Constitutional Disorders Research Articles

Copy Number Analysis of Whole Exome Sequencing Data

Abstract Background Genetic alterations implicated in constitutional disorders range in size from single nucleotide substitutions to losses and gains of millions of bases. Whole exome sequencing (WES) captures many clinically relevant variants, but an analytic pipeline optimized for detection of small substitutions, insertions, and deletions may miss pathogenic copy number variants (CNVs). Historically, our laboratory performed separate cytogenetic tests to evaluate for larger genomic imbalances. Addition of copy number analysis to our WES pipeline could improve the assay’s diagnostic yield. This study describes part of our strategy to validate copy number analysis of our constitutional whole exome sequencing data. Methods We identified 18 CNV specimens that underwent both chromosomal microarray analysis (CMA) and WES between 2017 and 2023. Variants included 13 deletions (size range 14Kb-2Mb) and 5 duplications (size range 325Kb-10Mb). The standard method of constitutional copy number analysis in our clinical laboratory is CMA using the Affymetrix Cytoscan HD Array and analysis software. CNVs identified on CMA were reviewed with reference to the Database of Genomic Variants and multiple academic hospital databases. WES was performed on an Illumina sequencing system following capture-based library enrichment with the Integrated DNA Technologies xGen Exome Research, CNV Backbone, Human mtDNA Research, and Human ID Research Panel probes. CNVs identified on WES were called using the Broad Institute’s Genomic Analysis Toolkit (GATK) with a quality score cutoff of 120. Whole exome sequencing data was visualized on the Integrative Genomics Viewer. Results 14 out of 18 CNVs reported on CMA were also detected on WES with a quality score at or above 120 (78%). This includes 10 out of 13 deletions (77%) and 3 out of 5 duplications (60%). For both deletions and duplications, the size ranges of detected variants at or above the cutoff score and undetected variants overlapped. One duplication reported on CMA was detected on WES with a quality score below 120. Visualization of whole exome sequencing data showed variation in probe density that may have affected CNV calling. Conclusions The majority of CNVs reported after CMA (14/18, 78%) were also detected on WES data using a GATK quality score cutoff of 120. The data does not suggest an association between CNV size and detection by WES, although interpretation is limited by the small number of cases. Data visualization may be required to identify some CNVs. Future steps to characterize the limitations of copy number analysis of WES data include assessments of variance in probe density and read depth across the exome. Copy number analysis of WES data from cases with normal CMA results will also be performed for assay validation.

Generalized lymphadenopathy as an initial presentation of systemic lupus erythematosus: case report and review of the literature

BackgroundSystemic lupus erythematosus (SLE) is a chronic multisystemic autoimmune disorder with a wide array of clinical manifestations. Lymphadenopathy is occasionally observed in patients with SLE, and although it is sometimes a feature of active disease, it is rarely the initial presenting feature at the time of diagnosis.Case presentationWe report the case of a 35-year-old woman with clinical features of generalized lymphadenopathy, fatigue, fever, and malaise of approximately 3 months duration with no other constitutional disorders or systemic involvement suggestive of SLE.Despite extensive laboratory, radiological, and histological evaluations for malignancies and infectious causes of generalized lymphadenopathy, all results returned normal. About a month later, she developed inflammatory polyarthritis, with a markedly elevated erythrocyte sedimentation rate (ESR) of 120 mm/h, and a subsequent serologic workup confirmed the diagnosis of SLE.Autoimmune serology testing revealed positive results for various antibodies, including antinuclear antibody (ANA) at a titre of 1:5120, homogenous and speckled pattern, anti-Sm, anti-dsDNA, U1-snRNP, Ku, SSA-AntiRo, SSB-AntiLa, anti-SM/RNP, anti-Histone, ribosomal-p protein, and anti-nucleosome antibodies. The diagnosis of SLE was established using the 2019 European League Against Rheumatism/American College of Rheumatology (EULAR/ ACR) criteria.She was on oral prednisolone, hydroxychloroquine, and calcium/vitamin D3 tablets. The patient improved remarkably, all swollen lymph nodes regressed spontaneously and she had no further need for a hematology review.ConclusionWhile similar cases have been reported in Europe and some parts of Asia, in Africa, a careful search of the literature revealed that such cases have been reported only from Egypt in North Africa and not from other parts of the continent; hence, this case report highlights the importance of recognizing atypical presentations of SLE to improve awareness and diagnosis in different regions.

