Pitfalls When Determining HNA-1 Genotypes and Finding Novel Alleles
Abstract
:1. Introduction
2. Results
2.1. In-House TaqMan PCR Genotyping Data
2.2. Commercial PCR-SSP Genotyping Dats
2.3. Multiplex Ligation-Dependent Probe Amplification Data
2.4. PCR-SBT Data
2.5. Long-Read Nanopore Sequencing Data
2.6. Novel Alleles
2.7. Segregation of Alleles through Family Studies
3. Discussion
4. Methods
4.1. Study Cohort
4.2. Validation Cohort
4.3. In-House TaqMan PCR and Commercial PCR-SSP Testing
4.4. Real-Time qPCR CN Investigation
4.5. Multiplex Ligation-Dependent Probe Amplification Testing
4.6. PCR-SBT Analysis
4.7. Long-Read Nanopore Sequencing
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
References
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New Nomenclature | c.108 | c.114 | c.194 | c.233 | c.244 | c.316 | STOP CODON | ||||
---|---|---|---|---|---|---|---|---|---|---|---|
Old Nomenclature | c.141 | c.147 | c.227 | c.266 | c.277 | c.349 | c.473 | c.505 | c.559 | c.641 | c.733 |
rs no. | rs200688856 | rs527909462 | rs448740 | rs5030738 | rs147574249 | rs2290834 | rs71632959 | rs71632958 | rs200215055 | rs758550229 | rs374752953 |
Pos (Hg38) | 161,629,989 | 161,629,983 | 161,629,903 | 161,629,864 | 161,629,853 | 161,629,781 | 161,626,282 | 161,626,250 | 161,626,196 | 161,624,609 | 161,624,517 |
Exons in FCGR3B | 3 | 4 | 5 | ||||||||
Amino acid change | Arg36Ser | 38Leu | Asn65Ser | Ala78Asp | Asp82Asn | Val106Ile | Val176Phe | ||||
FCGR3B*01 | G | C | A | C | G | G | A | C | G | C | T |
FCGR3B*02 | C | T | G | C | A | A | A | C | G | C | T |
FCGR3B*03 | C | T | G | A | A | A | A | C | G | C | T |
FCGR3B*04 (FCGR3B*01 var c.349G) | G | C | A | C | G | A | A | C | G | C | T |
FCGR3B*05 (FCGR3B*02 var c.244G) | C | T | G | C | G | A | A | C | G | C | T |
FCGR3B*01 var c.194G | G | C | G | C | G | G | A | C | G | C | T |
FCGR3B*02 var c.194G | C | T | A | C | A | A | A | C | G | C | T |
FCGR3A | G | C | G | C | G | A | G | T | T | T | C |
Method | Type | SNPs Included | CN Included | FCGR3B Specific | Detect Hemizygosity 1 | Type >2 Alleles | Detect Rare Variants | |||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
c.108 | c.114 | c.194 | c.233 | c.244 | c.316 | |||||||
In-house | TaqMan PCR | x | x | x | x | x | No | No | Yes/no 2 | No | Yes/no 3 | |
ERY Q | PCR-SSP/Gel | x | x | No | Yes | No | No | No | ||||
MLPA | Genescan | x | x | x | x | Yes | Yes | Yes | Yes | Yes/no 3 | ||
Sanger sequencing | PCR-SBT | Short fragment sequencing of exon 3 (526 bp) | No | No | No | No | No | |||||
Nanopore sequencing | Target specific amplification | Long-read sequencing (8262 bp) | Yes 4 | Yes | No 5 | Yes | Yes |
Commercial PCR-SSP 1 | qPCR | In-House TaqMan PCR | MLPA | |
---|---|---|---|---|
Father | c.194A(+) c.233C(+) c.233A(-) | CN = 2 | c.114C/T c.194A/G c.233C/C c.244G/A c.316G/A | CN = 2 c.114C/T c.194A/G c.233C/C c.316G/A |
FCGR3B*01/*02 | FCGR3B*01/*02 | FCGR3B*01/*02 | ||
Mother | c.194A(+) c.233C(-) c.233A(-) | CN = 1 | c.114C/C c.194A/G c.233C/C c.244G/G c.316G/A | CN = 1 c.114C c.194A c.233C c.316G |
FCGR3B*01 | FCGR3B*01/*Null 2 | FCGR3B*01/*Null | ||
