Table1_Contribution of Rare and Low-Frequency Variants to Multiple Sclerosis Susceptibility in the Italian Continental Population.XLSX

Genome-wide association studies identified over 200 risk loci for multiple sclerosis (MS) focusing on common variants, which account for about 50% of disease heritability. The goal of this study was to investigate whether low-frequency and rare functional variants, located in MS-established associated loci, may contribute to disease risk in a relatively homogeneous population, testing their cumulative effect (burden) with gene-wise tests. We sequenced 98 genes in 588 Italian patients with MS and 408 matched healthy controls (HCs). Variants were selected using different filtering criteria based on allelic frequency and in silico functional impacts. Genes showing a significant burden (n = 17) were sequenced in an independent cohort of 504 MS and 504 HC. The highest signal in both cohorts was observed for the disruptive variants (stop-gain, stop-loss, or splicing variants) located in EFCAB13, a gene coding for a protein of an unknown function (p < 10–4). Among these variants, the minor allele of a stop-gain variant showed a significantly higher frequency in MS versus HC in both sequenced cohorts (p = 0.0093 and p = 0.025), confirmed by a meta-analysis on a third independent cohort of 1298 MS and 1430 HC (p = 0.001) assayed with an SNP array. Real-time PCR on 14 heterozygous individuals for this variant did not evidence the presence of the stop-gain allele, suggesting a transcript degradation by non-sense mediated decay, supported by the evidence that the carriers of the stop-gain variant had a lower expression of this gene (p = 0.0184). In conclusion, we identified a novel low-frequency functional variant associated with MS susceptibility, suggesting the possible role of rare/low-frequency variants in MS as reported for other complex diseases.

全基因组关联研究（Genome-wide association studies）已针对多发性硬化（multiple sclerosis, MS）鉴定出200余个风险位点，相关研究聚焦于常见变异，这类变异约占该疾病遗传力的50%。本研究旨在探究定位于已明确与MS相关的风险位点内的低频与罕见功能变异，是否在相对同质化的人群中参与疾病发病风险，并通过基因水平检验（gene-wise tests）验证其累积变异负荷效应。我们对588例意大利籍MS患者与408例匹配健康对照（healthy controls, HC）的98个基因进行了测序。依据等位基因频率与计算机预测的功能影响，采用多套筛选标准挑选目标变异。在首次测序队列中显示出显著变异负荷的17个基因，我们在独立验证队列（包含504例MS患者与504例健康对照）中开展了二次测序验证。在两个队列中，定位于功能未明蛋白编码基因EFCAB13的破坏性变异（包括提前终止密码子变异、终止密码子丢失变异或剪接变异）的关联信号最强（p < 10⁻⁴）。其中，该基因的一个提前终止密码子变异的次要等位基因，在两个测序队列中均在MS患者群体中呈现显著高于健康对照的频率（分别为p=0.0093与p=0.025）；该结果在第三个独立队列（1298例MS患者与1430例健康对照）的单核苷酸多态性阵列（SNP array）检测结果的荟萃分析（meta-analysis）中得到进一步验证（p=0.001）。针对该变异的14例杂合携带者开展实时荧光定量PCR（real-time PCR）检测，未观测到提前终止密码子变异的等位基因存在，提示该变异可能通过无义介导的mRNA降解（non-sense mediated decay）引发转录本降解；同时我们发现，该提前终止密码子变异的携带者体内EFCAB13基因的表达水平显著降低（p=0.0184），进一步佐证了上述推测。综上，本研究鉴定出一个与MS易感性相关的新型低频功能变异，提示罕见/低频变异可能在MS的发病机制中发挥作用，这与其他复杂疾病的相关研究结论相符。