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钙化组织国际 2024;114(2): 157–170.在线发布 2023 Dec 3.doi： 10.1007/s00223-023-01162-4
PLS3 Mutations in X-Linked Osteoporosis: Clinical and Genetic Features in Five New Families
X连锁骨质疏松症中的PLS3突变：五个新家族的临床和遗传特征
1 Andreia Martins,1 Catarina Machado,1 Elena Lundberg,2 Ola Nilsson,3,4 Fan Wang,5 Alice Costantini,5 Symeon Tournis,6 Jakob Höppner,7,8 Corinna Grasemann,7 and Outi Mäkitie5,9,10,11阿德里安娜·科斯塔、
1 安德烈娅·马丁斯、 1 卡塔琳娜·马查多、 1 埃琳娜·伦德伯格、 2 奥拉·尼尔森、 3, 4 王凡、 5 爱丽丝·科斯坦蒂尼、 5 西蒙·图尔尼斯、 6 雅各布·霍普纳、 7, 8 科琳娜·格拉斯曼和 7 奥蒂·麦基蒂 5, 9, 10, 11 作者信息 文章注释 版权和许可信息 PMC 免责声明
Abstract 抽象
Childhood-onset osteoporosis is a rare but clinically significant condition. Studies have shown pathogenic variants in more than 20 different genes as causative for childhood-onset primary osteoporosis. The X-chromosomal PLS3, encoding Plastin-3, is one of the more recently identified genes. In this study, we describe five new families from four different European countries with PLS3-related skeletal fragility. The index cases were all hemizygous males presenting with long bone and vertebral body compression fractures. All patients had low lumbar spine bone mineral density (BMD). The age at the first clinical fracture ranged from 1.5 to 13 years old. Three of the identified PLS3 variants were stop-gain variants and two were deletions involving either a part or all exons of the gene. In four families the variant was inherited from the mother. All heterozygous women reported here had normal BMD and no bone fractures. Four patients received bisphosphonate treatment with good results, showing a lumbar spine BMD increment and vertebral body reshaping after 10 months to 2 years of treatment. Our findings expand the genetic spectrum of PLS3-related osteoporosis. Our report also shows that early treatment with bisphosphonates may influence the disease course and reduce the progression of osteoporosis, highlighting the importance of early diagnosis for prompt intervention and appropriate genetic counseling.
儿童期发病的骨质疏松症是一种罕见但具有临床意义的疾病。研究表明，20 多种不同基因的致病变异是儿童期发病的原发性骨质疏松症的致病因素。编码 Plastin-3 的 X 染色体 PLS3 是最近发现的基因之一。在这项研究中，我们描述了来自四个不同欧洲国家的五个新家庭，这些家庭具有与PLS3相关的骨骼脆性。指示病例均为半合子男性，表现为长骨和椎体压缩性骨折。所有患者腰椎骨密度（BMD）均较低。首次临床骨折的年龄范围为 1.5 至 13 岁。已鉴定的PLS3变异中，有3个是止损增益变异，2个是涉及该基因部分或全部外显子的缺失。在四个家庭中，该变异是从母亲那里遗传的。这里报告的所有杂合子女性骨密度正常，没有骨折。4例患者接受双膦酸盐治疗，治疗10个月至2年后，腰椎BMD增加，椎体重塑。我们的研究结果扩展了PLS3相关骨质疏松症的遗传谱。我们的报告还表明，双膦酸盐的早期治疗可能会影响病程并减少骨质疏松症的进展，这凸显了早期诊断对及时干预和适当遗传咨询的重要性。
关键词：早发性骨质疏松症，脆性骨折，儿童骨质疏松症，PLS3
Introduction 介绍
Osteoporosis results in bone fragility and has been typically divided into primary and secondary forms, with osteogenesis imperfecta (OI) representing the prototypical primary osteoporosis in childhood [1, 2]. Unlike secondary osteoporosis, which is often a consequence of a chronic disease, primary childhood-onset osteoporosis is much rarer and often a monogenic disorder that disrupts the normal synthesis and turnover of bone or cartilage [3]. An understanding of the causes of osteoporosis is important for its prevention, diagnosis, and treatment.
骨质疏松症导致骨脆性，通常分为原发性和继发性，成骨不全症（osteogenesis imperfecta， OI）是儿童期原发性骨质疏松症的原型[1,2]。与继发性骨质疏松症不同，继发性骨质疏松症通常是慢性疾病的结果，而原发性儿童期发病的骨质疏松症则更为罕见，通常是一种单基因疾病，会破坏骨骼或软骨的正常合成和更新[3]。了解骨质疏松症的病因对于骨质疏松症的预防、诊断和治疗非常重要。
Currently, the term early-onset osteoporosis (EOOP) refers to osteoporosis occurring in children and young adults. According to the International Society for Clinical Densitometry (ISCD) the diagnosis of EOOP in children requires a clinically significant fracture history and a bone mineral density (BMD) z-score at or below − 2.0 measured at the lumbar spine (LS) or total body. However, the presence of one or more spinal compression fractures in the absence of major trauma constitutes osteoporosis, even if the BMD is normal [4–6].
目前，早发性骨质疏松症（EOOP）一词是指发生在儿童和年轻人中的骨质疏松症。根据国际临床密度测量学会 （ISCD） 的说法，儿童 EOOP 的诊断需要有临床意义的骨折史和在腰椎 （LS） 或全身测量的骨矿物质密度 （BMD） z 评分等于或低于 -2.0。然而，即使骨密度正常，在没有严重创伤的情况下存在1例或多例脊柱压缩性骨折也构成骨质疏松症[4-6]。
In premenopausal women and males < 50 years the diagnosis of EOOP is based on a low BMD, defined as a BMD Z‐score ≤ − 2.0 or T‐score ≤ − 2.5 at the LS or femoral neck, when associating with either fragility fractures or an underlying chronic illness (secondary EOOP) [5, 6]. For men aged 50 and older and postmenopausal women osteoporosis is diagnosed with a DXA BMD T-score below − 2.5 [6].
绝经前女性和50岁<男性，EOOP的诊断基于低BMD，定义为LS或股骨颈的BMDZ评分≤-2.0或T评分≤-2.5，当伴有脆性骨折或基础慢性疾病（继发性EOOP）时[5,6]。对于50岁及以上的男性和绝经后女性，骨质疏松症的诊断为DXA BMD T评分低于−2.5 [ 6]。
During the last 10 years, studies have shown pathogenic variants in more than 20 different genes that can cause childhood-onset primary osteoporosis [7]. PLS3, encoding plastin-3, is one of the more recently identified genes underlying childhood-onset primary osteoporosis. PLS3 (OMIM 300131), which is located on chromosome Xq23 and has 16 exons, codes for the protein plastin-3, which is widely expressed in solid tissues and thought to be involved in cytoskeleton remodeling. Plastin-3 functions as an actin-binding protein participating in the dynamic assembly and disassembly of the actin cytoskeleton. In bone, plastin-3 has been suggested to either be part of the osteocytes’ mechanosensing apparatus or the mineralization process. Moreover, PLS3 has an important role in the development of neuromuscular junctions and some studies are revealing a potential role in muscle fibers [3, 7–11].
