In the last decade, more than 310 osteopetrotic patients from all over the world have been screened by our groups for genes responsible for human ARO. This allowed us to select 60 patients who did not have an identified genotype and could be investigated for mutations in the SNX10 gene.
The molecular analysis led to the identification of biallelic mutations in the SNX10 gene in 14 patients from 12 unrelated families, for a total of 9 novel mutations: 3 missense, 3 nonsense, and 3 splicing defects (Table 1).
Table 1. Molecular Findings in 14 New SNX10-Dependent Patients
|Patient#||Genomic changea||cDNA changeb||Predicted effect|
|1||g.72742G > T||c.212 + 1G > T||r.spl?c|
| ||g.72742G > T||c.212 + 1G > T||r.spl?c|
|2||g.72742G > T||c.212 + 1G > T||r.spl?c|
| ||g.72742G > T||c.212 + 1G > T||r.spl?c|
|3||g.72742G > T||c.212 + 1G > T||r.spl?c|
| ||g.72742G > T||c.212 + 1G > T||r.spl?c|
|4||g.69151A > C||c.95A > C||p.Tyr32Ser|
| ||g.69151A > C||c.95A > C||p.Tyr32Ser|
|5A-5B||g.69151A > C||c.95A > C||p.Tyr32Ser|
| ||g.69151A > C||c.95A > C||p.Tyr32Ser|
|6||g.72681G > C||c.152G > C||p.Arg51Prod|
| ||g.72681G > C||c.152G > C||p.Arg51Prod|
|7A-7B||g.72681G > C||c.152G > C||p.Arg51Prod|
| ||g.72681G > C||c.152G > C||p.Arg51Prod|
|8||g.69102C > T||c.46C > T||p.Arg16X|
| ||g.69102C > T||c.46C > T||p.Arg16X|
|9||g.69103G > T||c.47G > T||p.Arg16Leu|
| ||g.72713C > T||c.184C > T||p.Gln62X|
|10||g.73252G > T||c.311 + 1G > T||r.spl?c|
| ||g.73252G > T||c.311 + 1G > T||r.spl?c|
|11||g.69143C > A||c.87C > A||p.Tyr29X|
| ||g.69143C > A||c.87C > A||p.Tyr29X|
|12||g.69746G > C||c.111 + 5G > C||r.(spl?)e|
| ||g.69746G > C||c.111 + 5G > C||r.(spl?)e|
Three patients (patients 1, 2, and 3) were all found to be homozygous for the same nucleotide change at the donor splice site of exon 4 (c.212 + 1G > T).
Patient 4 was homozygous for a c.95A > C mutation predicted to cause a p.Tyr32Ser amino acid change, and her parents were shown to be heterozygous for the same nucleotide change. We found the same mutation in the homozygous state in 2 affected siblings (patients 5A and 5B) from an unrelated family; both their parents and their healthy sister bore the mutation in the heterozygous state.
Patient 6 was homozygous for a transversion (c.152G > C) predicted to lead to an amino acid substitution (p.Arg51Pro); both his consanguineous parents were heterozygous for this nucleotide change. The same mutation was found in the homozygous state in 2 affected siblings (patients 7A and 7B) from an unrelated family. Their consanguineous parents were heterozygous carriers while their unaffected sibling showed the wild-type codon.
Patient 8 was homozygous for a transition (c.46C > T) predicted to cause a nonsense mutation at codon 16 (p.Arg16X). Her parents' DNA was not available for analysis.
Patient 9 was compound heterozygous for 2 nucleotide changes, c.47G > T and c.184C > T, predicted to cause an amino acid substitution at codon 16 (p.Arg16Leu) and a stop at codon 62 (p.Gln62X), respectively. His mother was heterozygous for the first mutation and his father for the second mutation.
Patient 10 was homozygous for a nucleotide change at the donor splice-site of exon 5 (c.311 + 1G > T). The same mutation was found in the heterozygous state in his consanguineous parents and in 4 out of his 5 healthy siblings.
Patient 11 was homozygous for a transversion (c.87C > A) predicted to cause a stop at codon 29 (p.Tyr29X).
Patient 12 was homozygous for a nucleotide change close to the donor splice-site of exon 3 (c.111 + 5G > C). The same mutation was found in the heterozygous state in his consanguineous parents.
The missense substitutions (p.Arg16Leu, p.Tyr32Ser, and p.Arg51Pro) were not found in more than 100 chromosomes from healthy unrelated individuals from the same geographical areas, and were not present in dbSNP135; therefore, they are unlikely to be neutral polymorphisms. Alignment of SNX10 protein sequences from several species further supports this idea, because the mutated residues are strongly conserved in evolution (Supplementary Fig. 1).
