Identification of a De Novo UNC13D L1000P Mutation in a Familial Hemophagocytic Lymphohistiocytosis Patient

Abstract 4927

Familial hemophagocytic lymphohistiocytosis (FHL) is a rare autosomal recessive disorder characterized by massive infiltration of several organs by activated lymphocytes and macrophages. Four causative genes have been identified for this autosomal recessive disorder (PRF1, UNC13D, STX11 and STXBP2). Till now, gene mutations carried by FHL cases reported in literature were all identified to be inherited when pedigree analysis was performed. But the mutation types of these genes were diversified, when and how these different kinds of mutations occurred is yet to know. Here we report the first de novo UNC13D L1000P mutation identified in a FHL patient.

The patient was a 6 years old boy with fever (39-40.2°C), pancytopenia and hepatosplenomegaly. Blood test showed hemoglobin of 6.0g/dL, platelet count of 48×10⁹/L, and white blood cell count of 0.96×10⁹/L. ALT 91U/L, AST 68 IU/L, LDH 419IU/L, TP 67g/L, ALB 36g/L, Fibrinogen 2.03g/L, Serum ferrites 704ug/L. Ultrasound examination showed hepatosplenomegaly. Bone marrow morphological examination showed hemophagocytic phenomenon. Immunophenotyping of peripheral blood showed 68% of lymphocytes, no abnormal clonal cells were identified, the proportion of NK cells and perforin protein expression is normal. Herpes virus (HHV) type 1 to 8 screening showed EBV and HHV7 positive. Gene mutation screening for PRF1, UNC13D, STX11, STXBP2, SH2D1A and XIAP showed two UNC13D heterozygous mutations of c.1845_1847dupTGA/p.D615dup and c.2999T>C/p.L1000P, both mutations had never been reported in literature. The patient was diagnosed as FHL3 and got a remission by plasma exchange, antivirus, etoposide and dexamethasone treatment. And then he was performed allogeneic hematopoietic stem cell transplantation and soon got a complete implantation.

When pedigree analysis was performed, only the D615dup mutation was inherited from his mother, the L1000P mutation was not seen in either his parents. The genetic relationship between the patient and his parents was confirmed by 15 STR polymorphism analysis using AB Identifilier Kit, and then the hereditary relationship of UNC13D gene was further confirmed by analysis of common polymorphism site within the UNC13D gene. Further pedigree analysis confirmed that the D615dup was inherited from the maternal lineage, but none of his paternal lineage member (including his father and grandparents) carrying L1000P mutation. The oral mucosa, nail, hair follicle and semen samples from the patient's father were all identified to be negative for L1000P mutation, but the oral mucosa, nail and hair follicle samples from the patient were all identified to be carrying the heterozygous L1000P mutation. The cDNA fragment including both D615dup and L1000P mutations from the patient was amplified by PCR and clone sequenced. By cloning and sequencing, we identified that the two mutations were separately located on different UNC13D alleles. Through comprehensive analysis of the results showed above, we deemed that the UNC13D L1000P mutation carrying by the patient is a de novo mutation, it most likely occurred during the process of male gamete formation from his father.

It is known that there will be accidental event of de novo mutations during the process of gamete formation, and they are material basis of evolution and genetic disease. Due to the rarity of the incident, de novo mutation is hard to be observed in common Mendelian diseases and only have been reported in some very rare disease such as Proteus Syndrome. We are now reported the first observation of a de novo mutation in FHL patient, and we believe that the de novo UNC13D L1000P mutation and the hereditary UNC13D D615dup mutation each lead to defect of one UNC13D allele, and thus contribute to the genetic pathogenesis of the patient. We also believe this is evidence that the de novo mutation of UNC13D gene is a randomly and constantly accidental event in the general crowd.