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

doi:10.21203/rs.3.rs-3773173/v1

Download PDF

Research Article

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

https://doi.org/10.21203/rs.3.rs-3773173/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

We recently demonstrated that low-pass whole-genome sequencing (LP-WGS) with 1x coverage is equally sensitive and specific as chromosome microarray analysis (CMA) for detecting copy number variants (CNVs). Although in Europe and USA the first-tier tests have already transitioned to whole-genome or exome sequencing, even CMA is still unavailable for approximately 71% of the Brazilian population, who exclusively rely on public health system (~ 140 million people). LP-WGS, costing only a quarter of CMA's price, presents a more economical, faster, and technically simpler alternative for detecting CNVs. 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 fifteen 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.

copy number variants

CNV

structural variant

cytogenetics

postnatal diagnosis

low-pass whole genome sequencing

next generation sequencing (NGS)

genomic medicine

Copy number variants (CNVs) are regions in the human DNA associated to both population diversity and pathogenic conditions^1–3. Neurodevelopmental delay/intellectual disability (NDD/ID), autism spectrum disorders (ASD), and multiple congenital anomalies (MCA) are some conditions frequently associated to genomic disorders and clinically recognizable syndromes⁴. In the last decade, chromosomal microarray analysis (CMA) has been utilized as a first-tier diagnostic test to detect CNVs in the clinical setting⁵. Although the resolution of CMA depends on probe spacing and density, the test can be estimated in two orders of magnitude higher than karyotype, resulting in a diagnostic yield of 15–20% in patients with NDD/ID, ASD and/or MCA⁵. Nonetheless, due to the high cost of CMA, many patients remain undiagnosed in low- and middle-income countries.

In Brazil, CMA is available mostly in the private health sector, while more than 71% of the population rely exclusively on the Brazilian Unified Health System (Sistema Único de Saúde – SUS), one of the world’s largest single payer healthcare systems^6–8. A cost-effective alternative method to detect CNVs with similar resolution to CMA is the low-pass whole genome sequencing (LP-WGS), a high-throughput sequencing of the entire human genome at low-coverage^9–13. In a recent publication, we demonstrated that LP-WGS at an average depth of 1x is as sensitive and specific to detect microscopic and submicroscopic chromosome alterations as CMA¹⁴. LP-WGS detects CNVs at least 50 kb large and has a coverage suitable to detect the well-recognized microdeletion and microduplication syndromes, with an estimated cost of one quarter compared to CMA¹⁴. Therefore, this study was designed to evaluate, in a large cohort of patients from Nonprofit Organizations and University Centers, the efficiency and reliability of LP-WGS as a more affordable diagnostic option to replace CMA. We aimed to facilitate the introduction of LP-WGS in the public healthcare services in Brazil and, ultimately, to promote a solution for more equal access to genetic testing from individuals of all socioeconomic backgrounds in the country.

Casuistic

A total of 1363 patients from Nonprofit Organizations and University Centers in Brazil were enrolled for this study, between July 2021 and June 2022. Fifteen Institutions located in different states within the country referred patients with unexplained neurodevelopmental delay/intellectual disability, autism spectrum disorders, and/or multiple congenital anomalies for postnatal evaluation, following the same inclusion criteria recommended for CMA in a clinical routine⁵. Patients were excluded from this study if they had a clear diagnostic hypothesis (i.e., Down syndrome and Turner syndrome), or had a disorder not suspected to have a genetic origin. Figure 1 shows the geographical distribution of the Institutions that participated in this research.

Low-pass whole genome sequencing (LP-WGS)

Peripheral blood samples were collected in EDTA tubes and sent to DASA Genomic laboratory (São Paulo, SP) for DNA extraction, which was performed applying an automated methodology (QIAsymphony, Qiagen, Netherlands). Sequencing libraries were prepared using the Illumina DNA PCR-Free Library Prep, Tagmentation protocol (Illumina, USA), and quantified by qPCR assay using the KAPA Library Quantification Kit (Roche, Switzerland). LP-WGS experiments were performed on the NovaSeq 6000 equipment (Illumina, USA), utilizing the NovaSeq 6000 S4 Reagent Kit v1.5 (300 cycles), in a paired-read sequencing strategy, with 150 cycles from each end of the DNA fragment. For the simultaneous sequencing of 768 samples, NovaSeq XP 4-Lane Kit v1.5 (Illumina, USA) was used to separate the lanes of the flow cell, distributing 192 libraries in each of the four lanes. The sequencing results generated ~ 16 million paired-end reads of 150 bp per sample, corresponding to a 1x coverage. These reads were aligned to the human genome (GRCh38) using the BWA v0.7.17¹⁵ to generate the BAM files. Module MarkDuplicates from GATK¹⁶ was used to identify PCR duplicates and mark reads for exclusion in the downstream analysis.

