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A letter to the editor is presented regarding the case of a patient with RASopathy phenotype and germline KRAS mutation.

Neurofibromatosis type 1 results from loss-of-function NF1 pathogenic variants (PVs). Up to 30% of all NF1 PVs disrupt mRNA splicing, including deep intronic variants. Here, we retrospectively investigated the spectrum of NF1 deep intronic PVs in a cohort of 8,090 unrelated individuals from the University of Alabama at Birmingham (UAB) dataset with a molecularly confirmed neurofibromatosis type 1. All variants were identified through their effect on the NF1 transcript, followed by variant characterization at the DNA-level. A total of 68 distinct variants, which were ≥ 20 nucleotides away from the closest exon-intron junction, were identified in 2.5% unrelated individuals with NF1 (200/8,090). Nine different pathogenic splice variants, identified in 20 probands, led to exonization of different parts of intron 30 [23.2] or 31 [23a]. The two major NF1 transcript isoforms, distinguished by the absence (type I) or presence (type II) of the alternatively spliced cassette exon 31 [23a], are equally expressed in blood in control individuals without NF1 or NF1-affected individuals carrying their PV not in the introns flanking exon 31 [23a]. By fragment and cloning analysis we demonstrated that the exonization of intron 31 [23a] sequences due to deep intronic PV predominantly affects the NF1 isoform II. Seven additional (likely) pathogenic NF1 deep intronic variants not observed in the UAB dataset were found by classification of 36 variants identified by a literature search. Hence, the unique list of these 75 deep intronic (likely) PVs should be included in any comprehensive NF1 testing strategy., (© 2023. The Author(s).)

Missense variants in the NF2 gene result in variable NF2 disease presentation. Clinical classification of missense variants often represents a challenge, due to lack of evidence for pathogenicity and function. This study provides a summary of NF2 missense variants, with variant classifications based on currently available evidence. NF2 missense variants were collated from pathology-associated databases and existing literature. Association for Clinical Genomic Sciences Best Practice Guidelines (2020) were followed in the application of evidence for variant interpretation and classification. The majority of NF2 missense variants remain classified as variants of uncertain significance. However, NF2 missense variants identified in gnomAD occurred at a consistent rate across the gene, while variants compiled from pathology-associated databases displayed differing rates of variation by exon of NF2. The highest rate of NF2 disease-associated variants was observed in exon 7, while lower rates were observed toward the C-terminus of the NF2 protein, merlin. Further phenotypic information associated with variants, alongside variant-specific functional analysis, is necessary for more definitive variant interpretation. Our data identified differences in frequency of NF2 missense variants by exon between gnomAD population data and NF2 disease-associated variants, suggesting a potential genotype-phenotype correlation; further work is necessary to substantiate this., (© 2022 The Authors. Human Mutation published by Wiley Periodicals LLC.)

Constitutional LZTR1 or SMARCB1 pathogenic variants (PVs) have been found in ∼86% of familial and ∼40% of sporadic schwannomatosis cases. Hence, we performed massively parallel sequencing of the entire LZTR1, SMARCB1, and NF2 genomic loci in 35 individuals with schwannomas negative for constitutional first-hit PVs in the LZTR1/SMARCB1/NF2 coding sequences; however, with 22q deletion and/or a different NF2 PV in each tumor, including six cases with only one tumor available. Furthermore, we verified whether any other LZTR1/SMARCB1/NF2 (likely) PVs could be found in 16 cases carrying a SMARCB1 constitutional variant in the 3'-untranslated region (3'-UTR) c.*17C>T, c.*70C>T, or c.*82C>T. As no additional variants were found, functional studies were performed to clarify the effect of these 3'-UTR variants on the transcript. The 3'-UTR variants c.*17C>T and c.*82C>T showed pathogenicity by negatively affecting the SMARCB1 transcript level. Two novel deep intronic SMARCB1 variants, c.500+883T>G and c.500+887G>A, resulting in out-of-frame missplicing of intron 4, were identified in two unrelated individuals. Further resequencing of the entire repeat-masked genomics sequences of chromosome 22q in individuals negative for PVs in the SMARCB1/LZTR1/NF2 coding- and noncoding regions revealed five potential schwannomatosis-predisposing candidate genes, that is, MYO18B, NEFH, SGSM1, SGSM3, and SBF1, pending further verification., (© 2021 Wiley Periodicals LLC.)

