Variations of the Candidate SEZ6L2 Gene on Chromosome 16p11.2 in Patients with Autism Spectrum Disorders and in Human Populations

Background: Autism spectrum disorders (ASD) are a group of severe childhood neurodevelopmental disorders with still unknown etiology. One of the most frequently reported associations is the presence of recurrent de novo or inherited microdeletions and microduplications on chromosome 16p11.2. The analysis of rare variations of 8 candidate genes among the 27 genes located in this region suggested SEZ6L2 as a compelling candidate. Methodology/Principal Findings: We further explored the role of SEZ6L2 variations by screening its coding part in a group of 452 individuals, including 170 patients with ASD and 282 individuals from different ethnic backgrounds of the Human Genome Diversity Panel (HGDP), complementing the previously reported screening. We detected 7 previously unidentified non-synonymous variations of SEZ6L2 in ASD patients. We also identified 6 non-synonymous variations present only in HGDP. When we merged our results with the previously published, no enrichment of non-synonymous variation in SEZ6L2 was observed in the ASD group compared with controls. Conclusions/Significance: Our results provide an extensive ascertainment of the genetic variability of SEZ6L2 in human populations and do not support a major role for SEZ6L2 sequence variations in the susceptibility to ASD

Copy Number Variations in Autism Spectrum Disorders (identification and Characterization of New Candidate Genes (SEZ6L2 and CNTN3-6))

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Key role for gene dosage and synaptic homeostasis in autism spectrum disorders

International audienceAutism spectrum disorders (ASD) are characterized by impairments in reciprocal social communication, and repetitive, stereotyped verbal and non-verbal behaviors. Genetic studies have provided a relatively large number of genes that constitute a comprehensive framework to better understand this complex and heterogeneous syndrome. Based on the most robust findings, three observations can be made. First, genetic contributions to ASD are highly heterogeneous and most probably involve a combination of alleles with low and high penetrance. Second, the majority of the mutations apparently affect a single allele, suggesting a key role for gene dosage in susceptibility to ASD. Finally, the broad expression and function of the causative genes suggest that alteration of synaptic homeostasis could be a common biological process associated with ASD. Understanding the mechanisms that regulate synaptic homeostasis should shed new light on the causes of ASD and could provide a means to modulate the severity of the symptoms

Genetic variations of the melatonin pathway in patients with attention-deficit and hyperactivity disorders.

Melatonin is a powerful antioxidant and a synchronizer of many physiological processes. Alteration in melatonin signaling has been reported in a broad range of diseases, but little is known about the genetic variability of this pathway in humans. Here, we sequenced all the genes of the melatonin pathway -AA-NAT, ASMT, MTNR1A, MTNR1B and GPR50 - in 321 individuals from Sweden including 101 patients with attention-deficit/hyperactivity disorder (ADHD) and 220 controls from the general population. We could find several damaging mutations in patients with ADHD, but no significant enrichment compared with the general population. Among these variations, we found a splice site mutation in ASMT (IVS5+2T>C) and one stop mutation in MTNR1A (Y170X) - detected exclusively in patients with ADHD - for which biochemical analyses indicated that they abolish the activity of ASMT and MTNR1A. These genetic and functional results represent the first comprehensive ascertainment of melatonin signaling deficiency in ADHD

Genetic and Functional ascertainment of the Melatonin Pathway in Patients with Attention Deficit and Hyperactivity Disorders

Sleep wake cycles are frequently disturbed in patients with Attention Deficit and Hyperactivity Disorders. We hypothesized that the origin of the sleep problems may be the consequence of an abnormal circadian clock setting regulated by the melatonin pathway. Here, we sequenced all the genes of the melatonin pathway AA-NAT, ASMT, MTNR1A, MTNR1B and GPR50 in 328 individuals from Sweden including 108 patients with ADHD and 220 from the general population. Non-synonymous mutations were identified in all genes at a similar frequency in patients with ADHD and in controls. Among the functional variations, a splice site mutation (IVS5+2T>C) in ASMT and one stop mutation (Y170X) in MTNR1A were only detected in patients with ADHD. Biochemical analyses indicated that these mutations abolish the activity of ASMT and MTNR1A. We also identified clusters of SNPs within MTNR1B showing significant difference in the allelic frequency between ADHD patients and control (maximum signal at rs10830961 P=0.0002). Taken together, these genetic and functional results shed light on one new compelling candidate pathway for susceptibility to circadian rhythms alterations that could help clinicians for providing better treatments of patients with ADHD and sleep problems

Inherited 15q11–q13 CNVs identified in three ASD patients carrier of a <i>de novo</i> SHANK2 deletion.