CNV analysis and clinical interpretation

CNV data analysis was performed using the NxClinical software (BioDiscovery, California, USA), which calls copy number changes by comparing the number of reads of an experimental sample to an internal reference library. The data were normalized and the log₂ ratio Test/Reference was calculated. Following the same principle of microarray analysis, the theoretically expected log2 ratio value, when there are no changes in copy number, corresponds to zero (test/reference = 1). To identify CNVs, we used the SNP-FASST2 segmentation algorithm, based on the Hidden Markov Model, with a sensitivity threshold of 1.0 E-6. A genomic segment was considered duplicated or deleted when the log₂ ratio of a given region encompassing at least three targets was above 0.3 or below − 0.3, respectively^14,17,18.

Detected CNVs were classified according to their potential clinical impact as Pathogenic (P), Likely Pathogenic (LP), Variants of Uncertain Significance (VUS), Likely Benign (LB) and Benign (B), according to the European guidelines for constitutional cytogenomics analysis, American College of Medical Genetics (ACMG) and Clinical Genome Resource (ClinGen) guidelines^19,20. The common variants (LB/B) were not reported in this study.

Phenotype overview

Among the 1363 patients from our cohort, 63% (861/1363) were male and 37% (502/1363) female. Patients’ clinical features were documented using HPO terms and, in many cases, they present multiple phenotypic abnormalities (Supplementary Tables 1 and 2). Figure 2A presents a summary of the most common clinical features encountered in our cohort, grouped by HPO category, in which abnormality of the nervous system (n = 1291, 95%) followed by abnormality of the face (n = 763, 56%), cardiovascular system (n = 137, 10%), and the head (n = 92, 7%) represent the main reason of referral to genetic testing. Non-specific phenotypic abnormalities, often overlapped with one or more of these main HPO categories, were present in 54% of the cases (n = 741). These HPO categories were divided by groups, as presented in Fig. 2B.

Diagnostic yield

Out of the 1363 patients referred to genetic testing, 1059 had a negative result, and the remaining 304 patients either presented a disease-causing CNV (P/LP) or VUS; the corresponding frequency of P/LP CNVs and VUS was 16% and 6%, respectively. Considering only the P/LP CNVs, the overall diagnostic yield in our cohort was 16% (Fig. 3A); the main criteria for classification of P/LP CNVs and its corresponding frequency order is shown in Fig. 3B. Figure 4 illustrates some examples of CNVs detected by LP-WGS. The five most frequent syndromes observed in our cohort (detected at least twice), were 22q11.2 deletion (n = 13, 8.0%; MIM#192430), 16p11.2 deletion (n = 9, 5.6%; MIM#611913), Williams-Beuren syndrome (n = 8, 4.9%; MIM#194050), Wolf-Hirschhorn syndrome (n = 6, 3.7%; MIM#194190) and 15q11.2 deletion (n = 6, 3.7%; MIM#615656). The complete listing and the corresponding number of patients and frequencies of each syndrome is shown in Fig. 5.

The progress in genomic testing, such as CMA and whole exome or genome sequencing, has significantly reduced the diagnostic odyssey for genetic diseases in high-income countries^21–23. Despite all these scientific breakthroughs seen in the past decades, significant challenges persist in ensuing equal and inclusive access to these technological advancements particularly in low- and middle-income countries. There is an evident unequal participation of historically excluded populations in genomic field, which limits the ability to translate research discoveries that could potentially lead to improved health outcomes for society^24,25. In Brazil, for example, with an estimated population of 211 million (https://censo2022.ibge.gov.br/panorama/), sees 71% of its citizens exclusively reliant on the public healthcare system (https://www.gov.br/saude/pt-br), devoid of access to either CMA or sequencing technologies. It is estimated that approximately 3% of the population presents global developmental delay or intellectual disability, with or without other clinical features²⁶. Consequently, more than 6 million people affected by global developmental delay or intellectual disability in Brazil alone, remain without a molecular diagnosis. Among them, 15–20% could potentially uncover the causes of their conditions through DNA copy number changes investigations^5,8.

In our previous work, we showed that LP-WGS, also known as shallow sequencing, is equally sensitive and accurate as CMA in detecting copy number changes both in prenatal and postnatal samples¹⁴. LP-WGS is already widely applied in multiple international diagnostic centers for CNV detection in a variety of clinical applications, including NIPT²⁷, pre-implantation genetic testing²⁸, liquid biopsy²⁹, and solid tumor analysis³⁰. A recent extensive study revealed the widespread adoption of LP-WGS in Chinese hospitals for high-risk pregnancies, spontaneous abortions, couples with adverse pregnancy history, and children with congenital birth defects³¹. Their preferences for LP-WGS are rooted in its high-throughput nature and more even representation across the genome compared to CMA platforms. Further, the sequencing-based method provides possibility of automation, resulting in lower technical repeat rates and requiring lower input of DNA when compared to CMA. Nonetheless, the discussion in that survey did not cover CNV detection rate and the use of user-friendly commercial platforms.