Child 1 | c.194A(+) c.233C(-) c.233A(-) | CN = 1 | c.114C/C c.194A/G c.233C/C c.244G/G c.316G/A | CN = 1 c.114C c.194A c.233C c.316G |
FCGR3B*01 | FCGR3B*01/*Null 2 | FCGR3B*01/*Null | ||
Child 2 | c.194A(-) c.233C(+) c.233A(-) | CN = 1 | c.114T/C c.194G/G c.233C/C c.244A/G c.316A/A | CN = 1 c.114T c.194G c.233C c.316A |
FCGR3B*02 | FCGR3B*02/*Null 2 | FCGR3B*02/*Null |
Test | Genotype | c.114 | c.194 | c.233 | c.244 | c.316 | |
---|---|---|---|---|---|---|---|
Sample 1 | MLPA | FCGR3B*01/*01/*02 | CCT | AAG | CCC | GGA | GGA |
PCR-SBT | FCGR3B*02/*Null | CT | GG | CC | GA | AA | |
Sample 2 | MLPA | FCGR3B*01/*02/*02 | CTT | AGG | CCC | GAA | GAA |
PCR-SBT | FCGR3B*02/*Null | CT | GG | CC | GA | AA | |
Sample 3 | MLPA/Nanopore | FCGR3B*02/*03/*04 | TTC | GGA | CAC | AAG | AAA |
PCR-SBT | FCGR3B*04/*Null | CT | GG | CA | AG | AA |
Sample ID | CN FCGR3B 1 | c.108 | c.114 | c.194 | c.233 | c.244 | c.316 | FCGR3B Alleles |
---|---|---|---|---|---|---|---|---|
AAL_016 | 0 | A: 0% C: 0% G: 0% T: 0% | A: 0% C: 0% G: 0% T: 0% | A: 0% C: 0% G: 0% T: 0% | A: 0% C: 1% G: 0% T: 0% | A: 0% C: 0% G: 0% T: 0% | A: 0% C: 0% G: 0% T: 0% | *Null/*Null |
BRI_006 | 0 | A: 0% C: 2% G: 0% T: 0% | A: 2% C: 0% G: 2% T: 0% | A: 1% C: 3% G: 0% T: 0% | A: 2% C: 0% G: 2% T: 0% | A: 2% C: 0% G: 1% T: 0% | A: 4% C: 0% G: 0% T: 0% | *Null/*Null |
BRI_008 | 1 | G: 85% | C: 71% | A: 86% | C: 87% | G: 82% | G: 83% | *01/*Null |
AAL_001 | 2 | G: 43% C: 51% | T: 55% C: 40% | G: 47% A: 49% | C: 94% | G: 43% A: 53% | G: 44% A: 51% | *01/*02 |
AAL_011 | 2 | G: 42% C: 52% | T: 58% C: 37% | G: 48% A: 48% | C: 95% | G: 41% A: 54% | G: 43% A: 53% | *01/*02 |
BRI_001 | 2 | G: 42% C: 51% | T: 57% C: 37% | G: 48% A: 47% | C: 94% | G: 42% A: 53% | G: 43% A: 52% | *01/*02 |
BRI_004 | 2 | G: 41% C: 50% | T: 56% C: 36% | G: 47% A: 47% | C: 92% | G: 41% A: 52% | G: 42% A: 51% | *01/*02 |
BRI_005 | 2 | G: 41% C: 50% | T: 56% C: 36% | G: 47% A: 47% | C: 92% | G: 41% A: 52% | G: 42% A: 51% | *01/*02 |
BRI_003 | 2 | G: 43% C: 50% | T: 56% C: 37% | G: 46% A: 49% | C: 47% A: 47% | G: 42% A: 52% | G: 44% A: 50% | *01/*03 |
AAL_008 | 2 | C: 92% | T: 93% | G: 91% | C: 49% A: 48% | A: 93% | A: 96% | *02/*03 |
AAL_005 | 2 | C: 95% | T: 96% | G: 47% A: 52% | C: 97% | A: 98% | A: 97% | *02/*02 var c.194G |
AAL_004 | 2 | G: 43% C: 50% | T: 56% C: 38% | G: 46% A: 49% | C: 93% | G: 88% | G: 45% A: 50% | *01/*05 2 |
AAL_009 | 2 | C: 95% | T: 96% | G: 95% | C: 97% | G: 46% A: 52% | A: 97% | *02/*05 2 |
AAL_010 | 2 | C: 95% | T: 95% | G: 95% | C: 97% | G: 45% A: 53% | A: 97% | *02/*05 2 |
AAL_007 | 2 | G: 41% C: 51% | T: 57% C: 37% | G: 47% A: 47% | C: 93% | G: 84% | A: 93% | *04/*05 2 |
AAL_014 | 3 | G: 56% C: 36% | T: 44% C: 49% | G: 33% A: 61% | C: 60% A: 33% | G: 55% A: 39% | G: 28% A: 65% | *01/*03/*04 |
AAL_002 | 3 | G: 30% C: 65% | T: 68% C: 27% | G: 62% A: 35% | C: 63% A: 32% | G: 29% A: 67% | A: 96% | *02/*03/*04 |
AAL_006 | 3 | G: 28% C: 65% | T: 68% C: 25% | G: 61% A: 34% | C: 63% A: 31% | G: 28% A: 66% | A: 93% | *02/*03/*04 |
