The Broad Clinical Spectrum of Metatropic Dysplasia: A Case Series and Literature Review.

Objective To summarize the clinical, radiographic and genetic features of a family with metatropic dysplasia, in order to improve the level of understanding and diagnosis of this disease. Methods The proband, a one-year old boy, was diagnosed as metatropic dysplasia.His mother was 26 years old with mildly phenotype.Their clinical features and bone X-ray findings were analyzed.The DNA samples of the proband and his parents were collected.The coding exons and flanking introns regions of transient receptor potential vanilloid 4 (TRPV4) gene were amplified by polymerase chain reaction(PCR) and analyzed by DNA automatic detector.The pathology, diagnosis, treatment and prognosis were expounded. Results The symptoms of the boy were characterized by short extremities, a short trunk with progressive kyphoscoliosis, and craniofacial abnormalities that include a prominent forehead, midface hypoplasia, and a squared-off jaw.His motor development was slightly delayed.Mental development was normal.Bone X ray of the boy showed platyspondyly and severe metaphyseal enlargement with shortening of long bones and irregularities and delayed ossification of epiphysis.The patient and his mother were heterozygous for the nucleotide substitutions c. 2396>T (p.P799L) in TRPV4 gene. Conclusions The patient and his mother with metatropic dysplasia were diagnosed with TRPV4 gene analysis.The patient showed typical clinical features.His mother was mild.Metatropic dysplasia had signi-ficantly clinical heterogeneity.Gene analysis is helpful for the diagnosis. Key words: Spondyloepiphyseal dysplasia; Metatropic dysplasia; Transient receptor potential vanilloid 4 channel protein; Transient receptor potential vanilloid 4 gene mutation

Metatropic dysplasia (MD), is a rare skeletal dysplasia occurring predominantly in infants characterized by a distinctive long torso and short limbs; it is as a result of mutations in the TRPV4 gene. However, a clear distinction between various forms of skeletal dysplasias caused by the transient receptor potential vanilloid 4 (TRPV4) gene is difficult but could be achieved by a combination of gene sequencing, medical and radiological criteria. We hereby report a case of a 14‐month old girl who presented with an abnormal stature. The diagnosis of nonlethal MD was confirmed by X‐ray with dumbbell‐shaped long bones, platyspondyly, and delayed carpal ossification, as well as broadened pelvis with marginally widened ilia, epiphyseal plates, and slightly flattened acetabula. Furthermore, gene sequencing confirmed gene mutation on exon 15 of the TRPV4 gene with a heterozygous missense mutation (c.2396C > T), but no mutation was present in her parents. Our findings recorded metatropic dysplasia with the c.2396C > T mutation in the TRPV4 gene in China. This mutation caused changes in amino acid of TRPV4, which can induce growth retardation in children.

Rationale:To observe the natural history of the disease and the radiographic evolution of growth and development in patients with metatropic dysplasia (MD) and to complement the spectrum of mutations in the transient receptor potential vanilloid 4 (TRPV4) gene and the spectrum of MD phenotypes.Patient concerns:We report a patient with MD caused by a novel missense mutation in TRPV4, who possessed a mixed phenotype of both abnormal skeletal development and peripheral neuropathy. From 3 months to the age of 7 years, we observed the patient’s natural history and the imaging evolution of the patient’s growth and development.Diagnosis:The diagnosis of MD based on growth and developmental history, clinical presentation, imaging and mutation analysis of the TRPV4 gene.Interventions:She underwent posterior spinal osteotomy (T10, vertebral column resection), lateral kyphosis correction, internal fixation (T6-L3), and implant fusion. Surgical intervention can effectively delay the course of the disease.Outcomes:Sequencing analysis and family validation of the patient’s whole exon gene confirmed for the first time that the mutation in exon 11 of the TRPV4 gene was a heterozygous missense mutation (c.1811T > A) resulting in the mutation of isoleucine at position 604 to asparagine (p. I604N).Lessons:This study complements the spectrum of mutations in the TRPV4 gene and the spectrum of MD phenotypes and provides a reference for prenatal diagnosis, genetic counseling, mechanistic studies, and development of symptomatic treatment for this type of disease.

