A 25-year odyssey of genomic technology advances and structural variant discovery.

This perspective focuses on advances in genome technology over the last 25 years and their impact on germline variant discovery within the field of human genetics. The field has witnessed tremendous technological advances from microarrays to short-read sequencing and now long-read sequencing. Each technology has provided genome-wide access to different classes of human genetic variation. We are now on the verge of comprehensive variant detection of all forms of variation for the first time with a single assay. We predict that this transition will further transform our understanding of human health and biology and, more importantly, provide novel insights into the dynamic mutational processes shaping our genomes.

Trio-R: a script for assessing maternity and paternity in trio studies performed on Agilent chromosomal microarrays.

BACKGROUND: Trio studies, which involve the testing of samples from a proband and both parents, are often used by clinical laboratories to help with the classification of genetic variants, including copy number variants. In order for the results of the trio study to be valid, the mother and father must be the true biological parents of the proband. As such, non-paternity and sample mix-ups are potential sources of error. To address these potential issues, we developed a computer script to accurately assess maternity and paternity using single nucleotide polymorphism (SNP) data generated by Agilent chromosomal microarrays, a platform-of-choice for clinical copy number testing. RESULTS: We assessed the performance of the script on 10 putative trios tested at our laboratory, and found that the numbers and proportions of discordant SNPs were useful for determining parental relationships. The results of the assessment also confirmed maternity and paternity in the 10 trios tested, and by doing so essentially excluded pre-analytical sample switching in these 30 samples. CONCLUSIONS: Computational analysis of SNP data can be implemented as a quality control measure for trio testing performed on Agilent microarrays.

Microdeletion of 12q24.31: report of a girl with intellectual disability, stereotypies, seizures and facial dysmorphisms.

We provide a detailed clinical and molecular characterization of an 11-year-old female patient presenting with neurodevelopmental delay (NDD), intellectual disability (ID), seizures, stereotypies and dysmorphic features. Chromosomal microarrays analysis (CMA) detected a small, rare de novo deletion on chromosome 12q24.31 encompassing 31 protein-coding RefSeq genes and a microRNA. Phenotypic comparison with molecularly well-defined cases previously reported in the literature harboring an overlapping 12q24.31 microdeletion indicate that these patients shared common clinical features including neurodevelopmental delay, intellectual disability and behavioral problems. Also, seizures and dysmorphic features are frequent and a consistent pattern was recognized. Since there are remarkable resemblance between the patient described here and at least another one previously reported, our report is provides supportive evidence for the existence of an emerging syndrome caused by a microdeletion in 12q24.31. We propose a minimal region shared among patients contributing to the etiology of the common clinical features observed suggesting as candidate, for the first time, the gene SETD1B which is a component of a histone methyltransferase complex. In addition, we speculate on the possible contributive role of the MIR4304 to some clinical features observed in our patient. Evaluation of more patients with well-characterized deletions within 12q24.31, as well as careful clinical assessment of them, is needed to corroborate our hypothesis, to perform a more detailed genotype-phenotype correlation and, finally, to fully delineate this emerging microdeletion syndrome.

Preimplantation genetic risk reduction: a new dilemma in the era of chromosomal microarrays and exome sequencing.

New technologies are revealing genetic variants of unknown significance (VUS), raising questions about the indications that call for preimplanation genetic diagnosis (PGD). Two couples requesting PGD for VUS are presented. The first couple requested PGD for Lynch syndrome. Whole exome sequencing identified in a healthy male with a family history of Lynch-associated tumours, a MLH1 missense variant. The variant had not been reported as pathogenic, but was predicted as damaging by algorithms. The second couple had a child diagnosed with pervasive developmental disorder and intellectual disability, carrying a microduplication on chr:Xp.22.3, and a microdeletion on chr:17q21.31. The maternally inherited X linked microduplication was also present in the mother's healthy brother and daughter, whereas the chr17 microdeletion was a de-novo event. As chromosomal microarrays and whole-exome sequencing are becoming standard tests, couples are requesting PGD for these VUS. The risk of possible genetic diseases can be reduced by carrying out PGD for uncertain findings, yet will inevitably lead to the birth of affected children despite the transfer of embryos that are not carriers of the familial variants. Findings of unknown significance demand urgent discussion and guidelines for their use as a risk-reduction measure in the preimplantation setting.

A cohort study of neurodevelopmental disorders and/or congenital anomalies using high resolution chromosomal microarrays in southern Brazil highlighting the significance of ASD.

