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Context: Autosomal dominant inactivating sprouty-related EVH1 domain-containing protein 1 (SPRED1) mutations have recently been described in individuals presenting mainly with café au lait macules (CALMs), axillary freckling, and macrocephaly. The extent of the clinical spectrum of this new disorder needs further delineation.Objective: To determine the frequency, mutational spectrum, and phenotype of neurofibromatosis type 1-like syndrome (NFLS) in a large cohort of patients.Design, Setting, and Participants: In a cross-sectional study, 23 unrelated probands carrying a SPRED1 mutation identified through clinical testing participated with their families in a genotype-phenotype study (2007-2008). In a second cross-sectional study, 1318 unrelated anonymous samples collected in 2003-2007 from patients with a broad range of signs typically found in neurofibromatosis type 1 (NF1) but no detectable NF1 germline mutation underwent SPRED1 mutation analysis.Main Outcome Measures: Comparison of aggregated clinical features in patients with or without a SPRED1 or NF1 mutation. Functional assays were used to evaluate the pathogenicity of missense mutations.Results: Among 42 SPRED1-positive individuals from the clinical cohort, 20 (48%; 95% confidence interval [CI], 32%-64%) fulfilled National Institutes of Health (NIH) NF1 diagnostic criteria based on the presence of more than 5 CALMs with or without freckling or an NF1-compatible family history. None of the 42 SPRED1-positive individuals (0%; 95% CI, 0%-7%) had discrete cutaneous or plexiform neurofibromas, typical NF1 osseous lesions, or symptomatic optic pathway gliomas. In the anonymous cohort of 1318 individuals, 34 different SPRED1 mutations in 43 probands were identified: 27 pathogenic mutations in 34 probands and 7 probable nonpathogenic missense mutations in 9 probands. Of 94 probands with familial CALMs with or without freckling and no other NF1 features, 69 (73%; 95% CI, 63%-80%) had an NF1 mutation and 18 (19%; 95% CI, 12%-29%) had a pathogenic SPRED1 mutation. In the anonymous cohort, 1.9% (95% CI, 1.2%-2.9%) of individuals with the clinical diagnosis of NF1 according to the NIH criteria had NFLS.Conclusions: A high SPRED1 mutation detection rate was found in NF1 mutation-negative families with an autosomal dominant phenotype of CALMs with or without freckling and no other NF1 features. Among individuals in this study, NFLS was not associated with the peripheral and central nervous system tumors seen in NF1. [ABSTRACT FROM AUTHOR]

Spinal neurofibromas are found in up to 38% of NF1 patients. However, they cause clinical implications only in about 5% of the patients. In contrast, multiple symptomatic spinal neurofibromas are the main clinical finding in patients with familial spinal neurofibromatosis. Familial spinal neurofibromatosis has been considered to be a distinct clinical form of neurofibromatosiso Linkage analysis in two families and identification of a NF1 gene mutation in a third family strongly associate spinal neurofibromatosis with the NF1 gene. We describe a NF1 patient who satisfies the NIH diagnostic criteria and has severe spinal involvement with bilateral spinal root neurofibromas at every level. A recurrent splice site mutation (IVS19b3C > G) was identified in the NF1 gene in the patient. We discuss the possibility that the clinical picture of this patient represents an additional example of spinal neurofibromatosis. By comparison of the clinical expression of NF1 in this patient and that in another patient with the identical mutation the hypothesis that spinal neurofibromatosis is associated with a particular mutation is highly unlikely. The involvement of other genes linked to the NF1 gene or modifying genes is currently the most likely explanation for the clinical phenotype of spinal neurofibromatosis. [ABSTRACT FROM AUTHOR]

