Imbalanced Skeletal Muscle Mitochondrial Proteostasis Causes Bone Loss.

Although microgravity has been implicated in osteoporosis, the precise molecular mechanism remains elusive. Here, we found that microgravity might induce mitochondrial protein buildup in skeletal muscle, alongside reduced levels of LONP1 protein. We revealed that disruptions in mitochondrial proteolysis, induced by the targeted skeletal muscle-specific deletion of the essential mitochondrial protease LONP1 or by the acute inducible deletion of muscle LONP1 in adult mice, cause reduced bone mass and compromised mechanical function. Moreover, the bone loss and weakness phenotypes were recapitulated in skeletal muscle-specific overexpressing ΔOTC mice, a known protein degraded by LONP1. Mechanistically, mitochondrial proteostasis imbalance triggered the mitochondrial unfolded protein response (UPRmt) in muscle, leading to an up-regulation of multiple myokines, including FGF21, which acts as a pro-osteoclastogenic factor. Surprisingly, this mitochondrial proteostasis stress influenced muscle-bone crosstalk independently of ATF4 in skeletal muscle. Furthermore, we established a marked association between serum FGF21 levels and bone health in humans. These findings emphasize the pivotal role of skeletal muscle mitochondrial proteostasis in responding to alterations in loading conditions and in coordinating UPRmt to modulate bone metabolism.

Predicting postoperative mechanical complications with the ethnicity-adjusted global alignment and proportion score in degenerative scoliosis: does paraspinal muscle degeneration matter?

BACKGROUND: The global alignment and proportion (GAP) score was developed to predict mechanical complications (MCs) after adult spinal deformity surgery but showed limited sensitivity in the Asian population. Considering variations in sagittal parameters among different ethnic groups, our team developed the ethnicity-adjusted GAP score according to the spinopelvic parameters of 566 asymptomatic Chinese volunteers (C-GAP score). Notably, degenerative scoliosis (DS) patients with MCs following corrective surgery have more severe paraspinal muscle degeneration. For DS patients with various sagittal alignments, the unevenly distributed degeneration of paraspinal muscle may exert different influences on MC occurrence and largely affect the accuracy of the C-GAP score in clinical assessment. Therefore, incorporating paraspinal muscle degeneration indices within the C-GAP score may improve its accuracy in predicting MC occurrence. PURPOSE: We aimed to clarify the influence of paraspinal muscle degeneration on the C-GAP score predicting MC occurrence following DS surgery and modify the C-GAP score with paraspinal muscle degeneration parameters. STUDY DESIGN: A retrospective case-control study. SAMPLE SIZE: A total of 107 adult degenerative scoliosis patients. OUTCOME MEASURES: Demographic information, postoperative sagittal spinopelvic parameters, the GAP score, the C-GAP score, and paraspinal muscle degeneration parameters. METHODS: A total of 107 DS patients undergoing posterior spinal fusion surgery (≥4 vertebrae) with a minimum of 2 years follow-up (or experiencing MCs within 2 years) were retrospectively reviewed. Their C-GAP score was calculated based on our previous study and patients were divided into 3 C-GAP categories, "proportioned" (P), "moderately disproportioned" (MD), and "severely disproportioned" (SD). Relative cross-sectional area (cross-sectional area of muscle-disc ratio×100, rCSA) and fat infiltration rate, FI% at L1/2, L2/3, L3/4, and L4/5 discs were quantitatively evaluated using magnetic resonance imaging (MRI). In each C-GAP category, patients were additionally divided into the MC group and the non-MC group to analyze their paraspinal muscle degeneration. A multivariable logistic regression model consisting of the CSA-weighted average FI% (total FI%) and the C-GAP score, C-GAPM was constructed. The area under the curve (AUC) of the receiver operating characteristic (ROC) curves was used to evaluate the predictability of the GAP score, the C-GAP score, FI%, and C-GAPM. This project was supported by the National Natural Science Foundation of China (No.82272545) and Special Fund of Science and Technology Plan of Jiangsu Province (No.BE2023658). RESULTS: For all 107 patients, FI% at L1/2, L2/3, L3/4, and L4/5 discs and the total FI% of the MC group (n=32) were significantly higher than those of the non-MC group (n=75). The MC rates of 3 original GAP categories, P, MD, and SD categories were 25.00% (6/24), 27.03%(10/37), and 34.78% (16/46) (χ2=0.944, p=.624). Based on the C-GAP score, the MC rates of the P, MD, and SD categories were 11.90% (5/42), 34.69% (17/49), and 62.50% (10/16), showing significant differences (χ2=15.137, p=.001). In the C-GAP MD category, compared with the non-MC group (n=32), the MC group (n=17) has a higher total FI% (26.16(22.95, 34.00) vs. 22.67(16.39, 27.37)), p=.029). A similar trend was identified in the C-GAP SD category (34.79±11.56 vs. 19.00±5.17, p=.007), but not in the C-GAP P category (25.09(22.82, 32.66) vs. 24.66(17.36, 28.63), p=.361). The AUC of the GAP score, the C-GAP score, the total FI%, and C-GAPM were respectively 0.601, 0.722, 0.716, and 0.772. CONCLUSIONS: Paraspinal muscle degeneration exerts a significant effect on the occurrence of MC in the C-GAP MD, SD instead of P category. The integration of paraspinal muscle FI% with the C-GAP score (C-GAPM) enables a more accurate prediction of MCs following DS surgery. Surgeons should pay adequate attention to paraspinal muscle degeneration during surgical planning and postoperative management for patients in the C-GAP MD and SD categories.

