Roux-en-Y gastric bypass normalizes the blunted postprandial bile acid excursion associated with obesity.

BACKGROUND: Bile acids (BAs) are nutrient-responsive hormones that modulate energy balance through cell surface and nuclear receptors. Postprandial plasma BAs have been found to be decreased in obesity. OBJECTIVE: We aimed to determine whether meal-stimulated circulating BA levels are altered by Roux-en-Y gastric bypass (RYGB), an operation that modifies the neurohumoral determinants of food intake and energy expenditure to cause significant and durable weight loss. DESIGN: Longitudinal study measuring fasting and postprandial plasma BAs before and after RYGB. SUBJECTS: Five obese surgical patients and eight lean controls underwent frequent blood sampling after a standard liquid meal. Obese subjects were also tested at 1, 4 and 40 weeks after RYGB. Primary and secondary circulating BAs, as well as their glycine and taurine conjugates, were measured via reverse-phase high-performance liquid chromatography/mass spectroscopy. RESULTS: We found that postprandial excursion of conjugated BAs was 52.4% lower in obese than in lean individuals by area-under-the-curve (AUC) analysis (378 vs 793 µmol min l(-1), respectively, P<0.05). By 40 weeks after RYGB, the meal-induced rise in conjugated BAs increased by 55.5% to the level of healthy lean controls (378 pre-op vs 850 µmol min l(-) post-op by AUC analyses, P<0.05). In contrast, postprandial concentrations of unconjugated BAs were similar in lean and obese individuals and were not affected by surgery. CONCLUSION: In light of the growing evidence that BAs have key roles in glucose, lipid and energy homeostasis, the observation that RYGB normalizes the blunted postprandial circulating BA response in obesity suggests that BAs may contribute to the improvement in meal-related physiology seen after RYGB. Further studies are warranted to examine this hypothesis and to determine the degree to which an augmented BA response to nutrient ingestion may mediate the increased incretin response, brown adipose tissue activation and thermic effect of feeding that has been observed after this operation.

Molecular analysis of the retinoblastoma (RB1) gene in acute myeloid leukemia patients.

The pathogenesis of acute leukemia is still poorly understood. In the past few years several groups have reported deletion of the RB1 gene or altered pRB expression in certain hematologic malignancies, suggesting a possible role of RB1 gene inactivation in the process of leukemogenesis. Most studies regarding structural abnormalities of the RB1 gene indicate that gross deletions or rearrangements are present in a small percentage of patients with acute myeloid leukemia (AML), as is the case with retinoblastoma, where the majority of RB1 gene abnormalities are attributed to point mutations. To investigate if such point mutations in the RB1 gene may have a role in leukemogenesis in AML, we screened the RB1 gene of 36 AML patients using conformation-sensitive gel electrophoresis (CSGE). No point mutations were found in the 27 exons, their flanking intron regions or in the promoter region in any of the 36 patients. Thus, according to our findings, the susceptibility in these patients for developing AML does not appear to be related to point mutations in the RB1 gene. While screening for point mutations, we identified a number of new and previously noted neutral sequence variations indicating the efficiency and sensitivity of CSGE in identifying small changes in the RB1 gene.

Stickler syndrome. A mutation in the nonhelical 3' end of type II procollagen gene.

BACKGROUND: All of the mutations in the type II procollagen (COL2A1) gene that have been identified in families affected with Stickler syndrome have been located primarily in the triple helical region of the gene. We report what we believe is the first premature stop codon in the globular C-propeptide region encoded by the COL2A1 gene, in a family affected with Stickler syndrome. DESIGN: Genomic DNA from affected and unaffected family members of this three-generation family was amplified using the polymerase chain reaction. The polymerase chain reaction products were directly sequenced for DNA analysis. RESULTS: Direct sequencing showed a single base deletion in exon 50, resulting in a premature stop codon in exon 51 in the globular C-propeptide of COL2A1 gene in all affected members. CONCLUSIONS: These results implicate premature stop codons as a common cause of Stickler syndrome. The location of this premature stop codon in the far end of the nonhelical 3' end of the gene indicates that a truncated C-propeptide of at least 84 amino acid residues is inadequate for the functional gene product.

A possible hot spot in exon 21 of the retinoblastoma gene predisposing to a low penetrant retinoblastoma phenotype?

PURPOSE: To identify the mutation in the RB1 gene in a Syrian family showing incomplete penetrance of retinoblastoma (RB). METHODS: Genomic DNA was used as a template for the PCR reaction to amplify all exons as well as the promoter region of RB1 gene. These PCR products were screened by conformational sensitive gel electrophoresis and the 331-bp product containing exon 21 showing anomalous migration was sequenced directly to identify the mutation. RESULTS: We identified the missense point mutation in exon 21 of the RB1 gene converting a Cys-->Arg (codon 712) in one family with a low penetrant phenotype. The proband was unilaterally affected, whereas the paternal uncle was bilaterally affected and the mutation carrier father was unaffected. The T-->C substitution abolished a cleavage site for the Nde I restriction enzyme, enabling rapid detection of the mutant allele. CONCLUSION: Phenotypically, this family is different from the previously described low penetrant phenotype pedigree with the same mutation whose affected members all had unilateral tumors. These results suggest that codon 712 may represent a mutational 'hot spot' for the low penetrant phenotypes and that the mutation codes for retinoblastoma protein with an apparently residual tumor-suppressive function give rise to low penetrance. These results also raise the interesting question: what other factors influence the phenotype of mutation carriers in addition to the predisposing missense mutation.

