Paternal Low-Level Mosaicism-Caused SATB2-Associated Syndrome

Abstract

Mutations of SATB2 (OMIM#608148) gene at 2q33.1 have been associated with the autosomal dominant SATB2-associated syndrome (SAS), which is still short of comprehensive diagnosis technologies for small deletions and low-level mosaicism. In this Chinese Han family, single nucleotide polymorphism array identified a 4.9-kb deletion in the SATB2 gene in two consecutive siblings exhibiting obvious developmental delay and dental abnormalities but failed to find so in their parents. Prenatal diagnosis revealed that their third child carried the same deletion in SATB2 and the pregnancy was terminated. To determine the genetic causes behind the inheritance of SATB2 deletion, gap-PCR was performed on peripheral blood-derived genomic DNA of the family and semen-derived DNA from the father. Gap-PCR that revealed the deletions in the two affected siblings were inherited from the father, while the less intense mutant band indicated the mosaicism of this mutation in the father. The deletion was 3,013 bp in size, spanning from chr2: 200,191,313-200,194,324 (hg19), and covering the entire exon 9 and part of intron 8 and 9 sequences. Droplet digital PCR demonstrated mosaicism percentage of 13.2% and 16.7% in peripheral blood-derived genomic DNA and semen-derived DNA of the father, respectively. Hereby, we describe a family of special AT-rich sequence-binding protein 2-associated syndrome caused by paternal low-level mosaicism and provide effective diagnostic technologies for intragenic deletions.

Keywords: SATB2-associated syndrome; chromosome microarray analysis; droplet digital PCR; gap-PCR; mosaicism.

Figure 1

Pedigree of the family and the characterization of the deletion breakpoints by gap-PCR. (A) Pedigree of this family. The arrow refers to the proband. The solid square (male) and circle (female) represent the two affected siblings; (B) Gap-PCR of family members using the peripheral blood-derived genomic DNA and primer sets (4334-F/R). Lanes 1–7: Marker, II2, II1, III1, III2, II3, and I1. The expected SATB2 wild allele band of ∼4.3 kb is seen in members of II2, II1, III1, III2, II3, and I1. A lower band, consistent with the size of mutant allele, is seen in II1, III1, and III2 but not in II2 or I1. Notice that the father (II1) has a much less intense band of about 1.3 kb in size, which is the same size as his two children (III1 and III2). (C) Sequencing results of the ∼4.3-kb band and 1.3-kb band. The intragenic deletion border is chr2: 200,191,313-200,194,324 (hg19), which includes the entire exon 9 and its franking intronic sequences of the SATB2 gene. Blue arrows refer to gap-PCR primer set (4334-F/R) used to detect the breakpoints of the 3kb deletion.

Figure 2

Quantitative PCR (qPCR) of family members using the peripheral blood-derived genomic DNA. (A) Schematic diagram of location of qPCR primers. Blue arrows refer to qPCR primers; (B) Comparison of gene dosage ratios for the family members using the SATB2-exon 9 primers; (C) Comparison of gene dosage ratios for the family members using the SATB2-intron 8 primers; (D) Comparison of gene dosage ratios for the family members using the SATB2-intron 9 primers. F, normal female; C, control (normal male).

Figure 3

Droplet digital PCR analysis for the mutant frequencies of SATB2 gene. ROX-labeled probe detects the delete mutation of SATB2 gene (red dots). 6-FAM-labeled probe detects the reference gene PARP2 (blue dots). Green dots, ROX and 6-FAM double positive. Yellow dots, no amplification. (A) No-template control, NTC; no amplification on both fluorescence channels. (B) wild type, DNA from the proband’s mother; normal amplification on FAM channel, while no amplification on ROX channel. (C, D) heterozygosity, DNA from the proband and his sister, respectively; the ratios of ROX/FAM were close to 0.50. (E, F) mosaicism, DNA from the proband’s peripheral blood and semen of the father; the ratios of ROX/FAM were 13.16% and 16.68%, respectively.