X-Linked Osteogenesis Imperfecta Possibly Caused by a Novel Variant in PLS3.

Osteogenesis imperfecta (OI) represents a complex spectrum of genetic bone diseases that occur primarily due to mutations and deletions of the COL1A1 and COL1A2 genes. Recent molecular studies of the network of signaling pathways have contributed to a better understanding of bone remodeling and the pathogenesis of OI caused by mutations in many other genes associated with normal bone mineralization. In this paper, a case of a rare X-linked variant of OI with a change in the gene encoding plastin 3-a protein important for the regulation of the actin cytoskeleton, is presented. A 16-year-old patient developed ten bone fractures caused by minor trauma or injury, including a compression fracture of the second lumbar vertebra during his lifetime. Next-generation sequencing analysis did not show pathologically relevant deviations in the COL1A1 and COL1A2 genes. Targeted gene analyses (Skeletal disorder panel) of the patient, his father, mother and sister were then performed, detecting variants of uncertain significance (VUS) for genes PLS3, FN1 and COL11A2. A variant in the PLS3 gene were identified in the patient, his mother and sister. Since the PLS3 gene is located on the X chromosome, the mother and sister showed no signs of the disease. Although the variant in the PLS3 gene (c.685G>A (p.Gly229Arg)) has not yet been described in the literature, nor is its pathogenicity known, clinical findings combined with genetic testing showed that this variant may explain the cause of X-linked OI in our patient. This rare case of the PLS3 variant of X-linked OI might point to a novel target for personalized therapy in patients with this severe disease.

1. Introduction

Osteogenesis imperfecta (OI) is a clinically and genetically heterogeneous group of diseases that is inherited in an autosomal dominant, autosomal recessive, and X-linked manner [1,2]. This hereditary skeletal dysplasia manifests with three main clinical hallmarks: bone fragility ("brittle bone disease"), skeletal deformities and growth deficiency [2,3]. The clinical presentation of OI is variable in its severity. In addition to the skeletal findings, it can affect multiple organ systems and cause secondary complications. The most common secondary features include macrocephaly and dental abnormalities, blue sclerae, hearing loss, respiratory and cardiopulmonary complications [4]. The traditional types of OI (types I-IV) are inherited in an autosomal dominant manner and encompass about 80–85% of OI cases [2]. These cases are caused by pathogenic variants in exons of the genes that encode type I collagen, which is essential for normal extracellular matrix (ECM) function [5]. Previous studies have shown that in addition to pathogenic variants within exons, intronic region variants can cause splice-defective COL1A1 transcripts that also manifest with symptoms of OI [6,7]. Dysfunction of the molecular mechanisms that regulate bone mineralization, formation of ECM and normal osteoblast differentiation plays a key role in the pathogenesis of OI [2,8]. Osteocytes control bone turnover by regulating the activity of both osteoclasts and osteoblasts, and they enable constant remodeling of the extracellular matrix. The imbalance of osteoclast-mediated bone resorption and osteoblast-mediated bone formation can result in either loss or gain of bone mass [9]. In recent years, it has become clear that bone remodeling is regulated by several signaling pathways. The main pathways included in the regulation of bone formation are the Hedgehog (HH), parathyroid hormone-related peptide (PTHrP), fibroblast growth factor (FGF), C-type natriuretic peptide (CNP), transforming growth factor-beta (TGFβ), bone morphogenetic protein (BMP), Notch, WNT and osteocyte mechanosensing pathway [10,11,12]. Any disruption of genes whose protein products are involved in the molecular network of these signaling pathways can cause changes in bone remodeling and poor mineralization. Dysregulation of these pathways disrupts the mechanisms that control skeletal strength and integrity, leading to bone fragility associated with reduced bone mass [11].

Due to the discovery of a large number of new genes involved in the pathogenesis of OI, new classifications of this disease have been made based not only on the clinical but also on the molecular characteristics of OI. A classification by Marini et al. based on the molecular etiopathogenesis of the disease classifies OI by defects in collagen synthesis, bone mineralization, collagen modification and processing, and defects in osteoblast differentiation [2]. The majority of OI cases (type I-IV) are associated with reduced production of normal type I collagen or the synthesis of abnormal collagen as a result of pathogenic variants in COL1A1 and COL1A2 genes [3]. Pathogenic variants in the IFITM5 (OI type V) and SERPINF1 (OI type VI) genes are responsible for deficiencies in bone mineralization [13]. Among the genes responsible for OI with deficits in collagen modification and processing are CRTAP (type VII), LEPREI (type VIII), PPIB (type IX), SERPINH1 (type X), FKBP10 (type XI) and BMP1 (type XII). Pathogenic variants in genes SP7 (type XIII), TMEM38B (type XIV), WNT1 (type XV), CREB3L1 (type XVI), SPARC (type XVII) and MBTPS2 (type XVIII) cause defects in osteoblast differentiation [2].

