Clinical phenotype of the patients and patients already reported in the literature with SLC29A3 mutations.

<p>Clinical phenotype of the patients and patients already reported in the literature with SLC29A3 mutations.</p

Identification of a frameshift deletion in <i>SLC29A3</i>.

<p>(A) Pedigree of the family. The <i>SLC29A3</i> genotypes of the patients and the family members from whom DNA was available for sequence analysis are listed under their symbols. Genotyping was carried out twice. (B) Illumina sequencing reads displayed for patient P1. Reads overlapping the mutation in exon 2 of <i>SLC29A3</i> (bp position g.73,082,741–g.73,082,765; hg19; NCBI 37) show the homozygous deletion of one A leading to a frameshift (c.243delA). (C) Diagram of <i>SLC29A3.</i> Introns are represented by a straight line. 5′-UTR and 3′-UTR are represented by open rectangles. The coding region is represented by closed rectangles. Previously reported mutations are indicated at the corresponding locations. The mutation identified in P1 and P2 is shown in a red rectangle. (D–E) Abnormal expression of <i>SLC29A3</i> transcript variants 1, 2 and 3 in the patients' EBV-B cells. (D) <i>SLC29A3</i> exons 2–4 were amplified from cDNA obtained from the EBV-B cells of two controls (Ctrl 1 and Ctrl 2) and two patients (P1 and P2) and were ligated to the pCR2.1 vector. Clones containing <i>SLC29A3</i> transcripts were sequenced, and the frequency of each variant was calculated by dividing the number of clones containing the particular transcript by the total number of sequenced clones. For Ctrl1: variants 1 and 2: 65/81 and variant 3: 16/81. For Ctrl2: variants 1 and 2: 37/44 and variant 3: 7/44. For P1: variants 1 and 2: 31/85 and variant 3: 54/85. For P2: variants 1 and 2: 20/76 and variant 3: 56/76. Each variant is represented by a diagram, with numbers indicating the number of the exon. The red vertical line indicates the position of the c.243delA mutation. The closed rectangle below each transcript variant represents the corresponding translation products. Gray boxes indicate amino acids modified with respect to the WT form. (E) Levels of <i>SLC29A3</i> mRNA (variants 1 and 3 combined) were assessed by Q-PCR on EBV-B cells from four healthy controls (Ctrls), two parents (Het.), and the two patients. Threshold cycles (Ct) for <i>SLC29A3</i>, normalized with respect to those of GUS (ΔCt), are plotted as 2^−(ΔCt). Each dot represents the mean of three independent experiments for each individual. The horizontal bars indicate the mean for all individuals sharing the same genotype.</p

Whole-exome analysis results of P1.

<p>Coding variants include missense, nonsense, frameshift, in-frame deletions and insertions and readthrough variants.</p><p>Splice variants include all variants within 8 bp in the intron side, or 3 bp in the exon side of a splice junction.</p>a<p>: Both homozygous and heterozygous variations are included.</p>b<p>: Position coordinates for the markers correspond to the hg19, NCBI build 37.</p

Histology of nasal (A, B, F) and skin (C, D, E) biopsy specimens from P1 (A–E) and P2 (F).

<p>Most biopsy specimens contained vacuolated histiocytes suggestive of Mikulicz cells (A, E). Some specimens also contained large S100 protein-positive histiocytes enclosing lymphocytes (emperipolesis) (B, C, D, F). H&E staining (A, C, E) and immunohistochemical staining for S100 protein (B, D, F). Original magnification ×200 (A) and ×400 (B–F).</p

Expression and characteristics of hENT3-variant3-81fs.

<p>(A) Scheme of hENT3-variant1-WT based on the protein structure predicted by SVMtm or TMpred programs. Transmembrane domains are represented by long rectangles. The gray and red boxes highlight the amino acids that are not identical in hENT3-variant1-WT and hENT3-variant3-81fs. In the gray box: amino acids 81–100 that are replaced by 20 other amino acids in hENT3-variant3-81fs. In the red box: amino acids 101–128 that are deleted in hENT3-variant3-81fs. (B) Levels of hENT3 proteins were assessed by immunoblotting with an anti-V5-tag antibody. The V5 tag was located at the C-terminus of hENT3. (C) The graph shows the relative <i>SLC29A3</i> mRNA levels measured by Q-PCR for the same experiment with the V5-tagged constructs. Threshold cycles (Ct) for SLC29A3, normalized with respect to those of GUS (ΔCt), are plotted as 2^−(ΔCt). Immunoblotting and Q-PCR results representative of three independent experiments are shown. (D) Adenosine transport activity. Uptake of [³H]adenosine (0.026 µM) in <i>Xenopus</i> oocytes 48 h after the injection of Δ36hENT3-variant1-WT or other variants with or without the 81fs mutation as well as one H (349fs) and one PHID syndrome (314fs) mutant cRNAs. pH-dependent uptake at pH 5.5. The data shown are the means ± SEM from three independent experiments. ^*, <i>p</i><0.05, ^***, <i>p</i><0.001.</p

