Mutational analysis of OB gene in obese and type 2 diabetes affected subjects.

Peripheral blood DNA from 12 subjects affected by familial obesity and from 35 subjects affected by type 2 diabetes were analysed for mutations in the coding sequence of the OB gene. Mutational analysis, conducted using the single strand conformation polymorphism (SSCP) technique, followed by direct sequencing did not reveal the presence of nucleotide variants in the coding region of the OB gene. The lack of mutations in the coding sequence is consistent with previous data suggesting that mutations in the coding sequence of the OB gene are not common in human familial obesity. In 2 samples displaying a non-informative pattern of SSCP and in 8 additional samples the nucleotide sequence of portion of the intron 2 bordering the coding sequence of exon 2 identified a G in the positions +14IVS and +18IVS, according to a sequence reported previously, but in contrast with some others. All samples were homozygous for these intron variants.

Novel allele of the insulin receptor substrate-1 bearing two non-conservative amino acid substitutions in a patient with noninsulin-dependent diabetes mellitus. Mutations in brief no. 130. Online.

We analyzed by SSCP the complete IRS-1 coding sequence in NIDDM patient #25 D. Unique conformers corresponding to a Ser to Tyr substitution at codon 1043 (S1043Y), and to a Cys to Tyr substitution at codon 1095 (C1095Y) were detected in this patient. The results of sequential digestion with restriction enzymes indicated that the novel sequence variants segregate on the same allele. Relatives of patient #25 D were not available for study, to confirm segregation of the novel allele with NIDDM in the family. Several lines of evidence suggest that the non-conservative amino acid substitutions detected in NIDDM patient #25 D have the potential to affect IRS-1 functions and could play a pathogenic role in this patient. Both S1043Y and C1095Y occur in a highly conserved sequence from human skeletal muscle, human hepatoma, mouse, and rat IRS-1. Protein subsequence analysis revealed that the S1043Y substitution abolishes a consensus sequence for glycogen synthase kinase 3 phosphorylation. Furthermore, S1043Y and C1095Y are not common IRS-1 polymorphisms as they were detected only in 1/136 choromosomes from NIDDM patients (allele frequency in NIDDM patients = 0.0007) and in 0/120 chromosomes from control subjects.

Alterations of microsatellites in neurofibromas of von Recklinghausen's disease.

von Recklinghausen's disease, or type I neurofibromatosis, a common familial tumor syndrome, is characterized by the occurrence of multiple benign neoplasms of nerve sheath cells. The disease is caused by germ-line mutations of the NF1 gene, which encodes a member of the GTPase-activating superfamily of Ras regulatory proteins. We analyzed 5 dinucleotide repeat loci in DNAs from neurofibromas and matched normal skin from 16 NF1 patients. Eight cases (50%) manifested microsatellite alterations. Expansions or compressions of dinucleotide repeats were observed at one locus in four cases and at two loci in one case. Banding patterns compatible with the loss of a microsatellite allele were observed in four cases, including one that also presented microsatellite instability. The surprisingly high frequency of microsatellite alterations suggests that the NF1 gene or another gene(s) contributing to the pathogenesis of neurofibromas might be directly or indirectly implicated in the control of genomic integrity.