PMC6561467_01
Family pedigree of the proband and analysis of splice site mutations in AVPR2. A. Family pedigree of the proband (arrow), his 3 siblings and his parents. Unaffected (empty symbols), affected (filled symbols), carrier (half symbols), male (square), female (circle). B. RT-PCR was performed on peripheral blood lymphocyte cDNA from three controls (C1-C3), the affected patient (II-4) and his mother (I-2), with primers covering most of exon 2 and both sides of AVPR2 c. 276 A>G. A control PCR on the same samples was performed using primers for GAPDH. C. Sequencing of the AVPR2 exon 2 RT-PCR products of the patient and his mother where the locus of the mutation is highlighted (arrows). D. Minigene assay. cDNA from untransfected HEK293, empty vector-transfected HEK293 (EV), the AVPR2 wt and three mutants in pSPL3, and negative controls (reverse transcription negative controls without reverse transcriptase or without RNA, and RT-PCR negative control without cDNA). The arrow points to the mutation-specific band that was sequenced. E. Schematic of the RT-PCR product from the c.276 A>G mutant within the minigene assay lacking the 5' end of exon 2. The location of the SD6 and SA2 primers used in the minigene assay is indicated.
nihms-1026843-f0001.jpg
PMC6561467_02_nihms-1026843-f0001.jpg
F1
T>G (rs56689668; variant 2), both predicted in silico to affect mRNA splicing. The two affected children were hemizygous for these variants; the patients' mother (I-2) and sister (II-2) were heterozygous for these variants (Fig. 1A). RT-PCR products were present in all samples at the sizes expected for exon 2 without truncation (Fig. 1B). Sequencing of the RT-PCR products confirmed the presence of AVPR2 exon 2 in both the proband and the mother; the patient's mRNA contained only the c.276 A>G mutation whereas the mother's mRNA contained both the mutation and the wild-type (Fig. 1C), so we did not find evidence of alternative splicing in the lymphocyte mRNA of the patient. AVPR2, and exon 2 containing one or both variants together with the flanking intronic sequences, were cloned into the pSPL3 vector. After transfection into HEK293 human kidney cells, mRNA synthesis from the plasmids using the cells' own transcription and splicing machinery led to mRNA products containing exon 2 of AVPR2 flanked by two exons from the pSPL3 vector (Fig. 1E). Variant 1 leads to an approximately 900 bp product, consistent with a 5' truncated exon 2 lacking its first 251 bases (Fig. 1D), which was confirmed by sequencing of this band. The c.26 -6 T>G variant did not have an independent effect in this assay, and the construct containing both variants led to the same product as that seen with the c.276 A>G mutation alone (Fig. 1D). Fig. 1B and 1C).
PMC6561467_02
Family pedigree of the proband and analysis of splice site mutations in AVPR2. A. Family pedigree of the proband (arrow), his 3 siblings and his parents. Unaffected (empty symbols), affected (filled symbols), carrier (half symbols), male (square), female (circle). B. RT-PCR was performed on peripheral blood lymphocyte cDNA from three controls (C1-C3), the affected patient (II-4) and his mother (I-2), with primers covering most of exon 2 and both sides of AVPR2 c. 276 A>G. A control PCR on the same samples was performed using primers for GAPDH. C. Sequencing of the AVPR2 exon 2 RT-PCR products of the patient and his mother where the locus of the mutation is highlighted (arrows). D. Minigene assay. cDNA from untransfected HEK293, empty vector-transfected HEK293 (EV), the AVPR2 wt and three mutants in pSPL3, and negative controls (reverse transcription negative controls without reverse transcriptase or without RNA, and RT-PCR negative control without cDNA). The arrow points to the mutation-specific band that was sequenced. E. Schematic of the RT-PCR product from the c.276 A>G mutant within the minigene assay lacking the 5' end of exon 2. The location of the SD6 and SA2 primers used in the minigene assay is indicated.
nihms-1026843-f0001.jpg
PMC6561467_02_nihms-1026843-f0001.jpg
F1
T>G (rs56689668; variant 2), both predicted in silico to affect mRNA splicing. The two affected children were hemizygous for these variants; the patients' mother (I-2) and sister (II-2) were heterozygous for these variants (Fig. 1A). RT-PCR products were present in all samples at the sizes expected for exon 2 without truncation (Fig. 1B). Sequencing of the RT-PCR products confirmed the presence of AVPR2 exon 2 in both the proband and the mother; the patient's mRNA contained only the c.276 A>G mutation whereas the mother's mRNA contained both the mutation and the wild-type (Fig. 1C), so we did not find evidence of alternative splicing in the lymphocyte mRNA of the patient. AVPR2, and exon 2 containing one or both variants together with the flanking intronic sequences, were cloned into the pSPL3 vector. After transfection into HEK293 human kidney cells, mRNA synthesis from the plasmids using the cells' own transcription and splicing machinery led to mRNA products containing exon 2 of AVPR2 flanked by two exons from the pSPL3 vector (Fig. 1E). Variant 1 leads to an approximately 900 bp product, consistent with a 5' truncated exon 2 lacking its first 251 bases (Fig. 1D), which was confirmed by sequencing of this band. The c.26 -6 T>G variant did not have an independent effect in this assay, and the construct containing both variants led to the same product as that seen with the c.276 A>G mutation alone (Fig. 1D). Fig. 1B and 1C).