在过去10年中，研究表明，20多种不同基因的致病变异可导致儿童期发病的原发性骨质疏松症[7]。编码塑质素-3 的 PLS3 是最近发现的儿童发病原发性骨质疏松症的基发性基因之一。PLS3 （OMIM 300131） 位于染色体 Xq23 上，具有 16 个外显子，编码蛋白 plastin-3，该蛋白在实体组织中广泛表达，被认为参与细胞骨架重塑。Plastin-3 作为一种肌动蛋白结合蛋白，参与肌动蛋白细胞骨架的动态组装和拆卸。在骨骼中，塑质素-3被认为是骨细胞机械感应装置或矿化过程的一部分。此外，PLS3在神经肌肉接头的发育中起着重要作用，一些研究揭示了它在肌肉纤维中的潜在作用[3,7–11]。
Several families worldwide with variable forms of PLS3-related skeletal fragility have been reported. Most of these are frameshift and nonsense mutations, both likely to be followed by nonsense-mediated mRNA decay [1, 6, 7, 11]. Large intragenic deletions or duplications, leading to destroyed gene structure, and splice-site mutations, resulting in altered protein length, are also common [1, 12–16].
据报道，全世界有几个家族具有不同形式的 PLS3 相关骨骼脆性。其中大多数是移码和无义突变，两者都可能伴随着无义介导的mRNA衰变[1,6,7,11]。导致基因结构破坏的大基因内缺失或重复，以及导致蛋白质长度改变的剪接位点突变也很常见[1,12–16]。
In general, due to PLS3’s location on the X chromosome, loss of function affects males more severely than females. Heterozygous female carriers have a variable phenotype from normal to low BMD and may be symptomatic even in childhood. Males with PLS3-related X-linked dominant osteoporosis exhibit a skeletal disorder characterized by compromised bone strength, low bone formation, and defective mineralization [13–15]. This leads to prevalent peripheral and spinal fractures and loss of adult height, usually without the characteristic extra-skeletal manifestations of OI such as joint hyperlaxity, blue sclerae, or dentinogenesis imperfecta. Recent studies indicate that defective PLS3 function leads to severe abnormalities in vertebral morphology already in early childhood and to significant spinal pathology by early adulthood [7, 16].
一般来说，由于PLS3在X染色体上的位置，功能丧失对男性的影响比女性更严重。杂合子女性携带者的表型从正常到低 BMD 不等，甚至在儿童时期也可能出现症状。患有PLS3相关X连锁显性骨质疏松症的男性表现出骨骼疾病，其特征是骨强度受损、骨形成缓慢和矿化缺陷[13-15]。这导致普遍的外周和脊柱骨折以及成人身高下降，通常没有 OI 的特征性骨骼外表现，例如关节过度松弛、蓝色巩膜或牙本质发育不全症。最近的研究表明，PLS3功能缺陷导致儿童早期椎体形态严重异常，成年早期导致严重的脊柱病变[7,16]。
The investigation of rare Mendelian disorders with decreased BMD as a key diagnostic feature constitutes a strategy for identifying genetic determinants of osteoporosis. Moreover, after gene discoveries it is important to further explore the phenotypic features and the natural course of the identified genetic entities. Since relatively few families with PLS3 mutations have so far been described in the literature, the features and genetic variants of PLS3-related osteoporosis have not been fully characterized. In this report, we describe five new families with novel PLS3 variants leading to early-onset primary osteoporosis.
对罕见的孟德尔疾病的调查以BMD降低为关键诊断特征，构成了识别骨质疏松症遗传决定因素的策略。此外，在基因发现之后，进一步探索已鉴定的遗传实体的表型特征和自然过程也很重要。由于迄今为止文献中描述的具有 PLS3 突变的家族相对较少，因此 PLS3 相关骨质疏松症的特征和遗传变异尚未完全表征。在本报告中，我们描述了五个具有导致早发性原发性原发性骨质疏松症的新型 PLS3 变异的新家族。
Methods 方法
Subjects 科目
In this study we describe five new families with PLS3-linked skeletal fragility. The index cases with various PLS3 variants associating with primary osteoporosis were identified in hospitals in four different European countries (Sweden, Greece, Germany, and Portugal).
在这项研究中，我们描述了五个具有 PLS3 相关骨骼脆性的新家族。在四个不同欧洲国家（瑞典、希腊、德国和葡萄牙）的医院中发现了与原发性骨质疏松症相关的各种 PLS3 变体的指示病例。
All index subjects met the ISCD criteria for osteoporosis in children and young adults [4–6]. Secondary causes of osteoporosis were excluded by medical history, clinical examination, and appropriate laboratory analyses.
所有指标受试者均符合ISCD儿童和年轻人骨质疏松症标准[4-6]。骨质疏松症的继发性病因通过病史、临床检查和适当的实验室分析排除。
Data on patients’ clinical diagnoses, diagnostic investigations, and disease course were collected from hospital records. The information was collected separately by the team responsible for each family’s investigation. All index cases underwent clinical examination, blood sampling, skeletal radiography, bone densitometry, and genetic analyses.
从医院记录中收集有关患者临床诊断、诊断检查和病程的数据。这些信息是由负责每个家庭调查的小组单独收集的。所有指示病例均进行临床检查、血样、骨骼X线摄影、骨密度测定和基因分析。
The study was approved by the Research Ethics Committee of Stockholm, Sweden. A written informed consent was obtained from all participants and/or their legal representatives before reporting the data.
该研究得到了瑞典斯德哥尔摩研究伦理委员会的批准。在报告数据之前，已获得所有参与者和/或其法定代表人的书面知情同意书。
Genetic Analysis 基因分析
Various genetic methods were applied to identify the disease-causing gene variant in each family, according to local practices, including single-gene analysis by Sanger sequencing, gene panels using next-generation sequencing, and exome sequencing. Sanger sequencing was also used to confirm PLS3 variants in family members.
根据当地实践，应用各种遗传方法来鉴定每个家族的致病基因变异，包括通过Sanger测序进行单基因分析，使用下一代测序的基因panel和外显子组测序。Sanger 测序也用于确认家族成员中的 PLS3 变异。
Results 结果
Thanks to an international collaboration we identified altogether five families with different PLS3 variants leading to EOOP in one or more family members. We describe the clinical features of the five index patients and the genetic variants identified in altogether ten mutation-positive individuals. The data are also summarized in Table Table11.
