Additional file 1 of A rare mutation (p.F149del) of the NT5C3A gene is associated with pyrimidine 5′-nucleotidase deficiency
Creators: name: Bogusławska, Dżamila M.
name: Skulski, Michał
name: Bartoszewski, Rafał
name: Machnicka, Beata
name: Heger, Elżbieta
name: Kuliczkowski, Kazimierz
name: Sikorski, Aleksander F.
Publisher: name: figshare
Year: 2022
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 1: Supplementary figures and tables. This file contains supplementary materials: Figure S1.1 Constructs used to analysis of the post-transcriptional activity of the 3'-UTR regions of the human RBC’s specific nucleotidase isoform. Figure S1.2 A. Primer sequences for bisulfite sequencing PCR (BSP) used for detecting converted templates. B. Sequencing chromatogram for patient RK. Bisulfite converted DNA showing full conversion C → T in the CpG-rich fragment of the NT5C3A gene. The absence of a C-peak indicates the absence of 5-methylcytosine (5mC). Figure S1.3 Analysis of the workflow applied to prioritize variants found in the whole-exome sequencing (WES) data from the probands. Figure S1.4 The rare NT5C3A gene mutation (rs1227859962) is associated with pyrimidine-5′-nucleotidase deficiency. Fragment of sequencing traces of the NT5C3A gene in an affected patient (RK and MK) and asymptomatic family member (EK and AK). Figure S1.5 Functional analysis. A. Significant decline in enzymatic activity in HA patients’ erythrocyte hemolysate (erythrocytes) caused by lack of cytosolic pyrimidine 5ʹ-nucleotidase. B. Purine/pyrimidine ratio measurement in erythrocytes. HA patients demonstrated 2–2.5 times lower (1.26 ± 0.09) ratio than the control (Ctrl) group (3.04 ± 0.55). Figure S1.6 According to the AlphaFold Protein Structure Database (AF-A0A090N7U2-F1) the substrate binding site (marked in red) (NM_001166118.3:Ser152Ala153) and the phenyl-alanine crucial for both HA patients (NM_001166118.3:Phe149; marked in pink) are located in the β4 strand of the reticulocyte 5ʹ-nucleotidase (NM_001166118.3: 199-155aa; VFIFSAGI). Table S1.1 Blood test results of the studied HA patient (RK). Table S1.2 PCR primer sequences used for NT5C3A genotyping. Table S1.3 PCR primer sequences used for amplification of the transcript variant 4 of the NT5C3A gene. Table S1.4 Primer sequences for bisulfite sequencing PCR (BSP) and control PCR (primers for gDNA) used for detecting converted/unconverted templates. Both primer pairs were homologous to sequences with a similar chromosomal location. Table S1.5 Complete blood count (CBC) results of the studied family members. Table S1.6 Quality control of WES raw reads. Table S1.7 Summary of variants (mutations/polymorphisms) types of translation impact for all variants identified in both patients with HA. Table S1.8 Polymorphisms/mutations present in both brothers involved in RBC pathologies identified using the WES analysis of studied family members. Table S1.9 Comparison between quantity of mutated genes (mutations/polymorphisms present in both brothers) obtained from Exome-Seq analysis, and respective genes deposited in the UniGene NCBI databases: CD71++++ library (Lib.8975) and GPA++ library (Lib.11923). Table S1.10 List of variants whose frequency does not exceed 10% identified in the studied family using the WES analysis. Table S1.11 Number of variants potentially significant for the HA phenotype identified by WES and filtered by Ingenuity Variant Analysis tool in the studied family members. Table S1.12 Selectively enriched biological pathways were identified with gene set enrichment analysis. Table S1.13 List of variants identified in cDNA using Sanger sequencing. Table S1.14 Polymorphisms/mutations present in studied family members located in the NT5C3A gene identified using the Sanger method. Table S1.15 Polymorphisms/mutations present in both brothers located in genes encoding known 5′-nucleotidases involved in pyrimidine and purine metabolism identified using the WES analysis of studied family members. Table S1.16 List of genes encoding known 5′-nucleotidases involved in pyrimidine and purine metabolism and their biological function.