Cortical processing of speech and non-speech sounds in autism and Asperger syndrome

Autism and Asperger syndrome (AS) are neurodevelopmental disorders characterised by deficient social and communication skills, as well as restricted, repetitive patterns of behaviour. The language development in individuals with autism is significantly delayed and deficient, whereas in individuals with AS, the structural aspects of language develop quite normally. Both groups, however, have semantic-pragmatic language deficits. The present thesis investigated auditory processing in individuals with autism and AS. In particular, the discrimination of and orienting to speech and non-speech sounds was studied, as well as the abstraction of invariant sound features from speech-sound input. Altogether five studies were conducted with auditory event-related brain potentials (ERP); two studies also included a behavioural sound-identification task. In three studies, the subjects were children with autism, in one study children with AS, and in one study adults with AS. In children with autism, even the early stages of sound encoding were deficient. In addition, these children had altered sound-discrimination processes characterised by enhanced spectral but deficient temporal discrimination. The enhanced pitch discrimination may partly explain the auditory hypersensitivity common in autism, and it may compromise the filtering of relevant auditory information from irrelevant information. Indeed, it was found that when sound discrimination required abstracting invariant features from varying input, children with autism maintained their superiority in pitch processing, but lost it in vowel processing. Finally, involuntary orienting to sound changes was deficient in children with autism in particular with respect to speech sounds. This finding is in agreement with previous studies on autism suggesting deficits in orienting to socially relevant stimuli. In contrast to children with autism, the early stages of sound encoding were fairly unimpaired in children with AS. However, sound discrimination and orienting were rather similarly altered in these children as in those with autism, suggesting correspondences in the auditory phenotype in these two disorders which belong to the same continuum. Unlike children with AS, adults with AS showed enhanced processing of duration changes, suggesting developmental changes in auditory processing in this disorder.

Xanthine oxidoreductase in essential hypertension and metabolic syndrome : Experimental studies on rodent models

Hypertension, obesity, dyslipidemia and dysglycemia constitute metabolic syndrome, a major public health concern, which is associated with cardiovascular mortality. High dietary salt (NaCl) is the most important dietary risk factor for elevated blood pressure. The kidney has a major role in salt-sensitive hypertension and is vulnerable to harmful effects of increased blood pressure. Elevated serum urate is a common finding in these disorders. While dysregulation of urate excretion is associated with cardiovascular diseases, present studies aimed to clarify the role of xanthine oxidoreductase (XOR), i.e. xanthine dehydrogenase (XDH) and its post-translational isoform xanthine oxidase (XO), in cardiovascular diseases. XOR yields urate from hypoxanthine and xanthine. Low oxygen levels upregulate XOR in addition to other factors. In present studies higher renal XOR activity was found in hypertension-prone rats than in the controls. Furthermore, NaCl intake increased renal XOR dose-dependently. To clarify whether XOR has any causal role in hypertension, rats were kept on NaCl diets for different periods of time, with or without a XOR inhibitor, allopurinol. While allopurinol did not alleviate hypertension, it prevented left ventricular and renal hypertrophy. Nitric oxide synthases (NOS) produce nitric oxide (NO), which mediates vasodilatation. A paucity of NO, produced by NOS inhibition, aggravated hypertension and induced renal XOR, whereas NO generating drug, alleviated salt-induced hypertension without changes in renal XOR. Zucker fa/fa rat is an animal model of metabolic syndrome. These rats developed substantial obesity and modest hypertension and showed increased hepatic and renal XOR activities. XOR was modified by diet and antihypertensive treatment. Cyclosporine (CsA) is a fungal peptide and one of the first-line immunosuppressive drugs used in the management of organ transplantation. Nephrotoxicity ensue high doses resulting in hypertension and limit CsA use. CsA increased renal XO substantially in salt-sensitive rats on a high NaCl diet, indicating a possible role for this reactive oxygen species generating isoform in CsA nephrotoxicity. Renal hypoxia, common to these rodent models of hypertension and obesity, is one of the plausible XOR inducing factors. Although XOR inhibition did not prevent hypertension, present experimental data indicate that XOR plays a role in the pathology of salt-induced cardiac and renal hypertrophy.

