Transient modification of lin28b expression - Permanent effects on zebrafish growth

Recent genome-wide association studies and mouse models have identified LIN28B as a gene affecting several pubertal timing-related traits and vertebrate growth. However, the exact biological mechanisms underlying the associations remain unknown. We have explored the mechanisms linking LIN28B with growth regulation by combining human gene expression data with functional models. Specifically, we show that 1) pubertal timing-associated genetic variation correlates with LIN28B expression in the pituitary and hypothalamus, 2) downregulating lin28b in zebrafish embryos associates with aberrant development of kiss2-neurons, and 3) increasing lin28b expression transiently by synthetic mRNA injections during embryogenesis results in sustained enhancement of zebrafish growth. Unexpectedly, the mRNA injections resulted in advanced sexual maturation of female fish, suggesting that lin28b may influence pubertal timing through multiple developmental mechanisms. Overall, these results provide novel insight into LIN28B function in vertebrate growth regulation, emphasizing the importance of the gene and related genetic pathways for embryonic and juvenile development.Peer reviewe

Moving Beyond GWAS : Exploring the Function of the Gene LIN28B Associated With Pubertal Timing

Over the past decade, advances in genetics have led to identification of thousands of genetic loci in the human genome that contribute to complex traits such as the timing of pubertal onset. Following genome-wide association studies (GWAS), we currently know how a large proportion of the heritability of complex traits can be explained by common genetic variation in these loci. However, in many instances, we understand relatively little about the biology behind these genetic associations, and functional characterization of genetic variation has become a new bottleneck for genetic research. Prior to this thesis project, sequence variants nearby lin-28 homolog B (LIN28B) had become associated with pubertal timing in the general population. Remarkably, compared to all other common genetic variants, the variants in the LIN28B locus appeared to exert relatively large effects on pubertal timing: one pubertal timing advancing allele associating with ~1,5 months advancement in age at menarche (AAM) in females. Yet, the gene showed little evidence of affecting the established mechanisms behind pubertal onset. Exploring the molecular mechanisms by which LIN28B affects puberty therefore became the focus of this thesis, which is is founded on three original studies addressing the function of LIN28B. To move beyond the original GWAS results, we first utilized Finnish population cohorts to assess the potential pleiotropy of the gene in terms of body size and adult health. The second original study combined human gene expression data from the GTEx database with zebrafish models, evaluating the consequences of transient dysregulation of lin28b during embryogenesis. For the third study, we first created lin28b knockout zebrafish with CRISPR-Cas9 technology to evaluate the effects of permanent lin28b knockout, and utilized the GTEx data and the UK biobank resource to study whether LIN28B contributes to sex steroid signaling in humans. The results presented in this thesis suggest that LIN28B has pleiotropic actions on vertebrate phenotypes and may contribute to the timing and tempo of human growth in more complex ways than originally thought. Our studies demonstrate that the gene associates with variation in several body size parameters in adult humans, although showing little evidence of affecting metabolism. The results also suggest that the effects that LIN28B has on body size are evolutionarily conserved. Overexpression of lin28b during embryogenesis appears to stimulate zebrafish growth, and, intriguingly, lin28b knockout zebrafish show similar growth patterns as humans that carry sequence variants linked with lower LIN28B expression. Importantly, the data presented in this thesis indicates that the sequence variants associating with pubertal timing affect LIN28B expression mostly in the hypothalamus and the pituitary of adult humans. These changes in the LIN28B expression