<p>The aim of this thesis has been to identify genes and gene regions underlying four different disorders. In papers I-IV, positional cloning methods, such as linkage, association and haplotype analysis have been used for the identification of genomic regions associated with the ichthyosis prematurity syndrome (IPS), adult-onset autosomal dominant leukodystrophy (ADLD) and Kostmann disease. </p><p>IPS is a rare autosomal recessive skin disorder, which includes a premature birth of the affected child. We mapped the IPS locus to a region on chromosome 9q34, and within this region a haplotype is shared by IPS patients, which suggests a strong founder effect. The haplotype spans 76 kb, which includes four known genes. No sequence or mRNA expression alterations could be detected for the four genes in IPS patients. </p><p>A candidate region for an adult-onset leukodystrophy (ADLD) on chromosome 5 was investigated in a large Swedish family with ADLD. A significant multipoint LOD score of 9.45 was obtained for markers in the chromosome 5 region and fine-mapping of recombination events restricts a candidate gene region to 1.5 Mb. </p><p>Kostmann disease is an autosomal recessive form of severe congenital neutropenia. We have identified a 1.2 Mb region on chromosome 1q22 associated with the disease in the original Kostmann family. The region contains 37 genes.</p><p>In paper V, cDNA microarrays were used to asses the mRNA levels of 7,700 genes in lymphoblastoid cell lines derived from autistic and control samples. The <i>SEMA5A</i> gene, which is involved in axonal guidance, was found downregulated in the cells derived from autistic individuals, and this was confirmed by quantitative PCR. </p><p>In summary, candidate genes or gene regions have been identified for all four disorders and further studies are needed to confirm their roles in the pathogenesis of the disorders. </p>

Positional cloning is a method to identify genes from their position in the genome without prior knowledge about function. We used this approach to investigate the basis for three distinct genetic disorders; Obesity, Ichthyosis Prematurity Syndrome and Meniere's disease. Obesity appears when energy intake exceeds energy expenditure which leads to an abnormal accumulation of fat in the adipocyte tissue. We have studied a family with a balanced chromosomal translocation t(4;15) segregating with severe obesity. The chromosomal breakpoints create a fusion gene involving the gene for isoform 1 of RAR-related orphan receptor A (RORa1) which is implicated in the regulation of adipogenesis and lipoprotein metabolism. We hypothesize that the obesity in this family is caused by haploinsufficiency of this gene or a gain of function of the fusion gene. Ichthyosis prematurity syndrome (IPS) is a rare skin disorder belonging to a group of autosomal recessive congenital ichthyosis. We have mapped the locus for IPS to chromosome 9q34. Within the IPS locus, we identified a core haplotype with a high carrier frequency among affected, which indicate a possible founder mutation for the disease. The minimal shared region in affected patients contains seven genes which are candidates for IPS. Meniere's disease (MD) is characterised by spontaneous attacks of vertigo, fluctuating sensorineural low frequency hearing loss, aural fullness, and tinnitus. We mapped the MD locus to chromosome 12p13 using three Swedish families. The linked region is 463 kb, containing only one gene, a phosphoinositide-3-kinase (PIK3C2G). Involvement of phosphatidylinositol 3-kinases (PI-3K) in the intra cellular signalling cascades of cells in mammalian balance epithelia makes this gene a good candidate gene for MD.

The aim of this thesis has been to identify genes and gene regions underlying four different disorders. In papers I-IV, positional cloning methods, such as linkage, association and haplotype analysis have been used for the identification of genomic regions associated with the ichthyosis prematurity syndrome (IPS), adult-onset autosomal dominant leukodystrophy (ADLD) and Kostmann disease. IPS is a rare autosomal recessive skin disorder, which includes a premature birth of the affected child. We mapped the IPS locus to a region on chromosome 9q34, and within this region a haplotype is shared by IPS patients, which suggests a strong founder effect. The haplotype spans 76 kb, which includes four known genes. No sequence or mRNA expression alterations could be detected for the four genes in IPS patients. A candidate region for an adult-onset leukodystrophy (ADLD) on chromosome 5 was investigated in a large Swedish family with ADLD. A significant multipoint LOD score of 9.45 was obtained for markers in the chromosome 5 region and fine-mapping of recombination events restricts a candidate gene region to 1.5 Mb. Kostmann disease is an autosomal recessive form of severe congenital neutropenia. We have identified a 1.2 Mb region on chromosome 1q22 associated with the disease in the original Kostmann family. The region contains 37 genes. In paper V, cDNA microarrays were used to asses the mRNA levels of 7,700 genes in lymphoblastoid cell lines derived from autistic and control samples. The SEMA5A gene, which is involved in axonal guidance, was found downregulated in the cells derived from autistic individuals, and this was confirmed by quantitative PCR. In summary, candidate genes or gene regions have been identified for all four disorders and further studies are needed to confirm their roles in the pathogenesis of the disorders.

