16p11.2 600 kb Duplications confer risk for typical and atypical Rolandic epilepsy

Rolandic epilepsy (RE) is the most common idiopathic focal childhood epilepsy. Its molecular basis is largely unknown and a complex genetic etiology is assumed in the majority of affected individuals. The present study tested whether six large recurrent copy number variants at 1q21, 15q11.2, 15q13.3, 16p11.2, 16p13.11 and 22q11.2 previously associated with neurodevelopmental disorders also increase risk of RE. Our association analyses revealed a significant excess of the 600 kb genomic duplication at the 16p11.2 locus (chr16: 29.5-30.1 Mb) in 393 unrelated patients with typical (n = 339) and atypical (ARE; n = 54) RE compared with the prevalence in 65 046 European population controls (5/393 cases versus 32/65 046 controls; Fisher's exact test P = 2.83 × 10−6, odds ratio = 26.2, 95% confidence interval: 7.9-68.2). In contrast, the 16p11.2 duplication was not detected in 1738 European epilepsy patients with either temporal lobe epilepsy (n = 330) and genetic generalized epilepsies (n = 1408), suggesting a selective enrichment of the 16p11.2 duplication in idiopathic focal childhood epilepsies (Fisher's exact test P = 2.1 × 10−4). In a subsequent screen among children carrying the 16p11.2 600 kb rearrangement we identified three patients with RE-spectrum epilepsies in 117 duplication carriers (2.6%) but none in 202 carriers of the reciprocal deletion. Our results suggest that the 16p11.2 duplication represents a significant genetic risk factor for typical and atypical R

Evaluation of presumably disease causing SCN1A variants in a cohort of common epilepsy syndromes

Objective: The SCN1A gene, coding for the voltage-gated Na+ channel alpha subunit NaV1.1, is the clinically most relevant epilepsy gene. With the advent of high-throughput next-generation sequencing, clinical laboratories are generating an ever-increasing catalogue of SCN1A variants. Variants are more likely to be classified as pathogenic if they have already been identified previously in a patient with epilepsy. Here, we critically re-evaluate the pathogenicity of this class of variants in a cohort of patients with common epilepsy syndromes and subsequently ask whether a significant fraction of benign variants have been misclassified as pathogenic. Methods: We screened a discovery cohort of 448 patients with a broad range of common genetic epilepsies and 734 controls for previously reported SCN1A mutations that were assumed to be disease causing. We re-evaluated the evidence for pathogenicity of the identified variants using in silico predictions, segregation, original reports, available functional data and assessment of allele frequencies in healthy individuals as well as in a follow up cohort of 777 patients. Results and Interpretation: We identified 8 known missense mutations, previously reported as path

Evaluation of Presumably Disease Causing SCN1A Variants in a Cohort of Common Epilepsy Syndromes

A. Palotie on työryhmän jäsen.Objective The SCN1A gene, coding for the voltage-gated Na+ channel alpha subunit NaV1.1, is the clinically most relevant epilepsy gene. With the advent of high-throughput next-generation sequencing, clinical laboratories are generating an ever-increasing catalogue of SCN1A variants. Variants are more likely to be classified as pathogenic if they have already been identified previously in a patient with epilepsy. Here, we critically re-evaluate the pathogenicity of this class of variants in a cohort of patients with common epilepsy syndromes and subsequently ask whether a significant fraction of benign variants have been misclassified as pathogenic. Methods We screened a discovery cohort of 448 patients with a broad range of common genetic epilepsies and 734 controls for previously reported SCN1A mutations that were assumed to be disease causing. We re-evaluated the evidence for pathogenicity of the identified variants using in silico predictions, segregation, original reports, available functional data and assessment of allele frequencies in healthy individuals as well as in a follow up cohort of 777 patients. Results and Interpretation We identified 8 known missense mutations, previously reported as pathogenic, in a total of 17 unrelated epilepsy patients (17/448; 3.80%). Our re-evaluation indicates that 7 out of these 8 variants (p.R27T; p.R28C; p.R542Q; p.R604H; p.T1250M; p.E1308D; p.R1928G; NP_001159435.1) are not pathogenic. Only the p. T1174S mutation may be considered as a genetic risk factor for epilepsy of small effect size based on the enrichment in patients (P = 6.60 x 10(-4); OR = 0.32, fishers exact test), previous functional studies but incomplete penetrance. Thus, incorporation of previous studies in genetic counseling of SCN1A sequencing results is challenging and may produce incorrect conclusions.Peer reviewe

