Epigenomic, genomic, and transcriptomic landscape of schwannomatosis.

Schwannomatosis (SWNTS) is a genetic cancer predisposition syndrome that manifests as multiple and often painful neuronal tumors called schwannomas (SWNs). While germline mutations in SMARCB1 or LZTR1, plus somatic mutations in NF2 and loss of heterozygosity in chromosome 22q have been identified in a subset of patients, little is known about the epigenomic and genomic alterations that drive SWNTS-related SWNs (SWNTS-SWNs) in a majority of the cases. We performed multiplatform genomic analysis and established the molecular signature of SWNTS-SWNs. We show that SWNTS-SWNs harbor distinct genomic features relative to the histologically identical non-syndromic sporadic SWNs (NS-SWNS). We demonstrate the existence of four distinct DNA methylation subgroups of SWNTS-SWNs that are associated with specific transcriptional programs and tumor location. We show several novel recurrent non-22q deletions and structural rearrangements. We detected the SH3PXD2A-HTRA1 gene fusion in SWNTS-SWNs, with predominance in LZTR1-mutant tumors. In addition, we identified specific genetic, epigenetic, and actionable transcriptional programs associated with painful SWNTS-SWNs including PIGF, VEGF, MEK, and MTOR pathways, which may be harnessed for management of this syndrome.

Early-onset malignant phyllodes breast tumor in a patient with germline pathogenic variants in NF1 and BRCA1 genes.

We present a 24-year-old female patient affected by neurofibromatosis type 1 (NF1) who developed a malignant phyllodes tumor of the breast. The molecular studies showed that the patient carried a heterozygous inactivating deleterious variant in BRCA1 inherited from the father associated with a germline de novo pathogenic alteration in NF1; the tumor presented a biallelic inactivation of both genes. Therefore, tumor analyses helped to establish that the germline NF1 and BRCA1 variants were in cis on the paternal chromosome. This last information is important to provide adequate genetic counselling regarding the risk of recurrence in the offspring, as well as opportunity for early intervention. In conclusion, we present the first case of a malignant phyllodes tumor of the breast in patient carrying pathogenic variants in NF1 and BRCA1. Further studies will be necessary to understand if the phyllodes histotype represents a very rare component of NF1-associated breast cancer.

A novel CDKN2A in-frame deletion associated with pancreatic cancer-melanoma syndrome.

Pancreatic cancer-melanoma syndrome (PCMS) is an inherited condition in which mutation carriers have an increased risk of malignant melanoma and/or pancreatic cancer. About 30% of PCMS cases carry mutations in CDKN2A. This gene encodes several protein isoforms, one of which, known as p16, regulates the cell-cycle by interacting with CDK4/CDK6 kinases and with several non-CDK proteins. Herein, we report on a novel CDKN2A germline in-frame deletion (c.52_57delACGGCC) found in an Italian family with PCMS. By segregation analysis, the c.52_57delACGGCC was proven to segregate in kindred with cutaneous melanoma (CM), in kindred with CM and pancreatic cancer, and in a single case presenting only with pancreatic cancer. In the literature, duplication mapping in the same genic region has been already reported at the germline level in several unrelated CM cases as a variant of unknown clinical significance. A computational approach for studying the effect of mutational changes over p16 protein structure showed that both the deletion and the duplication of the c.52_57 nucleotides result in protein misfolding and loss of interactors' binding. In conclusion, the present results argue that the quantitative alteration of nucleotides c.52_57 has a pathogenic role in p16 function and that the c.52_57delACGGCC is associated with PCMS.

Evaluation of a Next-Generation Sequencing Assay for BRCA1 and BRCA2 Mutation Detection.

