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Introduction

Osteopetrosis refers to a heterogeneous group of rare bone disorders characterized by reduced osteoclast activity (1–3), which results in increased bone mass and decreased bone strength. The disease is classified into three major clinical subtypes according to the severity and mode of inheritance: Autosomal recessive osteopetrosis (ARO); intermediate ARO (IARO); and autosomal dominant osteopetrosis (ADO) (4). Mutations in the chloride channel 7 (CLCN7) gene locus on chromosome 16p13.3 have been reported to cause ARO, IARO and ADO type II (ADO-II) (5,6). To date, all reported cases of ADO-II are associated with mutations in the CLCN7 gene. The CLCN7 gene encodes the 803-amino acid chloride channel protein 7 (ClC-7), which provides the chloride conductance necessary for efficient proton pumping in the osteoclast ruffled membrane (4) and is involved in acidification of the resorption lacuna. A low-pH microenvironment is required during normal bone resorption (1). Osteoclasts are multinucleated giant cells responsible for bone resorption, which serve important roles during bone growth and tissue renewal (7). With the balance of bone modeling and remodeling, the normal mineralized matrix erosion activity of osteoclasts serves to maintain healthy and mechanically competent bone (8). Conversely, dysfunctional osteoclasts lead to osteopetrosis. The majority of patients are diagnosed with ADO, with an incidence of ~1 per 20,000 individuals (2,9).

ADO-II (OMIM 166600; www.omim.org), also known as Albers-Schönberg disease or marble bone disease, is a form of ADO comprising a clinical spectrum ranging from very mild to severe disease phenotypes, with a reported penetrance of 56–90% in different studies (1,10). The range of phenotypes observed under the ADO-II spectrum includes asymptomatic disease, osteosclerosis, a high rate of fractures, osteomyelitis, and hematological and neural defects (2). IARO is a milder form of ARO; patients with IARO manifest mandibular prognathism, occasional osteomyelitis, hepatosplenomegaly and a tendency to develop fractures (4). Radiographs of affected individuals reveal diffuse osteosclerosis and pathognomonic findings of ‘bone-within-bone’ or ‘endobones’ due to parallel bands of dense bone, which are often prominent in the pelvis, vertebrae and long bones (11). The thickening of the end plate in the vertebrae is termed ‘sandwich vertebrae’ or ‘rugger jersey spine’. Alterations in the metaphysis of long bones are termed ‘Erlenmeyer flask bone deformities’ (12,13).

At present, >23 mutations in CLCN7 have been identified in Chinese families with ADO-II (11,14–20). Among them, 14 mutations were reported in our previous studies (10,14,16,20). A few cases of IARO in China have been reported by Xue et al (17), Pang et al (19) and Zhang et al (16). In the present study, the clinical and molecular characterization of another five patients with ADO-II and two patients with IARO were reported. Additionally, four novel mutations were identified.

Materials and methods

Patients

The present study was approved by the Ethics Committee of the Shanghai Jiao Tong University Affiliated Sixth People's Hospital. All subjects who participated in the study were recruited by the Department of Osteoporosis and Bone Disease of the Shanghai Jiao Tong University Affiliated Sixth People's Hospital between December 2016 and December 2018, and all signed informed consent documents prior to entering the project. A total of seven families were included. All subjects were of Han ethnicity and had non-consanguineous parents. The pedigrees of the families with ADO-II and IARO are presented in Fig. 1.

Figure 1. Pedigrees of seven families with osteopetrosis analyzed in the present study. Circles indicate females; squares indicate males. Filled black shapes indicate patients with osteopetrosis. Shapes with a trunk line indicate non-penetrant carriers of ADO-II. Shapes with a dot indicate obligate carriers of IARO. Half-black shapes indicate obligate carriers with different heterozygous mutations. Arrows indicate probands. P indicates pregnant subjects. Diagonal lines represent deceased subjects.

