Background: Exon-targeted microarrays can detect small (<1000 bp) intragenic copy number variants... more Background: Exon-targeted microarrays can detect small (<1000 bp) intragenic copy number variants (CNVs), including those that affect only a single exon. This genome-wide high-sensitivity approach increases the molecular diagnosis for conditions with known disease-associated genes, enables better genotype-phenotype correlations, and facilitates variant allele detection allowing novel disease gene discovery. Methods: We retrospectively analyzed data from 63,127 patients referred for clinical chromosomal microarray analysis (CMA) at Baylor Genetics laboratories, including 46,755 individuals tested using exon-targeted arrays, from 2007 to 2017. Small CNVs harboring a single gene or two to five non-disease-associated genes were identified; the genes involved were evaluated for a potential disease association.
In original publication, the last row of Table 1 was incorrect. The correct Table 1 is given belo... more In original publication, the last row of Table 1 was incorrect. The correct Table 1 is given below: The online version of the original article can be found under
De novo copy number variants (dnCNVs) arising atĀ multiple loci in a personal genome have usually ... more De novo copy number variants (dnCNVs) arising atĀ 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. WeĀ 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 dis...
Impairment of ubiquitin-proteasome system activity involving ubiquitin ligase genes UBE3A, UBE3B,... more Impairment of ubiquitin-proteasome system activity involving ubiquitin ligase genes UBE3A, UBE3B, and HUWE1 and deubiquitinating enzyme genes USP7 and USP9X has been reported in patients with neurodevelopmental delays. To date, only a handful of single-nucleotide variants (SNVs) and copy-number variants (CNVs) involving TRIP12, encoding a member of the HECT domain E3 ubiquitin ligases family on chromosome 2q36.3 have been reported. Using chromosomal microarray analysis and whole-exome sequencing (WES), we have identified, respectively, five deletion CNVs and four inactivating SNVs (two frameshifts, one missense, and one splicing) in TRIP12. Seven of these variants were found to be de novo; parental studies could not be completed in two families. Quantitative PCR analyses of the splicing mutation showed a dramatically decreased level of TRIP12 mRNA in the proband compared to the family controls, indicating a loss-of-function mechanism. The shared clinical features include intellectua...
Degradation of proteins by the ubiquitin-proteasome system (UPS) is an essential biological proce... more Degradation of proteins by the ubiquitin-proteasome system (UPS) is an essential biological process in the development of eukaryotic organisms. Dysregulation of this mechanism leads to numerous human neurodegenerative or neurodevelopmental disorders. Through a multi-center collaboration, we identified six de novo genomic deletions and four de novo point mutations involving PSMD12, encoding the non-ATPase subunit PSMD12 (aka RPN5) of the 19S regulator of 26S proteasome complex, in unrelated individuals with intellectual disability, congenital malformations, ophthalmologic anomalies, feeding difficulties, deafness, and subtle dysmorphic facial features. We observed reduced PSMD12 levels and an accumulation of ubiquitinated proteins without any impairment of proteasome catalytic activity. Our PSMD12 loss-of-function zebrafish CRISPR/Cas9 model exhibited microcephaly, decreased convolution of the renal tubules, and abnormal craniofacial morphology. Our data support the biological import...
Chromosomal insertions are genomic rearrangements with a chromosome segment inserted into a non-h... more Chromosomal insertions are genomic rearrangements with a chromosome segment inserted into a non-homologous chromosome or a non-adjacent locus on the same chromosome or the other homologue, constituting~2% of nonrecurrent copy-number gains. Little is known about the molecular mechanisms of their formation. We identified 16 individuals with complex insertions among 56,000 individuals tested at Baylor Genetics using clinical array comparative genomic hybridization (aCGH) and fluorescence in situ hybridization (FISH). Custom high-density aCGH was performed on 10 individuals with available DNA, and breakpoint junctions were fine-mapped at nucleotide resolution by long-range PCR and DNA sequencing in 6 individuals to glean insights into potential mechanisms of formation. We observed microhomologies and templated insertions at the breakpoint junctions, resembling the breakpoint junction signatures found in complex genomic rearrangements generated by replication-based mechanism(s) with iterative template switches. In addition, we analyzed 5 families with apparently balanced insertion in one parent detected by FISH analysis and found that 3 parents had additional small copy-number variants (CNVs) at one or both sides of the inserting fragments as well as at the inserted sites. We propose that replicative repair can result in interchromosomal complex insertions generated through chromothripsis-like chromoanasynthesis involving two or three chromosomes, and cause a significant fraction of apparently balanced insertions harboring small flanking CNVs.
