Detection of Constitutional Structural Variants by Optical Genome Mapping: A Multisite Study of Postnatal Samples.

Optical genome mapping is a high-resolution technology that can detect all types of structural variations in the genome. This second phase of a multisite study compares the performance of optical genome mapping and current standard-of-care methods for diagnostic testing of individuals with constitutional disorders, including neurodevelopmental impairments and congenital anomalies. Among the 627 analyses in phase 2, 405 were of retrospective samples supplied by five diagnostic centers in the United States and 94 were prospective samples collected over 18 months by two diagnostic centers (June 2021 to October 2022). Additional samples represented a family cohort to determine inheritance (n = 119) and controls (n = 9). Full concordance of results between optical genome mapping and one or more standard-of-care diagnostic tests was 98.6% (618/627), with partial concordance in an additional 1.1% (7/627).

Multisite Assessment of Optical Genome Mapping for Analysis of Structural Variants in Constitutional Postnatal Cases.

This study compares optical genome mapping (OGM) performed at multiple sites with current standard-of-care (SOC) methods used in clinical cytogenetics. This study included 50 negative controls and 359 samples from individuals (patients) with suspected genetic conditions referred for cytogenetic testing. OGM was performed using the Saphyr system and Bionano Access software version 1.7. Structural variants, including copy number variants, aneuploidy, and regions of homozygosity, were detected and classified according to American College of Medical Genetics and Genomics guidelines. Repeated expansions in FMR1 and contractions in facioscapulohumeral dystrophy 1 were also analyzed. OGM results were compared with SOC for technical concordance, clinical classification concordance, intrasite and intersite reproducibility, and ability to provide additional, clinically relevant information. Across five testing sites, 98.8% (404/409) of samples yielded successful OGM data for analysis and interpretation. Overall, technical concordance for OGM to detect previously reported SOC results was 99.5% (399/401). The blinded analysis and variant classification agreement between SOC and OGM was 97.6% (364/373). Replicate analysis of 130 structural variations was 100% concordant. On the basis of this demonstration of the analytic validity and clinical utility of OGM by this multisite assessment, the authors recommend this technology as an alternative to existing SOC tests for rapid detection and diagnosis in postnatal constitutional disorders.

MEIS2 (15q14) gene deletions in siblings with mild developmental phenotypes and bifid uvula: documentation of mosaicism in an unaffected parent.

MEIS2 (Meis homeobox 2) encodes a homeobox protein in the three amino acid loop extension (TALE) family of highly conserved homeodomain-containing transcription regulators important for development. MEIS2 deletions/mutations have been associated with cleft lip/palate, dysmorphic facial features, cardiac defects, as well as intellectual disability at a variable severity. Here we report on one familial case that two affected siblings carry the same non-mosaic ~ 423 kb genomic deletion at 15q14 encompassing the entirety of CDIN1 and the last three exons (ex. 10, 11, 12) of the MEIS2 gene, while their unaffected father is mosaic for the same deletion in about 10% lymphocytes. Both siblings presented with mild developmental delay and bifid uvula, while no congenital cardiac abnormalities were identified. The elder sister also showed syncopal episodes and mild speech delay and the father had atrial septal defects. This is the first report showing multiple family members inherit a genomic deletion resulting in a MEIS2 partial truncation from a mosaic parent. Taken all together, this study has important implications for genetic counseling regarding recurrence risk and also points to the importance of offering MEIS2 gene tests covering both point mutations and microdeletions to individuals with milder bifid uvula and developmental delay.

Allelic and dosage effects of NHS in X-linked cataract and Nance-Horan syndrome: a family study and literature review.