CNNM2 Mutations Cause Impaired Brain Development and Seizures in Patients with

Intellectual disability and seizures are frequently associated with hypomagnesemia and have an important genetic component. However, to find the genetic origin of intellectual disability and seizures often remains challenging because of considerable genetic heterogeneity and clinical variability. In this study, we have identified new mutations in CNNM2 in five families suffering from mental retardation, seizures, and hypomagnesemia. For the first time, a recessive mode of inheritance of CNNM2 mutations was observed. Importantly, patients with recessive CNNM2 mutations suffer from brain malformations and severe intellectual disability. Additionally, three patients with moderate mental disability were shown to carry de novo heterozygous missense mutations in the CNNM2 gene. To elucidate the physiological role of CNNM2 and explain the pathomechanisms of disease, we studied CNNM2 function combining in vitro activity assays and the zebrafish knockdown model system. Using stable Mg2+ isotopes, we demonstrated that CNNM2 increases cellular Mg2+ uptake in HEK293 cells and that this process occurs through regulation of the Mg2+-permeable cation channel TRPM7. In contrast, cells expressing mutated CNNM2 proteins did not show increased Mg2+ uptake. Knockdown of cnnm2 isoforms in zebrafish resulted in disturbed brain development including neurodevelopmental impairments such as increased embryonic spontaneous contractions and weak touch-evoked escape behaviour, and reduced body Mg content, indicative of impaired renal Mg2+ absorption. These phenotypes were rescued by injection of mammalian wild-type Cnnm2 cRNA, whereas mammalian mutant Cnnm2 cRNA did not improve the zebrafish knockdown phenotypes. We therefore concluded that CNNM2 is fundamental for brain development, neurological functioning and Mg2+ homeostasis. By establishing the loss-of-function zebrafish model for CNNM2 genetic disease, we provide a unique system for testing therapeutic drugs targeting CNNM2 and for monitoring their effects on the brain and kidney phenotype. Author Summary Mental retardation affects 1-3% of the population and has a strong genetic etiology. Consequently, early identification of the genetic causes of mental retardation is of significant importance in the diagnosis of the disease, as predictor of the progress of the disease and for the determination of treatment. In this study, we identify mutations in the gene encoding for cyclin M2 (CNNM2) to be causative for mental retardation and seizures in patients with hypomagnesemia. Particularly, in patients with a recessive mode of inheritance, the intellectual disability caused by dysfunctional CNNM2 is dramatically severe and is accompanied by severely limited motor skills and brain malformations suggestive of impaired early brain development. Although hypomagnesemia has been associated to several neurological diseases, Mg2+ status is not regularly assessed in patients with seizures and mental disability. Our findings establish CNNM2 as an important protein for renal magnesium handling, brain development and neurological functioning, thus explaining the physiology of human disease caused by (dysfunctional) mutations in CNNM2. CNNM2 mutations should be taken into account in patients with seizures and mental disability, specifically in combination with hypomagnesemia

