Challenges in the diagnosis of neurofibromatosis type 1 (NF1) in young children facilitated by means of revised diagnostic criteria including genetic testing for pathogenic NF1 gene variants

Neurofibromatosis type 1 (NF1) is the most frequent disorder associated with multiple café-au-lait macules (CALM) which may either be present at birth or appear during the first year of life. Other NF1-associated features such as skin-fold freckling and Lisch nodules occur later during childhood whereas dermal neurofibromas are rare in young children and usually only arise during early adulthood. The NIH clinical diagnostic criteria for NF1, established in 1988, include the most common NF1-associated features. Since many of these features are age-dependent, arriving at a definitive diagnosis of NF1 by employing these criteria may not be possible in infancy if CALM are the only clinical feature evident. Indeed, approximately 46% of patients who are diagnosed with NF1 later in life do not meet the NIH diagnostic criteria by the age of 1 year. Further, the 1988 diagnostic criteria for NF1 are not specific enough to distinguish NF1 from other related disorders such as Legius syndrome. In this review, we outline the challenges faced in diagnosing NF1 in young children, and evaluate the utility of the recently revised (2021) diagnostic criteria for NF1, which include the presence of pathogenic variants in the NF1 gene and choroidal anomalies, for achieving an early and accurate diagnosis

Classification of NF1 microdeletions and its importance for establishing genotype/phenotype correlations in patients with NF1 microdeletions

An estimated 5–11% of patients with neurofibromatosis type-1 (NF1) harbour large deletions encompassing the NF1 gene and flanking regions. These NF1 microdeletions are subclassified into type 1, 2, 3 and atypical deletions which are distinguishable from each other by their extent and by the number of genes included within the deletion regions as well as the frequency of mosaicism with normal cells. Most common are type-1 NF1 deletions which encompass 1.4-Mb and 14 protein-coding genes. Type-1 deletions are frequently associated with overgrowth, global developmental delay, cognitive disability and dysmorphic facial features which are uncommon in patients with intragenic pathogenic NF1 gene variants. Further, patients with type-1 NF1 deletions frequently exhibit high numbers of neurofibromas and have an increased risk of malignant peripheral nerve sheath tumours. Genes located within the type-1 NF1 microdeletion interval and co-deleted with NF1 are likely to act as modifiers responsible for the severe disease phenotype in patients with NF1 microdeletions, thereby causing the NF1 microdeletion syndrome. Genotype/phenotype correlations in patients with NF1 microdeletions of different lengths are important to identify such modifier genes. However, these correlations are critically dependent upon the accurate characterization of the deletions in terms of their extent. In this review, we outline the utility as well as the shortcomings of multiplex ligation-dependent probe amplification (MLPA) to classify the different types of NF1 microdeletion and indicate the importance of high-resolution microarray analysis for correct classification, a necessary precondition to identify those genes responsible for the NF1 microdeletion syndrome

Emerging genotype-phenotype relationships in patients with large NF1 deletions.

The most frequent recurring mutations in neurofibromatosis type 1 (NF1) are large deletions encompassing the NF1 gene and its flanking regions (NF1 microdeletions). The majority of these deletions encompass 1.4-Mb and are associated with the loss of 14 protein-coding genes and four microRNA genes. Patients with germline type-1 NF1 microdeletions frequently exhibit dysmorphic facial features, overgrowth/tall-for-age stature, significant delay in cognitive development, large hands and feet, hyperflexibility of joints and muscular hypotonia. Such patients also display significantly more cardiovascular anomalies as compared with patients without large deletions and often exhibit increased numbers of subcutaneous, plexiform and spinal neurofibromas as compared with the general NF1 population. Further, an extremely high burden of internal neurofibromas, characterised by >3000 ml tumour volume, is encountered significantly, more frequently, in non-mosaic NF1 microdeletion patients than in NF1 patients lacking such deletions. NF1 microdeletion patients also have an increased risk of malignant peripheral nerve sheath tumours (MPNSTs); their lifetime MPNST risk is 16–26%, rather higher than that of NF1 patients with intragenic NF1 mutations (8–13%). NF1 microdeletion patients, therefore, represent a high-risk group for the development of MPNSTs, tumours which are very aggressive and difficult to treat. Co-deletion of the SUZ12 gene in addition to NF1 further increases the MPNST risk in NF1 microdeletion patients. Here, we summarise current knowledge about genotype–phenotype relationships in NF1 microdeletion patients and discuss the potential role of the genes located within the NF1 microdeletion interval whose haploinsufficiency may contribute to the more severe clinical phenotyp

