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Factor I deficiency due to homozygosity for a novel mutation in the CFI gene
Abstracts / Molecular Immunology 89 (2017) 147–151
variations in aHUS patients in whom no mutation was observed in the traditional disease-associated genes. http://dx.doi.org/10.1016/j.molimm.2017.06.100 067 Factor I deficiency due to homozygosity for a novel mutation in the CFI gene Lillemor Skattum 1,∗ , Åsa Hellberg 2 , Sólveig Óskarsdóttir 3 , Martin L. Olsson 2 1 Section of Microbiology, Immunology and Glycobiology, Department of Laboratory Medicine, Lund University, and Clinical Immunology and Transfusion Medicine, Laboratory Medicine, Office of Medical Services, Region Skåne, Lund, Sweden 2 Division of Hematology and Transfusion Medicine, Department of Laboratory Medicine, Lund University, and Clinical Immunology and Transfusion Medicine, Laboratory Medicine, Office of Medical Services, Region Skåne, Lund, Sweden 3 Queen Silvia Children’s Hospital, Sahlgrenska University Hospital, and Department of Pediatrics, The Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
Background: A girl, the third child of healthy but consanguineous parents from Iraq, presented with recurrent otitis media, appearing from the age of 6 weeks. This was explained by severe neutropenia found at 6 months of age. However, infections relapsed after normalization of neutrophil counts and at the age of 19 months she suffered the first of several invasive pneumococcal infections. Further immunological investigation was undertaken including complement deficiency screening. Materials and methods: Complement function was analyzed by ELISA. Plasma concentration of C3 was measured by nephelometry, while serum concentrations of factor I, properdin and factor H were measured by immunoelectrophoresis. Genetic analysis was performed by DNA sequencing of the 13 exons of CFI. Results and conclusions: Both alternative and classical complement activation pathway function was low by ELISA. Further investigation revealed low C3 concentration at 0.3 g/L (reference interval 0.77–1.38 g/L) and low to moderately low concentrations of properdin and factor H. Factor I was not detectable with the applied method; < 5% of a normal serum pool (reference interval 60–152%). Thus, factor I deficiency was suspected and family investigation showed moderately low factor I serum concentration in the patient’s mother (29%), father (35%) and two brothers (26 and 37%, respectively). Sequencing of CFI showed that the patient was homozygous for a novel mutation, c.1399T > C, in exon 11, resulting in a predicted amino acid change, p.Cys467Arg. Amino acid 467Cys is phylogenetically conserved and part of a cysteine bond likely to influence the tertiary structure of the factor I molecule. Both parents were heterozygous carriers of the same mutation. The patient was given prophylactic antibiotics to prevent further episodes of serious infections. This case illustrates the importance of recognizing complement deficiency as a cause of serious infections. http://dx.doi.org/10.1016/j.molimm.2017.06.101
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068 Analysis of complement gene variants in the clinical laboratory: Comparison of Next-Generation Sequencing (NGS) and Sanger methods Susan A. Lagerstedt 1,∗ , Dorottya Csuka 2 , Ágnes Szilágyi 2 , Zoltán Prohászka 2 , Roshini S. Abraham 1 1
Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA 2 Semmelweiss University, Budapest, Hungary Background: Complement genetic testing has conventionally used Sanger sequencing to identify pathogenic variants for atypical hemolytic uremic syndrome (aHUS) and/or thrombotic microangiopathy (TMA), and infections associated with complement deficiencies. Our laboratory has developed disease-targeted panels for complement defects using a clinically-validated NGS approach. The advantage of NGS for clinical use is the ability to evaluate several regions and samples simultaneously, which permits cost-effectiveness when multiple genes/exons are assessed. Materials and methods: To evaluate the sensitivity and specificity of NGS vs. Sanger, we used two cohorts of well-characterized disease samples. We compared variant calls in 10 independent patients with aHUS for 7 genes (C3, CD46 (MCP), CFB, CFH, CFHR5, CFI, and THBD). We also compared variant calls in 12 patients with complement deficiency for 6 genes (C4A/C4B, C2, F12, MBL2, and SERPING1). Results and conclusions: In the aHUS comparison, we observed 98 concordant variant occurrences (47 unique variants) and one discordant variant call. CFH c.3590 T > C in exon 22 was not detected by the NGS method, but was confirmed to be present by multiple Sanger reactions using different primer sets. CFH exon 22 lies in a region of homology with high potential for gene rearrangement and segmental duplication. We have selected several exons within CFH, CFHR1, CFHR3, and CFHR4 that will require supplemental Sanger sequencing. In the complement deficiency cohort, we observed 13 concordant variant occurrences (10 unique variants). One of these variants, C4A/C4B c.5026C > T was not displayed in the NGS variant calling file, however it was observed visually in the NGS BAM file and confirmed by supplemental Sanger sequencing. The homology of C4A and C4B genes requires supplemental analysis for variant detection. While Next Generation Sequencing (NGS) has revolutionized molecular diagnostics, many complex regions of DNA including homologous regions, gene hybrids, and structural variations still evade detection. Using a disease-targeted NGS approach, such areas can be identified and supplemented with alternate testing strategies. http://dx.doi.org/10.1016/j.molimm.2017.06.102
