Migraine Comorbidities : A Clinical and Molecular Genetic Study

Migraine is a highly prevalent disease, and despite several important breakthroughs there are still a many questions unanswered in the clinical, genetic and pathophysiological aspects of migraine research. Migraine has been linked to several other diseases such as epilepsy and stroke, but there are still unsolved issues concerning the true nature of these associations. Three genes predisposing to hemiplegic migraine and several loci associated to migraine have been identified, but so far no genes responsible for common forms of migraine have been recognized. Triptans have provided an important step in migraine treatment, but their usefulness in rare forms of migraine have been controversial. The Finnish Migraine Gene Project (FMGP) includes more than 1600 families and 7500 individuals. We evaluated comorbidity from 1000 consecutive subjects in the FMGP. To search for novel loci, we performed a genome-wide linkage scan in 36 families with high prevalences of migraine with visual aura. We collected 76 subjects from the FMGP who suffer from hemiplegic migraine and have used triptans. Finally, to study possible links between stroke and migraine we evaluated the prevalence of migraine in subjects with cervical artery dissection (CAD) and healthy controls. Migraine was associated with increased prevalence of allergy, hypotension and psychiatric diseases. Additionally, men suffering from migraine with aura had increased prevalence of epilepsy and stroke. Further evidence of association between migraine and epilepsy was found in our linkage study. The parametric two-point linkage analysis showed significant evidence of linkage between migraine aura and a locus on 9q21-q22. Interestingly, the same locus has been associated with occipitotemporal epilepsy. CAD seems to be a migraine risk factor, and therefore a link between stroke and migraine. Notably, CAD seems to alleviate migraine activity further indicating the association between these two conditions. Despite the contraindications of triptans, it seems that they are safe and effective in the abortive treatment of hemiplegic migraine.

Molecular Genetic Studies of Melanocortin Receptors in Morbid Obesity

The prevalence of obesity is increasing at an alarming rate in all age groups worldwide. Obesity is a serious health problem due to increased risk of morbidity and mortality. Although environmental factors play a major role in the development of obesity, the identification of rare monogenic defects in human genes have confirmed that obesity has a strong genetic component. Mutations have been identified in genes encoding proteins of the leptin-melanocortin signaling system, which has an important role in the regulation of appetite and energy balance. The present study aimed at identifying mutations and genetic variations in the melanocortin receptors 2-5 and other genes active on the same signaling pathway accounting for severe early-onset obesity in children and morbid obesity in adults. The main achievement of this thesis was the identification of melanocortin-4 receptor (MC4R) mutations in Finnish patients. Six pathogenic MC4R mutations (308delT, P299H, two S127L and two -439delGC mutations) were identified, corresponding to a prevalence of 3% in severe early-onset obesity. No obesity causing MC4R mutations were found among patients with adult-onset morbid obesity. The MC4R 308delT deletion is predicted to result in a grossly truncated nonfunctional receptor of only 107 amino acids. The C-terminal residues, which are important in MC4R cell surface targeting, are totally absent from the mutant 308delT receptor. In vitro functional studies supported a pathogenic role for the S127L mutation since agonist induced signaling of the receptor was impaired. Cell membrane localization of the S127L receptor did not differ from that of the wild-type receptor, confirming that impaired function of the S127L receptor was due to reduced signaling properties. The P299H mutation leads to intracellular retention of the receptor. The -439delGC deletion is situated at a potential nescient helix-loop-helix 2 (NHLH2) -binding site in the MC4R promoter. It was demonstrated that the transcription factor NHLH2 binds to the consensus sequence at the -439delGC site in vitro, possibly resulting in altered promoter activity. Several genetic variants were identified in the melanocortin-3 receptor (MC3R) and pro-opiomelanocortin (POMC) genes. These polymorphisms do not explain morbid obesity, but the results indicate that some of these genetic variations may be modifying factors in obesity, resulting in subtle changes in obesity-related traits. A risk haplotype for obesity was identified in the ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) gene through a candidate gene single nucleotide polymorphism (SNP) genotyping approach. An ENPP1 haplotype, composed of SNPs rs1800949 and rs943003, was shown to be significantly associated with morbid obesity in adults. Accordingly, the MC3R, POMC and ENPP1 genes represent examples of susceptibility genes in which genetic variants predispose to obesity. In conclusion, pathogenic mutations in the MC4R gene were shown to account for 3% of cases with severe early-onset obesity in Finland. This is in line with results from other populations demonstrating that mutations in the MC4R gene underlie 1-6% of morbid obesity worldwide. MC4R deficiency thus represents the most common monogenic defect causing human obesity reported so far. The severity of the MC4-receptor defect appears to be associated with time of onset and the degree of obesity. Classification of MC4R mutations may provide a useful tool when predicting the outcome of the disease. In addition, several other genetic variants conferring susceptibility to obesity were detected in the MC3R, MC4R, POMC and ENPP1 genes.

