Assessing the contribution of genes involved in monogenic bone disorders to the etiology of atypical femoral fractures

doi:10.21203/rs.3.rs-4467437/v1

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Assessing the contribution of genes involved in monogenic bone disorders to the etiology of atypical femoral fractures

https://doi.org/10.21203/rs.3.rs-4467437/v1

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published 15 Aug, 2024

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Background

Recent studies suggested that genetic variants associated with monogenic bone disorders were involved in the pathogenesis of atypical femoral fractures (AFF). Here, we aim to identify rare genetic variants by whole exome sequencing in genes involved in monogenic rare skeletal diseases in 12 women with AFF and 4 controls without any fracture.

Results

Out of 33 genetic variants identified in women with AFF, eleven (33.3%) were found in genes belonging to the Wnt pathway (LRP5, LRP6, DAAM2, WNT1, and WNT3A). One of them was rated as pathogenic (p.Pro582His in DAAM2), while all others were rated as variants of uncertain significance according to ClinVar and ACMG criteria.

Conclusions

Osteoporosis, rare bone diseases, and AFFs may share the same genes, thus making it even more difficult to identify unique risk factors.

Atypical femoral fracture

AFF

WES

monogenic bone disorder

Atypical femoral fractures (AFF) are low-impact fractures that differ from classic osteoporotic femoral fractures in that they occur in the subtrochanteric region or femoral diaphysis, usually in the setting of prolonged treatment with bisphosphonates (BP).While its physiopathology has not been completely clarified, genetic predisposition appears to be key given that the incidence of AFF is very low in the general population (5.9 per 10,000 person-years)(1), and specific patient populations appear to be at an increased risk of sustaining them including people with Asian ancestry and patients affected with specific monogenic bone disorders(2). A number of studies have tried to identify the genes involved in the AFF risk suggesting that AFF occur in the setting of a complex and heterogeneous genetic background where each affected individual could have their own genetic burden(3, 4). In a recent study by Zhou et al. (2023)(5), the authors suggested that genetic variants associated with monogenic bone disorders might play a role in the pathogenesis of AFF. In their study, 37 candidate genes involved in monogenic bone disorders were analyzed from whole-exome sequencing (WES) data in 60 AFF patients, with 95% having received bisphosphonates. Fifteen (25%) of the 60 AFF patients had clinical features of a monogenic bone disorder. In total, nine patients in their AFF cohort (15%) had a (likely) pathogenic variant, eight of whom fulfilled clinical criteria suggestive of monogenic bone disorders.

In the present study, we contribute new data on 12 women with AFF and 4 controls without any fracture, all of whom had received bisphosphonates for over 5 years due to a diagnosis of osteoporosis. All AFF patients and controls had been previously subjected to WES in search for rare genetic variants overrepresented in AFF patients and absent in controls(6). Here, we have explored in our samples the 37 candidate genes proposed by Zhou et al. and other additional genes involved in rare diseases with bone impairment(7).

Participants were previously described in a work published by our group(6). Briefly, twelve unrelated postmenopausal women with AFF (mean age ± SD of 74.5 ± 6.1) and four postmenopausal women without any fracture (controls) (mean age ± SD of 79 ± 7.2) were recruited in Hospital del Mar (Barcelona, Spain) and Hospital Universitario de La Princesa (Madrid, Spain). All of them received bisphosphonate (BP) treatment for > 5 years due to a diagnosis of osteoporosis. No patient had hypophosphatemia or suspicion of a monogenic disease. Half of the AFF patients had received corticosteroid therapy for more than one year due to polymyositis, rheumatoid arthritis, asthma or chronic bronchitis. None of the controls had receive glucocorticoids.

Whole exome sequencing (WES) was performed at the CNAG facilities (Barcelona, Spain). Capture was performed using Agilent Human All Exon 50Mb v5 and samples were sequenced at a coverage of 140x on a HiSeq 2000 sequencer. Pipeline of the WES is detailed in Garcia-Giralt et al. (2022).

We filtered for rare variants (MAF < 0.005) with CADD > 20 and the resulting variants were overlapped with our list of candidate genes (Table 1).

