16S rRNA Sequencing Data of Fecal Microbiota in an Italian Cohort of Patients with CDKL5 Deficiency Disorder

Summary of the study CDKL5 deficiency disorder (CDD) is a neurodevelopmental condition characterized by global developmental delay, early-onset seizures, intellectual disability, visual and motor impairments, distinct from Rett Syndrome (RTT) due to the absence of a clear regression period. Gastrointestinal (GI) disturbances and signs of subclinical immune dysregulation are common in CDD patients, yet the underlying causes are unknown. Recent studies hint at a possible link between neurological disorders and gut microbiota, an unexplored area in CDD. In this groundbreaking study, we examined fecal microbiota in CDD patients and their healthy relatives, revealing differences in bacterial diversity and composition. We further investigated microbiota changes based on various factors, including the severity of GI issues, seizure frequency, sleep disorders, food intake type, neuro-behavioral features (assessed through the RTT Behaviour Questionnaire – RSBQ), and ambulation capacity. Our findings suggest a potential connection between CDD, microbiota, and symptom severity. This study represents the first exploration of the gut-microbiota-brain axis in CDD patients, contributing to the growing body of research on the role of gut microbiota in neurodevelopmental disorders. It opens doors to potential interventions targeting intestinal microbes to enhance the well-being of individuals with CDD. Mehods The Dataset represent the raw data (.fastq) obtained from the sequencing of the fecal samples from 17 Italian Patients with CDD, and 17 Healthy Relatives (i.e. siblings or mother), collected at a single time-point. Samples from Patients affected by CDD are called CDD, samples from Healthy Relatives are called HC-CDD (i.e. healthy controls of patients affected by CDD). For details about the sample names see the “Explanation Table”. Bacterial DNA was extracted from fecal samples using the QIAmp Powerfexal DNA Kit (Qiagen, Germany) following the manufacturer's protocol. The 16S rRNA sequencing and analysis was performed by a service offered by Zymo Research (Germany). Targeted Library Preparation: The DNA samples were prepared for targeted sequencing with the Quick-16S™ NGS Library Prep Kit (Zymo Research). The primer sets used were Quick-16S™ Primer Set V3-V4 (Zymo Research). The sequencing library was prepared using an innovative library preparation process in which PCR reactions were performed in real-time PCR machines to control cycles and therefore limit PCR chimera formation. The final PCR products were quantified with qPCR fluorescence readings and pooled together based on equal molarity. The final pooled library was cleaned up with the Select-a-Size DNA Clean & Concentrator™, then quantified with TapeStation® (Agilent Technologies, Santa Clara, CA) and Qubit® (Thermo Fisher Scientific, Waltham, WA). Sequencing: The final library was sequenced on Illumina® MiSeq™ with a v3 reagent kit (600 cycles).研究概述 CDKL5缺乏症（CDKL5 deficiency disorder, CDD）是一种神经发育疾病，以全面发育迟缓、早发性癫痫、智力障碍、视觉与运动功能受损为核心特征，与雷特综合征（Rett Syndrome, RTT）的区别在于其无明确的退行期。CDD患者常出现胃肠道（Gastrointestinal, GI）紊乱与亚临床免疫失调的表现，但其潜在致病机制尚未阐明。近期研究提示神经系统疾病与肠道菌群（gut microbiota）之间可能存在关联，而这一领域在CDD相关研究中仍属未被探索的空白。在本项开创性研究中，我们对CDD患者及其健康亲属的粪便菌群开展了分析，揭示了两组样本在细菌多样性与群落组成上的显著差异。我们进一步基于多种因素探究了菌群变化特征，包括胃肠道症状严重程度、癫痫发作频率、睡眠障碍、食物摄入类型、神经行为特征（通过雷特综合征行为问卷——RSBQ评估）以及行走能力。 本研究结果提示CDD、肠道菌群与症状严重程度之间可能存在潜在关联。本研究是首次针对CDD患者开展的肠道菌群-脑轴相关探索，为肠道菌群在神经发育疾病中作用的相关研究补充了新的证据，同时为靶向调控肠道微生物以改善CDD患者健康状况的潜在干预手段开辟了研究方向。 研究方法 本数据集包含17名意大利CDD患者与17名健康亲属（即患者的兄弟姐妹或母亲）的粪便样本测序所得的原始数据（.fastq），所有样本均为单次采集。 CDD患者的样本命名为CDD，健康亲属的样本命名为HC-CDD（即CDD患者的健康对照）。样本名称的详细说明请参见"说明表"。 采用德国Qiagen公司的QIAmp Powerfexal DNA试剂盒，依照制造商说明书从粪便样本中提取细菌DNA。16S rRNA测序与分析由德国Zymo Research公司提供的测序服务完成。 靶向文库制备 使用Zymo Research公司的Quick-16S™ NGS文库制备试剂盒制备DNA样本以进行靶向测序，所用引物组为Quick-16S™ Primer Set V3-V4（Zymo Research）。本测序文库采用创新的文库制备流程构建：在实时荧光定量PCR仪中进行PCR反应，以控制循环次数，从而减少PCR嵌合体的形成。最终的PCR产物通过qPCR荧光读数进行定量，并基于等摩尔浓度进行混合。混合后的最终文库通过Select-a-Size DNA Clean & Concentrator™进行纯化，随后使用TapeStation®（美国安捷伦科技公司，加利福尼亚州圣克拉拉）与Qubit®（美国赛默飞世尔科技公司，马萨诸塞州沃尔瑟姆）进行定量。 测序 最终文库采用Illumina® MiSeq™测序平台，搭配v3试剂试剂盒（600个循环）进行测序。