A monoallelic UXS1 variant associated with short-limbed short stature.

BACKGROUND: Serine residues in the protein backbone of heavily glycosylated proteoglycans are bound to glycosaminoglycans through a tetrasaccharide linker. UXS1 encodes UDP-glucuronate decarboxylase 1, which catalyzes synthesis of UDP-xylose, the donor of the first building block in the linker. Defects in other enzymes involved in formation of the tetrasaccharide linker cause so-called linkeropathies, characterized by short stature, radio-ulnar synostosis, decreased bone density, congenital contractures, dislocations, and more. METHODS: Whole exome sequencing was performed in a father and son who presented with a mild skeletal dysplasia, as well as the father's unaffected parents. Wild-type and mutant UXS1 were recombinantly expressed in Escherichia coli and purified. Enzyme activity was evaluated by LC-MS/MS. In vivo effects were studied using HeparinRed assay and metabolomics. RESULTS: The son had short long bones, normal epiphysis, and subtle metaphyseal changes especially in his legs. The likely pathogenic heterozygous variant NM_001253875.1(UXS1):c.557T>A p.(Ile186Asn) detected in the son was de novo in the father. Purified Ile186Asn-UXS1, in contrast to the wild-type, was not able to convert UDP-glucuronic acid to UDP-xylose. Plasma glycosaminoglycan levels were decreased in both son and father. CONCLUSION: This is the first report linking UXS1 to short-limbed short stature in humans.

POU1F1/Pou1f1 c.143-83A > G Variant Disrupts the Branch Site in Pre-mRNA and Leads to Dwarfism.

POU Class 1 Homeobox1 (POU1F1/Pou1f1) is a well-established pituitary-specific transcription factor, and causes, when mutated, combined pituitary hormone deficiency in humans and mice. POU1F1/Pou1f1 has 2 isoforms: the alpha and beta isoforms. Recently, pathogenic variants in the unique coding region of the beta isoform (beta domain) and the intron near the exon-intron boundary for the beta domain were reported, although their functional consequences remain obscure. In this study, we generated mice carrying the Pou1f1 c.143-83A>G substitution that recapitulates the human intronic variant near the exon-intron boundary for the beta domain. Homozygous mice showed postnatal growth failure, with an average body weight that was 35% of wild-type littermates at 12 weeks, which was accompanied by anterior pituitary hypoplasia and deficiency of circulating insulin-like growth factor 1 and thyroxine. The results of RNA-seq analysis of the pituitary gland were consistent with reduction of somatotrophs, and this was confirmed immunohistochemically. Reverse transcription polymerase chain reaction of pituitary Pou1f1 mRNA showed abnormal splicing in homozygous mice, with a decrease in the alpha isoform, an increase in the beta isoform, and the emergence of the exon-skipped transcript. We further characterized artificial variants in or near the beta domain, which were candidate positions of the branch site in pre-mRNA, using cultured cell-basis analysis and found that only c.143-83A>G produced transcripts similar to the mice model. Our report is the first to show that the c.143-83A>G variant leads to splicing disruption and causes morphological and functional abnormalities in the pituitary gland. Furthermore, our mice will contribute understanding the role of POU1F1/Pou1f1 transcripts in pituitary development.

Cell-Dependent Pathogenic Roles of Filamin B in Different Skeletal Malformations.

Mutations of filamin B (FLNB) gene can lead to a spectrum of autosomal skeletal malformations including spondylocarpotarsal syndrome (SCT), Larsen syndrome (LRS), type I atelosteogenesis (AO1), type III atelosteogenesis (AO3), and boomerang dysplasia (BD). Among them, LRS is milder while BD causes a more severe phenotype. However, the molecular mechanism underlying the differences in clinical phenotypes of different FLNB variants has not been fully determined. Here, we presented two patients suffering from autosomal dominant LRS and autosomal recessive vitamin D-dependent rickets type IA (VDDR-IA). Whole-exome sequencing revealed two novel missense variants in FLNB, c.4846A>G (p.T1616A) and c.7022T>G (p.I2341R), which are located in repeat 15 and 22 of filamin B, respectively. The expression of FLNBI2341R in the muscle tissue from our LRS patient was remarkably increased. And in vitro studies showed that both variants led to a lack of filopodia and accumulation of the mutants in the perinuclear region in HEK293 cells. We also found that c.4846A>G (p.T1616A) and c.7022T>G (p.I2341R) regulated endochondral osteogenesis in different ways. c.4846A>G (p.T1616A) activated AKT pathways through inhibiting SHIP2, suppressed the Smad3 pathway, and impaired the expression of Runx2 in both Saos-2 and ATDC5 cells. c.7022T>G (p.I2341R) activated both AKT and Smad3 pathways and increased the expression of Runx2 in Saos-2 cells, while in ATDC5 cells it activated AKT pathways through inhibiting SHIP2, suppressed the Smad3 pathway, and reduced the expression of Runx2. Our study demonstrated the pathogenic mechanisms of two novel FLNB variants in two different clinical settings and proved that FLNB variants could not only directly cause skeletal malformations but also worsen skeletal symptoms in the setting of other skeletal diseases. Besides, FLNB variants differentially affect skeletal development which contributes to clinical heterogeneity of FLNB-related disorders.