level may have further consequences: LIN28B expression at the hypothalamic-pituitary (HP) axis seems to correlate positively with the expression of several hormonal genes like ESR1 and POMC. Highlighting the gene’s potential to contribute to sex steroid signaling, we finally associated LIN28B with the regulation of testosterone levels in adult humans, which might be relevant in terms of explaining many of the GWAS associations including pubertal timing. Overall, the results presented in this thesis offer novel insight into LIN28B function in pubertal timing, disease and development, the project simultaneously serving as an example for follow-up studies of GWAS loci in general.Edistysaskeleet genetiikan tutkimuksessa viimeisen kymmen vuoden aikana ovat johtaneet satojen sellaisten perimän kohtien tunnistamiseen, joissa esiintyvä vaihtelu vaikuttaa monitekijäisiin tauteihin ja ominaisuuksiin. Genominlaajuisten assosiaatiotutkimusten (GWAS) ansiosta tiedämme nyt kuinka merkittävä osuus monitekijäisten ominaisuuksien periytyvyydestä selittyy näiden kohtien yhteisvaikutuksella. Biologia näiden geneettisten assosiaatioiden taustalla on kuitenkin huonosti ymmärrettyä. Tarpeesta ymmärtää miten nämä perimän kohdat vaikuttavat eri ominaisuuksiin onkin muodostunut uusi pullonkaula geneettiselle tutkimukselle. Tämä väitöskirja sai alkunsa halusta ymmärtää murrosikään liittyvien GWAS-löydösten taustaa, keskittyen siihen miten LIN28B geenin läheisyydessä sijaitseva perinnöllinen vaihtelu vaikuttaa murrosiän ajoittumiseen ihmisillä. LIN28B valikoitui tutkimuksen kohteeksi erityisesti kahdesta syystä. Ensiksi, geenin lähellä sijaitseva geneettinen vaihtelu näyttää vaikuttavan murrosiän ajoittumiseen enemmän kuin mikään toinen yleinen muutos genomissa: esimerkiksi yksi puberteettia varhaistava alleeli assosioituu n. 1,5 kuukautta varhaisempaan kuukautisten alkamiseen. Toiseksi, geeni ei kuitenkaan näyttänyt kytkeytyvän niihin mekanismeihin, joiden oli osoitettu vaikuttavan murrosiän alkamisen taustalla. Tämä väitöskirja perustuu kolmeen osatyöhön. Ensimmäisessä tutkimuksessa hyödynsimme suomalaisia väestöaineistoja selvittääksemme voisiko LIN28B liittyä laajemminkin kehon koon säätelyyn ja aikuisterveyteen. Toisessa osatyössä yhdistimme ihmisten geeniekspressiodataa seeprakalamallien kanssa, tutkiaksemme miten GWAS tutkimuksissa löydetyt geenivariantit vaikuttivat LIN28B:n ilmentymiseen, ja kuinka sikiöaikainen muuntelu lin28b:n ekspressiomäärässä vaikuttaa kalojen kehitykseen. Kolmannessa osatyössä jatkoimme geeniekspressiodatan analyysiä ja loimme poistogeenisiä lin28b-seeprakalamalleja tutkiaksemme pysyvän lin28b:n hiljentämisen vaikutuksia. Tässä väitöskirjassa esitetyt tulokset osoittavat LIN28B:n vaikuttavan kasvuun ja kehitykseen monin eri tavoin: geeni näyttäisi liittyvän esimerkiksi ihmisten kehon koon säätelyyn luultua monimutkaisemmalla tavalla. Väitöskirjassa esitetyt tulokset tukevat käsitystä LIN28B evoluutiossa säilyneestä roolista selkärankaisten kasvun säätelyssä. Hetkellinen lin28b:n yliekpressio seeprakalan alkioissa näyttäisi stimuloivan kalojen kasvua, siinä missä pysyvä lin28b:n hiljentäminen johtaa samanlaisiin muutoksiin kasvussa kuin ihmisillä joilla on matalampaan LIN28B ekspressiotasoon liittyvä muoto geenistä. Tutkimuksemme osoittavat kuinka sama perimän vaihtelu joka liittyy murrosiän ajoittumiseen LIN28B geenin lähellä, vaikuttaa LIN28B:n ilmentymiseen erityisesti etenkin murrosiän kannalta tärkeissä kudoksissa, hypotalamuksessa ja aivolisäkkeessä. Tällä on mahdollisesti myös toiminnallisia seurauksia: LIN28B ilmentyminen näyttäisi korreloivan useiden hormonaalisten geenien ilmentymisen kanssa. Korostaen geenin mahdollista roolia sukupuolihormoneihin liittyvään signalointiin liittyen, väitöstutkimukseni nostaa esiin erityisesti ESR1- ja POMC-geenit, sekä linkittää LIN28B:n testosteronitasojen säätelyyn aikuisilla, joka voisi selittää osan LIN28B:hen liitetyistä fenotyypeistä. Kaiken kaikkiaan, tutkimusprojektit jotka esitellään kootusti tässä väitöskirjassa, ovat tuoneet lisätietoa siihen miten LIN28B vaikuttaa murrosikään, terveyteen ja yksilönkehitykseen, jonka lisäksi projektimme toimii myös yleisellä tasolla esimerkkinä keinoista joilla tutkia GWAS-löydösten biologista taustaa

