Differential aging of growth plate cartilage underlies differences in bone length and thus helps determine skeletal proportions

<div><p>Bones at different anatomical locations vary dramatically in size. For example, human femurs are 20-fold longer than the phalanges in the fingers and toes. The mechanisms responsible for these size differences are poorly understood. Bone elongation occurs at the growth plates and advances rapidly in early life but then progressively slows due to a developmental program termed “growth plate senescence.” This developmental program includes declines in cell proliferation and hypertrophy, depletion of cells in all growth plate zones, and extensive underlying changes in the expression of growth-regulating genes. Here, we show evidence that these functional, structural, and molecular senescent changes occur earlier in the growth plates of smaller bones (metacarpals, phalanges) than in the growth plates of larger bones (femurs, tibias) and that this differential aging contributes to the disparities in bone length. We also show evidence that the molecular mechanisms that underlie the differential aging between different bones involve modulation of critical paracrine regulatory pathways, including insulin-like growth factor (Igf), bone morphogenetic protein (Bmp), and Wingless and Int-1 (Wnt) signaling. Taken together, the findings reveal that the striking disparities in the lengths of different bones, which characterize normal mammalian skeletal proportions, is achieved in part by modulating the progression of growth plate senescence.</p></div

Disparities in chondrocyte proliferation, chondrocyte hypertrophy, and bone growth rate between shorter and longer bones.

<p>(A) Fluorescent images of proximal tibias, distal femurs, distal metacarpals, and proximal forelimb phalanges from mice at various postnatal ages. Rate of longitudinal bone growth was determined from the distance (vertical red bars) between the chondro-osseous junction (white dotted line) and the calcein-labeled (fluorescent green) bone. DAPI was used for counterstain. Scale bar, 200 μm. (B) Masson Trichrome–stained histological sections of hypertrophic zone of proximal tibias, distal femurs, distal metacarpals, and proximal forelimb phalanges, from C57BL/6 mice at E17.5 and various postnatal ages. Hypertrophic cell height diminished earlier in the metacarpals and phalanges. Scale bar, 30 μm. (C) Quantitative histological measurements of TH cell height (upper panel) and number of BrdU-labeled cells per column in the proliferative zone (middle panel), and rate of bone growth measured by calcein-labeling (lower panel). (D) Position-specific BrdU labeling indices of proliferative zone of proximal tibias, distal femurs, distal metacarpals, and proximal forelimb phalanges in 1- and 4-week-old mice. Cell position 1 denotes the proliferative chondrocyte closest to the resting zone, and black arrow indicates the cell position where the proliferative zone ends and the pre-hypertrophic region starts. Position-specific BrdU labeling indices at other time points are depicted in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005263#pbio.2005263.s004" target="_blank">S4 Fig</a>. Raw values for Fig 1C and 1D are available in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005263#pbio.2005263.s020" target="_blank">S1 Data</a>. BrdU, 5-bromo-2-deoxyuridine; E17.5, embryonic day 17.5; TH, terminal hypertrophic.</p

Differences in specific paracrine signaling pathways contribute to disparities in growth plate function between different bones.

<p>RNA-Seq was used to identify genes differentially expressed between proximal tibia and proximal phalanx (>2-fold, FDR < 0.05, both species, age 1 week). (A) Gene ontology and signaling pathway analyses showed enrichment for developmental-related functions and identified pathways previously implicated in growth plate biology. (B, C) Heatmaps were generated by hierarchical clustering of the 150 genes that showed the greatest differential expression between tibia and phalanx. Scale bar represents log₂ (fold differences). (D–F) Specific genes from IGF (panel D), BMP (panel E), and Wnt (panel F) signaling pathways that showed significant differential expression between 1-week tibia and phalanx by RNA-Seq were selected for validation by qPCR and to determine time course. (G, H) When cultured in monolayer, primary mouse chondrocytes isolated from tibias showed higher Igf2 expression (panel G, light green bars) and more rapid proliferation (panel H, open light green bars) than those from phalanges. Proliferation of phalangeal chondrocytes, but not tibial chondrocytes, responded positively to exogenous Igf1 (panel H, striped light green bars). When treated with siRNA against Igf2 (no exogenous Igf1; panel G, dark green bars), proliferation was inhibited (versus control) in tibial chondrocytes but not phalangeal chondrocytes (panel H, open dark green bars). This inhibition was partially reversed by exogenous Igf1 (panel H, striped dark green bar). (I) Western blot showed higher levels of p-SMAD1/5/9 in tibial/femoral growth plate chondrocytes than metacarpal/phalangeal chondrocytes, implying more active BMP signaling in the longer bones. (J, K) Neonatal mouse tibias and metatarsals were treated with Bmp2 or Noggin in culture for 3 days, followed by histological examination for chondrocyte hypertrophy. Scale bar, 50 μm. <i>N</i> = 8–10; horizontal line represents sample means. Raw values for Fig 4D–4H, and K are available in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005263#pbio.2005263.s020" target="_blank">S1 Data</a>. BMP, bone morphogenetic protein; FDR, false discovery rate; IGF, insulin-like growth factor; pSMAD1/5/9, phosphorylated SMAD1/5/9; qPCR, quantitative PCR; RNA-Seq, RNA sequencing; siRNA, small interfering RNA; Wnt, Wingless and Int-1.</p

Differences in bone length arise in part from modulation of the developmental program of growth plate senescence.