The efficiency of a novel targeted next-generation sequencing (NGS) test, the Devyser BRCA kit, for a comprehensive analysis of all 48 coding exons of the high-risk breast/ovarian cancer susceptibility genes BRCA1 and BRCA2 has been assessed. The new assay intended to detect nucleotide substitutions, small deletions/insertions, and large deletions/duplications. To document the false-negative and false-positive rates of the NGS assay in the hands of end users, 48 samples with previously identified 444 small variants and seven gross rearrangements were analyzed, showing 100% concordance with gold standards. Furthermore, all other 43 variants (42 single-nucleotide variation or insertion/deletion variation and one copy number variation, whose significance is or may be of clinical value), which were called by the NGS assay in a prospectively analyzed 179-sample set, were confirmed by Sanger sequencing or multiplex ligation probe amplification, according to their nature. We conclude that the Devyser BRCA kit performed satisfactorily for use in a clinical laboratory.

Double somatic SMARCB1 and NF2 mutations in sporadic spinal schwannoma.

In sporadic schwannomas, inactivation of both copies of the NF2 tumor suppressor gene on 22q is common. Constitutional mutations of SMARCB1 are responsible of schwannomatosis, an inherited tumor predisposition syndrome, characterized by the development of multiple schwannomas. We analysed the frequency of copy number changes on chromosome 22 and the mutation of NF2 and SMARCB1 in 26 sporadic schwannomas. We found two spinal schwannomas with an identical somatic missense mutation in SMARCB1 exon 9: p.(Arg377His). Both SMARCB1 mutated schwannomas had LOH of 22q and one of them harbored an inactivating mutation of NF2. The p.(Arg377His) change was not found in a series of 28 vestibular schwannomas. Our data indicate that mutations affecting SMARCB1 play a role in the development or progression of a small subset of spinal schwannomas and that biallelic inactivation of SMARCB1 may cooperate with deficiency of NF2 function in schwannoma tumorigenesis according to the "four-hit/three events" mechanism of tumorigenesis that we demonstrated in schwannomatosis-associated schwannomas.

A mosaic pattern of INI1/SMARCB1 protein expression distinguishes Schwannomatosis and NF2-associated peripheral schwannomas from solitary peripheral schwannomas and NF2-associated vestibular schwannomas.

BACKGROUND: The INI1/SMARCB1 gene protein product has been implicated in the direct pathogenesis of schwannomas from patients with one form of schwannomatosis [SWNTS1; MIM # 162091] showing a mosaic pattern of loss of protein expression by immunohistochemistry [93% in familial vs. 55% in sporadic cases]. AIM OF STUDY: To verify whether such INI1/SMARCB1 mosaic pattern could be extended to all schwannomas arising in the sporadic and familial schwannomatoses [i.e. to SMARCB1-related (SWNTS1) or LZTR1-related (SWNTS2) schwannomatosis or to SMARCB1/LZTR1-negative schwannomatosis] and whether it could be involved in classical NF2 or solitary peripheral schwannomas METHODS: We blindly analysed schwannoma samples obtained from a total of 22 patients including (a) 2 patients (2 males; aged 38 and 55 years) affected by non-familial SMARCB1-associated schwannomatosis (SWTNS1); (b) 1 patient (1 female; aged 33 years) affected by familial schwannomatosis (SWTNS1/ SMARCB1 germ line mutations); (c) 5 patients (3 males, 2 females; aged 33 to 35 years) affected by non-familial (sporadic) LZTR1-associated schwannomatosis (SWNTS2); (d) 3 patients (3 males; aged 35 to 47 years) affected by familial schwannomatosis (SWTNS2/ LZTR1 germ line mutations); (e) 2 patients (1 male, 1 female; aged 63 and 49 years, respectively) affected by non-familial schwannomatosis (SWTNS, negative for SMARCB1, LZTR1 and NF2 gene mutations); (f) 4 patients (3 males, 1 females; aged 15 to 24 years) affected by classical NF2 (NF2: harbouring NF2 germ line mutations; and (g) 5 patients (3 males, 2 females; aged 33 to 68 years) who had solitary schwannomas. [follow-up = 15-30 years; negative for constitutional/somatic mutation analysis for the SMARCB1, LZTR1 and NF2 genes] were (blindly) analyzed. The INI1/SMARCB1 immunostaining pattern was regarded as (1) diffuse positive nuclear staining [= retained expression] or (2) mosaic pattern [mixed positive/negative nuclei = loss of expression in a subset of tumour cells]. RESULTS: All solitary peripheral schwannomas and NF2-associated vestibular schwannomas showed diffuse nuclear INI1/SMARCB1 staining in 97-100% of neoplastic cells; schwannomas obtained from all cases of non-familial and familial schwannomatosis and NF2-associated non-vestibular schwannomas showed a mosaic pattern ranging from 10 to 70% of INI1/SMARCB1-positive expression. We did not record a complete lack of nuclear staining. CONCLUSIONS: The present data suggests that (a) mosaic loss of immunohistochemical INI1/SMARCB1 expression, despite the interlesional variability, is a reliable marker of schwannomatosis regardless of the involved gene and it might help in the differential diagnosis of schwannomatosis vs. solitary schwannomas and (b) INI1/SMARCB1 expression is not useful in the differential with mosaic NF2, since NF2-associated peripheral schwannomas show the same immunohistochemical pattern.

