Comparative mutation detection screening of the type VII collagen gene (COL7A1) using the protein truncation test, fluorescent chemical cleavage of mismatch, and conformation sensitive gel electrophoresis.

Mutations in the type VII collagen gene, COL7A1, give rise to the blistering skin disease, dystrophic epidermolysis bullosa. We have developed two new mutation detection strategies for the screening of COL7A1 mutations in patients with dystrophic epidermolysis bullosa and compared them with an established protocol using conformational sensitive gel electrophoresis. The first strategy consisted of an RNA based protein truncation test that amplified the entire coding region in only four overlapping nested reverse transcriptase-polymerase chain reaction assays. These fragments were transcribed and translated in vitro and analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We have used the protein truncation test procedure to characterize 15 truncating mutations in 13 patients with severe recessive dystrophic epidermolysis bullosa yielding a detection sensitivity of 58%. The second strategy was a DNA-based fluorescent chemical cleavage of mismatch (fl-CCM) procedure that amplified the COL7A1 gene in 21 polymerase chain reaction assays. Mismatches, formed between patient and control DNA, were identified using chemical modification and cleavage of the DNA. We have compared fl-CCM with conformational sensitive gel electrophoresis by screening a total of 50 dominant and recessive dystrophic epidermolysis bullosa patients. The detection sensitivity for fl-CCM was 81% compared with 75% for conformational sensitive gel electrophoresis (p = 0.37 chi2-test). Using a combination of the three techniques we have screened 93 dystrophic epidermolysis bullosa patients yielding an overall sensitivity of 87%, detecting 79 different mutations, 57 of which have not been reported previously. Comparing all three approaches, we believe that no single method is consistently better than the others, but that the fl-CCM procedure is a sensitive, semiautomated, high throughput system that can be recommended for COL7A1 mutation detection.

Allelic heterogeneity of dominant and recessive COL7A1 mutations underlying epidermolysis bullosa pruriginosa.

The inherited mechanobullous disease, dystrophic epidermolysis bullosa, is caused by type VII collagen gene (COL7A1) mutations. We studied six unrelated patients with a distinct clinical subtype of this disease, epidermolysis bullosa pruriginosa, characterized by pruritus, excoriated prurigo nodules, and skin fragility. Mutation analysis using polymerase chain reaction amplification of genomic DNA, heteroduplex analysis and direct nucleotide sequencing demonstrated pathogenetic COL7A1 mutations in each case. Four patients had a glycine substitution mutation on one COL7A1 allele (G1791E, G2242R, G2369S, and G2713R), a fifth was a compound heterozygote for a splice site mutation (5532 + 1G-to-A) and a single base pair deletion (7786delG), and a sixth patient was heterozygous for an out-of-frame deletion mutation (6863del16). This study shows that the molecular pathology in patients with the distinctive clinical features of epidermolysis bullosa pruriginosa is heterogeneous and suggests that other factors, in addition to the inherent COL7A1 mutation(s), may be responsible for an epidermolysis bullosa pruriginosa phenotype.

[Prevalence and morphology of pineal cysts discovered at pituitary MRI: review of 1844 examinations]. / Prévalence et morphologie des kystes épiphysaires: à propos de 1844 IRM hypophysaires.

PURPOSE: To evaluate the frequency, morphology and clinical long term evolution of pineal cysts depicted on MRI. PATIENTS AND METHODS: one thousand eight hundred and forty four (1 532 women and 126 men) MRI examinations were retrospectively reviewed. Coronal and sagittal spin echo T1 weighted sequences without and with gadolinium injection were performed, completed with spin echo T2 weighted images when a cystic sellar lesion was suspected. A pineal cyst was diagnosed as a rounded well defined lesion, with fluid signal in an enlarged pineal gland. Follow-up examinations were performed to evaluate the efficacy of the treatment of the sellar lesion. RESULTS: Twenty one epiphyseal cysts (1.27%) were diagnosed in 20 women (1.31%) and one man (0.79%). Their size was 1,2 +/- 0,4 cm (0,3 to 2 cm). They were asymptomatic. In 10 patients, follow-up MRI examinations did not show any change in size. During the clinical follow-up, these twenty-one patients remained asymptomatic (6 months to 5 years). CONCLUSION: The incidental detection of a pineal cyst at MRI is not exceptional. This lesion's pattern appears characteristic and their reputation of benignity is confirmed in our study.

