ClinVar Genomic variation as it relates to human health

The c.258+2T>C variant in SBDS is one of the most common pathogenic variants ide ntified in individuals with Shwachman-Diamond syndrome (Boocock 2003, https://ww w.ncbi.nlm.nih.gov/books/NBK1756/). It has also been identified in 0.9% (236/250 74) of Finnish chromosomes and 2 homozygotes by gnomAD (http://gnomad.broadinsti tute.org); however, this allele frequency may not be accurate due to the presenc e of a pseudogene (SBDSP). This variant usually occurs as the result of a gene c onversion event as the c.258+2C>T variant is present in the inactive pseudogene. It is predicted to result in a frameshift described as p.Cys84TyrfsX4 (Boocock 2003, Orelio 2011). This alteration is then predicted to lead to a truncated or absent protein. Loss of function of the SBDS gene is an established disease mech anism in autosomal recessive Shwachman-Diamond syndrome. In summary, this varian t meets criteria to be classified as pathogenic. ACMG/AMP Criteria applied: PVS1 , PM3_Very Strong, PS3.

The c.258+2T>C variant is a canonical splice donor variant in the SBDS gene. The variant is one of the most common pathogenic variants in SBDS (PMID: 20301722), and has been reported by multiple laboratories in the ClinVar as pathogenic (variant ID: 3196). Functional studies suggest that the c.258+2 T>C variant affects the protein's cellular localization and motility (PMID: 21695142). The c.258+2T>C variant is present in ExAC at 0.39% (474/120486), but it is not present in the homozygous state. Based on the combined evidence, the variant is classified as pathogenic.

A Heterozygous Splice site donor variant c.258+2T>C in Exon 2 of the SBDS gene that results in the amino acid substitution was identified. The observed variant has a minor allele frequency of 0.00388/0.00386 in gnomAD exomes and genomes, respectively. The severity of the impact of this variant on the protein is high, based on the effect of the protein and REVEL score . Rare Exome Variant Ensemble Learner (REVEL) is an ensembl method for predicting the pathogenicity of missense variants based on a combination of scores from 13 individual tools: MutPred, FATHMM v2.3, VEST 3.0, PolyPhen-2, SIFT, PROVEAN, MutationAssessor, MutationTaster, LRT, GERP++, SiPhy, phyloP, and phastCons. The REVEL score for an individual missense variant can range from 0 to 1, with higher scores reflecting greater likelihood that the variant is disease-causing. ClinVar has also classified this variant as Pathogenic/Likely Pathogenic [Variant ID: 3196]. This varaint is reported for Shwachman-Diamond syndrome and functional studies suggest that the c.258+2 T>C variant affects the protein's cellular localization and motility (Orelio C et . al., 2011). For these reasons, this variant has been classified as Pathogenic.

This variant affects the canonical splice donor site of intron 2 and is therefore predicted to interfere with splicing and result in loss of normal protein function through either protein truncation or nonsense-mediated mRNA decay (NMD). This is a common Pathogenic variant found in individuals with Shwachman-Diamond syndrome, which arises from a gene conversion event with a nearby SBDS pseudogene (*see note below) (PMID: 20301722, 12496757, 19222471). It has been previously reported as a compound heterozygous, homozygous, and, less frequently, as a heterozygous change in patients with Shwachman-Diamond syndrome (PMID: 12496757, 14749921, 15860664, 22935661). Functional studies suggest that the c.258+2 T>C variant affects the protein's cellular localization and motility (PMID: 21695142). It is present in the heterozygous state in the gnomAD population database at a frequency of 0.388% (1096/282568) and seen in the homozygous state in 2 individuals. Based on the available evidence, the c.258+2T>C variant is classified as Pathogenic. <b>*Note:</b> The SBDS gene has a highly homologous nonfunctional pseudogene called SBDSP. The majority of pathogenic variation in SBDS has been found to result from gene conversion (a process by which a small segment of the functional SBDS gene is replaced by a segment copied from the nonfunctional SBDSP pseudogene). The variants, c.258+2T>C and c.183_184delTAinsCT (p.Lys62Ter), are recurrent Pathogenic variants resulting from gene conversion. These variants have been reported in the compound heterozygous state (trans configuration) in multiple affected individuals. These variants have also been reported as a single complex allele, c.184A>T; 258+2T>C (cis configuration), which is then seen in the compound state with another Pathogenic variant on the second allele in affected individuals (PMID: 12496757, 29892551, 27290639, 28102861, 30109123).

