Identification of two novel human neuronal ceroid lipofuscinosis genes

The neuronal ceroid lipofuscinoses (NCLs) are a group of mostly autosomal recessively inherited neurodegenerative disorders. The aim of this thesis was to characterize the molecular genetic bases of these, previously genetically undetermined, NCL forms. Congenital NCL is the most aggressive form of NCLs. Previously, a mutation in the cathepsin D (CTSD) gene was shown to cause congenital NCL in sheep. Based on the close resemblance of the phenotypes between congenital NCLs in sheep and human, CTSD was considered as a potential candidate gene in humans as well. When screened for mutations by sequencing, a homozygous nucleotide duplication creating a premature stop codon was identified in CTSD in one family with congenital NCL. While in vitro the overexpressed truncated mutant protein was stable although inactive, the absence of CTSD staining in brain tissue samples of patients indicated degradation of the mutant CTSD in vivo. A lack of CTSD staining was detected also in another, unrelated family with congenital NCL. These results imply that CTSD deficiency underlies congenital NCL. While initially Turkish vLINCL was considered a distinct genetic entity (CLN7), mutations in the CLN8 gene were later reported to account for the disease in a subset of Turkish patients with vLINCL. To further dissect the genetic basis of the disease, all known NCL genes were screened for homozygosity by haplotype analysis of microsatellite markers and/or sequenced in 13 mainly consanguineous, Turkish vLINCL families. Two novel, family-specific homozygous mutations were identified in the CLN6 gene. In the remaining families, all known NCL loci were excluded. To identify novel gene(s) underlying vLINCL, a genomewide single nucleotide polymorphism scan, homozygosity mapping, and positional candidate gene sequencing were performed in ten of these families. On chromosome 4q28.1-q28.2, a novel major facilitator superfamily domain containing 8 (MFSD8) gene with six family-specific homozygous mutations in vLINCL patients was identified. MFSD8 transcript was shown to be ubiquitously expressed with a complex pattern of alternative splicing. Our results suggest that MFSD8 is a novel lysosomal integral membrane protein which, as a member of the major facilitator superfamily, is predicted to function as a transporter. Identification of MFSD8 emphasizes the genetic heterogeneity of Turkish vLINCL. In families where no MFSD8 mutations were detected, additional NCL-causing genes remain to be identified. The identification of CTSD and MFSD8 increases the number of known human NCL-causing genes to eight, and is an important step towards the complete understanding of the genetic spectrum underlying NCLs. In addition, it is a starting point for dissecting the molecular mechanisms behind the associated NCLs and contributes to the challenging task of understanding the molecular pathology underlying the group of NCL disorders.

The brain serotonin transporter binding in young adults; methodological considerations and association with Bulimia Nervosa and acquired obesity

The neurotransmitter serotonin (5-HT) modulates many functions important for life, e.g., appetite and body temperature, and controls development of the neural system. Disturbed 5-HT function has been implicated in mood, anxiety and eating disorders. The serotonin transporter (SERT) controls the amount of effective 5-HT by removing it from the extracellular space. Radionuclide imaging methods single photon emission tomography (SPET) and positron emission tomography (PET) enable studies on the brain SERTs. This thesis concentrated on both methodological and clinical aspects of the brain SERT imaging using SPET. The first study compared the repeatability of automated and manual methods for definition of volumes of interest (VOIs) in SERT images. The second study investigated within-subject seasonal variation of SERT binding in healthy young adults in two brain regions, the midbrain and thalamus. The third study investigated the association of the midbrain and thalamic SERT binding with Bulimia Nervosa (BN) in female twins. The fourth study investigated the association of the midbrain and hypothalamic/thalamic SERT binding and body mass index (BMI) in monozygotic (MZ) twin pairs. Two radioligands for SERT imaging were used: [123I]ADAM (studies I-III) and [123I]nor-beta-CIT (study IV). Study subjects included young adult MZ and dizygotic (DZ) twins screened from the FinnTwin16 twin cohort (studies I-IV) and healthy young adult men recruited for study II. The first study validated the use of an automated brain template in the analyses of [123I]ADAM images and proved automated VOI definition more reproducible than manual VOI definition. The second study found no systematic within-subject variation in SERT binding between scans done in summer and winter in either of the investigated brain regions. The third study found similar SERT binding between BN women (including purging and non-purging probands), their unaffected female co-twins and other healthy women in both brain regions; in post hoc analyses, a subgroup of purging BN women had significantly higher SERT binding in the midbrain as compared to all healthy women. In the fourth study, MZ twin pairs were divided into twins with higher BMI and co-twins with lower BMI; twins with higher BMI were found to have higher SERT binding in the hypothalamus/thalamus than their leaner co-twins. Our results allow the following conclusions: 1) No systematic seasonal variation exists in the midbrain and thalamus between SERT binding in summer and winter. 2) In a population-based sample, BN does not associate with altered SERT status, but alterations are possible in purging BN women. 3) The higher SERT binding in MZ twins with higher BMIs as compared to their leaner co-twins suggests non-genetic association between acquired obesity and the brain 5-HT system, which may have implications on feeding behavior and satiety.