The Sjögren-Larsson syndrome (SLS) is a genetically determined syndrome with autosomal recessive inheritance originally and comprehensively described from Sweden. It is characterized by the three cardinal signs congenital ichthyosis, spastic di- or tetraplegia and mental retardation.The present investigation covers all 35 SLS patients alive in Sweden in 1978 and the genetic study all 58 SLS patients born in Sweden in 1886-1977. Forty-five of these were bom in a restricted area in the northeast of Sweden and five more had ancestors from this area. This concentration is probably the result of a founder effect — transmission of the SLS gene mutation from early immigrants to this area — followed by little migration to and from this area. Ancestors of SLS patients have been traced back to an early immigrant to this area in the 14th century.The mean yearly incidences of SLS per 100.000 newborn during the years 1901-1977 were 0.6 in the whole of Sweden, 10.2 in the county of Västerbotten and 2.7 in the county of Norrbotten. The corresponding prevalence figures for SLS on 31 December 1978 were 0.4, 8.3 and 2.6 per 100.000 in these areas, respectively.The ichthyosis in the SLS was found to be congenital in the true sense of the word, as it was always seen at birth. The characteristic skin findings indicate that the use of replicas in ichthyotic patients may be a good complement to the clinical and histopathological examinations.All SLS patients had mental retardation and spastic di- or tetraplegia. The first observation of these symptoms was made between the age of 4 and 30 months.Small white glistening dots located in the ocular fundus were noted in all 30 SLS patients examined in this respect and seem to be pathognomonic for SLS.The fatty acid patterns of plasma phospholipids, cholesteryl esters, triglycerides and free fatty acids in SLS patients were examined. The results suggest that SLS is possibly a disorder of fatty acid metabolism leading to an altered composition of polyunsaturated fatty acids due to a disturbance in the A6-desaturation of essential fatty acids. / <p>Some photos have been censored.</p><p>En del foton har blivit censurerade.</p> / digitalisering@umu

