ABSTRACT
Bone marrow aspiration (BMA) smear analysis is essential for diagnosis, treatment, and monitoring of a variety of benign and neoplastic hematological conditions. Currently, this analysis is performed by manual microscopy. We conducted a multicenter study to validate a computational microscopy approach with an artificial intelligence-driven decision support system. A total of 795 BMA specimens (615 Romanowsky-stained and 180 Prussian blue-stained) from patients with neoplastic and other clinical conditions were analyzed, comparing the performance of the Scopio Labs X100 Full Field BMA system (test method) with manual microscopy (reference method). The system provided an average of 1,385 ± 536 (range, 0-3,131) cells per specimen for analysis. An average of 39.98 ± 19.64 fields of view (range, 0-140) per specimen were selected by the system for analysis, of them 87% ± 21% (range, 0%-100%) were accepted by the qualified operators. These regions were included in an average of 17.62 ± 7.24 regions of interest (range, 1-50) per specimen. The efficiency, sensitivity, and specificity for primary and secondary marrow aspirate characteristics (maturation, morphology, and count assessment), as well as overall interuser agreement, were evaluated. The test method showed a high correlation with the reference method for comprehensive BMA evaluation, both on Romanowsky- (90.85% efficiency, 81.61% sensitivity, and 92.88% specificity) and Prussian blue-stained samples (90.0% efficiency, 81.94% sensitivity, and 93.38% specificity). The overall agreement between the test and reference methods for BMA assessment was 91.1%. For repeatability and reproducibility, all standard deviations and coefficients of variation values were below the predefined acceptance criteria both for discrete measurements (coefficient of variation below 20%) and differential measurements (SD below 5%). The high degree of correlation between the digital decision support system and manual microscopy demonstrates the potential of this system to provide a high-quality, accurate digital BMA analysis, expediting expert review and diagnosis of BMA specimens, with practical applications including remote BMA evaluation and possibly new opportunities for the research of normal and neoplastic hematopoiesis.
ABSTRACT
Autoimmune myelofibrosis (AIMF) is a rare disorder characterized by cytopenias and autoimmunity, with characteristic bone marrow findings that include lymphocytic infiltration and fibrosis. AIMF is described predominantly in adult populations who have systemic lupus erythematosis (SLE), with scant pediatric cases described mainly in older adolescents with SLE. Here, we described the largest single-center pediatric experience of pediatric autoimmune myelofibrosis (PAIMF) series, demonstrating both similarities and distinctions from the adult experience. Patients overall respond well to steroid therapy, but these patients were significantly younger, infrequently carried a diagnosis of SLE, and causative genetic lesions were identified in many cases.
Subject(s)
Autoimmune Diseases , Leukopenia , Lupus Erythematosus, Systemic , Primary Myelofibrosis , Adult , Adolescent , Humans , Child , Primary Myelofibrosis/pathology , Autoimmune Diseases/diagnosis , Tertiary Care CentersABSTRACT
BACKGROUND: Mosaicism for chromosomal structural abnormalities, other than marker or ring chromosomes, is rarely inherited. METHODS: We performed cytogenetics studies and breakpoint analyses on a family with transmission of mosaicism for a derivative chromosome 8 (der(8)), resulting from an unbalanced translocation between the long arms of chromosomes 8 and 21 over three generations. RESULTS: The proband and his maternal half-sister had mosaicism for a der(8) cell line leading to trisomy of the distal 21q, and both had Down syndrome phenotypic features. Mosaicism for a cell line with the der(8) and a normal cell line was also detected in a maternal half-cousin. The der(8) was inherited from the maternal grandmother who had four abnormal cell lines containing the der(8), in addition to a normal cell line. One maternal half-aunt had the der(8) and an isodicentric chromosome 21 (idic(21)). Sequencing studies revealed microhomologies at the junctures of the der(8) and idic(21) in the half-aunt, suggesting a replicative mechanism in the rearrangement formation. Furthermore, interstitial telomeric sequences (ITS) were identified in the juncture between chromosomes 8 and 21 in the der(8). CONCLUSION: Mosaicism in the proband, his half-sister and half-cousin resulting from loss of chromosome 21 material from the der(8) appears to be a postzygotic event due to the genomic instability of ITS and associated with selective growth advantage of normal cells. The reversion of the inherited der(8) to a normal chromosome 8 in this family resembles revertant mosaicism of point mutations. We propose that ITS could mediate recurring revertant mosaicism for some constitutional chromosomal structural abnormalities.