Recently discovered cases of OI have been associated with pathogenic variants in an X-chromosome gene coding for plastin 3 (PLS3). The clinical presentation in hemizygous men matched the presentation of classical OI and was variable in heterozygous women [14]. The main role of PLS3 is in F-actin-binding, which consequently suggests that PLS3 participates in all processes dependent on F-actin dynamics, such as cell motility, cell division, focal adhesion, endocytosis, neurotransmission, vesicle trafficking, axonal local translation, and intracellular calcium PLS3-dependent processes [15,16]. Plastins are proteins with a single polypeptide chain composed of two tandem repeats of actin-binding domains (ABD1 and ABD2). Each ABD is assembled from two tandem calponin-homology (CH) domains (CH1 and CH2 in ABD1, and CH3 and CH4 in ABD2) [17]. The binding of each ABD to two separate actin filaments promotes the formation of a bundle resulting in distinct F-actin organization [16].

In the present study, a novel PLS3 variant in a nonconsanguineous family of a proband with X-linked OI was detected and potential link between this variant of plastin 3 and osteogenesis imperfecta was reviewed.

2. Materials and Methods

2.1. Subjects

A 16-year-old patient (proband), who complained of pain, pathological fractures and patellar subluxations that arose due to a moderate valgus of the distal femur, was admitted to St. Catherine’s Special Hospital. The patient was accompanied by his father, mother and sister, who had no symptoms related to diseases of the locomotor system and who were all included in the study. All participants involved in this study signed an informed consent form.

2.2. Clinical Examination

Clinical data were collected, including fracture history, height, weight, growth speed and family history. Additionally, the sclera, teeth, hearing and musculoskeletal system were checked upon clinical examination. Blood analysis was performed to determine serum calcium, inorganic phosphates, osteocalcin and vitamin D (25-OH) in all patients. Moreover, biochemical findings of creatinine and deoxypyridinoline were obtained from urine. The results of the analysis were interpreted according to the reference intervals of the Laboratory of the Special Hospital St. Catherine. Bone densitometry was performed in the whole family, which included the measurement of bone density of the proximal femur and spine, whose reference values were validated according to the Croatian population. The proband received high-dose vitamin D therapy (25,000 IU/day) for three months. To evaluate the effect of the therapy, we measured markers of bone remodeling and bone mineral density (BMD) measured by densitometry.

2.3. Genetic Testing

Genomic DNA was isolated from the patient’s blood sample and subjected to clinical next-generation sequencing using a multi-gene panel. Invitae Skeletal Disorders Panel includes sequence analysis and deletion/duplication testing of 320 genes was conducted (Table 1). All target genes were sequenced to a minimum depth of ≥50× and an average of 350×. Sequence reads were aligned with the reference genome (GRCh37) and single nucleotide variants (SNVs) were called from coding sequences and 20 bp of flanking intronic sequences. Promoters and other non-coding regions were not included. Exon-level copy number (deletions and duplications) and other types of non-SNV variants were identified using validated algorithms [18,19].

Table 1

A complete list of genes analyzed, including the relevant gene transcripts.