Purpose: The LZTR1 gene has been associated with schwannomatosis tumor predisposition and is located in a region that is deleted in the great majority (89%) of patients with 22q11.2 deletion syndrome (22q11.2DS). Since it is known that approximately 1 in 500 people in the general population will develop a sporadic schwannoma and there are no reports of the occurrence of schwannoma in 22q11.2DS, we investigated whether whole-gene deletion of LZTR1 occurs in schwannomatosis and assessed the risk of schwannoma in 22q11.2DS., Methods: We assessed the genetic testing results for LZTR1-associated schwannomatosis and the clinical phenotypes of patients with 22q11.2DS., Results: There were no reports of schwannoma in over 1,500 patients with 22q11.2DS. In addition, no patients meeting clinical diagnostic criteria for schwannomatosis had a whole-gene deletion in LZTR1. Only 1 patient in 110 with an apparently sporadic vestibular schwannoma had a constitutional whole-gene deletion of LZTR1., Conclusion: People with a large 22q11.2 deletion may have a reduced risk of developing a schwannoma compared to the general population., (© 2021. The Author(s).)

We report 281 individuals carrying a pathogenic recurrent NF1 missense variant at p.Met1149, p.Arg1276, or p.Lys1423, representing three nontruncating NF1 hotspots in the University of Alabama at Birmingham (UAB) cohort, together identified in 1.8% of unrelated NF1 individuals. About 25% (95% confidence interval: 20.5-31.2%) of individuals heterozygous for a pathogenic NF1 p.Met1149, p.Arg1276, or p.Lys1423 missense variant had a Noonan-like phenotype, which is significantly more compared with the "classic" NF1-affected cohorts (all p < .0001). Furthermore, p.Arg1276 and p.Lys1423 pathogenic missense variants were associated with a high prevalence of cardiovascular abnormalities, including pulmonic stenosis (all p < .0001), while p.Arg1276 variants had a high prevalence of symptomatic spinal neurofibromas (p < .0001) compared with "classic" NF1-affected cohorts. However, p.Met1149-positive individuals had a mild phenotype, characterized mainly by pigmentary manifestations without externally visible plexiform neurofibromas, symptomatic spinal neurofibromas or symptomatic optic pathway gliomas. As up to 0.4% of unrelated individuals in the UAB cohort carries a p.Met1149 missense variant, this finding will contribute to more accurate stratification of a significant number of NF1 individuals. Although clinically relevant genotype-phenotype correlations are rare in NF1, each affecting only a small percentage of individuals, together they impact counseling and management of a significant number of the NF1 population., (© 2019 The Authors. Human Mutation Published by Wiley Periodicals, Inc.)

Purpose: Neurofibromatosis type 1 (NF1) is characterized by a highly variable clinical presentation, but almost all NF1-affected adults present with cutaneous and/or subcutaneous neurofibromas. Exceptions are individuals heterozygous for the NF1 in-frame deletion, c.2970&#95;2972del (p.Met992del), associated with a mild phenotype without any externally visible tumors., Methods: A total of 135 individuals from 103 unrelated families, all carrying the constitutional NF1 p.Met992del pathogenic variant and clinically assessed using the same standardized phenotypic checklist form, were included in this study., Results: None of the individuals had externally visible plexiform or histopathologically confirmed cutaneous or subcutaneous neurofibromas. We did not identify any complications, such as symptomatic optic pathway gliomas (OPGs) or symptomatic spinal neurofibromas; however, 4.8% of individuals had nonoptic brain tumors, mostly low-grade and asymptomatic, and 38.8% had cognitive impairment/learning disabilities. In an individual with the NF1 constitutional c.2970&#95;2972del and three astrocytomas, we provided proof that all were NF1-associated tumors given loss of heterozygosity at three intragenic NF1 microsatellite markers and c.2970&#95;2972del., Conclusion: We demonstrate that individuals with the NF1 p.Met992del pathogenic variant have a mild NF1 phenotype lacking clinically suspected plexiform, cutaneous, or subcutaneous neurofibromas. However, learning difficulties are clearly part of the phenotypic presentation in these individuals and will require specialized care.

