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Objectives: To determine the frequency of clinically significant chromosomal abnormalities identified by chromosomal microarray in pregnancy losses at any gestational age and to compare microarray performance with that of traditional cytogenetic analysis when testing pregnancy losses., Methods: Among 535 fetal demise specimens of any gestational age, clinical microarray-based comparative genomic hybridization (aCGH) was performed successfully on 515, and a subset of 107 specimens underwent additional single nucleotide polymorphism (SNP) analysis., Results: Overall, clinically significant abnormalities were identified in 12.8% (64/499) of specimens referred with normal or unknown karyotypes. Detection rates were significantly higher with earlier gestational age. In the subset with normal karyotype, clinically significant abnormalities were identified in 6.9% (20/288). This detection rate did not vary significantly with gestational age, suggesting that, unlike aneuploidy, the contribution of submicroscopic chromosomal abnormalities to fetal demise does not vary with gestational age. In the 107 specimens that underwent aCGH and SNP analysis, seven cases (6.5%) had abnormalities of potential clinical significance detected by the SNP component, including female triploidy. aCGH failed to yield fetal results in 8.3%, which is an improvement over traditional cytogenetic analysis of fetal demise specimens., Conclusions: Both the provision of results in cases in which karyotype fails and the detection of abnormalities in the presence of a normal karyotype demonstrate the increased diagnostic utility of microarray in pregnancy loss. Thus, chromosomal microarray testing is a preferable, robust method of analyzing cases of pregnancy loss to better delineate possible genetic etiologies, regardless of gestational age., (Copyright © 2015 ISUOG. Published by John Wiley & Sons Ltd.)

Copy number variants (CNVs) are associated with many neurocognitive disorders; however, these events are typically large, and the underlying causative genes are unclear. We created an expanded CNV morbidity map from 29,085 children with developmental delay in comparison to 19,584 healthy controls, identifying 70 significant CNVs. We resequenced 26 candidate genes in 4,716 additional cases with developmental delay or autism and 2,193 controls. An integrated analysis of CNV and single-nucleotide variant (SNV) data pinpointed 10 genes enriched for putative loss of function. Follow-up of a subset of affected individuals identified new clinical subtypes of pediatric disease and the genes responsible for disease-associated CNVs. These genetic changes include haploinsufficiency of SETBP1 associated with intellectual disability and loss of expressive language and truncations of ZMYND11 in individuals with autism, aggression and complex neuropsychiatric features. This combined CNV and SNV approach facilitates the rapid discovery of new syndromes and genes involved in neuropsychiatric disease despite extensive genetic heterogeneity.

Background: While microarray testing can identify chromosomal abnormalities missed by karyotyping, its prenatal use is often avoided in low-risk pregnancies due to the possible identification of variants of uncertain significance (VOUS)., Methods: We tested 2,970 prenatal samples of all referral indications using a rapid BACs-on-Beads-based assay with probes for sex chromosomes, common autosomal aneuploidies, and 20 microdeletion/microduplication syndromes, designed as an alternative to microarray in low-risk pregnancies and an alternative to rapid aneuploidy testing in pregnancies also undergoing microarray analysis., Results: Interpretable results were obtained in 2,940 cases (99.0%), with 89% receiving results in 1 day. Aneuploidies were detected in 7.3% and partial chromosome abnormalities in 0.45% (n = 13), including 5 referred for maternal age, abnormal maternal serum screen, or isolated ultrasound markers. The added detection above karyotype was 1 in 745 in lower-risk cases with normal ultrasounds or isolated ultrasound markers/increased nuchal measurements and 1 in 165 for fetuses with structural/growth abnormalities. Neither false negatives nor false positives were found within test limitations. Female polyploidy could not be detected, while polyploidies with Y chromosomes were suspected and confirmed through additional analysis., Conclusion: When combined with karyotyping, this assay provides increased interrogation of specific chromosomal regions, while limiting VOUS identification.

A syndrome associated with 19q13.11 microdeletions has been proposed based on seven previous cases that displayed developmental delay, intellectual disability, speech disturbances, pre- and post-natal growth retardation, microcephaly, ectodermal dysplasia, and genital malformations in males. A 324-kb critical region was previously identified as the smallest region of overlap (SRO) for this syndrome. To further characterize this microdeletion syndrome, we present five patients with deletions within 19q12q13.12 identified using a whole-genome oligonucleotide microarray. Patients 1 and 2 possess deletions overlapping the SRO, and Patients 3-5 have deletions proximal to the SRO. Patients 1 and 2 share significant phenotypic overlap with previously reported cases, providing further definition of the 19q13.11 microdeletion syndrome phenotype, including the first presentation of ectrodactyly in the syndrome. Patients 3-5, whose features include developmental delay, growth retardation, and feeding problems, support the presence of dosage-sensitive genes outside the SRO that may contribute to the abnormal phenotypes observed in this syndrome. Multiple genotype-phenotype correlations outside the SRO are explored, including further validation of the deletion of WTIP as a candidate for male hypospadias observed in this syndrome. We postulate that unique patient-specific deletions within 19q12q13.1 may explain the phenotypic variability observed in this emerging contiguous gene deletion syndrome., (© 2013 Wiley Periodicals, Inc.)