[Perioperative cortical stimulation of language fields under local anesthesia in preparation to excision of tumors of the dominant hemisphere]. / La stimulation corticale per-opératoire des aires du langage sous anesthésie locale préalable à l'exérèse des tumeurs de l'hémisphère dominant.

OBJECTIVES: The aim of this study is to demonstrate the accuracy of direct cortical stimulation of language areas preparatory to the removal of infiltrating tumors of dominant hemisphere. MATERIAL AND METHODS: From June 1998 to March 2000 we included in our study 15 patients, aged from 30 to 75 years, harboring gliomas (14 high grades and 1 low grade) close to language-specific cortex. All patients had slight inaugural phasic troubles. They underwent craniotomy under local anesthesia for cortical stimulation language mapping, in conjunction with electrocorticography to identify the after-discharge threshold. Stimulation mapping covered the entire macroscopically tumor involved area, extending up to 3 cm away from the margins of the lesion, without searching to identify systematically the language areas. Therefore, the lesion was removed as completely as possible, respecting a security margin of at least 1 cm from the recognized language sites. RESULTS: We identified from 1 to 6 language sites for 14 of our patients, in different locations. A radical removal was achieved for all high grade gliomas, while the low grade was only partially removed. A patient died on the 16th postoperative day from pulmonary embolism; 2 patients deteriorated from peri- or immediate postoperative complications; 9 showed a transient neurological worsening that receded by the 2nd postoperative month, while 3 did not present any postoperative aggravation. At a follow-up of 1 month to 2 years (mean 8 months), 6 patients died and the 9 survivors maintain a good life quality. CONCLUSIONS: Cortical stimulation for language mapping is an accurate technique that allows the surgical morbidity of lesions in proximity to language areas to decrease and become comparable to the surgical morbidity for lesions in non eloquent areas.

Study of endolaryngeal structures by videolaryngoscopy after hanging: a new approach to understanding the physiopathogenesis.

PURPOSE: To evaluate laryngoscopic findings in hanging cases and to compare them with magnetic resonance imaging (MRI) and forensic autopsy results. MATERIALS AND METHODS: Postmortem nasolaryngofibroscopy and MRI of five people who died from hanging were performed. Three people who died from other causes than hanging were also examined with a flexible laryngofibroscope. The results were compared with injuries discovered during forensic autopsy. RESULTS: In all five hanging cases, laryngofibroscopic investigation showed a vocal fold position in complete adduction confirmed by MRI. This position did not seem to be influenced by the intensity of the forces applied to neck or postmortem delay and cadaveric phenomena. The vocal cords of the three non-hanging deceased were found in the intermediate position. These findings could suggest that pressure applied to the cervical nervous and cartilaginous structures or their elongation during hanging could lead to closure of the glottis with vocal cord adduction maintained after death. CONCLUSION: Laryngofibroscopic examination in hanging cases could be very useful in confirming the vital character of the hanging and understanding asphyxial phenomena in incomplete suspension without laryngeal crush.

[Intracranial spread of a melanoma of the face: the trigeminal pathway]. / Extension intracrânienne d'un mélanome de la face: la voie trigéminale.

INTRODUCTION: The intracranial evolution of cutaneous melanoma is usually due to metastases. Neurotropic invasion mainly concerns the trigeminal and the facial nerves. The melanoma is a rare entity among neurotropic tumors. OBSERVATION: A patient presented with a desmoplastic melanoma of the lower lid, complicated by a perineural extension to the cavernous sinus and the mandibular nerve via the maxillary nerve. DISCUSSION: This neurotropic evolution should be carefully monitored when close to a nerve. Extensive surgical excision including peripheral nerves in the vicinity of such tumors is recommended. MRI should be performed when a motor or sensitive disorder appears in the course of a desmoplastic melanoma.

The value of cortical stimulation applied to the surgery of malignant gliomas in language areas.

