Alessandro Cama

Mutations in Insulin-Receptor Gene in Insulin-Resistant Patients

Defects in insulin-receptor function have been associated with insulin-resistant states such as obesity and non-insulin-dependent diabetes mellitus (NIDDM). Several types of mutations in the insulin-receptor gene have been identified in patients with genetic syndromes of extreme insulin resistance. In some patients, insulin resistance results from a decrease in the number of insulin receptors on the cell surface. In one patient with leprechaunism (leprechaun/Minn-1), there is >90% decrease in the levels of insulin-receptor mRNA. This patient is a compound heterozygote for two mutations in the insulin-receptor gene, both of which act in a c/s-dominant fashion to decrease levels of mRNA transcribed from that allele. In one allele, there is a nonsense mutation at codon 897. All 22 exons of the other allele have a normal sequence, so that the mutation in this allele appears to map outside the coding sequence of the gene. Impaired insertion in the plasma membrane also causes insulin resistance. In two sisters (patients A-5 and A-8) with type A extreme insulin resistance, there is an 80–90% decrease in the number of insulin receptors expressed on the surface of their cells. Both sisters, whose parents are first cousins, are homozygous for a point mutation in which valine is substituted for phenylalanine at position 382 in the a-subunit of the insulin receptor. This mutation retards t posttranslational processing of the receptor and impairs the transport of receptors to the cell surface. Another patient with leprechaunism (leprechaun/Ark-1) is a compound heterozygote with two different mutant alleles of the insulin-receptor gene. In the allele derived from the father, there is a nonsense mutation at codon 672 that truncates the insulin receptor by deleting the COOH-terminal of the α-subunit and the entirβ-subunit. This truncated receptor, lacking a transmembrane domain, appears not to be expressed at the plasma membrane. In leprechaun/Ark-1, there is a missense mutation in the allele of the insulin-receptor gene derived from the mother. This point mutation results in substitution of glutamic acid for lysine at position 460 in the COOH-terminal half of the α-subunit. This mutation increases receptor affinity and impairs the ability of acid pH to dissociate insulin from the receptor within the endosome. There is a defect in recycling the receptor back to the plasma membrane associated with this defect. This results in an accelerated rate of receptor degradation and a consequent decrease in the number of receptors on the cell surface in vivo. Some patients have a normal number of receptors on their cell surface, but there is a defect in receptor function. A mutation has been described in the tetrabasic amino acid sequence separating the α- from the β-subunit. This mutation, identified in two sisters from a consanguineous pedigree, inhibits the cleavage of the receptor precursor into separate subunits. The uncleaved receptor has a decreased affinity to bind insulin. An insulin-resistant patient has been identified in whose insulin receptors there is a defect in tyrosine kinase activity due to a mutation that substitutes valine for glycine-1008, the third glycine in the conserved Gly-X-Gly-X-X-Gly motif in the ATP-binding domain of the β-subunit of the insulin receptor. The patient is heterozygous for this mutation. All of the mutations in the insulin-receptor gene that have been identified so far have been detected in patients with rare clinical syndromes associated with extreme insulin resistance. Recent advances in recombinant DNA technology have greatly simplified the task of detecting mutations. Thus, it should soon be possible to determine the prevalence of mutations in the insulin-receptor gene. These studies will also answer the question of whether mutations in the insulin-receptor gene contribute to the pathogenesis of insulin resistance with common forms of NIDDM.

Tyr(612) and Tyr(632) in human insulin receptor substrate-1 are important for full activation of insulin-stimulated phosphatidylinositol 3-kinase activity and translocation of GLUT4 in adipose cells.

To examine contributions of specific YXXM motifs in human insulin receptor substrate-1 (IRS-1) to mediating the metabolic actions of insulin, we studied IRS-1 mutants containing various substitutions of Phe for Tyr. In transfected NIH-3T3(IR) cells, insulin stimulation caused a 5-fold increase in phosphatidylinositol 3-kinase (PI3K) activity coimmunoprecipitated with wild-type IRS-1. No PI3K activity was associated with IRS1-F6 (Phe substituted for Tyr at positions 465, 612, 632, 662, 941, and 989). Adding back both Tyr(612) and Tyr(632) fully restored IRS-1-associated PI3K activity, whereas adding back either Tyr(612) or Tyr(632) alone was associated with intermediate PI3K activity. In rat adipose cells transfected with epitope-tagged GLUT4, insulin stimulation caused a 2-fold increase in cell surface GLUT4-HA. Cotransfection of cells with GLUT4-HA and either wild-type IRS-1 or IRS1-Y612/Y632 increased basal cell surface GLUT4-HA (in the absence of insulin) to approximately 80% of the levels seen in insulin-stimulated control cells, whereas overexpression of IRS1-F6 had no effect on the insulin dose-response curve. Overexpression of IRS1-Y612 or IRS1-Y632 caused intermediate effects. Thus, both Tyr(612) and Tyr(632) are important for IRS-1 to fully activate PI3K and mediate translocation of GLUT4 in response to insulin.