Purpose: To understand the ability of microarray-based comparative genomic hybridization to detect copy-number variation in the presence of maternal cell contamination., Methods: To simulate maternal cell contamination, normal female DNA was mixed at various levels with DNA carrying known copy-number variations. Mixtures were run on a whole-genome 135K oligonucleotide-based array. Data were analyzed with custom analysis software., Results: The array and software design allowed detection of larger copy-number variations at higher levels of maternal cell contamination than smaller copy-number variations. The smallest duplications and deletions were obscured at 22-31% and 55-58% maternal cell contamination, respectively. With male fetal samples, the sex chromosome ratios started showing observable shifts at ~10% maternal cell contamination., Conclusion: As knowledge of the maternal cell contamination level aids in interpretation of array results, we recommend concurrent, independent maternal cell contamination studies for all fetal samples for accurate and timely results. With male fetal samples in our laboratory, interfering levels of maternal cell contamination can be excluded when the sex chromosome plots appear normal. Thus, reportable male microarray-based comparative genomic hybridization results may be occasionally achieved without maternal cell contamination studies. Because the effects of maternal cell contamination on microarray results are dependent on array platforms, experimental techniques, and software algorithms, each laboratory should perform its own analysis to determine acceptable levels of maternal cell contamination for its assays.

Objective: To test the hypothesis that chromosomal microarray analysis frequently diagnoses conditions that require specific medical follow-up and that referring physicians respond appropriately to abnormal test results., Methods: A total of 46,298 postnatal patients were tested by chromosomal microarray analysis for a variety of indications, most commonly intellectual disability/developmental delay, congenital anomalies, dysmorphic features, and neurobehavioral problems. The frequency of detection of abnormalities associated with actionable clinical features was tallied, and the rate of physician response to a subset of abnormal tests results was monitored., Results: A total of 2088 diagnoses were made of more than 100 different disorders that have specific clinical features that warrant follow-up. The detection rate for these conditions using high-resolution whole-genome microarrays was 5.4%, which translates to 35% of all clinically significant abnormal test results identified in our laboratory. In a subset of cases monitored for physician response, appropriate clinical action was taken more than 90% of the time as a direct result of the microarray finding., Conclusions: The disorders diagnosed by chromosomal microarray analysis frequently have clinical features that need medical attention, and physicians respond to the diagnoses with specific clinical actions, thus arguing that microarray testing provides clinical utility for a significant number of patients tested.

Objective: To demonstrate the usefulness of microarray testing in prenatal diagnosis based on our laboratory experience., Methods: Prenatal samples received from 2004 to 2011 for a variety of indications (n = 5003) were tested using comparative genomic hybridization-based microarrays targeted to known chromosomal syndromes with later versions of the microarrays providing backbone coverage of the entire genome., Results: The overall detection rate of clinically significant copy number alterations (CNAs) among unbiased, nondemise cases was 5.3%. Detection rates were 6.5% and 8.2% for cases referred with abnormal ultrasounds and fetal demise, respectively. The overall rate of findings with unclear clinical significance was 4.2% but would reduce to 0.39% if only de novo CNAs were considered. In cases with known chromosomal rearrangements in the fetus or parent, 41.1% showed CNAs related to the rearrangements, whereas 1.3% showed clinically significant CNAs unrelated to the karyotype. Finally, 71% of the clinically significant CNAs found by microarray were below the resolution of conventional karyotyping of fetal chromosomes., Conclusions: Microarray analysis has advantages over conventional cytogenetics, including the ability to more precisely characterize CNAs associated with abnormal karyotypes. Moreover, a significant proportion of cases studied by array will show a clinically significant CNA even with apparently normal karyotypes., (© 2012 John Wiley & Sons, Ltd.)

Objective: The aim of this study is to understand the diagnostic utility of comparative genomic hybridization (CGH)-based microarrays for pregnancies with abnormal ultrasound findings., Methods: We performed a retrospective analysis of 2858 pregnancies with abnormal ultrasounds and normal karyotypes (when performed) tested in our laboratory using CGH microarrays targeted to known chromosomal syndromes with later versions providing backbone coverage of the entire genome. Abnormalities were stratified according to organ system involvement. Detection rates for clinically significant findings among these categories were calculated., Results: Clinically significant genomic alterations were identified in cases with a single ultrasound anomaly (n = 99/1773, 5.6%), anomalies in two or more organ systems (n = 77/808, 9.5%), isolated growth abnormalities (n = 2/76, 2.6%), and soft markers (n = 2/77, 2.6%). The following anomalies in isolation or with additional anomalies had particularly high detection rates: holoprosencephaly (n = 9/85, 10.6%), posterior fossa defects (n = 21/144, 14.6%), skeletal anomalies (n = 15/140, 10.7%), ventricular septal defect (n = 14/132, 10.6%), hypoplastic left heart (n = 11/68, 16.2%), and cleft lip/palate (n = 14/136, 10.3%)., Conclusions: Microarray analysis identified clinically significant genomic alterations in 6.5% of cases with one or more abnormal ultrasound findings; the majority were below the resolution of karyotyping. Larger data sets such as this allow for sub-stratification by specific anomalies to determine risks for genomic alterations detectable by microarray analysis., (© 2012 John Wiley & Sons, Ltd.)