variations in aHUS patients in whom no mutation was observed in the traditional disease-associated genes. http://dx.doi.org/10.1016/j.molimm.2017.06.100 067 Factor I deficiency due to homozygosity for a novel mutation in the CFI gene Lillemor Skattum 1,∗ , Åsa Hellberg 2 , Sólveig Óskarsdóttir 3 , Martin L. Olsson 2 1 Section of Microbiology, Immunology and Glycobiology, Department of Laboratory Medicine, Lund University, and Clinical Immunology and Transfusion Medicine, Laboratory Medicine, Office of Medical Services, Region Skåne, Lund, Sweden 2 Division of Hematology and Transfusion Medicine, Department of Laboratory Medicine, Lund University, and Clinical Immunology and Transfusion Medicine, Laboratory Medicine, Office of Medical Services, Region Skåne, Lund, Sweden 3 Queen Silvia Children’s Hospital, Sahlgrenska University Hospital, and Department of Pediatrics, The Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
Background: A girl, the third child of healthy but consanguineous parents from Iraq, presented with recurrent otitis media, appearing from the age of 6 weeks. This was explained by severe neutropenia found at 6 months of age. However, infections relapsed after normalization of neutrophil counts and at the age of 19 months she suffered the first of several invasive pneumococcal infections. Further immunological investigation was undertaken including complement deficiency screening. Materials and methods: Complement function was analyzed by ELISA. Plasma concentration of C3 was measured by nephelometry, while serum concentrations of factor I, properdin and factor H were measured by immunoelectrophoresis. Genetic analysis was performed by DNA sequencing of the 13 exons of CFI. Results and conclusions: Both alternative and classical complement activation pathway function was low by ELISA. Further investigation revealed low C3 concentration at 0.3 g/L (reference interval 0.77–1.38 g/L) and low to moderately low concentrations of properdin and factor H. Factor I was not detectable with the applied method; < 5% of a normal serum pool (reference interval 60–152%). Thus, factor I deficiency was suspected and family investigation showed moderately low factor I serum concentration in the patient’s mother (29%), father (35%) and two brothers (26 and 37%, respectively). Sequencing of CFI showed that the patient was homozygous for a novel mutation, c.1399T > C, in exon 11, resulting in a predicted amino acid change, p.Cys467Arg. Amino acid 467Cys is phylogenetically conserved and part of a cysteine bond likely to influence the tertiary structure of the factor I molecule. Both parents were heterozygous carriers of the same mutation. The patient was given prophylactic antibiotics to prevent further episodes of serious infections. This case illustrates the importance of recognizing complement deficiency as a cause of serious infections. http://dx.doi.org/10.1016/j.molimm.2017.06.101
149
068 Analysis of complement gene variants in the clinical laboratory: Comparison of Next-Generation Sequencing (NGS) and Sanger methods Susan A. Lagerstedt 1,∗ , Dorottya Csuka 2 , Ágnes Szilágyi 2 , Zoltán Prohászka 2 , Roshini S. Abraham 1 1
Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA 2 Semmelweiss University, Budapest, Hungary Background: Complement genetic testing has conventionally used Sanger sequencing to identify pathogenic variants for atypical hemolytic uremic syndrome (aHUS) and/or thrombotic microangiopathy (TMA), and infections associated with complement deficiencies. Our laboratory has developed disease-targeted panels for complement defects using a clinically-validated NGS approach. The advantage of NGS for clinical use is the ability to evaluate several regions and samples simultaneously, which permits cost-effectiveness when multiple genes/exons are assessed. Materials and methods: To evaluate the sensitivity and specificity of NGS vs. Sanger, we used two cohorts of well-characterized disease samples. We compared variant calls in 10 independent patients with aHUS for 7 genes (C3, CD46 (MCP), CFB, CFH, CFHR5, CFI, and THBD). We also compared variant calls in 12 patients with complement deficiency for 6 genes (C4A/C4B, C2, F12, MBL2, and SERPING1). Results and conclusions: In the aHUS comparison, we observed 98 concordant variant occurrences (47 unique variants) and one discordant variant call. CFH c.3590 T > C in exon 22 was not detected by the NGS method, but was confirmed to be present by multiple Sanger reactions using different primer sets. CFH exon 22 lies in a region of homology with high potential for gene rearrangement and segmental duplication. We have selected several exons within CFH, CFHR1, CFHR3, and CFHR4 that will require supplemental Sanger sequencing. In the complement deficiency cohort, we observed 13 concordant variant occurrences (10 unique variants). One of these variants, C4A/C4B c.5026C > T was not displayed in the NGS variant calling file, however it was observed visually in the NGS BAM file and confirmed by supplemental Sanger sequencing. The homology of C4A and C4B genes requires supplemental analysis for variant detection. While Next Generation Sequencing (NGS) has revolutionized molecular diagnostics, many complex regions of DNA including homologous regions, gene hybrids, and structural variations still evade detection. Using a disease-targeted NGS approach, such areas can be identified and supplemented with alternate testing strategies. http://dx.doi.org/10.1016/j.molimm.2017.06.102