APC gene mutations and colorectal adenomatosis in familial adenomatous polyposis

Correlations between germline APC mutation sites and colorectal pathophenotypes, as evaluated by the direct count of adenomas at colectomy, were investigated analysing colectomy specimens from 29 FAP patients carrying one mis-sense (codon 208) and 14 frame-shift or non-sense APC mutations (codons 232, 367, 437, 623, 876, 995, 1061, 1068, 1075, 1112, 1114, 1309, 1324, 1556). The mis-sense mutation at codon 208 was associated with a relatively mild colorectal pathophenotype. The mutation at codon 367, subject to alternative splicing, was associated with attenuated FAP. The mutation at codon 1309 was associated with the profuse colorectal adenomatosis. For 13 mutations, predicted to result in null alleles or truncated APC proteins, we correlated density and distribution of colorectal adenomas with the predicted functional effects of the mutation. The most severe colorectal pathophenotype was significantly associated with the truncating mutation at codon 1309, which is located downstream to the I β-catenin binding domain but upstream II β-catenin-binding domain. Mutations between codons 867 and 1114, which affect the I β-catenin binding domain, as well as mutations occurring in exons 6 and 9, predicted to result in null alleles, were associated with a less severe colorectal pathophenotype. Overall, the highest number of adenomas was detected in the right colon, followed by the left colon, transverse colon sigma and rectum. However, the highest density of adenomas was observed in the left colon, followed by the right colon, sigma, transverse colon and rectum. Colorectal carcinomas, observed in only five patients, were all in the left colon.

Subcellular localization of the BRCA1 gene product in mitotic cells

The product of the hereditary breast cancer susceptibility gene BRCA1 is a multifunctional protein involved in the maintenance of genomic integrity, in transcriptional coactivation, and in the control of cell growth. BRCA1‐deficient cells manifest chromosomal instability. During mitosis, BRCA1 is known to interact with γ‐tubulin in the centrosomes, key elements of the mitotic spindle. Using confocal microscopy and immunogold electron microscopy, we investigated the distribution of endogenous BRCA1 relative to mitotic spindle markers in breast cancer cells. By confocal analysis, BRCA1 and β‐tubulin colocalized to microtubules of the mitotic spindle and to the centrosomes. Immunogold electron microscopy confirmed these results and further revealed that BRCA1 and α‐tubulin codistributed to the walls of the centrioles and to pericentriolar fibers at centrosomes. During chromatid segregation, codistribution was also detected along individual spindle microtubules and at sites of insertion of microtubules on chromosomes. At cytokinesis, BRCA1 and α‐tubulin codistributed to the midbody. Coimmunoprecipitation supported the association of full‐length BRCA1 with α‐ and β‐tubulin. These results are consistent with an involvement of BRCA1 in the dynamics of the mitotic spindle and in the segregation of duplicated chromosomes.

Absence of insulin receptor gene mutations in three insulin-resistant women with the polycystic ovary syndrome

Women with polycystic ovary syndrome (PCOS) are markedly insulin-resistant, but the molecular mechanisms of these changes and their relationship to the hyperandrogenic state remain to be clarified. Mutations have recently been identified in the insulin receptor gene of patients with extreme forms of insulin resistance associated with hyperandrogenism (eg, type A insulin resistance), and these mutations account for the insulin resistance in such patients. We performed this study to determine whether mutations in the coding portion of the insulin receptor gene were responsible for insulin resistance in PCOS. Insulin binding studies using cultured skin fibroblasts of three obese (body mass index > 27 kg/m2) women with PCOS (ie, mild hyperandrogenemia and chronic anovulation of unknown etiology) and documented insulin resistance showed no apparent abnormalities in either the number or affinity of insulin binding sites. Direct sequencing of all 22 exons of the insulin receptor gene from two of the women with PCOS did not reveal any mutations. Furthermore, both alleles of the gene were expressed at equal levels. In a third insulin-resistant PCOS woman, there was no evidence for a mutation in the coding portion of the insulin receptor gene as determined by denaturing gradient gel electrophoresis (DGGE). We conclude that the insulin resistance in these PCOS women was caused by a defect extrinsic to the insulin receptor.

Mutations at coding mononucleotide repeats in gastric cancer with the microsatellite mutator phenotype

We analysed 50 gastric carcinomas (GCs) to verify whether mutations at coding repeats were associated with microsatellite instability (MSI). The tumors included: ten cases with no MSI, 14 cases with MSI=1 locus, 13 cases with MSI=two loci and 13 cases with MSI⩾3 loci. We investigated coding repeats within the TGF-β RII, IGFIIR, BAX, hMSH6, hMSH3 and BRCA2 genes. The TGF-β RII, IGFIIR, BAX, hMSH6 and hMSH3 repeats were altered in 11 (22%), five (10%), four (8%), 16 (32%) and five (10%) cases respectively. Mutations occurred only in MSI-positive (MSI+) tumors and correlated with increasing MSI levels. No alterations of the BRCA2 repeat were found. Mutations in genes other than hMSH6 were strongly associated to hMSH6 mutations, suggesting a key role of this gene. The non-coding BAT-26 and E-Cadherin 3′ UTR poly(A)8/(T)15 repeats were analysed in 44 of the 50 cases. Novel tumor-associated alleles were observed only in MSI-positive GCs and were in most cases associated with mutations at coding repeats. Further investigations with BAT-40 confirmed that four cases manifested mononucleotide repeat alterations restricted to hMSH6 and one case to TGF-β RII. A subset of tumors with MSI at two or more dinucleotide loci resulted negative for mutations at coding and non-coding mononucleotide repeats.