Purpose: Neurofibromatosis, type 1 (NF1) is an autosomal dominant disorder caused by mutations of the neurofibromin 1 (NF1) gene at 17q11.2. Approximately 5% of individuals with NF1 have a 1.4-Mb heterozygous 17q11.2 deletion encompassing NF1, formed through nonallelic homologous recombination (NAHR) between the low-copy repeats that flank this region. NF1 microdeletion syndrome is more severe than NF1 caused by gene mutations, with individuals exhibiting facial dysmorphisms, developmental delay (DD), intellectual disability (ID), and excessive neurofibromas. Although NAHR can also cause reciprocal microduplications, reciprocal NF1 duplications have been previously reported in just one multigenerational family and a second unrelated proband., Methods: We analyzed the clinical features in seven individuals with NF1 microduplications, identified among 48,817 probands tested in our laboratory by array-based comparative genomic hybridization., Results: The only clinical features present in more than one individual were variable DD/ID, facial dysmorphisms, and seizures. No neurofibromas were present. Three sets of parents were tested: one duplication was apparently de novo, one inherited from an affected mother, and one inherited from a clinically normal father., Conclusion: This is the first report comparing the phenotypes of nonrelated individuals with NF1 microduplications. This comparison will allow for further definition of this emerging microduplication syndrome.

Microdeletions of 1q43q44 result in a recognizable clinical disorder characterized by moderate to severe intellectual disability (ID) with limited or no expressive speech, characteristic facial features, hand and foot anomalies, microcephaly (MIC), abnormalities (agenesis/hypogenesis) of the corpus callosum (ACC), and seizures (SZR). Critical regions have been proposed for some of the more prominent features of this disorder such as MIC and ACC, yet conflicting data have prevented precise determination of the causative genes. In this study, the largest of pure interstitial and terminal deletions of 1q43q44 to date, we characterized 22 individuals by high-resolution oligonucleotide microarray-based comparative genomic hybridization. We propose critical regions and candidate genes for the MIC, ACC, and SZR phenotypes associated with this microdeletion syndrome. Three cases with MIC had small overlapping or intragenic deletions of AKT3, an isoform of the protein kinase B family. The deletion of only AKT3 in two cases implicates haploinsufficiency of this gene in the MIC phenotype. Likewise, based on the smallest region of overlap among the affected individuals, we suggest a critical region for ACC that contains ZNF238, a transcriptional and chromatin regulator highly expressed in the developing and adult brain. Finally, we describe a critical region for the SZR phenotype which contains three genes (FAM36A, C1ORF199, and HNRNPU). Although ~90% of cases in this study and in the literature fit these proposed models, the existence of phenotypic variability suggests other mechanisms such as variable expressivity, incomplete penetrance, position effects, or multigenic factors could account for additional complexity in some cases.

Fragile X E (FRAXE) is an X-linked form of intellectual disability characterized by mild to moderate cognitive impairment, speech delay, hyperactivity, and autistic behavior. The folate-sensitive fragile site FRAXE is located in Xq28 approximately 600 kb distal to the fragile X syndrome fragile site (FRAXA) and harbors an unstable GCC (CCG) triplet repeat adjacent to a CpG island in the 5' untranslated region of the AFF2 (FMR2) gene. The disorder results from amplification and methylation of the GCC repeat and resultant silencing of AFF2. Although chromosome abnormalities that disrupt AFF2 have been reported in two individuals with mild-moderate intellectual disability, microdeletions of Xq28 that delete only AFF2 have not been described as a potential cause of FRAXE-intellectual disability. We performed clinical and molecular characterization of two males with 240 and 499 kb deletions, respectively, at Xq28, both of which encompassed only one gene, AFF2. The 240 kb deletion in Patient 1 was intragenic and lead to the loss of 5' exons 2-4 of AFF2; the 499 kb deletion in Patient 2 removed the 5' exons 1-2 of AFF2 including approximately 350 kb upstream of the gene. Both individuals had developmental and speech delay, and one had mild dysmorphism. We predict disruption of AFF2 in these two patients is likely the cause of their overlapping phenotypes., (Copyright © 2011 Wiley Periodicals, Inc.)

Purpose: : Recently, molecular cytogenetic techniques have identified novel copy number variants in individuals with schizophrenia. However, no large-scale prospective studies have been performed to characterize the broader spectrum of phenotypes associated with such copy number variants in individuals with unexplained physical and intellectual disabilities encountered in a diagnostic setting., Methods: : We analyzed 38,779 individuals referred to our diagnostic laboratory for microarray testing for the presence of copy number variants encompassing 20 putative schizophrenia susceptibility loci. We also analyzed the indications for study for individuals with copy number variants overlapping those found in six individuals referred for schizophrenia., Results: : After excluding larger gains or losses that encompassed additional genes outside the candidate loci (e.g., whole-arm gains/losses), we identified 1113 individuals with copy number variants encompassing schizophrenia susceptibility loci and 37 individuals with copy number variants overlapping those present in the six individuals referred to our laboratory for schizophrenia. Of these, 1035 had a copy number variant of one of six recurrent loci: 1q21.1, 15q11.2, 15q13.3, 16p11.2, 16p13.11, and 22q11.2. The indications for study for these 1150 individuals were diverse and included developmental delay, intellectual disability, autism spectrum, and multiple congenital anomalies., Conclusion: : The results from our study, the largest genotype-first analysis of schizophrenia susceptibility loci to date, suggest that the phenotypic effects of copy number variants associated with schizophrenia are pleiotropic and imply the existence of shared biologic pathways among multiple neurodevelopmental conditions.

