Alyssa A. Tran

Generalized metabolic bone disease in Neurofibromatosis type I

Skeletal abnormalities are a recognized component of Neurofibromatosis type I (NF1) but a generalized metabolic bone defect in NF1 has not been fully characterized thus far. The purpose of this study was to characterize at the densitometric, biochemical and pathological level the bone involvement in NF1 patients. Using dual energy X-ray absorptiometry (DXA) we analyzed bone status in 73 unselected NF1 subjects, 26 males and 47 females, mainly children and adolescents (mean age: 16.6 years). In a subgroup of subjects with low bone mass, we measured indices of calcium-phosphate metabolism, bone turnover, and bone density before and after vitamin D and calcium treatment. We found statistically significant and generalized reduction in bone mass with the mean lumbar bone mineral density (BMD) z-score being -1.38+/-1.05 (CI 95% -1.62 to -1.13), and whole body bone mineral content (BMC) z-score -0.61+/-1.19 (CI 95% -0.94 to -0.29), both significantly reduced compared to normal controls (p<.001). PTH was moderately elevated and after 4 months of supplemental therapy with calcium and vitamin D, it decreased to the normal range. However, BMD z-scores did not significantly improve after 2 years of follow-up. Histological analysis of bone samples from NF1 patients revealed substantial alteration of bone microarchitecture due mainly to reduced trabecular bone. Our observations are consistent with a generalized bone metabolic defect due to loss of the function of neurofibromin. Early identification of patients with osteoporosis may permit more timely and aggressive treatments to prevent the likely substantial morbidity associated with increased fracture risk later in life.

RMRP mutations in cartilage‐hair hypoplasia

Cartilage hair hypoplasia (CHH) or McKusick type metaphyseal chondrodysplasia (MCD) (OMIM # 250250) is due to either the homozygous or compound heterozygous mutations in the nuclear encoded, non‐coding RNA gene RMRP. Twenty‐seven CHH patients were referred for molecular evaluation of the clinical diagnosis. RMRP mutations were found in 22 patients. The phenotype in one of the five mutation‐negative patients was fully congruent with the adopted case definition of CHH. In a second of these patients, the diagnosis of Schmid type MCD (OMIM # 156500) was made and confirmed by the detection of a mutation in the COL10A1 gene. The remaining patients most likely represent one or more MCDs hitherto not yet delineated. The pattern of cumulative growth in infancy and early childhood in the latter four patients was the single feature with greatest negative predictive power for CHH. Fourteen mutations detected here, had not been reported previously. In this ethnically heterogeneous population, we performed a retrospective study to compare the prevalence of clinical features compared to previous reports based mostly on more ethnically homogenous groups.

Assessment of bone mineral status in children with Marfan syndrome.

Marfan syndrome (MFS) is an autosomal dominant connective tissue disorder with skeletal involvement. It is caused by mutations in fibrillin1 (FBN1) gene resulting in activation of TGF‐β, which developmentally regulates bone mass and matrix properties. There is no consensus regarding bone mineralization in children with MFS. Using dual‐energy X‐ray absorptiometry (DXA), we evaluated bone mineralization in 20 children with MFS unselected for bone problems. z‐Scores were calculated based on age, gender, height, and ethnicity matched controls. Mean whole body bone mineral content (BMC) z‐score was 0.26 ± 1.42 (P = 0.41). Mean bone mineral density (BMD) z‐score for whole body was −0.34 ± 1.4 (P = 0.29) and lumbar spine was reduced at −0.55 ± 1.34 (P = 0.017). On further adjusting for stature, which is usually higher in MFS, mean BMC z‐score was reduced at −0.677 ± 1.37 (P = 0.04), mean BMD z‐score for whole body was −0.82 ± 1.55 (P = 0.002) and for lumbar spine was −0.83 ± 1.32 (P = 0.001). An increased risk of osteoporosis in MFS is controversial. DXA has limitations in large skeletons because it tends to overestimate BMD and BMC. By adjusting results for height, age, gender, and ethnicity, we found that MFS patients have significantly lower BMC and BMD in whole body and lumbar spine. Evaluation of diet, exercise, vitamin D status, and bone turnover markers will help gain insight into pathogenesis of the reduced bone mass. Further, larger longitudinal studies are required to evaluate the natural history, incidence of fractures, and effects of pharmacological therapy.

Decreased bone mineralization in children with Noonan syndrome: another consequence of dysregulated RAS MAPKinase pathway?

INTRODUCTION Noonan syndrome (NS) is a disorder of RAS- mitogen activated protein kinase (MAPK) pathway with clinical features of skeletal dysplasia. This pathway is essential for regulation of cell differentiation and growth including bone homeostasis. Currently, limited information exists regarding bone mineralization in NS. MATERIALS AND METHODS Using dual-energy X-ray absorptiometry (DXA), bone mineralization was evaluated in 12 subjects (mean age 8.7 years) with clinical features of NS. All subjects underwent genetic testing which showed mutations in PTPN11 gene (N=8) and SOS1 gene (N=1). In a subgroup of subjects with low bone mass, indices of calcium-phosphate metabolism and bone turnover were obtained. RESULTS 50% of subjects had low bone mass as measured by DXA. Z-scores for bone mineral content (BMC) were calculated based on age, gender, height, and ethnicity. Mean BMC z-score was marginally decreased at -0.89 {95% CI -2.01 to 0.23; p=0.1}. Mean total body bone mineral density (BMD) z-score was significantly reduced at -1.87 {95% CI -2.73 to -1.0; p=0.001}. Mean height percentile was close to - 2 SD for this cohort, thus total body BMD z-scores were recalculated, adjusting for height age. Adjusted mean total body BMD z-score was less reduced but still significant at -0.82 {95% CI -1.39 to -0.25; p=0.009}. Biochemical evaluation for bone turnover was unremarkable except serum IGF-I and IGF-BP3 levels which were low-normal for age. DISCUSSION Children with NS have a significantly lower total body BMD compared to age, gender, ethnicity and height matched controls. In addition, total BMC appears to trend lower in children with NS compared to controls. We conclude that the metabolic bone disease present resulted from a subtle variation in the interplay of osteoclast and osteoblast activity, without clear abnormalities being defined in the metabolism of either. Clinical significance of this finding needs to be validated by larger longitudinal studies. Also, histomorphometric analysis of bone tissue from NS patients and mouse model of NS may further elucidate the relationship between the RAS-MAPK pathway and skeletal homeostasis.

