Marinesco-Sjögrenin oireyhtymän molekyylitaustan selvittäminen

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.Marinesco-Sjögrenin oireyhtymä (MSS) on varhaisella lapsuusiällä alkava harvinainen peittyvästi periytyvä sairaus. MSS:n pääoireita ovat pikkuaivojen surkastumisesta johtuva ataksia, kaihi, lihasheikkous sekä liikunnallisen ja älyllisen suorituskyvyn heikkeneminen. Potilailla voi lisäksi esiintyä mm. hypogonadismia, luustomuutoksia, karsastusta sekä lyhyt aikuispituus. Harvinaisten, yhden perintötekijän aiheuttamien sairauksien tutkiminen voi olla apuna yleisempien keskushermostoa rappeuttavien sairauksien tautimekanismien selvittämistyössä. Tässä väitöstyössä selvitettiin Marinesco-Sjögrenin oireyhtymän taustalla oleva perintötekijä, sen mutaatiot sekä alustavasti molekyylitason tautimekanismia. Tutkimuksen tärkein löydös oli oireyhtymän taustalla olevan virheellisen SIL1-geenin tunnistaminen paikkaan perustuvaa kloonausstrategiaa käyttäen. Aikaisempien tutkimusten perusteella tiedetään, että SIL1 toimii saperoniproteiini GRP78:n nukleotidivaihtajana. Häiriintyneen saperoniproteiinien toiminnan on havaittu liittyvän perinnöllisen kaihin, tiettyjen lihastautien ja myös monien etenevien, aikuisiällä alkavien neurodegeneratiivisten sairauksien tautimekanismeihin. SIL1:n kanssa samankaltaisia proteiineja voidaankin pitää ehdokasgeeneinä muissa neurodegeneratiivisissa sairauksissa. Väitöstyössä tunnistettiin yhteensä kahdeksan SIL1-geenin mutaatiota eurooppalaisissa ja japanilaisissa perheissä. Kuusi löytämäämme mutaatiota johtaa joko suoraan tai lukukehyksen muuttumisen kautta tynkäproteiiniin, joka voi joko olla toiminnaltaan puutteellinen tai hajota. Yhden mutaation seurauksena valmiista proteiinista puuttuu joko 30 tai 64 aminohappoa, mikä viittaa siihen, että nämä aminohapot ovat oleellisia SIL1 proteiinin toiminnalle tai laskostumiselle. Lisäksi yksi mutaatio aiheuttaa valmiin proteiinin loppuosaan yhden aminohapon muutoksen, mikä johtaa virheellisen proteiinin kertymiseen solun sisään. MSS:ä aiheuttavien mutaatioiden löydyttyä uusien potilaiden diagnoosi voidaan varmistaa molekyyligeneettisin tutkimuksin. Väitöstyön tulokset antavat tärkeää tietoa MSS:n taudinkuvasta niillä potilailla, joilta löytyy mutaatio SIL1-geenissä. Kaikilta potilailta ei kuitenkaan löydy SIL1-geenin mutaatioita, ja oireyhtymän syytä näillä potilailla tutkittiin ehdokasgeeni-strategiaa käyttäen. Ehdokasgeeneiksi valittiin kolme geeniä, joilla on tunnettu tai todennäköinen interaktio SIL1-proteiinin kanssa ja jotka voisivat siten aiheuttaa samankaltaisen oirekuvan. Näiden joukosta ei kuitenkaan voitu osoittaa muita MSS:n taustalla olevia perintötekijöitä. Havaitsimme, että SIL1 ilmentyy monissa ihmisen kudoksissa ja mm. aivoissa usealla eri alueella. Hiiren kudoksissa SIL1 ja sen kanssa yhdessä toimiva GRP78 ilmenevät kehityksen aikana samankaltaisella tavalla, ja niiden ilmentyminen on runsainta pikkuaivojen purkinjensolukerroksessa, joka puuttuu MSS:ä sairastavilta potilailta lähes kokonaan. Hermosolun sisällä molemmat proteiinit sijaitsevat solulimakalvostossa. MSS:n tautimekanismin ymmärtäminen on perustana uusien hoitomuotojen kehittämiselle tulevaisuudessa ja auttaa myös muiden neurodegeneratiivisten sairauksien syiden selvittämisessä. Tutkimuksen tulokset antavat myös lisätietoa normaalin keskushermoston ja lihaskudoksen kehityksestä ja toiminnasta

Cost of providing cell-free DNA screening for Down syndrome in Finland using different strategies

