Investigation of parameters that affect the success rate of microarray-based allele-specific hybridization assays

Background

The development of microarray-based genetic tests for diseases that are caused by known mutations is becoming increasingly important. The key obstacle to developing functional genotyping assays is that such mutations need to be genotyped regardless of their location in genomic regions. These regions include large variations in G+C content, and structural features like hairpins.

Methods/Findings

We describe a rational, stable method for screening and combining assay conditions for the genetic analysis of 42 Phenylketonuria-associated mutations in the phenylalanine hydroxylase gene. The mutations are located in regions with large variations in G+C content (20–75%). Custom-made microarrays with different lengths of complementary probe sequences and spacers were hybridized with pooled PCR products of 12 exons from each of 38 individual patient DNA samples. The arrays were washed with eight buffers with different stringencies in a custom-made microfluidic system. The data were used to assess which parameters play significant roles in assay development.

Conclusions

Several assay development methods found suitable probes and assay conditions for a functional test for all investigated mutation sites. Probe length, probe spacer length, and assay stringency sufficed as variable parameters in the search for a functional multiplex assay. We discuss the optimal assay development methods for several different scenarios.     

How the RNA isolation method can affect microRNA microarray results

The quality of RNA is crucial in gene expression experiments. RNA degradation interferes in the measurement of gene expression, and in this context, microRNA quantification can lead to an incorrect estimation. In the present study, two different RNA isolation methods were used to perform microRNA microarray analysis on porcine brain tissue. One method is a phenol-guanidine isothiocyanate-based procedure that permits isolation of total RNA. The second method, miRVana? microRNA isolation, is column based and recovers the small RNA fraction alone. We found that microarray analyses give different results that depend on the RNA fraction used, in particular because some microRNAs appear very sensitive to the RNA isolation method. We conclude that precautions need to be taken when comparing microarray studies based on RNA isolated with different methods.     

Life Expectancy and Death by Diseases of the Circulatory System in Patients with Bipolar Disorder or Schizophrenia in the Nordic Countries

Objective

Excess mortality from diseases and medical conditions (natural death) in persons with psychiatric disorders has been extensively reported. Even in the Nordic countries with well-developed welfare systems, register based studies find evidence of an excess mortality. In recent years, cardiac mortality and death by diseases of the circulatory system has seen a decline in all the Nordic countries, but a recent paper indicates that women and men in Denmark, Finland, and Sweden, who had been hospitalised for a psychotic disorder, had a two to three-fold increased risk of dying from a cardiovascular disease. The aim of this study was to compare the mortality by diseases of the circulatory system among patients with bipolar disorder or schizophrenia in the three Nordic countries Denmark, Sweden, and Finland. Furthermore, the aim was to examine and compare life expectancy among these patients. Cause specific Standardized Mortality Rates (SMRs) were calculated for each specific subgroup of mortality. Life expectancy was calculated using Wiesler’s method.

Results

The SMR for bipolar disorder for diseases of the circulatory system was approximately 2 in all countries and both sexes. SMR was slightly higher for people with schizophrenia for both genders and in all countries, except for men in Denmark. Overall life expectancy was much lower among persons with bipolar disorder or schizophrenia, with life expectancy being from 11 to 20 years shorter.

Conclusion

Our data show that persons in the Nordic countries with schizophrenia or bipolar disorder have a substantially reduced life expectancy. An evaluation of the reasons for these increased mortality rates should be prioritized when planning healthcare in the coming years.     

Whey Protein Reduces Early Life Weight Gain in Mice Fed a High-Fat Diet

An increasing number of studies indicate that dairy products, including whey protein, alleviate several disorders of the metabolic syndrome. Here, we investigated the effects of whey protein isolate (whey) in mice fed a high-fat diet hypothesising that the metabolic effects of whey would be associated with changes in the gut microbiota composition. Five-week-old male C57BL/6 mice were fed a high-fat diet ad libitum for 14 weeks with the protein source being either whey or casein. Faeces were collected at week 0, 7, and 13 and the fecal microbiota was analysed by denaturing gradient gel electrophoresis analyses of PCR-derived 16S rRNA gene (V3-region) amplicons. At the end of the study, plasma samples were collected and assayed for glucose, insulin and lipids. Whey significantly reduced body weight gain during the first four weeks of the study compared with casein (P<0.001–0.05). Hereafter weight gain was similar resulting in a 15% lower final body weight in the whey group relative to casein (34.0±1.0 g vs. 40.2±1.3 g, P<0.001). Food intake was unaffected by protein source throughout the study period. Fasting insulin was lower in the whey group (P<0.01) and glucose clearance was improved after an oral glucose challenge (P<0.05). Plasma cholesterol was lowered by whey compared to casein (P<0.001). The composition of the fecal microbiota differed between high- and low-fat groups at 13 weeks (P<0.05) whereas no difference was seen between whey and casein. In conclusion, whey initially reduced weight gain in young C57BL/6 mice fed a high-fat diet compared to casein. Although the effect on weight gain ceased, whey alleviated glucose intolerance, improved insulin sensitivity and reduced plasma cholesterol. These findings could not be explained by changes in food intake or gut microbiota composition. Further studies are needed to clarify the mechanisms behind the metabolic effects of whey.     

