Contributed equally
Osteopetrosis is a heritable bone disorder that exhibits highly clinical and genetical heterogeneity, and is caused by defective osteoclastic resorption. The three main forms are the autosomal recessive severe (ARO), the intermediate autosomal and the autosomal dominant benign osteopetrosis forms. In the present study, the clinical, biochemical and radiological manifestations were described in a patient with osteopetrosis. Sequence analysis identified the compound heterozygous mutations, c.909C>A (p.Tyr303X) and c.2008C>T (p.Arg670X), in
Bone development and homeostasis is achieved by a strict balance between bone formation by osteoblasts and resorption by osteoclasts. Defective bone resorption results in osteopetrosis, an inherited disorder encompassing a clinical and genetical heterogenous group of conditions. Three main forms are classified on the basis of inheritance patterns, age of onset and severity. These are the autosomal recessive severe (ARO; MIM 259700), intermediate autosomal (IAO) and autosomal dominant benign osteopetrosis (ADO) forms (
Mutations in at least 10 genes have been identified as causative in various types of osteopetrosis cases in humans (
A 21-month-old male was admitted to Shanghai Children’s Medical Center (Shanghai, China) due to persistent anemia and a lack of dentition. The patient was full-term at birth, with a normal weight and length, and a subsequent weight and length of 12 kg (50th percentile) and 84.8 cm (between 25th and 50th percentile), respectively, at 21 months. A prominent forehead, enlarged abdomen with moderate hepatosplenomegaly and visual disturbance were noted during the physical examination. Laboratory tests revealed that the patient had moderate anemia, elevated levels of serum alkaline phosphatase, parathyroid hormone, creatine kinase and MB isoenzyme, and decreased serum Ca2+ levels. The levels of phosphonium, 1,25-dihydroxy vitamin D3, lactate dehydrogenase, thyroid hormone and thyroid stimulating hormone were within the normal ranges. Imaging examinations consisting of computerized tomography (CT) scans (LightSpeed 16 Slice CT; GE Healthcare, Fairfield, CT, USA) and X-rays (AMX IV Plus Portable X-Ray, GE Healthcare) revealed a general increase of bone density involving the skull, vertebrae and limbs (
Ethylenediaminetetraacetic acid-peripheral blood samples were obtained from the patient, his parents and 100 healthy individuals. Genomic DNA was isolated from the peripheral blood leukocytes using the QIAmp DNA Blood kit (Qiagen, Hilden, Germany). All the exons and exon-intron boundaries of the
The total RNA of the patient and the parents was isolated from their peripheral leukocytes using the QIAmp RNA Blood kit (Qiagen). The first-strand cDNA was synthesized using random primers, oligo primers and PrimeScript Reverse Transcriptase (Takara Biotechnology (Dalian) Co., Ltd., Dalian, China). The synthesized products were amplified with primer P1, 5′-TACGGGCTCACGGTGTCTG-3′, which is located in exon 17, and primer P2, 5′-GGTAGGCGTCTCGGAAGTC-3′, which is located in exon 23 of
The study was approved by the Ethics Committee of Shanghai Children’s Medical Center, Shanghai Jiao Tong University School of Medicine. Written informed consent was obtained from the parents and the 100 healthy individuals prior to blood sampling and DNA analysis.
Direct genomic DNA sequencing of the patient revealed compound heterozygous mutations, c.909C>A (p.Tyr303X) and c.2008C>T (p.Arg670X), in
RT-PCR performed on the RNA extracted from the father enabled the detection of one band of the expected 560 bp (from exon 17 to exon 22 of the
In the present study, the case of a patient with osteopetrosis was investigated. Molecular analysis of the patient revealed compound heterozygous nonsense mutations in
In order to determine the effect of
Mutations in the
According to previous studies, the compound heterozygous nonsense mutations of
In conclusion, a patient with ARO was studied. The compound heterozygous mutations, c.909C>A (p.Tyr303X) and c.2008C>T (p.Arg670X), in
The authors would like to thank all the members of the family for their participation in this study. This study was supported by the National Natural Science Foundation of China (grant nos. 81000207 and 81000346) and a foundation grant from Shanghai Science and Technology Commission for major issues (grant no. 11dz1950300).
Imaging examinations consisting of a CT scan and X-rays of the proband. A generalized increase in bone density involving (A) the skull, (B) the limbs and (C) the vertebrae was observed. CT, computed tomography.