Chromosomal microarray (CMA) testing, used to identify DNA copy number variations (CNVs), has hel... more Chromosomal microarray (CMA) testing, used to identify DNA copy number variations (CNVs), has helped advance knowledge about genetics of human neurodevelopmental disease and congenital anomalies. It has aided in discovering new CNV syndromes and uncovering disease genes. It has discovered CNVs that are not fully penetrant and/or cause a spectrum of phenotypes, including intellectual disability, autism, schizophrenia, and dysmorphisms. Such CNVs can pose challenges to genetic counseling. They also have helped increase knowledge of genetic risk factors for neurodevelopmental disease and raised awareness of possible shared etiologies among these variable phenotypes. Advances in CMA technology allow CNV identification at increasingly finer scales, improving detection of pathogenic changes, although these sometimes are difficult to distinguish from normal population variation. This paper confronts some of the challenges uncovered by CMA testing while reviewing advances in genetics and the clinical use of this test that has replaced standard karyotyping in most genetic evaluations.
ObjectiveāTo assess the additive value of prenatal chromosomal microarray analysis (CMA) for all ... more ObjectiveāTo assess the additive value of prenatal chromosomal microarray analysis (CMA) for all indications and the likelihood of detecting pathologic copy number variations (CNVs) based on specific indications. MethodsāA retrospective analysis was performed on amniocentesis and chorionic villi sampling results obtained between 2010 and 2014 in a single institution. A total of 3,314 consecutive patients undergoing invasive genetic testing for different indications were offered CMA in addition to standard karyotype. The prevalence of pathologic CNVs was compared between patients with low-risk indications and those with high-risk indications. Likewise, the prevalence of pathologic CNVs among patients with different sonographic abnormalities was calculated and compared with the low-risk group. Chi-square and Fisher exact tests were used for statistical analysis. ResultsāThe prevalence of pathologic CNVs was significantly higher in patients with high-risk indications and specifically those with sonographic abnormalities, compared with the low-risk group (2.8 and 5.9% vs. 0.4%, respectively; all pā&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;ā0.05). ConclusionāPrenatal CMA detected clinically relevant CNVs in fetuses with a normal karyotype. Major structural malformations and nuchal translucency (NT)āā„ā3.0āmm are associated with the highest risk for a CMA abnormality. Nevertheless, the prevalence of pathologic CNVs in the low-risk population was high enough (1:250) to consider genetic counseling in this group.
Genetics in medicine : official journal of the American College of Medical Genetics, Oct 25, 2016
We sought to investigate the utility of chromosomal microarray analysis (CMA) for prenatal diagno... more We sought to investigate the utility of chromosomal microarray analysis (CMA) for prenatal diagnosis of oral clefts, as compared with traditional chromosome analysis, for improved prenatal genetic counseling and discovery of a potential correlation between genotype and oral cleft. This retrospective analysis encompassed 270 prenatal oral cleft cases with documented detailed ultrasound findings and CMA results from four referral centers. Detection rates for pathogenic copy-number variants (CNVs) were calculated and compared with cases for which chromosome analysis was also performed. The overall detection rate was 14.8% (40/270) for pathogenic CNVs by CMA, 7.2% (9/125) for the nonsyndromic cases, and 21.4% (31/145) for the syndromic cases. Of the nonsyndromic cases with ultrasound soft markers, 20% (5/25) were identified with pathogenic CNVs. CMA showed an improved detection rate of 15.3% (29/190) compared with 10.5% (20/190) for chromosome analysis. This study not only highlights th...
