Molecular genetics of Meckel syndrome : Ciliary genes are defective in MKS

Meckel syndrome (MKS, MIM 249000) is a severe developmental disorder that leads to death already in utero or shortly after birth. MKS diagnosis can be established by a careful ultrasound examination already at 11-14 weeks of gestation. The main features of MKS are occipital meningoencephalocele, cystic kidney dysplasia and fibrotic changes of the liver. In addition, polydactyly is frequently reported in the cases. The aim of the study was to characterize the molecular and functional defects in MKS. In this study we were able to identify two major MKS mutations in Finnish population, which cover over 90% of the cases. The first mutation is a 29 bp intronic deletion in the MKS1 gene (c.1483-7_35del) that is found in 70% of the families and the second is a C>T substitution in the coding region of CC2D2A (c.1762C>T), that is found in 20% of the MKS families. Both of these mutations result in abnormal splicing. The discovery of the disease genes has revealed that MKS is caused by primary cilia dysfunction. MKS1 gene has a conserved B9 domain, and it is found in the predicted ciliary proteome. CC2D2A protein is also found in the predicted ciliary proteome and it has a Ca2+ binding domain. The number of genes behind MKS has increased rapidly in the past years and to date, mutations have been identified in five genes (MKS1, TMEM67/MKS3, CEP290/MKS4, RPGRIP1L/MKS5 and CC2D2A/MKS6). Identification of the disease genes mutations has also revealed that MKS is an allelic disorder with other syndromes with overlapping phenotypes. Disorders that are caused by primary cilia dysfunction are collectively known as ciliopathies. Sequence analysis of all the known MKS genes in Finnish and non-Finnish families available to us, where the mutation was still unknown, revealed mutations in 14 out of the 30 families included in the study. When we collected all the reported mutations in MKS genes in different syndromes we could see that there was clearly a genotype-syndrome correlation between the mutations and the syndromes, since the same pair of mutations has never been reported in different syndromes. The basic molecular events behind MKS will not only give us information of this syndrome, but also significant novel information on early fetal development in general.

Genotype-Phenotype Relationships in Long QT Syndrome: Role of Mental Stress, Adrenergic Activity and a Common KCNH2 Polymorphism

Long QT syndrome is a congenital or acquired arrhythmic disorder which manifests as a prolonged QT-interval on the electrocardiogram and as a tendency to develop ventricular arrhythmias which can lead to sudden death. Arrhythmias often occur during intense exercise and/or emotional stress. The two most common subtypes of LQTS are LQT1, caused by mutations in the KCNQ1 gene and LQT2, caused by mutations in the KCNH2 gene. LQT1 and LQT2 patients exhibit arrhythmias in different types of situations: in LQT1 the trigger is usually vigorous exercise whereas in LQT2 arrhythmia results from the patient being startled from rest. It is not clear why trigger factors and clinical outcome differ from each other in the different LQTS subtypes. It is possible that stress hormones such as catecholamines may show different effects depending on the exact nature of the genetic defect, or sensitivity to catecholamines varies from subject to subject. Furthermore, it is possible that subtle genetic variants of putative modifier genes, including those coding for ion channels and hormone receptors, play a role as determinants of individual sensitivity to life-threatening arrhythmias. The present study was designed to identify some of these risk modifiers. It was found that LQT1 and LQT2 patients show an abnormal QT-adaptation to both mental and physical stress. Furthermore, as studied with epinephrine infusion experiments while the heart was paced and action potentials were measured from the right ventricular septum, LQT1 patients showed repolarization abnormalities which were related to their propensity to develop arrhythmia during intense, prolonged sympathetic tone, such as exercise. In LQT2 patients, this repolarization abnormality was noted already at rest corresponding to their arrhythmic episodes as a result of intense, sudden surges in adrenergic tone, such as fright or rage. A common KCNH2 polymorphism was found to affect KCNH2 channel function as demonstrated by in vitro experiments utilizing mammalian cells transfected with the KCNH2 potassium channel as well as QT-dynamics in vivo. Finally, the present study identified a common β-1-adrenergic receptor genotype that is related a shorter QT-interval in LQT1 patients. Also, it was discovered that compound homozygosity for two common β-adrenergic polymorphisms was related to the occurrence of symptoms in the LQT1 type of long QT syndrome. The studies demonstrate important genotype-phenotype differences between different LQTS subtypes and suggest that common modifier gene polymorphisms may affect cardiac repolarization in LQTS. It will be important in the future to prospectively study whether variant gene polymorphisms will assist in clinical risk profiling of LQTS patients.

