VarSeq in Action: Deciphering Complex Genetic Puzzles in Immune Thrombocytopenia, Glaucoma, and Rare Syndromes

         December 31, 2023

In this blog, we explore the impactful applications of VarSeq in genetic research and diagnostics. Through case studies, we examine its role in diverse conditions like Immune Mediated Thrombocytopenia (ITP), glaucoma, and Cri du Chat syndrome. These studies demonstrate VarSeq’s capability in identifying genetic variants and influencing diagnosis and treatment across various medical scenarios, showcasing its crucial role in advancing personalized medicine.

High Frequency of Genetic Variants Influencing Diagnosis and Bleeding Pattern in Patients with Presumed Primary Immune Mediated Thrombocytopenia (ITP)

Immune mediated thrombocytopenia (ITP) is a platelet disorder in which the immune system attacks and destroys the body’s own platelets. Mechanisms leading to low platelet count in ITP are multifactorial, involving both increased peripheral platelet destruction and decreased platelet production. This is caused by autoantibodies targeting megakaryocytes and platelet-specific glycoproteins, as well as cytotoxic T cells directly acting on platelets. Although antibodies in some cases can be found in patients with ITP, there is no well-established diagnostic test and the diagnosis is thus, one of exclusion. While most cases of ITP resolve within 3 months with or without treatment, some patients do not respond to conventional treatment and develop a refractory ITP. It is likely that in the group of patients diagnosed with refractory ITP, some does not have an immune mediated thrombocytopenia, but other causes of low counts (PLC)s. With the development of high throughput sequencing and the increasing knowledge of inherited bleeding disorders, the possibility to identify the underlying cause of disease in these patients is steadily growing. In ITP, there is a high degree of interindividual differences in bleeding rates, where some patients have no bleeding symptoms despite of unmeasurable PLC, whereas other patients show severe bleeding symptoms at PLC > 30 x10 9 /L. The underlying etiology of these discrepancies is currently unknown and complicates the clinical care of these patients. We hypothesize that genetic defects influence the bleeding pattern in true ITP patients as well as being one of the underlying causes of thrombocytopenia in patients misdiagnosed with “refractory ITP”. The aim of this pilot study was to investigate whether upfront genetic screening of patients suspected of ITP could be a valuable diagnostic tool to determine presence of inherited thrombocytopenia, secondary thrombocytopenia and genetic variants that might affect bleeding pattern.

An in-silico gene panel consisting of 113 genes known to be associated with bleeding disorders as well as 86 genes associated with ITP (Using the VarSeq gene prioritisation algorithm phorank) was analysed and pathogenicity was classified according to the ACMG/AMP guidelines. 

Einar Ljungström Elmfors, Andreas Ørslev Rasmussen, Nadine G. Andersson, Annika Martensson, Eva Birgitte Leinoe, Maria Rossing, Eva Zetterberg, High Frequency of Genetic Variants Influencing Diagnosis and Bleeding Pattern in Patients with Presumed Primary Immune Mediated Thrombocytopenia (ITP), Blood, Volume 142, Supplement 1, 2023, Page 2584 ISSN 0006-4971,

In vivo identification of angle dysgenesis and its relation to genetic markers associated with glaucoma using artificial intelligence

Purpose: To predict the presence of angle dysgenesis on anterior-segment optical coherence tomography (ADoA) by using deep learning (DL) and to correlate ADoA with mutations in known glaucoma genes.

Participants: In total, 800 high-definition anterior-segment optical coherence tomography (AS-OCT) images were included, of which 340 images were used to build the machine learning (ML) model. Images used to build the ML model included 170 scans of primary congenital glaucoma (16 patients), juvenile-onset open-angle glaucoma (62 patients), and adult-onset primary open-angle glaucoma eyes (37 patients); the rest were controls (n = 85). The genetic validation dataset consisted of another 393 images of patients with known mutations that were compared with 320 images of healthy controls.

