Clinical and molecular characterization of five Chinese patients with autosomal recessive osteopetrosis

Abstract

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.

Keywords: CLCN7; TCIRG1; autosomal recessive osteopetrosis; cDNA sequencing; novel variant.

FIGURE 1

Patient X‐rays. Patient 1 (a) A significant increase in bone density was observed, and the vertebral endplate showed the typical “sandwich vertebrae” appearance. Patient 2 (b) The results showed that bone density increased, part of the medullary cavity disappeared, the vertebral endplate thickened, and the “sandwich vertebrae” appearance was observed. Patient 3A (c) A significant increase in bone density was evident. Patient 3B (d) Diffusely increased bone density was confirmed, and the long bones with obliterated marrow cavity showed the typical “bone in bone” appearance. Patient 4 (e) A diffuse increase in bone density was observed, and the marrow cavity of the long bones disappeared, showing the “bone in bone” appearance. Patient 5 (f) A generalized increase in bone density was evident, the marrow cavity of the long bones disappeared, and the vertebral endplate appeared thickened, having the “sandwich vertebrae” appearance

FIGURE 2

Family pedigrees and genetic sequencing findings. (a) The pedigrees of all patients. (b–f) Variants in the CLCN7 and TCIRG1 genes identified by WES were verified using Sanger sequencing. (b) We identified compound heterozygous variants c.1555C>T (p.L519F) and c.2299C>T (p.R767W) in the CLCN7 gene in Patient 1. (c) Patient 2 displayed compound heterozygous variants c.286‐9G>A and c.1025T>C in the CLCN7 gene. (d) Compound heterozygous variants c.1370del and c.1554+2T>C were identified in the TCIRG1 gene in Patients 3A and 3B. (e) Patient 4 harbored a compound heterozygous variant in the TCIRG1 gene (c.676G>T; p.E226* in exon 5 and c.1191del in exon 11). (f) Compound heterozygous variants c.909C>A (p.Y303*) and c.2008C>T (p.R670*) were identified in the TCIRG1 gene in Patient 5. All patients inherited the variants from their parents, respectively

FIGURE 3

In silico and in vitro analysis of the three novel variants in the CLCN7 gene. (a) The p.L342P and p.L519F variants are located in the voltage‐gated chloride channel domain of the CLCN7 protein, which is a pfam domain. (b and c) A cross‐species alignment of amino acid sequences showed that p.L342P and p.L519F variants occurred in a highly conserved region. (d) Electrophoresis of the RT‐PCR products from the cDNA samples obtained from patient 2 and her father showed two CLCN7 fragments of different sizes, whereas samples obtained from the patient's mother showed only one band. (e and f) Clone sequencing of RT‐PCR products revealed that the c.286‐9G>A variant leads to the retention of intron 3 and formation of a premature stop codon (p.E95Vfs*8), compared to the wild‐type sequence