Exposure to solar ultraviolet (UV) radiation is an established risk factor for skin cancer. Toll-like receptor-4 (TLR4)-mediated immune dysregulation has emerged as a key mechanism for the detrimental effects of acute and chronic UV exposure and skin cancer in mice. Single nucleotide polymorphisms (SNPs) on the TLR4 gene have been reported to increase or decrease susceptibility to various cancers in other organs. There is limited information on TLR4 SNPs and susceptibility to human keratinocyte carcinomas. The study's objective is to test the association between TLR4 SNPs and the risk of developing keratinocyte carcinomas. Skin cancer patients and controls at the University of Alabama at Birmingham completed a cross-sectional survey on personal and family history of skin cancer as well as on sunscreen use and tanning proneness. Peripheral blood samples were obtained from participants, and DNA was extracted to genotype the TLR4 SNPs. Descriptive analytics were used to describe the cohort. Multivariable logistic regression models were used to assess the association between TLR4 SNPs and skin cancer risk. The sample consisted of a cohort of 93 skin cancer patients over the age of 50 and 94 controls; 33.3% of cases and 44.7% of controls were females; 12.9% of cases and 17% of controls had a TLR4 SNP. The most common SNP was D299G/T399I in 9.7% of skin cancer patients and 13.8% of controls. We did not find a statistically significant association between the D299G/T399I SNP and skin cancer (odds ratio (OR) = 0.34, 95% CI: 0.11, 1.07, p = 0.065) adjusting for age, sex, eye color, actinic keratosis, sunscreen use and reapplication, and family history of skin cancer. Based on our findings from our limited cohort of participants, we found some protective effect for the TLR4 SNP for skin cancer, which was not statistically significant. Validation of these findings in a larger cohort is warranted.

Neurofibromatosis type 1 (NF1) is an autosomal dominant neurocutaneous disorder caused by loss-of-function variants in the NF1 gene. As of 20 November 2023, over 5000 distinct pathogenic or likely pathogenic variants have been reported in public databases. However, only a few NF1 genotype-phenotype correlations have been established so far. In this study, we present findings on 40 individuals with NF1, comprising 26 unrelated probands and 14 affected relatives, who carry one of nine NF1 heterozygous pathogenic splicing variants, all of which result in the in-frame skipping of exon 24 [19a] (NM_000267.3:r.3114_3197del, p.Asn1039_Arg1066del). These variants include c.3114-2A>G, c.3114-1G>A, c.3196A>G, c.3197G>A, c.3197G>T, c.3197+1G>A, c.3197+1G>T, c.3197+2T>C, and c.3197+3A>T. Among individuals with these variants, none exhibit externally visible plexiform neurofibromas, histopathologically confirmed cutaneous or subcutaneous neurofibromas, symptomatic spinal neurofibromas, or symptomatic optic pathway gliomas. The most prevalent, and sometimes sole, clinical feature observed in this cohort is multiple café-au-lait macules, with or without skinfold freckles: 85% and 60.5% of the individuals display six or more café-au-lait macules and freckles, respectively. In comparison to established NF1 genotype-phenotype correlations, these patients demonstrate highly similar clinical presentations to those associated with the NF1 pathogenic variant c.2970_2972del (p.Met992del), known for resulting in the mildest clinical features. Despite the generally mild phenotype, cognitive impairment, developmental delay, and/or learning difficulties are still observed in 33.3% of these patients, suggesting that learning challenges remain a prominent aspect of the phenotypic presentation in these individuals and necessitate specialized care. This newly established genotype-phenotype correlation will assist clinicians in improving the management of patients harboring NF1 exon 24 [19a] skipping variants and provide a new therapeutic target for NF1 treatment.

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).)

Uncovering frequent motives of action by which variants impair 3' splice site (3'ss) recognition and selection is essential to improve our understanding of this complex process. Through several mini-gene experiments, we demonstrate that the pyrimidine (Y) to purine (R) transversion NM_000267.3(NF1):c.1722-11T>G, although expected to weaken the polypyrimidine tract, causes exon skipping primarily by introducing a novel AG in the AG-exclusion zone (AGEZ) between the authentic 3'ss AG and the branch point. Evaluation of 90 additional noncanonical intronic NF1 3'ss mutations confirmed that 63% of all mutations and 89% (49/55) of the single-nucleotide variants upstream of positions -3 interrupt the AGEZ. Of these AGEZ-interrupting mutations, 24/49 lead to exon skipping suggesting that absence of AG in this region is necessary for accurate 3'ss selection already in the initial steps of splicing. The analysis of 91 noncanonical NF1 3'ss mutations also shows that 90% either introduce a novel AG in the AGEZ, cause a Y>R transversion at position -3 or remove ≥2 Ys in the AGEZ. We confirm in a validation cohort that these three motives distinguish spliceogenic from splice-neutral variants with 85% accuracy and, therefore, are generally applicable to select among variants of unknown significance those likely to affect splicing., (© 2020 The Authors. Human Mutation published by Wiley Periodicals, Inc.)

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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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.)