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NF1 germline mutation predisposes to breast cancer. NF1 mutations have also been proposed as oncogenic drivers in sporadic breast cancers. To understand the genomic and histologic characteristics of these breast cancers, we analyzed the tumors with NF1 germline mutations and also examined the genomic and proteomic profiles of unselected tumors. Among 14 breast cancer specimens from 13 women affected with neurofibromatosis type 1 (NF1), 9 samples (NF + BrCa) underwent genomic copy number (CN) and targeted sequencing analysis. Mutations of NF1 were identified in two samples and TP53 were in three. No mutation was detected in ATM, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, NBN, PALB2, PTEN, RAD50 , and STK11 HER2 (ErbB2) overexpression was detected by IHC in 69.2% (9/13) of the tumors. CN gain/amplification of ERBB2 was detected in 4 of 9 with DNA analysis. By evaluating HER2 expression and NF1 alterations in unselected invasive breast cancers in TCGA datasets, we discovered that among samples with ERBB2 CN gain/amplification, the HER2 mRNA and protein expression were much more pronounced in NF1 -mutated/deleted samples in comparison with NF1 -unaltered samples. This finding suggests a synergistic interplay between these two genes, potentially driving the development of breast cancer harboring NF1 mutation and ERBB2 CN gain/amplification. NF1 gene loss of heterozygosity was observed in 4 of 9 NF + BrCa samples. CDK4 appeared to have more CN gain in NF + BrCa and exhibited increased mRNA expression in TCGA NF1- -altered samples. Cancer Prev Res; 11(10); 655-64. ©2018 AACR ., (©2018 American Association for Cancer Research.)

High-throughput technologies generate considerable amount of data which often requires bioinformatic expertise to analyze. Here we present High-Throughput Tabular Data Processor (HTDP), a platform independent Java program. HTDP works on any character-delimited column data (e.g. BED, GFF, GTF, PSL, WIG, VCF) from multiple text files and supports merging, filtering and converting of data that is produced in the course of high-throughput experiments. HTDP can also utilize itemized sets of conditions from external files for complex or repetitive filtering/merging tasks. The program is intended to aid global, real-time processing of large data sets using a graphical user interface (GUI). Therefore, no prior expertise in programming, regular expression, or command line usage is required of the user. Additionally, no a priori assumptions are imposed on the internal file composition. We demonstrate the flexibility and potential of HTDP in real-life research tasks including microarray and massively parallel sequencing, i.e. identification of disease predisposing variants in the next generation sequencing data as well as comprehensive concurrent analysis of microarray and sequencing results. We also show the utility of HTDP in technical tasks including data merge, reduction and filtering with external criteria files. HTDP was developed to address functionality that is missing or rudimentary in other GUI software for processing character-delimited column data from high-throughput technologies. Flexibility, in terms of input file handling, provides long term potential functionality in high-throughput analysis pipelines, as the program is not limited by the currently existing applications and data formats. HTDP is available as the Open Source software (https://github.com/pmadanecki/htdp).

Neurofibromatosis type 1 (NF1), a common genetic disorder with a birth incidence of 1:2,000-3,000, is characterized by a highly variable clinical presentation. To date, only two clinically relevant intragenic genotype-phenotype correlations have been reported for NF1 missense mutations affecting p.Arg1809 and a single amino acid deletion p.Met922del. Both variants predispose to a distinct mild NF1 phenotype with neither externally visible cutaneous/plexiform neurofibromas nor other tumors. Here, we report 162 individuals (129 unrelated probands and 33 affected relatives) heterozygous for a constitutional missense mutation affecting one of five neighboring NF1 codons-Leu844, Cys845, Ala846, Leu847, and Gly848-located in the cysteine-serine-rich domain (CSRD). Collectively, these recurrent missense mutations affect ∼0.8% of unrelated NF1 mutation-positive probands in the University of Alabama at Birmingham (UAB) cohort. Major superficial plexiform neurofibromas and symptomatic spinal neurofibromas were more prevalent in these individuals compared with classic NF1-affected cohorts (both p < 0.0001). Nearly half of the individuals had symptomatic or asymptomatic optic pathway gliomas and/or skeletal abnormalities. Additionally, variants in this region seem to confer a high predisposition to develop malignancies compared with the general NF1-affected population (p = 0.0061). Our results demonstrate that these NF1 missense mutations, although located outside the GAP-related domain, may be an important risk factor for a severe presentation. A genotype-phenotype correlation at the NF1 region 844-848 exists and will be valuable in the management and genetic counseling of a significant number of individuals., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

