Copy number Variants (CNVs), which comprise deletions, insertions and inversions of genomic sequence, are a main form of genetic variation between individual genomes. CNVs are commonly present in the genomes of human and other species. However, they have not been extensively characterized as their ascertainment is challenging. I reviewed current CNV studies and CNV discovery methods, especially the algorithms which infer CNVs from whole genome Single Nucleotide Polymorphism (SNP) arrays and compared the performance of three analytical tools in order to identify the best method of CNV identification. Then I applied this method to identify CNV events in three European population isolates—the island of Vis in Croatia, the islands of Orkney in Scotland and villages in the South Tyrol in Italy - from Illumina genome-wide array data with more than 300,000 SNPs. I analyzed and compared CNV features across these three populations, including CNV frequencies, genome distribution, gene content, segmental duplication overlap and GC content. With the pedigree information for each population, I investigated the inheritance and segregation of CNVs in families. I also looked at association between CNVs and quantitative traits measured in the study samples. CNVs were widely found in study samples and reference genomes. Discrepancies were found between sets of CNVs called by different analytical tools. I detected 4016 CNVs in 1964 individuals, out of a total of 2789 participants from the three population isolates, which clustered into 743 copy number variable regions (CNVRs). Features of these CVNRs, including frequency and distribution, were compared and were shown to differ significantly between the Orcadian, South Tyrolean and Dalmatian population samples. Consistent with the inference that this indicated population-specific CNVR identity and origin, it was also demonstrated that CNV variation within each population can be used to measure genetic relatedness. Finally, I discovered that individuals who had extreme values of some metabolic traits possessed rare CNVs which overlapped with known genes more often than in individuals with moderate trait values.

Mental retardation (MR) is a very heterogeneous common neurodevelopmental disorder with a population prevalence of 2.5-3 %. The importance of genetic factors in the development of MR is high but in a significant number of cases the etiology remains unexplained. Recent studies using array methods pointed to frequent occurrence of copy number variants (CNVs) in patients with MR. Pathogenic CNVs were identified in 10-15 % patients with idiopathic MR and normal karyotype. The aim of our work was the analysis of genome-wide gains and losses of genetic material in a group of Czech patients with MR and a thorough bioinformatic analysis of the genetic changes identified aiming at the assessment of their clinical significance. We performed whole genome analysis using the HumanCytoSNP-12 BeadChips (Illumina) in 183 patients with idiopathic MR, normal karyotype and no FMR1 gene expansion. Data analysis was carried out using two independent programmes, GenomeStudio and QuantiSNP. The findings were subjected to two rounds of thorough bioinformatic analysis. Based on this analysis we classified the CNVs into 4 categories: pathogenic CNVs, probably pathogenic CNVs, CNVs with uncertain clinical significance and benign CNVs. With the exception of the benign variants, all CNVs were confirmed using an independent laboratory...

A perda auditiva é o defeito mais comum ao nascimento e cerca de 70 milhões de pessoas no mundo apresentam algum grau de perda auditiva. Além da alta incidência, as implicações da perda auditiva na linguagem, na cognição e no desenvolvimento emocional e social reforçam sua importância. No entanto, em grande parte dos pacientes, a causa da deficiência auditiva não é esclarecida. Nós usamos hibridação comparativa do genoma baseada em arrays (Array Comparative Genomic Hybridization aCGH) para investigar alterações no número de cópias de segmentos de DNA (Copy Number Variation CNV) em 31 indivíduos que apresentavam deficiência auditiva e sinais clínicos adicionais, mas que não puderam ser classificados em síndrome conhecida. A escolha de indivíduos sindrômicos se baseou no pressuposto de que, em média, apresentam alterações genômicas maiores e, portanto, mais provavelmente detectáveis com o uso de aCGH de 1 Mb, que era a plataforma disponível no início do projeto. CNVs não descrita em bancos de dados de indivíduos normais foram identificadas em oito pacientes, quatro delas ocorreram de novo enquanto as outras quatro foram herdadas de um genitor fenotipicamente normal. As alterações de novo definem segmentos cromossômicos que provavelmente contém genes relacionados à deficiência auditiva e sensíveis a dose, especificamente: 1q23.3-q25.2, 2q22q23, 6p25.3 e 11q13.2-q13.4. As alterações raras identificadas tanto nos pacientes quanto em um genitor normal poderiam ser um evento ao acaso, sem papel na deficiência auditiva; no entanto, a possibilidade de que essas alterações possam funcionar como fatores de predisposição não podem ser descartadas. Se considerarmos apenas as CNVs de novo como causativas dos fenótipos investigados, detectamos quatro pacientes portadores entre os 31 investigados (13%). Se considerarmos também as CNVs herdadas como possivelmente causativas, a taxa de desequilíbrios cromossômicos associados à surdez será de 26%. Esses resultados são provavelmente uma substimativa e esses números seriam possivelmente maiores com o uso de uma das plataformas de alta resolução disponíveis atualmente. Esses resultados, embora limitados, indicam que investigação por aCGH em pacientes com surdez sindrômica idiopática está entre os testes mais eficientes para detectar etiologia dos fenótipos, devendo ser incorporado à rotina no diagnóstico e aconselhamento genético. / Hearing loss is the most common congenital deficiency and about 70 million people worldwide present some degree of hearing impairment. In addition to its high incidence, hearing loss impacts language, cognition and social and emotional development. However, in a large proportion of patients, the cause of the hearing deficiency cannot be elucidated. We screened copy number changes by 1 Mb-array Comparative Genomic Hybridization (aCGH) in 31 individuals with syndromic hearing impairment whose clinical features were untypical for known disorders. The choice of evaluating syndromic rather than non-syndromic individuals was based on the assumption that they are more likely to carry larger genomic alterations which could be more easily detected by the comparatively low resolution 1 Mb aCCG, which was the available platform when this project started. Copy number changes (CNV) not documented in the database of normal individuals were detected in eight patients, four de novo imbalances and four inherited from a normal parent. The de novo alterations define candidate chromosome segments likely to harbor dosage sensitive genes related to hearing impairment, namely 1q23.3-q25.2, 2q22q23, 6p25.3 and 11q13.2- q13.4. The rare imbalances also present in normal parents might be casually associated with hearing impairment, but also have a possible role as a predisposition factor. When only the de novo CNVs were considered causative for the disease phenotypes, our study revealed relevant copy number changes in 4 patients (13%). If we also count the rare CNVs that had been inherited as possibly causative, the frequency of chromosome imbalances associated with syndromic deafness in our sample becomes 26%. These figures are probably underestimates and will probably become larger when high resolution oligoarray platforms are applied. These results indicate that aCGH is an efficient tool for defining the etiology of syndromic deafness and its use in routine diagnosis of hearing impairment and for genetic counseling is highly recommended.

