Evaluation of FGFR inhibitor ASP5878 as a drug candidate for achondroplasia.

Achondroplasia is caused by gain-of-function mutations in FGFR3 gene and leads to short-limb dwarfism. A stabilized analogue of C-type natriuretic peptide (CNP) is known to elongate bone by interacting with FGFR3 signals and thus is a promising drug candidate. However, it needs daily administration by percutaneous injection. FGFR inhibitor compounds are other drug candidates for achondroplasia because they directly fix the mutant protein malfunction. Although FGFR inhibitors elongate the bone of model mice, their adverse effects are not well studied. In this study, we found that a new FGFR inhibitor, ASP5878, which was originally developed as an anti-cancer drug, elongated the bone of achondroplasia model male mice at the dose of 300 µg/kg, which confers an AUC of 275 ng·h/ml in juvenile mice. Although ASP5878 was less effective in bone elongation than a CNP analogue, it is advantageous in that ASP5878 can be administered orally. The AUC at which minimal adverse effects were observed (very slight atrophy of the corneal epithelium) was 459 ng·h/ml in juvenile rats. The positive discrepancy between AUCs that brought efficacy and minimal adverse effect suggests the applicability of ASP5878 to achondroplasia in the clinical setting. We also analyzed effects of ASP5878 in a patient-specific induced pluripotent stem cell (iPSC) model for achondroplasia and found the effects on patient chondrocyte equivalents. Nevertheless, cautious consideration is needed when referring to safety data obtained from its application to adult patients with cancer in clinical tests.

Once-daily, subcutaneous vosoritide therapy in children with achondroplasia: a randomised, double-blind, phase 3, placebo-controlled, multicentre trial.

BACKGROUND: There are no effective therapies for achondroplasia. An open-label study suggested that vosoritide administration might increase growth velocity in children with achondroplasia. This phase 3 trial was designed to further assess these preliminary findings. METHODS: This randomised, double-blind, phase 3, placebo-controlled, multicentre trial compared once-daily subcutaneous administration of vosoritide with placebo in children with achondroplasia. The trial was done in hospitals at 24 sites in seven countries (Australia, Germany, Japan, Spain, Turkey, the USA, and the UK). Eligible patients had a clinical diagnosis of achondroplasia, were ambulatory, had participated for 6 months in a baseline growth study and were aged 5 to less than 18 years at enrolment. Randomisation was done by means of a voice or web-response system, stratified according to sex and Tanner stage. Participants, investigators, and trial sponsor were masked to group assignment. Participants received either vosoritide 15·0 µg/kg or placebo, as allocated, for the duration of the 52-week treatment period administered by daily subcutaneous injections in their homes by trained caregivers. The primary endpoint was change from baseline in mean annualised growth velocity at 52 weeks in treated patients as compared with controls. All randomly assigned patients were included in the efficacy analyses (n=121). All patients who received one dose of vosoritide or placebo (n=121) were included in the safety analyses. The trial is complete and is registered, with EudraCT, number, 2015-003836-11. FINDINGS: All participants were recruited from Dec 12, 2016, to Nov 7, 2018, with 60 assigned to receive vosoritide and 61 to receive placebo. Of 124 patients screened for eligibility, 121 patients were randomly assigned, and 119 patients completed the 52-week trial. The adjusted mean difference in annualised growth velocity between patients in the vosoritide group and placebo group was 1·57 cm/year in favour of vosoritide (95% CI [1·22-1·93]; two-sided p<0·0001). A total of 119 patients had at least one adverse event; vosoritide group, 59 (98%), and placebo group, 60 (98%). None of the serious adverse events were considered to be treatment related and no deaths occurred. INTERPRETATION: Vosoritide is an effective treatment to increase growth in children with achondroplasia. It is not known whether final adult height will be increased, or what the harms of long-term therapy might be. FUNDING: BioMarin Pharmaceutical.

Pharmacokinetics and safety after once and twice a day doses of meclizine hydrochloride administered to children with achondroplasia.