Objective: To develop a novel, rapid prenatal assay for pregnancies with high likelihood of normal karyotypes, using BACs-on-Beads(™) technology, a suspension array-based multiplex assay that employs Luminex(®) xMAP(®) technology, for the detection of gains and losses in chromosomal DNA., Methods: Fifteen relatively common microdeletions were selected that are not detectable, or may be missed, by karyotyping and usually do not present with abnormal ultrasound findings. Chromosomes 13, 18, 21, X, and Y were included. We validated the assay with 430 samples., Results: All microdeletions and aneuploidies were correctly identified, except for a 69,XXX incorrectly identified as a normal female and a male with ∼20% maternal cell contamination (MCC) that could not be distinguished from 69,XXY. MCC became apparent at 20 to 30%. Mosaicism was identified at 30 to 35% abnormal cells., Conclusion: We have developed an alternative to fluorescence in situ hybridization (FISH) aneuploidy screening and microarray analysis in otherwise normal pregnancies undergoing invasive testing. We demonstrated that the assay will detect all microdeletions and aneuploidies of regions covered on the assay. We developed analytical software that displays results for well-characterized syndromes but not abnormalities of unclear clinical significance. This assay is likely to be preferred by women seeking testing beyond routine karyotyping but who desire more information than provided by aneuploidy FISH., (Copyright © 2011 John Wiley & Sons, Ltd.)

Orofacial clefts of the lip and/or palate comprise one of the most common craniofacial birth defects in humans. Though a majority of cleft lip and/or cleft palate (CL/P) occurs as isolated congenital anomalies, there exist a large number of Mendelian disorders in which orofacial clefting is part of the clinical phenotype. Here we report on two individuals and one multi-generational family with microdeletions at 20p12.3 that include the bone morphogenetic protein 2 (BMP2) gene. In two propositi the deletion was almost identical at ∼600 kb in size, and BMP2 was the only gene deleted; the third case had a ∼5.5-Mb deletion (20p13p12.2) that encompassed at least 20 genes including BMP2. Clinical features were significant for cleft palate and facial dysmorphism in all three patients, including Pierre-Robin sequence in two. Microdeletion 20p13p12 involving BMP2 is rare and has been implicated in Wolff-Parkinson-White (WPW) syndrome with neurocognitive deficits and with Alagille syndrome when the deletion includes the neighboring JAG1 gene in addition to BMP2. Despite a significant role for the BMPs in orofacial development, heterozygous loss of BMP2 has not been previously reported in patients with syndromic clefting defects. Because BMP2 was the sole deleted gene in Patients 1 and 2 and one of the genes deleted in Patient 3, all of whom had clinical features in common, we suggest that haploinsufficiency for BMP2 is a crucial event that predisposes to cleft palate and additional anomalies. Lack of significant phenotypic components in family members of Patient 1 suggests variable expressivity for the phenotype., (Copyright © 2011 Wiley-Liss, Inc.)

Insertions occur when a segment of one chromosome is translocated and inserted into a new region of the same chromosome or a non-homologous chromosome. We report 71 cases with unbalanced insertions identified using array CGH and FISH in 4909 cases referred to our laboratory for array CGH and found to have copy-number abnormalities. Although the majority of insertions were non-recurrent, several recurrent unbalanced insertions were detected, including three der(Y)ins(Y;18)(q?11.2;p11.32p11.32)pat inherited from parents carrying an unbalanced insertion. The clinical significance of these recurrent rearrangements is unclear, although the small size, limited gene content, and inheritance pattern of each suggests that the phenotypic consequences may be benign. Cryptic, submicroscopic duplications were observed at or near the insertion sites in two patients, further confounding the clinical interpretation of these insertions. Using FISH, linear amplification, and array CGH, we identified a 126-kb duplicated region from 19p13.3 inserted into MECP2 at Xq28 in a patient with symptoms of Rett syndrome. Our results demonstrate that although the interpretation of most non-recurrent insertions is unclear without high-resolution insertion site characterization, the potential for an otherwise benign duplication to result in a clinically relevant outcome through the disruption of a gene necessitates the use of FISH to determine whether copy-number gains detected by array CGH represent tandem duplications or unbalanced insertions. Further follow-up testing using techniques such as linear amplification or sequencing should be used to determine gene involvement at the insertion site after FISH has identified the presence of an insertion.