Presence of compensatory curve predicts postoperative curve progression in congenital scoliosis after thoracolumbar hemivertebra resection and short fusion.

PURPOSE: To investigate the impact of preoperative compensatory curve on the postoperative curve progression in congenital scoliosis (CS) patients following thoracolumbar hemivertebra (HV) resection and short fusion. METHODS: This study retrospectively reviewed a consecutive cohort of patients with CS who underwent thoracolumbar HV resection and short fusion with a minimum of 2 years follow-up. According to the preoperative curve pattern, patients were divided into compensatory curve group non-compensatory curve group. Based on the postoperative coronal curve evolution, patients were further divided into the progressed group (Group P, with curve decompensation ≥ 20°) and the non-progressed group (Group NP, characterized by well-compensated curves). RESULTS: A total of 127 patients were included in this study, with 31 patients in the compensatory curve group and 96 patients in the non-compensatory curve group. The incidence of postoperative coronal curve progression was significantly higher in the compensatory curve group than that in non-compensatory curve group (35.5% vs. 13.5%, p = 0.007). In the compensatory curve group, patients who experienced postoperative curve progression showed fewer fusion segments (p = 0.001), greater preoperative UIV translation (p = 0.006), greater preoperative LIV tilt (p = 0.017), and larger postoperative UIV tilt (p < 0.001) compared with patients in group NP. Multiple logistic regression demonstrated that the shorter fusion segments and greater postoperative UIV tilt were two independent risk factors for postoperative curve progression. CONCLUSION: The presence of the compensatory curve was associated with a higher incidence of postoperative curve progression in patients with CS who underwent thoracolumbar HV resection and short fusion. Shorter fusion segments and greater postoperative UIV tilt were found to be the risk factors for postoperative curve progression.

Muscle-bone cross-talk through the FNIP1-TFEB-IGF2 axis is associated with bone metabolism in human and mouse.

Clinical evidence indicates a close association between muscle dysfunction and bone loss; however, the underlying mechanisms remain unclear. Here, we report that muscle dysfunction-related bone loss in humans with limb-girdle muscular dystrophy is associated with decreased expression of folliculin-interacting protein 1 (FNIP1) in muscle tissue. Supporting this finding, murine gain- and loss-of-function genetic models demonstrated that muscle-specific ablation of FNIP1 caused decreased bone mass, increased osteoclastic activity, and mechanical impairment that could be rescued by myofiber-specific expression of FNIP1. Myofiber-specific FNIP1 deficiency stimulated expression of nuclear translocation of transcription factor EB, thereby activating transcription of insulin-like growth factor 2 (Igf2) at a conserved promoter-binding site and subsequent IGF2 secretion. Muscle-derived IGF2 stimulated osteoclastogenesis through IGF2 receptor signaling. AAV9-mediated overexpression of IGF2 was sufficient to decrease bone volume and impair bone mechanical properties in mice. Further, we found that serum IGF2 concentration was negatively correlated with bone health in humans in the context of osteoporosis. Our findings elucidate a muscle-bone cross-talk mechanism bridging the gap between muscle dysfunction and bone loss. This cross-talk represents a potential target to treat musculoskeletal diseases and osteoporosis.

Gut Microbiota Alterations in Adolescent Idiopathic Scoliosis Are Associated with Aberrant Bone Homeostasis.

