June 2022 – Laboratory Genetics and Genomics and Molecular Pathology

A next-generation sequencing panel targeting genes associated with congenital neutropenia was ordered for a 2-month-old female infant with isolated persistent neutropenia. Two heterozygous pathogenic variants were identified in the SBDS gene (NM_016038.2): c.258+2T>C and c.183_184delinsCT. Representative reads are shown in Figure 1. Considering the presence of a highly homologous SBDSP1 pseudogene, long-range PCR using a SBDS-specific primer followed by Sanger sequencing were subsequently performed for variant confirmation (Figure 2).

Figure 1
Figure 2

Based on these results, which one of the following statements is the most appropriate interpretation for this case?

  • The SBDS variants are confirmed by Sanger sequencing and are in cis configuration. These findings are therefore less suggestive of Schwachman-Diamond syndrome; however, this individual is at least a carrier.
  • The SBDS variants are confirmed by Sanger sequencing and are in trans configuration. However, as results can be confounded by pseudogene interference, these findings remain inconclusive.
  • The SBDS variants are confirmed by Sanger sequencing, yet the phase is unclear. Parental testing is therefore indicated to determine phase.
  • The SBDS variants are confirmed by Sanger sequencing and are in trans configuration. These findings therefore support a diagnosis of Schwachman-Diamond syndrome.

The correct answer is ...

The SBDS variants are confirmed by Sanger sequencing and are in trans configuration. These findings therefore support a diagnosis of Schwachman-Diamond syndrome.

Schwachman-Diamond syndrome is an inherited bone marrow failure syndrome classically characterized by a triad of neutropenia, exocrine pancreatic insufficiency, and bony abnormalities. It is most often caused by biallelic pathogenic variants in the SBDS gene located at chromosome 7q11.21. The SBDS gene is involved in several processes such as ribosome biogenesis, mitotic spindle stabilization, DNA repair, and maintenance of stromal microenvironment.

Next-generation sequencing (NGS) approaches may be used, amongst others, for diagnostic investigation of suspected inherited bone marrow failure syndromes such as Schwachman-Diamond syndrome. In the presented case, NGS testing identified two pathogenic variants within the SBDS gene. While parental testing is often required to establish the phase of variants, in this case, visualization of the NGS reads demonstrated that the pathogenic variants identified are in trans configuration. Individual reads overlapping both variant loci and including the c.183_184delinsCT variant do not have the c.258+2T>C variant, and reads that include the c.258+2T>C variant do not have the c.183_184delinsCT variant. This finding indicates that the variants are in trans (on opposite alleles). (Figure 1).

The SBDS gene lies in close proximity with a highly homologous pseudogene SBDSP1. Variants leading to Schwachman-Diamond syndrome, such as those identified in this patient, most commonly arise from recombination or gene conversion events involving the SBDSP1 pseudogene. Gene conversion, which occurs in the setting of double-strand breaks, refers to a process in which genetic material is transferred from an initial DNA sequence and replaces a highly homologous receiver sequence, while leaving the initial sequence unchanged. Gene conversion events in which genetic material from a pseudogene replaces the sequence of a functional gene resulting in functional compromise of the receiver gene are involved in certain diseases such as Schwachman-Diamond syndrome.

From a diagnostic perspective, the highly homologous SBDSP1 pseudogene and gene conversion events raise diagnostic challenges related to ambiguous or erroneous alignment of NGS short reads with consequent risk for misdiagnosis. Accordingly, and as described in the case vignette, PCR amplification using SBDS-specific-primers which amplify the gene (and not the pseudogene) followed by Sanger sequencing of the amplicons may be used to confirm that identified variants lie within the SBDS gene and ensure accuracy of result interpretation. Figure 2 illustrates the electrophoregram of Sanger sequencing and provides confirmation that the variants are both present within the SBDS gene.

Therefore, these results support a diagnosis of Schwachman-Diamond syndrome, as the variants are in trans configuration and located in the SBDS gene.


  1. Yamada M, Uehara T, Suzuki H, et al. Shortfall of exome analysis for diagnosis of Shwachman-Diamond syndrome: Mismapping due to the pseudogene SBDSP1. Am J Med Genet A. 2020;182,7: 1631-1636. doi:10.1002/ajmg.a.61598
  2. Chen, JM., Cooper, D., Chuzhanova, N. et al. Gene conversion: mechanisms, evolution and human disease. Nat Rev Genet 8, 762–775 (2007). doi:10.1038/nrg2193
  3. Nelson A, Kasiani Myers. Shwachman-Diamond Syndrome. GeneReviews, edited by Margaret P Adam et. al., University of Washington, Seattle, 17 July 2008.
  4. Thompson AS, Giri N, Gianferante DM, et al. Shwachman Diamond syndrome: narrow genotypic spectrum and variable clinical features. Pediatr Res., 2022 March 23, doi:10.1038/s41390-022-02009-8.
  5. Dror Y, Donadieu J, Koglmeier J, et al. Draft consensus guidelines for diagnosis and treatment of Shwachman-Diamond syndrome. Ann N Y Acad Sci. 2011;1242:40-55. doi:10.1111/j.1749-6632.2011.06349.x
  6. Boocock GR, Morrison JA, Popovic M, et al. Mutations in SBDS are associated with Shwachman-Diamond syndrome. Nat Genet. 2003;33(1):97-101. doi:10.1038/ng1062

Marie-France Gagnon, M.D.

Fellow, Laboratory Genetics and Genomics
Mayo Clinic

Ann Moyer, M.D., Ph.D.

Ann Moyer, M.D., Ph.D.

Consultant, Laboratory Genetics and Genomics
Mayo Clinic
Associate Professor of Laboratory Medicine and Pathology
Mayo Clinic College of Medicine and Science

MCL Education

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