Whole Genome Sequencing Unravels New Genetic Determinants of Early-Onset Familial Osteoporosis and Low BMD in Malta

Abstract

Background: Osteoporosis is a skeletal disease with a strong genetic background. The study aimed to identify the genetic determinants of early-onset familial osteoporosis and low bone mineral density (BMD) in a two-generation Maltese family.

Methods: Fifteen relatives aged between 28-74 years were recruited. Whole genome sequencing was conducted on 12 relatives and shortlisted variants were genotyped in the Malta Osteoporotic Fracture Study (MOFS) for replication.

Results: Sequential variant filtering following a dominant inheritance pattern identified rare missense variants within SELP, TGF-β2 and ADAMTS20, all of which were predicted to be likely pathogenic and participate in osteoimmunology. TGF-β2 c.1136C>T was identified in five individuals from the MOFS in heterozygosity, four of whom had osteopenia/osteoporosis at the lumbar spine and hip, and/or had sustained a low-trauma fracture. Heterozygosity for the ADAMTS20 c.4090A>T was accompanied by lower total hip BMD (p = 0.018) and lower total serum calcium levels in MOFS (p < 0.01), recapitulating the findings from the family. Women carrying at least one copy of the alternative allele (TC/CC) for SELP c.2177T>C exhibited a tendency for lower lumbar spine BMD and/or wrist fracture history relative to women with TT genotype.

Conclusions: Our findings suggest that the identified variants, alone or in combination, could be causal factors of familial osteoporosis and low BMD, requiring replication in larger collections.

Keywords: ADAMTS20; BMD; SELP; TGF-β2; familial osteoporosis; whole genome sequencing.

Figure 1

Pedigree of the two-generation Maltese family. Roman numerals indicate generation, whereas Arabic numbers indicate the order. The arrow indicates the proband. Squares represent male relatives, females are depicted in circles, whereas diamonds signify a stillbirth with an unknown gender. Gestational age of the latter is unknown. A diagonal arrow indicates that the individual is deceased and no biological material was available for analyses. A half black symbol represents the presence of osteoporosis, whereas a quarter shading represents osteopenia or a low-trauma fracture history. Relatives with no bone mineral density (BMD) measurements are shaded in grey and marked as ‘unknown’.

Figure 2

Segregation of the three risk variants confirmed by Sanger sequencing in recruited relatives. The gene name represents the respective gene variants (i.e., SELP c.2177T>C, TGF-β2 c.1136C>T and ADAMTS20 c.4090A>T). The gene name (in italics) is followed by the genotype, with ‘-/-‘ representing a homozygous reference genotype, whereas ‘-/+‘ represents a heterozygous genotype for each respective variant.

Figure 3

Validation of the shortlisted gene variants. (a) Excerpt of the BAM file uploaded in IGV showing the heterozygous change for all three variants, (b) Sanger sequencing traces showing a confirmed homozygous reference (top) and heterozygous (bottom) sequence for each of the three risk variants, (c) protein structure of P-selectin, TGF-β2 and ADAMTS20 with a focus on the position of the variant within the protein according to AlphaFold Protein Structure Database [57,58].

Figure 4

The interaction network highlighting the interaction of P-selectin, TGF-β2 and ADAMTS20 (shown in red) with TNF-α and pro-inflammatory cytokines using Ingenuity Pathway Analysis^®.