Supplementary Material for: Cytogenomic Characterization of a Novel de novo Balanced Reciprocal Translocation t(1;12) by Genome Sequencing Leading to Fusion Gene Formation of <b><i>EYA3/EFCAB4b</i></b>

<b><i>Introduction:</i></b> The accurate detection of breakpoint regions of disease-associated chromosomal rearrangements helps understand the molecular mechanisms and identify the risks involved with disrupted genes. <b><i>Methods:</i></b> In this study, a girl with growth retardation is characterized using positional cloning and genome sequencing. The techniques include fluorescence in situ hybridization (FISH) with paint (WCP) and bacterial-artificial chromosomes (BAC) probes, PCR, real-time PCR, and short and long-read sequencing. <b><i>Results:</i></b> The translocation was identified by GTG banding and confirmed by WCP FISH. Microarray ruled out the involvement of other copy number variations except for 6 homozygous regions which are not disease-causing variants. Fine mapping with FISH showed split signals with BAC clone RP11-312A3. Genome sequencing of short-read with an average 30× depth and long-read sequencing technology with a 3.8× coverage identified both breakpoints, confirmed by Sanger sequencing, that showed microhomology. The breakpoint at 1p and 12p regions disrupted <i>EYA3</i> and <i>EFCAB4B</i> genes. Expression analysis of <i>EYA3</i> showed a 7-fold increase, suggesting the formation of a fusion gene with <i>EFCAB4B</i>. <i>EYA3</i> is involved in skeleton development, and <i>EFCAB4B</i> plays a role in calcium metabolism, which may be relevant for the patient’s phenotype. <b><i>Conclusion:</i></b> The systematic application of genome techniques to translocations and their advantages is discussed.<b><i>引言：</i></b> 精准识别疾病相关染色体重排的断裂点区域，有助于解析其分子机制，并评估受累基因相关的患病风险。<b><i>方法：</i></b> 本研究通过位置克隆与基因组测序技术，对1例生长迟缓女童进行特征分析。所用技术包括：采用全染色体涂染（whole chromosome painting, WCP）探针与细菌人工染色体（bacterial-artificial chromosomes, BAC）探针的荧光原位杂交（fluorescence in situ hybridization, FISH）、聚合酶链式反应（PCR）、实时荧光定量PCR、短读长测序与长读长测序。<b><i>结果：</i></b> 经GTG显带技术鉴定出该易位，并通过WCP-FISH予以验证。基因芯片检测排除了其他拷贝数变异的参与，仅发现6个非致病的纯合子区域。利用FISH进行精细定位时，观察到BAC克隆RP11-312A3出现分裂信号。平均测序深度为30×的短读长测序，与覆盖度为3.8×的长读长测序技术共同定位到两条断裂点，经桑格测序验证，二者存在微同源序列。1号染色体短臂（1p）与12号染色体短臂（12p）区域的断裂点分别破坏了<i>EYA3</i>与<i>EFCAB4B</i>基因。对<i>EYA3</i>的表达分析显示其表达量上调7倍，提示其与<i>EFCAB4B</i>形成融合基因。<i>EYA3</i>参与骨骼发育过程，<i>EFCAB4B</i>则在钙代谢中发挥作用，二者可能与该患者的表型相关。<b><i>结论：</i></b> 本研究探讨了基因组技术在易位分析中的系统性应用及其优势。