Biochemical measurements

Fasting peripheral blood samples were collected from each subject during a clinical visit and were analyzed in the central laboratory of Shanghai Jiao Tong University Affiliated Sixth People's Hospital. Full blood count, including haemoglobin and platelets, and phosphate, calcium, alkaline phosphatase (ALP), creatinine, alanine transaminase, aspartate transaminase, lactate dehydrogenase (LDH), creatine kinase (CK) and CK-MB levels were measured using a Hitachi 7600 automatic biochemical analyzer (Hitachi, Ltd., Tokyo, Japan). Serum parathyroid hormone (PTH) concentrations, and Serum 25-hydroxyvitamin D (25OHD) levels were measured using an ECLIA Elecsys autoanalyzer (E170; Roche Diagnostic GmbH, Mannheim, Germany).

Measurement of radiographs and bone mineral density (BMD)

Radiographic studies were performed at the Department of Radiology of Shanghai Jiao Tong University Affiliated Sixth People's Hospital. X-rays of the skull, thoracic and lumbar vertebrae, distal femoral and proximal tibiae, and pelvis were acquired to detect bone abnormalities.

The BMD (g/cm2) of the lumbar spine (L1-4) and the left proximal femur, including the femoral neck and total hip, were measured using dual-energy X-ray absorptiometry densitometers at the Department of Osteoporosis and Bone Disease of Shanghai Jiao Tong University Affiliated Sixth People's Hospital.

Molecular genetics analyses

All fasting blood samples were collected in the morning for laboratory tests and DNA analyses. Genomic DNA was isolated from peripheral white blood cells from 2 ml blood using a DNA extraction kit (Shanghai Laifeng Biotechnology Co., Ltd., Shanghai, China; http://www.lifefeng.com) according to the manufacturer's protocol. The CLCN7 gene was screened for mutations in the probands from the seven families. A database was analysed and additional mutation sites were identified in other family members and 250 healthy ethnically matched controls (125 males and 125 females) (14). All 25 exons of the CLCN7 gene, including the exon-intron boundaries, were amplified via polymerase chain reaction (PCR) using 16 pairs of primers: CLCN7E1 forward (F): 5′-CGACCGGGCCTCGGTGGTT-3′, CLCN7E1 reverse (R): 5′-GCGGCCTCCGAAGACTCCAGACC-3′, CLCN7E2 F: 5′-AGCCGGATCAGTTCTGCTT-3′, CLCN7E2 R: 5′-CACTCCTCCGTCTGAGAGCA-3′, CLCN7E3_4 F: 5′-CCCCATGTGCAGTTCTCTTG-3′, CLCN7E3_4 R: 5′-AGCAGCCTTCTTGGTTACGG-3′, CLCN7E5_6 F: 5′-CACTGGGCCCTTCATAATCC-3′, CLCN7E5_6 R: 5′-TTCTAAAAGTGCCCGGGTTG-3′, CLCN7E7 F: 5′-GACGTGTGTGCTGCTCTTCC-3′, CLCN7E7 R: 5′-CAAACTGAAAGCGGGAAACTG-3′, CLCN7E8_9 F: 5′-CCCAGCCACTCTGCCTGATC-3′, CLCN7E8_9 R: 5′-CGCCAGGCTGTCCTCAGAT-3′, CLCN7E10_11 F: 5′-AGCCCCTTCCCCTGCACAGC-3′, CLCN7E10_11 R: 5′-CGATGGGTGGCCCCAAGGTG-3′, CLCN7E12 F: 5′-CTTCCCCTCTTGCTCTCCACT-3′, CLCN7E12 R: 5′-CTATCGATGGCACGGAGAGTC-3′, CLCN7E13_14 F: 5′-GGTGGTCCTTGAGTTTCAGCA-3′, CLCN7E13_14 R: 5′-TAAACCCCATTCCACCACGTC-3′, CLCN7E15 F: 5′-CAGTGTCCTCCATCAGGGACT-3′, CLCN7E15 R: 5′-CATTTCTCCTGGGTCCACATC-3′, CLCN7E16 F: 5′-GTGTCCTCCTTGCCCTCTGT-3′, CLCN7E16 R: 5′-GATCCTCCTGCCTTGGTCTCT-3′, CLCN7E17 F: 5′-CTCCCCATGGGATCCTTTTAG-3′, CLCN7E17 R: 5′-CCTGGTCCAGACTCCACACAT-3′, CLCN7E18_19 F: 5′-CAGCAGGGTGTACTGTGCTAGG-3′, CLCN7E18_19 R: 5′-CAGAAACCCTGAGCCTACCC-3′, CLCN7E20_21 F: 5′-GGGGTAGGCTCAGGGTTTCT-3′, CLCN7E20_21 R: 5′-ATGGCTGCACACTCAGCTTC-3′, CLCN7E22_23 F: 5′-GAGGCTGGTGTGAGCAGGTAG-3′, CLCN7E22_23 R: 5′-CAGAGTCACCGAGTCCTCTCC-3′, CLCN7E24_25 F: 5′-TCGGTGACTCTGTCTCCTGTG-3′, CLCN7E24_25 R: 5′-ACTGCTGGGGAGCATGGTT-3′. The PCR was performed using the HotStar Taq polymerase (Takara Bio, Inc., Otsu, Japan). The thermocycling conditions were the following: 95°C for 2 min, followed by 11 cycles of 94°C for 20 sec, 64°C −0.5°C/cycle for 40 sec and 72°C for 1 min, followed by 24 cycles of 94°C for 20 sec, 58°C for 30 sec and 72°C for 1 min, the final extension was performed at 72°C for 2 min for all primer pairs except for the except for CLCN7E10_11. The thermocycling conditions used to amplify the genomic region corresponding to CLCN7E10_11 were the following: Initial denaturation at 95°C for 2 min, followed by 35 cycles of 96°C for 10 sec and 68°C for 1 min. Direct sequencing was performed using the BigDye Terminator Cycle Sequencing Ready Reaction kit, v.3.1 (Applied Biosystems; Thermo Fisher Scientific, Inc.), and the resulting PCR products were directly sequenced using an automated ABI PRISM 3130 sequencer (Applied Biosystems; Thermo Fisher Scientific, Inc.). Basic Local Alignment Search Tool (www.ncbi.nlm.nih.gov/tools/cobalt/cobalt.cgi?link_loc=BlastHomeAd) was used to perform homology analysis of the R286W, G793R, Y746D, Y99C, E313K and P470L sites in eight vertebrate species. The potential causal effects of the R286W, G793R, Y746D, Y99C, E313K and P470L missense mutations were predicted using PolyPhen-2 software (genetics.bwh.harvard.edu/pph2) (21).