Multiplex ligation-dependent probe amplification (MLPA) has been widely used to identify copy-number variations (CNVs), but MLPA's sensitivity and specificity in mosaic CNV detection are largely unknown. Here, we present two mosaic deletions identified by MLPA as NF1 deletion of exons 17-21 and NF2 deletion of exons 9-10. Through cDNA analysis, genomic breakpoint-spanning PCR and Sanger sequencing, we found however both NF1 and NF2 deletions are each composed of two consecutive deletions, which cannot be differentiated by MLPA. Importantly, these consecutive deletions are most likely originating from a single genomic rearrangement and have been preserved independently in different populations of cells.

Neurofibromatosis type 1 (NF1) is one of the most frequent genetic disorders, affecting 1:3,000 worldwide. Identification of genotype-phenotype correlations is challenging because of the wide range clinical variability, the progressive nature of the disorder, and extreme diversity of the mutational spectrum. We report 136 individuals with a distinct phenotype carrying one of five different NF1 missense mutations affecting p.Arg1809. Patients presented with multiple café-au-lait macules (CALM) with or without freckling and Lisch nodules, but no externally visible plexiform neurofibromas or clear cutaneous neurofibromas were found. About 25% of the individuals had Noonan-like features. Pulmonic stenosis and short stature were significantly more prevalent compared with classic cohorts (P < 0.0001). Developmental delays and/or learning disabilities were reported in over 50% of patients. Melanocytes cultured from a CALM in a segmental NF1-patient showed two different somatic NF1 mutations, p.Arg1809Cys and a multi-exon deletion, providing genetic evidence that p.Arg1809Cys is a loss-of-function mutation in the melanocytes and causes a pigmentary phenotype. Constitutional missense mutations at p.Arg1809 affect 1.23% of unrelated NF1 probands in the UAB cohort, therefore this specific NF1 genotype-phenotype correlation will affect counseling and management of a significant number of patients., (© 2015 The Authors. **Human Mutation published by Wiley Periodicals, Inc.)

Genomic rearrangements can cause both Mendelian and complex disorders. Currently, several major mechanisms causing genomic rearrangements, such as non-allelic homologous recombination (NAHR), non-homologous end joining (NHEJ), fork stalling and template switching (FoSTeS), and microhomology-mediated break-induced replication (MMBIR), have been proposed. However, to what extent these mechanisms contribute to gene-specific pathogenic copy-number variations (CNVs) remains understudied. Furthermore, few studies have resolved these pathogenic alterations at the nucleotide-level. Accordingly, our aim was to explore which mechanisms contribute to a large, unique set of locus-specific non-recurrent genomic rearrangements causing the genetic neurocutaneous disorder neurofibromatosis type 1 (NF1). Through breakpoint-spanning PCR as well as array comparative genomic hybridization, we have identified the breakpoints in 85 unrelated individuals carrying an NF1 intragenic CNV. Furthermore, we characterized the likely rearrangement mechanisms of these 85 CNVs, along with those of two additional previously published NF1 intragenic CNVs. Unlike the most typical recurrent rearrangements mediated by flanking low-copy repeats (LCRs), NF1 intragenic rearrangements vary in size, location, and rearrangement mechanisms. We propose the DNA-replication-based mechanisms comprising both FoSTeS and/or MMBIR and serial replication stalling to be the predominant mechanisms leading to NF1 intragenic CNVs. In addition to the loop within a 197-bp palindrome located in intron 40, four Alu elements located in introns 1, 2, 3, and 50 were also identified as intragenic-rearrangement hotspots within NF1., (Copyright © 2015 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

For the rabphillin-3A-like (RPH3AL) gene, a putative tumor suppressor, the clinical significance of genetic alterations in breast cancers was evaluated. DNA and RNA were extracted from formalin-fixed, paraffin-embedded (FFPE) cancers and matching normal tissues. DNA samples were assessed for loss of heterozygosity (LOH) at the 17p13.3 locus of RPH3AL and the 17p13.1 locus of the tumor suppressor, TP53. RPH3AL was sequenced, and single nucleotide polymorphisms (SNPs) were genotyped. RNA samples were evaluated for expression of RPH3AL, and FFPE tissues were profiled for its phenotypic expression. Alterations in RPH3AL were correlated with clinicopathological features, LOH of TP53, and patient survival. Of 121 cancers, 80 had LOH at one of the RPH3AL locus. LOH of RHP3AL was associated with nodal metastasis, advanced stage, large tumor size, and poor survival. Although ~50% were positive for LOH at the RPH3AL and TP53 loci, 19 of 105 exhibited LOH only at the RPH3AL locus. Of these, 12 were non-Hispanic Caucasians (Whites), 15 had large tumors, and 12 were older (>50 years). Patients exhibiting LOH at both loci had shorter survival than those without LOH at these loci (log-rank, P = 0.014). LOH at the TP53 locus alone was not associated with survival. Analyses of RPH3AL identified missense point mutations in 19 of 125 cases, a SNP (C>A) in the 5'untranslated region at -25 (5'UTR-25) in 26 of 104, and a SNP (G>T) in the intronic region at 43 bp downstream to exon-6 (intron-6-43) in 79 of 118. Genotype C/A or A/A of the SNP at 5'UTR-25 and genotype T/T of a SNP at intron-6-43 were predominantly in Whites. Low levels of RNA and protein expression of RPH3AL were present in cancers relative to normal tissues. Thus, genetic alterations in RPH3AL are associated with aggressive behavior of breast cancers and with short survival of patients.