NF1 mutations predispose to neurofibromatosis type 1 (NF1) and women with NF1 have a moderately elevated risk for breast cancer, especially under age 50. Germline genomic analysis may better define the risk so screening and prevention can be applied to the individuals who benefit the most. Survey conducted in several neurofibromatosis clinics in the United States has demonstrated a 17.2% lifetime risk of breast cancer in women affected with NF1. Cumulated risk to age 50 is estimated to be 9.27%. For genomic profiling, fourteen women with NF1 and a history of breast cancer were recruited and underwent whole exome sequencing (WES), targeted genomic DNA based and RNA-based analysis of the NF1 gene. Deleterious NF1 pathogenic variants were identified in each woman. Frameshift mutations because of deletion/duplication/complex rearrangement were found in 50% (7/14) of the cases, nonsense mutations in 21% (3/14), in-frame splice mutations in 21% (3/14), and one case of missense mutation (7%, 1/14). No deleterious mutation was found in the following high/moderate-penetrance breast cancer genes: ATM, BRCA1, BRCA2, BARD1, BRIP1, CDH1, CHEK2, FANCC, MRE11A, NBN, PALB2, PTEN, RAD50, RAD51C, TP53, and STK11. Twenty-five rare or common variants in cancer related genes were discovered and may have contributed to the breast cancers in these individuals. Breast cancer predisposition modifiers in women with NF1 may involve a great variety of molecular and cellular functions., (© 2017 Wiley Periodicals, Inc.)

A 28-year-old female with PIK3CA-related segmental overgrowth presented with headaches. She also had a unilateral vestibular schwannoma (VS), as well as 3 small (<2 cm) meningiomas, which according to the Manchester consensus diagnostic criteria for neurofibromatosis 2 (NF2) is sufficient for a clinical diagnosis. Analysis of blood revealed a mosaic PIK3CA c.2740G>A (p.Gly914Arg) mutation, confirming the diagnosis of PIK3CA-related overgrowth, but no mutations in NF2 were detected. Although VS has not previously been reported in PIK3CA-related segmental overgrowth, meningiomas have, raising the question of whether this patient's VS and meningiomas represent coincidental NF2 or phenotypic extension of her overgrowth syndrome. Genetic analysis of the VS revealed a heterozygous NF2 mutation c.784C>T (p.Arg262Ter) and loss of a portion of 22q, including NF2, SMARCB1, and LZTR1 genes. These results suggest that the patient has 2 different mosaic disorders, NF2 and PIK3CA-related overgrowth. The PIK3CA mutation was also present in the VS. Confirmation of the clinical diagnosis of mosaic NF2 in this patient has implications for monitoring and highlights the possibility of co-occurrence of mosaicism for multiple rare disorders in a single patient., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Although isolated cafe-au-lait macules (CALMs) are a common skin finding, they are an early feature of neurofibromatosis type 1 (NF1)., Objective: We sought to develop an algorithm determining the risk of children with CALMs to have constitutional NF1., Methods: We conducted a retrospective study of patients with isolated CALMs. Diagnosis of NF1 was based on detecting NF1 mutation in blood or fulfilling clinical criteria., Results: In all, 170 of 419 (41%) and 21 of 86 (24%) children with isolated CALMs who underwent molecular testing and clinical follow-up, respectively, were given a diagnosis of NF1. Presence of fewer than 6 CALMs at presentation or atypical CALMs was associated with not having NF1 (P < .001). An algorithm based on age, CALMs number, and presence of atypical macules predicted NF1 in both cohorts. According to the algorithm, children older than 29 months with at least 1 atypical CALM or less than 6 CALMs have a 0.9% (95% confidence interval 0%-2.6%) risk for constitutional NF1 whereas children younger than 29 months with 6 or more CALMs have a high risk (80.4%, 95% confidence interval 74.6%-86.2%)., Limitations: The study was designed to detect constitutional NF1 and not NF1 in mosaic form., Conclusions: A simple algorithm enables categorization of children with isolated CALMs as being at low or high risk for having NF1., (Copyright © 2017 American Academy of Dermatology, Inc. Published by Elsevier Inc. All rights reserved.)

