Entomotoxicology in burnt bodies: a case of maternal filicide-suicide by fire.

One of the most common methods of maternal filicide is by fire. In this case study, a 40-year-old female and her children were found completely burned in a burnt out car. All bodies showed a degree of destruction by fire consisting to a level 3 of the Crow-Glassman Scale (CGS) and early stage of insect activity. Toxicological analyses were performed on soft tissues and body fluids still available. The results were positive for diazepam and its metabolites only for children with blood concentrations consistent with therapeutic doses of benzodiazepines. Home video surveillance cameras confirmed sedation prior to death recording the mother while administering some drops of sedative drugs in a soft drink to the children just a couple of hours before setting fire to the car. Based on autopsy findings, all victims were still alive at the time of fire. The cause of death was determined as carbon monoxide poisoning and fatal thermal injuries by fire. This case study has a special focus on the entomotoxicology and the potential role of insects in death investigations of burnt bodies, supposed to be an inadequate substratum for insect colonization. It demonstrates that in burnt bodies, arthropod colonization can be quite immediate after fire is extinguished. Toxicological analyses performed on larvae actively feeding on the children's bodies were positive for diazepam and its metabolites in small amount compared with blood concentrations, whereas the larvae collected from the mother's body were totally negative. These data, according to the autopsy findings and the toxicological results from the victim's blood and tissues, supported the suspect of a non-lethal sedation prior to death, which is a common behaviour in maternal filicide.

A novel PAX1 null homozygous mutation in autosomal recessive otofaciocervical syndrome associated with severe combined immunodeficiency.

Otofaciocervical syndrome (OFCS) is a rare disorder characterized by facial anomalies, cup-shaped low-set ears, preauricular fistulas, hearing loss, branchial defects, skeletal anomalies, and mild intellectual disability. Autosomal dominant cases are caused by deletions or point mutations of EYA1. A single family with an autosomal recessive form of OFCS and a homozygous missense mutation in PAX1 gene has been described. We report whole exome sequencing of 4 members of a consanguineous family in which 2 children, showing features of OFCS, expired from severe combined immunodeficiency (SCID). To date, the co-occurrence of OFCS and SCID has never been reported. We found a nonsense homozygous mutation in PAX1 gene in the 2 affected children. In mice, Pax1 is required for the formation of specific skeletal structures as well as for the development of a fully functional thymus. The mouse model strongly supports the hypothesis that PAX1 depletion in our patients caused thymus aplasia responsible for SCID. This report provides evidence that bi-allelic null PAX1 mutations may lead to a multi-system autosomal recessive disorders, where SCID might represent the main feature.

Creation of an international registry to support discovery in schwannomatosis.

Schwannomatosis is a tumor suppressor syndrome that causes multiple tumors along peripheral nerves. Formal diagnostic criteria were first published in 2005. Variability in clinical presentation and a relative lack of awareness of the syndrome have contributed to difficulty recognizing affected individuals and accurately describing the natural history of the disorder. Many critical questions such as the mutations underlying schwannomatosis, genotype-phenotype correlations, inheritance patterns, pathologic diagnosis of schwannomatosis-associated schwannomas, tumor burden in schwannomatosis, the incidence of malignancy, and the effectiveness of current, or new treatments remain unanswered. A well-curated registry of schwannomatosis patients is needed to facilitate research in field. An international consortium of clinicians and scientists across multiple disciplines with expertise in schwannomatosis was established and charged with the task of designing and populating a schwannomatosis patient registry. The International Schwannomatosis Registry (ISR) was built around key data points that allow confirmation of the diagnosis and identification of potential research subjects to advance research to further the knowledge base for schwannomatosis. A registry with 389 participants enrolled to date has been established. Twenty-three additional subjects are pending review. A formal process has been established for scientific investigators to propose research projects, identify eligible subjects, and seek collaborators from ISR sites. Research collaborations have been created using the information collected by the registry and are currently being conducted. The ISR is a platform from which multiple research endeavors can be launched, facilitating connections between affected individuals interested in participating in research and researchers actively investigating a variety of aspects of schwannomatosis. © 2016 Wiley Periodicals, Inc.