Tumour necrosis factor receptor-associated periodic syndrome (TRAPS) : Identification of novel TNFRSF1A mutations and intracellular signalling defects

The systemic autoinflammatory disorders are a group of rare diseases characterized by periodically recurring episodes of acute inflammation and a rise in serum acute phase proteins, but with no signs of autoimmunity. At present eight hereditary syndromes are categorized as autoinflammatory, although the definition has also occasionally been extended to other inflammatory disorders, such as Crohn s disease. One of the autoinflammatory disorders is the autosomally dominantly inherited tumour necrosis factor receptor-associated periodic syndrome (TRAPS), which is caused by mutations in the gene encoding the tumour necrosis factor type 1 receptor (TNFRSF1A). In patients of Nordic descent, cases of TRAPS and of three other hereditary fevers, hyperimmunoglobulinemia D with periodic fever syndrome (HIDS), chronic infantile neurologic, cutaneous and articular syndrome (CINCA) and familial cold autoinflammatory syndrome (FCAS), have been reported, TRAPS being the most common of the four. Clinical characteristics of TRAPS are recurrent attacks of high spiking fever, associated with inflammation of serosal membranes and joints, myalgia, migratory rash and conjunctivitis or periorbital cellulitis. Systemic AA amyloidosis may occur as a sequel of the systemic inflammation. The aim of this study was to investigate the genetic background of hereditary periodically occurring fever syndromes in Finnish patients, to explore the reliability of determining serum concentrations of soluble TNFRSF1A and metalloproteinase-induced TNFRSF1A shedding as helpful tools in differential diagnostics, as well as to study intracellular NF-κB signalling in an attempt to widen the knowledge of the pathomechanisms underlying TRAPS. Genomic sequencing revealed two novel TNFRSF1A mutations, F112I and C73R, in two Finnish families. F112I was the first TNFRSF1A mutation to be reported in the third extracellular cysteine-rich domain of the gene and C73R was the third novel mutation to be reported in a Finnish family, with only one other TNFRSF1A mutation having been reported in the Nordic countries. We also presented a differential diagnostic problem in a TRAPS patient, emphasizing for the clinician the importance of differential diagnostic vigiliance in dealing with rare hereditary disorders. The underlying genetic disease of the patient both served as a misleading factor, which possibly postponed arrival at the correct diagnosis, but may also have predisposed to the pathologic condition, which led to a critical state of the patient. Using a method of flow cytometric analysis modified for the use on fresh whole blood, we studied intracellular signalling pathways in three Finnish TRAPS families with the F112I, C73R and the previously reported C88Y mutations. Evaluation of TNF-induced phosphorylation of NF-κB and p38, revealed low phosphorylation profiles in nine out of ten TRAPS patients in comparison to healthy control subjects. This study shows that TRAPS is a diagnostic possibility in patients of Nordic descent, with symptoms of periodically recurring fever and inflammation of the serosa and joints. In particular in the case of a family history of febrile episodes, the possibility of TRAPS should be considered, if an etiology of autoimmune or infectious nature is excluded. The discovery of three different mutations in a population as small as the Finnish, reinforces the notion that the extracellular domain of TNFRSF1A is prone to be mutated at the entire stretch of its cysteine-rich domains and not only at a limited number of sites, suggesting the absence of a founder effect in TRAPS. This study also demonstrates the challenges of clinical work in differentiating the symptoms of rare genetic disorders from those of other pathologic conditions and presents the possibility of an autoinflammatory disorder as being the underlying cause of severe clinical complications. Furthermore, functional studies of fresh blood leukocytes show that TRAPS is often associated with a low NF-κB and p38 phosphorylation profile, although low phosphorylation levels are not a requirement for the development of TRAPS. The aberrant signalling would suggest that the hyperinflammatory phenotype of TRAPS is the result of compensatory NF-κB-mediated regulatory mechanisms triggered by a deficiency of the innate immune response.

Irritable bowel syndrome in the general population : epidemiology, comorbidity and societal costs