Conventional Cytogenetic Analysis of Constitutional Abnormalities: A 20-Year Review of Proficiency Test Results From the College of American Pathologists/American College of Medical Genetics and Genomics Cytogenetics Committee.

The joint College of American Pathologists/American College of Medical Genetics and Genomics Cytogenetics Committee works to ensure competency and proficiency of clinical cytogenetics testing laboratories through proficiency testing programs for various clinical tests offered by such laboratories, including the evaluation of constitutional abnormalities. To review and analyze 20 years of constitutional chromosome analysis proficiency testing results (2003-2022), primarily utilizing G-banded karyograms. A retrospective review of results from 2003 through 2022 was performed, identifying challenges addressing constitutional disorders. The chromosomal abnormalities and overall performance were evaluated. A total of 184 cases from 161 proficiency testing challenges were administered from 2003 through 2022. Challenges consisted of metaphase images and accompanying clinical history for evaluation of numerical and/or structural abnormalities. Of the 184 cases, only 2 (1%) failed to reach an 80% grading consensus for recognition of the abnormality. Both cases illustrated the limitations of correctly characterizing some chromosomal abnormalities, including recombinant chromosomal abnormalities and isochromosome identification. In addition, 2 cases failed to reach a consensus for nomenclature reporting: 1 with an isochromosome and another with a duplication. This 20-year review illustrates the high rate of competency and proficiency of cytogenetic laboratories in the correct identification of constitutional chromosome abnormalities.

Mixed connective tissue disease with juvenile onset: results of a retrospective single-center study

Mixed connective tissue disease (MCTD) is one of the very rare systemic autoimmune diseases; it accounts for 0.1–0.6% of cases in pediatric rheumatologists' practices. MCTD is characterized by a broad spectrum of clinical manifestations and a high frequency of extremely unspecific symptoms at the onset, with the overall picture of the disease forming slowly and gradually. The diagnosis is often delayed and confirmed only at an advanced stage of organ dysfunction with the development of irreversible changes.Objective: to identify a group of patients fulfilling the criteria for MCTD in an open, single-center, continuous retrospective study among anti-ribonucleoprotein (anti-RNP) antibody-positive patients and to analyze their demographic, clinical and laboratory characteristics and therapy.Material and methods. All anti-RNP-positive patients admitted to the pediatric department of V.A. Nasonova Research Institute of Rheumatology from 2019 to 2023 and meeting at least one of the variants of the MCTD criteria (Kasukawa, Alarcуn-Segovia, Kahn and Sharp criteria) were included in the study.Results and discussion. 18 (56.25%, 17 girls and 1 boy) of 32 anti-RNP-positive patients fulfilled criteria for MCTD. Patients most frequently fulfilled a combination of criteria – Sharp and Kahn (n=8) or Alarcуn-Segovia and Kahn (n=8). The median age of onset of MCTD was 12.2 [9.7; 13.9] years. The most common clinical manifestations were arthritis (100%), various skin lesions (94.4 %), Raynaud's phenomenon (88.9%), lymphadenopathy (72.2%) and general constitutional disorders (50%). Sjögren's syndrome (SS) was diagnosed in 17 (94.4%) patients. All patients had antinuclear factor (ANF) 1/1280, and the anti-RNP level was >200 U/ml. There were also antibodies against double-stranded DNA (n=5), Ro- (n=4) and Sm- (n=5) antigens. An IgM rheumatoid factor was detected in 6 patients and hypergammaglobulinemia in 10 patients. Capillaroscopic changes in the nailfold with predominant scleroderma type were found in 77.8% of patients. The most common combination was of Raynaud's phenomenon, arthritis, SS, lymphadenopathy and hypergammaglobulinemia (50%). All patients received glucocorticoids, 9 – hydroxychloroquine, 8 – methotrexate, 3 – mycophenolate mofetil, 1 – cyclophosphamide, 1 – azathioprine. Biologic DMARDs (bDMARDs) were prescribed to 12 (66.7%) patients: 3 – rituximab, 8 – abatacept, 1 – belimumab, with an acceptable safety profile and initial efficacy.Conclusion. Most patients in the study met the Kahn criteria. Only 2 patients met all variants of the criteria, which indicates the need to use a combination of criteria when a MCTD is suspected. A combination of Raynaud's phenomenon, arthritis, SS, lymphadenopathy and hypergammaglobulinemia was observed in half of patients with MCTD. The presence of Raynaud's phenomenon and high ANF titer in children with rheumatic diseases, especially with a polymorphic clinical picture, requires the inclusion of MCTD in differential diagnosis. Preliminary results indicate the safety of the use of biologic drugs in children with MCTD.

