Genome sequencing identifies a large non-coding region deletion of SNX10 causing autosomal recessive osteopetrosis

A 4-year-old male child of a second-degree consanguineous couple (Fig. 1A) was examined. He was born at term by normal vaginal delivery with a birth weight of 3.5 kg (−0.10 SD). At the age of 3 years, he was hospitalized for fever. At 4 years of age, he was 98 cm (−1 SD) tall, weighed 14.0 kg (−1.4 SD), and had a head circumference of 51 cm (+0.38 SD). Blood investigations revealed anaemia, thrombocytopenia and elevated lactate dehydrogenase (1047 U/L; reference value: 225–460 U/L). Physical examination revealed a large skull, frontal bossing and pectus carinatum (Fig. 1B). These observations were similar to those of affected individuals described in the previous reports [7, 8]. His hearing was normal, and the other developmental stages were also age-appropriate. We did not observe any visual impairment in him at this age. However, at the age of 5 years and 2 months, he developed a vision problem and bilateral optic atrophy was diagnosed. Hepatosplenomegaly and mesenteric lymphadenopathy were also noted. We found no neurologic abnormalities in the proband. Blood tests revealed a low haemoglobin level and decreased white blood cells and platelet counts. Further details of the biochemical investigations are given in Table 2.

Fig. 1
figure 1

A Pedigree of the 4-year-old boy born to a second degree consanguineously married couple. B Radiographs showing that the proband has large skull, frontal bossing and pectus carinatum. CG Radiographs at 4 years of age showing – (C) increased bone density at the outer cortex of the skull, (D, E) increase in bone mineral density at epiphyses and metaphyses. Irregular bone mineral density was noted at diaphysis, (E, F) increased thickness of vertebrae showing “sandwich” appearance of the vertebral plates

Table 2 Biochemical investigations of the proband

Complete skeletal examination revealed increased bone density at the outer cranial cortex, epiphyses, and metaphyses. A moderate increase in bone density and irregular transparency of the diaphysis were noted at the tibia. The proband was found to have an increased width of the ribs, and radiographs of the spine showed “sandwich vertebrae” and a bone-in-bone appearance (Fig. 1C–G).

Exome sequencing revealed no significant disease-causing variants. Genome sequencing analysis using the in-house variant prioritization strategy revealed a large homozygous deletion of 72,012 bp, which was confirmed by manual analysis of the BAM file using Integrative Genomics Viewer. This deletion includes upstream region of SNX10, the noncoding exon 1, and a portion of intron 1 (Fig. 2A). GAP PCR and Sanger sequencing confirmed the deletion of 72,012 bp along with the insertion of two nucleotides (NC_000007.14:g.26263639_26335651delinsCA) in the proband (Fig. 2B). Both parents were carriers of the indel. This variant is not included in any of the control population databases.

Fig. 2
figure 2

A Schematic representation showing ~72 kb indel variant upstream of SNX10. Screen capture of the BAM files analysis using the Integrative Genomics Viewer for the SNX10 indel (g.26263639_26335651delinsCA) was found in the proband (B) Sanger chromatogram showing homozygous deletion and insertion of two nucleotides in the proband. parents are carriers of the identified variant

Further mRNA analysis using RT-PCR with the primer set for exon 1 and7 of SNX10 showed the expected band size (559 bp) in the control, the absence of PCR product in the proband, while the parents showed normal DNA band size (559 bp) due to a normal allele in them. Another primer set including only exon 6 and 7 of SNX10 showed a very weak band (105 bp) in the proband, indicating a lower amount of SNX10 transcript compared to the control and healthy parents. RT-PCR for GAPDH was used for control normalization (Fig. 3).

Fig. 3
figure 3

A Agarose gel electrophoretic separation of reverse transcriptase PCR (RT-PCR) amplicons encompassing exon 1 and 7 and exon 6 and 7 performed on cDNA samples of proband, carriers (parents) and control. Proband showed absence of SNX10 transcript in the exon 1–7 primer set but lesser amount of transcript was observed in the exon 6–7 primer set; GAPDH represents control. B Densitometric quantification of the DNA bands obtained from RT-PCR with primers for exon 6 and 7 of SNX10

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