Use of whole-genome sequencing for diagnosis of suspected connective tissue disorder in canine patient

Background: Whole genome sequencing (WGS) is an underutilized tool for
diagnosis of suspected genetic diseases, particularly in canines.
Objective: Utilize genetic analysis following WGS of an affected canine
patient to pinpoint a potential genetic basis for disease. Animals: One
client-owned, ten-month-old Golden Retriever presenting with signs of
possible connective tissue disorder. Blood and DNA samples from a
non-affected German Shepherd-Beagle mix and seven Golden Retrievers
previously tested for ichthyosis were used as controls. Methods: A
physical exam, blood draw, and radiographs were performed on the patient
while at the hospital. WGS was performed on the DNA of the affected
patient derived from EDTA blood, and genetic analysis focused on SNVs,
small indels, and structural variants was performed to detect potential
disease-causing mutations within the genome. PCR amplification and Sanger
sequencing of a suspected region was performed to confirm presence of
deletion. Results: The patient presented for a cardiology consultation and
displayed clinical signs of cardiac enlargement, pneumothorax, and joint
hypermobility. Following genetic analysis, a roughly four kilobase
deletion encompassing exon 24 of the FBN1 gene was detected, likely
causing an in-frame deletion in the coding sequence. Conclusions: Given
the estimated impact of the mutation and the patient’s associated clinical
signs, we suspect Marfan syndrome

背景：全基因组测序（Whole genome sequencing, WGS）是用于疑似遗传性疾病诊断的未被充分挖掘的工具，在犬类临床中应用尤为不足。
研究目标：通过对患病犬只实施全基因组测序后的遗传分析，明确该疾病的潜在遗传致病基础。
受试动物：1只由饲主饲养的10月龄金毛寻回犬，表现出疑似结缔组织疾病的临床症状。本研究以1只非患病的德国牧羊犬-比格犬混血犬，以及7只此前已完成鱼鳞病检测的金毛寻回犬的血液与DNA样本作为对照。
实验方法：对就诊患犬开展体格检查、血液采集及X光影像学检查。采集患犬EDTA抗凝血以提取DNA并进行全基因组测序，同时针对单核苷酸变异（Single Nucleotide Variants, SNVs）、小型插入缺失变异（insertions-deletions, indels）及结构变异开展遗传分析，以检测基因组内潜在的致病突变。此外，针对疑似致病区域实施聚合酶链式反应（PCR）扩增与桑格测序（Sanger sequencing），以确认该缺失变异的存在。
实验结果：该患犬因心脏科会诊就诊，表现出心脏增大、气胸及关节过度活动的临床症状。经遗传分析后，研究人员检测到一段约4千碱基对（kb）的缺失变异，该变异覆盖原纤维蛋白1基因（FBN1）的第24号外显子，大概率会导致编码序列出现框内缺失。
研究结论：结合该突变的预估影响与患犬的相关临床症状，本研究怀疑该犬罹患马方综合征（Marfan syndrome）。