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.
Novel methodologies for detection of chromosomal abnormalities have been made available in the recent years but their clinical utility in prenatal settings is still unknown. We have conducted a comparative study of currently available methodologies for detection of chromosomal abnormalities after invasive prenatal sampling. A multicentric collection of a 1-year series of fetal samples with indication for prenatal invasive sampling was simultaneously evaluated using three screening methodologies: (1) karyotype and quantitative fluorescent polymerase chain reaction (QF-PCR), (2) two panels of multiplex ligation-dependent probe amplification (MLPA), and (3) chromosomal microarray-based analysis (CMA) with a targeted BAC microarray. A total of 900 pregnant women provided informed consent to participate (94% acceptance rate). Technical performance was excellent for karyotype, QF-PCR, and CMA (~1% failure rate), but relatively poor for MLPA (10% failure). Mean turn-around time (TAT) was 7 days for CMA or MLPA, 25 for karyotype, and two for QF-PCR, with similar combined costs for the different approaches. A total of 57 clinically significant chromosomal aberrations were found (6.3%), with CMA yielding the highest detection rate (32% above other methods). The identification of variants of uncertain clinical significance by CMA (17, 1.9%) tripled that of karyotype and MLPA, but most alterations could be classified as likely benign after proving they all were inherited. High acceptability, significantly higher detection rate and lower TAT, could justify the higher cost of CMA and favor targeted CMA as the best method for detection of chromosomal abnormalities in at-risk pregnancies after invasive prenatal sampling.
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.
Chromosomal imbalances are a major cause of intellectual disability and multiple congenital anomalies (MR/MCA). With the advent of the array-based comparative genomic hybridization (array CGH) method, increasingly smaller and more complex rearrangements are detected in patients with MR/MCA. Among the numerous copy-number variants (CNVs) of various sizes identified when testing such patients, de novo large (i.e., >1 Mb) CNVs are relatively rare events [Miller et al., 2010]. When identified in a patient, such events have a high likelihood for being responsible for the abnormal phenotype. In the next generation, the genomic imbalance is transmitted following the classical Mendelian rules and, based on the current literature, is not supposed to increase the likelihood of the occurrence of a second de novo event. Here, we observed three de novo chromosomal rearrangements on the same chromosome arm in a three-generation family. The proposita (Patient III-1) presented with craniofacial anomalies (Fig. 1), moderate intellectual disability, and behavior disturbance. Her father (Patient II-2) had no physical anomalies but expressive language delay which required speech therapy. He could not complete normal school and was the only member of the family who had language delay and school difficulties. Supplementary clinical information is available online (See Supporting Information). A: Pedigree of the present family. Normal: no imbalance identified by array CGH. The arrow indicates the proposita. B: Photographs showing facial dysmorphic features with prominent forehead, high frontal hairline, low-set, and posteriorly rotated ears in Patient III-1. The karyotypes performed on the child (III-1) and her father (II-2) were both normal: 46,XX and 46,XY at ISCN +550 bands, respectively. A 44K oligoarray CGH performed on Patient III-1 led to the identification of a ∼1.43 Mb duplication in the 7q11.23 region and a ∼1.92 Mb deletion in the 7q22 region (see Methods in Supporting Information online). No other imbalance was identified. To fine-map the aberrations, 400K oligoarray was performed on Patient III-1. The 7q11.23 duplication was confirmed while the 7q22 deletion was more complex than previously thought, with two close deleted regions (Fig. 2A–C). A 1.34 Mb deletion was followed by a 361 kb two-copies (diploid) region and a 214 kb deletion. The parents' DNAs of Patient III-1 were analyzed with 44K arrays and showed normal results in the mother (II-3) while the father (II-2) only carried the duplication. These parental analyses demonstrated that the two 7q22 deletions occurred de novo in Patient III-1 while the 7q11.23 duplication was paternally inherited. We next performed 44K arrays in the phenotypically normal grandparents (individuals I-1 and I-2). Both grandparents