OBJECTIVE: Low bone mineral density is the major prognostic factor for adolescent idiopathic scoliosis (AIS), but the underlying mechanisms remain unclear. Accumulating evidence suggests that gut microbiota (GM) have the potential to affect bone development, and the GM signatures are altered in AIS patients. However, the effect of GM alterations on aberrant bone homeostasis in AIS remains unclear. This study aims to investigate the GM profile in AIS patients with different bone mineral density (BMD) and explore the association between GM, osteopenia, and aberrant bone turnover. METHODS: A total of 126 patients with AIS who received surgical treatment were retrospectively included in this study. We analyzed the composition of the GM by 16S rRNA sequencing and BMD by dual X-ray absorptiometry. Based on the BMD of the femur neck, the patients were divided into the osteopenia group (OPN) if the Z score < -1, and the normal (NOR) group if the Z score ≥ -1 SD compared to the healthy control. For the 16S rRNA sequencing, the raw reads were filtered to remove low-quality reads, and operational taxonomic units were identified with the Uparse program. Weighted UniFrac distance matrix for the beta-diversity metrics and principal coordinate analysis (PCoA) was performed, and the statistical comparisons were made with permutational multivariate analysis of variance (PERMANOVA) and analysis of similarity (ANONISM). Linear discriminant analysis effect size (LEfSe) was used to identify the enriched species in two groups. The "Random forest" was applied to determine the optimal biomarker for OPN according to the mean decrease in Gini value. The metabolic function was predicted by the Tax4Fun analysis. The Pearson correlation coefficient was used to evaluate the associations between GM species, bone turnover markers, and BMD. RESULTS: The serum ß-CTX was increased in the OPN group (n = 67) compared to the NOR group (n = 59). Patients in OPN groups showed significantly decreased α diversity indicated by the Shannon index. Principal coordinate analysis (PCoA) analysis showed significant clustering of GM between OPN and NOR groups. At genus level, the Escherichia-Shigella and Faecalibacterium were significantly enriched in the OPN group compared to that in the NOR group (p < 0.05), whereas the abundance of Prevotella was significantly decreased (p = 0.0012). The relative abundance of Megamonas and Prevotella was positively correlated with the femur BMD. The abundance of Escherichia-Shigella was negatively correlated with femur BMD and positively correlated with serum ß-CTX levels. Functional analysis revealed significant differences in starch and sucrose metabolism, pyruvate and cysteine, and methionine metabolism between NOR and OPN groups. CONCLUSION: The alterations of GM in AIS patients are correlated with osteopenia. The association between enriched species, BMD, and bone turnover markers provides novel diagnostic and therapeutic targets for the clinical management of AIS.

Development of ethnicity-adjusted global alignment and proportion score to predict the risk of mechanical complications following corrective surgery for adult spinal deformity.