Microdeletions of 1q41q42 have recently been classified as a syndrome. Features include significant developmental delay and characteristic dysmorphic features as well as cleft palate, clubfeet, seizures, and short stature in some individuals, with a clinical diagnosis of Fryns syndrome in two individuals with congenital diaphragmatic hernia at the severe end of the spectrum. The gene DISP1, which is involved in sonic hedgehog signaling, has been proposed as a candidate for the midline defects in this syndrome. We undertook a genotype-phenotype analysis of seven previously unreported individuals with deletions of 1q41q42 that range from 777 kb to 6.87 Mb. Three of the individuals in our cohort do not display the major features of the syndrome and have more proximal deletions that only overlap with the previously described 1q41q42 smallest region of overlap (SRO) at DISP1. One individual with several features of the syndrome has a more distal deletion that excludes DISP1. The three remaining individuals have larger deletions that include the entire SRO and demonstrate features of the microdeletion syndrome. Confounding genotype-phenotype correlations, one of the small deletions involving DISP1 was inherited from a phenotypically normal parent. DISP1 haploinsufficiency may not be solely responsible for the major features of 1q41q42 microdeletion syndrome, and other genes in the SRO likely play a role in the phenotype. Additionally, some features present in a minority of individuals, such as Pelger-Huët anomaly, may be attributed to deletions of genes outside of the SRO., (Copyright © 2010 Elsevier Masson SAS. All rights reserved.)

Purpose: Autism spectrum disorders represent a range of neurodevelopmental disorders that have been shown to have a strong genetic etiological component. Microarray-based comparative genomic hybridization and other molecular cytogenetic techniques are discovering an increasing number of copy number variations in individuals with autism spectrum disorder., Methods: We examined the yield of abnormal microarray-based comparative genomic hybridization findings in our laboratory for individuals referred for testing for autism spectrum disorder. We also examined the presence of autistic features among 151 additional individuals who were referred for microarray-based comparative genomic hybridization testing for indications other than autism spectrum disorder but had genomic alterations overlapping those found in cases referred for autism spectrum disorder., Results: We identified 1461 individuals referred for testing for autism spectrum disorder, with likely significant abnormalities reported in approximately 11.6% of individuals analyzed with whole-genome arrays. These abnormalities include alterations that encompass novel candidate genes such as SNTG2, SOX5, HFE, and TRIP38. A minority of individuals with overlapping abnormalities (19%) had autistic features, and many of the copy number variations identified in our study are inherited (69% among those found in individuals with autism spectrum disorder)., Conclusions: Our results suggest these copy number variations are one of multiple factors contributing to the development of an autism spectrum disorder phenotype. Additionally, the broad phenotypic spectrum of the patients with these copy number variations suggests that these copy number variations are not autism spectrum disorder-specific but likely more generally impair neurodevelopment.

Background: Subtelomeric deletions of the long arm of chromosome 20 are rare, with only 11 described in the literature. Clinical features of individuals with these microdeletions include severe limb malformations, skeletal abnormalities, growth retardation, developmental and speech delay, mental retardation, seizures and mild, non-specific dysmorphic features., Methodology/principal Findings: We characterized microdeletions at 20q13.33 in six individuals referred for genetic evaluation of developmental delay, mental retardation, and/or congenital anomalies. A comparison to previously reported cases of 20q13.33 microdeletion shows phenotypic overlap, with clinical features that include mental retardation, developmental delay, speech and language deficits, seizures, and behavior problems such as autistic spectrum disorder. There does not appear to be a clinically recognizable constellation of dysmorphic features among individuals with subtelomeric 20q microdeletions., Conclusions/significance: Based on genotype-phenotype correlation among individuals in this and previous studies, we discuss several possible candidate genes for specific clinical features, including ARFGAP1, CHRNA4 and KCNQ2 and neurodevelopmental deficits. Deletion of this region may play an important role in cognitive development.

Prune belly syndrome is a rare congenital disorder characterized by deficiency of abdominal wall muscles, cryptorchidism, and urinary tract anomalies. We have had the opportunity to study a baby with prune belly syndrome associated with an apparently de novo 1.3-megabase interstitial 17q12 microdeletion that includes the hepatocyte nuclear factor-1-beta gene at 17q12. One previous patient, an adult, has been reported with prune belly syndrome and a hepatocyte nuclear factor-1-beta microdeletion. Hepatocyte nuclear factor-1-beta is a widely expressed transcription factor that regulates tissue-specific gene expression and is expressed in numerous tissues including mesonephric duct derivatives, the renal tubule of the metanephros, and the developing prostate of the mouse. Mutations in hepatocyte nuclear factor-1-beta cause the "renal cysts and diabetes syndrome," isolated renal cystic dysplasia, and a variety of other malformations. Based on its expression pattern and the observation of two affected cases, we propose that haploinsufficiency of hepatocyte nuclear factor-1-beta may be causally related to the production of the prune belly syndrome phenotype through a mechanism of prostatic and ureteral hypoplasia that results in severe obstructive uropathy with urinary tract and abdominal distension., (Copyright Thieme Medical Publishers.)

