Small supernumerary marker chromosomes (sSMC): why do they break, where they break and how to distinguish harmful from harmless sSMC?

Small supernumerary marker chromosomes (sSMC) are defined as additional centric chromosome fragments too small to be identified or characterized unambiguously by banding cytogenetics alone. Even though certain sSMC were associated with specific clinical pictures and syndromes, for most of the sSMC only first steps towards genotype-phenotype correlations were achieved. Therefore sSMC are still a problem in clinical cytogenetics and can be harmful due to different mechanisms like induction of genomic imbalances and/or uniparental disomy of the sSMC’s sister chromosomes. This study had the aim to provide new insights into the questions (i) if and why sSMC include specific breakpoints and (ii) how to distinguish harmful from harmless sSMC. Thus, several approaches for better sSMC characterization (HCM-FISH) and/or, characterization of sSMC breakpoints were developed (PCL-FISH; 1MB sets spanning the transitions of dosage-sensitive and dosage-insensitive pericentric regions) and established. sSMC breakpoints were characterized in detail using these new approaches, but also by microdissection based array-comparative genomic hybridization. First hints were obtained that breakpoints involved in sSMC formation are situated preferentially in gene- poor regions of the pericentric regions. Concerning genotype-phenotype correlation of sSMC the present study further identified one new “complex sSMC” associated syndrome: the der(13 or 21)t(13 or 21;18) syndrome, which is associated with a mild clinical phenotype irrespective of partial trisomy 18p. Finally, influence of mosaicism on sSMC-related phenotypes was studied in detail. In conclusion, the present study provided important new data for genotype-phenotype correlation and biological understanding of sSMC.Kleine überzählige Marker-Chromosomen (engl. small supernumerary marker chromosomes = sSMC) sind definiert als Chromosomen, die zusätzlich zum normalen Chromosomensatz des Menschen vorliegen können, welche aber zu klein sind um allein mittels Zytogenetik eindeutig charakterisiert zu werden. Auch wenn bestimmte sSMC schon mit spezifischen Syndromen assoziiert werden konnten, wurde für die meisten der sSMC bisher nur erste Schritte bezüglich einer Genotyp-Phänotyp-Korrelationen erreicht. Daher sind sSMC immer noch ein Problem in der klinischen Zytogenetik. Sie können sich durch verschiedene Mechanismen ungünstig auf den Phänotyp des Trägers auswirken, z.B. durch das Hervorrufen genomischer Imbalancen und/oder die Induktion einer uniparentalen Disomie der sSMC-Schwesterchromosomen. Die hier vorliegende Studie hatte das Ziel neue Einsichten in die Fragen zu erarbeiten (1) ob und warum sSMC spezifische Bruchpunkte haben, und (2) wie schädliche von harmlosen sSMC zu unterscheiden sind. Daher wurden zunächst mehrere neue Sondensets für eine bessere sSMC-Charakterisierung entwickelt. sSMC Bruchpunkte wurden mit diesen neuen Ansätzen, aber auch durch mikrodissektions-basierende array-komparative genomische Hybridisierung genauer bestimmt. Hierdurch ergaben sich erste Hinweise darauf, dass die sSMC-Bruchpunkte bevorzugt in genarmen Bereichen des Genoms liegen. Bezüglich der Frage nach einer verbesserten Genotyp-Phänotyp-Korrelation von sSMC wurde in der vorliegenden Studie u.a. ein neues mit einem sog. "komplexen sSMC" verbundenes Syndrom definiert: das der(13 or 21)t(13 or 21;18)-Syndrom, welches mit einem milden klinischen Phänotyp trotz einer partiellen Trisomie 18p verbunden ist. Schließlich wurde der Einfluss von zellulären Mosaiken in sSMC-basierenden Syndromen auf den Phänotyp im Detail untersucht. Insgesamt liefert die vorliegende Studie neue Daten für das biologische Verständnis von sSMC und deren klinische Auswirkungen auf deren Träger