In this study, we applied LP-WGS in a large multicentric Brazilian cohort in order to evaluate the cost-effectiveness of this approach as a diagnostic alternative to replace CMA and provide more equal access to genetic testing in our country. Notably, the diagnostic yield (P/LP CNVs) in this cohort (16%) is comparable to the 15–20% determined for CMA and the CNV detection rate is highly efficient, costing only a quarter of CMA¹⁴. Regarding the frequency of VUS, it is estimated in approximately 6% from postnatal microarrays³², similar to our findings with LP-WGS. Moreover, our data revealed that clinically recognizable syndromes, characterized for having recurrent breakpoints, can be effectively diagnoses by LP-WGS. This emphasizes that the sequencing-based method is accurate for mapping the critical region of diseases and identifies the precise boundaries of dosage-sensitive genes. Whereas most centers develop their custom bioinformatic pipelines for detecting copy number changes from sequence reads, we employed a commercial software solution that offers CNV calls, displays, and annotations, which certainly accelerated testing and implementing LP-WGS in a routine diagnostic setting.

In summary, our findings demonstrate that LP-WGS is a high-throughput technology that serves as a cost-effective and reliable alternative to CMA, potentially revolutionizing genetic diagnosis in low- and middle-income countries.

ETHICAL APPROVAL AND CONSENT TO PARTICIPATE

This study was approved by the Ethics Committee from Institute of Bioscience - University of São Paulo (CAAE: 53093821.3.0000.5464), and informed consent was obtained from the patients or patient’s parents for genetic testing.

CONSENT FOR PUBLICATION

Not applicable.

AVAILABILITY OF DATA AND MATERIALS

All data generated or analyzed during this study are included in this published article.

COMPETING INTERESTS

P.C.M. and C.R. declare that they serve as genomic analyst and consultant for DASA, respectively. All other authors report no conflict of interest relevant to this article.

FUNDING

This study was supported by a FAPESP grant (2013/08028-1) and resources from DASA.

AUTHORS' CONTRIBUTIONS

Conceptualization, P.C.M., D.V., C.R.; Methodology, M.G.P., M.C.B., G.G.G.F., R.G., M.R-G., D.B., G.L.Y., M.R.P-B., G.S.C., A.C.D.M., J.F.M., E.P., R.M.Z-C., N.M.K-N., T.P.V., C.E.S., V.L.G.D.S.L., D.K.R.V., R.B., J.M.P-N.; Validation, P.C.M., C.R., P.M.P., A.B., L.G.D.A.; Formal analysis, P.C.M.; Data curation, P.C.M., A.C.V.K., C.R.; Writing - Review & Editing, P.C.M., D.V., A.C.V.K., C.R.; Supervision, C.R.; Project administration, C.R., F.M.; Funding acquisition, C.R., C.S-N., A.G.O.

ACKNOWLEDGEMENTS

We thank all the patients, family members and health care providers from all the Institutions that participated in this study.