由于国际合作，我们总共确定了五个具有不同 PLS3 变体的家族，这些家族导致一个或多个家族成员的 EOOP。我们描述了五名指示患者的临床特征以及在总共十名突变阳性个体中发现的遗传变异。表11也汇总了数据。
Table 1 表1
Clinical characteristics, fracture history, BMD scores at the time of first evaluation from the index patients of five families with a pathogenic variant in the gene encoding plastin-3 (PLS3)
临床特征、骨折史、首次评估时 BMD 评分，来自编码质体蛋白 3 （PLS3） 基因致病变异的 5 个家庭的指示患者
| Index patient | PLS3 mutation | Inheritance pattern | Age at first fracture (years) | Height/BMI (z-score, SD) | Long bone fratures (n) | VCFs | Laboratory and other evaluations | BMD before therapy (z-score, SD) | BMD after therapy (z-score, SD) | Other clinical findings |
|---|---|---|---|---|---|---|---|---|---|---|
| Family 1 | Hemizygous frameshift variant c.1543del (p.Asp515Metfs*11) | XL | 2 | + 2/+ 1 | 8 (Ulna, radius, humerus, tibia, and metacarpals) | T5 C3–C5 | Dental examination noted poor trabecular bone in the jaw bone, but was otherwise normal General endocrine/metabolic laboratory and ophthalmology assessments have been unremarkable | LS − 1.6 (0.538 g/cm2) Total body less head − 0.9 (0.548 g/cm2) | ? (Pamidronate) | Pectus excavatum, flat feet, broad and short thumbs and digit 4–5 syndactyly |
| Family 2 | Hemizygous nonsense variant c.827G>A (pTrp276*) | XL | 13 | − 2/− O.5 | 2 (radius) | L1 | Full laboratory investigations were unremarkable | LS − 3.7 (0.748 g/cm2) FN − 2.9 (0.665 g/cm2) TH − 3.0 (0.678 g/cm2) Radius ultradistal − 5.0 (0.274 g/cm2) | FN − 2.8 (0.681 g/cm2) TH − 2.7 (0.706 g/cm2) (10 months teriparatide) LS − 4.9 (0.612 g/cm2) (15 months teriparatide) | – |
| Family 3 家庭房 3 | Hemizygous nonsense variant c.994_995delGA (p.Asp332*) 半合子无义变异c.994_995delGA （p.Asp332*） | De novo variant | 5 | − 1/− 1 − 1/− 1 | 2 (Tibia, femur) 2（胫骨、股骨） | Multiple vertebral bodies 多个椎体 | Laboratory parameters of bone metabolism were repeatedly within age-appropriate norms 骨代谢的实验室参数反复在适合年龄的标准范围内 | LS − 2.9 | LS − 2.6 (4 months pamidronate) | Pectus carinatum, Growth hormone deficiency |
| Family 4 家庭房 4 | Hemizygous deletion X:114771600-114890428 半合子缺失 X：114771600-114890428 | XL | 1.5 | − 1/− 2 − 1/− 2 | 7 (Forearm, wrist, elbow, and clavicle) 7（前臂、手腕、肘部和锁骨） | L2 | Increased alkaline phosphatase and slightly increased PTH (67 ng/l; normal 15–65 ng/l) with vitamin D deficiency (18.4 μg/l; normal 30–70 μg/l) 碱性磷酸酶升高，PTH略有升高（67 ng/l;正常值15-65 ng/l），维生素D缺乏症（18.4 μg/l;正常值30-70 μg/l） | LS − 3 | LS − 2.67 (2 years pamidronate) | Pectus carinatum, joint hypermobility |
| Family 5 家庭房 5 | Hemizygous deletion X:114644041-115418576 半合子缺失 X：114644041-115418576 | XL | 8 | 0/+ 2 | 0 | From C7 to L5, it spares D1, D2, D10, and L1 从 C7 到 L5，它保留了 D1、D2、D10 和 L1 | Bone resorption marker type 1 collagen N telopeptides in 24-h urine were greatly increased (1194 nM BCE/mM creatinine). General, endocrine/metabolic laboratory investigation were unremarkable 24 小时尿液中骨吸收标志物 1 型胶原 N 端肽显着增加 （1194 nM BCE/mM 肌酐）。一般内分泌/代谢实验室检查无明显异常 | LS − 5 (0.299 g/cm2) FN − 5.6 (0.406 g/cm2) TH − 6.7 (0.320 g/cm2) | LS + 0.7 FN + 0.3 TH − 4.1 (1 year pamidronate) | – |
All index patients are males. According to ISCD criteria, only clinically significant fracture history was considered (minor fractures, such as fingers, were excluded). The reference sequence for the variants described in families 1–3 is NM_005032.6
所有指示患者均为男性。根据 ISCD 标准，仅考虑有临床意义的骨折史（排除手指等轻微骨折）。家族 1-3 中描述的变异的参考序列为 NM_005032.6。
BMI body mass index, BMD bone mineral density, FN femoral neck, LS lumbar spine, TH total hip, VCF vertebral compression fractures, XL X-linked
BMI 体重指数、BMD 骨矿物质密度、FN 股骨颈、LS 腰椎、TH 全髋关节、VCF 椎体压缩性骨折、XL X 连锁
Family 1 家庭房 1
The proband is an 8-year-old Swedish boy referred for investigation due to multiple low-energy fractures. At 2 years of age, he had his first fracture after minimal trauma. By 8 years, he had had eight fractures after minor trauma (ulna, radius, humerus, tibia, and metacarpals).
先证者是一名 8 岁的瑞典男孩，因多处低能量骨折被转诊检查。2岁时，他在轻微创伤后第一次骨折。到 8 岁时，他在轻微创伤后有 8 处骨折（尺骨、桡骨、肱骨、胫骨和掌骨）。
He is the fifth out of six children of healthy European parents and had an unremarkable neonatal and childhood period. None of the parents have a history of skeletal disease. The patient’s maternal uncle, age 38 years, has a history of multiple fractures and severe back pain requiring analgesic medication since young age (Fig. 1), having a clinical diagnosis of Osteoporosis pseudoglioma syndrome since 11 years old.
他是健康欧洲父母的六个孩子中的第五个，新生儿和童年时期平淡无奇。父母均无骨骼疾病史。患者舅舅，38岁，自年轻时有多处骨折和严重背痛，需要镇痛药物治疗（图1），11 岁起临床诊断为骨质疏松症假性胶质瘤综合征。
Families pedigrees: Pedigrees of the five families presented with mutation of the PLS3 gene
家族谱系：出现PLS3基因突变的五个家族的系谱
On physical examination at 8 years, the proband’s height was 142.5 cm (+ 2.0 SD) and body mass index (BMI) 17.9 kg/m2 (+ 1.0 SD). He had pectus excavatum, flat feet, broad and short thumbs and digits 4–5 syndactyly bilaterally. Dental examination with radiography showed poor trabecular bone in the jaw bone, but was otherwise normal.
在 8 岁时体格检查时，先证者的身高为 142.5 cm （+ 2.0 SD），体重指数 （BMI） 为 17.9 kg/m 2 （+ 1.0 SD）。他有漏斗胸、扁平足、宽而短的拇指和双侧并指 4-5 的手指。牙科X线检查显示颌骨小梁骨较差，但其他方面正常。
The BMD measurements showed a Z-score of − 1.6 at lumbar spine (LS). Spine x-ray showed reduced height (approximately 40%) of T5 and slightly wedged vertebral bodies C3–C5 (Fig. 2A).
BMD 测量显示腰椎 （LS） 的 Z 评分为 -1.6。脊柱 X 线显示 T5 高度降低（约 40%），椎体 C3-C5 略微楔形（图 2A）。
Spinal images: collapse and a reduction of height of multiple vertebral bodies: A Family 1 index patient’s x-ray; B Family 2 index patient’s CT; C Family 3 index patient’s MRI; D Family 4 index patient’s MRI; E Family 5 index patient’s MRI
脊柱图像：多个椎体塌陷和高度降低：家庭 1 指数患者的 X 光片;B 家族 2 指数患者的 CT;C 家族 3 指数患者的 MRI;D 家族 4 指数患者的 MRI;E Family 5 指标患者的 MRI
Genetic analysis with a gene panel (Blueprint Genetics) identified a hemizygous frameshift variant in PLS3 [c.1543del (p.Asp515Metfs*11)] in the proband (Fig. (Fig.3).3). This variant was classified as likely pathogenic and was inherited from the unaffected mother who also harbored the same PLS3 variant. This variant is predicted to cause loss of normal protein function through protein truncation and/or nonsense-mediated mRNA decay. The variant is absent in gnomAD and has not been described in the medical literature or reported in any disease-related variant database. The DNA sample of the mother’s brother, who also had osteoporosis, was not available for testing.