<p>(A) Senescent changes in gene expression in the growth plate, including genes encoding many paracrine signaling molecules, are more advanced in the shorter bones. (B) Age-independent differences in gene expression within key regulatory pathways also contribute to differences in growth rate. (C) Consequently, the developmental program of growth plate senescence, including structural involution and declines in proliferation and hypertrophic cell size, is more advanced in shorter bones. (D) Differences in proliferation rate and hypertrophic cell size result in disparities in the rate of bone elongation and thus cumulative bone length. Bmp, bone morphogenetic protein; GP, growth plate; IGF, insulin-like growth factor; Igfbp, IGF-binding protein; Wif, Wnt inhibitory factor; Wnt, Wingless and Int-1.</p

Growth plate senescence-associated changes in gene expression are more advanced in the phalangeal growth plates than in tibial growth plates.

<p>RNA-Seq was performed on laser capture micodissected PZ or HZ of mouse or rat proximal tibias (at age 1 and 4 weeks) or proximal phalanges (at 1 week). (A, B) Positive correlation (mouse PZ, <i>p</i> < 1 × 10⁻⁴⁴; mouse HZ, <i>p</i> < 1 × 10⁻¹³⁹; rat PZ, <i>p</i> < 1 × 10⁻²⁵⁴; rat HZ, <i>p</i> < 1 × 10⁻²⁸¹; Pearson’s correlation) between senescent changes (log₂[fold differences]) in tibial growth plate (1 week versus 4 weeks, >4-fold; FDR < 0.05) and differential expression between bones at 1 week (tibia versus phalanx >4-fold; FDR < 0.05), in PZ (panel A) and HZ (panel B) of both species. (C–F) Venn diagram depicting numbers of genes with significant changes (>4-fold; FDR < 0.05) in expression between 1 and 4 weeks of age in tibial growth plates and/or significant differences between tibia and phalanx at 1 week of age. Overlaps were greater than expected by chance (chi-squared, <i>p</i> < 0.0001) in both zones of mouse (panel C and E) and rat (panel D and F). (G, H) Heatmap of genes showing senescence-associated changes in expression (1 versus 4 weeks in tibia; >2-fold, FDR < 0.05, in both species; 200 genes in PZ, 129 genes in HZ). The senescence-associated changes correlated with differential expression between 1-week phalanx and tibia, suggesting that senescence-associated changes are more advanced in phalanges than tibias (<i>R</i>, Pearson’s correlation coefficient). Scale bar represents log₂(fold differences). (I, J) qPCR in a subset of genes showed that changes in gene expression (mRNA normalized to 18S RNA) began before 1 week of age and confirmed that changes in the phalanges tended to be more advanced than in tibias. <i>p</i>-Values for age and type of bone. Raw values are available in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005263#pbio.2005263.s020" target="_blank">S1 Data</a>. exp, number of overlapping genes expected by chance; FDR, false discovery rate; HZ hypertrophic zone; NS, not significant; obs, number of observed overlapping genes; P, phalanx; PZ, proliferative zone; qPCR, quantitative PCR; RNA-Seq, RNA sequencing; T, tibia; w, week.</p

Effect of Genetic Diagnosis on Patients with Previously Undiagnosed Disease

BackgroundMany patients remain without a diagnosis despite extensive medical evaluation. The Undiagnosed Diseases Network (UDN) was established to apply a multidisciplinary model in the evaluation of the most challenging cases and to identify the biologic characteristics of newly discovered diseases. The UDN, which is funded by the National Institutes of Health, was formed in 2014 as a network of seven clinical sites, two sequencing cores, and a coordinating center. Later, a central biorepository, a metabolomics core, and a model organisms screening center were added.MethodsWe evaluated patients who were referred to the UDN over a period of 20 months. The patients were required to have an undiagnosed condition despite thorough evaluation by a health care provider. We determined the rate of diagnosis among patients who subsequently had a complete evaluation, and we observed the effect of diagnosis on medical care.ResultsA total of 1519 patients (53% female) were referred to the UDN, of whom 601 (40%) were accepted for evaluation. Of the accepted patients, 192 (32%) had previously undergone exome sequencing. Symptoms were neurologic in 40% of the applicants, musculoskeletal in 10%, immunologic in 7%, gastrointestinal in 7%, and rheumatologic in 6%. Of the 382 patients who had a complete evaluation, 132 received a diagnosis, yielding a rate of diagnosis of 35%. A total of 15 diagnoses (11%) were made by clinical review alone, and 98 (74%) were made by exome or genome sequencing. Of the diagnoses, 21% led to recommendations regarding changes in therapy, 37% led to changes in diagnostic testing, and 36% led to variant-specific genetic counseling. We defined 31 new syndromes.ConclusionsThe UDN established a diagnosis in 132 of the 382 patients who had a complete evaluation, yielding a rate of diagnosis of 35%. (Funded by the National Institutes of Health Common Fund.)