Microdeletion and Microduplication Analysis of Chinese Conotruncal Defects Patients with Targeted Array Comparative Genomic Hybridization

Objective

The current study aimed to develop a reliable targeted array comparative genomic hybridization (aCGH) to detect microdeletions and microduplications in congenital conotruncal defects (CTDs), especially on 22q11.2 region, and for some other chromosomal aberrations, such as 5p15-5p, 7q11.23 and 4p16.3.

Methods

Twenty-seven patients with CTDs, including 12 pulmonary atresia (PA), 10 double-outlet right ventricle (DORV), 3 transposition of great arteries (TGA), 1 tetralogy of Fallot (TOF) and one ventricular septal defect (VSD), were enrolled in this study and screened for pathogenic copy number variations (CNVs), using Agilent 8 x 15K targeted aCGH. Real-time quantitative polymerase chain reaction (qPCR) was performed to test the molecular results of targeted aCGH.

Results

Four of 27 patients (14.8%) had 22q11.2 CNVs, 1 microdeletion and 3 microduplications. qPCR test confirmed the microdeletion and microduplication detected by the targeted aCGH.

Conclusion

Chromosomal abnormalities were a well-known cause of multiple congenital anomalies (MCA). This aCGH using arrays with high-density coverage in the targeted regions can detect genomic imbalances including 22q11.2 and other 10 kinds CNVs effectively and quickly. This approach has the potential to be applied to detect aneuploidy and common microdeletion/microduplication syndromes on a single microarray.     

A child with mosaicism for deletion (14)(q11.2q13)

In this case report we describe a child with a de novo deletion in the (q11.2q13) region of chromosome 14. The child presented with dysmorphic features - anophthalmia, microcephaly, and growth retardation. Cytogenetic studies showed mosaicism. The karyotype was 46,XX,del(14)(q11.2;q13) [16] /46,XX [9]. We compared the features observed in this child with that of others with the same deletion reported in scientific literature and found that this is the first report of a child mosaic for this deletion. It is also the first time it has been reported in association with anophthalmia.     

Analysis of 17p11.2 chromosome region rearrangements in CMT1 patients from Ukraine

Two intercomplementary methods of 17p11.2 duplication/deletion identification have been elaborated: STR allelic variants analysis and direct PMP22 gene dosage measuring by means of quantitative Real-Time PCR. It has been carried out detection and analysis of 17p11.2 chromosome region rearrangements in CMT1 patients from Ukraine. It has been registered the high level of de novo cases with 17p11.2-duplication. It has been shown the 17p11.2 chromosome region duplication/deletion association with CMT1A and HNPP clinical phenotypes which may be used in differential diagnosis of this type of CMT polyneuropathy. The article is published in the original.     

Ring chromosome 15: characterization by array CGH

Ring chromosome 15 [r(15)] is an uncommon finding with less than 50 patients reported. Precise genotype–phenotype correlations are problematic because of the difficulties in determining the extent of euchromatic loss, the level of mosaicism, and the influence of the timing of ascertainment. We report two discordant examples of r(15) patients. In the first case, prenatal diagnosis of a de novo r(15) was made during the second trimester: mos 46,XX,r(15)(p11.2q26)[32]/45,XX,-15[13]/47,XX,r(15)(p11.2q26)x2[3]/46,XX,dic r(15)(p11.2q26p11.2q26[1]/46,XX[2]. Postnatal follow-up revealed extremely small stature, heart defects, and developmental delay. Patient 2 was a 31-year-old short-statured female who was living independently: 46,XX,r(15)(p11q26). Both cases showed loss of the 15q subtelomeric region by fluorescence in situ hybridization (FISH). To investigate the discordance in phenotypes between the two patients, we undertook array comparative genomic hybridization (array CGH) analyses to more fully characterize the deletions associated with these otherwise structurally indistinguishable r(15) chromosomes from conventional cytogenetic analyses and fluorescence in situ hybridization (FISH) studies. By array CGH, patient 1 showed deletion of multiple contiguous clones predicting an approximately 6 Mb deletion of distal 15q. In contrast, patient 2 showed loss of just the 15q subtelomeric clone and an interstitial clone by array CGH confirming that the severity of the phenotype correlated with the size of the deletion at the molecular level. These cases illustrate the utility of array CGH characterization for determining the size of the associated deletion in ring chromosomes and for facilitating phenotype–genotype correlations.     