Phenotypic variability among individuals with neurofibromatosis type 1 (NF1) has long been a challenge for clinicians and an enigma for researchers. Members of the same family and even identical twins with NF1 often demonstrate variable disease expression. Many mechanisms for this variability have been proposed. We have performed an exploratory study of copy number variants (CNVs) as a possible source of phenotypic variability in NF1. We enrolled 11 pairs of monozygotic (MZ) twins with NF1 and their parents, catalogued their clinical characteristics, and utilized a single nucleotide polymorphism (SNP) microarray to identify CNVs in blood and saliva. The 11 twin pairs showed high concordance for presence and number of café-au-lait spots, cutaneous neurofibromas, IQ, and ADHD. They were more likely to be discordant for optic pathway glioma, plexiform neurofibromas, skeletal manifestations, and malignancy. Microarray analysis identified a total of 81 CNVs meeting our conservative criteria, 37 of which overlap known genes. Of interest, three CNVs were previously unreported. Microarray analysis failed to ascertain any CNV differences within twin pairs, between twins and parents, or between tissues in any one individual. Results of this small pilot study did not demonstrate any de novo CNV events in our MZ twin pairs, nor were de novo CNVs overrepresented in these individuals with NF1. A much larger sample size would be needed to form any conclusions about the role of CNVs in NF1 variable expressivity. Alternative explanations for discordant phenotypes include epigenetic changes, smaller genetic alterations, or environmental factors. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Neurofibromatosis type 1 (NF1) is a common genetic disorder characterized by the occurrence of nerve sheath tumors and considerable clinical heterogeneity. Some translational studies have been limited by the lack of animal models available for assessing patient-specific mutations. In order to test therapeutic approaches that might restore function to the mutated gene or gene product, we developed mice harboring NF1 patient-specific mutations including a nonsense mutation (c.2041C>T; p.Arg681*) and a missense mutation (c.2542G>C; p.Gly848Arg). The latter is associated with the development of multiple plexiform neurofibromas along spinal nerve roots. We demonstrate that the human nonsense NF1(Arg681*) and missense NF1(Gly848Arg) mutations have different effects on neurofibromin expression in the mouse and each recapitulates unique aspects of the NF1 phenotype, depending upon the genetic context when assessed in the homozygous state or when paired with a conditional knockout allele. Whereas the missense Nf1(Gly848Arg) mutation fails to produce an overt phenotype in the mouse, animals homozygous for the nonsense Nf1(Arg681*) mutation are not viable. Mice with one Nf1(Arg681*) allele in combination with a conditional floxed Nf1 allele and the DhhCre transgene (Nf1(4F/Arg681*); DhhCre) display disorganized nonmyelinating axons and neurofibromas along the spinal column, which leads to compression of the spinal cord and paralysis. This model will be valuable for preclinical testing of novel nonsense suppression therapies using drugs to target in-frame point mutations that create premature termination codons in individuals with NF1., (© 2016. Published by The Company of Biologists Ltd.)

Neurofibromatosis type 1 (NF1) is a common autosomal dominant neurologic condition characterized by significant clinical heterogeneity, ranging from malignant cancers to cognitive deficits. Recent studies have begun to reveal rare genotype-phenotype correlations, suggesting that the specific germline NF1 gene mutation may be one factor underlying disease heterogeneity. The purpose of this study was to define the impact of the germline NF1 gene mutation on brain neurofibromin function relevant to learning. Herein, we employ human NF1-patient primary skin fibroblasts, induced pluripotent stem cells and derivative neural progenitor cells (NPCs) to demonstrate that NF1 germline mutations have dramatic effects on neurofibromin expression. Moreover, while all NF1-patient NPCs exhibit increased RAS activation and reduced cyclic AMP generation, there was a neurofibromin dose-dependent reduction in dopamine (DA) levels. Additionally, we leveraged two complementary Nf1 genetically-engineered mouse strains in which hippocampal-based learning and memory is DA-dependent to establish that neuronal DA levels and signaling as well as mouse spatial learning are controlled in an Nf1 gene dose-dependent manner. Collectively, this is the first demonstration that different germline NF1 gene mutations differentially dictate neurofibromin function in the brain., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

We report on a pair of normally conceived monochorionic/dizygotic (MC/DZ) sex discordant twins. The comparison of blood and skin genotypes revealed that the chimerism was also present in the skin. We conjecture about the developmental origins of this case., (© 2015 Wiley Periodicals, Inc.)