In silico studies
All the mutations identified fall in the Phox-homology (PX) domain of SNX10, which is the only functional domain.12 Modeling of the PX domain of SNX10 protein showed that this region is folded into a globular shape, composed of 3 exposed beta-strands followed by 3 alpha-helices that well overlie the alpha-helices and beta-strands of the chosen template Grd19p. The model also showed the electropositive basic pocket that is responsible for binding to the negatively charged phosphate groups (Fig. 1A).
Figure 1. Mutations in relation to the SNX10 protein structure. (A) Three-dimensional model of PX domain of human SNX10 protein (in blue), determined by homology modeling using the protein structure of yeast Grd19p (1OCU, in gray) as a template. The three residues affected by missense mutations in our patients are depicted in magenta; putative functional residues are depicted in green. (B) Alignment of PX domains of human SNX10 (Q9Y5X0-1), Grd19p (PDB ID: 1OCU), and human SNX3 (O60493-1). Secondary structure alignment was obtained from the structure of Grd19p.11 Numbers on the left hand side of each sequence denote the full-length sequence position. Amino acid colors as in A.
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SNX10 mutations occurred at residues not involved in phosphatidylinositol binding and located in the three beta-strands at the opposite side of the binding pocket, so they could not directly affect the binding activity.
Mutation of the exposed positively charged residue Arg16 into Leucine might disrupt contacts to other proteins and therefore affect SNX10 function.
Tyr32 occupies a buried position. Mutation p.Tyr32Ser might locally destabilize the internal packing and affect the secondary structure of the neighboring beta-strand; this could indirectly impair binding to other proteins. Alternatively, it could affect phosphatidylinosytol-3-phosphate binding by altering the side chain conformation of the conserved Arg94 that is known to directly bind the phosphate group.13
Arg51 makes contact to the neighboring beta-strand and might be important for local main chain conformation. Mutation p.Arg51Pro could either distort the main chain conformation and/or affect binding to another protein.
Grd19p is the yeast homologue of human SNX3, which is another member of the PX-only family. Alignment of the PX domain of human SNX10 protein, Grd19p and human SNX3 showed that Tyr32 and Arg51 are conserved, whereas Arg16 was not (Fig. 1B). However, multispecies alignment of SNX10 proteins highlighted that this residue is maintained, suggesting a possible role specific to SNX10 and not shared by other sorting nexins (Supplementary Fig. 1).
Clinical evaluation of patients
We carefully reviewed the clinical history of these patients in order to identify the specific features of SNX10-dependent ARO (Table 2).
Table 2. Clinical Features at Diagnosis and HSCT Outcome in SNX10-Dependent ARO
|Patient#||Age at onset||Bone fractures||Neurological defects||Hematological defects||Other features||Age at HSCT||HSC origin; donor||Outcome and follow-up|
|1||3 months||No||Hydrocephalus, nystagmus, impaired vision||Anemia (Hb 10.2 g/dL)||—||33 months||Bone marrow; MUD||Bone rescue; alive and well at 11 years; vision partially maintained|
|2||2.5 months||No||Strabismus||Anemia (Hb 7.9 g/dL)||Hypertelorism, frontal bossing, upper airway obstruction||4 months||Bone marrow; MUD||Dead shortly after HSCT due to VOD and pulmonary hypertension|
|3||Early infancy||No||Blindness, hydrocephalus|| ||Upper airway obstruction||Not done||—||Dead at 10 years due to bacterial infection|
|4||3 months||No||Blindness||Anemia (Hb 8.7 g/dL)||Stridor, airway difficulties||Not done||—||Dead at 20 months|
|5A||4 months||No||Impaired vision||Anemia (Hb 11.3 g/dL after transfusion at 6 months)a||Fever, decreased urine output, failure to thrive||7 months||Bone marrow; MRD||Engrafted; dead at 1 year due to multiple infections and pulmonary hemorrhage|
|5B||At birth||No||Impaired vision||Hepatosplenomegaly||Hypocalcaemia, frontal bossing||6 months||Peripheral blood; partially matched RD||Engrafted; bone improvement; dead at 18 months due to multisystem organ failure|