MCT1-, MCT4- ja CD147-proteiinit kehittyvässä kilin pötsissä

Nuoren märehtijän alkaessa syödä kiinteää ravintoa, etumahojen suhteellinen osuus mahoista kasvaa ja niiden seinämän epiteeli alkaa kehittyä mahdollistaakseen ravintoaineiden tehokkaan imeytymisen. Märehtijöillä rehun hiilihydraatit hajoavat pötsissä haihtuviksi rasvahapoiksi, ja monokarboksylaattikuljettajien uskotaan avustavan haihtuvien rasvahappojen imeytymisessä pötsin seinämän läpi. Pötsin seinämässä on todettu olevan ainakin MCT1- ja MCT4 –isoformeja. Nämä tarvitsevat toimiakseen CD147 -proteiinin (myös OX-47, EMMPRIN, HT7 ja basigin), joka on on glykosyloitu integraalinen membraaniproteiini. Tämän tutkimuksen tarkoituksena oli selvittää MCT1-, MCT4- ja CD147 –proteiinien muutoksia pötsin toiminnan kehittymisen aikana. Toisena tavoitteena oli selvittää, voidaanko näytteenä käyttää solukalvojen sijasta pötsin seinämästä tehtyä homogenaattia. Tutkimuksessa käytettiin eri ikäisinä lopetetuista kileistä kerättyjä näytteitä. Kilejä oli yhteensä 31, joista 7 oli 3-21 tunnin ikäisiä, 7 viikon ikäisiä, 7 kahden viikon ikäisiä, 1 kolmen viikon ikäinen, 2 neljän viikon ikäistä, sekä 7 kahdeksan viikon ikäistä. Pötsin seinämästä otettiin näyte, josta valmistettiin homogenaatti ja eristettiin solukalvot eli membraanit. Pötsinäytteistä löydettiin MCT1- ja CD147 –proteiineja, mutta MCT4- isoformia ei ollut havaittavissa. Membraaninäytteissä havaittiin MCT1 -isoformin pitoisuuksien kasvavan iän mukana, paitsi kahdeksan viikon ikäisillä kileillä, joilla MCT1 –isoformin määrät vähenivät merkitsevästi. CD147 –proteiinia oli havaittavissa jo vastasyntyneiden kilien pötsinäytteissä. Membraaninäytteissä CD147 -proteiinin määrä kasvoi lineaarisesti iän mukana ja CD147- proteiinin ja MCT1 –isoformin välillä havaittiin tilastollisesti merkitsevä korrelaatio. Homogenaattinäytteissä MCT1 -isoformin määrissä ei havaittu korrelaatiota iän kanssa. MCT1- ja MCT4 -isoformien solukalvolle siirtymisessä avustavan CD147 –proteiinin ei myöskään havaittu korreloivan koe-eläinten iän tai MCT1 -isoformin kanssa. Membraani- ja homogenaattinäytteistä mitattujen MCT1- ja CD147 -määrien välillä ei ollut korrelaatiota. Haihtuvien rasvahappojen muodostus alkaa, kun eläin aloittaa kiinteän ravinnon syömisen. Tästä seuraa, että haihtuvia rasvahappoja kuljettavia proteiineja tarvitaan epiteelisolujen pinnalle. Tutkimuksessa havaittiin haihtuvia rasvahappoja kuljettavan MCT1 –proteiinin ja sen apuproteiinien määrän lisääntyminen iän myötä. N. 8-11 viikon iässä, jolloin pötsin toiminta on kehittynyt aikuisen eläimen tasolle, MCT1 –proteiinin määrä oli merkitsevästi vähäisempi kuin 4 viikon iässä. Tulosten perusteella homogenaatti ei ole hyvä tapa mitata membraaniproteiinien määrää.