Background: Irritable bowel syndrome (IBS) is a common functional gastrointestinal (GI) disorder characterised by abdominal pain and abnormal bowel function. It is associated with a high rate of healthcare consumption and significant health care costs. The prevalence and economic burden of IBS in Finland has not been studied before. The aims of this study were to assess the prevalence of IBS according to various diagnostic criteria and to study the rates of psychiatric and somatic comorbidity in IBS. In addition, health care consumption and societal costs of IBS were to be evaluated. Methods: The study was a two-phase postal survey. Questionnaire I identifying IBS by Manning 2 (at least two of the six Manning symptoms), Manning 3 (at least three Manning symptoms), Rome I, and Rome II criteria, was mailed to a random sample of 5 000 working age subjects. It also covered extra-GI symptoms such as headache, back pain, and depression. Questionnaire II, covering rates of physician visits, and use of GI medication, was sent to subjects fulfilling Manning 2 or Rome II IBS criteria in Questionnaire I. Results: The response rate was 73% and 86% for questionnaires I and II. The prevalence of IBS was 15.9%, 9.6%, 5.6%, and 5.1% according to Manning 2, Manning 3, Rome I, and Rome II criteria. Of those meeting Rome II criteria, 97% also met Manning 2 criteria. Presence of severe abdominal pain was more often reported by subjects meeting either of the Rome criteria than those meeting either of the Manning criteria. Presence of depression, anxiety, and several somatic symptoms was more common among subjects meeting any IBS criterion than by controls. Of subjects with depressive symptoms, 11.6% met Rome II IBS criteria compared to 3.7% of those with no depressiveness. Subjects meeting any IBS criteria made more physician visits than controls. Intensity of GI symptoms and presence of dyspeptic symptoms were the strongest predictors of GI consultations. Presence of dyspeptic symptoms and a history of abdominal pain in childhood also predicted non-GI visits. Annual GI related individual costs were higher in the Rome II group (497 ) than in the Manning 2 group (295 ). Direct expenses of GI symptoms and non GI physician visits ranged between 98M for Rome II and 230M for Manning 2 criteria. Conclusions: The prevalence of IBS varies substantially depending on the criteria applied. Rome II criteria are more restrictive than Manning 2, and they identify an IBS population with more severe GI symptoms, more frequent health care use, and higher individual health care costs. Subjects with IBS demonstrate high rates of psychiatric and somatic comorbidity regardless of health care seeking status. Perceived symptom severity rather than psychiatric comorbidity predicts health care seeking for GI symptoms. IBS incurs considerable medical costs. The direct GI and non-GI costs are equivalent to up to 5% of outpatient health care and medicine costs in Finland. A more integral approach to IBS by physicians, accounting also for comorbid conditions, may produce a more favourable course in IBS patients and reduce health care expenditures.

Shwachman-Diamond syndrome : Clinical, genetic and radiological study

Shwachman-Diamond syndrome (SDS) is a rare autosomal recessive disorder in which the cardinal symptoms arise from exocrine pancreatic insufficiency and bone marrow dysfunction. Previous studies have suggested increased risk of fatal complications among Finnish SDS infants. The genetic defect responsible for the disease was recently identified; the SBDS gene is located at chromosome 7q11 and encodes a protein that is involved in ribosome biosynthesis. The discovery of the SBDS gene has opened new insights into the pathogenesis of this multi-organ disease. This study aimed to assess phenotypic and genotypic features of Finnish patients with SDS. Seventeen Finnish patients with a clinical diagnosis of SDS were included in the study cohort. Extensive clinical, biochemical and imaging assessments were performed to elucidate the phenotypic features, and the findings were correlated with the SBDS genotype. Imaging studies included abdominal magnetic reso-nance imaging (MRI), brain MRI, cardiac echocardiography including tissue Doppler examination, and cardiac MRI. The skeletal phenotype was assessed by dual-energy X-ray absorptiometry and bone histomorphometry. Twelve patients had mutations in the SBDS gene. In MRI, a characteristic pattern of fat-replaced pancreas with occasional enhancement of scattered parenchymal foci and of pancreatic duct was noted in the SBDS mutation-positive patients while the mutation-negative patients did not have pancreatic fat accumulation. The patients with SBDS mutations had significantly reduced bone mineral density associated with low-energy peripheral fractures and vertebral compression fractures. Bone histomorphometry confirmed low-turnover osteoporosis. The patients with SBDS mutations had learning difficulties and smaller head size and brain volume than control subjects. Corpus callosum, cerebellar vermis, and pos-terior fossa structures were significantly smaller in SDS patients than in controls. Patients with SDS did not have evidence of clinical heart disease or myocardial fibrosis. However, subtle diastolic changes in the right ventricle and exercise-induced changes in the left ventricle contractile reserve were observed. This study expanded the phenotypic features of SDS to include primary low-turnover osteoporosis and structural alterations in the brain. Pancreatic MRI showed characteristic changes in the SBDS mutation-positive patients while these were absent in the mutation-negative patients, suggesting that MRI can be used to differentiate patients harbouring SBDS mutations from those without mutations. No evidence for clinical cardiac manifestations was found, but imaging studies revealed slightly altered myocardial function that may have clinical implications. These findings confirm the pleiotropic nature of SDS and underscore the importance of careful multidisciplinary follow-up of the affected individuals.