使用基因panel（Blueprint Genetics）进行遗传分析，在先证者PLS3 [c.1543del （p.Asp515Metfs*11）]中鉴定出半合移码变异（图1）。（图 3）.3）。该变异被归类为可能致病性，并遗传自未受影响的母亲，该母亲也携带相同的 PLS3 变异。预计该变体会通过蛋白质截断和/或无义介导的 mRNA 衰变导致正常蛋白质功能丧失。该变异在 gnomAD 中不存在，并且尚未在医学文献中描述或在任何与疾病相关的变异数据库中报告。母亲的兄弟也患有骨质疏松症，其DNA样本无法用于检测。
PLS3 variants: Schematic representation of PLS3 the RNA-splicing form NM_005032.6 showing the location of the 5 identified variants. E1-16 = exons 1–16
PLS3 变体：PLS3 的示意图，即 RNA 剪接形式 NM_005032.6，显示了 5 个已鉴定变体的位置。E1-16 = 外显子 1–16
The patient has been treated with bisphosphonates (pamidronate monthly) for the last 10 months and has not experienced any new fractures during this period.
患者在过去 10 个月中一直接受双膦酸盐（每月一次帕米膦酸钠）治疗，在此期间没有出现任何新的骨折。
Family 2 家庭房 2 （Family 2）
This 26-year-old European man was referred due to unstable fracture at L1 vertebra after a low impact trauma, managed with posterior spinal fusion (Fig. 2B). He had a past history of radius fractures, at the age of 13 and 15 years, following sports injury. The physical examination was unremarkable (height 162 cm − 2.0 SD, BMI 23 kg/m2 + 0.5 SD).
这名 26 岁的欧洲男性因 L1 椎骨不稳定骨折在低冲击创伤后被转诊，通过脊柱后路融合术进行治疗（图 2B）。他既往有桡骨骨折史，分别在 13 岁和 15 岁时因运动损伤。体格检查无异常（身高 162 cm − 2.0 SD，BMI 23 kg/m 2 + 0.5 SD）。
He was born from healthy non-consanguineous parents. There was a family history of multiple fractures in his grandfather and his uncle, from his mother’s family arm (Fig. 1).
他出生于健康的非血缘父母。他的祖父和叔叔有多处骨折的家族史，来自他母亲的家族手臂（图1）。
The BMD Z-score (GE, Lunar Prodigy Pro) at the LS was − 3.7. BMD measurements of the family members revealed that his brother, aged 20 years, had low BMD at LS (Z-score − 3.1), FN (Z-score − 2.4), and Radius 33% (Z-score − 3.0). His father had osteopenia at LS (T-score − 2.3) and FN (T-score − 1.5). His sister aged 21 years and his mother aged 46 years had normal BMD, LS Z-score − 0.8 and − 1.7, respectively.
LS的BMD Z值（GE，Lunar Prodigy Pro）为−3.7。家庭成员的 BMD 测量显示，他的兄弟，20 岁，在 LS（Z 评分 -3.1）、FN（Z 评分-2.4）和半径 33%（Z 评分-3.0）时 BMD 较低。他的父亲在LS（T评分-2.3）和FN（T-评分-1.5）时患有骨质减少症。他21岁的姐姐和46岁的母亲BMD正常，LS Z评分分别为-0.8和-1.7。
Sanger sequencing revealed a novel nonsense variant in PLS3, c.827G>A (pTrp276T*), in the index case (RefSeq NM_005032) (Fig. 3). This variant was maternally inherited and the index’s brother also harbored the same variant.
Sanger测序显示，在指示病例（RefSeq NM_005032）中，PLS3中出现了一种新的无义变异，c.827G>A （pTrp276T*）（图3）。该变异是母系遗传的，该指数的兄弟也携带相同的变异。
The proband was started on teriparatide treatment along with calcium and vitamin D. Ten months after teriparatide, BMD Z-score of the FN was − 2.8 and TH − 2.7, increased by 2.4% and 4.1%, respectively, while radius BMD did not change. BMD at LS (0.579 g/cm2/ Z-score − 5.1) decreased by 22%. No fractures or adverse events occurred during the 10-month follow-up. One possible explanation for the substantial decline at the LS (as opposed to the improvement at the hip) is the trajectory of BMD following spinal fusion surgery. Indeed, 5 months later, BMD at the LS increased by 5.7% (Z-score − 4.9).
先证者开始与钙和维生素D一起接受特立帕肽治疗，特立帕肽治疗后10个月，FN的BMD Z评分为-2.8和TH-2.7，分别增加2.4%和4.1%，而半径BMD没有变化。LS的BMD（0.579 g/cm 2 / Z-score − 5.1）下降了22%。在 10 个月的随访期间未发生骨折或不良事件。LS大幅下降（与髋关节改善相反）的一个可能解释是脊柱融合手术后BMD的轨迹。事实上，5 个月后，LS 的 BMD 增加了 5.7%（Z 评分 -4.9）。
Family 3 家庭房 3
The patient is a now 20-year-old man, the only child of a healthy non-consanguineous couple of Russian descent. The parents are of average height without a fracture history (Fig. 1). He presented to pediatric endocrinology at the age of 7 years for evaluation of short stature. At that time, he had suffered a finger fracture after a fall, at the age of 5 years, followed by a fracture of the left femur and of the right tibia due to minor falls. A diagnosis of growth hormone (GH) deficiency was established based on insufficient GH levels in two GH stimulation tests at 9 years of age and he was started on recombinant human GH. GH treatment was discontinued at 17 years when the annual growth rate fell below 3 cm. The final height is 176 cm (− 0.6 SD), which falls in the range of the parental target height and he developed a mild pectus carinatum.
病人是一名现年20岁的男子，是一对健康的俄罗斯血统非血缘夫妇的独生子。父母身高中等，无骨折史（图1）。他在 7 岁时到儿科内分泌科就诊，以评估身材矮小。当时，他在5岁时摔倒后手指骨折，随后因轻微跌倒导致左股骨和右胫骨骨折。根据 9 岁时两次 GH 刺激试验中 GH 水平不足，诊断为生长激素 （GH） 缺乏症，并开始接受重组人 GH 治疗。生长激素治疗在17岁时停止，年生长率降至3厘米以下。最终身高为 176 厘米（-0.6 标准差），落在父母目标身高的范围内，并且他出现了轻度隆突胸。
BMD by DXA at 9 years revealed a Z-score of − 2.8 at the LS. Following the initiation of GH therapy, the Z-score improved to − 2.5 at age 13, but at 16 years, the Z-score had dropped to − 2.9. Magnetic resonance imaging (MRI) revealed collapse of multiple vertebral bodies (Fig. 2C).
9 岁时 DXA 的 BMD 显示 LS 的 Z 评分为 -2.8。在开始GH治疗后，Z评分在13岁时提高到-2.5，但在16岁时，Z评分下降到-2.9。磁共振成像（MRI）显示多个椎体塌陷（图2C）。
Genetic testing showed an X-linked inherited osteoporosis caused by a hemizygous mutation in the PLS3 gene [c.994_995delGA (p.Asp332*)]. This diagnosis was established at the age of 18 years (Fig. 3).
基因检测显示 PLS3 基因半合突变引起的 X 连锁遗传性骨质疏松症 [c.994_995delGA （p.Asp332*）]。该诊断是在 18 岁时确定的（图 3）。
Treatment was started with vitamin D and calcium supplementation and intravenous bisphosphonate (pamidronate). In the 1st year he only received 3 mg/kg body weight of pamidronate. It was intended to administer 9 mg/kg BW for the 1st year. However, due to the onset of the coronavirus disease pandemic in 2020 the follow up visits had to be canceled. Treatment was switched to zoledronate and he received two doses of 4 mg in 6 months’ intervals. At 18 years (after the low dose of pamidronate), the LS BMD had mildly improved with a Z-score − 2.6. At the same time an X-ray of the spine showed normal height of the vertebrae of the thoracic and lumbar spine. During follow-up no fractures occurred.