This study analyzes the utility of peroperative cortical language mapping applied to the surgery of high-grade gliomas situated within or in close vicinity to speech areas. Fifteen consecutive patients harboring high-grade gliomas located in the dominant hemisphere, causing regressive or minor language troubles, underwent awake craniotomy in our institution between June 1998 and April 2000. The technique of cortical stimulation under local anesthesia for language mapping, initially described by Ojemann and colleagues, was applied with some modifications. All patients tolerated awake craniotomy except one, who was intubated after the mapping procedure. Mapping results confirmed a high variability in location of language sites. It was possible to achieve a gross total tumor removal in all cases. Nine patients (60%) exhibited a transient postoperative aggravation. Two patients (13%) presented permanent phasic aggravation. One patient died 16 days after surgery from pulmonary embolism. Five patients died for tumor progression, with a mean survival time of 16.4 months and a median high-quality survival period of 14.2 months. With a mean follow-up of 9.9 months (range, 18-6 months), the 9 survivors are recurrence-free and reveal no significant change in linguistic abilities. This technique is well tolerated and consents to maximize the extent of surgical removal while minimizing the risks of permanent postoperative deficits. This results in an improvement of survival and quality of life.

A recurrent COL7A1 mutation, R2814X, in British patients with recessive dystrophic epidermolysis bullosa.

Mutations in the type VII collagen gene, COL7A1, underlie all forms of dystrophic epidermolysis bullosa (DEB). The identification of COL7A1 mutations in DEB is complicated because the COL7A1 gene contains 118 distinct exons and most mutations are specific to individual families. In an attempt to simplify mutation screening procedures we searched for recurrent mutations in genomic DNA from 38 British patients with recessive DEB using polymerase chain reaction (PCR), heteroduplex analysis and direct nucleotide sequencing. We identified a recurrent premature termination codon, R2814X, on three out of 76 alleles. Previously we identified the COL7A1 mutations R578X and 7786delG as other frequent molecular abnormalities in British recessive DEB patients. Taken together, these three mutations account for approximately 25% of the molecular pathology of this disease in our population discovered thus far and we recommend initial screening for these mutations by PCR and restriction analysis before undertaking more exhaustive COL7A1 gene analysis. Such an approach is likely to reveal underlying COL7A1 mutations in a significant number of cases.

Three cases of de novo dominant dystrophic epidermolysis bullosa associated with the mutation G2043R in COL7A1.

In the absence of a positive family history, it is often difficult to determine whether a single case of mild-to-moderately severe dystrophic epidermolysis bullosa (DEB) represents autosomal recessive or de novo dominant disease. Recent molecular analyses of the type VII collagen gene, COL7A1, have established that the vast majority of such cases are recessive in nature. Nevertheless, a small number of de novo dominant patients have been documented. In this report, we describe three further examples of de novo dominant disease. In each case the COL7A1 mutation comprised the same glycine substitution, G2043R. This mutation has previously been reported in both dominant DEB pedigrees and as a de novo phenomenon and is the most common COL7A1 mutation in dominant DEB throughout the world. These cases emphasize the importance of molecular analysis in providing accurate genetic counselling in this genodermatosis.

A recurrent frameshift mutation in exon 19 of the type VII collagen gene (COL7A1) in Mexican patients with recessive dystrophic epidermolysis bullosa.

Dystrophic epidermolysis bullosa (DEB) is an inherited blistering skin disorder caused by mutations in the type VII collagen gene (COL7A1). In this study, we determined the molecular basis of autosomal recessive DEB in a 19-year-old Hispanic Mexican woman by PCR amplification of genomic DNA, heteroduplex analysis, and automated sequencing of heteroduplex bandshifts. This approach revealed a homozygous frameshift mutation, 2470insG, in exon 19 of COL7A1 and resulted in attenuated basement membrane zone expression of type VII collagen, a reduced number of anchoring fibrils at the dermal-epidermal junction, and a sub-lamina densa level of blister formation. Clinically, the patient had widespread trauma-induced skin fragility and complete loss of the nails, but had less pseudosyndactyly of the fingers and toes and milder mucosal involvement compared to most patients with the generalized form of this genodermatosis. We also screened 7 other Hispanic-Mexican patients with recessive DEB, none of whom were known to be related to this individual, for the mutation 2470insG using heteroduplex analysis and direct sequencing and detected this mutation on 7/14 alleles. Haplotype analysis using intragenic COL7A1 and flanking polymorphisms and microsatellite markers revealed that all the mutant alleles had arisen on similar allelic backgrounds, consistent with propagation of a common Hispanic Mexican ancestral haplotype. In view of the high allelic frequency of the mutation 2470insG in the patients studied, we recommend initial screening for this mutation when attempting to identify the molecular pathology of recessive DEB in Hispanic Mexican patients.