AAL_012 | 3 | G: 29% C: 62% | T: 65% C: 26% | G: 59% A: 34% | C: 63% A: 30% | G: 29% A: 64% | A: 94% | *02/*03/*04 |
BRI_002 | 3 | G: 29% C: 64% | T: 67% C: 26% | G: 60% A: 35% | C: 64% A: 30% | G: 29% A: 66% | A: 92% | *02/*03/*04 |
BRI_009 | 3 | G: 30% C: 64% | T: 67% C: 27% | G: 60% A: 36% | C: 64% A: 31% | G: 30% A: 66% | A: 94% | *02/*03/*04 |
BRI_010 | 3 | G: 34% C: 61% | T: 66% C: 30% | G: 57% A: 40% | C: 73% A: 24% | G: 34% A: 63% | A: 83% | *02/*03/*04 |
AAL_015 | 4 | G: 44% C: 50% | T: 56% C: 39% | G: 46% A: 50% | C: 71% A: 24% | G: 43% A: 52% | G: 24% A: 72% | *01/*02/*03/*04 |
Sample ID | CN FCGR3B 1 | c.108 | c.114 | c.194 | c.197 2 | c.233 | c.244 | c.316 | FCGR3B alleles |
---|---|---|---|---|---|---|---|---|---|
AAL_003 | 3 | G: 61% C: 33% | T: 39% C: 55% | G: 60% A: 36% | A: 95% | C: 65% A: 30% | G: 30% A: 65% | A: 95% | *03/*04/Novel (GCGCAA 3) |
AAL_013 | 2 | G: 45% C: 51% | T: 57% C: 40% | G: 95% | A: 95% | C: 97% | A: 97% | A: 97% | *02/Novel (GCGCAA 3) |
BRI_007 | 1 | G: 83% | C: 70% | G: 83% | C: 79% | C: 85% | G: 80% | A: 85% | *Null/Novel (GCGCGA 3 + novel SNP c.197C) |
Nucleotide | c.108 | c.114 | c.194 | c.197 1 | c.233 | c.244 | c.316 | STOP CODON | ||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Pos on chr.1 (Hg38) | 161,629,989 | 161,629,983 | 161,629,903 | 161,629,900 | 161,629,864 | 161,629,853 | 161,629,781 | 161,626,282 | 161,626,250 | 161,626,196 | 161,624,609 | 161,624,517 |
rs no. in FCGR3B | rs200688856 | rs527909462 | rs448740 | rs761007181 | rs5030738 | rs147574249 | rs2290834 | rs71632959 | rs71632958 | rs200215055 | rs758550229 | rs374752953 |
Exons in FCGR3B | 3 | 4 | 5 | |||||||||
Amino acid change | Arg36Ser | 38Leu | Asn65Ser | Leu66Arg | Ala78Asp | Asp82Asn | Val106Ile | |||||
FCGR3B*01 | G | C | A | A | C | G | G | A | C | G | C | T |
FCGR3B*02 | C | T | G | A | C | A | A | A | C | G | C | T |
FCGR3B*03 | C | T | G | A | A | A | A | A | C | G | C | T |
FCGR3B*04 | G | C | A | C | G | A | G | A | C | G | C | T |
FCGR3B*05 | C | T | G | A | C | G | A | A | C | G | C | T |
FCGR3B*01 var c. 194G | G | C | G | A | C | G | G | A | C | G | C | T |
FCGR3B*02 var c. 194A | C | T | A | A | C | A | A | A | C | G | C | T |
Novel 1 (AAL_003, AAL_013) | G | C | G | A | C | A | A | A | C | G | C | T |
Novel 2 (BRI_007) | G | C | G | C | C | G | A | A | C | G | C | T |
FCGR3A | G | C | G | A | C | G | A | G | T | T | T | C |
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Kløve-Mogensen, K.; Browne, T.; Haunstrup, T.M.; Steffensen, R. Pitfalls When Determining HNA-1 Genotypes and Finding Novel Alleles. Int. J. Mol. Sci. 2024, 25, 9127. https://doi.org/10.3390/ijms25169127
Kløve-Mogensen K, Browne T, Haunstrup TM, Steffensen R. Pitfalls When Determining HNA-1 Genotypes and Finding Novel Alleles. International Journal of Molecular Sciences. 2024; 25(16):9127. https://doi.org/10.3390/ijms25169127
Chicago/Turabian StyleKløve-Mogensen, Kirstine, Tom Browne, Thure Mors Haunstrup, and Rudi Steffensen. 2024. "Pitfalls When Determining HNA-1 Genotypes and Finding Novel Alleles" International Journal of Molecular Sciences 25, no. 16: 9127. https://doi.org/10.3390/ijms25169127