Microarray-based technology to conduct array comparative genomic hybridization (aCGH) has made a ... more Microarray-based technology to conduct array comparative genomic hybridization (aCGH) has made a significant impact on the diagnosis of human genetic diseases. Such diagnoses, previously undetectable by traditional G-banding chromosome analysis, are now achieved by identifying genomic copy number variants (CNVs) using the microarray. Not only can hundreds of well-characterized genetic syndromes be detected in a single assay, but new genomic disorders and disease-causing genes can also be discovered through the utilization of aCGH technology. Although other platforms such as single nucleotide polymorphism (SNP) arrays can be used for detecting CNVs, in this chapter we focus on describing the methods for performing aCGH using Agilent oligonucleotide arrays for both prenatal (e.g., amniotic fluid and chorionic villus sample) and postnatal samples (e.g., blood).
Endosomal protein recycling is a fundamental cellular process important for cellular homeostasis,... more Endosomal protein recycling is a fundamental cellular process important for cellular homeostasis, signaling, and fate determination that is implicated in several diseases. WASH is an actin-nucleating protein essential for this process, and its activity is controlled through K63-linked ubiquitination by the MAGE-L2-TRIM27 ubiquitin ligase. Here, we show that the USP7 deubiquitinating enzyme is an integral component of the MAGE-L2-TRIM27 ligase and is essential for WASH-mediated endosomal actin assembly and protein recycling. Mechanistically, USP7 acts as a molecular rheostat to precisely fine-tune endosomal F-actin levels byĀ counteracting TRIM27 auto-ubiquitination/degradation and preventing overactivation of WASH through directly deubiquitinating it. Importantly, we identify de novo heterozygous loss-of-function mutations of USP7 in individuals with a neurodevelopmental disorder, featuring intellectual disability and autism spectrum disorder. These results provide unanticipated insi...
To the Editor: Cell-free DNA-based noninvasive prenatal screening (which has also been called non... more To the Editor: Cell-free DNA-based noninvasive prenatal screening (which has also been called noninvasive prenatal diagnosis and noninvasive prenatal testing) is now offered by several companies that use various analytic methods. This screening has been reported to have higher detection rates for trisomy 21 (Down's syndrome) (99.0%), trisomy 18 (96.8%), and trisomy 13 (92.1%) than previous noninvasive prenatal screening methods.(1) However, evidence of the actual performance of noninvasive prenatal screening in the general population is limited. Furthermore, discordant results between noninvasive prenatal screening and traditional cytogenetic analysis have been observed,(2) despite the high sensitivity and specificity of the assay. We .Ā .Ā .
Background: Int22h1/int22h2-mediated Xq28 duplication syndrome is caused by~0.5 Mb chromosomal du... more Background: Int22h1/int22h2-mediated Xq28 duplication syndrome is caused by~0.5 Mb chromosomal duplications mediated by nonallelic homologous recombination between intron 22 homologous region 1 (int22h1) and 2 (int22h2), which, in addition to int22h3, are also responsible for inversions disrupting the F8 gene in hemophilia A. This syndrome has recently been described in 9 males with cognitive impairment, behavioral problems, and distinctive facial features; and 6 females with milder phenotypes. The reciprocal deletion was previously reported in a mother and daughter. It was suggested that this deletion may not have phenotypic effects in females because of skewed chromosome X inactivation, but may be embryonic lethal in males. Methods: Array comparative genomic hybridization analyses were performed using oligonucleotide-based chromosomal microarray. Chromosome X inactivation studies were performed at the AR (androgen receptor) and FMR1 (fragile X mental retardation 1) loci. Results: We present here 5 males and 6 females with int22h1/int22h2-mediated Xq28 duplication syndrome. The males manifested cognitive impairment, behavioral problems, and distinctive facial features. Two of the six females manifested mild cognitive impairment. This duplication was maternally inherited, and skewed chromosome X inactivation was observed in the majority of females carrying the duplication. We also report the reciprocal deletion in a mother and daughter with overweight, but normal cognition. In addition, we present the first case of a prenatally diagnosed de novo int22h1/int22h2-mediated deletion in a healthy female infant. We reviewed individuals previously reported with similar or overlapping rearrangements and evaluated the potential roles of genes in the rearrangement region. Conclusions: The similarity of clinical features among individuals with the int22h1/int22h2-mediated Xq28 duplication supports the notion that this duplication causes a recognizable syndrome that affects males with females exhibiting milder phenotypes. It is suggested that the observed cognitive impairment in this syndrome results from increased dosage of RAB39B gene located within the duplicated region. Increased dosage of CLIC2 may also contribute to the phenotype. The reciprocal deletion results in skewed chromosome X inactivation and no clinical phenotype in females. Review of overlapping deletions suggests that hemizygous loss of VBP1 may be the cause for the proposed male lethality associated with this deletion.
