropsychiatric disorders according to the familial and twin studies [3, 4]. A growing list of disease-associated genetic factors has been found through cytogenetics, genome-wide association studies, linkage analysis, and exome sequencing. Functional studies following these genetic analysis and pathophysiological studies have implicated some underlying X. Xu The Reproductive Medicine Hospital of the First Hospital of Lanzhou University, Lanzhou, Gansu, China X. Zou Department of Pediatrics, No. 3 Hospital of the Sun Yat-sen University, Guangzhou, Guangdong, China K. Xia Key Laboratory of Medical Information Research, Central South University, Changsha, Hunan, China
J. Ou J. Zhao Mental Health Institute, The Second Xiangya Hospital, Central South University, Changsha, Hunan, China
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mechanisms, including abnormal synaptogenesis, neurite growth, negative changes of neural plasticity [5, 6], neuronal cell loss and neurodegeneration [7], metabolic abnormality [8], and mitochondrial dysfunction [9, 10]. We have recently reported a genome-wide association study of autism in Chinese population [11]. Three risk single nucleotide polymorphisms, rs926938, rs6537835, and rs1877455, were identified, and related haplotypes at AMPD1-NRAS-CSDE1, TRIM33, and TRIM33-BCAS2 associated with autism were mapped to a previously reported autism linkage region 1p13.2 [12–14]. The adenosine monophosphate deaminase (AMPD, EC 3.5.4.6) catalyzes AMP hydrolyzing into IMP and NH3. The proteins of AMPD family include three members. AMPD1, mapped on chromosome 1p13.2, encodes AMP deaminase 1, which plays an important role in the purine nucleotide cycle [15]. To further find out the genetic evidence of AMPD1 for autism, we performed the mutation analysis on AMPD1 gene in autism patients of Han Chinese population in additional independent sample, and the possible underlying mechanism was also examined.
Materials and methods Subjects The study sample included 830 patients (males 731, females 99, the average age was 6, range from 3 to 18 years old) recruited from Elim Autism Training Department of the Qingdao Municipal Autism Research Institute, Qingdao, Shandong Province, China, and Psychiatric Department of the Second Xiangya Hospital of Central South University, Changsha, Hunan Province, China. All enrolled probands and their parents were of Chinese Han ancestry. Patients were diagnosed according to International classification of Diseases, Diagnostic and Statistical Manual of Mental Disorders-IV criteria, and autistic disorder and the Chinese Society of Psychiatry’s Chinese Classification of Mental Disorders, by two independent senior psychiatrists from the Psychiatric department of the Second Xiangya Hospital. Karyotyping analysis was performed on all patients and their parents to rule out those with chromosomal abnormalities. Moreover, Patients with fragile X syndrome, tuberous sclerosis, dysmorphic features, or any other neurological/medical condition suspected to autism were excluded. In order to rule out possible unreported polymorphism in our patients, unrelated controls was collected randomly, which included 514 unrelated Han Chinese healthy volunteers (males 248, females 266, the average age is 22, range from 3 to 46 years old). All patients, parents and controls were not involved in our previous study [11].
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Written informed consent was obtained from all the patients along with their guardians and controls. The research was approved by the Human Ethics Committee of The State Key Laboratory of Medical Genetics, Central South University, and is compliant with the Code of Ethics of the World Medical Association [16]. Sequencing and mutant analysis Genomic DNA was extracted from peripheral blood lymphocytes of all individuals by means of the standard phenol–chloroform method. The primer spanning all coding regions (Genbank accession number RefSeq NM_000036.2), splicing junctions, and UTR of AMPD1 were designed by the online tool Primer3 (http://frodo.wi. mit.edu/). PCR reactions were carried out in a thermocycler (GeneAmp 2720, Applied Biosystems Inc., Foster City, CA, USA). The primer sequences and PCR conditions are available upon request. PCR products were verified by 6 % polyacrylamide gel electrophoresis and silver staining. Sanger sequencing of all the 830 affected cases along with the 514 unaffected controls was performed on an ABI3100/ 3130 automated sequencer (Applied Biosystems Inc., Foster City, CA, USA) according to the manufacturer’s instructions. The parents whose children carry novel variants were further sequenced on the mutated points to detect inheritance status. Conservative analyses of nonsynonymous variants were performed by multiple sequence alignment tools clustalX, phylogenetic P values (Phylop), and genomic evolutionary rate profiling (GERP). To perform functional prediction, we used the software SIFT, PolyPhen, and MutationTaster to score these variants. Mitochondrial complex activity assay Primary B lymphocytes from patients and control were transformed to lymphoblast cell lines (LCLs) using Epstein-Barr virus and cyclosporin A [17]. LCLs of 5 patients (p.C483S, p.R500C, p.P572S, p.S626C, and p.T681I) and 3 controls cultured in T25 flasks were centrifuged at 2509g for 5 min to collect cells. Mitochondrial complex activities were measured using following kits according to manufacturer’s instructions: Complex I activity was evaluated by following Mitochondrial Complex I Activity Assay Kit (Novagen, Merck KGaA, Darmstadt, Germany); succinate-coenzyme Q reductase activity was measured by Mitochondrial Complex II Activity Assay Kit (Novagen, Merck KGaA, Darmstadt, Germany); Complex IV activity assay was performed using Mitochondrial Complex IV (Human) Activity Assay Kit (Novagen, Merck KGaA, Darmstadt, Germany); Complex V was evaluated by following ATPase activity through
Eur Arch Psychiatry Clin Neurosci
Mitochondrial Complex V (ATP Synthase) Activity Assay Kit(Novagen, Merck KGaA, Darmstadt, Germany); and MitoToxTM OXPHOS Complex III Activity Kit (Abcam, Cambridge, UK) was used to evaluate Complex II and Complex III activities. For mitochondrial dysfunction can often be caused by the simple decrease in numbers, mitochondrial number should be normalized by the activity of citrate synthase, which is thought to be relatively stable and proportional to the mitochondrial numbers [18]. The activity of citrate synthase in LCLs was evaluated by Citrate Synthase Assay Kit (Sigma-Aldrich Co., St. Louis, MO, USA). All the measurements were performed on BioTek SynergyTM 2 microplate reader (BioTek U.S., Winooski, VT, USA). Each assay was repeated at least three times. LDH release assay Lymphoblast cell lines were cultured in 6-well plates for 24 h ahead of test. LCLs of 5 patients (p.C483S, p.R500C, p.P572S, p.S626C, and p.T681I) and 3 controls were collected separately by centrifuged at 2509g for 5 min. Cell culture medium was also collected and centrifuged at 5,0009g for 10 min to remove cell debris. LCLs were lysed in a lysis buffer (0.1 % triton X-100 in PBS) for 10 min at 4 °C. LDH in the medium and cell lysates were measured using CytoTox 96Ò Non-Radioactive Cytotoxicity Assay Kit (Promega, Madison, WI, USA) according to manufacturer’s instruction. The rate of LDH release was given by the following: %LDH release = medium A492/total A492 = medium A492/(medium A492 ? cell lysate A492). The measurements were performed on BioTek SynergyTM 2 microplate reader (BioTek U.S., Winooski, VT, USA). Each experiment was repeated for three times. Statistic analysis The data of each patient were compared with the 95 % CIs of the control values. And the values above the highest limit of the 95 % CI or outside the lowest limit of the 95 % CI were considered different. The data of patients were presented as mean ± SEM. Fisher’s exact test was used to compare the allelic and genotypic distributions between patients and controls and to calculate the odds ratio and 95 % confidence intervals (95 % CI). Statistical analyses were conducted using the SPSS 13.0 statistical software package (SPSS Inc., Chicago, IL, USA). A P value \ 0.05 (two-tailed) was considered significant.