利用即時聚合酶連鎖反應及酵素免疫連結分析法評估黃連素對BALB/c小鼠脾臟細胞分泌細胞激素之影響

In Chinese medicine, bitter foods are described as “heat-removing” agents for reducing fever and have been widely used in therapeutic application. The major bitter compounds in bitter foods are alkaloids, such as aloperine, amygdalin, berberine, crotaline and naringenin. The bitter compounds are suggested to have multiple physiological functions, including anti-pyretic, anti-nociceptive and anti-tumor activities. However, there are few discussions on immunomodulatory effects of bitter compounds. Therefore, the objective of this study was to investigate the anti-inflammatory effects of five selected bitter compounds including aloperine, amygdalin, berberine, crotaline and naringenin administrations on cytokine secretions by BALB/c mouse splenocytes under four different in vitro experimental models. The potent bitter compound, berberine, was further selected to evaluate its immunomodulatory mechanism using real time polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) methods. In this study, there were four different in vitro experimental models used to detect the immunomodulatory effects of bitter compound administrations on BALB/c primary splenocytes. In the spontaneous splenocyte culture model, the primary splenocytes were just co-cultured with samples. In the inflammatory model, the lipopolysaccharide (LPS)-stimulated splenocytes were co-cultured with samples. In the preventive model, splenocyte cultures were first incubated with samples, then washed out samples, and finally stimulated with LPS. In the repair model, splenocyte cultures were first stimulated with LPS, then washed out LPS, and finally treated with samples. In the first part, the results showed that five selected bitter compounds including aloperine, amygdalin, berberine, crotaline and naringenin, significantly (P<0.05) decreased the productions of pro-inflammatory cytokines by spontaneous splenocytes, such as interleukin (IL)-6 or tumor necrosis factor alpha (TNF-α). The levels of IL-6 and TNF-α significantly decreased in the inflammatory and preventive models at moderate concentrations of the selected bitter compounds expect crotaline. In addition, the ratios of pro-/anti- inflammatory cytokine, displayed as either IL-6/IL-10 or TNF-α/IL-10, also significantly diminished in both inflammatory and preventive models by the selected bitter compounds expect crotaline. It is suggested that the selected bitter compounds expect crotaline might have anti-inflammatory effects in vitro. Among the five selected bitter compounds, berberine has a significant effect on inhibiting the inflammation induced by LPS. Therefore, berberine was further administered to splenocyte cultures to detect its regulation on gene expression. In the second part, an investigation was further done to clarify the effect of berberine on cytokine mRNA expressions of primary splenocyte. Results showed that berberine had no significant effect on the