Approximately 5% of all patients with neurofibromatosis type-1 (NF1) exhibit large deletions of the NF1 gene region. To date, only nine unrelated cases of large NF1 duplications have been reported, with none of the affected patients exhibiting multiple café au lait spots (CALS), Lisch nodules, freckling, or neurofibromas, the hallmark signs of NF1. Here, we have characterized two novel NF1 duplications, one sporadic and one familial. Both index patients with NF1 duplications exhibited learning disabilities and atypical CALS. Additionally, patient R609021 had Lisch nodules, whereas patient R653070 exhibited two inguinal freckles. The mother and sister of patient R609021 also harbored the NF1 duplication and exhibited cognitive dysfunction but no CALS. The breakpoints of the nine NF1 duplications reported previously have not been identified and hence their underlying generative mechanisms have remained unclear. In this study, we performed high-resolution breakpoint analysis that indicated that the two duplications studied were mediated by nonallelic homologous recombination (NAHR) and that the duplication breakpoints were located within the NAHR hotspot paralogous recombination site 2 (PRS2), which also harbors the type-1 NF1 deletion breakpoints. Hence, our study indicates for the first time that NF1 duplications are reciprocal to type-1 NF1 deletions and originate from the same NAHR events., (© 2014 WILEY PERIODICALS, INC.)

Palindromic sequences can form hairpin structures or cruciform extrusions, which render them susceptible to genomic rearrangements. A 197-bp long palindromic AT-rich repeat (PATRR17) is located within intron 40 of the neurofibromatosis type 1 (NF1) gene (17q11.2). Through comprehensive NF1 analysis, we identified six unrelated patients with a rearrangement involving intron 40 (five deletions and one reciprocal translocation t(14;17)(q32;q11.2)). We hypothesized that PATRR17 may be involved in these rearrangements thereby causing NF1. Breakpoint cloning revealed that PATRR17 was indeed involved in all of the rearrangements. As microhomology was present at all breakpoint junctions of the deletions identified, and PATRR17 partner breakpoints were located within 7.1 kb upstream of PATRR17, fork stalling and template switching/microhomology-mediated break-induced replication was the most likely rearrangement mechanism. For the reciprocal translocation case, a 51 bp insertion at the translocation breakpoints mapped to a short sequence within PATRR17, proximal to the breakpoint, suggesting a multiple stalling and rereplication process, in contrast to previous studies indicating a purely replication-independent mechanism for PATRR-mediated translocations. In conclusion, we show evidence that PATRR17 is a hotspot for pathogenic intragenic deletions within the NF1 gene and suggest a novel replication-dependent mechanism for PATRR-mediated translocation., (© 2014 WILEY PERIODICALS, INC.)

Purpose: "Jaffe-Campanacci syndrome" describes the complex of multiple nonossifying fibromas of the long bones, mandibular giant cell lesions, and café-au-lait macules in individuals without neurofibromas. We sought to determine whether Jaffe-Campanacci syndrome is a distinct genetic entity or a variant of neurofibromatosis type 1., Methods: We performed germline NF1, SPRED1, and GNAS1 (exon 8) mutation testing on patients with Jaffe-Campanacci syndrome or Jaffe-Campanacci syndrome-related features. We also performed somatic NF1 mutation testing on nonossifying fibromas and giant cell lesions., Results: Pathogenic germline NF1 mutations were identified in 13 of 14 patients with multiple café-au-lait macules and multiple nonossifying fibromas or giant cell lesions ("classical" Jaffe-Campanacci syndrome); all 13 also fulfilled the National Institutes of Health diagnostic criteria for neurofibromatosis type 1. Somatic NF1 mutations were detected in two giant cell lesions but not in two nonossifying fibromas. No SPRED1 or GNAS1 (exon 8) mutations were detected in the seven NF1-negative patients with Jaffe-Campanacci syndrome, nonossifying fibromas, or giant cell lesions., Conclusion: In this study, the majority of patients with café-au-lait macules and nonossifying fibromas or giant cell lesions harbored a pathogenic germline NF1 mutation, suggesting that many Jaffe-Campanacci syndrome cases may actually have neurofibromatosis type 1. We provide the first proof of specific somatic second-hit mutations affecting NF1 in two giant cell lesions from two unrelated patients, establishing these as neurofibromatosis type 1-associated tumors.