Results Through sequencing the exons, splicing junctions, and 50 UTR of AMPD1 gene in 830 patients, we identified 14
unreported variants in the coding sequence (Table 1), including 11 missense variants and 3 synonymous mutations. Several SNPs were detected as well (Supplemental Table 1). Among the 11 non-synonymous variants, 10 missense variants (sequence results were shown in Supplemental Fig. 1) were neither presented in 514 normal control subjects nor the 1,000 Genomes data (http://browser.1000genomes.org/). Only 1 non-synonymous variant p.R108L was detected in both 1 patient and 2 controls. We analyzed the allelic and genotypic distribution frequencies of the p.R108L variant; as shown in Supplemental Table 2, the P values of genotypic frequencies and allelic frequencies for p.R108L in 830 autism patients and 514 control subjects were 0.310575 and 0.310846, respectively, and odds ratio of the p.R108L allele was 3.234 (95 % CI = 0.293–35.711; P = 0.310783). Since there was lack of a statistically significant association of AMPD1 p.R108L with the risk of autism, it was excluded in following analysis. Besides, 6 control-specific variants were also detected (Table 1). To see whether these 10 patient-specific missense mutations were de novo, we also sequenced the parental DNA samples. The p.P572S mutation was a de novo mutation, whereas the rest were inherited no bias between maternal versus paternal transmission (Table 1). The 3 synonymous mutations, p.F213F, p.P234P, and p.T523T, were not to be observed in controls, and we excluded them in further analysis. To investigate possible influence of the 10 patient-specific missense mutations on AMPD1 protein function, we performed the conservation prediction by using ClustalX, Phylop, and GERP, and function consequences by using MutatinTaster, Polyphen-2, and SIFT on these mutants. ClustalX result demonstrated asterisks in the amino acid positions 500, 572, 626, and 681 (Table 1 and Supplemental Fig. 2), which revealed highly conserved amino acid on these sites, and the dot on position 483 suggested moderate conserved amino acid. As shown in Table 2, p.D203V, p.D276N, p.C483S, p.R500C, p.P572S, p.S626C, and p.T681I were predicted conserved nucleotide substitutions by Phylop for the scores that were close to 1. Nucleotide substitutions in p.D276N, p.C483S, p.R500C, p.P572S, p.S626C, and p.T681I revealed damaging among GERP analysis. Considering the analysis results above, the amino acid positions 483, 500, 572, 626, and 681 were quite conservative in vertebrates AMPD1. MutationTaster predicted p.I202M, p.D276N, p.R500C, p.P572S, p.S626C, and p.T681I as disease causing nucleotide substitutions. Both Polyphen-2 and SIFT results predicted that p.D276N, p.R500C, p.P572S, p.S626C, and p.T681I were probably damaging nucleotide variations (Table 2). The results of three functional analysis tools demonstrated that p.D276N, p.R500C, p.P572S, p.S626C, and p.T681I mutations
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Eur Arch Psychiatry Clin Neurosci Table 1 Variants identified in the AMPD1 coding regions
Location
Nucleotide change
Amino acid change
Patients (n = 830, het/homo)
Controls (n = 514, het/homo)
Inheritance statusa
Nonsynonymous variants
N/A data were not available, het/ homo homozygous change/ heterozygous change, Pat paternal, Mat maternal a
Whether the variant was inherited from the parents or not
b
The 2 mutants were carried by the same patient
c
The control-specific variants
Exon1
c.31C[G
p. Q11E
1/0
0/0
Mat
Exon1
c.70G[A
p. A24T
1/0
0/0
Pat
Exon4
c.323G[T
p. R108L
1/0
2/0
N/A
Exon5b
c.606T[G
p. I202M
1/0
0/0
Mat
Exon5b
c.608A[T
p. D203V
1/0
0/0
Mat
Exon6c
c.693C[G
p. D231E
0/0
1/0
N/A
Exon6c
c.753T[A
p. Y251X
0/0
1/0
N/A
Exon6
c.826G[A
p. D276N
1/0
0/0
Pat
Exon10
c.1449C[G
p. C483S
1/0
0/0
Pat
Exon10c Exon11
c.1460C[T c.1498C[T
p. R487L p. R500C
0/0 2/0
1/0 0/0
N/A Pat
Exon12c
c.1641T[G
p. D547E
0/0
1/0
N/A De novo
Exon12
c.1714C[T
p. P572S
1/0
0/0
Exon13c
c.1783C[T
p. R595X
0/0
1/0
N/A
Exon13
c.1877C[G
p. S626C
1/0
0/0
Mat
Exon13c
c.1879C[A
p. H627L
0/0
1/0
N/A
Exon14
c.2042C[T
p. T681I
1/0
0/0
Mat N/A
Synonymous variants Exon5
c.639C[T
p. F213F
1/0
0/0
Exon6
c.702T[A
p. P234P
1/0
0/0
N/A
Exon11
c.1569C[T
p.T523T
1/0
0/0
N/A
Table 2 Conservative and functional analysis on 10 missense mutations in AMPD1 Mutant
Protein domain
Conservative analysis
Functional analysis
Phylop
GERP
ClustalX
MutationTaster
PolyPhen-2
SIFT
p.Q11E
N/A
0.022, N
0.46, T
N
Polymorphism
0.001, B
0.95, T
p.A24T
N/A
0.017, N
0.67, T
N
Polymorphism
0.077, B
0.17, T
p.I202M
Myosin binding domain
0.027, N
0.83, T
N
Disease causing
0.049, B
0.24, T
p.D203V
Myosin binding domain
0.998, C
0.93, T
N
Disease causing
0.000, B
0.27, T
p.D276N
Myosin binding domain
0.999, C
1, D
N
Disease causing
0.999, P
0.03, D
p.C483S
N/A
0.972, C
1, D
MC
Disease causing
0.000, B
1, T
p.R500C
N/A
0.999, C
1, D
C
Disease causing
1.000, P
0, D
p.P572S
N/A
0.999, C
1, D
C
Disease causing
1.000, P
0, D
p.S626C
N/A
0.999, C
1, D
C
Disease causing
1.000, P
0.05, D
p.T681I
Catalytic site
0.999, C
1, D
C
Disease causing
1.000, P
0, D
C conserved change, MC moderate conserved, N non-conserved, T tolerable change, B benign, P probably damaging, D damaging, N/A no exact domain information
probably impact the structure and function of human AMPD1 protein. It was reported that children with autism were more likely to suffer from mitochondrial dysfunction (mainly in complexes I and IV), mtDNA over-replication, and mtDNA deletions than controls [10]. Thus, we assayed the activities of electron transport chains (ETC) in LCLs from
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patients with variants p.C483S, p.R500C, p.P572S, p.S626C, and p.T681I to see whether there is any mitochondrial defect in these patients. As shown in Table 3, p.P572S and p.S626C mutations resulted in significant reduction (0.0014 ± 0.0003 OD/min/lg protein and 0.0016 ± 0.0002 OD/min/lg protein) in the activity of Complex I compared to control (0.0019–0.0029 OD/min/
Eur Arch Psychiatry Clin Neurosci Table 3 Mitochondrial Activities in the LCLs Cell line with mutation
Mean (SEM) OD/min/lg cell lysate a
Affected complexes
ComplexV
Complex I
Complex II
Complex III
Complex IV
p.C483S
0.0017 (0.0015, 0.0019)
0.00082 (0.00070, 0.0093)
0.016 (0.015, 0.017)
0.00068 (0.00048, 0.00087)
0.0048 (0.0043, 0.0054)
N
p.R500C
0.0024 (0.0023, 0.0024)
0.00093 (0.00085, 0.00102)
0.017 (0.015, 0.019)
0.00064 (0.00042, 0.00086)
0.0043 (0.0038, 0.0047)
N
p.P572S
0.0014 (0.0011, 0.0017)
0.00073 (0.00060, 0.00086)
0.017 (0.016, 0.018)
0.00068 (0.00049, 0.00086)
0.0039 (0.0038, 0.0040)
Ia
p.S626C
0.0016 (0.0014, 0.0018)
0.00080 (0.00070, 0.00090)
0.020 (0.017, 0.023)
0.00080 (0.00062, 0.00098)
0.0048 (0.0043, 0.0053)
Ia
p.T681I
0.0017 (0.0014, 0.0020)
0.00063 (0.00070, 0.00090)
0.018 (0.015, 0.022)
0.00070 (0.00051, 0.00089)
0.0044 (0.0039, 0.0050)
N
0.00068 (0.00018, 0.00185)
0.018 (0.012, 0.024)
0.00058 (0.00016, 0.00099)
0.0038 (0.0026, 0.0050)
Mean activities (95 % CI) Control (n = 3)
0.0024 (0.0019, 0.0029)
The table was established by evaluating the activity of each segment of the electron transport chain and then comparing them with the 95 % CIs obtained with control values N mitochondrial activities were not affected a
Values were outside the lowest limit of the 95 % CI
lg protein, mean = 0.0024 OD/min/lg protein). And all these 5 mutations showed no differences on other four mitochondria complex activities (Fig. 1). Necrosis is the major form of cell death observed in pathological situation. A key signature for necrotic cells is the permeabilization of the plasma membrane, which can be quantified by measuring the release of the intracellular enzyme lactate dehydrogenase (LDH) [19]. We measured the LDH release in culture medium of the LCLs from patients with variants in AMPD1 gene. As shown in Table 4, most of these variants (p.C483S, p.R500C, p.S626C, and p.T681I) revealed a tendency that LDH release was higher than control. But a significant increase in LDH release (28.33 ± 2.9 %) in culture medium of LCLs was only identified in p.S626C mutation compared to control (14.89–17.77 %, mean = 16.33 %), implying this mutation may result in certain damaging in cells.