BACKGROUND CONTEXT: Surgery for degenerative scoliosis (DS) is a complex procedure with high complication and revision rates. Based on the concept that pelvic incidence (PI) is a constant parameter, the global alignment and proportional (GAP) score was developed from sagittal alignment data collected in the Caucasian populations to predict mechanical complications. However, the PI varies among different ethnic groups, and the GAP score may not apply to Chinese populations. Thus, this study aims to assess the predictability of the GAP score for mechanical complications in the Chinese populations and develop an ethnicity-adjusted GAP score. PURPOSE: To test the predictability of the original GAP score in the Chinese population and develop a Chinese ethnicity-tailored GAP scoring system. STUDY DESIGN/SETTINGS: Retrospective cohort study. PATIENT SAMPLE: A total of 560 asymptomatic healthy volunteers were enrolled to develop Chinese ethnicity-tailored GAP (C-GAP) score and a total of 114 DS patients were enrolled to test the predictability of original GAP score and C-GAP score. OUTCOME MEASURES: Demographic information, sagittal spinopelvic parameters of healthy volunteers and DS patients were collected. Mechanical complications were recorded at a minimum of 2-year follow-up after corrective surgery for DS patients. METHODS: A total of 560 asymptomatic healthy volunteers with a mean age of 61.9±14.1 years were enrolled to develop ethnicity-adjusted GAP score. Besides, 114 surgically trated DS patients (M/F=10/104) with a mean age of 60.7±7.1 years were retrospectively reviewed. Demographic data and radiological parameters of both groups, including PI, lumbar lordosis (LL), sacral slope (SS), the sagittal vertical axis (SVA), and global tilt (GT) were collected. Ideal LL, SS, and GT were obtained by calculating their correlation with PI of healthy volunteers using linear regression analysis. Relative pelvic version (RPV), relative lumbar lordosis (RLL), lordosis distribution index (LDI), and relative spinopelvic alignment (RSA) were obtained using the ideal parameters, and the Chinese population adjusted GAP score (C-GAP) was developed based on these values. The predictability of original and C-GAP for mechanical failure was evaluated using clinical and radiological data of DS patients by evaluating the area under the curve (AUC) using receiver operating characteristic curve. This study was supported the National Natural Science Foundation of China (NSFC) (No. 82272545), ($ 8,000-10,000) and the Jiangsu Provincial Key Medical Center, and the China Postdoctoral Science Foundation (2021M701677), Level B ($ 5,000-7,000). RESULTS: Ideal SS=0.53×PI+9 (p=.002), ideal LL=0.48×PI+22 (p=.023) and ideal GT=0.46 × PI-9 (p=.011). were obtained by correlation analysis using sagittal parameters from those healthy volunteers, and RPV, RLL, RSA, and LDI were calculated accordingly. Then, the ethnicity-adjusted C-GAP score was developed by summing up the numeric value of calculated RPV, RLL, RSA, and LDI. The AUC was classified as ''no or low discriminatory power'' for the original GAP score in predicting mechanical complications (AUC=0.592, p=.078). Similarly, the original GAP score did not correlate with mechanical complications in DS patients. According to the C-GAP score, the sagittal parameters were proportional in 25 (21.9%) cases, moderately disproportional in 68 (59.6%), and severely disproportional in 21% (18.5%) cases. The incidence of mechanical complications was statistically different among proportioned and moderately disproportional and severely disproportional portions of the C-GAP score (p=.03). The predictability of the C-GAP score is high with an AUC=0.773 (p<.001). In addition, there is a linear correlation between mechanical complication rate and C-GAP score (χ=0.102, p=.02). CONCLUSION: The Ethnicity-adjusted C-GAP score system developed in the current study provided a more accurate and reliable for predicting the risk of mechanical complications after corrective surgery for adult spinal deformity.

FNIP1 abrogation promotes functional revascularization of ischemic skeletal muscle by driving macrophage recruitment.

Ischaemia of the heart and limbs attributable to compromised blood supply is a major cause of mortality and morbidity. The mechanisms of functional angiogenesis remain poorly understood, however. Here we show that FNIP1 plays a critical role in controlling skeletal muscle functional angiogenesis, a process pivotal for muscle revascularization during ischemia. Muscle FNIP1 expression is down-regulated by exercise. Genetic overexpression of FNIP1 in myofiber causes limited angiogenesis in mice, whereas its myofiber-specific ablation markedly promotes the formation of functional blood vessels. Interestingly, the increased muscle angiogenesis is independent of AMPK but due to enhanced macrophage recruitment in FNIP1-depleted muscles. Mechanistically, myofiber FNIP1 deficiency induces PGC-1α to activate chemokine gene transcription, thereby driving macrophage recruitment and muscle angiogenesis program. Furthermore, in a mouse hindlimb ischemia model of peripheral artery disease, the loss of myofiber FNIP1 significantly improved the recovery of blood flow. Thus, these results reveal a pivotal role of FNIP1 as a negative regulator of functional angiogenesis in muscle, offering insight into potential therapeutic strategies for ischemic diseases.

Selecting the Vertebra above Sagittal Stable Vertebra as the Distal Fusion Level in Scheuermann's Kyphosis: A Prospective Study with a Minimum of 2-Year Follow-Up.