The molecular pathogenesis of schwannomatosis, a paradigm for the co-involvement of multiple tumour suppressor genes in tumorigenesis

Schwannomatosis is characterized by the predisposition to develop multiple schwannomas and, less commonly, meningiomas. Despite the clinical overlap with neurofibromatosis type 2 (NF2), schwannomatosis is not caused by germline NF2 gene mutations. Instead, germline mutations of either the SMARCB1 or LZTR1 tumour suppressor genes have been identified in 86% of familial and 40% of sporadic schwannomatosis patients. In contrast to patients with rhabdoid tumours, which are due to complete loss-of-function SMARCB1 mutations, individuals with schwannomatosis harbour predominantly hypomorphic SMARCB1 mutations which give rise to the synthesis of mutant proteins with residual function that do not cause rhabdoid tumours. Although biallelic mutations of SMARCB1 or LZTR1 have been detected in the tumours of patients with schwannomatosis, the classical two-hit model of tumorigenesis is insufficient to account for schwannoma growth, since NF2 is also frequently inactivated in these tumours. Consequently, tumorigenesis in schwannomatosis must involve the mutation of at least two different tumour suppressor genes, an occurrence frequently mediated by loss of heterozygosity of large parts of chromosome 22q harbouring not only SMARCB1 and LZTR1 but also NF2. Thus, schwannomatosis is paradigmatic for a tumour predisposition syndrome caused by the concomitant mutational inactivation of two or more tumour suppressor genes. This review provides an overview of current models of tumorigenesis and mutational patterns underlying schwannomatosis that will ultimately help to explain the complex clinical presentation of this rare disease

Non-coding RNA ANRIL and the number of plexiform neurofibromas in patients with NF1microdeletions

BACKGROUND: Neurofibromatosis type-1 (NF1) is caused by mutations of the NF1 gene at 17q11.2. In 95% of non-founder NF1 patients, NF1 mutations are identifiable by means of a comprehensive mutation analysis. 5-10% of these patients harbour microdeletions encompassing the NF1 gene and its flanking regions. NF1 is characterised by tumours of the peripheral nerve sheaths, the pathognomonic neurofibromas. Considerable inter- and intra-familial variation in expressivity of the disease has been observed which is influenced by genetic modifiers unrelated to the constitutional NF1 mutation. The number of plexiform neurofibromas (PNF) in NF1 patients is a highly heritable genetic trait. Recently, SNP rs2151280 located within the non-coding RNA gene ANRIL at 9p21.3, was identified as being strongly associated with PNF number in a family-based association study. The T-allele of rs2151280, which correlates with reduced ANRIL expression, appears to be associated with higher PNF number. ANRIL directly binds to the SUZ12 protein, an essential component of polycomb repressive complex 2, and is required for SUZ12 occupancy of the CDKN2A/CDKN2B tumour suppressor genes as well as for their epigenetic silencing. METHODS: Here, we explored a potential association of PNF number and PNF volume with SNP rs2151280 in 29 patients with constitutional NF1 microdeletions using the exact Cochran-Armitage test for trends and the exact Mann–Whitney–Wilcoxon test. Both the PNF number and total tumour volume in these 29 NF1 patients were assessed by whole-body MRI. The NF1 microdeletions observed in these 29 patients encompassed the NF1 gene as well as its flanking regions, including the SUZ12 gene. RESULTS: In the 29 microdeletion patients investigated, neither the PNF number nor PNF volume was found to be associated with the T-allele of rs2151280. CONCLUSION: Our findings imply that, at least in patients with NF1 microdeletions, PNF susceptibility is not associated with rs2151280. Although somatic inactivation of the NF1 wild-type allele is considered to be the PNF-initiating event in NF1 patients with intragenic mutations and patients with NF1 microdeletions, both patient groups may differ with regard to tumour progression because of the heterozygous constitutional deletion of SUZ12 present only in patients with NF1 microdeletions

