A Rare Mutation in SMAD9 Associated With High Bone Mass Identifies the SMAD‐Dependent BMP Signaling Pathway as a Potential Anabolic Target for Osteoporosis

ABSTRACT Novel anabolic drug targets are needed to treat osteoporosis. Having established a large national cohort with unexplained high bone mass (HBM), we aimed to identify a novel monogenic cause of HBM and provide insight into a regulatory pathway potentially amenable to therapeutic intervention. We investigated a pedigree with unexplained HBM in whom previous sequencing had excluded known causes of monogenic HBM. Whole exome sequencing identified a rare (minor allele frequency 0.0023), highly evolutionarily conserved missense mutation in SMAD9 (c.65T>C, p.Leu22Pro) segregating with HBM in this autosomal dominant family. The same mutation was identified in another two unrelated individuals both with HBM. In silico protein modeling predicts the mutation severely disrupts the MH1 DNA‐binding domain of SMAD9. Affected individuals have bone mineral density (BMD) Z‐scores +3 to +5, mandible enlargement, a broad frame, torus palatinus/mandibularis, pes planus, increased shoe size, and a tendency to sink when swimming. Peripheral quantitative computed tomography (pQCT) measurement demonstrates increased trabecular volumetric BMD and increased cortical thickness conferring greater predicted bone strength; bone turnover markers are low/normal. Notably, fractures and nerve compression are not found. Both genome‐wide and gene‐based association testing involving estimated BMD measured at the heel in 362,924 white British subjects from the UK Biobank Study showed strong associations with SMAD9 (PGWAS = 6 × 10−16; PGENE = 8 × 10−17). Furthermore, we found Smad9 to be highly expressed in both murine cortical bone–derived osteocytes and skeletal elements of zebrafish larvae. Our findings support SMAD9 as a novel HBM gene and a potential novel osteoanabolic target for osteoporosis therapeutics. SMAD9 is thought to inhibit bone morphogenetic protein (BMP)‐dependent target gene transcription to reduce osteoblast activity. Thus, we hypothesize SMAD9 c.65T>C is a loss‐of‐function mutation reducing BMP inhibition. Lowering SMAD9 as a potential novel anabolic mechanism for osteoporosis therapeutics warrants further investigation. © 2019 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research.


Ethical Approvals
Written informed consent was collected for all participants in line with the Declaration of Helsinki (8). The UK HBM study was approved by the Bath Multi-centre Research Ethics Committee (REC: 05/Q2001/78) and at each NHS Local REC.
The AOGC study was approved by the Queensland Office of Human Research Ethics Committee (Ref:2008/018), the University of Queensland (Ref:200800376) and/or relevant research ethics authorities at each participating center. Directly recruited participants gave written, informed consent. Some participants were recruited through genetic and/or clinical studies (all with ethical approval) but also provided written informed consent to contribute to collaborative genetic studies (13)(14)(15). DNA was obtained from peripheral venous blood or from saliva through standard methods.  (19,20) and annotated by ANNOVAR (21). Further analysis of sequence data was performed with custom R and Bioconductor scripts. Good-quality SNPs, excluding those with a genotype quality score <60 were retained (determined by the GATK algorithm; range 0 [poor] to 100 [excellent]). Platform related artefacts were identified as variants where the allele count was 6 SDs higher than that expected from the maximum population minor allele frequency (MAF) under the binomial approximation. Remaining SNPs and indels were assessed according to prediction of potentially damaging consequence (''nonsynonymous SNV,'' ''splicing,'' ''frameshift substitution,'' ''stopgain SNV,'' ''stoploss SNV'') by using both RefSeq and UCSC transcripts. Further filtering excluded SNPs with a MAF <0.05 (observed in NCBI dbSNP (GRCh37/ Hg19), 1000 Genomes (22), ExAC (http://exac.broadinstitute.org/), or internal databases from >3000 exomes), 1000 Genomes small indels (called with DINDEL (23)). Variants not present in any database were considered novel. Genotype Quality scores for the two additional isolated HBM cases were 99 and read depths were 15,14 and 21,19.