LIN28B affects gene expression at the hypothalamic-pituitary axis and serum testosterone levels

Genome-wide association studies (GWAS) have recurrently associated sequence variation nearby LIN28B with pubertal timing, growth and disease. However, the biology linking LIN28B with these traits is still poorly understood. With our study, we sought to elucidate the mechanisms behind the LIN28B associations, with a special focus on studying LIN28B function at the hypothalamic-pituitary (HP) axis that is ultimately responsible for pubertal onset. Using CRISPR-Cas9 technology, we first generated lin28b knockout (KO) zebrafish. Compared to controls, the lin28b KO fish showed both accelerated growth tempo, reduced adult size and increased expression of mitochondrial genes during larval development. Importantly, data from the knockout zebrafish models and adult humans imply that LIN28B expression has potential to affect gene expression in the HP axis. Specifically, our results suggest that LIN28B expression correlates positively with the expression of ESR1 in the hypothalamus and POMC in the pituitary. Moreover, we show how the pubertal timing advancing allele (T) for rs7759938 at the LIN28B locus associates with higher testosterone levels in the UK Biobank data. Overall, we provide novel evidence that LIN28B contributes to the regulation of sex hormone pathways, which might help explain why the gene associates with several distinct traits.Peer reviewe

Dissecting the Role of Trisomy 21 in Childhood Acute Lymphoblastic Leukaemia

Children with Down’s Syndrome (DS) have a twenty fold increased risk of developing acute lymphoblastic leukaemia (ALL) and a five hundred fold increased chance of developing acute megakaryoblastic leukaemia (AMKL). This suggests a role for trisomy 21 as a ‘first hit’ event, predisposing these children to leukaemogenesis. Childhood leukaemias differ from adult leukaemias in that they have an increased incidence, improved prognosis and have a distinct mutational spectrum. I hypothesized that T21 distorts DS foetal hematopoiesis inducing a predisposition to B-ALL and that DS iPSCs could recapitulate these effects providing an in- vitro approach to identify likely target cells for 2nd hits. The objective of this project is to utilise human DS induced pluripotent stem cells (DS-iPSCs) to model B-cell development in children with DS and to elucidate the effects of trisomy 21 on B-cell differentiation in-vitro and relate this to the characteristics of DS-ALL. In-vitro B-cell differentiation of isogenic DS iPSCs produced both pro and preB cells and an immature cellular compartment termed the LM cell immunophenotypically identified as CD34+ CD38- CD33hi CD45Rahi that displayed misexpression of the immature myeloid marker CD33. I hypothesised misexpression of CD33 induced a failure in lineage resolution and a partial block in B cell commitment at the LM cell. Transcriptomic analysis of LM, pro and preB compartments revealed enhanced stem, myeloid, cell cycle and protein synthesis gene expression pathways in the T21 LM progenitor suggestive of pre-leukaemic development. This suggests a DS hematopoietic hierarchical model in which a failure of lineage resolution in the T21 LM progenitor results in impaired B-cell commitment and conflicting lympho-myeloid signature observed in the B- cell compartment. I propose the LM cell is a potential in-utero target cell for DS pre- leukaemic initiation and that this DS-iPSC system offers a platform for targeting potential therapeutic candidates of DS-ALL

Influence and regulation of PCBP2 and YTHDF2 RNA-binding proteins during self-renewal and differentiation of human induced pluripotent stem cells