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).

Optical Genome Mapping for Genome-Wide Structural Variation Analysis in Hematologic Malignancies: A Prospective Study Demonstrates Additional Findings Compared to Standard-of-Care (SOC) Cytogenomic Methods

Genomic structural variants (SVs) are key in understanding the pathogenesis of hematologic malignancies. The current standard-of-care (SOC) cytogenomic methods, including chromosome karyotyping (KT) and fluorescence in situ hybridization (FISH), have inherent limitations in identifying SVs, while next-generation sequencing technologies have limited ability to detect most SVs. Optical Genome Mapping (OGM) is a high-resolution cytogenomic technique that overcomes the limitations of SOC cytogenomic methods and detects all classes of SVs. Recent studies, mostly retrospective, have demonstrated OGM's analytic validity and clinical utility for detecting SVs in both hematologic malignancies and constitutional genetic disorders. This prospective study, using OGM as a validated laboratory developed test (LDT) for detection of SVs, included 112 patients with a wide variety of hematologic malignancies undergoing clinical evaluation and management and demonstrates OGM as superior to SOC cytogenomic methods. Patient blood or bone marrow samples were subject to the Bionano OGM-Dx™ HemeOne assay. OGM analysis was successful for 105/112 (94%) samples with 7/112 (6%) samples failing analysis. Of the 105 analyzed samples, 74 (70%) had abnormal and 31 (30%) had normal findings by OGM analysis, with 49% of abnormal OGM findings classified as complex. At initial diagnosis, 49 cases had guideline and SOC result-based disease risk stratification with 18/49 (37%) graded as high risk, 15/49 (31%) as intermediate risk, and 16/49 (32%) as low risk. Additionally, 60 cases had SOC cytogenomic analysis (KT or FISH) results available; 37% with KT and FISH, 27% with only KT, and 35% with only FISH. OGM cases with normal findings were concordant with KT in 9/10 (90%) of cases and FISH in 8/12 (67%) of cases. For those cases that had abnormal findings by KT and/or FISH, OGM detected additional cytogenomic findings in 79% of cases. A change of risk stratification was made in 12/42 (29%) of cases with additional findings by OGM analysis compared to KT and/or FISH; 9/12 (75%) with upgraded risk and 3/12 (25%) with downgraded risk stratification. In 10/42 (24%) of cases, OGM findings resulted in a change in therapeutic management recommendations. This is exemplified by an acute lymphoblastic leukemia case: KT detected a sole t(9;22) abnormality however, OGM detected t(9;22) and additional findings of gains at chromosomes 1q, 17q, and 19p and losses at chromosomes 5q, 7p, and 11q (see figure 1), changing the risk stratification from intermediate to adverse and directly leading to a change in therapeutic management. The impact of OGM on disease risk stratification and case management is in alignment with previously published studies with OGM analysis demonstrating its power to detect additional findings not seen by SOC cytogenomic analysis. The prospective study outcomes suggest a significant role for OGM in delivering comprehensive results for improved diagnostic assessment and significant prognostic information for better case management and care compared to SOC cytogenomic methods.

SAT274 Multilocus Imprinting Disorder Due To Genome-wide Paternal Uniparental Disomy Associated With Adrenocortical Tumors