Potential applications of reproductive and molecular genetic technologies in the selective breeding of aquaculture species

The use of reproductive and genetic technologies can increase the efficiency of selective breeding programs for aquaculture species. Four technologies are considered, namely: marker-assisted selection, DNA fingerprinting, in-vitro fertilization, and cryopreservation. Marker-assisted selection can result in greater genetic gain, particularly for traits difficult or expensive to measure, than conventional selection methods, but its application is currently limited by lack of high density linkage maps and by the high cost of genotyping. DNA fingerprinting is most useful for genetic tagging and parentage verification. Both in-vitro fertilization and cryopreservation techniques can increase the accuracy of selection while controlling accumulation of inbreeding in long-term selection programs. Currently, the cost associated with the utilization of reproductive and genetic techniques is possibly the most important factor limiting their use in genetic improvement programs for aquatic species.

Molecular genetic studies on voltage-gated ion channels

Several different methods have been employed in the study of voltage-gated ion channels. Electrophysiological studies on excitable cells in vertebrates and molluscs have shown that many different voltage-gated potassium (K⁺) channels and sodium channels may coexist in the same organism. Parallel genetic studies in Drosophila have identified mutations in several genes that alter the properties of specific subsets of physiologically identified ion channels. Chapter 2 describes molecular studies that identify two Drosophila homologs of vertebrate sodium-channel genes. Mutations in one of these Drosophila sodium-channel genes are shown to be responsible for the temperature-dependent paralysis of a behavioural mutant para^ts. Evolutionary arguments, based on the partial sequences of the two Drosophila genes, suggest that subfamilies of voltage-gated sodium channels in vertebrates remain to be identified.

In Drosophila, diverse voltage-gated K⁺ channels arise from alternatively spliced mRNAs generated at the Shaker locus. Chapter 3 and the Appendices describe the isolation and characterization of several human K⁺-channel genes, similar in sequence to Shaker. Each of these human genes has a highly conserved homolog in rodents; thus, this K⁺-channel gene family probably diversified prior to the mammalian radiation. Functional K⁺ channels encoded by these genes have been expressed in Xenopus oocytes and their properties have been analyzed by electrophysiological methods. These studies demonstrate that both transient and noninactivating voltage-gated K⁺ channels may be encoded by mammalian genes closely related to Shaker. In addition, results presented in Appendix 3 clearly demonstrate that independent gene products from two K⁺-channel genes may efficiently co-assemble into heterooligomeric K⁺ channels with properties distinct from either homomultimeric channel. This finding suggests yet another molecular mechanism for the generation of K⁺-channel diversity.

A molecular genetic investigation of the population structure of Atlantic menhaden (Brevoortia tyrannus)

Atlantic menhaden (Brevoortia tyrannus), through landings, support one of the largest commercial fisheries in the United States. Recent consolidation of the once coast-wide reduction fishery to waters within and around Chesapeake Bay has raised concerns over the possibility of the loss of unique genetic variation resulting from concentrated fishing pressure. To address this question, we surveyed variation at the mitochondrial cytochrome c oxidase subunit I (COI) gene region and seven nuclear microsatellite loci to evaluate stock structure of Atlantic menhaden. Samples were collected from up to three cohorts of Atlantic menhaden at four geographic locations along the U.S. Atlantic coast in 2006 and 2007, and from the closely related Gulf menhaden (B. patronus) in the Gulf of Mexico. Genetic divergence between Atlantic menhaden and Gulf menhaden, based on the COI gene region sequences and microsatellite loci, was more characteristic of conspecific populations than separate species. Hierarchical analyses of molecular variance indicated a homogeneous distribution of genetic variation within Atlantic menhaden. No significant variation was found between young-of-the-year menhaden (YOY) collected early and late in the season within Chesapeake Bay, between young-of-the-year and yearling menhaden collected in the Chesapeake Bay during the same year, between YOY and yearling menhaden taken in Chesapeake Bay in successive years, or among combined YOY and yearling Atlantic menhaden collected in both years from the four geographic locations. The genetic connectivity between the regional collections indicates that the concentration of fishing pressure in and around Chesapeake Bay will not result in a significant loss of unique genetic variation.