Mouse and Human CRKL Is Dosage Sensitive for Cardiac Outflow Tract Formation

The human chromosome 22q11.2 region is susceptible to rearrangements during meiosis leading to velo-cardio-facial/DiGeorge/22q11.2 deletion syndrome (22q11DS) characterized by conotruncal heart defects (CTDs) and other congenital anomalies. The majority of individuals have a 3 Mb deletion whose proximal region contains the presumed disease-associated gene TBX1 (T-box 1). Although a small subset have proximal nested deletions including TBX1, individuals with distal deletions that exclude TBX1 have also been identified. The deletions are flanked by low-copy repeats (LCR22A, B, C, D). We describe cardiac phenotypes in 25 individuals with atypical distal nested deletions within the 3 Mb region that do not include TBX1 including 20 with LCR22B to LCR22D deletions and 5 with nested LCR22C to LCR22D deletions. Together with previous reports, 12 of 37 (32%) with LCR22B–D deletions and 5 of 34 (15%) individuals with LCR22C–D deletions had CTDs including tetralogy of Fallot. In the absence of TBX1, we hypothesized that CRKL (Crk-like), mapping to the LCR22C–D region, might contribute to the cardiac phenotype in these individuals. We created an allelic series in mice of Crkl, including a hypomorphic allele, to test for gene expression effects on phenotype. We found that the spectrum of heart defects depends on Crkl expression, occurring with analogous malformations to that in human individuals, suggesting that haploinsufficiency of CRKL could be responsible for the etiology of CTDs in individuals with nested distal deletions and might act as a genetic modifier of individuals with the typical 3 Mb deletion.     

High rate of detection of 13q14 deletion mosaicism among retinoblastoma patients (using more extensive methods)

Summary Using a cell population with a high proportion of early mitotic cells and by examining more cells derived from peripheral lymphocytes, we found three cases with a 13q14 deletion mosaicism among fifteen retinoblastoma patients; one with a de novo 13/18 balanced translocation, and another with a monosomy 13(q13»q21.2 or 21.3). The three patients with a 13q14 deletion mosaicism had sporadic retinoblastoma (two had bilateral and one unilateral retinoblastoma). The results indicate that 13q14 deletion mosaicism plays a major role in the etiology of this tumor.     

A distinctive phenotype associated with an interstitial deletion 6q14 contained within a de novo pericentric inversion 6 (p11.2q15)

This report describes a nearly 25-year-old female with an interstitial deletion of band 14 in the long arm of one chromosome 6 (6q14). The deletion is contained within a de novo pericentric inversion with breakpoints in 6p11.2 and 6q15 (Karyotype 46,XX, del(6)(q13q15),inv(6)(p11.2q15). The distal breakpoint of the deletion and the pericentric inversion at 6q15 are the same, but the proximal breakpoints differ. Since cells with other chromosomal findings were not detected in cultured lymphocytes and fibroblasts, chromosome mosaicism seems unlikely. Thus, it is assumed that the inversion and the deletion originated from the same event. The development of a distinctive phenotype in the patient was observed over a period of 22 years. It includes characteristic dysmorphic facial features such as ocular hypertelorism, flat nasal bridge, prominent zygomatic bones, and a depressed glabella. A striking, non-progressive deficit of motor control is manifest in an inability to use her hands properly and a broad-based slow-motion-like gait. Although severely deficient in abstract mental abilities and speech development, she is well adapted to family life and to a school for retarded individuals. Normal height and head circumference, and reduced sensitivity to pain are noteworthy. Presumably the deletion caused the phenotype and the distinct behavioral pattern. This patient probably represents a novel chromosomal phenotype that results from aggregate haploinsufficiency of gene loci in the deleted region.     