The SBDS c.258+2T>C variant is predicted to disrupt the GT donor site and interfere with normal splicing. This variant has been reported in the homozygous and compound heterozygous states in individuals with Shwachman-Diamond Syndrome (Boocock et al. 2003. PubMed ID: 12496757; Myers et al. 2014. PubMed ID: 24388329). This variant has also been observed in the compound heterozygous state with a second pathogenic variant in an individual undergoing inherited bone marrow failure panel testing at our laboratory (Internal Data, PreventionGenetics). This variant is reported in 0.94% of alleles in individuals of European (Finnish) descent in gnomAD. Variants that disrupt the consensus splice donor site in SBDS are expected to be pathogenic. This variant is interpreted as pathogenic.

This variant was determined to be pathogenic according to ACMG Guidelines, 2015 [PMID:25741868].

Canonical splice site variant in a gene for which loss-of-function is a known mechanism of disease; Results from a conversion event with a nearby SBDS pseudogene, an inactive gene with numerous pathogenic variants (Boocock et al., 2003); Published functional studies demonstrate that truncating variants, including c.258+2 T>C, may affect the protein's cellular localization and motility (Austin et al., 2005; Orelio et al., 2011); This variant is associated with the following publications: (PMID: 27127007, 15942154, 29620724, 16007594, 30109123, 12496757, 15860664, 21695142, 22934832, 14749921, 19148133, 22935661, 24629175, 25525159, 24426364, 26762974, 26866830, 17376717, 27153395, 26822237, 27431290, 17478638, 26492932, 15342903, 27519942, 25844324, 27290639, 15701631, 29716638, 25729736, 29146883, 23125299, 28102861, 28485484, 29375851, 30664904, 15474150, 30198570, 17920346, 30308536, 30105119, 27617157, 31321910, 31732620, 30894704, 32098966, 31965297, 32434641, 31019026, 32293785, 31980526, 32552793, 32581362, 32412173, 31589614, 32868804, 32888943)

PVS1, PP2, PP5

This variant was determined to be pathogenic according to ACMG Guidelines, 2015 [PMID:25741868].

The SBDS c.258+2T>C variant, also described as p.Cys84fsTer3, impacts the canonical splice donor site and results in an 8-bp deletion consistent with the use of a cryptic splice site and causing a frameshift and premature termination of the protein (Boocock et al. 2003). This variant, which occurs as a result of a gene conversion event with the nearby and highly homologous pseudogene SBDSP, is one of the most common disease-associated alleles in the SBDS gene (Nelson and Myers 2018). Across a small selection of the available literature, the c.258+2T>C variant was identified in 195 individuals with a confirmed or probable diagnosis of Shwachman-Diamond syndrome, including in a homozygous state in 13 individuals and in a compound heterozygous state in 182 individuals, 119 of whom carried the c.183_184delTAinsCT (p.Lys62Ter) variant, another common gene conversion variant, as the second variant (Boocock et al. 2003; Woloszynek et al. 2004; Myers et al. 2014; Cho et al. 2015; Ipatova et al. 2019). The c.258+2T>C variant was absent from 148 control individuals (Boocock et al. 2003; Woloszynek et al. 2004) but is reported at a frequency of 0.009412 in the European (Finnish) population of the Genome Aggregation Database (version 2.1.1). This population also includes two individuals who appear to be homozygous for this variant, though there are no homozygous individuals reported in a more recent version of the database (version 3.1.1). The presence of the SBDSP pseudogene may complicate the accurate reporting of the variant frequency in the population (Nelson and Myers 2018). In a functional study, Orelio et al. (2011) transiently transfected HeLa cells with GFP constructs containing full length or truncated protein due to the c.258+2T>C variant. The authors observed that the full-length protein was localized in both the nucleus and cytoplasm, while the truncated protein was mainly detected in the nucleus. The presence of the variant also affected nuclear import of SBDS proteins. Based on the available evidence, the c.258+2T>C variant is classified as pathogenic for Shwachman-Diamond syndrome.

This variant was identified as homozygous._x000D_ Criteria applied: PVS1, PS3, PS4

The identified mutation changes the splicing process of SBDS gene.

ACMG classification criteria: PVS1 strong, PS3 supporting, PM3 very strong

SBDS NM_016038.3 exon 2 c.258+2T>C: This variant has been reported in the literature in several individuals with Shwachman-Diamond syndrome as homozygous or compound heterozygous (most often found in trans with c.183_184delinsCT) and is one of the most common pathogenic variants for this gene (Boocock 2003 PMID:12496757, Nakashima 2004 PMID:14749921, Austin 2005 PMID:15860664, Andolina 2013 PMID:22935661, Myers 2014 PMID:20301722). This variant is present in 0.3% (468/126574) of European alleles in the Genome Aggregation Database (http://gnomad.broadinstitute.org/rs113993993). This variant is present in ClinVar, with several labs classifying this variant as pathogenic (Variation ID:3196). Evolutionary conservation and computational predictive tools for this variant are limited or unavailable. In addition, functional studies have shown a deleterious effect of this variant (Orelio 2011 PMID:21695142). Of note, this variant alters the consensus splice sequence (+/- 1,2) which is predicted to result in an absent or abnormal protein. Loss of function has been reported as a disease mechanism for this gene and disease (Woloszynek 2004 PMID:15284109). In summary, this variant is classified as pathogenic.