Molecular genetic basis for the rhino mouse from Chinese KM subcolony

Both the rhino mouse and hairless mouse resulted from hairless gene mutation, but they show different phenotypes of skin physiology. The rhino mouse has more similar histological characters to human papular alopecia. Therefore rhino mouse is a good experimental animal model for human papular alopecia. This study reports a hairless mouse named rhino KIZ, arose from KM colony in Kunming Institue of Zoology, by systematic studies on morphology, skin histopathology, gene sequence, pedigree and protein domain analysis. The results demonstrate that a C-to-T transition in exon 11 of hr gene (The mutant gene has been applied for a Chinese patent (patent No. 03135280)) results in the rhino KIZ. The rhino KIZ with clear genetic mechanism will be a useful animal model.

Study of inter species diversity and population structure by molecular genetic method in Iranian Artemia

Artemia is a small crustacean that adapted to live in brine water and has been seen in different brine water sources in Iran. Considering the importance of genetic studies manifest inter population differences in species, to estimate genetic structure, detect difference at molecular level and separate different Artemia populations of Iran, also study of phylogenic relationships among them, samples of Artemia were collected from nine region: Urmia lake in West Azerbaijan, Shoor and Inche-Borun lakes in Golestan, Hoze-Soltan and Namak lakes in Qom, Maharloo and Bakhteghan lakes in Fars, Nough pool in Kerman and Mighan pool in Markazi and DNA extracted by phenol-chloroform method. Primers designed on a ribosomal fragment (16s rRNA) of mt DNA sequence and PCR was done. Digestion of the 1566 bp segment PCR product by 10 restriction endonuclease (Alu I, EcoR I, Eco47 I, Hae III, Hind III, Hinf I, Mbo I, Msp I, Rsa I, TaqI) showed 25 different haplotypes: 9 in Urmia, 4 in Shoor and Inche- Borun, 1 in Namak and Hoze-Soltan, 3 in Mighan, 1 in Bakhtegan Maharlo, 3 in Maharloo and 4 in Nough. Measurement of haplotype and nucleotide diversity intra population and nucleotide diversity and divergence inter populations and evolutionary distance between haplotypes showed a high diversity in mitochondrial genome of Artemia in studied regions whose results are similar to those explained for highly geographic expansion organism. In addition, results showed considerable heterogeneity between different populations and there are enough evidences in haplotypic level for separation of studied samples and division of Iranian Artemia to seven populations including Urmia, Shoor and Inche-Borun, Hoze-Soltan and Namak, Maharloo, Bakhteghan, Nough and Mighan. Phylogenetic analysis of the 16S rRNA data set resulted strict consensus and neighbor joining distance trees, demonstrated that all samples were monophyletic and parthenogenetic form derivation from bisexual populations and genetically high resemblance to those of A. urmiana. Study of 270 specimens from different region showed the genus Artemia in Iran clustered into three clades including: 1- Shoor, Inche-Burun, Hoze-Soltan, Namak, Bakhtegan and Maharloo 2- Nough and Mighan 3- Urmia. Totally, obtained results indicated to ability of used techniques for study of inter species diversity, population structure, reveal of phylogenic relationship and dividing of different populations of Artemia in Iran.

