Background Disorders of the Ras/MAPK pathway have an overlapping skeletal phenotype (eg. scoliosis, osteopenia). The Ras proteins regulate cell proliferation and differentiation and NF1 individuals have osteoclast hyperactivity and increased bone resorption as measured by urine pyridinium crosslinks [pyridinoline (Pyd) and deoxypyridinoline (Dpd)]. Pyd and Dpd are hydroxylysine derived cross-links of collagen found in bone and cartilage and excreted in the urine. Dpd is most abundant in bone. The aim of this study was to evaluate if other syndromes of the Ras/MAPK pathway have increased bone resorption, which may impact the skeletal phenotype. Methods and Results Participants: [Noonan syndrome (n=14), Costello syndrome (n=21), and cardiofaciocutaneous (CFC) syndrome (n=14)]. Pyridinium cross-links from two consecutive first morning urines were extracted after acid hydrolysis and analyzed by High Performance Liquid Chromotography. Three separate analyses of covariance (ANCOVA) were performed to compare Pyd, Dpd, and Dpd/Pyd ratio of each group to controls after controlling for age. Data were compared to 99 healthy controls. Conclusions The Dpd and the Dpd/Pyd ratio were elevated (p<0.0001) in all 3 conditions compared to controls suggesting that collagen degradation was predominantly from bone. The data suggest that the Ras/MAPK signal transduction pathway is important in bone homeostasis.

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Newborn screening allows the diagnosis of congenital adrenal hyperplasia (CAH) before symptoms appear, preventing the severe and potentially life-threatening crisis associated with this disease in infancy. Traditional screening by enzyme immunoassay results in a large number of false positives. To reduce the number of unnecessary tests, anxiety to families and physicians, and the burden to the newborn screening follow-up program, we implemented a second-tier test for CAH using steroid profiling by an ultra-performance liquid chromatography-tandem mass spectrometry. We measured three steroids: 17-hydroxyprogesterone, androstenedione, and cortisol and correlated them with the age of infant at the time of sample collection and birth weight. Both age at collection and birth weight affected the levels of adrenal steroids, but the use of appropriate cut offs and analyte ratios allowed the identification of infants with CAH. This approach was effective in identifying infants with CAH, with both salt-wasting and simple virilizing forms, while reducing the false-positive rate from 2.6 to 0.09%.

Although neurofibromatosis type 1 (NF1) is a neurocutaneous disorder, skeletal abnormalities such as long-bone dysplasia, scoliosis, sphenoid wing dysplasia, and osteopenia are observed. To investigate the role of bone resorption as a mechanism for the bony abnormalities, we selected urinary pyridinium crosslinks (collagen degradation products excreted in urine) as a measure of bone resorption in NF1. Bone resorption was evaluated by quantitative assessment of the urinary excretion of pyridinium crosslinks [pyridinoline (Pyd) and deoxypyridinoline (Dpd)]. Total (free plus peptide-bound) pyridinium crosslinks from the first morning urines from 59 NF1 children (ages 5-19) were extracted and analyzed (17 children with a localized skeletal dysplasia, and 42 without). The data were compared with a healthy reference population without NF1 (n ϭ 99). Multivariate analyses, controlling for age showed statistically significant increases for Dpd (p Ͻ 0.001) and the Dpd/Pyd ratio (p Ͻ 0.001) in NF1 individuals with and without a skeletal dysplasia. NF1 children have an increase in the urinary excretion of pyridinium crosslinks, reflecting increased bone resorption. The effects of NF1 haploinsufficiency likely contribute to abnormal bone remodeling, either directly or indirectly by aberrant Ras signaling, potentially predisposing NF1 individuals to localized skeletal defects. (Pediatr Res 63: 697-701, 2008) N eurofibromatosis type 1 (NF1), a common autosomal dominant disorder affecting Ϸ1/3500 individuals worldwide, has variable expressivity. Clinical manifestations include café-au-lait macules, intertriginous freckling, Lisch nodules, neurofibromas, optic pathway tumors, and distinctive osseous lesions (1-3). The prototypical skeletal manifestations of NF1 are proportionate short stature, macrocephaly, longbone dysplasia, progressive scoliosis, and sphenoid wing dysplasia. Long-bone dysplasia most often affects the tibia and presents with anterolateral bowing often leading to fracture and nonunion or pseudarthrosis (4,5). The long-bone dysplasia in NF1 is very distinctive, and the presence of tibial pseudarthrosis alone should raise the potential diagnosis of NF1, as 50 -80% of individuals with pseudarthrosis have NF1 (6 -8).Scoliosis is the most common orthopedic manifestation in NF1 with reports documenting between 10 and 33% of NF1 individuals having scoliosis (9). Other osseous manifestations of NF1 include bone cysts, spinal canal widening, vertebral body narrowing, rib-penciling, vertebral scalloping with dural ectasias, and decreased bone mineral density. In isolation, each skeletal abnormality associated with NF1 is rare, but, as a whole, the osseous defects are relatively frequent.The NF1 gene, located on the long arm of chromosome 17, encodes the protein neurofibromin, which is a Ras-GAP protein (10). The "tumor suppressor" properties of this protein do not easily explain the mesodermally derived osseous manifestations observed in NF1. The frequent association of osseous dysplasias seen in NF1 suggests, ...

Skeletal abnormalities including scoliosis, tibial dysplasia, sphenoid wing dysplasia, and decreased bone mineral density (BMD) are associated with neurofibromatosis type 1 (NF1). We report the cellular phenotype of NF1 human-derived osteoclasts and compare the in vitro findings with the clinical phenotype.Functional characteristics (e.g. osteoclast formation, migration, adhesion, resorptive capacity) and cellular mechanistic alterations (e.g. F-actin polymerization, MAPK phosphorylation, RhoGTPase activity) from osteoclasts cultured from peripheral blood of individuals with NF1 (N=75) were assessed. Osteoclast formation was compared to phenotypic, radiologic, and biochemical data.NF1 osteoprogenitor cells demonstrated increased osteoclast forming capacity. Human NF1-derived osteoclasts demonstrated increased migration, adhesion, and in vitro bone resorption. These activities coincided with increased actin belt formation and hyperactivity in MAPK and RhoGTPase pathways. Although osteoclast formation was increased, no direct correlation of osteoclast formation with BMD, markers of bone resorption, or the clinical skeletal phenotype was observed suggesting that osteoclast formation in vitro cannot directly predict NF1 skeletal phenotypes.While NF1 haploinsufficiency produces a generalized osteoclast gain-in-function and may contribute to increased bone resorption, reduced BMD, and focal skeletal defects associated with