Achondroplasia (ACH) is the most common short-limbed skeletal dysplasia caused by activating mutations in the fibroblast growth factor receptor 3 (FGFR3) gene. We identified that meclizine hydrochloride inhibited FGFR3 signaling in various chondrocytic cells and promoted longitudinal bone growth in mouse model of ACH. Meclizine has safely been used for more than 50 years, but it lacks the safety data for repeated administration and pharmacokinetics (PK) when administered to children. We performed a phase Ia study to evaluate the PK and safety of meclizine administered orally to ACH children. Twelve ACH children aged from 5 to younger than 11 years were recruited, and the first 6 subjects received once a day of meclizine in the fasted condition, subsequent 6 subjects received twice a day of meclizine in the fed condition. Meclizine was well tolerated in ACH children with no serious adverse events. The mean Cmax, Tmax, AUC0-24h, t1/2 during 24 hours in the fasted condition were 130 ng/mL, 1.7 hours, 761 ng·h/mL, and 8.5 hours respectively. The simulation of repeated administration of meclizine for 14 days demonstrated that plasma concentration apparently reached steady state around 10 days after the first dose both at once a day and twice a day administration. The AUC0-10h of the fasting and fed condition were 504 ng·h/mL and 813 ng·h/mL, respectively, indicating exposure of meclizine increased with the diet. Although higher drug exposure was confirmed in ACH children compared to adults, a single administration of meclizine seemed to be well tolerated.

Optimal non-invasive diagnosis of fetal achondroplasia combining ultrasonography with circulating cell-free fetal DNA analysis.

OBJECTIVES: To assess the performance of non-invasive prenatal testing (NIPT) for achondroplasia using high-resolution melting (HRM) analysis, and to propose an optimal diagnostic strategy combining ultrasound examination and cell-free fetal DNA (cffDNA) analysis. METHODS: In this prospective multicenter study, cffDNA was extracted from blood of pregnant women at risk for fetal achondroplasia (owing to paternal achondroplasia, previous affected child or suspected rhizomelic shortening) and of pregnant low-risk controls. The presence of either one of the two main fibroblast growth factor receptor 3 gene (FGFR3) mutations was determined using HRM combined with confirmation by SNaPshot minisequencing. Results were compared with phenotypes obtained using three-dimensional computed tomography or postnatal examination, and/or molecular diagnosis by an invasive procedure. Fetal biometry (head circumference and femur length) was analyzed in order to develop a strategy in which cffDNA analysis for diagnosis of achondroplasia is offered only in selected cases. RESULTS: Eighty-six blood samples from women at risk for fetal achondroplasia and 65 from controls were collected. The overall sensitivity and specificity of NIPT were 1.00 (95% CI, 0.87-1.00) and 1.00 (95% CI, 0.96-1.00), respectively. Critical reduction in femur length of affected fetuses could be observed from 26 weeks' gestation. CONCLUSIONS: HRM combined with SNaPshot minisequencing is a reliable method for NIPT for achondroplasia. Its implementation in routine clinical care combined with ultrasonography is an efficient strategy for the non-invasive diagnosis of achondroplasia. Copyright © 2018 ISUOG. Published by John Wiley & Sons Ltd.

Multiplex PCR in noninvasive prenatal diagnosis for FGFR3-related disorders.

Thanatophoric dysplasia and achondroplasia are allelic disorders caused by a constitutively active mutation in the FGFR3 gene. Because thanatophoric dysplasia is a lethal disorder and achondroplasia is non-lethal, they need to be distinguished after ultrasound identification of fetal growth retardation with short limbs. Accordingly, we have developed a noninvasive prenatal test using cell-free fetal DNA in the maternal circulation to distinguish thanatophoric dysplasia and achondroplasia. A multiplex PCR system encompassing five mutation hotspots in the FGFR3 gene allowed us to efficiently identify the responsible mutation in cell-free DNA in all examined pregnancies with a suspected thanatophoric dysplasia or achondroplasia fetus. This system will be helpful in the differential diagnosis of thanatophoric dysplasia and achondroplasia in early gestation and in couples concerned about the recurrence of thanatophoric dysplasia due to germinal mosaicism.

Decreased Plasma COMP and Increased Plasma CTX-II Levels in a Chinese Pseudoachondroplasia Family with Novel COMP Mutation.