Orientador: Maria Beatriz Puzzi / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-07T22:49:13Z (GMT). No. of bitstreams: 1 Souto_MariamPatriciaAuada_D.pdf: 7783740 bytes, checksum: ba776e2125f132647d619bed067b9da5 (MD5) Previous issue date: 2006 / Resumo: A síndrome de Sjögren-Larsson (SSL) é uma ictiose congênita com alterações neurológicas decorrentes da deficiência enzimática da enzima aldeído graxo desidrogenase (FALDH). Mutações no gene ALDH3A2 são responsáveis pela doença. A proposta foi estudar os aspectos clínicos, histológicos, estruturais e moleculares de dez pacientes com SSL. Métodos: Foram selecionados dez pacientes com SSL de quatro famílias distintas. Foi realizada ressonância magnética cerebral (RM) em oito pacientes e, em sete destes, também espectroscopia de prótons; os resultados foram comparados com a espectroscopia de sete voluntários sadios. Foram coletadas biópsias de pele de cada paciente e pele sã de 17 voluntários. Os espécimes foram corados com hematoxilina-eosina e imagens digitais foram adquiridas a partir do exame histológico de rotina; a luminância em escala de cinza foi analisada utilizando método quantitativo de análise de textura de imagem digital: transformada rápida de Fourier, calculada a partir dos valores do momento de inércia (função harmônica) das frequências espaciais nas direções vertical e horizontal da camada espinhosa da epiderme. Amostras de pele de nove pacientes com SSL também foram obtidas para cultura de fibroblastos utilizando método padrão para dosagem da enzima FALDH. Para a extração de DNA genômico foi coletado sangue total periférico de nove pacientes. Os fragmentos foram amplificados por PCR. Os resultados obtidos foram analisados e comparados com a seqüência normal de DNA. Resultados: A mutação encontrada foi a 1108-1G?C no intron 7/exon 8. Os pacientes exibiram diferenças na presença e intensidade dos sinais e sintomas da doença (prurido, retardo mental, espasticidade, fotofobia e alterações da retina). No exame de RM dos pacientes com SSL foi observada desmielinização da substância branca profunda; na espectroscopia, presença de pico anormal de lípídios. O estudo da textura da camada espinhosa da epiderme, sob a Transformada de Fourier, mostrou amplitudes de função harmônica mais largas no grupo com SSL na direção vertical e não na direção horizontal, correspondendo a núcleos e nucléolos maiores e ao espessamento focal da carioteca dos ceratinócitos. Conclusões: As manifestações clínicas e histológicas da SLS não são exclusivamente decorrentes da mutação c.1108-1G>C, mas também expressão de outras modificações genéticas e influências ambientais. A mutação descrita pode ser classificada como uma causa comum da doença no Brasil. Os achados de textura podem ser explicados pela maior síntese de DNA na epiderme na SSL, tendo sido descrita, também, uma produção mais rápida da camada córnea e uma renovação celular 3,5 vezes o normal / Abstract: Background: Sjögren-Larsson syndrome (SLS) is an autosomal recessive disorder characterized by ichthyosis, mental retardation, and spasticity. Various mutations in the ALDH3A2 gene that codes for fatty aldehyde dehydrogenase (FALDH) are responsible for the disease but the genotype-phenotype relationships are undefined. Objectives: The purpose of this study was to describe ten individuals with Sjögren-Larsson syndrome from four families representing the first cohort of Brazilian patients defined at the molecular level and investigate whether image analysis of routine H&E skin sections using fast Fourier transformation (FFT) could detect structural alterations in SLS. Patients and methods: A 5-mm punch biopsy for histologic analysis was obtained under local anesthesia from each patient. Similar biopsies were obtained from age- and anatomical site-matched from 17 controls. The samples were stained with haematoxylin and eosin and observed under a light microscope. Digital images of routine histologic sections were taken and their gray scale luminance was analyzed by FFT. The inertia values were determined for different ranges of the spatial frequencies in the vertical and horizontal directions. Skin biopsies were also obtained from nine patients with SLS for establishing fibroblast cultures using standard methods to determine FALDH enzyme activity. MR imaging was performed in eight patients. Singel-voxel proton MR spectra were acquired from cerebral white matter in seven patients with SLS and in seven controls. Total genomic DNA was extracted from peripheral blood of nine patients using standard methods. ALDH3A2 exons and their flanking sequences were amplified by PCR and sequenced. Results: All patients were homozygous for a unique c.1108-1G>C splice-site mutation and displayed a profound reduction in fatty aldehyde dehydrogenase enzyme activity. The patients exhibited a moderate-to-severe form of Sjögren-Larsson syndrome with respect to mental retardation, spastic diplegia and ichthyosis. Within this phenotypic context, patients varied, even within a family, in the presence of pruritus, thickness of the epidermal granular layer, retinal glistening white dots and photophobia. Brain MR imaging revealed retardation of myelination. Proton MR spectroscopy of white matter revealed abnormal high-lipid peak. The study of epidermal spinous layer texture by Fast Fourier Transform showed higher amplitudes in the vertical direction in SLS patients, corresponding to greater nuclei and nucleoli, higher number of nucleoli and focal thickening of keratinocytes¿ carioteca. Conclusion: We concluded that the latter clinical features are not strictly determined by the c.1108-1G>C mutation, but are subject to modification by other genetic/environmental factors. The c.1108-1G>C mutation may be classified as a common cause of Sjögren-Larsson syndrome in Brazil. The textural findings are in keeping with higher DNA synthesis and a 3.5x turn over that were demonstrated in SLS epidermis / Doutorado / Clinica Medica / Doutor em Clínica Médica

Monogenic disorders, i.e., disorders caused by mutations in a single gene, are rare and clinically heterogeneous conditions. Identification of the genetic cause of monogenic traits can bring new insights into molecular pathways and disease mechanisms. The aims of the present study were to identify the mutant genes in two autosomal recessive skin disorders and to characterize the functions of the mutated genes.  In order to identify candidate genes for the two disorders whole-genome SNP analysis, homozygosity mapping and gene sequencing were used. Autosomal recessive congenital ichthyosis (ARCI) is a group of disorders characterized by extensive scaling and redness of the skin.  A subgroup of ARCI patients (n=27) was selected based on specific ultrastructural aberrations in their skin, revealed by electron microscopy. Mutations were identified in the Ichthyin gene in 93% of the selected patients, indicating a strong association between mutant Ichthyin and the specific morphological abnormalities. Ichthyin mRNA levels were shown to increase during keratinocyte differentiation in cells from healthy and affected individuals. Electron microscopy revealed a localization of ichthyin protein to keratins and desmosomes in epidermis. Staining of epidermal lipids identified aberrant lipid aggregates in skin sections of patients with Ichthyin mutations, indicating a role for Ichthyin in epidermal lipid metabolism. In twelve KLICK syndrome patients with ichthyosis, palmoplantar keratoderma and keratotic striae on joints, a single-nucleotide deletion was identified in the 5’ region of the proteasome maturation protein (POMP) gene.  The deletion caused an increase in the proportion of POMP transcripts with long 5’ UTR’s in patient keratinocytes.  Immunohistochemical analysis of differentiated skin cell layers revealed aberrant expression of POMP, proteasome subunits and the skin protein filaggrin in patients. CHOP expression, associated with endoplasmic reticulum stress, was increased in the same layers. siRNA silencing of POMP in cell cultures reduced proteasome subunit levels and induced expression of CHOP.  The results indicate that the mutation in KLICK patients causes POMP and proteasome insufficiency with subsequent cellular stress. This study conclusively contributes to the understanding of epidermal physiology and the pathogenesis of two inherited skin diseases.

