Osteopetrosis is a genetically heterogenous, fatal bone disorder characterized by increased bone density. Globally, various genetic causes are reported for osteopetrosis with all forms of inheritance patterns. A precise molecular diagnosis is necessary for prognosis and for prescribing treatment paradigms in osteopetrosis. Here we report on thirteen individuals diagnosed with infantile malignant osteopetrosis coming from ten unrelated Pakistani families; nine of whom are consanguineous. We performed whole exome sequencing and Sanger sequencing in all families and identified homozygous variants in genes previously reported for autosomal recessive inheritance of osteopetrosis. All the identified variants are expected to affect the stability or length of gene products except one nonsynonymous missense variant. TCIRG1 was found as a candidate causal gene in majority of the families. We report six novel variants; four in TCIRG1 and one each in CLCN7 and OSTM1. Our combined findings will be helpful in molecular diagnosis and genetic counselling of patients with osteopetrosis particularly in populations with high consanguinity.

BACKGROUND: Osteopetrosis is characterized by increased bone density and bone marrow cavity stenosis due to a decrease in the number of osteoclasts or the dysfunction of their differentiation and absorption properties usually caused by biallelic variants of the TCIRG1 and CLCN7 genes.
METHODS: In this study, we describe five Chinese children who presented with anemia, thrombocytopenia, hepatosplenomegaly, repeated infections, and increased bone density. Whole-exome sequencing identified five compound heterozygous variants of the CLCN7 and TCIRG1 genes in these patients.
RESULTS: Patient 1 had a novel variant c.1555C>T (p.L519F) and a previously reported pathogenic variant c.2299C>T (p.R767W) in CLCN7. Patient 2 harbored a novel missense variant (c.1025T>C; p.L342P) and a novel splicing variant (c.286-9G>A) in CLCN7. Patients 3A and 3B from one family displayed the same compound heterozygous TCIRG1 variant, including a novel frameshift variant (c.1370del; p.T457Tfs*71) and a novel splicing variant (c.1554+2T>C). In Patient 4, two novel variants were identified in the TCIRG1 gene: c.676G>T; p.E226* and c.1191del; p.P398Sfs*5. Patient 5 harbored two known pathogenic variants, c.909C>A (p.Y303*) and c.2008C>T (p.R670*), in TCIRG1. Analysis of the products obtained from the reverse transcription-polymerase chain reaction revealed that the c.286-9G>A variant in CLCN7 of patient 2 leads to intron 3 retention, resulting in the formation of a premature termination codon (p.E95Vfs*8). These five patients were eventually diagnosed with autosomal recessive osteopetrosis, and the three children with TCIRG1 variants received hematopoietic stem cell transplantation.
CONCLUSIONS: Our results expand the spectrum of variation of genes related to osteopetrosis and deepen the understanding of the relationship between the genotype and clinical characteristics of osteopetrosis.

Infantile malignant osteopetrosis (IMO) is a rare autosomal recessive disease characterized by a higher bone density in bone marrow caused by the dysfunction of bone resorption. Clinically, IMO can be diagnosed with medical examination, bone mineral density test and whole genome sequencing.
We present the case of a 4-month-old male infant with abnormal skull development, hypocalcemia and premature closure of the cranial sutures. Due to the hyper bone density showed by his radiographic examination, which are characteristic patterns of IMO, we speculated that he might be an IMO patient. In order to confirm this diagnosis, a high-precision whole exome sequencing of the infant and his parents was performed. The analysis of high-precision whole exome sequencing results lead to the identification of two novel heterozygous mutations c.504-1G > C (a splicing site mutation) and c.1371delC (p.G458Afs*70, a frameshift mutation) in gene TCIRG1 derived from his parents. Therefore, we propose that there is a close association between these two mutations and the onset of IMO.
To date, these two novel mutations in gene TCIRG1 have not been reported in the reference gene database of Chinese population. These variants have likewise not been reported outside of China in the Genome Aggregation Database (gnomAD). Our case suggests that the use of whole exome sequencing to detect these two mutations will improve the identification and early diagnosis of IMO, and more specifically, the identification of homozygous individuals with TCIRG1 gene mutation. We propose that these mutations in gene TCIRG1 could be a novel therapeutic target for the IMO in the future.