Ziel der Habilarbeit war die molekulargenetische und -zytogenetische Charakterisierung von Kopf-Hals-Karzinomen, um das biologische Verhalten dieser Tumoren besser einschätzen zu können als mit Hilfe der bekannten klinisch-pathologischen Tumorklassifikation. Dazu wurden 100 primäre Plattenepithelkarzinome des Kopf-Hals-Bereiches, 27 Fälle syn- oder metachroner Metastasen und 10 Fälle von Zweitkarzinomen mit Hilfe der Comparativen Genomischen Hybridisierung (CGH) analysiert. Alle untersuchten Tumoren stammten von Patienten, die primär im Zeitraum zwischen 1994 und 1998 in der HNO-Klinik der Charité operiert wurden. Die Auswertung der analysierten Tumoren brachte die nachfolgenden wesentlichen Ergebnisse: Plattenepithelkarzinome des Kopf-Hals-Bereiches sind durch Deletionen der Chromosomen 3p, 4p/q, 5q, 6q, 8p, 9p, 11p/q, 13q, 18q und 21q sowie DNA-Gewinne im Bereich von 1p, 3q, 5p, 8q, 9q, 11q13, 16p, 17q, 19, 20q und 22q. Gut differenzierte Tumoren sind durch Deletionen auf Chromosom 3p und 9p kombiniert mit einem DNA-Gewinn auf 3q gekennzeichnet. Die schlecht differenzierten Malignome zeigen zusätzlich die Deletionen von Chromosom 4, 5q, 8p, 11, 13q, 18q sowie 21q und die DNA-Gewinne von 8q, 11q13 sowie 20q.Der metastatische Phänotyp ist bei Kopf-Hals-Karzinomen durch Deletionen im Bereich der Chromosomen 10q und 11 charakterisiert.Als wichtigstes Ergebnis konnten zwei unabhängige genetische Prognosemarker für Kopf-Hals-Karzinome charakterisiert werden - die 11q13- und 3q-Amplifikation. In der multivariaten Cox-Regression zeigte sich, daß der Einfluß beider Parameter, besonders aber der 11q13-Amplifikation für die Einschätzung des rezidiv- und metastasenfreien sowie des Gesamtüberlebens der Patienten größer ist als der aller klinisch-pathologischen Parameter, insbesondere der TNM-Klassifikation. / Comparative Genomic Hybridization (CGH) was performed on 100 primary head and neck squamous cell carcinomas, 27 cases of lymph node metastases and 10 second primaries. The main results are summarized as follows: The entity of head and neck squamous cell carcinomas is characterized by a pattern of chromosomal alterations involving deletions chromosomes 3p, 4p/q, 5q, 6q, 8p, 9p, 11p/q, 13q, 18q and 21q combined with overrepresentations of chromosomes 1p, 3q, 5p, 8q, 9q, 11q13, 16p, 17q, 19, 20q and 22q.Well differentiated carcinomas (G1) carry deletions on chromosomes 3p and 9p together with the overrepresentation of 3q indicating early tumor development. Accordingly, the undifferentiated tumors (G3) were charaterized by addtional deletions on chromosomes 4, 5q, 8p, 11, 13q, 18q, 21q and overrepresentations on 8q, 11q13 and 20q suggesting that these changes are preferentially associated with tumor progression.The metastatic phenotype of head and neck suqamous cell carcinomas is significantly associated with deletions of chromosomes 10q and 11.The overall survival time as well as the recurrence free survival time were significantly lower in patients who's tumors showed amplifications of the chromosomal region 11q13 and/or overrepresentations of chromosome 3q (tested for significance by the log rank test p=0.0008 / p=0.0299). Multivariate analysis (Cox's proportional hazards model) revealed both chromosomal alterations as most important independent prognostic factors in HNSCC prior to the established TNM staging system.