GENE	TRANSCRIPT	GENE	TRANSCRIPT	GENE	TRANSCRIPT	GENE	TRANSCRIPT
ACAN	NM_013227.3	CDKN1C	NM_000076.2	DHCR24	NM_014762.3	C2CD3	NM_015531.5
ACP5	NM_001111035.2	CDT1	NM_030928.3	DIP2C	NM_014974.2	CA2	NM_000067.2
ACVR1	NM_001105.4	CENPJ	NM_018451.4	DLL3	NM_016941.3	CANT1	NM_138793.3
ADAMTS10	NM_030957.3	CEP120	NM_153223.3	DLX3	NM_005220.2	CASR	NM_000388.3
ADAMTS17	NM_139057.3	CEP135	NM_025009.4	DMRT2	NM_006557.6	CCDC8	NM_032040.4
AFF4	NM_014423.3	CEP152	NM_014985.3	DNA2	NM_001080449.2	CDC45	NM_001178010.2
AGA	NM_000027.3	CEP63	NM_025180.3	DONSON	NM_017613.3	CDC6	NM_001254.3
AGPS	NM_003659.3	CFAP410	NM_004928.2	DVL1	NM_004421.2	FN1	NM_212482.2
AIFM1	NM_004208.3	CHST14	NM_130468.3	DVL3	NM_004423.3	FTO	NM_001080432.2
ALPL	NM_000478.5	CHST3	NM_004273.4	DYM	NM_017653.3	FUCA1	NM_000147.4
AMER1	NM_152424.3	CHUK	NM_001278.4	DYNC2H1	NM_001080463.1	FZD2	NM_001466.3
ANKH	NM_054027.4	CLCN7	NM_001287.5	DYNC2LI1	NM_016008.3	GALNS	NM_000512.4
ANO5	NM_213599.2	COG1	NM_018714.2	EBP	NM_006579.2	GALNT3	NM_004482.3
ARCN1	NM_001655.4	COL10A1	NM_000493.3	EIF2AK3	NM_004836.6	GDF5	NM_000557.4
ARSB	NM_000046.3	COL11A1	NM_001854.3	ESCO2	NM_001017420.2	GDF6	NM_001001557.2
ARSE	NM_000047.2	COL11A2	NM_080680.2	EVC	NM_153717.2	GHR	NM_000163.4
ASCC1	NM_001198800.2	COL1A1	NM_000088.3	EVC2	NM_147127.4	GHRHR	NM_000823.3
ASPM	NM_018136.4	COL1A2	NM_000089.3	EXOC6B	NM_001321729.1	GHSR	NM_198407.2
ATR	NM_001184.3	COL27A1	NM_032888.3	EXOSC2	NM_014285.6	CSGALNACT1	NM_001130518.1
B3GALT6	NM_080605.3	COL2A1	NM_001844.4	EXT1	NM_000127.2	CSPP1	NM_024790.6
B3GAT3	NM_012200.3	COL9A1	NM_001851.4	EXT2	NM_207122.1	CTSA	NM_000308.3
B4GALT7	NM_007255.2	COL9A2	NM_001852.3	EXTL3	NM_001440.3	CTSK	NM_000396.3
BGN	NM_001711.5	COL9A3	NM_001853.3	FAM20C	NM_020223.3	CUL7	NM_014780.4
BMP1	NM_006129.4	COMP	NM_000095.2	FAM46A	NM_017633.2	CWC27	NM_005869.3
BMP2	NM_001200.3	CREB3L1	NM_052854.3	FAR1	NM_032228.5	DDR2	NM_006182.2
BMPER	NM_133468.4	CRTAP	NM_006371.4	FBN1	NM_000138.4	DDRGK1	NM_023935.2
BMPR1B	NM_001203.2	CSF1R	NM_005211.3	FGF23	NM_020638.2	IFT43	NM_052873.2
IFT52	NM_001303458.2	IFT122	NM_052985.3	LIG4	NM_002312.3	MYH3	NM_002470.3
IFT57	NM_018010.3	IFT140	NM_014714.3	LMNA	NM_170707.3	MYO18B	NM_032608.6
IFT74	NM_001099222.1	IFT172	NM_015662.2	LMX1B	NM_002316.3	NAGLU	NM_000263.3
IFT80	NM_020800.2	IDUA	NM_000203.4	LONP1	NM_004793.3	NANS	NM_018946.3
IFT81	NM_014055.3	IFITM5	NM_001025295.2	LOXL3	NM_032603.3	NBAS	NM_015909.3
IGF1	NM_000618.4	IFT122	NM_052985.3	LRP4	NM_002334.3	NEK1	NM_012224.2
IGF2	NM_000612.5	IFT140	NM_014714.3	LRP5	NM_002335.3	NEU1	NM_000434.3
IHH	NM_002181.3	IFT172	NM_015662.2	LRRK1	NM_024652.4	NKX3-2	NM_001189.3
IMPAD1	NM_017813.4	IDUA	NM_000203.4	LTBP2	NM_000428.2	NOG	NM_005450.4
FGF9	NM_002010.2	IFITM5	NM_001025295.2	LTBP3	NM_001130144.2	NOTCH2	NM_024408.3
FGFR1	NM_023110.2	PCYT1A	NM_005017.3	MAFB	NM_005461.4	NPPC	NM_024409.3
FGFR2	NM_000141.4	PDE4D	NM_001104631.1	MAN2B1	NM_000528.3	NPR2	NM_003995.3
FGFR3	NM_000142.4	PEX5	NM_001131025.1	MANBA	NM_005908.3	NPR3	NM_000908.3
FIG4	NM_014845.5	PEX7	NM_000288.3	MAP3K7	NM_145331.2	NSDHL	NM_015922.2