Nance-Horan syndrome (NHS) is a rare X-linked dominant disorder caused by mutation in the NHS gene on chromosome Xp22.13. (OMIM 302350). Classic NHS manifested in males is characterized by congenital cataracts, dental anomalies, dysmorphic facial features and occasionally intellectual disability. Females typically have a milder presentation. The majority of reported cases of NHS are the result of nonsense mutations and small deletions. Isolated X-linked congenital cataract is caused by non-recurrent rearrangement-associated aberrant NHS transcription. Classic NHS in females associated with gene disruption by balanced X-autosome translocation has been infrequently reported. We present a familial NHS associated with translocation t(X;19) (Xp22.13;q13.1). The proband, a 28-year-old female, presented with intellectual disability, dysmorphic features, short stature, primary amenorrhea, cleft palate, and horseshoe kidney, but no NHS phenotype. A karyotype and chromosome microarray analysis (CMA) revealed partial monosomy Xp/partial trisomy 19q with the breakpoint at Xp22.13 disrupting the NHS gene. Family history revealed congenital cataracts and glaucoma in the patient's mother, and congenital cataracts in maternal half-sister and maternal grandmother. The same balanced translocation t(X;19) was subsequently identified in both the mother and maternal half-sister, and further clinical evaluation of the maternal half-sister made a diagnosis of NHS. This study describes the clinical implication of NHS gene disruption due to balanced X-autosome translocations as a unique mechanism causing Nance-Horan syndrome, refines dose effects of NHS on disease presentation and phenotype expressivity, and justifies consideration of karyotype and fluorescence in situ hybridization (FISH) analysis for female patients with familial NHS if single-gene analysis of NHS is negative.

Disruption of normal patterns of FOXF1 expression in a lethal disorder of lung development.

BACKGROUND: Alveolar capillary dysplasia with misalignment of the pulmonary veins (ACDMPV) is a lethal disorder of lung development. ACDMPV is associated with haploinsufficiency of the transcription factor FOXF1, which plays an important role in the development of the lung and intestine. CNVs upstream of the FOXF1 gene have also been associated with an ACDMPV phenotype, but mechanism(s) by which these deletions disrupt lung development are not well understood. The objective of our study is to gain insights into the mechanisms by which CNVs contribute to an ACDMPV phenotype. METHODS: We analysed primary lung tissue from an infant with classic clinical and histological findings of ACDMPV and harboured a 340 kb deletion on chromosome 16q24.1 located 250 kb upstream of FOXF1. RESULTS: In RNA generated from paraffin-fixed lung sections, our patient had lower expression of FOXF1 than age-matched controls. He also had an abnormal pattern of FOXF1 protein expression, with a dramatic loss of FOXF1 expression in the lung. To gain insights into the mechanisms underlying these changes, we assessed the epigenetic landscape using chromatin immunoprecipitation, which demonstrated loss of histone H3 lysine 27 acetylation (H3K27Ac), an epigenetic mark of active enhancers, in the region of the deletion. CONCLUSIONS: Together, these data suggest that the deletion disrupts an enhancer responsible for directing FOXF1 expression in the developing lung and provide novel insights into the mechanisms underlying a fatal developmental lung disorder.

Assessing copy number aberrations and copy neutral loss of heterozygosity across the genome as best practice: An evidence based review of clinical utility from the cancer genomics consortium (CGC) working group for myelodysplastic syndrome, myelodysplastic/myeloproliferative and myeloproliferative neoplasms.