Inactivation of Ihh in Sp7-Expressing Cells Inhibits Osteoblast Proliferation, Differentiation, and Bone Formation, Resulting in a Dwarfism Phenotype with Severe Skeletal Dysplasia in Mice.

Indian hedgehog (Ihh) is an indispensable paracrine factor for proper tissue patterning, skeletogenesis, and cellular proliferation. Recent genetic studies have revealed critical roles of chondrocyte-derived Ihh in regulating chondrocyte proliferation, hypertrophy and cartilage ossification. However, the functions of Sp7-expressing cell-derived Ihh in osteoblast differentiation and bone formation remain unclear. Sp7 is an essential transcription factor for osteoblast differentiation. In the current study, we generated Sp7-iCre; Ihhfl/fl mice, in which the Ihh gene was specifically deleted in Sp7-expressing cells to investigate the roles of Ihh. Ihh ablation in Sp7-expressing cells resulted in a dwarfism phenotype with severe skeletal dysplasia and lethality at birth, but with normal joint segmentation. Sp7-iCre; Ihhfl/fl mice had fewer osteoblasts, almost no cortical and trabecular bones, smaller skulls, and wider cranial sutures. Additionally, the levels of osteogenesis- and angiogenesis-related genes, and of major bone matrix protein genes were significantly reduced. These results demonstrated that Ihh regulates bone formation in Sp7-expressing cells. Ihh deficiency in primary osteoblasts cultured in vitro inhibited their proliferation, differentiation, and mineralization ability, and reduced the expression of osteogenesis-related genes. Moreover, the deletion of Ihh also attenuated the Bmp2/Smad/Runx2 pathway in E18.5 tibial and primary osteoblasts. The activity of primary osteoblasts in mutant mice was rescued after treatment with rhBMP2. In summary, our data revealed that Ihh in Sp7-expressing cells plays an indispensable role in osteoblast differentiation, mineralization, and embryonic osteogenesis, further implicated that its pro-osteogenic role may be mediated through the canonical Bmp2/Smad/Runx2 pathway.

Early Life Interventions Can Shape Aging.

It is well documented that the environment of the developing fetus, including availability of nutrients and presence of toxins, can have major impact on adult phenotype, age-related traits and risk of chronic disease. There is also accumulating evidence that postnatal environment can impact adult characteristics related to evolutionary fitness, health, and aging. To determine whether early life hormonal interventions can alter trajectory of aging, we have examined the effects of early life growth hormone (GH) replacement therapy in Prop1df (Ames dwarf) mice which are GH deficient and remarkably long lived. Twice-daily GH injections between the ages of two and eight weeks completely normalized ("rescued") a number of adult metabolic characteristics believed to contribute to extended longevity of these mutants. Importantly, longevity of Ames dwarf mice was reduced by early life GH treatment. This was associated with histone H3 modifications. We conclude that the trajectory of mammalian aging can be modified by early life interventions. Mechanistic links among interventions during postnatal development, adult metabolic characteristics, aging, and longevity, apparently involve epigenetic phenomena.

Deletion of Six3 in post-proliferative neurons produces weakened SCN circadian output, improved metabolic function, and dwarfism in male mice.