Cutaneous foetal injuries related to amniocentesis.

Amniocentesis is one of the most important prenatal diagnostic procedures available to assess congenital abnormalities. It is performed worldwide due to its simplicity of execution and lack of risk. The most frequent known accidents in amniocentesis are abortion, oligohydramnios, amniositis and placental abruption, while direct fetal injuries produced by contact with the needle are rarely seen. The injuries produced are extremely variable in severity, but the most frequent is skin wounds, which usually heal as small, round depressed scars. The cases we describe concern the occurrence of iatrogenic cutaneous wound lesions to a fetus during amniocentesis. The medical-legal analysis of the cases required dermatological expertise in order to exclude a different pathogenesis for the skin injuries to the child and were assigned by the court, in order to assess the administrative compensation due to the parents of the child as a result of medical malpractice.

Multiple spinal ganglioneuromas in a patient harboring a pathogenic NF1 mutation.

Ganglioneuroma is a rare benign tumor originating from autonomic ganglia and is considered the benign counterpart of neuroblastoma. Ganglioneuromas may be present as an isolated finding and, rarely, in association with neurofibromatosis type 1 (NF1). However, ganglioneuromas of the cervical spine with intradural extension and multiple locations are extremely rare. We describe a 32-year-old woman with multiple ganglioneuromas of the cervical, dorsal and lumbar spine associated with a few café-au-lait spots and subcutaneous nodules. The patient lacked other NF1 stigmata, such as freckling, Lisch nodules and cutaneous neurofibromas. Although our patient did not fulfill the NF1 diagnostic criteria, molecular diagnosis revealed a pathogenic mutation in the NF1 gene. Approximately 30 patients affected by NF1 and ganglioneuromas have been reported: in all these individuals, NF1 diagnosis was made according to the clinical diagnostic criteria and no patients have molecular diagnosis. Therefore, this is the first case with multiple spinal ganglioneuromas associated with a pathogenic NF1 mutation.

Fatal malonyl CoA decarboxylase deficiency due to maternal uniparental isodisomy of the telomeric end of chromosome 16.

Malonic aciduria is a rare autosomal recessive disorder caused by deficiency of malonyl-CoA decarboxylase, encoded by the MLYCD gene. We report on a patient with clinical presentation in the neonatal period. Metabolic investigations led to a diagnosis of malonyl-CoA decarboxylase deficiency, confirmed by decreased activity in cultured fibroblasts. High doses of carnitine and a diet low in lipids led to a reduction in malonic acid excretion, and to an improvement in his clinical conditions, but at the age of 4 months he died suddenly and unexpectedly. No autopsy was performed. Molecular analysis of the MLYCD gene performed on the proband's RNA and genomic DNA identified a previously undescribed mutation (c.772-775delACTG) which was homozygous. This mutation was present in his mother but not in his father; paternity was confirmed by microsatellite analysis. A hypothesis of maternal uniparental disomy (UPD) was investigated using fourteen microsatellite markers on chromosome 16, and the results confirmed maternal UPD. Maternal isodisomy of the 16q24 region led to homozygosity for the MLYCD mutant allele, causing the patient's disease. These findings are relevant for genetic counselling of couples with a previously affected child, since the recurrence risk in future pregnancies is dramatically reduced by the finding of UPD. In addition, since the patient had none of the clinical manifestations previously associated with maternal UPD 16, this case provides no support for the existence of maternally imprinted genes on chromosome 16 with a major effect on phenotype.

The location of constitutional neurofibromatosis 2 (NF2) splice site mutations is associated with the severity of NF2.

Neurofibromatosis 2 (NF2) patients with constitutional splice site NF2 mutations have greater variability in disease severity than NF2 patients with other types of mutations; the cause of this variability is unknown. We evaluated genotype-phenotype correlations, with particular focus on the location of splice site mutations, using mutation and clinical information on 831 patients from 528 NF2 families with identified constitutional NF2 mutations. The clinical characteristics examined were age at onset of symptoms of NF2 and number of intracranial meningiomas, which are the primary indices of the severity of NF2. Two regression models were used to analyse genotype-phenotype correlations. People with splice site mutations in exons 1-5 had more severe disease than those with splice site mutations in exons 11-15. This result is compatible with studies showing that exons 2 and 3 are required for self-association of the amino terminal of the NF2 protein in vitro, and that deletions of exons 2 and 3 in transgenic and knockout mouse models of NF2 cause a high prevalence of Schwann cell derived tumours.

