Example use cases

In this section, we provide illustrative examples of how AION can help identify causative variants in patients.

To run these cases, simply follow these steps:

1. Download the VCF files to your computer

2. Go to AION → Submit data and follow the instructions here Creating a case for how to submit your data correctly

3. Run analysis

Then follow the usual results analysis procedure.

Case 1

Case 1 is a patient affected by a very specific ophthalmological disease. A variant in CNGB1 was strongly prioritized in Rank #1 due to a very strong match between the candidate disease and patient's phenotype, and also due to being an homozygous variant in a gene associated to autosomal recessive inheritance. Furthermore, this variant was also correctly prioritized thanks to its description in Clinvar, even if it was annotated as "Conflicting interpretations" (as some submitters annotated it as Pathogenic, others as Likely Pathogenic), showing how AION correctly leverages information from databases.

Sample VCF:

• Patient sample (Female, affected): aion_case1.vcf

Reference genome: hg19

HPO terms:

• HP:0000510, HP:0000618

  • HP:0000510 (Rod-cone dystrophy)

  • HP:0000618 (Blindness)

Causative variant:

• CNGB1 - chr16:57935275:T>A (homozygous)

Case 2

Case 2 is a patient affected by neurodevelopmental delay. A variant in KIF1A was prioritized in rank #1, not only due to phenotype matching between the patient and the candidate disease, but also due to AION's ML score prediction of pathogenicity (91%). After performing variant segregation in the parents, this variant was shown to be de novo (absent from the parents), thus confirming its pathogenicity. Interestingly, this variant was later annotated as Likely Pathogenic in ClinVar due to description in other patients. This case illustrates how AION machine learning algorithms can perform correct predictions of pathogenicity in cases where the variant has not been previously described in databases!

Sample VCF:

• Patient sample (Female, affected): aion_case2.vcf

Reference genome: hg19

HPO terms:

• HP:0012758, HP:0000639, HP:0003477

  • HP:0012758 (Neurodevelopmental delay)

  • HP:0000639 (Nystagmus)

  • HP:0003477 (Peripheral axonal neuropathy)

Causative variant:

• KIF1A - ch2:241723208:A>G (p.Leu249Pro, heterozygous)

Case 3

Case 3 is a patient affected by a multisystemic disease including delayed bone growth. Very accurate phenotypic description enabled the identification of 2 previously undescribed variants in ADAMTSL2, which have later been confirmed as pathogenic . Interestingly, these 2 variants are in compound heterozygosis, exemplifying how AION is able to identify and prioritize compatible compound heterozygous variants, even when none of them have been previously described. Of note, these 2 variants were also prioritized on top ranks (#1 and #2) even if this trio case is run without parents. Nevertheless, running trio samples helps to shorten “diagnostic odyssey” because having segregation information prevents geneticists to perform additional tests (e.g. Sanger sequencing) to confirm variant cosegregation in parents.

Sample VCF:

• Patient sample (Male, affected): aion_case3_proband.vcf.gz

• Father sample: aion_case3_father.vcf.gz

• Mother sample: aion_case3_mother.vcf.gz

Reference genome: hg19

HPO terms:

• HP:0002750, HP:0001263, HP:0000233, HP:0003097, HP:0001156, HP:0004322, HP:0010049, HP:0003022, HP:0001488, HP:0003066, HP:0000240, HP:0009803, HP:0001561, HP:0002205, HP:0001377, HP:0000343

  • HP:0002750 (Delayed skeletal maturation)

  • HP:0001263 (Global developmental delay)

  • HP:0000233 (Thin vermilion border)

  • HP:0003097 (Short femur)

  • HP:0001156 (Brachydactyly)

  • HP:0004322 (Short stature)

  • HP:0010049 (Short metacarpal)

  • HP:0003022 (Hypoplasia of the ulna)

  • HP:0001488 (Bilateral ptosis)

  • HP:0003066 (Limited knee extension)

  • HP:0000240 (Abnormality of skull size)

  • HP:0009803 (Short phalanx of finger)

  • HP:0001561 (Polyhydramnios)

  • HP:0002205 (Recurrent respiratory infections)

  • HP:0001377 (Limited elbow extension)

  • HP:0000343 (Long philtrum)

Causative variants:

• ADAMTSL2 - chr9:136435570:G>T (p.Glu845Ter, heterozygous)

• ADAMTSL2 - chr9:136412195:G>A (p.Gly267Ser, heterozygous)

Case 4

Case 4 is a patient affected by a neurodevelopmental disorder with very characteristic facial features. In this case, a missense variant in NIPBL was strongly prioritized in Rank #1 due to a very strong match between the candidate disease and patient's phenotype. This variant was shown to be de novo thanks to trio sequencing; nevertheless, when running this case as a singleton, this variant was also prioritized in rank #1, proving how AION is able to prioritize causative variants with little or no information from databases. While in the trio scenario prioritization would be driven by our implementation of ACMG criteria (tagging this variant as Pathogenic), in the singleton scenario we can see that prioritization is also driven by AION's ML score prediction of pathogenicity (ML 96%), based on our in-house machine learning model and annotation from >100 different databases.

