PMC6856949_01
Photographs of warts present in the patient's feet.
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F1
A 45-year-old Caucasian female born to first-cousin parents, with two healthy children and without any relevant family history record of prior diseases, was seen at the service of Dermatology (University of Coimbra, Coimbra, Portugal) in March 2014 because of persistent extensive, skin-colored, exuberant, and disfiguring warts in both feet and hands since the age of 10 years (Figure 1). In addition, expanded DN T-cells showed a polyclonal TCRVbeta repertoire with a distribution per TCR family evaluated, fully consistent with reference TCRVbeta repertoire values observed in HLA class II-restricted CD4+ T-cells from healthy donors (Supplementary Figure 1). In addition, CD8+ TCRalphabeta+ T-cells also showed a normal polyclonal TCRVbeta repertoire (vs. normal age-matched reference values, shown in Supplementary Figure 1) and a normal cytokine production profile in response to PMA and CMV (Table 4).
Schematic representation of the CD4 protein molecule (A) and CD4 surface membrane expression in the patient's (vs. healthy donor) blood CD8- TCRgammadelta- T-cells, monocytes, and dendritic cells (B). Panel (A) shows a schematic representation of the CD4 protein molecule including the localization of epitopes identified by the distinct antibody clones used in this study. Amino acid positions that conform each domain are indicated between brackets. Black arrows depict the domain that contains those epitopes that the antibody clones are directed to. Panel (B) shows CD4 surface membrane expression levels for CD8- TCRgammadelta- T-cells, monocytes, and dendritic cells for the different anti-CD4 antibody clones tested in the patient (black histogram) compared to a representative healthy donor (gray histogram) and an isotype control (red dash line), and the staining for a negative population (CD8+ T-cells) in the patient (green line) and the healthy control (blue line). DCs, dendritic cells.
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F2
These eight CD4 MoAb clones were directed against five different epitopes located in two distinct domains of the CD4 protein (Supplementary Material; Figure 2A), as confirmed in competitive staining inhibition experiments (data not shown), in line with previous data in the literature. Detectable levels of either surface membrane (sm) or intracytoplasmic (cy) CD4 expression were observed in none of the cell lineages that usually express this protein in blood of healthy controls, such as T-cells, monocytes, and DCs (Figure 2B).
Nucleotide sequences of CD4 PCR products for the regions of interest and their localization in the CD4 gene. Panel (A) shows a representative sequence of CD4 focused on the region of interest. Gray underlining shows the nucleotide sequence affected by the major deletion (NM_000616: r.1157_1278del), and orange top line shows the nucleotide sequence affected by the minor deletion (NM_000616: r.1157_1185del). Panels (B,C) show mRNA electropherograms obtained from analysis of the minor (NM_000616: r.1157_1185del) and major (NM_000616: r.1157_1278del) deletions detected in the patient's CD4 cDNA, respectively, and the patient's relatives (C). Panels (D-F) show genomic DNA electropherograms from: (D), wild type CD4 gene; (E), patient's homozygous mutated CD4 gene; and (F), heterozygous mutated CD4 gene from the patient's parents and children. Panels (G,H) display a schematic representation of the localization in wild type CD4 protein of the mutated (green) nucleotide bases and the deleted (red) amino acid sequences, including the minor (G) and major (H) deletions, observed in the truncated CD4 cDNA molecules detected in the CD4null patient here reported. Panel (I) shows the family pedigree (men are represented as squares and women as circles), including the key clinical manifestations and, between brackets, the results of CD4 DNA analysis in a coded/graphic format: half-shaded circles and squares, heterozygous DNA; fully shaded circles and squares, homozygous mutated DNA; and unshaded circles and squares, wild type DNA.
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F3
Both truncated forms of CD4 RNA presented with a frameshift deletion starting at the juxtamembrane region at the first bp of exon 8 and a premature stop codon associated with a truncated protein with normal extracellular domains in the absence of the anchoring domain to the membrane (Figure 3). In turn, the second frameshift deletion produced a 430-amino-acid protein because of (only) a 29 bp deletion (NM_000616: c.1157_1185del) (Figure 3) that ended just before a 5 bp combination (TGCAG), homologous to the sequence observed at the end of the 7-8 intron (Figure 3). Further cDNA sequencing from her children and her parents revealed two CD4 mRNA sequences, one carrying the large deletion observed in the patient (NM_000616: c.1156_1278del) and another with the unmutated (wild type) CD4 allele sequence (Figure 3).
PMC6856949_02