Results

Clinical manifestations of patients

Table I presents detailed clinical parameters, biochemical results and BMDs for the patients. Fig. 2 presents radiographs from patients with ADO-II and IARO. The patients with ADO-II were diagnosed at 5–47 years old; five were male (II1 in family 1, I1 and II5 in family 3, and I1 and II1 in family 6) and four were female (III1 in family 1, II1 in family 4, and II5 and II7 in family 5). Patients with ADO-II: One patient exhibited a notably below-average height (II7 in family 5); one patient exhibited pigeon chest (II1 in family 6); three patients experienced fractures of the rib, femur, tibia and radius (II5 in family 3, II1 in family 4, II1 in family 6); one patient exhibited dental abnormalities (II5 in family 3); three patients exhibited anemia (III1 in family 1, II1 in family 4, II7 in family 5); and one patient, the proband of family 4, was diagnosed with thrombocytopenia and splenomegaly. Additionally, the proband of family 4 exhibited a visual impairment from birth.

Figure 2. Radiology results for the patients (X-rays of skull of A, pelvis, tibia and fibula of C are taken from the proband of family 6. X-rays of vertebrae of A and C are taken from the proband of family 5. X-rays of B and D are taken from the proband of family 2). (A and C) X-rays from a patient with ADO-II. Note the generalized increase in the bone density of the skull, vertebrae, pelvis, tibia and fibula. Radiology revealed vertebral endplate thickening (the black arrow in A indicates a sandwich vertebrae sign) and typical iliac wings (the black arrow of C indicates bone-in-bone appearance). (B and D) X-rays from a patient with IARO. Note the diffuse sclerosis of the skull, vertebrae, pelvis and femur. Radiology revealed tooth destruction (white arrow in B), hip deformity (black arrow in D) and the Erlenmeyer flask deformity (white arrow in D) of the distal femur. ADO-II, autosomal dominant osteopetrosis type II; IARO, intermediate autosomal recessive osteopetrosis.

