Novel 9q34.11 gene deletions encompassing combinations of four Mendelian disease genes: STXBP1, SPTAN1, ENG, and TOR1A.

PURPOSE: A number of genes in the 9q34.11 region may be haploinsufficient. However, studies analyzing genotype-phenotype correlations of deletions encompassing multiple dosage-sensitive genes in the region are lacking. METHODS: We mapped breakpoints of 10 patients with 9q34.11 deletions using high-resolution 9q34-specific array comparative genomic hybridization (CGH) to determine deletion size and gene content. RESULTS: The 9q34.11 deletions range in size from 67 kb to 2.8 Mb. Six patients exhibit intellectual disability and share a common deleted region including STXBP1; four manifest variable epilepsy. In five subjects, deletions include SPTAN1, previously associated with early infantile epileptic encephalopathy, infantile spasms, intellectual disability, and hypomyelination. In four patients, the deletion includes endoglin (ENG), causative of hereditary hemorrhagic telangiectasia. Finally, in four patients, deletions involve TOR1A, of which molecular defects lead to early-onset primary dystonia. Ninety-four other RefSeq genes also map to the genomic intervals investigated. CONCLUSION: STXBP1 haploinsufficiency results in progressive encephalopathy characterized by intellectual disability and may be accompanied by epilepsy, movement disorders, and autism. We propose that 9q34.11 genomic deletions involving ENG, TOR1A, STXBP1, and SPTAN1 are responsible for multisystemic vascular dysplasia, early-onset primary dystonia, epilepsy, and intellectual disability, therefore revealing cis-genetic effects leading to complex phenotypes.

Novel mutation and three other sequence variants segregating with phenotype at keratoconus 13q32 susceptibility locus.

Keratoconus (KTCN), a non-inflammatory corneal disorder characterized by stromal thinning, represents a major cause of corneal transplantations. Genetic and environmental factors have a role in the etiology of this complex disease. Previously reported linkage analysis revealed that chromosomal region 13q32 is likely to contain causative gene(s) for familial KTCN. Consequently, we have chosen eight positional candidate genes in this region: MBNL1, IPO5, FARP1, RNF113B, STK24, DOCK9, ZIC5 and ZIC2, and sequenced all of them in 51 individuals from Ecuadorian KTCN families and 105 matching controls. The mutation screening identified one mutation and three sequence variants showing 100% segregation under a dominant model with KTCN phenotype in one large Ecuadorian family. These substitutions were found in three different genes: c.2262A>C (p.Gln754His) and c.720+43A>G in DOCK9; c.2377-132A>C in IPO5 and c.1053+29G>C in STK24. PolyPhen analyses predicted that c.2262A>C (Gln754His) is possibly damaging for the protein function and structure. Our results suggest that c.2262A>C (p.Gln754His) mutation in DOCK9 may contribute to the KTCN phenotype in the large KTCN-014 family.

Multiallelic synthetic quality control material: lessons learned from the cystic fibrosis external quality assessment scheme.

AIM: With the arrival of increasingly complex molecular tests, we are obliged to create new ways to monitor and troubleshoot the underperformance of these multiplex assays. A synthetic multiallelic quality control material has been designed to augment genomic DNA controls. We aimed to evaluate the control on a large scale, testing it on a wide variety of oligonucleotide ligation assays, test protocols, and analysis software. In addition, we investigated how laboratories treat untried and complex materials. METHODS: The synthetic control monitored 32 cystic fibrosis transmembrane conductance regulator mutations and polymorphisms simultaneously. Participants of a cystic fibrosis external quality assessment scheme were invited to analyze the quality control. RESULTS: In total, 58 laboratories participated in this study. Twenty-seven (47%) laboratories detected 32 variants; another 27 laboratories (47%) detected from 31 to 4 variants and 4 participants reported no variants (6%). The main observations included administrative errors when indicating variants on a checklist, errors caused by misreading the instructions for use of the control or assay, and technical problems related to the assay used. CONCLUSION: Synthetic quality control materials proved to be valuable in troubleshooting underperforming assays and complement existing genomic controls. The study also revealed a strong need for increased quality control in the postanalytical phase of testing.