mRNA expressions of IL-6, IL-10 and TNF-α, suggesting that berberine administration might affect cytokine expression via post-transcriptional regulation. However, berberine administration could down-regulate the gene expression ratio of Th2 (IL-5)/Th1 (IL-2) cytokines in the inflammatory model. The results suggested that berberine administration might have potential effects on modulating the Th2/Th1 immune balance toward the Th1 pole in vitro. In conclusion, berberine administration at moderate concentrations may significantly decrease the inflammation induced by LPS in vitro, via decreasing the ratio of pro-/anti-inflammatory cytokine (IL-6/IL-10 or TNF-α/IL-10). Besides, it is suggested that berberine could modulate immune responses through down-regulating the gene expression ratio of Th2/Th1 cytokines.傳統中醫普遍認為苦味食物具有良好的”退火”功效，已經被廣泛運用在食療保健上。苦豆鹼(aloperine)、苦杏仁苷(amygdalin)、黃連素(berberine)、野百合鹼(crotaline)以及柚苦苷(naringenin)等，為苦味食物中主要的苦味生物鹼，研究證實其具有許多生理活性，包含退燒、鎮痛以及抗癌等活性，但對苦味物質的免疫調節功能，至今仍無完整研究，因此本篇主要研究目的為，選取五種苦味物質：苦豆鹼、苦杏仁苷、黃連素、野百合鹼以及柚苦苷，在動物體外四種不同實驗模式下，探討其抗發炎活性，並從其中選出黃連素，利用即時聚合酶連鎖反應及酵素免疫連結分析法評估苦味物質黃連素對BALB/c小鼠脾臟細胞分泌細胞激素之影響，以探討其免疫調節功能。本研究使用四種不同體外實驗來評估苦味物質對BALB/c小鼠初代脾臟細胞之免疫調節作用，實驗設計如下：單獨添加樣品與初代脾臟細胞共同培養(單獨添加樣品模式)、以內毒素脂多醣(lipopolysaccharide, LPS)刺激脾臟細胞同時添加樣品與細胞共同培養(發炎模式)、樣品先與脾臟細胞培養，洗去樣品，再加入LPS刺激脾臟細胞(預防模式)以及先以LPS刺激脾臟細胞，洗去LPS，再加入樣品與脾臟細胞培養(恢復模式)。 第一部分研究結果顯示，單獨添加五種苦味物質苦豆鹼、苦杏仁苷、黃連素、野百合鹼以及柚苦苷與脾臟細胞共同培養，可顯著抑制促發炎細胞激素介白質(interleukin, IL)-6或腫瘤壞死因子-α (tumor necrosis factor alpha, TNF-α)分泌量。脾臟細胞在發炎及預防的模式下，添加苦豆鹼、苦杏仁苷、黃連素以及柚苦苷可顯著抑制促發炎細胞激素分泌量，並調節促發炎IL-6 (TNF-α)/抗發炎細胞激素IL-10分泌量的比值，顯示苦味物質具有抗發炎的潛力，而五種苦味物質中又以黃連素抗發炎的能力最顯著，故以黃連素探討其對初代脾臟細胞細胞激素基因表現量之影響。 第二部分研究黃連素在不同細胞培養模式下對脾臟細胞細胞激素mRNA表現量的影響，發現黃連素對IL-6、IL-10及TNF-α mRNA的表現無顯著調控作用，推測黃連素對脾臟細胞細胞激素分泌之調控，可能屬於轉錄後調節作用(post-transcriptional regulation)。另外，在發炎模式下，黃連素可以下調Th2 (IL-5)/Th1 (IL-2)細胞激素mRNA表現量的比值，顯示黃連素具有調節免疫的功能，可使免疫平衡傾向Th1反應。 綜合本實驗結果發現，黃連素可以藉由降低促發炎/抗發炎細胞激素(IL-6/IL-10或TNF-α/IL-10)分泌量的比值，而減緩LPS所誘導的發炎反應。除此之外，黃連素可以下調Th2/Th1細胞激素mRNA表現量的比值，進而調節小鼠脾臟細胞的免疫反應變化。總目錄 .......................................................... i 表目錄 .......................................................... vi 圖目錄 .......................................................... ix 縮寫對照表 ...................................................... x 中文摘要 ........................................................ xi 英文摘要 ........................................................ xiii 第一章 緒言 ................................................ 1 第二章 文獻回顧 ............................................ 2 第一節 免疫介紹 ............................................ 2 一、 免疫系統 ........................................ 2 (一) 先天性免疫反應 ................................. 3 (二) 適應性免疫反應 ................................. 5 二、 發炎反應 ........................................ 7 三、 輔助型T細胞第一型及第二型之平衡 ................ 8 四、 細胞激素簡介 .................................... 8 五、 細胞激素基因表現與蛋白質生成量的關係 ............ 10 第二節 苦味物質介紹 ........................................ 11 一、 五種苦味物質之相關研究與介紹 .................... 