FKBP10	NM_021939.3	PGM3	NM_001199917.1	MATN3	NM_002381.4	NSMCE2	NM_173685.2
FLNA	NM_001456.3	PISD	NM_001326411.1	MBTPS2	NM_015884.3	NXN	NM_022463.4
FLNB	NM_001457.3	PKDCC	NM_138370.2	SH3PXD2B	NM_001017995.2	OBSL1	NM_015311.2
MCM5	NM_006739.3	PLK4	NM_014264.4	SLC17A5	NM_012434.4	OCRL	NM_000276.3
MCPH1	NM_024596.4	PLOD2	NM_182943.2	SLC26A2	NM_000112.3	ORC1	NM_004153.3
MEOX1	NM_004527.3	PLS3	NM_005032.6	SLC35D1	NM_015139.2	ORC4	NM_002552.4
MESP2	NM_001039958.1	POC1A	NM_015426.4	SLC39A13	NM_152264.4	ORC6	NM_014321.3
MGP	NM_000900.3	POLR1A	NM_015425.4	SLCO2A1	NM_005630.2	OSTM1	NM_014028.3
MMP13	NM_002427.3	POP1	NM_015029.2	SLCO5A1	NM_030958.2	P3H1	NM_022356.3
MMP14	NM_004995.3	POR	NM_000941.2	SMAD4	NM_005359.5	P4HB	NM_000918.3
MMP2	NM_004530.5	PPIB	NM_000942.4	SMARCAL1	NM_014140.3	PAM16	NM_016069.9
MMP9	NM_004994.2	PPP3CA	NM_000944.4	SNRPB	NM_198216.1	PAPSS2	NM_001015880.1
MNX1	NM_005515.3	PRKAR1A	NM_002734.4	SNX10	NM_001199835.1	PCGF2	NM_007144.2
GJA1	NM_000165.4	PTDSS1	NM_014754.2	SOX9	NM_000346.3	PCNT	NM_006031.5
GLB1	NM_000404.2	PTH1R	NM_000316.2	SP7	NM_001173467.2	TRMT10A	NM_152292.4
GMNN	NM_015895.4	PTHLH	NM_198965.1	SPARC	NM_003118.3	TRPS1	NM_014112.4
GNAS	NM_000516.5	PTPN11	NM_002834.3	SQSTM1	NM_003900.4	TRPV4	NM_021625.4
GNE	NM_001128227.2	PYCR1	NM_006907.3	SRCAP	NM_006662.2	TTC21B	NM_024753.4
GNPAT	NM_014236.3	RAB33B	NM_031296.2	SUCO	NM_014283.4	TUBGCP6	NM_020461.3
GNPTAB	NM_024312.4	RBBP8	NM_002894.2	SULF1	NM_001128205.1	TYROBP	NM_003332.3
GNPTG	NM_032520.4	RECQL4	NM_004260.3	TAB2	NM_015093.5	VAC14	NM_018052.3
GNS	NM_002076.3	RIPPLY2	NM_001009994.2	TAPT1	NM_153365.2	VPS33A	NM_022916.4
GORAB	NM_152281.2	RMRP	NR_003051.3	TBCE	NM_003193.4	WDR19	NM_025132.3
GPC6	NM_005708.3	RNU4ATAC	NR_023343.1	TBX15	NM_152380.2	WDR34	NM_052844.3
GPX4	NM_001039848.2	SFRP4	NM_003014.3	TBX3	NM_005996.3	WDR35	NM_001006657.1
GSC	NM_173849.2	INPPL1	NM_001567.3	TBX5	NM_000192.3	WDR60	NM_018051.4
GUSB	NM_000181.3	JAG1	NM_000214.2	TBX6	NM_004608.3	WISP3	NM_003880.3
GZF1	NM_022482.4	KAT6B	NM_012330.3	TBXAS1	NM_001061.4	WNT1	NM_005430.3
HES7	NM_032580.3	KIAA0586	NM_001244189.1	TCIRG1	NM_006019.3	WNT3	NM_030753.4
HGSNAT	NM_152419.2	KIAA0753	NM_014804.2	TCTEX1D2	NM_152773.4	WNT3A	NM_033131.3
HPGD	NM_000860.5	KIF22	NM_007317.2	TCTN3	NM_015631.5	WNT5A	NM_003392.4
HSPG2	NM_005529.6	KL	NM_004795.3	TGFB1	NM_000660.5	XRCC4	NM_022406.3
HYAL1	NM_153281.1	KMT2A	NM_001197104.1	TMEM165	NM_018475.4	XYLT1	NM_022166.3
IARS2	NM_018060.3	LARP7	NM_016648.3	TMEM38B	NM_018112.2	XYLT2	NM_022167.3
ICK	NM_016513.4	LBR	NM_002296.3	TNFRSF11A	NM_003839.3	SGSH	NM_000199.3
IDS	NM_000202.6	LEMD3	NM_014319.4	TNFRSF11B	NM_002546.3	ROR2	NM_004560.3
IDUA	NM_000203.4	LFNG	NM_001040167.1	TNFSF11	NM_003701.3	RSPRY1	NM_133368.2
IFITM5	NM_001025295.2	LIFR	NM_002310.5	MSX2	NM_002449.4	RTTN	NM_173630.3
SEC24D	NM_014822.3	TRAPPC2	NM_001011658.3	SERPINH1	NM_001235.3	RUNX2	NM_001024630.3
TRIP11	NM_004239.4	TREM2	NM_018965.3	SERPINF1	NM_002615.6	SC5D	NM_006918.4
SETBP1	NM_015559.2	TRIM37	NM_015294.4	ZMPSTE24	NM_005857.4