Accelerated osteoblastic differentiation in patient-derived dental pulp stem cells carrying a gain-of-function mutation of TRPV4 associated with metatropic dysplasia

Polycystic kidney disease (PKD), a disease characterized by the enlargement of the kidney through cystic growth is the fourth leading cause of end-stage kidney disease world-wide. Transient receptor potential Vanilloid 4 (TRPV4), a calcium-permeable TRP, channel participates in kidney cell physiology and since TRPV4 forms complexes with another channel whose malfunction is associated to PKD, TRPP2 (or PKD2), we sought to determine whether patients with PKD, exhibit previously unknown mutations in TRPV4. Here, we report the presence of mutations in the TRPV4 gene in patients diagnosed with PKD and determine that they produce gain-of-function (GOF). Mutations in the sequence of the TRPV4 gene have been associated to a broad spectrum of neuropathies and skeletal dysplasias but not PKD, and their biophysical effects on channel function have not been elucidated. We identified and examined the functional behavior of a novel E6K mutant and of the previously known S94L and A217S mutant TRVP4 channels. The A217S mutation has been associated to mixed neuropathy and/or skeletal dysplasia phenotypes, however, the PKD carriers of these variants had not been diagnosed with these reported clinical manifestations. The presence of certain mutations in TRPV4 may influence the progression and severity of PKD through GOF mechanisms. PKD patients carrying TRVP4 mutations are putatively more likely to require dialysis or renal transplant as compared to those without these mutations.

Transient receptor potential vanilloid 4 (TRPV4) is a mechanosensitive ion channel highly expressed in chondrocytes that supports cartilage development and homeostasis. Mutations in the channel can cause skeletal dysplasias, including the gain-of-function mutations V620I and T89I, which lead to brachyolmia and metatropic dysplasia, respectively. These mutations suppress hypertrophic differentiation, but the mechanisms by which they alter chondrocyte response to mechanical load remain to be elucidated. To determine the effect of these mutations on chondrocyte mechanotransduction, tissue-engineered cartilage was derived from differentiated clustered regularly interspaced short palindromic repeats (CRISPR)-edited human-induced pluripotent stem cells (hiPSCs) harboring the moderate V620I or severe T89I TRPV4 mutations. Wild-type and mutant tissue-engineered hiPSC-derived cartilage contructs were subjected to compressive mechanical loading at physiological levels, and transcriptomic signatures were assessed by RNA-sequencing. Our results demonstrate that the V620I and T89I mutations diminish the mechanoresponsiveness of chondrocytes, as evidenced by reduced gene expression downstream of TRPV4 activation, including those involved in endochondral ossification. Changes in the expression of genes involved in extracellular matrix production and organization were found to contribute toward the phenotype in V620I mutant chondrocytes, whereas dysregulated retinoic acid signaling was linked to T89I, and disrupted proliferation was common to both. Our findings suggest that dysfunctional mechanotransduction due to V620I and T89I mutations in TRPV4 contribute to the developmental phenotypes, supporting TRPV4 modulation as a potential pharmacologic target.NEW & NOTEWORTHY Gain-of-function mutations in TRPV4, a mechano- and osmosensitive ion channel, are linked to skeletal dysplasias, but their effects on chondrocyte mechanotransduction remain unknown. Using human iPSCs harboring skeletal dysplasia-causing mutations, we developed and mechanically loaded tissue-engineered cartilage. Our findings show that V620I and T89I mutations reduce chondrocyte mechanoresponsiveness, evidenced by decreased gene expression downstream of TRPV4 activation, providing insight into TRPV4-related skeletal disorders and potential pharmacological targets.

G.P.238: Phenotypic spectrum of three patients affected by TRPV4 mutations

Novel TRPV4 mutation in a large Chinese family with congenital distal spinal muscular atrophy, skeletal dysplasia and scaly skin

Metatropic dysplasia is a congenital skeletal dysplasia characterized by severe platyspondyly, dumbbell-like deformity of long tubular bones, and progressive kyphoscoliosis with growth. It is caused by mutations in the gene TRPV4, encoding the transient receptor potential vanilloid 4, which acts as a calcium channel. Many heterozygous single base mutations of this gene have been associated with the disorder, showing autosomal dominant inheritance. Although abnormal endochondral ossification has been observed by histological examination of bone in a patient with lethal metatropic dysplasia, the etiology of the disorder remains largely unresolved. As dental pulp stem cells (DPSCs) are mesenchymal stem cells that differentiate into bone lineage cells, DPSCs derived from patients with congenital skeletal dysplasia might be useful as a disease-specific cellular model for etiological investigation. The purpose of this study was to clarify the pathological association between TRPV4 mutation and chondrocyte differentiation by analyzing DPSCs from a patient with non-lethal metatropic dysplasia. We identified a novel heterozygous single base mutation, c.1855C>T in TRPV4. This was predicted to be a missense mutation, p.L619F, in putative transmembrane segment 5. The mutation was repaired by CRISPR/Cas9 system to obtain isogenic control DPSCs for further analysis. The expression of stem cell markers and fibroblast-like morphology were comparable between patient-derived mutant and control DPSCs, although expression of TRPV4 was lower in mutant DPSCs than control DPSCs. Despite the lower TRPV4 expression in mutant DPSCs, the intracellular Ca2+ level was comparable at the basal level between mutant and control DPSCs, while its level was markedly higher following stimulation with 4α-phorbol 12,13-didecanoate (4αPDD), a specific agonist for TRPV4, in mutant DPSCs than in control DPSCs. In the presence of 4αPDD, we observed accelerated early chondrocyte differentiation and upregulated mRNA expression of SRY-box 9 (SOX9) in mutant DPSCs. Our findings suggested that the novel missense mutation c.1855C>T of TRPV4 was a gain-of-function mutation leading to enhanced intracellular Ca2+ level, which was associated with accelerated chondrocyte differentiation and SOX9 upregulation. Our results also suggest that patient-derived DPSCs can be a useful disease-specific cellular model for elucidating the pathological mechanism of metatropic dysplasia.