Mitochondrial Malfunction in Tumor Formation and Neuromuscular Diseases : Consequences of defects in fumarate hydratase and in the respiratory chain

The mitochondrion is an organelle of outmost importance, and the mitochondrial network performs an array of functions that go well beyond ATP synthesis. Defects in mitochondrial performance lead to diseases, often affecting nervous system and muscle. Although many of these mitochondrial diseases have been linked to defects in specific genes, the molecular mechanisms underlying the pathologies remain unclear. The work in this thesis aims to determine how defects in mitochondria are communicated within - and interpreted by - the cells, and how this contributes to disease phenotypes. Fumarate hydratase (FH) is an enzyme of the citrate cycle. Recessive defects in FH lead to infantile mitochondrial encephalopathies, while dominant mutations predispose to tumor formation. Defects in succinate dehydrogenase (SDH), the enzyme that precedes FH in the citrate cycle, have also been described. Mutations in SDH subunits SDHB, SDHC and SDHD are associated with tumor predisposition, while mutations in SDHA lead to a characteristic mitochondrial encephalopathy of childhood. Thus, the citrate cycle, via FH and SDH, seems to have essential roles in mitochondrial function, as well as in the regulation of processes such as cell proliferation, differentiation or death. Tumor predisposition is not a typical feature of mitochondrial energy deficiency diseases. However, defects in citrate cycle enzymes also affect mitochondrial energy metabolism. It is therefore necessary to distinguish what is specific for defects in citrate cycle, and thus possibly associated with the tumor phenotype, from the generic consequences of defects in mitochondrial aerobic metabolism. We used primary fibroblasts from patients with recessive FH defects to study the cellular consequences of FH-deficiency (FH-). Similarly to the tumors observed in FH- patients, these fibroblasts have very low FH activity. The use of primary cells has the advantage that they are diploid, in contrast with the aneuploid tumor cells, thereby enabling the study of the early consequences of FH- in diploid background, before tumorigenesis and aneuploidy. To distinguish the specific consequences of FH- from typical consequences of defects in mitochondrial aerobic metabolism, we used primary fibroblasts from patients with MELAS (mitochondrial encephalopathy with lactic acidosis and stroke-like episodes) and from patients with NARP (neuropathy, ataxia and retinitis pigmentosa). These diseases also affect mitochondrial aerobic metabolism but are not known to predispose to tumor formation. To study in vivo the systemic consequences of defects in mitochondrial aerobic metabolism, we used a transgenic mouse model of late-onset mitochondrial myopathy. The mouse contains a transgene with an in-frame duplication of a segment of Twinkle, the mitochondrial replicative helicase, whose defects underlie the human disease progressive external ophthalmoplegia. This mouse model replicates the phenotype in the patients, particularly neuronal degeneration, mitochondrial myopathy, and subtle decrease of respiratory chain activity associated with mtDNA deletions. Due to the accumulation of mtDNA deletions, the mouse was named deletor. We first studied the consequences of FH- and of respiratory chain defects for energy metabolism in primary fibroblasts. To further characterize the effects of FH- and respiratory chain malfunction in primary fibroblasts at transcriptional level, we used expression microarrays. In order to understand the in vivo consequences of respiratory chain defects in vivo, we also studied the transcriptional consequences of Twinkle defects in deletor mice skeletal muscle, cerebellum and hippocampus. Fumarate accumulated in the FH- homozygous cells, but not in the compound heterozygous lines. However, virtually all FH- lines lacked cytoplasmic FH. Induction of glycolysis was common to FH-, MELAS and NARP fibroblasts. In deletor muscle glycolysis seemed to be upregulated. This was in contrast with deletor cerebellum and hippocampus, where mitochondrial biogenesis was in progress. Despite sharing a glycolytic pattern in energy metabolism, FH- and respiratory chain defects led to opposite consequences in redox environment. FH- was associated with reduced redox environment, while MELAS and NARP displayed evidences of oxidative stress. The deletor cerebellum had transcriptional induction of antioxidant defenses, suggesting increased production of reactive oxygen species. Since the fibroblasts do not represent the tissues where the tumors appear in FH- patients, we compared the fibroblast array data with the data from FH- leiomyomas and normal myometrium. This allowed the determination of the pathways and networks affected by FH-deficiency in primary cells that are also relevant for myoma formation. A key pathway regulating smooth muscle differentiation, SRF (serum response factor)-FOS-JUNB, was found to be downregulated in FH- cells and in myomas. While in the deletor mouse many pathways were affected in a tissue-specific basis, like FGF21 induction in the deletor muscle, others were systemic, such as the downregulation of ALAS2-linked heme synthesis in all deletor tissues analyzed. However, interestingly, even a tissue-specific response of FGF21 excretion could elicit a global starvation response. The work presented in this thesis has contributed to a better understanding of mitochondrial stress signalling and of pathways interpreting and transducing it to human pathology.