Subject(s)
Mosaicism , Ring Chromosomes , Humans , Chromosomes, Human, Pair 8/genetics , Karyotyping , In Situ Hybridization, Fluorescence , Chromosome Aberrations , Translocation, Genetic/genetics , Germ CellsABSTRACT
Acute myeloid leukemia (AML) with NUP98 rearrangement (AML-NUP98) has been uncommonly reported in adults, and its incidence in our institution is â¼2.5%. There were four men and five women with a median age of 49 years, among which six cases were de novo AML and three were therapy-related. Five cases were AML with minimal differentiation or without maturation, followed by four with monocytic differentiation. NUP98 rearrangement was confirmed in all cases by FISH, and five cases showed cryptic translocations. The median overall survival (OS) was 13 months, shorter than that of AML-NPM1 (p < 0.05), and similar to that in AML-KMT2A patients in our institution. The unfavorable OS was further confirmed by comparing to AML patients in TCGA database. In conclusion, adult AML-NUP98 is associated with cryptic translocations and an unfavorable outcome. Our study suggests that incorporating the NUP98 probe into AML FISH panels are warranted to improve clinical management.
Subject(s)
Leukemia, Myeloid, Acute , Chromosome Aberrations , Female , Gene Rearrangement , Humans , Leukemia, Myeloid, Acute/diagnosis , Leukemia, Myeloid, Acute/genetics , Male , Middle Aged , Nuclear Pore Complex Proteins/genetics , Translocation, GeneticABSTRACT
De novo copy number variants (dnCNVs) arising at multiple loci in a personal genome have usually been considered to reflect cancer somatic genomic instabilities. We describe a multiple dnCNV (MdnCNV) phenomenon in which individuals with genomic disorders carry five to ten constitutional dnCNVs. These CNVs originate from independent formation incidences, are predominantly tandem duplications or complex gains, exhibit breakpoint junction features reminiscent of replicative repair, and show increased de novo point mutations flanking the rearrangement junctions. The active CNV mutation shower appears to be restricted to a transient perizygotic period. We propose that a defect in the CNV formation process is responsible for the "CNV-mutator state," and this state is dampened after early embryogenesis. The constitutional MdnCNV phenomenon resembles chromosomal instability in various cancers. Investigations of this phenomenon may provide unique access to understanding genomic disorders, structural variant mutagenesis, human evolution, and cancer biology.
Subject(s)
Chromosome Aberrations , DNA Copy Number Variations , Genetic Diseases, Inborn/embryology , Genetic Diseases, Inborn/genetics , Genomic Instability , Mutation , Chromosome Breakpoints , Chromosome Duplication , DNA Replication , Embryonic Development , Female , Gametogenesis , Humans , MaleABSTRACT
Dermatofibrosarcoma protuberans (DFSP) is a low-to-intermediate grade infiltrative dermal neoplasm with a predilection for the trunk and extremities. DFSP in the vulvar region is extremely rare, with fewer than 50 cases reported to date in the literature. The histologic diagnosis of this neoplasm is facilitated by the characteristic storiform pattern of spindle cells with infiltration into the subcutaneous fat in a "honeycomb" pattern. However, morphologic variants including the very rare myxoid DFSP have been recognized that pose significant diagnostic difficulties, especially when they occur at unusual sites. The authors describe a case of myxoid DFSP of the vulva in a 44-year-old woman that was initially misdiagnosed as a neurofibroma. Subsequent excision led to significant challenges in diagnosis due to lack of typical morphology and unusual immunohistochemical staining pattern. Presence of peripheral adipose tissue trapping was noted focally that led to suspicion of DFSP. The diagnosis was confirmed by the detection of the characteristic COL1A1/PDGFB fusion transcript by reverse-transcription polymerase chain reaction. This case underscores the diagnostic challenge presented by variants of DFSP presenting in unusual locations and the value of molecular confirmation of the diagnosis.