Abstract Disclosure: E.M. Pinto: None. E. Lalli: None. G. Wu: None. R. Ribeiro: None. G. Zambetti: None. Pediatric adrenocortical tumor (ACT) is an uncommon malignancy frequently associated with Li-Fraumeni syndrome, a familial cancer predisposition disorder caused by germline mutations in the tumor suppressor TP53. ACT is also associated with other genetic constitutional disorders, such as Beckwith-Wiedemann syndrome (BWS) which results from the deregulation of a gene cluster on chromosome 11p15. We report here the identification of three pediatric patients with ACT and additional tumors, two of which received a clinical diagnosis of BWS. Family histories of these two probands include multiple unsuccessful pregnancies. All patients have wild-type TP53 sequence and no evidence for germline chromosome 11p15 alterations. Molecular analyses by whole genome sequencing revealed genome-wide copy neutral loss of heterozygosity (cnLOH) in all tumors. Microsatellite STR and MS-MLPA confirmed genome-wide cnLOH with the selective loss of all maternal chromosomes and duplication of paternal chromosomes in each tumor. Analysis of skin fibroblasts showed mosaic paternal uniparental disomy with partial LOH for all chromosomes, while no LOH was detected in blood DNA. The most prominent clinical feature of genome-wide cnLOH is an unbalanced monoallelic and parent-of-origin specific expression of imprinted genes known as multilocus imprinting disorder (MID). MID has been associated with maternal-effect variants that alter the maintenance of methylation at germline-derived differentially methylated regions (DMRs) in early embryogenesis. In addition, causative trans-acting factors in imprinting disorders, including the subcortical maternal complex (SCMC), have the potential to affect multiple DMRs across the genome, resulting in a multilocus imprinting disorder. Our study identifies three pediatric patients who developed ACT and other tumors that have undergone genome wide cnLOH that specifically selected against all maternal chromosomes. None of three patients presented with germline alterations in TP53 or 11p15, typical of pediatric ACT. WGS data revealed a germline mutation (p.M573T) in the histone methyltransferase NSD1 in the mother of patient #2. NSD1 mutations are reportedly associated with recurrent miscarriages, tissue overgrowth and increased cancer risk. Alterations in other epigenetic regulators, such as SETD1B (p.R226C), a component of a histone methyltransferase complex and TNRC18 (p.P454L), that enables chromatin binding activity were observed in both mother and child in each family. Further analysis of maternal-effect variants associated with abnormal pregnancies and MID will be important for understanding the mechanisms underlying genome-wide cnLOH. Our results support the molecular testing of multiple loci in atypical clinical presentations of patients with pediatric ACT. Presentation: Saturday, June 17, 2023

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.

The International Consensus Classification (ICC) of hematologic neoplasms with germline predisposition, pediatric myelodysplastic syndrome, and juvenile myelomonocytic leukemia.

Updating the classification of hematologic neoplasia with germline predisposition, pediatric myelodysplastic syndrome (MDS), and juvenile myelomonocytic leukemia (JMML) is critical for diagnosis, therapy, research, and clinical trials. Advances in next-generation sequencing technology have led to the identification of an expanding group of genes that predispose to the development of hematolymphoid neoplasia when mutated in germline configuration and inherited. This review encompasses recent advances in the classification of myeloid and lymphoblastic neoplasia with germline predisposition summarizing important genetic and phenotypic information, relevant laboratory testing, and pathologic bone marrow features. Genes are organized into three major categories including (1) those that are not associated with constitutional disorder and include CEBPA, DDX41, and TP53; (2) those associated with thrombocytopenia or platelet dysfunction including RUNX1, ANKRD26, and ETV6; and (3) those associated with constitutional disorders affecting multiple organ systems including GATA2, SAMD9, and SAMD9L, inherited genetic mutations associated with classic bone marrow failure syndromes and JMML, and Down syndrome. A provisional category of germline predisposition genes is created to recognize genes with growing evidence that may be formally included in future revised classifications as substantial supporting data emerges. We also detail advances in the classification of pediatric myelodysplastic syndrome (MDS), expanding the definition of refractory cytopenia of childhood (RCC) to include early manifestation of MDS in patients with germline predisposition. Finally, updates in the classification of juvenile myelomonocytic leukemia are presented which genetically define JMML as a myeloproliferative/myelodysplastic disease harboring canonical RAS pathway mutations. Diseases with features overlapping with JMML that do not carry RAS pathway mutations are classified as JMML-like. The review is based on the International Consensus Classification (ICC) of Myeloid and Lymphoid Neoplasms as reported by Arber et al. (Blood 140(11):1200-1228, 2022).