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.

Clinical characterization of individuals with deletions of genes in holoprosencephaly pathways by aCGH refines the phenotypic spectrum of HPE.

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.

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.

Impact of genotype-first diagnosis: the detection of microdeletion and microduplication syndromes with cancer predisposition by aCGH.

BACKGROUND: The use of microarray-based comparative genomic hybridization has allowed the genetic diagnosis of some conditions before their full clinical presentation. This "genotype-first" diagnosis has the most clinical implications for genomic alterations that confer an elevated risk of cancer. In these cases, diagnosis before the manifestation of the patient's full phenotype dramatically impacts genetic counseling, clinical management, and eventual prognosis and survivability. METHODS: Using microarray-based comparative genomic hybridization, we tested 18,437 individuals with indications such as developmental disabilities and congenital anomalies. RESULTS: We identified 34 (0.18%) individuals with DNA copy number gains or losses that encompassed gene regions associated with recognized genetic conditions with an increased risk for cancer. Three of the 34 individuals (8.8%) had a previously abnormal cytogenetic study which microarray-based comparative genomic hybridization confirmed and/or further characterized. Seven of the 34 individuals (20.6%) either had the correct disease specified in the clinical indication for study or had clinical features highly indicative of that syndrome. The remaining 24 patients (70.6%) had indications for study that were not specific to the diagnosed syndrome, such as "developmental delay" or "dysmorphic features." CONCLUSIONS: The ability of microarray-based comparative genomic hybridization to rapidly and objectively interrogate the genome for chromosomal imbalances has led to the opportunity to optimize medical management and outcome. This has an even more profound impact and clinical utility in conditions associated with cancer predisposition syndromes.

aCGH detects partial tetrasomy of 12p in blood from Pallister-Killian syndrome cases without invasive skin biopsy.

Pallister-Killian syndrome (PKS) is a genetic disorder characterized by mental retardation, seizures, streaks of hypo- or hyperpigmentation and dysmorphic features. PKS is associated with tissue-limited mosaic partial tetrasomy of 12p, usually caused by an isochromosome 12p. The mosaicism is usually detected in cultured skin fibroblasts or amniotic cells and rarely in phytohemagluttinin-stimulated lymphocytes, which suggests stimulation of T-lymphocytes may distort the percentage of abnormal cells. We recently reported on the identification by microarray-based comparative genomic hybridization (aCGH) of a previously unsuspected case of partial tetrasomy of 12p caused by an isochromosome 12p. Here we report on seven additional individuals with partial tetrasomy of 12p characterized by our laboratory. All individuals were referred for mental retardation/developmental delay and/or dysmorphic features. In each case, aCGH using genomic DNA extracted from whole peripheral blood detected copy-number gain for all clones for the short arm of chromosome 12. In all but one case, FISH on metaphases from cultured lymphocytes did not detect the copy-number gain; in the remaining case, metaphase FISH on cultured lymphocytes showed an isochromosome in 10% of cells. However, interphase FISH using probes to 12p on peripheral blood smears showed additional hybridization signals in 18-70% of cells. Microarray and FISH analysis on cultured skin biopsies from four individuals confirmed the presence of an isochromosome 12p. Our results demonstrate the usefulness of aCGH with genomic DNA from whole peripheral blood to detect chromosome abnormalities that are not present in stimulated blood cultures and would otherwise require invasive skin biopsies for identification.

Human anterior chamber angle development without cell death or macrophage involvement.

