Novel TCIRG1 Mutations Causing Congenital Neutropenia

Background: Severe congenital neutropenia (SCN) is a bone marrow failure disorder leading to severe neutropenia related infections and leukemia. More than 30 different genes associated with this disorder have been identified. We previously identified a novel heterozygous missense mutation (R736S) of TCIRG1 as the probable cause for SCN in a large multigenerational family through exome sequencing (Makaryan et al; Hum Mutat. 2014 Jul;35(7):824-7). We also identified a statistically significant correlation of rare missense variants of TCIRG1 with lower ANC in three cohorts from the NHLBI Grand Opportunity Exome Sequencing Project (GO ESP; Rosenthal et al; Genet Epidemiol. 2016 Sep;40(6):470-4). Recently, we have discovered a novel TCIRG1 variant (R736C) at the same position in a new family with congenital neutropenia. TCIRG1 encodes subunit a3, a 116-kD component (OC116) of V-ATPases, vacuolar proton pumps which mediate H+ transport through membranes. Homozygous loss-of-function mutations of TCIRG1 are the most common cause of autosomal recessive osteopetrosis (ARO). Mutations R736S and R736C occur at an amino acid residue critical for proton translocation. There are no reports in the literature of individuals with heterozygous mutations at this locus. This is in contrast to the numerous healthy individuals with various heterozygous variants throughout the TCIRG1 gene. Parents of ARO patients harboring homozygous TCIRG1 mutations are considered healthy with no musculoskeletal or hematopoietic abnormalities. Pathogenesis of TCIRG1-associated neutropenia is largely unknown.

Hypothesis: We hypothesize that certain heterozygous TCIRG1 variants, like R736S and R736C, may cause SCN through specific molecular pathways.

Methods: Family and medical history, differential WBC counts and whole exome sequencing/Identity by Decent (IBD) analysis were used to identify the genetic basis of neutropenia in the previously reported family. Sanger sequencing was used to confirm the findings of exome sequencing and to identify other affected individuals. The R736C mutation was identified by whole exome sequencing with further site-specific analysis of 35 genes. Affected relatives were identified by Sanger sequencing. The Polyphen 2 online prediction tool was used to predict the impact of both substitutions on TCIRG1 function.

Results: Discovery and details of the original family were described in our previous report: (Makaryan et al; Hum Mutat. 2014 Jul;35(7):824-7). The second family was discovered in Taiwan, where the primary physician contacted SCNIR about a young patient with neutropenia who had a novel TCIRG1 mutation. Shortly thereafter, we identified two more affected family members. In both families the severity of recurrent bacterial infections is variable, roughly correlating with the severity of the neutropenia. For example, the grandmother of the index patient in the second family was not aware that she had neutropenia. The novel R736C TCIRG1 variant is located in the same position as the previous R736S variant. Arginine in this position is evolutionary highly conserved and both these substitutions cause a major change in amino acid size and properties. Polyphen-2 analysis predicts the effects of both mutations "probably damaging” (score 1.0).

Conclusions: Understanding the genetic, epidemiological and molecular basis of TCIRG1-associated neutropenia is important for improving the diagnosis and treatment of this form of congenital neutropenia. The pathogenesis of TCIRG1-associated neutropenia is unknown. It is very important to emphasize that carriers of pathogenic variants from both families have neutropenia with variable severity. The report of this new family suggests that there may be other patients and families with neutropenia caused by mutations in TCIRG1. Population based genome studies, careful family histories and whole genome/exome sequencing will be highly valuable for identifying and helping new patients with this disorder.

Kelley:X4 Pharmaceuticals, Inc.: Research Funding. Bolyard:X4 Pharmaceuticals, Inc.: Research Funding.