Familial Hemophagocytic Lymphohistiocytosis-Type 3 (FHL3) via the UNC13D Inversion

Hemophagocytic Lymphohistiocytosis (HLH) is a rapidly progressing, hyperinflammatory condition in which activated T cells and macrophages infiltrate numerous organs. Clinical manifestations include fever, hepatosplenomegaly, pancytopenia, hemophagocytosis, severely attenuated or absent NK cell function, elevated iron levels, and elevated soluble CD25 (Henter et al. 2007. PubMed ID: 16937360). Familial (primary) HLH (FHL) and sporadic (secondary) HLH are clinically similar. Both types may be triggered by viral infections (Epstein-Barr virus, EBV), rheumatic disorders, and malignancies. The incidence of FHL is approximately 1 in 50,000 live births with 70-80% of patients showing clinical symptoms during infancy (Aricò et al. 1996. PubMed ID: 8637226; Janka. 1983. PubMed ID: 6354720). Though rare, cases of late-onset, adult FHL have been reported in patients ranging in age from their twenties to sixties (Allen et al. 2001. PubMed ID: 11410413; Clementi et al. 2002. PubMed ID: 12229880; Nagafuji et al. 2007. PubMed ID: 17606450). Males with HLH may have X-linked Lymphoproliferative Disorder (XLP) that may include gammaglobulinemia, and lymphoma (Coffey et al. 1998. PubMed ID: 9771704; Arico et al. 2001. PubMed ID: 11159547; Rigaud et al. 2006. PubMed ID: 17080092; Booth et al. 2011. PubMed ID: 20926771).

FHL is primarily an autosomal recessive or X-linked disorder, though a few autosomal dominant conditions are associated with FHL. Over 75% of FHL cases can be attributed to pathogenic variants in one of six genes: PRF1 (FHL Type 2), UNC13D (FHL Type 3), STX11 (FHL Type 4), STXBP2 (FHL Type 5), and the XIAP and SH2D1A genes (both are associated with X-linked FHL). Pathogenic variants in PRF1 account for 20-40% of all FHL cases and pathogenic variants in UNC13D, STX11, and STXBP2 account for approximately 20-25%, 14%, and 10% of FHL cases, respectively (Gholam et al. 2011. PubMed ID: 21303357). Around 60% of X-linked FHL is caused by pathogenic variants in the SH2D1A gene (Coffey et al. 1998. PubMed ID: 9771704; Gilmour et al. 2000. PubMed ID: 10898506; Yin et al. 1999. PubMed ID: 10598819; Nichols et al. 1998. PubMed ID: 9811875). Most of the FHL genes encode proteins that help regulate lytic granule targeting, docking, and membrane fusion during CTL and NK cell degranulation. PRF1 encodes a primary component of lytic granule cargo involved directly in membrane disruption of target cells at the immunological synapse (Gholam et al. 2011. PubMed ID: 21303357).  

Pathogenic variants are located throughout the UNC13D gene and comprise primarily missense/nonsense variants, splice-site variants, and small insertions and deletions. Most variants are inherited from parental carriers; de novo variants are rare. Large copy number variants are rare with only one large duplication having been reported to date (Feldmann et al. 2003. PubMed ID: 14622600; Yamamoto et al. 2004. PubMed ID: 15466010; Zur Stadt et al. 2006. PubMed ID: 16278825; Hiejima et al. 2018. PubMed ID: 29596912).

UNC13D encodes the Munc13-4 protein which regulates SNARE-mediated lytic granule exocytosis in CTLs and NK cells (Feldmann et al. 2003. PubMed ID: 14622600). UNC13D has been cited as a nonessential gene for growth of human tissue culture cells (Online Gene Essentiality, ogee.medgenius.info).

Mouse models in which Unc13d is absent have shown a complete loss of platelet dense granule secretion and attenuated platelet aggregation.  Mice lacking Unc13d also had prolonged bleeding times which is consistent with the Unc13d gene product serving a critical role during proper platelet function (Ren et al. 2010. PubMed ID: 20435885).

This test involves analysis of a known pathogenic inversion involving the UNC13D gene that was observed in 7% of FHL patients in one report (Meeths et al. 2011. PubMed ID: 21931115; Qian et al. 2014. PubMed ID: 24470399). This test employs custom PCR reactions to detect a 253-kb inversion involving Alu elements in intron 30 and in the 3' region downstream of the UNC13D gene. This inversion abolishes UNC13D protein expression and is known to cause disease when present with another pathogenic UNC13D gene variant. 

The UNC13D inversion was identified in 7% of FHL patients in one report (Meeths et al. 2011. PubMed ID: 21931115; Qian et al. 2014. PubMed ID: 24470399).

This test is performed using custom PCR primer probesets to amplify regions within and surrounding the 253-kb inversion sequence. Appropriate positive and negative controls are used in order to confirm the presence of the inversion in carriers and when present in the homozygous state in affected individuals.