The mitochondrion is a tiny organelle that is the power supplier of the cell and vital to the functioning of the body organs. Additionally it contains a small circular genome of about 16 kb, present in many copies which makes the mitochondrial DNA more viable than nuclear DNA. Mitochondrial DNA is also maternally inherited and thus provides a direct link to maternal relatives. These two properties are of particular use for forensic samples, which only contain limited or degraded amounts of DNA, and for historical samples (ancient DNA). This thesis presents work on the mitochondrial DNA in the hypervariable regions (HV) I and II, in both contemporary and historical samples. Forensic genetics makes use of mitochondrial DNA analysis in court as circumstantial evidence, and population databases are used for the calculation of evidence value. Population samples (299) across Sweden have been analysed in order to enrich the EDNAP mtDNA database (EMPOP) (paper I). The application of mitochondrial DNA analysis allowed for analysis of historical skeletal remains: Copernicus, 1473-1543 (paper II), Karin Göring, 1888-1931 (paper III) and Medieval bones, 880-1000 AD, from a mass grave found in Sigtuna, Sweden (paper IV). The thesis also includes analyses of bones and teeth from the shipwrecked crew of the Vasa warship, 1628, samples from the Vasa museum, Stockholm, Sweden (paper V). Overall, the varying age of the samples and the different conservation environments (soil and water) accounted for variations in quality, but still allowed for successful DNA analysis.

Abstract Cellular energy is produced by the mitochondria via oxidative phosphorylation. In addition to nuclear DNA; the mitochondrion contains circular mitochondrial DNA (mtDNA) molecules. MtDNA is maternally inherited and encodes 37 genes that are crucial for the energy production of the cell. Mutations in the mtDNA cause mitochondrial diseases that manifest as maternally inherited energy metabolism disorders. Common symptoms include diabetes mellitus, myopathy, sensorineural hearing impairment, eye and vision problems, epilepsy and brain manifestations (encephalopathy). Mitochondrial mutations are often heteroplasmic; cells and tissues contain a mix of healthy and mutated mtDNA. The percentage of mutated mtDNA contributes to the severity of symptoms. Mitochondrial DNA also contains numerous polymorphisms; some of which have been reported to be non-neutral, thus contributing to the occurrence of common diseases. Whole mtDNA sequences were obtained from patients with diabetes mellitus (64), epilepsy (79) and unknown mitochondrial disease (66) using conformation-sensitive gel electrophoresis and direct sequencing. Whole mtDNA sequences of a Finnish family with ataxia were also obtained. Restriction fragment length analysis and cloning were used for heteroplasmy quantification. Whole mitochondrial genomes were organized into phylogenetic trees. All nonsynonymous mutations were analyzed with pathogenicity predicting algorithms (SNAP, PolyPhen-2, PMut, SIFT Blink). Non-neutral risk mutations were identified in diabetes mellitus and epilepsy patients. These patients had maternal relatives with diabetes, epilepsy and/or sensorineural hearing impairment. M. 3010A>G and m.16189T>C were found in increased frequency in diabetics and the haplogroup U5b variant m.15218A>G was detected more often among patients with epilepsy. These mutations were predicted to be deleterious in effect. Mitochondrial haplogroup V was found in increased frequency in matrilineal diabetes mellitus patients. We identified an m.8993T>C mutation in a Finnish family with ataxia. This mutation caused an adult-onset ataxic phenotype; previous studies have reported only juvenile onset phenotypes. Novel and rare mtDNA mutations were discovered in patients with an unspecified mitochondrial disease phenotype; these included an insertion m.7585insT and a novel MTTT mutation m. 15933G>A. This thesis emphasizes the importance of full mtDNA sequencing in patients with a suspected mitochondrial disease; novel mutations remain undetected if only the most common mutations are screened. In addition, the increasing importance of non-neutral mtDNA risk variants is supported by the findings of this thesis. In the future, individualized genetics and information on personal risk alleles will become even more important for maintaining health on a personal level. / Tiivistelmä Mitokondriot ovat energiaa tuottavia soluelimiä. Mitokondrioissa on oma rengasmainen mitokondriaalinen DNA (mtDNA), joka esiintyy solussa useana kopiona. MtDNA periytyy vain äidin kautta, joten kaikille lapsille periytyy sama mitokondriaalinen DNA. MtDNA koodaa 37:ää geeniä, jotka ovat tärkeitä solun energiantuotannolle. Geenimuutos mtDNA:ssa voi aiheuttaa äidiltä periytyvän mitokondriotaudin. Mitokondriotaudit ovat energia-aineenvaihdunnan sairauksia, joissa tavallisia oireita ovat diabetes mellitus, lihasoireet (esimerkiksi lihasten ennenaikainen väsymys, myopatia), sydänlihasoireet, maksaoireet, silmä- ja näköoireet, aistimistyyppinen kuulovika sekä aivo-oireet, kuten epilepsia. Oireet vaihtelevat huomattavasti, ja sama mutaatio voi aiheuttaa hyvin erilaisia taudinkuvia. Vakavimmillaan mitokondriotauti voi johtaa kuolemaan jo varhaislapsuudessa. Mutaation prosenttiosuus eli heteroplasmia-aste on usein oireiden vakavuutta määrittelevä tekijä. Mitokondriaalinen DNA