The importance of gaining insight into the mechanisms underlying uterine quiescence and contractility is highlighted by the absence of an effective strategy to prevent or treat preterm labor, the greatest cause of perinatal mortality and morbidity worldwide. Although current evidence suggests that in myometrial smooth muscle cells (mSMCs) calcium homeostasis is modulated near term to promote uterine contractility, the efficacy of blocking voltage-operated calcium channels is limited by dose-related cardiovascular side effects. Thus, we considered whether uterine contractility might be modulated by calcium entry via transient receptor potential vanilloid 4 (TRPV4) channels. In mSMC, TRPV4 gene and protein expression increased with gestation, and TRPV4-mediated Ca(2+) entry and contractility were increased in mSMC from pregnant compared to nonpregnant rats. Cell membrane TRPV4 expression was specifically increased, whereas the expression of β-arrestin-1 and β-arrestin-2, molecules that can sequester TRPV4 in the cytoplasm, decreased. Physical interaction of β-arrestin-2 and TRPV4 was apparent in nonpregnant, but absent in pregnant, mouse uterus. Moreover, direct pharmacologic activation of TRPV4 increased uterine contraction, but oxytocin-induced myometrial contraction was blocked by pharmacologic inhibition of TRPV4 and decreased in mice with global deletion of TRPV4. Finally, TRPV4 channel blockade prolonged pregnancy in two distinct in vivo murine models of preterm labor, whereas the absence of either β-arrestin-1 or β-arrestin-2 increased susceptibility to preterm labor. These data suggest that TRPV4 channel activity modulates uterine contractility and might represent a therapeutic target to address preterm labor.

Mutations in the transient receptor potential vanilloid 4 (TRPV4) gene have been associated with autosomal dominant skeletal dysplasias and peripheral nervous system syndromes (PNSS). PNSS include Charcot–Marie–Tooth disease (CMT) type 2C, congenital spinal muscular atrophy and arthrogryposis and scapuloperoneal spinal muscular atrophy. We report the clinical, electrophysiological and muscle biopsy findings in two unrelated patients with two novel heterozygous missense mutations in the TRPV4 gene. Whole exome sequencing was carried out on genomic DNA using Illumina TruseqTM 62Mb exome capture. Patient 1 harbours a de novo c.805C > T (p.Arg269Cys) mutation. Clinically, this patient shows signs of both scapuloperoneal spinal muscular atrophy and skeletal dysplasia. Patient 2 harbours a novel c.184G > A (p.Asp62Asn) mutation. While the clinical phenotype is compatible with CMT type 2C with the patient's muscle harbours basophilic inclusions. Mutations in the TRPV4 gene have a broad phenotypic variability and disease severity and may share a similar pathogenic mechanism with Heat Shock Protein related neuropathies.

The transient receptor potential vanilloid 4 (TRPV4) cation channel, a member of the TRP vanilloid subfamily, is expressed in a broad range of tissues where it participates in the generation of Ca2+ signals and/or depolarization of the membrane potential. Regulation of TRPV4 abundance at the cell surface is critical for osmo- and mechanotransduction. Defects in TRPV4 are the cause of several human diseases, including brachyolmia type 3 (MIM:113500) (also known as brachyrachia or spondylometaphyseal dysplasia Kozlowski type [MIM:118452]), and metatropic dysplasia (MIM:156530) (also called metatropic dwarfism or parastremmatic dwarfism [MIM:168400]). These bone dysplasia mutants are characterized by severe dwarfism, kyphoscoliosis, distortion and bowing of the extremities, and contractures of the large joints. These diseases are characterized by a combination of decreased bone density, bowing of the long bones, platyspondyly, and striking irregularities of endochondral ossification with areas of calcific stippling and streaking in radiolucent epiphyses, metaphyses, and apophyses. In this review, we discuss the potential effect of the mutation on the regulation of TRPV4 functions, which are related to human diseases through deviated function. In particular, we emphasize how the constitutive active TRPV4 mutant affects endochondral ossification with a reduced number of hypertrophic chondrocytes and the presence of cartilage islands within the zone of primary mineralization. In addition, we summarize current knowledge about the role of TRPV4 in the pathogenesis of several diseases.