Sudmant PH et al. Global diversity, population stratification, and selection of human copy-number variation. Sci (1979) 349, (2015).
Carvalho CMB, Lupski JR. Mechanisms underlying structural variant formation in genomic disorders. Nature Reviews Genetics Preprint at https://doi.org/10.1038/nrg.2015.25 (2016).
Lupski JR. Genomic disorders: Structural features of the genome can lead to DNA rearrangements and human disease traits. Trends Genet. 1998;14:417–22.
Asadollahi R, et al. The clinical significance of small copy number variants in neurodevelopmental disorders. J Med Genet. 2014;51:677–88.
Miller DT, et al. Consensus Statement: Chromosomal Microarray Is a First-Tier Clinical Diagnostic Test for Individuals with Developmental Disabilities or Congenital Anomalies. Am J Hum Genet. 2010;86:749–64.
Castro MC et al. Brazil’s unified health system: the first 30 years and prospects for the future. The Lancet vol. 394 345–356 Preprint at https://doi.org/10.1016/S0140-6736(19)31243-7 (2019).
Lewis M, Penteado E, Malik AM. Brazil’s Mixed Public and Private Hospital System. World Hosp Health Serv. 2015;51:22–6.
Krepischi ACV et al. Chromosomal microarray analyses from 5778 patients with neurodevelopmental disorders and congenital anomalies in Brazil. Sci Rep 12, (2022).
Chaubey A, et al. Low-Pass Genome Sequencing: Validation and Diagnostic Utility from 409 Clinical Cases of Low-Pass Genome Sequencing for the Detection of Copy Number Variants to Replace Constitutional Microarray. J Mol Diagn. 2020;22:823–40.
Dong Z, et al. Low-pass whole-genome sequencing in clinical cytogenetics: A validated approach. Genet Sci. 2016;18:940–8.
Dong Z et al. Copy-number variants detection by low-pass whole-genome sequencing. Curr Protoc Hum Genet 2017, 8.17.1–8.17.16 (2017).
Wang H et al. Low-pass genome sequencing versus chromosomal microarray analysis: implementation in prenatal diagnosis. Genet Sci 10.1038/s41436-019.
Dong Z, et al. Low-pass genome sequencing–based detection of absence of heterozygosity: validation in clinical cytogenetics. Genet Sci. 2021;23:1225–33.
Mazzonetto PC, et al. Low-pass whole genome sequencing is a reliable and cost-effective approach for copy number variant analysis in the clinical setting. Ann Hum Genet. 2023. 10.1111/ahg.12532.
Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 2009;25:1754–60.
Depristo MA, et al. A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat Genet. 2011;43:491–501.
Villela D, Costa SS, Vianna-Morgante AM, Krepischi ACV, Rosenberg C. Efficient detection of chromosome imbalances and single nucleotide variants using targeted sequencing in the clinical setting. Eur J Med Genet. 2017;60:667–74.
Villela D, et al. Detection of mosaicism for segmental and whole chromosome imbalances by targeted sequencing. Ann Hum Genet. 2021;85:18–26.
Silva M, et al. European guidelines for constitutional cytogenomic analysis. Eur J Hum Genet. 2019;27:1–16.
Riggs ER, et al. Technical standards for the interpretation and reporting of constitutional copy-number variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics (ACMG) and the Clinical Genome Resource (ClinGen). Genet Sci. 2020;22:245–57.
van der Sanden BPGH, et al. The performance of genome sequencing as a first-tier test for neurodevelopmental disorders. Eur J Hum Genet. 2023;31:81–8.
Srivastava S et al. Meta-analysis and multidisciplinary consensus statement: exome sequencing is a first-tier clinical diagnostic test for individuals with neurodevelopmental disorders and the NDD Exome Scoping Review Work Group. 10.1038/s41436.
Nisar H et al. Whole-genome sequencing as a first-tier diagnostic framework for rare genetic diseases. Experimental Biology and Medicine vol. 246 2610–2617 Preprint at https://doi.org/10.1177/15353702211040046 (2021).
Khoury MJ et al. Health equity in the implementation of genomics and precision medicine: A public health imperative. Genetics in Medicine vol. 24 1630–1639 Preprint at https://doi.org/10.1016/j.gim.2022.04.009 (2022).
Rebbeck TR et al. A Framework for Promoting Diversity, Equity, and Inclusion in Genetics and Genomics Research. JAMA Health Forum vol. 3 Preprint at https://doi.org/10.1001/jamahealthforum.2022.0603 (2022).
Shevell M. Practice parameter: Evaluation of the child with global developmental delay Report of the Quality Standards Subcommittee of the American Academy of Neurology and The Practice Committee of the Child Neurology Society. http://www.aan.com/ (2003).
Lo YMD. Non-invasive prenatal diagnosis by massively parallel sequencing of maternal plasma DNA. Open Biol 2, (2012).
Wells D, et al. Clinical utilisation of a rapid low-pass whole genome sequencing technique for the diagnosis of aneuploidy in human embryos prior to implantation. J Med Genet. 2014;51:553–62.
Lenaerts L, et al. Genomewide copy number alteration screening of circulating plasma DNA: Potential for the detection of incipient tumors. Ann Oncol. 2019;30:85–95.
Kader T et al. Copy number analysis by low coverage whole genome sequencing using ultra low-input DNA from formalin-fixed paraffin embedded tumor tissue. Genome Med 8, (2016).
Zheng Y et al. Experience of Low-Pass Whole-Genome Sequencing-Based Copy Number Variant Analysis: A Survey of Chinese Tertiary Hospitals. Diagnostics 12, (2022).
Perovic D et al. Chromosomal microarray in postnatal diagnosis of congenital anomalies and neurodevelopmental disorders in Serbian patients. J Clin Lab Anal 36, (2022).

Competing interest reported. P.C.M. and C.R. declare that they serve as genomic analyst and consultant for DASA, respectively. All other authors report no conflict of interest relevant to this article.

Download PDF

Version 1

posted

You are reading this latest preprint version

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

Status:

Version 1

Abstract

Figures

INTRODUCTION

PATIENTS AND METHODS

Casuistic

Low-pass whole genome sequencing (LP-WGS)

CNV analysis and clinical interpretation

RESULTS

Phenotype overview

Diagnostic yield

DISCUSSION

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1