were normal by array CGH, demonstrating that the 7q11.23 occurred de novo in Patient II-2. The Agilent 400K array-derived profile of chromosomes 7 and FISH analyses performed on Patient III-1. Black, red, and green dots indicate the log2-transformed fluorescence intensity ratios for normal, duplicated, or deleted (respectively) oligonucleotide probe falling outside the threshold setting used (ADM-2 algorithm at a threshold of 6.0 using CGH Analytics version 4.0). A: The ideogram and the fluorescence intensity ratio for each oligonucleotide probe of the entire chromosome 7. B: Detailed view of the duplication. C: Detailed view of the complex deletion. The horizontal axis indicates the log2-transformed fluorescence intensity ratio for each oligonucleotide probe and the vertical axis indicates their physical position on chromosome 7. The aberrant area is indicated by grey rectangle. Italic symbols indicate gene names. D–F: Dual-color FISH analyses performed on metaphases and interphase nuclei of Patient III-1. FISH with RP11-193P05 BAC clone located in the centromeric 7q22 deletion and RP11-667P12 BAC clone located in the 7q11.23 duplication showed strong red signals on metaphases and two close red signals on interphase nuclei and no green signal suggesting that the duplication and the centromeric 7q22 deletion were located on the same chromosome (probes were labeled with SpectrumGreen and SpectrumOrange, respectively). G: FISH with RP11-193P05 BAC clone located in the centromeric 7q22 deletion and G248P88412A8 fosmid located in the telomeric 7q22 deletion showed no red and green signals on the same chromosome demonstrating that both 7q22 deletions were located on the same chromosome (probes were labeled with SpectrumGreen and SpectrumOrange, respectively). A subtelomeric 7p probe labeled in green was used as control probe. The arrows mark the deletions observed on metaphases. FISH performed in Patients II-2 and III-1 and individuals I-1 and I-2 confirmed the array CGH results (Fig. 2D–F). In addition, interphase and metaphase FISH performed in Patient III-1 with a combination of probes located in the deleted and in the duplicated regions showed that the two deletions and the duplication occurred on the same haplotype (Fig. 2E–G). A target 60K custom oligoarray CGH experiment was performed on Patient III-1 to refine the locations of the breakpoints of the 7q22 deletions (data not shown). Next, by long-range PCR (TaKaRa LA Taq, TAKARA) and direct sequencing (primer sequences are available upon request), we characterized the breakpoints at a molecular level for the two 7q22 deletions. Sequence analyses showed microhomologies at the breakpoint junctions for both deletions (see Supplementary Fig. 1 in Supporting Information online). However, BLAST comparison with regions of 5 kb flanking and within the two 7q22 deletions as template did not reveal significant homologies among them or with sequences within or surrounding the WBS locus. Analysis of single-copy STR markers within the WBS locus revealed that Patient II-2 carried three distinct alleles at loci AFMb055xe5, D7S489B, CR16T, and D7S1870, two of them of paternal origin in all informative positions. Thus, a de novo 7q11.23 duplication in Patient II-2 was confirmed, generated by interchromosomal exchange in meiosis I in grandfather's (I-1) germ cells. Patient III-1 inherited the same alleles from her father and paternal grandfather. The analysis of two multi-copy STRs (BASTR1 and BBSTR1), which are present in the different blocks of segmental duplications, revealed a gain of one block type in Patients II-2 and III-1, with normal copy number of the other blocks, and normal results in all other family members (see Supplementary Fig. 2 in Supporting Information online). These data indicate that the duplication is ∼1.55 Mb in size, the exact reciprocal of the most common WBS deletion, generated by non-allelic homologous recombination (NAHR) between specific blocks of segmental duplications. We then used indirect assays based on site-specific nucleotide dosage analysis to infer the location of the breakpoint and the relative orientation of the WBS locus duplication [Somerville et al., 2005]. Comparing Patient II-2 with respect to his parents, relative gains of pseudogene-type NCF1 copy and medial-type copy at the second site were observed, suggesting that the site of exchange between chromosomes had occurred between the medial centromeric blocks B, in a position proximal to the NCF1 gene (see Supplementary Fig. 2 in Supporting Information online). Therefore, the WBS locus duplication is most likely located in tandem and was