Table 1

List of candidate genes involved in monogenic diseases with bone impairment
Candidate genes by Zhou et al. (2023)	Other genes involved in bone phenotypes (7)*
ALPL	AHNAK
BMP1	ANKH
CA2 (CAII)	ANO5
CLCN7	ATP6V0A2
COL1A1	B4GALT7
COL1A2	BMP5
CREB3L1	CASR
CRTAP	CLCN5
CTSK	CUL7
DKK1	CYP19A1
FKBP10	DMP1
IFITM5 (BRIL)	DAAM2
LRP5	ENPP1
MBTPS2	ESR1
OSTM1	FAM20A
P3H1 (LEPRE1)	FAM20C
P4HB	FBXW5
PHEX	FGF23
PLEKHM1	FGFR1
PLOD2	GALNT3
PLS3	GNAS
PPIB (CYPB)	GORAB
SEC24D	IDUA
SERPINF1	IFIH1
SERPINH1	KDM5B
SGMS2	KLOTHO
SNX10	KREMEN
SP7	LGR4
SPARC	LMNA
TAPT1	LRP4
TCIRG1	MAFB
TENT5A (FAM46A)	MMP2
TMEM38B	NOTCH2
TNFRSF11A (RANK)	PTH1R
TNFSF11	PTHLH
WNT1	PTK7
WNT3A	PYCR1
	RAS
	RIN3
	RUNX2
	SEM1
	SLC34A3
	SMAD6
	SNZ10
	SOST
	SOX9
	TNFRSF11B
	TNXB
	XYLT2
	ZMPSTE24
*taken from Suppl Table 12 ((7))

A total of 41 genetic variants were detected in genes belonging to the list of candidate genes (Table 2). Only one of the variants identified was rated as pathogenic according to ClinVar or ACMG guidelines (p.Pro582His in DAAM2(8)), while all others were rated as variants of uncertain significance (VUS) according to ACMG criteria.

Table 2

Genetic variants found in AFF patients and controls detected by WES
ID Patient	Gene	Genetic variant	CADD Score	ALT frequency	CLINVAR	ACMG classification
AFF2	FAM20C	p.Ser410Thr	25.8	0.003385	Benign/Likely benign	VUS
AFF2	LMNA	p.Arg401Cys	34	0.00003295	VUS	VUS
AFF2	TNXB	p.Val1831Met	25.1	0.00005775	VUS	VUS
AFF2	TNXB	p.Asp677Gly	23.1	0.002365	VUS/likely benign	VUS
AFF3	LRP6	p.Ser1385Cys;p.Ser1476Cys;p.Ser1521Cys	26.2	0.000008236	No inf	VUS
AFF3	WNT3A	p.Tyr260Phe	23.4	No inf	No inf	VUS
AFF4	DAAM2	p.Arg990Leu	27.1	0.001183	No inf	VUS
AFF4	WNT1	p.Gly259_Gly262dup	21.9	0.000002787	No inf	VUS
AFF4	ZMPSTE24	p.Ser27Phe	23.4	0.00002471	No inf	VUS
AFF5	LRP5	p.Arg258Cys	34	0.00001648	VUS	VUS
AFF5	PTH1R	p.Tyr221Cys	26.2	0.000008238	VUS	VUS
AFF6	LRP5	p.Ser1482Leu	35.35	0.00003	VUS	VUS
AFF6	LRP5	p.Arg1036Gln	24.3	0.002487	VUS/likely benign	VUS
AFF6	PLEKHM1	p.Arg103His;p.Arg1047His;p.Arg958His	34	0.0004942	No inf	VUS
AFF6	SLC34A3	p.Lys298del	No inf	No inf	No inf	VUS
AFF7	SLC34A3	p.Arg468Gln	29.2	0.00004129	No inf	VUS
AFF8	LRP5	p.Pro1504Leu	34.34	0.0004	VUS/likely benign	VUS
AFF8	MMP2	p.Glu166Lys	24	0.001688	Benign/Likely benign	VUS
AFF9	AHNAK	p.Lys1438Asn	22.9	0.0002965	No inf	VUS
AFF9	DAAM2	p.Pro582His	22.8	0.001	Pathogenic	likely pathogenic
AFF10	DAAM2	p.Lys776Thr	28,2	0.0008113	No inf	VUS
AFF10	ANKH	p.Arg36Trp	34	0.00001647	No inf	VUS
AFF11	AHNAK	p.Glu5dup	No inf	No inf	No inf	VUS
AFF11	ATP6V0A2	p.Arg141Leu	25.6	0.0002883	VUS/likely benign	VUS
AFF11	BMP1	p.Arg371His	33	0.003706	Benign/Likely benign	VUS
AFF11	CUL7	p.Arg183Gln	24.4	0.00005765	VUS	VUS
AFF11	DAAM2	p.Pro555Leu/p.Pro555Leu*	24.7	0.0008105	No inf	VUS
AFF11	FKBP10	p.Ile436Thr	28.6	0.002	VUS/likely benign	VUS
AFF11	PTH1R	p.Pro581Arg	25.7	0.00008237	VUS	VUS
AFF11	SLC34A3	p.Arg568Cys	24.1	0.00003038	VUS	VUS
AFF11	TNXB	p.Arg2889Trp	21.9	0.0001573	VUS	VUS
AFF12	CUL7	p.Gly872Ser	34	0.001392	VUS/likely benign	VUS
AFF12	PYCR1	c.-112C > T	34	0.0005564	No inf	VUS
Control2	COL1A1	p.Pro417Ser	22.6	0.00008238	VUS/likely benign	VUS
Control2	IFIH1	c.1641 + 1G > C	25.9	0.006483	VUS/Benign	VUS
Control2	NOTCH2	p.Arg1048His	22.8	0.00005765	VUS	VUS
Control2	XYLT2	p.Ala614Val	21.7	0.0006342	VUS	VUS
Control3	ANO5	p.Glu185Gln	26.3	0.0002471	VUS	VUS
Control3	COL1A2	p.Arg906His	27.8	0.0001235	VUS	VUS
Control3	TNXB	p.Val1482Met	25.9	0.001581	Benign/Likely benign	VUS
Control4	FKBP10	p.Gly286Arg	30	0.000008236	VUS	VUS
Abbreviations: ALT, alternative allele; ACMG, American College of Medical Genetics and Genomics; VUS, Variant of Uncertain Significance; AFF, atypical femoral fracture. *AFF11 is homozygous for the variant p.Pro555Leu