2.4. Bioinformatics Analysis of Gene Variants of Unknown Significance

Bioinformatics software Sorting Intolerant to Tolerant (SIFT), Polymorphism Phenotyping v2 (PolyPhen-2) and Align-GVGD were used to predict the possible pathogenicity of the gene variants found in proband’s sample. SIFT (https://sift.bii.a-star.edu.sg/) is a tool that predicts the possible impact of an amino acid substitution based on sequence homology and the physical properties of amino acids. PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/) predicts whether an amino acid substitution affects protein function by comparing the physical and chemical properties of amino acids. Align-GVGD (http://agvgd.hci.utah.edu/) is a freely available program that combines the biophysical characteristics of amino acids and protein multiple sequence alignment to predict possible pathogenicity of gene variants.

3. Results

3.1. Phenotypes of the Patients

The proband, a 15-year-old boy (164 cm, 65 kg, BMI = 24.2 kg/m2), was the first child of a nonconsanguineous family. He was delivered vaginally at term with a birth weight of 3500 g. In the maternity ward, he had neonatal jaundice and phototherapy was performed. His psychomotor development was normal. He suffered his first low-trauma fracture (fifth metatarsal bone of his right foot) at the age of 2 years and ten months. Since then, he had experienced a total of 10 fractures, including a fracture of the neck of the left humerus, the radius metaphysis and a compression fracture of the L2 vertebra (Figure 1).

Figure 1

(a) Profile lumbar spine radiograph showing a compression fracture of the L2 vertebral body. The superior endplate is compressed posteriorly, with minor loss of vertebral body height corresponding to grade 1 by Genant classification. (b) Anteroposterior radiograph of the left upper arm showing a spiral fracture of the proximal third of the humerus.

During the physical examination, impaired growth was determined (3rd–15th percentile), while sclerae, dentition and joint laxity were normal. Although the hearing loss was initially observed at age 3, the most recent hearing test showed a normal tympanogram and tonal audiogram. Radiographic images showed inadequate bone mineralization, while densitometry revealed reduced bone density in favor of osteoporosis. Laboratory findings showed decreased levels of vitamin D (25-OH). The serum concentration was normalized after administering an increased dose of vitamin D (25,000 IU). The proband had normal inorganic phosphates, creatinine, ALP, ALT and deoxypyridinoline concentrations with an increased serum concentration of osteocalcin and calcium. He had low BDMs at the lumbar spine of 0.587 g/cm2 (L1-L4 T score −4.3) and left hip of 0.604 g/cm2 (T score −2.8). The proband’s 14-year-old sister from the same parents was healthy (163 cm, 89 kg, BMI = 33.5 kg/m2) with normal hearing, sclerae, dentition and joint laxity. Laboratory findings showed normal serum calcium and osteocalcin levels with significantly reduced inorganic phosphates and vitamin D (25-OH). She had BDMs at the lumbar spine of 0.990 g/cm2 (L1-L4 T score −0.5) and left hip of 0.971 g/cm2 (T score 0.2), which indicate initial osteoporotic changes. The mother of the proband was healthy (155 cm, 70 kg, BMI = 29.1kg/m2). She had normal hearing, sclerae, dentition and joint laxity. Laboratory findings showed normal serum calcium levels, osteocalcin and inorganic phosphates with significantly reduced vitamin D (25-OH). She had BDMs at the lumbar spine of 0.897 g/cm2 (L1-L4 T score −1.4) and left hip of 0.903 g/cm2 (T score −0.3), which indicate initial osteoporotic changes. The mother and her siblings had not yet experienced bone fractures. The proband’s father and his parents were healthy with no history of fractures. More clinical features of the whole family with results of bone remodeling markers measured on 1 September 2021, are shown in Table 2.

Table 2

A complete list of clinical features of the whole family.

	Proband	Sister	Mother	Father
Age (years)	15	14	42	41
Height (cm)	164	163	155	177
Weight (kg)	65	89	70	107
Vertebral compression fractures	1	No	No	No
Long-bone fractures	10	No	No	No
Sclerae	White	White	White	White
Subluxation of the joints	3	No	No	No
Dentinogenesis imperfecta	No	No	No	No
Hearing loss	No	No	No	No
F1 BDM (g/cm2)	0.604	/	/	/
F1 BDM T-score	−2.8	/	/	/
F2 BDM (g/cm2)	0.689	0.971	0.903	1.163
F2 BDM T-score	−2.3	0.2	−0.3	0.9
S1 BDM (g/cm2)	0.587	/	/	/
S1 BDM T-score	−4.6	/	/	/
S2 BDM (g/cm2)	0.622	0.990	0.897	0.916
S2 BDM T-score	−4.3	−0.5	−1.4	−1.6
Ca (mmol/L)	2.67 *	2.61	2.43	2.58 *
Inorganic phosphates (mmol/L)	1.39	0.84 *	1.22	1.07
Osteocalcin (µg/L)	34.0 *	18.3	7.91	3.92
vitamin D (25-OH) (nmol/L)	83	36 *	39 *	75
Creatinine (mmol/L)	12.3	4.9	13.4	15.6
Deoxypyridinoline (nM/mM of creatinine)	9.1	9.9	4.4	5.0

F1—proximal femur densitometry finding on May 20, 2021; F2—proximal femur densitometry finding on 1 September 2021; S1—spine densitometry finding on May 20, 2021; S2—spine densitometry finding on 1 September 2021; *—values outside the reference interval.

