Therapeutic Development in Neurofibromatosis

Although neurofibromatosis (NF) was initially recognized in the nineteenth century, only in the past two decades we have witnessed a paradigm shift in therapeutics. This progress is driven by the increasing understanding of the natural history of the NF-associated tumors and understanding of the molecular landscape of these disorders. Multiple clinical trials have been launched evaluating non-surgical treatment modalities and more studies are in the pipeline. Recently, the NF community has adopted standardized endpoints recommended by the Response Evaluation in Neurofibromatosis and Schwannomatosis (REiNS) International Collaboration established in 2011. Such collaborations among academic, regulatory and supporting communities are crucial for providing the infrastructure needed for advancing the therapeutic development in the field of NF

Restoration of Normal NF1 Function with Antisense Morpholino Treatment of Recurrent Pathogenic Patient-Specific Variant c.1466A>G; p.Y489C

Neurofibromatosis type 1 (NF1) is an autosomal dominant genetic disorder with almost 3000 different disease-causing variants within the NF1 gene identified. Up to 44% of these variants cause splicing errors to occur within pre-mRNA. A recurrent variant in exon 13, c.1466A>G; p.Y489C (Y489C) results in the creation of an intragenic cryptic splice site, aberrant splicing, a 62 base pair deletion from the mRNA, and subsequent frameshift. We investigated the ability of phosphorodiamidate morpholino oligomers (PMOs) to mask this variant on the RNA level, thus restoring normal splicing. To model this variant, we have developed a human iPS cell line homozygous for the variant using CRISPR/Cas9. PMOs were designed to be 25 base pairs long, and to cover the mutation site so it could not be read by splicing machinery. Results from our in vitro testing showed restoration of normal splicing in the RNA and restoration of full length neurofibromin protein. In addition, we observe the restoration of neurofibromin functionality through GTP-Ras and pERK/ERK testing. The results from this study demonstrate the ability of a PMO to correct splicing errors in NF1 variants at the RNA level, which could open the door for splicing corrections for other variants in this and a variety of diseases

Statins, bone, and neurofibromatosis type 1

Neurofibromatosis type 1 (NF1) is a dominantly inherited multi-system disorder. Major features include pigmentary abnormalities, benign tumors of the nerve sheath (neurofibromas), malignant tumors, learning disabilities, and skeletal dysplasia. The NF1 gene functions as a tumor suppressor, but haploinsuffiency probably accounts for some aspects of the non-tumor phenotype. The protein product, neurofibromin, is a Ras GTPase-activating protein, and various Ras pathway inhibitors are being tested in preclinical models and clinical trials for effectiveness in treating NF1 complications. This month in BMC Medicine, a paper by Kolanczyk et al describes a preclinical mouse model for tibial dysplasia and provides evidence that the drug lovastatin – in use to treat cardiovascular disease – may be beneficial, opening the door to clinical trials in humans

Sequencing the transcriptome of milk production: milk trumps mammary tissue

Background: Studies of normal human mammary gland development and function have mostly relied on cell culture, limited surgical specimens, and rodent models. Although RNA extracted from human milk has been used to assay the mammary transcriptome non-invasively, this assay has not been adequately validated in primates. Thus, the objectives of the current study were to assess the suitability of lactating rhesus macaques as a model for lactating humans and to determine whether RNA extracted from milk fractions is representative of RNA extracted from mammary tissue for the purpose of studying the transcriptome of milk-producing cells. Results: We confirmed that macaque milk contains cytoplasmic crescents and that ample high-quality RNA can be obtained for sequencing. Using RNA sequencing, RNA extracted from macaque milk fat and milk cell fractions more accurately represented RNA from mammary epithelial cells (cells that produce milk) than did RNA from whole mammary tissue. Mammary epithelium-specific transcripts were more abundant in macaque milk fat, whereas adipose or stroma-specific transcripts were more abundant in mammary tissue. Functional analyses confirmed the validity of milk as a source of RNA from milk-producing mammary epithelial cells. Conclusions: RNA extracted from the milk fat during lactation accurately portrayed the RNA profile of milk-producing mammary epithelial cells in a non-human primate. However, this sample type clearly requires protocols that minimize RNA degradation. Overall, we validated the use of RNA extracted from human and macaque milk and provided evidence to support the use of lactating macaques as a model for human lactation

Return of non-ACMG recommended incidental genetic findings to pediatric patients: Considerations and opportunities from experiences in genomic sequencing

