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Somatic and germline FOXP3 mosaicism in the mother of a boy with IPEX syndrome

2018; Wiley; Volume: 48; Issue: 5 Linguagem: Inglês

10.1002/eji.201747445

1521-4141

Yunting Lin, Aijing Xu, Chunhua Zeng, Jing Cheng, Na Li, Huilin Niu, Li Liu, Xiuzhen Li,

Immune Cell Function and Interaction

Confirmatory Sanger sequencing of whole exome sequencing first identified a somatic and germline FOXP3 mosaicism with two different mutational events of c.210 + 1G > T and c.210 + 1G > A in the mother of a boy with IPEX syndrome. Immunodysregulation, polyendocrinopathy, and enteropathy, X-linked (IPEX) syndrome is a rare monogenic disease caused by mutations of FOXP3 gene located on Xp11.23 with an X-linked recessive inheritance pattern. As the FOXP3 gene is essential for the function of regulatory T cells, its deficiency leads to multisystem autoimmune manifestations 1-3. In the absence of curative treatment with hematopoietic stem cell transplantation, IPEX syndrome is a severe life-threatening disorder, especially for the first 2 years of life 4, 5. Therefore, pinpointing the origin of the pathogenic mutations is particularly important to preventing the recurrence of this disease. Genetic mutations are due to DNA errors or parental inheritance; thus, the involvement of the parents in the molecular survey for an affected patient will benefit precise diagnosis and genetic counseling for the families. However, uncommonly, the transmission of deleterious variants from germline mosaic alleles in a phenotypically normal parent will challenge and obstruct the accurate diagnosis and efficient prevention of those diseases 6, 7. Here we describe an unusual pedigree of a patient with IPEX syndrome (Fig. 1A). The proband began to present symptoms of recurrent cough, asthma, and edema at 19 months of age. During his clinical course to 4 years of age, other manifestations of nephropathy, bronchopneumonia, enteropathy, gastric volvulus, hepatosplenomegaly, diabetes mellitus, ketoacidosis, atrial septal defect, nasosinusitis, cataract, auditory abnormality, elevated serum immunoglobulin E, urinary infection, bacteremia, conjunctivitis, and failure to thrive emerged subsequently. The above diagnoses were made by physical and radiological examinations and laboratory tests (Fig. 1B–E and Supporting Information Table 1). Renal biopsy and blood tests revealed autoimmune responses (Fig. 1D and E, and Supporting Information Table 1), and the patient was suspected to suffer from IPEX syndrome. To identify the phenotype-producing mutations and further exclude other overlapping disorders, the proband-parent trios were enrolled for high-throughput whole exome sequencing (WES) using genomic DNA from whole blood with an average depth over 100X and coverage of 99.5%. A "de novo" substitution of guanine (G) with thymine (T) at the c.210 + 1 nucleotide position of intron 2 within the FOXP3 gene (NG_007392.1, NM_014009.3) was identified using an established medical re-sequencing analysis pipeline (MERAP) 8. Because the c.210 + 1G > T is a known pathogenic replacement resulting in abnormal splicing 9 and no other autoimmune disorder-associated pathogenic variant was identified, a definite diagnosis of IPEX syndrome for the proband was made. Confirmatory Sanger sequencing was subsequently employed. The c.210 + 1G > T hemizygous variant was confirmed in the proband but absent in the father. However, the mother show a markedly repressed T allele compared with the wild-type G base. Rather than a noise signal, the identical mutation of the proband was indeed shared by his mother as a probable mosaicism in view of both sense and antisense sequencing chromatograms after re-extraction and retesting of retained blood samples. Interestingly, another known deleterious adenine (A) peak 10, though almost below the detection level, at the same position was not ruled out in terms of the Sanger diagrams in the mother (Fig. 1F). To verify the mosaicism hypothesis, the 6-year-old healthy sister was tested. An unquestionable heterozygous mutation of c.210 + 1G > T was shown in the sister by classic Sanger method (Fig. 1F), enabling us to demonstrate that the mutated T allele of the siblings was vertically transmitted from their mother. Therefore, the mother was genetically confirmed as a mosaic. To further investigate the mosaic status and