Introduction A financial analysis is carried out to assess costs and benefits of providing cell-free DNA screening in Finland, using different strategies. Materials and methods Three cell-free DNA screening strategies are considered: Primary, all women; Secondary, those with positive Combined test; and Contingent, the 10-30% with the highest Combined test risks. Three costs are estimated: additional cost for 10,000 pregnancies compared with the Combined test; 'marginal' cost of avoiding a Down syndrome birth which occurs in a pregnancy that would have been false-negative using the Combined test; and marginal cost of preventing the iatrogenic loss of a non-Down syndrome birth which occurs in a pregnancy that would have been false-positive. Results Primary cell-free DNA will require additional funds of euro250,000. The marginal cost per Down syndrome birth avoided is considerably less than the lifetime medical and indirect cost; the marginal cost per unaffected iatrogenic fetal loss prevented is higher than one benefit measure but lower than another. If the ultrasound component of the Combined test is retained, as would be in Finland, the additional funds required rise to euro992,000. Secondary cell-free DNA is cost-saving as is a Contingent strategy with 10% selected but whilst when 20-30% costs rise they are much less than for the Primary strategy and are cost-beneficial. Conclusions When considering the place of cell-free DNA screening it is important to make explicit the additional and marginal costs of different screening strategies and the associated benefits. Under most assumptions the balance is favorable for Contingent screening.Peer reviewe

Alkiodiagnostiikan nykytilanne

English summaryPeer reviewe

Constitutional mosaicism for aBRCA2mutation as a cause of early-onset breast cancer

Germline mutations in theBRCA1andBRCA2genes cause hereditary breast and ovarian cancer syndrome (HBOC). Mutations in these genes are usually inherited, and reports ofde novo BRCA1/2mutations are rare. To date, only one patient with low-levelBRCA1mutation mosaicism has been published. We report on a breast cancer patient with constitutional somatic mosaicism of aBRCA2mutation.BRCA2mutation c.9294C>G, p.(Tyr3098Ter) was detected in 20% of reads in DNA extracted from peripheral blood using next-generation sequencing (NGS). TheBRCA2mutation was subsequently observed at similar levels in normal breast tissue, adipose tissue, normal right fallopian tube tissue and ovaries of the patient, suggesting that this mutation occurred early in embryonic development. This is the first case to report constitutional mosaicism for aBRCA2mutation and shows thatBRCA2mosaicism can underlie early-onset breast cancer. NGS forBRCA1/2should be considered for patients whose tumors harbor aBRCA1/2mutation and for individuals suggestive of genetic predisposition but without a family history of HBO.Peer reviewe

Multi-exon COL5A1 deletion in a child with classical Ehlers-Danlos syndrome : A case report expanding the allelic spectrum and showing evidence of parental gonosomal mosaicism

Classical Ehlers-Danlos syndrome (cEDS) is a rare inherited autosomal dominant connective tissue disorder with core clinical features including skin hyperextensibility, abnormal scarring, and generalized joint hypermobility. Classical EDS is predominantly caused by small pathogenic variants in the genes COL5A1 and COL5A2 and occasionally by a COL1A1 point mutation p.(Arg312Cys), while gross deletions or duplications are uncommon. Gonosomal mosaicism is thought to be exceedingly rare with only two cases reported in the literature. We report a child with cEDS due to a rare gross deletion of exons 2-65 in the COL5A1 gene, inherited from an unaffected mosaic father. The level of mosaicism in the father was approximately 43% in leucocyte cells and 30% in DNA extracted from skin. Our results expand the allelic spectrum of cEDS variants and suggest that parental mosaicism needs to be considered in patients with suspected cEDS, given its implication for genetic counseling.Peer reviewe

Neonatal Alexander Disease : Novel GFAP Mutation and Comparison to Previously Published Cases

Alexander disease (AxD) is a genetic leukodystrophy caused by GFAP mutations leading to astrocyte dysfunction. Neonatal AxD is a rare phenotype with onset in the first month of life. The proband, belonging to a large pedigree with dominantly inherited benign familial neonatal epilepsy (BFNE), had a phenotype distinct from the rest of the family, with hypotonia and macrocephaly in addition to drug-resistant neonatal seizures. The patient deteriorated and passed away at 6 weeks of age. The pathological and neuroimaging data were consistent with the diagnosis of AxD. Genetic analysis of the proband identified a novel de novo GFAP missense mutation and a KCNQ2 splice site mutation segregating with the BFNE phenotype in the family. The GFAP mutation was located in the coil 2B region of GFAP protein, similar to most neonatal-onset AxD cases with an early death. The clinical and neuroradiological features of the previously published neonatal AxD patients are presented. This study further supports the classification of neonatal-onset AxD as a distinct phenotype based on the age of onset.Peer reviewe

A patient with pontocerebellar hypoplasia type 6 : Novel RARS2 mutations, comparison to previously published patients and clinical distinction from PEHO syndrome