Table I. Clinical characteristics, biochemical results and BMD values of patients with osteopetrosis and family members.

Table I.

Clinical characteristics, biochemical results and BMD values of patients with osteopetrosis and family members.

Family	Member	Sex	Age (years)	Height (cm)	Weight (kg)	LDH (U/l)	CK (U/l)	ALP (U/l)	Hb (g/l)	PLT (109/l)	L1-4 (Z score)	FN (Z score)	TH (Z score)	Dental problem	Bone fracture	Other clinical characteristics
1	Proband	F	15	153.3	61	248	179	79	124	255	7.7	4.8	6.6	No	No	No
	Father	M	42	174.7	92	NA	NA	NA	NA	NA	0.5	0.2	0.7	No	No	No
2	Proband	M	24	153.0	45	205	139	150	86	49	14.1	14.4	14.6	Yes	Yes	Hepatosplenomegaly, gallstone, secondary hyperparathyroidism
3	Proband	M	28	170.5	68	190	156	54	149	217	11.1	11.1	9.1	Yes	Yes	No
4	Proband	F	32	153.0	67	256	86	100	71	83	11.5	NA	NA	No	Yes	Splenomegaly, visual impairment, tinnitus
5	Proband	F	42	148.5	48	NA	NA	46	108	249	9.6	6.0	6.0	No	No	Secondary hyperparathyroidism
6	Proband	M	10	143.7	41	210	207	170	146	273	6.8	5.4	6.0	No	Yes	Pigeon chest
	Father	M	41	160.5	69	NA	NA	80	165	211	0.3	−0.3	−0.3	No	No	No
7	Proband	F	37	148.2	49	274	NA	67	62	350	16.9	17.6	14.1	Yes	Yes	Visual and audile impairment, pigeon chest, cyst, arthralgia, ankylosis, deformity
	Father	M	63	170.0	65	NA	NA	NA	NA	NA	0.4	0.5	−0.3	No	No	No
	Mother	F	63	160.0	56	NA	NA	77	126	225	−0.8	−1.1	−1.3	No	No	No

[i] BMDs in probands in family 1 and 7 were measured using a Hologic DXA densitometer, and the Z scores for L1-4, FN and TH of this proband were calculated by comparison with Caucasian women. The BMDs of the other patients were measured using a Lunar DXA densitometer, and the Z scores for L1-4, FN and TH were calculated by comparison with the age-specific BMD reference values for Chinese children and adolescents. ALP, alkaline phosphate (reference range: 15–112 U/l, 75–344 U/l for children <10 years); BMD, bone mineral density; CK, creatine kinase (reference range: 21–190 U/l); DXA, dual-energy X-ray absorptiometry; FN, femoral neck BMD; Hb, hemoglobin (reference range: 130–175 g/l); L1-4, lumbar spine BMD; LDH, lactate dehydrogenase (reference range: 114–240 U/l); NA, not analyzed; PLT, platelets (reference range: 125–350×10⁹/l); TH, total hip BMD.

In family 1, a 15-year-old female (proband, III1), the only daughter of nonconsanguineous parents, was born at term with a normal height and weight. The height and weight of the proband at the time of assessment were 153.3 cm and 61.0 kg, respectively. At ~1 month prior to evaluation, the proband complained of back pain. X-rays revealed an increased bone density in vertebrae, with the sandwich vertebrae sign. Laboratory data revealed an elevation in LDH levels and a minor reduction in hemoglobin (Hb) levels. Subsequently, her parents visited our department, and presented with normal BMDs.