discern if the c.210 + 1G > A mutation was authentic, eight different tissues of the mother were obtained. Specifically, we collected ectoderm-derived hair, oral epithelium, saliva (mixture of exfoliated oral epithelium and leukocytes from salivary glands), nail and cuticle, and mesoderm-derived whole blood, urine (including renal and bladder exfoliated epithelial cells), and menstrual blood (containing blood and mucosal tissue from the inner lining of the uterus). To our surprise, both G > T and G > A detrimental mutations were displayed with diverse frequencies (Fig. 1G). For instance, an apparently higher T peak than A peak was present in the hair, whereas, inversely, replacement of the wild-type G allele by an A base was predominant in the nail. Because the default lower cut-off value of heterozygous variant was 20% in the MERAP pipeline, the estimated frequency of eight implicated tissues ranging from 1.72 to 17.24% was below the threshold of detection, and thus led to a "de novo" mutation recommended by WES after the filter process. A further retrospective survey of the original electronic read data confirmed the G > T mutation on the c.210 + 1 site of FOXP3 gene with a frequency of 12.5%, but no G > A replacement was displayed as only 32 reads mapped to this site (data not shown). Given that mosaic status existed in multi tissues, we speculated a spontaneous mutagenesis in the mother during early embryonic development on account of her asymptomatic parents based on family history. A two-hit hypothesis was suggested as two different mutations arose in the identical site. The heterogeneity of mutated base frequency among diverse samples might result from the timing of mutation events and imbalanced assignment during germ layer differentiation. Although the endoderm-derived tissues were not available, we deduced an earlier occurrence of G > T substitution during the 4-cell stage of zygote cleavage according to the existing data, while the G > A event probably occurred in the 8-cell stage as there are two X chromosomes in females. Moreover, a DNA-damage-repair scenario was also taken into account. A potential rescue response was triggered by the G > T replacement, but unfortunately, a mispairing of A was introduced as a secondary mutation. In addition, as both the menstrual blood histologically close to the intrinsic germ cells and other ectoderm-derived tissues were measured and showed two different FOXP3 mosaic mutations, the mother was then defined as a somatic and germline mosaic carrying both c.210 + 1G > T and c.210 + 1G > A disease-causing splicing variants of IPEX syndrome, indicating an intrafamilial recurrence risk. The fact that only the c.210 + 1G > T mosaic allele was transmitted to both the proband and the sister might be due to the imbalance of X-inactivation or just a rare occurrence. In conclusion, this report describes a FOXP3 mosaicism for the first time, either in male patient or female carrier. This mosaicism case and two mutation events open the possibilities in the genetic basis of IPEX syndrome. Our study provided an accurate diagnosis of a pathogenic mutation and its inheritance for the proband, predicted the timing of two different mutation events in the mother, and addressed a valuable aspect for genetic counseling of recurrent risks. Our report also alerts clinicians and geneticists to be cautious in molecular findings to avoid omissions of mosaic alleles and prevent intrafamilial recurrences of identical genetic abnormalities. This study was approved by the Institutional Review Board of Guangzhou Women and Children's Medical Center (Guangzhou, China). The informed consents were obtained from all subjects or their guardians. We thank the enrolled family for participation in this study and thank Dr. Gendie Lash and Dr. Yuxia Zhang for correcting the language. This work was supported by the Natural Science Foundation of Guangdong Province, China for Y.L. (2016A030313500) and the Municipal Healthcare Joint-Innovation Major Project of Guangzhou for L.L. (201604020020). The authors declare no commercial or financial conflict of interest. X.L., L.L., and Y.L. designed the study. X.L., L.L., A.X., C.Z., and J.C. collected the medical records. Y.L., A.X., and H.N. performed the experiments. Y.L. and N.L. analyzed the data. X.L., L.L., and Y.L. wrote the paper. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.