Discussion In this study, fourteen novel variants in the coding sequence of AMPD1 in Han Chinese samples of autism patients were identified, eleven of which were missense. p.R108L was not statistically significant on the P values of genotypic frequencies and allelic frequencies in patients and controls, so the variant would not be associated with autism risk. Among the rest ten missense variants, nine were inherited except for the de novo variation p.P572S. Traditionally, variants passed from parents to children were considered to be less effective in autism pathology. Autism
Table 4 LDH release in culture medium of the LCLs Cell line with mutation
LDH release rate (%) Mean (SEM)
Affected situationa
p.C483S
18.67 (15.61, 21.72)
N
p.R500C
23.00 (17.71, 28.29)
N
p.P572S
16.67 (5.97, 27.36)
N
p.S626C
28.33 (23.30, 33.37)
Y
p.T681I
27.33 (12.36, 42.31)
N
LDH release rate (%) (95 % CI) Control (n = 3)
16.33 (14.89, 17.77)
The table was established by evaluating the LDH release rate and then comparing them with the 95 % CI obtained with control values Y increased LDH release rate;N LDH release rate was not affected a
Values were above the highest limit of the 95 % CI
is a complex disorder and etiologically multi-factorial such as some environmental factors. Besides, different genetic backgrounds of patients would contribute to the autism pathology together with these AMPD1 mutants. In this sense, these nine inherited mutants should also be considered in the pathology of autism. To further figure out the possible roles of these ten missense mutants in autism, conservative and functional prediction was performed. It reveals that these missense mutants would have a chance of impacting the normal function of AMPD1 protein, especially p.R500C, p.P572S, p.S626C, and p.T681I, which were predicted to be both conservative and functional damaging. The T681I mutation was located in the catalytic site (Table 2), a highly conserved SLSTDDP motif [20].
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A reduced expression of several ETC genes in the anterior cingulate gyrus, motor cortex, and thalamus was observed in autism brains, implying a possible contribution of mitochondrial dysfunction in the etiology of autism [9]. Our study demonstrated that p.P572S and p.S626C mutations resulted in dysfunction of mitochondrial Complex I as assayed in the LCLs from patients. Nevertheless, it is still unknown how mutations in AMPD1 lead to mitochondrial dysfunction. One of the consequences of mitochondrial dysfunction is necrosis, especially under the situations of the uncoupling of the respiratory chain and hyperproduction of reactive oxygen species [21]. It is demonstrated that S626C mutant resulted in increased LDH release, underscoring the possible contribution of necrosis to the etiology of autism. AMPD1 catalyzes the hydrolytic removal of an amino group from the 6 position of the adenine nucleotide ring and plays a central role in maintaining the adenylate energy charge and in controlling flux through the purine nucleotide cycle. Aberrant AMPD would lead to the purine metabolism imbalance as well as breaking the equilibrium with ATP and AMP; then, the normal energy current in cells will be damaged [15]. AMPD1 activity deficiency was conventionally considered as the cause of myopathy (MIM 102770) [22], and the role of purine nucleotide in the etiology of autism has been reported. Deficiency in adenylosuccinase, a key enzyme in the purine nucleotide cycle, reveals variable degrees of developmental delay, often accompanied by autistic phenotype and seizures [23]. Moreover, the polymorphism on adenosine deaminase also confers susceptibility to autism [24]. Recently, Akizu et al. reported that mutations in AMPD2 result in an early-onset neurodegenerative disease-pontocerebellar hypoplasia [25]. AMPD2 dysfunction destroyed the cellular guanine nucleotide pools by regulating the feedback inhibition of adenosine derivatives during de novo purine synthesis process. As a result, it leads to GTPdependent translation initiation deficiency and then degeneration in the brain. Autism is a neuronal developmental disorder. However, accumulating evidences indicate that the neuronal development and neurodegeneration share many molecular and cellular mechanisms [26–28], and we thus speculate that deficiency in AMPD1 may affect similar pathways and then results in neuronal dysfunction. Substantial evidences suggest that a large proportion of autism patients also have problems with other organs, including the intestines [29], muscle, and the immune system [30]. Wide range motor abnormalities have been reported in autism, including coordination, gait [31], and impaired facial movement and voice [32]. As AMPD1 is highly expressed in muscle, it is intriguing to examine the contribution of AMPD1 deficiency in these non-neuronal phenotypes.
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In conclusion, potentially deleterious variants p.R500C, p.P572S, p.S626C, and p.T681I in AMPD1 were observed in our autism patients, and p.P572S and p.S626C lead to deteriorate mitochondria function and necrosis of cells. Our data indicated that AMPD1 is highly associated with the etiology of autism, warranting further genetic screening in large population. Acknowledgments We would like to thank our patients and their family for participating in the study, and thank Prof. Jia da Li and Fengyu Zhang for providing comments that improve manuscript. We also thank laboratory colleagues for the help and advices during the experiment. This work was supported by National Basic Research Program of China (2012CB517902), National Natural Science Foundation of China (81330027, 81161120544) and National Key Technology R&D Program of China (2012BAI03B02). Conflict of interest of interest.
