A novel missense mutation in the CLCN7 gene linked to benign autosomal dominant osteopetrosis: a case series

Osteopetrosis (OP) is a clinically and genetically heterogeneous group of diseases characterized by a symmetrical increase in bone density[1, 2]. The sclerosis of bone is due to the absence of or to a defect in osteoclast bone resorption. The overall incidence of these conditions, dependent on the disease forms, varies from 1:20,000 to 1:250,000[1‐3].

The severity of the presentation of OP in infancy varies from asymptomatic to fatal and can be grouped into autosomal recessive (ARO) and autosomal dominant (ADO)[1‐3]. The severest forms tend to have autosomal recessive inheritance, whereas the mildest forms are inherited in an autosomal dominant manner[3, 4]. The malignant and intermediate form of ARO is a life-threatening condition and the disease is manifested in infancy and early childhood[1, 3]. The benign ADO typically has an onset in late childhood or adolescence[1, 3] and can be subdivided into three subclasses: benign type I (ADO-I), benign type II (ADO-II), and benign type III (ADO-III)[1, 5, 6]. ADO-I is generally very mild with a diffuse sclerosis without alterations in the bone turnover. Genetic mutations of Low-density lipoprotein receptor-related protein 5 gene were identified to be responsible for ADO-I[7, 8], thus causing an increased bone formation ‘high bone density’ rather than a decreased bone resorption[7, 8]. This phenotype is not associated with an increased fracture rate and is reported to be fully penetrant[9], whereas ADO-II has an extremely heterogeneous course ranging from an asymptomatic to a severe phenotype. ADO-II is the most common form; it is characterized by thickness of the vertebral end plates (sandwich vertebrae or rugger jersey spine) and the bone within the bone, and is most commonly noted in the pelvis, vertebrae and at the ends of long bones. ADO-II is associated with diffuse pain, hematological and neural failure, osteomyelitis and frequent pathological fractures. Early death in patients with ADO-II is rare, but some patients can experience a very poor quality of life[10]. In 1904, Albers-Schönberg was the first to describe a case of OP[11], which is an identical OP phenotype to ADO-II symptoms. ADO-III or centrifugal OP has been described in a Vietnamese family; this type is predominantly characterized by sclerosis of the distal appendicular skeleton and the skull, but with minor involvement of the axial skeleton[12].

The families of Chloride channel (CLC) proteins have received considerable attention, often with surprising developments, over the past five years[13], and their physiological roles are impressively illustrated by various inherited human diseases and knockout mouse models[14]. The Chloride channel 7 (CLCN7) gene is a member of the mammalian CLC gene family[14]. In osteoclasts, the Chloride channel 7 (CLC7) protein resides in the late endocytotic–lysosomal pathway of the ruffled membrane borders and is involved in the acidification of the resorption lacunae. The physiological function of the CLC7 protein was unclear until Kornak showed that the disruption of the CLCN7 gene in mice causes severe OP, and that the CLC7 protein played an essential role in the acidification of the extracellular resorption lacunae, which is very important for osteoclast-mediated resorption of mineralized bone[15]. In humans, mutations in the CLCN7 gene give rise to the complete spectrum of OP phenotypes, ranging from malignant and intermediate forms of ARO to ADO-II and even asymptomatic ADO-II cases[16‐21]. Indeed, the complete clinical and molecular analysis of several patients found novel mutations, including missense, frameshift, nonsense, deletion and splice-site defects[20‐22].

The first successful bone marrow (BM) transplantation for OP patients was in 1977[23, 24]. At that time, the biological origin of osteoclasts and osteoblasts was unknown. The OP patients were successfully treated by bone BM transplantation, from the availability of compatible donors of osteoclasts and a mixture of hematopoietic lineages. This was later confirmed experimentally[25]. BM transplantation is a drastic treatment and it is applied only to children severely affected by OP. If it is successful, it will save the life of a child who would otherwise die from the disease progression, which results in growth retardation and neurological defects. Development of hematopoietic stem cell transplantation (HSCT) is a promising treatment for this disease due to the elimination of rejection risks[26]. In the future, an alternative approach that holds the potential for the permanent cure of OP is in utero prenatal HSTC (for review see[27]).

The scleroses of bone in patients are often asymptomatic at an early stage and the precise diagnosis is clinical and largely depends on the radiographic appearance of the skeleton. The radiographic appearance exhibits a diffused sclerosis in skull, spine, pelvis and appendicular bones, and a funnel-like appearance and characteristic lucent bands at the metaphysis of the long bones that result from defects of bone remodeling[3]. Other features are also observed, such as bone-in-bone appearance, and focal sclerosis of the skull base, pelvis and vertebral end plates[3]. In other cases the diagnosis may be reached by chance[28]. In the absence of typical radiographic findings, raised creatine kinase BB isoenzyme and tartrate-resistant acid phosphatase (TRAP) can be helpful in making a diagnosis of ADO[29‐31]. However, by systematic investigations, in our case, we have diagnosed the patients’ manifestations as related to ADO-II. Our diagnosis was based on clinical symptoms, radiographic appearance and laboratory analysis. The laboratory data (Table1) showed normal range of serum sugar, urea, creatinine, phosphorus, calcium, bilirubin, alanine transaminase, aspartate transaminase, lactate dehydrogenase and parathyroid hormone for all the family members, except a slight elevation in serum alkaline phosphatase for the brother (Case 2) who has milder symptoms (Table1). However, we did not analyze creatine kinase BB isoenzyme and TRAP because of the absence of these assays in our locality. The index patient (Case 1) showed lower values for Hb and WBC, and her platelet count was diminished by a factor of 10 (Table1). Therefore, the index patient (Case 1) showed anemia and thrombocytopenia, whereas the blood examination was normal for other family members (Table1).