The proband (II2) of family 2, a 24-year-old male, was the second child of nonconsanguineous parents. The proband exhibited growth retardation. The weight and height of the patient were 45 kg and 153 cm, respectively. He complained of discomfort in the abdomen and were diagnosed with osteopetrosis with anemia and hepatosplenomegaly at 3 months. He only had 20 teeth, and exhibited yellowish skin and a swollen abdomen. Additionally, he experienced a cementoma 6 years previously; there was no deterioration. Fractures occurred in the neck of the femur when he was 4 years old and in the distal radius when he was 16 years old due to a non-violent etiology. He experienced anemia with thrombocytopenia for the majority of his life, and received a red cell suspension >3 times due to severe anemia. X-rays revealed diffuse sclerosis (Fig. 2). Laboratory data revealed increased ALP levels, and reduced Hb and PLT levels. The proband's parents exhibited normal clinical features as assessed by accessory examinations, including radiographs and BMD. The proband's older brother succumbed at the age of 3 years and was also diagnosed with osteopetrosis.

In family 3, the proband (II5), a 28-year-old male, was the youngest child of nonconsanguineous parents. The proband came to our department as a result of abnormal skeletal radiological manifestations. The weight and height of the proband were 68.0 kg and 170.5 cm, respectively. He complained of shoulder pain 10 days prior to the visit. A rib fracture with a non-violent etiology occurred during childhood. Skeletal radiography revealed an increase in bone density in the vertebrae with the sandwich vertebrae sign. Laboratory data were normal. Of the seven members of this family, including the proband's parents and other relatives, no abnormal clinical manifestations were observed, and they refused to undergo radiography or BMD assessments.

In family 4, the proband (II1), a 32-year-old female, was the first child of nonconsanguineous parents. She was born at term with normal delivery. The height and weight of the proband at the time of the examination were 153.0 cm and 67.0 kg, respectively. She was blind from birth. Fractures with a non-violent etiology occurred in the left femur twice when she was 1 and 20 years old, and in the right femur when she was 10 years old. Tinnitus and splenomegaly had developed in recent years. X-rays revealed an increase in the bone density of vertebrae with the sandwich sign and the pelvis with the bone-in-bone sign. Laboratory data revealed elevated LDH levels, and reduced Hb and PLT levels. Of the six members of the proband's family, including her parents and other relatives, no abnormal clinical manifestations or BMD were observed.

In family 5, the proband (II7), a 42-year-old female, was the sixth child of nonconsanguineous parents. She came to our department as a result of abnormal skeletal radiological manifestations. Her weight and height were 48 kg and 148.5 cm, respectively. X-rays revealed a diffuse increase in bone density with evidence of the sandwich sign in vertebrae and bone-in-bone sign in the pelvis. Laboratory data revealed reduced Hb levels. The same signs were observed in X-rays from the proband's twin sister. The clinical manifestations, laboratory data, radiographs and BMDs of the proband's sons were normal.

In family 6, the proband (II1), a 10-year-old male, the only child of nonconsanguineous parents, was born at term with a normal length and weight. The proband's height and weight at the time of examination were 143.7 cm and 41.0 kg, respectively. He had suffered from recurrent influenza since childhood. Fractures with a non-violent etiology occurred in the left tibia when he was 3 years old and in the radius when he was 10. Pigeon chest was observed. Laboratory data revealed elevated CK levels. Their radiograph exhibited a typical appearance. The clinical manifestations, laboratory data, radiographs and BMDs of the proband's parents were normal.

The proband (II1) of family 7, a 37-year-old female, the first child of nonconsanguineous parents, experienced growth retardation. Her height and weight at the time of examination were 148.2 cm and 49.0 kg, respectively. She exhibited cysts and recurrent infections of the right knee joint during the previous 10 years. She suffered arthralgia, ankylosis and deformity of the right knee with claudication. Amblyopia of the left eye and amblyacousia of the left ear had occurred since childhood. She also experienced dental problems, including misalignment, loose teeth, tooth loss and dental cavities. A fracture of the right radius with a non-violent etiology occurred when she was 17 years old. She fainted numerous times as a result of anemia. X-rays revealed a diffuse increase in bone density. Laboratory data revealed increased LDH levels and a reduction in Hb levels. The three members of the proband's family, including her parents and sister, did not display abnormal clinical manifestations, laboratory data, radiographs or BMDs.