Delineation of the frequency and boundary of chromosomal copy number variations in paediatric neuroblastoma

Neuroblastoma, the most common solid tumour in early childhood, is characterized by very frequent chromosomal copy number variations (CNVs). While chromosome 2p amplification, 17q gain, 1p and 11q deletion in human neuroblastoma tissues are well-known, the exact frequencies and boundaries of the chromosomal CNVs have not been delineated. We analysed the publicly available single nucleotide polymorphism (SNP) array data which were originally generated by the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative, defined the frequencies and boundaries of chromosomes 2p11.2 – 2p25.3 amplification, 17q11.1-17q25.3 gain, 1p13.3-1p36.33 deletion and 11q13.3-11q25 deletion in neuroblastoma tissues, and identified chromosome 7q14.1 (Chr7:38254795-38346971) and chromosome 14q11.2 (Chr14:21637401-22024617) deletion in blood and bone marrow samples from neuroblastoma patients, but not in tumour tissues. Kaplan Meier analysis showed that double deletion of Chr7q14.1 and Chr14q11.2 correlated with poor prognosis in MYCN gene amplified neuroblastoma patients. In conclusion, the oncogenes amplified or gained and tumour suppressor genes deleted within the boundaries of chromosomal CNVs in tumour tissues should be studied for their roles in tumourigenesis and as therapeutic targets. Focal deletions of Chr7q14.1 and Chr14q11.2 together in blood and bone marrow samples from neuroblastoma patients can be used as a marker for poorer prognosis and more aggressive therapies.     

Cytogenetic findings in a prospective series of patients with DiGeorge anomaly.

High-resolution cytogenetics analysis of peripheral blood lymphocytes was done prospectively on 27 of 28 patients with features of DiGeorge anomaly. Twenty-two patients (81%) had normal chromosome studies with no detectable deletion in chromosome 22. Five patients (18%) had demonstrable chromosome abnormalities. Three patients had monosomy 22q11, one due to a 4q;22q translocation, one due to a 20q;22q translocation, and one due to an interstitial deletion of 22q11. One patient had monosomy 10p13, and one patient had monosomy 18q21.33, although the latter had subsequent resolution of T-cell defects. These findings are consistent with the heterogeneity of DiGeorge anomaly but confirm the association with monosomy 22q11 in some cases. However, monosomy 10p13 may also lead to this phenotype. Because of these associated chromosome findings, cytogenetic analyses should be done on patients with suspected DiGeorge anomaly. This is particularly important since many of the abnormalities involving chromosome 22 are translocations that can be familial with a higher recurrence risk. Since only one subtle, interstitial deletion of chromosome 22 was observed, it is not clear whether high-resolution cytogenetic analysis is cost beneficial for all such patients.     

Genomic findings in patients with clinical suspicion of 22q11.2 deletion syndrome

Chromosome 22q11.2 deletion syndrome, one of the most common human genomic syndromes, has highly heterogeneous clinical presentation. Patients usually harbor a 1.5 to 3 Mb hemizygous deletion at chromosome 22q11.2, resulting in pathognomic TBX1, CRKL and/or MAPK1 haploinsufficiency. However, there are some individuals with clinical features resembling the syndrome who are eventually diagnosed with genomic disorders affecting other chromosomal regions. The objective of this study was to evaluate the additive value of high-resolution array-CGH testing in the cohort of 41 patients with clinical features of 22q11.2 deletion syndrome and negative results of standard cytogenetic diagnostic testing (karyotype and FISH for 22q11.2 locus). Array-CGH analysis revealed no aberrations at chromosomes 22 or 10 allegedly related to the syndrome. Five (12.2 %) patients were found to have other genomic imbalances, namely 17q21.31 microdeletion syndrome (MIM#610443), 1p36 deletion syndrome (MIM#607872), NF1 microduplication syndrome (MIM#613675), chromosome 6pter-p24 deletion syndrome (MIM#612582) and a novel interstitial deletion at 3q26.31 of 0.65 Mb encompassing a dosage-dependent gene NAALADL2. Our study demonstrates that the implementation of array-CGH into the panel of classic diagnostic procedures adds significantly to their efficacy. It allows for detection of constitutional genomic imbalances in 12 % of subjects with negative result of karyotype and FISH targeted for 22q11.2 region. Moreover, if used as first-tier genetic test, the method would provide immediate diagnosis in ～40 % phenotypic 22q11.2 deletion subjects.     