3.2. Genetic Findings

Multiplex ligation-dependent probe amplification (MLPA) analysis and next-generation sequencing analysis performed on the COL1A1 and COL1A2 genes excluded pathogenic variants. Subsequent analysis performed using a multi-gene skeletal disorder panel on the proband’s blood sample identified variants of uncertain significance (VUS) in PLS3, FN1 and COL11A2 (Figure 2). These variants were not present in the Genome Aggregation Database (gnomAD), the Exome Aggregation Consortium (ExAC), or Invitae’s in-house variant database.

Figure 2

The pedigree of the family with X-linked osteogenesis imperfecta (OI) and variants of unknown significance (VUS) for the PLS3, NF1, and COL11A2 genes. The proband is marked with an arrow. The proband’s sister, mother and her siblings had no history of fractures. Created with BioRender.com (accessed on 10 November 2021).

The c.4418G>A (p.Arg1473Gln) variant in exon 28 of the FN1 gene was heterozygous in both the mother and the patient, thus reducing the likelihood that it is the cause of the disease. Additionally, we found an amplification of exons 41–66 of the COL11A2 gene in the proband and his healthy father. The exact location of this copy-number change is unknown. The analysis from Invitae suggests that the 5’ breakpoint is likely in intron 40, but the 3’ boundary is difficult to determine as it likely exists beyond the end of the gene.

A c.685G>A (p.Gly229Arg) variant in the X-linked PLS3 gene was identified in the patient, his mother and his sister. This variant has not yet been described in the literature, nor is its pathogenicity known. Furthermore, we confirmed the absence of this variant in the proband’s maternal grandfather, suggesting that the variant either occurred de novo in the proband’s mother or she may have inherited it from the proband’s grandmother.

Along with these observations, our clinical findings indicate that this variant may explain the X-linked OI in the proband. Since the PLS3 gene is located on the X chromosome, the mother and sister were not expected to show pathological fractures if this variant were definitively pathogenic. The results of the genetic analysis performed in this family has led to the discovery of an apparently novel variant in the PLS3 gene, for which the available evidence indicates a favorable likelihood of pathogenicity (Figure 2).

3.3. Bioinformatics Analysis

Algorithms developed to predict the effect of missense changes on protein structure and function showed results in favor of the pathogenicity of the newly discovered variant of the PLS3 gene (Figure 3).

Figure 3

Molecular structure of the PLS3 protein with an indicated mutation in exon 7, which is a part of the CH1 (calponin-homology 1) domain. Created with BioRender.com (accessed on 10 November 2021).

The new variant in the PLS3 gene was characterized as “deleterious” by SIFT software, which predicts whether amino acid substitution affects protein function. A similar result was shown by the bioinformatics tool PolyPhen-2, which classified the change of PLS3 gene (c.685G>A (p.Gly229Arg)) as “probably harmful”.

Align-GVGD, a web-based program that combines the biophysical characteristics of amino acids and protein multiple sequence alignments, classified the new variant of the PLS3 gene as “Class C15”. Prediction groups form a spectrum from C0 to C65, with C0 least likely to interfere with protein function and C65 most likely.

3.4. The Effect of Vitamin D Treatment

Hypovitaminosis was successfully corrected, and there was an improvement in bone density after three months of therapy with increased doses of vitamin D (25,000 IU/day). Bone densitometry confirmed the increase in BMD measured on 1 September 2021, compared to BMD on 20 May 2021 on both the femur (F1 BDM = 0.604; F2 BDM = 0.689) and the spine (S1 BDM = 0.587; S2 BDM = 0.622) (Table 2).

4. Discussion

We identified a Croatian family with X-linked OI caused by a novel missense variant in the PLS3 gene (c.685G>A (p.Gly229Arg)). The proband presented with low bone mass, frequent pathological fractures and occasional subluxations of the patella. His mother and sister were healthy without previous fractures; however, the densitometry findings revealed initial osteoporotic changes. All of the family members had normal hearing, sclerae, dentition and joint laxity. These findings suggest that the variant in PLS3, described now for the first time, may have an impact on the process of bone formation or mineralization, while its role in odontogenesis and the processes associated with the formation of other connective tissue is not clinically noticeable. Interestingly, Hu et al. in their case of X-linked OI showed an entire family that had characteristic blue sclera, while the proband and his family did not have the stated characteristic. Additionally, in their study, it was stated that the mother, as the carrier of the mutation, had normal BMD, while in this case the proband’s sister and mother had reduced BMD [14]. Densitometric findings indicated initial osteoporotic changes visible on the bones of the spine and proximal femur. Such findings suggest a diverse range of clinical phenotypes in women as the process of X chromosome inactivation is random and leads to mosaicism [20].