OBJECTIVE: The increasing prevalence of obesity makes it important to increase the understanding of the maturation and function of the neuronal integrators and regulators of metabolic function. METHODS: Behavioral, molecular, and physiological analyses of transgenic mice with Sine oculis 3 (Six3) deleted in mature neurons using the Synapsincreallele. RESULTS: Conditional deletion of the homeodomain transcription factor Six3 in mature neurons causes dwarfism and weakens circadian wheel-running activity rhythms but increases general activity at night, and improves metabolic function, without impacting pubertal onset or fertility in males. The reduced growth in 6-week-old Six3fl/fl:Synapsincre (Six3syn) males correlates with increased somatostatin (SS) expression in the hypothalamus and reduced growth hormone (GH) in the pituitary. In contrast, 12-week-old Six3syn males have increased GH release, despite an increased number of the inhibitory SS neurons in the periventricular nucleus. GH is important in glucose metabolism, muscle function, and bone health. Interestingly, Six3syn males have improved glucose tolerance at 7, 12, and 18 weeks of age, which, in adulthood, is associated with increased % lean mass and increased metabolic rates. Further, 12-week-old Six3syn males have reduced bone mineralization and a lower bone mineral density, indicating that reduced GH levels during early life cause a long-term reduction in bone mineralization. CONCLUSION: Our study points to the novel role of Six3 in post-proliferative neurons to regulate metabolic function through SS neuron control of GH release.

Biallelic cGMP-dependent type II protein kinase gene (PRKG2) variants cause a novel acromesomelic dysplasia.

BACKGROUND: C-type natriuretic peptide (CNP), its endogenous receptor, natriuretic peptide receptor-B (NPR-B), as well as its downstream mediator, cyclic guanosine monophosphate (cGMP) dependent protein kinase II (cGKII), have been shown to play a pivotal role in chondrogenic differentiation and endochondral bone growth. In humans, biallelic variants in NPR2, encoding NPR-B, cause acromesomelic dysplasia, type Maroteaux, while heterozygous variants in NPR2 (natriuretic peptide receptor 2) and NPPC (natriuretic peptide precursor C), encoding CNP, cause milder phenotypes. In contrast, no variants in cGKII, encoded by the protein kinase cGMP-dependent type II gene (PRKG2), have been reported in humans to date, although its role in longitudinal growth has been clearly demonstrated in several animal models. METHODS: Exome sequencing was performed in two girls with severe short stature due to acromesomelic limb shortening, brachydactyly, mild to moderate platyspondyly and progressively increasing metaphyseal alterations of the long bones. Functional characterisation was undertaken for the identified variants. RESULTS: Two homozygous PRKG2 variants, a nonsense and a frameshift, were identified. The mutant transcripts are exposed to nonsense-mediated decay and the truncated mutant cGKII proteins, partially or completely lacking the kinase domain, alter the downstream mitogen activation protein kinase signalling pathway by failing to phosphorylate c-Raf 1 at Ser43 and subsequently reduce ERK1/2 activation in response to fibroblast growth factor 2. They also downregulate COL10A1 and upregulate COL2A1 expression through SOX9. CONCLUSION: In conclusion, we have clinically and molecularly characterised a new acromesomelic dysplasia, acromesomelic dysplasia, PRKG2 type (AMDP).

TMEM165 a new player in proteoglycan synthesis: loss of TMEM165 impairs elongation of chondroitin- and heparan-sulfate glycosaminoglycan chains of proteoglycans and triggers early chondrocyte differentiation and hypertrophy.

TMEM165 deficiency leads to skeletal disorder characterized by major skeletal dysplasia and pronounced dwarfism. However, the molecular mechanisms involved have not been fully understood. Here, we uncover that TMEM165 deficiency impairs the synthesis of proteoglycans by producing a blockage in the elongation of chondroitin-and heparan-sulfate glycosaminoglycan chains leading to the synthesis of proteoglycans with shorter glycosaminoglycan chains. We demonstrated that the blockage in elongation of glycosaminoglycan chains is not due to defect in the Golgi elongating enzymes but rather to availability of the co-factor Mn2+. Supplementation of cell with Mn2+ rescue the elongation process, confirming a role of TMEM165 in Mn2+ Golgi homeostasis. Additionally, we showed that TMEM165 deficiency functionally impairs TGFß and BMP signaling pathways in chondrocytes and in fibroblast cells of TMEM165 deficient patients. Finally, we found that loss of TMEM165 impairs chondrogenic differentiation by accelerating the timing of Ihh expression and promoting early chondrocyte maturation and hypertrophy. Collectively, our results indicate that TMEM165 plays an important role in proteoglycan synthesis and underline the critical role of glycosaminoglycan chains structure in the regulation of chondrogenesis. Our data also suggest that Mn2+ supplementation may be a promising therapeutic strategy in the treatment of TMEM165 deficient patients.