BACKGROUND: The uptake of exome/genome sequencing has introduced unexpected testing results (incidental findings) that have become a major challenge for both testing laboratories and providers. While the American College of Medical Genetics and Genomics has outlined guidelines for laboratory management of clinically actionable secondary findings, debate remains as to whether incidental findings should be returned to patients, especially those representing pediatric populations. METHODS: The Sequencing Analysis and Diagnostic Yield working group in the Clinical Sequencing Evidence-Generating Research Consortium has collected a cohort of pediatric patients found to harbor a genomic sequencing-identified non-ACMG-recommended incidental finding. The incidental variants were not thought to be associated with the indication for testing and were disclosed to patients and families. RESULTS: In total, 23 non-ACMG-recommended incidental findings were identified in 21 pediatric patients included in the study. These findings span four different research studies/laboratories and demonstrate differences in incidental finding return rate across study sites. We summarize specific cases to highlight core considerations that surround identification and return of incidental findings (uncertainty of disease onset, disease severity, age of onset, clinical actionability, and personal utility), and suggest that interpretation of incidental findings in pediatric patients can be difficult given evolving phenotypes. Furthermore, return of incidental findings can benefit patients and providers, but do present challenges. CONCLUSIONS: While there may be considerable benefit to return of incidental genetic findings, these findings can be burdensome to providers and present risk to patients. It is important that laboratories conducting genomic testing establish internal guidelines in anticipation of detection. Moreover, cross-laboratory guidelines may aid in reducing the potential for policy heterogeneity across laboratories as it relates to incidental finding detection and return. However, future discussion is required to determine whether cohesive guidelines or policy statements are warranted

A YWHAZ Variant Associated With Cardiofaciocutaneous Syndrome Activates the RAF-ERK Pathway

Cardiofaciocutaneous (CFC) syndrome is a genetic disorder characterized by distinctive facial features, congenital heart defects, and skin abnormalities. Several germline gain-of-function mutations in the RAS/RAF/MEK/ERK pathway are associated with the disease, including KRAS, BRAF, MEK1, and MEK2. CFC syndrome thus belongs to a group of disorders known as RASopathies, which are all caused by pathogenic mutations in various genes encoding components of the RAS pathway. We recently identified novel variants in YWHAZ, a 14-3-3 family member, in individuals with a phenotype consistent with CFC that may potentially be deleterious and disease-causing. In the current study, we take advantage of the vertebrate model Xenopus laevis to analyze the functional consequence of a particular YWHAZ variant, S230W, and investigate the molecular mechanisms underlying its activity. We show that compared with wild type YWHAZ, the S230W variant induces severe embryonic defects when ectopically expressed in early Xenopus embryos. The S230W variant also rescues the defects induced by a dominant negative FGF receptor more efficiently and enhances Raf-stimulated Erk phosphorylation to a higher level than wild type YWHAZ. Although neither YWHAZ nor the variant promotes membrane recruitment of Raf proteins, the variant binds to more Raf and escapes phosphorylation by casein kinase 1a. Our data provide strong support to the hypothesis that the S230W variant of YWHAZ is a gain-of-function mutation in the RAS-ERK pathway and may underlie a CFC phenotype

Cutaneous neurofibromas Current clinical and pathologic issues

ObjectiveTo present the current terminology and natural history of neurofibromatosis 1 (NF1) cutaneous neurofibromas (cNF).MethodsNF1 experts from various research and clinical backgrounds reviewed the terms currently in use for cNF as well as the clinical, histologic, and radiographic features of these tumors using published and unpublished data.ResultsNeurofibromas develop within nerves, soft tissue, and skin. The primary distinction between cNF and other neurofibromas is that cNF are limited to the skin whereas other neurofibromas may involve the skin, but are not limited to the skin. There are important cellular, molecular, histologic, and clinical features of cNF. Each of these factors is discussed in consideration of a clinicopathologic framework for cNF.ConclusionThe development of effective therapies for cNF requires formulation of diagnostic criteria that encompass the clinical and histologic features of these tumors. However, there are several areas of overlap between cNF and other neurofibromas that make distinctions between cutaneous and other neurofibromas more difficult, requiring careful deliberation with input across the multiple disciplines that encounter these tumors and ultimately, prospective validation. The ultimate goal of this work is to facilitate accurate diagnosis and meaningful therapeutics for cNF