not mediated by a parental inversion polymorphism. Although we were not able to confirm the orientation of the duplication in Patients II-2 and III-1, three color interphase FISH (using BACs RP11-552B12, RP11-329B05, and RP11-746H03 as probes) confirmed that the WBS locus was not inverted in individual I-1 (data not shown). Lack of paternal inheritance in Patient III-1 at STR loci D7S501 and D7S2453 further confirmed that both deletions were de novo, of paternal origin, and occurring on the same grandpaternal chromosome 7 carrying the duplication. By haplotyping, no recombination was observed in the paternal chromosome 7 inherited by PIII-1, indicating an intrachromosomal origin for both 7q22 deletions (see Supplementary Fig. 3 in Supporting Information online). Since all rearrangements (WBS duplication and complex 7q22 deletions) had originated in the same chromosome from the grandfather (I-1), we further searched for putative inversion polymorphism at the 7q22 edge of the rearranged intervals. We performed multicolor interphase FISH with BAC probes located within the centromeric deletion (RP11-193P05) and in normal copy-number regions: between the two deletions (RP11-768J05) and telomeric to the deletions (RP11-698M09). Multicolor interphase FISH did not reveal any abnormal probe order in the grandparental (I-1) or paternal (II-2) samples, ruling out inversions, while confirmed the deletion of RP11-193P05 in Patient III-1 (see Supplementary Fig. 4 in Supporting Information online). Interstitial deletions of 7q11.23 cause Williams–Beuren syndrome (OMIM #194050) while the reciprocal duplication is responsible for 7q11.23 duplication syndrome (OMIM #609757) [Somerville et al., 2005]. A series of 14 patients with the latter condition showed that cognitive abilities ranged from normal to moderate intellectual disability but variable speech delay was a constant finding [Van der Aa et al., 2009]. Autism was also associated with this duplication [Sanders et al., 2011]. These data are consistent with Patient II-2's phenotype who carried a 7q11.23 duplication and only presented with speech delay and learning disability. In addition to the 7q11.23 duplication, Patient III-1 carried two close 7q22 deletions. Few patients with intellectual disability and a cytogenetically visible deletion including the 7q22 region have been reported [Franceschini et al., 1978; Serup, 1980; Abuelo and Padre-Mendoza, 1982]. However, these deletions were much larger and cannot be compared with the interstitial deletions that we describe. An additional patient carrying a de novo 7q22 deletion (chr7:103,887,195-106,790,044; hg18) has been uploaded in the DECIPHER database (Patient #4470; http://decipher.sanger.ac.uk/). The deletion of our patient is smaller (1.9 vs. 2.9 Mb) and entirely included in the deletion of the DECIPHER's patient. This patient shared intellectual disability with our patient. He also presented with overgrowth features (tall stature, macrocephaly, large tongue, and high birth weight) while Patient III-1 of our study presented with measurements relatively above the mean [aged 7 years and 1 month, her weight, height, and OFC were 31 kg (+3 SD), 126 cm (+1.5 SD), 54 cm (+2.25 SD), respectively]. Several genomic variants of small size located in the 7q22 region have been identified in individuals from the general population (http://projects.tcag.ca/variation/) but none of them significantly overlapped with the deletion observed in Patient III-1. A two-hit model for severe developmental delay has been recently proposed [Girirajan et al., 2010]. Within this model, a parent carries a microdeletion/microduplication which predisposes to learning disability and/or neuropsychiatric phenotypes but is not sufficient for being responsible for severe developmental delay. In the next generation, a child carries the parental event and a second event. Such co-occurrence is responsible for severe developmental delay. The present family illustrates such a two-hit model. The father only presented with learning disability and a single event (i.e., a 7q11.23 duplication). In contrast, his intellectually disabled child presented with two CNVs: the paternally inherited 7q11.23 duplication and a complex 7q22 deletion. The chromosome 7q11.23 microdeletion/duplication occurs because of the unique genomic architecture in this region. This region is flanked by highly homologous clusters of genes and pseudogenes organized into low-copy-repeat blocks known as duplicons. The high degree of sequence homology among these flanking duplicons, as well as their proximity to each other, predisposes to recurrent