A perda auditiva é o defeito mais comum ao nascimento e cerca de 70 milhões de pessoas no mundo apresentam algum grau de perda auditiva. Além da alta incidência, as implicações da perda auditiva na linguagem, na cognição e no desenvolvimento emocional e social reforçam sua importância. No entanto, em grande parte dos pacientes, a causa da deficiência auditiva não é esclarecida. Nós usamos hibridação comparativa do genoma baseada em arrays (Array Comparative Genomic Hybridization aCGH) para investigar alterações no número de cópias de segmentos de DNA (Copy Number Variation CNV) em 31 indivíduos que apresentavam deficiência auditiva e sinais clínicos adicionais, mas que não puderam ser classificados em síndrome conhecida. A escolha de indivíduos sindrômicos se baseou no pressuposto de que, em média, apresentam alterações genômicas maiores e, portanto, mais provavelmente detectáveis com o uso de aCGH de 1 Mb, que era a plataforma disponível no início do projeto. CNVs não descrita em bancos de dados de indivíduos normais foram identificadas em oito pacientes, quatro delas ocorreram de novo enquanto as outras quatro foram herdadas de um genitor fenotipicamente normal. As alterações de novo definem segmentos cromossômicos que provavelmente contém genes relacionados à deficiência auditiva e sensíveis a dose, especificamente: 1q23.3-q25.2, 2q22q23, 6p25.3 e 11q13.2-q13.4. As alterações raras identificadas tanto nos pacientes quanto em um genitor normal poderiam ser um evento ao acaso, sem papel na deficiência auditiva; no entanto, a possibilidade de que essas alterações possam funcionar como fatores de predisposição não podem ser descartadas. Se considerarmos apenas as CNVs de novo como causativas dos fenótipos investigados, detectamos quatro pacientes portadores entre os 31 investigados (13%). Se considerarmos também as CNVs herdadas como possivelmente causativas, a taxa de desequilíbrios cromossômicos associados à surdez será de 26%. Esses resultados são provavelmente uma substimativa e esses números seriam possivelmente maiores com o uso de uma das plataformas de alta resolução disponíveis atualmente. Esses resultados, embora limitados, indicam que investigação por aCGH em pacientes com surdez sindrômica idiopática está entre os testes mais eficientes para detectar etiologia dos fenótipos, devendo ser incorporado à rotina no diagnóstico e aconselhamento genético. / Hearing loss is the most common congenital deficiency and about 70 million people worldwide present some degree of hearing impairment. In addition to its high incidence, hearing loss impacts language, cognition and social and emotional development. However, in a large proportion of patients, the cause of the hearing deficiency cannot be elucidated. We screened copy number changes by 1 Mb-array Comparative Genomic Hybridization (aCGH) in 31 individuals with syndromic hearing impairment whose clinical features were untypical for known disorders. The choice of evaluating syndromic rather than non-syndromic individuals was based on the assumption that they are more likely to carry larger genomic alterations which could be more easily detected by the comparatively low resolution 1 Mb aCCG, which was the available platform when this project started. Copy number changes (CNV) not documented in the database of normal individuals were detected in eight patients, four de novo imbalances and four inherited from a normal parent. The de novo alterations define candidate chromosome segments likely to harbor dosage sensitive genes related to hearing impairment, namely 1q23.3-q25.2, 2q22q23, 6p25.3 and 11q13.2- q13.4. The rare imbalances also present in normal parents might be casually associated with hearing impairment, but also have a possible role as a predisposition factor. When only the de novo CNVs were considered causative for the disease phenotypes, our study revealed relevant copy number changes in 4 patients (13%). If we also count the rare CNVs that had been inherited as possibly causative, the frequency of chromosome imbalances associated with syndromic deafness in our sample becomes 26%. These figures are probably underestimates and will probably become larger when high resolution oligoarray platforms are applied. These results indicate that aCGH is an efficient tool for defining the etiology of syndromic deafness and its use in routine diagnosis of hearing impairment and for genetic counseling is highly recommended.