The cytogenetic evaluation of 30 cultured primary prostatic cancer specimens obtained during radi... more The cytogenetic evaluation of 30 cultured primary prostatic cancer specimens obtained during radical prostatectomies of patients with relatively early stage disease is reported. The majority of specimens examined showed a normal male karyotype, 46,XY. Nine samples contained clonally abnormal populations including five specimens which were hyperdiploid (modal range, 65-92 chromosomes), one specimen containing double minute chromosomes, and three containing structural aberrations. Loss of the Y chromosome and a partial trisomy for chromosome 4 was observed in a sample from one patient. Another sample showed a translocation between the long arms of chromosomes 5 and 7. The only tumor obtained from a previously irradiated patient contained no normal cells, a modal chromosome number of 45, loss of chromosomes 2 and Y, and multiple structural rearrangements. The appearance of any clonal cytogenetic abnormality correlated in general with a poorly differentiated state of cancer. A survey of...
Array Comparative Genomic Hybridization (a-CGH) is a powerful molecular cytogenetic tool to detec... more Array Comparative Genomic Hybridization (a-CGH) is a powerful molecular cytogenetic tool to detect genomic imbalances and study disease mechanism and pathogenesis. We report our experience with the clinical implementation of this high resolution human genome analysis, referred to as Chromosomal Microarray Analysis (CMA). CMA was performed clinically on 2513 postnatal samples from patients referred with a variety of clinical phenotypes. The initial 775 samples were studied using CMA array version 4 and the remaining 1738 samples were analyzed with CMA version 5 containing expanded genomic coverage. Overall, CMA identified clinically relevant genomic imbalances in 8.5% of patients: 7.6% using V4 and 8.9% using V5. Among 117 cases referred for additional investigation of a known cytogenetically detectable rearrangement, CMA identified the majority (92.5%) of the genomic imbalances. Importantly, abnormal CMA findings were observed in 5.2% of patients (98/1872) with normal karyotypes/FIS...
Curation and interpretation of copy number variants identified by genome-wide testing is challeng... more Curation and interpretation of copy number variants identified by genome-wide testing is challenged by the large number of events harbored in each personal genome. Conventional determination of phenotypic relevance relies on patterns of higher frequency in affected individuals versus controls; however, an increasing amount of ascertained variation is rare or private to clans. Consequently, frequency data have less utility to resolve pathogenic from benign. One solution is disease-specific algorithms that leverage gene knowledge together with variant frequency to aid prioritization. We used large-scale resources including Gene Ontology, protein-protein interactions and other annotation systems together with a broad set of 83 genes with known associations to epilepsy to construct a pathogenicity score for the phenotype. We evaluated the score for all annotated human genes and applied Bayesian methods to combine the derived pathogenicity score with frequency information from our diagno...
New human mutations are thought to originate in germ cells, thus making a recurrence of the same ... more New human mutations are thought to originate in germ cells, thus making a recurrence of the same mutation in a sibling exceedingly rare. However, increasing sensitivity of genomic technologies has anecdotally revealed mosaicism for mutations in somatic tissues of apparently healthy parents. Such somatically mosaic parents might also have germline mosaicism that can potentially cause unexpected intergenerational recurrences. Here, we show that somatic mosaicism for transmitted mutations among parents of children with simplex genetic disease is more common than currently appreciated. Using the sensitivity of individual-specific breakpoint PCR, we prospectively screened 100 families with children affected by genomic disorders due to rare deletion copy-number variants (CNVs) determined to be de novo by clinical analysis of parental DNA. Surprisingly, we identified four cases of low-level somatic mosaicism for the transmitted CNV in DNA isolated from parental blood. Integrated probabilis...