CYTOGENETIC ANALYSIS OF 2,205 CASES OF HEMATOLOGICAL NEOPLASIA AND GENETIC DISEASES: A SINGLE CENTER EXPERIENCE

Cytogenetics is defined as the study of chromosome morphology, pathology function, and behavior. Cytogenetics is important to define clonality for bone marrow disorders and for diagnosing a lot of constitutional disorders. We analyzed a ten-year experience of bone marrow and peripheral blood cytogenetics for defining clonal and not clonal disorders at the Cancer Cytogenomics Laboratory of the Federal University of Ceará. All bone marrow and peripheral blood samples were submitted to cell culture (for 24 hours and 72 hours, respectively), after which all were processed, and G-banding performed using trypsin and Wright-Giemsa staining. The karyotype results of 2,205 patients were analyzed including additional information such as age, sex, requesting unit and suspected diagnosis. Among these, 1744 (79.1%) were bone marrow samples and 461 (20.9%) were peripheral blood samples used to investigate constitutional changes. The female gender (54.4%) stood out in the Bone Marrow samples, aged between 71 and 80 years (20.2%). Among the bone marrow results, 726 of 1744 (42%) were normal and 431 of them (24.7%) were abnormal karyotypes. The main etiological suspicion was Myelodysplastic Syndrome (54.2% of cases), and 42.1% were normal. Among the chromosomal abnormalities, the main alterations found were: 26% cases with isolated del(5q) and/or other alterations, 10% of cases were complex, 10% were isolated del(11q) and/or with other alterations, 9% of cases with del (7q) isolated and/or with other alterations and 7% cases with +8 isolated and/or with other alterations, 2% cases had del(17q) isolated and/or with other alterations, 1% of cases had del(17p) and one case with i(17q). A total of 127 cases were suspected as AML, with 34.6% altered karyotypes, 34.4% normal karyotypes. Among the abnormal karyotypes: 20,5% cases with t(15;17) and 13,6% were complex karyotypes, 6,8% were del(11q23), 4,5% cases with t(8;21), 1 case with del(17p) and another with i(17q). A total of 161 cases were suspected as CML, among which: 49,7% abnormal karyotype, 24.2% were normal. Among the chromosomal abnormalities, 65% were t(9;22) and 2,2% were t(9;22) and other alterations and/or variants of t(9;22). In this way, this study shows that cytogenetic was important for diagnostic purposes of patients principally for diseases such as Myelodysplastic Syndrome, Acute Myeloid Leukemia and Chronic Myeloid Leukemia. Cytogenetic analysis is also fundamental for analysis of constitutional and somatic genetic disorders. Regarding the constitutional results, 176/461 (38%) showed chromosomal abnormalities with 66 (37.5%) showing trisomy 21 followed by trisomy 18 (14.7%) and androgen insensitivity syndrome (11.3%). Identification of constitutional or malignance chromosomal anomalies can provide important information for genetic medical counseling. Cytogenetic analysis of hematological neoplasms and for constitutional disorders is helpful for diagnosis and monitoring the effects of treatment, being still very important in the new molecular era.

Long-read sequencing for molecular diagnostics in constitutional genetic disorders.

Long-read sequencing (LRS) has been around for more than a decade, but widespread adoption of the technology has been slow due to the perceived high error rates and high sequencing cost. This is changing due to the recent advancements to produce highly accurate sequences and the reducing costs. LRS promises significant improvement over short read sequencing in four major areas: (1) better detection of structural variation (2) better resolution of highly repetitive or nonunique regions (3) accurate long-range haplotype phasing and (4) the detection of base modifications natively from the sequencing data. Several successful applications of LRS have demonstrated its ability to resolve molecular diagnoses where short-read sequencing fails to identify a cause. However, the argument for increased diagnostic yield from LRS remains to be validated. Larger cohort studies may be required to establish the realistic boundaries of LRS's clinical utility and analytical validity, as well as the development of standards for clinical applications. We discuss the limitations of the current standard of care, and contrast with the applications and advantages of two major LRS platforms, PacBio and Oxford Nanopore, for molecular diagnostics of constitutional disorders, and present a critical argument about the potential of LRS in diagnostic settings.

