TCIRG1 Associated Congenital Neutropenia

Severe congenital neutropenia (SCN) is a rare autosomal dominant hematopoietic disorder with an estimated incidence of 1 in 200,000 for individuals of European descent. Although several causal genes have been identified, the genetic basis for >30% of cases remains unknown. We report finding a novel single nucleotide variant (SNV) R736S identified in TCIRG1, utilizing SNP chip analysis, whole exome sequencing and confirmatory Sanger sequencing, in a multi-generation family (median blood neutrophil counts 0.486 x 10⁹/L, range 0.074 to 1.235 x 10⁹/L for 11 affected members and 3.808 x 10⁹/L, range 1.548 to 6.488 x 10⁹/L for 7 unaffected members). There is a perfect cosegregation of the variant with neutropenia in this family; all 11 affected, but none of the unaffected individuals share this novel variant.

TCIRG1 is located at 11q13 and encodes the vacuolar H+-ATPase a3 subunit, a 116-kD component of V-ATPases called OC116, and is essential for bone tissue maintenance and homeostasis. Homozygous or compound heterozygous mutations in this gene cause autosomal recessive malignant osteopetrosis (arOP) characterized by bone resorption defect due to osteoclast malfunction. Interestingly, the same gene, through an alternative splicing and usage of an alternative initiation codon in exon 7, gives rise to another protein, i.e., TIRC7, which is a T cell-specific membrane protein demonstrated to play an essential role for T-lymphocyte activation and immune response. Recent publications suggest the existence of additional splice variants expressed in various tissues in normal volunteers. SCN due to mutations in TCIRG1 have not been previously described.

The arginine (R) to serine (S) substitution is a critical change because arginine at position 736 is essential for proton translocation and is evolutionarily highly conserved with a Genomic Evolutionary Rate Profiling (GERP) score = 3.78. Although direct structural data on TCIRG1’s protein products are lacking, the effect of this mutation on the protein structure is predicted to be “probably damaging” by Polyphen (an automatic tool for prediction of possible impact of an amino acid substitution on the structure and function of the protein).

Western blot analysis of protein products of this gene performed using peripheral blood mononuclear cells show reduced expression of TCIRG1 protein in the affected family members from this family utilizing a commercially available polyclonal antibody developed against the N-terminal cytoplasmic domain of human OC116. The ∼45kDa TCIRG1 product was down-regulated by 26%, 49%, and 35% in the 3 affected individuals, compared to 2 normal controls. Another antibody, raised against the TCIRG1 fragment 121aa-220aa, detects a ∼120 kDa protein band with similar expression levels in both affected individuals and healthy controls. These assays confirm the multi-product nature of TCIRG1 and suggest deregulatory involvement of this protein in the pathogenesis of the neutropenia in this family.

Sanger sequencing of additional 20 unrelated SCN patients known to be negative for mutations in the known causal genes, revealed two different SNVs in TCIRG1 in two unrelated SCN patients, based on Minor Allele Frequency < 0.5% and high conservation among mammals (GERP score > 3). The first variant, g.16065 G>A, (IVS 14 -1G>A), is located in the acceptor splice site of the intron 14, and predicted to alter the splicing. The second variant, c.479 G>A (p.GLY160GLU), is located in exon 5 and predicted to be possibly damaging by Polyphen. No other SNV’s predicted to alter protein structure have been found.

We conclude that mutations in TCIRG1 are a rare but important cause of autosomal dominant congenital neutropenia. Further studies are in progress to define the pathophysiological mechanisms for neutropenia and the importance of the vATPases in maintaining the integrity of neutrophil production and deployment.