Balkan Journal of Medical Genetics

VALUE OF OPTICAL GENOME MAPPING (OGM) FOR DIAGNOSTICS OF RARE DISEASES: A FAMILY CASE REPORT
Kovanda A1,2, Miljanović O3, Lovrečić L1,2, Maver A1,2, Hodžić A1,2, Peterlin B1,2,*
*Corresponding Author: *Corresponding Author: Prof. Borut Peterlin, Clinical Institute of Genomic Medicine, University Medical Centre Ljubljana, Šlajmerjeva 4, 1000 Ljubljana, Slovenia. borut.peterlin@kclj.si
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MATERIALS AND METHODS

Patients Two undiagnosed male siblings with an overlapping clinical presentation of thrombocytopenia, sacro-coccy- geal teratoma, hydronephrosis/reflux vesicoureteral and obesity, who were referred to the CGMI, CCM, Monte- negro, were enrolled in this family case-report. Clinical data was collected during the patients’ in- person appointments and evaluation by clinical geneticist at the CGMI, CCM, and all specialist examinations, were performed as part of standard routine clinical care. Before genetic testing, pre-test genetic counseling was provided by a clinical geneticist, followed by obtaining written parental consent at the CGMI, CCM. All procedures in the study were conducted according to the routine standard of care and in accordance with the principles of the Declaration of Helsinki. Karyotyping was performed at the CGMI, CCM, while mi- croarray analyses, exome sequencing and optical genome mapping were performed at the CIGM, UMCL, Slovenia. Karyotype Chromosome analysis was performed for both pro- bands, by using G-bended karyotyping (bend resolution 400 – 470, according to ISCN), after 72 hours of peripheral blood cultivation. Microarray analyses Microarray analysis was initially performed on the probands and their parents by using oligonucleotide ar- ray Agilent Technologies 4×180K (AMADID:035689), according to the manufacturer’s instructions. Agilent Cy- toGenomics 5.1.2.1 software was used to visualize and report the CNVs, as previously described 12. Exome sequencing Exome sequencing of proband 1 and proband 2 with parents in trio setup was performed as previously described 13,14, and included the analysis of a total of >2000 genes associated with the clinical phenotype of the probands. The full list of genes for each of the included gene panels is available in the Supplement. Optical genome mapping Optical genome mapping was performed as previ- ously described12. Briefly, high-weight molecular DNA was extracted from 1.5 million lymphocytes from whole blood (EDTA collected) using the SP Blood & Cell Culture DNA Isolation Kit or the SP-G2 Blood & Cell Culture DNA Isolation Kit following manufacturer instructions (Bionano Genomics Inc., San Diego USA). The follow- ing day, DNA molecules were labeled with the DLE-1 enzyme using the Direct Label and Stain (DLS) Kit or Direct Label and Stain-G2 (GLS-G2) kit (Bionano Genom- ics Inc.). Labeled DNA was loaded on the three-flowcell Saphyr Chip® G2.2 or G2.3 (Bionano Genomics Inc.) and ran on the Saphyr instrument (Bionano Genomics Inc.) to reach a minimum yield of 500 Gbp (DLE-1 label, [GRCh38] reference genome). The de novo assembly and Variant Annotation Pipeline were executed on Bionano Solve3.7_20221013_25 while reporting and direct visual- ization of SVs was done on Bionano Access 1.7.2. Variant interpretation We reported only those genomic variants that have statistical support based on the adequate genomic cover- age and chosen analysis type for the detection of CNV, duplications, deletions, and other SVs such as insertions, inversion, intra- and inter-chromosomal translocations, as determined by internal Access QC parameters. The method does not enable the analysis of regions that do not contain DLE-1 labeling sites (centromeres, telomeres, and other heterochromatin regions). According to the ACMG and ClinGen guidelines15, CNV variants are classified into one of five classes of pathogenicity based on the sum of points in each category of assessment, and were classi- fied by comparison with their overlap with SV and CNV variants contained in the DGV (Database of Genomic Variants - http://dgv.tcag.ca/gb2/gbrowse/dgv2_hg19/ )16, gnomAD (genome Aggregation Database - https://gno- mad.broadinstitute.org/), OGM (Bionano Genomics Inc. internal Access® database), ClinGen (Clinical Genome Resource Consortium) (https://dosage.clinicalgenome. org/), DECIPHER (https://www.deciphergenomics.org/), and/or ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/) public databases and the CIGM genomic variant database. OGM results are given according to the genome mapping nomenclature as specified in the ISCN guidelines17. Visualization and figure preparation Figure 1 was prepared from original visualizations generated by Bionano Access 1.7.2 software (Bionano), segregation, and optical genome mapping, respectively. The final composite Figure 1 was technically prepared in terms of size, layout, format, and type of file with no modification to the original data, from the original vi- sualizations, by using GIMP 2.1018. The pedigree was constructed and drawn using (Progeny Clinical Version N/Progeny Lab Version N) (Progeny Genetics LLC, Aliso Viejo, CA, www.progenygenetics.com).

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