Role of cytokine and Toll-like receptor genes in pathogenesis of inborn heart disease

Sporadic congenital heart disease (CHD) may result from immune disorders in the mother – embryo system and/or constitutional disorders in regulatory systems, including those associated with TLR receptors, cytokines and their receptors. The aim of our study was to investigate associations between cytokine and TLR genes and sporadic congenital heart disease in children. In the main group, 188 children with sporadic (without family history) congenital heart defects were examined. Separate groups of CHD were identified: septal CHD – 98 children; valvular heart disease – 17 children; Fallot tetralogy – 15 children; aorta coarctation – 10 children; fetal drains – 32 children; single ventricle affection – 9 children, and anomalous drainage of v. pulmonalis was diagnosed in 7 children. The control group included 103 age- and sex-matched healthy children. We have determined gene polymorphisms of five genes encoding cytokines and their receptors (IL6 rs1800796, IL6 rs2069827, IL6R rs2228145, IL6R rs2229238, IL8 rs4073, IL10 rs1800871, IL10 rs1800896, IL10 rs1800872, TNF rs1800629, TNF rs361525, TNF rs1799964), four genes Toll-like receptors (TLR: TLR1 rs5743611, TLR1 rs5743551, TLR2 rs5743708, TLR2 rs3804099, TLR4 rs4986791, TLR4 rs4986790, TLR6 rs3775073, TLR6 rs5743810). The dbSNP, SNPinfo, SNPnexus databases were used to select and design test systems. Stepwise logistic regression was the main method of statistical analysis. Clinical diagnosis of congenital heart defects is associated with immune regulatory genes. In particular, the missense mutation TLR6 rs5743810, which was a predictor of congenital valvular heart disease, is of particular importance. Development of congenital heart valve defects and aortic coarctation is associated with intergenic interactions of TLR2 rs5743708 with TLR6 rs5743810, and TLR2 rs5743708 with TLR6 rs3775073, respectively. For congenital heart valve defects, such polymorphic regions are as follows: IL6 rs2069827, IL6R rs2229238, and IL8 rs4073, for aortic coarctation – IL6R rs2228145, IL8 rs4073. Development of septal congenital heart defects is associated with general contribution of polymorphic variants of the TLR genes and cytokines to this pathology. A missense mutation of the TLR4 rs4986790 gene and a TNF rs1799964 mutation leading to increased synthesis of the TNFα molecule, may have a combined effect on this process. In general, contribution of TLR and cytokine genes interactions to the CHD development seems to be not significant.

EP388: Optical genome mapping capability expanded to enable detection of absence of heterozygosity

Optical genome mapping (OGM) is a technique able to detect all classes of structural variants (SVs), including deletions, insertions, duplications, inversions, chromosomal aneuploidies, and translocations, as well as complex rearrangements across the whole genome that are undetectable by traditional methods such as genome sequencing and cytogenetics. OGM is now also able to detect copy-neutral absence of heterozygosity (AOH), which has traditionally been identified using chromosomal microarray and microsatellite analysis. Copy neutral absence of heterozygosity refers to a specific type of genetic condition where both alleles are the same and this can be a result of uniparental disomy or consanguinity. One consequence of AOH can be an increased susceptibility to recessive disease. Here we describe a method for AOH analysis based on optical genome mapping from the Bionano Genomics Saphyr system. Regions of AOH are identified by a consistent decrease in heterozygous SV calls across a genomic region in a case sample compared to the level observed in controls. DNA was processed using the Bionano Genomics Saphyr system and analyzed using the Bionano Access software. After filtering SV calls for high-quality, informative sites, AOH events were simulated by splicing together SV calling datasets from 153 controls and 4 double haploid samples, where AOH events were represented by regions derived from double haploid genomes, or regions derived from chromosome X in males. AOH regions were determined using a Hidden Markov Model (HMM), which was used to model the spatial dependence between neighboring SVs of a given zygosity: the hidden state was whether an SV belongs to an AOH or background region, and the observable states were whether the SV was called as homozygous or heterozygous. Model parameters were estimated by fitting the model to the simulated dataset, and performance was evaluated on newly simulated samples and additional samples with known AOH events that were previously identified using chromosomal microarray analysis. In simulated data, our method achieved high sensitivity and precision in detecting large AOH regions above 25 Mbp, with 92% sensitivity and 97% precision. Using a small cohort of samples with known AOH events, we have confirmed the utility of this method and were able to detect constitutional AOH events. In the current implementation, AOH events are detected if present in all cells; lower variant allele frequency events are not assessed. Our results show that it is possible to detect AOH regions using optical genome mapping alone. The performance for AOH detection by OGM is sufficient to detect the most relevant AOH events associated with constitutional disorders and adds an important new dimension to OGM technology. The new AOH algorithm is now available as part of Bionano Access version 1.7.