A Founder Effect for the HGD G360R Mutation in Italy: Implications for a Regional Screening of Alkaptonuria.

We sought to establish rapid and specific genotyping methods for G360R mutation and for seven tightly linked markers in the homogentisate dioxygenase gene to address the question of whether G360R is a mutational hot spot or the result of a founder effect, as it has been repeatedly found in alkaptonuric patients from a geographic isolate in Italy.For G360R and single nucleotide polymorphism genotyping, high-resolution melting analysis was performed. Microsatellites were analysed by multiplex PCR and capillary electrophoresis. To investigate the natural history of the G360R mutation, we genotyped markers in 52 controls and in 8 unrelated patients from the UK and USA, who also segregated the G360R mutation, and calculated its age using DMLE+2.3 software.A distinct G360R-bearing haplotype was identified in all patients of Caucasian descent. Estimated mutation age was 545 generations (95% credible set, 402-854), suggesting that G360R arose in an ancestor who lived 8,000-10,000 years BC. Archaeological, historical and demographic data support that a G360R carrier has settled the remote valley where present-day population might have a heterozygote frequency of at least 6%.Given the late health-threatening complications of alkaptonuria and a cure within reach, inhabitants of this isolate would benefit from screening and genetic counselling.

A Systematic Assessment of Accuracy in Detecting Somatic Mosaic Variants by Deep Amplicon Sequencing: Application to NF2 Gene.

The accurate detection of low-allelic variants is still challenging, particularly for the identification of somatic mosaicism, where matched control sample is not available. High throughput sequencing, by the simultaneous and independent analysis of thousands of different DNA fragments, might overcome many of the limits of traditional methods, greatly increasing the sensitivity. However, it is necessary to take into account the high number of false positives that may arise due to the lack of matched control samples. Here, we applied deep amplicon sequencing to the analysis of samples with known genotype and variant allele fraction (VAF) followed by a tailored statistical analysis. This method allowed to define a minimum value of VAF for detecting mosaic variants with high accuracy. Then, we exploited the estimated VAF to select candidate alterations in NF2 gene in 34 samples with unknown genotype (30 blood and 4 tumor DNAs), demonstrating the suitability of our method. The strategy we propose optimizes the use of deep amplicon sequencing for the identification of low abundance variants. Moreover, our method can be applied to different high throughput sequencing approaches to estimate the background noise and define the accuracy of the experimental design.

Broadening the spectrum of SMARCB1-associated malignant tumors: a case of uterine leiomyosarcoma in a patient with schwannomatosis.