Genome-wide association study in Dachshund: identification of a major locus affecting intervertebral disc calcification

Intervertebral disc calcification and herniation commonly affects Dachshund where the predisposition is caused by an early onset degenerative process resulting in disc calcification. A continuous spectrum of disc degeneration is seen within and among dog breeds, suggesting a multifactorial etiology. The number of calcified discs at 2 years of age determined by a radiographic evaluation is a good indicator of the severity of disc degeneration and thus serves as a measure for the risk of developing intervertebral disc herniation. The aim of the study was to identify genetic variants associated with intervertebral disc calcification in Dachshund through a genome-wide association (GWA) study. Based on thorough radiographic examinations, 48 cases with ≥ 6 disc calcifications or surgically treated for disc herniation and 46 controls with 0-1 disc calcifications were identified. GWA using the Illumina CanineHD BeadChip identified a locus on chromosome 12 from 36.8 to 38.6 Mb with 36 markers reaching genome-wide significance (P(genome) = 0.00001-0.026). This study suggests that a major locus on chromosome 12 harbors genetic variations affecting the development of intervertebral disc calcification in Dachshund.     

Lethal contractural syndrome type 3 (LCCS3) is caused by a mutation in PIP5K1C, which encodes PIPKI gamma of the phophatidylinsitol pathway

Lethal congenital contractural syndrome (LCCS) is a severe form of arthrogryposis. To date, two autosomal recessive forms of the disease (LCCS and LCCS2) have been described and mapped to chromosomes 9q34 and 12q13, respectively. We now describe a third LCCS phenotype (LCCS3)--similar to LCCS2 yet without neurogenic bladder. Using 10K single-nucleotide-polymorphism arrays, we mapped the disease-associated gene to 8.8 Mb on chromosome 19p13. Further analysis using microsatallite markers narrowed the locus to a 3.4-Mb region harboring 120 genes. Of these genes, 30 candidates were sequenced, which identified a single homozygous mutation in PIP5K1C. PIP5K1C encodes phosphatidylinositol-4-phosphate 5-kinase, type I, gamma (PIPKI gamma ), an enzyme that phophorylates phosphatidylinositol 4-phosphate to generate phosphatidylinositol-4,5-bisphosphate (PIP(2)). We demonstrate that the mutation causes substitution of aspartic acid with asparagine at amino acid 253 (D253N), abrogating the kinase activity of PIPKI gamma . Thus, a defect in the phosphatidylinositol pathway leading to a decrease in synthesis of PIP(2), a molecule active in endocytosis of synaptic vesicle proteins, culminates in lethal congenital arthrogryposis.     

Lethal congenital contractural syndrome type 2 (LCCS2) is caused by a mutation in ERBB3 (Her3), a modulator of the phosphatidylinositol-3-kinase/Akt pathway

Lethal congenital contractural syndrome type 2 (LCCS2) is an autosomal recessive neurogenic form of arthrogryposis that is associated with atrophy of the anterior horn of the spinal cord. We previously mapped LCCS2 to 6.4 Mb on chromosome 12q13 and have now narrowed the locus to 4.6 Mb. We show that the disease is caused by aberrant splicing of ERBB3, which leads to a predicted truncated protein. ERBB3 (Her3), an activator of the phosphatidylinositol-3-kinase/Akt pathway--regulating cell survival and vesicle trafficking--is essential for the generation of precursors of Schwann cells that normally accompany peripheral axons of motor neurons. Gain-of-function mutations in members of the epidermal growth-factor tyrosine kinase-receptor family have been associated with predilection to cancer. This is the first report of a human phenotype resulting from loss of function of a member of this group.