Comprehensive analysis of NRG1 common and rare variants in Hirschsprung patients

Hirschsprung disease (HSCR, OMIM 142623) is a developmental disorder characterized by the absence of ganglion cells along variable lengths of the distal gastrointestinal tract, which results in tonic contraction of the aganglionic gut segment and functional intestinal obstruction. The RET proto-oncogene is the major gene for HSCR with differential contributions of its rare and common, coding and noncoding mutations to the multifactorial nature of this pathology. Many other genes have been described to be associated with the pathology, as NRG1 gene (8p12), encoding neuregulin 1, which is implicated in the development of the enteric nervous system (ENS), and seems to contribute by both common and rare variants. Here we present the results of a comprehensive analysis of the NRG1 gene in the context of the disease in a series of 207 Spanish HSCR patients, by both mutational screening of its coding sequence and evaluation of 3 common tag SNPs as low penetrance susceptibility factors, finding some potentially damaging variants which we have functionally characterized. All of them were found to be associated with a significant reduction of the normal NRG1 protein levels. The fact that those mutations analyzed alter NRG1 protein would suggest that they would be related with HSCR disease not only in Chinese but also in a Caucasian population, which reinforces the implication of NRG1 gene in this pathology.     

Localization of enkephalin- and neurotensin-like immunoreactivities in cat adrenal medulla

Enkephalin (ENK)- and neurotensin (NT)-immunoreactivities (IR) were localized in cat adrenal medulla using immunocytochemistry. ENK was localized mainly in adrenaline cells, 70%-80% of which were heavily labelled. About 5%-10% of the noradrenaline cells were ENK-immunoreactive (IR). A dense network of ENK-IR nerves was localized among adrenaline cells. NT was localized only in the noradrenaline cells, 60%-70% of which were immunoreactive. NT- and ENK-IR were localized in separate noradrenaline cells. A dense network of NT-IR nerves was restricted among noradrenaline cells. The present findings confirm that ENK- and NT-like peptides are localized in separate adrenal medullary cells and demonstrate that also the preganglionic nerves are divided in subpopulations according to their neuropeptide content. These nerves have a selective localization among adrenaline and noradrenaline cells and may have physiological significance in the control of secretion of catecholamines and neuropeptides from adrenal medulla.     

Down-regulation of serum/glucocorticoid regulated kinase 1 in colorectal tumours is largely independent of promoter hypermethylation

Background

We have previously shown that serum/glucocorticoid regulated kinase 1 (SGK1) is down-regulated in colorectal cancers (CRC) with respect to normal tissue. As hyper-methylation of promoter regions is a well-known mechanism of gene silencing in cancer, we tested whether the SGK1 promoter region was methylated in colonic tumour samples.

Methodology/Principal Findings

We investigated the methylation profile of the two CpG islands present in the promoter region of SGK1 in a panel of 5 colorectal cancer cell lines by sequencing clones of bisulphite-treated DNA samples. We further confirmed our findings in a panel of 10 normal and 10 tumour colonic tissue samples of human origin. We observed CpG methylation only in the smaller and more distal CpG island in the promoter region of SGK1 in both normal and tumour samples of colonic origin. We further identified a single nucleotide polymorphism (SNP, rs1743963) which affects methylation of the corresponding CpG.

Conclusions/Significance

Our results show that even though partial methylation of the promoter region of SGK1 is present, this does not account for the different expression levels seen between normal and tumour tissue.     

Four new loci associations discovered by pathway-based and network analyses of the genome-wide variability profile of Hirschsprung’s disease

Finding gene associations in rare diseases is frequently hampered by the reduced numbers of patients accessible. Conventional gene-based association tests rely on the availability of large cohorts, which constitutes a serious limitation for its application in this scenario. To overcome this problem we have used here a combined strategy in which a pathway-based analysis (PBA) has been initially conducted to prioritize candidate genes in a Spanish cohort of 53 trios of short-segment Hirschsprung’s disease. Candidate genes have been further validated in an independent population of 106 trios. The study revealed a strong association of 11 gene ontology (GO) modules related to signal transduction and its regulation, enteric nervous system (ENS) formation and other HSCR-related processes. Among the preselected candidates, a total of 4 loci, RASGEF1A, IQGAP2, DLC1 and CHRNA7, related to signal transduction and migration processes, were found to be significantly associated to HSCR. Network analysis also confirms their involvement in the network of already known disease genes. This approach, based on the study of functionally-related gene sets, requires of lower sample sizes and opens new opportunities for the study of rare diseases.     

