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Contains fulltext : 110038.pdf (Publisher’s version ) (Closed access) Potocki-Shaffer syndrome (PSS) is a contiguous gene disorder due to the interstitial deletion of band p11.2 of chromosome 11 and is characterized by multiple exostoses, parietal foramina, intellectual disability (ID), and craniofacial anomalies (CFAs). Despite the identification of individual genes responsible for multiple exostoses and parietal foramina in PSS, the identity of the gene(s) associated with the ID and CFA phenotypes has remained elusive. Through characterization of independent subjects with balanced translocations and supportive comparative deletion mapping of PSS subjects, we have uncovered evidence that the ID and CFA phenotypes are both caused by haploinsufficiency of a single gene, PHF21A, at 11p11.2. PHF21A encodes a plant homeodomain finger protein whose murine and zebrafish orthologs are both expressed in a manner consistent with a function in neurofacial and craniofacial development, and suppression of the latter led to both craniofacial abnormalities and neuronal apoptosis. Along with lysine-specific demethylase 1 (LSD1), PHF21A, also known as BHC80, is a component of the BRAF-histone deacetylase complex that represses target-gene transcription. In lymphoblastoid cell lines from two translocation subjects in whom PHF21A was directly disrupted by the respective breakpoints, we observed derepression of the neuronal gene SCN3A and reduced LSD1 occupancy at the SCN3A promoter, supporting a direct functional consequence of PHF21A haploinsufficiency on transcriptional regulation. Our finding that disruption of PHF21A by translocations in the PSS region is associated with ID adds to the growing list of ID-associated genes that emphasize the critical role of transcriptional regulation and chromatin remodeling in normal brain development and cognitive function.

The mechanisms involved in the formation of subtelomeric rearrangements are now beginning to be elu- cidated. Breakpoint sequencing analysis of 1p36 rear- rangements has made important contributions to this line of inquiry. Despite the unique architecture of segmental duplications inherent to human subtelomeres, no common mechanism has been identiWed thus far and diVerent non- exclusive recombination-repair mechanisms seem to predominate. In order to gain further insights into the mechanisms of chromosome breakage, repair, and stabilization mediating subtelomeric rearrangements in humans, we investigated the constitutional rearrangements of 1p36. Cloning of the breakpoint junctions in a complex rear- rangement and three non-reciprocal translocations revealed similarities at the junctions, such as microhomology of up to three nucleotides, along with no signiWcant sequence iden- tity in close proximity to the breakpoint regions. All the breakpoints appeared to be unique and their occurrence was limited to non-repetitive, unique DNA sequences. Several recombination- or cleavage-associated motifs that may promote non-homologous recombination were observed in close proximity to the junctions. We conclude that NHEJ is likely the mechanism of DNA repair that generates these rearrangements. Additionally, two appar- ently pure terminal deletions were also investigated, and the reWnement of the breakpoint regions identiWed two dis- tinct genomic intervals »25-kb apart, each containing a series of 1p36 speciWc segmental duplications with 90-98% identity. Segmental duplications can serve as substrates for ectopic homologous recombination or stimulate geno- mic rearrangements.

We demonstrate that satellite III (SatIII) DNA subfamilies cloned from human acrocentric chromosomes arose in the Hominoidea superfamily. Two groups, distinguished by sequence composition, evolved nonconcurrently, with group 2 evolving 16-23 million years ago (MYA) and the more recent group 1 sequences emerging approximately 4.5 MYA. We also show the relative order of emergence of each group 2 subfamily in the various primate species. Our results show that each SatIII subfamily is an independent evolutionary unit, that the rate of evolution is not uniform between species, and that the evolution within a species is not uniform between chromosomes.

Although approximately 1 in 500 individuals carries a reciprocal translocation, little is known about the mechanisms that result in their formation. We analyzed the sequences surrounding the breakpoints in three unbalanced translocations of 1p and 9q, all of which were designated t(1;9)(p36.3;q34), to investigate the presence of sequence motifs that might mediate nonhomologous end joining (NHEJ). The breakpoint regions were unique in all individuals. Two of three translocations demonstrated insertions and duplications at the junctions, suggesting NHEJ in the formation of the rearrangements. No homology was identified in the breakpoint regions, further supporting NHEJ. We found translin motifs at the breakpoint junctions, suggesting the involvement of translin in the joining of the broken chromosome ends. We propose a model for balanced translocation formation in humans similar to transposition in bacteria, in which staggered nicks are repaired resulting in duplications and insertions at the translocation breakpoints.