Next, we performed genetic analysis for the whole family, including an aunt and an uncle, in order to link the disease-type with a possible gene(s) mutation and to find out the origin of the inherited condition. We wanted to address possible risks for the development of the disease in children born within the family in the future because the parents are cousins, and because there is the possibility of other consanguineous marriages involving the aunt and the uncle. With these notations, we decided to help the family to perform genetic counseling in this particular case.

To this end, we performed systematic sequencing of the genes most commonly associated with OP: TCIRG1, CLCN7, OSTM1, RANK and RANKL. The six members of the family, including the parents of the minors, gave written informed consent to participate in this analysis. From the genomic DNA, the coding regions of the above five genes were amplified using the polymerase chain reaction (PCR) method. Complete DNA sequencing on each PCR product was performed and a missense mutation was found at exon 15 of the CLCN7 gene (Figure2). In the index patient, sequencing of the other associated genes revealed coding sequences for wild-type protein. Therefore, other family members were exclusively screened for mutation at exon 15 of the CLCN7 gene (Figure2).

When the whole coding regions of the CLCN7 gene were analyzed for the index patient (daughter), a unique heterozygous missense mutation (C GG>T GG) in exon 15 (c.1225C>T) was detected (Figure2), which changes the amino acid position 409 from arginine to tryptophan of the CLC7 protein (p.R409W). Although the mother and the aunt displayed a wild-type (p.Arg409) amino acid for the CLC7 protein, all the other family members had the heterozygous mutation p.Arg409Trp (Figure2 and Table3). The missense mutation (p.Arg409Trp) found in this work is unique and has not yet been described in the published literature. It has previously been found that the heterozygous mutation in the CLCN7 gene is linked to benign ADO (ADO-II) symptoms[20, 21], because mild symptoms appear in early childhood and worsen over time with life-threatening complications. These features have been historically experienced within the family: one child died at age 10 years, another child (index patient) has ADO-II progression at the age of 12 years, whereas their 16-year-old brother had no obvious symptoms at early childhood but clinical features have now started to appear in relation to ADO-II. Our genetic findings of the mutation CLCN7 gene confirm that the brother is also at risk of developing ADO-II complications.

Molecular and clinical findings have shown that various mutations in the CLCN7 gene lead to the development of various types of OP, from asymptomatic to severe forms[15, 17, 20, 21, 32]. Mice with a disruption of the corresponding gene CLCN7 showed severe OP, retinal degeneration, and they only live for between five and seven weeks[33]. In humans it was also noted that homozygous mutation in the CLCN7 gene causes malignant infantile ARO[15], whereas patients heterozygous for dominant-negative mutations have a less severe form of ADO-II[32]. It is known that ADO-II symptoms are not homogenous and have wide clinical manifestations from asymptomatic to severe[31, 34]. Large scale genotype–phenotype correlation analysis of CLCN7-dependent ARO cases and ADO-II cases probably does not support the haploinsufficiency mechanism, rather the hypothesis has to be validated experimentally with biochemical assays[20]. This suggests to us that either the father or the uncle might develop ADO-II symptoms in late adulthood, or they might live a normal life without any obvious ADO-II phenotype. The genetic findings in this work allows us to perform pedigree analysis, indicating the origin of inheritance to be common for the father and uncle (Figure3).

In this study, we report a case of a Kurdish family affected by an OP phenotype with broad severity, and diagnosed using complete clinical management, laboratory analysis, and treatment in our locality. We have identified a novel missense mutation (p.R409W) in exon 15 of the CLCN7 gene as responsible for ADO-II in the 12-year-old index patient and her 16-year old brother; their father is a seemingly healthy carrier of the mutation. Our results show that the uncle (father’s brother) is also a carrier indicating that another possible consanguineous marriage between the aunt (mother’s sister) and the uncle is not recommended. We believe that it is very probable that their progeny would develop ADO-II because they will carry an inherited mutated allele of the CLCN7 gene. Although unidentified genetic variants might exist, a marriage between the uncle-carrier and an unrelated woman could also develop risks similar to those of his marriage to the non-carrier aunt. However, the complete genotype–phenotype correlation remains obscure, even though one allele of the CLCN7 gene was found to be mutated. Discovery of a specific biochemical assay marker is required to quantify the actual CLC activity of the CLC7 protein that might support diagnosis more precisely to correlate the CLC function to the disease forms.

Written informed consent was obtained from the parents to report the case together with any accompanying images. A scanned-original copy of the written consent is available for review by the Editor-in-Chief of this journal.