Sample VCF:

• Patient sample (Male, affected): aion_case4_proband.vcf.gz

• Father sample: aion_case4_father.vcf.gz

• Mother sample: aion_case4_mother.vcf.gz

Reference genome: hg19

HPO terms:

• HP:0000750, HP:0000219, HP:0000252, HP:0005280, HP:0008897, HP:0000343, HP:0000294, HP:0002342, HP:0000664, HP:0007665

  • HP:0000750 (Delayed speech and language development)

  • HP:0000219 (Thin upper lip vermilion)

  • HP:0000252 (Microcephaly)

  • HP:0005280 (Depressed nasal bridge)

  • HP:0008897 (Postnatal growth retardation)

  • HP:0000343 (Long philtrum)

  • HP:0000294 (Low anterior hairline)

  • HP:0002342 (Intellectual disability, moderate)

  • HP:0000664 (Synophrys)

  • HP:0007665 (Curly eyelashes)

Causative variant:

• NIPBL - chr5:37044760:G>T (p.Cys2091Phe, heterozygous)

Case 5

Case 5 is a patient affected by a severe immunological disorder. Partly thanks to accurate laboratory-related HPOs (e.g. Reduced granulocyte CD18 level), an homozygous loss-of-function variant in FERMT3 was strongly prioritized in Rank #1. This case exemplifies how important is an accurate phenotypic characterization, even of HPOs that are not related to visible physical symptoms.

Sample VCF:

• Patient sample (Male, affected): aion_case5_proband.vcf.gz

• Father sample: aion_case5_father.vcf.gz

• Mother sample: aion_case5_mother.vcf.gz

Reference genome: hg19

HPO terms:

• HP:0002716, HP:0032455, HP:0001433, HP:0010701

  • HP:0002716 (Lymphadenopathy)

  • HP:0032455 (Reduced granulocyte CD18 level)

  • HP:0001433 (Hepatosplenomegaly)

  • HP:0010701 (Abnormal immunoglobulin level)

Causative variant:

• FERMT3 chr11:63978208:C>T (p.Gln96Ter, homozygous)

Case 6

Case 6 is a patient affected by a congenital gastrointestinal disorder. In this case, AION was decisive to provide a quick diagnosis to a newborn patient. By identifying these 2 variants in SLC9A3 (coding Na(+)/H(+) antiporter 3, NHE3 protein) in compound heterozygosis, described in ClinVar, in a case with a very strong phenotypic match with the candidate disease, a fast diagnosis of this case was possible, enabling a better managing of this patient since a very young age and genetic counselling.

Sample VCF:

• Patient sample (Male, affected): aion_case6_proband.vcf.gz

• Father sample: aion_case6_father.vcf.gz

• Mother sample: aion_case6_mother.vcf.gz

Reference genome: hg19

HPO terms:

• HP:0032368, HP:0001518, HP:0032484, HP:0001561, HP:0001510, HP:0005208, HP:0003270, HP:0012604, HP:0032487

  • HP:0032368 (Acidemia)

  • HP:0001518 (Small for gestational age)

  • HP:0032484 (Elevated fecal sodium)

  • HP:0001561 (Polyhydramnios)

  • HP:0001510 (Growth delay)

  • HP:0005208 (Secretory diarrhea)

  • HP:0003270 (Abdominal distention)

  • HP:0012604 (Hyponatriuria)

  • HP:0032487 (Reduced fecal osmolality)

Causative variants:

• SLC9A3, chr5:483385:C>T (p.Arg382Gln, heterozygous)

• SLC9A3, chr5:477461:AG>A (p.Ser582Leufs, heterozygous)

Case 7

Case 7 is a patient affected by nonsyndromic intellectual disability, speech impairments and motor delay, described in more detail this paper (SRR11604298). Patients data has been analysed with Nostos DRAGEN secondary analysis pipeline for small variants, structural variants (SVs) and copynumber variants (CNVs).

The causative variant, a large duplication in chrX has been strongly prioritised in Rank #1 thanks to correct ACMG classification powered by annotation of overlapping known pathogenic CNV in dbVar, as well as strong clinical overlap between the patient and the diseases linked to associated genes.

Sample VCFs:

• Patient small variant sample (affected , male): aion_case7_proband.vcf.gz

• Patient CNV sample: aion_case7_cnv.vcf.gz

• Patient SV sample: aion_case7_SV.vcf.gz

Reference genome: hg19

HPO terms:

• HP:0001249, HP:0000750, HP:0001270

  • HP:0001249 (Intellectual disability)

  • HP:0000750 (Delayed speech and language development)

  • HP:0001270 (Motor delay)

Causative variant:

• chrX:53357561-53645092 duplication affecting the following disease associated genes: HSD17B10, HUWE1, SMC1A