Genetic analysis

A total of one homozygous and four heterozygous missense mutations, one splice mutation and one compound heterozygous mutation in the CLCN7 gene were identified in the probands (Table II; Fig. 3). The heterozygous mutation (Y746D), the splice mutation (c.2232-2A>G), the homozygous mutation (G793R) and the compound heterozygous mutation (P470L and K217X) were novel. Additionally, six missense mutations, c.856C>T (R286W), c.2236T>G (Y746D), c.296A>G (Y99C), c.937G>A (E313K), c.2377G>C (G793R) and c.1409C>T (P470L), occurred at a highly conserved position, according to a comparison of the protein sequences from eight vertebrates. The missense mutations in the CLCN7 gene (R286W, G793R, Y746D, Y99C, E313K and P470L) were predicted by PolyPhen-2 to exhibit pathogenic effects (Table II). Based on the genetic analysis, the probands in families 1, 3, 4, 5 and 6 with heterozygous mutations were diagnosed with ADO-II, whereas the proband in family 2 with the homozygous mutation and the proband in family 7 with the compound heterozygous mutation were diagnosed with IARO.

Figure 3. Analyses of mutations in the chloride channel 7 gene in patients with osteopetrosis. (A) c.856C>T mutation in exon 9. (B) c.2377G>C mutation in exon 24. (C) c.2232-2A>G mutation in intron 22. (D) c.937G>A mutation in exon 10. (E) c.2236T>G mutation in exon 22. (F) c.296A>G mutation in exon 3. (G) c.1409C>T mutation in exon 16. (H) c.647_648 dupTG mutation in exon 7.

Table II. Summary of the findings of the molecular analysis of patients with osteopetrosis.

Table II.

Summary of the findings of the molecular analysis of patients with osteopetrosis.

Family no.	Patient/sex	Mutation site	Mutation type	Protein level	DNA level	PolyPhen-2 score	Status
1	III1/F	EXON9	Missense	p.Arg286Trp	c.856C>T	1.000	Heterozygous
	II1/M	EXON9	Missense	p.Arg286Trp	c.856C>T	1.000	Heterozygous
2	II2/M	EXON24	Missense	p.Gly793Arg	c.2377G>C	0.751	Homozygous
	I2/F	EXON24	Missense	p.Gly793Arg	c.2377G>C	0.751	Heterozygous
3	II5/M	INTRON22	Putative aberrant splicing	–	c.2232-2A>G	–	Heterozygous
	I1/M	INTRON22	Putative aberrant splicing	–	c.2232-2A>G	–	Heterozygous
4	II1/F	EXON10	Missense	p.Glu313Lys	c.937G>A	1.000	Heterozygous
5	II7/F	EXON22	Missense	p.Tyr746Asp	c.2236T>G	1.000	Heterozygous
6	II1/M	EXON3	Missense	p.Tyr99Cys	c.296A>G	1.000	Heterozygous
	I1/M	EXON3	Missense	p.Tyr99Cys	c.296A>G	1.000	Heterozygous
7	II1/F	EXON16	Missense	p.Pro470Leu	c.1409C>T	1.000	Compound heterozygous
		EXON7	Insertion	p.Lys217X	c.647_648dupTG	–
	I1/M	EXON16	Missense	p.Pro470Leu	c.1409C>T	1.000	Heterozygous
	I1/F	EXON7	Insertion	p.Lys217X	c.647_648dupTG	–	Heterozygous
	II3/F	EXON7	Insertion	p.Lys217X	c.647_648dupTG	–	Heterozygous