Les chromosomes marqueurs surnuméraires (CMS) sont définis comme des chromosomes de structure anormale qui ne peuvent pas être identifiés ni caractérisés de façon non ambigüe par les techniques de cytogénétique conventionnelle seules et qui sont de taille égale ou plus petits qu’un chromosome 20 de la même métaphase. La prévalence de cette anomalie chromosomique est estimée à 0,071% en post-natal, 0,075% en diagnostic prénatal et 0,288% chez les patients atteints de retard mental et/ou du développement. Chez les patients infertiles, la fréquence des CMS est estimée à 0,122% et varie selon le sexe. Les CMS sont sans conséquence clinique dans 70% des cas. Dans un tiers des cas, ils peuvent être responsables de nombreuses anomalies du développement et de la reproduction humaine. A ce jour, il existe très peu d’études de caractérisation des CMS permettant une cartographie précise des gènes présents. Dans ce travail, nous avons étudié une série de huit CMS par cytogénétique conventionnelle, FISH (fluorescent in situ hybridization) et CGH array (array comparative genomic hybridization). Nous avons établi une cartographie des gènes présents dans ces CMS. L’étude de la relation génotype-phénotype des patients nous a permis de proposer l’implication de certains gènes candidats dans des anomalies du développement et de la reproduction humaine. Notre étude de l’implication des CMS dans les anomalies du développement humain s’est basée sur l’étude cytogénétique de trois fœtus. Les deux premiers fœtus étaient porteurs d’un CMS(20) en anneau. Le sujet 1 présentait un retard de croissance intra-utérin (RCIU) et une dysmorphie cranio-faciale. Le sujet 2 n’avait pas d’anomalies particulières à part une obésité diagnostiquée à l’âge de quatre mois. La taille de ces CMS(20) était de 13,6 Mb pour le sujet 1 et 4,8 Mb pour le sujet 2. Le gène SSTR4 présent sur le CMS(20) du sujet 2 code pour un récepteur de la somatostatine. Cette hormone joue un rôle dans le comportement alimentaire. Le troisième fœtus présentait un hygroma kystique et un RCIU associé à un CMS(13) néocentromérique. Les explorations par CGH array ont révélé un gain chromosomique de la région 13q21.1qter de 39 Mb contenant 80 gènes dont GPC5, GPC6, SPRY2, EFNB2, SOX1 et DZIP1. La modification d’expression de ces gènes est susceptible d’être responsable du phénotype des sujets étudiés.Notre étude de l’implication des CMS dans les anomalies de la reproduction humaine s’est basée sur l’étude cytogénétique de cinq patients qui présentaient des troubles de la fertilité (anomalies de la spermatogenèse, insuffisance ovarienne prématurée, syndrome des ovaires polykystiques et fausses couches spontanées). Les CMS explorés par CGH array correspondaient aux régions chromosomiques 15q11.2 (3,6 Mb), 21p11.2 (0,266 Mb), 6p11.2q12 (9 Mb) et 20p11.21 (3,3 Mb). Le CMS d’une des patientes ne contenait pas d’euchromatine et une autre patiente était porteuse de deux CMS d’origines chromosomiques différentes. Plusieurs gènes candidats (POTE B, BAGE et THBD) ont pu être identifiés. La modification de leur expression ainsi que des effets mécaniques ou biochimiques perturbant la méiose et la maturation des gamètes pourraient être responsables des troubles de la fertilité observés chez ces patients. L’étude des CMS par CGH array nous a permis de caractériser précisément les points de cassure des CMS, leur taille et leur composition génétique afin de cartographier les gènes présents dans les CMS et d’établir des relations entre le génotype et le phénotype des patients. / Small supernumerary marker chromosomes (sSMC) are defined as structurally abnormal chromosomes which cannot be unambiguously identified or characterized by conventional banding cytogenetic techniques alone and are generally equal in size or smaller than a chromosome 20 of the same metaphase spread. sSMC frequency is estimated at 0.071% in postnatal cases, 0.075% in prenatal cases, and 0.288% for mentally and/or development retarded patients. In infertile patients cases, sSMC frequency is estimated at 0.122% and is different in male (0.165%) and female infertility (0.022%). sSMC have no clinical consequences in 70% of the cases. In one third of the cases, they can be responsible for various human development and reproduction anomalies. To date, only a few studies precisely characterizing the sSMC contents have been performed.In this study, we used conventional cytogenetics, FISH (fluorescent in situ hybridization) and array CGH (array comparative genomic hybridization) to characterize eight sSMC and to precisely localize the genes included. The study of the genotype-phenotype correlations of the patients led us to suppose the implication of some candidate genes in human development and reproduction anomalies.Our study of the implication of sSMC in human development anomalies was based on the cytogenetic study of three fetuses. The first two fetuses carried a ring sSMC(20). Case 1 presented with intrauterine growth retardation and craniofacial dysmorphism. Case 2 had a normal phenotype except for obesity diagnosed at the age of four