CD177 expression in neutrophils.

<p><b>A.</b> Neutrophils of 5 subjects were gated on CD66b⁺ cells and analysed for surface expression of CD177. Three neutrophil subsets defined by CD177 expression were shown: negative (neg) in black, intermediate (int) in orange and high (hi) in pink, percentages of each subset were indicated as numbers in the same colour. <b>B</b>. Atypical CD177 expression with CD177^int subset >20% of neutrophils. <b>C.</b> A dot plot showing percentages of CD177^hi versus CD177^neg of total neutrophils in healthy subjects, each dot represents one subject. Three major phenotypic groups were marked as CD177^hi, CD177^hi/neg, and CD177^null. <b>D.</b> Contour plots showing neutrophils from a CD177^null subject (left) and a tri-model CD177 expressing donor (right), co-stained with two CD177 specific monoclonal antibodies (MEM-166 and REA258).</p

<i>CD177</i> g.7497A allele frequencies correlate to neutrophil CD177 expression.

<p><b>A—C</b>. Association of reference allele frequency (g.7497A) with neutrophil CD177 expression measured by geometric mean fluorescence intensity (MFI) of CD177 on the cell surface of total neutrophils (<b>A</b>), percentages of CD177^hi neutrophils (<b>B</b>), and CD177^neg neutrophils (<b>C</b>) in the blood of cohort 2. <b>D</b>. Heat map of variant allele frequency (determined by deep sequencing) and neutrophil phenotypes (determined by flow cytometry) in cohort 1.</p

Models for <i>CD177</i> allelic arrangement and genotype-phenotype relation.

<p><b>A</b>. A proposed allelic arrangement model to account for <i>CD177</i> variant read frequencies. <i>CD177P1</i>-derived segments are shown in blue boxes and g.7497T in red lines. <i>CD177</i> genes are in yellow and g.7497A in green. Prevalence of each arrangement in cohort 2 is shown, together with calculated Hardy-Weinberg ratio. <b>B</b>. Model to account for observed CD177 genotype-phenotype relation.</p

Two exons of enriched SNP density and a novel stop codon variation in <i>CD177</i> gene.

<p><b>A</b>. Summary of deep sequencing of <i>CD177</i> gene (located at 43.8Mb, band q13.2, of the forward strand of chromosome 19 in human genome 19, 43.3 Mb and q13.31 for hg38). Amplicons covering the entire coding sequence of 9 exons were sequenced. 100% coverage was obtained at indicated read depths. 24 low frequency SNPs were found in exon adjacent non-coding regions and 17 polymorphisms were identified in <i>CD177</i> coding sequences. Representative allelic frequencies of nine SNPs in exon 5 and 7 are displayed according to CD177 phenotypes. Graphs were generated from Integrative Genomics Viewer from Broad Institute referenced on hg19. <b>B</b>. SNV frequency of 41 variants (called from hg19) in cohort 1. Each dashed line represents genotypes from a single individual. A schematic <i>CD177</i> gene structure is shown of variants within <i>CD177</i>. <b>C</b>. Sanger sequencing of <i>CD177</i> exon 7 in DNA isolated from neutrophils (upper panel) and saliva (lower panel) from three individuals same as shown in <b>A</b>. Reference (Ref) and variant (Var) read frequencies derived from deep sequencing and CD177 neutrophil phenotypes for each subject were indicated on the top of each panel.</p

Ectopic and allelic <i>CD177P1</i> exon 7 conversion.