PURPOSE: The iridocorneal angle in the mammalian eye including the trabecular meshwork (TM) develops from undifferentiated mesenchyme/neural crest between the iris root and cornea. The precise mechanisms underlying anterior angle development are unclear, and the contribution of cell death and phagocytic resorption by macrophages in angle development is controversial. In this study, we examined the human anterior chamber angle during various stages of development for evidence of cell death and phagocytic resorption. METHODS: Eyes from the human fetus (F) of 7, 8, 9, 10, 11, 13, 15, 18, 19, 21, 22, 23, and 27 weeks as well as eyes from 5- and 11-month-old children and donors 24, 48, and 67 years of age were obtained. Formalin-fixed and paraffin-embedded sections were examined by hematoxylin and eosin (H&E) staining. Immunohistochemistry was performed using polyclonal antibodies against CD68. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) labeling was also performed to evaluate cell death. RESULTS: By light microscopy, the development of human angle structures appeared to progress as previously described. Histological evidence of cellular death or resorption by macrophages was not observed. Furthermore, the chamber angle tissues did not stain with CD68 at any stage of development. Few CD68 positive cells were observed in the iris stroma and the anterior ciliary body between fetal weeks 10 and 18 (F10w and F18w). TUNEL labeled nuclei were not detected in the anterior chamber angle in any fetal or infant eyes. By contrast, TUNEL positive nuclei in TM cells were observed in the examined adult donor specimens. CONCLUSIONS: The results suggest that at the time points examined, neither cell death nor phagocytic resorption with macrophages appear to play a role in the development of the human anterior chamber angle.

Clinical utility of contemporary molecular cytogenetics.

The development of microarray-based comparative genomic hybridization (array CGH) methods represents a critical new advance in molecular cytogenetics. This new technology has driven a technical convergence between molecular diagnostics and clinical cytogenetics, questioned our naïve understanding of the complexity of the human genome, revolutionized the practice of medical genetics, challenged conventional wisdom related to the genetic bases of multifactorial and sporadic conditions, and is poised to impact all areas of medicine. The use of contemporary molecular cytogenetic techniques in research and diagnostics has resulted in the identification of many new syndromes, expanded our knowledge about the phenotypic spectrum of recognizable syndromes, elucidated the genomic bases of well-established clinical conditions, and refined our view about the molecular mechanisms of some chromosomal aberrations. Newer methodologies are being developed, which will likely lead to a new understanding of the genome and its relationship to health and disease.

Williams syndrome in a preterm infant with phenotype of Alagille syndrome.

We report on a preterm infant born at 31 weeks of gestation with a phenotype suggestive of Alagille syndrome, yet microarray analysis identified a deletion on 7q11.23 at the Williams syndrome locus. The infant died on day 18 of life with overwhelming sepsis. This case illustrates the importance of microarray analysis in diagnosing genetic conditions, especially in preterm babies whose facial and other clinical manifestations have not fully developed.

Design, development, validation, and use of synthetic nucleic acid controls for diagnostic purposes and application to cystic fibrosis testing.

We have designed, tested, and validated synthetic DNA molecules that may be used as reference standard controls in the simultaneous detection of mutations in one or more genes. These controls consist of a mixture of oligonucleotides (100 to 120 bases long) each designed for the detection of one or more disease-causing mutation(s), depending on the proximity of the mutations to one another. Each control molecule is identical to 80 to 100 bases that span the targeted mutations. In addition, each oligonucleotide is tagged at the 5' and 3' ends with distinct nucleic acid sequences that allow for the design of complementary primers for polymerase chain reaction amplification. We designed the tags to amplify control molecules comprising 32 CFTR mutations, including the American College of Medical Genetics minimum carrier screening panel of 23, with one pair of primers in a single tube. We tested the performance of these controls on many platforms including the Applied Biosystems/Celera oligonucleotide ligation assay and the Tm Bioscience Tag-It platforms. All 32 mutations were detected consistently. This simple methodology allows for maximum flexibility and rapid implementation. It has not escaped our notice that the design of these molecules makes possible the production of similar controls for virtually any mutation or sequence of interest.

Comparative genomic hybridization by microarray for the detection of cytogenetic imbalance.

Chromosomal abnormalities often result in the improper dosage of genes in a particular chromosome or chromosome segment, which may cause specific and complex clinical phenotypes. Comparative genomic hybridization by microarray (array CGH) is a high-throughput and high-resolution method for the detection of microscopic and submicroscopic chromosome abnormalities, some of which may not be detectable by conventional cytogenetic techniques. In addition, with the human genome sequenced and publicly available, array CGH allows for the direct correlation between chromosomal anomalies and genomic sequence. Properly constructed, microarrays have the potential to be a valuable tool for the detection of chromosomal abnormalities in cancer and genetic disease.

