The molecular basis of Marinesco-Sjögren syndrome

Marinesco-Sjögren syndrome (MSS) is a rare autosomal recessive neurodegenerative disorder characterized by cerebellar ataxia due to cerebellar cortical atrophy, infantile- or childhood-onset bilateral cataracts, progressive myopathy, and mild to severe mental retardation. Additional features include hypergonadotropic hypogonadism, various skeletal abnormalities, short stature, and strabismus. The neuroradiologic hallmarks are hypoplasia of both the vermis and cerebellar hemispheres. The histopathologic findings include severe cerebellar atrophy and loss of Purkinje and granule cells. The common pathologic findings in muscle biopsy are variation in muscle fiber size, atrophic fibers, fatty replacement, and rimmed vacuole formation. The presence of marked cerebellar atrophy with myopathy distinguishes MSS from another rare syndrome, the congenital cataracts, facial dysmorphism, and neuropathy syndrome (CCFDN). Previously, work by others had resulted in the identification of an MSS locus on chromosome 5q31. A subtype of MSS with myoglobinuria and neuropathy had been linked to the CCFDN locus on chromosome 18qter, at which mutations in the CTDP1 gene had been identified. We confirmed linkage to the previously identified locus on chromosome 5q31 in two Finnish families with eight affected individuals, reduced the critical region by fine-mapping, and identified SIL1 as a gene underlying MSS. We found a common homozygous founder mutation in all Finnish patients. The same mutation was also present in patient samples from Norway and Sweden. Altogether, we identified eight mutations in SIL1, including nonsense, frameshift, splice site alterations, and one missense mutation. SIL1 encodes a nucleotide exchange factor for the endoplasmic reticulum (ER) resident heat-shock protein 70 chaperone GRP78. GRP78 functions in protein synthesis and quality control of the newly synthesized polypeptides. It senses and responds to stressful cellular conditions. We showed that in mice, SIL1 and GRP78 show highly similar spatial and temporal tissue expression in developing and mature brain, eye, and muscle. Studying endogenous proteins in mouse primary hippocampal neurons, we found that SIL1 and GRP78 colocalize and that SIL1 localizes to the ER. We studied the subcellular localization of two mutant proteins, a missense mutant found in two patients and an artificial mutant lacking the ER retrieval signal, and found that both mutant proteins formed aggregates within the ER. Well in line with our findings and the clinical features of MSS, recent work by Zhao et al. showed that a truncation of SIL1 causes ataxia and cerebellar Purkinje cell loss in the naturally occurring woozy mutant mouse. Prior to Purkinje cell degeneration, the unfolded protein response is initiated and abnormal protein accumulations are present. MSS thus joins the group of protein misfolding and accumulation diseases. These findings highlight the importance of SIL1 and the role of the ER in neuronal function and survival. The results presented in this thesis provide tools for the molecular genetic diagnostics of MSS and give a basis for future studies on the molecular pathogenesis of MSS. Understanding the mechanisms behind this pleiotropic syndrome may provide insights into more common forms of ataxia, myopathy, and neurodegeneration.

Differentiation of neural stem cells in fragile X syndrome

Multipotent stem cells can self-renew and give rise to multiple cell types. One type of mammalian multipotent stem cells are neural stem cells (NSC)s, which can generate neurons, astrocytes and oligodendrocytes. NSCs are likely involved in learning and memory, but their exact role in cognitive function in the developing and adult brain is unclear. We have studied properties of NSCs in fragile X syndrome (FXS), which is the most common form of inherited mental retardation. FXS is caused by the lack of functional fragile X mental retardation protein (FMRP). FMRP is involved in the regulation of postsynaptic protein synthesis in a group I metabotropic glutamate receptor 5 (mGluR5)-dependent manner. In the absence of functional FMRP, the formation of functional synapses is impaired in the forebrain which results in alterations in synaptic plasticity. In our studies, we found that FMRP-deficient NSCs generated more neurons and less glia than control NSCs. The newborn neurons derived from FMRP-deficient NSCs showed an abnormally immature morphology. Furthermore, FMRP-deficient NSCs exhibited aberrant oscillatory Ca2+ responses to glutamate, which were specifically abolished by an antagonist of the mGluR5 receptor. The data suggested alterations in glutamatergic differentiation of FMRP-deficient NSCs and were further supported by an accumulation of cells committed to glutamatergic lineage in the subventricular zone of the embryonic Fmr1-knockout (Fmr1-KO) neocortex. Postnatally, the aberrant cells likely contributed to abnormal formation of the neocortex. The findings suggested a defect in the differentiation of distinct glutamatergic mGluR5 responsive cells in the absence of functional FMRP. Furthermore, we found that in the early postnatal Fmr1-KO mouse brain, the expression of mRNA for regulator of G-protein signalling-4 (RGS4) was decreased which was in line with disturbed G-protein signalling in NSCs lacking FMRP. Brain derived neurotrophic factor (BDNF) promotes neuronal differentiation of NSCs as the absence of FMRP was shown to do. This led us to study the effect of impaired BDNF/TrkB receptor signaling on NSCs by overexpression of TrkB.T1 receptor isoform. We showed that changes in the relative expression levels of the full-length and truncated TrkB isoforms influenced the replication capacity of NSCs. After the differentiation, the overexpression of TrkB.T1 increased neuronal turnover. To summarize, FMRP and TrkB signaling are involved in normal differentiation of NSCs in the developing brain. Since NSCs might have potential for therapeutic interventions in a variety of neurological disorders, our findings may be useful in the design of pharmacological interventions in neurological disorders of learning and memory.