OBJECTIVE: The proper selection of the lower instrumented vertebra (LIV) remains controversial in the surgical treatment of Scheuermann's disease and there is a paucity of studies investigating the clinical outcomes of fusion surgery when selecting the vertebra one level proximal to the sagittal stable vertebra (SSV-1) as LIV. The purpose of this study is to investigate whether SSV-1 could be a valid LIV for Scheuermann kyphosis (SK) patients with different curve patterns. METHODS: This was a prospective study on consecutive SK patients treated with posterior surgery between January 2018 and September 2020, in which the distal fusion level ended at SSV-1. The LIV was selected at SSV-1 only in patients with Risser >2 and with LIV translation less than 40 mm. All of the patients had a minimum of 2-year follow-up. Patients were further grouped based on the sagittal curve pattern as thoracic kyphosis (TK, n = 23) and thoracolumbar kyphosis (TLK, n = 13). Radiographic parameters including global kyphosis (GK), lumbar lordosis (LL), sagittal vertical axis (SVA), LIV translation, pelvic incidence (PI), pelvic tilt (PT), and sacral slope (SS) were measured preoperatively, postoperatively, and at the latest follow-up. The intraoperative and postoperative complications were recorded. The Scoliosis Research Society (SRS)-22 scores were performed to evaluate clinical outcomes. RESULTS: A total of 36 patients were recruited in this study, with 23 in the TK group and 13 in the TLK group. In TK group, the GK was significantly decreased from 80.8° ± 10.1° to 45.4° ± 7.7° after surgery, and was maintained at 45.3° ± 8.6° at the final follow-up. While in the TLK group, GK was significantly decreased from 70.7° ± 9.2° to 39.1° ± 5.4° after surgery (p < 0.001) and to 39.3° ± 4.5° at the final follow-up. Meanwhile, despite presenting with different sagittal alignment, significant improvement was observed in LL, SVA, and LIV translation for both TK and TLK groups (p < 0.05). Self-reported scores of pain and self-image in TK group and scores of self-image and function in TLK group showed significant improvement at the final follow-up (all p < 0.05). Distal junctional kyphosis (DJK) was observed in two patients (8.7%) in TK group, and one patient (7.7%) in TLK group. No revision surgery was performed. CONCLUSION: Selecting SSV-1 as LIV can achieve satisfactory radiographic and clinical outcomes for SK patients with different curve patterns without increasing the risk of DJK. This selection strategy could be a favorable option for SK patients with Risser sign >2 and LIV translation less than 40 mm.

The role of paraspinal muscle degeneration in coronal imbalance in patients with degenerative scoliosis.

Background: Coronal imbalance in degenerative scoliosis is common and is highly correlated with health-related quality of life. Paraspinal muscle is critical to spine stability, but little is known about its contribution to coronal imbalance in degenerative scoliosis. This study aims to investigate the relationship between paraspinal muscle (PSM) degeneration and coronal imbalance in patients with degenerative scoliosis (DS). Methods: This is a retrospective cohort study. A total of 117 patients with DS were retrospectively reviewed. Parameters of PSM (bilateral cross-sectional area, CSA; fat infiltration rate, FI%) at the apical disc and adjacent levels were quantitatively evaluated using MRI. Standing whole-spine radiograph was used to evaluate the coronal (Cobb angle, CA; coronal balance distance, CBD) and sagittal (thoracic kyphosis, TK; lumbar lordosis, LL; sagittal vertical axis, SVA) parameters. Patients were divided into 3 groups: coronal balanced (Type A), coronal imbalanced shifting to concavity (Type B), and coronal imbalanced shifting to convexity (Type C). Results: Based on our criteria, 56 patients were assigned to Type A, 34 patients to Type B, and 27 patients to Type C. There was no significant difference on the sagittal profiles and CSA between the groups. However, Type A showed significantly lower FI% than Type B and Type C on both concavity and convexity (Concave side: Type A vs. Type B vs. Type C, 30.8±8.1 vs. 45.1±7.7 vs. 38.7±12.5, P=0.001; Convex side: Type A vs. Type B vs. Type C, 32.6±10.9 vs. 46.3±7.3 vs. 40.7±11.8, P=0.004). Specifically, Cobb angle was negatively correlated with CSA, mainly at convexity (R=-0.415, P=0.008). Similarly, the increase of CBD significantly correlated with FI% at concavity (R=0.491, P=0.001) and convexity (R=0.354, P=0.025). Conclusions: DS patients with coronal imbalance demonstrated a worse PSM degeneration when compared with those without coronal imbalance. Besides, PSM degeneration strongly correlated with coronal imbalance, which implies that PSM degeneration may contribute to the coronal imbalance in patients with DS.

The Establishment of a Mouse Model for Degenerative Kyphoscoliosis Based on Senescence-Accelerated Mouse Prone 8.