The authors declare that they have no conflict
References 1. Waterhouse L, Morris R, Allen D, Dunn M, Fein D, Feinstein C, Rapin I, Wing L (1996) Diagnosis and classification in autism. J Autism Dev Disord 26(1):59–86 2. Blumberg SBM, Kogan MD, Schieve LA, Jones, JR. (2013) Changes in prevalence of parent-reported autism spectrum disorder in school-aged U.S. Children: 2007 to 2011–2012. National Health Statistics Reports 65 3. Piven J, Palmer P, Jacobi D, Childress D, Arndt S (1997) Broader autism phenotype: evidence from a family history study of multiple-incidence autism families. Am J Psychiatry 154(2):185–190 4. Bailey A, Le Couteur A, Gottesman I, Bolton P, Simonoff E, Yuzda E, Rutter M (1995) Autism as a strongly genetic disorder: evidence from a British twin study. Psychol Med 25(1):63–77 5. Walsh CA, Morrow EM, Rubenstein JL (2008) Autism and brain development. Cell 135(3):396–400. doi:10.1016/j.cell.2008.10. 015 6. Kelleher RJ 3rd, Bear MF (2008) The autistic neuron: troubled translation? Cell 135(3):401–406. doi:10.1016/j.cell.2008.10.017 7. Courchesne E, Pierce K, Schumann CM, Redcay E, Buckwalter JA, Kennedy DP, Morgan J (2007) Mapping early brain development in autism. Neuron 56(2):399–413. doi:10.1016/j.neuron. 2007.10.016 8. Page T (2000) Metabolic approaches to the treatment of autism spectrum disorders. J Autism Dev Disord 30(5):463–469 9. Anitha A, Nakamura K, Thanseem I, Matsuzaki H, Miyachi T, Tsujii M, Iwata Y, Suzuki K, Sugiyama T, Mori N (2013) Downregulation of the expression of mitochondrial electron transport complex genes in autism brains. Brain Pathol 23(3):294–302. doi:10.1111/bpa.12002 10. Giulivi C, Zhang YF, Omanska-Klusek A, Ross-Inta C, Wong S, Hertz-Picciotto I, Tassone F, Pessah IN (2010) Mitochondrial dysfunction in autism. JAMA 304(21):2389–2396. doi:10.1001/ jama.2010.1706 11. Xia K, Guo H, Hu Z, Xun G, Zuo L, Peng Y, Wang K, He Y, Xiong Z, Sun L, Pan Q, Long Z, Zou X, Li X, Li W, Xu X, Lu L, Liu Y, Hu Y, Tian D, Long L, Ou J, Zhang L, Pan Y, Chen J, Peng H, Liu Q, Luo X, Su W, Wu L, Liang D, Dai H, Yan X, Feng Y, Tang B, Li J, Miedzybrodzka Z, Xia J, Zhang Z, Zhang
Eur Arch Psychiatry Clin Neurosci
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
X, StClair D, Zhao J, Zhang F (2013) Common genetic variants on 1p13.2 associate with risk of autism. Mol Psychiatry. doi:10. 1038/mp.2013.146 Auranen M, Nieminen T, Majuri S, Vanhala R, Peltonen L, Jarvela I (2000) Analysis of autism susceptibility gene loci on chromosomes 1p, 4p, 6q, 7q, 13q, 15q, 16p, 17q, 19q and 22q in Finnish multiplex families. Mol Psychiatry 5(3):320–322 Risch N, Spiker D, Lotspeich L, Nouri N, Hinds D, Hallmayer J, Kalaydjieva L, McCague P, Dimiceli S, Pitts T, Nguyen L, Yang J, Harper C, Thorpe D, Vermeer S, Young H, Hebert J, Lin A, Ferguson J, Chiotti C, Wiese-Slater S, Rogers T, Salmon B, Nicholas P, Petersen PB, Pingree C, McMahon W, Wong DL, Cavalli-Sforza LL, Kraemer HC, Myers RM (1999) A genomic screen of autism: evidence for a multilocus etiology. Am J Hum Genet 65(2):493–507. doi:10.1086/302497 Allen-Brady K, Miller J, Matsunami N, Stevens J, Block H, Farley M, Krasny L, Pingree C, Lainhart J, Leppert M, McMahon WM, Coon H (2009) A high-density SNP genome-wide linkage scan in a large autism extended pedigree. Mol Psychiatry 14(6):590–600. doi:10.1038/mp.2008.14 Van den Berghe G, Bontemps F, Vincent MF, Van den Bergh F (1992) The purine nucleotide cycle and its molecular defects. Prog Neurobiol 39(5):547–561 Dale O, Salo M (1996) The Helsinki declaration, research guidelines and regulations: present and future editorial aspects. Acta Anaesthesiol Scand 40(7):771–772 Anderson MA, Gusella JF (1984) Use of cyclosporin A in establishing Epstein-Barr virus-transformed human lymphoblastoid cell lines. In Vitro 20(11):856–858 Bernier FP, Boneh A, Dennett X, Chow CW, Cleary MA, Thorburn DR (2002) Diagnostic criteria for respiratory chain disorders in adults and children. Neurology 59(9):1406–1411 Chan FK, Moriwaki K, De Rosa MJ (2013) Detection of necrosis by release of lactate dehydrogenase activity. Methods Mol Biol 979:65–70. doi:10.1007/978-1-62703-290-2_7 Gross M, Morisaki H, Morisaki T, Holmes EW (1994) Identification of functional domains in AMPD1 by mutational analysis. Biochem Biophys Res Commun 205(2):1010–1017. doi:10.1006/ bbrc.1994.2767 Kroemer G, Dallaporta B, Resche-Rigon M (1998) The mitochondrial death/life regulator in apoptosis and necrosis. Annu Rev Physiol 60:619–642. doi:10.1146/annurev.physiol.60.1.619 Sabina RL, Swain JL, Patten BM, Ashizawa T, O’Brien WE, Holmes EW (1980) Disruption of the purine nucleotide cycle. A
23.
24.
25.
26.
27. 28.
29. 30.
31.
32.
potential explanation for muscle dysfunction in myoadenylate deaminase deficiency. J Clin Invest 66(6):1419–1423. doi:10. 1172/JCI109995 Spiegel EK, Colman RF, Patterson D (2006) Adenylosuccinate lyase deficiency. Mol Genet Metab 89(1–2):19–31. doi:10.1016/j. ymgme.2006.04.018 Persico AM, Militerni R, Bravaccio C, Schneider C, Melmed R, Trillo S, Montecchi F, Palermo MT, Pascucci T, Puglisi-Allegra S, Reichelt KL, Conciatori M, Baldi A, Keller F (2000) Adenosine deaminase alleles and autistic disorder: case–control and family-based association studies. Am J Med Genet 96(6): 784–790. doi:10.1002/1096-8628(20001204)96:6\784:AID-AJMG 18[3.0.CO;2-7 Akizu N, Cantagrel V, Schroth J, Cai N, Vaux K, McCloskey D, Naviaux RK, Van Vleet J, Fenstermaker AG, Silhavy JL, Scheliga JS, Toyama K, Morisaki H, Sonmez FM, Celep F, Oraby A, Zaki MS, Al-Baradie R, Faqeih EA, Saleh MA, Spencer E, Rosti RO, Scott E, Nickerson E, Gabriel S, Morisaki T, Holmes EW, Gleeson JG (2013) AMPD2 regulates GTP synthesis and is mutated in a potentially treatable neurodegenerative brainstem disorder. Cell 154(3):505–517. doi:10.1016/j.cell.2013.07.005 Yang J, Weimer RM, Kallop D, Olsen O, Wu Z, Renier N, Uryu K, Tessier-Lavigne M (2013) Regulation of axon degeneration after injury and in development by the endogenous calpain inhibitor calpastatin. Neuron. doi:10.1016/j.neuron.2013.08.034 Glynn P (2000) Neural development and neurodegeneration: two faces of neuropathy target esterase. Prog Neurobiol 61(1):61–74 Wong M (2013) Mammalian target of rapamycin (mTOR) pathways in neurological diseases. Biomed J 36(2):40–50. doi:10. 4103/2319-4170.110365 White JF (2003) Intestinal pathophysiology in autism. Exp Biol Med (Maywood) 228(6):639–649 Depino AM (2013) Peripheral and central inflammation in autism spectrum disorders. Mol Cell Neurosci 53:69–76. doi:10.1016/j. mcn.2012.10.003 Jansiewicz EM, Goldberg MC, Newschaffer CJ, Denckla MB, Landa R, Mostofsky SH (2006) Motor signs distinguish children with high functioning autism and Asperger’s syndrome from controls. J Autism Dev Disord 36(5):613–621. doi:10.1007/ s10803-006-0109-y Al Abdulmohsen T, Kruger TH (2011) The contribution of muscular and auditory pathologies to the symptomatology of autism. Med Hypotheses 77(6):1038–1047. doi:10.1016/j.mehy. 2011.08.044
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ORIGINAL PAPER
AMPD1 functional variants associated with autism in Han Chinese population Lusi Zhang • Jianjun Ou • Xiaojuan Xu • Yu Peng • Hui Guo • Yongcheng Pan Jingjing Chen • Tianyun Wang • Hao Peng • Qiong Liu • Di Tian • Qian Pan • Xiaobin Zou • Jingping Zhao • Zhengmao Hu • Kun Xia
•
Received: 24 January 2014 / Accepted: 9 August 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Autism is a childhood neurodevelopmental disorder with high heterogeneity. Following our genomewide associated loci with autism, we performed sequencing analysis of the coding regions, UTR and flanking splice junctions of AMPD1 in 830 Chinese autism individuals as well as 514 unrelated normal controls. Fourteen novel variants in the coding sequence were identified, including 11 missense variants and 3 synonymous mutations. Among these missense variants, 10 variants were absent in 514 control subjects, and conservative and functional prediction was carried out. Mitochondria activity and lactate dehydrogenase assay were performed in 5 patients’ lymphoblast cell lines; p.P572S and p.S626C showed decreased mitochondrial complex I activity, and p.S626C increased lactate dehydrogenase release in medium. Conclusively, our data suggested that mutational variants in AMPD1 contribute to autism risk in Han Chinese population, uncovering the Electronic supplementary material The online version of this article (doi:10.1007/s00406-014-0524-6) contains supplementary material, which is available to authorized users. L. Zhang X. Xu Y. Peng H. Guo Y. Pan J. Chen T. Wang H. Peng Q. Liu D. Tian Q. Pan J. Zhao Z. Hu (&) K. Xia (&) State Key Laboratory of Medical Genetics, Central South University, 110, Xiangya Road, Changsha Hunan, China e-mail: [email protected] K. Xia e-mail: [email protected] L. Zhang Y. Peng H. Guo K. Xia School of Life Sciences, Central South University, Changsha, Hunan, China