<p><b>A</b>. CD177^hi and CD177^neg neutrophils were sorted from a single donor with bimodal CD177 expression (<i>left</i>). Genomic variant allele frequencies were determined by deep sequencing (<i>top</i>), and compared with sequence variations in cDNA from CD177^hi (<i>middle</i>) and CD177^neg neutrophils (<i>bottom</i>). Two <i>CD177</i> transcripts were found in CD177^neg neutrophils. SNPs present in gDNA and cDNA of CD177^neg subsets but absent from CD177^hi cells are labelled (*). <b>B</b>. Schematic summaries of <i>CD177</i> vs <i>CD177P1</i> gDNA variations and CD177 mRNA in two neutrophil subsets. <i>CD177P1</i> derived nucleotides in exon 4, 5 and 7 are outlined, suggesting one <i>CD177</i> allele partially supplied by <i>CD177P1</i> exon 7. <b>C</b>&<b>E</b>. Genomic sequence traces in indicated loci within exon 4, 5 and 7 of two CD177^null individuals. Only <i>CD177P1</i> exon 7 sequences are detected in CD177^null subjects, who harbour both <i>CD177</i> and <i>CD177P1</i> upstream elements, i.e., exon 4 (<b>C</b>) and exon 5 (<b>E</b>). <b>D</b>&<b>F</b>. Schematic genomic <i>CD177</i>/<i>CD177P1</i> structures of CD177^null individuals as shown in <b>C</b>&<b>E</b>. <b>G</b>. Confirmation of ectopic and allelic <i>CD177P1</i> exon 7 conversion in three subjects by MLPA. The plots show the peak ratio of probes for indicated loci of <i>CD177</i> and <i>CD177P1</i> genes. Exon 2 is used as a reference read out of the <i>CD177</i> gene only; probes for exon 4, 5, 7 and 9 bind to both <i>CD177</i> and <i>CD177P1</i>, whereas probes labelled as (P1) are specific to <i>CD177P1</i> exon 5 and 7. The graph shows one copy duplication of <i>CD177P1</i> exon 7 in blue in the same subject as shown in <b>A</b> & <b>B</b>; and two copies duplication (allelic conversion) in the two CD177^null subjects (red & orange) as shown in <b>C-F</b>. The subject in red also shows duplication of <i>CD177P1</i> exon 5 in concordant to <b>C</b> & <b>D</b>.</p

List of 17 protein-coding variations showed their incidence, genomic location, polymorphisms and alteration in amino acids (A.A.) in GRCh 37 (hg19) and updated version GRCh38 (hg38).

<p>List of 17 protein-coding variations showed their incidence, genomic location, polymorphisms and alteration in amino acids (A.A.) in GRCh 37 (hg19) and updated version GRCh38 (hg38).</p

<i>CD177</i> and <i>CD177P1</i> variations.

<p><b>A</b>. <i>CD177</i> locus on human chromosome 19 and a schematic comparison of <i>CD177</i> and <i>CD177P1</i> genes. <b>B.</b> Current annotation of three copy number variations of <i>CD177</i> and <i>CD177P1</i> gene polymorphisms. <b>C</b>. <i>CD177</i> reference allele frequencies of two polymorphisms, g.1991C in exon 4 and g.7497A in exon 7, of cohort 1. Each dot represents one of 40 tested subjects. 15 out of 40 subjects displayed allele frequencies of g.1991C and g.7497A simultaneously at 50%. 14/40 subjects harboured similarly 50% g.7497A allele but 75% g.1991C. <b>D</b>. Proposed <i>CD177/CD177P1</i> haplotypes in two loci of exon 4 (C/G) and exon 7 (A/T). <i>CD177</i> gene in black line and <i>CD177P1</i> in grey. The most frequent genotype is highlighted.</p

Analysis of <i>CD177</i> variations and <i>CD177P1</i> divergence from gDNA and cDNA sequence.

<p><b>A</b>. CD177^neg and CD177^hi cells were sorted from an individual who predominantly express CD177^hi in neutrophils (<i>left</i>). Sequencing traces showed <i>CD177</i> codon variations at indicated loci, comparing results from gDNA deep sequencing (top), CD177 mRNA isolated from purified CD177^hi cells (middle) and CD177^neg neutrophils (bottom). The reference nucleotide sequences were labelled in colour letters below. The two SNPs present in gDNA and cDNA of both cell subsets were labelled (*). <b>B</b>. A schematic summary of the variations in gDNA and mRNA in neutrophils of this subject. Sequence variations were indicated on the top of the gene/transcript and exons were indicated (E2, E4 etc) below. <i>CD177P1</i> nucleotides in exon 4, 5 and 7 were outlined. CD177P1 transcripts were shown in grey signifying expected NMD with the stop codon variation g.7497T in red.</p

An inheritable phenotype of CD177 determined by ratio of <i>CD177/CD177P1</i> alleles.

<p><b>A-D</b>. Pedigrees of 4 unrelated families with different CD177 geno- and phenotypes, all exhibit Mendelian inheritance. ⊕: female, ⊠: male; filled: <i>CD177</i> exon 7, blank: <i>CD177P1</i> exon 7. Genotypes were also shown in schematic structures, <i>CD177</i> in orange and <i>CD177P1</i> in blue, with g.7497A/T polymorphisms labelled in both alleles at <i>CD177</i> and <i>CD177P1</i> loci. Chromatograms showed genomic sequence trace of <i>CD177</i> g.7497 labelled with ratio of Ref:Var in each subject. Histograms showed CD177 phenotypes, numbers in corresponding colours indicating percentages of neutrophil subsets, CD177^neg in black, CD177^int in orange, CD177^hi in pink.</p

MLPA probes.

<p>MLPA probes.</p