Constitutional SMARCB1 mutations at 22q11.23 have been found in ∼50% of familial and <10% of sporadic schwannomatosis cases. We sequenced highly conserved regions along 22q from eight individuals with schwannomatosis whose schwannomas involved somatic loss of one copy of 22q, encompassing SMARCB1 and NF2, with a different somatic mutation of the other NF2 allele in every schwannoma but no mutation of the remaining SMARCB1 allele in blood and tumor samples. LZTR1 germline mutations were identified in seven of the eight cases. LZTR1 sequencing in 12 further cases with the same molecular signature identified 9 additional germline mutations. Loss of heterozygosity with retention of an LZTR1 mutation was present in all 25 schwannomas studied. Mutations segregated with disease in all available affected first-degree relatives, although four asymptomatic parents also carried an LZTR1 mutation. Our findings identify LZTR1 as a gene predisposing to an autosomal dominant inherited disorder of multiple schwannomas in ∼80% of 22q-related schwannomatosis cases lacking mutation in SMARCB1.

Neurofibromatosis type 1 (NF1) is one of the most common autosomal dominant disorders in humans. NF1 is caused by mutations of the NF1 gene. Mutation detection is complex owing to the large size of the NF1 gene, the presence of pseudogenes, and the great variety of mutations. Also, few probable genotype-phenotype correlations have been found in NF1. In this study 78 Korean patients from 60 families were screened for NF1 mutations. Mutation analysis of the entire coding region and flanking splice sites was carried out and included the use of a combination of reverse transcription polymerase chain reaction, multiplex ligation probe amplification, or fluorescence in situ hybridization. Mutation spectrum and genotype-phenotype relationship were assessed. Fifty-two distinct NF1 mutations were identified in 60 families. The mutations included 30 single base substitutions (12 missense and 18 nonsense), 11 missplicing mutations, seven small insertion or deletions, and four gross deletions. Sixteen (30.8%) mutations were novel; c.1A>G, c.2033&#95;2034insC, c.2540T>C, c.4537C>T, c.5546G>A, c.6792C>A, and c.6792C>G were recurrently identified. The mutations were evenly distributed across exon 1 through intron 47 of NF1, and no mutational hot spots were found. A genotype-phenotype analysis suggests that there is no clear relationship between specific mutations and clinical features. This analysis revealed a wide spectrum of NF1 mutations in Korean patients. As technologies advance in molecular genetics, the mutation detection rate will increase. Considering that 30.8% of detected mutations were novel, exhaustive mutation analysis of NF1 may be an important tool in early diagnosis and genetic counseling., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Piebaldism is an autosomal dominant disorder characterized by congenital hypopigmented patches of skin and hair and has been found to be associated with mutations in the KIT or SLUG genes. Café-au-lait macules (CALM) may occasionally be seen in piebaldism. There are four reports describing six patients who were said to have both piebaldism and neurofibromatosis type 1 (NF1) due to the presence of multiple CALM and intertriginous freckling, but none of these patients had undergone comprehensive NF1 mutation analysis. We describe a large family with piebaldism in which two members meet diagnostic criteria for NF1 based on the presence of >5 CALM and intertriginous freckling. Interestingly, only these two family members are of mixed race, which could be of importance. A novel complex mutation in the KIT gene was identified in several family members affected with piebaldism; the proband meeting diagnostic criteria for NF1 also underwent comprehensive NF1 and SPRED1 testing with no mutations detected. These findings suggest that piebaldism may occasionally include CALM and intertriginous freckling, which may create diagnostic confusion especially in the absence of a family history of piebaldism. However, careful clinical evaluation and molecular testing if necessary should distinguish these two disorders., (Copyright © 2012 Wiley Periodicals, Inc.)

Here we report on a patient with an interstitial deletion on the long(q) arm of chromosome 1 who presents with a unique constellation of anomalies including brachydactyly type E, Müllerian agenesis, growth hormone deficiency, as well as other abnormalities. We present the clinical details of this patient's presentation, the skeletal findings, and provide characterization of the deletion at the molecular level. We postulate that these skeletal anomalies are distinctive to 1q deletions involving the 1q24q25 region., ((c) 2008 Wiley-Liss, Inc.)