Deletion of the distal band of the short arm of chromosome 1 (monosomy 1p36) is the most common terminal deletion syndrome, occurring in about 1 in 5000 newborns. Of the 121 subjects ascertained for our study to date, 12 (9.9%) have interstitial deletions, three of which are complex rearrangements showing more than one deletion. Herein we report the characterization of a complex rearrangement with two interstitial deletions in the same chromosome 1p36.33-p36.23. We narrowed and analyzed the breakpoints and junctions between the sequence fragments involved in the rearrangement to determine the structure of this deleted chromosome 1. The analyses of the DNA sequence at the junctions showed additional complexity: an inversion and a third de-novo interstitial deletion. We reconstructed this complex rearrangement of 1p36 to understand the mechanism of formation. Analysis of the breakpoint junctions revealed that three of the four breakpoints each interrupted a gene. Alignments of the junctions showed the lack of any sequence similarity between the breakpoints, suggesting the involvement of non-homologous end joining (NHEJ) in the ligation of broken ends following deletion. The identification of translin recognition sites in the breakpoints suggests translin involvement in the repair of broken chromosomes. This report is one of the first to examine constitutional chromosomal rearrangements at the DNA sequence level. The discovery of cryptic events in seemingly simple chromosome rearrangements may provide the basis for proposing mechanisms of formation.

Potocki-Shaffer syndrome (PSS) is a contiguous gene deletion syndrome that results from haploinsufficiency of at least two genes within the short arm of chromosome 11[del(11)(p11.2p12)]. The clinical features of PSS can include developmental delay, mental retardation, multiple exostoses, parietal foramina, enlarged anterior fontanel, minor craniofacial anomalies, ophthalmologic anomalies, and genital abnormalities in males. We constructed a natural panel of 11p11.2-p13 deletions using cell lines from 10 affected individuals, fluorescence in situ hybridization (FISH), microsatellite analyses, and array-based comparative genomic hybridization (array CGH). We then compared the deletion sizes and clinical features between affected individuals. The full spectrum of PSS manifests when deletions are at least 2.1 Mb in size, spanning from D11S1393 to D11S1385/D11S1319 (44.6-46.7 Mb from the 11p terminus) and encompassing EXT2, responsible for multiple exostoses, and ALX4, causing parietal foramina. Yet one subject with parietal foramina whose deletion does not include ALX4 indicates that ALX4 in this subject may be rendered functionally haploinsufficient by a position effect. Based on comparative deletion mapping of eight individuals with the full PSS syndrome including mental retardation and two PSS families with no mental retardation, at least one gene related to mental retardation is likely located between D11S554 and D11S1385/D11S1319, 45.6-46.7 Mb from the 11p terminus.

Interchromosomal insertional translocations are rare chromosome rearrangements with an incidence of about 1:80,000 live births. We report on the clinical and cytogenetic findings of a newborn baby with partial trisomy 10q22-10q24 due to a maternal insertional translocation 15;10. Partial trisomy of the long arm of chromosome 10 is a distinctive chromosome aberration characterized by prenatal-onset growth retardation and craniofacial, skeletal, and other somatic anomalies. Most cases are unbalanced products from reciprocal chromosome translocations, and insertional translocations are rarely involved. The proband was initially referred because of severe intrauterine growth retardation, and fluorescence in situ hybridization (FISH) using painting probes confirmed the maternal balanced (15;10) insertion.

Approximately one in 500 individuals carries a reciprocal translocation. Balanced translocations are usually associated with a normal phenotype unless the translocation breakpoints disrupt a gene(s) or cause a position effect. We investigated breakpoint junctions at the sequence level in phenotypically normal balanced translocation carriers. Eight breakpoint junctions derived from four nonrelated subjects with apparently balanced translocation t(1;22)(p36;q13) were examined. Additions of nucleotides, deletions, duplications, and a triplication identified at the breakpoints demonstrate high complexity at the breakpoint junctions and indicate involvement of multiple mechanisms in the DNA breakage and repair process during translocation formation. Possible detailed nonhomologous end-joining scenarios for t(1;22) cases are presented. We propose that cryptic imbalances in phenotypically normal, balanced translocation carriers may be more common than currently appreciated.

Deletion of chromosome 1p36 is the most commonly observed terminal deletion in humans with a frequency of 1 in 5,000 in the general population. In contrast, 22q13 duplications are rare and only a few cases have been reported. Unbalanced translocations resulting in monosomy 1p36 and a trisomy of 22q13.3 are, thus far, unreported in the literature. Here we present the clinical data and the results of array CGH and FISH analysis of four patients with unbalanced translocations t(1;22)(p36;q13) inherited from unrelated balanced translocation carrier parents. The sizes of the imbalances ranged from 0.12 Mb to nearly 10 Mb. One balanced translocation carrier parent had disruption of the period homolog 3 (PER3) gene and reported sleep disturbances. Overall, patients tended to have more features consistent with deletion of 1p36 than duplication of 22q.