months. The size of these sSMC(20) was approximately 13,6 Mb for case 1 and 4,8 Mb for case 2. The SSTR4 gene located on the case 2 sSMC(20) is coding for one of the somatostatin receptor. This hormone has multiple effects on variable cells and is implicated in the regulation of food behavior, which could explain the obesity of case 2. Case 3 presented with intrauterine growth retardation and a cystic hygroma associated with a neocentric sSMC(13). Array CGH investigations showed a 32.9 Mb gain from 13q31.1 to 13qter region containing 80 genes. Among these genes, six genes could be involved in the phenotype of the proband (GPC5, GPC6, SPRY2, EFNB2, SOX1 and DZIP1). The expression modification of these genes could be responsible for the phenotype observed.Our study of the implication of sSMC in human reproduction anomalies was based on the cytogenetic study of five patients presenting fertility troubles (spermatogenesis impairment, ovarian insufficiency, polycystic ovary syndrome and repeated abortions). The sSMC explored by array CGH corresponded to the 15q11.2 region (3.6 Mb), the 21p11.2 region (0.266 Mb), the 6p11.2q12 region (9 Mb) and 20p11.21 region (3.3 Mb). The sSMC of one of the patients did not contain euchromatin and one patient carried two sSMC derived from two different chromosomes. Among the genes present on the sSMC, some candidate genes (POTE B, BAGE and THBD) have been identified. The modification of their expression and mechanical or biochemical effects of the sSMC impeding meiosis could be directly responsible for the fertility trouble observed in these patients. A detailed molecular cytogenetic investigation using array CGH allowed us to precisely characterize the chromosomal breakpoints, the size and genomic constitution of sSMC. This study may be helpful to address genotype–phenotype correlations and for medical and genetic counseling.

Les Carcinomes Papillaires du Rein (pRCC) représentent la seconde forme histologique de cancers du rein . Ils correspondent à une entité hétérogène de tumeurs subdivisées en type I et type II sur leurs caractéristiques anatomopathologiques. Leur pronostic au stade métastatique est inferieur à celui des carcinomes à cellules claires. Les caractéristiques biologiques des pRCC sont mal connues et n’ont pas permis de développer jusqu'à ce jour de thérapeutiques spécifiques.Ce travail propose, en première partie, une synthèse des données disponibles biologiques, anatomo-pathologiques, thérapeutiques et pronostiques des pRCC. Cette synthèse a fait l’objet d’une publication. ( Albiges et al. The Oncologist 2012)Le second volet est dédié à l’analyse de la place du proto-oncogène MET au sein des pRCC de type I et II et plus particulièrement les différentes modalités d’activation de ce gène. Cette analyse (i) caractérise les anomalies quantitatives de l’ADN du gène MET (CGH array pour les pRCC de type II et CGMA pour les pRCC de type I) et leur corrélation au niveau d’expression génique; (ii) recherche l’existence de mutations activatrices du domaine tyrosine kinase par séquençage du gène MET chez les pRCC de type I; et (iii) analyse également les niveaux d’expression du ligand et des co-activateurs de ce récepteur MET. Ces résultats sont en cours de publication (Albiges et al. Clinical Cancer Research)Le troisième et dernier volet de ce travail vise à identifier des pistes biologiques propres aux pRCC par l’analyses de sous groupes distinguable en termes de profils d’expression génique et surtout par l’analyses des anomalies de l’ADN identifiées par CGH array des pRCC de type II, couplées aux données de transcriptome. / Papillary renal cell carcinomas (pRCC) are the second most common form of Renal Carcinomas and belongs to the non clear cell carcinomas family. This tumour type is an heterogeneous group of tumours usually subdivided in type I and type II according to pathological features. The prognosis of pRCC in the metastatic setting is worse to clear cell carcinoma’s prognosis. Biological characteristics of pRCC are poorly known and did not allow the development of specific targeted therapies.This work first presents a synthesis of published data regarding biology, pathology, therapeutics and prognosis of pRCC. This review has been published. (Albiges et al. The Oncologist 2012)Second part is dedicated to the analysis of MET proto-oncogene across pRCC. The main focus is to assess MET activation drivers. This analysis (i) characterises MET gene DNA copy number alterations (CGH array for type II pRCC and CGMA approach for Type I pRCC) and their correlation with gene expression profiling; (ii) assess activating mutations within the tyrosine kinase of MET gene in the type I pRCC; and (iii) investigate expression level of ligand and co-activators of MET receptor. This analysis is under publication. (Albiges et al. Clinical Cancer Research)Third and last part of this work aims at identifing new biological pathway specific to pRCC using clustering of gene expression profiling and DNA abnormalities assessed by CGHarray inthe type II pRCC subtypes with matching gene expression data.

