Rare Copy Number Variants in Array-Based Comparative Genomic Hybridization in Early-Onset Skeletal Fragility

Abstract

Early-onset osteoporosis is characterized by low bone mineral density (BMD) and fractures since childhood or young adulthood. Several monogenic forms have been identified but the contributing genes remain inadequately characterized. In search for novel variants and novel candidate loci, we screened a cohort of 70 young subjects with mild to severe skeletal fragility for rare copy-number variants (CNVs). Our study cohort included 15 subjects with primary osteoporosis before age 30 years and 55 subjects with a pathological fracture history and low or normal BMD before age 16 years. A custom-made high-resolution comparative genomic hybridization array with enriched probe density in >1,150 genes important for bone metabolism and ciliary function was used to search for CNVs. We identified altogether 14 rare CNVs. Seven intronic aberrations were classified as likely benign. Five CNVs of unknown clinical significance affected coding regions of genes not previously associated with skeletal fragility (ETV1-DGKB, AGBL2, ATM, RPS6KL1-PGF, and SCN4A). Finally, two CNVs were pathogenic and likely pathogenic, respectively: a 4 kb deletion involving exons 1–4 of COL1A2 (NM_000089.3) and a 12.5 kb duplication of exon 3 in PLS3 (NM_005032.6). Although both genes have been linked to monogenic forms of osteoporosis, COL1A2 deletions are rare and PLS3 duplications have not been described previously. Both CNVs were identified in subjects with significant osteoporosis and segregated with osteoporosis within the families. Our study expands the number of pathogenic CNVs in monogenic skeletal fragility and shows the validity of targeted CNV screening to potentially pinpoint novel candidate loci in early-onset osteoporosis.

PLS3 Mutations Cause Severe Age and Sex-Related Spinal Pathology

“…Results were compared with congruent findings in 13 mutationnegative individuals from the same families. We have previously described that mutations in the X-chromosomally inherited PLS3 lead to a skeletal disorder predominantly in males characterized by compromised bone strength, low rate of bone formation, defective mineralization, and consequently prevalent fractures and loss of adult height (5,8,12). Findings in the present study indicate that defective PLS3 function leads to severe ageand sex-related abnormalities in vertebral morphology already in early childhood and to significant spinal pathology by early adulthood.…”

“…We have previously identified four unrelated Finnish families with PLS3 osteoporosis due to different PLS3 mutations (Figure 1) (5,8,12). Family A has an intronic PLS3 splice site mutation c.73-24T>A (p.Asp25Alafs * 17) as previously described (5).…”

“…Family A has an intronic PLS3 splice site mutation c.73-24T>A (p.Asp25Alafs * 17) as previously described (5). Family B has a 12.5 kb tandem duplication spanning intron 2 to 3 of PLS3, identified by array-comparative genomic hybridization and predicted to lead to loss of function (12). Family C includes a single subject with a de novo heterozygous missense mutation c.1424A>G (p.Asn446Ser), identified through Sanger sequencing of the whole gene (8).…”

“…We performed genetic validations on DNA extracted from peripheral blood, as previously described (5,8,12). We screened all samples for the families' pertinent, previously identified PLS3 mutation (NCBI Reference Sequence NM_005032).…”

“…To date, we have identified four unrelated Finnish families with PLS3 osteoporosis due to different PLS3 mutations (5,8,12). Granted the access to this unique cohort of PLS3 mutation-positive subjects, we set out to evaluate their spinal pathology using magnetic resonance imaging (MRI).…”