Mutation update of spinal muscular atrophy in Spain: molecular characterization of 745 unrelated patients and identification of four novel mutations in the SMN1 gene

Spinal muscular atrophy (SMA) is caused by mutations in the SMN1 gene. We have studied the molecular pathology of SMA in 745 unrelated Spanish patients using PCR-RFLP, SMN gene dosage analysis, linkage studies, long-range PCR and direct sequencing. Our systematic approach allowed us to complete genetic testing and risk assessment in 736 SMA patients (98.8%). Females were more frequently affected by the acute form of the disease (type I), whereas chronic forms (type II–III) predominated in males (p < 0.008). Absence of the SMN1 gene was detected in 671 patients (90%), and hybrid SMN1–SMN2 genes were observed in 37 cases (5%). Furthermore, we detected 13 small mutations in 28 patients (3.8%), four of which were previously identified in other populations (c.91dupT; c.770_780dup11; p.Tyr272Cys and p.Thr274Ile), while five mutations were found to date only in Spanish patients (c.399_402delAGAG, p.Ile116Phe, p.Gln136Glu, c.740dupC and c.834+2T>G). The c.399_402delAGAG mutation accounted for 1.9% of all Spanish SMA patients. Finally, we discovered four novel mutations: c.312dupA, c.411delT, p.Trp190X and p.Met263Thr. Our results confirm that most SMA cases are due to large genetic rearrangements in the repetitive region of the SMA locus, resulting in absence-dysfunction of the SMN1 gene. By contrast, ancestrally inherited small mutations are responsible for only a small number of cases. Four prevalent changes in exons 3 and 6 (c.399_402delAGAG; c.770_780dup11; p.Tyr272Cys; p.Thr274Ile) accounted for almost 70% of our patients with these subtle mutations. An SMN–SMN dimer model featuring tight hydrophobic-aromatic interactions is proposed to explain the impact of mutations at the C-terminal end of the protein.     

Mitochondrial DNA polymorphisms associated with longevity in a Finnish population

Sequence variation in mitochondrial DNA (mtDNA) may cause slight differences both in the functioning of the respiratory chain and in free radical production, and an association between certain mtDNA haplogroups and longevity has been suggested. In order to determine further the role of mtDNA in longevity, we studied the frequencies of mtDNA haplogroups and haplogroup clusters among elderly subjects and controls in a Finnish population. Samples were obtained from 225 persons aged 90-91 years (Vitality 90+) and from 400 middle-aged controls and 257 infants. MtDNA haplogroups were determined by restriction fragment length polymorphism. The haplogroup frequencies of the Vitality 90+ group differed from both those of the middle-aged controls ( P=0.01) and the infants ( P=0.00005), haplogroup H being less frequent than among the middle-aged subjects ( P=0.001) and infants ( P=0.00001), whereas haplogroups U and J were more frequent. Haplogroup clusters also differed between Vitality 90+ and both the middle-aged subjects ( P=0.002) and infants ( P=0.00001), the frequency of haplogroup cluster HV being lower in the former and that of UK and WIX being higher. These data suggest an association between certain mtDNA haplogroups or haplogroup clusters and longevity. Furthermore, our data appear to favour the presence of advantageous polymorphisms and support a role for mitochondria and mtDNA in the degenerative processes involved in ageing.     

Localization of enkephalin- and neurotensin-like immunoreactivities in cat adrenal medulla

Summary Enkephalin (ENK)- and neurotensin (NT)-immunoreactivities (IR) were localized in cat adrenal medulla using immunocytochemistry. ENK was localized mainly in adrenaline cells, 70%–80% of which were heavily labelled. About 5%–10% of the noradrenaline cells were ENK-immunoreactive (IR). A dense network of ENK-IR nerves was localized among adrenaline cells. NT was localized only in the noradrenaline cells, 60%–70% of which were immunoreactive. NT- and ENK-IR were localized in separate noradrenaline cells. A dense network of NT-IR nerves was restricted among noradrenaline cells.The present findings confirm that ENK- and NT-like peptides are localized in separate adrenal medullary cells and demonstrate that also the preganglionic nerves are divided in subpopulations according to their neuropeptide content. These nerves have a selective localization among adrenaline and noradrenaline cells and may have physiological significance in the control of secretion of catecholamines and neuropeptides from adrenal medulla.     

Comparative study on cerebrovascular injuries by three chlorophenoxyacetic acids (2,4-D, 2,4,5-T and MCPA)

At toxic doses in rats, 2,4-dichlorophenoxyacetic acid (2,4-D) and 2-methyl-4-chlorophenoxyacetic acid (MCPA) caused injuries in blood vessels in the brain. 2. 2,4-D caused extravasations of the circulating Evans blue-albumin complex also in the spinal cord. 3. By contrast, potency in damaging cerebral vessels was almost non-existent with 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). 4. None of the three chlorophenoxyacetic acids caused such cerebrovascular injuries in mice, guinea pigs, Syrian hamsters, rabbits and chickens.     

Localization of enkephalins in adrenaline cells and the nerves innervating adrenaline cells in rat adrenal medulla

Summary The coexistence of met5- and leu5-enkephalinlike immunoreactivities with catecholamines in the rat adrenal medulla was studied with combined fluorescence microscopy and immunocytochemistry. Both met5- and leu5-enkephalin-like immunoreactivities were localized in few heavily stained adrenaline cells and in a population of nerves innervating adrenaline cells and as well as ganglion cells among the adrenaline cells. Only occasionally single noradrenaline cells exhibited light immunostaining for both enkephalins but no positive fibers could be found around the noradrenaline cells. In electron microscope the immunoreaction was seen in the granules of the adrenaline cells and in the large synaptic vesicles of the nerve terminals around the adrenaline cells. The present findings suggest that enkephalin-like immunoreactivity coexists mainly with adrenaline in rat adrenal medulla and that the enkephalin immunoreactive terminals regulate secretion of adrenaline from rat adrenal medulla.