Studies indicate that pathogenic variants in the PLS3 gene, which encodes plastin 3, play a major role in bone metabolism and lead to severe early osteoporosis [21]. Different variants in PLS3, which is ubiquitously expressed in solid tissues, lead to decreased bone mineral density [22]. Previous findings suggest that the majority of the OI-linked PLS3 pathogenic variants are either loss-of-function changes (nonsense or frameshift varaints) which rarely result in translated protein products due to nonsense-mediated mRNA decay [16,23]. Separately, a rare single nucleotide polymorphism of the PLS3 gene was reported in association with osteoporosis in postmenopausal women [24]. The current identified X-linked PLS3 actin bundling-deficient mutation (L478P) that produces a full-length protein disables actin-binding in the ABD2 and thus prevents F-actin bundling. The bundling-deficient PLS3 fails to co-localize with any F-actin structures in cells despite preserved F-actin binding through a non-mutation-bearing ABD [25]. Our results indicate that the Gly229Arg missense change in exon 7, which encodes a CH1 domain that is a key part of ABD1, may thus cause actin-binding disorders. Based on literature reports, we believe that disease-causing variants in the PLS3 gene are generally loss-of-function. However, there is currently not enough evidence available to determine whether p.Gly229Arg is a loss-of-function variant.

Different variants in PLS3 have shown differences in distribution between lamellipodia and focal adhesions [16]. Studies on the chicken homolog of the PLS3 gene have shown that the function of its protein product can be linked to mechanosensitivity of osteocytes [26]. Dendrites are the most mechanosensitive part of the osteocyte and they are indicators of overall osteocyte mechanosensitivity [27]. Increased PLS3 expression was observed during osteoblast maturation and within osteocyte dendritic processes indicating its role in bone morphogenesis and remodeling [25]. Although other examined mutations that produce a full-length protein have fully retained F-actin bundling ability, it is shown that they have defects in Ca2+ sensitivity. While wild-type PLS3 was distributed equally in lamellipodia and focal adhesions, the Ca2+-hyposensitive PLS3 was localized exclusively at focal adhesions. On the other hand, the Ca2+-hypersensitive PLS3 mutants were bound to lamellipodia. These findings unveiled that severe osteoporosis can be caused by a mutational disruption of the Ca2+-controlled PLS3’s cycling between lamellipodia and focal adhesions [16]. Additionally, it is possible that the PLS3 mutation we found in exon 7 (c.685G>A), which replaces the amino acid glycine with arginine, changes the conformation of PLS3 itself and consequently leads to hypersensitivity or hyposensitivity of the PLS3 protein to calcium. Such a change in calcium sensitivity would lead to misregulation of actin cytoskeleton remodeling and consequently to OI symptoms as found in our patient. Although the exact mechanism of pathogenesis of the novel PLS3 variant we have described here remains unknown, the genetic analysis in the family, absence in population genomic databases, and the in silico predictions suggest that it is very likely a pathogenic variant that causes X-linked OI unrelated to collagenopathies.

Discoveries in the field of bone development and actin cytoskeleton reorganization provide a better understanding of mechanisms by which plastin 3 causes OI [28]. The leading hypotheses include insufficient mineralization by osteoblasts, dysregulation of osteocyte mechanosensing, and increased bone resorption by osteoclasts [16,23,27,29,30]. Previous findings showed that PLS3 actin-bundling activity, as well as finely tuned Ca2+ regulation, are essential for proper bone formation [16]. Different localization of PLS3 within cells is altered by sensitivity to Ca2+, which suggests that fine regulation of PLS3 by Ca2+ is critical for bone formation, as its imbalance in either direction results in OI [25]. Studies on animal models have indicated the importance of PLS3 in bone tissue development and preservation of bone architecture [26]. The mouse knockout model for PLS3 showed decreased bone strength and osteoporosis, while PLS3 knockdown in zebrafish manifests in muscular and skeletal abnormalities [28,30]. In contrast to classical OI caused by COL1A1 and COL1A2 variants or a mutation in the IFITM5 gene (OI type V), which result in hypermineralized bone matrix, defects in PLS3 cause significant hypomineralization of the bone matrix [31,32,33]. Such findings were confirmed by densitometry in our patients (mother, sister and proband), whose bones showed a loss of bone density in support of the diagnosis of osteoporosis. A recent study has proposed a role for PLS3 in osteoclast activity through the regulation of podosomes by nuclear factor κB (NFκB) signaling [30]. Receptor activator of nuclear factor κB ligand (RANKL) signaling inhibits osteoblastic differentiation mainly through activating NFκB as well as inhibiting the β-catenin synthesis and promoting osteoclastogenesis [12]. Since PLS3 represents the major plastin isoform in osteocytes, it could contribute to both osteogenesis and osteolysis [34]. The findings that Ca2+ is involved in the redistribution of PLS3 from focal adhesions to the leading edge represent a strong link between the activities of PLS3 and the machinery thought to drive bone mechanosensing and reorganization [25].