Pathophysiological factors of irritable bowel syndrome, and the effects of probiotic supplementation

Gastrointestinal symptoms and impaired quality of life caused by irritable bowel syndrome (IBS) affect up to 20% of the adult population worldwide. The exact aetiology and pathophysiology of IBS are incompletely understood. Clinical studies suggest that supplementation with certain probiotics may be beneficial in IBS, but there is not enough evidence to make general recommendations. The aim of this thesis was to investigate microbiota- and mucosa-associated pathophysiological factors of IBS, and to evaluate the long-term effects of multispecies probiotic supplementation on symptoms, quality of life, intestinal microbiota and systemic inflammatory markers in IBS. The intestinal microbiota composition in IBS patients and healthy control subjects was analysed by quantitative polymerase chain reaction (qPCR). Significantly lower counts for the Clostridium coccoides and the Bifidobacterium catenulatum groups were found in IBS compared to controls. Quantitative differences also appeared in subgroup analysis based on the predominant bowel habit: diarrhoea patients harboured significantly lower numbers of Lactobacillus spp. than the constipation-predominant patients, while higher counts for Veillonella spp. were detected in constipation-predominant patients compared to healthy controls. Analysis of mucosal biopsies by a metabolomic approach revealed multiple differences between patients and controls. The most prominent finding was an upregulation of specific lipid species, principally lysophosphatidylcholines and ceramides, in IBS. The effects of multispecies probiotic supplementation with Lactobacillus rhamnosus GG, Lactobacillus rhamnosus Lc705, Propionibacterium freudenreichii subsp. shermanii JS, and Bifidobacterium breve Bb99 or Bifidobacterium animalis subsp. lactis Bb12 was evaluated in two, randomised, double-blind, placebo-controlled trials. Compared to placebo, the probiotic supplementation significantly reduced the total symptoms of IBS. No effects on bowel habit were seen. Health-related quality of life (HRQOL) is reduced in patients with IBS in comparison with the Finnish population on the whole. The probiotic supplementation improved one IBS-specific domain of quality of life (bowel symptoms), whereas no other effects on HRQOL were seen. The probiotics had no major effects on the predominant microbiota as measured by qPCR, but a microarray-based analysis suggested that the probiotic consumption stabilised the microbiota. No effects on serum sensitive-CRP or cytokines were detected. In conclusion, alterations in the microbiota composition and in the mucosal metabolite profile are potential pathophysiological factors of IBS. Multispecies probiotic supplementation alleviates the gastrointestinal symptoms of IBS, and improves the bowel symptoms domain of HRQOL. Probiotic supplementation in IBS is associated with a stabilisation of microbiota, but it does not influence systemic inflammatory markers.