Pseudoachondroplasia (PSACH) is an autosomal dominant osteochondrodysplasia caused by mutations in the gene encoding cartilage oligomeric matrix protein (COMP). Accurate clinical diagnosis of PSACH is sometimes difficult. Here, we identified a novel COMP mutation (c.1675G>A, p.Glu559Lys) in a Chinese PSACH family. We detected the plasma levels of COMP and type II collagen (CTX-II) in the four affected individuals. The results showed the levels of plasma COMP significantly decreased and plasma CTX-II significantly increased in the three PSACH patients with COMP mutation. However, both plasma levels of COMP and CTX-II were not to have found significant difference between the presymptomatic carrier and the age-matched subjects. In vitro analysis and immunofluorescence displayed wild type COMP homogenously expressed in cytoplasm, but mutant proteins were irregularly accumulated inside the HEK-293 cells. Western blot revealed that the quantity of the mutant COMP was more compared to wild type COMP in cells after transfection for 12 hours and 24 hours. Subsequently, 3D structural analysis showed three changes have taken place in secondary structure of the mutant COMP. In conclusion, the novel mutation of COMP may result in intracellular accumulation of the mutant protein. Decreased plasma COMP and increased plasma CTX-II may potentially serve as diagnostic markers of PSACH but may not be applicable in the presymptomatic carrier.

Droplet digital PCR combined with minisequencing, a new approach to analyze fetal DNA from maternal blood: application to the non-invasive prenatal diagnosis of achondroplasia.

BACKGROUND: Achondroplasia is generally detected by abnormal prenatal ultrasound findings in the third trimester of pregnancy and then confirmed by molecular genetic testing of fetal genomic DNA obtained by aspiration of amniotic fluid. This invasive procedure presents a small but significant risk for both the fetus and mother. Therefore, non-invasive procedures using cell-free fetal DNA in maternal plasma have been developed for the detection of the fetal achondroplasia mutations. METHODS: To determine whether the fetus carries the de novo mis-sense genetic mutation at nucleotide 1138 in FGFR3 gene involved in >99% of achondroplasia cases, we developed two independent methods: digital-droplet PCR combined with minisequencing, which are very sensitive methods allowing detection of rare alleles. RESULTS: We collected 26 plasmatic samples from women carrying fetus at risk of achondroplasia and diagnosed to date a total of five affected fetuses in maternal blood. The sensitivity and specificity of our test are respectively 100% [95% confidence interval, 56.6-100%] and 100% [95% confidence interval, 84.5-100%]. CONCLUSIONS: This novel, original strategy for non-invasive prenatal diagnosis of achondroplasia is suitable for implementation in routine clinical testing and allows considering extending the applications of these technologies in non-invasive prenatal diagnosis of many other monogenic diseases. © 2016 John Wiley & Sons, Ltd.

C-type natriuretic peptide plasma levels are elevated in subjects with achondroplasia, hypochondroplasia, and thanatophoric dysplasia.

CONTEXT: C-type natriuretic peptide (CNP) is a crucial regulator of endochondral bone growth. In a previous report of a child with acromesomelic dysplasia, Maroteaux type (AMDM), caused by loss-of-function of the CNP receptor (natriuretic peptide receptor-B [NPR-B]), plasma levels of CNP were elevated. In vitro studies have shown that activation of the MAPK kinase (MEK)/ERK MAPK pathway causes functional inhibition of NPR-B. Achondroplasia, hypochondroplasia, and thanatophoric dysplasia are syndromes of short-limbed dwarfism caused by activating mutations of fibroblast growth factor receptor-3, which result in overactivation of the MEK/ERK MAPK pathway. OBJECTIVE: The purpose of this study was to determine whether these syndromes exhibit evidence of CNP resistance as reflected by increases in plasma CNP and its amino-terminal propeptide (NTproCNP). DESIGN: This was a prospective, observational study. SUBJECTS: Participants were 63 children and 20 adults with achondroplasia, 6 children with hypochondroplasia, 2 children with thanatophoric dysplasia, and 4 children and 1 adult with AMDM. RESULTS: Plasma levels of CNP and NTproCNP were higher in children with achondroplasia with CNP SD scores (SDSs) of 1.0 (0.3-1.4) (median [interquartile range]) and NTproCNP SDSs of 1.4 (0.4-1.8; P < .0005). NTproCNP levels correlated with height velocity. Levels were also elevated in adults with achondroplasia (CNP SDSs of 1.5 [0.7-2.1] and NTproCNP SDSs of 0.5 [0.1-1.0], P < .005). In children with hypochondroplasia, CNP SDSs were 1.3 (0.7-1.5) (P = .08) and NTproCNP SDSs were 1.9 (1.8-2.3) (P < .05). In children with AMDM, CNP SDSs were 1.6 (1.4-3.3) and NTproCNP SDSs were 4.2 (2.7-6.2) (P < .01). CONCLUSIONS: In these skeletal dysplasias, elevated plasma levels of proCNP products suggest the presence of tissue resistance to CNP.

Non-invasive prenatal detection of achondroplasia using circulating fetal DNA in maternal plasma.