Methods: ADoA was defined as the absence of Schlemm’s canal, the presence of hyperreflectivity over the region of the trabecular meshwork, or a hyperreflective membrane. DL was used to classify a given AS-OCT image as either having angle dysgenesis or not. ADoA was then specifically looked for on AS-OCT images of patients with mutations in the known genes for glaucoma.

Results: The final prediction, which was a consensus-based outcome from the three optimized DL models, had an accuracy of >95%, a specificity of >97%, and a sensitivity of >96% in detecting ADoA in the internal test dataset. Among the patients with known gene mutations, ( MYOC, CYP1B1, FOXC1 , and LTBP2 ) ADoA was observed among all the patients in the majority of the images, compared to only 5% of the healthy controls.

Conclusion: ADoA can be objectively identified using models built with DL.

The variant call files (VCFs) generated were analyzed using Golden Helix VarSeq Software v. 1.2.1 (Bozeman, MT). VarSeq variants with read depth <15 and genotype quality score <20 were excluded. To identify rare mutations, variant frequency databases were used to remove variants that were present at high frequencies among large population groups.

Gupta V, Birla S, Varshney T, Somarajan BI, Gupta S, Gupta M, Panigrahi A, Singh A, Gupta D. In vivo identification of angle dysgenesis and its relation to genetic markers associated with glaucoma using artificial intelligence. Indian J Ophthalmol. 2023 Dec 26. doi: 10.4103/IJO.IJO_1456_23. Epub ahead of print. PMID: 38146977.

Esophageal Cancer with Early Onset in a Patient with Cri du Chat Syndrome

Background: In Cri du Chat (CdC), cancer as comorbidity is extremely rare. In databases from Denmark, Spain, Australia, New Zealand, and Japan, no cancer was reported; in Italy and Germany, four cancers were identified out of 321 CdCs. Methods: In a 29-year-old CdC patient, clinical investigations following hematemesis led to the diagnosis of esophageal adenocarcinoma (EAC). A high pain threshold was also observed. Conventional and molecular cytogenetic defined the size of the deletion, and exome analysis on the trio completed the molecular work. Results: Cytogenetic analysis showed a de novo chromosomal alteration: 46,XY,ishdel(5)(p14.3)(D5S28-) and arr[GRCh37] 5p15.33p14.3(1498180_19955760)x1. A quantitative sensory test demonstrated a high heat threshold. A 18f-fluorodeoxyglucose PET/TC scan of the brain failed to detect reduction of metabolism in the somatosensory area or insular cortex. Exome analysis in the trio (patient and parents) failed to identify variants to be interpreted as a likely risk factor for EAC. Conclusion: We conclude that the presence of well-known risk factors (maleness, obesity, gastroesophageal reflux, and Barrett’s metaplasia) in a patient with very limited capability of expressing discomfort or referring clinical symptoms have been the main risk factors for developing EAC. At present, based on the available data, there is no evidence of any increased risk of developing cancer in CdC patients.

“After quality-filtration, the reads were aligned to the reference human genome sequence (GRCh37/hg19) with an ISAAC aligner (Raczy, C. et al. Isaac: ultra-fast whole-genome secondary analysis on Illumina sequencing platforms. Bioinformatics 29, 2041–3, 2013). Variant annotation (limited to variants with a minimum quality score of 20 and a minimum read depth of 30×) was performed with VarSeq v1.4.5 (Golden Helix, Inc., Bozeman, MT, USA). Variants with a population frequency of > 5% (gnomAD) were excluded. For the variants analyzed, the effect on protein function (damaging or tolerated) was reported as in SIFT and Polyphen.”

Danesino, C.; Gualtierotti, M.; Origi, M.; Cistaro, A.; Malacarne, M.; Massidda, M.; Bencardino, K.; Coviello, D.; Albani, G.; Schiera, I.G.; et al. Esophageal Cancer with Early Onset in a Patient with Cri du Chat Syndrome. Diseases 2024, 12, 9.

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