A novel microdeletion syndrome with loss of the MSH2 locus and hereditary non-polyposis colorectal cancer.

Constitutional chromosome deletions can predispose to the development of cancer with the phenotypic characteristics of inherited cancer syndromes, when the deleted region encompasses a tumour suppressor gene. Examples of such conditions are represented by the cytogenetic deletions associated with retinoblastoma, Wilms tumour and familial adenomatous polyposis. So far, no constitutional deletions involving the genes implicated in hereditary non-polyposis colorectal cancer (HNPCC) have been identified. This may be at least partially because of the lack of distinctive phenotypic manifestations in HNPCC. We describe the first case of a constitutional microdeletion associated with HNPCC. Suspicion of a microdeletion was prompted by the association of mental retardation, postnatal growth deficiency, minor congenital anomalies and early onset (37 years) sporadic colon cancer. The patient was found to harbour a microdeletion within chromosome 2p16-p21, including the MSH2 gene. Since there are very few reports of deletions of the 2p16-p21 region, our observation sets the grounds for the definition of a novel multiple congenital anomaly/mental retardation/cancer microdeletion syndrome.

High resolution deletion analysis of constitutional DNA from neurofibromatosis type 2 (NF2) patients using microarray-CGH.

Neurofibromatosis type 2 (NF2) is an autosomal dominant disorder whose hallmark is bilateral vestibular schwannoma. It displays a pronounced clinical heterogeneity with mild to severe forms. The NF2 tumor suppressor (merlin/schwannomin) has been cloned and extensively analyzed for mutations in patients with different clinical variants of the disease. Correlation between the type of the NF2 gene mutation and the patient phenotype has been suggested to exist. However, several independent studies have shown that a fraction of NF2 patients with various phenotypes have constitutional deletions that partly or entirely remove one copy of the NF2 gene. The purpose of this study was to examine a 7 Mb interval in the vicinity of the NF2 gene in a large series of NF2 patients in order to determine the frequency and extent of deletions. A total of 116 NF2 patients were analyzed using high-resolution array-comparative genomic hybridization (CGH) on an array covering at least 90% of this region of 22q around the NF2 locus. Deletions, which remove one copy of the entire gene or are predicted to truncate the schwannomin protein, were detected in 8 severe, 10 moderate and 6 mild patients. This result does not support the correlation between the type of mutation affecting the NF2 gene and the disease phenotype. This work also demonstrates the general usefulness of the array-CGH methodology for rapid and comprehensive detection of small (down to 40 kb) heterozygous and/or homozygous deletions occurring in constitutional or tumor-derived DNA.

Neurofibromatosis type 2 attributable to gonosomal mosaicism in a clinically normal mother, and identification of seven novel mutations in the NF2 gene.

Neurofibromatosis type 2 (NF2) is an autosomal dominant cancer syndrome that predisposes to the development of bilateral vestibular schwannomas sometimes associated with schwannomas at other locations, meningiomas, ependymomas and juvenile posterior subcapsular lenticular opacities. This disease is caused by inactivating mutations in the NF2 tumour-suppressor gene, located in 22q12. Recently, somatic mosaicism has been demonstrated in some "de novo" NF2 patients. We here report the genetic study of 33 NF2 patients from 33 unrelated Italian families. Twelve mutations were characterised, including seven newly identified mutations and five recurrent ones. Furthermore, we describe one patient with an inactivating mutation that lies in exon 13 but that is present in only a portion of the lymphocytes and, more importantly, a clinically normal individual carrying a somatic/germinal mosaicism for a nonsense mutation in exon 10 of the NF2 gene. Our results confirm the relatively high percentage of mosaicism for mutations in the NF2 gene and establish the importance of evaluating genomic DNA from several tissues, in addition to lymphocytes, so as to identify mosaicism in "de novo" NF2 patients and their relatives. In addition, the demonstration of somatic and/or gonadal mosaicism is an important tool for accurate genetic counselling in families with sporadic cases of NF2.