contribution of mutant protein to disease development that operates via mitochondria dysfunction and cell necrosis. Keywords
Autism AMPD1 Mutation Mitochondria
Introduction Autism is a broadly defined childhood neurodevelopmental disorder with onset prior to 3 years of age. The core symptoms were characterized by impairment in language and social communication, and repetitive and restrictive behaviors [1]. Autism has emerged as a major public health concern with an estimated prevalence of 1:50 in USA [2] and is considered to be the most inheritable neuropsychiatric disorders according to the familial and twin studies [3, 4]. A growing list of disease-associated genetic factors has been found through cytogenetics, genome-wide association studies, linkage analysis, and exome sequencing. Functional studies following these genetic analysis and pathophysiological studies have implicated some underlying X. Xu The Reproductive Medicine Hospital of the First Hospital of Lanzhou University, Lanzhou, Gansu, China X. Zou Department of Pediatrics, No. 3 Hospital of the Sun Yat-sen University, Guangzhou, Guangdong, China K. Xia Key Laboratory of Medical Information Research, Central South University, Changsha, Hunan, China
J. Ou J. Zhao Mental Health Institute, The Second Xiangya Hospital, Central South University, Changsha, Hunan, China
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mechanisms, including abnormal synaptogenesis, neurite growth, negative changes of neural plasticity [5, 6], neuronal cell loss and neurodegeneration [7], metabolic abnormality [8], and mitochondrial dysfunction [9, 10]. We have recently reported a genome-wide association study of autism in Chinese population [11]. Three risk single nucleotide polymorphisms, rs926938, rs6537835, and rs1877455, were identified, and related haplotypes at AMPD1-NRAS-CSDE1, TRIM33, and TRIM33-BCAS2 associated with autism were mapped to a previously reported autism linkage region 1p13.2 [12–14]. The adenosine monophosphate deaminase (AMPD, EC 3.5.4.6) catalyzes AMP hydrolyzing into IMP and NH3. The proteins of AMPD family include three members. AMPD1, mapped on chromosome 1p13.2, encodes AMP deaminase 1, which plays an important role in the purine nucleotide cycle [15]. To further find out the genetic evidence of AMPD1 for autism, we performed the mutation analysis on AMPD1 gene in autism patients of Han Chinese population in additional independent sample, and the possible underlying mechanism was also examined.
Materials and methods Subjects The study sample included 830 patients (males 731, females 99, the average age was 6, range from 3 to 18 years old) recruited from Elim Autism Training Department of the Qingdao Municipal Autism Research Institute, Qingdao, Shandong Province, China, and Psychiatric Department of the Second Xiangya Hospital of Central South University, Changsha, Hunan Province, China. All enrolled probands and their parents were of Chinese Han ancestry. Patients were diagnosed according to International classification of Diseases, Diagnostic and Statistical Manual of Mental Disorders-IV criteria, and autistic disorder and the Chinese Society of Psychiatry’s Chinese Classification of Mental Disorders, by two independent senior psychiatrists from the Psychiatric department of the Second Xiangya Hospital. Karyotyping analysis was performed on all patients and their parents to rule out those with chromosomal abnormalities. Moreover, Patients with fragile X syndrome, tuberous sclerosis, dysmorphic features, or any other neurological/medical condition suspected to autism were excluded. In order to rule out possible unreported polymorphism in our patients, unrelated controls was collected randomly, which included 514 unrelated Han Chinese healthy volunteers (males 248, females 266, the average age is 22, range from 3 to 46 years old). All patients, parents and controls were not involved in our previous study [11].
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Written informed consent was obtained from all the patients along with their guardians and controls. The research was approved by the Human Ethics Committee of The State Key Laboratory of Medical Genetics, Central South University, and is compliant with the Code of Ethics of the World Medical Association [16]. Sequencing and mutant analysis Genomic DNA was extracted from peripheral blood lymphocytes of all individuals by means of the standard phenol–chloroform method. The primer spanning all coding regions (Genbank accession number RefSeq NM_000036.2), splicing junctions, and UTR of AMPD1 were designed by the online tool Primer3 (http://frodo.wi. mit.edu/). PCR reactions were carried out in a thermocycler (GeneAmp 2720, Applied Biosystems Inc., Foster City, CA, USA). The primer sequences and PCR conditions are available upon request. PCR products were verified by 6 % polyacrylamide gel electrophoresis and silver staining. Sanger sequencing of all the 830 affected cases along with the 514 unaffected controls was performed on an ABI3100/ 3130 automated sequencer (Applied Biosystems Inc., Foster City, CA, USA) according to the manufacturer’s instructions. The parents whose children carry novel variants were further sequenced on the mutated points to detect inheritance status. Conservative analyses of nonsynonymous variants were performed by multiple sequence alignment tools clustalX, phylogenetic P values (Phylop), and genomic evolutionary rate profiling (GERP). To perform functional prediction, we used the software SIFT, PolyPhen, and MutationTaster to score these variants. Mitochondrial complex activity assay Primary B lymphocytes from patients and control were transformed to lymphoblast cell lines (LCLs) using Epstein-Barr virus and cyclosporin A [17]. LCLs of 5 patients (p.C483S, p.R500C, p.P572S, p.S626C, and p.T681I) and 3 controls cultured in T25 flasks were centrifuged at 2509g for 5 min to collect cells. Mitochondrial complex activities were measured using following kits according to manufacturer’s instructions: Complex I activity was evaluated by following Mitochondrial Complex I Activity Assay Kit (Novagen, Merck KGaA, Darmstadt, Germany); succinate-coenzyme Q reductase activity was measured by Mitochondrial Complex II Activity Assay Kit (Novagen, Merck KGaA, Darmstadt, Germany); Complex IV activity assay was performed using Mitochondrial Complex IV (Human) Activity Assay Kit (Novagen, Merck KGaA, Darmstadt, Germany); Complex V was evaluated by following ATPase activity through
Eur Arch Psychiatry Clin Neurosci
Mitochondrial Complex V (ATP Synthase) Activity Assay Kit(Novagen, Merck KGaA, Darmstadt, Germany); and MitoToxTM OXPHOS Complex III Activity Kit (Abcam, Cambridge, UK) was used to evaluate Complex II and Complex III activities. For mitochondrial dysfunction can often be caused by the simple decrease in numbers, mitochondrial number should be normalized by the activity of citrate synthase, which is thought to be relatively stable and proportional to the mitochondrial numbers [18]. The activity of citrate synthase in LCLs was evaluated by Citrate Synthase Assay Kit (Sigma-Aldrich Co., St. Louis, MO, USA). All the measurements were performed on BioTek SynergyTM 2 microplate reader (BioTek U.S., Winooski, VT, USA). Each assay was repeated at least three times. LDH release assay Lymphoblast cell lines were cultured in 6-well plates for 24 h ahead of test. LCLs of 5 patients (p.C483S, p.R500C, p.P572S, p.S626C, and p.T681I) and 3 controls were collected separately by centrifuged at 2509g for 5 min. Cell culture medium was also collected and centrifuged at 5,0009g for 10 min to remove cell debris. LCLs were lysed in a lysis buffer (0.1 % triton X-100 in PBS) for 10 min at 4 °C. LDH in the medium and cell lysates were measured using CytoTox 96Ò Non-Radioactive Cytotoxicity Assay Kit (Promega, Madison, WI, USA) according to manufacturer’s instruction. The rate of LDH release was given by the following: %LDH release = medium A492/total A492 = medium A492/(medium A492 ? cell lysate A492). The measurements were performed on BioTek SynergyTM 2 microplate reader (BioTek U.S., Winooski, VT, USA). Each experiment was repeated for three times. Statistic analysis The data of each patient were compared with the 95 % CIs of the control values. And the values above the highest limit of the 95 % CI or outside the lowest limit of the 95 % CI were considered different. The data of patients were presented as mean ± SEM. Fisher’s exact test was used to compare the allelic and genotypic distributions between patients and controls and to calculate the odds ratio and 95 % confidence intervals (95 % CI). Statistical analyses were conducted using the SPSS 13.0 statistical software package (SPSS Inc., Chicago, IL, USA). A P value \ 0.05 (two-tailed) was considered significant.