|6||4 months||No||Impaired vision, deafness||Hepatosplenomegaly anemia (Hb 9.5 g/dL)||—||Not done|| ||Alive at 4 years|
|7A||3 months||No||Mild strabismus||Hepatosplenomegaly, anemia (Hb 6.7 g/dL), thrombocytopenia (86.000/µL)||Frontal bossing, upper airway obstruction||7 months||Bone marrow; MRD||Engrafted; alive and well at 22 years, normal bone, impaired vision|
|7B||3 months||No||Impaired vision, developmental delay||Hepatosplenomegaly, anemia (Hb 9.4 g/dL)||Dolichocephalous, coxa vara, scoliosis||4 months||Bone marrow; MRD||25% chimerism; alive at 18 years, intermediate osteopetrosis, blind|
|8||6 months||No||Impaired vision||Anemia (Hb 8.9 g/dL)||Frontal bossing||Not reported||—||Lost to follow-up|
|9||4 months||No||Hydrocephalus||Anemia (Hb 7.9 g/dL at 6 months)a||—||20 months||Bone marrow; MRD||Alive and reasonably well at 30 months|
|10||1 year||Several||Impaired vision||Anemia (Hb 10.4 g/dL)||—||Not done|| ||Alive at 22 years, wheelchair-bound due to recurrent non-healing fractures|
|11||7 months||No||Impaired vision||Anemia (Hb 8.1 g/dL)||—||1st at 15 months; 2nd at 81 months||1st peripheral blood, 2nd bone marrow; MUD||Alive and well at 20 years|
|12||3.5 months||No||Blindness||Splenomegaly, anemia (Hb 9 g/dL)||Upper airway obstruction||9 months||Peripheral blood; MMRD||Alive and well at 3.5 years|
Patients 1, 2, and 3 were born in unrelated families from the same region in Northern Sweden (Fig. 2A) with no evidence for parental consanguinity. Patient 1 came to attention because of a large skull at 3 months of age raising the suspicion of hydrocephalus. Nystagmus, diminished visual acuity, and late dentition were also present. Radiological investigation performed at 2 years of age showed a generalized increase in bone density (Fig. 2B, C; left panel), thus leading to the diagnosis of osteopetrosis, Västerbottenian type. A moderate anemia (hemoglobin 10.2 g/dL) was also present; therefore, at 33 months of age the patient received an HSCT from a human leukocyte antigen (HLA)-matched unrelated donor, after conditioning according to a protocol recently recommended by the European Group for Bone Marrow Transplantation-European Society for Immunodeficiencies (EBMT-ESID) (www.esid.org/downloads/OPGuidelines-2011). In the post-HSCT period, veno-occlusive disease (VOD) with cytomegalovirus (CMV) reactivation and hypercalcemia occurred and were treated with no further complications. He achieved full engraftment and an almost complete rescue of his sclerotic bone phenotype (Fig. 2D). Two months after transplantation he was back home in reasonably good conditions. Vision was partially maintained; at 11 years he is alive and well (Fig. 2C; right panel), attending school with no specific assistance.
Figure 2. (A) Map of Sweden with the Province of Västerbotten in dark brown. The patients with the “Västerbottenian form” come from a small village north of the Province capital Umeå. (B) X-rays of patient 1 showing a diffuse increase in bone density, signe du masque, and bone-in-bone appearance. (C) Pictures of patient 1. Before HSCT (left panel) frontal bossing is particularly pronounced, whereas after HSCT (right panel) the patient displays a normal facies. (D) X-rays of a lower limb of patient 1 before (left panel) and after HSCT (right panel), showing a clear reduction in bone density 1 year after transplantation.
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Patient 2 was diagnosed at 2.5 months due to hypertelorism, frontal bossing, upper airway obstruction, strabismus, severe anemia (hemoglobin 7.9 g/dL), and a dense sclerotic skeleton. She received a transplant at 4 months of age with an HLA-matched unrelated donor, after conditioning according to EBMT-ESID guidelines. Full donor chimerism was achieved; however, the transplant was complicated by VOD and pulmonary hypertension, leading to her death.
Patient 3 displayed manifestations of the disease early in infancy: she presented with upper airway obstruction, blindness, and hydrocephalus requiring a ventriculoperitoneal (VP) shunt; in addition, delayed tooth eruption was noted at 30 months. An X-ray of the skull clearly demonstrated an osteopetrotic phenotype. The family refused HSCT and she died at 10 years of age due to a bacterial infection.