Background: Neurofibromatosis type 1 (NF1) is a common disorder of dysregulated tissue growth secondary to mutations in the tumor suppressor gene NF1. Pulmonary arterial hypertension (PAH) in patients with NF1 is hypothesized to be secondary to an underlying vasculopathy., Methods: We describe the entity we term NF1-associated PAH (NF1-PAH) in four new patients and update the data on four previously published reports of patients with PAH and NF1. We performed genetic testing of the bone morphogenic protein receptor 2 (BMPR2) gene, which mutated in 70% of patients with familial PAH and approximately 25% of patients with idiopathic PAH. We report, for the first time, pathologic findings in the autopsy-obtained lung of one patient with NF1-PAH., Results: Patients with NF1-PAH have a generally poor long-term prognosis. In four patients, we observed the mosaic pattern of lung attenuation on a CT scan of the chest, a radiographic finding that can be consistent with an underlying vasculopathy. No mutations or rearrangements in the BMPR2 gene were found. We observed complex plexiform lesions in the one available autopsy specimen. Similar lesions are a hallmark of plexogenic pulmonary arteriopathy and are associated with several severe types of PAH. (Plexiform lesions should not be confused with plexiform neurofibromas, which are distinctive tumors seen in NF1.), Conclusions: Our findings suggest that NF1 should be considered as being "associated with PAH as outlined in the Revised Clinical Classification of Pulmonary Hypertension. Understanding the mechanism of PAH in NF1 may inform the pathogenesis of PAH, NF1-PAH itself, and other NF1-associated vasculopathies. The pulmonary vasculature should now be included among the arterial beds affected by NF1 vasculopathy.

Osseous abnormalities, including long-bone dysplasia with pseudarthrosis (PA), are associated with neurofibromatosis type 1 (NF1). Prospectively acquired tissue from the PA site of two individuals with NF1 was used for immunohistochemical characterization and genotype analysis of the NF1 locus. Typical immunohistochemical features of neurofibroma were not observed. Genotype analysis of PA tissue with use of four genetic markers (D17S1863, GXALU, IN38, and 3NF1-1) spanning the NF1 locus demonstrated loss of heterozygosity. These results are the first to document double inactivation of NF1 in PA tissue and suggest that the neurofibromin-Ras signal transduction pathway is involved in this bone dysplasia in NF1.

The expression of a gene requires not only a normal coding sequence but also intact regulatory regions, which can be located at large distances from the target genes, as demonstrated for an increasing number of developmental genes. In previous mutation studies of the role of FOXL2 in blepharophimosis syndrome (BPES), we identified intragenic mutations in 70% of our patients. Three translocation breakpoints upstream of FOXL2 in patients with BPES suggested a position effect. Here, we identified novel microdeletions outside of FOXL2 in cases of sporadic and familial BPES. Specifically, four rearrangements, with an overlap of 126 kb, are located 230 kb upstream of FOXL2, telomeric to the reported translocation breakpoints. Moreover, the shortest region of deletion overlap (SRO) contains several conserved nongenic sequences (CNGs) harboring putative transcription-factor binding sites and representing potential long-range cis-regulatory elements. Interestingly, the human region orthologous to the 12-kb sequence deleted in the polled intersex syndrome in goat, which is an animal model for BPES, is contained in this SRO, providing evidence of human-goat conservation of FOXL2 expression and of the mutational mechanism. Surprisingly, in a fifth family with BPES, one rearrangement was found downstream of FOXL2. In addition, we report nine novel rearrangements encompassing FOXL2 that range from partial gene deletions to submicroscopic deletions. Overall, genomic rearrangements encompassing or outside of FOXL2 account for 16% of all molecular defects found in our families with BPES. In summary, this is the first report of extragenic deletions in BPES, providing further evidence of potential long-range cis-regulatory elements regulating FOXL2 expression. It contributes to the enlarging group of developmental diseases caused by defective distant regulation of gene expression. Finally, we demonstrate that CNGs are candidate regions for genomic rearrangements in developmental genes.

