Unilateral vestibular schwannoma and meningiomas in a patient with PIK3CA-related segmental overgrowth: Co-occurrence of mosaicism for 2 rare disorders.

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

Gonosomal mosaicism for an NF1 deletion in a sperm donor: evidence of the need for coordinated, long-term communication of health information among relevant parties.

BACKGROUND: Screening of gamete donors can reduce but cannot eliminate the risks for medical problems in donor-conceived offspring. We present a case of gonosomal mosaicism discovered in an anonymous sperm donor after receiving two reports of neurofibromatosis type 1 (NF1) in donor-conceived offspring, to illustrate that long-term, systematic investigation of health issues in donors and offspring can be invaluable to the welfare of these individuals. METHODS: A repeat physical evaluation and ophthalmology examination were performed on the donor. DNA samples were examined by RTPCR fragment analysis, multiplex ligation-dependent probe amplification (MLPA) and targeted array-comparative genomic hybridization (aCGH). RESULTS: Gonosomal mosaicism for a deletion mutation in the NF1 gene was identified in 20% of sperm and a smaller percentage of lymphocytes. CONCLUSIONS: Long-term communication of medical information among donors, recipients and donor-conceived offspring is beneficial for the health management of all parties. Development of a secure, coordinated data system is critical to achieving this goal. Recommendations are provided for management and communication of critical information based on this experience.

Deletions involving long-range conserved nongenic sequences upstream and downstream of FOXL2 as a novel disease-causing mechanism in blepharophimosis syndrome.

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

Exhaustive mutation analysis of the NF1 gene allows identification of 95% of mutations and reveals a high frequency of unusual splicing defects.

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.

Exon 10b of the NF1 gene represents a mutational hotspot and harbors a recurrent missense mutation Y489C associated with aberrant splicing.

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.

Chondrodysplasia punctata with multiple congenital anomalies: a new syndrome?

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

Qualitative and quantitative analysis of tumour invasion in vivo and in vitro.

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