Late results after paediatric cardiac surgery

Background: Congenital heart defects include a wide range of inborn malformations. Depending on the defect, the life expectancy of a newborn with cardiac anomaly varies from a few days to a normal life span. In most instances surgery, is the only treatment available. The late results of surgery have not been comprehensively investigated. Aims: Mortality, morbidity and the life situation of all Finnish patients who had been operated on for congenital heart defect during childhood were investigated. Methods: Patient and surgical data were gathered from all hospitals that had performed heart surgeries on children. Late mortality and survival data were obtained from the population registry, and the causes of deaths from Statistics Finland. Morbidity of patients operated on during 1953-1989 was assessed by the usage of medicines. The pharmacotherapy data of patients and controls were obtained from the Social Insurance Institute. The life situation of patients was surveyed by mailed questionnaire. Survival, causes of deaths and life situation of patients were compared with those of the general population. Results: A total of 7240 cardiac operations were performed on 6461 children during the first 37 years of cardiac surgery (1953-1989). The number of procedures constantly rose during this period, and the increase continued in later years. The patient material varied over time, as more defects became surgically treatable. During 1953-1989 the operative mortality (death within 30 days of surgery) was 6.9%. In the 1990s a slight rise occurred in early mortality, as increasingly complicated patients were surgically treated. During 2000-2003 practically no defects were beyond the operative range. Thus, the operative mortality of 4.4% was excellent, decreasing even further to 2.0% in 2004-2007. The overall 45-year survival of patients operated on in 1953-1989 was 78%, and the corresponding figure for the general population was 93%. Survival depended on the defect, being worst among patients with univentricular heart. Late survival was also better during the 1990s and at the beginning of the 21st century. Of the 6028 early survivors, 592 died late (>30 days) after surgery. A total of 397 deaths (67%) were related and 185 (31%) unrelated to congenital heart defect. The cause of death was unknown in 10 cases. Of those 5774 patients who survived their first operation and had complete follow-up, 16% were operated on several times. Seventeen percent of patients used medicines for cardiac symptoms (heart failure, arrhythmia, hypertension and coronary disease). Patients risk of using cardiac medicines was 2.16 (Cl 1.97-2.37) times higher than that of controls. Patients also had more genetic syndromes and mental retardation and more often used medicines for asthma and epilepsy. Adult patients who had been operated on as children had coped surprisingly well with their defects. Their level of education was similar and their employment level even higher than expected, and they were living in a steady relationship as often as the general population. Conclusions: Cardiac surgery developed rapidly, and nowadays practically all defects can be treated. The overall survival of all operated patients was 78%, 16% less than that of the general population. However, it was significantly better than the anticipated natural survival. However, many patients had health problems; 16% needed reoperations and 17% cardiac medicines to maintain their condition. Most of the patients assessed their general health as good and lived a normal life.