Pontocerebellar hypoplasia type 6 (PCH6) is a rare infantile-onset progressive encephalopathy caused by biallelic mutations in RARS2 that encodes the mitochondrial arginine-tRNA synthetase enzyme (mtArgRS). The clinical presentation overlaps that of PEHO syndrome (Progressive Encephalopathy with edema, Hypsarrhythmia and Optic atrophy). The proband presented with severe intellectual disability, epilepsy with varying seizure types, optic atrophy, axial hypotonia, acquired microcephaly, dysmorphic features and progressive cerebral and cerebellar atrophy and delayed myelination on MRI. The presentation had resemblance to PEHO syndrome but sequencing of ZNHIT3 did not identify pathogenic variants. Subsequent whole genome sequencing revealed novel compound heterozygous variants in RARS2, a missense variant affecting a highly conserved amino acid and a frameshift variant with consequent degradation of the transcript resulting in decreased mtArgRS protein level confirming the diagnosis of PCH6. Features distinguishing the proband's phenotype from PEHO syndrome were later appearance of hypotonia and elevated lactate levels in blood and cerebrospinal fluid. On MRI the proband presented with more severe supratentorial atrophy and lesser degree of abnormal myelination than PEHO syndrome patients. The study highlights the challenges in clinical diagnosis of patients with neonatal and early infantile encephalopathies with overlapping clinical features and brain MRI findings.Peer reviewe

Urine microRNA Profiling Displays miR-125a Dysregulation in Children with Fragile X Syndrome

A triplet repeat expansion leading to transcriptional silencing of the FMR1 gene results in fragile X syndrome (FXS), which is a common cause of inherited intellectual disability and autism. Phenotypic variation requires personalized treatment approaches and hampers clinical trials in FXS. We searched for microRNA (miRNA) biomarkers for FXS using deep sequencing of urine and identified 28 differentially regulated miRNAs when 219 reliably identified miRNAs were compared in dizygotic twin boys who shared the same environment, but one had an FXS full mutation, and the other carried a premutation allele. The largest increase was found in miR-125a in the FXS sample, and the miR-125a levels were increased in two independent sets of urine samples from a total of 19 FXS children. Urine miR-125a levels appeared to increase with age in control subjects, but varied widely in FXS subjects. Should the results be generalized, it could suggest that two FXS subgroups existed. Predicted gene targets of the differentially regulated miRNAs are involved in molecular pathways that regulate developmental processes, homeostasis, and neuronal function. Regulation of miR-125a has been associated with type I metabotropic glutamate receptor signaling (mGluR), which has been explored as a treatment target for FXS, reinforcing the possibility that urine miR-125a may provide a novel biomarker for FXS

Urine microRNA Profiling Displays miR-125a Dysregulation in Children with Fragile X Syndrome

A triplet repeat expansion leading to transcriptional silencing of the FMR1 gene results in fragile X syndrome (FXS), which is a common cause of inherited intellectual disability and autism. Phenotypic variation requires personalized treatment approaches and hampers clinical trials in FXS. We searched for microRNA (miRNA) biomarkers for FXS using deep sequencing of urine and identified 28 differentially regulated miRNAs when 219 reliably identified miRNAs were compared in dizygotic twin boys who shared the same environment, but one had an FXS full mutation, and the other carried a premutation allele. The largest increase was found in miR-125a in the FXS sample, and the miR-125a levels were increased in two independent sets of urine samples from a total of 19 FXS children. Urine miR-125a levels appeared to increase with age in control subjects, but varied widely in FXS subjects. Should the results be generalized, it could suggest that two FXS subgroups existed. Predicted gene targets of the differentially regulated miRNAs are involved in molecular pathways that regulate developmental processes, homeostasis, and neuronal function. Regulation of miR-125a has been associated with type I metabotropic glutamate receptor signaling (mGluR), which has been explored as a treatment target for FXS, reinforcing the possibility that urine miR-125a may provide a novel biomarker for FXS

Terveydenhoitotyön asiantuntijuus : Vain joka toinen kuolee tupakkaan -tupakkateemapäivä