Dispersal affects both social behavior and population structure and is therefore a key determinant of long-term population persistence. However, dispersal strategies and responses to spatial habitat alteration may differ between sexes. Here we analyzed spatial and temporal variation in ten polymorphic microsatellite DNA loci of male and female Cabanis's greenbuls (Phyllastrephuscabanisi), a cooperative breeder of Afrotropical rainforest, to quantify rates of gene flow and fine-grained genetic structuring within and among fragmented populations. We found genetic evidence for female-biased dispersal at small spatial scales, but not at the landscape level. Local autocorrelation analysis provided evidence of positive genetic structure within 300 m distance ranges, which is consistent with behavioral observations of short-distance natal dispersal. At a landscape scale, individual-based autocorrelation values decreased over time while levels of admixture increased, possibly indicating increased gene flow over the past decade.

Although the prognostic value of p53 abnormalities in Stage III microsatellite stable (MSS) colorectal cancers (CRCs) is known, the gene expression profiles specific to the p53 status in the MSS background are not known. Therefore, the current investigation has focused on identification and validation of the gene expression profiles associated with p53 mutant phenotypes in MSS Stage III CRCs. Genomic DNA extracted from 135 formalin-fixed paraffin-embedded tissues, was analyzed for microsatellite instability (MSI) and p53 mutations. Further, mRNA samples extracted from five p53-mutant and five p53-wild-type MSS-CRC snap-frozen tissues were profiled for differential gene expression by Affymetrix Human Genome U133 Plus 2.0 arrays. Differentially expressed genes were further validated by the high-throughput quantitative nuclease protection assay (qNPA), and confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) and by immunohistochemistry (IHC). Survival rates were estimated by Kaplan-Meier and Cox regression analyses. A higher incidence of p53 mutations was found in MSS (58%) than in MSI (30%) phenotypes. Both univariate (log-rank, P = 0.025) and multivariate (hazard ratio, 2.52; 95% confidence interval, 1.25-5.08) analyses have demonstrated that patients with MSS-p53 mutant phenotypes had poor CRC-specific survival when compared to MSS-p53 wild-type phenotypes. Gene expression analyses identified 84 differentially expressed genes. Of 49 down-regulated genes, LPAR6, PDLIM3, and PLAT, and, of 35 up-regulated genes, TRIM29, FUT3, IQGAP3, and SLC6A8 were confirmed by qNPA, qRT-PCR, and IHC platforms. p53 mutations are associated with poor survival of patients with Stage III MSS CRCs and p53-mutant and wild-type phenotypes have distinct gene expression profiles that might be helpful in identifying aggressive subsets.

Long interspersed (L1) and Alu elements are actively amplified in the human genome through retrotransposition of their RNA intermediates by the -100 still retrotranspositionally fully competent L1 elements. Retrotransposition can cause inherited disease if such an element is inserted near or within a functional gene. Using direct cDNA sequencing as the primary assay for comprehensive NF1 mutation analysis, we uncovered in 18 unrelated index patients splicing alterations not readily explained at the genomic level by an underlying point-mutation or deletion. Improved PCR protocols avoiding allelic drop-out of the mutant alleles uncovered insertions of fourteen Alu elements, three L1 elements, and one poly(T) stretch to cause these splicing defects. Taken together, the 18 pathogenic L1 endonuclease-mediated de novo insertions represent the largest number of this type of mutations characterized in a single human gene. Our findings show that retrotransposon insertions account for as many as -0.4% of all NF1 mutations. Since altered splicing was the main effect of the inserted elements, the current finding was facilitated by the use of RNA-based mutation analysis protocols, resulting in improved detection compared to gDNA-based approaches. Six different insertions clustered in a relatively small 1.5-kb region (NF1 exons 21(16)-23(18)) within the 280-kb NF1 gene. Furthermore, three different specific integration sites, one of them located in this cluster region, were each used twice, i.e. NM&#95;000267.3(NF1):c.1642-1&#95;1642 in intron 14(10c), NM&#95;000267.3(NF1):c.2835&#95;2836 in exon 21(16), and NM&#95;000267.3(NF1):c.4319&#95;4320 in exon 33(25). Identification of three loci that each served twice as integration site for independent retrotransposition events as well as 1.5-kb cluster region harboring six independent insertions supports the notion of non-random insertion of retrotransposons in the human genome. Currently, little is known about which features make sites particularly vulnerable to L1 EN-mediated insertions. The here identified integration sites may serve to elucidate these features in future studies., Competing Interests: The authors have declared that no competing interests exist.