2019 Fall.Includes bibliographical references.Embryonic stem cells (ESCs) are able to self-renew or differentiate into any cell type in the body, a property known as pluripotency that enables them to initiate early growth and development. However, the ethical implications of harvesting and manipulating ESCs hinders their use in basic research and the clinical applications. Thus, the discovery that somatic cells can be exogenously reprogrammed into induced pluripotent stem cells (iPSCs) offers new and exciting possibilities for gene therapy, personalized medicine and basic research. However, more research is needed into the mechanisms involved in regulating pluripotency in order for iPSCs to reach their full potential in the research lab and clinic. To maintain a state of self-renewal, yet also be able to rapidly differentiate in response to external signals, pluripotent stem cells need to exert tight control over gene expression through transcriptional and post-transcriptional mechanisms. There are several notable transcriptional networks that regulate pluripotency, but the post-transcriptional mechanisms remain poorly characterized. mRNA decay is one form of post-transcriptional regulation that can help to both maintain the steady-state of a transcriptome or facilitate its rapid remodeling. To this end, degradation rates are influenced by the elements contained in an mRNA and the RNA-binding proteins (RBPs) they associate with. Previous reports have indicated the RNA modification N6-methyladenosine (m6A) and C-rich sequence elements (CREs) can affect mRNA decay in pluripotent stem cells. Therefore, we sought to further understand the roles of m6A and CREs in mRNA decay in stem cells by characterizing the expression and mRNA targets of two RBPs that recognize these elements, YTHDF2 and PCBP2, respectively. In this thesis, I report YTHDF2 is differentially regulated in pluripotent and differentiated cells and that YTHDF2 contributes to pluripotency by targeting a group of mRNAs encoding factors important for neural development. The down-regulation of YTHDF2 during neural differentiation is consistent with increased expression of neural factors during this time. Moreover, YTHDF2 expression is regulated at the level of translation via elements located in the first 300 nucleotides of the 3' untranslated region of YTHDF2 mRNA. Based on these results, I propose that stem cells are primed for rapid differentiation by transcribing low levels of mRNAs encoding neural factors that are subsequently targeted for degradation, in part by YTHDF2, until differentiation is induced. On the other hand, PCBP2 is up-regulated upon differentiation of pluripotent stem cells and regulates several mRNAs associated with pluripotency and development, including LIN28B. Notably, expression of long non-coding RNAs (lncRNAs) that contain human endogenous retrovirus element H (HERV-H) is influenced by PCBP2. HERV-H lncRNAs are almost exclusively expressed in stem cells and play a role in maintaining a pluripotent state, although their functions are not fully understood. Intriguingly, some HERV-lncRNAs can also regulate PCBP2 expression, as altering the expression of LINC01356 or LINC00458 effects PCBP2 protein levels. Based on these results, I propose the reciprocal regulation of PCBP2 and HERV-H lncRNAs influences whether stem cells maintain a state of self-renewal or differentiate. Taken together, these findings demonstrate that YTHDF2 and PCBP2 post-transcriptionally regulate gene expression in stem cells and influence pluripotency