Subject(s)
Dermatofibrosarcoma/pathology , Skin Neoplasms/pathology , Vulvar Neoplasms/pathology , Adult , Biomarkers, Tumor/analysis , Diagnostic Errors , Female , Humans , Immunohistochemistry , Neurofibroma/diagnosis , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
In clinical diagnostics, both array comparative genomic hybridization (array CGH) and single nucleotide polymorphism (SNP) genotyping have proven to be powerful genomic technologies utilized for the evaluation of developmental delay, multiple congenital anomalies, and neuropsychiatric disorders. Differences in the ability to resolve genomic changes between these arrays may constitute an implementation challenge for clinicians: which platform (SNP vs array CGH) might best detect the underlying genetic cause for the disease in the patient? While only SNP arrays enable the detection of copy number neutral regions of absence of heterozygosity (AOH), they have limited ability to detect single-exon copy number variants (CNVs) due to the distribution of SNPs across the genome. To provide comprehensive clinical testing for both CNVs and copy-neutral AOH, we enhanced our custom-designed high-resolution oligonucleotide array that has exon-targeted coverage of 1860 genes with 60,000 SNP probes, referred to as Chromosomal Microarray Analysis - Comprehensive (CMA-COMP). Of the 3240 cases evaluated by this array, clinically significant CNVs were detected in 445 cases including 21 cases with exonic events. In addition, 162 cases (5.0%) showed at least one AOH region >10 Mb. We demonstrate that even though this array has a lower density of SNP probes than other commercially available SNP arrays, it reliably detected AOH events >10 Mb as well as exonic CNVs beyond the detection limitations of SNP genotyping. Thus, combining SNP probes and exon-targeted array CGH into one platform provides clinically useful genetic screening in an efficient manner.
Subject(s)
Comparative Genomic Hybridization/methods , DNA Copy Number Variations/genetics , Genotyping Techniques/methods , Polymorphism, Single Nucleotide/genetics , Genome, Human , Genomics , Heterozygote , Humans , Oligonucleotide Array Sequence AnalysisABSTRACT
BACKGROUND: Deafness, onychodystrophy, osteodystrophy, mental retardation, and seizures (DOORS) syndrome is a rare autosomal recessive disorder of unknown cause. We aimed to identify the genetic basis of this syndrome by sequencing most coding exons in affected individuals. METHODS: Through a search of available case studies and communication with collaborators, we identified families that included at least one individual with at least three of the five main features of the DOORS syndrome: deafness, onychodystrophy, osteodystrophy, intellectual disability, and seizures. Participants were recruited from 26 centres in 17 countries. Families described in this study were enrolled between Dec 1, 2010, and March 1, 2013. Collaborating physicians enrolling participants obtained clinical information and DNA samples from the affected child and both parents if possible. We did whole-exome sequencing in affected individuals as they were enrolled, until we identified a candidate gene, and Sanger sequencing to confirm mutations. We did expression studies in human fibroblasts from one individual by real-time PCR and western blot analysis, and in mouse tissues by immunohistochemistry and real-time PCR. FINDINGS: 26 families were included in the study. We did exome sequencing in the first 17 enrolled families; we screened for TBC1D24 by Sanger sequencing in subsequent families. We identified TBC1D24 mutations in 11 individuals from nine families (by exome sequencing in seven families, and Sanger sequencing in two families). 18 families had individuals with all five main features of DOORS syndrome, and TBC1D24 mutations were identified in half of these families. The seizure types in individuals with TBC1D24 mutations included generalised tonic-clonic, complex partial, focal clonic, and infantile spasms. Of the 18 individuals with DOORS syndrome from 17 families without TBC1D24 mutations, eight did not have seizures and three did not have deafness. In expression studies, some mutations abrogated TBC1D24 mRNA stability. We also detected Tbc1d24 expression in mouse phalangeal chondrocytes and calvaria, which suggests a role of TBC1D24 in skeletogenesis. INTERPRETATION: Our findings suggest that mutations in TBC1D24 seem to be an important cause of DOORS syndrome and can cause diverse phenotypes. Thus, individuals with DOORS syndrome without deafness and seizures but with the other features should still be screened for TBC1D24 mutations. More information is needed to understand the cellular roles of TBC1D24 and identify the genes responsible for DOORS phenotypes in individuals who do not have a mutation in TBC1D24. FUNDING: US National Institutes of Health, the CIHR (Canada), the NIHR (UK), the Wellcome Trust, the Henry Smith Charity, and Action Medical Research.