Prognostic significance of microsatellite instability in sporadic mucinous colorectal cancers.

We investigated the prognostic significance of microsatellite instability (MI) in 50 consecutive patients with sporadic mucinous colorectal cancer who had undergone only surgery. We evaluated MI and the pathological features with a possible prognostic value for each tumor, and the effect of the examined parameters on patients' outcome was statistically analyzed (univariate and multivariate analysis). All patients were followed-up for a minimum of 72 months or until death; in evaluating survival, only deaths of colorectal cancer were considered. DNA extracted from tumor sections and the corresponding normal tissue was analyzed by polymerase chain reaction at six microsatellite loci: D2S123, D3S1611, D3S49, D5S107, BAT26, BAT40. Alterations at two or more loci were detected in 36% of cases (MI+ tumors). MI+ and MI- cancers differed significantly in the pattern of growth, and most MI+ tumors showed an expanding type of growth (72.2%, P = .005). At univariate analysis, improved survival rate was significantly associated with MI, as well as with the following parameters: expanding cancer growth, Dukes stage, and absence of venous invasion. Nevertheless, at multivariate analysis, only the pattern of cancer growth and Dukes stage were independent prognostic factors, whereas the effect on survival of MI and venous invasion was found to be negligible. In our study, MI+ and MI- cancers differ only on the pattern of growth; therefore, our data suggest that the better survival rate in mucinous cancers with genomic instability is strictly related to their less aggressive type of growth.

Microsatellite instability in sporadic mucinous colorectal carcinomas: relationship to clinico-pathological variables.

A series of 44 sporadic mucinous colorectal carcinomas was analysed for microsatellite instability; 30 consecutive sporadic non-mucinous colorectal cancers served as controls. Mucinous carcinomas showed microsatellite instability more frequently than non-mucinous cancers: 26/44 and 8/30, respectively (P = 0.005); the difference was higher for cancers with two or more microsatellite alterations: 12 of the 44 mucinous carcinomas versus one of the 30 non-mucinous carcinomas (P = 0.007). On comparing the clinico-pathological features of mucinous carcinomas with and without microsatellite instabilities, no differences were found with respect to the following variables; sex ratio, tumour localization, tumour size, peritumoural lymphocytic infiltration, Crohn's-like lymphoid reaction, peritumoural fibrosis, Dukes' stage, and relationship with adenoma. Mucinous cancers with DNA replication errors were characterized by three features: onset in younger patients (P < 0.05); exophytic gross shape (P = 0.03); and an expanding pattern of growth (P = 0.003). Of the 12 mucinous carcinomas with instability in two or more microsatellites, ten (83.3 per cent) exhibited an expanding pattern of growth, while mucinous cancers with instability in one microsatellite or without genomic instability showed no distinctive growth pattern. This study confirms the relationship between microsatellite instabilities and mucin production in colorectal carcinomas, but shows that replication error RER-positive and RER-negative mucinous cancers differ in few clinico-pathological features. These differences are only in part similar to those previously reported in RER-positive colorectal carcinomas. These data indicate that mucinous carcinoma of the large bowel could represent a histological subset separate from other histotypes.

Expression of epidermal growth factor, transforming growth factor-alpha and their receptor in the human oesophagus.

Increasing evidence indicates that epidermal growth factor and transforming growth factor-alpha are involved in the maintenance of oesophageal mucosal integrity. However, their cellular origin and the exact localization of their receptor in the oesophagus are still unclear. Therefore, we examined the expression of the two growth factors and their shared receptor in the normal human oesophagus at both mRNA and protein level, by immunohistochemistry, in situ hybridization and reverse transcription-polymerase chain reaction. In addition to being expressed in the proliferative compartment of the oesophageal epithelium, the receptor was found in a variety of cells, including smooth muscle cells, submucosal gland cells and the epithelium lining their ducts. Immunohistochemically, the pattern of distribution of epidermal growth factor paralleled that of its receptor. In situ hybridization demonstrated epidermal growth factor mRNA expression in the oesophageal epithelium and submucosal glands. Additionally, amplified transcripts of predicted size were detected by reverse transcription-polymerase chain reaction, thus confirming that authentic transcripts of the growth factor exist in the normal human oesophagus. Transforming growth factor-alpha mRNA and protein expression, while similar to that of epidermal growth factor, predominated in the more differentiated cell layers of the stratified squamous epithelium. These results demonstrate that the normal oesophagus can synthesize both growth factors. Moreover, the peculiar distribution of these peptides and the concomitant expression of their receptor in multiple cell types suggest that the two growth factors may exert diverse physiological functions in the oesophagus and participate in defence and reparative events following mucosal injury.