Rearranjos cromossômicos estruturais estão potencialmente associados ao desenvolvimento de doenças genéticas devido à disrupção, inativação ou alteração da dosagem gênica. O objetivo deste projeto foi realizar a caracterização genômica de duas pacientes e seus familiares portadores de rearranjo cromossômico estrutural envolvendo o braço curto do cromossomo 4 e o braço longo do cromossomo 12, associando técnicas de citogenética clássica (bandamento GTG), citogenética molecular (FISH) e citogenômica (array-CGH), para definição diagnóstica e maior conhecimento sobre os fatores envolvidos na correlação cariótipo-genótipo-fenótipo. Foram avaliados seis indivíduos, duas pacientes, primas em primeiro grau que apresentavam alterações fenotípicas, assim como seus familiares, portadores de translocação aparentemente equilibrada e fenótipo normal. Apesar das duas pacientes apresentarem alteração cromossômica comum, derivativo do cromossomo 4 [der(4)], foram observados achados fenotípicos distintos. A investigação permitiu a definição do diagnóstico de deleção 4p16 e trissomia 12qter para as duas pacientes com fenótipo alterado e cariótipo 46,XX,der(4)t(4;12)(p16;q24.3), a definição precisa dos pontos de quebra em 4p16.3 e 12q24.31->q24.33, assim como a determinação da origem parental do rearranjo e a definição do diagnóstico citogenético final de quatro portadores de translocação aparentemente equilibrada e cariótipo t(4;12)(4pter->4p16.3::2q24.31->12qter;12qter->12q24.31::4p16.3->4pter),direcionando o aconselhamento genético para a família. Nas duas pacientes, a técnica de array-CGH (Plataforma 2x400K, Agilent®) detectou uma diferença sutil de tamanho entre as perdas e ganhos referentes aos cromossomos envolvidos no rearranjo, sendo diagnosticado em P1 uma perda de 2.707.221 pb na citobanda 4p16.3, além de um ganho de 12.405.205 pb em 12q24.31->q24.33. A paciente 2 apresentou uma perda de 2.710.969 pb em 4p16.3 e um ganho de 12.393.885 pb em 12q24.31->q24.33. Ambas as regiões de desequilíbrio genômico incluem genes que podem ser relevantes para manifestação fenotípica observada nas pacientes, entre eles: WHSC1, NELFA, LETM1, FGFRL1 e SPON2. Os resultados da investigação citogenômica indicaram, ainda, a presença de translocação equilibrada nos quatro indivíduos portadores, não sendo detectadas perdas e/ou ganhos genômicos nas regiões dos pontos de quebra cromossômica. Os resultados obtidos na investigação do padrão de metilação dos genes FGFRL1 e SPON2 não permitiram afirmar que uma provável repressão da expressão gênica devido ao imprinting materno e paterno esteja associada às características fenotípicas distintas observadas nas duas pacientes. Embora tenha sido possível a indicação de genes correlacionados ao fenótipo das pacientes, a correlação entre a alteração genética e o fenótipo das mesmas pode depender da ação sinérgica dos mais de 190 genes envolvidos neste rearranjo cromossômico estrutural familiar. / Structural chromosomal rearrangements are potentially associated with the development of genetic disorders due to disruption, inactivation or gene dosage alterations. The objective of this project was to perform the genomic characterization of a familial structural chromosomal rearrangement involving the short arm of chromosome 4 and the long arm of chromosome 12 in two patients and carriers. The experimental approach involved using a combination of classical cytogenetic techniques (GTG banding), molecular cytogenetics (FISH) and cytogenomics (array-CGH), to provide a diagnostic definition and a better understanding of how changes in the karyotype and genotype may be associated with the phenotype. Six individuals were evaluated, two patients with phenotypic abnormalities, as well as the carriers of an apparently balanced 4p;12q translocation with normal phenotypes. Although the two patients showed a common chromosomal abnormality, the derivative chromosome 4 [der (4)], they presented distinct phenotypic findings. The investigation provided a definition of the diagnosis of 4p16 deletion and trisomy 12qter for the two patients with abnormal phenotypes and a karyotype 46,XX,der(4)t(4;12)(p16;q24.3). In addition a precise definition of the breakpoints at 4p16.3 and 12q24.31->q24.33, and the parental origin