Patient 4 was born from healthy nonconsanguineous parents of Mexican descent. At 3 months of age she presented with stridor and airway difficulties; therefore, radiological examination was performed, leading to the diagnosis. The patient also showed anemia (hemoglobin 8.7 g/dL) and narrowing of the optic canals with significant visual impairment. She underwent a surgery for the unroofing of the optic canal at 4 months, but despite this she progressively lost all her visual capacity and by 6 months she was essentially blind with documented optic atrophy. At 15 months of age she developed hydrocephalus requiring a VP shunt and hypocalcemia, treated with calcitriol without benefit. At 20 months of age, during preparation for transplantation, she displayed mild to moderate developmental delay, both in regard to language and to gross motor milestones, frontal bossing, and roving eye movements. Ear, nose, and throat (ENT) assessment revealed severe apnea due to choanal obstruction, whereas hearing was normal; in addition, she developed a right VII nerve palsy. Severe anemia was noted (hemoglobin 6.2 g/dL) as well as thrombocytopenia (platelet count 96,000/µL). At this point, just prior to the admission for transplantation, she acutely arrested and died, probably due to her airway compromise.
Patient 5A was the second child of healthy, unrelated, Spanish-speaking American parents. She was diagnosed at 4 months of age, when she was admitted to the local hospital for fever, decreased urine output, and failure to thrive. Radiological examination revealed increased bone density with skeletal changes suggestive of osteopetrosis, whereas neurological investigation showed impaired vision. She presented to the transplant center at 6 months of age with failure to thrive (4.8 kg and 59.9 cm), anemia, thrombocytopenia, and obstructive sleep apnea. Prior to being admitted for transplantation, she had a tracheostomy tube placed due to the severity of sleep apnea. At 7 months of age she received a T-replete bone marrow graft from an HLA-matched family donor, after conditioning with busulfan and cyclophosphamide, and cyclosporine for graft-versus-host disease (GVHD) prophylaxis. She achieved full engraftment and donor chimerism. Post-HSCT complications included colonization with multiple viruses, such as adenovirus (tracheal, day +18 through day +29; and stool, day +71 through day +115) and CMV (tracheal, day +93 through day +98). Haemophilus influenzae was cultured from the tracheostomy tube on day –6 through day +4. On day +82, she began having hematemesis that may have been related to diffuse alveolar hemorrhage, diagnosed on day +90. She did not recover and died on day +135.
Patient 5B was the younger brother of patient 5A. He started treatment with calcitriol soon after birth due to hypocalcemia and was diagnosed with osteopetrosis at approximately 2 months of age. He displayed frontal bossing, impaired vision, and hepatosplenomegaly. Prior to HSCT, he underwent a tonsillectomy for apnea. At 6 months of age he received an HLA-partially matched, granulocyte colony-stimulating factor (G-CSF)-mobilized, and CD3-depleted family donor transplant after a reduced intensity preparative regimen consisting of fludarabine, thiotepa, melphalan, OKT3, rituximab, and cyclosporine. He achieved myeloid engraftment on day +17 and platelet engraftment on day +15. Posttransplantation complications included acute skin GVHD (grade 2), beginning at day +41 and lasting for a week. He later developed limited chronic skin GVHD. He achieved full donor chimerism and a reduction in bone mineral density by almost 50% was seen by 6 months post-HSCT by quantitative computed tomography. In addition, while pre-HSCT auditory brainstem response (ABR) showed moderate conductive hearing loss bilaterally, the 3- and 6-month post-HSCT ABRs were normal with bilateral middle ear dysfunction. Unfortunately, he developed methicillin resistant Staphylococcus aureus sepsis and died on day +353 of multisystem organ failure.
Patient 6 was born from healthy consanguineous parents (first degree consanguinity) of Turkish origin. At 4 months he displayed generalized osteosclerosis, visual impairment, deafness, anemia (hemoglobin 9.5 g/dL), and hepatosplenomegaly. HSCT has not yet been performed due to lack of a suitable donor. At present, he is alive at 4 years of age with anemia and thrombocytopenia.
Patients 7A and 7B are the second and third child of healthy consanguineous Turkish parents (second degree consanguinity). Patient 7A was admitted to the clinic at 3 months of age due to upper airway obstruction, massive hepatosplenomegaly, anemia (hemoglobin 6.7 g/dL), and thrombocytopenia (platelet count 86,000/µL). The diagnosis of osteopetrosis was confirmed by X-rays of extremities. At the age of 7 months he received a bone marrow transplant from an HLA-identical family donor after receiving myeloablative conditioning with busulfan and cyclophosphamide combined with fractionated radiation of the enlarged spleen (cumulative dose 7 Gy). After prolonged aplasia, complete donor engraftment was achieved. He experienced acute skin GVHD (grade 1-2) and limited chronic GVHD. The patient is alive and well at 21 years after HSCT with full donor chimerism; however, his vision is impaired with bilateral optical nerve atrophy.