Helsingin kaupunki toteuttaa Savuton Helsinki - Tupakoinnin ehkäisy- ja vähentämisohjelman Helsingissä vuosina 2007-2015. Ohjelman tavoitteena on tukea lasten ja nuorten tupakoimattomuutta sekä saada helsinkiläiset vähentämään tupakointia. Kouluterveyskyselyjen tuloksissa runsaasti tupakoivana ryhmänä nousivat esiin ammatillisten oppilaitosten opiskelijat. Suunnittelimme teemapäivän yhteistyötahojen kanssa kahteen ammatilliseen oppilaitokseen kevään ja syksyn 2008 aikana. Tarkoituksenamme oli pitää teemapäivä tupakoinnin haitoista ja vaikutuksista terveyteen toiminnallisia keinoja hyväksi käyttäen. Terveyden edistämisen tutkimusten mukaan pelkän tiedon jakamisen on todettu olevan tehoton tapa vaikuttaa nuoren päätökseen olla tupakoimatta. Toteutimme teemapäivät Suomen liikemiesten kauppaopistossa Pasilassa 28.10.2008 ja Helsingin liiketalousopistossa Malmilla 30.10.2008. Teemapäivän tarkoituksena oli saada opiskelijat pohtimaan tupakoimistaan ja mahdollista lopettamista sekä vahvistaa tupakoimattomien opiskelijoiden päätöstä olla tupakoimatta. Tarjosimme nuorille mahdollisuuden tutustua tupakoinnin terveysriskeihin eri aisteja hyödyntäen. Tarjosimme ajatussätkiä eli paperikääröjä, joihin oli kirjoitettu tupakointiin liittyviä kantaaottavia lauseita. Eri aistipisteissä nuorilla oli mahdollisuus kuunnella kuulokkeista tupakkayskää, haistaa pahvilaatikossa olevan tuhkakupin avulla, miltä tupakoitsija haisee ja samaistua keuhkoahtaumatautiin sairastuneen hengitykseen hengittämällä ohuen pillin läpi. Havainnollistimme tupakoinnin yhteyttä erektiohäiriöön laatikkoon asetetun tekopeniksen ja jauholla täytetyn kondomin avulla. Tupakan vaikutusta makuaistiin nuoret saivat kokeilla juomalla laimeaa mehua. Olimme tehneet myös ihmisfiguurin ja postereita, joissa kerroimme tupakoinnin haitoista terveyteen. Kaikilla teemapäivään osallistuneilla oli mahdollisuus osallistua väittämävisailukilpailuun. Tilassa oli esillä aiheeseen liittyviä julisteita ja muuta jaettavaa materiaalia. Teemapäivää varten tuottamamme materiaali jää Helsingin kaupungin opiskeluterveydenhuollon käyttöön. Lyhyellä aikavälillä teemapäivän vaikuttavuuden arviointi on vaikeaa. Pitkän aikavälin tavoitteena on kannustaa nuoria savuttomuuteen. Palautteen perusteella kokemuksellisuus on hyvä tapa välittää tietoa tupakoinnin haitoista. Katsomme onnistuneemme tavoitteessamme pysäyttää nuoret hetkeksi pohtimaan tupakoinnin mielekkyyttä. Tämänkaltainen teemapäivä ei yksin riitä tukemaan nuoria savuttomiksi, vaan teemapäivä tulisi niveltää osaksi koulun kokonaissuunnitelmaa tupakoinnin vähentämiseksi.This final thesis is a project executed in co-operation with Finnish Cancer Organisations, City of Helsinki School and Student Health Care, Haaga-Helia University of applied sciences, Helsinki and Finnish Business College, Helsinki. Our purpose was to develop a new approach to a anti-smoking campaign among young people and especially second level students at two vocational schools in Helsinki, where up to 20 per cent of students smoke at 16 years of age. Previous research indicated that just putting out information is in fact not effective when it comes to making a decision to stay smokefree, so we decided to take on a more functional method of educating the students. The theme day offered the participants a chance to become acquainted with the dangers of smoking in a new method using various senses. We had come up with for example “thought cigarettes”, paper rolls that resemble cigarettes but only contain snide remarks about smoking; an odour box, a cardboard box from which one can actually smell the “sweet smell of tobacco”; we offered everyone a glass of extra-mild juice to exemplify what smoking does to one’s sense of flavour; one could try breathing through a straw to get a feel as to what it’s like to have chronic obstructive pulmonary disease (COPD); we even had a cardboard box with two fake penises inside – one stiff and the other a condom filled with flour – to clarify the fact that smoking causes impotence. We also had posters and a quiz for all the participants to fill. The results were encouraging. Mostly the feedback we got from the participants was positive. Based on this experience we concluded that making good use of one’s senses is indeed a good method of educating young people on the hazards of smoking. Then again it seemed that we got a better response from the students when their participation was not based on volunteering. It is quite hard to assess the immediate effect of our theme day, but in the long run our objective was to give the students information and education. Thus we think they now have some new tools for making the decision themselves to cease smoking. However, such a theme day would most likely be more effective if it was combined with an overall antismoking policy at the school it was held at.Henna Leppälä, Jenna Meriläinen, Heini Murtonen, Paula Nylander, Minna Osmala, Tanja Seppänen. (Kaikki tekijät eivät mahtuneet tekijäkenttään.