of the rearrangement was determined. A precise definition of the cytogenetic diagnosis of four carriers with an apparently balanced translocation and karyotype t(4;12)(4pter->4p16.3::2q24.31->12qter; 12qter 12q24.31->4pter::4p16.3), facilitated the genetic counseling for the family. In both patients, the array-CGH technique (2x400K Platform, Agilent®) detected a subtle difference in size between losses and gains in the chromosomal regions involved in the rearrangement. Patient 1 presented a loss of 2,707,221 bp in the cytoband 4p16.3, and a gain of 12,405,205 bp in 12q24.31->q24.33. Patient 2 had a loss of 2,710,969 bp in 4p16.3 and a gain of 12,393,885 bp in 12q24.31 -> q24.33. Both regions of genomic imbalance included genes that may be relevant to phenotypic findings observed in our patients, including: WHSC1, NELFA, LETM1, FGFRL1 and SPON2. Genomic findings also confirmed the presence of a balanced translocation in four carriers, with no genomic losses and/or gains in the regions of chromosome breakpoints. The results of the investigation of the methylation pattern of FGFRL1 and SPON2 genes could not demonstrate that repression of gene expression due to maternal and paternal imprinting was associated with the distinct phenotypes observed in the two patients. Although it has been possible to indicate genes related to the phenotype of the patients, the correlation between the genetic alteration and phenotype may depend on the synergistic action of multiple genes from more than the 190 involved in this familial chromosomal rearrangement.

A doença de Alzheimer (DA) é a causa mais comum de demência na população, sendo responsável por cerca de 50 a 60% dos casos. Embora o diagnóstico clínico da doença na maioria das vezes seja acurado, a confirmação da DA só é feita post mortem através principalmente da caracterização dos dois tipos principais de lesões neurais: depósitos extracelulares de placas de β amiloide e emaranhados de proteína tau hiperfosforilada. Até o momento, o envolvimento de apenas quatro genes foi confirmado na etiologia da DA, três deles (APP, PSEN1 e PSEN2) associados à forma familial de herança mendeliana, que corresponde a um tipo raro e grave. No entanto, apesar de inúmeros trabalhos de associação genômica, (Genome wide association studies- GWAS) sugerirem uma possível participação de vários outros genes na suscetibilidade à manifestação da forma multifatorial da DA, o gene APOE, ainda é o único consistente e reproduzivelmente associado à doença. As descobertas derivadas dos GWAS investigando o papel de SNPs coletivamente explicam somente uma pequena porcentagem da variação herdada que contribui para o risco de desenvolver a DA. Atualmente, há novas abordagens para investigar a base genética do restante da variabilidade fenotípica herdada e que pode influenciar a suscetibilidade ao desenvolvimento de doenças complexas. O papel da variação do número de cópias de segmentos de DNA (Copy Number Variation - CNV) na genética de doenças complexas foi demonstrado por diversos estudos nos últimos anos e evidencia que desequilíbrios genômicos também podem contribuir significantemente para a resistência ou susceptibilidade a várias patologias. Outro aspecto que vem assumindo crescente importância é a análise de modificações epigenéticas que podem constituir um mecanismo molecular básico e contribuir diretamente para a patogênese da DA. Logo, este trabalho teve como objetivo principal investigar dois aspectos relacionados à DA: (1) a identificação de CNVs que podem estar contribuindo para o desenvolvimento da forma multifatorial da DA, usando a técnica de array-CGH, e (2) a análise de alterações do padrão global de metilação do DNA no córtex frontal de indivíduos com a forma multifatorial da DA, usando um microarranjo que interroga o status de metilação de 450.000 sítios CpGs. Em nossa investigação sobre desequilíbrios genômicos na DA, identificamos 6 CNVs raras com conteúdo gênico relevante para o fenótipo investigado. Dois indivíduos distintos do grupo DA apresentam microduplicações em genes que codificam diferentes subunidades do mesmo tipo de canal de Ca2+ dependente de voltagem, o tipo L. Além disso, dos outros genes selecionados como especialmente interessantes, 4 estão envolvidos em