11 (一) 苦豆鹼(aloperine) ................................ 11 (二) 苦杏仁苷(amygdalin) ............................. 11 (三) 黃連素(berberine) ................................ 12 (四) 野百合鹼(crotaline) ............................... 13 (五) 柚苦苷(naringenin) ............................... 14 第三節 研究動機及目的 ...................................... 14 第四節 實驗設計 ............................................ 16 第三章 材料與方法 ......................................... 18 第一節 五種苦味物質樣品之製備與其對初代脾臟細胞體外免疫調節 功能之研究 .......................................... 18 一、 五種苦味物質樣品之製備 .......................... 18 二、 免疫初代細胞之取得與培養 ........................ 20 (一) 免疫初代細胞之來源 ............................ 20 (二) 脾臟細胞之取得與培養 .......................... 21 三、 五種苦味物質對小鼠初代脾臟細胞存活率之評估 ...... 22 四、 不同實驗模式下五種苦味物質對小鼠脾臟細胞分泌細胞 激素之影響 ...................................... 24 (一) 模式A：單獨添加樣品對細胞的影響 .............. 24 (二) 模式B：模擬在發炎模式下內毒素脂多醣與樣品同時 存在對細胞的影響 ............................. 24 (三) 模式C：模擬在預防模式下樣品對細胞的影響 ..... 24 (四) 模式D：模擬在恢復模式下樣品對細胞的影響 ..... 25 (五) 脾臟細胞細胞激素之測定 ....................... 25 五、 統計分析 ........................................ 27 第二節 小鼠脾臟細胞培養不同時間其TNF-α, IL-6, IL-10, IL-4, IL-5 與IL-2 mRNA表現的變化 ............................. 27 一、 初代脾臟細胞自發性及脂多醣刺激下之培養 .......... 27 (一) 初代脾臟細胞之自發性培養 ..................... 27 (二) 初代脾臟細胞以脂多醣誘導發炎狀態之培養 ....... 28 二、 以real-time PCR (RT-PCR)方法分析初代脾臟RNA表現量 28 (一) 總RNA之抽取 ................................ 28 (二) RNA電泳 .................................... 29 (三) RNA轉成cDNA (Reverse transcription reaction) ..... 31 (四) Real time polymerase chain reaction (RT-PCR) ....... 33 三、 統計分析 ........................................ 38 第三節 黃連素對小鼠脾臟細胞細胞激素mRNA表現量之影響 ..... 38 一、 不同實驗模式下黃連素對初代脾臟細胞細胞激素mRNA 表現量之影響 .................................... 38 (一) 模式a：單獨添加黃連素對脾臟細胞的影響 ........ 38 (二) 模式b：模擬在發炎模式下脂多醣與黃連素同時存在對 細胞的影響 .................................... 38 (三) 模式c：模擬在預防模式下添加黃連素對細胞的影響.. 39 (四) 模式d：模擬在恢復模式下添加黃連素對細胞的影響.. 39 二、 統計分析 ......................................... 39 第四章 結果 ................................................. 40 第一節 五種苦味物質對初代脾臟細胞體外免疫調節功能之研究 .... 40 一、 五種苦味物質對BALB/c小鼠初代脾臟細胞存活率之影響 40 二、 不同實驗模式下五種苦味物質對小鼠脾臟細胞分泌細胞激 素之影響 ......................................... 47 (一) 模式A：單獨添加樣品對脾臟細胞的影響 .......... 47 (二) 模式B：模擬在發炎狀態下脂多醣與樣品同時存在對 脾臟細的影響 ................................. 53 (三) 模式C：模擬在預防模式下樣品對脾臟細胞的影響... 60 (四) 模式D：模擬在恢復模式下樣品對脾臟細胞的影響... 66 第二節 小鼠脾臟細胞培養不同時間其TNF-α, IL-6, IL-10, IL-4, IL-5 與IL-2 mRNA表現的變化 ............................. 73 第三節 黃連素對小鼠脾臟細胞細胞激素mRNA表現量之影響 ..... 75 (一) 添加黃連素在實驗模式a下對BALB/c小鼠脾臟細胞 細胞激素mRNA表現量之影響 .................. 75 (二) 添加黃連素在實驗模式b下對BALB/c小鼠脾臟細胞 細胞激素mRNA表現量之影響 .................. 77 (三) 添加黃連素在實驗模式c下對BALB/c小鼠脾臟細胞 細胞激素mRNA表現量之影響 .................. 79 (四) 添加黃連素在實驗模式d下對BALB/c小鼠脾臟細胞 細胞激素mRNA表現量之影響 .................. 81 第五章 討論 ................................................. 83 一、 五種苦味物質對初代脾臟細胞體外免疫調節功能之研究 ..... 83 二、 黃連素對小鼠脾臟細胞細胞激素mRNA表現量之影響 ...... 87 第六章 結論 ................................................. 92 第七章 參考文獻 ............................................. 9