治疗开始于补充维生素 D 和钙以及静脉注射双膦酸盐（帕米膦酸盐）。在第 1 年，他只接受了 3 mg/kg 体重的帕米膦酸钠。它打算在第 1 年施用 9 mg/kg BW。然而，由于 2020 年冠状病毒病大流行的爆发，后续访问不得不取消。治疗改用唑来膦酸钠，每隔 6 个月接受两剂 4 mg。在 18 岁时（低剂量帕米膦酸钠后），LS BMD 轻度改善，Z 评分 − 2.6。同时，脊柱的X射线显示胸椎和腰椎的椎骨高度正常。随访期间未发生骨折。
Family 4 家庭房 4
The index patient is a currently 17-year-old adolescent of Polish descent treated for X-linked juvenile osteoporosis and a pectus carinatum in the endocrine and bone health clinic of a tertiary university hospital. His height is 170.7 cm (− 1 SD) and the BMI is 17.8 kg/m2 (− 2 SD).
指示患者是一名目前 17 岁的波兰裔青少年，在一所三级大学医院的内分泌和骨骼健康诊所接受 X 连锁青少年骨质疏松症和隆胸治疗。他的身高是 170.7 厘米 （-1 SD），BMI 是 17.8 kg/m 2 （-2 SD）。
The first fracture occurred at the age of 1.5 years and by 8 years of age, six additional fractures (forearm, wrist, elbow -twice- and clavicle) had occurred. At 13 years of age a fall resulted in a vertebral fracture of L2, which required spinal fusion surgery (Fig. 2D) and he was presented to a specialized bone clinic for suspected OI. Physical examination revealed marked hypermobility of the joints and a mild thoracic asymmetry.
第一次骨折发生在 1.5 岁时，到 8 岁时，又发生了 6 处骨折（前臂、手腕、肘部 - 两次 - 和锁骨）。13岁时，一次跌倒导致L2椎体骨折，需要进行脊柱融合手术（图2D），他因疑似OI被送往骨科专科门诊。体格检查显示关节明显过度活动和轻度胸廓不对称。
Family history revealed back pain in the mother, but fractures had never occurred. The patient's maternal grandfather had suffered from fractures sporadically, but due to adequate trauma. The patient has a healthy sister (Fig. 1).
家族史显示母亲背痛，但从未发生过骨折。病人的外祖父曾有过零星的骨折，但创伤足够严重。患者有一个健康的妹妹（图1）。
A DXA scan at 12 years revealed a LS Z-score of − 2.9. A follow up at age 13.5 years showed a Z-score of − 3.0 after the osteosynthesis material was removed. Molecular genetic testing revealed a hemizygous deletion starting before the PLS3 gene and spanning into intron 3 of the same gene (chromosomal coordinates X:114771600-114890428) (Fig. 3). Genetic testing of the mother revealed that she is a heterozygous carrier of the deletion (DXA with LS T-score of − 1).
12 岁时的 DXA 扫描显示 LS Z 评分为 -2.9。13.5 岁时的随访显示，去除合成骨材料后的 Z 评分为 -3.0。分子遗传学检测显示，从PLS3基因之前开始，到同一基因的内含子3（染色体坐标X：114771600-114890428）的半合子缺失（图3）。对母亲的基因检测显示，她是缺失的杂合子携带者（DXA，LS T 评分为 − 1）。
Due to the multiple fractures and reported back pain an intravenous therapy with pamidronate (9 mg/kg BW in the 1st year, 5 mg/kg BW subsequently) was started at the age of 13.5 years. A thoracic x-ray at age 15.5 years showed a loss of height at thoracic vertebra Τ12. The corresponding DXA measurement revealed a LS Z-score of − 3.5 (− 2.7 after adjusting for height). Even though the DXA Z-score did not improve with the therapy, there have been no further fractures since the treatment with bisphosphonates was started.
由于多处骨折和报告的背痛，在 13.5 岁时开始静脉注射帕米膦酸钠（第 1 年为 9 mg/kg BW，随后为 5 mg/kg BW）。15.5 岁时的胸部 X 线检查显示胸椎 Τ12 高度下降。相应的 DXA 测量结果显示 LS Z 分数为 -3.5（调整高度后为 -2.7）。尽管 DXA Z 评分没有随着治疗而改善，但自双膦酸盐治疗开始以来，没有进一步的骨折。
Family 5 家庭房 5
An 11-year-old Portuguese boy, the only child of a healthy Portuguese couple (Fig. 1), has a past history of two finger fractures after a sports injury at the age of 8 years and obesity. At 11 years-old, he was 144.9 cm tall (− 0.07 SD) with a weight of 60.9 kg (+ 2 SD) and BMI of 29 kg/m2 (+ 2 SD). He was admitted to the hospital due to a 3-month progressive worsening of lumbar pain with increasing difficulty to walk due to multiple spinal fractures. There was no history of trauma. The physical examination was unremarkable. His 36-year-old father had lumbar spine osteoporosis but no fractures. The mother is healthy, has no history of bone fractures and has a normal BMD.
一名11岁的葡萄牙男孩是一对健康的葡萄牙夫妇的独生子（图1），在8岁时因运动损伤和肥胖而有两根手指骨折的既往史。11 岁时，他身高 144.9 厘米（− 0.07 SD），体重 60.9 kg （+ 2 SD），BMI 为 29 kg/m 2 （+ 2 SD）。他因腰痛 3 个月进行性加重，多处脊柱骨折导致行走困难增加，入院治疗。没有外伤史。体格检查无异常。他36岁的父亲患有腰椎骨质疏松症，但没有骨折。母亲身体健康，无骨折史，骨密度正常。
An MRI of the spine revealed multiple vertebral body fractures (from C7 to L5, only sparing T1, T2, T10, and L1) with collapse and a height reduction of 25–50% (Fig. 2E). Full body X-ray scan revealed diffuse osteopenia but did not find any other fractures. The DXA revealed a Z-score of − 5.0 in LS. Neurological, cardiological, ENT, and ophthalmological evaluations were normal.
脊柱 MRI 显示多处椎体骨折（从 C7 到 L5，仅保留 T1、T2、T10 和 L1），伴有塌陷和高度降低 25-50%（图 2E）。全身 X 线扫描显示弥漫性骨质减少，但未发现任何其他骨折。DXA 显示 LS 的 Z 分数为 − 5.0。神经、心脏、耳鼻喉科和眼科评估结果正常。
Next-generation sequencing (Illumina) of genomic DNA for 26 genes, including PLS3, was performed (Human All Exon V6, Agilent Technologies) and led to the identification of a novel hemizygous deletion removing the whole PLS3 gene (Fig. 3). Array comparative genomic hybridization, CytoScan 750 K, confirmed the presence of a 775 kb deletion on chromosome X (coordinates X:114644041-115418576) removing the entire PLS3 gene as well as AGTR2, encoding type-2 angiotensin II receptor.
对包括PLS3在内的26个基因的基因组DNA进行了下一代测序（Illumina）（人类全外显子V6，Agilent Technologies），并鉴定出一种新的半合子缺失，去除了整个PLS3基因（图3）。阵列比较基因组杂交 CytoScan 750 K 证实 X 染色体上存在 775 kb 缺失（坐标 X：114644041-115418576），去除了整个 PLS3 基因以及编码 2 型血管紧张素 II 受体的 AGTR2。
A heterozygous variant of unknown significance (VUS) in LRP5 was also identified [NM_002335.3:c.3443C>T(p.Thr1148Ile)]. The patient inherited the PLS3 deletion from his unaffected mother and the LRP5 variant from his father with spinal osteoporosis but no fractures.
