Famà Rosella, Borroni Ester, Zanolini Diego, Merlin Simone, Bruscaggin Valentina, Walker Gillian E, Olgasi Cristina, Babu Deepak, Agnelli Giacchello Jacopo, Valeri Federica, Giordano Mara, Borchiellini Alessandra, Follenzi Antonia
Department of Health Sciences, Università del Piemonte Orientale, Novara, Italy.
Hemostasis and Thrombosis Unit, Città Della Salute e Della Scienza, Molinette, Turin, Italy.
J Thromb Haemost. 2020 May;18(5):1050-1064. doi: 10.1111/jth.14779.
We have identified a synonymous F8 variation in a severe hemophilia A (HA) patient who developed inhibitors following factor VIII (FVIII) prophylaxis. The unreported c.6273 G > A variant targets the consensus splicing site of exon 21.
To determine the impact of c.6273 G > A nucleotide substitution on F8 splicing and its translated protein.
Patient peripheral blood mononuclear cells were isolated and differentiated into monocyte-derived macrophages (MDMs). FVIII distribution in cell compartments was evaluated by immunofluorescence. The splicing of mutated exon 21 was assessed by exon trapping. Identified FVIII splicing variants were generated by site-directed mutagenesis, inserted into a lentiviral vector (LV) to transduce Chinese hamster ovary (CHO) cells, and inject into B6/129 HA-mice. FVIII activity was assessed by activated partial thromboplastin time, whereas anti-FVIII antibodies and FVIII antigen, by ELISA.
HA-MDMs demonstrated a predominant retention of FVIII around the endoplasmic reticulum. Exon trapping revealed the production of two isoforms: one retaining part of intron 21 and the other skipping exon 21. These variants, predicted to truncate FVIII in the C1 domain, were detected in the patient. CHO cells transduced with the two FVIII transcripts confirmed protein retention and absence of the C2 domain. HA mice injected with LV carrying FVIII mutants, partially recovered FVIII activity without the appearance of anti-FVIII antibodies.
Herein, we demonstrate the aberrant impact of a FVIII synonymous mutation on its transcription, activity, and pathological outcomes. Our data underline the importance of increasing the knowledge regarding the functional consequences of F8 mutations and their link to inhibitor development and an effective replacement therapy.
我们在一名重度甲型血友病（HA）患者中发现了一个同义F8变异，该患者在接受凝血因子VIII（FVIII）预防治疗后产生了抑制剂。未报告的c.6273 G>A变异靶向第21外显子的共有剪接位点。
确定c.6273 G>A核苷酸替换对F8剪接及其翻译蛋白的影响。
分离患者外周血单个核细胞并将其分化为单核细胞衍生的巨噬细胞（MDM）。通过免疫荧光评估FVIII在细胞区室中的分布。通过外显子捕获评估突变的第21外显子的剪接。通过定点诱变产生鉴定出的FVIII剪接变体，将其插入慢病毒载体（LV）中以转导中国仓鼠卵巢（CHO）细胞，并注射到B6/129 HA小鼠中。通过活化部分凝血活酶时间评估FVIII活性，而通过ELISA评估抗FVIII抗体和FVIII抗原。
HA-MDM显示FVIII主要保留在内质网周围。外显子捕获显示产生了两种异构体：一种保留了部分第21内含子，另一种跳过了第21外显子。预计这些变体将在C1结构域中截断FVIII，在患者中检测到了这些变体。用两种FVIII转录本转导的CHO细胞证实了蛋白保留且没有C2结构域。注射携带FVIII突变体的LV的HA小鼠部分恢复了FVIII活性，且未出现抗FVIII抗体。
在此，我们证明了FVIII同义突变对其转录、活性和病理结果的异常影响。我们的数据强调了增加对F8突变的功能后果及其与抑制剂发展和有效替代疗法的联系的认识的重要性。