Computational analysis and refinement of sequence structure on chromosome 22q11.2 region: application to the development of quantitative real-time PCR assay for clinical diagnosis

The low-copy repeat (LCR) is a new class of repetitive DNA element and has been implicated in many human disorders, including DiGeorge/velocardiofacial syndrome (DGS/VCFS). It is now recognized that nonallelic homologous recombination (NAHR) through LCRs flanking the chromosome 22q11.2 region leads to genome rearrangements and results in the DGS/VCFS. To refine the structure and content of chromosome 22q11.2 LCRs, we applied computational analysis to dissect region-specific LCRs using publicly available sequences. Nine distinct duplicons between 1.6 and 65 kb long and sharing >95% sequence identity were identified. The presence of these sequence motifs supports the NAHR mechanism. Further sequence analysis suggested that the previously defined 3-Mb deletion may actually comprise two deletion intervals of similar size close to each other and thus indistinguishable when using fluorescence in situ hybridization (FISH) analysis. The differentially deleted regions contain several hypothetical proteins and UniGene clusters and may partially explain the clinical heterogeneity observed in DGS/VCFS patients with the 3-Mb common deletion. To implement further sequence information in molecular medicine, we designed a real-time quantitative PCR assay and validated the method in 122 patients with suspected DGS/VCFS. The assay detected 28 patients with chromosome 22q11.2 deletion later confirmed using FISH. Our results indicated that the developed assay is reliable as well as time and cost effective for clinical diagnosis of chromosome 22q11.2 deletion. They also suggest that this methodology can be applied to develop a molecular approach for clinical detection and diagnosis of other genomic disorders.     

Screening of patients at risk for 22q11 deletion

The aim of this study was to determine whether deletion 22q11.2 studies should become apart of a standardized diagnostic workup for selected groups of at risk patients. We prospectively investigated four cohorts of unselected patients referred because of 1) congenital heart defect (CHD), 2) palatal anomalies, 3) hypocalcaemia, 4) dysmorphic features suggestive of del 22q11.2. Fluorescence in situ hybridization analysis revealed deletion 22q11.2 in 9.4% (6/64) patients with CHD. From 18 patients referred because of the hypocalcaemia, six (33.3%) had 22q11.2 deletion. In the group of 31 children with dysmorphic traits, the diagnosis was confirmed in two (6.4%) patients. None of the 58 children with palatal anomalies showed evidence of 22q11.2 deletion. Conclusions: Testing for the 22q11.2 microdeletion can be recommended in all patients with conotruncal heart defects and in patients with hypocalcaemia. It should be also considered in patients presenting only with dysmorphic traits suggestive of del 22q11.2, while screening in patients with cleft palate is not warranted.     

22q11.2 distal deletion: a recurrent genomic disorder distinct from DiGeorge syndrome and velocardiofacial syndrome

Microdeletions within chromosome 22q11.2 cause a variable phenotype, including DiGeorge syndrome (DGS) and velocardiofacial syndrome (VCFS). About 97% of patients with DGS/VCFS have either a common recurrent ~3 Mb deletion or a smaller, less common, ~1.5 Mb nested deletion. Both deletions apparently occur as a result of homologous recombination between nonallelic flanking low-copy repeat (LCR) sequences located in 22q11.2. Interestingly, although eight different LCRs are located in proximal 22q, only a few cases of atypical deletions utilizing alternative LCRs have been described. Using array-based comparative genomic hybridization (CGH) analysis, we have detected six unrelated cases of deletions that are within 22q11.2 and are located distal to the ~3 Mb common deletion region. Further analyses revealed that the rearrangements had clustered breakpoints and either a ~1.4 Mb or ~2.1 Mb recurrent deletion flanked proximally by LCR22-4 and distally by either LCR22-5 or LCR22-6, respectively. Parental fluorescence in situ hybridization (FISH) analyses revealed that none of the available parents (11 out of 12 were available) had the deletion, indicating de novo events. All patients presented with characteristic facial dysmorphic features. A history of prematurity, prenatal and postnatal growth delay, developmental delay, and mild skeletal abnormalities was prevalent among the patients. Two patients were found to have a cardiovascular malformation, one had truncus arteriosus, and another had a bicuspid aortic valve. A single patient had a cleft palate. We conclude that distal deletions of chromosome 22q11.2 between LCR22-4 and LCR22-6, although they share some characteristic features with DGS/VCFS, represent a novel genomic disorder distinct genomically and clinically from the well-known DGS/VCF deletion syndromes.     