Schwannomatosis is a tumor predisposition syndrome characterized by development of multiple intracranial, spinal, and peripheral schwannomas. Constitutional alterations in either SMARCB1 or LZTR1 on 22q are responsible of the phenotype. We describe a 34-year-old woman who developed multiple benign peripheral sheath tumors and a uterine leiomyosarcoma. The patient carried a de novo constitutional alteration in exon 8 of SMARCB1, c.1118G > A, which destroyed the splice donor site of intron 8. Two schwannomas and the leiomyosarcoma of the patient retained the SMARCB1 mutation; in addition, the tumors showed loss of the normal chromosome 22. In conclusion, our findings enlarged the spectrum of SMARCB1-predisposing tumors and demonstrated, for the first time, the association of a malignant smooth muscle tumor to schwannomatosis. Therefore, clinicians should definitely be aware that a constitutional SMARCB1 mutation, which mainly predisposes to benign nerve sheath tumors, may also predispose to aggressive neoplasms throughout life, within an unexpected spectrum.

Expanding the mutational spectrum of LZTR1 in schwannomatosis.

Schwannomatosis is characterized by the development of multiple non-vestibular, non-intradermal schwannomas. Constitutional inactivating variants in two genes, SMARCB1 and, very recently, LZTR1, have been reported. We performed exome sequencing of 13 schwannomatosis patients from 11 families without SMARCB1 deleterious variants. We identified four individuals with heterozygous loss-of-function variants in LZTR1. Sequencing of the germline of 60 additional patients identified 18 additional heterozygous variants in LZTR1. We identified LZTR1 variants in 43% and 30% of familial (three of the seven families) and sporadic patients, respectively. In addition, we tested LZTR1 protein immunostaining in 22 tumors from nine unrelated patients with and without LZTR1 deleterious variants. Tumors from individuals with LZTR1 variants lost the protein expression in at least a subset of tumor cells, consistent with a tumor suppressor mechanism. In conclusion, our study demonstrates that molecular analysis of LZTR1 may contribute to the molecular characterization of schwannomatosis patients, in addition to NF2 mutational analysis and the detection of chromosome 22 losses in tumor tissue. It will be especially useful in differentiating schwannomatosis from mosaic Neurofibromatosis type 2 (NF2). However, the role of LZTR1 in the pathogenesis of schwannomatosis needs further elucidation.

MLH1 constitutional and somatic methylation in patients with MLH1 negative tumors fulfilling the revised Bethesda criteria.

Lynch syndrome (LS) is a tumor predisposing condition caused by constitutional defects in genes coding for components of the mismatch repair (MMR) apparatus. While hypermethylation of the promoter of the MMR gene MLH1 occurs in about 15% of colorectal cancer samples, it has also been observed as a constitutional alteration, in the absence of DNA sequence mutations, in a small number of LS patients. In order to obtain further insights on the phenotypic characteristics of MLH1 epimutation carriers, we investigated the somatic and constitutional MLH1 methylation status of 14 unrelated subjects with a suspicion of LS who were negative for MMR gene constitutional mutations and whose tumors did not express the MLH1 protein. A novel case of constitutional MLH1 epimutation was identified. This patient was affected with multiple primary tumors, including breast cancer, diagnosed starting from the age of 55 y. Investigation of her offspring by allele specific expression revealed that the epimutation was not stable across generations. We also found MLH1 hypermethylation in cancer samples from 4 additional patients who did not have evidence of constitutional defects. These patients had some characteristics of LS, namely early age at onset and/or positive family history, raising the possibility of genetic influences in the establishment of somatic MLH1 methylation.

Application of COLD-PCR for improved detection of NF2 mosaic mutations.