Sanger sequencing validation of pedigree based HBM mutation
Polymerase chain reaction (PCR) amplification of identified exons was performed on 50ng genomic DNA in a reaction mix consisting; 10X Immolase reaction buffer, 10mM dNTPs, 50mM MgCl2, 5µM each primer, 0.5 U Immolase polymerase Taq (Bioline Reagents Ltd, London), and water to final volume of 25µl. PCR cycling conditions and primer sequences are shown below. Samples were Sanger sequenced using standard techniques (BigDye v3.1 chemistry, Life Technologies Corporation, California), and capillary sequenced (3130 Genetic Analyzer, Life Technologies Corporation, California).

SMAD9_Ex2 Primers
Fwd primer: CACCCTGTTCAAGGGCTTAG The 55-year-old mother, with BMD Z-Scores +3.3 at the total hip and at L1, had never sustained a fracture. Six years earlier she had had a right calcaneal spur surgically removed. She had widespread joint pains affecting ankles, knees (with previous arthroscopy), hips, shoulders, hands and feet, limiting mobility to 50 yards, with a diagnosis of fibromyalgia prompting high analgesic use. She had a history of asthma, hypertension, hypercholesterolaemia and depression with corresponding medications, with normal menopause at 53. She had no visual or auditory impairments, or dental history of note. She was tall and obese (BMI 41.4), with above average shoe size, a broad frame, enlarged mandible but no tori. She had a full range of movement in all joints, bilateral knee crepitus, bilateral pes planus and no signs of nerve compression.

III.2: Half-sister to index case (c.65T>C, p.Leu22Pro)
The 22-year-old half-sister, with BMD Z-Scores +4.8 at the total hip and +2.6 at L1, had not fractured. She had had sciatica for five years, lumbar back pain and fronto-temporal headaches for 11 years, with a diagnosis of migraine. She had no significant dental history, no visual or auditory impairments, but was unable to float. She had had a normal menarche at 12. She used inhalers for asthma and medication for depression and anxiety. She was tall and obese (BMI 43), with above average shoe size, a broad frame, enlarged mandible, a torus palatinus in the midline of her hard palate (3cm x 7mm), normal joint movement and no signs of nerve compression.

I.2: Grandmother of index case (wild-type)
The 75-years-old grandmother, who did not have HBM (BMD Z-Scores +0.1 at the total hip and +0.8 at L1) had never sustained a fracture. She had osteoarthritis affecting knees, hips, lumbar spine, fingers, left elbow, limiting mobility to 3 metres and a wheelchair. She had had auditory impairment since school and had had cataracts removed. She was overweight (BMI 28.1) with normal shoe size, a normal frame, mandible and no tori, with reduced extension of the right elbow and left knee, with bilateral crepitus of her knees. She died aged 81 of pneumonia secondary to advanced Emphysema.

Isolated HBM case (c.65T>C, p.Leu22Pro) from the UK
This 55-year-old female, with BMD Z-Scores +5.0 at the total hip and +4.7 at L1, had never fractured and reported difficulty floating. Her adult left upper cuspid tooth had never erupted; wisdom teeth had been extracted for overcrowding. She had noticed her own mandible enlargement. She had a congenital astigmatism of her left eye with poor vision, and congenital bilateral pes planus. She had no auditory impairment. She took tamoxifen postsurgery for breast cancer. She was obese (BMI 35) with a broad frame, mandible enlargement, but no tori. She had normal joints and no signs of nerve compression.  Filtering step restricted sample to individuals who were unrelated, Caucasian ancestry, with complete weight and height data, enough high-quality DNA available for WES, and were able to be sequenced within our financial constraints. BMD restriction ensured all HBM cases had TH or LS Z-score ≥+2.5, and all LBM Cases a TH Z-Score < -1.5 and LS Z-Score ≤-0.5.      This research has been conducted using the UK Biobank Resource (accession IDs: 12703). We would like to thank the Wolfson Bioimaging Facility at the University of Bristol, UK, for confocal microscope access and imaging support and Jessica Harris and Sharon Song at the Queensland University of Technology for their help with genotyping.