Background: Microarray-based comparative genomic hybridization (aCGH) is a powerful diagnostic tool for the detection of DNA copy number gains and losses associated with chromosome abnormalities, many of which are below the resolution of conventional chromosome analysis. It has been presumed that whole-genome oligonucleotide (oligo) arrays identify more clinically significant copy-number abnormalities than whole-genome bacterial artificial chromosome (BAC) arrays, yet this has not been systematically studied in a clinical diagnostic setting., Results: To determine the difference in detection rate between similarly designed BAC and oligo arrays, we developed whole-genome BAC and oligonucleotide microarrays and validated them in a side-by-side comparison of 466 consecutive clinical specimens submitted to our laboratory for aCGH. Of the 466 cases studied, 67 (14.3%) had a copy-number imbalance of potential clinical significance detectable by the whole-genome BAC array, and 73 (15.6%) had a copy-number imbalance of potential clinical significance detectable by the whole-genome oligo array. However, because both platforms identified copy number variants of unclear clinical significance, we designed a systematic method for the interpretation of copy number alterations and tested an additional 3,443 cases by BAC array and 3,096 cases by oligo array. Of those cases tested on the BAC array, 17.6% were found to have a copy-number abnormality of potential clinical significance, whereas the detection rate increased to 22.5% for the cases tested by oligo array. In addition, we validated the oligo array for detection of mosaicism and found that it could routinely detect mosaicism at levels of 30% and greater., Conclusions: Although BAC arrays have faster turnaround times, the increased detection rate of oligo arrays makes them attractive for clinical cytogenetic testing.

Holoprosencephaly (HPE) is the most common developmental forebrain anomaly in humans. Both environmental and genetic factors have been identified to play a role in the HPE phenotype. Previous studies of the genetic bases of HPE have taken a phenotype-first approach by examining groups of patients with HPE for specific mutations or deletions in known or candidate HPE genes. In this study, we characterized the presence or absence of HPE or a microform in 136 individuals in which microarray-based comparative genomic hybridization (aCGH) identified a deletion of one of 35 HPE loci. Frank holoprosencephaly was present in 11 individuals with deletions of one of the common HPE genes SHH, ZIC2, SIX3, and TGIF1, in one individual with a deletion of the HPE8 locus at 14q13, and in one individual with a deletion of FGF8, whereas deletions of other HPE loci and candidate genes (FOXA2 and LRP2) expressed microforms of HPE. Although individuals with deletions of other HPE candidates (DISP1, LSS, HHIP, SMO, BMP4, CDON, CDC42, ACVR2A, OTX2, and WIF1) had clinically significant features, none had frank HPE or a microform. A search for significant aCGH findings in individuals referred for testing for HPE revealed a novel association of a duplication involving GSK3B at 3q13.33 with HPE or a microform, seen in two unrelated individuals.

Background: Deletions that encompass 2q31.1 have been proposed as a microdeletion syndrome with common clinical features, including intellectual disability/developmental delay, microcephaly, cleft palate, growth delay, and hand/foot anomalies. In addition, several genes within this region have been proposed as candidates for split hand-foot malformation 5 (SHFM5). Methods: To delineate the genotype-phenotype correlation between deletions of this region, we identified 14 individuals with deletions at 2q31.1 detected by microarray analysis for physical and developmental disabilities. Results: All subjects for whom detailed clinical records were available had neurological deficits of varying degree. Seven subjects with deletions encompassing the HOXD cluster had hand/foot anomalies of varying severity, including syndactyly, brachydactyly, and ectrodactyly. Of 7 subjects with deletions proximal to the HOXD cluster, 5 of which encompassed DLX1/DLX2, none had clinically significant hand/foot anomalies. In contrast to previous reports, the individuals in our study did not display a characteristic gestalt of dysmorphic facial features. Conclusion: The absence of hand/foot anomalies in any of the individuals with deletions of DLX1/DLX2 but not the HOXD cluster supports the hypothesis that haploinsufficiency of the HOXD cluster, rather than DLX1/DLX2, accounts for the skeletal abnormalities in subjects with 2q31.1 microdeletions.

Objective: To determine the detection rates of whole-genome microarray technology compared to targeted microarray analysis for chromosome abnormalities in prenatal samples submitted for diagnostic testing., Methods: Microarray analysis using either whole-genome bacterial artificial chromosome (BAC)-based and oligonucleotide (oligo)-based microarrays or targeted BAC microarrays was performed on 182 and 62 prenatal cases, respectively, from North American healthcare providers without previously known chromosome abnormalities or family history of a parent with a known chromosome rearrangement., Results: Microarray analysis identified clinically significant chromosome alterations in 7 out of 182 (3.8%) prenatal specimens, two of which each had two unrelated abnormalities. After excluding two of the cases in which the abnormality would have been identified by routine karyotyping, the diagnostic yield of clinically significant findings was 5 out of 182 (2.7%). One case had a finding of unclear significance (0.5%) and 16 cases had benign copy number variants (CNVs) (8.8%). Targeted microarray analysis combined with previously published data demonstrated detection rates of 0.9% for clinically significant results, 0.5% for results of unclear significance, and 8.0% for benign CNVs., Conclusions: Whole-genome prenatal aCGH detected clinically significant submicroscopic chromosome abnormalities in addition to chromosome abnormalities that could be identified by concurrent karyotyping without an increase in unclear results or benign CNVs compared to targeted aCGH.