Objective: Degenerative kyphoscoliosis (DKS) is a complex spinal deformity associated with degeneration of bones, muscles, discs, and facet joints. The aim of this study was to establish an animal model of degenerative scoliosis that recapitulates key pathological features of DKS and to validate the degenerative changes in senescence-accelerated mouse prone 8 (SAMP8) mice. Methods: Thirty male mice were divided into 2 groups: 10 bipedal C57BL/6J mice were used as the control group, and 20 bipedal SAMP8 mice were used as the experimental group. Mice were bipedalized under general anesthesia. The incidence of scoliosis and bone quality was determined using radiographs and in vivo micro-CT images 4, 8, and 12 weeks after surgery, respectively. Histomorphological studies of muscle samples were performed after sacrifice at 12 weeks after surgery. Results: On the 12th week, the incidence rates of kyphosis in C57BL/6J and SAMP8 groups were 50% and 100%, respectively. Overall, the incidence and angle of kyphosis were significantly higher in the bipedal SAMP8 group compared to the C57BL/6J group (44.7°± 6.2° vs. 84.3°± 10.3°, P<0.001). Based on 3D reconstruction of the entire spine, degeneration of the intervertebral disc was observed in bipedal SAMP8 mice, including the reduction of disc height and the formation of vertebral osteophytes. The bone volume ratio (BV/TV) was significantly suppressed in the bipedal SAMP8 group compared with the bipedal C57BL/6J group. In addition, HE staining and Mason staining of the paraspinal muscle tissue showed chronic inflammation and fibrosis in the muscles of the bipedal SAMP8 group. Conclusions: The SAMP8 mouse model can be taken as a clinically relevant model of DKS, and accelerated aging of the musculoskeletal system promotes the development of kyphosis.

Selection of the optimal distal fusion level for Scheuermann kyphosis with different curve patterns: when can we stop above the sagittal stable vertebra?

PURPOSE: To investigate the optimal lowest instrumented vertebra (LIV) in the treatment of Scheuermann kyphosis (SK) with different curve patterns. METHODS: Fifty-two SK patients who underwent posterior surgery between January 2010 and December 2017 with a minimum follow-up of 2 years were retrospectively reviewed. Patients were divided into two groups based on the curve pattern: the Scheuermann thoracic kyphosis (STK group) or Scheuermann thoracolumbar kyphosis (STLK group). Based on the relationship between the sagittal stable vertebra (SSV) and LIV, both groups were further divided into the SSV group and SSV-1 group. Radiographic parameters, distal junctional kyphosis (DJK) incidence and SRS-22 questionnaire scores were evaluated. RESULTS: In STK and STLK groups, there were no significant differences in most pre- and postoperative radiographic assessments between SSV and SSV-1 subgroups. DJK incidence showed no significant differences between groups during follow-up (P > 0.05). LIV-PSVL was significantly more negative in the SSV-1 group than that in the SSV group (P < 0.001). Within the SSV-1 group, patients with DJK showed a more negative LIV-PSVL (P = 0.039). Moderate correlation was observed between preoperative LIV-PSVL and DJK with a Spearman coefficient of - 0.474 (P = 0.035). Receiver operative characteristic curve analysis showed that the threshold value of preoperative LIV-PSVL to predict DJK was - 37.35 mm (area under the curve 0.882). CONCLUSION: Shorter fusion stopping at SSV-1 achieved comparable clinical outcomes and did not increase the risk of DJK for both STK and STLK patients. For patients whose preoperative LIV-PSVL < - 37.35 mm, extending fusion to SSV is an acceptable solution to prevent DJK.

A Novel FLCN Intragenic Deletion Identified by NGS in a BHDS Family and Literature Review.

Birt-Hogg-Dubé syndrome (BHDS, MIM #135150), caused by germline mutations of FLCN gene, is a rare autosomal dominant inherited disorder characterized by skin fibrofolliculomas, renal cancer, pulmonary cysts and spontaneous pneumothorax. The syndrome is considered to be under-diagnosed due to variable and atypical manifestations. Herein we present a BHDS family. Targeted next generation sequencing (NGS) and multiplex ligation-dependent probe amplification (MLPA) revealed a novel FLCN intragenic deletion spanning exons 10-14 in four members including the proband with pulmonary cysts and spontaneous pneumothorax, one member with suspicious skin lesions and a few pulmonary cysts, as well as two asymptomatic family members. In addition, a linkage analysis further demonstrated one member with pulmonary bullae to be a BHDS-ruled-out case, whose bullae presented more likely as an aspect of paraseptal emphysema. Furthermore, the targeted NGS and MLPA data including our previous and present findings were reviewed and analyzed to compare the advantages and disadvantages of the two methods, and a brief review of the relevant literature is included. Considering the capability of the targeted NGS method to detect large intragenic deletions as well as determining deletion junctions, and the occasional false positives of MLPA, we highly recommend targeted NGS to be used for clinical molecular diagnosis in suspected BHDS patients.