LRP5 中还鉴定出一个意义不明的杂合变异 （VUS） [NM_002335.3：c.3443C>T（p.Thr1148Ile）]。患者从未受影响的母亲那里继承了 PLS3 缺失，从患有脊髓骨质疏松症但没有骨折的父亲那里继承了 LRP5 变体。
The patient started quarterly intravenous pamidronate cycles (1 mg/kg/day for 3 days), calcium and vitamin D. After 1 year of treatment the DXA revealed a general increase in all BMD values (Z-score + 0.7 in LS) and regression of all symptoms, with no new fractures.
患者开始每季度静脉注射帕米膦酸盐周期（1 mg/kg/天，持续 3 天）、钙和维生素 D。治疗 1 年后，DXA 显示所有 BMD 值普遍增加（Ls 中 Z 评分 + 0.7）和所有症状消退，无新骨折。
Discussion 讨论
Several PLS3 variants have been reported to date [1–3, 13] as summarized in Table Table2.2. In this article, we identified a total of five pathogenic PLS3 variants in five families with osteoporosis and osteoporotic fractures manifesting already in childhood. Due to PLS3’s location on the X chromosome, PLS3 loss-of-function variants affect males more than female. Of the 28 previously reported index patients (Table (Table2),2), only one was a woman. The phenotype in affected hemizygous males was comparable in all reported patients in included multiple peripheral and vertebral body fractures.
迄今为止，已经报道了几种PLS3变体[1–3,13]，如表2.2所示。在这篇文章中，我们在五个家庭中确定了总共五种致病性PLS3变异，这些家族的骨质疏松症和骨质疏松性骨折已经在儿童时期表现出来。由于 PLS3 位于 X 染色体上，PLS3 功能丧失变异对男性的影响大于女性。在先前报告的28例指示患者中（表（表2），2），只有1例为女性。受累半合子男性的表型在所有报告的患者中具有可比性，包括多发性外周和椎体骨折。
Table 2 表2
Review of genetic and clinical presentation of PLS3 linked early-onset osteoporosis cases reported in literature
文献报道的PLS3相关早发性骨质疏松症病例的遗传和临床表现回顾
| Number of family and References | PLS3 mutation | Age at first fracture/age at study time (years) | Long bone fractures (n) | VCFs | BMD before therapy (z-score, SD) | BMD after therapy (z-score, SD) | Other clinical findings |
|---|---|---|---|---|---|---|---|
| 1 Dijk et al. [1] (6 patients were identified in this family) 1 Dijk et al. [ 1] （该家族共发现 6 例患者） | Single deleterious hemizygous frameshift c.235delT;p.(Tyr79Ilefs*6), in exon 3 外显子 3 中的单个有害半合子移码 c.235delT;p.（Tyr79Ilefs*6） | 2/41 | 13 | Almost all vertebrae 几乎所有的椎骨 | LS − 5.5, FN − 3.4 LS − 5,5，FN − 3,4 | LS − 4.6, FN − 3.1 (2 years bisphosphonate) LS − 4.6， FN − 3.1 （2年双膦酸盐） | None |
| 2 Dijk et al. [1] (2 male patients were identified in this family) 2 Dijk et al. [ 1] （该家族中发现 2 例男性患者） | Nonsense mutation, c.1471C → T;p.(Gln491*), in exon 13 无义突变，c.1471C → T;p.（Gln491*），外显子 13 | ?/34 | 13 | Multiple 倍数 | LS − 3.4/FN − 3.4 LS−3.4/FN-3.4 | – | None |
| 3 Dijk et al. [1] 3 Dijk等人[ 1] | c.321T → A variant in exon 4 c.321T → 外显子 4 的变体 | ?/13 | 6 | – | LS − 1.7, FN − 3.2, TB − 3.7 LS - 1.7，FN - 3.2，TB - 3.7 | – | Split-hand/split-foot malformation type 3 (10q24 duplication); joint hyperlaxity; hearing loss; severe osteoarthritis of mandibular condyles |
| 4 Dijk et al. [1] 4 Dijk等人[ 1] | Splice-site mutation c.748+1G>A in exon 7 外显子 7 中的剪接位点突变 c.748+1G>A | – | – | Yes | – | – | None |
| 5 Dijk et al. [1] 5 Dijk等人[ 1] | Insertion mutation c.759_760 insAAT in exon 8 插入突变 c.759_760 insAAT 在外显子 8 中 | – | Yes | Yes | LS − 2.5 光源 − 2.5 | – | None |
| 6 Dijk et al. [1] 6 Dijk等人[ 1] | Frameshift PLS3 mutation c.1647delC in exon 15 外显子 15 中的移码 PLS3 突变 c.1647delC | – | Yes | Yes | LS − 2.8 光源 − 2.8 | – | None |
| 7 Fahiminiya et al. [17] (2 brothers) 7 Fahiminiya et al. [ 17] （2 兄弟） | Hemizygous frameshift deletion in exon 10 (c.994_995delGA) 外显子 10 中的半合子移码缺失 （c.994_995delGA） | 2–2/7–3 | 4/1 | Multiple thoracic 多胸 | LS − 3.5/− 1.7 LS−3.5/−1.7 | LS − 0.8/LS + 0.03 (6 years alendronate) | Brother 1: clumsy gait and mild spastic cerebral palsy |
| 8 Fahiminiya et al. [17] (2 brothers) 8 Fahiminiya et al. [ 17] （2 兄弟） | Hemizygous missense variant in exon 13 (c.1433T>C) 外显子 13 中的半合子错义变异 （c.1433T>C） | 5–4/6–6 | 2/1 | Multiple thoracic 多胸 | LS − 3.4/− LS 3.3 LS − 3.4/− LS 3.3 | Refuse treatment | None |
| 9 Laine et al. [15] (2 brothers) 9 Laine et al. [ 15] （2 兄弟） | Hemizygous ;splice site mutation (c.73-24T>A) 半合子 ;剪接位点突变 （c.73-24T>A） | 8–8/12–9 | 4/2 | Several thoracic 几个胸腔 | LS − 2.7, TH − 2/LS − 3.1, TH − 1.8 LS−2.7，TH−2/LS−3.1，TH−1.8 | – | None |
| 10 Nishi et al. [18] (2 brothers) | Missense mutation c. 1103C>A in exon 10 (Heterozygous missense variants also in OTOG and USH2A) | 6/11 | Recurrent fractures/1 | – | TB − 5.6/− 4.2 | – | Developmental delay; deafness; inguinal/umbilical hernia; facial dysmorphisms; blue sclerae; small joint laxity |
| 11 Kämpe et al. [7] | Hemizygous nonsense variant (c.766C>T; pArg256*) in exon 8 | 10/30 | 6 | Multiple | 18 ;years old: LS − 4.1, FN − 3.3 | LS − 3.8, FN − 2.8 (1-year zoledronic acid) | Blue sclerae; yellow teeth and loss of enamel; joint hyperlaxity; soft skin; minor aortic valve regurgitation; asthma |
| 12 Kämpe et al. [7] | Missense variant in exon 12 (c.1424A>G; p.N446S) | 6/10 | 3 | 3 | LS − 6.6, TB − 3.5 | No more fractures after start treatment. Increase BMD (1 year pamidronate + zoledronic acid 6/6 months) | Joint hyperlaxity |
| 13 Kämpe et al. [13] (2 brothers) | Deletion of exons 4–16 | 11–7/11–7 | 0 | Multiple in lumbar and thoracic spine | LS − 3.4/LS − 3.4 | – | Brother 1: waddling gait (muscular hypotonia) and facial dysmorphism Brother 2: waddling gait (muscular hypotonia); facial dysmorphism; small joint laxity; opalescent teeth |
