PMC5322879_01
Pedigree of a Chinese family with risk for recurrence of immunodeficiency.. Arrow indicates the proband. Alive family members were subjected to genetic analysis.
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The pedigree comprised 9 individuals spanning three generations (Fig 1). Both variant analysis and chromosomal karyotyping were performed with amniotic fluid fetal cells (Fig 1, III-3).
Identification of a novel SH2D1A pathogenic variant in the pregnant woman.. (A) Integrative Genomics Viewer (IGV) snapshot of the sequence data at SH2D1A for the pregnant (II-1) sample, and the corresponding calls made from the data. Each horizontal gray bar represents one read, the short line in the gray bar indicates bases deletion. (B) Sanger sequencing verified the NGS result. The arrows indicate the deletion of TTTCA in mutant allele which leads to frameshift and introduces a premature termination codon (p.Ile84Serfs*18).
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Date priority filtering revealed that the pregnant woman was heterozygous for a novel deletion variant NM_002351.4(SH2D1A):c.[251_255delTTTCA];[251_255TTTCA=] (Fig 2A). NGS results, the variant was detected in the pregnant woman (Fig 2B). Notably, the variant was not detected in other alive family members, including the parents of the pregnant women. Both variant analysis and chromosomal karyotyping were performed with amniotic fluid fetal cells (Fig 1, III-3).
RNA assay of the SH2D1A mRNA.. (A) RT-PCR analysis of SH2D1A mRNA from peripheral blood mononuclear cells (PBMCs) of the pregnant carrier (II-1) and a healthy female control. The cDNA fragments of 213-bp, which corresponded to the predicted wild-type transcript, were identified in both the pregnant carrier and the control. (B) cDNA sequencing revealed that SH2D1A transcripts of the pregnant carrier were heterozygous for c.251_255delTTTCA. (C) Sequencing analysis results of the pGM-T clones with the cDNA amplicons.
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The same splicing pattern was found between the wild-type control and the c.251_255delTTTCA heterozygous pregnant woman (Fig 3A). Further cDNA sequencing revealed that SH2D1A transcripts of the pregnant carrier were heterozygous for c.251_255delTTTCA (Fig 3B). Sequence analysis showed an approximately 1:1 proportion of wild-type and c.251_255delTTTCA clones (16:18) (Fig 3C). Both variant analysis and chromosomal karyotyping were performed with amniotic fluid fetal cells (Fig 1, III-3).
Prenatal testing of the fetus.. (A) Sanger sequencing revealed that the fetus was unaffected. (B) Karyotyping result of the fetus was consistent with a normal 46,XY chromosomal constitution.
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Sanger sequencing and karyotyping results demonstrated that the fetus (III-3) was unaffected (NM_002351.4(SH2D1A):c.[251_255delTTTCA];[0]) with normal male karyotype (Fig 4).