Purpose: Pitt-Hopkins syndrome is characterized by severe mental retardation, characteristic dysmorphic features, and susceptibility to childhood-onset seizures and intermittent episodes of hyperventilation. This syndrome is caused by haploinsufficiency of TCF4, which encodes a basic helix-loop-helix transcription factor. Missense, nonsense, splice-site mutations, and gene deletions have been found in individuals with Pitt-Hopkins syndrome. Previous reports have suggested that the Pitt-Hopkins syndrome phenotype is independent of mutation or deletion type., Methods: We screened 13,186 individuals with microarray-based comparative genomic hybridization. We also conducted a review of the literature and statistical analysis of the phenotypic features for all individuals with confirmed mutations or deletions of TCF4., Results: We identified seven individuals with TCF4 deletions. All patients have features consistent with Pitt-Hopkins syndrome, although only three have breathing anomalies, and none has seizures. Our review of previously reported cases with TCF4 mutations and deletions showed that all patients with Pitt-Hopkins syndrome reported to date have severe psychomotor retardation, the onsets of seizures and hyperventilation episodes are limited to the first decade in most reported patients with Pitt-Hopkins syndrome, hyperventilation episodes are more common than seizures and are seen in the oldest patients, and individuals with missense TCF4 mutations are more likely to develop seizures., Conclusions: On the basis of an analysis of published cases, we propose a genotype-phenotype correlation of increased seizure activity with missense TCF4 mutations.

Background: Of the fewer than 100 cases reported within the literature of constitutional deletions involving the long arm of chromosome 6, only five have been characterized using high-resolution microarray analysis. Reported 6q deletion patients show a high incidence of mental retardation, ear anomalies, hypotonia, and postnatal growth retardation., Results: We report a 16-month-old male presenting with developmental delay and dysmorphic features who was found by array-based comparative genomic hybridization (aCGH) to have a ~2.16 Mb de novo deletion within chromosome band 6q16.1 that encompasses only two genes. Expression studies of the mouse homologue of one of the genes, the ephrin receptor 7 gene (EPHA7), have shown the gene functions during murine embryogenesis to form cortical domains, determine brain size and shape, and play a role in development of the central nervous system (CNS)., Discussion: Our results suggest that deletion of EPHA7 plays a role in the neurologic and dysmorphic features, including developmental delay, hypotonia, and ear malformations, observed in some 6q deletion patients.

Region-specific probes developed for the diagnosis of specific syndrome, can be adapted to elucidate the exact nature of certain chromosomal structural anomalies. We describe the use of FISH probes in characterizing a prenatally diagnosed chromosome rearrangement. An abnormal chromosome 21 was detected during amniocentesis for maternal age indication, and a similar appearing chromosome 21 was found in the mother. The exact nature of the rearrangement was not immediately evident from G-banded karyotypes. FISH was performed using a whole chromosome painting probe, as well as the region-specific probes D21S65 (21q21-22.1), D21S55 (21q22.3) and D21S1219/D21S1220 (21q22.3-qter) (Oncor). Results showed an interstitial paracentric inversion, with breakpoints in bands 21q22.1 and 21q22.3, which was identical in the mother and the fetus: 46,XX,?inv(21)(q).ish inv(21)(q22.1q22.3)(wcp+.D21S65 mv, D21S55 mv, D21S1219/D21S1220 st). In this case, FISH using region-specific probes was helpful in characterizing the inversion and aided in the genetic counselling of risk assessment for the family.

We describe the cloning and characterization of the NTT gene (noncoding transcript in T cells), identified by differential display RT-PCR based on the differential presence of its transcript in a subset of activated, human CD4+ T-cell clones. The full-length cDNA and genomic sequences were cloned and found to produce a 17-kb transcript that is polyadenylated, but is not spliced. Consistent with the transcript's nuclear predominance, NTT has no open reading frame larger than 270 bp. It is transcribed in a select subset of CD4+ T-cell clones propagated in vitro. Its transcription can also be induced in freshly isolated T cells by in vitro activation with PHA or with PMA and ionomycin. In vivo, NTT transcripts are found only in activated, but not resting, T cells. Transcripts were absent in a variety of lymphoid cell lines and transformed lines from other tissues. NTT is a new member of the small group of genes including XIST (X-specific transcript), H19, and IPW (imprinted gene in the Prader-Willi syndrome region), which are transcribed but not translated, and may have a role in the regulation of neighboring genes. XIST, H19, and IPW exhibit monoallelic expression, but both NTT alleles are expressed in CD4+ T-cell clones. Southern blot and fluorescence in situ hybridization analyses show that NTT is a single-copy gene residing in chromosome 6q23-q24, near the interferon-gamma receptor gene (IFN-gamma R). Their proximity and shared expression pattern suggest a possible functional relationship.