| 14 Kämpe et al. [13] | Deletion of the entire gene—the span of the deletion was determined to be ~ 600 kb, affecting two protein-coding genes, PLS3 and AGTR2 and two non-coding RNA genes DANT2 and PLS3-AS1 | 4/12 | 7 | Multiple (all spine) | LS − 3.6 | LS − 0.2, FN − 1.9, and TB − 1.4 (After bisphosphonates—time?) | None |
| 15 Lv et al. [19] | Deletion exon10-exon16 (deletion from intron 9 to 3′UTR) | 2/10 | Multiple | Multiple thoracic and lumbar | LS − 3, FN − 3.4 | LS 2.1, FN − 0.4 (2 years zoledronic acid) | Kyphosis |
| 16 Kannu et al. [3] | 3.411-MB deletion in chromosome region Xq23 (112,419,139–115,830,286) involved 17 RefSeq genes including PLS3 gene | 4/4 | 4 | T4, T9, T12, L2 | LS − 2.1 | LS − 0.1 (2 years pamidronate) | None |
| 17 Kannu et al. [3] | c.1730dup mutation | 2/2 | 2 | T7, L1 | LS − 4 | LS − 2 (2 years pamidronate) | – |
| 18 Costantini et al. [20] (2 patients in the family) | Duplication mutation g.114,848,381_114,860,880dup | ?/21 | 10 metatarsal fractures | Multiple | LS − 3.1 | Improvement in vertebral shape (2 years bisphosphonate) | – |
| 19 Costantini et al. [20] | Frameshift c.1096_1100delAACTT in exon 10 | 2.5/8 | 1 | T5, T6, and T8 | LS − 3.5, TB − 2.2 | LS − 2.3, TB − 0.8 (1 year alendronate) | Blue sclerae; joint hyperlaxity; kyphosis; facial dysmorphism (epicanthic folds, narrow external ear canals, micrognathia, and a high-arched palate) |
| 20 Collet et al. [21] | Frameshift c.1206dup in exon 11 | 13/? | Yes | Yes | LS − 2.3 | – | – |
| 21 Collet et al. [21] | Missense c.1876G>A in exon 18 | 18/? | Yes | Yes | LS − 3.9 | – | – |
| 22 Balasubramania et al. [22] | Hemizygous frameshift mutation deletion, c.1765del in exon 16 | 13/40 | ? | Almost all lower thoracic and lumbar vertebrae | LS − 4.8, TH − 3.6 | – | None |
| 23 Balasubramania et al. [22] | Hemizygous nonsense mutation c.1295T>A in exon 12 | 2/12 | 3 | 7 vertebral lumbar-thoracic | LS − 2.7, TB − 2.6 | LS − 1.4, TB − 1.7 (6 months pamidronate) LS + 1.4 (2.5 years Pamidronate/Zoledronic acid) | None |
| 24 Chen et al. [23] | Nonsense mutation in exon 7 (c.745G>T) | 6/11 | 2 | Multiple thoracic vertebral compression | LS − 1.2, FN − 2.1 | – | Blue sclera |
| 25 Cao et al. [24] | Splice-site mutation c.892-1G>A in intron 8 | 4/11 | 5 | 0 | LS − 1.8, FN − 2.3 | – | None |
| 26 Hu et al. [9] (2 brothers) | Frameshift mutation c.1106_1107insGAAA in exon 10 | 4/12 | 1 | Multiple vertebral | LS − 2, FN − 3.2 | LS + 1, FN + 1 (24 months bisphosphonate) | Blue sclera and kyphosis |
| 27 Wang et al. [2] (3 family members affected) | Nonsense mutation c.244C>T in exon 4 | 7/14 | Yes | No | LS − 2.6, TH − 2.1, TB − 2.3 | – | None |
| 28 Wu et al. [12] (2 brothers) | Hemizygous splicing mutation, c.892-2A>G in intron 8 | 4–4/5–16 | 3 | Multiple lumbar | LS − 3.3, FN − 4.5, TH − 4.5/LS − 1.3, FN − 3.2, TH − 2.4 | Brother 1: LS − 3 (6-month bisphosphonate) | Scoliosis |
Data are presented considering the index patient of each family. All index patients were males, except the proband in family 12
考虑到每个家庭的指示患者，提供数据。除家庭12中的先证者外，所有指示患者均为男性
BMD bone mineral density, FN femoral neck, LS lumbar spine, TB total body, TH total hip, VCF vertebral compression fractures
BMD 骨密度、FN 股骨颈、LS 腰椎、TB 全身、TH 全髋关节、VCF 椎体压缩性骨折
Our report expands the genetic spectrum of PLS3-related osteoporosis. Four of the variants in the present study correspond to novel PLS3 mutations in four different families with skeletal abnormalities. Additionally, the hemizygous nonsense PLS3 variant c.994_995delGA (p.Asp332*) identified in one of the families was previously described in case reports in familiar cases of osteoporosis related to PLS3 mutations [17]. The index cases were all hemizygous males presenting with vertebral body compression fractures. Only one index patient (family number 5) did not present long bone fractures. The phenotype of these patients was comparable to what has been reported in other studies [8, 9, 25]. All patients had low LS BMD and the age at the first clinical fracture ranged from 1.5 to 13 years, as described in Table Table11.
我们的报告扩展了PLS3相关骨质疏松症的遗传谱。本研究中的四个变体对应于四个不同骨骼异常家族中的新型 PLS3 突变。此外，在其中一个家族中发现的半合子无义PLS3变异体c.994_995delGA（p.Asp332*）先前在与PLS3突变相关的骨质疏松症病例的病例报告中曾有过描述[17]。指示病例均为半合子男性，表现为椎体压缩性骨折。只有 1 例指示患者（第 5 家族）没有出现长骨折。这些患者的表型与其他研究报道的表型相当[8,9,25]。所有患者的LS BMD较低，首次临床骨折的年龄为1.5-13岁，如表11所示。
The index patient in family 5 at the age of 8 years had the most severe phenotype showing compression fractures of almost all vertebral bodies with a LS Z-score of − 5. In this case, variants were identified in two different genes, LRP5 and PLS3. A possibility of digenic inheritance should be considered and more studies, namely in other family members, need to be performed to determine the significance of the LRP5 variant, which was classified as a variant of unknown significance.
家庭 5 中 8 岁的指示患者具有最严重的表型，显示几乎所有椎体的压缩性骨折，LS Z 评分为 − 5。在这种情况下，在两个不同的基因LRP5和PLS3中鉴定出变异。应考虑二元遗传的可能性，并需要进行更多的研究，即在其他家庭成员中，以确定 LRP5 变异的意义，LRP5 变异被归类为意义未知的变异。
All heterozygous women reported here had normal bone density and no bone fractures. However, other studies have reported symptomatic osteoporosis and significant spinal compression fractures also in women with heterozygous PLS3 variants [16]. Occasionally, heterozygous females may present with more severe childhood-onset osteoporosis [7]. Therefore, it seems important that females with heterozygous PLS3 variants are detected early in order to carry out an adequate follow up and early medical intervention.