Where genotype is not predictive of phenotype: towards an understanding of the molecular basis of reduced penetrance in human inherited disease

Some individuals with a particular disease-causing mutation or genotype fail to express most if not all features of the disease in question, a phenomenon that is known as ‘reduced (or incomplete) penetrance’. Reduced penetrance is not uncommon; indeed, there are many known examples of ‘disease-causing mutations’ that fail to cause disease in at least a proportion of the individuals who carry them. Reduced penetrance may therefore explain not only why genetic diseases are occasionally transmitted through unaffected parents, but also why healthy individuals can harbour quite large numbers of potentially disadvantageous variants in their genomes without suffering any obvious ill effects. Reduced penetrance can be a function of the specific mutation(s) involved or of allele dosage. It may also result from differential allelic expression, copy number variation or the modulating influence of additional genetic variants in cis or in trans. The penetrance of some pathogenic genotypes is known to be age- and/or sex-dependent. Variable penetrance may also reflect the action of unlinked modifier genes, epigenetic changes or environmental factors. At least in some cases, complete penetrance appears to require the presence of one or more genetic variants at other loci. In this review, we summarize the evidence for reduced penetrance being a widespread phenomenon in human genetics and explore some of the molecular mechanisms that may help to explain this enigmatic characteristic of human inherited disease

Pronounced maternal parent-of-origin bias for type-1 NF1 microdeletions

Neurofibromatosis type 1 (NF1) is caused, in 4.7–11% of cases, by large deletions encompassing the NF1 gene and its flanking regions within 17q11.2. Different types of large NF1 deletion occur which are distinguishable by their breakpoint location and underlying mutational mechanism. Most common are the type-1 NF1 deletions of 1.4 Mb which exhibit recurrent breakpoints caused by nonallelic homologous recombination (NAHR), also termed unequal crossover. Here, we analyzed 37 unrelated families of patients with de novo type-1 NF1 deletions by means of short tandem repeat (STR) profiling to determine the parental origin of the deletions. We observed that 33 of the 37 type-1 deletions were of maternal origin (89.2% of cases; p < 0.0001). Analysis of the patients’ siblings indicated that, in 14 informative cases, ten (71.4%) deletions resulted from interchromosomal unequal crossover during meiosis I. Our findings indicate a strong maternal parent-of-origin bias for type-1 NF1 deletions. A similarly pronounced maternal transmission bias has been reported for recurrent copy number variants (CNVs) within 16p11.2 associated with autism, but not so far for any other NAHR-mediated pathogenic CNVs. Region-specific genomic features are likely to be responsible for the maternal bias in the origin of both the 16p11.2 CNVs and type-1 NF1 deletions

Atypical NF1 microdeletions: challenges and opportunities for Genotype/Phenotype correlations in patients with large NF1 deletions

Patients with neurofibromatosis type 1 (NF1) and type 1 NF1 deletions often exhibit more severe clinical manifestations than patients with intragenic NF1 gene mutations, including facial dysmorphic features, overgrowth, severe global developmental delay, severe autistic symptoms and considerably reduced cognitive abilities, all of which are detectable from a very young age. Type 1 NF1 deletions encompass 1.4 Mb and are associated with the loss of 14 protein-coding genes, including NF1 and SUZ12. Atypical NF1 deletions, which do not encompass all 14 protein-coding genes located within the type 1 NF1 deletion region, have the potential to contribute to the delineation of the genotype/phenotype relationship in patients with NF1 microdeletions. Here, we review all atypical NF1 deletions reported to date as well as the clinical phenotype observed in the patients concerned. We compare these findings with those of a newly identified atypical NF1 deletion of 698 kb which, in addition to the NF1 gene, includes five genes located centromeric to NF1. The atypical NF1 deletion in this patient does not include the SUZ12 gene but does encompass CRLF3. Comparative analysis of such atypical NF1 deletions suggests that SUZ12 hemizygosity is likely to contribute significantly to the reduced cognitive abilities, severe global developmental delay and facial dysmorphisms observed in patients with type 1 NF1 deletions