Testicular function in adolescent boys with Klinefelter syndrome

Klinefelter syndrome (KS) is the most frequent karyotype disorder of male reproductive function. Since its original clinical description in 1942 and the identification of its chromosomal basis 47,XXY in 1959, the typical KS phenotype has become well recognized, but the mechanisms behind the testicular degeneration process have remained unrevealed. This prospective study was undertaken to increase knowledge about testicular function in adolescent KS boys. It comprised a longitudinal follow-up of growth, pubertal development, and serum reproductive hormone levels in 14 prepubertal and pubertal KS boys. Each boy had a testicular biopsy that was analyzed with histomorphometric and immunohistochemical methods. The KS boys had sufficient testosterone levels to allow normal onset and progression of puberty. Their serum testosterone levels remained within the low-normal range throughout puberty, but from midpuberty onwards, findings like a leveling-off in testosterone and insulin-like factor 3 (INSL3) concentrations, high gonadotropin levels, and exaggerated responses to gonadotropin-releasing hormone stimulation suggest diminished testosterone secretion. We also showed that the Leydig cell differentiation marker INSL3 may serve as a novel marker for onset and normal progression of puberty in boys. In the KS boys the number of germ cells was already markedly lower at the onset of puberty. The pubertal activation of the pituitary-testicular axis accelerated germ cell depletion, and germ cell differentiation was at least partly blocked at the spermatogonium or early primary spermatocyte stages. The presence of germ cells correlated with serum reproductive hormone levels. The immature Sertoli cells were incapable of transforming to the adult type, and during puberty the degeneration of Sertoli cells increased markedly. The older KS boys displayed an evident Leydig cell hyperplasia, as well as fibrosis and hyalinization of the interstitium and peritubular connective tissue. Altered immunoexpression of the androgen receptor (AR) suggested that in KS boys during puberty a relative androgen deficiency develops at testicular level. The impact of genetic features of the supernumerary X chromosome on the KS phenotype was also studied. The present study suggests that parental origin of the supernumerary X chromosome and the length of the CAG repeat of the AR gene influence pubertal development and testicular degeneration. The current study characterized by several means the testicular degeneration process in the testes of adolescent KS boys and confirmed that this process accelerates at the onset of puberty. Although serum reproductive hormone levels indicated no hypogonadism during early puberty, the histological analyses showed an already markedly reduced fertility potential in prepubertal KS boys. Genetic features of the X chromosome affect the KS phenotype.

Real-Time PCR : A Molecular Approach to Investigate the Role of Intestinal Microbiota in the Pathophysiology of Irritable Bowel Syndrome

Irritable bowel syndrome (IBS) is a common multifactorial functional intestinal disorder, the pathogenesis of which is not completely understood. Increasing scientific evidence suggests that microbes are involved in the onset and maintenance of IBS symptoms. The microbiota of the human gastrointestinal (GI) tract constitutes a massive and complex ecosystem consisting mainly of obligate anaerobic microorganisms making the use of culture-based methods demanding and prone to misinterpretation. To overcome these drawbacks, an extensive panel of species- and group-specific assays for an accurate quantification of bacteria from fecal samples with real-time PCR was developed, optimized, and validated. As a result, the target bacteria were detectable at a minimum concentration range of approximately 10 000 bacterial genomes per gram of fecal sample, which corresponds to the sensitivity to detect 0.000001% subpopulations of the total fecal microbiota. The real-time PCR panel covering both commensal and pathogenic microorganisms was assessed to compare the intestinal microbiota of patients suffering from IBS with a healthy control group devoid of GI symptoms. Both the IBS and control groups showed considerable individual variation in gut microbiota composition. Sorting of the IBS patients according to the symptom subtypes (diarrhea, constipation, and alternating predominant type) revealed that lower amounts of Lactobacillus spp. were present in the samples of diarrhea predominant IBS patients, whereas constipation predominant IBS patients carried increased amounts of Veillonella spp. In the screening of intestinal pathogens, 17% of IBS samples tested positive for Staphylococcus aureus, whereas no positive cases were discovered among healthy controls. Furthermore, the methodology was applied to monitor the effects of a multispecies probiotic supplementation on GI microbiota of IBS sufferers. In the placebo-controlled double-blind probiotic intervention trial of IBS patients, each supplemented probiotic strain was detected in fecal samples. Intestinal microbiota remained stable during the trial, except for Bifidobacterium spp., which increased in the placebo group and decreased in the probiotic group. The combination of assays developed and applied in this thesis has an overall coverage of 300-400 known bacterial species, along with the number of yet unknown phylotypes. Hence, it provides good means for studying the intestinal microbiota, irrespective of the intestinal condition and health status. In particular, it allows screening and identification of microbes putatively associated with IBS. The alterations in the gut microbiota discovered here support the hypothesis that microbes are likely to contribute to the pathophysiology of IBS. The central question is whether the microbiota changes described represent the cause for, rather than the effect of, disturbed gut physiology. Therefore, more studies are needed to determine the role and importance of individual microbial species or groups in IBS. In addition, it is essential that the microbial alterations observed in this study will be confirmed using a larger set of IBS samples of different subtypes, preferably from various geographical locations.