Investigation of The Genetic Regulation of Delayed Puberty

PhDThe genetic control of puberty remains an important but mostly unanswered question. Late pubertal timing affects over 2% of adolescents and is associated with adverse health outcomes. Self-limited delayed puberty (DP) segregates in an autosomal dominant pattern and is highly heritable; however, its neuroendocrine pathophysiology and genetic regulation remain unclear. The genetic control of puberty remains an important but mostly unanswered question. Late pubertal timing affects over 2% of adolescents and is associated with adverse health outcomes. Self-limited delayed puberty (DP) segregates in an autosomal dominant pattern and is highly heritable; however, its neuroendocrine pathophysiology and genetic regulation remain unclear. Our large, accurately phenotyped cohort of patients with familial self-limited DP is a unique resource with a relatively homogeneous genetic composition. I have utilised this cohort to investigate the genetic variants segregating with the DP trait in these pedigrees. Whole exome sequencing in eighteen probands and their relatives, and subsequent targeted sequencing in an extended subgroup of the cohort, has revealed potential novel genetic regulators of pubertal timing. In ten unrelated probands, I identified rare mutations in IGSF10, a gene that is strongly expressed in the nasal mesenchyme during embryonic migration of gonadotropin-releasing hormone (GnRH) neurons. IGSF10 knockdown both in vitro and in a transgenic zebrafish model resulted in perturbed GnRH neuronal migration. Loss-of-function mutations in IGSF10 were also identified in five patients with absent puberty due to hypogonadotropic hypogonadism (HH). Additionally, I have identified and investigated one rare, pathogenic mutation in HS6ST1 – a gene known to cause HH - in one family with DP, and two rare variants in FTO – a gene implicated in the timing of menarche in the general population - in 3 families. Further potentially pathogenic variants have emerged from investigating candidate genes identified from microarray studies (LGR4, SEMA6A and NEGR1) and from related clinical phenotypes (IGSF1). Our large, accurately phenotyped cohort of patients with familial self-limited DP is a unique resource with a relatively homogeneous genetic composition. I have utilised this cohort to investigate the genetic variants segregating with the DP trait in these pedigrees. Whole exome sequencing in eighteen probands and their relatives, and subsequent targeted sequencing in an extended subgroup of the cohort, has revealed potential novel genetic regulators of pubertal timing. In ten unrelated probands, I identified rare mutations in IGSF10, a gene that is strongly expressed in the nasal mesenchyme during embryonic migration of gonadotropin-releasing hormone (GnRH) neurons. IGSF10 knockdown both in vitro and in a transgenic zebrafish model resulted in perturbed GnRH neuronal migration. Loss-of-function mutations in IGSF10 were also identified in five patients with absent puberty due to hypogonadotropic hypogonadism (HH). Additionally, I have identified and investigated one rare, pathogenic mutation in HS6ST1 – a gene known to cause HH - in one family with DP, and two rare variants in FTO – a gene implicated in the timing of menarche in the general population - in 3 families. Further potentially pathogenic variants have emerged from investigating candidate genes identified from microarray studies (LGR4, SEMA6A and NEGR1) and from related clinical phenotypes (IGSF1).Clinical Training Fellowship from Barts and the London Charity (grant number 417/1551), Clinical Research Training Fellowship from the Wellcome Trust (grant number 102745) and the Rosetrees Trust (grant number M222)

Emerging Roles of Epigenetics in the Control of Reproductive Function: Focus on Central Neuroendocrine Mechanisms

Reproduction is an essential function for perpetuation of the species. As such, it is controlled by sophisticated regulatory mechanisms that allow a perfect match between environmental conditions and internal cues to ensure adequate pubertal maturation and achievement of reproductive capacity. Besides classical genetic regulatory events, mounting evidence has documented that different epigenetic mechanisms operate at different levels of the reproductive axis to finely tune the development and function of this complex neuroendocrine system along the lifespan. In this mini-review, we summarize recent evidence on the role of epigenetics in the control of reproduction, with special focus on the modulation of the central components of this axis. Particular attention will be paid to the epigenetic control of puberty and Kiss1 neurons because major developments have taken place in this domain recently. In addition, the putative role of central epigenetic mechanisms in mediating the influence of nutritional and environmental cues on reproductive function will be discussed

