Bassem A. Bejjani

Genomic and Genic Deletions of the FOX Gene Cluster on 16q24.1 and Inactivating Mutations of FOXF1 Cause Alveolar Capillary Dysplasia and Other Malformations

Alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV) is a rare, neonatally lethal developmental disorder of the lung with defining histologic abnormalities typically associated with multiple congenital anomalies (MCA). Using array CGH analysis, we have identified six overlapping microdeletions encompassing the FOX transcription factor gene cluster in chromosome 16q24.1q24.2 in patients with ACD/MPV and MCA. Subsequently, we have identified four different heterozygous mutations (frameshift, nonsense, and no-stop) in the candidate FOXF1 gene in unrelated patients with sporadic ACD/MPV and MCA. Custom-designed, high-resolution microarray analysis of additional ACD/MPV samples revealed one microdeletion harboring FOXF1 and two distinct microdeletions upstream of FOXF1, implicating a position effect. DNA sequence analysis revealed that in six of nine deletions, both breakpoints occurred in the portions of Alu elements showing eight to 43 base pairs of perfect microhomology, suggesting replication error Microhomology-Mediated Break-Induced Replication (MMBIR)/Fork Stalling and Template Switching (FoSTeS) as a mechanism of their formation. In contrast to the association of point mutations in FOXF1 with bowel malrotation, microdeletions of FOXF1 were associated with hypoplastic left heart syndrome and gastrointestinal atresias, probably due to haploinsufficiency for the neighboring FOXC2 and FOXL1 genes. These differences reveal the phenotypic consequences of gene alterations in cis.

Mutations in CYP1B1, the Gene for Cytochrome P4501B1, Are the Predominant Cause of Primary Congenital Glaucoma in Saudi Arabia

The autosomal recessive disorder primary congenital glaucoma (PCG) is caused by unknown developmental defect(s) of the trabecular meshwork and anterior chamber angle of the eye. Homozygosity mapping with a DNA pooling strategy in three large consanguineous Saudi PCG families identified the GLC3A locus on chromosome 2p21 in a region tightly linked to PCG in another population. Formal linkage analysis in 25 Saudi PCG families confirmed both significant linkage to polymorphic markers in this region and incomplete penetrance, but it showed no evidence of genetic heterogeneity. For these 25 families, the maximum combined two-point LOD score was 15.76 at a recombination fraction of .021, with the polymorphic marker D2S177. Both haplotype analysis and homozygosity mapping in these families localized GLC3A to a 5-cM critical interval delineated by markers D2S2186 and D2S1356. Sequence analysis of the coding exons for cytochrome P4501B1 (CYP1B1) in these 25 families revealed three distinctive mutations that segregate with the phenotype in 24 families. Additional clinical and molecular data on some mildly affected relatives showed variable expressivity of PCG in this population. These results should stimulate a study of the genetic and environmental events that modify the effects of CYP1B1 mutations in ocular development. Furthermore, the small number of PCG mutations identified in this Saudi population makes both neonatal and population screening attractive public health measures.

Detection of low-level mosaicism by array CGH in routine diagnostic specimens.

The advent of microarray‐based comparative genomic hybridization (array CGH) promises to revolutionize clinical cytogenetics because of its ability to rapidly screen the genome at an unprecedented resolution. Yet, the ability of array CGH to detect and evaluate low‐level mosaicism is not known. Our laboratory has analyzed over 3,600 clinical cases with the SignatureChip® which we developed for the detection of microdeletions, microduplications, aneuploidy, unbalanced translocations, and subtelomeric and pericentromeric copy number alterations. Here, we report 18 cases of mosaicism detected by array CGH in a routine diagnostic setting, 14 of which were not known to us at the time of the analysis. These 14 cases represent ∼8% of all abnormal cases identified in our laboratory. For each case, fluorescence in situ hybridization (FISH) analysis was performed on PHA‐stimulated cultures after mosaic chromosome abnormalities were suspected by array CGH. In all cases, FISH confirmed the mosaic chromosome abnormalities which included a variety of marker chromosomes, autosomal trisomies, terminal and interstitial deletions, and derivative chromosomes. Interestingly, confirmatory FISH analyses on direct blood smears indicated that the percentage of abnormal cells in unstimulated cultures was in some cases different than that found in PHA‐stimulated cells. We also report the detection of a previously unsuspected case of an isochromosome 12p (associated with Pallister–Killian syndrome) by array CGH using genomic DNA extracted from peripheral blood. These results support a growing body of data that suggests that stimulated peripheral blood cultures likely distort the percentage of abnormal cells and may, for some chromosome abnormalities, make their detection unlikely by conventional analysis. Thus, array CGH, which is based on genomic DNA extracted directly from uncultured peripheral blood, may be more likely to detect low‐level mosaicism for unbalanced chromosome abnormalities than traditional cytogenetic techniques.