Molecular analysis of the
Transcription experiment of the
PCR primers used for amplification of
Primer | Sequence (5′ to 3′) | Size (bp) |
---|---|---|
EXON1 F | TCAACCTCTCCCAGACTTCC | 320 |
EXON1 R | CTGAGCTGCATTCACGGAG | |
EXON2 F | TCAGTGAGTGAAGGTGCACAG | 319 |
EXON2 R | GTTCAAATGGGGCCAGG | |
EXON3 F | TCCACACCTTTCTGGAGGAG | 266 |
EXON3 R | TTTCAGATCAAACTTGGCCC | |
EXON4+EXON5 F | GAGTTTGGGGCAGCAGG | 564 |
EXON4+EXON5 R | CACTGGACAAGGAGTCGGAG | |
EXON6+EXON7 F | GAGGCCTCCTGCCTTCC | 561 |
EXON6+EXON7 R | GGCCAGAAGGACACAGCTAC | |
EXON8 F | CCTATCGTGACTCCTCCCC | 262 |
EXON8 R | ACCTCCTGCACCCACCTC | |
EXON9 F | GAGGTGGGTGCAGGAGG | 372 |
EXON9 R | CTGGAAGTGAGGCAGAAACG | |
EXON10 F | ATCTCCAGCTGGGCCTG | 301 |
EXON10 R | CCTCAGGCTCACACCCAC | |
EXON11+EXON12 F | AAGTGATGGGTTCTTGACTGC | 570 |
EXON11+EXON12 R | AGGAATGCATCACTGCGG | |
EXON13 F | AGTCTGGCTGGAGGTGAGG | 291 |
EXON13 R | CACACAGGAGTGCTCAGCG | |
EXON14+EXON15 F | GGGAAAACAGGGTGGTGAG | 597 |
EXON14+EXON15 R | GATCTTGCAGCTCCCAGTG | |
EXON16+EXON17 F | CGTGACTGCTGTGACTCAGG | 604 |
EXON16+EXON17 R | GCAGAACTCGATGGTGTGG | |
EXON18 F | GCCTGGATGATGAAGAGGAG | 307 |
EXON18 R | AACTGAGGCCCAGAGAGAAG | |
EXON19+EXON20 F | AAGTGGGACTGTCCAAGGAG | 613 |
EXON19+EXON20 R | TCCCAGATCCTACACCATGC |
PCR, polymerase chain reaction; F, forward; R, reverse.
PCR primers used for amplification of
Primer | Sequence (5′ to 3′) | Size (bp) |
---|---|---|
Promoter F (rs960467) | GGAAGCCTCCACTCCGACCC | 475 |
Promoter R (rs960467) | GTGATGAGCGACGGCGACCA | |
Exon1 F | CGTTGCAGGTCACATGGTC | 470 |
Exon1 R | GCCTCCGAAGACTCCAGAC | |
Exon2 F | CGGATCAGTTCTGCTTCCAG | 511 |
Exon2 R | CATGCTGTCACTGCTGTCCT | |
Exon3+Exon4 F | TGCTGGGATTGTAGGTGTCA | 629 |
Exon3+Exon4 R | GAGCAGCCTTCTTGGTTACG | |
Exon5+Exon6 F | CACACTGGGCCCTTCATAAT | 810 |
Exon5+Exon6 R | TCTGCTCCTCCTGAGGTTGT | |
Exon7 F | GTGTCTGCTGCTCTCCTCAG | 243 |
Exon7 R | GCTCCTGAACCAGCAAAGAG | |
Exon8+Exon9 F (VNTR in intron 8) | GCTTGGCTGCTGTTTAGCTC | 764 |
Exon8+Exon9 R (VNTR in intron 8) | AAGCCCATCTCCCTGAGTG | |
Exon10+Exon11 F | GTGCTGACCCTGCTGTCTCT | 797 |
Exon10+Exon11 R | AGGACCAAGGCCTGACAGA | |
Exon12 F | CACTGGCAAGTCCAGAGAGG | 559 |
Exon12 R | GCAGCAACTGTGTGACATCC | |
Exon13 F | CCAGTGTGTTTCTCCCCTGT | 443 |
Exon13 R | CTGTGGTTTTTGCCAACAGA | |
Exon14 F | ATTGCTCTGCTGGACACCTT | 551 |
Exon14 R | GCAGGGCCTCACTTCCTAC | |
Exon15 F (rs12926089, rs12926669) | CAGTGTCCTCCATCAGGGACT | 401 |
Exon15 R (rs12926089, rs12926669) | CTCTGAGATCTGGGTGGACAG | |
Exon16 F | CTCCCAACGTGTGCTCTCTC | 306 |
Exon16 R | ATCCTCCTGCCTTGGTCTCT | |
Exon17 F | TGAGAACAGGGAGCCTTCTG | 432 |
Exon17 R | AGGTGCGACACTTTTGTCCT | |
Exon18+Exon19 F | GGTGACTGTGCCCTCTGC | 730 |
Exon18+Exon19 R | CCCAGAAACCCTGAGCCTAC | |
Exon20+Exon21 F | CTGTGAGCCTCCAAACAGC | 717 |
Exon20+Exon21 R | GTCCACACAGCCCTCCAT | |
Exon22+Exon23 F | AGGCTGGTGTGAGCAGGTAG | 638 |
Exon22+Exon23 R | GCCCCTTGACTTCAGCTCTA | |
Exon24+Exon25 F | CTGAAGTCAAGGGGCTGAGG | 806 |
Exon24+Exon25 R | AGACCACTGCCCACAACAG |
PCR, polymerase chain reaction; CLCN7, chloride channel 7 gene; F, forward; R, reverse.
Molecular analysis of the
Subject | Nucleotide change of |
Nucleotide change of |
Genotypes | |||
---|---|---|---|---|---|---|
| ||||||
rs960467 | rs12926089 | rs12926669 | Intron 8 VNTR | |||
Patient | c.909C>A | c.1798-1G>T | G/A | G | T | Three repeat units |
c.2008C>T | ||||||
Mother | c.909C>A | c.1798-1G>T | G/A | G | T | Three repeat units |
Father | c.2008C>T | - |
G | G | T | Three repeat units |
Accession number of the
Accession number of the
rs960467,
No nucleotide change. VNTR, variable number tandem repeat.