Approximately one in 500 individuals carries a reciprocal translocation. Of the 121 monosomy 1p36 subjects ascertained by our laboratory, three independent cases involved unbalanced translocations of 1p and 9q, all of which were designated t(1;9)(p36.3;q34). These derivative chromosomes were inherited from balanced translocation carrier parents. To understand better the causes and consequences of chromosome breakage and rearrangement in the human genome, we characterized each derivative chromosome at the DNA sequence level and identified the junctions between 1p36 and 9q34. The breakpoint regions were unique in all individuals. Insertions and duplications were identified in two balanced translocation carrier parents and their unbalanced offspring. Sequence analyses revealed that the translocation breakpoints disrupted genes. This study demonstrates that apparently balanced reciprocal translocations in phenotypically normal carriers may have cryptic imbalance at the breakpoints. Because disrupted genes were identified in the phenotypically normal translocation carriers, caution should be exercised when interpreting data on phenotypically abnormal carriers with apparently balanced rearrangements that disrupt putative candidate genes.

Structural chromosome abnormalities have aided in gene identification for over three decades. Delineation of the deletion sizes and rearrangements allows for phenotype/genotype correlations and ultimately assists in gene identification. In this report, we have delineated the precise rearrangements in four subjects with deletions, duplications, and/or triplications of 1p36 and compared the regions of imbalance to two cases recently published. Fluorescence in situ hybridization (FISH) analysis revealed the size, order, and orientation of the duplicated/triplicated segments in each subject. We propose a premeiotic model for the formation of these complex rearrangements in the four newly ascertained subjects, whereby a deleted chromosome 1 undergoes a combination of multiple breakage-fusion-bridge (BFB) cycles and inversions to produce a chromosome arm with a complex rearrangement of deleted, duplicated and triplicated segments. In addition, comparing the six subjects' rearrangements revealed a region of overlap that when triplicated is associated with craniosynostosis and when deleted is associated with large, late-closing anterior fontanels. Within this region are the MMP23A and -B genes. We show MMP23 gene expression at the cranial sutures and we propose that haploinsufficiency results in large, late-closing anterior fontanels and overexpression results in craniosynostosis. These data emphasize the important role of cytogenetics in investigating and uncovering the etiologies of human genetic disease, particularly cytogenetic imbalances that reveal potentially dosage-sensitive genes.

Patients with t(11;19) leukemias have one of two different translocations resulting in gene rearrangement with the MLL gene on 11q23. The ELL (eleven-nineteen lysine-rich leukemia) gene at 19p13.1, is rearranged in individuals with t(11;19) and acute myeloid leukemia, while the ENL (eleven-nineteen leukemia) gene at 19p13.3, is rearranged most frequently in those patients with t(11;19) and B-cell acute lymphoid leukemia. However, AML and rare T-cell ALL patients have been reported with the ENL gene rearrangement. At the cytogenetic level of resolution, it is not always possible to distinguish which of the two genes is involved in these translocations and precise molecular classification may be necessary for appropriate chemotherapy regimens to be devised and administered. We therefore developed a dual-color FISH (fluorescent in situ hybridization) assay using BACs (bacterial artificial chromosomes) for ELL and ENL. ELL is directly labeled in spectrum orange and ENL is directly labeled in spectrum green and the probes are co-hybridized. The strategy for the assay is to assess the interphase nuclei of patients for gain of an orange signal, indicating an ELL gene rearrangement (3O2G signal pattern), or gain of a green signal, indicating an ENL gene rearrangement (3G2O signal pattern). The gain of signal is due to splitting of the respective locus during the translocation of distal 19p to 11q23. As constructed, the probes showed a 100% specificity for the respective translocations, based on metaphase and interphase analysis of ELL and ENL t(11;19) positive patients; all of the patients were diagnosed with myeloid leukemia. In addition, further comparative validation was performed using the commercially available dual-color breakapart MLL probe (Vysis). Analysis of the ELL positive AML patients with the MLL probe showed the expected abnormal interphase signal pattern (1O1G1Fusion), confirming MLL gene rearrangement. However, the MLL pattern for the single patient with an ENL gene rearrangement and AML showed an alternate interphase pattern, indicating an alternate MLL breakpoint. Analysis of additional patients is required to determine if those with AML and ENL gene rearrangements routinely have a different MLL breakpoint; this difference could potentially contribute to a myeloid disease presentation. In order to establish clinically valid cut-offs for residual disease analysis, interphase validation was performed. Probe specificity was 100%, with a sensitivity of greater than 97% for both loci. This results in a normal cut-off (plus 2 S.D.) of 0.9/200 abnormal cells for ENL gene rearrangement and 2.18/200 abnormal cells for ELL gene rearrangement. In this laboratory’s use, the probe set is slightly more sensitive for disease detection than the commercially available MLL gene probe (Vysis), which has a sensitivity of 96% and a normal cut-off of 1.84/200 cells. This probe set allows quick distinction between ELL and ENL gene rearrangements in patients with acute leukemia.