The Philadelphia story: the 22q11.2 deletion: report on 250 patients

A submicroscopic deletion of chromosome 22q11.2 has been identified in the majority of patients with the DiGeorge, velocardiofacial, and conotruncal anomaly face syndromes, and in some patients with the Opitz G/BBB and Cayler cardiofacial syndromes. We have been involved in the analysis of DiGeorge syndrome and related diagnoses since 1982 and have evaluated a large number of patients with the deletion. We describe our cohort of 250 patients whose clinical findings help to define the extremely variable phenotype associated with the 22q11.2 deletion and may assist clinicians in providing genetic counseling and guidelines for clinical management based on these findings.     

22q11.2 Microduplication in a patient with 19p13.12–13.13 deletion

Background

The chromosome 22q11.2 region microduplication has been described in patients with variable phenotypes. Here we present a 3-month-old girl with both 22q11.2 microduplication and 19p13.12–13.13 deletion. The presence of both genomic imbalances in one patient has not been previously reported in literature.

Methods

A routine G-banding karyotype analysis was performed using peripheral lymphocytes. Chromosome microarray analysis (CMA) was done using Affymetrix CytoScan™ HD array.

Results

The result of karyotyping showed that the patient is 46,XX,t(12;19)(q24.3;p13.1), but CMA detected a 2.8 Mb microduplication within the region 22q11.2 (chr22: 18,648,866–21,465,659) and a 1.2 Mb deletion on the chromosome 19at band p13.12–p13.13 (chr19: 13,107,938–14,337,347) in her genome, while no abnormalities were identified on 12q24.3. The 3-month-old girl presented with microcephaly, cleft palate, low set and retroverted ears, and facial dysmorphism which consisted of the following: a long narrow face, widely spaced eyes, downslanting palpebral fissures, broad nasal base, short philtrum, thin upper lip, and micro/retrognathia. She also had a congenital right pulmonary artery sling and tracheal stenosis and suffered from significant hypotonia and partial bilateral mixed hearing loss.

Conclusions

We report a case of 22q11.2 duplication syndrome with 19p13.12–13.13 deletion. Synergistic effect from the two genomic imbalances is likely responsible for the complicated clinical features observed in this patient.     

Transgenic Expression of MicroRNA-185 Causes a Developmental Arrest of T Cells by Targeting Multiple Genes Including Mzb1

miR-185 is a microRNA (miR) that targets Bruton''s tyrosine kinase in B cells, with reductions in miR-185 linked to B cell autoantibody production. In hippocampal neurons, miR-185 targets both sarcoplasmic/endoplasmic reticulum calcium ATPase 2 and a novel Golgi inhibitor. This miR is haploinsufficient in 90–95% of individuals with chromosome 22q11.2 deletion syndrome, patients who can present with immune, cardiac, and parathyroid problems, learning disorders, and a high incidence of schizophrenia in adults. The reduced levels of miR-185 in neurons cause presynaptic neurotransmitter release. Many of the 22q11.2 deletion syndrome patients have a thymic hypoplasia, which results in a peripheral T cell lymphopenia and unusual T helper cell skewing. The molecular targets of miR-185 in thymocytes are unknown. Using an miR-185 T cell transgenic approach, increasing levels of miR-185 attenuated T cell development at the T cell receptor β (TCRβ) selection checkpoint and during positive selection. This caused a peripheral T cell lymphopenia. Mzb1, Nfatc3, and Camk4 were identified as novel miR-185 targets. Elevations in miR-185 enhanced TCR-dependent intracellular calcium levels, whereas a knockdown of miR-185 diminished these calcium responses. These effects concur with reductions in Mzb1, an endoplasmic reticulum calcium regulator. Consistent with their haploinsufficiency of miR-185, Mzb1 levels were elevated in thymocyte extracts from several 22q11.2 deletion syndrome patients. Our findings indicate that miR-185 regulates T cell development through its targeting of several mRNAs including Mzb1.     