Subject(s)
Carrier Proteins/genetics , Craniofacial Abnormalities/genetics , Exome/genetics , Hand Deformities, Congenital/genetics , Hearing Loss, Sensorineural/genetics , Intellectual Disability/genetics , Internationality , Nails, Malformed/genetics , Phenotype , Sequence Analysis, DNA/methods , Adolescent , Carrier Proteins/chemistry , Child , Child, Preschool , Craniofacial Abnormalities/diagnosis , Female , GTPase-Activating Proteins , Hand Deformities, Congenital/diagnosis , Hearing Loss, Sensorineural/diagnosis , Humans , Infant , Intellectual Disability/diagnosis , Male , Membrane Proteins , Nails, Malformed/diagnosis , Nerve Tissue Proteins , Young AdultABSTRACT
BACKGROUND: Genomic rearrangements usually involve one of the two chromosome homologues. Homozygous microdeletion/duplication is very rare. The chromosome 22q11.2 region is prone to recurrent rearrangements due to the presence of low-copy repeats. A common 3 Mb microdeletion causes the well-characterised DiGeorge syndrome (DGS). The reciprocal duplication is associated with an extremely variable phenotype, ranging from apparently normal to learning disabilities and multiple congenital anomalies. METHODS AND RESULTS: We describe duplications of the DGS region on both homologues in five patients from three families, detected by array CGH and confirmed by both fluorescence in situ hybridisation and single nucleotide polymorphism arrays. The proband in the first family is homozygous for the common duplication; one maternally inherited and the other a de novo duplication that was generated by nonallelic homologous recombination during spermatogenesis. The 22q11.2 duplications in the four individuals from the other two families are recurrent duplications on both homologues, one inherited from the mother and the other from the father. The phenotype in the patients with a 22q11.2 tetrasomy is similar to the features seen in duplication patients, including cognitive deficits and variable congenital defects. CONCLUSIONS: Our studies that reveal phenotypic variability in patients with four copies of the 22q11.2 genomic segment, demonstrate that both inherited and de novo events can result in the generation of homozygous duplications, and further document how multiple seemingly rare events can occur in a single individual.
Subject(s)
Chromosome Duplication , Chromosomes, Human, Pair 10/genetics , DiGeorge Syndrome/genetics , Adult , Chromosome Deletion , Chromosomes, Human, Pair 22/genetics , DNA Copy Number Variations , Female , Humans , In Situ Hybridization, Fluorescence , Infant , Male , Phenotype , PregnancyABSTRACT
Prader-Willi syndrome is caused by the lack of paternal contribution for the imprinted 15q11-q13 region that originates through a number of mechanisms such as paternal deletion of 15q11-q13, maternal uniparental disomy, or by an imprinting defect due to epimutations in the paternal imprinting center. In the present report, we describe a female patient with complex maternal uniparental trisomy for the 15q11-q13 Prader-Willi syndrome critical region due to a de novo interstitial duplication of 15q11-q13 region that is present in one of the maternal homologs. As a result, the patient has three maternally derived copies of the Prader-Willi syndrome critical region and absence of paternal 15 contribution and thus, presents with a Prader-Willi syndrome phenotype with risk for developing additional phenotypes (e.g., autism and psychiatric phenotypes) characteristic of maternally derived duplications of this region. We suggest that this is a rather unique mechanism leading to Prader-Willi syndrome that has not been previously reported.
Subject(s)
Chromosomes, Human, Pair 15/genetics , DNA Copy Number Variations/genetics , Prader-Willi Syndrome/genetics , Trisomy/genetics , Uniparental Disomy/genetics , Adult , Female , Humans , Infant , Phenotype , Prader-Willi Syndrome/physiopathologyABSTRACT
Interstitial deletions of the short arm of chromosome 6 are rare and have been associated with developmental delay, hypotonia, congenital anomalies, and dysmorphic features. We used array comparative genomic hybridization in a South Carolina Autism Project (SCAP) cohort of 97 subjects with autism spectrum disorders (ASDs) and identified an ~ 5.4 Mb deletion on chromosome 6p22.3-p23 in a 15-year-old patient with intellectual disability and ASDs. Subsequent database queries revealed five additional individuals with overlapping submicroscopic deletions and presenting with developmental and speech delay, seizures, behavioral abnormalities, heart defects, and dysmorphic features. The deletion found in the SCAP patient harbors ATXN1, DTNBP1, JARID2, and NHLRC1 that we propose may be responsible for ASDs and developmental delay.
