Olaya Villa

Mosaic Uniparental Disomies and Aneuploidies as Large Structural Variants of the Human Genome

Mosaicism is defined as the coexistence of cells with different genetic composition within an individual, caused by postzygotic somatic mutation. Although somatic mosaicism for chromosomal abnormalities is a well-established cause of developmental and somatic disorders and has also been detected in different tissues, its frequency and extent in the adult normal population are still unknown. We provide here a genome-wide survey of mosaic genomic variation obtained by analyzing Illumina 1M SNP array data from blood or buccal DNA samples of 1991 adult individuals from the Spanish Bladder Cancer/EPICURO genome-wide association study. We found mosaic abnormalities in autosomes in 1.7% of samples, including 23 segmental uniparental disomies, 8 complete trisomies, and 11 large (1.5-37 Mb) copy-number variants. Alterations were observed across the different autosomes with recurrent events in chromosomes 9 and 20. No case-control differences were found in the frequency of events or the percentage of cells affected, thus indicating that most rearrangements found are not central to the development of bladder cancer. However, five out of six events tested were detected in both blood and bladder tissue from the same individual, indicating an early developmental origin. The high cellular frequency of the anomalies detected and their presence in normal adult individuals suggest that this type of mosaicism is a widespread phenomenon in the human genome. Somatic mosaicism should be considered in the expanding repertoire of inter- and intraindividual genetic variation, some of which may cause somatic human diseases but also contribute to modifying inherited disorders and/or late-onset multifactorial traits.

Autism-specific copy number variants further implicate the phosphatidylinositol signaling pathway and the glutamatergic synapse in the etiology of the disorder

Autism spectrum disorders (ASDs) constitute a group of severe neurodevelopmental conditions with complex multifactorial etiology. In order to explore the hypothesis that submicroscopic genomic rearrangements underlie some ASD cases, we have analyzed 96 Spanish patients with idiopathic ASD after extensive clinical and laboratory screening, by array comparative genomic hybridization (aCGH) using a homemade bacterial artificial chromosome (BAC) array. Only 13 of the 238 detected copy number alterations, ranging in size from 89 kb to 2.4 Mb, were present specifically in the autistic population (12 out of 96 individuals, 12.5%). Following validation by additional molecular techniques, we have characterized these novel candidate regions containing 24 different genes including alterations in two previously reported regions of chromosome 7 associated with the ASD phenotype. Some of the genes located in ASD-specific copy number variants act in common pathways, most notably the phosphatidylinositol signaling and the glutamatergic synapse, both known to be affected in several genetic syndromes related with autism and previously associated with ASD. Our work supports the idea that the functional alteration of genes in related neuronal networks is involved in the etiology of the ASD phenotype and confirms a significant diagnostic yield for aCGH, which should probably be included in the diagnostic workup of idiopathic ASD.

Small supernumerary marker chromosome causing partial trisomy 6p in a child with craniosynostosis.

We report on a child with a small supernumerary marker chromosome (sSMC) causing partial trisomy 6p. The child showed a phenotype consisting of neonatal craniosynostosis, microcephaly, and borderline developmental delay. By molecular techniques the sSMC has been shown to contain ∼16 Mb of genomic DNA from 6p21.1 to 6cen, being de novo and of maternal origin.

Blast cells with nuclear extrusions in the form of micronuclei are associated with MYC amplification in acute myeloid leukemia

We report three cases of acute myeloid leukemia without maturation [AML-M1 subtype according to the French-American-British classification (FAB)] with the presence of MYC oncogene amplification in form of double minutes (dmin) or homogeneously staining region (hsr). Blasts cells showed a particular morphology with extrusion of chromatin material. We observed by FISH the phenomenon of MYC aggregation in interphase cells and the formation of micronuclei excluded from the nucleus. The appearance of chromatin extrusion in cytological analysis should draw attention of the presence of dmin aggregation and possible MYC amplification.

Direct tandem duplication in chromosome 19q characterized by array CGH.

Partial trisomy of the long arm of chromosome 19 is a rare aneusomy. Only six cases of pure duplications have been previously reported, two of which were prenatally detected. Here we describe the clinical manifestations in a 15-month-old girl with a de novo dup(19)(q12q13.2) and the application of array-based comparative genomic hybridization with a resolution of approximately 1 Mb to characterize the duplicated segment. Seven clones were found duplicated, and the size of the fragment was determined to be 10.8 Mb. The scarce number of patients reported and the difficulty of accurately defining the duplicated segment when conventional cytogenetic methods are applied hamper the delineation of a clinical phenotype for duplication of chromosome 19q. To our knowledge this is the fifth live born reported with a pure dup(19), and the first report in which the duplicated segment has been accurately characterized by means of array CGH.

Methotrexate resistance in vitro is achieved by a dynamic selectionprocess of tumor cell variants emerging during treatment.

Genetic instability leads to tumor heterogeneity, which in turn provides a source of cell variants responsible for drug resistance. However, the source of resistant cells during the process of acquired resistance is poorly understood. Our aim has been to characterize the mechanism by which acquired resistance to methotrexate emerges during the course of cancer cell treatment in vitro. We recently demonstrated that, in vitro, HT‐29 colon cancer cells become transiently sensitive to methotrexate by depleting the extracellular milieu of survival factors; on the other hand, the cell population under treatment can reversibly adapt to grow below a critical cell density in the presence of the drug. Here, we show that this adapted cell population gives rise to permanent resistant populations through repeated cycles of cell death and growth. This increased cell turnover, but not merely cell proliferation, is required for the appearance of increasing degrees of stable resistance that are progressively selected by drug pressure. Such a process, taking place in multiple steps, is here designated “dynamic selection.” The analysis of sensitive and resistant HT‐29 cell populations revealed that methotrexate induces genomic instability—characterized by centrosome amplification and aberrant chromosome recombination—leading to a low‐level amplification of the 5q chromosome arm as one of the earliest genetic events selected during treatment. Therefore, this model provides a mechanism by which a tumor cell population lacking resistant subpopulations before treatment is able to acquire the genetic changes required for stable drug resistance.