Small supernumerary marker chromosomes derived from chromosome 14 and/or 22

Small supernumerary marker chromosomes (sSMCs) are additional derivative chromosomes present in an otherwise numerically and structurally normal karyotype. They may derive from each of the 24 human chromosomes, and most contain a normal centromeric region with an alphoid sequence from a single chromosome. The majority of human chromosomes have a unique centromeric DNA-sequence enabling their indubitable characterization. However, chromosomes 14 and 22 share a common centromeric sequence D14/22Z1, and sSMCs with this DNA-stretch can derive from either chromosome. Euchromatin-carrying sSMCs(14 or 22) may be further characterized by molecular cytogenetics. However, in most diagnostic laboratories, heterochromatic sSMCs cannot be differentiated between chromosomes 14 or 22 derivation and are often reported as der(14 or 22). Still, heterochromatic sSMC(14 or 22) can be distinguished from each other using the D22Z4 probe (non-commercial) localized to 22p11.2. Herein, 355 sSMC(14 or 22) analyzed in the authors’ laboratory during the last ~ 20 years are summarized to address the questions: (1) What are the true frequencies of chromosome 14- and chromosome 22- derived sSMCs within D14/22Z1-positive cases? (2) Does sub-characterization of sSMC(14) and sSMC(22) make a difference in routine diagnostics? These questions could be answered as follows: (ad 1) within the studied group of sSMCs ~ 40% are derived from chromosome 14 and ~ 60% from chromosome 22; (ad 2) the knowledge on exact sSMC origin can help to save costs in routine diagnostics; i.e. in a clinically abnormal person with sSMC(14) a test for uniparental disomy is indicated, which is not necessary if a chromosome 22 origin for the sSMC was determined

XLIII Conferências de Genética Doutor Jacinto Magalhães: resumo das comunicações

Suplemento da revista Nascer & Crescer com os resumos dos Posters e Comunicações orais apresentadas durante as XLIII Conferências de Genética Doutor Jacinto Magalhães que decorreu no dia 28 de março de 2014, na cidade do Porto, Portuga