这里报告的所有杂合子女性骨密度正常，没有骨折。然而，其他研究也报道了杂合子PLS3变异型女性的症状性骨质疏松症和显著的脊柱压缩性骨折[16]。有时，杂合子女性可能表现为更严重的儿童期发病的骨质疏松症[7]。因此，早期发现具有杂合子 PLS3 变异的女性似乎很重要，以便进行充分的随访和早期医疗干预。
Regarding treatment, four patients were treated with pamidronate and zoledronate and only one received teriparatide. These differences reflect different clinical practices and experiences, but also the fact that teriparatide is not used to treat pediatric osteoporosis.
在治疗方面，4例患者接受帕米膦酸钠和唑来膦酸钠治疗，仅1例患者接受特立帕肽治疗。这些差异反映了不同的临床实践和经验，但也反映了特立帕肽不用于治疗小儿骨质疏松症的事实。
Considering the increase of BMD after 15 months of teriparatide treatment in the index patient of family 2, we may conclude that PLS3 mutation-related osteoporosis can respond to teriparatide treatment. This is in line with what was shown by Valimaki et al. who demonstrated that after 24-month treatment with teriparatide, all PLS3 mutation-positive patients showed a minor increase in BMD without new clinical fractures [26]. Nevertheless, treatment results are variable, and only very little evidence exists. More studies and long-term follow-up are needed.
考虑到 2 家庭指示患者特立帕肽治疗 15 个月后 BMD 的增加，我们可以得出结论，PLS3 突变相关骨质疏松症对特立帕肽治疗有反应。这与Valimaki等人的研究结果一致，他们证明，在特立帕肽治疗24个月后，所有PLS3突变阳性患者的BMD均略有增加，无新的临床骨折[26]。然而，治疗结果是可变的，只有很少的证据存在。需要更多的研究和长期随访。
Early treatment with bisphosphonates may also positively influence the course of the diseases, since no fractures occurred once patients were started on pamidronate/zoledronate, although the BMD improved only slightly in most cases. Furthermore, as long as vertebral reshaping is partially explained by stabilization of BMD, we may hypothesize that this treatment might be an important factor in vertebral body reshaping in growing patients, as it was possible to see in vertebra imaging progress after treatment in the index patient of family three. A more complete study with a long-term follow-up would be important to understand this phenomenon. More studies are needed as well as long-term evaluation, in order to understand the efficiency of bisphosphonates treatment in PLS3 osteoporosis patients [8].
早期使用双膦酸盐治疗也可能对疾病的进程产生积极影响，因为一旦患者开始使用帕米膦酸钠/唑来膦酸钠，就不会发生骨折，尽管在大多数情况下 BMD 仅略有改善。此外，只要椎体重塑部分由BMD的稳定来解释，我们就可以假设这种治疗可能是成长中患者椎体重塑的重要因素，因为在第三家庭的指示患者治疗后，椎骨成像进展是可能的。进行更完整的研究并进行长期随访对于理解这一现象非常重要。需要更多的研究和长期评估，以了解双膦酸盐治疗PLS3骨质疏松症患者的效率[8]。
Although these observations are in line with the previous reports [11], data still remain scarce and require further studies on the molecular mechanisms leading to severely compromised bone tissue properties in PLS3 mutation carriers. Importantly, our study shows that a genetic diagnosis in the index case often prompted additional investigations in the family, leading to the identification of additional affected relatives. This underscores the importance of timely genetic diagnosis and extensive family history.
尽管这些观察结果与之前的报道一致[11]，但数据仍然稀缺，需要进一步研究导致PLS3突变携带者骨组织特性严重受损的分子机制。重要的是，我们的研究表明，指示病例的基因诊断通常会促使对家庭进行额外的调查，从而识别出其他受影响的亲属。这凸显了及时的基因诊断和广泛的家族史的重要性。
In conclusion, hemizygous mutations in PLS3 cause a monogenic form of X-linked osteoporosis. Affected males often have bone pain and a history of multiple low-impact long bone and vertebral fractures, as well as thorax deformities at diagnosis. Early diagnosis is of utmost importance to prevent fractures, deformities, pain and disability, and requires a careful family history, clinical and radiological evaluation, and timely genetic testing.
总之，PLS3 中的半合子突变会导致单基因形式的 X 连锁骨质疏松症。受累男性在诊断时通常有骨痛和多处低冲击长骨和椎体骨折史，以及胸部畸形。早期诊断对于预防骨折、畸形、疼痛和残疾至关重要，需要仔细的家族史、临床和放射学评估以及及时的基因检测。
Our findings expand the genetic spectrum of PLS3-related osteoporosis and highlight the importance of early diagnosis and early intervention with bisphosphonates for the affected individuals.
我们的研究结果扩展了PLS3相关骨质疏松症的遗传谱，并强调了双膦酸盐早期诊断和早期干预对受影响个体的重要性。
Author Contributions 作者贡献
All authors contributed to the study conception. AC designed the study and prepared the first draft of the paper. AM, CM, EL, ON, FW, AC, ST, JH, CG, OM contributed to the acquisition, analysis and interpretation of data for the work. All authors revised the paper critically for intellectual content and approved the final version. All authors agree to be accountable for the work and to ensure that any questions relating to the accuracy and integrity of the paper are investigated and properly resolved.
所有作者都对研究构思做出了贡献。AC设计了这项研究，并编写了论文的初稿。AM、CM、EL、ON、FW、AC、ST、JH、CG、OM 为这项工作的数据获取、分析和解释做出了贡献。所有作者都对论文进行了批判性的学术内容修改，并批准了最终版本。所有作者都同意对工作负责，并确保与论文的准确性和完整性有关的任何问题都得到调查和妥善解决。
Funding 资金
Open access funding provided by FCT|FCCN (b-on). This study was funded by the ESPE Research Unit Grant, the Academy of Finland, The Sigrid Jusélius Foundation, The Swedish Research Council, Konung Gustaf V:s och Drottning Victorias Frimurarestiftelse, and the Stockholm County Council (OM).
FCT提供的开放获取资金|FCCN （b-on）。这项研究由 ESPE 研究单位资助、芬兰科学院、西格丽德·尤塞利乌斯基金会、瑞典研究委员会、Konung Gustaf V：s och Drottning Victorias Frimurarestiftelse 和斯德哥尔摩县议会 （OM） 资助。
Declarations 声明
Adriana Costa, Andreia Martins, Catarina Machado, Elena Lundberg, Ola Nilsson, Fan Wang, Alice Costantini, Symeon Tournis, Jakob Höppner, Corinna Grasemann, and Outi Mäkitie have no financial or non-financial interests to disclose that are related to this publication.
阿德里安娜·科斯塔、安德烈娅·马丁斯、卡塔琳娜·马查多、埃琳娜·伦德伯格、奥拉·尼尔森、王凡、爱丽丝·科斯坦蒂尼、西蒙·图尔尼斯、雅各布·霍普纳、科琳娜·格拉斯曼和奥蒂·麦基蒂没有与本出版物相关的财务或非财务利益需要披露。
The study was approved by the Research Ethics Committee of Stockholm, Sweden.
该研究得到了瑞典斯德哥尔摩研究伦理委员会的批准。
A written informed consent was obtained from all participants and/or their legal representatives before reporting the data.
在报告数据之前，已获得所有参与者和/或其法定代表人的书面知情同意书。
The authors state that this research was conducted in accordance with the Helsinki Declaration as revised in 2008. No research animals were involved in the study.
作者指出，这项研究是根据2008年修订的《赫尔辛基宣言》进行的。该研究没有涉及研究动物。
Footnotes 脚注
Publisher's Note 出版商注
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
施普林格·自然（Springer Nature）对已出版地图和机构隶属关系中的管辖权主张保持中立。
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