Somatic mosaicism represents the coexistence of two or more cell populations with different genotypes in one person, and it is involved in >30 monogenic disorders. Somatic mosaicism characterizes approximately 25% to 33% of patients with de novo neurofibromatosis type 2 (NF2). The identification of mosaicism is crucial to patients and their families because the clinical course of the disease and its transmission risk is influenced by the degree and distribution of mutated cells. Moreover, in NF2, the capability of discriminating patients with mosaicism is especially important to make differential diagnosis with schwannomatosis. However, the identification of mosaic variants is considerably difficult, and the development of specific molecular techniques to detect low levels of unknown molecular alterations is required. Co-amplification at lower denaturation temperature (COLD)-PCR has been described as a powerful method to selectively amplify minority alleles from mixtures of wild-type and mutation-containing sequences. Here, we applied COLD-PCR to molecular analysis of patients with NF2 mosaicism. With the use of COLD-PCR, followed by direct sequencing, we were able to detect NF2 mutations in blood DNA of three patients with NF2 mosaicism. Our study has shown the capability of COLD-PCR in enriching low-represented mutated allele in blood DNA sample, making it usable for molecular diagnosis of patients with mosaicism.

Novel neurofibromatosis type 2 mutation presenting with status epilepticus.

Neurofibromatosis type 2 (NF2) is a dominantly inherited syndrome caused by mutations of the tumour-suppressor NF2, which encodes the merlin protein. Mutations are associated with a predisposition to development of benign tumours in the central nervous system. Even though cerebral cortical lesions are frequently associated with seizures, epilepsy is rarely described in NF2. Here, we describe an adult case of NF2 in which the onset of symptoms was characterised by status epilepticus. In this patient, we identified the novel c.428_430delCTTdel mutation in NF2, involving the amino-terminal FERM domain, which is fundamental for the correct tumour suppressor function of the protein. Bioinformatic analyses revealed an important structural perturbation of the FERM domain, with a predicted impairment of the anti-tumour activity.

High resolution melting analysis for a rapid identification of heterozygous and homozygous sequence changes in the MUTYH gene.

BACKGROUND: MUTYH-associated polyposis (MAP) is an autosomal recessive form of intestinal polyposis predisposing to colorectal carcinoma. High resolution melting analysis (HRMA) is a mutation scanning method that allows detection of heterozygous sequence changes with high sensitivity, whereas homozygosity for a nucleotide change may not lead to significant curve shape or melting temperature changes compared to homozygous wild-type samples. Therefore, HRMA has been mainly applied to the detection of mutations associated with autosomal dominant or X-linked disorders, while applications to autosomal recessive conditions are less common. METHODS: MUTYH coding sequence and UTRs were analyzed by both HRMA and sequencing on 88 leukocyte genomic DNA samples. Twenty-six samples were also examined by SSCP. Experiments were performed both with and without mixing the test samples with wild-type DNA. RESULTS: The results show that all MUTYH sequence variations, including G > C and A > T homozygous changes, can be reliably identified by HRMA when a condition of artificial heterozygosity is created by mixing test and reference DNA. HRMA had a sensitivity comparable to sequencing and higher than SSCP. CONCLUSIONS: The availability of a rapid and inexpensive method for the identification of MUTYH sequence variants is relevant for the diagnosis of colorectal cancer susceptibility, since the MAP phenotype is highly variable.

The use of COLD-PCR and high-resolution melting analysis improves the limit of detection of KRAS and BRAF mutations in colorectal cancer.

Fast and reliable tests to detect mutations in human cancers are required to better define clinical samples and orient targeted therapies. KRAS mutations occur in 30-50% of colorectal cancers (CRCs) and represent a marker of clinical resistance to cetuximab therapy. In addition, the BRAF V600E is mutated in about 10% of CRCs, and the development of a specific inhibitor of mutant BRAF kinase has prompted a growing interest in BRAF (V600E) detection. Traditional methods, such as PCR and direct sequencing, do not detect low-level mutations in cancer, resulting in false negative diagnoses. In this study, we designed a protocol to detect mutations of KRAS and BRAF(V600E) in 117 sporadic CRCs based on coamplification at lower denaturation temperature PCR (COLD-PCR) and high-resolution melting (HRM). Using traditional PCR and direct sequencing, we found KRAS mutations in 47 (40%) patients and BRAF(V600E) in 10 (8.5%). The use of COLD-PCR in apparently wild-type samples allowed us to identify 15 newly mutated CRCs (10 for KRAS and 5 for BRAF (V600E)), raising the percentage of mutated CRCs to 48.7% for KRAS and to 12.8% for BRAF (V600E). Therefore, COLD-PCR combined with HRM permits the correct identification of less represented mutations in CRC and better selection of patients eligible for targeted therapies, without requiring expensive and time-consuming procedures.

