Biomedical subjects

I Celli

Publications and source records attributed to I Celli.

3 recordsLinked to original sources

PubMed1998

A method for point mutation analysis that links SSCP and dye primer fluorescent sequencing.

A method is presented for mutation detection directly from single-strand conformational polymorphism (SSCP) variants. This approach is based on: (i) amplification of the exons to be analysed by SSCP using the forward primer with an eight-base tail to form a universal SSCP cassette; (ii) excision from the gel of the shifted silver-stained bands; (iii) reamplification of the eluted DNAs using, as the forward primer, a 26-base universal adaptor primer corresponding to the 18-base-21M13 sequence plus the eight nucleotides of the universal SSCP cassette; and (iv) direct sequencing of the purified products using the standard-21M13 fluorescent primer. This procedure presents several advantages including the avoidance of a cloning step which leads to significant time reduction, while maintaining comparable accuracy at relatively low costs. In conclusion, the presence of the universal SSCP cassette and subsequent reamplification with the same adaptor primer for direct sequencing makes the method universal for scanning and identification of gene alterations.

Base Sequence↗

PubMed1997

Frequency of RET mutations in long- and short-segment Hirschsprung disease.

Hirschsprung disease, or congenital aganglionic megacolon, is a genetic disorder of neural crest development affecting 1:5,000 newborns. Mutations in the RET proto-oncogene, repeatedly identified in the heterozygous state in both long- and short-segment Hirschsprung patients, lead to loss of both transforming and differentiating capacities of the activated RET through a dominant negative effect when expressed in appropriate cellular systems. The approach of single-strand conformational polymorphism analysis established for all the 20 exons of the RET proto-oncogene, and previously used to screen for point mutations in Hirschsprung patients allowed us to identify seven additional mutations among 39 sporadic and familial cases of Hirschsprung disease (detection rate 18%). This relatively low efficiency in detecting mutations of RET in Hirschsprung patients cannot be accounted by the hypothesis of genetic heterogeneity, which is not supported by the results of linkage analysis in the pedigrees analyzed so far. Almost 74% of the point mutations in our series, as well as in other patient series, were identified among long segment patients, who represented only 25% of our patient population. The finding of a C620R substitution in a patient affected with total colonic aganglionosis confirms the involvement of this mutation in the pathogenesis of different phenotypes (i.e., medullary thyroid carcinoma and Hirschsprung). Finally the R313Q mutation identified for the first time in homozygosity in a child born of consanguineous parents is associated with the most severe Hirschsprung phenotype (total colonic aganglionosis with small bowel involvement).

DNA Mutational Analysis↗

PubMed1997

A Cys634Gly substitution of the RET proto-oncogene in a family with recurrence of multiple endocrine neoplasia type 2A and cutaneous lichen amyloidosis.

Germ-line mutations of the RET proto-oncogene, involving five cysteine residues at codons 609, 611, 618, 620 and 634, are associated with two variants of the inherited cancer syndrome multiple endocrine neoplasia type 2: type 2A and familial medullary thyroid carcinoma. The association of multiple endocrine neoplasia type 2A with the dermatological disorder cutaneous lichen amyloidosis has already been reported, and mutations in the Cys634 have been identified in different families. We describe here an additional pedigree in which multiple endocrine neoplasia type 2A and cutaneous lichen amyloidosis cosegregate. A Cys634Gly was identified by direct sequencing of the RET proto-oncogene exon 11 in the affected individuals. The mutation creates a new HaeIII site, and restriction analysis performed on all family members rules out the presence of the altered allele in two children and consequently the risk of developing thyroid tumors. These results emphasize the role of molecular analysis of the RET proto-oncogene in diagnosing presymptomatically those individuals at risk of inheriting the disease allele.

Adolescent↗