Chromosomal microarray (CMA) is the reference in evaluation of copy number variations (CNVs) in individuals with neurodevelopmental disorders (NDDs), such as intellectual disability (ID) and/or autism spectrum disorder (ASD), which affect around 3-4% of the world's population. Modern platforms for CMA, also include probes for single nucleotide polymorphisms (SNPs) that detect homozygous regions in the genome, such as long contiguous stretches of homozygosity (LCSH). These regions result from complete or segmental chromosomal homozygosis and may be indicative of uniparental disomy (UPD), inbreeding, population characteristics, as well as replicative DNA repair events. In this retrospective study, we analyzed CMA reading files requested by geneticists and neurologists for diagnostic purposes along with available clinical data. Our objectives were interpreting CNVs and assess the frequencies and implications of LCSH detected by Affymetrix CytoScan HD (41%) or 750K (59%) platforms in 1012 patients from the south of Brazil. The patients were mainly children with NDDs and/or congenital anomalies (CAs). A total of 206 CNVs, comprising 132 deletions and 74 duplications, interpreted as pathogenic, were found in 17% of the patients in the cohort and across all chromosomes. Additionally, 12% presented rare variants of uncertain clinical significance, including LPCNVs, as the only clinically relevant CNV. Within the realm of NDDs, ASD carries a particular importance, owing to its escalating prevalence and its growing repercussions for individuals, families, and communities. ASD was one clinical phenotype, if not the main reason for referral to testing, for about one-third of the cohort, and these patients were further analyzed as a sub-cohort. Considering only the patients with ASD, the diagnostic rate was 10%, within the range reported in the literature (8-21%). It was higher (16%) when associated with dysmorphic features and lower (7%) for "isolated" ASD (without ID and without dysmorphic features). In 953 CMAs of the whole cohort, LCSH (≥ 3 Mbp) were analyzed not only for their potential pathogenic significance but were also explored to identify common LCSH in the South Brazilians population. CMA revealed at least one LCSH in 91% of the patients. For about 11.5% of patients, the LCSH suggested consanguinity from the first to the fifth degree, with a greater probability of clinical impact, and in 2.8%, they revealed a putative UPD. LCSH found at a frequency of 5% or more were considered common LCSH in the general population, allowing us to delineate 10 regions as potentially representing ancestral haplotypes of neglectable clinical significance. The main referrals for CMA were developmental delay (56%), ID (33%), ASD (33%) and syndromic features (56%). Some phenotypes in this population may be predictive of a higher probability of indicating a carrier of a pathogenic CNV. Here, we present the largest report of CMA data in a cohort with NDDs and/or CAs from the South of Brazil. We characterize the rare CNVs found along with the main phenotypes presented by each patient and show the importance and usefulness of LCSH interpretation in CMA results that incorporate SNPs, as well as we illustrate the value of CMA to investigate CNV in ASD.

The evolution of comprehensive genetic analysis in neurology: Implications for precision medicine.

Technological advancements have facilitated the availability of reliable and thorough genetic analysis in many medical fields, including neurology. In this review, we focus on the importance of selecting the appropriate genetic test to aid in the accurate identification of disease utilizing currently employed technologies for analyzing monogenic neurological disorders. Moreover, the applicability of comprehensive analysis via NGS for various genetically heterogeneous neurological disorders is reviewed, revealing its efficiency in clarifying a frequently cloudy diagnostic picture and delivering a conclusive and solid diagnosis that is essential for the proper management of the patient. The feasibility and effectiveness of medical genetics in neurology require interdisciplinary cooperation among several medical specialties and geneticists, to select and perform the most relevant test according to each patient's medical history, using the most appropriate technological tools. The prerequisites for a comprehensive genetic analysis are discussed, highlighting the utility of appropriate gene selection, variant annotation, and classification. Moreover, genetic counseling and interdisciplinary collaboration could improve diagnostic yield further. Additionally, a sub-analysis is conducted on the 1,502,769 variation records with submitted interpretations in the Clinical Variation (ClinVar) database, with a focus on neurology-related genes, to clarify the value of suitable variant categorization. Finally, we review the current applications of genetic analysis in the diagnosis and personalized management of neurological patients and the advances in the research and scientific knowledge of hereditary neurological disorders that are evolving the utility of genetic analysis towards the individualization of the treatment strategy.

Optical Genome Mapping: A Promising New Tool to Assess Genomic Complexity in Chronic Lymphocytic Leukemia (CLL).