LKB1 tumor suppression in Peutz-Jeghers Syndrome

Kasvainten, ajatellaan syntyvän yksittäisen solun perimän mutaatioista, jonka seurauksena tuon solun kasvu häiriintyy. Ruoansulatuskanavan polyyppien syntyä käytetään usein mallina siitä, miten nämä epiteelisoluun kerääntyvät mutaatiot aiheuttavat asteittain pahenevan kasvuhäiriön. Peutz–Jeghersin oireyhtymä (PJS) on perinnöllinen polypoosisyndrooma, jossa oireita aiheuttavat erityisesti maha-suolikanavan hamartomatoottiset polyypit. Noin puolella PJS potilaista havaitaan mutaatioita LKB1 kasvunrajoite geenissä. Hiirille joilta toinen Lkb1 alleeli on poistettu (Lkb1+/-) kehittyy PJS-tyypin maha-suolikanavan polyyppeja, joissa on epiteelin liikakasvun lisäksi merkittävä sileälihaskomponentti, aivan kuten PJS polyypeissa. Kuten myös muissa ruoansulatuskanavan polypooseissa, sekä PJS että hiirten polyypeissa Cyclo-oxygenaasi-2:n (COX-2) määrä on usein kohonnut. PJS-polyyppien kehittymisen molekulaarinen mekanismi on kuitenkin selvittämättä. Koska vain osa PJS potilaista kantaa LKB1 mutaatioita, mutaatiot jossakin toisessa lokuksessa saattaisivat selittää osan PJS tapauksista. Jotta PJS:n geneettinen tausta selviäisi, seulottiin kolmen LKB1:n kanssa interaktoivan proteiinin (BRG1, STRADα ja MO25α) geenit PJS potilaista joilla ei ole havaittu LKB1 mutaatioita. Yhdessäkään tutkituista geeneistä ei havaittu tautia aiheuttavia mutaatioita. Näiden kolmen geenin pois sulkeminen, ja uusien menetelmien ansiosta kasvanut havaittujen Lkb1 mutaatioden määrä viittaavat LKB1:n olevan useimpien PJS tapausten taustalla. COX-2:n estäjien käyttö on tehokkaasti vähentänyt polyyppien määrää familiaarisessa adenomatoottisessa polypoosissa. Tästä johtuen COX-2:n eston tehokkuutta tutkittiin PJS polypoosissa. PJS-tyypin polypoosin havaittin pienenevän merkittävästi Lkb1+/- hiirissä, joilta oli lisäksi poistettu toinen tai molemmat COX-2:n alleeleista. Lisäksi farmakologinen COX-2:n esto Celecoxib:lla vähensi polypoosia tehokkaasti. Näin ollen COX-2:n eston tehokkuutta tutkittiin seuraavaksi PJS potilaissa. Kuuden kuukauden Celecoxib hoidon jälkeen polypoosin havaittiin vähentyneen merkittävästi osalla potilaista (2/6). Nämä tulokset osoittavat COX-2:n roolin PJS-polyyppien kehityksessä, ja viittaavat COX-2:n eston vähentävän polypoosia. Kasvunrajoitegeenin klassisen määritelmän mukaan kasvaimen kehitys vaatii perinnöllisen mutaation lisäksi geenin toisenkin alleelin mutaation, mutta PJS-polyyppien häiriintyneestä epiteelistä ei kuitenkaan systemaattisesti löydy toista LKB1:n mutaatiota. Havainto johti tutkimukseen, jossa selvitettiin voisiko LKB1:n kasvun rajoitus välittyäkin epäsuorasti tukikudokseksi ajatelluista sileälihassoluista. Tätä tutkittiin kehittämällä poistogeeninen hiirimalli jossa Lkb1 on mutatoitunut vain sileälihassoluissa. Näille hiirille kehittyi polyyppeja, jotka ovat kaikin tavoin PJS-polyyppien kaltaisia. Lkb1:n menettäneiden solujen havaittiin tuottavan vähemmän transformoivaa kasvutekijä beetaa (TGFß), joka aiheutti solujen välisen viestinnän heikentymisen ja mahdollisesti viereisten epiteelisolujen liikakasvun. Vastaava häiriö havaittiin myös PJS-potilaiden polyypeissa, mikä viittaa siihen, että potilaillakin sileälihassolujen häiriö on polyyppien taustalla. Havainto suuntaa täten hoitokohteiden etsintää ja osoittaa että LKB1 toimii kasvunrajoittajana epätyypillisellä tavalla pitäen naapurisolujen kasvun kurissa.

Management of 'Tough' fish syndrome (TFS) in tropical Saddletail Snapper to re-instill market confidence

Successful identification of these factors influence upon TFS will empower stakeholders to make informed decisions as to how to best utilise the resource, boost consumer confidence thus ensuring the improved profitability of the fishery into the future.

Rituximab for chronic fatigue syndrome?