Intrachromosomal 3p Insertion as a Cause of Reciprocal Pure Interstitial Deletion and Duplication in Two Siblings: Further Delineation of the Emerging Proximal 3p Deletion Syndrome

Very few cases of constitutional interstitial deletions of the proximal short arm of chromosome 3 have been reported; however, the proximal 3p deletion is emerging as a clinically recognizable syndrome. We present an intrachromosomal insertion of 3p12.3p14.1 in a phenotypic normal man (46,XY,ins(3)(p25p12.3p14.1)) which is responsible for the unbalanced karyotype in 2 affected offspring, one with a 3p12.3p14.1 interstitial deletion and the other with a reciprocal duplication. The exceptionality of these 2 reciprocal recombinants contributes to a better definition of the proximal 3p deletion syndrome and its duplication counterpart.

Severe ipsilateral musculoskeletal involvement in a Cornelia de Lange patient with a novel NIPBL mutation.

Cornelia de Lange Syndrome (CdLS) is a congenital autosomal dominant (NIPBL, SMC3 and RAD21) or X-linked (SMC1A and HDAC8) disorder characterized by facial dysmorphism, pre and postnatal growth retardation, developmental delay and/or intellectual disability, and multiorgan involvement. Musculoskeletal malformations are usually bilateral and affect mainly the upper limbs; the range goes from brachyclinodactyly to severe reduction defects. Instead lower extremities are usually less and mildly involved. Here, we report on a 3-year-old Senegalese boy with typical craniofacial CdLS features, pre and postnatal growth retardation, atrial septal defect, developmental delay and right ipsilateral limb malformations, consistent with oligodactyly of the 3rd and 4th fingers, tibial agenesis and fibula hypoplasia. Exome sequencing and Sanger sequencing showed a novel missense mutation in NIPBL gene (c.6647A>G; p.(Tyr2216Cys)), which affects a conserved residue located within NIPBL HEAT repeat elements. Pyrosequencing analysis of NIPBL gene, disclosed similar levels of wild-type and mutated alleles in DNA and RNA samples from all tissues analyzed (oral mucosa epithelial cells, peripheral blood leukocytes and fibroblasts). These findings indicated the absence of somatic mosaicism, despite of the segmental asymmetry of the limbs, and confirmed biallelic expression for NIPBL transcripts, respectively. Additionally, conditions like Split-hand/foot malformation with long-bone deficiency secondary to duplication of BHLHA9 gene have been ruled out by the array-CGH and MLPA analysis. To our knowledge, this is the first CdLS patient described with major ipsilateral malformations of both the upper and lower extremities, that even though this finding could be due to a random event, expands the spectrum of limb reduction defects in CdLS.

ACTB Loss-of-Function Mutations Result in a Pleiotropic Developmental Disorder

ACTB encodes β-actin, an abundant cytoskeletal housekeeping protein. In humans, postulated gain-of-function missense mutations cause Baraitser-Winter syndrome (BRWS), characterized by intellectual disability, cortical malformations, coloboma, sensorineural deafness, and typical facial features. To date, the consequences of loss-of-function ACTB mutations have not been proven conclusively. We describe heterozygous ACTB deletions and nonsense and frameshift mutations in 33 individuals with developmental delay, apparent intellectual disability, increased frequency of internal organ malformations (including those of the heart and the renal tract), growth retardation, and a recognizable facial gestalt (interrupted wavy eyebrows, dense eyelashes, wide nose, wide mouth, and a prominent chin) that is distinct from characteristics of individuals with BRWS. Strikingly, this spectrum overlaps with that of several chromatin-remodeling developmental disorders. In wild-type mouse embryos, β-actin expression was prominent in the kidney, heart, and brain. ACTB mRNA expression levels in lymphoblastic lines and fibroblasts derived from affected individuals were decreased in comparison to those in control cells. Fibroblasts derived from an affected individual and ACTB siRNA knockdown in wild-type fibroblasts showed altered cell shape and migration, consistent with known roles of cytoplasmic β-actin. We also demonstrate that ACTB haploinsufficiency leads to reduced cell proliferation, altered expression of cell-cycle genes, and decreased amounts of nuclear, but not cytoplasmic, β-actin. In conclusion, we show that heterozygous loss-of-function ACTB mutations cause a distinct pleiotropic malformation syndrome with intellectual disability. Our biological studies suggest that a critically reduced amount of this protein alters cell shape, migration, proliferation, and gene expression to the detriment of brain, heart, and kidney development.

Three-Year Follow-Up of a Prenatally Ascertained Apparently Non-Mosaic sSMC(10): Delineation of a Non-Critical Region

Small supernumerary marker chromosomes (sSMC) originating from chromosome 10 are rare and usually found in mosaic form. We present a de novo apparently non-mosaic sSMC(10) prenatally diagnosed in amniotic fluid and postnatally confirmed in peripheral blood. Characterization by array-CGH showed a pericentromeric duplication of 7.1 Mb of chromosome 10. The fetus did not show ultrasound abnormalities, and a normal female phenotype was observed during a 3-year postnatal follow-up. The absence of phenotypic abnormalities in the present case provides evidence of a non-critical pericentromeric region in 10p11.21q11.1 (hg19 35,355,570-42,448,569) associated with a duplication.