Purpose: To investigate the genetic basis of autosomal dominant vitreoretinochoroidopathy (ADVIRC), a rare, inherited retinal dystrophy that may be associated with defects of ocular development, including nanophthalmos., Methods: A combination of linkage analysis and DNA sequencing in five families was used to identify disease-causing mutations in VMD2. The effect of these mutations on splicing was assessed using a minigene system., Results: Three pathogenic sequence alterations in VMD2 were identified in five families with nanophthalmos associated with ADVIRC. All sequences showed simultaneous missense substitutions and exon skipping., Conclusions: VMD2 encodes bestrophin, a transmembrane protein located at the basolateral membrane of the RPE, that is also mutated in Best macular dystrophy. We support that each heterozygous affected individual produces three bestrophin isoforms consisting of the wild type and two abnormal forms: one containing a missense substitution and the other an in-frame deletion. The data showed that VMD2 mutations caused defects of ocular patterning, supporting the hypothesized role for the RPE, and specifically VMD2, in the normal growth and development of the eye.

Neurofibromatosis type 1 (NF1) is one of the most common autosomal dominant disorders and is caused by mutations in the NF1 gene. Mutation detection is complex due to the large size of the NF1 gene, the presence of pseudogenes and the great variety of possible lesions. Although there is no evidence for locus heterogeneity in NF1, mutation detection rates rarely exceed 50%. We studied 67 unrelated NF1 patients fulfilling the NIH diagnostic criteria, 29 familial and 38 sporadic cases, using a cascade of complementary techniques. We performed a protein truncation test starting from puromycin-treated EBV cell lines and, if no mutation was found, continued with heteroduplex, FISH, Southern blot and cytogenetic analysis. We identified the germline mutation in 64 of 67 patients and 32 of the mutations are novel. This is the highest mutation detection rate reported in a study of typical NF1 patients. All mutations were studied at the genomic and RNA level. The mutational spectrum consisted of 25 nonsense, 12 frameshift, 19 splice mutations, six missense and/or small in-frame deletions, one deletion of the entire NF1 gene, and a translocation t(14;17)(q32;q11.2). Our data suggest that exons 10a-10c and 37 are mutation-rich regions and that together with some recurrent mutations they may account for almost 30% of the mutations in classical NF1 patients. We found a high frequency of unusual splice mutations outside of the AG/GT 5 cent and 3 cent splice sites. As some of these mutations form stable transcripts, it remains possible that a truncated neurofibromin is formed., (Copyright 2000 Wiley-Liss, Inc.)

Purpose: To analyze the spectrum and frequency of NF1 mutations in exon 10b., Methods: Mutation and sequence analysis was performed at the DNA and cDNA level., Results: We identified nine exon 10b mutations in 232 unrelated patients. Some mutations were recurrent (Y489C and L508P), others were unique (1465-1466insC and IVS10b+2delTAAG). Surprisingly, at the RNA level, Y489C causes skipping of the last 62 nucleotides of exon 10b. Another recurrent mutation, L508P, is undetectable by the Protein Truncation Test., Conclusion: As exon 10b shows the highest mutation rate yet found in any of the 60 NF1 exons, it should be implemented with priority in mutation analysis.

We report a male neonate with craniofacial dysmorphic features, multiple congenital anomalies and an unusual form of chondrodysplasia punctata. Radiographic examination revealed punctate epiphyses and coronal clefting of the thoracic spine. The hand radiographs showed some similarities to the brachytelephalangic type of chondrodysplasia punctata. However, the disorder did not fit well with any known entity of chondrodysplasia punctata or other condition characterized by punctate epiphyses.

Qualitative and quantitative methods for the analysis of invasion in 'natural' and in experimental tumours in vivo and in vitro are reviewed. In human tumours the functional consequences of invasion were evaluated histologically through staging on the basis of depths of invasion and through the presence of tumour cells inside vessels. Antibodies against components of the basement membrane have facilitated the definition of minimal invasion. With new probes derived from oncogene research the search for molecular differences between invasive and non-invasive parts of the tumour has begun. Since the same methods as those used for analysis of natural tumours also apply to experimental tumours in vivo, the major advantage of the latter is the possibility of manipulation. We have described a new mesenterium assay that may permit the selection of invasive cells from non-invasive ones in transfection experiments. Invasion relative to growth as a function of time was quantified in the kidney invasion test. In three-dimensional confrontations between embryonic chick heart fragments and invasive cells, we have used both a subjective grading and a qualitative computer-assisted image analysis of serial histological sections to score invasion. In two-dimensional confrontations supplementary methods could be applied, since such confrontations permitted direct observations on living cultures. In a variety of natural and experimental tumours, ultrastructural analysis, transmigration in two-compartment chambers, and release of metabolic label have demonstrated the role of motility and of lytic activity in tumour invasion.