Modification of ventricular repolarization in silent long QT syndrome mutation carriers

Congenital long QT syndrome (LQTS) is a familial disorder characterized by ventricular repolarization that makes carriers vulnerable to malignant ventricular tachycardia and sudden cardiac death. The three main subtypes (LQT1, LQT2 and LQT3) constitute 95% of cases. The disorder is characterized by a prolonged QT interval in electrocardiograms (ECG), but a considerable portion are silent carriers presenting normal (QTc < 440 ms) or borderline (QTc < 470 ms) QT interval. Genetic testing is available only for 60-70% of patients. A number of pharmaceutical compounds also affect ventricular repolarization, causing a clinically similar disorder called acquired long QT syndrome. LQTS carriers - who already have impaired ventricular repolarization - are especially vulnerable. In this thesis, asymptomatic genotyped LQTS mutation carriers with non-diagnostic resting ECG were studied. The body surface potential mapping (BSPM) system was utilized for ECG recording, and signals were analyzed with an automated analysis program. QT interval length, and the end part of the T wave, the Tpe interval, was studied during exercise stress testing and an epinephrine bolus test. In the latter, T wave morphology was also analyzed. The effect of cetirizine was studied in LQTS carriers and also with supra- therapeutic dose in healthy volunteers. At rest, LQTS mutation carriers had a slightly longer heart rate adjusted QTc interval than healthy subjects (427 ± 31 ms and 379 ± 26 ms; p<0.001), but significant overlapping existed. LQT2 mutation carriers had a conspicuously long Tpe-interval (113 ± 24 ms; compared to 79 ± 11 ms in LQT1, 81 ± 17 ms in LQT3 and 78 ± 10 ms in controls; p<0.001). In exercise stress tests, LQT1 mutation carriers exhibit a long QT interval at high heart rates and during recovery, whereas LQT2 mutation carriers have a long Tpe interval at the beginning of exercise and at the end of recovery at low heart rates. LQT3 mutation carriers exhibit prominent shortening of both QT and Tpe intervals during exercise. A small epinephrine bolus revealed disturbed repolarization, especially in LQT2 mutation carriers, who developed prolonged Tpe intervals. A higher epinephrine bolus caused abnormal T waves with a different T wave profile in LQTS mutation carriers compared to healthy controls. These effects were seen in LQT3 as well, a group that may easily escape other provocative tests. In the cetirizine test, the QT and Tpe intervals were not prolonged in LQTS mutation carriers or in healthy controls. Subtype-specific findings in exercise test and epinephrine bolus test help to diagnose silent LQTS mutation carriers and to guide subtype-specific treatments. The Tpe interval, which signifies the repolarization process, seems to be a sensitive marker of disturbed repolarization along with the QT interval, which signifies the end of repolarization. This method may be used in studying compounds that are suspected to affect repolarization. Cetirizine did not adversely alter ventricular repolarization and would not be pro-arrhythmic in common LQT1 and LQT2 subtypes when used at its recommended doses.

Lower Gastrointestinal Microbiota in Health and Irritable Bowel Syndrome : Characterisation and Effect of Probiotic Intervention