Sporadic childhood hepatoblastomas show activation of β-catenin , mismatch repair defects and p53 mutations

Hepatoblastoma, a rare embryonic tumor that may arise sporadically or in the context of hereditary syndromes (familial adenomatous polyposis and Beckwith–Wiedemanns) is the most frequent liver cancer of childhood. Deregulation of the APC/β-catenin pathway occurs in a consistent fraction of hepatoblastomas, with mutations in the APC and β-catenin genes implicated in familial adenomatous polyposis-associated and sporadic hepatoblastomas, respectively. Alterations in other cancer-related molecular pathways have not been reported. We investigated a series of 21 sporadic paraffin-embedded hepatoblastoma cases for mutations in the p53 (exons 5–8) and β-catenin (exon 3) genes, loss of heterozygosity at APC, microsatellite instability and immunohistochemical expression of β-catenin and of the two main mismatch repair proteins, MLH1 and MSH2. No loss of heterozygosity at APC was detected. We found mutations in β-catenin and p53 in 4/21 (19%) and 5/21 (24%) cases respectively, β-catenin protein accumulation in 14/21 cases (67%), microsatellite instability in 17/21 cases (81%), of which eight resulted positive for high-level of microsatellite instability (in four cases associated with loss of MLH1/MSH2 immunostaining). No correlations between involved molecular pathway(s) and hepatoblastoma histotype(s) emerged. This study confirms that β-catenin deregulation is involved in sporadic hepatoblastoma and also suggests that mismatch repair defects and p53 mutations contribute to this rare liver cancer. Sporadic hepatoblastoma appears to be molecularly and phenotypically heterogeneous and may reflect different pathways of liver carcinogenesis.

Detection of Mutations in Insulin Receptor Gene by Denaturing Gradient Gel Electrophoresis

Denaturing gradient gel electrophoresis (DGGE) has been used to screen for mutations in the insulin receptor gene. Each of the 22 exons was amplified by the polymerase chain reaction (PCR). For each exon, one of the two PCR primers contained a guanine-cytosine (GC) clamp at its 5′ end. The DNA was analyzed by electrophoresis through a polyacrylamide gel containing a gradient of denaturants. Two geometries for the gels were compared; the gradient of denaturants was oriented either parallel or perpendicular to the electric field. The sensitivity of the technique was evaluated by determining whether DGGE succeeded in detecting known mutations and polymorphisms in the insulin receptor gene. With parallel gels, 12 of 16 sequence variants were detected. The use of perpendicular gels increased the sensitivity of detection so that all 16 sequence variants were successfully detected when DNA was analyzed by a combination of perpendicular and parallel gels. Furthermore, DGGE was used to investigate a patient with leprechaunism whose insulin receptor genes had not previously been studied. Two mutant alleles were identified in this patient. The allele inherited from the father had a mutation substituting alanine for Val-28; in the allele inherited from the mother, arginine was substituted for Gly-366.

Analysis of adenomatous polyposis coli gene in thyroid tumours

Familial adenomatous polyposis (FAP) is known to be associated with neoplasia of various tissues, including thyroid carcinoma. Germline mutations of the tumour-suppressor gene APC, responsible for the predisposition to FAP, may therefore be involved in the pathogenesis of these tumours. In this report the structure of the APC gene has been investigated in 26 thyroid tumours, at different stages of dedifferentiation, that were surgically excised from patients with a negative history of FAP. Approximately 35% of the APC gene coding region, where most of the mutations are clustered, has been analysed by a combination of single-strand conformation polymorphism and direct sequencing. No significant alterations could be demonstrated in any sample examined. It is concluded that, at least in patients not affected by FAP, APC gene abnormalities do not seem to play a relevant role in the pathogenesis of thyroid carcinoma.

Genetic evidence that juvenile nasopharyngeal angiofibroma is an integral FAP tumour

Juvenile nasopharyngeal angiofibroma (JNA) is a rare locally invasive neoplasm composed of cavernous vascular channels set in an abundant myxoid stroma of fibroblasts and myofibroblasts.1,2 The histological similarity to erectile tissue, the almost exclusive occurrence in pubescent males, and expression of multiple steroid receptors suggest that JNA growth is stimulated by male sex hormones.1,3 The frequency of JNA is significantly increased in male familial adenomatous polyposis (FAP) patients, suggesting that it may arise through alterations of the adenomatous polyposis coli ( APC )/ β-catenin gene pathway.4 This was supported by the high frequency of recurrent β-catenin gene mutations detected in sporadic JNA, but no APC mutations have thus far been found.5–7 We analysed the sequence of the APC gene and the presence of recurrent β-catenin mutations in matched blood and tumour …