diferentes processos inflamatórios e 1 é responsável por codificar a enzima nicotinamida fosforibosiltransferase, participante importante da via de biossíntese da molécula nicotinamida adenina dinucleotídeo (NAD). A implicação de um possível envolvimento de mediadores da sinalização celular do Ca2+ e da via de biossíntese da NAD na etiologia da DA também foi reforçada pelos nossos resultados sobre o padrão de metilação do DNA na DA. Dois genes importantes para a homeostasia intracelular do Ca2+ e via de biossíntese da NAD apresentaram sítios CpGs diferenciamente metilados nos sujeitos com DA / Alzheimer\'s disease (AD) is the most common form of dementia in the population, corresponding to 50-60% of all cases. Although clinical diagnosis seems to be accurate, the definitive diagnosis of the disease can only be made by a post mortem neuropathological exam that certifies the presence of the two hallmarks of AD: the accumulation of extracellular senile plaques containing β-amyloid (Aβ) and the intracellular neurofibrillary tangles containing hyperphosphorylated tau protein. Four genes are known to be involved in the etiology of AD, three of them (APP, PSEN1 and PSEN2) are associated to the familial form of the disease, which show autosomal dominant inheritance and correspond to the more severe and rare type of AD. Despite many genome wide association studies (GWAS), APOE still remains the only unequivocal genetic risk factor associated to the multifactorial form of AD. The discoveries from GWAS using SNPs collectively explain only a small percentage of heritable variation that may contribute in AD risk. Currently, new approaches have been used to investigate the genetic basis of the phenotypical variability inheritance that can influence the susceptibility of complex diseases. The important role of DNA copy number variation (CNV) has been demonstrated by several studies over the last years and shows that genomic imbalances may also significantly contribute to resistance or susceptibility to various complex diseases. Additionally, there is now increasing interest in exploring how epigenetic modifications, in particular DNA methylation, could influence complex diseases etiology. Thus, the major aim of this work were to investigate two aspects related to the multifactorial form of AD: (1) identification of rare CNVs, using array-CGH, that could contribute to the development of the disease, and (2) analysis of the DNA methylation pattern in frontal cortex of individuals with AD. In our study, we identified 6 rare CNVs with relevant gene content to the investigated phenotype. Two distinct subjects with AD from our casuistic presented microduplications in genes that encode different subunits of the same type of Ca2+ voltage channel, the L-type. Furthermore, among the other selected genes, four are involved in different inflammatory process and one encodes the nicotinamide phosphoribosyltransferase enzyme, important mediator of nicotinamide adenine dinucleotide (NAD) biosynthesis. The implication of a possible involvement of Ca2+ intracellular signaling mediators and NAD biosynthesis pathway in the etiology of AD was also reinforced by our analysis of DNA methylation pattern. Interestingly, two important genes, one to intracellular Ca2+ homeostasis and the other to NAD biosynthesis pathway presented CpGs sites differently methylated in the AD subjects

A infertilidade afeta aproximadamente 15% dos casais, sendo atualmente reconhecido o envolvimento de fatores masculinos em metade dos casos. Alterações nas análises seminais são detectadas na maioria dos homens inférteis e a mais frequente é a baixa concentração de espermatozoides no ejaculado, conhecida como oligozoospermia. Vários estudos mostram uma forte relação entre fatores genéticos e a infertilidade, incluindo alterações cromossômicas e microdeleções do cromossomo Y, porém as causas da oligozoospermia ainda permanecem obscuras. O desenvolvimento de novas tecnologias de investigação vem possibilitando a detecção de alterações a nível genômico, como mutações e variações no número de cópias (CNVs). O presente trabalho teve por objetivo a caracterização genômica de homens com oligozoospermia sem causa definida, visando estabelecer correlação entre alterações no