The human gastrointestinal (GI) microbiota is a complex ecosystem that lives in symbiosis with its host. The growing awareness of the importance of the microbiota to the host as well as the development of culture-free laboratory techniques and computational methods has enormously expanded our knowledge of this microbial community. Irritable bowel syndrome (IBS) is a common functional bowel disorder affecting up to a fifth of the Western population. To date, IBS diagnosis has been based on GI symptoms and the exclusion of organic diseases. The GI microbiota has been found to be altered in this syndrome and probiotics can alleviate the symptoms, although clear links between the symptoms and the microbiota have not been demonstrated. The aim of the present work was to characterise IBS related alterations in the intestinal microbiota, their relation to IBS symptoms and their responsiveness to probiotic theraphy. In this thesis research, the healthy human microbiota was characterised by cloning and sequencing 16S rRNA genes from a faecal microbial community DNA pool that was first profiled and fractionated according to its guanine and cytosine content (%G+C). The most noticeable finding was that the high G+C Gram-positive bacteria (the phylum Actinobacteria) were more abundant compared to a corresponding library constructed from the unfractionated DNA pool sample. Previous molecular analyses of the gut microbiota have also shown comparatively low amounts of high G+C bacteria. Furthermore, the %G+C profiling approach was applied to a sample constructed of faecal DNA from diarrhea-predominant IBS (IBS-D) subjects. The phylogenetic microbial community comparison performed for healthy and IBS-D sequence libraries revealed that the IBS-D sample was rich in representatives of the phyla Firmicutes and Proteobacteria whereas Actinobacteria and Bacteroidetes were abundant in the healthy subjects. The family Lachnospiraceae within the Firmicutes was especially prevalent in the IBS-D sample. Moreover, associations of the GI microbiota with intestinal symptoms and the quality of life (QOL) were investigated, as well as the effect of probiotics on these factors. The microbial targets that were analysed with the quantitative real-time polymerase chain reaction (qPCR) in this study were phylotypes (species definition according to 16S rRNA gene sequence similarity) previously associated with either health or IBS. With a set of samples, the presence or abundance of a phylotype that had 94% 16S rRNA gene sequence similarity to Ruminococcus torques (R. torques 94%) was shown to be associated with the severity of IBS symptoms. The qPCR analyses for selected phylotypes were also applied to samples from a six-month probiotic intervention with a mixture of Lactobacillus rhamnosus GG, L. rhamnosus Lc705, Propionibacterium freudenreichii ssp. shermanii JS and Bifidobacterium breve Bb99. The intervention had been previously reported to alleviate IBS symptoms, but no associations with the analysed microbiota representatives were shown. However, with the phylotype-specific assays applied here, the abundance of the R. torques 94% -phylotype was shown to be lowered in the probiotic-receiving group during the probiotic supplementation, whereas a Clostridium thermosuccinogenes 85% phylotype, previously associated with a healthy microbiota, was found to be increased compared to the placebo group. To conclude, with the combination of methods applied, higher abundance of Actinobacteria was detected in the healthy gut than found in previous studies, and significant phylum-level microbiota alterations could be shown in IBS-D. Thus, the results of this study provide a detailed overview of the human GI microbiota in healthy subjects and in subjects with IBS. Furthermore, the IBS symptoms were linked to a particular clostridial phylotype, and probiotic supplementation was demonstrated to alter the GI microbiota towards a healthier state with regard to this and an additional bacterial phylotype. For the first time, distinct phylotype-level alterations in the microbiota were linked to IBS symptoms and shown to respond to probiotic therapy.

Molecular genetics of Usher syndrome -inherited deafness and blindness

Usher syndrome (USH) is an inherited blindness and deafness disorder with variable vestibular dysfunction. The syndrome is divided into three subtypes according to the progression and severity of clinical symptoms. The gene mutated in Usher syndrome type 3 (USH3), clarin 1 (CLRN1), was identified in Finland in 2001 and two mutations were identified in Finnish patients at that time. Prior to this thesis study, the two CLRN1 gene mutations were the only USH mutations identified in Finnish USH patients. To further clarify the Finnish USH mutation spectrum, all nine USH genes were studied. Seven mutations were identified: one was a previously known mutation in CLRN1, four were novel mutations in myosin VIIa (MYO7A) and two were a novel and a previously known mutation in usherin (USH2A). Another aim of this thesis research was to further study the structure and function of the CLRN1 gene, and to clarify the effects of mutations on protein function. The search for new splice variants resulted in the identification of eight novel splice variants in addition to the three splice variants that were already known prior to this study. Studies of the possible promoter regions for these splice variants showed the most active region included the 1000 bases upstream of the translation start site in the first exon of the main three exon splice variant. The 232 aa CLRN1 protein encoded by the main (three-exon) splice variant was transported to the plasma membrane when expressed in cultured cells. Western blot studies suggested that CLRN1 forms dimers and multimers. The CLRN1 mutant proteins studied were retained in the endoplasmic reticulum (ER) and some of the USH3 mutations caused CLRN1 to be unstable. During this study, two novel CLRN1 sequence alterations were identified and their pathogenicity was studied with cell culture protein expression. Previous studies with mice had shown that Clrn1 is expressed in mouse cochlear hair cells and spiral ganglion cells, but the expression profile in mouse retina remained unknown. The Clrn1 knockout mice display cochlear cell disruption/death, but do not have a retinal phenotype. The zebrafish, Danio rerio, clrn1 was found to be expressed in hair cells associated with hearing and balance. Clrn1 expression was also found in the inner nuclear layer (INL), photoreceptor layer and retinal pigment epithelium layer (RPE) of the zebrafish retina. When Clrn1 production was knocked down with injected morpholino oligonucleotides (MO) targeting Clrn1 translation or correct splicing, the zebrafish larvae showed symptoms similar to USH3 patients. These larvae had balance/hearing problems and reduced response to visual stimuli. The knowledge this thesis research has provided about the mutations in USH genes and the Finnish USH mutation spectrum are important in USH patient diagnostics. The extended information about the structure and function of CLRN1 is a step further in exploring USH3 pathogenesis caused by mutated CLRN1 as well as a step in finding a cure for the disease.

