PLS3 sequencing in childhood-onset primary osteoporosis identifies two novel disease-causing variants

Cohort 1 (“primary osteoporosis”) consisted of 31 patients (17 boys and 14 girls), who had been referred to the Metabolic Bone Clinic, Children’s Hospital, Helsinki during the years 2003–2013 for investigation for childhood-onset primary osteoporosis but where the investigation did not reveal a molecular cause of their disease. The inclusion criteria were (1) exclusion of type I collagen-related OI, either clinically or by genetic testing, and (2) exclusion of secondary osteoporosis with biochemical and individually determined clinical evaluations. Further, all had been screened and found negative for pathogenic variants in WNT1 and LRP5 and several had undergone more extensive genetic testing before inclusion. At the time of referral, all the patients were between the ages of 4 and 17 years, displayed clinically an osteoporotic phenotype, and several of them had similarly affected family members. Most of the patients (25/31) fulfilled the ISCD criteria [2] for pediatric osteoporosis. Six patients only had a history of increased non-spinal fractures and a BMD Z-score >−2.0 but had other additional features (e.g., osteopenic appearance on skeletal radiographs, fragile bone on orthopedic surgery) suggesting osteoporosis.

Cohort II (“fracture prone”) consisted of 64 otherwise healthy children who had sustained multiple fractures (43 boys and 21 girls), all recruited at the Children’s Hospital, Helsinki, Finland during a prospective epidemiological study [20]. Over a 12-month period, all children (n = 1412) aged 4–15 years who had been treated for an acute and radiographically confirmed fracture were assessed for fracture history. The trauma mechanisms and previous medical histories were available for 1361 (96%) of the children. The inclusion criteria for cohort II were (1) age 4–15 years, (2) ≥2 low-energy long bone fractures before age 10 years, (3) ≥3 low-energy long bone fractures before age 16 years, or (4) ≥1 low-energy vertebral fracture (loss of ≥20% vertebral height). Children with a diagnosis or suspicion of OI were excluded, as well as children with an underlying disease that could explain their bone fragility. Altogether 71 patients of the >1400 children fulfilled these criteria; DNA from peripheral blood was available for 64 of them and they comprised cohort II. Data on blood biochemistry, spinal radiographs, and DXA measurements were collected for all participants.

As expected, children in cohort I displayed in general lower BMD Z-scores at the lumbar spine and a higher prevalence of vertebral compression fractures (p < 0.001 and p < 0.004, respectively) than children in cohort II (Fig. 1). Data for BMD at the lumbar spine and femoral neck and information about vertebral compression fractures were not available for four, six, and three children, respectively, in cohort I. In cohort II, 61% (n = 39) of the children had sustained at least three significant fractures, and of them 54% (n = 21) had sustained at least four significant fractures. Twenty percent (n = 13) had sustained at least three significant fractures before the age of 10 years, and of them 38% (n = 5) had sustained at least four significant fractures before the age of 10 years.

Genomic DNA was extracted with the Puragene DNA Purification kit (Gentra), Archive Pure DNA Blood Kit (5Prime), or QIAamp DNA Blood Maxi Kit (Qiagen). Primers for PCR amplification were constructed using Primer3Plus and were derived from the canonical ensemble transcript (ENST00000355899). Sanger sequencing was performed using BigDye® technology on a 3730 ABI sequencer, and electropherograms were later interpreted using the Staden package (version 2.0.0b10). Primers are available upon request.

Patient 2 in cohort I together with her nuclear family was subjected to whole exome sequencing. Sequencing was performed using an Illumina HiSeq 2500 at Science for Life Laboratory, Stockholm, Sweden. The Agilent SureSelect XT All Exon V4 target enrichment kit was used for whole exome capture. All analysis computations were performed on resources provided by SNIC through Uppsala Multidisciplinary Center for Advanced Computational Science (UPPMAX) [21]. For a full description of the data processing and analysis, see Supplemental appendix and Supplemental Fig. 1 (Online Resource).

Iliac crest bone biopsies were taken as part of normal diagnostic evaluation for both patients in cohort I who were deemed to have disease-causing variants in PLS3. For bone turnover assessments, the patients were pre-treated with oral tetracycline for 2 × 2 days with a 10-day treatment-free interval. The bone biopsies were analyzed by an experienced histomorphometrist using a semiautomatic image analyzer (Bioquant Osteo; Bioquant Image Analysis Corp., Nashville, TN, USA). The recommendations of the American Society for Bone and Mineral Research regarding abbreviations and nomenclature were followed [22]. Age-specific reference values and Z-scores were calculated for all parameters [23, 24].

The statistical analyses were performed using R version 3.3.1. Because of the distribution of the data, the Mann-Whitney U test was used to compare BMD between the groups. The chi-square test was used to test categorical data. A p value <0.05 was considered significant.

Sanger sequencing of PLS3 in cohort I identified two patients (6.5%) with novel variants in PLS3 that were deemed to be damaging and causative of their phenotypes (described below). In the remaining 29 patients in cohort I, a total of 7 single nucleotide variants (SNVs) and 1 small deletion were found (Table 1). Three of the SNVs were found in coding regions, all were synonymous and with an allele frequency of at least 4% in the general Finnish population (SISu database); the other four variants were intronic, all previously reported. One of the synonymous variants, rs140121121, has previously been associated with osteoporosis [12], but this SNV was not enriched in our cohort compared with the normal Finnish population (allele frequency 0.067 vs 0.051). Outside the coding region, we found one 13 bp deletion (rs201765481) situated 190 bp upstream of exon 6 of PLS3. Its allele frequency was higher than expected in our cohort (0.043 vs 0.009 (dbSNP)), but when we sequenced this region for 96 healthy Finnish control samples, the frequency was found to be similar (0.059) as in our cohort, suggesting that the deletion is not pathogenic. The other non-coding variants were regarded as not being of significance in this context, either because of their high allele frequencies or location far from canonical splice sites.