Desequilíbrios genómicos nas patologias do desenvolvimento e do comportamento

Tese de doutoramento em Ciências da Saúde (Pré-Bolonha), ramo de Ciências Biomédicas, apresentada à Faculdade de Medicina da Universidade de CoimbraA citogenética convencional desde a década de 1970 que se tornou uma técnica fundamental no diagnóstico de alterações cromossómicas numéricas e estruturais. Esta área da genética clínica proporciona um diagnóstico em 10-15% dos pacientes com perturbações do neurodesenvolvimento, em particular o défice intelectual (DI), as perturbações do espectro do autismo (PEA) e malformações congénitas (MC). Contudo, a sua capacidade de resolução não vai além das 3-10 Mb, mesmo com bandas de alta resolução. Com a introdução das técnicas de citogenética molecular, a Fluorescence in situ Hybridization (FISH) e mais tarde a Multiplex Ligation-dependent Probe Amplification (MLPA), foi possível alargar o leque de diagnósticos a um maior nível de resolução, aumentando o número de desequilíbrios genómicos identificáveis em aproximadamente 5% dos pacientes, com particular relevância no caso das síndromes de microdeleção e microduplicação e nos rearranjos das regiões subteloméricas. Nos últimos anos a introdução das metodologias de array-based Comparative Genomic Hybridization (array-CGH) tornou possível fazer a análise de todo o genoma numa só experiência mas com uma resolução até 100 vezes superior à citogenética convencional, dependendo da capacidade de resolução da plataforma utilizada. Surge assim uma nova escala de detecção dos desequilíbrios genómicos, passando-se do número de cópias de um cromossoma (aneuploidia) ou da duplicação e deleção de segmentos cromossómicos microscópicos para a detecção de desequilíbrios envolvendo segmentos genómicos submicroscópicos, que podem ser menores que 100 Kb e denominados de Copy Number Variants (CNVs). Abriram-se assim novas perspectivas para a procura da etiologia dos distúrbios do neurodesenvolvimento, como o DI e o PEA, assim como para as malformações congénitas. Com a aplicação das técnicas de array-CGH em grandes grupos de pacientes tem sido possível estabelecer recentemente novas síndromes de microdeleção e microduplicação, construir bases de dados para recolha, organização e comparação das CNVs identificadas e do seu significado clínico. Os desafios e dilemas de interpretação da causalidade de algumas cromossomopatias estenderam-se à escala das CNVs, sendo imperativo interpretar com clareza, determinando as que constituem variantes benignas ou polimórficas, as que são patogénicas e as que permanecem de significado clínico incerto (SCI). Para atingir este objectivo é determinante que o laboratório tenha acesso a uma caracterização clínica detalhada, que permita uma boa correlação genótipo-fenótipo. Neste trabalho um grupo de 147 pacientes com DI, PEA e/ou MC, foi avaliado utilizando uma plataforma de array-CGH com oligonucleótidos, determinando-se a prevalência de CNVs benignas (polimórficas), patogénicas ou de significado clínico incerto. Estabeleceu-se um algoritmo com critérios claros de avaliação e interpretação do significado clínico das CNVs identificadas. Em seis pacientes com desequilíbrios microscópicos, diagnosticados pela citogenética convencional, a caracterização por array-CGH revelou-se fundamental na identificação do conteúdo génico envolvido, na reclassificação do rearranjo, em dois pacientes, e detectou uma nova CNV patogénica envolvida no fenótipo em outros dois. Nos outros 141 pacientes, 22 revelaram uma CNV submicroscópica classificada como patogénica (15,6%) e em 34 pacientes apenas se observaram CNVs de SCI (24,1%). No grupo das CNVs patogénicas submicroscópicas, 50% correspondem a microdeleções ou microduplicações recorrentes em regiões de síndromes já estabelecidas, a outra metade foi classificada como patogénica pelo seu conteúdo génico e ou pacientes sobreponíveis já reportados. Com este trabalho sai reforçada a estratégia de aplicação da array-CGH como teste de primeira linha no diagnóstico de pacientes com distúrbios do neurodesenvolvimento e MC, quando não existe uma orientação clínica específica. Inverte-se a estratégia laboratorial passando a FISH e a MLPA ou mesmo a citogenética convencional para uma segunda etapa de caracterização do rearranjo e/ou avaliação dos progenitores por vezes portadores equilibrados. As novas técnicas de sequenciação denominadas por Next Generation Sequencing (NGS) irão eventualmente substituir a array-CGH no diagnóstico dos distúrbios do neurodesenvolvimento e MC. A sequenciação massiva tem a capacidade de detectar mutações pontuais assim como deleções, duplicações mas também rearranjos equilibrados como translocações e inversões. No presente, sequenciar todo o genoma é exequível, mas muito dispendioso, em poucos anos será financeiramente comportável e talvez mais aplicável em larga escala. Contudo, será necessária uma evolução ao nível bioinformático de modo a resolver alguns impasses relacionados com a aplicação da NGS. A interpretação do grande número de novas variantes