Non-canonical splice site variant demonstrated to result in loss of function (Bowling et al., 2017); This variant is associated with the following publications: (PMID: 26931382, 28554332, 32190976)

Clinical genome sequencing revealed 3 variants of uncertain significance in the ALG1 gene. Multiple lines of computational evidence support a deleterious effect of these variants on the gene or gene product. cDNA experiments performed on a research basis demonstrated that the paternally inherited variant (c.1187+3A>G) is damaging. The maternally inherited variants are rare and are not present in the 1000 Genomes Project nor the Exome Aggregate Consortium (ExAC) databases. Based on these findings, the clinical team elevated these variants to likely pathogenic.

The homozygous c.1187+3A>G variant in ALG1 was identified by our study in one individual with congenital disorder of glycosylation. The c.1187+3A>G variant in ALG1 has been reported in 1 individual with congenital disorder of glycosylation (PMID: 26931382), and has been identified in 0.01906% (24/125942) of European (non-Finnish) chromosomes by the Genome Aggregation Database (gnomAD, http://gnomad.broadinstitute.org; dbSNP rs369160589). Although this variant has been seen in the general population, its frequency is low enough to be consistent with a recessive carrier frequency and the presence of this variant in combination with a reported pathogenic variant and in an individual with congenital disorder of glycosylation increases the likelihood that the c.1187+3A>G variant is pathogenic (PMID: 26931382; Variation ID: 4724). This variant is located in the 5' splice region. Computational tools do suggest an impact to splicing and cDNA analysis by the NIH Undiagnosed Diseases Network demonstrated this variant has a damaging effect (Variation ID: 224118). A biomarker for loss of function in ALG1, xeno-tetrasaccharide, was detected in the serum of an individual with this variant in the compound heterozygous state (PMID: 26931382). Loss of function of the ALG1 gene is an established disease mechanism in autosomal recessive congenital disorder of glycosylation and there is evidence that this gene is necessary for survival (PMID: 26931382). However, these types of assays may not accurately represent biological function and this information is not predictive enough to determine pathogenicity. This variant has also been reported as likely pathogenic in ClinVar by multiple submitters (Variation ID: 224118). Two additional variants in this splice region have been reported pathogenic in ClinVar, raising the possibility that this splice region is a mutational hot spot (Variation ID: 95934, 194107). In summary, this variant meets criteria to be classified as pathogenic for congenital disorder of glycosylation in an autosomal recessive manner based on the predicted impact of the variant and our findings in the literature and ClinVar. ACMG/AMP Criteria applied: PM2, PM3, PS3, PVS1, PM1_Supporting (Richards 2015).

VARIANT INTERPRETATION: The c.1187+3A>G variant in ALG1 has been reported in three individuals with congenital disorders of glycosylation. One is homozygous and two are compound heterozygous with the p.Ser258Leu variant in ALG1 (Ng 2016 26931382, Bowling 2017 28554332, SCV001164452.1). It has also been identified in 0.02% (23/112692) of European chromosomes by gnomAD (http://gnomad.broadinstitute.org) and is reported in ClinVar (Variation ID: 224118). This variant is located in the 5' splice region. In vitro qPCR analysis demonstrates a retention of exon 11 and a stop gain after the addition of 84 nucleotides, consistent with pathogenicity (Bowling 2017 28554332). In summary, this variant meets criteria to be classified as pathogenic for autosomal recessive Congenital disorders of glycosylation. ACMG/AMP Criteria applied: PS3, PM3_Strong.

The heterozygous c.1187+3A>G variant in ALG1 was identified by our study, in the compound heterozygous state with a variant of uncertain significance (ClinVar Variation ID: 1483545), in one individual with intrauterine growth restriction,microcephaly, brain abnormalities, and hypertonia. Trio exome analysis revealed that this variant was in trans with a variant of uncertain significance (ClinVar Variation ID: 1483545). The c.1187+3A>G variant in ALG1 has been reported in two individuals with congenital disorder of glycosylation type I (PMID: 26931382, SCV001164452.1), but has been identified in 0.02% (16/68040) of European (non-Finnish) chromosomes by the Genome Aggregation Database (gnomAD, http://gnomad.broadinstitute.org; dbSNP ID: rs369160589). Although this variant has been seen in the general population in a heterozygous state, its frequency is low enough to be consistent with a recessive carrier frequency. This variant has also been reported in ClinVar (Variation ID: 224118) and has been interpreted as pathogenic or likely pathogenic by multiple submitters. Of the two affected individuals previously reported, one was a homozygote (SCV001164452.1) and one was a compound heterozygote who carried a reported likely pathogenic variant in trans (PMID: 26931382, PMID: 28554332, ClinVar Variation ID: 4724), which increases the likelihood that the c.1187+3A>G variant is pathogenic. RT-PCR analysis performed on affected tissue showed evidence of intron retention before exon 12, leading to a premature stop codon after addition of 84bp (PMID: 28554332). This variant is located in the 5’ splice region. Computational tools do suggest an impact to splicing. However, this information is not predictive enough to determine pathogenicity. In summary, although additional studies are required to fully establish its clinical significance, this variant is likely pathogenic for autosomal recessive congenital disorder of glycosylation type I. ACMG/AMP Criteria applied: PS3_Moderate, PM3, PM2_Supporting, PP4 (Richards 2015).