<p>Deletions (del) and duplications (dup) are indicated in red and blue, respectively. Paternally and maternally imprinted genes are indicated in yellow and pink, respectively. Genes altered by the CNVs are indicated in blue or red. The bottom part of the figure indicates the location of the deletions/duplications previously associated with neuropsychiatric disorders <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002521#pgen.1002521-Miller1" target="_blank">[43]</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002521#pgen.1002521-deKovel1" target="_blank">[61]</a>. BP, breakpoint; Inh_M, inherited by mother; Inh_F, inherited by father; AS, Angelman syndrome; ASD, Autism spectrum disorders; ADHD, attention deficit-hyperactivity disorder; BP, bipolar disorder; DD: developmental delay; DBD, disruptive behavior disorder; EPI, epilepsy; GAD, generalized anxiety disorder; OCD, obsessive-compulsive disorder; ID, intellectual disability; PWS, Prader-Willi syndrome; SCZ, schizophrenia.</p

Characterization of CNVs in three patients carrying a <i>de novo</i> deletion of <i>SHANK2</i>.

<p>Paternally or maternally inherited CNVs are indicated by squares and circles, respectively. <i>De novo</i> CNVs are indicated by stars. Deletions and duplications are indicated in red and blue, respectively. CNVs hitting exons or only introns are filled with grey and white, respectively. Squares and circles within star represent <i>de novo</i> CNV of paternal or maternal origin; circles within squares represent CNV inherited by father or mother. ABCC6, ATP-binding cassette, sub-family C, member 6 pseudogene 2; ADAM, ADAM metallopeptidase; AMY1, amylase (salivary); AMY2A, amylase (pancreatic); ARHGAP11B, Rho GTPase activating protein 11B; CAMSAP1L1, calmodulin regulated spectrin-associated protein 1-like 1; CHRNA7, cholinergic receptor, nicotinic, alpha 7; CNTN4, contactin 4; CTNNA3, catenin (cadherin-associated protein), alpha 3; CYFIP1, cytoplasmic FMR1 interacting protein 1; DUSP22, dual specificity phosphatase 22; GALM, galactose mutarotase; GCNT2, glucosaminyl (N-acetyl) transferase 2; GOLGA, golgi autoantigen, golgin subfamily a; GSTT1, glutathione S-transferase theta 1; HLA-DRB, major histocompatibility complex, class II, DR beta; LAMA4, laminin, alpha 4; NIPA, non imprinted in Prader-Willi/Angelman syndrome; NLGN1, neuroligin 1; NME7, non-metastatic cells 7; OR, olfactory receptor; PCDHA, protocadherin alpha; RFPL4B, ret finger protein-like 4B; RHD, Rh blood group, D antigen; SFMBT1, Scm-like with four mbt domains 1; SHANK2, SH3 and multiple ankyrin repeat domains 2; SMC2, structural maintenance of chromosomes 2; TNS3, tensin 3; TUBGCP5, tubulin, gamma complex associated protein 5; UGT2B17, UDP glucuronosyltransferase 2 family, polypeptide B17.</p

Characterization of the functional impact of <i>SHANK2</i> mutations in cultured neuronal cells.

<p>A. The colocalization of <i>ProSAP1A/SHANK2</i>-EGFP (postsynaptic marker) and Bassoon (presynaptic marker) indicated that the mutations did not disturb the formation of SHANK2 clusters at excitatory synapses along the dendrites. B. The quantification of synapse density was performed on 20 transfected hippocampal neurons per construct from at least three independent experiments. The majority of the <i>ProSAP1A</i> variants affecting a conserved amino acid among SHANK proteins reduced significantly the synaptic density compared with the variants that affect amino acid non conserved among SHANK proteins (Mann-Whitney U-test: n_WT = 20, n_mut = 20; U_S557N = 82.5, p_S557N = 0.001; U_R569H = 124, p_R569H = 0.04; U_L629P = 149, p_L629P = 0.17; U_V717F = 114, p_V717F = 0.02; U_A729T = 73, p_A729T = 0.000; U_K780Q = 154, p_K780Q = 0.221; U_R818H = 108, p_R818H = 0.012; U_A822T = 154.5, p_A822T = 0.224; U_V823M = 129, p_V823M = 0.056; U_Y967C = 134, p_Y967C = 0.076; U_G1170R = 78, p_G1170R = 0.001; U_R1290W = 142, p_R1290W = 0.121; U_Q1308R = 162, p_Q1308R = 0.314; U_D1535N = 97, p_D1535N = 0.005; U_P1586L = 137, p_P1586L = 0.910; U_L1722P = 79, p_L1722P = 0.001, *p<0.05, **p<0.01, ***p<0.001). <b>C.</b> Effect of the variants on synaptic density. The y-axis represents −log P compared to WT (P obtained with Mann-Whitney test). After Bonferroni correction for 16 tests, only P values<0.003 were considered as significant. Variants represented in red were specific to ASD, in orange were shared by ASD and controls, and in green were specific to the controls. Open circles and filled circles represent non conserved and conserved amino acids, respectively. Prim, primary; second, secondary.</p

<i>SHANK2</i> mutations in patients with ASD.