Survey of exist specifications morphometric, meristic and inter species molecular genetic on Bream (Abramis brava orientalis) in Anzali wetland, southern coast of Caspian Sea, Aras Dam Lake and Azerbijan Republic

Bream (Abramis brava orientalis) is one of Cyprindae the Caspian Sea and its basin which has a special ecological, biological and economical role. Stock of this fish in the Caspian Sea has reduced during several years for different reason the over fishing, different industrial, agriculture, urban pollution and destroy of the spawning habitat. So that fishery company decided to recover the stock of this fish by the way of artificial reproduction of a Bream couple hunted from south coast of the Caspian Sea (Iran) and setting the fingerling to the rivers and inflow wetlands of the Caspian Sea.This activity has due to 20 tons Bream annual fishing in the Iranian South coast of the Caspian Sea (Gilan province coast and Anzali wetland), The artificial reproduction has decreased Bream population diversity of Caspian sea and Anzali wetland.So it has been declined to improve Braem population diversity by the entrance of Azerbijan republic Bream and encounter to the Caspian sea Bream. Meanwhile there is Bream in the Aras Dam Lake which had been forgotten by the Fishery Company of Iran .For this reason specifications morphometric, meristic and inter species Molecular Genetic have been surveyed in Anzali wetland,Southern coast of Caspian Sea ,Aras Darn Lake and Azerbijan republic during 2003-2005. According to the research on specifications of Morphometric and Meristic of Anzali wetland(120 species),Southern coast of Caspian Sea(90 species), Aras Dam Lake(110 species) and Azerbijan Republic(125 species)has Morphometric and Meristic differences. So that average weight and total length of Anzali wetland Bream respectively was 167 g and 23/76 cm, 102 g and 27/62 cm in Caspian Sea , 461 g and 3 5/38 cm in Aras Darn Lake and 3 4189 g and 15/21 cm in Azerbijan republic (We forced to use 1 year Bream of artificial reproduction in Iran). Also variation coefficient average Morphometric, Morphometric specification Ration and meristic in Anzali wetland Bream was 17/45, 21/56 and 4/63, in Caspian Sea bream 22/58, 15/27 and 3124, in Aras Dam lake Lake 17145. 1.5/27 and 3/57 and Azerbaijan republic Bream 22/29, 19/66 and 4/22. Also Bream of these four regions in general status had Morphometric significant differences based on One Way ANOVA Analysis. Meanwhile Anzali wetland Bream with Caspian Sea Bream from 41 Morphometric surveyed factors in 33 factors, with Aras Darn Lake Bream in 41 factors, with Azerbkjan republic Bream in 41 factors,Caspian Sea Bream with Aras Darn Lake Bream in 36 factors,with Azerbijan republic B ream in 40 factors and A ras Dam L ake Bream with Azerbijan republic Bream in 38 factors had significant statistical differences. These four regions Bream had differences according to the Morphomertric specification ration based on One Way ANOVA Analysis. Also Anzali wetland Bream was surveyed with Caspian Sea Bream from 37 factors i n 27 factors, Anzali wetland Bream with Aras Dam 1ake in 37 factors Anzali wetland Bream with Azerbijan republic Bream in 32 factors,Caspian sea bream with Arsa Dam Lake Bream in 26 factors, Caspian Sea Bream with Azerbijan republic Bream in 29 factors and Aras Dam Lake Bream with Azerbijan republic Bream in 34 factor had significant statistical differences. Based on Meristic factor of four regions bream in 16 surveyed factors in 10 factors had meaningful differences according to the One Way ANOVA Analysis. While Anzali wetland Bream was surveyed with Caspian Sea Bream from in 3 factors,Anzali wetland Bream with Aras Dam lake in 8 factors,Anzali wetland Bream with Azerbijan republic B ream in 6 factors,Caspian Sea bream with Arsa Dam Lake Bream in 6 factors,Caspian sea Bream with Azerbijan republic Bream in 3 factors and Aras Dam Lake Bream with Azerijan republic Bream in 8 factor had significant statistical differences.Meanwihle based on Factor Analysis and Discriminant Breams had differences. Also according to the resrarchs Anzali wetland Bream in 0+ age group till 5+ (6 age groups),Caspian Sea bream in 1+ - 5+(5 age groups),Aras Darn Lake Bream in 1+ - 7+ (7 age groups) and Azerbijan republic Bream for Morphometric and Meristic studies in 1+age group and for molecular Genetic reaserch were in 8+and 9+ age groups. According to the research 4 ecosystems Bream in status of same age, Aras lake Bream were bigger according to weight and length.Also in this research genetic diversity between four population was researched by PCR-RFLP technic on a piece of mitochondrion genome with the length of 3500bp contain of tRNA-leu,tRNA-glu,ND5/6,Cytb. Between 17 used enzyme. 4 enzyme, Dral, Bc11, Haefll and Banff showed diversity in totally 6 composite haplotype was detected. Maximum nucleotide diversity by the value% 0/58 in Azerbijan republic Bream by all haplotype. Aras darn Lake Bream had 2 haplotype and nucleotide diversity of %0/35.Anzali wetland and Caspian Sea Bream had no diversity. Statistical analysis by the usage of Monte Carlo with 1000 repeat showed significant differences between Azerbaijan Bream and other Bream(P<0/0001) but there was no significant difference between 3 regions Bream(P>0/5).