Novel treatments in chronic lymphocytic leukemia (CLL) have generated interest regarding the clinical impact of genomic complexity, currently assessed by chromosome banding analysis (CBA) and chromosomal microarray analysis (CMA). Optical genome mapping (OGM), a novel technique based on imaging of long DNA molecules labeled at specific sites, allows the identification of multiple cytogenetic abnormalities in a single test. We aimed to determine whether OGM is a suitable alternative to cytogenomic assessment in CLL, especially focused on genomic complexity. Cytogenomic OGM aberrations from 42 patients were compared with CBA, FISH, and CMA information. Clinical−biological characteristics and time to first treatment (TTFT) were analyzed according to the complexity detected by OGM. Globally, OGM identified 90.3% of the known alterations (279/309). Discordances were mainly found in (peri-)centromeric or telomeric regions or subclonal aberrations (<15−20%). OGM underscored additional abnormalities, providing novel structural information on known aberrations in 55% of patients. Regarding genomic complexity, the number of OGM abnormalities had better accuracy in predicting TTFT than current methods (C-index: 0.696, 0.602, 0.661 by OGM, CBA, and CMA, respectively). A cut-off of ≥10 alterations defined a complex OGM group (C-OGM, n = 12), which included 11/14 patients with ≥5 abnormalities by CBA/CMA and one patient with chromothripsis (Kappa index = 0.778; p < 0.001). Moreover, C-OGM displayed enrichment of TP53 abnormalities (58.3% vs. 3.3%, p < 0.001) and a significantly shorter TTFT (median: 2 vs. 43 months, p = 0.014). OGM is a robust technology for implementation in the routine management of CLL patients, although further studies are required to define standard genomic complexity criteria.

Analytical and clinical performance of chromosomal microarrays compared with FISH panel and conventional karyotyping in patients with chronic lymphocytic leukemia.

In this single center retrospective analysis on 102 CLL patients, we assessed analytical and clinical performance of CMA against a targeted FISH panel (ATM, TP53, CEP12, D13S319 and LAMP1 loci) and karyotyping. CMA yielded additional information compared to karyotype in 39 cases (38 %). On the other hand, while CMA detected aberrations were also detected by FISH in all 31 cases (30 %), aberrations with low clonal size (<30 %) detected by FISH were missed by CMA. When evaluated with National Cancer Center Network (NCCN) guidelines, the capture rate of prognostic relevant cytogenetic information for FISH only, FISH + Chromosomes and FISH + CMA analyses were 95, 96 and 100 % respectively. With Cancer Cytogenomics Consortium (CGC) Criteria, these figures for FISH only, FISH + Chromosomes and FISH + CMA were 88 %, 92 and 100 % respectively. In conclusion, CMA provides additional analytical information to FISH and karyotyping, but this information has a clinical utility only in a small number of patients. Limit of detection (LOD) issues preclude replacement of FISH by CMA, but CMA may be a viable alternative to karyotyping. Further research is warranted.

The Phenotypic Spectrum of 15q13.3 Region Duplications: Report of 5 Patients.

Chromosome 15q13.3 microduplications are associated with a wide spectrum of clinical presentations ranging from normal to different neuropsychiatric conditions, such as developmental delay (DD), intellectual disability (ID), epilepsy, hypotonia, autism spectrum disorders (ASD), attention-deficit hyperactivity disorder, and schizophrenia. The smallest region of overlap for 15q13.3 duplications encompasses the Cholinergic Receptor Nicotinic Alpha 7 Subunit (CHRNA7) gene, a strong candidate for the behavioral abnormalities. We report on a series of five patients with 15q13.3 duplications detected by chromosomal microarray. The size of the duplications ranged from 378 to 537 kb, and involved the CHRNA7 gene in all patients. The most common clinical features, present in all patients, were speech delay, autistic behavior, and muscle hypotonia; DD/ID was present in three patients. One patient presented epileptic seizures; EEG anomalies were observed in three patients. No consistent dysmorphic features were noted. Neuroimaging studies revealed anomalies in two patients: Dandy-Walker malformation and a right temporal cyst. 15q13.3 duplications are associated with various neuropsychiatric features, including speech delay, hypotonia, ASD, and ID, also present in our patient group. Our study brings detailed clinical and molecular data from five ASD patients with 15q13.3 microduplications involving the CHRNA7 gene, contributing to the existing knowledge about the association of 15q13.3 duplications with neuropsychiatric phenotypes.

Genetic Screening in Patients with Craniofacial Malformations.

Craniofacial malformations include a variety of anomalies, including cleft lip with or without cleft palate, craniosynostosis, microtia, and hemifacial microsomia. All of these anomalies can be either isolated or part of a defined genetic syndrome. A clinical geneticist or genetic counselor should be a member of the craniofacial team to help determine which patients have isolated anomalies and which are likely to have a syndrome. They would then arrange for the appropriate genetic testing to confirm the diagnosis of the specific syndrome. The identification of the specific syndrome is important for the overall care of the patient (as it identifies risk for other medical problems such as congenital heart defect) that will have to be taken into account in the care of the craniofacial malformation. In addition, knowing the specific syndrome will allow the family to understand how this happened to their child and the recurrence risk for future pregnancies. With the advent of new technologies, there are now many types of genetic testing available (including, karyotype, fluorescence in situ hybridization, chromosomal microarrays, and next generation sequencing) and the medical geneticist and genetic counselor can determine which specific testing is needed for a given patient.

