Table 2.

Classification of diagnoses (n=240) and case examples describing UDN-driven investigations leading to diagnoses.

Classification of diagnosis		Example Case Description	Diagnosis	UDN-driven investigations to establish diagnosis
Clinical diagnosesa (n=16, 7%)		Case 1: 22y male with episodic thrombocytopenia, lymphopenia, neutropenia, recurrent fevers	Autoimmune thrombocytopenic purpura	Complementation/supplementation of prior clinical data (aggregate evidence led to diagnosis): B cell flow cytometry and Fas mediated apoptosis normal. Intermittent anti-platelet antibodies. Recurrent severe episodes of thrombocytopenia, (neutropenia possibly secondary to treatment), responded to anti-CD20 therapy but complicated by serum sickness, ultimately stabilized after bone marrow transplant on NIH protocol treating refractory autoimmune disease. This is a non-genetic diagnosis.
		Case 2: 60y male with periodic fevers, monthly episodes lasting 7–10 days	Waldenstrom macroglobulinemia	Complementation/supplementation of prior clinical data (aggregate evidence led to diagnosis): IgM elevation mild but bone marrow aspirate flow cytometry showed 1% clonal population of kappa-restricted plasmacytoid CD38/CD138+ cells. This is a non-genetic diagnosis.
Diagnoses due to phenotype-directed testingb (n=29, 12%)	Non-genetic laboratory testing (n=12)	Case 3: 26y female with progressive limb girdle weakness	Anti-HMGCR myopathy	Complementation/supplementation of prior clinical data (neurology evaluation) and subsequent targeted testing: Intermittent weakness was consistent with autoimmune myopathy: anti-HMGCR and anti-MDA5 antibodies identified on myositis panel (not included in myositis panel prior to UDN). This is a non-genetic diagnosis.
		Case 4: 29y male with acquired transfusion-dependent dyserythropoietic anemia.	Autoimmune dyserythropoietic anemia	Complementation/supplementation of prior clinical data (bone marrow aspirate) and subsequent targeted testing: Bone marrow aspirate pathology led to molecular study: increased population of gamma-delta T cells likely mediating cytotoxic immune response against erythroid precursors. Responded to cyclosporine. This is a non-genetic diagnosis.
	Single gene testing (n=17)	Case 5: 4y female with neurodevelopmental regression and non-diagnostic pre-UDN ES	PLA2G6-associated Neurodegeneration with brain iron accumulation 2B (MIM#256600)	Complementation/supplementation of prior clinical data (neurology evaluation) and subsequent targeted testing : Neurological phenotype consistent with NBIA: MLPA for PLA2G6 revealed novel homozygous deletion encompassing noncoding exon 1, with RT-PCR demonstrating absent PLA2G6 expression.
		Case 6: 39y male with cognitive decline, personality changes, cerebral volume loss, and non-diagnostic pre-UDN ES	Frontotemporal dementia (MIM#105550)	Complementation/supplementation of prior clinical data (consultation with neurology expert at another UDN clinical site) and subsequent targeted testing: Progressive phenotype was consistent with C9orf72 repeat expansion disease; testing showed >44 repeats, not detectable on ES
Diagnoses stemming from ES/GS and downstream analysesc (n=190, 79%)	Exome sequencing (n=116)	Case 7: 6m male with severe congenital hypotonia and fine tremor and non-diagnostic pre-UDN ES	Congenital myopathy with tremor (MIM# 618524), due to de novo variant in MYBPC1 NM 002465.3:c.776T>C (p.Leu259Pro)	Innovative analysis of genomic data (reanalysis of pre-UDN non-diagnostic ES) and collaborations outside network (case-matching and functional assays): Detected on UDN reanalysis of raw ES data. Variant was not reported on pre-UDN ES due to perceived poor phenotypic fit. Three other similarly affected cases identified, leading to new disease association for MYBPC1.
		Case 8: 20m male with infantile spasms, microcephaly, lamellar cataracts, developmental delays and non-diagnostic pre-UDN ES	Neurodevelopmental disorder with epilepsy, cataracts, feeding difficulties and delayed brain myelination (MIM#617393), due to de novo variant in NACC1 NM_052876.3:c.892C>T (p.Arg298Trp)	Innovative analysis of genomic data (reanalysis of pre-UDN sequences) and collaborations within and outside network (case-matching): Reanalysis of pre-UDN ES data prioritized a de novo variant in NACC1. Additional individuals identified through GeneMatcher and the UDN patient webpage, leading to new disease gene identification.
