A family with seizures and minor features of tuberous sclerosis and a novel TSC2 mutation.

The authors studied nine members of a family that demonstrated a limited form of tuberous sclerosis complex (TSC). Cutaneous findings were limited to hypopigmented macules in four patients. Five family members had recurrent seizures, and three of these had migrational defects of the cerebral mantle. Mutational analysis of TSC2 indicated the presence of the novel missense change 3106T-->C, 1036S-->P in all family members with seizures. The findings suggest that this mild variant form of TSC is due to a novel TSC2 mutation.

Denaturing high-performance liquid chromatography (DHPLC) is a highly sensitive, semi-automated method for identifying mutations in the TSC1 gene.

Sensitive and automated methods for the detection of DNA sequence variation are required for a wide variety of genetic studies. Diagnostic testing in human genetic disorders is one application of such methods. Tuberous sclerosis complex (TSC) is an autosomal dominant familial tumor syndrome characterized by the development of benign tumors (hamartomas) in multiple organs (OMIM # 19110, #191092). There is a high frequency of sporadic cases and significant demand from patients and families for genetic testing information. Two TSC genes have been identified (TSC1 and TSC2) and together account for all cases [1,2]. Here we report our methods for DHPLC analysis of the TSC1 gene and demonstrate the high sensitivity of this method in a blinded analysis of 21 TSC patients with known TSC1 mutations. In this series, DHPLC detected 27/28 (96%) known TSC1 sequence variations. The only sequence variation not identified by DHPLC in this study is a mosaic case.

Mutational analysis in a cohort of 224 tuberous sclerosis patients indicates increased severity of TSC2, compared with TSC1, disease in multiple organs.

Tuberous sclerosis (TSC) is a relatively common hamartoma syndrome caused by mutations in either of two genes, TSC1 and TSC2. Here we report comprehensive mutation analysis in 224 index patients with TSC and correlate mutation findings with clinical features. Denaturing high-performance liquid chromatography, long-range polymerase chain reaction (PCR), and quantitative PCR were used for mutation detection. Mutations were identified in 186 (83%) of 224 of cases, comprising 138 small TSC2 mutations, 20 large TSC2 mutations, and 28 small TSC1 mutations. A standardized clinical assessment instrument covering 16 TSC manifestations was used. Sporadic patients with TSC1 mutations had, on average, milder disease in comparison with patients with TSC2 mutations, despite being of similar age. They had a lower frequency of seizures and moderate-to-severe mental retardation, fewer subependymal nodules and cortical tubers, less-severe kidney involvement, no retinal hamartomas, and less-severe facial angiofibroma. Patients in whom no mutation was found also had disease that was milder, on average, than that in patients with TSC2 mutations and was somewhat distinct from patients with TSC1 mutations. Although there was overlap in the spectrum of many clinical features of patients with TSC1 versus TSC2 mutations, some features (grade 2-4 kidney cysts or angiomyolipomas, forehead plaques, retinal hamartomas, and liver angiomyolipomas) were very rare or not seen at all in TSC1 patients. Thus both germline and somatic mutations appear to be less common in TSC1 than in TSC2. The reduced severity of disease in patients without defined mutations suggests that many of these patients are mosaic for a TSC2 mutation and/or have TSC because of mutations in an as-yet-unidentified locus with a relatively mild clinical phenotype.

The effects of hepes buffer on clotting tests, assay of factors V and VIII and on the hydrolysis of esters by thrombin and thrombokinase.

Shorter clotting times were found in the presence of 50 mM Hepes (N-2-hydroxyethylpiperazine-N1-2-ethanesulfonic acid) buffer than of 50mM Imidazole buffer in one-stage assays of factors V and VIII, in modified APTT and PT tests and in tests of the clotting of human plasma by purified human thrombin. All tests were performed at ionic strength 0.155 in the presence of either Hepes. NaOH or Imidazole. HCl buffer, pH 7.4 at 37 degrees. The faster clotting in the presence of Hepes buffer, therefore, is probably due, at least in part, to acceleration by Hepes of thrombin's enzymatic action on fibrinogen and/or of the polymerization of the fibrin monomers. Hepes may also have effects of other blood clotting reactions. Rates of hydrolysis of TAME or BAME (p-toluenesulfonyl- or benzoyl-L-arginine methyl ester) at pH 7.4 37 degrees by purified human bovine thrombin were essentially the same in 200 mM Hepes as in 250 mM Tris. HCl buffer (rates in Hepes. NaOH or Hepes. KOH buffers were compared with those in Tris. HCl plus NaCl for KCl). However, with purified bovine thrombokinase, rates of TAME hydrolysis in Hepes buffer were accelerated and rates of BAME hydrolysis slightly inhibited. Hepes, therefore, reacts with thrombokinase but whether this accelerates (or inhibits) the rate of converting prothrombin to thrombin remains to be determined. In addition, Hepes has an inhibitory effect on clotting since increasing the concentration of Hepes from 50 mM to 200 mM inhibits clotting in the PT, APTT and bovine thrombin-human plasma tests. Hepes buffer is being added to some plasmas and to some reagents used in clotting tests. It is, therefore, important to realize that its concentration must be monitored closely or erroneous results may be obtained in clotting tests and assays of clotting factors. The clotting times were the same in the presence of 50 mM Tris. HCl as in Imidazole. HCl buffers in APTT tests at three ionic strengths but they differed slightly in plasma-thrombin tests. Depending upon the ionic strength, 17 mM Barbital Sodium. HCl buffer inhibited APTT tests but accelerated plasma-thrombin tests. All the buffers tested, therefore, have individual effects on the clotting tests.

The effects of biguanides on thrombokinase, thrombin and trypsin.

A purified preparation of bovine thrombokinase (activated Factor X) loses the ability to hydrolyze TAME (p-toluenesulfonyl-L-arginine methyl ester) when it is incubated at 37 degrees in 0.25 M Tris. HCl buffer, pH 7.4 with lauroxypropyl biguanide, N1, N5-dimethyl, N1-lauroxypropyl biguanide, N1-p-chlorophenethyl, N5-phenethyl biguanide, or N1-methyl, N1-p-chlorobenzyl, N5-o,p-dichlorobenzyl biguanide. Activity is lost much more slowly when 0.15 M NaCl is also present. Lauroxypropyl biguanide is the most potent of the compounds tested, 0.22 mM causing thrombokinase to lose almost all of its activity in about 30 minutes at 37 degrees in pH 7.4 buffered saline. Topical bovine thrombin also loses activity when incubated with either of the lauroxypropyl biguanides but not with the diphenethyl or the dibenzyl compound. Instead, the latter biguanides accelerate thrombin's hydrolysis of TAME. The percent acceleration is not affected or only slightly decreased by the presence of 0.15 M NaCl or KCl, and it is also unaffected by incubating the enzyme with the compounds in buffered saline for 4 to 120 minutes. Purified bovine trypsin is stabilized by both lauroxypropyl and the diphenethyl biguanide when incubated at 37 degrees in pH 7.4 buffered saline for the 60 minute test period but neither compounds has any effect on its rate of hydrolysis of TAME. It is postulated that the enzymes first react rapidly and reversibly with all of the test biguanides and, depending upon the enzyme and the substrate, the rate of hydrolysis of the substrate is unaffected, accelerated or inhibited. The lauroxypropyl biguanides also undergo a second, slower reaction with both thrombokinase and thrombin that produces loss of enzymatic activity. The dibenzyl and diphenethyl biguanides also undergo this second slow reaction with thrombokinase but not with thrombin, and none of the biguanides undergo this second reaction with trypsin.