Analysis of the neurofibromatosis type 2 gene in different human tumors of neuroectodermal origin.

The autosomal dominant syndrome neurofibromatosis type 2 (NF2) is characterized by the development of bilateral vestibular schwannomas, meningiomas, ependymomas and gliomas. The NF2 gene, recently isolated from chromosome 22, is mutated in both sporadic and NF2 tumors such as schwannomas, meningiomas and ependymomas. Mutations of the gene have been described not only in the neoplasms usually associated with NF2, but also in 30% of the melanomas and 41 % of the mesotheliomas analyzed. In particular, the finding of mutations in melanomas supports the hypothesis that the NF2 gene is involved in the genesis of several tumor types that arise from the embryonic neural crest. In this study we examined, by single-strand conformational polymorphism (SSCP) analysis, 41 tumors of the central nervous system (11 schwannomas and 30 gliomas), 19 melanomas and 15 Merkel cell carcinoma specimens for mutations in the coding sequence of the NF2 gene. We found three inactivating mutations of the NF2 gene in schwannomas. No alterations of the gene were detected by SSCP analysis of the other tumors. These results confirm the role of NF2 in pathogenesis of schwannomas, but do not define its significance in the genesis of the other neuroectodermal tumors studied.

Screening for mutations in the neurofibromatosis type 2 (NF2) gene in sporadic meningiomas.

Meningiomas are benign tumors of the central nervous system. They are usually sporadic but can also occur associated with the neurofibromatosis type 2 (NF2) syndrome. The gene responsible for NF2, recently isolated from chromosome 22, encodes a membrane-organizing protein that shows high sequence homology to a protein family thought to link the cytoskeleton with membrane proteins. Mutations of the NF2gene have been described in sporadic meningiomas, exclusively in tumors that show loss of heterozygosity (LOH) of 22q. These preliminary results indicate that the NF2 gene is involved in the pathogenesis of at least a subset of meningiomas, where it does indeed behave as a tumor suppressor gene. In order to characterize better the role of the NF2 gene in the genesis of meningiomas we have examined the entire coding sequence of the gene in 125 meningiomas by single-strand conformational polymorphism analysis; furthermore, LOH analysis for markers of 22q has been carried out. Inactivating mutations were identified in 30% of our samples, all of which also showed LOH of 22q. The majority of mutations identified were frameshifts and nonsense mutations, which are predicted to produce a truncated or nonfunctional protein. We also found two missense and three in-frame deletions that may pinpoint specific regions of the protein critical to its function. Furthermore, the distribution of mutations throughout the gene, suggested that exons 2, 3, 5, 11 and 13 are more frequently involved. Our results reconfirm the importance of the NF2 gene in the pathogenesis of meningiomas and also suggest that there may be a nonrandom clustering of mutations throughout the gene.

Somatic mutations in the neurofibromatosis type 2 gene in sporadic meningiomas.