Multiple studies have demonstrated the utility of chromosomal microarray (CMA) testing to identify clinically significant copy number alterations (CNAs) and copy-neutral loss-of-heterozygosity (CN-LOH) in myeloid malignancies. However, guidelines for integrating CMA as a standard practice for diagnostic evaluation, assessment of prognosis and predicting treatment response are still lacking. CMA has not been recommended for clinical work-up of myeloid malignancies by the WHO 2016 or the NCCN 2017 guidelines but is a suggested test by the European LeukaemiaNet 2013 for the diagnosis of primary myelodysplastic syndrome (MDS). The Cancer Genomics Consortium (CGC) Working Group for Myeloid Neoplasms systematically reviewed peer-reviewed literature to determine the power of CMA in (1) improving diagnostic yield, (2) refining risk stratification, and (3) providing additional genomic information to guide therapy. In this manuscript, we summarize the evidence base for the clinical utility of array testing in the workup of MDS, myelodysplastic/myeloproliferative neoplasms (MDS/MPN) and myeloproliferative neoplasms (MPN). This review provides a list of recurrent CNAs and CN-LOH noted in this disease spectrum and describes the clinical significance of the aberrations and how they complement gene mutation findings by sequencing. Furthermore, for new or suspected diagnosis of MDS or MPN, we present suggestions for integrating genomic testing methods (CMA and mutation testing by next generation sequencing) into the current standard-of-care clinical laboratory testing (karyotype, FISH, morphology, and flow).

Novel 1.3 Mb germline duplication in chromosome 8q21.11 by microarray comparative genomic hybridization plus single nucleotide polymorphism analysis in an adult patient with pancytopenia and urinary bladder complications.

We present the case of a 30-year-old woman with a history of perinatal complications as well as bladder and urinary disease through her childhood and adult life. Microarray comparative genomic hybridization (aCGH) analysis revealed a 1.3 megabase duplication at chromosome 8q21.11 encompassing the CASC9 and HNF4G genes.

Combined comparative genomic hybridization and single-nucleotide polymorphism array detects cryptic chromosomal lesions in both myelodysplastic syndromes and cytopenias of undetermined significance.

The diagnosis of myelodysplastic syndrome (MDS) can be challenging, and may be facilitated by correlation with cytogenetic testing. Microarray analysis using comparative genomic hybridization and/or single-nucleotide polymorphism array can detect chromosomal abnormalities not seen by standard metaphase cytogenetics. We examined the ability of combined comparative genomic hybridization and single-nucleotide polymorphism analysis (hereafter referred to as 'combined array') to detect changes among 83 patients with unexplained cytopenias undergoing pathologic evaluation for MDS and compared results with 18 normal bone marrow controls. Thirty-seven patients (45%) were diagnosed with MDS, 12 patients (14%) were demonstrated to have 'indeterminate dyspoiesis' (insufficient for classification of MDS), 27 (33%) were essentially normal, and 7 patients (8%) had alternative pathologic diagnoses. Twenty-one MDS patients (57% of diagnoses) had effectively normal metaphase cytogenetics, but combined array showed that 5 of these (13% of MDS patients) harbored major cryptic chromosomal aberrations. Furthermore, nearly half of patients with 'indeterminate dyspoiesis' and 1 with normal morphology had clonal cytopenia(s) of undetermined significance by combined array analysis. Cryptic array findings among MDS patients and those with clonal cytopenias(s) included large-scale copy-neutral loss of heterozygosity (up to 118 Mb) and genomic deletion of loci implicated in MDS pathogenesis (eg, TET2 (4q22) and NUP98 (11p15)). By comparison, in MDS patients with abnormal metaphase cytogenetics, microarray mostly recapitulated findings seen by routine karyotype. Combined array analysis has considerable diagnostic yield in detecting cryptic chromosomal aberrations in MDS and in demonstrating aberrant clonal hematopoiesis in cytopenic patients with indeterminate morphologic dysplasia.

Pathogenetics of alveolar capillary dysplasia with misalignment of pulmonary veins.

Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a lethal lung developmental disorder caused by heterozygous point mutations or genomic deletion copy-number variants (CNVs) of FOXF1 or its upstream enhancer involving fetal lung-expressed long noncoding RNA genes LINC01081 and LINC01082. Using custom-designed array comparative genomic hybridization, Sanger sequencing, whole exome sequencing (WES), and bioinformatic analyses, we studied 22 new unrelated families (20 postnatal and two prenatal) with clinically diagnosed ACDMPV. We describe novel deletion CNVs at the FOXF1 locus in 13 unrelated ACDMPV patients. Together with the previously reported cases, all 31 genomic deletions in 16q24.1, pathogenic for ACDMPV, for which parental origin was determined, arose de novo with 30 of them occurring on the maternally inherited chromosome 16, strongly implicating genomic imprinting of the FOXF1 locus in human lungs. Surprisingly, we have also identified four ACDMPV families with the pathogenic variants in the FOXF1 locus that arose on paternal chromosome 16. Interestingly, a combination of the severe cardiac defects, including hypoplastic left heart, and single umbilical artery were observed only in children with deletion CNVs involving FOXF1 and its upstream enhancer. Our data demonstrate that genomic imprinting at 16q24.1 plays an important role in variable ACDMPV manifestation likely through long-range regulation of FOXF1 expression, and may be also responsible for key phenotypic features of maternal uniparental disomy 16. Moreover, in one family, WES revealed a de novo missense variant in ESRP1, potentially implicating FGF signaling in the etiology of ACDMPV.

A 0.7 Mb de novo duplication at 7q21.3 including the genes DLX5 and DLX6 in a patient with split-hand/split-foot malformation.

Split-hand/split-foot malformation (SHFM1) has been reported to be caused by deletions, duplications or rearrangements involving the 7q21.3 region harboring DSS1, DLX5, and DLX6. We report on a female patient with unilateral syndactyly of the third and fourth fingers of the right hand and overgrowth and lateral deviation of the right great toe. There was a split foot malformation on the right, with absent fifth toe. The left hand was apparently normal and left foot was intact. The patient has no hearing loss. We performed conventional G-banding karyotype analysis, array comparative genomic hybridization (aCGH) and fluorescence in situ hybridization (FISH). G-banding karyotype result was normal 46,XX. However, a duplication of 719 kb (96,303,736-97,022,335; NCBI build36/hg18, March 2006) was identified at the 7q21.3 region by aCGH. The array result was also confirmed by FISH analysis. The duplicated region harbors only DLX5 and DLX6, which are known for their role in SHFM1. Additionally, FISH analysis of parental samples showed de novo origin of this abnormality in the patient. This is the first report that highlights the duplication of 719 kb at 7q21.3, harboring only DLX5 and DLX6 associated with the SHFM1 phenotype.

Significance of genome-wide analysis of copy number alterations and UPD in myelodysplastic syndromes using combined CGH - SNP arrays.

Genetic information is an extremely valuable data source in characterizing the personal nature of cancer. Chromosome instability is a hallmark of most cancer cells. Chromosomal abnormalities are correlated with poor prognosis, disease classification, risk stratification, and treatment selection. Copy number alterations (CNAs) are an important molecular signature in cancer initiation, development, and progression. Recent application of whole-genome tools to characterize normal and cancer genomes provides the powerful molecular cytogenetic means to enumerate the multiple somatic, genetic and epigenetic alterations that occur in cancer. Combined array comparative genomic hybridization (aCGH) with single nucleotide polymorphism (SNP) array is a useful technique allowing detection of CNAs and loss of heterozygosity (LOH) or uni-parental disomy (UPD) together in a single experiment. It also provides allelic information on deletions, duplications, and amplifications. UPD can result in an abnormal phenotype when the chromosomes involved are imprinted. Myelodysplastic syndromes (MDS) are the most common clonal stem cell hematologic malignancy characterized by ineffective hematopoiesis, which leads to rapid progression into acute myeloid leukemia. UPD that occurs without concurrent changes in the gene copy number is a common chromosomal defect in hematologic malignancies, especially in MDS. Approximately 40-50% of MDS patients do not have karyotypic abnormalities that are detectable using classical metaphase cytogenetic techniques (MC) because of inherent limitations of MC, low resolution and the requirement of having dividing cells. In this review, we highlight advances in the clinical application of microarray technology in MDS and discuss the clinical potential of microarray.

Radiation effects on human leptomeningeal cell response to cerebrospinal fluid and PDGF-BB.