Molecular Genetics of Rare Growth and Puberty Disorders in Finland

Growth and pubertal development are complex and interconnected processes, disruption of which leads to abnormal development of the adult height or secondary sexual characteristics or both, and often causes notable distress and even adverse health effects for the individual. Growth and pubertal development are both dependent on hormones secreted from the pituitary gland. Growth hormone (GH), secreted from the pituitary somatotropes, is required for growth of the bones and cartilage and achievement of the adult height. Formation of GH-secreting pituitary tumors, somatotropinomas, leads to excessive GH secretion and acromegaly or gigantism, which are both characterized by exaggerated growth, either at peripheral body parts or at the long bones depending on the onset of GH excess. In a proportion of cases, a germline gene defect can predispose to somatotropinoma formation. The ability to reproduce is achieved in puberty once the sex organs and other sexual characteristics mature into the adult form. The onset of pubertal development occurs upon the reactivation of the hypothalamic-pituitary-gonadal axis after quiescency following the previous activation phase in infancy. In the pubertal reactivation, increased gonadotropin-releasing hormone (GnRH) secretion from the hypothalamus triggers the secretion of gonadotropins from the pituitary. Premature activation of gonadotropin secretion leads to central precocious puberty (CPP), where the pubertal development begins before the age of eight in girls or the age of nine in boys. In turn, deficient gonadotropin secretion leads to congenital hypogonadotropic hypogonadism (CHH), characterized by delayed, absent, or partial puberty. If defective sense of smell co-occurs with CHH, the condition is named Kallmann syndrome (KS). Despite multiple genes are implicated in the disorders of pituitary hormone secretion, a great proportion of patients miss a molecular genetic diagnosis. The aim of this thesis was to discover defects in specific genes and evaluate their roles in disorders of growth and pubertal development, which originate from aberrant GH or gonadotropin secretion. The disorders were gigantism, acromegaly, CPP, and CHH. Variants in the potassium channel gene KCNQ1 have been previously implicated in growth hormone deficiency, and co-expression of the mutated KCNQ1 with the potassium channel subunit KCNE2 has decreased adrenocorticotropin secretion from a pituitary tumor cell line. KCNQ1 and KCNE2 were screened for germline variants in 49 Finnish patients and four patients of other ethnicities with acromegaly, who represented the phenotypic model opposite to growth hormone deficiency. In KCNQ1, deep intronic and common synonymous variants were identified, and one heterozygous variant with unknown significance in KCNE2 was found in three patients. The frequency of the KCNE2 variant was significantly higher among the patients compared to controls. Two Polish and one Finnish CPP patient as well as their family members were screened for variants in MKRN3, a maternally imprinted gene suggested to function as a pubertal brake. Novel, deleterious variants segregating with CPP in a paternally inherited manner were identified in both families. The first MKRN3 variant in Finnish CPP patients and the first long-term effects of a variant in this gene in a boy with CPP are described. Twenty-four Finnish patients with normosmic CHH or KS were screened for variants in the microRNA genes MIR7-3, MIR141, MIR429, and MIR200A-C, which were predicted to regulate CHH-related genes based on evidence from animal models, literature, or bioinformatic analyses or all. A common, heterozygous variant in MIR200A was detected in one patient. The genetic basis of KS in a Finnish patient with a de novo 2.38 Mb deletion in 9q31.2 and no likely pathogenic variants in a KS gene targeted sequencing panel was investigated with whole genome linked-read sequencing, whole exome sequencing, and RNA sequencing. In the whole genome linked-read sequencing, the deletion was found to encompass six protein-coding genes, including ZNF462, consistent with his Weiss-Kruszka syndrome. The deletion did not cover the nearby KS candidate gene PALM2AKAP2, expression of which was not suppressed