Most people complain about being tired and wish they could sleep in for a few more hours instead of going to work. However, 'being tired' has a whole different meaning for people living with chronic fatigue syndrome, which is also known as myalgic encephalomyelitis.

Identification of the Meckel syndrome gene (MKS1) exposes a novel ciliopathy

Meckel syndrome (MKS, MIM 249000) is an autosomal recessive developmental disorder causing death in utero or shortly after birth. The hallmarks of the disease are cystic kidney dysplasia and fibrotic changes of the liver, occipital encephalocele with or without hydrocephalus and polydactyly. Other anomalies frequently seen in the patients are incomplete development of the male genitalia, club feet and cleft lip or palate. The clinical picture has been well characterized in the literature while the molecular pathology underlying the disease has remained unclear until now. In this study we identified the first MKS gene by utilizing the disease haplotypes in Finnish MKS families linked to the MKS1 locus on chromosome 17q23 (MKS1) locus. Subsequently, the genetic heterogeneity of MKS was established in the Finnish families. Mutations in at least four different genes can cause MKS. These genes have been mapped to the chromosomes 17q23 (MKS1), 11q13 (MKS2), 8q22 (MKS3) and 9q33 (MKS4). Two of these genes have been identified so far: The MKS1 gene (this work) and the MKS3 gene. The identified MKS1 gene was initially a novel human gene which is conserved among species. We found three different MKS mutations, one of them being the Finnish founder mutation. The information available from MKS1 orthologs in other species convinced us that the MKS1 gene is required for normal ciliogenesis. Defects of the cilial system in other human diseases and model organisms actually cause phenotypic features similar to those seen in MKS patients. The MKS3 (TMEM67) gene encodes a transmembrane protein and the gene maps to the syntenic Wpk locus in the rat, which is a model with polycystic kidney disease, agenesis of the corpus callosum and hydrocephalus. The available information from these two genes suggest that MKS1 would encode a structural component of the centriole required for normal ciliary functions, and MKS3 would be a transmembrane component most likely required for normal ciliary sensory signaling. The MKS4 locus was localized to chromosme 9q32-33 in this study by using an inbred Finnish family with two affected and two healthy children. This fourth locus contains TRIM32 gene, which is associated to another well characterized human ciliopathy, Bardet Biedl syndrome (BBS). Future studies should identify the MKS4 gene on chromosome 9q and confirm if there are more than two genes causing MKS Finnish families. The research on critical signaling pathways in organogenesis have shown that both Wnt and Hedgehog pathways are dependent on functional cilia. The MKS gene products will serve as excellent model molecules for more detailed studies of the functional role of cilia in organogenesis in more detail.

Molecular mechanisms of cancer predisposition in HNPCC/Lynch syndrome

Hereditary non-polyposis colorectal carcinoma (HNPCC; Lynch syndrome) is among the most common hereditary cancers in man and a model of cancers arising through deficient DNA mismatch repair (MMR). It is inherited in a dominant manner with predisposing germline mutations in the MMR genes, mainly MLH1, MSH2, MSH6 and PMS2. Both copies of the MMR gene need to be inactivated for cancer development. Since Lynch syndrome family members are born with one defective copy of one of the MMR genes in their germline, they only need to acquire a so called second hit to inactivate the MMR gene. Hence, they usually develop cancer at an early age. MMR gene inactivation leads to accumulation of mutations particularly in short repeat tracts, known as microsatellites, causing microsatellite instability (MSI). MSI is the hallmark of Lynch syndrome tumors, but is present in approximately 15% of sporadic tumors as well. There are several possible mechanisms of somatic inactivation (i.e. the second hit ) of MMR genes, for instance deletion of the wild-type copy, leading to loss of heterozygosity (LOH), methylation of promoter regions necessary for gene transcription, or mitotic recombination or gene conversion. In the Lynch syndrome tumors carrying germline mutations in the MMR gene, LOH was found to be the most frequent mechanism of somatic inactivation in the present study. We also studied MLH1/MSH2 deletion carriers and found that somatic mutations identical to the ones in the germline occurred frequently in colorectal cancers and were also present in extracolonic Lynch syndrome-associated tumors. Chromosome-specific marker analysis implied that gene conversion, rather than mitotic recombination or deletion of the respective gene locus accounted for wild-type inactivation. Lynch syndrome patients are predisposed to certain types of cancers, the most common ones being colorectal, endometrial and gastric cancer. Gastric cancer and uroepithelial tumors of bladder and ureter were observed to be true Lynch syndrome tumors with MMR deficiency as the driving force of tumorigenesis. Brain tumors and kidney carcinoma, on the other hand, were mostly MSS, implying the possibility of alternative routes of tumor development. These results present possible implications in clinical cancer surveillance. In about one-third of families suspected of Lynch syndrome, mutations in MMR genes are not found, and we therefore looked for alternative mechanisms of predisposition. According to our results, large genomic deletions, mainly in MSH2, and germline epimutations in MLH1, together explain a significant fraction of point mutation-negative families suspected of Lynch syndrome and are associated with characteristic clinical and family features. Our findings have important implications in the diagnosis and management of Lynch syndrome families.