We have previously reported functional disomy for X-linked genes in females with tiny ring X chromosomes and a phenotype significantly more abnormal than Turner syndrome. In such cases the disomy results from failure of these X chromosomes to inactivate because they lack DNA sequences essential for cis X inactivation. Here we describe a novel molecular mechanism for functional X disomy that is associated with maternal isodisomy. In this case, the severe mental retardation and multiple congenital abnormalities in a female with a mosaic 45,X/ 46,X,del(X)(q21.3-qter)/ 46X,r(X) karyotype are associated with overexpression of the genes within Xpter to Xq21.31 in many of her cells. Her normal X, ring X, and deleted linear X chromosomes originate from the same maternal X chromosome, and all are transcriptionally active. None expresses X inactive specific transcript (XIST), although the locus and region of the putative X inactivation center (XIC) are present on both normal and linear deleted X chromosomes. To our knowledge, this is the first report of a functional maternal X isodisomy, and the largest X chromosome to escape inactivation. In addition, these results (1) show that cis inactivation does not invariably occur in human females with two X chromosomes, even when the XIC region is present on both of them; (2) provide evidence for a critical time prior to the visible onset of X inactivation in the embryo when decisions about X inactivation are made; and (3) support the hypothesis that the X chromosome counting mechanism involves chromosomal imprinting, occurs prior to the onset of random inactivation, and is required for subsequent inactivation of the chromosome.

We have recently reported results of DNA replication analysis of three X-linked loci (FRAXA, F8C and XIST) on the X chromosomes in male and female fibroblasts using fluorescence in situ hybridization (FISH) (1). Although our findings that XIST replicates later on the active X than on the inactive X are similar to those of Boggs & Chinault (2) based on a FISH assay in female lymphoblasts, they are the opposite of observations recently reported by Hansen et al. (3) using a different technique. Because our conclusions about the inactive X were deduced from the behavior of the active X in male cells, we reexamined the time when these loci replicate on the human inactive X chromosome isolated from its homolog in somatic cell hybrids. We also studied the same chromosome as an active X in related hybrids. The results provide direct evidence that the expressed XIST locus on the inactive X replicates earlier than its repressed homolog on the active X and earlier than the FRAXA locus which is repressed on this chromosome. The silent XIST locus on the active X replicates late along with F8C which is also not transcribed in these cells. Possible reasons for the different results obtained by Hansen et al. (3) are discussed.

Small ring X chromosomes were first described in mosaic karyotypes of females with the relatively benign phenotype of Turner syndrome. The presence of these rings in association with more severe phenotypes including mental retardation has raised the possibility that they lack sequences necessary for X chromosome inactivation, specifically genes within the X inactivation center (XIC) essential for cis X-inactivation. We recently showed that ring X chromosomes ascertained because of the severe phenotype do not express XIST, a candidate for the relevant gene, and that they are in fact active chromosomes. We now report studies of the genetic content of 11 of these ring X chromosomes (9 associated with severe phenotypes). Our results indicate that these chromosomes contain contiguous segments of DNA and have variable proximal and distal breakpoints and some include mainly long arm or mainly short arm sequences. As expected for ring chromosomes, they lack telomeric sequences. Many of the ring chromosomes lack the XIST locus, consistent with XIST being necessary for cis inactivation. However, the breakpoints in four ring chromosomes that have XIST sequences but do not express XIST suggest that other sequences within the XIC distal to XIST as it is now defined are also needed.

Considerable evidence suggests that the X inactive transcript gene, XIST/Xist, has a role in the initial steps of X chromosome inactivation in the female mammalian embryo. It is transcribed exclusively from inactive X chromosomes, and its noncoding transcript seems to be essential for cis inactivation. Unexpected for a developmental gene, XIST continues to be expressed in adult somatic cells. To determine the effect of reversal of inactivation on the expression of XIST, we studied human X chromosomes that had been induced to reverse X inactivation by hybridization of chorionic villi cells from term placentas with mouse A9 cells. In nine hybrids with a reactivated X chromosome, XIST was either not expressed or expressed much less than the locus on the inactive X chromosome in the chorionic villi cells from which they were derived. The repressibility of XIST by reversal of inactivation in these placental cells mirrors events that occur during the ontogeny of oocytes and indicates that the locus is subject to regulation in somatic cells long after inactivation is established in the embryo. The small residual XIST activity from these active chromosomes suggests that low levels of XIST expression do not interfere with chromosome activity and raises the possibility that the induction of cis inactivation requires a certain level of XIST transcription. The chorionic villi hybrids provide an experimental system to study the developmental regulation of XIST.

The relationship between the transcriptional state of a locus and the time when it replicates during DNA synthesis is increasingly apparent. Active autosomal genes tend to replicate early, whereas inactive ones are more permissive and frequently replicate later. Although the inactive X chromosome replicates later than its active homologue, little is known about the replication of X-linked genes. We have used FISH to examine the replication of loci on the active X chromosome that are not transcribed, either because the tissue analyzed was not the expressing tissue (F8C), because the locus is silent on all active X chromosomes (XIST), or because it has been mutated by expansion and methylation of a CpG island (FMR1). In this assay, an unreplicated locus is characterized by a single hybridization signal, and a replicated locus is characterized by a doublet hybridization signal. The percentage of doublets is used as a measure of relative time of replication in S phase. The validity of this approach has been established elsewhere, since results compare favorably with those obtained using traditional methods for studying DNA replication. Our results show that the FMR1 gene replicates relatively later in fragile X (fraX) males with the full mutation than in normal males, irrespective of the probe used. The F8C locus is late replicating in both normal and fraX males and replicates at nearly the same time on active and inactive X in females. The XIST locus replicates late in all the males studied and asynchronously in female cells.(ABSTRACT TRUNCATED AT 250 WORDS)