case_images: PMC11792323_01
case_images: Identification and description of the patient along with the SMARCC2 variant. (A) Pedigree of the family. Males are indicated by squares and females are indicated by circles. Generations are labeled using Roman numerals (I, II). Solid symbols indicate affected individuals. (B) Photograph to highlight facial anomalies, including small left eye, epicanthus, low-set ears, wide nose and thick lower lip, and scoliosis in the proband. (C) The data analysis algorithm used for filtering all single nucleotide variants identified using whole exome sequencing, with the number of remaining variants after each filtering step. On filtering and prioritization, heterozygous variant of the SMARCC2 gene was identified as the top candidate. AD, autosomal dominant; AR, autosomal recessive; MAF, minor allele frequency; OMIM: Online Mendelian Inheritance in Man. (D) Sanger sequencing to confirm the c.1496 + 1G > T variant in the SMARCC2 gene in this family
case_images: 13023_2024_3510_Fig1_HTML.jpg
case_images: PMC11792323_02_13023_2024_3510_Fig1_HTML.jpg
case_images: Fig1
case_images: Finally, WES identified a heterozygous splicing variant (c.1496 + 1G > T) in the SMARCC2 gene, which has not been reported previously and has not been included in any public database (Fig. 1C). Additionally, Sanger sequencing confirmed that both the proband and his elder brother harbored the c.1496 + 1G > T variant, while the allele of their parents was wild-type, suggesting de novo status of the variant in the twins (Fig. 1D).
case_images: PMC11792323_02
case_images: Identification and description of the patient along with the SMARCC2 variant. (A) Pedigree of the family. Males are indicated by squares and females are indicated by circles. Generations are labeled using Roman numerals (I, II). Solid symbols indicate affected individuals. (B) Photograph to highlight facial anomalies, including small left eye, epicanthus, low-set ears, wide nose and thick lower lip, and scoliosis in the proband. (C) The data analysis algorithm used for filtering all single nucleotide variants identified using whole exome sequencing, with the number of remaining variants after each filtering step. On filtering and prioritization, heterozygous variant of the SMARCC2 gene was identified as the top candidate. AD, autosomal dominant; AR, autosomal recessive; MAF, minor allele frequency; OMIM: Online Mendelian Inheritance in Man. (D) Sanger sequencing to confirm the c.1496 + 1G > T variant in the SMARCC2 gene in this family
case_images: 13023_2024_3510_Fig1_HTML.jpg
case_images: PMC11792323_02_13023_2024_3510_Fig1_HTML.jpg
case_images: Fig1
case_images: Finally, WES identified a heterozygous splicing variant (c.1496 + 1G > T) in the SMARCC2 gene, which has not been reported previously and has not been included in any public database (Fig. 1C). Additionally, Sanger sequencing confirmed that both the proband and his elder brother harbored the c.1496 + 1G > T variant, while the allele of their parents was wild-type, suggesting de novo status of the variant in the twins (Fig. 1D).
case_images: Electrophoresis and TA-clone assays for the c.1496 + 1G > T variant. (A) The schematic diagram to show the position of the c.1496 + 1G > T variant and the primers location that were used to amplify the cDNA fragments of the SMARCC2. (B) Electrophoresis of the RT-PCR products to show the aberrant splicing caused by the c.1496 + 1G > T variant. HC, healthy control (I-1); P, proband (II-2). (C) TA-clone assays showed that half of the sequenced clones had abnormal splicing events, including 80% harboring 126 bases retention in intron 16 (c.1496_1497insttgggtcttgtggctgaggggaagggggtacatgaggatgcctgcctatggatgtgttttggcaacagtagagatgccagcaaggaacacagaggagcagacagaaaaaagcacctctagagaaag, p.Met498_Arg499insSWVLWLRGRGYMRMPAYGCVLATVEMPARNTEEQTEKSTSRE) and 20% resulting in exon 16 skipping (c.1383_1496del, p.Ile461_Arg499delinsMet). (D) Schematic representation of the wild-type (WT) mRNA transcript. In the WT, splicing occurred at the authentic splice site and the cDNA can be spliced normally. (E) Schematic representation of the mutant mRNA transcripts caused by the c.1496 + 1G > T variant
case_images: 13023_2024_3510_Fig2_HTML.jpg
case_images: PMC11792323_02_13023_2024_3510_Fig2_HTML.jpg
case_images: Fig2
case_images: T variant locates in intron 16 of SMARCC2, where the adjacent exon region encodes the key SWIRM domain (Fig. 2A). T variant was highly likely to cause loss of the donor splice site (score = 1.0); however, there was also a moderate possibility of obtaining a new donor splice site (score = 0.52). To assess the effects of the c.1496 + 1G > T variant on splicing, SMARCC2 cDNA fragments were obtained from the proband and his healthy father. Surprisingly, only two bands were detected in the normal control sample (Fig. 2B). TA-clone sequencing showed that 75% of the clones were 654-bp products, and the other 25% were novel transcripts with an extra 93 bp from intron 18 (ggccgccaggttgatgctgataccaaggctgggcgaaagggcaaagagctggatgacctggtgccagagacggctaagggcaagccagagctg). Compared to the control sample, the mutant SMARCC2 generated four bands of increased size (Fig. 2B). TA clone sequencing showed that both the wild-type and novel transcripts were present in the patient, accounting for 50% of the total clones (Fig. 2C and D). The remaining 20% resulted from the skipping of exon 16 (c.1383_1496del, p.Ile461_Arg499delinsMet) (Fig. 2C and E).
case_images: Three-dimensional molecular structure modeling of the SMARCC2 protein. (A) The crystal structure of the wild-type (WT) SMARCC2 shows that the Ile461 to Arg499 residues locate in the region composed of alpha-helices. (B, C) Both the p.Met498_Arg499ins42aa and p.Ile461_Arg499delinsMet variants result in the disruption of alpha-helices
case_images: 13023_2024_3510_Fig3_HTML.jpg
case_images: PMC11792323_02_13023_2024_3510_Fig3_HTML.jpg
case_images: Fig3
case_images: Ile461 to Arg499 amino acid residues form multiple alpha helix structures (Fig. 3A); however, insertion of 42 amino acids between Met498 and Arg499 or Ile461_Arg499delinsMet can cause defects in the formation of such important structures (Fig. 3B and C). It is well-established that the region spanning from Ile461 to Arg499 is situated within the