Germline characterization of early-aged onset of hereditary non-polyposis colorectal cancer.

OBJECTIVES: Hereditary non-polyposis colorectal cancer (HNPCC) is characterized by the early onset of colorectal cancer (approximately 40 years). Adolescent colorectal cancer is unusual in HNPCC families. We speculated that some DNA mismatch repair germline mutations might be associated with early onset of disease. STUDY DESIGN: Genomic DNA was extracted from members of a kindred with virulent HNPCC fitting the Amsterdam Criteria for HNPCC and sequenced for 2 DNA mismatch repair genes, hMSH2 and hMLH1. A sigmoid adenocarcinoma from the 14-year-old proband was analyzed for highfrequency microsatellite instability and immunostained for DNA mismatch repair gene expression. RESULTS: A germline mutation was identified at nucleotide 676 (codon 226) of the hMLH1 gene. The C to T transition created a nonsense mutation, truncating the hMLH1 protein. This mutation also alters the splice donor sequence, because nucleotide 676 is 2 base pairs from the 3' end of the exon 8. The proband's tumor demonstrated high-frequency microsatellite instability and displayed loss of hMLH1 expression, indicating bi-allelic inactivation of hMLH1. CONCLUSIONS: A complex mutation of hMLH1 at codon 226 is associated with adolescent onset of colorectal cancer in an HNPCC family. Genetic screening of other suspected HNPCC families with unusually young members with cancer might reveal certain genotypes with particularly virulent forms of this disease.

A paired tracer microinjection technique designed for assessment of single-nephron glucose-calcium interactions in the anesthetized rat.

The first part of this study evaluates a new paired microinjection technique for studying single-nephron permeability (in this case to calcium) following injection of 5-10 nL of a Ringer solution into a superficial proximal tubule. The mean difference in fractional 45Ca recovery from two identical microinjections into the same nephron site was 2.2 +/- 0.2% for 89 paired microinjections. Individual nephrons therefore normally show differences in calcium permeability with time. However, moment-to-moment variations in ion transport in any one nephron are in a random direction; differences cancel one another out if enough experiments are performed. The technique thus appears well suited to studies where comparisons are made between the acute nephron responses to two test solutions. It specifically overcomes problems of nephron heterogeneity seen in some other micropuncture techniques. The second part of this study uses the new technique to investigate the effects of a raised intratubular D-glucose concentration on single-nephron calcium transport. Urinary 45Ca recoveries from late proximal microinjections were significantly higher when D- (as opposed to L-) glucose was included in the injectate (6.87 +/- 0.88 vs. 5.24 +/- 0.50%; p < .02). The ability of D-glucose to depress tubular calcium reabsorption at distal nephron sites may contribute to the observed hypercalciuria following systemic D-glucose loading. It may also be relevant to the acute renal failure accompanying renal stone disease, where a relationship between hypercalciuria, urolithiasis, and the consumption of refined carbohydrates has been proposed.

Effects of D-glucose, L-glucose and D-mannitol on renal calcium handling and general renal function in the rat.

Standard renal clearance techniques were used to compare the effects of D-glucose, L-glucose and D-mannitol on renal calcium handling and general renal function in the anaesthetized rat. A significant (P < 0.01) calciuresis was seen in animals infused with D-glucose. This resulted from a decreased tubular reabsorption of calcium. A similar response was not seen with L-glucose or D-mannitol. The contrasting actions of the three sugars on tubular calcium transport may relate to their different transport characteristics, since only D-glucose is actively reabsorbed. Responses of the three sugars also differed with respect to fractional fluid and sodium reabsorption which were significantly (P < 0.001) lower in animals infused with L-glucose and D-mannitol, and plasma insulin concentrations which were significantly (P < 0.01) elevated only in the D-glucose group. An unexpected finding was a highly significant (P < 0.001) and progressive fall in glomerular filtration rate (GFR) in animals infused with L-glucose. The reasons for this are not known.

Renal micropuncture study of the effects of D-glucose tubular calcium handling in the anaesthetized rat.

Tracer microinjection studies were used to grossly locate the nephron site of altered calcium transport following D-glucose loading. Superficial proximal and distal nephrons were injected with a solution containing 45Ca and either D-glucose or L-glucose (as control). Tubule calcium transport was assessed from the recovery of radioactivity in the final urine. 45Ca recoveries from early proximal microinjections were comparable in the two groups (2.63 +/- 0.68 vs. 3.70 +/- 1.19%). However, 45Ca recoveries from late proximal microinjections were significantly higher than D-glucose was included in the injectate (7.39 +/- 1.34 vs. 2.83 +/- 0.67%, P < 0.05). 45Ca recoveries from distal microinjections were also significantly higher in the D-glucose group (87.6 +/- 2.43 vs. 71.0 +/- 2.92%, P < 0.001). These data suggest that the effects of D-glucose on renal calcium handling are mediated at the level of the distal tubule, thick ascending limb of the loop of Henle and/or pars recta. The precise mechanisms involved are not known.