PURPOSE: To perform a reliable non-invasive detection of the fetal achondroplasia using maternal plasma. METHODS: We developed a quantitative fluorescent-polymerase chain reaction (QF-PCR) method suitable for detection of the FGFR3 mutation (G1138A) causing achondroplasia. This method was applied in a non-invasive detection of the fetal achondroplasia using circulating fetal-DNA (cf-DNA) in maternal plasma. Maternal plasmas were obtained at 27 weeks of gestational age from women carrying an achondroplasia fetus or a normal fetus. RESULTS: Two percent or less achondroplasia DNA was reliably detected by QF-PCR. In a woman carrying a normal fetus, analysis of cf-DNA showed only one peak of the wild-type G allele. In a woman expected an achondroplasia fetus, analysis of cf-DNA showed the two peaks of wild-type G allele and mutant-type A allele and accurately detected the fetal achondroplasia. CONCLUSIONS: The non-invasive method using maternal plasma and QF-PCR may be useful for diagnosis of the fetal achondroplasia.

Acanthosis nigricans and insulin sensitivity in patients with achondroplasia and hypochodroplasia due to FGFR3 mutations.

BACKGROUND AND AIMS: Acanthosis nigricans (AN) has been reported in association with severe skeletal dysplasias due to activating mutations in FGFR3, including thanatophoric dysplasia, severe achondroplasia (ACH) with developmental delay and AN (SADDAN syndrome), and Crouzon syndrome with AN. There are isolated reports of patients with ACH and AN. In this series, we report clinical and biochemical data on five male patients, four with ACH and one with hypochondroplasia (HCH), who developed AN without SADDAN. METHODS AND RESULTS: We compared the results of a 1.75 g/kg oral glucose tolerance test performed in patients with ACH/HCH and AN with age-, sex-, and puberty-matched short children. Three of the patients were treated with recombinant human GH (dose range, 45-50 microg/kg/d), one patient had discontinued treatment 6 months before presentation, and one had never been treated. All patients had a fasting plasma glucose of less than 6 mmol/liter, and no patient had a plasma glucose greater than 7.8 mmol/liter at 2 h after ingestion of a glucose load. Although body mass index was higher in patients with skeletal dysplasia (28.9 +/- 7.3 vs. 20 +/- 0.6 kg/m(2); P = 0.01), mean fasting plasma insulin concentration was greater in controls (14.4 +/- 4.8 vs. 6.0 +/- 4.5 mU/liter; P = 0.03), as was homeostasis assessment index for insulin resistance (2.5 +/- 0.9 vs. 1.17 +/- 0.8; P = 0.05). CONCLUSION: Our findings suggest that the development of AN in patients with ACH/HCH is not due to insulin insensitivity either on its own or secondary to treatment with recombinant human GH. Whether the AN is due to altered melanocyte function in these individuals remains to be established.

Improved prenatal detection of a fetal point mutation for achondroplasia by the use of size-fractionated circulatory DNA in maternal plasma--case report.

INTRODUCTION: The efficacious analysis of fetal loci involving point mutations from circulatory fetal DNA in maternal plasma is hindered by the preponderance of maternal DNA. It has recently been shown that the size difference between fetal and maternal DNA species can be used for the selective enrichment of circulatory fetal DNA in maternal plasma. We have now tested this approach for the detection of a fetal point mutation in the fibroblast growth factor receptor 3 (FGFR3) gene that causes achondroplasia. METHODS: Circulatory DNA was extracted from maternal plasma and size-fractionated by agarose gel electrophoresis. The fraction with a size less than 300 bp was examined by a touchdown PCR assay specific for the FGFR3 gene, and the mutation was identified by SfcI restriction analysis. RESULT: Our analysis indicated that although the fetal mutation was discernible in the analysis of total plasma DNA, the result using size-fractionated DNA was much more evident. CONCLUSION: The enrichment of circulatory fetal DNA in maternal plasma by size-fractionation facilitates the detection of subtle feto-maternal genetic differences, such as those involving point mutations. This approach can easily be extended for the non-invasive prenatal determination of other fetal loci.

Detection of achondroplasia G380R mutation from PCR amplicons by using inosine modified carbon electrodes based on electrochemical DNA chip technology.