Heterozygous variants in ACTL6A, encoding a component of the BAF complex, are associated with intellectual disability

Pathogenic variants in genes encoding components of the BRG1‐associated factor (BAF) chromatin remodeling complex have been associated with intellectual disability syndromes. We identified heterozygous, novel variants in ACTL6A, a gene encoding a component of the BAF complex, in three subjects with varying degrees of intellectual disability. Two subjects have missense variants affecting highly conserved amino acid residues within the actin‐like domain. Missense mutations in the homologous region in yeast actin were previously reported to be dominant lethal and were associated with impaired binding of the human ACTL6A to β‐actin and BRG1. A third subject has a splicing variant that creates an in‐frame deletion. Our findings suggest that the variants identified in our subjects may have a deleterious effect on the function of the protein by disturbing the integrity of the BAF complex. Thus, ACTL6A gene mutation analysis should be considered in patients with intellectual disability, learning disabilities, or developmental language disorder.

Corner fracture type spondylometaphyseal dysplasia: Overlap with type II collagenopathies

Spondylometaphyseal dysplasia (SMD) corner fracture type (also known as SMD “Sutcliffe” type, MIM 184255) is a rare skeletal dysplasia that presents with mild to moderate short stature, developmental coxa vara, mild platyspondyly, corner fracture‐like lesions, and metaphyseal abnormalities with sparing of the epiphyses. The molecular basis for this disorder has yet to be clarified. We describe two patients with SMD corner fracture type and heterozygous pathogenic variants in COL2A1. These two cases together with a third case of SMD corner fracture type with a heterozygous COL2A1 pathogenic variant previously described suggest that this disorder overlaps with type II collagenopathies. The finding of one of the pathogenic variants in a previously reported case of spondyloepimetaphyseal dysplasia (SEMD) Strudwick type and the significant clinical similarity suggest an overlap between SMD corner fracture and SEMD Strudwick types.

Neonatal fractures as a presenting feature of LMOD3-associated congenital myopathy

Nemaline myopathy is a rare inherited disorder characterized by weakness, hypotonia, and depressed deep tendon reflexes. It is clinically and genetically heterogeneous, with the most severe phenotype presenting as perinatal akinesia, severe muscle weakness, feeding difficulties and respiratory failure, leading to early mortality. Pathogenic variants in 12 genes, encoding components of the sarcomere or factors related to myogenesis, have been reported in patients affected with the disorder. Here, we describe an early, lethal presentation of decreased fetal movements, hypotonia, muscle weakness, and neonatal respiratory failure requiring ventilator support in three siblings from a consanguineous family. All exhibited perinatal fractures, and thus, a skeletal dysplasia was considered as possibly contributing to the phenotype. However, whole exome sequencing revealed a homozygous, loss‐of‐function pathogenic variant in LMOD3, which has recently been associated with nemaline myopathy and, in a subset of patients, perinatal fractures. This case demonstrates the importance of considering congenital neuromuscular disorders in the differential diagnosis of perinatal fractures.

Whole-Exome Sequencing Identifies an Intronic Cryptic Splice Site in SERPINF1 Causing Osteogenesis Imperfecta Type VI: WES FINDS INTRONIC CRYPTIC SPLICE SITE IN SERPINF1 CAUSING OI TYPE VI

The heritable disorder osteogenesis imperfecta (OI) is characterized by bone fragility and low bone mass. OI type VI is an autosomal recessive form of the disorder with moderate to severe bone fragility. OI type VI is caused by mutations in the serpin peptidase inhibitor, clade F, member 1 (SERPINF1), the gene coding for pigment epithelium‐derived factor (PEDF). Here, we report a patient with OI type VI caused by a novel homozygous intronic variant in SERPINF1 identified by whole‐exome sequencing (WES). The mutation was not identified using a low bone mass gene panel based on next‐generation sequencing. This variant creates a novel consensus splice donor site (AGGC to AGGT) in intron 4. Analysis of cDNA generated from fibroblasts revealed retention of a 32‐bp intronic fragment between exons 4 and 5 in the cDNA, a result of alternative splicing from the novel splice‐donor site. As a result, the aberrant insertion of this intronic fragment generated a frameshift pathogenic variant and induced nonsense‐mediated decay. Furthermore, gene expression by quantitative PCR showed SERPINF1 expression was dramatically reduced in patient fibroblasts, and PEDF level was also significantly reduced in the patients plasma. In conclusion, we report a novel homozygous variant that generates an alternative splice‐donor in intron 4 of SERPINF1 which gives rise to severe bone fragility. The work also demonstrates clinical utility of WES analysis, and consideration of noncoding variants, in the diagnostic setting of rare bone diseases.