Ising model on a random network with annealed or quenched disorder

We study the equilibrium properties of an Ising model on a disordered random network where the disorder can be quenched or annealed. The network consists of fourfold coordinated sites connected via variable length one-dimensional chains. Our emphasis is on nonuniversal properties and we consider the transition temperature and other equilibrium thermodynamic properties, including those associated with one-dimensional fluctuations arising from the chains. We use analytic methods in the annealed case, and a Monte Carlo simulation for the quenched disorder. Our objective is to study the difference between quenched and annealed results with a broad random distribution of interaction parameters. The former represents a situation where the time scale associated with the randomness is very long and the corresponding degrees of freedom can be viewed as frozen, while the annealed case models the situation where this is not so. We find that the transition temperature and the entropy associated with one-dimensional fluctuations are always higher for quenched disorder than in the annealed case. These differences increase with the strength of the disorder up to a saturating value. We discuss our results in connection to physical systems where a broad distribution of interaction strengths is present.

UK guidance on the initial evaluation of an infant or an adolescent with a suspected disorder of sex development

It is paramount that any child or adolescent with a suspected disorder of sex development (DSD) is assessed by an experienced clinician with adequate knowledge about the range of conditions associated with DSD. If there is any doubt, the case should be discussed with the regional team. In most cases, particularly in the case of the newborn, the paediatric endocrinologist within the regional DSD team acts as the first point of contact. The underlying pathophysiology of DSD and the strengths and weaknesses of the tests that can be performed should be discussed with the parents and affected young person and tests undertaken in a timely fashion. This clinician should be part of a multidisciplinary team experienced in management of DSD and should ensure that the affected person and parents are as fully informed as possible and have access to specialist psychological support. Finally, in the field of rare conditions, it is imperative that the clinician shares the experience with others through national and international clinical and research collaboration. © 2011 Blackwell Publishing Ltd.

Pancreatic and adrenal development and function in an ovine model of polycystic ovary syndrome.

Polycystic Ovary Syndrome (PCOS) is a complex disorder encompassing reproductive and metabolic dysfunction. Ovarian hyperandrogenism is an endocrine hallmark of human PCOS. In animal models, PCOS-like abnormalities can be recreated by in utero over-exposure to androgenic steroid hormones. This thesis investigated pancreatic and adrenal development and function in a unique model of PCOS. Fetal sheep were directly exposed (day 62 and day 82 of gestation) to steroidal excesses - androgen excess (testosterone propionate - TP), estrogen excess (diethylstilbestrol - DES) or glucocorticoid excess (dexamethasone - DEX). At d90 gestation there was elevated expression of genes involved in β- cell development and function: PDX-1 (P<0.001), and INS (P<0.05), INSR (P<0.05) driven by androgenic excess only in the female fetal pancreas. β- cell numbers (P<0.001) and in vitro insulin secretion (P<0.05) were also elevated in androgen exposed female fetuses. There was a significant increase in insulin secreting β-cell numbers (P<0.001) and in vivo insulin secretion (glucose stimulated) (P<0.01) in adult female offspring, specifically associated with prenatal androgen excess. At d90 gestation, female fetal adrenal gene expression was perturbed by fetal estrogenic exposure. Male fetal adrenal gene expression was altered more dramatically by fetal glucocorticoid exposure. In female adult offspring from androgen exposed pregnancies there was increased adrenal steroidogenic gene expression and in vivo testosterone secretion (P<0.01). This highlights that the adrenal glands may contribute towards excess androgen secretion in PCOS, but such effects might be secondary to other metabolic alterations driven by prenatal androgen exposure, such as excess insulin secretion Thus there may be dialogue between the pancreas and adrenal gland, programmed during early life, with implications for adult health Given both hyperinsulinaemia and hyperandrogenism are common features in PCOS, we suggest that their origins may be at least partially due to altered fetal steroidal environments, specifically excess androgenic stimulation

Mapping the gene for autosomal dominant restless legs syndrome in an Irish family