Distinct effects of growth hormone deficiency and disruption of hypothalamic kisspeptin system on reproduction of male mice.

Growth hormone (GH) deficiency is a common cause of late sexual maturation and fertility issues. To determine whether GH-induced effects on reproduction are associated with alterations in hypothalamic kisspeptin system, we studied the male reproduction in two distinct GH deficiency mouse models. In the first model, mice present GH deficiency secondary to arcuate nucleus of the hypothalamus (ARH) lesions induced by posnatal monosodium glutamate (MSG) injections. MSG-induced ARH lesions led to significant reductions in hypothalamic Ghrh mRNA expression and consequently growth. Hypothalamic Kiss1 mRNA expression and Kiss1-expressing cells in the ARH were disrupted in the MSG-treated mice. In contrast, kisspeptin immunoreactivity remained preserved in the anteroventral periventricular and rostral periventricular nuclei (AVPV/PeN) of MSG-treated mice. Importantly, ARH lesions caused late sexual maturation and infertility in male mice. In our second mouse model, we studied animals profound GH deficiency due to a loss-of-function mutation in the Ghrhr gene (Ghrhrlit/lit mice). Interestingly, although Ghrhrlit/lit mice exhibited late puberty onset, hypothalamic Kiss1 mRNA expression and hypothalamic kisspeptin fiber density were normal in Ghrhrlit/lit mice. Despite presenting dwarfism, the majority of Ghrhrlit/lit male mice were fertile. These findings suggest that spontaneous GH deficiency during development does not compromise the kisspeptin system. Furthermore, ARH Kiss1-expressing neurons are required for fertility, while AVPV/PeN kisspeptin expression is sufficient to allow maturation of the hypothalamic-pituitary-gonadal axis in male mice.

Phenotypic Characterization of Immortalized Chondrocytes from a Desbuquois Dysplasia Type 1 Mouse Model: A Tool for Studying Defects in Glycosaminoglycan Biosynthesis.

The complexity of skeletal pathologies makes use of in vivo models essential to elucidate the pathogenesis of the diseases; nevertheless, chondrocyte and osteoblast cell lines provide relevant information on the underlying disease mechanisms. Due to the limitations of primary chondrocytes, immortalized cells represent a unique tool to overcome this problem since they grow very easily for several passages. However, in the immortalization procedure the cells might lose the original phenotype; thus, these cell lines should be deeply characterized before their use. We immortalized primary chondrocytes from a Cant1 knock-out mouse, an animal model of Desbuquois dysplasia type 1, with a plasmid expressing the SV40 large and small T antigen. This cell line, based on morphological and biochemical parameters, showed preservation of the chondrocyte phenotype. In addition reduced proteoglycan synthesis and oversulfation of glycosaminoglycan chains were demonstrated, as already observed in primary chondrocytes from the Cant1 knock-out mouse. In conclusion, immortalized Cant1 knock-out chondrocytes maintained the disease phenotype observed in primary cells validating the in vitro model and providing an additional tool to further study the proteoglycan biosynthesis defect. The same approach might be extended to other cartilage disorders.

Hormone resistance and short stature: A journey through the pathways of hormone signaling.

Hormone resistances have been described in association with growth disorders, the majority involving the growth hormone (GH)/insulin-like growth factor 1(IGF-1) axis or hormones with specific paracrine-autocrine actions in the growth plate. Defects in hormone receptors or in proteins involved in intracellular signal transduction (post-receptor defects) are the main mechanisms of hormone resistance leading to short stature. The characteristic phenotypes of each of these hormonal resistances are very distinct and bring with them important insights into the role of each hormone and its signaling pathway. In this review, we discuss the molecular and clinical aspects of the main hormone resistances associated with short stature in humans.

Differential Diagnosis of the Short IGF-I-Deficient Child with Apparently Normal Growth Hormone Secretion.

