Malignant Recessive Osteopetrosis Research Articles

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. Disclosures:No relevant conflicts of interest to declare.

Familial malignant osteopetrosis in children: a case report

The clinical, radiological, pathological and laboratory findings of two brothers with autosomal recessive malignant osteopetrosis are presented. Our findings are similar to characteristics previously reported in the literature about patients with osteopetrosis. The 6-year-old male patient was pale and had petechiae on his arms and legs. He also had macrocephalia, splenomegaly, severe pancytopenia, genu valgus, hypocalcemia, amaurosis, cessation of physical development, generalized bone sclerosis and recurrent infections with a history of multiple incidences of acute otitis media. Generalized bone sclerosis and irregular sclerosis of the maxilla and mandible were seen on radiographs. The oral mucosa was apparently normal but permanent tooth eruption was delayed although there was early loss of deciduous teeth. The recommended treatment was blood transfusion and therapy with antibiotics when necessary; a bone marrow transplant was not possible due to lack of a compatible donor.

Chloride channel 7 (CLCN7) gene mutations in intermediate autosomal recessive osteopetrosis.

Osteopetrosis is a heterogeneous group of inherited disorders that includes a malignant autosomal recessive form, an intermediate autosomal recessive form and autosomal dominant forms of the disease. Most malignant osteopetroses have been ascribed to mutations in the OC116 gene encoding the human a3 subunit of vacuolar H(+)-ATPase. Few cases of autosomal recessive malignant osteopetrosis have been ascribed to mutations in the chloride channel 7 gene (CLCN7), which accounts for all autosomal dominant type II cases reported to date. Up until now, however, nothing has been known regarding the molecular basis of the intermediate form of osteopetrosis (IARO). Our study of two Portuguese IARO families shows that homozygosity for ClCN7 mutations also accounts for this form of osteopetrosis. The two patients presented with spontaneous fractures in the first years of life and generalised increase of bone density. Direct sequencing of the ClCN7 gene in both patients revealed homozygosity for two mutations (G203D and P470Q). We conclude therefore that ClCN7 mutations not only account for some dominant and malignant forms but also for intermediate forms of osteopetrosis.

Human osteopetrosis and other sclerosing disorders: recent genetic developments.

Osteopetroses are rare human genetic disorders due to markedly decreased bone resorption. To date, the only gene whose inactivation was known to be responsible for human osteopetroses was that encoding carbonic anhydrase type II. Recessive malignant osteopetrosis is linked to decreased osteoclast function, unlike several osteopetroses in rodents that are caused by the inactivation of genes stimulating osteoclast differentiation. Recent advances in genetics have shown that some patients affected with recessive malignant osteopetrosis have inactivating mutations in a subunit of the vacuolar proton pump that is actively produced in the osteoclast brush border, but not in the lysosomes of other cells. The same gene is mutated in osteopetrotic oc/oc mice. The genes responsible for autosomal dominant osteopetrosis (ADO) have not yet been identified. Also, different localizations have been observed for ADO II, the type with sandwich vertebrae, and ADO I, presenting with diffuse osteocondensation. Less data than in malignant osteopetrosis are avaible on the cellular mechanism of decreased bone resorption in ADO but there is also genetic heterogeneity in ADO II. Pycnodysostosis is also due to a decreased resorption, and is quite close to osteopetrosis. Pycnodysostosis is linked to an inactivating mutation in the gene encoding cathepsin K, which is required for osteoclastic resorption. Decreased bone resorption is not the only defect inducing osteosclerosis, and Camurati-Engelmann disease is due to increased bone formation. Recently it has been shown that it is associated with a mutation in the propeptide of TGFbeta1. Thus, human osteosclerosing disorders have a wide range of phenotypes and genotypes and knowledge of them will contribute to a better understanding of the remodeling of normal bone.

Prenatal diagnosis of malignant osteopetrosis in Bedouin families by linkage analysis

Autosomal recessive malignant osteopetrosis (MOP) is a lethal disease, unless bone marrow is successfully transplanted. Yet a donor may not always be available, and even when there is one transplantation results are far from optimal. The difficulty in obtaining conclusive results by sonographic and X-ray evaluation of the fetus makes prenatal molecular diagnosis highly desirable. Subsequent to the chromosomal localization of the MOP gene in Arab-Bedouin families from the Negev region in Israel, linkage analysis was used for the prenatal diagnosis of this disease in Bedouin families at risk. Twelve cases were diagnosed, three fetuses were found to be affected, and one of the pregnancies was terminated. The other two pregnancies continued to term and the diagnosis of osteopetrosis was confirmed by X-ray immediately after birth. This is the first report on prenatal diagnosis of autosomal recessive osteopetrosis by linkage analysis.