Results Through sequencing the exons, splicing junctions, and 50 UTR of AMPD1 gene in 830 patients, we identified 14
unreported variants in the coding sequence (Table 1), including 11 missense variants and 3 synonymous mutations. Several SNPs were detected as well (Supplemental Table 1). Among the 11 non-synonymous variants, 10 missense variants (sequence results were shown in Supplemental Fig. 1) were neither presented in 514 normal control subjects nor the 1,000 Genomes data (http://browser.1000genomes.org/). Only 1 non-synonymous variant p.R108L was detected in both 1 patient and 2 controls. We analyzed the allelic and genotypic distribution frequencies of the p.R108L variant; as shown in Supplemental Table 2, the P values of genotypic frequencies and allelic frequencies for p.R108L in 830 autism patients and 514 control subjects were 0.310575 and 0.310846, respectively, and odds ratio of the p.R108L allele was 3.234 (95 % CI = 0.293–35.711; P = 0.310783). Since there was lack of a statistically significant association of AMPD1 p.R108L with the risk of autism, it was excluded in following analysis. Besides, 6 control-specific variants were also detected (Table 1). To see whether these 10 patient-specific missense mutations were de novo, we also sequenced the parental DNA samples. The p.P572S mutation was a de novo mutation, whereas the rest were inherited no bias between maternal versus paternal transmission (Table 1). The 3 synonymous mutations, p.F213F, p.P234P, and p.T523T, were not to be observed in controls, and we excluded them in further analysis. To investigate possible influence of the 10 patient-specific missense mutations on AMPD1 protein function, we performed the conservation prediction by using ClustalX, Phylop, and GERP, and function consequences by using MutatinTaster, Polyphen-2, and SIFT on these mutants. ClustalX result demonstrated asterisks in the amino acid positions 500, 572, 626, and 681 (Table 1 and Supplemental Fig. 2), which revealed highly conserved amino acid on these sites, and the dot on position 483 suggested moderate conserved amino acid. As shown in Table 2, p.D203V, p.D276N, p.C483S, p.R500C, p.P572S, p.S626C, and p.T681I were predicted conserved nucleotide substitutions by Phylop for the scores that were close to 1. Nucleotide substitutions in p.D276N, p.C483S, p.R500C, p.P572S, p.S626C, and p.T681I revealed damaging among GERP analysis. Considering the analysis results above, the amino acid positions 483, 500, 572, 626, and 681 were quite conservative in vertebrates AMPD1. MutationTaster predicted p.I202M, p.D276N, p.R500C, p.P572S, p.S626C, and p.T681I as disease causing nucleotide substitutions. Both Polyphen-2 and SIFT results predicted that p.D276N, p.R500C, p.P572S, p.S626C, and p.T681I were probably damaging nucleotide variations (Table 2). The results of three functional analysis tools demonstrated that p.D276N, p.R500C, p.P572S, p.S626C, and p.T681I mutations
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Eur Arch Psychiatry Clin Neurosci Table 1 Variants identified in the AMPD1 coding regions
Location
Nucleotide change
Amino acid change
Patients (n = 830, het/homo)
Controls (n = 514, het/homo)
Inheritance statusa
Nonsynonymous variants
N/A data were not available, het/ homo homozygous change/ heterozygous change, Pat paternal, Mat maternal a
Whether the variant was inherited from the parents or not
b
The 2 mutants were carried by the same patient
c
The control-specific variants
Exon1
c.31C[G
p. Q11E
1/0
0/0
Mat
Exon1
c.70G[A
p. A24T
1/0
0/0
Pat
Exon4
c.323G[T
p. R108L
1/0
2/0
N/A
Exon5b
c.606T[G
p. I202M
1/0
0/0
Mat
Exon5b
c.608A[T
p. D203V
1/0
0/0
Mat
Exon6c
c.693C[G
p. D231E
0/0
1/0
N/A
Exon6c
c.753T[A
p. Y251X
0/0
1/0
N/A
Exon6
c.826G[A
p. D276N
1/0
0/0
Pat
Exon10
c.1449C[G
p. C483S
1/0
0/0
Pat
Exon10c Exon11
c.1460C[T c.1498C[T
p. R487L p. R500C
0/0 2/0
1/0 0/0
N/A Pat
Exon12c
c.1641T[G
p. D547E
0/0
1/0
N/A De novo
Exon12
c.1714C[T
p. P572S
1/0
0/0
Exon13c
c.1783C[T
p. R595X
0/0
1/0
N/A
Exon13
c.1877C[G
p. S626C
1/0
0/0
Mat
Exon13c
c.1879C[A
p. H627L
0/0
1/0
N/A
Exon14
c.2042C[T
p. T681I
1/0
0/0
Mat N/A
Synonymous variants Exon5
c.639C[T
p. F213F
1/0
0/0
Exon6
c.702T[A
p. P234P
1/0
0/0
N/A
Exon11
c.1569C[T
p.T523T
1/0
0/0
N/A
Table 2 Conservative and functional analysis on 10 missense mutations in AMPD1 Mutant
Protein domain
Conservative analysis
Functional analysis
Phylop
GERP
ClustalX
MutationTaster
PolyPhen-2
SIFT
p.Q11E
N/A
0.022, N
0.46, T
N
Polymorphism
0.001, B
0.95, T
p.A24T
N/A
0.017, N
0.67, T
N
Polymorphism
0.077, B
0.17, T
p.I202M
Myosin binding domain
0.027, N
0.83, T
N
Disease causing
0.049, B
0.24, T
p.D203V
Myosin binding domain
0.998, C
0.93, T
N
Disease causing
0.000, B
0.27, T
p.D276N
Myosin binding domain
0.999, C
1, D
N
Disease causing
0.999, P
0.03, D
p.C483S
N/A
0.972, C
1, D
MC
Disease causing
0.000, B
1, T
p.R500C
N/A
0.999, C
1, D
C
Disease causing
1.000, P
0, D
p.P572S
N/A
0.999, C
1, D
C
Disease causing
1.000, P
0, D
p.S626C
N/A
0.999, C
1, D
C
Disease causing
1.000, P
0.05, D
p.T681I
Catalytic site
0.999, C
1, D
C
Disease causing
1.000, P
0, D
C conserved change, MC moderate conserved, N non-conserved, T tolerable change, B benign, P probably damaging, D damaging, N/A no exact domain information
probably impact the structure and function of human AMPD1 protein. It was reported that children with autism were more likely to suffer from mitochondrial dysfunction (mainly in complexes I and IV), mtDNA over-replication, and mtDNA deletions than controls [10]. Thus, we assayed the activities of electron transport chains (ETC) in LCLs from
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patients with variants p.C483S, p.R500C, p.P572S, p.S626C, and p.T681I to see whether there is any mitochondrial defect in these patients. As shown in Table 3, p.P572S and p.S626C mutations resulted in significant reduction (0.0014 ± 0.0003 OD/min/lg protein and 0.0016 ± 0.0002 OD/min/lg protein) in the activity of Complex I compared to control (0.0019–0.0029 OD/min/
Eur Arch Psychiatry Clin Neurosci Table 3 Mitochondrial Activities in the LCLs Cell line with mutation
Mean (SEM) OD/min/lg cell lysate a
Affected complexes
ComplexV
Complex I
Complex II
Complex III
Complex IV
p.C483S
0.0017 (0.0015, 0.0019)
0.00082 (0.00070, 0.0093)
0.016 (0.015, 0.017)
0.00068 (0.00048, 0.00087)
0.0048 (0.0043, 0.0054)
N
p.R500C
0.0024 (0.0023, 0.0024)
0.00093 (0.00085, 0.00102)
0.017 (0.015, 0.019)
0.00064 (0.00042, 0.00086)
0.0043 (0.0038, 0.0047)
N
p.P572S
0.0014 (0.0011, 0.0017)
0.00073 (0.00060, 0.00086)
0.017 (0.016, 0.018)
0.00068 (0.00049, 0.00086)
0.0039 (0.0038, 0.0040)
Ia
p.S626C
0.0016 (0.0014, 0.0018)
0.00080 (0.00070, 0.00090)
0.020 (0.017, 0.023)
0.00080 (0.00062, 0.00098)
0.0048 (0.0043, 0.0053)
Ia
p.T681I
0.0017 (0.0014, 0.0020)
0.00063 (0.00070, 0.00090)
0.018 (0.015, 0.022)
0.00070 (0.00051, 0.00089)
0.0044 (0.0039, 0.0050)
N
0.00068 (0.00018, 0.00185)
0.018 (0.012, 0.024)
0.00058 (0.00016, 0.00099)
0.0038 (0.0026, 0.0050)
Mean activities (95 % CI) Control (n = 3)
0.0024 (0.0019, 0.0029)
The table was established by evaluating the activity of each segment of the electron transport chain and then comparing them with the 95 % CIs obtained with control values N mitochondrial activities were not affected a
Values were outside the lowest limit of the 95 % CI
lg protein, mean = 0.0024 OD/min/lg protein). And all these 5 mutations showed no differences on other four mitochondria complex activities (Fig. 1). Necrosis is the major form of cell death observed in pathological situation. A key signature for necrotic cells is the permeabilization of the plasma membrane, which can be quantified by measuring the release of the intracellular enzyme lactate dehydrogenase (LDH) [19]. We measured the LDH release in culture medium of the LCLs from patients with variants in AMPD1 gene. As shown in Table 4, most of these variants (p.C483S, p.R500C, p.S626C, and p.T681I) revealed a tendency that LDH release was higher than control. But a significant increase in LDH release (28.33 ± 2.9 %) in culture medium of LCLs was only identified in p.S626C mutation compared to control (14.89–17.77 %, mean = 16.33 %), implying this mutation may result in certain damaging in cells.
