Clinical And Genetic Study Of Two Infantile Malignant Osteopetrosis Patients

Osteopetrosis is a group of heterogeneous disorders characterized by dysfunction of osteoclasts. Depending on the clinical manifestations, the disease is classified into 3 major phenotypic groups: autosomal dominant osteopetrosis（ADO）, the mildest type; infantile malignant osteopetrosis（IMO）, the most severe type and the intermediate autosomal osteopetrosis（IAO） with the phenotypes between ADO and IMO. IMO is a fatal condition. IMO patients usually die before the age of three years old. To date, 12 genes are known to be causally related to the disease. Among these,TCIRG1 and CLCN7 are the most common gene. The inheritance patterns of CLCN7-related osteopetrosis can be autosomal dominant or autosomal recessive, but TCIRG1-related osteopetrosis is usually autosomal recessive. To date, only six osteopetrosis patients caused by heterozygous TCIRG1 mutations have been reported,and none is Chinese in ethnicity.We have performed clinical and genetic investigations of two IMO patients. Wealso followed them up for 4-5 years. The age-at-onset of patient 1 and patient 2 were7 months and 2 years old, respectively. The prominent clinical phenotype is the generally increased bone density. X-ray examination showed “sandwich vertebrae”,“bone in bone” appearance of pelvis, the flask deformity of long bones. They also exhibited hematopoietic dysfunction, hepatosplenomegaly, neurodegeneration and other complications. What is special about patient 2 is that he survives longer than most IMO patients. Our last visit to the case was at the age of 7 years old.Genomic DNA of the patients and their parents was extracted from peripheral blood. Primers were designed to amplify exons of CLCN7 and TCIRG1 genes. PCR and Sanger sequencing were performed. Bioinformatics analysis was conducted to predict the biological effects of the mutations. For family 2, we constructed haplotypes using SNPs and microsatellites that flanking TCIRG1 gene. We also detected copy number variations（CNV） status by using chromosomal microarray analysis（CMA）.In family 1, a novel c.285+1G>A（IVS3+1G>A） mutation and a known c.896C>T（p.Ala299Val） mutation was identified in CLCN7 gene of the patient. The c.285+1G>A mutation was located in the splice donor of the third intron. It was predicted to interfere with normal splicing between exon 3 and 4 by bioinformatics analysis. It is predicted to truncate seven hundred and eleven amino acids from the C terminus of the wild-type protein, which contains all of the functional domains. The pathogenicity of c.896C>T mutation was clear for it was once reported in an osteopetrosis patient. The c.285+1G>A mutation was identified in the patient’s father and c.896C>T mutation in the mother. Sequencing of the patient’s TCIRG1 gene did not reveal any pathogenic mutation.In family 2, a novel four bases heterozygous mutation c.449₄52delAGAG（p.Gln149Glnfs16） was detected in the fifth exon of TCIRG1 gene of the patient. This altered the reading frame in the wild-type gene, a stop codon was introduced at the sixteenth codon at the 3’ of the mutation. This truncates six hundred and sixty six amino acids from the C terminus of the encoded protein, which contains the entire ATPase V0 complex domain. This mutation was also found in the patient’s father,who was clinically normal. No pathogenic mutation was identified in CLCN7 gene of the patient. Haplotype construction showed the patient and his father shared the same mutation bearing haplotype. CMA results of the patient and his mother revealed normal CNV status of TCIRG1 and CLCN7 genes. Three CNV were detected in the genome of the patient, but all were believed benign by the database. As the father was clinically unaffected by the mutation, this discrepancy may be explained by incomplete penetrance.We have performed clinical and genetic investigations in two osteopetrosis patients and their families. Two novel and one reported mutations were identified. Our results provide more data for the mutational spectrum of TCIRG1 and CLCN7 genes.In family 2, we demonstrated that heterozygous mutation of TCIRG1 gene may lead to IMO. This is the first report among Chinese ethnicity that heterozygous mutation of TCIRG1 gene may cause IMO.