Paternally inherited microdeletion at 15q11.2 confirms a significant role for the SNORD116 C/D box snoRNA cluster in Prader–Willi syndrome

Prader–Willi syndrome (PWS) is a neurobehavioral disorder manifested by infantile hypotonia and feeding difficulties in infancy, followed by morbid obesity secondary to hyperphagia. It is caused by deficiency of paternally expressed transcript(s) within the human chromosome region 15q11.2. PWS patients harboring balanced chromosomal translocations with breakpoints within small nuclear ribonucleoprotein polypeptide N (SNRPN) have provided indirect evidence for a role for the imprinted C/D box containing small nucleolar RNA (snoRNA) genes encoded downstream of SNRPN. In addition, recently published data provide strong evidence in support of a role for the snoRNA SNORD116 cluster (HBII-85) in PWS etiology. In this study, we performed detailed phenotypic, cytogenetic, and molecular analyses including chromosome analysis, array comparative genomic hybridization (array CGH), expression studies, and single-nucleotide polymorphism (SNP) genotyping for parent-of-origin determination of the 15q11.2 microdeletion on an 11-year-old child expressing the major components of the PWS phenotype. This child had an ∼236.29 kb microdeletion at 15q11.2 within the larger Prader–Willi/Angelman syndrome critical region that included the SNORD116 cluster of snoRNAs. Analysis of SNP genotypes in proband and mother provided evidence in support of the deletion being on the paternal chromosome 15. This child also met most of the major PWS diagnostic criteria including infantile hypotonia, early-onset morbid obesity, and hypogonadism. Identification and characterization of this case provide unequivocal evidence for a critical role for the SNORD116 snoRNA molecules in PWS pathogenesis. Array CGH testing for genomic copy-number changes in cases with complex phenotypes is proving to be invaluable in detecting novel alterations and enabling better genotype–phenotype correlations.

Use of targeted array-based CGH for the clinical diagnosis of chromosomal imbalance: Is less more?

Chromosome analysis is an important component to the diagnosis of congenital anomalies, developmental delay, and mental retardation. Routine chromosome analysis identifies aneuploidy and structural rearrangements greater than 5 Mb but cannot identify abnormalities of the telomeric regions or microdeletions reliably. Molecular cytogenetic techniques were developed to overcome these limitations. High‐resolution comparative genomic hybridization (CGH)‐based microarrays (array CGH) were developed to increase the resolution of chromosomal studies and to provide a comprehensive assay by using large‐insert clones as the target for analysis. We constructed a microarray for the clinical diagnosis of medically significant and relatively common chromosomal alterations. Nine hundred six bacterial artificial chromosome (BAC) clones were chosen, the chromosomal locations of which were confirmed by fluorescence in situ hybridization (FISH). FISH‐testing showed that 7% of the clones were mismapped based on map locations obtained from two publicly available databases (58 mapped to the wrong chromosome and three mapped to a different locus on the same chromosome), 16% cross‐hybridized to other chromosomes, and 12% did not hybridize or showed poor hybridization signals under uniform FISH conditions. Thus, from a total of 906 BAC clones that were evaluated, only 589 (65%) were deemed adequate for arraying on this clinical device. The performance of this array was tested in a set of blinded experiments on a cohort of phenotypically normal individuals and on individuals with known chromosome abnormalities. The array identified deletion/duplication polymorphisms not seen by FISH in the phenotypically normal individuals and detected single copy dosage differences in all of the cases with known chromosomal abnormalities. All abnormalities detected by the array were confirmed by FISH with BACs from the appropriate loci. Our data demonstrate that the rigorous assessment of BACs and their use in array CGH is especially important when the microarray is used for clinical diagnosis. In addition, this study illustrates that when constructed carefully with proper attention to the quality of the BACs that are arrayed, array CGH is an effective and efficient tool for delineating chromosomal aberrations and an important adjunct to FISH and conventional cytogenetics.