ABSTRACT
We report on a consanguineous couple with two affected sons who presented with primary microcephaly and moderate to severe intellectual disabilities. A SNP array uncovered two overlapping regions of copy-neutral absence of heterozygosity (AOH) in both sibs. This led to sequencing of WDR62, a gene that codes for a spindle pole protein recently identified as a cause of primary microcephaly. A homozygous missense mutation in WDR62, p.E400K, was found in both boys and segregated with the condition in this family. WDR62 is one of seven genes responsible for autosomal recessive primary microcephaly (MCPH), and appears to be one of the most frequently involved in MCPH following ASPM. Studies of ASPM and WDR62 should perhaps be pursued in all cases of primary microcephaly with or without gross brain malformations.
Subject(s)
Consanguinity , Microcephaly/genetics , Mutation, Missense , Nerve Tissue Proteins/genetics , Cell Cycle Proteins , Female , Homozygote , Humans , Infant , Male , Pedigree , Polymorphism, Single NucleotideABSTRACT
Mitochondrial DNA (mtDNA) depletion syndrome encompasses a heterogeneous group of disorders characterized by a reduction in the mtDNA copy number. We identified two patients with clinical presentations consistent with mtDNA depletion syndrome (MDS), who were subsequently found to have apparently homozygous point mutations in TYMP and DGUOK, two of the nine nuclear genes commonly associated with these disorders. Further sequence analyses of parents indicated that in each case only one parent; the mother of the first and the father of the second, was a heterozygous carrier of the mutation identified in the affected child. The presence of underlying deletions was ruled out by use of a custom target array comparative genomic hybridization (CGH) platform. A high-density single-nucleotide polymorphism (SNP) array analysis revealed that the first patient had a region of copy-neutral absence of heterozygosity (AOH) consistent with segmental isodisomy for an 11.3 Mb region at the long-arm terminus of chromosome 22 (including the TYMP gene), and the second patient had results consistent with complete isodisomy of chromosome 2 (where the DGUOK gene is located). The combined sequencing, array CGH and SNP array approaches have demonstrated the first cases of MDS due to uniparental isodisomy. This diagnostic scenario also demonstrates the necessity of comprehensive examination of the underlying molecular defects of an apparently homozygous mutation in order to provide patients and their families with the most accurate molecular diagnosis and genetic counseling.
Subject(s)
DNA, Mitochondrial/genetics , Genes, Recessive , Mitochondrial Diseases/genetics , Uniparental Disomy/diagnosis , Adult , Base Sequence , Chromosomes, Human, Pair 2 , Chromosomes, Human, Pair 22 , Comparative Genomic Hybridization , Fatal Outcome , Female , Follow-Up Studies , Homozygote , Humans , Infant , Male , Mutation , Pedigree , Polymorphism, Single Nucleotide , Syndrome , Thymidine Phosphorylase/genetics , Young AdultABSTRACT
Complex genomic rearrangements (CGRs) consisting of two or more breakpoint junctions have been observed in genomic disorders. Recently, a chromosome catastrophe phenomenon termed chromothripsis, in which numerous genomic rearrangements are apparently acquired in one single catastrophic event, was described in multiple cancers. Here, we show that constitutionally acquired CGRs share similarities with cancer chromothripsis. In the 17 CGR cases investigated, we observed localization and multiple copy number changes including deletions, duplications, and/or triplications, as well as extensive translocations and inversions. Genomic rearrangements involved varied in size and complexities; in one case, array comparative genomic hybridization revealed 18 copy number changes. Breakpoint sequencing identified characteristic features, including small templated insertions at breakpoints and microhomology at breakpoint junctions, which have been attributed to replicative processes. The resemblance between CGR and chromothripsis suggests similar mechanistic underpinnings. Such chromosome catastrophic events appear to reflect basic DNA metabolism operative throughout an organism's life cycle.