Cytochrome P450 (CYP) family 4 constitutes monoxygenases responsible for hydroxylation of fatty acids and other lipids. For example, CYP4F3 metabolizes leukotrienes and CYP4F8 prostaglandin H. Importantly, six of the twelve CYP4 enzymes are orphans, i.e., with an unknown biological function. The catalytic activity of the enzyme CYP4F8 is known in seminal vesicles, but not in skin or psoriatic lesions, where CYP4F8 is highly expressed. The orphan CYP4F22 is also expressed in skin, and mutations in its gene has been linked to the rare skin disease lamellar ichthyosis, together with, inter alia, mutations in the genes of 12R-LOX and eLOX3. These enzymes appear to constitute a pathway producing hydroperoxides and epoxyalcohols from arachidonic acid. CYP4F22 is hypothesized to act in a consecutive step within this pathway. The aim of this thesis was to develop analytical methods to prepare and analyze hydroperoxides and epoxyalcohols derived from fatty acids by LC-MS/MS, and to investigate the catalytic performance of CYP4F8 and CYP4F22 for these substrates. The 12R-hydroperoxide of arachidonic acid (12R-HPETE) was prepared by autoxidation and separated from other hydroperoxides by chiral HPLC. MS/MS analysis showed that the hydroperoxides were unstable within the ion trap, but were stabilized by an increase in the isolation width. From the hydroperoxides, epoxyalcohols were generated by hematin treatment, and separated by normal phase HPLC. MS/MS spectra of several epoxyalcohols, derived both from arachidonic acid and linoleic acid, were characterized with aid of [2H]isotopomers and MS3 analysis. Apart from metabolic studies the thesis also include detailed information on MS/MS analysis of several oxygenated fatty acids, with proposed fragmentation mechanisms. The open reading frame of CYP4F22 was expressed in a recombinant yeast system, and LC-MS/MS analysis revealed that CYP4F22 catalyzed ω3 hydroxylation of arachidonic acid, but not any of the tested epoxyalcohols. In contrast, CYP4F8 metabolizes an epoxyalcohol derived from 12R-HPETE, 11R,12R-epoxy-10-hydroxyeicosatrienoic acid, to the ω3 hydroxy metabolite. Conclusively, it was demonstrated that LC-MS/MS could be used for the analysis and separation of hydroperoxides and epoxyalcohols for metabolic studies.

Epidermolysis bullosa simplex (EBS) and epidermolytic ichthyosis (EI) are rare skin fragility diseases characterized by intra-epidermal blistering due to autosomal dominant-negative mutations in basal (KRT5 or KRT14) and suprabasal (KRT1 or KRT10) keratin genes,  respectively. Despite vast knowledge in the disease pathogenesis, the pathomechanisms are not fully understood, and no effective remedies exist. The purpose of this work was to search for keratin gene mutations in EBS patients, to develop in vitro models for studying EBS and EI, and to investigate novel pharmacological approaches for both diseases. We identified both novel and recurrent KRT5 mutations in all studied EBS patients but one which did not show any pathogenic keratin mutations. Using cultured primary keratinocytes from EBS patients, we reproduced a correlation between clinical severity and cytoskeletal instability in vitro. Immortalized keratinocyte cell lines were established from three EBS and three EI patients with different phenotypes using HPV16-E6E7. Only cell lines derived from severely affected patients exhibited spontaneous keratin aggregates under normal culture conditions. However, heat stress significantly induced keratin aggregates in all patient cell lines. This effect was more dramatic in cells from patients with a severe phenotype. In organotypic cultures, the immortalized cells were able to differentiate and form a multilayered epidermis reminiscent of those observed in vivo. Addition of two molecular chaperones, trimethylamine N-oxide dihydrate (TMAO) and sodium 4-phenylbutyrate (4-PBA), reduced the keratin aggregates in both stressed and unstressed EBS and EI keratinocytes, respectively. The mechanism of action of TMAO and 4-PBA was shown to involve the endogenous chaperone system (Heat shock proteins e.g. Hsp70). Besides, MAPK signaling pathways also seemed to be incriminated in the pathogenesis of EBS. Furthermore, depending on which type of keratin is mutated, 4-PBA up-regulated Hsp70 and KRT4 (possibly compensating for mutated KRT1/5), and down-regulated KRT1 and KRT10, which could further assist in protecting EBS and EI cells against stress. In conclusion, novel and recurrent pathogenic keratin mutations have been identified in EBS. Immortalized EBS and EI cell lines that functionally reflect the disease phenotype were established. Two pharmacologic agents, TMAO and 4-PBA, were shown to be promising candidates as novel treatment of heritable keratinopathies in this in vitro model.