que serão identificadas será um desafio enorme para cientistas e clínicos, com a necessidade de integração concertada do genótipo com o fenótipo. Palavras-chave: array-CGH, citogenética, défice intelectual, neurodesenvolvimentoConventional karyotyping has been used as the standard technique since the 1970s in the diagnosis of numerical and structural chromosomal aberrations. This area of clinical genetics gives a diagnosis in 10-15 % of the patients with neurodevelopment disorders, including intellectual disability (ID), autism spectrum disorders (ASD) and congenital malformations (CM). However, conventional karyotyping usually only detects rearrangements larger than 3-10 Mb, even in high resolution banding. With the development of molecular cytogenetics, the Fluorescence in situ Hybridization (FISH), and the Multiplex Ligation-dependent Probe Amplification (MLPA), it was possible to detect a larger number of genomic imbalances with a high resolution, increasing the detection rate in 5 % of the patients, particularly in the diagnosis of microdeletion and microduplication syndromes and subtelomeric rearrangements. In recent years with the introduction of array-based Comparative Genomic Hybridization (array-CGH) techniques it became possible to screen the whole human genome in a single experiment with an increasing resolution that can be 100 times higher than conventional karyotyping, depending on the resolution of the array. Genomic imbalances can be detected with a higher resolution, starting in the copy number of a chromosome (aneuploidy) or the duplication and deletion of microscopic chromosome segments and achieving the detection of submicroscopic genomic imbalances, even smaller than 100 kb, the Copy Number Variants (CNVs). The etiology of neurodevelopment disorders including ID, ASD and CM, has new perspectives of diagnosis. Since the application of array-CGH in large cohorts of patients, it was possible in recent years to establish new microdeletion and microduplication syndromes and to construct databases with the collection of the identified CNVs allowing the comparison of its clinical significance. The challenges and dilemmas of the interpretation of genomic variation in cytogenetics extend to the resolution of the CNVs size, being essential to determine which CNVs are benign or polymorphic, which are pathogenic and which remain unclassified variants with uncertain clinical significance. To achieve this goal it is very important that it is provided to the laboratory a detailed clinical characterization, for an ascertain genotype-phenotype correlation. In the present work a group of 147 patients with ID, ASD and CM, was evaluated using oligonucleotide array-CGH, in order to ascertain the prevalence of benign (or polymorphic) CNVs, pathogenic or CNVs of uncertain clinical significance. An algorithm with clear criteria of evaluation and interpretation of the clinical significance of the identified CNVs was established. In six patients with previous identified microscopic rearrangements, the characterization by array-CGH was essential to identify the genes involved, leading to the re-classification of the type of rearrangement in two of the patients and disclosing a new pathogenic CNV involved in the phenotype in two other ones. In the other 141 patients, 22 revealed a submicroscopic CNV classified as pathogenic (15,6%) and in 34 patients were only identified CNVs of uncertain clinical significance (24,1%). In the group of the submicroscopic pathogenic CNVs, 50% were recurrent microdeletions or microduplications in regions of previous established syndromes, the other half were classified as pathogenic because of its genic content and/or previous overlapping reports. With this work the use of array-CGH as the first tier diagnostic technique of patients with neurodevelopment disorders and CM, when there isn’t a specific clinical orientation, is reinforced. The strategy of the laboratory investigations is now inverted, FISH and MLPA techniques or even conventional karyotyping could be a second step in the investigation when it is needed the characterization of the rearrangement involved and the screening of the parents that could be balanced carriers. Next Generation Sequencing (NGS) technologies will eventually replace the array-CGH in the genetic diagnosis of neurodevelopment disorders and CM. Massive sequencing will be able to detect mutations as well as deletions, duplications, and balanced rearrangements like inversions and translocations. At the moment sequencing the whole genome is feasible, but an expensive strategy, probably in a few years it will be affordable and maybe suitable for a large scale. However, several bioinformatics advances will be necessary to solve some issues related with NGS. The interpretation of a large number of new variants will be a huge challenge for scientists and clinicians, that will need to collaborate to achieve a good integration of the genotype and the phenotype. Keywords: array-CGH, cytogenetics, intellectual disability, neurodevelopmen