Restless Legs Syndrome (RLS) is a common neurological disorder affecting nearly 15% of the general population. Ironically, RLS can be described as the most common condition one has never heard of. It is usually characterised by uncomfortable, unpleasant sensations in the lower limbs inducing an uncontrollable desire to move the legs. RLS exhibits a circadian pattern with symptoms present predominantly in the evening or at night, thus leading to sleep disruption and daytime somnolence. RLS is generally classified into primary (idiopathic) and secondary (symptomatic) forms. Primary RLS includes sporadic and familial cases of which the age of onset is usually less than 45 years and progresses slowly with a female to male ratio of 2:1. Secondary forms often occur as a complication of another health condition, such as iron deficiency or thyroid dysfunction. The age of onset is usually over 45 years, with an equal male to female ratio and more rapid progression. Ekbom described the familial component of the disorder in 1945 and since then many studies have been published on the familial forms of the disorder. Molecular genetic studies have so far identified ten loci (5q, 12q, 14p, 9p, 20p, 16p, 19p, 4q, 17p). No specific gene within these loci has been identified thus far. Association mapping has highlighted a further five areas of interest. RLS6 has been found to be associated with SNPs in the BTBD9 gene. Four other variants were found within intronic and intergenic regions of MEIS1, MAP2K5/LBXCOR1, PTPRD and NOS1. The pathophysiology of RLS is complex and remains to be fully elucidated. Conditions associated with secondary RLS, such as pregnancy or end-stage renal disease, are characterised by iron deficiency, which suggests that disturbed iron homeostasis plays a role. Dopaminergic dysfunction in subcortical systems also appears to play a central role. An ongoing study within the Department of Pathology (University College Cork) is investigating the genetic characteristics of RLS in Irish families. A three generation RLS pedigree RLS3002 consisting of 11 affected and 7 unaffected living family members was recruited. The family had been examined for four of the known loci (5q, 12q, 14p and 9p) (Abdulrahim 2008). The aim of this study was to continue examining this Irish RLS pedigree for possible linkage to the previously described loci and associated regions. Using informative microsatellite markers linkage was excluded to the loci on 5q, 12q, 14p, 9p, 20p, 16p, 19p, 4q, 17p and also within the regions reported to be associated with RLS. This suggested the presence of a new unidentified locus. A genome-wide scan was performed using two microsatellite marker screening sets (Research Genetics Inc. Mapping set and the Applied Biosystems Linkage mapping set version 2.5). Linkage analysis was conducted under an autosomal dominant model with a penetrance of 95% and an allele frequency of 0.01. A maximum LOD score of 3.59 at θ=0.00 for marker D19S878 indicated significant linkage on chromosome 19p. Haplotype analysis defined a genetic region of 6.57 cM on chromosome 19p13.3, corresponding to 2.5 Mb. There are approximately 100 genes annotated within the critical region. Sequencing of two candidate genes, KLF16 and GAMT, selected on the assumed pathophysiology of RLS, did not identify any sequence variant. This study provides evidence of a novel RLS locus in an Irish pedigree, thus supporting the picture of RLS as a genetically heterogeneous trait.

Altered ultrasonic vocalization and impaired learning and memory in Angelman syndrome mouse model with a large maternal deletion from Ube3a to Gabrb3.

Angelman syndrome (AS) is a neurobehavioral disorder associated with mental retardation, absence of language development, characteristic electroencephalography (EEG) abnormalities and epilepsy, happy disposition, movement or balance disorders, and autistic behaviors. The molecular defects underlying AS are heterogeneous, including large maternal deletions of chromosome 15q11-q13 (70%), paternal uniparental disomy (UPD) of chromosome 15 (5%), imprinting mutations (rare), and mutations in the E6-AP ubiquitin ligase gene UBE3A (15%). Although patients with UBE3A mutations have a wide spectrum of neurological phenotypes, their features are usually milder than AS patients with deletions of 15q11-q13. Using a chromosomal engineering strategy, we generated mutant mice with a 1.6-Mb chromosomal deletion from Ube3a to Gabrb3, which inactivated the Ube3a and Gabrb3 genes and deleted the Atp10a gene. Homozygous deletion mutant mice died in the perinatal period due to a cleft palate resulting from the null mutation in Gabrb3 gene. Mice with a maternal deletion (m-/p+) were viable and did not have any obvious developmental defects. Expression analysis of the maternal and paternal deletion mice confirmed that the Ube3a gene is maternally expressed in brain, and showed that the Atp10a and Gabrb3 genes are biallelically expressed in all brain sub-regions studied. Maternal (m-/p+), but not paternal (m+/p-), deletion mice had increased spontaneous seizure activity and abnormal EEG. Extensive behavioral analyses revealed significant impairment in motor function, learning and memory tasks, and anxiety-related measures assayed in the light-dark box in maternal deletion but not paternal deletion mice. Ultrasonic vocalization (USV) recording in newborns revealed that maternal deletion pups emitted significantly more USVs than wild-type littermates. The increased USV in maternal deletion mice suggests abnormal signaling behavior between mothers and pups that may reflect abnormal communication behaviors in human AS patients. Thus, mutant mice with a maternal deletion from Ube3a to Gabrb3 provide an AS mouse model that is molecularly more similar to the contiguous gene deletion form of AS in humans than mice with Ube3a mutation alone. These mice will be valuable for future comparative studies to mice with maternal deficiency of Ube3a alone.