chromosomal rearrangements through NAHR mechanism during meiosis [Lupski, 1998] Although genomic structural polymorphisms, including paracentric inversions and copy number variation in the flanking segmental duplications, have been reported as susceptibility alleles for germline rearrangements at the WBS locus [Bayés et al., 2003; Cuscó et al., 2008], we did not find them in the grandfather's sample (I-1). In addition, molecular testing and breakpoint mapping suggested a tandem orientation of the duplicated interval, being the exact reciprocal of the WBS most common deletion and mediated by interchromosomal NAHR. In contrast, several mechanisms, including non-homologous end-joining (NHEJ) [Lieber, 2008] or the recently described replication-based mechanisms of fork stalling and template switching/microhomology-mediated break induced replication (FoSTeS) [Lee et al., 2007] can be responsible for non-recurrent rearrangements without the need for a homologous template or only requiring microhomology [Zhang et al., 2009]. Sequence analyses performed in Patient III-1 showed microhomologies at the breakpoint junctions for both 7q22 deletions. These data suggest that the deletions may have been mediated by NHEJ or FoSTeS mechanisms, and both through intrachromosomal rearrangements on the same chromosome arm harboring the WBS locus duplication, as shown by haplotype analysis. Chromosomes rearrangements (i.e., deletions and duplications) are thought to be stable through generations. This assumption is fundamental to current guidelines for parental follow-up studies for cytogenetically visible alterations, FISH and array CGH abnormalities. However, this assumption is not always true. Size expansions of a deletion from a normal parent to his affected child have been reported in two independent families [Faravelli et al., 2007; South et al., 2008]. The awareness that inherited deletions are not always stable challenges current algorithms for family studies. Furthermore, it is assumed that a genomic imbalance does not increase the likelihood of the occurrence of a second de novo event elsewhere in the genome. Therefore, the two chromosomal rearrangements observed in the intellectually disabled child of the present family may have occurred by chance. However, since de novo large CNVs are relatively rare events and that both duplication and deletions occurred on the same chromosome 7, we speculate that the duplication may have predisposed to the occurrence of the second de novo events. In theory, the duplication could alter chromosome territories in the nuclei, prompting to the formation of abnormal chromatin loops than could facilitate additional rearrangements [Cremer and Cremer, 2001]. Proximity of chromosomes or chromosomal loci during cell division, the so called chromosome kissing, has been shown to correlate with the frequency of recurrent translocations and other rearrangements [Cavalli, 2007]. If such predisposition was further confirmed, this finding would challenge our current algorithms for family studies. Indeed, in this family, if the father carrying a 7q11.23 duplication had asked for a prenatal diagnosis during a pregnancy, only a specific FISH probe targeting the 7q11.23 region would have been used. In such case, the de novo 7q22 deletions would have been missed in the child. Additional observations and further studies are needed to determine whether the occurrence of these abnormalities is coincidental or whether the presence of an initial CNV predisposes to the recurrence of additional de novo genomic imbalances. We are grateful to the family who participated in this study and to Rémi Houlgatte and Catherine Chevalier from Biogenouest de Nantes, France. Additional Supporting Information may be found in the online version of this article. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
We present a case of acute myeloblastic leukemia (AML-M2) with a complex t(8;21) translocation and additional acquired chromosomes yielding a hyperdiploid karyotype. AML1/ETO transcript was observed by reverse transcription-polymerase chain reaction. Fluorescence in situ hybridization (FISH), spectral karyotyping (SKY), and comparative genomic hybridization (CGH) were performed to further identify the chromosomes observed by G banding. The patient was treated according to our current protocol for AML. He remains in complete remission +11 months from diagnosis. Further follow-up of this patient and the analysis of a larger number of children are needed to define whether the gains of the specific extra chromosomes modify the good prognosis that t(8;21) confers to this subgroup of AML.