número de cópias e perdas de heterozigosidade (LOHs) e o fenótipo de infertilidade. Foram selecionados 18 pacientes após rigorosa avaliação clínica e investigação do histórico reprodutivo, sendo excluídos pacientes portadores de alterações cromossômicas e portadores de microdeleções do cromossomo Y. Seis homens comprovadamente férteis foram selecionados para o grupo controle. A investigação genômica de ambos os grupos, amostral e controle, foi realizada pela técnica de hibridação genômica comparativa em microarranjos (aCGH) utilizando a plataforma de resolução 180K (Agilent®,US), analisada pelo software Nexus 8.0. Foram detectadas alterações possivelmente patogênicas no cromossomo Y, no cromossomo X e em autossomos. Um ganho na região de AZFc envolvendo apenas os genes DAZ1 e DAZ4 foi detectado em nove pacientes e em quatro controles, sendo classificado como alteração benigna. Porém, alterações na região de AZFc possivelmente relacionadas ao fenótipo de oligozoospermia foram detectadas em três pacientes e incluíram extensas duplicações e deleções envolvendo, entre outros genes, as quatro cópias do gene DAZ. Após comparação de regiões selecionadas com a literatura e com diferentes bancos de dados genéticos, sugerimos que os genes PLEC, SPATC1, COL1A1, MOV10L1, SYCE3 e ODF3B possam estar associados a alterações na produção espermática. Adicionalmente, entre os doze miRNAs presentes em regiões de LOH possivelmente relacionadas ao fenótipo de infertilidade, dez têm como alvo genes com funções relacionadas à espermatogênese e reprodução humana. Estudos adicionais a nível de expressão e sequenciamento gênico são necessários para confirmar a correlação entre o genótipo e o fenótipo de oligozoospermia. / Infertility affects about 15% of the couples, and it is currently recognized, that male factors are involved in about 50% of cases. Changes in seminal parameters are detected in most infertile men and the most common alteration, known as oligozoospermia, is a low concentration of sperm in the ejaculate. Several studies show a strong relationship between genetic factors and infertility, including chromosomal abnormalities and microdeletions of Y chromosome, however, the causes of oligozoospermia remain unclear. The development of new research technologies has allowed the detection of changes at genomic levels, such as mutations and copy number variations (CNVs). This study aimed to perform a genomic characterization of patients with idiopathic oligozoospermia to determine whether there is a correlation between changes of copy number and losses of heterozygosity (LOHs) in relation to the phenotype of infertility. Eighteen patients were selected for the cases after rigorous clinical examination and investigation of their reproductive history. Patients with chromosomal abnormalities or microdeletions of the Y chromosome were excluded. Six proven fertile men comprised the control group. Genomic investigation of both groups was performed by microarray comparative genomic hybridization (aCGH) using 4X180K platform (Agilent, US) analysed by Nexus 8.0 software. Potential pathogenic changes were detected on Y chromosome, as well as on the X and autosome chromosomes. A gain in AZFc region involving only DAZ1 and DAZ4 genes was detected in nine patients and four controls, and was considered as benign. However, changes in AZFc region, that could be related to the oligozoospermia phenotype were detected in three patients. These changes included extensive duplications and deletions involving the four copies of the DAZ gene together with copy number changes affecting other genes. After comparing the selected regions with the literature and with different databases, we suggest that changes such as LOH affecting PLEC, SPATC1, COL1A1, MOV10L1, SYCE3 and ODF3B genes may influence sperm production. Our analysis indicates that, ten out of the twelve miRNAs present in LOH regions could be involved in the infertility phenotype and could have target genes with functions related to spermatogenesis and human reproduction. Additional studies involving gene sequencing and expression analysis are needed to confirm the the correlation between the genotype and oligozoospermia phenotype.

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