Molecular Genetic Background of Tumours in Hereditary Leiomyomatosis and Renal Cell Cancer Syndrome

Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC) is a hereditary tumour predisposition syndrome. Its phenotype includes benign cutaneous and uterine leiomyomas (CLM, ULM) with high penetrance and rarer renal cell cancer (RCC), most commonly of papillary type 2 subtype. Over 130 HLRCC families have been identified world-wide but the RCC phenotype seems to concentrate in families from Finland and North America for unknown reasons. HLRCC is caused by heterozygous germline mutations in the fumarate hydratase (FH) gene. FH encodes the enzyme fumarase from mitochondrial citric acid cycle. Fumarase enzyme activity or type or site of the FH mutation are unassociated with disease phenotype. The strongest evidence for tumourigenesis mechanism in HLRCC supports a hypoxia inducible factor driven process called pseudohypoxia resulting from accumulation of the fumarase substrate fumarate. In this study, to assess the importance of gene- or exon-level deletions or amplifications of FH in patients with HLRCC-associated phenotypes, multiplex ligation-dependent probe amplification (MLPA) method was used. One novel FH mutation, deletion of exon 1, was found in a Swedish male patient with an evident HLRCC phenotype with CLM, RCC, and a family history of ULM and RCC. Six other patients with CLM and 12 patients with only RCC or uterine leiomyosarcoma (ULMS) remained FH mutation-negative. These results suggest that copy number aberrations of FH or its exons are an infrequent cause of HLRCC and that only co-occurrence of benign tumour types justifies FH-mutation screening in RCC or ULMS patients. Determination of the genomic profile of 11 HLRCC-associated RCCs from Finnish patients was performed by array comparative genomic hybridization. The most common copy number aberrations were gains of 2, 7, and 17 and losses of 13q12.3-q21.1, 14, 18, and X. When compared to aberrations of sporadic papillary RCCs, HLRCC-associated RCCs harboured a distinct DNA copy number profile and lacked many of the changes characterizing the sporadic RCCs. The findings suggest a divergent molecular pathway for tumourigenesis of papillary RCCs in HLRCC. In order to find a genetic modifier of RCC risk in HLRCC, genome-wide linkage and identical by descent (IBD) analysis studies were performed in Finnish HLRCC families with microsatellite marker mapping and SNP-array platforms. The linkage analysis identified only one locus of interest, the FH gene locus in 1q43, but no mutations were found in the genes of the region. IBD analysis yielded no convincing haplotypes shared by RCC patients. Although these results do not exclude the existence of a genetic modifier for RCC risk in HLRCC, they emphasize the role of FH mutations in the malignant tumourigenesis of HLRCC. To study the benign tumours in HLRCC, genome-wide DNA copy number and gene expression profiles of sporadic and HLRCC ULMs were defined with modern SNP- and gene-expression array platforms. The gene expression array suggests novel genes involved in FH-deficient ULM tumourigenesis and novel genes with putative roles in propagation of sporadic ULM. Both the gene expression and copy number profiles of HLRCC ULMs differed from those of sporadic ULMs indicating distinct molecular basis of the FH-deficient HLRCC tumours.