Subject(s)
Chromosome Aberrations , DNA Repair , Developmental Disabilities/genetics , Neoplasms/genetics , Base Sequence , Child , Child, Preschool , Chromosome Breakage , Comparative Genomic Hybridization , DNA Replication , Female , Humans , In Situ Hybridization, Fluorescence , Infant , Male , Molecular Sequence DataABSTRACT
The ability of chromosome microarray analysis (CMA) to detect submicroscopic genetic abnormalities has revolutionized the clinical diagnostic approach to individuals with intellectual disability, neurobehavioral phenotypes, and congenital malformations. The recognition of the underlying copy number variant (CNV) in respective individuals may allow not only for better counseling and anticipatory guidance but also for more specific therapeutic interventions in some cases. The use of CMA technology in prenatal diagnosis is emerging and promises higher sensitivity for several highly penetrant, clinically severe microdeletion and microduplication syndromes. Genetic counseling complements the diagnostic testing with CMA, given the presence of CNVs of uncertain clinical significance, incomplete penetrance, and variable expressivity in some cases. While oligonucleotide arrays with high-density exonic coverage remain the gold standard for the detection of CNVs, single-nucleotide polymorphism (SNP) arrays allow for detection of consanguinity and most cases of uniparental disomy and provide a higher sensitivity to detect low-level mosaic aneuploidies.
Subject(s)
DNA Copy Number Variations , Microarray Analysis/methods , Nervous System Diseases/diagnosis , Nervous System Diseases/genetics , Polymorphism, Single Nucleotide , Chromosomes , HumansABSTRACT
Mutations in DGUOK result in mitochondrial DNA (mtDNA) depletion and may present as neonatal liver failure. Neonatal hemochromatosis (NH(1)) is a liver disorder of uncertain and varied etiology characterized by hepatic and non-reticuloendothelial siderosis. To date, deoxyguanosine kinase (dGK(2)) deficiency has not been formally recognized in cases of NH. We report an African American female neonate with clinical and autopsy findings consistent with NH, and mtDNA depletion due to a homozygous mutation in DGUOK. This report highlights hepatocerebral mtDNA depletion in the differential of neonatal tyrosinemia, advocates considering dGK deficiency in cases of NH, and posits mitochondrial oxidative processes in the pathogenesis of NH.
Subject(s)
Hemochromatosis/enzymology , Phosphotransferases (Alcohol Group Acceptor)/deficiency , Autopsy , DNA, Mitochondrial/genetics , Fatal Outcome , Female , Hemochromatosis/diagnosis , Hemochromatosis/genetics , Hemochromatosis/pathology , Hemochromatosis/therapy , Homozygote , Humans , Infant, Newborn , Liver/pathology , Mutation/genetics , Phenotype , Phosphotransferases (Alcohol Group Acceptor)/geneticsABSTRACT
Genomic instability is a feature of the human Xp22.31 region wherein deletions are associated with X-linked ichthyosis, mental retardation and attention deficit hyperactivity disorder. A putative homologous recombination hotspot motif is enriched in low copy repeats that mediate recurrent deletion at this locus. To date, few efforts have focused on copy number gain at Xp22.31. However, clinical testing revealed a high incidence of duplication of Xp22.31 in subjects ascertained and referred with neurobehavioral phenotypes. We systematically studied 61 unrelated subjects with rearrangements revealing gain in copy number, using multiple molecular assays. We detected not only the anticipated recurrent and simple nonrecurrent duplications, but also unexpectedly identified recurrent triplications and other complex rearrangements. Breakpoint analyses enabled us to surmise the mechanisms for many of these rearrangements. The clinical significance of the recurrent duplications and triplications were assessed using different approaches. We cannot find any evidence to support pathogenicity of the Xp22.31 duplication. However, our data suggest that the Xp22.31 duplication may serve as a risk factor for abnormal phenotypes. Our findings highlight the need for more robust Xp22.31 triplication detection in that such further gain may be more penetrant than the duplications. Our findings reveal the distribution of different mechanisms for genomic duplication rearrangements at a given locus, and provide insights into aspects of strand exchange events between paralogous sequences in the human genome.