Discussion In this study, fourteen novel variants in the coding sequence of AMPD1 in Han Chinese samples of autism patients were identified, eleven of which were missense. p.R108L was not statistically significant on the P values of genotypic frequencies and allelic frequencies in patients and controls, so the variant would not be associated with autism risk. Among the rest ten missense variants, nine were inherited except for the de novo variation p.P572S. Traditionally, variants passed from parents to children were considered to be less effective in autism pathology. Autism
Table 4 LDH release in culture medium of the LCLs Cell line with mutation
LDH release rate (%) Mean (SEM)
Affected situationa
p.C483S
18.67 (15.61, 21.72)
N
p.R500C
23.00 (17.71, 28.29)
N
p.P572S
16.67 (5.97, 27.36)
N
p.S626C
28.33 (23.30, 33.37)
Y
p.T681I
27.33 (12.36, 42.31)
N
LDH release rate (%) (95 % CI) Control (n = 3)
16.33 (14.89, 17.77)
The table was established by evaluating the LDH release rate and then comparing them with the 95 % CI obtained with control values Y increased LDH release rate;N LDH release rate was not affected a
Values were above the highest limit of the 95 % CI
is a complex disorder and etiologically multi-factorial such as some environmental factors. Besides, different genetic backgrounds of patients would contribute to the autism pathology together with these AMPD1 mutants. In this sense, these nine inherited mutants should also be considered in the pathology of autism. To further figure out the possible roles of these ten missense mutants in autism, conservative and functional prediction was performed. It reveals that these missense mutants would have a chance of impacting the normal function of AMPD1 protein, especially p.R500C, p.P572S, p.S626C, and p.T681I, which were predicted to be both conservative and functional damaging. The T681I mutation was located in the catalytic site (Table 2), a highly conserved SLSTDDP motif [20].
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A reduced expression of several ETC genes in the anterior cingulate gyrus, motor cortex, and thalamus was observed in autism brains, implying a possible contribution of mitochondrial dysfunction in the etiology of autism [9]. Our study demonstrated that p.P572S and p.S626C mutations resulted in dysfunction of mitochondrial Complex I as assayed in the LCLs from patients. Nevertheless, it is still unknown how mutations in AMPD1 lead to mitochondrial dysfunction. One of the consequences of mitochondrial dysfunction is necrosis, especially under the situations of the uncoupling of the respiratory chain and hyperproduction of reactive oxygen species [21]. It is demonstrated that S626C mutant resulted in increased LDH release, underscoring the possible contribution of necrosis to the etiology of autism. AMPD1 catalyzes the hydrolytic removal of an amino group from the 6 position of the adenine nucleotide ring and plays a central role in maintaining the adenylate energy charge and in controlling flux through the purine nucleotide cycle. Aberrant AMPD would lead to the purine metabolism imbalance as well as breaking the equilibrium with ATP and AMP; then, the normal energy current in cells will be damaged [15]. AMPD1 activity deficiency was conventionally considered as the cause of myopathy (MIM 102770) [22], and the role of purine nucleotide in the etiology of autism has been reported. Deficiency in adenylosuccinase, a key enzyme in the purine nucleotide cycle, reveals variable degrees of developmental delay, often accompanied by autistic phenotype and seizures [23]. Moreover, the polymorphism on adenosine deaminase also confers susceptibility to autism [24]. Recently, Akizu et al. reported that mutations in AMPD2 result in an early-onset neurodegenerative disease-pontocerebellar hypoplasia [25]. AMPD2 dysfunction destroyed the cellular guanine nucleotide pools by regulating the feedback inhibition of adenosine derivatives during de novo purine synthesis process. As a result, it leads to GTPdependent translation initiation deficiency and then degeneration in the brain. Autism is a neuronal developmental disorder. However, accumulating evidences indicate that the neuronal development and neurodegeneration share many molecular and cellular mechanisms [26–28], and we thus speculate that deficiency in AMPD1 may affect similar pathways and then results in neuronal dysfunction. Substantial evidences suggest that a large proportion of autism patients also have problems with other organs, including the intestines [29], muscle, and the immune system [30]. Wide range motor abnormalities have been reported in autism, including coordination, gait [31], and impaired facial movement and voice [32]. As AMPD1 is highly expressed in muscle, it is intriguing to examine the contribution of AMPD1 deficiency in these non-neuronal phenotypes.
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In conclusion, potentially deleterious variants p.R500C, p.P572S, p.S626C, and p.T681I in AMPD1 were observed in our autism patients, and p.P572S and p.S626C lead to deteriorate mitochondria function and necrosis of cells. Our data indicated that AMPD1 is highly associated with the etiology of autism, warranting further genetic screening in large population. Acknowledgments We would like to thank our patients and their family for participating in the study, and thank Prof. Jia da Li and Fengyu Zhang for providing comments that improve manuscript. We also thank laboratory colleagues for the help and advices during the experiment. This work was supported by National Basic Research Program of China (2012CB517902), National Natural Science Foundation of China (81330027, 81161120544) and National Key Technology R&D Program of China (2012BAI03B02). Conflict of interest of interest.
