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Case Reports
. 2014 Jun 4;15:64.
doi: 10.1186/1471-2350-15-64.

Structural Variation and Missense Mutation in SBDS Associated With Shwachman-Diamond Syndrome

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Free PMC article
Case Reports

Structural Variation and Missense Mutation in SBDS Associated With Shwachman-Diamond Syndrome

Claudia M B Carvalho et al. BMC Med Genet. .
Free PMC article

Abstract

Background: Shwachman-Diamond syndrome (SDS) is an autosomal recessive ribosomopathy caused mainly by compound heterozygous mutations in SBDS. Structural variation (SV) involving the SBDS locus has been rarely reported in association with the disease. We aimed to determine whether an SV contributed to the pathogenesis of a case lacking biallelic SBDS point mutations.

Case presentation: Whole exome sequencing was performed in a patient with SDS lacking biallelic SBDS point mutations. Array comparative genomic hybridization and Southern blotting were used to seek SVs across the SBDS locus. Locus-specific polymerase chain reaction (PCR) encompassing flanking intronic sequence was also performed to investigate mutation within the locus. RNA expression and Western blotting were performed to analyze allele and protein expression. We found the child harbored a single missense mutation in SBDS (c.98A > C; p.K33T), inherited from the mother, and an SV in the SBDS locus, inherited from the father. The missense allele and SV segregated in accordance with Mendelian expectations for autosomal recessive SDS. Complementary DNA and western blotting analysis and locus specific PCR support the contention that the SV perturbed SBDS protein expression in the father and child.

Conclusion: Our findings implicate genomic rearrangements in the pathogenesis of some cases of SDS and support patients lacking biallelic SBDS point mutations be tested for SV within the SBDS locus.

Figures

Figure 1
Figure 1
Clinical features of SDS proband. a. Transverse abdominal ultrasound image obtained at 5 years 11 months of age revealing normal size, contour, and echogenicity of the pancreas, indicated by arrow. b. Axial conventional spin echo T1-weighted MR image obtained at 5 years 11 months of age showing normal signal intensity of the pancreatic parenchyma with no evidence of atrophy or fatty infiltration of the pancreas, indicated by arrow. c. Axial contrast-enhanced CT image obtained at 6 years 5 months of age demonstrating normal attenuation of the pancreatic parenchyma with no evidence of atrophy or fatty infiltration of the pancreas, indicated by arrow. d. The proband’s growth curve showing markedly short stature. The bottom-most curve normal curve represents the 5th percentile for girls.
Figure 2
Figure 2
Segregation of private mutations spanning SBDS in family HOU1479. Missense mutation c.98A > C was maternally inherited by all four children whereas the SV is present only in the father and the patient with SDS. a. Top: Pedigree of family HOU1479 with information about carrier status of both mutations in each family member. Bottom: SBDS allele status with corresponding chromatograms of exon 1 Sanger sequenced using genomic DNA (gDNA) or cDNA prepared from RNA extracted from LCLs. b. Western blot analysis of SBDS in LCL whole cell extracts prepared from an unrelated, unaffected control (CTL), the proband and her family members as indicated. Beta-actin blotting was performed for a protein loading control. c. Genomic organization of SBDS and SBDSP1 with identification of the region of the probe used for Southern blot in d. d. Southern blotting of SBDS and SBDSP1 in samples from family HOU1479 and three unrelated, unaffected controls (CTL1-3) using KpnI restriction enzyme.
Figure 3
Figure 3
Southern blotting of SBDS and SBDSP1 in samples from family HOU1479 using Xba I restriction enzyme.
Figure 4
Figure 4
Additional studies of the genomic segment that includes SBDS supports the presence of an insertion of unknown origin within the paternal allele. a. Agarose gel analysis of a 13.1 kb long-range PCR product spanning SBDS and flanking region, the region targeted by the Southern blotting studies (Figures 2 and 3). DNA from patient (BAB3762), parents (BAB3763- mother, BAB3764-father) and a normal controls (CTL1 and CTL2) were amplified using primers DelFb + KpnR, and then digested with either KpnI or SacI. KpnI did not digest the 13.1 kb PCR product, consistent with lack of amplification of the paternal allele. Consistently, SacI digestion of the 13.1 kb PCR product showed an identical pattern in samples and controls. b. Sanger sequencing of intron 2 amplified along with exon 2 using short range PCR revealed inconsistent segregation of a paternal genotype for two polymorphic SNPs in the patient (BAB3762). *Non-digested PCR product.
Figure 5
Figure 5
Approximate location of the inherited SV at the SBDS locus based on Southern blotting assays using KpnI and XbaI restriction enzymes. a. Size and location of the expected segments obtained using KpnI and XbaI restriction enzymes based on the reference genome (blue arrows) and extra segments obtained exclusively in SV carriers (BAB3762 and BAB3764 - purple arrows). The nature of the SV is currently unknown although the Southern blotting results are consistent with a genomic insertion of at least 2.8 kb (represented by a crosshatch red box) somewhere between SBDS 5’flanking region and intron 3 (represented by a red box with an arrow on top). Red box: Southern blotting probe. Yellow rectangle highlights a common probe target region between the reference genome and the genome of the SV carriers. b. An alternative hypothesis of SV present within the pseudogene. Genomic coordinates as of GRCh37/ hg19 to SBDS and SBDSP1 loci are represented on top of each region. CEN: centromere; TEL: telomere.

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