Schwannomatosis associated with multiple meningiomas due to a familial SMARCB1 mutation.

Schwannomatosis (MIM 162091) is a condition predisposing to the development of central and peripheral schwannomas; most cases are sporadic without a clear family history but a few families with a clear autosomal dominant pattern of transmission have been described. Germline mutations in SMARCB1 are associated with schwannomatosis. We report a family with multiple schwannomas and meningiomas. A SMARCB1 germline mutation in exon 1 was identified. The mutation, c.92A>T (p.Glu31Val), occurs in a highly conserved amino acid in the SMARCB1 protein. In addition, in silico analysis demonstrated that the mutation disrupts the donor consensus sequence of exon 1. RNA studies verified the absence of mRNA transcribed by the mutant allele. This is the first report of a SMARCB1 germline mutation in a family with schwannomatosis characterized by the development of multiple meningiomas.

Genomic rearrangements of the CDKN2A locus are infrequent in Italian malignant melanoma families without evidence of CDKN2A/CDK4 point mutations.

Predisposition to familial cutaneous malignant melanoma has been associated with mutations in the CDKN2A and CDK4 genes. However, only a small subgroup of melanoma pedigrees harbour CDKN2A or CDK4 germline mutations. It is possible that other types of CDKN2A rearrangements, not detectable by routine PCR-based approaches, are involved in a fraction of melanoma cases negative for point sequence changes. In order to gain insights on the possible role of CDKN2A large deletions or duplications in melanoma susceptibility in the Italian population, we screened a series of 124 cutaneous malignant melanoma families referred to five national medical/cancer genetics centres. All probands were negative for point mutations in CDKN2A and CDK4. All samples were tested by MLPA (multiplex ligation-dependent probe amplification), and the results were confirmed by real-time quantitative PCR in a subset of 53 cases. No genomic rearrangements were detected in this series, one of the largest so far investigated. These data suggest that large deletions/duplications in the CDKN2A locus are infrequently involved in the development of familial melanoma in the Italian population. Based on these results, routine search for these rearrangements in CDKN2A- and CDK4-mutation negative melanoma families is not warranted, although it would be reasonable to pursue it in selected cases with very strong family history and/or showing linkage to 9p21.

High-resolution melting analysis for rapid detection of KRAS, BRAF, and PIK3CA gene mutations in colorectal cancer.

High-resolution melting analysis (HRMA) provides a valid approach to efficiently detect DNA genetic and somatic mutations. In this study, HRMA was used for the screening of 116 colorectal cancers (CRCs) to detect hot-spot mutations in the KRAS and BRAF oncogenes. Mutational hot spots on the PIK3CA gene, exons 9 and 20, were also screened. Direct sequencing was used to confirm and characterize HRMA results. HRMA revealed abnormal melting profiles in 65 CRCs (56.0%), 16 of them harboring mutations in 2 different genes simultaneously. The frequency of mutations was 17.2% for PIK3CA (11.2% in exon 9 and 6.0% in exon 20), 43.1% for KRAS exon 2, and 9.5% in exon 15 of the BRAF gene. We found a significant association between PIK3CA and KRAS mutations (P = .008), whereas KRAS and BRAF mutations were mutually exclusive (P = .001). This report describes a novel approach for the detection of PIK3CA somatic mutations by HRMA.