Habitat fragmentation can restrict geneflow, reduce neighbourhood effective population size, and increase genetic drift and inbreeding in small, isolated habitat remnants. The extent to which habitat fragmentation leads to population fragmentation, however, differs among landscapes and taxa. Commonly, researchers use information on the current status of a species to predict population effects of habitat fragmentation. Such methods, however, do not convey information on species-specific responses to fragmentation. Here, we compare levels of past population differentiation, estimated from microsatellite genotypes, with contemporary dispersal rates, estimated from multi-strata capture-recapture models, to infer changes in mobility over time in seven sympatric, forest-dependent bird species of a Kenyan cloud forest archipelago. Overall, populations of sedentary species were more strongly differentiated and clustered compared to those of vagile ones, while geographical patterning suggested an important role of landscape structure in shaping genetic variation. However, five of seven species with broadly similar levels of genetic differentiation nevertheless differed substantially in their current dispersal rates. We conclude that post-fragmentation levels of vagility, without reference to past population connectivity, may not be the best predictor of how forest fragmentation affects the life history of forest-dependent species. As effective conservation strategies often hinge on accurate prediction of shifts in ecological and genetic relationships among populations, conservation practices based solely upon current population abundances or movements may, in the long term, prove to be inadequate., (© 2011 Blackwell Publishing Ltd.)

Cutaneous neurofibromas are the hallmarks of neurofibromatosis type 1 (NF1). They are composed of multiple cell types, and traditionally they are believed to arise from small nerve tributaries of the skin. A key finding in the context of this view has been that subpopulations of tumor Schwann cells harbor biallelic inactivation of the NF1 gene (NF1(-/-)). In the present study, our aim was to clarify further the pathogenesis of cutaneous neurofibromas. First, we detected cells expressing multipotency-associated biomarkers in cutaneous neurofibromas. Second, we developed a method for isolating and expanding multipotent neurofibroma-derived precursor cells (NFPs) from dissociated human cutaneous neurofibromas and used it to analyze their growth and differentiation potential. In analogy to solitary cells resident in neurofibromas, NFPs were found to express nestin and had the potential to differentiate to, at least, Schwann cells, neurons, epithelial cells, and adipocytes. Mutation analysis of the NFPs revealed that their genotype was NF1(+/-). The results led us to speculate that the development of cutaneous neurofibromas includes the recruitment of multipotent NF1(+/-) precursor cells. These cells may be derived from the multipotent cells of the hair roots, which often are intimately associated with microscopic neurofibromas., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Objective: Since the anti-tumor activity of 5-fluorouracil (5-FU) is due to induction of apoptosis, we assessed the value of expression of key apoptotic molecules (Bax, Bcl-2 and p53) in predicting the efficacy of 5-FU therapy for colorectal adenocarcinomas (CRCs)., Methods: Archival tissues of CRCs from 56 patients who received a complete regimen of 5-FU-based chemotherapy after surgery, and 56 patients matched for age, gender, ethnicity, tumor stage, tumor location, and tumor differentiation who had undergone only surgery (without any pre- or post-surgery therapy), were evaluated for immunophenotypic expression of Bax, Bcl-2, and p53. Also, these CRCs were evaluated for Bax mutations. The predictive capacity or prognostic value of these markers was assessed by estimating overall survival., Results: The majority of low Bax expressing CRCs have exhibited mutations at the G (8) tract. There was no significant difference in overall survival rates between the categories of surgery alone and 5-FU-treated patients. However, a better survival was observed for patients who received chemotherapy when their CRCs had low Bax/Bcl2 ratio (HR, 1.55; 95% CI: 1.46-31.00). Patients who received surgery alone and whose CRCs lacked Bax expression had 5.33 times higher mortality than those with high Bax expression (95% CI: 1.78-15.94), when controlled for tumor stage and other confounders. Bcl-2 and nuclear p53 accumulation had no predictive value in either patient group., Conclusion: These findings are the first to demonstrate that high Bax expression is a good prognosticator for patients who underwent surgery alone, and that patient with low Bax/Bcl-2 expression ratio benefit from 5-FU-based adjuvant therapies.