Causal variants screened by whole exome sequencing in a patient with maternal uniparental isodisomy of chromosome 10 and a complicated phenotype.

Uniparental disomy (UPD), which is the abnormal situation in which both copies of a chromosomal pair have been inherited from one parent, may cause clinical abnormalities by affecting genomic imprinting or causing autosomal recessive variation. Whole Exome Sequencing (WES) and chromosomal microarray analysis (CMA) are powerful technologies used to search for underlying causal variants. In the present study, WES was used to screen for candidate causal variants in the genome of a Chinese pediatric patient, who had been shown by CMA to have maternal uniparental isodisomy of chromosome 10. This was associated with numerous severe medical problems, including bilateral deafness, binocular blindness, stunted growth and leukoderma. A total of 13 rare homozygous variants of these genes were identified on chromosome 10. These included a classical splice variant in the HPS1 gene (c.398+5G>A), which causes Hermansky-Pudlak syndrome type 1 and may explain the patient's ocular and dermal disorders. In addition, six likely pathogenic genes on other chromosomes were found to be associated with the subject's ocular and aural disorders by phenotypic analysis. The results of the present study demonstrated that WES and CMA may be successfully combined in order to identify candidate causal genes. Furthermore, a connection between phenotype and genotype was established in this patient.

Genomic aberrations in myeloid sarcoma without blood or bone marrow involvement: characterization of formalin-fixed paraffin-embedded samples by chromosomal microarrays.

Myeloid sarcoma (MS) is a presentation of acute myeloid leukemia (AML) as a tumor mass outside of the bone marrow. Viable cells from MS are frequently unavailable for cytogenetic studies. We therefore investigated whether chromosomal microarray analysis (CMA) using formalin-fixed paraffin-embedded (FFPE) tissues can detect clinically important genetic abnormalities in MS. CMA successfully identified genomic aberrations in six cases of MS, and in two cases it revealed multiple abnormalities equivalent to a complex karyotype, thus predicting a poor outcome. CMA using FFPE material is therefore a feasible and clinically applicable approach for detection of prognostically significant genomic abnormalities in MS.

Molecular characterization of complex chromosomal rearrangement: first report of novel t(7;12) (q11;q22) as part of a complex karyotype in de novo AML-M2 case.

The strong association of diagnostic karyotype with clinical outcome has made cytogenetics one of the most valuable diagnostic and prognostic tools for acute myeloid leukemia (AML) till today. Complex chromosomal findings are reported to be seen in nearly 10-15% of adult AMLs and are generally associated with poor outcome. In the current report, we present the results of hematologic, immunophenotypic, cytogenetic, chromosomal microarray and molecular analyses of a 60-year-old female patient diagnosed with AML-M2. Cytogenetic analysis revealed complex chromosomal findings involving seven different chromosomes. However, cytogenetic analyses were not able to precisely unveil all karyotypic changes, hence chromosomal microarray was used for further characterization. The most interesting observation was identification of a t(7;12) (q11;q22) as part of this complex karyotype. To the best of our knowledge, this is the first report of identification of novel t(7;12) (q11;q22) as part of a complex karyotype in de novo AML-M2.

Prenatal management of the fetus with isolated congenital diaphragmatic hernia in the era of the TOTAL trial.

Congenital diaphragmatic hernia (CDH) may be isolated or associated with other structural anomalies, the latter with poor prognosis. The defect allows viscera to herniate through the defect into the chest, competing for space with the developing lungs. At birth, pulmonary hypoplasia leads to respiratory insufficiency and persistent pulmonary hypertension that is lethal in up to 30% of patients. When isolated, survival chances can be predicted by antenatal measurement of lung size and liver herniation. Chromosomal microarrays and exome sequencing contribute to understanding genetic factors underlying isolated CDH. Prenatal intervention aims at stimulating lung development, clinically achieved by percutaneous fetal endoscopic tracheal occlusion (FETO) under local anesthesia. The Tracheal Occlusion To Accelerate Lung growth trial (www.totaltrial.eu) is an international randomized trial investigating the role of fetal therapy for severe and moderate pulmonary hypoplasia. Despite an apparent increase in survival following FETO, the search for lesser invasive and more potent prenatal interventions must continue.

Cytogenetic evaluation: a primer for pediatric nurse practitioners.

Patients with genetic disorders require specific types of cytogenetic testing for accurate diagnosis and prognosis followed by prompt treatment. This primer will serve as a guide for pediatric nurse practitioners on the use of various cytogenetic testing for the diagnosis of genetic disorders. Knowledge of the latest cytogenetic technologies will facilitate diagnosis and counseling related to genetic abnormalities such as inherited disorders, mental retardation, developmental delay, and autism. This reference will enable pediatric nurse practitioners to help identify patients with various inherited genetic disorders and provide subsequent monitoring and treatment.