Discovery of a previously unrecognized microdeletion syndrome of 16p11.2–p12.2

We have identified a recurrent de novo pericentromeric deletion in 16p11.2–p12.2 in four individuals with developmental disabilities by microarray-based comparative genomic hybridization analysis. The identification of common clinical features in these four individuals along with the characterization of complex segmental duplications flanking the deletion regions suggests that nonallelic homologous recombination mediated these rearrangements and that deletions in 16p11.2–p12.2 constitute a previously undescribed syndrome.

Expanding the clinical phenotype of the 3q29 microdeletion syndrome and characterization of the reciprocal microduplication

BackgroundInterstitial deletions of 3q29 have been recently described as a microdeletion syndrome mediated by nonallelic homologous recombination between low-copy repeats resulting in an ~1.6 Mb common-sized deletion. Given the molecular mechanism causing the deletion, the reciprocal duplication is anticipated to occur with equal frequency, although only one family with this duplication has been reported.ResultsIn this study we describe 14 individuals with microdeletions of 3q29, including one family with a mildly affected mother and two affected children, identified among 14,698 individuals with idiopathic mental retardation who were analyzed by array CGH. Eleven individuals had typical 1.6-Mb deletions. Three individuals had deletions that flank, span, or partially overlap the commonly deleted region. Although the clinical presentations of individuals with typical-sized deletions varied, several features were present in multiple individuals, including mental retardation and microcephaly. We also identified 19 individuals with duplications of 3q29, five of which appear to be the reciprocal duplication product of the 3q29 microdeletion and 14 of which flank, span, or partially overlap the common deletion region. The clinical features of individuals with microduplications of 3q29 also varied with few common features. De novo and inherited abnormalities were found in both the microdeletion and microduplication cohorts illustrating the need for parental samples to fully characterize these abnormalities.ConclusionOur report demonstrates that array CGH is especially suited to identify chromosome abnormalities with unclear or variable presentations.

The identification of microdeletion syndromes and other chromosome abnormalities: Cytogenetic methods of the past, new technologies for the future

Chromosome analysis is an important diagnostic tool in the identification of causes of mental retardation, developmental delay, and other developmental disabilities. Cytogenetic approaches have revealed the chromosomal basis of a large number of genetic syndromes. The recent use of microarray‐based comparative genomic hybridization (array CGH) has accelerated the identification of novel cytogenetic abnormalities. We present the results of array CGH in 8,789 clinical cases submitted for a variety of developmental problems. Of these cases, 6.9% showed clinically relevant abnormalities, 1.2% showed benign copy‐number variants (polymorphisms), 2.5% showed recurrent alterations of unclear clinical significance—many of which are likely to be polymorphisms—and 1.4% showed novel alterations of unclear relevance. Although cytogenetic methods, including array CGH, have great potential for identifying novel chromosomal syndromes, this high‐resolution analysis may also result in diagnostic challenges imposed on laboratories and clinicians regarding findings of unclear clinical significance.

A multicenter study of the frequency and distribution of GJB2 and GJB6 mutations in a large North American cohort