PURPOSE: The pathogenesis of meningiomas remains largely unknown, but there is evidence that exposure radiation increases the risk of meningiomas. We therefore evaluated whether radiation stimulates human leptomeningeal cell proliferation and activates growth regulatory pathways. MATERIALS AND METHODS: Cultures from six human primary leptomeningeal cells were established and irradiated with 0.5, 1 or 2 Gy. Cells were subsequently maintained for approximately 3-6 months and then treated with the mitogen, platlet-derived growth factor-BB (PDGF-BB), or human cerebrospinal fluid (CSF) obtained from patients with no neurological disease. Cell proliferation was evaluated by CyQUANT, and changes in the regulatory activation levels of signal transducer and activator of transcription (STAT3), mitogen-activated protein kinase kinase 1/2 (MEK1/2), mitogen activated protein kinase (MAPK 44/42), Akt, and mammalian target of rapamycin (mTOR) were assessed by Western blot. RESULTS: At both 3 and 6 months post-irradiation, CSF stimulated cell proliferation in all six leptomeningeal cultures. At 3 months, radiation was associated with an increase in STAT3, MEK 1/2, p44/42 MAPK and variable Akt activation, and at 6 months, STAT3, p44/42 MAPK, variable Akt and mTOR activation was observed in response to PDGF-BB and CSF. CONCLUSIONS: Irradiation of fetal leptomeningeal cells was associated with loss of heterozygosity of 1p36 and persistent sensitization leading to upregulation in growth pathways in response to PDGF and CSF. These findings suggest a mechanistic basis for radiation's role in meningioma initiation.

Renal failure associated with APECED and terminal 4q deletion: evidence of autoimmune nephropathy.

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a rare autosomal recessive disorder caused by mutations in the autoimmune regulator gene (AIRE). Terminal 4q deletion is also a rare cytogenetic abnormality that causes a variable syndrome of dysmorphic features, mental retardation, growth retardation, and heart and limb defects. We report a 12-year-old Saudi boy with mucocutaneous candidiasis, hypoparathyroidism, and adrenocortical failure consistent with APECED. In addition, he has dysmorphic facial features, growth retardation, and severe global developmental delay. Patient had late development of chronic renal failure. The blastogenesis revealed depressed lymphocytes' response to Candida albicans at 38% when compared to control. Chromosome analysis of the patient revealed 46,XY,del(4)(q33). FISH using a 4p/4q subtelomere DNA probe assay confirmed the deletion of qter subtelomere on chromosome 4. Parental chromosomes were normal. The deleted array was further defined using array CGH. AIRE full gene sequencing revealed a homozygous mutation namely 845_846insC. Renal biopsy revealed chronic interstitial nephritis with advanced fibrosis. In addition, there was mesangial deposition of C3, C1q, and IgM. This is, to the best of our knowledge, the first paper showing evidence of autoimmune nephropathy by renal immunofluorescence in a patient with APECED and terminal 4q deletion.

Interstitial del(20)(q11.2q12) - clinical and molecular cytogenetic characterization.

A 2-year-old male patient with dysmorphic facial features and multiple congenital anomalies suggestive of a chromosome syndrome is presented. The facial features consisted of a large and high forehead, mild metopic ridging, a small triangular face, depressed nasal bridge, microphthalmia (right more than the left), protruding ears, and mildly prominent anteverted nose with long and smooth philtrum. Cytogenetic analysis showed 46,XY,del(20)(q11.2q12). Parental karyotypes were normal. Molecular characterization of del(20)(q11.2q12) by high-resolution microarray comparative genomic hybridization (arrayCGH) showed an approximately 6.8 Mb deletion. To our knowledge this is the first report of a de novo interstitial del(20)(q11.2q12) characterized by arrayCGH.

Isodicentric Philadelphia chromosomes in imatinib mesylate (Gleevec)-resistant patients.