Dominant mutations in the calcium-permeable ion channel TRPV4 (transient receptor potential vanilloid 4) cause diverse and largely distinct channelopathies, including inherited forms of neuromuscular disease, skeletal dysplasias and arthropathy. Pathogenic TRPV4 mutations cause gain of ion channel function and toxicity that can be rescued by small molecule TRPV4 antagonists in cellular and animal models, suggesting that TRPV4 antagonism could be therapeutic for patients. Numerous variants in TRPV4 have been detected with targeted and whole exome/genome sequencing, but for the vast majority, their pathogenicity remains unclear. Here, we used a combination of clinical information and experimental structure-function analyses to evaluate 30 TRPV4 variants across various functional protein domains. We report clinical features of seven patients with TRPV4 variants of unknown significance and provide extensive functional characterization of these and an additional 17 variants, including structural position, ion channel function, subcellular localization, expression level, cytotoxicity and protein-protein interactions. We find that gain-of-function mutations within the TRPV4 intracellular ankyrin repeat domain target charged amino acid residues important for RhoA interaction, whereas ankyrin repeat domain residues outside of the RhoA interface have normal or reduced ion channel activity. We further identify a cluster of gain-of-function variants within the intracellular intrinsically disordered region that may cause toxicity via altered interactions with membrane lipids. In contrast, assessed variants in the transmembrane domain and other regions of the intrinsically disordered region do not cause gain of function and are likely benign. Clinical features associated with gain of function and cytotoxicity include congenital onset of disease, vocal cord weakness and motor-predominant disease, whereas patients with likely benign variants often demonstrated late-onset and sensory-predominant disease. These results provide a framework for assessing additional TRPV4 variants with respect to likely pathogenicity, which will yield critical information to inform patient selection for future clinical trials for TRPV4 channelopathies.

Disorders of water balance are among the most common and morbid of the electrolyte disturbances, and are reflected clinically as abnormalities in the serum sodium concentration. The transient receptor potential vanilloid 4 (TRPV4) channel is postulated to comprise an element of the central tonicity-sensing mechanism in the mammalian hypothalamus, and is activated by hypotonic stress in vitro. A nonsynonymous polymorphism in the TRPV4 gene gives rise to a Pro-to-Ser substitution at residue 19. We show that this polymorphism is significantly associated with serum sodium concentration and with hyponatremia (serum sodium concentration < or =135 mEq/L) in 2 non-Hispanic Caucasian male populations; in addition, mean serum sodium concentration is lower among subjects with the TRPV4(P19S) allele relative to the wild-type allele. Subjects with the minor allele were 2.4-6.4 times as likely to exhibit hyponatremia as subjects without the minor allele (after inclusion of key covariates). Consistent with these observations, a human TRPV4 channel mutated to incorporate the TRPV4(P19S) polymorphism showed diminished response to hypotonic stress (relative to the wild-type channel) and to the osmotransducing lipid epoxyeicosatrienoic acid in heterologous expression studies. These data suggest that this polymorphism affects TRPV4 function in vivo and likely influences systemic water balance on a population-wide basis.

The functional contribution of transient receptor potential vanilloid 4 (TRPV4) expression in maintaining human corneal endothelial cells (HCEC) homeostasis is unclear. Accordingly, we determined the effects of TRPV4 gene and protein overexpression on responses modulating the viability and survival of HCEC. Q-PCR, Western blot, FACS analyses and fluorescence single-cell calcium imaging confirmed TRPV4 gene and protein overexpression in lentivirally transduced 12V4 cells derived from their parent HCEC-12 line. Although TRPV4 overexpression did not alter the baseline transendothelial electrical resistance (TEER), its cellular capacitance (Ccl) was larger than that in its parent. Scanning electron microscopy revealed that only the 12V4 cells developed densely packed villus-like protrusions. Stimulation of TRPV4 activity with GSK1016790A (GSK101, 10 µmol/L) induced larger Ca2+ transients in the 12V4 cells than those in the parental HCEC-12. One to ten nmol/L GSK101 decreased 12V4 viability, increased cell death rates and reduced the TEER, whereas 1 µmol/L GSK101 was required to induce similar effects in the HCEC-12. However, the TRPV4 channel blocker RN1734 (1 to 30 µmol/L) failed to alter HCEC-12 and 12V4 morphology, cell viability and metabolic activity. Taken together, TRPV4 overexpression altered both the HCEC morphology and markedly lowered the GSK101 dosages required to stimulate its channel activity.