Mutation spectrum and genotype-phenotype analyses in Cowden disease and Bannayan-Zonana syndrome, two hamartoma syndromes with germline PTEN mutation

The tumour suppressor gene PTEN , which maps to 10q23.3 and encodes a 403 amino acid dual specificity phosphatase (protein tyrosine phosphatase; PTPase), was shown recently to play a broad role in human malignancy. Somatic PTEN deletions and mutations were observed in sporadic breast, brain, prostate and kidney cancer cell lines and in several primary tumours such as endometrial carcinomas, malignant melanoma and thyroid tumours. In addition, PTEN was identified as the susceptibility gene for two hamartoma syndromes: Cowden disease (CD; MIM 158350) and Bannayan-Zonana (BZS) or Ruvalcaba-Riley-Smith syndrome (MIM 153480). Constitutive DNA from 37 CD families and seven BZS families was screened for germline PTEN mutations. PTEN mutations were identified in 30 of 37 (81%) CD families, including missense and nonsense point mutations, deletions, insertions, a deletion/insertion and splice site mutations. These mutations were scattered over the entire length of PTEN , with the exception of the first, fourth and last exons. A 'hot spot' for PTEN mutation in CD was identified in exon 5 that contains the PTPase core motif, with 13 of 30 (43%) CD mutations identified in this exon. Seven of 30 (23%) were within the core motif, the majority (five of seven) of which were missense mutations, possibly pointing to the functional significance of this region. Germline PTEN mutations were identified in four of seven (57%) BZS families studied. Interestingly, none of these mutations was observed in the PTPase core motif. It is also worthy of note that a single nonsense point mutation, R233X, was observed in the germline DNA from two unrelated CD families and one BZS family. Genotype-phenotype studies were not performed on this small group of BZS families. However, genotype-phenotype analysis inthe group of CD families revealed two possible associations worthy of follow-up in independent analyses. The first was an association noted in the group of CD families with breast disease. A correlation was observed between the presence/absence of a PTEN mutation and the type of breast involvement (unaffected versus benign versus malignant). Specifically and more directly, an association was also observed between the presence of a PTEN mutation and malignant breast disease. Secondly, there appeared to be an interdependent association between mutations upstream and within the PTPase core motif, the core motif containing the majority of missense mutations, and the involvement of all major organ systems (central nervous system, thyroid, breast, skin and gastrointestinal tract). However, these observations would need to be confirmed by studying a larger number of CD families.      

Identification of three novel RB1 mutations in Brazilian patients with retinoblastoma by "exon by exon" PCR mediated SSCP analysis

AIMS: To carry out a retrospective study, screening for mutations of the entire coding region of RB1 and adjacent intronic regions in patients with retinoblastoma. METHODS: Mutation screening in DNA extracts of formalin fixed, paraffin wax embedded tissues of 28 patients using combined "exon by exon" polymerase chain reaction mediated single strand conformational polymorphism analysis, followed by DNA sequencing. RESULTS: Eleven mutations were found in 10 patients. Ten mutations consisted of single base substitutions; 10 were localised in exonic regions (eight nonsense, one missense, and one frameshift) and another one in the intron-exon splicing region. Three novel mutations were identified: a 2 bp insertion in exon 2 (g.5506-5507insAG, R73fsX77), a G to A transition affecting the last invariant nucleotide of intron 13 (g.76429G>A), and a T to C transition in exon 20 (g.156795T>C, L688P). In addition, eight C to T transitions, resulting in stop codons, were found in five different CGA codons (g.64348C>T, g.76430C>T, g.78238C>T, g.78250C>T, and g.150037C>T). Although specific mutation hotspots have not been identified in the literature, eight of the 11 mutations occurred in CGA codons and seven fell within the E1A binding domains (codons 393-572 and 646-772), whereas five were of both types-in CGA codons within E1A binding domains. CONCLUSIONS: CGA codons and E1A binding domains are apparently more frequent mutational targets and should be initially screened in patients with retinoblastoma. Paraffin wax embedded samples proved to be valuable sources of DNA for retrospective studies, providing useful information for genetic counselling.     

Classroom aeroallergen exposure in Arkansas head start centers.