by the deletion. The patient carried no rare variants in thirty-two known KS genes in the whole exome sequencing and displayed no abnormal splicing of fifteen KS genes expressed in peripheral blood leukocytes. He is the first reported patient with a 9q31.2 deletion, KS, and Weiss-Kruszka syndrome. Screening of sixteen other Finnish KS patients for variants in PALM2AKAP2 revealed no likely pathogenic defects in this gene. In conclusion, the thesis produced new information on the association of KCNQ1, KCNE2, and the selected microRNA gene variants with disorders of aberrant pituitary hormone secretion. The results demonstrate that germline variants in KCNQ1 or KCNE2 do not seem to account for somatotropinoma formation and that variants in the microRNA genes are unlikely causes of CHH. In turn, deletions in 9q31.2 appear to underlie KS, but based on the results, variants in the KS candidate gene PALM2AKAP2 do not seem to contribute to the condition in the investigated cohort. In addition, novel variants in MKRN3 were identified, and they were found to underlie CPP in Finnish patients for the first time. Finally, an interesting finding was that male carriers of MKRN3 variants may reach their target height without treatment.Väitöstyön tavoitteena oli selvittää tiettyjen geenien vaihtelun merkitystä akromegaliassa, ennenaikaisessa murrosiässä ja synnynnäisessä hypogonadotrooppisessa hypogonadismissa. Kasvu ja murrosiän kehitys ovat riippuvaisia aivolisäkkeen erittämistä hormoneista. Joskus ituradan geenivirhe voi käynnistää kasvuhormonia erittävän aivolisäkkeen kasvaimen muodostumisen, mikä edelleen johtaa hormonin liikaeritykseen ja akromegaliaan. Murrosiän kehitykseen tarvitaan gonadotropiineja, joiden erityksen käynnistyminen liian varhain johtaa ennenaikaiseen murrosikään eli murrosiän alkamiseen tytöillä alle 8-vuotiaana ja pojilla alle 9-vuotiaana. Gonadotropiinien puutteellinen eritys taas johtaa synnynnäiseen hypogonadotrooppiseen hypogonadismiin (HH), joka ilmenee viivästyneenä, puuttuvana tai osittaisena murrosikänä. Hypogonadotrooppista hypogonadismia kutsutaan Kallmannin syndroomaksi (KS), jos potilaalla on myös puutteellinen hajuaisti. Kolmessa ensimmäisessä osatyössä KCNQ1- ja KCNE2-geenit tutkittiin 53 akromegaliapotilaalta, mikroRNA-geenit MIR7-3, MIR141, MIR429 ja MIR200A-C yhteensä 24 suomalaiselta HH- tai KS-potilaalta ja MKRN3-geeni puolalaiselta ja suomalaiselta perheeltä, joilla esiintyi ennenaikaista murrosikää. KCNQ1:stä löydettiin todennäköisesti harmittomia variantteja ja KCNE2:sta kolmelta akromegaliapotilaalta merkitykseltään tuntematon variantti, joka oli merkittävästi yleisempi tutkittujen potilaiden kuin kontrollien joukossa. Todennäköisesti harmiton variantti MIR200A:ssa löydettiin yhdeltä KS-potilaalta ja MKRN3:sta löytyi molemmilta perheiltä ennen raportoimaton ja ennenaikaista murrosikää isältä perittynä aiheuttava variantti. Myös MKRN3:n variantin pitkäaikaisvaikutukset pojalla, jolla on ennenaikainen murrosikä, raportoidaan. Neljäs osatyö selvitti suomalaisen KS-potilaan sairauden geneettistä perustaa. Hänellä olleen 9q31.2-kromosomin deleetion havaittiin kattavan kuusi geeniä, muun muassa ZNF462:n, jonka puutos selitti hänen Weiss-Kruszkan syndroomansa. Deleetio ei vaikuttanut sen lähellä sijaitsevan KS:n ehdokasgeeni PALM2AKAP2:n ilmentymiseen. Potilaalta ei löytynyt harvinaisia variantteja 32 tunnetussa KS-geenissä, ja silmukointianalyysin tulos oli tutkittujen geenien osalta normaali. Potilas on ensimmäinen, jolla on raportoitu 9q31.2-kromosomin deleetio sekä Kallmannin ja Weiss-Kruszkan syndroomat. PALM2AKAP2:sta ei 16 muulla suomalaisella KS-potilaalla löytynyt todennäköisesti tautia aiheuttavia variantteja. Väitöstyö tuotti uutta tietoa KCNQ1-, KCNE2- ja tutkittujen mikroRNA-geenien varianttien yhteydestä poikkeaviin aivolisäkehormonien erityksen sairauksiin. Tulosten perusteella KCNQ1- ja KCNE2-geenien variantit ovat epätodennäköisiä akromegalian ja tutkittujen mikroRNA-geenien variantit HH:n aiheuttajia. Kuitenkin deleetiot 9q31.2-kromosomissa näyttävät voivan aiheuttaa Kallmannin syndroomaa, mutta ehdokasgeeni PALM2AKAP2:n variantit eivät liittyneet tautiin tutkituilla. Työssä löydettiin myös uusia MKRN3-geenin variantteja, joiden havaittiin aiheuttavan ennenaikaista murrosikää Suomessa ensi kertaa