<p>A. A heterozygous deletion of <i>SHANK2</i> was identified with the Illumina Human 1M-Duo SNP array in a patient with autism (AU038_3). The deletion spans 421 kb on chromosome 11q13.3, covers twelve exons of the human <i>SHANK2</i> and is not present in the parents. Each dot shows Log R Ratio (LRR; in red) and B allele frequency (BAF; in green). QuantiSNP score is represented with a blue line and indicates the deletion size. B. Location of the CNV and sequence variants (from this study and Berkel <i>et al.</i> 2010) along the SHANK2 protein: in red the variations specific to ASD, in orange the variations shared by ASD and controls and in green the variations specific to controls <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002521#pgen.1002521-Berkel1" target="_blank">[26]</a>. The breakpoints of the <i>SHANK2</i> deletion in AU038_3 are represented with a dotted line on the protein. Stars indicate the variants affecting conserved amino acids. C. A total of 40 variants were identified and variants affecting conserved amino acids in other SHANK proteins are enriched in patients with ASD (n_conserved = 12 and n_{non-conserved} = 3) compared with controls (n_conserved = 6 and n_{non-conserved} = 11) (Fisher's exact test 1-sided, P = 0.013, OR = 6.83, 95% IC = 1.19–53.40). D. The percentage of carriers of <i>SHANK2</i> variants in patients with ASD and Controls. Variants affecting a conserved amino acid among the SHANK proteins are enriched in patients with ASD (n_conserved = 29 and n_{non-conserved} = 822) compared with controls (n_conserved = 16 and n_{non-conserved} = 1074) (Fisher's exact test 1-sided, P = 0.004, OR = 2.37, 95% CI = 1.23–4.70). Open squares and filled squares represent the non-conserved and conserved amino acids, respectively. ANK, Ankyrin repeat domain; SH3, Src homology 3 domain; PDZ, postsynaptic density 95/Discs large/zona occludens-1 homology domain; SAM, sterile alpha motif domain; BSR, brain specific region; H, homer binding site; D, dynamin binding site; C, cortactin binding site. The proline-rich region is represented as a horizontal gray line.</p

Genomic structure, isoforms, and expression of human <i>SHANK2</i>.

<p>A. Genomic structure of the human <i>SHANK2</i> gene. Transcription of <i>SHANK2</i> produces four main mRNA from three distinct promoters: <i>SHANK2E</i> (<i>AB208025</i>), <i>ProSAP1A</i> (<i>AB208026</i>), <i>ProSAP1</i> (<i>AB208027</i>) and <i>AF141901</i>. There are three translation starts: in exon 2 for <i>SHANK2E</i>, in exon1b for <i>ProSAP1A</i>, and in exon1c for <i>ProSAP1</i> and <i>AF141901</i>; and two independent stop codons: in exon 22b for <i>AF141901</i> and in exon 25 for <i>SHANK2E</i>, <i>ProSAP1A</i> and <i>ProSAP1</i>. Conserved domains of protein interaction or protein binding site are represented in color: ANK (red), SH3 (orange), PDZ (blue) and SAM (green), H (pink), D, (dark blue) and C (purple). Black stars identify the alternative spliced exons (‘brain-specific exons’ in turquoise: 19, 20 and 23). B. RT-PCRs of <i>SHANK2</i> isoforms on RNA from different human control tissues (Clontech), and different brain regions of four controls (2 males and 2 females). The amplified regions specific to each isoform of <i>SHANK2</i> are indicated by gray boxes. C. Alternative splicing of human <i>SHANK2</i>; exons 19, 20 and 23 are specific to the brain. ANK, ankyrin; SH3, Src homology 3; PDZ, PSD95/DLG/ZO1; SAM, sterile alpha motif; He, heart; Li, liver; B, brain; SM, skeletal muscle; Pl, placenta; K, kidney; Lu, lung; Pa, pancreas; FC, frontal cortex; Hi, hippocampus; TC, temporal cortex; T, thalamus; OC, occipital cortex; Ce, cerebellum; Cx, whole cortex; BLCL, B lymphoblastoid cell lines; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; BSR, brain specific region; H, homer binding site; D, dynamin binding site; C, cortactin binding site. The ages of the two males and the two females studied were 74, 42, 55, and 36 years with a post-mortem interval of 10, 21, 24, and 2 h, respectively.</p