The heterozygous c.258+2T>C variant in SBDS was identified by our study, in the compound heterozygous state along with a pathogenic variant (ClinVar Variation ID: 265256), in one individual with Swachman-Diamond syndrome. This individual also carried a pathogenic variant (ClinVar Variation ID: 265256), however the phase of these variants are unknown at this time. The c.258+2T>C variant in SBDS has been reported in over 108 unrelated individuals with Swachman-Diamond syndrome (PMID: 15769891, PMID: 15860664, PMID: 12496757) but has been identified in 0.8% (88/10582) of European (Finnish) chromosomes by the Genome Aggregation Database (gnomAD, http://gnomad.broadinstitute.org; dbSNP ID: rs113993993); however, this allele frequency may not be accurate due to the presence of a pseudogene (SBDSP). This variant has also been reported in ClinVar (Variation ID: 3196) and has been interpreted as pathogenic by multiple submitters. Of these 108 individuals, 9 were homozygotes (PMID: 15860664, PMID: 12496757, PMID: 15769891) and 97 were compound heterozygotes who carried pathogenic or likely pathogenic variants in trans (PMID: 15769891, ClinVar Variation ID: 3195, ClinVar Variation ID: 21539; PMID: 15860664, ClinVar Variation ID: 929404, ClinVar Variation ID: 3195, ClinVar Variation ID: 265256, PMID: 12496757, ClinVar Variation ID: 3195, ), which increases the likelihood that the c.258+2T>C variant is pathogenic. RT-PCR analysis performed on affected tissue shows evidence of altered splicing of exon 2, with an 8bp deletion, frameshift, and premature protein truncation (PMID: 12496757); in vitro assays suggest that the resulting prematurely truncated protein is unstable (PMID: 17478638). This variant is located in the 5' splice region. Computational tools predict a splicing impact, though this information is not predictive enough to determine pathogenicity. Loss of function is an established disease mechanism of autosomal recessive Swachman-Diamond syndrome. In summary, this variant meets criteria to be classified as pathogenic for autosomal recessive Swachman-Diamond syndrome. ACMG/AMP Criteria applied: PVS1, PS3_Moderate, PM3_VeryStrong (Richards 2015).

PM1, PM3, PS3, PS4, PVS1

Variant summary: SBDS c.258+2T>C is located in a canonical splice-site and is predicted to affect mRNA splicing resulting in a significantly altered protein due to either exon skipping, shortening, or inclusion of intronic material. Several computational tools predict a significant impact on normal splicing: Three predict the variant abolishes the canonical 5' splicing donor site and three predict the variant creates/strengthens a cryptic 5' donor site. At least one publication reports experimental evidence that this variant indeed affects mRNA splicing and results in the skipping of exon 2 (e.g. Peretto_2023). The variant allele was found at a frequency of 0.0039 in 251238 control chromosomes in the gnomAD database, including 2 homozygotes. However, due to the presence of a SBDS pseudogene, this frequency may be inaccurate, allowing no conclusion about variant significance. c.258+2T>C has been reported in the literature in many individuals affected with Shwachman-Diamond Syndrome 1 and is considered a common disease variant (e.g. Furutani_2022). These data indicate that the variant is very likely to be associated with disease. The following publications have been ascertained in the context of this evaluation (PMID: 36835434, 34758064). ClinVar contains an entry for this variant (Variation ID: 3196). Based on the evidence outlined above, the variant was classified as pathogenic.

The c.258+2T>C intronic pathogenic mutation results from a T to C substitution two nucleotides after coding exon 2 in the SBDS gene. This mutation has been described in multiple individuals with Shwachman-Diamond syndrome; it has been reported in homozygous individuals and is also known to occur as part of a complex allele: c.[183_184delTAinsCT;258+2T>C] (Boocock GR et al. Nat. Genet., 2003 Jan;33:97-101). In a study of seven individuals with c.258+2T>C in trans with a second mutation, each had a history of pancreatic insufficiency, four had skeletal abnormalities, and two had hematological malignancies (Kawakami T et al. Tohoku J. Exp. Med., 2005 Jul;206:253-9). RT-PCR identified a deletion of 8 base pairs at the end of exon 2 in some affected individuals; gene sequencing revealed the c.258+2T>C alteration in these individuals, which would be consistent with the use of an upstream cryptic splice donor predicted to result in an 8 base pair deletion leading to a frameshift and premature protein truncation (p.C84Yfs*4; Boocock GR et al. Nat. Genet., 2003 Jan;33:97-101). In addition to the clinical data presented in the literature, alterations that disrupt the canonical splice site are expected to cause aberrant splicing, resulting in an abnormal protein or a transcript that is subject to nonsense-mediated mRNA decay. As such, this alteration is classified as pathogenic.