Interestingly, variants in COL1A1 and COL1A2 were detected in control individuals, suggesting their putative involvement in the underlying osteoporosis phenotype. The same could be proposed for the FKBP10 and TNXB genes, which were found mutated in controls and AFF patients. Out of 33 genetic variants identified in women with AFF, eleven (33.3%) were found in genes belonging to the Wnt pathway (LRP5, LRP6, DAAM2, WNT1, and WNT3A). Moreover, the AFF11 woman was homozygous for a mutation in DAAM2. It is difficult to discern whether these genes play a role in the pathophysiology of AFF in addition to their known role in low bone mass.

Subject AFF1 did not carry any mutations from our list of candidate genes while all other AFF patients were carriers of variants in more than one gene. For example, subject AFF11 accumulated up to 9 rare genetic variants, one of them in homozygosity (DAAM2). Unfortunately, there is no information about this latter variant in the ClinVar database. Interestingly, Daam2 KO mice showed a marked reduction in bone strength, despite minimal changes in bone morphology and mineral content, indicating an abnormal bone composition and structure explained in part by cortical impairment(7).

While none of our patients could be confidently diagnosed of any monogenic bone disorder, like most of the Dutch AFF cohort(5), all shared the common feature of severe postmenopausal osteoporosis that required long-term BP treatment and, in some cases, also denosumab. Noteworthy, fifty percent of the women with AFF in our cohort and none of the controls had been on long-term glucocorticoid treatment which is a recognized risk factor for AFF(9). When comparing both cohorts according to the gene candidate list proposed by Zhou et al. (see Table 1) and considering all AFF patients with or without a clinical suspicion of monogenic bone disorders, a similar number of carriers of variants in Mendelian bone disease genes (46% in Zhou et al. vs 50% in this cohort) was detected.

Encompassing all of our findings we conclude that in our cohort we did not detect a major gene involved in AFF pathophysiology. We speculate that AFF development is probably the result of the sum of genetic variants together with other structural, physiological and environmental factors. It is also important to note that osteoporosis, rare bone diseases, and AFFs can share the same genes, therefore making the identification of unique risk factors even more challenging.

These results warrant further studies of genes related to monogenic bone disorders in the setting of severe osteoporosis, in addition to their potential role in AFF pathogenesis.

Ethics approval and consent to participate

The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Clinical Research Ethics Committee of Parc de Salut Mar (CEIC; 2017/7717/I).

Consent for publication

Not applicable

Availability of data and materials

The datasets generated and/or analysed during the current study are not publicly available due extensive genomic information but are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

This research was funded by Centro de Investigación Biomédica en Red de Fragilidad y Envejecimiento Saludable (grant number CB16/10/00245); Fundación Española de Investigación Ósea y del Metabolismo Mineral (FEIOMM); Fondo de Investigación en Salud (grant number PI19/00033) from Instituto de Salud Carlos III (ISCIII) and European Union Fund. The research was also supported by MCIN/AEI/10.13039/501100011033, Project PID2019-107188RB-C21, and AGAUR. DO is recipient of a Miquel Servet grant from ISCIII.

Authors' contributions

Conceptualization: N.G.G., D.G., S.B. and R.R.; Patient recruiting and phenotyping: D.O., S.C. and X.N.; Methodology: N.G.G., D.G., S.B., and R.R.; Validation: N.G.G. and R.R.; Formal Analysis: N.G.G. and R.R.; Investigation: N.G.G., D.G., S.B., and R.R.; Data Curation: all authors; Writing—Original Draft Preparation, N.G.G. and D.O.; Writing—Review & Editing: All authors; Supervision: S.B., D.G., X.N. and R.R.; Project Administration: N.G.G.; Funding Acquisition: N.G.G., X.N., S.C., S.B. and D.G. All authors have reviewed and agreed to the published version of the manuscript.

Acknowledgements

Not applicable

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No competing interests reported.

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You are reading this latest preprint version

Assessing the contribution of genes involved in monogenic bone disorders to the etiology of atypical femoral fractures

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