Patient 7B is the younger sister of patient 7A. Osteopetrosis was diagnosed at the age of 3 months due to frontal bossing, anemia (hemoglobin 9.4 g/dL), liver enlargement, and abnormal eye movements. At the age of 4 months she received a bone marrow transplant from a matched related donor and initially showed the expected radiological improvement. However, after 5 years she again developed an osteopetrotic phenotype (Supplementary Fig. 2) with hydrocephalus and multiple fractures, and indeed the overall donor chimerism was determined to be 25% with predominance of donor T-cells and complete lack of donor macrophages. The patient is alive at 18.5 years of age with mental and growth retardation (height at 3rd centile), coxa vara, scoliosis, blindness, epilepsy, and behavioral problems.
Patient 8 was the sixth child of healthy consanguineous parents of Pathan descent. He was diagnosed at 6 months of age due to frontal bossing, impaired vision, and anemia (hemoglobin 8.9 g/dL); an elder sister had expired at 7 years of age with the same diagnosis. Soon after, the family moved and the patient was lost to follow-up.
Patient 9 was born from healthy unrelated parents from Belgium. At the age of 4 months he presented with a gradual increase in head circumference, resulting from hydrocephalus, for which he underwent a third ventriculostomy procedure. From the age of 6 months, a decreased rate of growth was noted. Because of failure to thrive and persistent fever, he was hospitalized; anemia was documented (hemoglobin 7.9 g/dL) as well as increased bone density on radiography of the thorax. Further radiographic examination confirmed generalized osteopetrosis, “bone-in-bone,” and sandwich vertebrae. Bone biopsy at 15 months of age showed the presence of osteoclasts (Supplementary Fig. 3). Physical examination at the time of diagnosis revealed scaphocephaly with frontal bossing. A small fontanel could be palpated. Horizontal nystagmus was present, although vision was thought to be preserved. Abdominal palpation and ultrasound did not reveal hepatosplenomegaly. Magnetic resonance imaging (MRI) of the brain revealed atrophy of the optic nerves, stenosis of the petrosal canal, and narrowing of the internal acoustic canal. He received an HSCT from an HLA-identical parental donor at 20 months of age. The most important posttransplantation complications were a CMV reactivation and acute skin GVHD (grade 3), which was successfully treated with prednisone and tacrolimus. Presently, at 30 months of age, he is very active with normal neuromotor development. Hearing and visual capacity also remains normal.
Patient 10 was born from healthy Muslim consanguineous parents (first-degree consanguinity) from Pakistan. At 12 months of age he presented with increased bone density, several fractures, impaired vision, and mild anemia (hemoglobin 10.4 g/dL). He did not receive HSCT because there was no donor available. Currently he has impaired but functional vision and normal blood counts. He has had recurrent non-healing fractures of the femurs and is therefore wheelchair-bound. He is alive at 22 years of age without HSCT.
Patient 11 has been already described (patient 4 in Mazzolari and colleagues, 2009).14 Briefly, she was born from healthy nonconsanguineous parents of Italian origin. She was diagnosed at 7 months due to a severe osteopetrotic phenotype, received 2 HSCTs (the second when she was almost 7 years old), and is now alive and well, 13 years after transplantation. Another affected sibling was born to this family 10 years later; she's described in the same article (Patient 10 in Mazzolari and colleagues, 2009)14; the molecular analysis could be performed only on the former sibling, due to lack of sample from the latter.
Patient 12 was the first child of healthy, related, German-speaking Sinti parents (second-degree consanguinity). At the age of 3.5 months he was admitted for hematuria, upper airway infection, and gastroenteritis. Hepatosplenomegaly, thrombocytopenia, anemia (hemoglobin 9 g/dL), as well as binocular nystagmus were noted and a CMV infection and Norovirus enteritis were diagnosed. At the age of 6.5 months a bone marrow evaluation was performed because of persisting anemia and thrombocytopenia, which excluded leukemia. Because of the clinical presentation, osteopetrosis was within the differential diagnosis and confirmed by X-rays. He presented to the transplant center at 7 months of age with anemia (hemoglobin 9.2 g/dL), splenomegaly, frontal bossing, upper airway obstruction, and blindness. Bone biopsy was performed and showed the presence of osteoclasts (data not shown). At 9 months of age he received a CD34+-selected peripheral stem cell graft from his HLA-haploidentical father, after conditioning according to the EBMT-ESID guidelines. The transplant course was uneventful; he achieved engraftment and full donor chimerism and is alive and well 3 years after HSCT.