The authors declare that they have no conflict
References 1. Waterhouse L, Morris R, Allen D, Dunn M, Fein D, Feinstein C, Rapin I, Wing L (1996) Diagnosis and classification in autism. J Autism Dev Disord 26(1):59–86 2. Blumberg SBM, Kogan MD, Schieve LA, Jones, JR. (2013) Changes in prevalence of parent-reported autism spectrum disorder in school-aged U.S. Children: 2007 to 2011–2012. National Health Statistics Reports 65 3. Piven J, Palmer P, Jacobi D, Childress D, Arndt S (1997) Broader autism phenotype: evidence from a family history study of multiple-incidence autism families. Am J Psychiatry 154(2):185–190 4. Bailey A, Le Couteur A, Gottesman I, Bolton P, Simonoff E, Yuzda E, Rutter M (1995) Autism as a strongly genetic disorder: evidence from a British twin study. Psychol Med 25(1):63–77 5. Walsh CA, Morrow EM, Rubenstein JL (2008) Autism and brain development. Cell 135(3):396–400. doi:10.1016/j.cell.2008.10. 015 6. Kelleher RJ 3rd, Bear MF (2008) The autistic neuron: troubled translation? Cell 135(3):401–406. doi:10.1016/j.cell.2008.10.017 7. Courchesne E, Pierce K, Schumann CM, Redcay E, Buckwalter JA, Kennedy DP, Morgan J (2007) Mapping early brain development in autism. Neuron 56(2):399–413. doi:10.1016/j.neuron. 2007.10.016 8. Page T (2000) Metabolic approaches to the treatment of autism spectrum disorders. J Autism Dev Disord 30(5):463–469 9. Anitha A, Nakamura K, Thanseem I, Matsuzaki H, Miyachi T, Tsujii M, Iwata Y, Suzuki K, Sugiyama T, Mori N (2013) Downregulation of the expression of mitochondrial electron transport complex genes in autism brains. Brain Pathol 23(3):294–302. doi:10.1111/bpa.12002 10. Giulivi C, Zhang YF, Omanska-Klusek A, Ross-Inta C, Wong S, Hertz-Picciotto I, Tassone F, Pessah IN (2010) Mitochondrial dysfunction in autism. JAMA 304(21):2389–2396. doi:10.1001/ jama.2010.1706 11. Xia K, Guo H, Hu Z, Xun G, Zuo L, Peng Y, Wang K, He Y, Xiong Z, Sun L, Pan Q, Long Z, Zou X, Li X, Li W, Xu X, Lu L, Liu Y, Hu Y, Tian D, Long L, Ou J, Zhang L, Pan Y, Chen J, Peng H, Liu Q, Luo X, Su W, Wu L, Liang D, Dai H, Yan X, Feng Y, Tang B, Li J, Miedzybrodzka Z, Xia J, Zhang Z, Zhang
Eur Arch Psychiatry Clin Neurosci
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
X, StClair D, Zhao J, Zhang F (2013) Common genetic variants on 1p13.2 associate with risk of autism. Mol Psychiatry. doi:10. 1038/mp.2013.146 Auranen M, Nieminen T, Majuri S, Vanhala R, Peltonen L, Jarvela I (2000) Analysis of autism susceptibility gene loci on chromosomes 1p, 4p, 6q, 7q, 13q, 15q, 16p, 17q, 19q and 22q in Finnish multiplex families. Mol Psychiatry 5(3):320–322 Risch N, Spiker D, Lotspeich L, Nouri N, Hinds D, Hallmayer J, Kalaydjieva L, McCague P, Dimiceli S, Pitts T, Nguyen L, Yang J, Harper C, Thorpe D, Vermeer S, Young H, Hebert J, Lin A, Ferguson J, Chiotti C, Wiese-Slater S, Rogers T, Salmon B, Nicholas P, Petersen PB, Pingree C, McMahon W, Wong DL, Cavalli-Sforza LL, Kraemer HC, Myers RM (1999) A genomic screen of autism: evidence for a multilocus etiology. Am J Hum Genet 65(2):493–507. doi:10.1086/302497 Allen-Brady K, Miller J, Matsunami N, Stevens J, Block H, Farley M, Krasny L, Pingree C, Lainhart J, Leppert M, McMahon WM, Coon H (2009) A high-density SNP genome-wide linkage scan in a large autism extended pedigree. Mol Psychiatry 14(6):590–600. doi:10.1038/mp.2008.14 Van den Berghe G, Bontemps F, Vincent MF, Van den Bergh F (1992) The purine nucleotide cycle and its molecular defects. Prog Neurobiol 39(5):547–561 Dale O, Salo M (1996) The Helsinki declaration, research guidelines and regulations: present and future editorial aspects. Acta Anaesthesiol Scand 40(7):771–772 Anderson MA, Gusella JF (1984) Use of cyclosporin A in establishing Epstein-Barr virus-transformed human lymphoblastoid cell lines. In Vitro 20(11):856–858 Bernier FP, Boneh A, Dennett X, Chow CW, Cleary MA, Thorburn DR (2002) Diagnostic criteria for respiratory chain disorders in adults and children. Neurology 59(9):1406–1411 Chan FK, Moriwaki K, De Rosa MJ (2013) Detection of necrosis by release of lactate dehydrogenase activity. Methods Mol Biol 979:65–70. doi:10.1007/978-1-62703-290-2_7 Gross M, Morisaki H, Morisaki T, Holmes EW (1994) Identification of functional domains in AMPD1 by mutational analysis. Biochem Biophys Res Commun 205(2):1010–1017. doi:10.1006/ bbrc.1994.2767 Kroemer G, Dallaporta B, Resche-Rigon M (1998) The mitochondrial death/life regulator in apoptosis and necrosis. Annu Rev Physiol 60:619–642. doi:10.1146/annurev.physiol.60.1.619 Sabina RL, Swain JL, Patten BM, Ashizawa T, O’Brien WE, Holmes EW (1980) Disruption of the purine nucleotide cycle. A
23.
24.
25.
26.
27. 28.
29. 30.
31.
32.
potential explanation for muscle dysfunction in myoadenylate deaminase deficiency. J Clin Invest 66(6):1419–1423. doi:10. 1172/JCI109995 Spiegel EK, Colman RF, Patterson D (2006) Adenylosuccinate lyase deficiency. Mol Genet Metab 89(1–2):19–31. doi:10.1016/j. ymgme.2006.04.018 Persico AM, Militerni R, Bravaccio C, Schneider C, Melmed R, Trillo S, Montecchi F, Palermo MT, Pascucci T, Puglisi-Allegra S, Reichelt KL, Conciatori M, Baldi A, Keller F (2000) Adenosine deaminase alleles and autistic disorder: case–control and family-based association studies. Am J Med Genet 96(6): 784–790. doi:10.1002/1096-8628(20001204)96:6\784:AID-AJMG 18[3.0.CO;2-7 Akizu N, Cantagrel V, Schroth J, Cai N, Vaux K, McCloskey D, Naviaux RK, Van Vleet J, Fenstermaker AG, Silhavy JL, Scheliga JS, Toyama K, Morisaki H, Sonmez FM, Celep F, Oraby A, Zaki MS, Al-Baradie R, Faqeih EA, Saleh MA, Spencer E, Rosti RO, Scott E, Nickerson E, Gabriel S, Morisaki T, Holmes EW, Gleeson JG (2013) AMPD2 regulates GTP synthesis and is mutated in a potentially treatable neurodegenerative brainstem disorder. Cell 154(3):505–517. doi:10.1016/j.cell.2013.07.005 Yang J, Weimer RM, Kallop D, Olsen O, Wu Z, Renier N, Uryu K, Tessier-Lavigne M (2013) Regulation of axon degeneration after injury and in development by the endogenous calpain inhibitor calpastatin. Neuron. doi:10.1016/j.neuron.2013.08.034 Glynn P (2000) Neural development and neurodegeneration: two faces of neuropathy target esterase. Prog Neurobiol 61(1):61–74 Wong M (2013) Mammalian target of rapamycin (mTOR) pathways in neurological diseases. Biomed J 36(2):40–50. doi:10. 4103/2319-4170.110365 White JF (2003) Intestinal pathophysiology in autism. Exp Biol Med (Maywood) 228(6):639–649 Depino AM (2013) Peripheral and central inflammation in autism spectrum disorders. Mol Cell Neurosci 53:69–76. doi:10.1016/j. mcn.2012.10.003 Jansiewicz EM, Goldberg MC, Newschaffer CJ, Denckla MB, Landa R, Mostofsky SH (2006) Motor signs distinguish children with high functioning autism and Asperger’s syndrome from controls. J Autism Dev Disord 36(5):613–621. doi:10.1007/ s10803-006-0109-y Al Abdulmohsen T, Kruger TH (2011) The contribution of muscular and auditory pathologies to the symptomatology of autism. Med Hypotheses 77(6):1038–1047. doi:10.1016/j.mehy. 2011.08.044
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