Background: Exon-targeted microarrays can detect small (<1000 bp) intragenic copy number variants... more Background: Exon-targeted microarrays can detect small (<1000 bp) intragenic copy number variants (CNVs), including those that affect only a single exon. This genome-wide high-sensitivity approach increases the molecular diagnosis for conditions with known disease-associated genes, enables better genotype-phenotype correlations, and facilitates variant allele detection allowing novel disease gene discovery. Methods: We retrospectively analyzed data from 63,127 patients referred for clinical chromosomal microarray analysis (CMA) at Baylor Genetics laboratories, including 46,755 individuals tested using exon-targeted arrays, from 2007 to 2017. Small CNVs harboring a single gene or two to five non-disease-associated genes were identified; the genes involved were evaluated for a potential disease association.
In original publication, the last row of Table 1 was incorrect. The correct Table 1 is given belo... more In original publication, the last row of Table 1 was incorrect. The correct Table 1 is given below: The online version of the original article can be found under
De novo copy number variants (dnCNVs) arising atĀ multiple loci in a personal genome have usually ... more De novo copy number variants (dnCNVs) arising atĀ 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. WeĀ 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 dis...
Impairment of ubiquitin-proteasome system activity involving ubiquitin ligase genes UBE3A, UBE3B,... more Impairment of ubiquitin-proteasome system activity involving ubiquitin ligase genes UBE3A, UBE3B, and HUWE1 and deubiquitinating enzyme genes USP7 and USP9X has been reported in patients with neurodevelopmental delays. To date, only a handful of single-nucleotide variants (SNVs) and copy-number variants (CNVs) involving TRIP12, encoding a member of the HECT domain E3 ubiquitin ligases family on chromosome 2q36.3 have been reported. Using chromosomal microarray analysis and whole-exome sequencing (WES), we have identified, respectively, five deletion CNVs and four inactivating SNVs (two frameshifts, one missense, and one splicing) in TRIP12. Seven of these variants were found to be de novo; parental studies could not be completed in two families. Quantitative PCR analyses of the splicing mutation showed a dramatically decreased level of TRIP12 mRNA in the proband compared to the family controls, indicating a loss-of-function mechanism. The shared clinical features include intellectua...
Degradation of proteins by the ubiquitin-proteasome system (UPS) is an essential biological proce... more Degradation of proteins by the ubiquitin-proteasome system (UPS) is an essential biological process in the development of eukaryotic organisms. Dysregulation of this mechanism leads to numerous human neurodegenerative or neurodevelopmental disorders. Through a multi-center collaboration, we identified six de novo genomic deletions and four de novo point mutations involving PSMD12, encoding the non-ATPase subunit PSMD12 (aka RPN5) of the 19S regulator of 26S proteasome complex, in unrelated individuals with intellectual disability, congenital malformations, ophthalmologic anomalies, feeding difficulties, deafness, and subtle dysmorphic facial features. We observed reduced PSMD12 levels and an accumulation of ubiquitinated proteins without any impairment of proteasome catalytic activity. Our PSMD12 loss-of-function zebrafish CRISPR/Cas9 model exhibited microcephaly, decreased convolution of the renal tubules, and abnormal craniofacial morphology. Our data support the biological import...
Chromosomal insertions are genomic rearrangements with a chromosome segment inserted into a non-h... more Chromosomal insertions are genomic rearrangements with a chromosome segment inserted into a non-homologous chromosome or a non-adjacent locus on the same chromosome or the other homologue, constituting~2% of nonrecurrent copy-number gains. Little is known about the molecular mechanisms of their formation. We identified 16 individuals with complex insertions among 56,000 individuals tested at Baylor Genetics using clinical array comparative genomic hybridization (aCGH) and fluorescence in situ hybridization (FISH). Custom high-density aCGH was performed on 10 individuals with available DNA, and breakpoint junctions were fine-mapped at nucleotide resolution by long-range PCR and DNA sequencing in 6 individuals to glean insights into potential mechanisms of formation. We observed microhomologies and templated insertions at the breakpoint junctions, resembling the breakpoint junction signatures found in complex genomic rearrangements generated by replication-based mechanism(s) with iterative template switches. In addition, we analyzed 5 families with apparently balanced insertion in one parent detected by FISH analysis and found that 3 parents had additional small copy-number variants (CNVs) at one or both sides of the inserting fragments as well as at the inserted sites. We propose that replicative repair can result in interchromosomal complex insertions generated through chromothripsis-like chromoanasynthesis involving two or three chromosomes, and cause a significant fraction of apparently balanced insertions harboring small flanking CNVs.