muuntuu nopeasti evoluution aikana, joten siinä esiintyy paljon normaalia vaihtelua (polymorfioita). Osa näistä polymorfioista on kuitenkin todettu lievästi haitallisiksi, ja ne lisäävät riskiä sairastua kansanterveydellisesti yleisiin sairauksiin, kuten diabetekseen. Kartoitimme koko mitokondriogenomin muutokset eri potilasryhmiltä, joihin kuului diabetesta, epilepsiaa ja ataksiaa sairastavia potilaita. Lisäksi tutkittiin potilaita, joilla epäiltiin mitokondriotautia. Keskeiset käytetyt menetelmät olivat DNA:n rakenteellisia muutoksia havaitseva geelielektroforeesi ja sekvensointi. Määritimme heteroplasmian käyttäen restriktioentsyymianalyysia sekä kloonausta bakteerisoluihin. Järjestimme potilaiden mtDNA-sekvenssit fylogeneettisiksi puiksi ja kaikki proteiinin koodausta muuttavat geenimuutokset analysoimme haitallisuutta ennustavilla tietokoneohjelmilla (SNAP, PolyPhen-2, PMut, SIFT BLink). Diabetesta sairastavilla potilailla, joilla myös äidinpuoleisessa suvussa esiintyy diabetesta, havaitsimme useammin m.3010A>G- ja m.16189T>C-geenimuutoksia kuin väestöllä keskimäärin. Tutkimustulos tukee aikaisemmin julkaistuja tutkimustuloksia m.16189T>C-geenimuutoksen haitallisuudesta. Epilepsiapotilailta löytyi m.15218A>G-geenimuutos kahdessa U5a1-haploryhmän alatyypissä. Patogeenisyysanalyysien mukaan nämä geenimuutokset olivat haitallisia. Mitokondriaalinen haploryhmä V havaittiin useammin diabetesta sairastavilla kuin terveillä henkilöillä. Tutkimukseen valittiin potilaita, joiden äidinpuoleisilla lähisukulaisilla esiintyi yhtä tai useampaa seuraavista: diabetes, epilepsia tai aistimistyyppinen kuulovika. Väitöstutkimuksessa todetaan lisäksi, että m.8993T>C-mutaatio aiheuttaa aikuisiällä alkavaa ataksia-oireistoa. Kyseinen mutaatio on aiemmin yhdistetty vain lapsuusiän taudinkuviin. Kuvasimme uuden insertiomutaation (m.7585insT) kardiomyopatiasuvussa sekä uuden MTTT-geenin mutaation (m.15933G>A) tuntematonta mitokondriotautia sairastavalla potilaalla. Väitöskirjatutkimuksen tulokset osoittavat, että on tärkeää tutkia koko mitokondriogenomi, kun kyseessä on tuntemattomaksi jäänyt mitokondriaalinen taudinkuva. Uudet, tautia aiheuttavat, mtDNA:n geenimuutokset voivat jäädä tunnistamatta, jos tutkitaan ainoastaan raportoidut, tunnetut, mutaatiot. Lisäksi voi todeta, että mitokondriogenomissa esiintyy lievästi haitallisia geenimuutoksia tai niiden yhdistelmiä, jotka saattavat lisätä riskiä sairastua kansanterveydellisesti merkittäviin sairauksiin, kuten diabetekseen.

Recent research demonstrated that besides a role in sex determination and male fertility, the Y chromosome is involved in additional functions including prostate cancer, sex-specific effects on the brain and behaviour, graft-versus-host disease, nociception, aggression and autoimmune diseases. The results presented in this thesis include an analysis of sex-biased genes encoded on the X and Y chromosomes of rodents. Expression data from six different somatic tissues was analyzed and we found that the X chromosome is enriched in female biased genes and depleted of male biased ones. The second study described copy number variation (CNV) patterns in a world-wide collection of human Y chromosome samples. Contrary to expectations, duplications and not deletions were the most frequent variations. We also discovered novel CNV patterns of which some were significantly overrepresented in specific haplogroups. A substantial part of the thesis focuses on analysis of spatial expression of two Y-encoded brain-specific genes, namely PCDH11Y and NLGN4Y. The perhaps most surprising discovery was the observation that X and Y transcripts of both gene pairs are mostly expressed in different cells in human spinal cord and medulla oblongata. Also, we detected spatial expression differences for the PCDH11X gene in spinal cord. The main focus of the spatial investigations was to uncover genetically coded sexual differences in expression during early development of human central nervous system (CNS). Also, investigations of the expression profiles for 13 X and Y homolog gene pairs in human CNS, adult brain, testes and still-born chimpanzee brain samples were included. Contrary to previous studies, we found only three X-encoded genes from the 13 X/Y homologous gene pairs studied that exhibit female-bias. We also describe six novel non-coding RNAs encoded in the human MSY, some of which are polyadenylated and with conserved expression in chimpanzee brain. The description of dimorphic cellular expression patterns of X- and Y-linked genes should boost the interest in the human specific gene PCDH11Y, and draw attention to other Y-encoded genes expressed in the brain during development. This may help to elucidate the role of the Y chromosome in sex differences during early CNS development in humans. / <p>chinese, finnish, norwegian, schizophrenia, bipolar, bipolar disorder, msy, male specific region Y, PAR1, PAR2, pseudoautosomal, male-biased, female-biased, male biased, female biased, ashkenazi population, structure, variants, YHRD, Elena Jazin, Björn Reinius, Per Ahlberg, brain, hjärna, CNS, central nervous system, IR2, inverted repeat 2, isodicentric, genetics, genetik, padlock, rolling circle, amplification, PCR, sY1191, sY1291, STS, DDX3Y, DAZ, AZFa, AZFb, AZFc, AZF, Repping, haplogroup J, rearrangements, DE-M145, I-M170, E-M96, Q-M242, R-M207, O-M175, G-M201, D-M174, C-M130, NO-M214, N-M231, poland</p>