In family 1, a known heterozygous missense mutation, c.856C>T, in exon 9 of the CLCN7 gene was identified in the proband (III1) and her father (II1), resulting in an arginine-to-tryptophan substitution at p.286. In family 2, a novel homozygous missense mutation, c.2377G>C, in exon 24 of the CLCN7 gene was identified in the proband (II2), and a heterozygous mutation was detected in his mother (I2), resulting in a glycine-to-arginine substitution at p.793. In family 3, a novel heterozygous splice mutation, c.2232-2A>G, leading to truncated protein in intron 22 was identified in the proband (II5) and his father (I1). In family 4, a known heterozygous missense mutation, c.937G>A, in exon 10 of the CLCN7 gene was identified in the proband (II1), resulting in a glutamate-to-lysine substitution at p.313. In family 5, the novel heterozygous missense mutation c.2236T>G in exon 22 was identified in the proband (II7) and her twin sister (II5), resulting in a tyrosine-to-aspartate substitution at p.746. In family 6, a known heterozygous missense mutation, c.296A>G, in exon 3 of the CLCN7 gene was identified in the proband (II1) and his father (I1), resulting in a tyrosine-to-cysteine substitution at p.99. In family 7, a novel insertion mutation (c.647_648 dupTG) in exon 7 was identified in the proband (II1), and her mother (I2) and sister (II3), resulting in a lysine-to-stop codon substitution at p.217 that produced a truncated protein. Additionally, a missense c.1409C>T mutation in exon 16 of the CLCN7 gene was identified in the proband (II1) and her father (I1), resulting in a proline-to-leucine substitution at p.470. The eight mutations were not detected in the 250 healthy controls.

Discussion

In the present study, direct sequencing of the CLCN7 gene in the seven families revealed four heterozygous missense mutations (R286W, Y746D, Y99C and E313K), one homozygous missense mutations (G793R), one splice mutation (c.2232-2A>G) and one compound heterozygous mutation (P470L, K217X) in the probands. The heterozygous missense mutations (R286W, Y99C and E313K) and the compound heterozygous mutation (P470L) have been reported previously (10,15,18,19). The mutations (R286W, Y746D, Y99C, G793R, E313K and P470L) all occurred at highly conserved positions among eight vertebrate species and were predicted to exert a pathogenic effect by PolyPhen-2. The CLCN7 gene is expressed at high levels in the osteoclast ruffled membrane; ClC-7 provides the chloride conductance required for efficient proton pumping (1,4–5). CLC chloride channels are homodimers composed of two subunits, each containing 18 intramembranous α-helices, four highly conserved Cl− binding sites and two cystathionine β synthase (CBS) domains (CBS1 and CBS2) located near the carboxyl terminus of the topological domain (19). Cleiren et al (5) first identified mutations in the CLCN7 gene as causing ADO-II. The mutations (R286W, Y99C, E313K and P470L) in the CLCN7 gene have been hypothesized to be located in the intramembrane α-helices, creating a positive electrical potential to prevent the fast flux of Cl− at the binding site (19). The Y746D, G793R and c.2232-2A>G mutations were hypothesized to be located in CBS2. Y746D and G793R are hypothesized to disturb protein sorting and interfere with the localization of the protein in the ruffled-border formation, which is required for normal lysosomal function and bone resorption (22,23). The novel splicing mutation (c.2232-2A>G) in the CLCN7 gene occurred at the splice acceptor site, potentially interfering with the splicing of exon 23 with exon 24 and thereby truncating the functional domains. The novel insertion mutation (c.647_648 dupTG) in exon 7 resulted in a lysine-to-stop codon substitution at p.217, leading to the synthesis of a truncated protein. The novel CLCN7 mutations identified in the present study may increase understanding of the spectrum of CLCN7 mutations in this disorder.

Osteopetrosis is a rare inherited metabolic disease. ADO-II was first described by Albers-Schönberg in 1904 (5). The onset of ADO-II may occur in adulthood or childhood (16,23). Patients with ADO-II generally experience symptoms including osteosclerosis, fractures, bone pain, osteomyelitis, osteoarthritis, rickets, deafness and blindness (19). CLCN7-dependent IARO exhibits an autosomal recessive inheritance profile and is milder than CLCN7-associated ARO (1). A few cases of IARO have been previously reported (16–17,19). The onset usually occurs in the first year of life; patients frequently reach adulthood, compared with the <3-year life expectancy of patients with ARO (24).