Abstracts from the 50th European Society of Human Genetics Conference: Posters

International audienc

Frameshift mutations at the C-terminus of HIST1H1E result in a specific DNA hypomethylation signature

BACKGROUND: We previously associated HIST1H1E mutations causing Rahman syndrome with a specific genome-wide methylation pattern. RESULTS: Methylome analysis from peripheral blood samples of six affected subjects led us to identify a specific hypomethylated profile. This "episignature" was enriched for genes involved in neuronal system development and function. A computational classifier yielded full sensitivity and specificity in detecting subjects with Rahman syndrome. Applying this model to a cohort of undiagnosed probands allowed us to reach diagnosis in one subject. CONCLUSIONS: We demonstrate an epigenetic signature in subjects with Rahman syndrome that can be used to reach molecular diagnosis

Avaliação citogenômica em indivíduos com cardiopatias congênitas conotruncais

As cardiopatias congênitas (CCs) são o grupo mais comum de defeitos congênitos graves, afetando 4 a 12 em cada 1.000 nascimentos, sendo uma importante causa de defeitos congênitos associados à mortalidade infantil. Vias biológicas moleculares e celulares complexas estão envolvidas no desenvolvimento do coração, e pouco se sabe sobre os mecanismos subjacentes das CCs. Os defeitos conotruncais, malformações com significativa morbidade, representam cerca de 20% de todos os casos de CCs. Apesar dos avanços no tratamento médico e cirúrgico, a etiologia das CCs ainda não é totalmente compreendida. Com mais crianças com CC sobrevivendo até a idade adulta e começando a formar famílias, torna-se ainda mais crítico a compreensão das bases genéticas das CCs. Estudos clássicos indicaram que a origem da CC é multifatorial, devido tanto à predisposição genética quanto às influências ambientais. Os desequilíbrios genômicos que levam à variação do número de cópias parecem ter uma influência muito maior sobre o desenvolvimento de vários tipos de CCs do que previsto anteriormente. Neste trabalho, utilizamos hibridização genômica comparativa baseada em microarranjos (array-CGH) para estudar retrospectivamente 60 indivíduos com defeitos conotruncais e identificar desequilíbrios genômicos. As variações no número de cópias de DNA (CNVs) detectadas foram comparadas com dados de bancos de dados genômicos, e seu significado clínico foi avaliado. Detectamos em 38,3% (23/60) dos casos de CCs desequilíbrios genômicos. Em 8,3% (5/60) destes casos, os desequilíbrios foram causais; em 8,3% (5/60), CNVs de significado desconhecido foram identificadas; e, em 21,6% (13/60), foram detectadas variantes comuns. Concluimos que, a interpretação dos resultados deve ser refinada, e embora ainda não exista um consenso a respeito dos tipos de CCS que devem ser avaliados por uma análise citogenômica, a identificação da variação do número de cópias em indivíduos com cardiopatias congênitas conotruncais pode, potencialmente, ajudar na avaliação e manejo desta condição. O uso prospectivo ou retrospectivo do array-CGH como uma ferramenta diagnóstica beneficiaria as famílias afetadas ao fornecer um diagnóstico mais preciso, influenciando o manejo global da doença em um número significativo de casos. Além disso, os resultados desse estudo ressaltam a importância crescente do uso de análises genômicas amplas para identificar CNVs em pacientes com CCs, aumentando assim a informação disponível sobre variações genômicas associadas a esta condição.Congenital heart defects (CHDs) are the commonest group of major birth defects, affecting four to twelve per 1,000 total births, being an important cause of birth defects associated infant mortality. Complex molecular and cell biological pathways are involved in heart development, and little is known about the underlying mechanisms of CHDs. The conotruncal defects, malformations with significant morbidity and mortality, represent about 20% of all CHD cases. Despite advances in medical and surgical care, the etiology of CHD is still not completely understood. With more children with CHD surviving to adulthood and starting families, it becomes even more critical to understand the genetic bases of CHD. Classic studies have found that CHD is multifactorial, due to both genetic predisposition and environmental influences. Genomic imbalances leading to copy number changes seems to have a much greater influence on the development of various types of CHD than previously predicted. We used array-comparative genomic hybridization (array-CGH) to retrospectively study 60 subjects with conotruncal defects and identify genomic imbalances. The DNA copy number variations (CNVs) detected were matched with data from genomic databases, and their clinical significance was evaluated. We found that 38.3% (23/60) of CHD cases possessed genomic imbalances. In 8.3% (5/60) of these cases, the imbalances were causal; in 8.3% (5/60), CNVs of unknown significance were identified; and in 21.6% (13/60), common variants were detected. In conclusion, although the interpretation of the results must be refined, and although there is not yet a consensus regarding which types of CHD should be evaluated by cytogenomic analysis, the identification of copy number changes in subjects with conotruncal congenital heart defects can potentially help in the evaluation and management of this condition. The use of retrospective or prospective array-CGH as a diagnostic tool would benefit families by providing a more accurate diagnosis and would affect overall disease management in a significant number of cases. Furthermore, the results of such studies emphasize the growing importance of the use of genome-wide assays to identify CNVs in subjects with CHD, thereby increasing the available information about of genomic variation associated to this condition