Molecular diagnosis of developmental disorders

ABSTRACT Idiopathic developmental disorders (DDs) affect ~1% of the population worldwide. This being a considerable amount, efforts are being made to elucidate the disease mechanisms. One or several genetic factors cause 30-40% of DDs, and only 10% are caused by environmental factors. The remaining 50% of DD patients go undiagnosed, mostly due to a lack of diagnostic techniques. The cause in most undiagnosed cases is though to be a genetic factor or a combination of genetic and environmental factors. Despite the surge of new technologies entering the market, their implementation into diagnostic laboratories is hampered by costs, lack of information about the expected diagnostic yield, and the wide range of selection. This study evaluates new microarray methods in diagnosing idiopathic DDs, providing information about their added diagnostic value. Study I analysed 150 patients by array comparative genomic hybridization (array CGH, 44K and 244K), with a subsequent 18% diagnostic yield. These results are supported by other studies, indicating an enourmous added diagnostic value of array CGH, compared with conventional cytogenetic analysis. Nevertheless, 80% of the patients remained undiagnosed in Study I. In an effort to diagnose more patients, in Study IV the resolution was increased from 8.9 Kb of the 244K CGH array to 0.7 Kb, by using a single-nucleotide polymorphism (SNP) array. However, no additional pathogenic changes were detected in the 35 patients assessed, and thus, for diagnostic purposes, an array platform with ca 9 Kb resolution appears adequate. The recent vast increase in reports of detected aberrations and associated phenotypes has enabled characterization of several new syndromes first based on a common aberration and thereafter by delineation of common clinical characteristics. In Study II, a familial deletion at 9q22.2q22.32 with variable penetrance was described. Despite several reports of aberrations in the adjacent area at 9q associated with Gorlin syndrome, the patients in this family had a unique phenotype and did not present with the syndrome. In Study III, a familial duplication of chromosome 6p22.2 was described. The duplication caused increased expression of an important enzyme of the γ-aminobutyric acid (GABA) degradation pathway, causing oxidative stress of the brain, and thus, very likely, the mild mental retardation of these patients. These two case studies attempted to pinpoint candidate genes and to resolve the pathogenic mechanism causing the clinical characteristics of the patients. Presenting rare genetic and clinical findings to the international science and medical community enables interpretation of similar findings in other patients. The added value of molecular karyotyping in patients with idiopathic DD is evident. As a first line of testing, arrays with a median resolution of at least 9 Kb should be considered and further characterization of detected aberrations undertaken when possible. Diagnostic whole-exome sequencing may be the best option for patients who remain undiagnosed after high-resolution array analysis.

Medicines information sources and services for consumers : A special focus on the Internet and people with depression

The range of consumer health and medicines information sources has diversified along with the increased use of the Internet. This has led to a drive to develop medicines information services and to better incorporate the Internet and e-mail into routine practice in health care and in community pharmacies. To support the development of such services more information is needed about the use of online information by consumers, particularly of those who may be the most likely to use and to benefit from the new sources and modes of medicines communication. This study explored the role and utilization of the Internet-based medicines information and information services in the context of a wider network of information sources accessible to the public in Finland. The overall aim was to gather information to develop better and more accessible sources of information for consumers and services to better meet the needs of consumers. Special focus was on the needs and information behavior among people with depression and using antidepressant medicines. This study applied both qualitative and quantitative methods. Consumer medicines information needs and sources were identified by analyzing the utilization of the University Pharmacy operated national drug information call center (Study I) and surveying Finnish adults (n=2348) use of the different medicines information sources (Study II). The utilization of the Internet as a source of antidepressant information among people with depression was explored by focus group discussions among people with depression and with current or past use of the antidepressant(s) (n=29, Studies III & IV). Pharmacy response to the needs of consumers in term of providing e-mail counseling was assessed by conducting a virtual pseudo customer study among the Finnish community pharmacies (n=161, Study V). Physicians and pharmacists were the primary sources of medicines information. People with mental disorders were more frequent users of telephone- and Internet-based medicines information sources and patient information leaflets than people without mental disorders. These sources were used to complement rather than replace information provided face-to-face by health professionals. People with depression used the Internet to seek facts about antidepressants, to share experiences with peers, and for the curiosity. They described that the access to online drug information was empowering. Some people reported lacking the skills necessary to assess the quality of online information. E-mail medication counseling services provided by community pharmacies were rare and varied in quality. Study results suggest that rather than discouraging the use of the Internet, health professionals should direct patients to use accurate and reliable sources of online medicines information. Health care providers, including community pharmacies should also seek to develop new ways of communicating information about medicines with consumers. This study determined that people with depression and using antidepressants need services enabling interactive communication not only with health care professionals, but also with peers. Further research should be focused on developing medicines information service facilitating communication among different patient and consumer groups.