In the present study, five families with ADO-II and two families with IARO were reported. The proband of family 4 (II1) diverged from other patients with ADO-II due to her impaired vision, increased tinnitus, bone marrow failure and splenomegaly secondary to extramedullary hematopoiesis. Waguespack et al (25) reported the rate of severe visual loss in subjects with ADO, with an overall prevalence of 19%, and a rare rate (~2%) of bone marrow failure. The proband suffered from vision loss from birth, consistent with previous reports (19,25). The mechanism by which visual loss occurs remains unclear.

In family 2, the proband (II2), a 24-year-old male from nonconsanguineous parents, was diagnosed with IARO caused by a homozygous mutation in the CLCN7 gene, and presented with early-onset bone marrow failure (anemia and thrombocytopenia) and splenomegaly. The proband inherited one mutation from his mother; the peripheral blood DNA of his father was normal. Therefore, two possibilities existed to explain the presence of the other mutation: A germ cell mutation or germ cell mosaicism in the gonads of the proband's father, or a mutation in the fertilized egg itself. The proband's older brother (II1), who was diagnosed with osteopetrosis, succumbed at 3 years of age due to bone marrow failure and massive hepatosplenomegaly; the genotype of the brother is not known.

In family 7, the proband (II1), a 37-year-old female from nonconsanguineous parents, was diagnosed with IARO caused by a compound heterozygous mutation in the CLCN7 gene, and presented with early-onset hearing impairments, unilateral vision impairments and pigeon chest. Notably, the P470L mutation was previously reported by Xue et al (17) to cause IARO when homozygous; however, the heterozygous mutation P470L, inherited from the patient's father, was not predicted to cause disease. Conversely, the inheritance of an insertion mutation (resulting in a lysine-to-stop codon substitution at p.217 and truncated protein) from the patient's mother resulted in another type of IARO due to the lack of compensatory ClC-7 protein. The patient manifested notably more severe symptoms than the patients with ADO-II.

In the present study, seven symptomatic patients (including family members) with ADO-II presented classical clinical manifestations and radiographic findings. The father (II1) in family 1 and the father (I1) in family 6 were unaffected gene carriers with normal biochemical measurements, radiographs and BMDs. The penetrance of ADO-II was ~66%, with a highly variable phenotype. The association between genotype and phenotype was difficult to determine in the present study. Modifier genes, DNA methylation or intrinsic osteoclast factors may serve roles in the manifestation of this disease. Further studies should be performed to determine the mechanism of incomplete penetrance.

There were a number of limitations in our study. The relatively small sample size, including only two cases of IARO, were insufficient to characterize all the properties of CLCN7-dependent osteopetrosis or determine phenotype-genotype associations. In addition, investigations into the mechanisms underlying the effects of these novel mutations were not determined, and further in-depth research is required.

In conclusion, various mutations (R286W, Y746D, Y99C, G793R, E313K, c.2232-2A>G, P470L and K217X) in the CLCN7 gene were identified in six patients (the probands in family 1, 2, 3, 5, 6, 7) with familial osteopetrosis and one patient (the proband in family 4) with sporadic osteopetrosis. Furthermore, the phenotypes of patients with ADO-II and IARO were characterized. The findings from the present study increase the spectrum of reported CLCN7 gene mutations and improve the present understanding of osteopetrosis. Additionally, it may aid further studies aiming to dissect the heterogeneity of ADO-II and IARO.

Acknowledgements

Not applicable.

Funding

The present study was supported by grants from the National Basic Research Program of China (grant no. 2014CB942903), the National Natural Science Foundation of China (grant nos. 81570794 and 81770871 to Z.-L.Z.) and the Shanghai Municipal Commission of Health and Family Planning (grant no. 20164Y0062).

Availability of data and materials

All data generated or analyzed during the present study are included in this published article.

Authors' contributions

ZZ and HY designed and conceived the present study and analyzed the genetic test results. LL and SL analyzed the clinical information, analyzed the genetic test results and drafted the manuscript. CW collected and analyzed data. All authors read and approved the final manuscript.

Ethics approval and consent to participate

The present study was approved by the Ethics Committee of the Shanghai Jiao Tong University Affiliated Sixth People's Hospital. All participants provided written informed consent.

Patient consent for publication

All patients within this study provided consent for the publication of their data.

Competing interests

The authors declare that they have no competing interests.

References