Subject(s)
Chromosomes, Human, X/genetics , DNA Copy Number Variations/genetics , Gene Duplication/genetics , Gene Rearrangement/genetics , Base Sequence , Chromosome Breakage , Chromosome Mapping , Comparative Genomic Hybridization , Female , Gene Order , Humans , Male , Molecular Sequence Data , Phenotype , Segmental Duplications, Genomic/genetics , Sequence AlignmentABSTRACT
Four unrelated families with the same unbalanced translocation der(4)t(4;11)(p16.2;p15.4) were analyzed. Both of the breakpoint regions in 4p16.2 and 11p15.4 were narrowed to large â¼359-kb and â¼215-kb low-copy repeat (LCR) clusters, respectively, by aCGH and SNP array analyses. DNA sequencing enabled mapping the breakpoints of one translocation to 24 bp within interchromosomal paralogous LCRs of â¼130 kb in length and 94.7% DNA sequence identity located in olfactory receptor gene clusters, indicating nonallelic homologous recombination (NAHR) as the mechanism for translocation formation. To investigate the potential involvement of interchromosomal LCRs in recurrent chromosomal translocation formation, we performed computational genome-wide analyses and identified 1143 interchromosomal LCR substrate pairs, >5 kb in size and sharing >94% sequence identity that can potentially mediate chromosomal translocations. Additional evidence for interchromosomal NAHR mediated translocation formation was provided by sequencing the breakpoints of another recurrent translocation, der(8)t(8;12)(p23.1;p13.31). The NAHR sites were mapped within 55 bp in â¼7.8-kb paralogous subunits of 95.3% sequence identity located in the â¼579-kb (chr 8) and â¼287-kb (chr 12) LCR clusters. We demonstrate that NAHR mediates recurrent constitutional translocations t(4;11) and t(8;12) and potentially many other interchromosomal translocations throughout the human genome. Furthermore, we provide a computationally determined genome-wide "recurrent translocation map."
Subject(s)
Chromosomes, Human, Pair 11/genetics , Chromosomes, Human, Pair 4/genetics , Recombination, Genetic , Translocation, Genetic , Chromosome Breakage , Chromosome Disorders/genetics , Chromosome Disorders/pathology , Chromosome Mapping/methods , Comparative Genomic Hybridization , Family , Female , Humans , Male , Molecular Sequence Data , Multigene Family , Oligonucleotide Array Sequence Analysis , Phenotype , Polymerase Chain Reaction/methods , Receptors, Odorant/genetics , Segmental Duplications, Genomic/genetics , Sequence Analysis, DNAABSTRACT
We demonstrate the utility of an exon coverage microarray platform in detecting intragenic deletions: one in exons 24-27 of the EP300 gene and another in exons 27 and 28 of the CREBBP gene in two patients with Rubinstein-Taybi syndrome (RSTS). RSTS is a heterogeneous disorder in which approximately 45-55% of cases result from deletion or mutations in the CREBBP gene and an unknown portion of cases result from gene changes in EP300. The first case is a 3-year-old female with an exonic deletion of the EP300 gene who has classic facial features of RSTS without the thumb and great toe anomalies, consistent with the milder skeletal phenotype that has been described in other RSTS cases with EP300 mutations. In addition, the mother of this patient also had preeclampsia during pregnancy, which has been infrequently reported. The second case is a newborn male who has the classical features of RSTS. Our results illustrate that exon-targeted array comparative genomic hybridization (aCGH) is a powerful tool for detecting clinically significant intragenic rearrangements that would be otherwise missed by aCGH platforms lacking sufficient exonic coverage or sequencing of the gene of interest.
Subject(s)
CREB-Binding Protein/genetics , E1A-Associated p300 Protein/genetics , Exons/genetics , Gene Deletion , Rubinstein-Taybi Syndrome/genetics , Child, Preschool , Comparative Genomic Hybridization/methods , Female , Humans , Infant, Newborn , Male , Oligonucleotide Array Sequence Analysis , PregnancyABSTRACT
Array comparative genomic hybridization (aCGH) is a powerful tool for the molecular elucidation and diagnosis of disorders resulting from genomic copy-number variation (CNV). However, intragenic deletions or duplications--those including genomic intervals of a size smaller than a gene--have remained beyond the detection limit of most clinical aCGH analyses. Increasing array probe number improves genomic resolution, although higher cost may limit implementation, and enhanced detection of benign CNV can confound clinical interpretation. We designed an array with exonic coverage of selected disease and candidate genes and used it clinically to identify losses or gains throughout the genome involving at least one exon and as small as several hundred base pairs in size. In some patients, the detected copy-number change occurs within a gene known to be causative of the observed clinical phenotype, demonstrating the ability of this array to detect clinically relevant CNVs with subkilobase resolution. In summary, we demonstrate the utility of a custom-designed, exon-targeted oligonucleotide array to detect intragenic copy-number changes in patients with various clinical phenotypes.