Replication asynchrony between homologs 15q11.2: Cytogenetic evidence for genomic imprinting

Summary Replication kinetics of the Prader-Willi syndrome critical region (15q11.2) was investigated in seven normal healthy adult females using RBG replication bands. Replication asynchrony between homologs 15q11.2 was identified consistently in about 40% of cells in all individuals. It was limited to the stages in which Xp22, Xp11, Xq13 and Xq24/26 were visible in the late-replicating X chromosome. This asynchrony suggested that replication timing overlapped between 15q11.2 and the early replicating R-bands of the late X chromosome in some cells, and that the difference in replication timing between homologs was probably related to genomic imprinting; the latter has been suggested as a pathogenetic basis of Prader-Willi syndrome. As a result of an analysis of the proportions of asynchronous and synchronous cells in each replication stage, two types of cells were deduced providing 11 methylation mosaicism of genomic imprinting was assumed. The first type was composed of cells with normal replication in one homolog and delayed replication in the other. The second type was composed of cells with normal replication in both homologs. Our results provide cytogenetic evidence of methylation mosaicism for mammalian genomic imprinting.     

Mosaic and non-mosaic trisomy 15q2

Two unrelated patients are presented. In the first mosaicism with normal cells and cells trisomic for the distal long arm (q2) of chromosome 15 was found. The 15q2 trisomy was due to a chromosome 14, to the long arm of which an extra 15q2 region was attached (14pter----14q32::15q22----15qter). In the second trisomy 15q2 was present as a consequence of a balanced t(7;15)(p22;q15) translocation in the mother.     

22q11 deletion in DGS/VCFS monozygotic twins with discordant phenotypes

22q11.2 deletion is a common genetic disorder characterised by a wide spectrum of clinical manifestations. To date no simple genotype-phenotype correlation has been established. Moreover, several reports have mentioned phenotypic discordance between monozygotic twins. No definite mechanism has been demonstrated and mosaicism, a postzygotic second hit, environmental effects and chance events have been proposed. The twinning process itself has been suspected in two cases (11, 23). We report the case of monozygous twins with a 22q11.2 deletion who are discordant for a heart defect. We found no arguments for mosaicism or twin-to-twin transfusion syndrome. The frequent discordance for heart defects in DiGeorge/Velo-cardio-facial syndromes (DGS/VCFS) does not favour the hypothesis of somatic mutations contributing to the phenotypic variation, but rather a complex interaction between genetic and environmental systems.     

Unusual chromosomal mosaicism as a cause of mental retardation and congenital malformations in a familial reciprocal translocation carrier, t(17;22)(q24.2;q11.23)

Familial reciprocal translocations are generally without phenotypic effect, although there is some evidence for a small excess of mental retardation and congenital malformations (MR/CM) in children carrying familial reciprocal translocations. Possible mechanisms whereby such translocations could have a phenotypic effect include cryptic unbalanced rearrangements, uniparental disomy, and disruption of putative genes at the breakpoints, unmasking recessive alleles on the normal homologs. Mosaicism for a supernumerary derivative chromosome in a carrier of a familial reciprocal translocation has not yet been described. We report a boy presenting with MR/CM and a familial reciprocal translocation, t(17;22)(q24.2;q11.23), inherited from the mother. Cytogenetic analysis of peripheral blood lymphocytes showed a balanced karyotype in all 32 analyzed metaphase spreads. Molecular genetic analysis was consistent with biparental origin of the normal homologs. In metaphase spreads from skin fibroblasts a supernumerary chromosome was found in all 24 cells analyzed and could be identified as der(22)t(17;22)(q24.2;q11.23). Several possible segregation modes at meiosis I followed by meiosis II or postzygotic nondisjunction of the der(22) might have led to this unusual chromosomal mosaicism. We propose hidden mosaicism as a possible cause for MR/CM in patients who apparently carry a balanced familial reciprocal translocation.