The detection of Achondroplasia G380R mutation from real samples was performed by monitoring the oxidation signal of guanine. Inosine-substituted 12-mer capture probes related to the homozygous or heterozygous alleles of Achondroplasia G380R mutation were adsorbed onto carbon paste electrode (CPE) surface. No guanine signal was obtained from the capture probes, since the inosine bases were electroinactive. Then, these probes were hybridized with the denatured PCR samples on the CPE surface. The hybridization between the probe and target sequences was determined by using the oxidation signal of guanine in connection with differential pulse voltammetry (DPV). The oxidation signal of guanine was observed as a result of the specific hybridization between the probe and PCR sample. The changes in the peak height of the guanine signal provided the information whether the PCR sample contained heterozygous allele or homozygous allele. Numerous factors affecting the hybridization and nonspecific binding events were optimized to detect down to 41.24 fmol/ml target DNA. The electrochemical detection of Achondroplasia G380R mutation from PCR samples greatly shortened and simplified the diagnosis for the homozygous mutant type, which is lethal for the newborn.

Effects and serum levels of thrombopoietin in a case of chronic thrombocytopenia with achondroplasia.

Thrombopoietin (TPO), produced mainly in the liver, is a major regulator of platelet production. Serum TPO levels are generally increased in thrombocytopenia. We report a case of a 12-year-old boy with chronic severe thrombocytopenia, achondroplasia and nephritis. Severe chronic thrombocytopenia was found at 9 months of age. It was resistant to any treatment. Studies on megakaryocytic colonies in vitro revealed that the marrow cells responded well to TPO and no plasma inhibitor was found. Although hepatic function test results were normal, serum TPO levels in the patient (0.94 fmol/ml) were consistent with those in age-matched children (0.49-1.75 fmol/ml). Chronic thrombocytopenia requires individual evaluation before clinical trials with TPO.

Hemoglobin level is linked to growth hormone-dependent proteins in short children.

Erythropoiesis was investigated in 32 children wih short stature and in eight children with skeletal dysplasia by studying blood hemoglobin in relation to growth and to serum concentrations of insulin-like growth factor I (IGF-I), IGF binding protein-3 (IGFBP-3), and erythropoietin (EPO) before, during, and after 12 months of recombinant human growth hormone (GH) treatment. Blood hemoglobin concentration was positively correlated with relative body height and with serum IGF-I and IGFBP-3 levels (P = .001 to .02), but not with the concentrations of EPO. The normal age-dependency of hemoglobin was lacking. Hemoglobin levels and their responses to GH treatment were similar in the patients with GH deficiency and those with normal GH secretion. Treatment with GH accelerated growth and elevated the concentrations of hemoglobin, IGF-I, and IGFBP-3. In the eight patients with skeletal dysplasia, body mass increased similarly, but gain in height was less than in the other patients, and the increase in hemoglobin was markedly pronounced. In this group, the correlations between hemoglobin, IGF-I, and IGFBP-3 were extremely close (r = 0.80 to 0.85, P = .031 to .008). These findings are in accord with earlier observations from in vitro and animal studies, and suggest that the GH-IGF axis is involved in the physiologic elevation of hemoglobin levels during childhood.

Growth hormone (GH) treatment in achondroplasia.

Achondroplasia is one of the most commonly known types of skeletal dysplasia in the adult leading to short stature. Before beginning growth hormone (GH) treatment of short stature in patients with achondroplasia, we evaluated their growth pattern and their hypothalamic-pituitary function, including GH secretion. We studied 22 patients with achondroplasia (7 males and 15 females: age range, 3 to 12 years). The z-score of their height at admission was -5.4 +/- 1.2 (mean +/- SD), and that of their annual height gain before admission was -3.1 +/- 1.3 (mean +/- SD). GH response to provocative tests was normal in all patients except five: four showed subnormal (< 10 ng/ml) response to L-Dopa stimuli, and one patient showed subnormal (< 20 ng/ml) response to GRF stimuli. The mean GH concentration during sleep was found to be low (< 5 ng/ml) in three patients. These three patients were suspected to have latent GH deficiency, as they also showed a markedly low IGF-1 level and marked delay of bone age. LH, FSH, TSH, and cortisol response to provocative tests were normal in all the patients. We treated this group of patients with recombinant human GH (1 IU/kg/week). In 18 patients who were treated with GH for more than 6 months, height velocity during GH therapy was significantly increased compared to that before GH therapy (4.1 +/- 0.8 cm/year vs 7.2 +/- 1.4 cm/year). We conclude that parameters reflecting hypothalamic-pituitary function, particularly GH secretion, should be examined in achondroplasia patients, and that GH treatment may be beneficial in the treatment of short stature in achondroplasia.