The splicesite variant has been reported previously in homozygous and compound heterozygous state in individuals with Shwachman-Diamond syndrome Cho WK, et al., 2015. Functional studies suggest that the c.258+2 T>C variant affects theprotein's cellular localization and motility Orelio C, et al., 2011. The variant is reported with 0.3% allele frequency in gnomADExomes and is novel not in any individuals in 1000 Genomes. This variant has been reported to the ClinVar database as LikelyPathogenic/ Pathogenic multiple submissions. The variant affects the GT donor splice site downstream of exon 2. This variant is predicted to cause loss of normal protein function through protein truncation. Loss of function variants have been previouslyreported to be disease causing. For these reasons, this variant has been classified as Pathogenic.

SBDS: PVS1, PS4:Moderate

Based on the classification scheme VCGS_Germline_v1.3.4, this variant is classified as Pathogenic. Following criteria are met: 0102 - Loss of function is a known mechanism of disease in this gene and is associated with Shwachman-Diamond syndrome (MIM#260400). (I) 0106 - This gene is associated with autosomal recessive disease. (I) 0115 - Variants in this gene are known to have variable expressivity. A phenotypic spectrum with variable severity was observed in a cohort study. In addition, two unrelated asymptomatic individuals were later diagnosed with mild Shwachmann-Diamond syndrome following genetic investigations due to family history and clinical follow-ups (PMID: 24388329). (I) 0210 - Splice site variant proven to affect splicing of the transcript with a known effect on protein sequence. RT-PCR analysis of patient cells showed an 8bp deletion at the end of exon 2 consistent with the use of an upstream cryptic splice donor site. The deletion results in a frameshift and a premature termination codon, p.(Cys84Tyrfs*4) (PMID: 12496757, 15860664). (SP) 0252 - This variant is homozygous. (I) 0304 - Variant is present in gnomAD (v2) <0.01 for a recessive condition (1092 heterozygotes, 2 homozygotes). (SP) 0311 - Alternative nucleotide changes at the same canonical splice site are present in gnomAD (v2 & v3) (highest allele count: 3 heterozygotes, 0 homozygotes). (I) 0801 - This variant has very strong previous evidence of pathogenicity in unrelated individuals. It has been previously reported as pathogenic in many patients with Shwachman-Diamond syndrome and is one of the most common pathogenic variants in cases of SBDS [ClinVar, PMIDs: 12496757, 32150944, Nelson and Myers (2018)]. (SP) 1201 - Heterozygous variant detected in trans with a second pathogenic heterozygous variant, NM_016038.2(SBDS):c.183_184delinsCT; p.(Lys62*), in a recessive disease. (SP) 1209 - This variant has been shown to be both maternally and paternally inherited (biallelic) (Sanger analysis). (I) Legend: (SP) - Supporting pathogenic, (I) - Information, (SB) - Supporting benign

This pathogenic sequence change has previously been described in individuals with Shwachman- Diamond syndrome in the homozygous or compound heterozygous state with other pathogenic changes (PMIDs 12496757, 27127007, 21695142,15284109, 15942154). This sequence change was also identified in 4 individuals in the heterozygous state with apparently acquired aplastic anemia and short telomeres in granulocytes (PMID: 17478638); however further studies with larger populations are required to establish the association. Functional studies have shown that this sequence change results in a non-expressed product and impairs SBDS function (PMID 17478638). These collective evidences indicate that this sequence change is pathogenic.

This variant has been previously reported as disease-causing. It would be pathogenic in a recessive state; heterozygotes would be carriers for the condition. It was found once in our study heterozygous in a 12-year-old male with pilocytic astrocytoma.

NG_007277.1(NM_016038.2):c.258+2T>C in the SBDS gene has an allele frequency of 0.009 in European (Finnish) subpopulation in the gnomAD database. Andolina JR et al. identified compound heterozygous mutations c183_184 TA>CT and c.258+2 T>C in two patients with Shwachman-Diamond syndrome (PMID: 22935661). Taken together, we interprete this variant as Pathogenic/Likely pathogenic. ACMG/AMP Criteria applied: PVS1; PM3; PP4.