Signal ratios for three assay targets by antibody response category.

Signal ratios were calculated by dividing the mean fluorescent intensity of the target by the mean fluorescent intensity of the calibrators. Vaccination and infection combined produced the strongest antibody response against all three assay targets compared to infection or vaccination alone. Participants whose antibody response was derived from infection only had greater signal ratios for the RBD target compared to participants with vaccination-derived antibody response only. ns = not significant (p-value>0.05); **p-value<0.001.</p

SARS-CoV-2 seropositivity by participant demographics and SARS-CoV-2 exposures and risk behaviors.

SARS-CoV-2 seropositivity by participant demographics and SARS-CoV-2 exposures and risk behaviors.</p

Participant demographics, SARS-CoV-2 exposures and risk behaviors, and ANC test results.

Participant demographics, SARS-CoV-2 exposures and risk behaviors, and ANC test results.</p

SARS-CoV-2 antibody response categorization of study participants by enrollment month and district.

No evident antibody response was defined as testing negative for anti-RBD IgG. Infection-derived antibody response was defined as positive anti-RBD IgG AND no reported COVID-19 vaccination. Vaccination and infection-derived hybrid antibody response was defined as positive anti-RBD IgG AND positive anti-nucleocapsid IgG AND reported COVID-19 vaccination. Vaccination-derived antibody response was defined as positive anti-RBD IgG AND reported COVID-19 vaccination AND negative anti-nucleocapsid IgG. Equivocal IgG responses, unknown COVID-19 vaccination status, and unknown or Sinopharm vaccine type were categorized as indeterminate.</p

Inclusivity in global research.

SARS-CoV-2 serosurveys help estimate the extent of transmission and guide the allocation of COVID-19 vaccines. We measured SARS-CoV-2 seroprevalence among women attending ANC clinics to assess exposure trends over time in Zambia. We conducted repeated cross-sectional SARS-CoV-2 seroprevalence surveys among pregnant women aged 15–49 years attending their first ANC visits in four districts of Zambia (two urban and two rural) during September 2021-September 2022. Serologic testing was done using a multiplex bead assay which detects IgG antibodies to the nucleocapsid protein and the spike protein receptor-binding domain (RBD). We calculated monthly SARS-CoV-2 seroprevalence by district. We also categorized seropositive results as infection alone, infection and vaccination, or vaccination alone based on anti-RBD and anti-nucleocapsid test results and self-reported COVID-19 vaccination status (vaccinated was having received ≥1 dose). Among 8,304 participants, 5,296 (63.8%) were cumulatively seropositive for SARS-CoV-2 antibodies from September 2021 through September 2022. SARS-CoV-2 seroprevalence primarily increased from September 2021 to September 2022 in three districts (Lusaka: 61.8–100.0%, Chongwe: 39.6–94.7%, Chipata: 56.5–95.0%), but in Chadiza, seroprevalence increased from 27.8% in September 2021 to 77.2% in April 2022 before gradually dropping to 56.6% in July 2022. Among 5,906 participants with a valid COVID-19 vaccination status, infection alone accounted for antibody responses in 77.7% (4,590) of participants. Most women attending ANC had evidence of prior SARS-CoV-2 infection and most SARS-CoV-2 seropositivity was infection-induced. Capturing COVID-19 vaccination status and using a multiplex bead assay with anti-nucleocapsid and anti-RBD targets facilitated distinguishing infection-induced versus vaccine-induced antibody responses during a period of increasing COVID-19 vaccine coverage in Zambia. Declining seroprevalence in Chadiza may indicate waning antibodies and a need for booster vaccines. ANC clinics have a potential role in ongoing SARS-CoV-2 serosurveillance and can continue to provide insights into SARS-CoV-2 antibody dynamics to inform near real-time public health responses.</div