Gene synteny comparisons between different vertebrates provide new insights into breakage and fusion events during mammalian karyotype evolution

<p>Abstract</p> <p>Background</p> <p>Genome comparisons have made possible the reconstruction of the eutherian ancestral karyotype but also have the potential to provide new insights into the evolutionary inter-relationship of the different eutherian orders within the mammalian phylogenetic tree. Such comparisons can additionally reveal (i) the nature of the DNA sequences present within the evolutionary breakpoint regions and (ii) whether or not the evolutionary breakpoints occur randomly across the genome. Gene synteny analysis (E-painting) not only greatly reduces the complexity of comparative genome sequence analysis but also extends its evolutionary reach.</p> <p>Results</p> <p>E-painting was used to compare the genome sequences of six different mammalian species and chicken. A total of 526 evolutionary breakpoint intervals were identified and these were mapped to a median resolution of 120 kb, the highest level of resolution so far obtained. A marked correlation was noted between evolutionary breakpoint frequency and gene density. This correlation was significant not only at the chromosomal level but also sub-chromosomally when comparing genome intervals of lengths as short as 40 kb. Contrary to previous findings, a comparison of evolutionary breakpoint locations with the chromosomal positions of well mapped common fragile sites and cancer-associated breakpoints failed to reveal any evidence for significant co-location. Primate-specific chromosomal rearrangements were however found to occur preferentially in regions containing segmental duplications and copy number variants.</p> <p>Conclusion</p> <p>Specific chromosomal regions appear to be prone to recurring rearrangement in different mammalian lineages ('breakpoint reuse') even if the breakpoints themselves are likely to be non-identical. The putative ancestral eutherian genome, reconstructed on the basis of the synteny analysis of 7 vertebrate genome sequences, not only confirmed the results of previous molecular cytogenetic studies but also increased the definition of the inferred structure of ancestral eutherian chromosomes. For the first time in such an analysis, the opossum was included as an outgroup species. This served to confirm our previous model of the ancestral eutherian genome since all ancestral syntenic segment associations were also noted in this marsupial.</p

Fine mapping of meiotic NAHR-associated crossovers causing large NF1 deletions

Large deletions encompassing the NF1 gene and its flanking regions belong to the group of genomic disorders caused by copy number changes that are mediated by the local genomic architecture. Although nonallelic homologous recombination (NAHR) is known to be a major mutational mechanism underlying such genomic copy number changes, the sequence determinants of NAHR location and frequency are still poorly understood since few high-resolution mapping studies of NAHR hotspots have been performed to date. Here, we have characterized two NAHR hotspots, PRS1 and PRS2, separated by 20 kb and located within the low-copy repeats NF1-REPa and NF1-REPc, which flank the human NF1 gene region. High-resolution mapping of the crossover sites identified in 78 type 1 NF1 deletions mediated by NAHR indicated that PRS2 is a much stronger NAHR hotspot than PRS1 since 80% of these deletions exhibited crossovers within PRS2, whereas 20% had crossovers within PRS1. The identification of the most common strand exchange regions of these 78 deletions served to demarcate the cores of the PRS1 and PRS2 hotspots encompassing 1026 and 1976 bp, respectively. Several sequence features were identified that may influence hotspot intensity and direct the positional preference of NAHR to the hotspot cores. These features include regions of perfect sequence identity encompassing 700 bp at the hotspot core, the presence of PRDM9 binding sites perfectly matching the consensus motif for the most common PRDM9 variant, specific pre-existing patterns of histone modification and open chromatin conformations that are likely to facilitate PRDM9 binding