Multiplex ligation-dependent probe amplification for genetic screening in autism spectrum disorders: efficient identification of known microduplications and identification of a novel microduplication in ASMT.
Multiplex ligation-dependent probe amplification for genetic screening in autism spectrum disorders: efficient identification of known microduplications and identification of a novel microduplication in ASMT.
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DOI：
10.1186/1755-8794-1-50
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发表时间：
2008-10-16
影响因子：
2.7
通讯作者：
Buxbaum, Joseph D.
中科院分区：
文献类型：
--
作者：
Cai, Guiqing;Edelmann, Lisa;Goldsmith, Juliet E.;Cohen, Ninette;Nakamine, Alisa;Reichert, Jennifer G.;Hoffman, Ellen J.;Zurawiecki, Danielle M.;Silverman, Jeremy M.;Hollander, Eric;Soorya, Latha;Anagnostou, Evdokia;Betancur, Catalina;Buxbaum, Joseph D.
此前已有研究表明，特定的微缺失和微重复（其中许多也与认知障碍（CI）相关）可能会出现自闭症谱系障碍（ASD）。多重连接依赖性探针扩增 (MLPA) 是筛选此类复发性微缺失和微重复的有效方法。在当前的研究中，使用 MLPA 对总共 279 名确定患有 ASD 的无关受试者进行了与 CI 相关的基因组疾病筛查。荧光原位杂交 (FISH)、定量聚合酶链式反应 (Q-PCR) 和/或直接 DNA 测序用于验证潜在的微缺失和微重复。甲基化敏感的 MLPA 用于表征 Prader-Willi/Angelman (PWA) 区域重复的个体。 MLPA 显示两名受试者具有典型的 ASD 相关母体 15q11-q13 PWA 区域间质重复。另外两名受试者显示出先前未曾描述过的 PWA 区域较小的从头重复。这两个新重复中的基因包括一种情况下的 GABRB3 和 ATP10A，另一种情况下的基因包括 MKRN3、MAGEL2 和 NDN。此外，两名受试者表现出 22q11/DiGeorge 综合征区域的重复。一名个体被发现在 17p13 上的一个 ASPA 基因拷贝中携带 12 kb 的缺失，当两个等位基因发生突变时，就会导致卡纳万病。两名受试者在 Xp11.4 上显示出 TM4SF2 基因的部分重复，该基因先前与 X 连锁非特异性智力低下有关，但在我们随后的分析中，在对照中也发现了此类变异。 ASMT 基因位于性染色体的假常染色体区域 1 (PAR1)，之前被认为与 ASD 易感性有关，在 6% 至 7% 的病例中观察到了部分重复，但只有 2% 的对照组出现了部分重复（P = 0.003）。 MLPA 被证明是筛查染色体异常的有效方法。我们发现 15q11-q13 和 22q11 中的重复，包括新的从头小重复，可能通过增加责任和/或加剧症状而导致当前样本中的 ASD。我们的数据表明，TM4SF2 中的重复与表型无关，因为它们存在于对照中。 PAR1/PAR2 的结果是对这些区域基因剂量的首次大规模研究，ASMT 基因座的发现表明需要对 ASMT 基因的重复进行进一步研究，以深入了解其与 ASD 的潜在参与。我们的研究还发现了 MLPA 的一些局限性，其中探针结合序列中的单碱基变化会改变结果。总之，我们的研究表明，如果通过其他方法验证 MLPA 识别的缺失，MLPA 重点关注公认的医学遗传条件，可能是一种廉价的检测 ASD 患者微缺失和微重复的方法，用于遗传咨询。
It has previously been shown that specific microdeletions and microduplications, many of which also associated with cognitive impairment (CI), can present with autism spectrum disorders (ASDs). Multiplex ligation-dependent probe amplification (MLPA) represents an efficient method to screen for such recurrent microdeletions and microduplications. In the current study, a total of 279 unrelated subjects ascertained for ASDs were screened for genomic disorders associated with CI using MLPA. Fluorescence in situ hybridization (FISH), quantitative polymerase chain reaction (Q-PCR) and/or direct DNA sequencing were used to validate potential microdeletions and microduplications. Methylation-sensitive MLPA was used to characterize individuals with duplications in the Prader-Willi/Angelman (PWA) region. MLPA showed two subjects with typical ASD-associated interstitial duplications of the 15q11-q13 PWA region of maternal origin. Two additional subjects showed smaller, de novo duplications of the PWA region that had not been previously characterized. Genes in these two novel duplications include GABRB3 and ATP10A in one case, and MKRN3, MAGEL2 and NDN in the other. In addition, two subjects showed duplications of the 22q11/DiGeorge syndrome region. One individual was found to carry a 12 kb deletion in one copy of the ASPA gene on 17p13, which when mutated in both alleles leads to Canavan disease. Two subjects showed partial duplication of the TM4SF2 gene on Xp11.4, previously implicated in X-linked non-specific mental retardation, but in our subsequent analyses such variants were also found in controls. A partial duplication in the ASMT gene, located in the pseudoautosomal region 1 (PAR1) of the sex chromosomes and previously suggested to be involved in ASD susceptibility, was observed in 6–7% of the cases but in only 2% of controls (P = 0.003). MLPA proves to be an efficient method to screen for chromosomal abnormalities. We identified duplications in 15q11-q13 and in 22q11, including new de novo small duplications, as likely contributing to ASD in the current sample by increasing liability and/or exacerbating symptoms. Our data indicate that duplications in TM4SF2 are not associated with the phenotype given their presence in controls. The results in PAR1/PAR2 are the first large-scale studies of gene dosage in these regions, and the findings at the ASMT locus indicate that further studies of the duplication of the ASMT gene are needed in order to gain insight into its potential involvement in ASD. Our studies also identify some limitations of MLPA, where single base changes in probe binding sequences alter results. In summary, our studies indicate that MLPA, with a focus on accepted medical genetic conditions, may be an inexpensive method for detection of microdeletions and microduplications in ASD patients for purposes of genetic counselling if MLPA-identified deletions are validated by additional methods.
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