Purpose: The aim of the study was to determine the actual GJB2 and GJB6 mutation frequencies in North America after several years of generalized testing for autosomal recessive nonsyndromic sensorineural hearing loss to help guide diagnostic testing algorithms, especially in light of molecular diagnostic follow-up to universal newborn hearing screening.Methods: Mutation types, frequencies, ethnic distributions, and genotype-phenotype correlations for GJB2 and GJB6 were assessed in a very large North American cohort.Results: GJB2 variants were identified in 1796 (24.3%) of the 7401 individuals examined, with 399 (5.4%) homozygous and 429 (5.8%) compound heterozygous. GJB6 deletion testing was performed in 12.0% (888/7401) of all cases. The >300-kb deletion was identified in only nine individuals (1.0%), all of whom were compound heterozygous for mutations in GJB2 and GJB6. Among a total of 139 GJB2 variants identified, 53 (38.1%) were previously unreported, presumably representing novel pathogenic or benign variants.Conclusions: The frequency and distribution of sequence changes in GJB2 and GJB6 in North America differ from those previously reported, suggesting a considerable role for loci other than GJB2 and GJB6 in the etiology of autosomal recessive nonsyndromic sensorineural hearing loss, with minimal prevalence of the GJB6 deletion.Purpose: To determine short–term effects of intravitreal bevacizumab for subfoveal choroidal neovascularization (CNV) in pathologic myopia. Methods: In this prospective interventional case series, patients were treated with 2.5 mg of intravitreal bevacizumab and followed for 3 months. Best-corrected visual acuity (BCVA), optical coherence tomography (OCT), and fluorescein angiography (FA) were recorded. Indications for retreatment were active leaking CNV shown by FA and presence of subretinal fluid by OCT in combination with visual disturbances. Results: Fourteen patients were included, with a mean age of 53.86 ± 16.26 years (range 29–85). Mean spherical equivalent was −13.87 ± 3.68 diopters (−7.25 to −20.50). Minimum follow-up was 3 months. There were no adverse events. The mean initial visual acuity was 20/200 improving to 20/100 at 2 weeks, 20/80 at 4 weeks, and 20/60 at 8 and 12 weeks (P = 0.007; P = 0.001; P = 0.005; P = 0.001, respectively). Initial foveal thickness improved from 385.43 &mgr;m ± 125.83 &mgr;m to 257.64 ± 76.6 &mgr;m and 194.54 ± 54.35 &mgr;m after the first and third month, respectively (P = 0.001). Conclusions: Initial treatment results of patients with CNV due to pathologic myopia did not reveal any short-term safety concerns. Intravitreal bevacizumab resulted in a significant decrease in foveal thickness and improvement in visual acuity. These favorable initial results support further larger and long-term studies.

The discovery of microdeletion syndromes in the post-genomic era: review of the methodology and characterization of a new 1q41q42 microdeletion syndrome

Purpose: The advent of molecular cytogenetic technologies has altered the means by which new microdeletion syndromes are identified. Whereas the cytogenetic basis of microdeletion syndromes has traditionally depended on the serendipitous ascertainment of a patient with established clinical features and a chromosomal rearrangement visible by G-banding, comparative genomic hybridization using microarrays has enabled the identification of novel, recurrent imbalances in patients with mental retardation and apparently nonspecific features. Compared with the “phenotype-first” approach of traditional cytogenetics, array-based comparative genomic hybridization has enabled the detection of novel genomic disorders using a “genotype-first” approach. We report as an illustrative example the characterization of a novel microdeletion syndrome of 1q41q42.Methods: We tested more than 10,000 patients with developmental disabilities by array-based comparative genomic hybridization using our targeted microarray. High-resolution microarray analysis was performed using oligonucleotide microarrays for patients in whom deletions of 1q41q42 were identified. Fluorescence in situ hybridization was performed to confirm all 1q deletions in the patients and to exclude deletions or other chromosomal rearrangements in the parents.Results: Seven cases were found with de novo deletions of 1q41q42. The smallest region of overlap is 1.17 Mb and encompasses five genes, including DISP1, a gene involved in the sonic hedgehog signaling pathway, the deletion of which has been implicated in holoprosencephaly in mice. Although none of these patients showed frank holoprosencephaly, many had other midline defects (cleft palate, diaphragmatic hernia), seizures, and mental retardation or developmental delay. Dysmorphic features are present in all patients at varying degrees. Some patients showed more severe phenotypes and carry the clinical diagnosis of Fryns syndrome.Conclusions: This new microdeletion syndrome with its variable clinical presentation may be responsible for a proportion of Fryns syndrome patients and adds to the increasing number of new syndromes identified with array-based comparative genomic hybridization. The genotype-first approach to identifying recurrent chromosome abnormalities is contrasted with the traditional phenotype-first approach. Targeting developmental pathways in a functional approach to diagnostics may lead to the identification of additional microdeletion syndromes.