Modification of standard proteinase K/phenol method for DNA isolation to improve yield and purity from frozen blood.

Research in medical genetics may frequently involve freezing of large numbers of peripheral blood samples. This is a convenient method for storing blood for subsequent DNA isolation and analysis. An area of potential concern is the low yield of DNA from blood samples that have been frozen. Here we report a modification of the widely used standard proteinase K/phenol DNA isolation method for improving the yield and purity of DNA from frozen blood samples, by an initial trypsinisation of whole blood before cell lysis to obtain lymphocytic nuclei and subsequent DNA purification. We report an increased total yield of DNA with pretrypsinised blood as well as improved purity. These results indicate that trypsinisation of thawed whole blood helps the deproteinisation process, reducing the amount of protein associated with the nuclear pellet. This modification to improve yield and purity of DNA from frozen blood samples should be useful to laboratories performing DNA based diagnostic work or studying molecular genetic mechanisms of disease.

PCR assay confirms diagnosis in syndrome with variably expressed phenotype: mutation detection in Stickler syndrome.

Stickler syndrome is an autosomal dominant disease with ocular (severe myopia, vitreal degeneration, and retinal detachment) and other systemic manifestations (hearing loss, cleft palate, epiphyseal dysplasia, and premature osteoarthritis). As with other dominantly inherited conditions, the clinical phenotype of Stickler syndrome varies considerably. To date, all mutations have been located in the type II procollagen (COL2A1) gene. Analysis of a C-->T mutation we had identified previously, in COL2A1 gene in exon 40, in a three generation pedigree showed the loss of a cleavage site for the TaqI restriction enzyme. We designed a rapid PCR based restriction enzyme assay to detect this mutation and used it to establish the diagnosis in a neonate from the same pedigree, presenting with the first occurrence of the Pierre-Robin sequence in the family and minimal ocular findings. These results underline the potential diagnostic value of many as yet undetected DNA mutations in families affected with Stickler syndrome, since the variability of the phenotype can impede accurate diagnosis, appropriate genetic counselling, and effective intervention and prophylactic treatment for affected people.

Stop codon in the procollagen II gene (COL2A1) in a family with the Stickler syndrome (arthro-ophthalmopathy).

Linkage analysis with restriction fragment length polymorphisms for the gene for type II procollagen (COL2A1) was carried out in a family with the Stickler syndrome, or arthro-ophthalmopathy, an autosomal dominant disorder that affects the eyes, ears, joints, and skeleton. The analysis demonstrated linkage of the disease and COL2A1 with a logarithm-of-odds score of 1.51 at zero recombination. A newly developed procedure for preparing cosmid clones was employed to isolate the allele for type II procollagen that was linked to the disease. Analysis of over 7000 nucleotides of the gene revealed a single base mutation that altered a CG dinucleotide and converted the codon CGA for arginine at amino acid position alpha 1-732 to TGA, a stop codon. From previous work on procollagen biosynthesis, it is apparent that the truncated polypeptide synthesized from an allele with a stop codon at alpha 1-732 cannot participate in the assembly of type II procollagen, and therefore that the mutation would decrease synthesis of type II procollagen. It was not apparent, however, why the mutation produced marked changes in the eye, which contains only small amounts of type II collagen, but relatively mild effects on the many cartilaginous structures of the body that are rich in the same protein.

A second mutation in the type II procollagen gene (COL2AI) causing stickler syndrome (arthro-ophthalmopathy) is also a premature termination codon.

Genetic linkage analyses suggest that mutations in type II collagen may be responsible for Stickler syndrome, or arthro-ophthalmopathy (AO), in many families. In the present study oligonucleotide primers were developed to amplify and directly sequence eight of the first nine exons of the gene for type II procollagen (COL2A1). Analysis of the eight exons in 10 unrelated probands with AO revealed that one had a single-base mutation in one allele that changed the codon of -CGA- for arginine at amino acid position alpha 1-9 in exon 7 to a premature termination signal for translation. The second mutation found to cause AO was, therefore, similar to the first in that both created premature termination signals in the COL2A1 gene. Since mutations producing premature termination signals have not previously been detected in genes for fibrillar collagens, the results raise the possibility that such mutations in the COL2A1 gene are a common cause of AO.

Detection of sequence variants in the gene for human type II procollagen (COL2A1) by direct sequencing of polymerase chain reaction-amplified genomic DNA.

The direct sequencing of the human type II procollagen (COL2A1) gene from polymerase chain reaction (PCR)-amplified genomic DNA is described. Thirty-two regions of the COL2A1 gene were asymmetrically amplified with intron primers which were specifically chosen to amplify a region spanning 500 to 800 bp of sequence encoding one or more exons and their accompanying intervening sequences. Primers for dideoxynucleotide sequencing of the PCR products were then designed to provide complete exon sequence information and to insure that intron:exon splice junction sequence data would be obtained. Amplification and sequencing reactions were performed on an automated workstation to facilitate the handling of multiple DNA templates. The procedure allowed efficient sequencing of over 25,000 bp of each allele of the COL2A1 gene per diploid genome. We used this method for the comparative analyses of COL2A1 sequences in DNA isolated from the blood of 42 unrelated individuals and we identified 21 neutral sequence variants in the gene. The sequence variations were confirmed by independent assays, including restriction enzyme digestion. The sequence variants described here will be important for identifying haplotypes of the type II procollagen gene that will be useful in defining a genetic etiology for diseases of cartilaginous tissues.