Veitia Reiner A, Cowles Jamie D, Caburet Sandrine
Department of Life Sciences, Université Paris Cité, CNRS, Institut Jacques Monod, CNRS UMR7592, Paris, France.
Department of Life Sciences, Université Paris Saclay, Gif-sur-Yvette, France.
Hum Reprod. 2025 Jun 1;40(6):1220-1233. doi: 10.1093/humrep/deaf058.
How updated expression and genomic data combined with a disease/disorder-specific classification system can be used to correct a gene model for a better evaluation of the pathogenicity of variants found in patients?
By combining available genomic and transcriptomic data from several species and a quantitative classification framework with primary ovarian insufficiency (POI)-adjusted parameters, we correct the human NOBOX (newborn ovary homeobox) gene model and provide a reclassification of variants previously reported in POI cases.
The NOBOX gene, encoding a gonad-specific transcription factor with a crucial role in early folliculogenesis and considered a major gene involved in POI, is currently described as being expressed as four transcripts, the longest one considered canonical. All the variants identified in POI cases have been evaluated according to this canonical transcript, and the various functional tests have been performed using the corresponding predicted protein.
STUDY DESIGN, SIZE, DURATION: We refined and corrected the NOBOX gene model using available genomic and RNAseq data in human and 16 other mammalian species. Expression data were selected for tissue specificity, strand specificity, and coverage. The analysis of RNAseq data from different ovarian fetal stages allows for a time-course description of NOBOX isoforms. Literature was scanned to retrieve NOBOX variants reported in POI cases, and NOBOX variants present in ClinVar and GnomAD 4 databases were also retrieved.
PARTICIPANTS/MATERIALS, SETTING, METHODS: Strand-specific RNAseq data from human fetal ovaries and human adult testes were analysed to infer the correct human NOBOX gene isoforms. The conservation of the gene structure was verified by combining the aligned genomic sequences from 17 mammalian species covering a wide phylogenetic range and the relevant RNAseq data. As changing a gene model implies a reclassification of variants, we set up a quantitative framework with updated variant frequencies from GnomAD4 and POI-adjusted parameters following the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines. Using this framework, we reclassified 44 NOBOX variants reported in POI patients and families, 117 NOBOX variants reported in ClinVar, and 2613 NOBOX variants present in GnomAD4.
The corrected NOBOX gene model proposes the invalidation of two transcripts, including the canonical one. The two correct isoforms were present in fetal ovarian samples, and only one was detected in adult testes. Only 14 variants remained as possibly causative for POI. Furthermore, this re-evaluation strongly suggests that NOBOX biallelic variants are the most likely cause of POI.
Large tables are provided as supplementary data sets on the Zenodo repository.
LIMITATIONS, REASONS FOR CAUTION: The proposed gene model is robust but relies on available transcriptomic data covering a range of time points and tissues. Our scoring system was manually adjusted and other laboratories can implement it with different parameters.
For the NOBOX variants that cannot be considered pathogenic or causative anymore, the genome/exome sequencing data of the corresponding patients should be reanalysed. Furthermore, the functional studies performed using the obsolete coding sequence should be reconsidered. The corrected gene model should be taken into account when evaluating novel NOBOX variants identified in POI patients. Our results highlight the importance of the careful assessment of the most updated expression data for validating a gene model, enabling a correct evaluation of the pathogenicity of variants found in patients. The proposed quantitative framework developed here can be used for the classification of variants in other genes underlying POI. Furthermore, the global approach based on quantitatively adjusting the ACMG/AMP guidelines could be extended to other inherited pathologies.
STUDY FUNDING/COMPETING INTEREST(S): This project was not funded. All the authors have no conflict of interest to disclose.
如何将最新的表达和基因组数据与疾病/病症特异性分类系统相结合，以校正基因模型，从而更好地评估在患者中发现的变异的致病性？
通过整合来自多个物种的可用基因组和转录组数据以及一个带有原发性卵巢功能不全（POI）校正参数的定量分类框架，我们校正了人类NOBOX（新生儿卵巢同源框）基因模型，并对先前在POI病例中报道的变异进行了重新分类。
NOBOX基因编码一种性腺特异性转录因子，在早期卵泡发生中起关键作用，被认为是参与POI的主要基因，目前被描述为表达为四种转录本，其中最长的一种被视为标准转录本。根据这个标准转录本对在POI病例中鉴定出的所有变异进行了评估，并使用相应的预测蛋白进行了各种功能测试。
研究设计、规模、持续时间：我们利用人类和其他16种哺乳动物物种的可用基因组和RNA测序数据，对NOBOX基因模型进行了优化和校正。选择表达数据时考虑了组织特异性、链特异性和覆盖范围。对来自不同胎儿卵巢阶段的RNA测序数据进行分析，以描述NOBOX异构体的时间进程。检索文献以获取在POI病例中报道的NOBOX变异，同时也检索了ClinVar和GnomAD 4数据库中存在的NOBOX变异。
参与者/材料、设置、方法：分析来自人类胎儿卵巢和成人睾丸的链特异性RNA测序数据，以推断正确的人类NOBOX基因异构体。通过结合来自17种哺乳动物物种的比对基因组序列和相关RNA测序数据，验证了基因结构的保守性。由于改变基因模型意味着对变异进行重新分类，我们根据美国医学遗传学与基因组学学会/分子病理学协会（ACMG/AMP）指南，建立了一个包含来自GnomAD4的更新变异频率和POI校正参数的定量框架。利用这个框架，我们对在POI患者和家系中报道的44个NOBOX变异、在ClinVar中报道的117个NOBOX变异以及在GnomAD4中存在的2613个NOBOX变异进行了重新分类。
校正后的NOBOX基因模型表明两种转录本无效，包括标准转录本。这两种正确的异构体存在于胎儿卵巢样本中，而在成人睾丸中仅检测到一种。只有14个变异仍可能是POI的病因。此外，这种重新评估强烈表明NOBOX双等位基因变异最有可能是POI的病因。
大型表格作为补充数据集提供在Zenodo存储库中。
局限性、注意事项：所提出的基因模型是可靠的，但依赖于涵盖一系列时间点和组织的可用转录组数据。我们的评分系统是手动调整的，其他实验室可能会以不同参数实施。
对于那些不再被认为是致病或病因性的NOBOX变异，应重新分析相应患者的基因组/外显子组测序数据。此外，应重新考虑使用过时编码序列进行的功能研究。在评估POI患者中鉴定出的新型NOBOX变异时，应考虑校正后的基因模型。我们的结果强调了仔细评估最新表达数据以验证基因模型的重要性，从而能够正确评估在患者中发现的变异的致病性。这里开发的所提出的定量框架可用于对POI相关其他基因中的变异进行分类。此外，基于对ACMG/AMP指南进行定量调整的整体方法可扩展到其他遗传性疾病。
研究资金/利益冲突：本项目未获资助。所有作者均无利益冲突需要披露。