Retinoic acid (RA) induces differentiation of neuroblastoma cells in vitro and is used with variable success to treat aggressive forms of this disease. This variability in clinical response to RA is enigmatic, as no mutations in components of the RA signaling cascade have been found. Using a large-scale RNAi genetic screen, we identify crosstalk between the tumor suppressor NF1 and retinoic acid-induced differentiation in neuroblastoma. Loss of NF1 activates RAS-MEK signaling, which in turn represses ZNF423, a critical transcriptional coactivator of the retinoic acid receptors. Neuroblastomas with low levels of both NF1 and ZNF423 have extremely poor outcome. We find NF1 mutations in neuroblastoma cell lines and in primary tumors. Inhibition of MEK signaling downstream of NF1 restores responsiveness to RA, suggesting a therapeutic strategy to overcome RA resistance in NF1-deficient neuroblastomas., (Copyright 2010 Elsevier Inc. All rights reserved.)

The recently identified human ortholog of the Rabphillin-3A-Like (RPH3AL) gene, located at the 17p13.3 locus, has been assessed for its mutational status and clinical significance in colorectal adenocarcinoma (CRC). Prospectively collected 95 frozen CRCs and their matching benign colonic epithelial tissues were evaluated for mutations and mRNA expression. Since, we observed a higher incidence of a single nucleotide polymorphism (SNP) at the -25 position in the 5'untranslated region (5'UTR-25) of RPH3AL, we performed the genotyping analysis of this SNP in a retrospective CRC cohort (n=134) to assess their clinical importance. Univariate and multivariate outcome analyses were performed. The cDNA analysis has detected point mutations in 6 CRCs, coding region SNPs in 14 CRCs, and non-coding region SNPs in 38 CRCs. Combined analyses of both cohorts has demonstrated that the incidence of SNP at 5'UTR-25 was 41% (95 of 229), and its A/A genotype (9%, 20 of 229) was observed exclusively in non-Hispanic Caucasians, and 19 of these cases were diagnosed with nodal metastasis. Patients who exhibited homozygous for A or C alleles had a significantly decreased levels of mRNA expression, increased risk of CRC recurrence and mortality. Therefore, these findings have significant clinical implications in assessing the aggressiveness of CRC.

Neurofibromatosis type 1 (NF1), the most common tumor-predisposing disorder in humans, is caused by defects in the NF1 tumor-suppressor gene. Comprehensive mutation analysis applying RNA-based techniques complemented with FISH analysis achieves mutation detection rates of approximately 95% in NF1 patients. The majority of mutations are minor lesions, and approximately 5% are total gene deletions. We found 13 single- and/or multiexon deletions/duplications out of 1,050 detected mutations using our RNA-based approach in a cohort of 1,100 NF1 patients and confirmed these changes using multiplex ligation-dependent probe amplification (MLPA). With MLPA, we found another 12 novel multiexon deletion/duplications in 55 NF1 patients for whom analysis with multiple assays had not revealed a NF1 mutation, including 50 previously analyzed comprehensively. The extent of the 22 deletions and 3 duplications varied greatly, and there was no clustering of breakpoints. We also evaluated the sensitivity of MLPA in identifying deletions in a mosaic state. Furthermore, we tested whether the MLPA P122 NF1 area assay could distinguish between type I deletions, with breakpoints in low-copy repeats (NF1-LCRs), and type II deletions, caused by aberrant recombination between the JJAZ gene and its pseudogene. Our study showed that intragenic deletions and/or duplications represent only approximately 2% of all NF1 mutations. Although MLPA did not substantially increase the mutation detection rate in NF1 patients, it was a useful first step in a comprehensive mutation analysis scheme to quickly pinpoint patients with single- or multiexon deletions/duplications as well as patients with a total gene deletion who will not need full sequencing of the complete coding region.