Quo vadis Britannia? Davida Hume’a rozważania nad angielską konstytucją

The reign of the Hanoverian dynasty was a period of stability and prosperity in Great Britain, until the 1770s. The perfect symbiosis between the Whig oligarchy and the Crown was the cause of exceptional political stability. During this period, however, there were also neither major constitutional disorders nor sophisticated systematic analyses. According to the author, the most important attempt to describe British political mechanics is the political reflection of David Hume. David Hume, the most eminent British philosopher of the 18th century, is the author of an original and penetrating analysis of British political institutions. Arguing without regard to party sympathies, he broke down Whig and Tory conceptions of political systems, and instead proposed a dynamic vision of Great Britain’s political system, from its past as a barbarian monarchy, through a civilised monarchy, to a mixed modern monarchy. He sees this evolution as a dialectical struggle between power and liberty, the two most important forces that govern political systems. This article presents an attempt to reconstruct Hume’s coherent constitutional theory based on historical, political, and philosophical aspects of his argument. According to the author, all three perspectives form a coherent argument that makes Hume one of the classics of English constitutionalism. The author develops these ideas on the basis of a detailed analysis of Hume’s works, starting with the History of England.

Prolactinoma and cardiovascular diseases – an interdisciplinary problem

Prolactin-secreting pituitary tumour is a rare disease, in which excess prolactin causes significant functional and constitutional disorders of the whole body. Hyperprolactinaemia is associated with changes in body composition and metabolic disorders. The long-term prognosis and quality of life in patients with prolactinoma are mainly influenced by cardiovascular disorders, which, if left untreated, increase the cardiovascular risk and limit the treatment options for secondary organ complications. Cardiovascular mortality in patients with prolactinoma is several times higher than in the general population. Early diagnosis of prolactin-releasing pituitary tumour and a thorough morphological and functional cardiovascular assessment at each stage of the disease are necessary for risk stratification. Patients with prolactinoma should be put on combined treatment based on both serum prolactin control, and reduction of cardiovascular risk factors. Normalisation of prolactin levels and reduction of the tumour mass, achieved mainly through effective pharmacotherapy, reduce mortality and the risk of cardiovascular complications; therefore, the earliest possible diagnosis of prolactin pituitary tumour and implementation of appropriate treatment as well as active diagnosis and therapy of coexisting organ complications should be set as a goal in these patients. It seems that a thorough cardiological assessment of patients with prolactinoma should be obligatory, regardless of their age or the time of diagnosis. The aim of this study was to present the complexity of clinical problems in patients with prolactin secreting pituitary tumours, who require special interdisciplinary care.

Chromosomal microarray analysis, including constitutional and neoplastic disease applications, 2021 revision: a technical standard of the American College of Medical Genetics and Genomics (ACMG)

Chromosomal microarray technologies, including array comparative genomic hybridization and single-nucleotide polymorphism array, are widely applied in the diagnostic evaluation for both constitutional and neoplastic disorders. In a constitutional setting, this technology is accepted as the first-tier test for the evaluation of chromosomal imbalances associated with intellectual disability, autism, and/or multiple congenital anomalies. Furthermore, chromosomal microarray analysis is recommended for patients undergoing invasive prenatal diagnosis with one or more major fetal structural abnormalities identified by ultrasonographic examination, and in the evaluation of intrauterine fetal demise or stillbirth when further cytogenetic analysis is desired. This technology also provides important genomic data in the diagnosis, prognosis, and therapy of neoplastic disorders, including both hematologic malignancies and solid tumors. To assist clinical laboratories in the validation of chromosomal microarray methodologies for constitutional and neoplastic applications, the American College of Medical Genetics and Genomics (ACMG) Laboratory Quality Assurance Committee has developed these updated technical laboratory standards, which replace the ACMG technical standards and guidelines for microarray analysis in constitutional and neoplastic disorders previously published in 2013.