The ALG1 c.1187+3A>G variant is predicted to interfere with splicing. This variant has been reported in the compound heterozygous state in individuals with congenital disorder of glycosylation (Ng et al. 2016. PubMed ID: 26931382; Bowling et al. 2017. PubMed ID: 28554332; Burdick et al. 2020. PubMed ID: 32190976). RT-PCR analysis indicated this variant leads to retention of intron 11, resulting in addition of 84 nucleotides (28 codons) and a stop gain (Bowling et al. 2017. PubMed ID: 28554332; González-Domínguez et al. 2021. PubMed ID: 34567092). An additional genome study also confirmed that this variant disrupted splicing (Burdick et al. 2020. PubMed ID: 32190976). This variant is reported in 0.020% of alleles in individuals of European (Non-Finnish) descent in gnomAD (http://gnomad.broadinstitute.org/variant/16-5132677-A-G). This variant is interpreted as likely pathogenic.

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 congenital disorder of glycosylation, type Ik (MIM#608540). (I) 0106 - This gene is associated with autosomal recessive disease. (I) 0210 - Splice site variant proven to affect splicing of the transcript with a known effect on protein sequence. Quantitative PCR from a patient's blood RNA showed the variant led to increased retention of intron 11, which is predicted to result in a premature stop codon and nonsense-mediated decay (NMD) of RNA (PMID: 28554332). (SP) 0251 - This variant is heterozygous. (I) 0304 - Variant is present in gnomAD (v2) <0.01 for a recessive condition (25 heterozygotes, 0 homozygotes). (SP) 0600 - Variant is located in the annotated glycosyl transferases group 1 domain (DECIPHER). (I) 0702 - Other premature termination variants comparable to the one identified in this case have strong previous evidence for pathogenicity. Many NMD-predicted variants have been reported as likely pathogenic/pathogenic in ClinVar. (SP) 0801 - This variant has strong previous evidence of pathogenicity in unrelated individuals. It has been reported as likely pathogenic/pathogenic multiple times in ClinVar, and as compound heterozygous with p.(Ser258Leu) in an affected individual with type I congenital disorder of glycosylation (PMIDs: 26931382, 28554332). (SP) 1206 - This variant has been shown to be paternally inherited (by trio analysis). (I) Legend: (SP) - Supporting pathogenic, (I) - Information, (SB) - Supporting benign

Variant summary: ALG1 c.1187+3A>G alters a conserved nucleotide located close to a canonical splice site and therefore could affect mRNA splicing, leading to a significantly altered protein sequence. Several computational tools predict a significant impact on normal splicing: Three predict the variant abolishes a 5 prime splicing donor site. One predict the variant weakens a 5 prime donor site. At least one publication reports experimental evidence that this variant affects mRNA splicing (Bowling_2017). The variant allele was found at a frequency of 0.0001 in 249520 control chromosomes. This frequency is not significantly higher than estimated for a pathogenic variant in ALG1 causing Congenital Disorder Of Glycosylation Type 1K (0.0001 vs 0.0011), allowing no conclusion about variant significance. c.1187+3A>G has been reported in the literature in individuals affected with Congenital Disorder Of Glycosylation Type 1K. These data indicate that the variant may be associated with disease. The following publications have been ascertained in the context of this evaluation (PMID: 28554332, 32190976, 34567092, 26931382, 38736633). ClinVar contains an entry for this variant (Variation ID: 224118). Based on the evidence outlined above, the variant was classified as likely pathogenic.

This sequence change falls in intron 11 of the ALG1 gene. It does not directly change the encoded amino acid sequence of the ALG1 protein. RNA analysis indicates that this variant induces altered splicing and may result in an absent or altered protein product. This variant is present in population databases (rs369160589, gnomAD 0.02%). This variant has been observed in individual(s) with congenital disorders of glycosylation (PMID: 26931382, 32190976). In at least one individual the data is consistent with being in trans (on the opposite chromosome) from a pathogenic variant. ClinVar contains an entry for this variant (Variation ID: 224118). Variants that disrupt the consensus splice site are a relatively common cause of aberrant splicing (PMID: 17576681, 9536098). Studies have shown that this variant results in retention of intron 11 of the ALG1 gene, and produces a non-functional protein and/or introduces a premature termination codon (PMID: 28554332). For these reasons, this variant has been classified as Pathogenic.

PS4_moderate, PP3_supporting, PS3_moderate

Variant classified as Pathogenic and reported on 07-27-2021 by Invitae. Assertions are reported exactly as they appear on the patient provided laboratory report. GenomeConnect does not attempt to reinterpret the variant. The IDDRC-CTSA National Brain Gene Registry (BGR) is a study funded by the U.S. National Center for Advancing Translational Sciences (NCATS) and includes 13 Intellectual and Developmental Disability Research Center (IDDRC) institutions. The study is led by Principal Investigator Dr. Philip Payne from Washington University. The BGR is a data commons of gene variants paired with subject clinical information. This database helps scientists learn more about genetic changes and their impact on the brain and behavior. Participation in the Brain Gene Registry requires participation in GenomeConnect. More information about the Brain Gene Registry can be found on the study website - https://braingeneregistry.wustl.edu/.