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Human overgrowth disorders are characterized by excessive prenatal and/or postnatal growth of various tissues. These disorders often present with tall stature, macrocephaly, and/or abdominal organomegaly and are sometimes associated with additional phenotypic abnormalities such as intellectual disability and increased cancer risk. As the genetic etiology of these disorders have been elucidated, a surprising pattern has emerged. Multiple monogenic overgrowth syndromes result from variants in epigenetic regulators: variants in histone methyltransferases NSD1and EZH2cause Sotos syndrome and Weaver syndrome, respectively, variants in DNA methyltransferase DNMT3Acause Tatton-Brown-Rahman syndrome, and variants in chromatin remodeler CHD8cause an autism spectrum disorder with overgrowth. In addition, very recently, a variant in histone reader protein SPIN4was identified in a new X-linked overgrowth disorder. In this review, we discuss the genetics of these overgrowth disorders and explore possible common underlying mechanisms by which epigenetic pathways regulate human body size.

Recombinant human growth hormone (GH) is commonly used to treat short stature in children. However, GH treatment has limited efficacy, particularly in severe, non-GH-deficient conditions such as chondrodysplasias, and potential off-target effects. Because short stature results from decreased growth plate chondrogenesis, we developed a cartilage-targeting single-chain human antibody fragment (CaAb) aiming to deliver therapeutic molecules to the growth plate, thereby increasing treatment efficacy while minimizing adverse effects on other tissues. To this end, we created fusion proteins of these CaAbs conjugated with insulin-like growth factor 1 (IGF-1), an endocrine and/or paracrine factor that positively regulates chondrogenesis. These CaAb-IGF-1 fusion proteins retained both cartilage binding and IGF-1 biological activity, and they were able to stimulate bone growth in an organ culture system. Using a GH-deficient (lit) mouse model, we found that subcutaneous injections of these CaAb-IGF-1 fusion proteins increased overall growth plate height without increasing proliferation in kidney cortical cells, suggesting on-target efficacy at the growth plate and less off-target effect on the kidney than IGF-1 alone. Alternate-day injections of these fusion proteins, unlike IGF-1 alone, were sufficient to produce a therapeutic effect. Our findings provide proof of principle that targeting therapeutics to growth plate cartilage can potentially improve treatment for childhood growth disorders.

Weaver syndrome is characterized by tall stature, advanced bone age, characteristic facies, and variable intellectual disability. It is caused by heterozygous mutations in enhancer of zeste homolog 2 (EZH2), a histone methyltransferase responsible for histone H3 at lysine 27 (H3K27) trimethylation. However, no early truncating mutations have been identified, suggesting that null mutations do not cause Weaver syndrome.

Growth plate chondrocytes undergo sequential differentiation to form the resting zone, the proliferative zone (PZ), and the hypertrophic zone (HZ). The important role of microRNAs (miRNAs) in the growth plate was previously revealed by cartilage-specific ablation of Dicer, an enzyme essential for biogenesis of many miRNAs. To identify specific miRNAs that regulate differentiation of PZ chondrocytes to HZ chondrocytes, we microdissected individual growth plate zones from juvenile rats and performed miRNA profiling using a solution hybridization method and miRNA sequencing. Thirty-four miRNAs were differentially expressed between the PZ and the HZ, and we hypothesized that some of the miRNAs that are preferentially expressed in the PZ may promote proliferation and inhibit hypertrophic differentiation. Consistent with this hypothesis, transfection of inhibitors for four of these miRNAs (mir-369-3p, mir-374-5p, mir-379-5p, and mir-503-5p) decreased proliferation in primary epiphyseal chondrocytes. The inhibitors for three of these miRNAs (mir-374-5p, mir-379-5p, and mir-503-5p) also increased expression of multiple genes that are associated with chondrocyte hypertrophic differentiation. We next hypothesized that preferential expression of these miRNAs in the PZ is driven by the parathyroid hormone-related protein (PTHrP) concentration gradient across the growth plate. Consistent with this hypothesis, treatment of primary chondrocytes with a parathyroid hormone (PTH)/PTHrP receptor agonist, PTH1-34, increased expression of mir-374-5p, mir-379-5p, and mir-503-5p. Taken together, our findings suggest that the PTHrP concentration gradient across the growth plate induces differential expression of mir-374-5p, mir-379-5p, and mir-503-5p between the PZ and the HZ. In the PZ, the higher expression levels of these miRNAs promote proliferation and inhibit hypertrophic differentiation. In the HZ, downregulation of these miRNAs inhibits proliferation and promotes hypertrophic differentiation.

Body growth is rapid in infancy but subsequently slows and eventually ceases due to a progressive decline in cell proliferation that occurs simultaneously in multiple organs. We previously showed that this decline in proliferation is driven in part by postnatal down-regulation of a large set of growth-promoting genes in multiple organs. We hypothesized that this growth-limiting genetic program is orchestrated by microRNAs (miRNAs). Bioinformatic analysis identified target sequences of the miR-29 family of miRNAs to be overrepresented in age–down-regulated genes. Concomitantly, expression microarray analysis in mouse kidney and lung showed that all members of the miR-29 family, miR-29a, -b, and -c, were strongly up-regulated from 1 to 6 weeks of age. Real-time PCR confirmed that miR-29a, -b, and -c were up-regulated with age in liver, kidney, lung, and heart, and their expression levels were higher in hepatocytes isolated from 5-week-old mice than in hepatocytes from embryonic mouse liver at embryonic day 16.5. We next focused on 3 predicted miR-29 target genes (Igf1, Imp1, and Mest), all of which are growth-promoting. A 3′-untranslated region containing the predicted target sequences from each gene was placed individually in a luciferase reporter construct. Transfection of miR-29 mimics suppressed luciferase gene activity for all 3 genes, and this suppression was diminished by mutating the target sequences, suggesting that these genes are indeed regulated by miR-29. Taken together, the findings suggest that up-regulation of miR-29 during juvenile life drives the down-regulation of multiple growth-promoting genes, thus contributing to physiological slowing and eventual cessation of body growth.

Background:In juvenile mammals, the epiphyses of long bones grow by chondrogenesis within the articular cartilage. A better understanding of the molecular mechanisms that regulate the growth of articular cartilage may give insight into the antecedents of joint disease, such as osteoarthritis.Methods:We used laser capture microdissection to isolate chondrocytes from the superficial, middle, and deep zones of growing tibial articular cartilage in the 1-wk-old mouse and then investigated expression patterns by microarray. To identify molecular markers for each zone of the growing articular cartilage, we found genes showing zone-specific expression and confirmed by real-time PCR and in situ hybridization.Results:Bioinformatic analyses implicated ephrin receptor signaling, Wnt signaling, and bone morphogenetic protein signaling in the spatial regulation of chondrocyte differentiation during growth. Molecular markers were identified for superficial (e.g., Cilp, Prg4), middle (Cxcl14, Tnn), and deep zones (Sfrp5, Frzb). Comparison between juvenile articular and growth plate cartilage revealed that the superficial-to-deep zone transition showed similarity with the hypertrophic-to-resting zone transition.Conclusion:Laser capture microdissection combined with microarray analysis identified novel signaling pathways that are spatially regulated in growing mouse articular cartilage and revealed similarities between the molecular architecture of the growing articular cartilage and that of growth plate cartilage.