Amplification/duplication of the BCR-ABL gene has been found to be one of the key factors leading to drug resistance to imatinib mesylate (IM). In the present study, we used G-banding to identify the presence of de novo identical isodicentric chromosomes in IM-resistant patients. Fluorescence in situ hybridization (FISH) analysis on interphase nuclei confirmed the heterogeneity and amplification of the fused BCR-ABL gene. FISH analysis superimposed on G-banding confirmed the presence of isodicentric Philadelphia chromosomes. The impact of the isodicentric Philadelphia chromosomes on genomic instability, heterogeneity, and amplification of the Philadelphia chromosomes in IM-resistant patients is discussed.

Complex de novo cryptic subtelomeric rearrangements in a fetus with multiple ultrasonographic abnormalities and a normal karyotype at amniocentesis.

OBJECTIVE: Prenatal diagnosis is usually offered to the majority of pregnancies with fetal structural abnormalities detected by prenatal ultrasound; however, only a small proportion show an abnormal karyotype. We wanted to detect cryptic subtelomeric rearrangements (CSTR) in a fetus with multiple abnormal ultrasonographic findings that revealed a normal karyotype at amniocentesis. METHODS: Fetal chromosome analysis was performed from amniotic fluid cells. Parental chromosome analysis was done on PHA stimulated lymphocyte cultures. For fluorescence in situ hybridization (FISH) analysis, ToTelVysion multicolor DNA probe mixture was used to hybridize the p and q telomeres of each chromosome. RESULTS: The amniotic fluid chromosome analysis revealed an apparently normal 46,XY karyotype. A follow-up FISH analysis showed three apparently balanced complex translocations involving (1) the chromosome 4p and 22q telomeres (2) 4q and 11q telomeres and (3) 8p, 20p and 20q telomeres. Parental chromosome and subtelomere FISH analysis was found to be normal. CONCLUSION: To our knowledge, this is the first report of complex de novo cryptic translocations in an abnormal fetus. These CSTR identified by FISH with subtelomere-specific probes are not detected by other cytogenetic and/or molecular cytogenetic approaches. However, to confirm the balanced nature of CSTR, array-CGH can be helpful. Further studies are in progress to determine the frequency of CSTR and its significance in the etiology of fetal abnormalities.

Cytogenetics and etiology of ambiguous genitalia in 120 pediatric patients.

BACKGROUND: A newborn with ambiguous genitalia needs prompt evaluation to detect life-threatening conditions (e.g., salt-losing crisis in congenital adrenal hyperplasia [CAH]) and gender assignment. Sex assignment in these children continues to be a challenging diagnostic and therapeutic problem. We studied the causes and characteristics of ambiguous genitalia in children who were referred to a cytogenetic laboratory. PATIENTS AND METHODS: We retrospectively reviewed a total of 120 medical records of patients with a primary indication of ambiguous genitalia that were referred to the cytogenetic lab for karyotyping during the period of 1989 to 1999. Diagnosis was based on a clinical impression from the primary physician, who was primarily a staff pediatrician, endocrinologist and/or pediatric urologist. RESULTS: CAH was the underlying cause of ambiguous genitalia in 41 of 63 patients with ambiguity due to endocrine causes; 39 of these patients showed a 46,XX karyotype and 2 cases were 46,XY (both the 46,XY patients had 3 beta-hydroxylase deficiency). In 57 patients, ambiguous genitalia were due to congenital developmental defects. The most common endocrine case of ambiguous genitalia was 21-OH deficiency. Seven patients were classified as idiopathic with six showing the 46,XY and one the 46,XX karyotype. Gender was reassigned at birth or at diagnosis in 15 patients. CONCLUSION: The etiology of ambiguous genitalia is variable. The physician managing these families could minimize the trauma of having a child with unidentified sex by providing appropriate genetic counseling so that the parents can make an early decision. Prenatal DNA testing in at-risk families should be considered and appropriate therapy offered to minimize or prevent genital ambiguity.