Chromosomal microarray (CMA) testing, used to identify DNA copy number variations (CNVs), has hel... more Chromosomal microarray (CMA) testing, used to identify DNA copy number variations (CNVs), has helped advance knowledge about genetics of human neurodevelopmental disease and congenital anomalies. It has aided in discovering new CNV syndromes and uncovering disease genes. It has discovered CNVs that are not fully penetrant and/or cause a spectrum of phenotypes, including intellectual disability, autism, schizophrenia, and dysmorphisms. Such CNVs can pose challenges to genetic counseling. They also have helped increase knowledge of genetic risk factors for neurodevelopmental disease and raised awareness of possible shared etiologies among these variable phenotypes. Advances in CMA technology allow CNV identification at increasingly finer scales, improving detection of pathogenic changes, although these sometimes are difficult to distinguish from normal population variation. This paper confronts some of the challenges uncovered by CMA testing while reviewing advances in genetics and the clinical use of this test that has replaced standard karyotyping in most genetic evaluations.
ObjectiveāTo assess the additive value of prenatal chromosomal microarray analysis (CMA) for all ... more ObjectiveāTo assess the additive value of prenatal chromosomal microarray analysis (CMA) for all indications and the likelihood of detecting pathologic copy number variations (CNVs) based on specific indications. MethodsāA retrospective analysis was performed on amniocentesis and chorionic villi sampling results obtained between 2010 and 2014 in a single institution. A total of 3,314 consecutive patients undergoing invasive genetic testing for different indications were offered CMA in addition to standard karyotype. The prevalence of pathologic CNVs was compared between patients with low-risk indications and those with high-risk indications. Likewise, the prevalence of pathologic CNVs among patients with different sonographic abnormalities was calculated and compared with the low-risk group. Chi-square and Fisher exact tests were used for statistical analysis. ResultsāThe prevalence of pathologic CNVs was significantly higher in patients with high-risk indications and specifically those with sonographic abnormalities, compared with the low-risk group (2.8 and 5.9% vs. 0.4%, respectively; all pā&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;ā0.05). ConclusionāPrenatal CMA detected clinically relevant CNVs in fetuses with a normal karyotype. Major structural malformations and nuchal translucency (NT)āā„ā3.0āmm are associated with the highest risk for a CMA abnormality. Nevertheless, the prevalence of pathologic CNVs in the low-risk population was high enough (1:250) to consider genetic counseling in this group.
Genetics in medicine : official journal of the American College of Medical Genetics, Oct 25, 2016
We sought to investigate the utility of chromosomal microarray analysis (CMA) for prenatal diagno... more We sought to investigate the utility of chromosomal microarray analysis (CMA) for prenatal diagnosis of oral clefts, as compared with traditional chromosome analysis, for improved prenatal genetic counseling and discovery of a potential correlation between genotype and oral cleft. This retrospective analysis encompassed 270 prenatal oral cleft cases with documented detailed ultrasound findings and CMA results from four referral centers. Detection rates for pathogenic copy-number variants (CNVs) were calculated and compared with cases for which chromosome analysis was also performed. The overall detection rate was 14.8% (40/270) for pathogenic CNVs by CMA, 7.2% (9/125) for the nonsyndromic cases, and 21.4% (31/145) for the syndromic cases. Of the nonsyndromic cases with ultrasound soft markers, 20% (5/25) were identified with pathogenic CNVs. CMA showed an improved detection rate of 15.3% (29/190) compared with 10.5% (20/190) for chromosome analysis. This study not only highlights th...