Patients with typical features of neurofibromatosis type 1 (NF1) limited to a specific body segment are usually referred to as having "segmental NF1", which is generally assumed to be the result of somatic mosaicism for a NF1 mutation. Mosaicism has also been demonstrated at the molecular level in some sporadic cases with phenotypically classic NF1. In the present report, we describe a patient with NF1 disease manifestations throughout the whole body, but leaving a few sharply delineated segments of the skin unaffected, suggestive of revertant mosaicism. A large intragenic deletion was found by mutation analysis using long-range RT-PCR. The intra-exonic breakpoints were characterized in exon 13 and exon 28, resulting in a deletion of 99,571 bp at the genomic level. The presence of two genetically distinct cell populations, confirming mosaicism for this NF1 mutation, was shown by analysis of several tissues. Revertant mosaicism was excluded by demonstrating heterozygosity for markers residing in the deletion region. The findings in this patient demonstrate two things: (1) although the entire body is affected, mosaicism can still be suspected at clinical examination and proven by DNA analysis and skin biopsies; (2) long-range RT-PCR is a feasible method for demonstrating large intragenic deletions in NF1.

Background: Mutation analysis of the neurofibromatosis type 1 (NF1) gene has shown that about 30% of NF1 patients carry a splice mutation resulting in the production of one or several shortened transcripts. Some of these transcripts were also found in fresh lymphocytes of healthy individuals, albeit typically at a very low level. Starting from this initial observation, we were interested to gain further insight into the complex nature of NF1 mRNA processing., Results: We have used a RT-PCR plasmid library based method to identify novel NF1 splice variants. Several transcripts were observed with specific insertions/deletions and a survey was made. This large group of variants detected in one single gene allows to perform a comparative analysis of the factors involved in splice regulation. Exons that are prone to skipping were systematically analysed for 5' and 3' splice site strength, branch point strength and secondary structure., Conclusion: Our study revealed a complex splicing pattern, generating a great diversity in NF1 transcripts. We found that, on average, exons that are spliced out in part of the mRNA have significantly weaker acceptor sites. Some variants identified in this study could have distinct roles and might expand our knowledge of neurofibromin.

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.

In mice, tissue-specific alternative splicing of the myosin V gene in the C-terminal tail domain is well documented with exclusion of exon F from brain transcripts, but present in skin, particularly in melanocytes. As alternative splicing of the myosin V C-terminal tail domain in human tissues is undocumented, we studied the presence of myosin V splice forms in different types of human cultured normal skin cells, i.e., dermal fibroblasts, melanocytes, and keratinocytes as well as in human blood leukocytes, using an RT-PCR-based method. In all cell types studied, several different length transcripts were present containing different exon combinations, including exon F. This is the first report showing the presence of exon F as well as alternative splicing in the human myosin V tail domain. As the full-length transcript is most abundant in melanocytes and leukocytes and both cell types are involved in Griscelli syndrome, a tissue-specific role of this particular transcript needs further investigation., (Copyright 1998 Academic Press.)

Neurofibromatosis type 1 (NF1), characterised by peripheral neurofibromas, café-au-lait spots and iris Lisch nodules, is one of the most common inherited disorders. We have analysed exons 35 to 49 in 21 unrelated NF1 patients using reverse transcription-polymerase chain reaction and protein truncation analysis. In two unrelated patients we found skipping of exon 37 at the cDNA level. Sequence analysis of genomic DNA revealed the presence of a C6792A transversion in one patient and a C6792G transversion in a second patient; both transversions change the codon for tyrosine to a nonsense codon. Sequencing of the exonic sequences as well as the branch sites, and the 3' and 5' splice sites of exons 36, 37 and 38 did not reveal any additional sequence abnormality. This is the first report showing that nonsense mutations in the NF1 gene can induce the skipping of a constitutive exon.

Hereditary protein S deficiency is a risk factor for developing recurrent venous thromboembolic disease and is caused by a defect in the protein S 1 (PROS1) gene. Identification of the mutation in the PROS1 gene can overcome diagnostic uncertainty in family members with borderline protein S levels. We describe a novel nonisotopic method for molecular diagnosis of protein S deficiency, using fluorescein-labeled amplification and sequencing primers. As a first step, all exons of the PROS1 gene are selectively amplified, and heteroduplex analysis is performed. As a second step, all exons are analyzed by direct sequencing. Using this method, we have characterized the molecular defect in two Belgian families with hereditary protein S deficiency type I: a frameshift mutation in exon XIV (1881insTC) and a missense mutation caused by a T-to-C transition, resulting in substitution of Leu405 by Pro (L405P).