La prédiction de la zygotie à partir du génotype le plus probable en la comparant à la PCR-SSP, n’était pas fiable liée à la possibilité d’avoir d’autres génotypes possibles pour le même phénotype. En effet, la fréquence très proche de l’haplotype R0 et r dans la population tunisienne rend la déduction du génotype le plus probable très aléatoire. Dans un deuxième temps, l’évaluation de la méthode moléculaire la plus convenable à la détermination de la zygotie RHD a été réalisée par comparaison des trois techniques moléculaires (PCR-SSP, PCR-RFLP et Q-PCR) et par l’analyse des résultats discordants par séquençage du gène RHD et des « boîtes Rhésus ». L’analyse de 370 échantillons RH:1 à l'aide de ces trois tests moléculaires a montré que 81,9% des résultats étaient en concordance alors que 18,1% en discordance. L’analyse des cas discordants a montré que notre cohorte se compose de 193 sujets dizygotes et 145 hémizygotes et 32 dont la zygotie reste inconnue. Cette étude a révélé 19 nouveaux polymorphismes des « boîtes Rhésus » et a permis aussi de décrire trois nouveaux allèles RHD: RHD(Trp185Stop), RHD(Ala176Thr) et RHD(Ile342Ile). Cette étude a également mis en valeur l’hétérogénéité des « boîtes Rhésus » et la complexité des allèles RHD en décrivant les nouveaux polymorphismes obtenus, ce qui met en évidence les limites des approches moléculaires de la détermination de la zygotie. La Q-PCR a été la méthode la plus adaptée à la détermination de la zygotie, mais en raison des contraintes économiques locales, la PCR-RFLP pourrait être une alternative malgré l’hétérogénéité des « boîtes Rhésus » et la complexité du gène RHD. / Determination of paternal RHD zygosity can help the clinician to assess the risk of HDN. It was determined initially by both assignment of the most probable genotype and PCR-SSP. The prediction of zygosity based on the most probable genotype was not reliable due to the possibility of other genotypes for the same phenotype. In fact, the frequencies of R0 and r haplotypes in the Tunisian population are approached and make the deduction of the most probable genotype very aleatory. Secondly, the evaluation of the most convenient molecular method for RHD zygosity determination was realized by comparison of three molecular techniques (PCR-SSP, PCR-RFLP and RQ-PCR) and analysis of discordant results by sequencing of the RHD gene and Rhesus boxes. Analysis of 370 RH:1 samples by these three molecular tests showed concordant results in 81.9% and discordant results in 18.1%. Molecular investigations revealed that our cohort consists of 193 dizygous and 145 hemizygous samples and 32 which zygosity remains unknown. This study revealed 19 novel Rhesus boxes polymorphisms, and described 3 novel RHD alleles: RHD(Trp185Stop), RHD(Ala176Thr) and RHD(Ile342Ile). This study also underlined Rhesus boxes heterogeneity and RHD alleles complexity by describing of new polymorphisms which showed the limits of molecular approaches for RHD zygosity determination. RQ-PCR is the most convenient method for first intension paternal RHD zygosity determination in Tunisians. However taking into account local economic constraints PCR-RFLP could be an alternative despite the Rhesus boxes heterogeneity and RHD complexity.

Abstract The use of 3D imaging of craniofacial soft tissue has increased in medical science, and imaging technology has been developed greatly in recent years. 3D models are quite accurate and with imaging devices based on stereophotogrammetry, capturing the data is a quick and easy operation for the subject. However, analyzing 3D models of the face or head can be challenging and there is a growing need for efficient quantitative methods. In this thesis, new mathematical methods and tools for measuring craniofacial structures are developed. The thesis is divided into three parts. In the first part, facial 3D data of Lithuanian twins are used for the determination of zygosity. Statistical pattern recognition methodology is used for classification and the results are compared with DNA testing. In the second part of the thesis, the distribution of surface normal vector directions of a 3D infant head model is used to analyze skull deformation. The level of flatness and asymmetry are quantified by functionals of the kernel density estimate of the normal vector directions. Using 3D models from infants at the age of three months and clinical ratings made by experts, this novel method is compared with some previously suggested approaches. The method is also applied to clinical longitudinal research in which 3D images from three different time points are analyzed to find the course of positional cranial deformation and associated risk factors. The final part of the thesis introduces a novel statistical scale space method, SphereSiZer, for exploring the structures of a probability density function defined on the unit sphere. The tools developed in the second part are used for the implementation of SphereSiZer. In SphereSiZer, the scale-dependent features of the density are visualized by projecting the statistically significant gradients onto a planar contour plot of the density function. The method is tested by analyzing samples of surface unit normal vector data of an infant head as well as data from generated simulated spherical densities. The results and examples of the study show that the proposed novel methods perform well. The methods can be extended and developed in further studies. Cranial and facial 3D models will offer many opportunities for the development of new and sophisticated analytical methods in the future. / Tiivistelmä Pään ja kasvojen pehmytkudoksen 3D-kuvantaminen on yleistynyt lääketieteessä, ja siihen tarvittava teknologia on kehittynyt huomattavasti viime vuosina. 