	Genome sequencing (n=74)	Case 9: 10y female with congenital neuropathy, muscle weakness, calf atrophy, and abnormal gait, and non-diagnostic pre-UDN ES	Charcot-Marie-Tooth disease, axonal, type 2S (MIM#616155), caused by biallelic variants in IGHMBP2 NM_002180.2:c.1730T>C (p.Leu577Pro) and c.1235+894C>A	Innovative analysis of genomic data (dual analysis of UDN sequence data by clinical site) and functional assay at clinical site Maternal missense pathogenic variant reported on pre-UDN ES. Clinical site reanalysis of UDN GS data revealed a paternally inherited cryptic intronic splice site acceptor. Direct sequencing of IGHMBP2 cDNA with and without a nonsense mediated decay (NMD) inhibitor confirmed inclusion of a mini pseudoexon in mRNA. The abnormal transcript was subject to NMD
		Case 10: 31y female with adult-onset, slowly progressive distal asymmetric myopathy with scapular winging, mild facial weakness and non-diagnostic pre-UDN ES	Multisystem proteinopathy 3 (MIM#615424), caused by a de novo heterozygous 500 base pair deletion encompassing exon 10 of HNRNPA1 NM_031157.3:c.1063+15_*5-68del	Innovative analysis of genomic data (dual analysis of UDN sequence data by clinical site + RNASeq) and collaborations within network (between the clinical site and the sequencing core) Collaborative analysis of UDN GS data by the clinical site and sequencing core identified the deletion which was confirmed by qPCR. RNASeq on patient fibroblasts confirmed that the aberrant transcript escapes NMD and results in truncation of the M9 protein domain.
		Case 11: 6y female with macrocephaly, developmental delay and dysmorphic features, and non-diagnostic ES	Kleefstra syndrome type 2, caused by a heterozygous 127 kb deletion of 7q36.1 involving exons 8-55 of KMT2C	Innovative analysis of genomic data (dual analysis of UDN sequence data and manual curation by clinical site): Not reported as diagnostic due to size and presence of deletions in normal individuals; however, manual curation of KMT2C isoforms by the clinical site revealed that this distal deletion occurred in the isoform most expressed in brain tissue, whereas the deletions in healthy individuals in DGV were proximal and not in the brain expressed isoform.
Diagnoses on non-sequencing, genome-wide diagnostic assay: Chromosome microarray (CMA)d (n=5, 2%)		Case 12: 10y female with severe developmental delay, absent speech, joint laxity, tethered cord, and non-diagnostic pre-UDN ES	Wieacker-Wolff syndrome (MIM#314580), due to a de novo heterozygous 95 kb deletion involving exon 1 of ZC4H2	Innovative analysis of genomic data (CMA performed on more advanced platform): Deletion was detected on a high density CMA. Pre-UDN SNP-based CMA did not detect the deletion, likely due to being below the level of resolution. Reason for nondetection in UDN GS unclear.

^aClinical diagnoses: Defined as diagnosis made on clinical grounds, including aggregate assessment of non-specific test results: also conferred when clinical diagnostic criteria were met, or pathognomonic signs were present

^bDiagnoses due to phenotype-directed testing: When the clinical manifestations were suggestive of a disorder or group of disorders, further targeted genetic or non-genetic laboratory tests were performed or reviewed and led to diagnoses. These were further classified into “non-genetic laboratory testing” and “single gene testing”

^cDiagnoses stemming from ES or GS and downstream analyses: These included diagnoses with first-pass ES or GS, or with further innovative genomics and network and outside collaborations involving animal modeling and functional assays.

^dDiagnoses on non-sequencing, genome-wide diagnostic assay: Chromosome microarray (CMA): CMA performed with more sensitive platform, or when it had not been obtained prior to the UDN

HMGCR = 3-hydroxy-3-methylglutaryl-coenzyme A reductase; MDA5= melanoma differentiation-associated gene 5; ES=exome sequencing; GS= genome sequencing; NBIA= Neurodegeneration with brain iron accumulation; MLPA= Multiplex ligation-dependent probe amplification; DGV= Database of Genomic Variants