Application of array-based comparative genomic hybridization to clinical diagnostics.

Microarray-based comparative genomic hybridization (array CGH) is a revolutionary platform that was recently adopted in the clinical laboratory. This technology was first developed as a research tool for the investigation of genomic alterations in cancer. It allows for a high-resolution evaluation of DNA copy number alterations associated with chromosome abnormalities. Array CGH is based on the use of differentially labeled test and reference genomic DNA samples that are simultaneously hybridized to DNA targets arrayed on a glass slide or other solid platform. In this review, we examine the technology and its transformation from a research tool into a maturing diagnostic instrument. We also evaluate the various approaches that have shaped the current platforms that are used for clinical applications. Finally, we discuss the advantages and shortcomings of "whole-genome" arrays and compare their diagnostic use to "targeted" arrays. Depending on their design, microarrays provide distinct advantages over conventional cytogenetic analysis because they have the potential to detect the majority of microscopic and submicroscopic chromosomal abnormalities. This new platform is poised to revolutionize modern cytogenetic diagnostics and to provide clinicians with a powerful tool to use in their increasingly sophisticated diagnostic capabilities.

Of mice and men: tyrosinase modification of congenital glaucoma in mice but not in humans.

PURPOSE: Primary congenital glaucoma (PCG) is an autosomal recessive ocular trait caused by mutations in the gene for cytochrome P4501B1 (CYP1B1). Although PCG is often considered to be fully penetrant, the disease shows 50% penetrance in some Saudi Arabian families. The familial segregation of the nonpenetrance suggests a genetic modifier. Recently, tyrosinase (Tyr) deficiency was found to worsen the drainage structure/ocular dysgenesis phenotype of Cyp1b1-/- mice, suggesting that Tyr is a modifier of the phenotype. In the current study, tyrosinase (TYR) was investigated in human PCG. METHODS: A genome-wide screen, a single nucleotide polymorphism (SNP) analysis in the TYR chromosomal region 11q13-q21, and sequencing of the TYR gene was performed with individuals from Saudi Arabian families with multiple, clinically confirmed, molecularly proven, nonpenetrant members. RESULTS: The study outcome did not support TYR as a modifier of the PCG phenotype in this population. The sequencing data showed no TYR mutations in the nonpenetrant family members and no difference in polymorphism frequencies between nonpenetrant or fully penetrant families. CONCLUSIONS: TYR is not a modifier of the CYP1B1-associated PCG phenotype in the Saudi Arabian population.

Detecting sex chromosome anomalies and common triploidies in products of conception by array-based comparative genomic hybridization.

OBJECTIVES: In recent years, array-based comparative genomic hybridization (array CGH) has moved to the forefront of molecular cytogenetics with its ability to rapidly characterize chromosome abnormalities at resolutions much higher than routine chromosome banding. However, array CGH, like all CGH procedures, has heretofore been deemed unable to detect ploidy, a major cause of fetal demise and spontaneous miscarriage. METHOD: We recently developed a CGH microarray that is designed for detecting aneuploidy and unbalanced chromosome rearrangements. Here, we introduce the use of a Klinefelter male cell line (47,XXY) as a control for array CGH analyses on products of conception (POCs). RESULTS: This approach facilitates the detection of common trisomies and monosomies of the sex chromosomes by reducing the analysis to the identification of single copy gains or losses. Furthermore, in a blinded study, careful interpretation of the microarray results with particular attention to the sex chromosome ratios between the patient sample and the control allowed for the detection of some common triploidies. CONCLUSION: These results suggest that using a chromosomally abnormal cell line in array CGH analysis can be applied to other CGH platforms and that array CGH, when properly performed and analyzed, is a powerful tool that can detect most chromosomal abnormalities observed in a clinical setting including some polyploidies.