The current differential diagnosis for a short child with low insulin-like growth factor I (IGF-I) and a normal growth hormone (GH) peak in a GH stimulation test (GHST), after exclusion of acquired causes, includes the following disorders: (1) a decreased spontaneous GH secretion in contrast to a normal stimulated GH peak ("GH neurosecretory dysfunction," GHND) and (2) genetic conditions with a normal GH sensitivity (e.g., pathogenic variants of GH1 or GHSR) and (3) GH insensitivity (GHI). We present a critical appraisal of the concept of GHND and the role of 12- or 24-h GH profiles in the selection of children for GH treatment. The mean 24-h GH concentration in healthy children overlaps with that in those with GH deficiency, indicating that the previously proposed cutoff limit (3.0-3.2 µg/L) is too high. The main advantage of performing a GH profile is that it prevents about 20% of false-positive test results of the GHST, while it also detects a low spontaneous GH secretion in children who would be considered GH sufficient based on a stimulation test. However, due to a considerable burden for patients and the health budget, GH profiles are only used in few centres. Regarding genetic causes, there is good evidence of the existence of Kowarski syndrome (due to GH1 variants) but less on the role of GHSR variants. Several genetic causes of (partial) GHI are known (GHR, STAT5B, STAT3, IGF1, IGFALS defects, and Noonan and 3M syndromes), some responding positively to GH therapy. In the final section, we speculate on hypothetical causes.

Circular RNA circANAPC2 mediates the impairment of endochondral ossification by miR-874-3p/SMAD3 signalling pathway in idiopathic short stature.

Idiopathic short stature (ISS) is a main reason for low height among children. Its exact aetiology remains unclear. Recent findings have suggested that the aberrant expression of circRNAs in peripheral blood samples is associated with many diseases. However, to date, the role of aberrant circRNA expression in mediating ISS pathogenesis remains largely unknown. The up-regulated circANAPC2 was identified by circRNA microarray analysis and RT-qPCR. Overexpression of circANAPC2 inhibited the proliferation of human chondrocytes, and cell cycle was arrested in G1 phase. The expressions of collagen type X, RUNX2, OCN and OPN were significantly down-regulated following circANAPC2 overexpression. Moreover, Von Kossa staining intensity and alkaline phosphatase activity were also decreased. Luciferase reporter assay results showed that circANAPC2 could be targeted by miR-874-3p. CircANAPC2 overexpression in human chondrocytes inhibits the expression of miR-874-3p. The co-localization of circANAPC2 and miR-874-3p was confirmed in both human chondrocytes and murine femoral growth plates via in situ hybridization. The rescue experiment demonstrated that the high expression of miR-874-3p overexpression antagonized the suppression of endochondral ossification, hypertrophy and chondrocyte growth caused by circANAPC2 overexpression. A high-throughput screening of mRNA expression and RT-qPCR verified SMAD3 demonstrated the highest different expressions following overcircANAPC2. Luciferase reporter assay results indicated that miR-874-3p could be targeted by Smad3, thus down-regulating the expression of Smad3. Subsequent rescue experiments of SMAD3 further confirmed that circANAPC2 suppresses endochondral ossification, hypertrophy and chondrocyte growth through miR-874-3p/Smad3 axis. The present study provides evidence that circANAPC2 can serve as a promising target for ISS treatment.

Dmrt2 promotes transition of endochondral bone formation by linking Sox9 and Runx2.

Endochondral bone formation is fundamental for skeletal development. During this process, chondrocytes undergo multiple steps of differentiation and coordinated transition from a proliferating to a hypertrophic stage, which is critical to advance skeletal development. Here, we identified the transcription factor Dmrt2 (double-sex and mab-3 related transcription factor 2) as a Sox9-inducible gene that promotes chondrocyte hypertrophy in pre-hypertrophic chondrocytes. Epigenetic analysis further demonstrated that Sox9 regulates Dmrt2 expression through an active enhancer located 18 kb upstream of the Dmrt2 gene and that this enhancer's chromatin status is progressively activated through chondrocyte differentiation. Dmrt2-knockout mice exhibited a dwarf phenotype with delayed initiation of chondrocyte hypertrophy. Dmrt2 augmented hypertrophic chondrocyte gene expression including Ihh through physical and functional interaction with Runx2. Furthermore, Dmrt2 deficiency reduced Runx2-dependent Ihh expression. Our findings suggest that Dmrt2 is critical for sequential chondrocyte differentiation during endochondral bone formation and coordinates the transcriptional network between Sox9 and Runx2.

Improvement of the skeletal phenotype in a mouse model of diastrophic dysplasia after postnatal treatment with N-acetylcysteine.