Switching Between Discrete and Continuous Process Models to Predict Molecular Genetic Activity

Two kinds of process models have been used in programs that reason about change: Discrete and continuous models. We describe the design and implementation of a qualitative simulator, PEPTIDE, which uses both kinds of process models to predict the behavior of molecular energetic systems. The program uses a discrete process model to simulate both situations involving abrupt changes in quantities and the actions of small numbers of molecules. It uses a continuous process model to predict gradual changes in quantities. A novel technique, called aggregation, allows the simulator to switch between theses models through the recognition and summary of cycles. The flexibility of PEPTIDE's aggregator allows the program to detect cycles within cycles and predict the behavior of complex situations.

Molecular genetic analysis of DNA alterations in Irish colorectal cancer

Colorectal cancer is the most common cause of death due to malignancy in nonsmokers in the western world. In 1995 there were 1,757 cases of colon cancer in Ireland. Most colon cancer is sporadic, however ten percent of cases occur where there is a previous family history of the disease. In an attempt to understand the tumorigenic pathway in Irish colon cancer patients, a number of genes associated with colorectal cancer development were analysed in Irish sporadic and HNPCC colon cancer patients. The hereditary forms of colon cancer include Familial adenomatous polyposis coli (FAP) and Hereditary Non-Polyposis Colon Cancer (HNPCC). Genetic analysis of the gene responsible for FAP, (the APC gene) has been previously performed on Irish families, however the genetic analysis of HNPCC families is limited. In an attempt to determine the mutation spectrum in Irish HNPCC pedigrees, the hMSH2 and hMLHl mismatch repair genes were screened in 18 Irish HNPCC families. Using SSCP analysis followed by DNA sequencing, five mutations were identified, four novel and a previously reported mutation. In families where a mutation was detected, younger asyptomatic members were screened for the presence of the predisposing mutation (where possible). Detection of mutations is particularly important for the identification of at risk individuals as the early diagnosis of cancer can vastly improve the prognosis. The sensitive and efficient detection of multiple different mutations and polymorphisms in DNA is of prime importance for genetic diagnosis and the identification of disease genes. A novel mutation detection technique has recently been developed in our laboratory. In order to assess the efficacy and application of the methodology in the analysis of cancer associated genes, a protocol for the analysis of the K-ras gene was developed and optimised. Matched normal and tumour DNA from twenty sporadic colon cancer patients was analysed for K-ras mutations using the Glycosylase Mediated Polymorphism Detection technique. Five mutations of the K-ras gene were detected using this technology. Sequencing analysis verified the presence of the mutations and SSCP analysis of the same samples did not identify any additional mutations. The GMPD technology proved to be highly sensitive, accurate and efficient in the identification of K-ras gene mutations. In order to investigate the role of the replication error phenomenon in Irish colon cancer, 3 polyA tract repeat loci were analysed. The repeat loci included a 10 bp intragenic repeat of the TGF-β-RII gene. TGF-β-RII is involved in the TGF-β epithelial cell growth pathway and mutation of the gene is thought to play a role in cell proliferation and tumorigenesis. Due to the presence of a repeat sequence within the gene, TGFB-RII defects are associated with tumours that display the replication error phenomenon. Analysis of the TGF-β-RII 10 bp repeat failed to identify mutations in any colon cancer patients. Analysis of the Bat26 and Bat 40 polyA repeat sequences in the sporadic and HNPCC families revealed that instability is associated with HNPCC tumours harbouring mismatch repair defects and with 20 % of sporadic colon cancer tumours. No correlation between K-ras gene mutations and the RER+ phenotype was detected in sporadic colon cancer tumours.