DEPDC5

Recent studies reported DEPDC5 loss-of-function mutations in different focal epilepsy syndromes. Here we identified 1 predicted truncation and 2 missense mutations in 3 children with rolandic epilepsy (3 of 207). In addition, we identified 3 families with unclassified focal childhood epilepsies carrying predicted truncating DEPDC5 mutations (3 of 82). The detected variants were all novel, inherited, and present in all tested affected (n=11) and in 7 unaffected family members, indicating low penetrance. Our findings extend the phenotypic spectrum associated with mutations in DEPDC5 and suggest that rolandic epilepsy, albeit rarely, and other nonlesional childhood epilepsies are among the associated syndromes

DEPDC5 Mutations in Genetic Focal Epilepsies of Childhood

Recent studies reported DEPDC5 loss-of-function mutations in different focal epilepsy syndromes. Here we identified 1 predicted truncation and 2 missense mutations in 3 children with rolandic epilepsy (3 of 207). In addition, we identified 3 families with unclassified focal childhood epilepsies carrying predicted truncating DEPDC5 mutations (3 of 82). The detected variants were all novel, inherited, and present in all tested affected (n=11) and in 7 unaffected family members, indicating low penetrance. Our findings extend the phenotypic spectrum associated with mutations in DEPDC5 and suggest that rolandic epilepsy, albeit rarely, and other nonlesional childhood epilepsies are among the associated syndromes

CNNM2 mutations impair Mg²⁺ uptake in HEK293 cells.

<p>(A) Time curve of ²⁵Mg²⁺ uptake in mock, wild-type CNNM2 and mutant CNNM2 transfected cells. Symbols indicate cells transfected with the vector empty (• mock) or containing <i>Cnnm2</i> sequences encoding for wild-type or mutant CNNM2 proteins (▪ CNNM2, ▴ CNNM2-p.Glu122Lys, ▾ CNNM2-p.Ser269Trp, ⧫ CNNM2-p.Leu330Phe, ○ CNNM2-p.Glu357Lys, □ CNNM2-p.Thr568Ile). Each data point represent the mean of 3 independent experiments ± SEM. * indicates significant differences compared to mock (<i>P</i><0.05). (B) Representative immunoblots showing that p.Glu122Lys and p.Ser269Trp mutations reduce CNNM2 membrane expression (upper blot) and a CNNM2 expression control (lower blot). Quantification of cell surface expression of wild-type (WT) and mutant CNNM2 proteins corrected for total protein expression. Results are the mean ± SEM of 3 independent experiments. * indicate significant differences compared to WT CNNM2 transfected cells (<i>P</i><0.05).</p

Clinical and biochemical data of patients.

<p>Clinical and biochemical data of patients.</p

Knockdown of <i>cnnm2a</i> results in Mg wasting in zebrafish larvae (5 dpf).

<p>(A) mRNA expression of <i>cnnm2a</i> in developing zebrafish. Expression patterns were analysed by RT-qPCR (n = 6 per time point). (B) Survival curve at 5 dpf (n = 3 per experimental condition). The dose of zero represents injection with control-MO. (C) Morphological phenotypes in zebrafish larvae (5 dpf) in <i>cnnm2a</i> knockdown experiments. (D) Distribution of morphological phenotypes in zebrafish larvae (5 dpf) untreated (wild-type) or injected with different doses of <i>cnnm2a</i>-MO or control-MO. Numbers on top of the bars indicate the number of animals in each experimental condition. (E) Distribution of morphological phenotypes in zebrafish larvae at 5 dpf in rescue experiments. The wild-type phenotype (class I) was restored in morphants by co-injection of <i>cnnm2a</i>-MO (2 ng MO/embryo) with wild-type (WT) CNNM2 cRNA (50 pg cRNA/embryo), but not with mutant (MT, p.Glu357Lys) CNNM2 cRNA (50 pg cRNA/embryo). (F) Magnesium content in zebrafish injected with different doses of <i>cnnm2a</i>-MO, the dose of zero represents injection with control-MO (n = 10 per experimental condition except in 8 ng MO-injected zebrafish where n = 5). (G) Rescue of Mg wasting in morphant zebrafish by co-injection of <i>cnnm2a</i>-MO (2 ng MO/embryo) with cRNA encoding for wild-type (WT) CNNM2 (50 pg cRNA/embryo). Co-injection with cRNA encoding for mutant (MT, p.Glu357Lys) CNNM2 (50 pg cRNA/embryo) did not restore Mg levels (n = 10 per experimental condition). Data are presented as mean ± SEM. Different letters indicate significant differences between mean values in experimental groups (<i>P</i><0.05).</p