Cardiovascular risk in patients with Cushing’s disease – an interdisciplinary problem

Cushing’s disease is a chronic endogenous hypercortisolaemia associated with overproduction of adrenocorticotropic hormone by a pituitary adenoma, leading to multiple systemic complications that significantly increase morbidity and mortality, as well as reduce the quality of life as a result of prolonged tissue exposure to excess cortisol. Hypercortisolaemia in Cushing’s disease is associated with significant functional and constitutional disorders of the entire body. The consequences of chronic hypercortisolaemia include haemodynamic disorders associated with excessive vascular contraction and increased blood pressure, obesity, carbohydrate metabolism disorders, dyslipidaemia, and coagulopathies, which may contribute to significant cardiovascular remodelling. Cardiovascular disorders have a particular impact on long-term prognosis and quality of life in Cushing’s disease. If left untreated, Cushing’s disease significantly increases the cardiovascular risk and limits the treatment options for secondary organ complications. Cardiovascular mortality (myocardial infarction, heart failure, stroke) is several times higher in patients with Cushing’s disease than in the general population. Early diagnosis of the corticotropic pituitary tumour, as well as a thorough morphological and functional cardiovascular assessment seem essential in risk stratification. Normalisation of cortisol levels after combined neurosurgical and/or pharmacological treatment reduces mortality and the risk of cardiovascular and respiratory complications. The aim of this study is to present the complexity of clinical problems in patients with Cushing’s disease, who are in a particular need of interdisciplinary care.

Clinical utility and cost‐effectiveness analysis of chromosome testing concomitant with chromosomal microarray of patients with constitutional disorders in a U.S. academic medical center

Chromosomal microarray (CMA) is now widely used as first-tier testing for the detection of copy number variants (CNVs) and absence of heterozygosity (AOH) in patients with multiple congenital anomalies (MCA), autism spectrum disorder (ASD), developmental delay (DD), and/or intellectual disability (ID). Chromosome analysis is commonly used to complement CMA in the detection of balanced genomic aberrations. However, the cost-effectiveness and the impact on clinical management of chromosome analysis concomitant with CMA were not well studied, and there is no consensus on how to best utilize these two tests. To assess the clinical utility and cost-effectiveness of chromosome analysis concomitant with CMA in patients with MCA, ASD, DD, and/or ID, we retrospectively analyzed 3,360 postnatal cases for which CMA and concomitant chromosome analysis were performed in the Colorado Genetic Laboratory (CGL) at the University Of Colorado School Of Medicine. Chromosome analysis alone yielded a genetic diagnosis in two patients (0.06%) and contributed additional information to CMA results in 199 (5.92%) cases. The impact of abnormal chromosome results on patient management was primarily related to counseling for reproductive and recurrence risks assessment (101 cases, 3.01%) while a few (5 cases, 0.15%) led to changes in laboratory testing and specialist referral (25 cases, 0.74%). The incremental cost-effectiveness ratio (ICER) of combined testing demonstrated the cost of each informative chromosome finding was significantly higher for patients with clinically insignificant (CI) CMA findings versus clinically significant (CS) CMA results. Our results suggest that a stepwise approach with CMA testing with reflex to chromosome analysis on cases with CS CMA findings is a more cost-effective testing algorithm for patients with MCA, ASD, and/or DD/ID.

EP436 - Identification of structural variation in constitutional disorders by optical genome mapping

eP436 - Identification of structural variation in constitutional disorders by optical genome mapping

Next-generation sequencing for inborn errors of immunity

Inborn errors of immunity (IEIs) include several hundred gene defects affecting various components of the immune system. As with other constitutional disorders, next-generation sequencing (NGS) is a powerful tool for the diagnosis of these diseases. While NGS can provide molecular confirmation of disease in a patient with a suspected or classic phenotype, it can also identify new molecular defects of the immune system, expand gene-disease phenotypes, clarify mechanism of disease, pattern of inheritance or identify new gene-disease associations. Multiple clinical specialties are involved in the diagnosis and management of patients with IEI, and most have no formal genetic training or expertise. To effectively utilize NGS tools and data in clinical practice, it is relevant and pragmatic to obtain a modicum of knowledge about genetic terminology, the variety of platforms and tools available for high-throughput genomic analysis, the interpretation and implementation of such data in clinical practice. There is considerable variability not only in the technologies and analytical tools used for NGS but in the bioinformatics approach to variant identification and interpretation. The ability to provide a molecular basis for disease has the potential to alter therapeutic management and longer-term treatment of the disease, including developing personalized approaches with molecularly targeted therapies. This review is intended for the clinical specialist or diagnostic immunologist who works in the area of inborn errors of immunity, and provides an overview of the need for genetic testing in these patients (the “why” aspect), the various technologies and analytical approaches, bioinformatics tools, resources, and challenges (the “how” aspect), and the clinical evidence for identifying which patients might be best served by such testing (the “when” aspect).