Patient 1 is a presently 30-year-old Finnish male, who was found to have a novel hemizygous nonsense variant (c.766C>T; pArg256*) in exon 8 of PLS3. This variant is not found in dbSNP, SISu, ExAC, or gnomAD databases and classified as pathogenic according to the American Collage of Medical Genetics and Genomics (ACMG) guidelines for interpreting sequence variants [25]. He has also been sequenced for a core panel of bone fragility genes (Blueprint Genetics, Helsinki), including COL1A1 and COL1A2, without pathogenic findings. The mother of patient 1 was confirmed to be heterozygous for the variant, while the brother, father, and three maternal relatives were negative for the variant. Patient 1 has a history of multiple fractures since early childhood. Between the ages of 9 and 10 years, he fractured both femurs in two separate low-energy traumas. At 10 years, he was diagnosed with multiple vertebral compression fractures, and at 13 years, he sustained two humeral fractures at two separate occasions. A bone biopsy at the age of 11 years confirmed the diagnosis of trabecular osteoporosis, low bone turnover, and normal mineralization (Fig. 2 and Supplemental Table 1; Online Resource). He had considerably low DXA measurements; at 18 years, the BMD Z-score for the lumbar spine was −4.1 and for the femoral neck −3.3. He then received a 1-year treatment with zoledronic acid, and by age 20, a slight improvement was seen with BMD Z-scores of −3.8 and −2.8 for lumbar spine and femoral neck (Fig. 3). The patient also displays some extraskeletal features such as slightly blue sclerae, slightly yellow teeth and loss of enamel, generalized joint hyperlaxity, soft skin, minor aortic valve regurgitation, and asthma. In other respects, his pubertal development and adult height (175 cm) were normal. Measurements for calcium, phosphate, and alkaline phosphate were normal and there was no hypercalciuria, but urinary NTX was low (normal creatinine). Taken together, his biochemical profile was normal except for a mild vitamin D deficiency (serum 25-OH-vitamin D 35 nmol/L). The mother, heterozygous for the variant, had osteopenia on DXA scan (total body Z-score −1.4 at 46 years) and had sustained one radius fracture after a fall at 35 years. The mother also had joint hyperlaxity and slightly blue sclerae but was otherwise healthy.

Patient 2 whose variant was deemed disease-causing is a 10-year-old Finnish girl with no family history of osteoporosis. She proved to be heterozygous for a novel de novo missense variant in exon 12 (c.1424A>G; p.N446S) that was absent in her parents and healthy sister (Supplemental Fig. 2; Online Resource). The variant was not found in either dbSNP, SISu, ExAC, or gnomAD databases. The amino acid in this position is highly conserved over different species, and the missense variant has a scaled CADD score of 21.5 and is predicted deleterious by both SIFT and MutationTaster. According to the ACMG guidelines [25], this variant is classified as likely pathogenic, which means that the variant is considered to be pathogenic at a level of ≥90% certainty. However, since the phenotype was significantly more severe than anticipated for a female with a heterozygous missense variant in PLS3, we investigated the possibility of other causative variants. An array-CGH detected no significant gene dosage imbalances; her other PLS3 allele was also intact. We also performed whole exome sequencing for patient 2 and her nuclear family (healthy parents and healthy sister). Since the parents were completely healthy, the analysis focused on recessively inherited and de novo variants. However, apart from the de novo variant in PLS3, no other potential disease-causing variants were found. Importantly, no other damaging variants were found in any other genes associated with OI. The genes COL1A1 and COL1A2 had also previously been Sanger sequenced without pathogenic findings. Moreover, previously reported female patients heterozygous for pathogenic variants in PLS3 show variable expressivity [12, 19] (Supplemental Table 2; Online Resource). Based on these findings, the heterozygous PLS3 variant was regarded as the most likely cause for the phenotype.

Patient 2 has a history of multiple long bone and vertebral compression fractures and remarkably low BMD. By the age of 6 years, she had sustained three low-energy long bone fractures and one finger fracture and was then referred to the Metabolic Bone Clinic, Children’s Hospital, Helsinki for further investigations. Her BMD Z-scores for the lumbar spine, proximal femur, and total body were −6.6, −4.5, and −3.5, respectively. Spinal radiographs showed three asymptomatic vertebral compression fractures. Extra-skeletal manifestations were seen in the form of joint hyperlaxity with hyperextension in elbows and knees, but she did not have blue sclerae. Secondary causes of osteoporosis were excluded. Serum calcium, phosphate, alkaline phosphatase, PTH, and vitamin D were normal. A bone biopsy confirmed the diagnosis of trabecular osteoporosis, low bone turnover, and normal mineralization (Fig. 2 and Supplemental Table 1; Online Resource). Treatment with pamidronate was started at the age of 6 years with a cumulative dose of 9 mg/kg the first year and continued with zoledronic acid 0.025 mg/kg every 6 months thereafter. Follow-up measurements at 17, 29, and 40 months showed a good treatment response and she has not experience new fractures thereafter (Fig. 4).

In cohort II, Sanger sequencing of the PLS3 gene showed in total 8 SNVs and 1 small deletion in the 64 patients with fractures (Table 1). All variants have been previously described, and most of them corresponded to the findings in cohort I. One coding variant was unique to cohort II, a rare missense variant (rs140968059, p.I309V) found in heterozygous form in one girl. However, the substitution was predicted benign by both SIFT and MutationTaster, and both isoleucine and valine are branched-chain amino acids with very similar chemical structures, making a substitution between the two less likely to be damaging. Thus, none of the variants found in the 64 fracture-prone children were considered causative of their skeletal fragility.