Immunolocalization of CYP1B1 in normal, human, fetal and adult eyes.

CYP1B1 is a cytochrome P450 enzyme implicated in autosomal recessive primary congenital glaucoma (PCG). The mechanism and function of CYP1B1 in the development of the PCG phenotype is unknown. Previously, investigators have reported detection of Cyp1b1 mRNA in the ciliary body and epithelium and neuroepithelium in the developing mouse eye, employing in situ hybridization techniques. Similarly, additional investigators have detected CYP1B1 mRNA in the iris, ciliary body, non-pigmented ciliary epithelial line, cornea, retinal-pigment epithelium, and retina in the human adult eye, using Northern blotting. This study was designed to immunolocalize CYP1B1 protein in the various ocular structures of normal, human fetal and adult eyes. Normal fetal and adult eyes were immunolabeled with a polyclonal antibody against human CYP1B1 using indirect immunofluorescence, and then compared with appropriate controls. The intensity of immunolabeling of the various ocular structures was assessed by qualitative and semi-quantitative techniques. In the anterior segment anti-CYP1B1 immunoreactivity (IR) was detected early in fetal development in the primitive ciliary epithelium. As well, the most intense CYP1B1 IR was in the non-pigmented ciliary epithelium. In addition, CYP1B1 IR was also present in the corneal epithelium and keratocytes, both layers of the iris pigmented epithelium, and retina. However, CYP1B1 IR was absent in the trabecular meshwork in all of the samples. In general, CYP1B1 immunolabeling in the human fetal eyes was more intense when compared to adult eyes. CYP1B1 IR was primarily immunolocalized to the non-pigmented ciliary epithelium and early in fetal development. In addition, CYP1B1 IR was not detected in the trabecular meshwork. These findings suggest that the abnormalities in the development of the trabecular meshwork in PCG may result from diminished or absent metabolism of important endogenous substrates in the ciliary epithelium due to non-functional CYP1B1 enzyme.

Array-based comparative genomic hybridization in clinical diagnosis.

The sequencing of the human genome and development of high-throughput microarray technologies have enhanced the detection of copy number alterations in cancer research and the study of constitutional chromosomal abnormalities. Microarray-based comparative genomic hybridization (array CGH) has integrated molecular and traditional cytogenetics and has begun to impact the clinician's approach to medical genetics. Clinical applications of array CGH may define new genetic syndromes, expand the phenotype of existing syndromes and characterize a genomic signature of some cancers. As array CGH becomes the initial diagnostic approach for the investigation of constitutional and acquired chromosomal abnormalities, the combination of bioinformatics, robotics and microarray technology will set the stage for a new generation of high-resolution and high-throughput tools for genetic analysis, diagnosis and gene discovery.

Expanding the phenotype of alveolar capillary dysplasia (ACD).

OBJECTIVES: To define the phenotype of congenital alveolar capillary dysplasia (ACD) as a first step toward mapping the responsible gene(s). STUDY DESIGN: Analysis of pathology reports and microscopic slides of 23 subjects with ACD and sequence analysis of two candidate genes. RESULTS: Our review of the pre- and postmortem records delineates both the natural history of this condition and the associated anomalies. Our collection of families corroborates the likely autosomal recessive nature of this condition in some families and provides additional data for genetic and prenatal counseling. Anomalies of many organ systems were detected either in the prenatal period or during the hospital course. However, some major anomalies were not detected until postmortem examination. Left-right asymmetry and gastrointestinal malrotation emerge as important, previously recognized but underappreciated phenotypic features of ACD. Finally, we used sequence analysis to exclude mutations in the coding region of two candidate genes, bone morphogenetic protein type II receptor (BMPR2) and endothelial monocyte-activating polypeptide II (EMAP II), as candidates for ACD. CONCLUSIONS: Understanding the clinical spectrum of ACD and the cloning of an "ACD gene" both have implications for counseling, for prenatal testing, and for understanding the molecular pathophysiology of ACD and other organ malformations that are associated with this condition.