BACKGROUND: The impact of preschool environmental conditions on classroom aeroallergen concentrations is not fully understood. OBJECTIVE: To examine the relationship between school environmental conditions and classroom aeroallergen concentrations in the Pulaski County Head Start (HS) Program. METHODS: Thirty-three HS centers in Pulaski County, Arkansas, underwent a detailed environmental evaluation. Classroom settled dust samples were analyzed for the presence of common indoor allergens. RESULTS: Classroom eating (70%), wall-to-wall carpeting (58%), and water damage (33%) were common. Median classroom allergen levels were as follows: dust mite (Der p 1 and Der f 1), 0.6 microg/g; Fel d 1, 0.4 microg/g; Can f 1, 1.7 microg/g; cockroach, below detection; Mus m 1, 0.18 microg/g; and mold spores, 17,800 CFU/g. Can f 1 and Mus m 1 allergens were detected in 100% of HS centers. Facilities with carpeting, increased humidity, and single-use facilities showed trends toward increased dust mite concentrations. Detectable cockroach allergen was more common in classrooms cleaned by teachers than by professional housekeepers. CONCLUSIONS: Aeroallergens were commonly detected in Pulaski County HS center classrooms, with dog and mouse allergens detected in 100% of centers. Median classroom allergen concentrations were low, and classroom characteristics were not strongly predictive of increased allergen exposure.     

A dinucleotide mutation in the endothelin-B receptor gene is associated with lethal white foal syndrome (LWFS); a horse variant of Hirschsprung disease

Lethal white foal syndrome (LWFS) is a congenital anomaly of horses characterized by a white coat colour and aganglionosis of the bowel, which is similar to Hirschsprung disease (HSCR). We decided to investigate possible mutations of the endothelin-B receptor gene ( EDNRB ) in LWFS as recent studies in mutant rodents and some patients have demonstrated EDNRB defects. First, we identified a full-length cDNA for horse EDNRB . This cDNA fragment contained a 1329 bp open reading frame which encoded 443 amino acid residues. The predicted amino acid sequence was 89, 91 and 85% identical to human, bovine and mouse as well as rat EDNRB respectively, but only 55% identical to the human, bovine and rat endothelin A receptor (EDNRA). Secondly, sequence analysis, together with allele-specific PCR and the amplification- created restriction site (ACRS) technique, revealed a dinucleotide TC-- >AG mutation, which changed isoleucine to lysine in the predicted first transmembrane domain of the EDNRB protein. This was associated with LWFS when homozygous and with the overo phenotype when heterozygous.      

Myopathic alterations in extraocular muscle of rats subchronically fed pyridostigmine bromide

To determine if alterations in extraocular muscle morphology occur after subchronic oral administration of pyridostigmine bromide, rats were continuously fed 90 mg/kg in meal and examined at 1, 2, 4, 7, and 15 days. Within the first day, blood acetylcholinesterase activity was reduced by 87% and remained inhibited by 74-91% during the study. Light microscopy demonstrated that by day 1 approximately 3% of the extraocular myofibers were shrunken and invaded by inflammatory cells. The most severe degenerative changes consisting of vacuoles and inflammatory cell infiltration occurred at day 1 with progressively less severe changes at days 2 and 4. At days 7 and 15, 1.3-4.5% of the myofibers still exhibited damage. Ultrastructurally, all presynaptic areas were normal but the postsynaptic areas of affected myofibers at days 1, 2, and 4 showed myofilament and Z-band dissolution, mitochondrial inclusions, subneural fold and T-tubule/sarcoplasmic reticulum vacuolization and subneural fold depth reduction. By days 7 and 15, these changes were diminished in some cases and in others alterations appeared similar to day 1. We conclude that subchronic feeding of pyridostigmine bromide induces myopathic rather than neurogenic changes in rat extraocular muscle and that the myopathy is different in these muscles than in the diaphragm from the the same rats.     

Identification of MEN1 gene mutations in sporadic carcinoid tumors of the lung

Lung carcinoids occur sporadically and rarely in association with multiple endocrine neoplasia type 1 (MEN1). There are no well defined genetic abnormalities known to occur in these tumors. We studied 11 sporadic lung carcinoids for loss of heterozygosity (LOH) at the locus of the MEN1 gene on chromosome 11q13, and for mutations of the MEN1 gene using dideoxy fingerprinting. Additionally, a lung carcinoid from a MEN1 patient was studied. In four of 11 (36%) sporadic tumors, both copies of the MEN1 gene were inactivated. All four tumors showed the presence of a MEN1 gene mutation and loss of the other allele. Observed mutations included a 1 bp insertion, a 1 bp deletion, a 13 bp deletion and a single nucleotide substitution affecting a donor splice site. Each mutation predicts truncation or potentially complete loss of menin. The remaining seven tumors showed neither the presence of a MEN1 gene mutation nor 11q13 LOH. The tumor from the MEN1 patient showed LOH at chromosome 11q13 and a complex germline MEN1 gene mutation. The data implicate the MEN1 gene in the pathogenesis of sporadic lung carcinoids, representing the first defined genetic alteration in these tumors.