Diastrophic dysplasia (DTD) is a recessive chondrodysplasia caused by mutations in the SLC26A2 gene encoding for a sulfate/chloride transporter. When SLC26A2 is impaired intracellular level of sulfate is reduced leading to the synthesis of undersulfated proteoglycans. In normal chondrocytes, the main source of intracellular sulfate is the extracellular uptake through SLC26A2, but a small amount comes from the catabolism of sulfur-containing amino acids and other thiols. Here N-acetylcysteine (NAC), an extensively used drug, is proposed as alternative source of intracellular sulfate in an animal model of DTD (dtd mouse). Mutant and wild type mice were treated twice a day with hypodermic injections of 250 mg NAC/kg body weight for one week after birth. At the end of the treatment, an improvement trend in cartilage proteoglycan sulfation and in the skeletal phenotype of treated dtd mice were observed. Thus, a longer treatment lasted three weeks starting from birth was performed. Treated mutant mice showed a significant increase of cartilage proteoglycan sulfation and a relevant improvement of the skeletal phenotype based on measurements of several bony elements and bone quality by DEXA and micro CT. Moreover, the amelioration of the overall growth plate morphology in treated dtd mice suggested a partial rescue of the endochondral ossification process. Overall, the results prove that NAC is an effective source of intracellular sulfate for dtd mice in the postnatal period. This finding paves the way for a potential pharmacological treatment of DTD patients taking advantage from a drug repositioning strategy.

The histone H3-lysine 4-methyltransferase Mll4 regulates the development of growth hormone-releasing hormone-producing neurons in the mouse hypothalamus.

In humans, inactivating mutations in MLL4, which encodes a histone H3-lysine 4-methyltransferase, lead to Kabuki syndrome (KS). While dwarfism is a cardinal feature of KS, the underlying etiology remains unclear. Here we report that Mll4 regulates the development of growth hormone-releasing hormone (GHRH)-producing neurons in the mouse hypothalamus. Our two Mll4 mutant mouse models exhibit dwarfism phenotype and impairment of the developmental programs for GHRH-neurons. Our ChIP-seq analysis reveals that, in the developing mouse hypothalamus, Mll4 interacts with the transcription factor Nrf1 to trigger the expression of GHRH-neuronal genes. Interestingly, the deficiency of Mll4 results in a marked reduction of histone marks of active transcription, while treatment with the histone deacetylase inhibitor AR-42 rescues the histone mark signature and restores GHRH-neuronal production in Mll4 mutant mice. Our results suggest that the developmental dysregulation of Mll4-directed epigenetic control of transcription plays a role in the development of GHRH-neurons and dwarfism phenotype in mice.

De Novo Variants in LMNB1 Cause Pronounced Syndromic Microcephaly and Disruption of Nuclear Envelope Integrity.

Lamin B1 plays an important role in the nuclear envelope stability, the regulation of gene expression, and neural development. Duplication of LMNB1, or missense mutations increasing LMNB1 expression, are associated with autosomal-dominant leukodystrophy. On the basis of its role in neurogenesis, it has been postulated that LMNB1 variants could cause microcephaly. Here, we confirm this hypothesis with the identification of de novo mutations in LMNB1 in seven individuals with pronounced primary microcephaly (ranging from -3.6 to -12 SD) associated with relative short stature and variable degree of intellectual disability and neurological features as the core symptoms. Simplified gyral pattern of the cortex and abnormal corpus callosum were noted on MRI of three individuals, and these individuals also presented with a more severe phenotype. Functional analysis of the three missense mutations showed impaired formation of the LMNB1 nuclear lamina. The two variants located within the head group of LMNB1 result in a decrease in the nuclear localization of the protein and an increase in misshapen nuclei. We further demonstrate that another mutation, located in the coil region, leads to increased frequency of condensed nuclei and lower steady-state levels of lamin B1 in proband lymphoblasts. Our findings collectively indicate that de novo mutations in LMNB1 result in a dominant and damaging effect on nuclear envelope formation that correlates with microcephaly in humans. This adds LMNB1 to the growing list of genes implicated in severe autosomal-dominant microcephaly and broadens the phenotypic spectrum of the laminopathies.

MITOL dysfunction causes dwarfism with anterior pituitary hypoplasia.