Meningiomas are benign tumors of the central nervous system. Although usually sporadic, they can occur in patients affected by the autosomal dominant syndrome, neurofibromatosis type 2 (NF2). The NF2 gene has recently been isolated from chromosome 22. The presence of germline mutations in NF2 patients and the loss of heterozygosity (LOH) on 22q in NF2 tumors support the hypothesis that the NF2 gene acts as a tumor suppressor. Cytogenetic and LOH studies have suggested that the gene responsible for the development of meningiomas is located in the region of 22q in which the NF2 gene maps. The meningiomas gene could therefore be the NF2 gene itself. Recently, somatic mutations of the NF2 gene have been identified in sporadic meningiomas, thus supporting the hypothesis that the NF2 gene is also important in meningioma pathogenesis. In this study, we analyzed sixty-one sporadic meningiomas for LOH of 22q and for mutations in the NF2 gene. LOH was detected in 36 of the 60 informative tumors. Single-strand conformational polymorphism analysis was used to identify nine mutations in five of the eight exons of the NF2 gene studied. The nine tumors with an altered NF2 gene also showed LOH for 22q markers. These results further support the hypothesis that mutations in the NF2 gene are a critical pathogenetic event in at least some meningiomas.

The neurofibromatosis type 2 gene is inactivated in schwannomas.

Schwannomas are tumors arising from schwann cells surrounding peripheral nerves. Although most schwannomas are sporadic, they are seen in approximately 90% of individuals with neurofibromatosis type 2 (NF2), an autosomal dominantly inherited disease with an incidence of 1:40000 live births. The NF2 gene has recently been isolated on chromosome 22 and encodes a putative membrane organizing protein named schwannomin. It is believed to act as a tumor suppressor gene based on the high frequency of loss of heterozygosity (LOH) on this autosome in both sporadic and NF2 associated schwannomas and meningiomas and the identification of inactivating mutation in NF2 patients. In this study we examined 61 schwannomas including 48 sporadic schwannomas (46 of which are vestibular schwannomas) and 12 schwannomas obtained from NF2 patients, for mutations in 10 of the 16 coding exons of the NF2 gene. Twelve inactivating mutations were identified, 8 in sporadic tumours and 4 in tumors from people with NF2. These results support the hypothesis that loss of function of schwannomin is a frequent and fundamental event in the genesis of schwannomas.

Types, stability, and phenotypic consequences of chromosome rearrangements leading to interstitial telomeric sequences.

Using in situ hybridisation, we identified interstitial telomeric sequences in seven chromosomal translocations present in normal and in syndromic subjects. Telomeric sequences were also found at the centromeric ends of a 4p and a 4q caused by centric fission of one chromosome 4. We found that rearrangements leading to interstitial telomeric sequences were of three types: (1) termino-terminal rearrangements with fusion of the telomeres of two chromosomes, of which we report one case; (2) rearrangements in which an acentric fragment of one chromosome fuses to the telomere of another chromosome. We describe four cases of Prader-Willi syndrome with the 15q1-qter transposed to the telomeric repeats of different recipient chromosomes; (3) telomere-centromere rearrangements in which telomeric sequences of one chromosome fuse with the centromere of another chromosome. We describe two examples of these rearrangements in which not only telomeric sequences but also remnants of alphoid sequences were found at the fusion point. Instability at the fusion point of the derivative chromosome was found in the Prader-Willi translocations but we were unable to correlate this instability with culture conditions. The two subjects with the termino-terminal rearrangement and the centric fission respectively have normal phenotypes. The two patients with telomere-centromere fusions were unbalanced for the short arm of an acrocentric chromosome and had failure to thrive; one of them also had dysmorphic facies. We postulate that these phenotypes could be the result of uniparental disomy.

Germ-line mutations of the RET proto-oncogene in multiple endocrine neoplasia type 2A.

Multiple endocrine neoplasia type 2A (MEN 2A) is a dominantly inherited cancer syndrome that affects tissues derived from neural ectoderm. It is characterized by medullary thyroid carcinoma (MTC) and phaeochromocytoma. The MEN2A gene has recently been localized by a combination of genetic and physical mapping techniques to a 480-kilobase region in chromosome 10q11.2 (refs 2,3). The DNA segment encompasses the RET proto-oncogene, a receptor tyrosine kinase gene expressed in MTC and phaeochromocytoma and at lower levels in normal human thyroid. This suggested RET as a candidate for the MEN2A gene. We have identified missense mutations of the RET proto-oncogene in 20 of 23 apparently distinct MEN 2A families, but not in 23 normal controls. Further, 19 of these 20 mutations affect the same conserved cysteine residue at the boundary of the RET extracellular and transmembrane domains.