Microarray-based technology to conduct array comparative genomic hybridization (aCGH) has made a ... more Microarray-based technology to conduct array comparative genomic hybridization (aCGH) has made a significant impact on the diagnosis of human genetic diseases. Such diagnoses, previously undetectable by traditional G-banding chromosome analysis, are now achieved by identifying genomic copy number variants (CNVs) using the microarray. Not only can hundreds of well-characterized genetic syndromes be detected in a single assay, but new genomic disorders and disease-causing genes can also be discovered through the utilization of aCGH technology. Although other platforms such as single nucleotide polymorphism (SNP) arrays can be used for detecting CNVs, in this chapter we focus on describing the methods for performing aCGH using Agilent oligonucleotide arrays for both prenatal (e.g., amniotic fluid and chorionic villus sample) and postnatal samples (e.g., blood).
Endosomal protein recycling is a fundamental cellular process important for cellular homeostasis,... more Endosomal protein recycling is a fundamental cellular process important for cellular homeostasis, signaling, and fate determination that is implicated in several diseases. WASH is an actin-nucleating protein essential for this process, and its activity is controlled through K63-linked ubiquitination by the MAGE-L2-TRIM27 ubiquitin ligase. Here, we show that the USP7 deubiquitinating enzyme is an integral component of the MAGE-L2-TRIM27 ligase and is essential for WASH-mediated endosomal actin assembly and protein recycling. Mechanistically, USP7 acts as a molecular rheostat to precisely fine-tune endosomal F-actin levels byĀ counteracting TRIM27 auto-ubiquitination/degradation and preventing overactivation of WASH through directly deubiquitinating it. Importantly, we identify de novo heterozygous loss-of-function mutations of USP7 in individuals with a neurodevelopmental disorder, featuring intellectual disability and autism spectrum disorder. These results provide unanticipated insi...
To the Editor: Cell-free DNA-based noninvasive prenatal screening (which has also been called non... more To the Editor: Cell-free DNA-based noninvasive prenatal screening (which has also been called noninvasive prenatal diagnosis and noninvasive prenatal testing) is now offered by several companies that use various analytic methods. This screening has been reported to have higher detection rates for trisomy 21 (Down's syndrome) (99.0%), trisomy 18 (96.8%), and trisomy 13 (92.1%) than previous noninvasive prenatal screening methods.(1) However, evidence of the actual performance of noninvasive prenatal screening in the general population is limited. Furthermore, discordant results between noninvasive prenatal screening and traditional cytogenetic analysis have been observed,(2) despite the high sensitivity and specificity of the assay. We .Ā .Ā .
Background: Int22h1/int22h2-mediated Xq28 duplication syndrome is caused by~0.5 Mb chromosomal du... more Background: Int22h1/int22h2-mediated Xq28 duplication syndrome is caused by~0.5 Mb chromosomal duplications mediated by nonallelic homologous recombination between intron 22 homologous region 1 (int22h1) and 2 (int22h2), which, in addition to int22h3, are also responsible for inversions disrupting the F8 gene in hemophilia A. This syndrome has recently been described in 9 males with cognitive impairment, behavioral problems, and distinctive facial features; and 6 females with milder phenotypes. The reciprocal deletion was previously reported in a mother and daughter. It was suggested that this deletion may not have phenotypic effects in females because of skewed chromosome X inactivation, but may be embryonic lethal in males. Methods: Array comparative genomic hybridization analyses were performed using oligonucleotide-based chromosomal microarray. Chromosome X inactivation studies were performed at the AR (androgen receptor) and FMR1 (fragile X mental retardation 1) loci. Results: We present here 5 males and 6 females with int22h1/int22h2-mediated Xq28 duplication syndrome. The males manifested cognitive impairment, behavioral problems, and distinctive facial features. Two of the six females manifested mild cognitive impairment. This duplication was maternally inherited, and skewed chromosome X inactivation was observed in the majority of females carrying the duplication. We also report the reciprocal deletion in a mother and daughter with overweight, but normal cognition. In addition, we present the first case of a prenatally diagnosed de novo int22h1/int22h2-mediated deletion in a healthy female infant. We reviewed individuals previously reported with similar or overlapping rearrangements and evaluated the potential roles of genes in the rearrangement region. Conclusions: The similarity of clinical features among individuals with the int22h1/int22h2-mediated Xq28 duplication supports the notion that this duplication causes a recognizable syndrome that affects males with females exhibiting milder phenotypes. It is suggested that the observed cognitive impairment in this syndrome results from increased dosage of RAB39B gene located within the duplicated region. Increased dosage of CLIC2 may also contribute to the phenotype. The reciprocal deletion results in skewed chromosome X inactivation and no clinical phenotype in females. Review of overlapping deletions suggests that hemizygous loss of VBP1 may be the cause for the proposed male lethality associated with this deletion.
