Low-pass whole genome sequencing is a reliable and cost-effective approach for copy number variant analysis in the clinical setting.

INTRODUCTION: Next generation sequencing technology has greatly reduced the cost and time required for sequencing a genome. An approach that is rapidly being adopted as an alternative method for CNV analysis is the low-pass whole genome sequencing (LP-WGS). Here, we evaluated the performance of LP-WGS to detect copy number variants (CNVs) in clinical cytogenetics. MATERIALS AND METHODS: DNA samples with known CNVs detected by chromosomal microarray analyses (CMA) were selected for comparison and used as positive controls; our panel included 44 DNA samples (12 prenatal and 32 postnatal), comprising a total of 55 chromosome imbalances. The selected cases were chosen to provide a wide range of clinically relevant CNVs, the vast majority being associated with intellectual disability or recognizable syndromes. The chromosome imbalances ranged in size from 75 kb to 90.3 Mb, including aneuploidies and two cases of mosaicism. RESULTS: All CNVs were successfully detected by LP-WGS, showing a high level of consistency and robust performance of the sequencing method. Notably, the size of chromosome imbalances detected by CMA and LP-WGS were compatible between the two different platforms, which indicates that the resolution and sensitivity of the LP-WGS approach are at least similar to those provided by CMA. DISCUSSION: Our data show the potential use of LP-WGS to detect CNVs in clinical diagnosis and confirm the method as an alternative for chromosome imbalances detection. The diagnostic effectiveness and feasibility of LP-WGS, in this technical validation study, were evidenced by a clinically representative dataset of CNVs that allowed a systematic assessment of the detection power and the accuracy of the sequencing approach. Further, since the software used in this study is commercially available, the method can easily be tested and implemented in a routine diagnostic setting.

Low-pass whole genome sequencing as a cost-effective alternative to chromosomal microarray analysis for low- and middle-income countries.

Low-pass whole genome sequencing (LP-WGS) has been applied as alternative method to detect copy number variants (CNVs) in the clinical setting. Compared with chromosomal microarray analysis (CMA), the sequencing-based approach provides a similar resolution of CNV detection at a lower cost. In this study, we assessed the efficiency and reliability of LP-WGS as a more affordable alternative to CMA. A total of 1363 patients with unexplained neurodevelopmental delay/intellectual disability, autism spectrum disorders, and/or multiple congenital anomalies were enrolled. Those patients were referred from 15 nonprofit organizations and university centers located in different states in Brazil. The analysis of LP-WGS at 1x coverage (>50kb) revealed a positive testing result in 22% of the cases (304/1363), in which 219 and 85 correspond to pathogenic/likely pathogenic (P/LP) CNVs and variants of uncertain significance (VUS), respectively. The 16% (219/1363) diagnostic yield observed in our cohort is comparable to the 15%-20% reported for CMA in the literature. The use of commercial software, as demonstrated in this study, simplifies the implementation of the test in clinical settings. Particularly for countries like Brazil, where the cost of CMA presents a substantial barrier to most of the population, LP-WGS emerges as a cost-effective alternative for investigating copy number changes in cytogenetics.

BRCA1 and BRCA2 germline mutation analysis from a cohort of 1267 patients at high risk for breast cancer in Brazil.

We determined the frequency and mutational spectrum of BRCA1 and BRCA2 in a series of patients at high risk for developing breast cancer from Brazil. A total of 1267 patients were referred for BRCA genetic testing, and no obligation of fulfilling criteria of mutation probability methods for molecular screening was applied. Germline deleterious mutations in BRCA1/2 (i.e., pathogenic/likely pathogenic variants) were identified in 156 out of 1267 patients (12%). We confirm recurrent mutations in BRCA1/2, but we also report three novel mutations in BRCA2, not previously reported in any public databases or other studies. Variants of unknown significance (VUS) represent only 2% in this dataset and most of them were detected in BRCA2. The overall mutation prevalence in BRCA1/2 was higher in patients diagnosed with cancer at age > 35 years old, and with family history of cancer. The present data expand our knowledge of BRCA1/2 germline mutational spectrum, and it is a valuable clinical resource for genetic counseling and cancer management programs in the country.

Congenital chromoanagenesis in the routine postnatal chromosomal microarray analyses.

Chromosomal microarray analyses (CMA) have greatly increased both the yield and diagnostic accuracy of postnatal analysis; it has been used as a first-tier cytogenetic test in patients with intellectual disability, autism spectrum disorder, and multiple congenital abnormalities. During the last 15 years, we performed CMA in approximately 8,000 patients with neurodevelopmental and/or congenital disorders, of which 13 (0.16%) genetically catastrophic complex chromosomal rearrangements were identified. These ultrarare rearrangements showed clustering of breakpoints, characteristic of chromoanagenesis events. Al1 13 complex events display underlying formation mechanisms, originating either by a synchronization of the shattering of clustered chromosome regions in which regional asynchrony of DNA replication may be one of the main causes of disruption. We provide an overview of the copy number profiling in these patients. Although several previous studies have suggested that chromoanagenesis is often a genetic disease source in postnatal diagnostic screening, due to either the challenge of clinical interpretation of these complex rearrangements or the limitation of microarray resolution relative to the small size and complexity of chromogenic induced chromosome abnormalities, bringing further attention and to study its occurrence in the clinical setting is extremely important.

Chromosomal microarray analyses from 5778 patients with neurodevelopmental disorders and congenital anomalies in Brazil.

Chromosomal microarray analysis (CMA) has been recommended and practiced routinely since 2010 both in the USA and Europe as the first-tier cytogenetic test for patients with unexplained neurodevelopmental delay/intellectual disability, autism spectrum disorders, and/or multiple congenital anomalies. However, in Brazil, the use of CMA is still limited, due to its high cost and complexity in integrating the results from both the private and public health systems. Although Brazil has one of the world's largest single-payer public healthcare systems, nearly all patients referred for CMA come from the private sector, resulting in only a small number of CMA studies in Brazilian cohorts. To date, this study is by far the largest Brazilian cohort (n = 5788) studied by CMA and is derived from a joint collaboration formed by the University of São Paulo and three private genetic diagnostic centers to investigate the genetic bases of neurodevelopmental disorders and congenital abnormalities. We identified 2,279 clinically relevant CNVs in 1886 patients, not including the 26 cases of UPD found. Among detected CNVs, the corresponding frequency of each category was 55.6% Pathogenic, 4.4% Likely Pathogenic and 40% VUS. The diagnostic yield, by taking into account Pathogenic, Likely Pathogenic and UPDs, was 19.7%. Since the rational for the classification is mostly based on Mendelian or highly penetrant variants, it was not surprising that a second event was detected in 26% of those cases of predisposition syndromes. Although it is common practice to investigate the inheritance of VUS in most laboratories around the world to determine the inheritance of the variant, our results indicate an extremely low cost-benefit of this approach, and strongly suggest that in cases of a limited budget, investigation of the parents of VUS carriers using CMA should not be prioritized.