In mitochondrial disorders, short stature and growth failure are common symptoms, but their underlying mechanism remains unknown. In this study, we examined the cause of growth failure of mice induced by nestin promoter-driven knockout of the mitochondrial ubiquitin ligase MITOL (MARCH5), a key regulator of mitochondrial function. MITOL-knockout mice have congenital hypoplasia of the anterior pituitary caused by decreased expression of pituitary transcript factor 1 (Pit1). Consistently, both mRNA levels of growth hormone (GH) and prolactin levels were markedly decreased in the anterior pituitary of mutant mice. Growth failure of mutant mice was partly rescued by hypodermic injection of recombinant GH. To clarify whether this abnormality was induced by the primary effect of MITOL knockdown in the anterior pituitary or a secondary effect of other lesions, we performed lentiviral-mediated knockdown of MITOL on cultured rat pituitary GH3 cells, which secrete GH. GH production was severely compromised in MITOL-knockdown GH3 cells. In conclusion, MITOL plays a critical role in the development of the anterior pituitary; therefore, mice with MITOL dysfunction exhibited pituitary dwarfism caused by anterior pituitary hypoplasia. Our findings suggest that mitochondrial dysfunction is commonly involved in the unknown pathogenesis of pituitary dwarfism.

Investigation of the role of ß-TrCP in growth hormone transduction defect (GHTD).

Background Growth hormone(GH) and epidermal growth factor (EGF) stimulate cell growth and differentiation, and crosstalking between their signaling pathways is important for normal cellular development. Growth hormone transduction defect (GHTD) is characterized by excessive GH receptor (GHR) degradation, due to over-expression of the E3 ubiquitin ligase, cytokine inducible SH2-containing protein (CIS). GH induction of GHTD fibroblasts after silencing of messenger RNA (mRNA) CIS (siCIS) or with higher doses of GH restores normal GH signaling. ß-Transducing-repeat-containing protein (ß-TrCP), another E3 ubiquitin ligase, also plays a role in GHR endocytosis. We studied the role of ß-TrCP in the regulation of the GH/GHR and EGF/EGF receptor (EGFR) pathways in normal and GHTD fibroblasts. Materials and methods Fibroblast cultures were developed from gingival biopsies of a GHTD (P) and a control child (C). Protein expression and cellular localization of ß-TrCP were studied by Western immunoblotting and immunofluorescence, respectively, after: (1) GH 200 µg/L human GH (hGH) induction, either with or without silence CIS (siCIS), and (2) inductions with 200 µg/L GH or 1000 µg/L GH or 50 ng/mL EGF. Results After induction with: (1) GH200/siCIS, the protein expression and cytoplasmic-membrane localization of ß-TrCP were increased in the patient, (2) GH200 in the control and GH1000 in the patient, the protein and cytoplasmic-membrane localization of ß-TrCP were increased and (3) EGF, the protein expression and cytoplasmic-membrane localization of ß-TrCP were increased in both the control and the patient. Conclusions (1) ß-TrCP appears to be part of the negative regulatory mechanism of the GH/GHR and EGF/EGFR pathways. (2) There appears to be a negative correlation between ß-TrCP and CIS. (3) In the control and GHTD patient, ß-TrCP increases when CIS is suppressed, possibly as a compensatory inhibitor of the GH/GHR pathway.

The association of microcephaly protein WDR62 with CPAP/IFT88 is required for cilia formation and neocortical development.

WDR62 mutations that result in protein loss, truncation or single amino-acid substitutions are causative for human microcephaly, indicating critical roles in cell expansion required for brain development. WDR62 missense mutations that retain protein expression represent partial loss-of-function mutants that may therefore provide specific insights into radial glial cell processes critical for brain growth. Here we utilized CRISPR/Cas9 approaches to generate three strains of WDR62 mutant mice; WDR62 V66M/V66M and WDR62R439H/R439H mice recapitulate conserved missense mutations found in humans with microcephaly, with the third strain being a null allele (WDR62stop/stop). Each of these mutations resulted in embryonic lethality to varying degrees and gross morphological defects consistent with ciliopathies (dwarfism, anophthalmia and microcephaly). We find that WDR62 mutant proteins (V66M and R439H) localize to the basal body but fail to recruit CPAP. As a consequence, we observe deficient recruitment of IFT88, a protein that is required for cilia formation. This underpins the maintenance of radial glia as WDR62 mutations caused premature differentiation of radial glia resulting in reduced generation of neurons and cortical thinning. These findings highlight the important role of the primary cilium in neocortical expansion and implicate ciliary dysfunction as underlying the pathology of MCPH2 patients.