3D-mallit ovat melko tarkkoja, ja kuvaus stereofotogrammetriaan perustuvalla laitteella on nopea ja helppo tilanne kuvattavalle. Kasvojen ja pään 3D-mallien analysointi voi kuitenkin olla haastavaa, ja tarve tehokkaille kvantitatiivisille menetelmille on kasvanut. Tässä väitöskirjassa kehitetään uusia matemaattisia kraniofakiaalisten rakenteiden mittausmenetelmiä ja -työkaluja. Työ on jaettu kolmeen osaan. Ensimmäisessä osassa pyritään määrittämään liettualaisten kaksosten tsygositeetti kasvojen 3D-datan perusteella. Luokituksessa hyödynnetään tilastollista hahmontunnistusta, ja tuloksia verrataan DNA-testituloksiin. Toisessa osassa analysoidaan pään epämuodostumia imeväisikäisten päiden 3D-kuvista laskettujen pintanormaalivektorien suuntiin perustuvan jakauman avulla. Tasaisuuden ja epäsymmetrian määrää mitataan normaalivektorien suuntakulmien ydinestimaatin funktionaalien avulla. Kehitettyä menetelmää verrataan joihinkin aiemmin ehdotettuihin lähestymistapoihin mittaamalla kolmen kuukauden ikäisten imeväisten 3D-malleja ja tarkastelemalla asiantuntijoiden tekemiä kliinisiä pisteytyksiä. Menetelmää sovelletaan myös kliiniseen pitkittäistutkimukseen, jossa tutkitaan pään epämuodostumien ja niihin liittyvien riskitekijöiden kehitystä kolmena eri ajankohtana otettujen 3D-kuvien perusteella. Viimeisessä osassa esitellään uusi tilastollinen skaala-avaruusmenetelmä SphereSiZer, jolla tutkitaan yksikköpallon tiheysfunktion rakenteita. Toisessa osassa kehitettyjä työkaluja sovelletaan SphereSiZerin toteutukseen. SphereSiZer-menetelmässä tiheysfunktion eri skaalojen piirteet visualisoidaan projisoimalla tilastollisesti merkitsevät gradientit tiheysfunktiota kuvaavalle isoviivakartalle. Menetelmää sovelletaan imeväisikäisen pään pintanormaalivektoridataan ja simuloituihin, pallotiheysfunktioihin perustuviin otoksiin. Tulosten ja esimerkkien perusteella väitöskirjassa esitetyt uudet menetelmät toimivat hyvin. Menetelmiä voidaan myös kehittää edelleen ja laajentaa jatkotutkimuksissa. Pään ja kasvojen 3D-mallit tarjoavat paljon mahdollisuuksia uusien ja laadukkaiden analyysityökalujen kehitykseen myöhemmissä tutkimuksissa.

Prenatal assessment of fetal health is routinely offered throughout pregnancy to ensure that the most effective management can be provided to maintain fetal and maternal well-being. Currently, invasive testing is used for definitive diagnosis of fetal aneuploidy, which is associated with a 1% risk of iatrogenic fetal loss. Developing non-invasive prenatal testing (NIPT) is a key area of research and methods to increase the level of cell-free fetal DNA (cffDNA) within the maternal circulation have been discussed to improve accuracy of such tests. In this study, three strategies; co-amplification at lower denaturation temperature polymerase chain reaction (COLD-PCR), inverse-PCR and Pippin Prep™ gel electrophoresis, were analysed to identify a novel approach to selectively enrich shorter cffDNA fragments from larger maternal cell-free DNA (cfDNA). The sensitivity of droplet digital PCR (ddPCR) against real-time PCR (qPCR) was compared for fetal sex and RHD genotyping. In addition RHD zygosity testing was carried out for non-maternal samples. Consequently, Pippin Prep™ gel electrophoresis was combined with ddPCR analysis for the NIPD of Down Syndrome (DS) in pseudo-maternal samples. The results revealed that the Pippin Prep™ gel electrophoresis enrichment approach successfully demonstrated 2-fold to 5-fold increases in the cffDNA fraction. However, further optimisation assays of COLD-PCR and inverse-PCR using actual maternal samples were required. The spike experiments for DS detection revealed that with the present assay IV overrepresentation of the chromosome 21 target could be significantly detected for samples with ≥15% ‘cffDNA fraction’. In conjunction with the Pippin Prep™ enrichment method, this would have enabled assessment of all 10 maternal samples. Alternatively, fetal sex and RHD genotyping results determined that ddPCR provides a more sensitive platform compared to qPCR approaches, particularly for samples that express low cffDNA fractions (<2%). The ddPCR platform also proved to be a rapid and accurate system for the determination of RHD zygosity. This study highlights that ddPCR could be used as opposed to qPCR for accurate determination of fetal sex and RHD status. While sequencing approaches currently provide the most sensitive platforms for NIPT of fetal aneuploidy, high costs (>£400) prevent universal application. The combination of cffDNA enrichment with ddPCR analysis could provide a cheaper and more widely available platform for NIPD. However, further large scale validation studies using actual maternal samples are required.