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Cytogenetic and molecular identification of translocation t(10; 17)(q11.2; p13)dn. ( A ) Part of the karyotype shows chromosomes 10 and 17 of patients with PFAPA and t index (10; 17). The normal chromosomes 10 and 17 are shown on the left side of each pair. The derived chromosomes 10 and 17 of t(10; 17) (q11.2; p13) are shown on the right side of each pair. ( B ) A schematic diagram of the analysis results of the translocation sequence. The top and bottom ideograms of normal chromosomes 10 (gray) and 17 (purple). The middle panel shows the molecular connections at the left der (10) connection and the right der (17) connection. The deletion on the two chromosomes in the breakpoint region (middle part) resulted in the loss of most of the SPAG7 gene from chromosome 17. Family Video, The numbers give the coordinates of the breakpoint mapping in the base pair. The gene (blue) is indicated in the corresponding exon-intron structure, and the arrow indicates the direction of transcription. ( C ) Confirm the breakpoint connection point by Sanger sequencing. The middle part shows derivative chromosomes 10 and 17 with annotated breakpoints. The upper and lower images show electropherograms from Sanger sequencing on derived chromosomes, where the sequence is derived and the breakpoints indicated.

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In order to exclude submicroscopic chromosomal imbalances related or not related to chromosomal translocations, we performed an array comparative genomic hybridization with a functional resolution of 0.1Mb. Except for the well-known copy number variation, no imbalance was detected at the applied resolution. In particular, we did not detect any imbalance around the designated breakpoint in cytogenetics. In addition, pathogenic sequence variants in genes related to periodic fever syndrome or autoinflammatory diseases, namely MEFV, NLRP3, NLRP12, ELA2, TNFRSF1A, MVK and CARD15, were excluded as causes or modifications of the phenotype.

Given the changes in the balance of cytogenetics and molecular cytogenetics and the lack of mutations in genes associated with periodic fever syndrome, we wondered whether translocations might cause destruction or otherwise relax genes potentially associated with PFAPA. Therefore, our goal is to clone the fusion between chromosomes 10 and 17 on the derived chromosomes der (10) and der (17). To this end, we generated a paired-end 18 (paired) library with a long insert size, with a 4600 bp insert size, and then sequenced the library using a 36 bp read.F M TV, After mapping and quality filtering, 24.1 million non-redundant read pairs remain, resulting in 38.7 times the physical genome coverage. The high-confidence structural variation of the inter-chromosomal variation was determined and filtered against the germline variants identified in the samples of the 1,000 genome project19 and additional whole-genome sequencing studies. In this way, it is inferred that the two derived chromosomes are fused in a high-confidence balance between chromosomes 10 and 17 near the designated breakpoint of cytogenetics. The connection was amplified by PCR using different primer combinations, and verified by Sanger sequencing (Figure 1). This leads to a precise definition of the fusion breakpoint: on the der (10) chromosome, chr10: 43, 444, 710 and chr17: 4, 861, 900 are fused. On der (17), chr17: 4, 867, 779 and chr10: 43, 450, 287 (all hg19, see Figure 1) merge. At the two junctions, there are two chromosomes with the same base. It is worth noting that at the molecular level, the translocation is not balanced, but results in the loss of the genetic material of the affected chromosomes, namely the deletion of 5577bp on chromosome 10 and 5879bp on chromosome 17. These losses can also be analyzed in the read depth of short-read data (Figure 2), indicating that the corresponding genomic material is not integrated into other parts of the genome.

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The reading depth map shows the deletion of the breakpoint junction in chromosomes 10 and 17. The dark gray bars represent the number of sequencing reads mapped between the breakpoints on chromosome 10 (5577bp region) and chromosome 17 (5879bp region). The light gray bars indicate the number of sequencing reads mapped to adjacent regions of the same size upstream and downstream of the breakpoint. The lower reads at the breakpoint compared to the surrounding area support the loss of heterozygosity on both chromosomes.

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Although the breakpoints and related deletions on chromosome 10 do not affect the gene, the deletion on chromosome 17 results in the deletion of exons 2-7, thus leading to the loss of most of the coding region of the SPAG7 gene (Figure 1). To investigate whether the second allele of SPAG7 was also changed in the patient USA ,we sequenced all the exons and exon-intron boundaries of the gene in the patient. Except for known polymorphisms, no sequence variants were detected. Therefore, the lack of haploid SPAG7 may be related to PFAPA in the author.

SPAG7 encodes sperm related antigen 7 (alias ACRP, FSA-1, MGC20134; MIM * 610056). This gene has been identified by sequencing cDNA from CD34+ hematopoietic stem cells as a homolog of fox sperm acrosome-1. 21, 22 It is highly conserved and encodes a protein with 227 amino acids (NP_004881), which is widely expressed. Given the typical manifestations of PFAPA syndrome, nuclear expression in lymph nodes and tonsil cells is worth noting (http://www.proteinatlas.org). The SPAG7 protein contains two nuclear localization signals (AA35-51 and 122-139) and the R3H domain (AA46-109), which are named as the most conserved characteristic interval of arginine and histidine residues. This domain is expected to bind ssDNA or ssRNA in a sequence-specific manner. 23 This may indicate that the protein has a potential effect on the response of viruses or free (cellular) DNA or RNA. In this case, analysis based on array and qPCR showed that SPAG7 was significantly overexpressed in RNA from 57 kinds of parvovirus B19 seropositive peripheral blood mononuclear cells, and 13 kinds of parvovirus B19-seronegative donors Body comparison shows that it is responding to viral infection. twenty four

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