The cytogenetic evaluation of 30 cultured primary prostatic cancer specimens obtained during radi... more The cytogenetic evaluation of 30 cultured primary prostatic cancer specimens obtained during radical prostatectomies of patients with relatively early stage disease is reported. The majority of specimens examined showed a normal male karyotype, 46,XY. Nine samples contained clonally abnormal populations including five specimens which were hyperdiploid (modal range, 65-92 chromosomes), one specimen containing double minute chromosomes, and three containing structural aberrations. Loss of the Y chromosome and a partial trisomy for chromosome 4 was observed in a sample from one patient. Another sample showed a translocation between the long arms of chromosomes 5 and 7. The only tumor obtained from a previously irradiated patient contained no normal cells, a modal chromosome number of 45, loss of chromosomes 2 and Y, and multiple structural rearrangements. The appearance of any clonal cytogenetic abnormality correlated in general with a poorly differentiated state of cancer. A survey of...
Array Comparative Genomic Hybridization (a-CGH) is a powerful molecular cytogenetic tool to detec... more Array Comparative Genomic Hybridization (a-CGH) is a powerful molecular cytogenetic tool to detect genomic imbalances and study disease mechanism and pathogenesis. We report our experience with the clinical implementation of this high resolution human genome analysis, referred to as Chromosomal Microarray Analysis (CMA). CMA was performed clinically on 2513 postnatal samples from patients referred with a variety of clinical phenotypes. The initial 775 samples were studied using CMA array version 4 and the remaining 1738 samples were analyzed with CMA version 5 containing expanded genomic coverage. Overall, CMA identified clinically relevant genomic imbalances in 8.5% of patients: 7.6% using V4 and 8.9% using V5. Among 117 cases referred for additional investigation of a known cytogenetically detectable rearrangement, CMA identified the majority (92.5%) of the genomic imbalances. Importantly, abnormal CMA findings were observed in 5.2% of patients (98/1872) with normal karyotypes/FIS...
Curation and interpretation of copy number variants identified by genome-wide testing is challeng... more Curation and interpretation of copy number variants identified by genome-wide testing is challenged by the large number of events harbored in each personal genome. Conventional determination of phenotypic relevance relies on patterns of higher frequency in affected individuals versus controls; however, an increasing amount of ascertained variation is rare or private to clans. Consequently, frequency data have less utility to resolve pathogenic from benign. One solution is disease-specific algorithms that leverage gene knowledge together with variant frequency to aid prioritization. We used large-scale resources including Gene Ontology, protein-protein interactions and other annotation systems together with a broad set of 83 genes with known associations to epilepsy to construct a pathogenicity score for the phenotype. We evaluated the score for all annotated human genes and applied Bayesian methods to combine the derived pathogenicity score with frequency information from our diagno...
New human mutations are thought to originate in germ cells, thus making a recurrence of the same ... more New human mutations are thought to originate in germ cells, thus making a recurrence of the same mutation in a sibling exceedingly rare. However, increasing sensitivity of genomic technologies has anecdotally revealed mosaicism for mutations in somatic tissues of apparently healthy parents. Such somatically mosaic parents might also have germline mosaicism that can potentially cause unexpected intergenerational recurrences. Here, we show that somatic mosaicism for transmitted mutations among parents of children with simplex genetic disease is more common than currently appreciated. Using the sensitivity of individual-specific breakpoint PCR, we prospectively screened 100 families with children affected by genomic disorders due to rare deletion copy-number variants (CNVs) determined to be de novo by clinical analysis of parental DNA. Surprisingly, we identified four cases of low-level somatic mosaicism for the transmitted CNV in DNA isolated from parental blood. Integrated probabilis...
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