In recent years, the genetic range of diseases associated with osteoporosis has expanded widely and, so far, at least 24 genes have been identified to cause OI [11]. Mechanistic studies in vitro and preclinical mouse models have demonstrated defects in type I collagen processing and crosslinking, post-translational modifications, folding, procollagen transport from rough ER to the Golgi or collagen secretion and structure [11,35]. Moreover, some forms of OI associated with collagen type I deposition and mineralization are caused by mutations in SERPINF1 or IFITM5, while mutations in WNT1 or SP7 are linked to inhibition of chondrocyte differentiation and stimulation of osteoblast differentiation [36,37,38,39]. Other diseases associated with disordered bone formation, in which collagen processing is not affected, have disrupted cellular signaling via the WNT, the RANKL-RANK and the NOTCH2 signaling pathways, which are important in the regulation of bone resorption and formation [11,40,41,42]. The understanding of these pathways through the study of rare bone diseases has opened up new possibilities of specific therapeutic agents for the treatment of common osteoporosis. Many rare fragility disorders remain insufficiently understood and hence drug targets remain undiscovered for future drug development. Acquired bone fragility conditions such as cytokine and glucocorticoid-induced as well as postmenopausal osteoporosis are far more common and new, pathway-specific treatments are still needed [11].

Today, there are numerous therapeutic options for the treatment of OI, including bisphosphonates, denosumab, teriparatide, sclerostin inhibitory antibody, transforming growth factor-beta inhibition, orthopedic management of OI and, among the latest therapies, the use of stem cells [4,43]. Bisphosphonates are the basis of pharmacological treatment and act by inhibiting osteoclast activity and enhancing bone resorption. Current evidence demonstrates that bisphosphonates increase bone mineral density in children and adults with OI and also reduce the risk of fractures [44]. They are most commonly used in pediatric patients because, during growth, they favorably affect the fusion of the vertebrae after compression fractures. However, when using them, care should be taken to avoid side effects that include the acute phase of the infusion reaction and transient hypocalcemia [4,45]. In our case, we showed that high doses of vitamin D improved bone density after three months as evidenced by densitometry findings performed on the proximal femur and lumbar spine. Among the drugs for OI, biological drugs like denosumab are being investigated today. This drug is an antibody for RANKL and inhibits osteoclast differentiation and function [46]. Additionally, like bisphosphonates, it acts on osteoclasts in order to inhibit bone resorption. Several studies have shown that denosumab treatment improves bone mineral density in patients with OI [4]. Another inhibitory antibody (sclerostin) has an inhibitory effect on bone formation via the canonical WNT signaling pathway. Dysregulation of sclerostin expression causes skeletal disorders characterized by loss of bone mass [47]. Romosozumab is a humanized monoclonal antibody that inhibits bone resorption by inhibiting sclerostin and promoting bone formation [48]. The parathyroid hormone analog (teriparatide) induces bone anabolism and stimulates bone formation before it enhances bone resorption in adults with type I OI [49]. Drugs that inhibit transforming growth factor-beta (TGFβ) act on TGFβ signaling, which is extremely important for the formation of the skeleton [50]. Fresolimumab is one of the TGFβ inhibitors and is currently being studied in adult patients with OI [4]. By performing a complex two-part operation in the severe form of OI type III, Jeleč et al. demonstrated that, aside from drug therapy, personalized surgical treatment has an important role in treating OI patients [51]. Recently, mesenchymal stem cells (MSCs) have been suggested as an ideal tool for bone and cartilage regeneration [52]. Research on the treatment of OI is also developing in the direction of MSC transplants. This form of therapy is a personalized treatment that starts before birth or as soon as possible after birth. In this way, it is possible to prevent fractures, which is not possible with any other therapy available today [29].

The limitation of the study was the small number of people we could test for this variant of the PLS3 gene and relate to the clinical phenotype since we found a novel variant never before described in the literature.

5. Conclusions

The normal process of bone remodeling and mineralization is extremely important for the formation of sufficiently strong bones. To improve the treatment of patients with bone dysplasia, further studies of the molecular substrate involved in the regulation of signaling pathways that control bone remodeling are needed. Our discovery of a novel missense variant in the PLS3 gene that is segregating with disease in a Croatian family and present in a hemizygous state in an affected male proband suggests a link to X-linked OI. The protein product of PLS3 participates in the reorganization of the actin cytoskeleton contributes to a better understanding of the involvement of this gene in the pathogenesis of X-linked OI. Future research of the role of plastin 3 in the function of bone remodeling regulation by osteoblasts and osteoclasts will shed light on potential molecular targets in personalized therapy.