Case Report: Two Novel Frameshift Mutations in SLC20A2 and One Novel Splice Donor Mutation in PDGFB Associated With Primary Familial Brain Calcification

Primary familial brain calcification (PFBC, OMIM#213600), also known as Fahr's disease, is characterized by bilateral and symmetric brain calcification in the basal ganglia (globus pallidus, caudate nucleus, and putamen), thalamus, subcortical white matter, and cerebellum. PFBC can be caused by loss-of-function mutations in any of the six known causative genes. The most common clinical manifestations include movement disorders, cognitive impairment, and neuropsychiatric signs that gradually emerge in middle-aged patients. To broaden the PFBC mutation spectrum, we examined nine members of a family with PFBC and two sporadic cases from clinical departments, and sequenced all PFBC-causative genes in the index case. Two novel frameshift mutations in SLC20A2 [NM_001257180.2; c.806delC, p.(Pro269Glnfs*49) and c.1154delG, p.(Ser385Ilefs*70)] and one novel splice donor site mutation (NM_002608.4, c.456+1G>C, r.436_456del) in PDGFB were identified in the patient cohort. c.806delC co-segregated with brain calcification and led to SLC20A2 haploinsufficiency among the affected family members. The c.456+1G>C mutation in PDGFB resulted in aberrant mRNA splicing, thereby forming mature transcripts containing an in-frame 21 base pair (bp) deletion, which might create a stably truncated protein [p.(Val146_Gln152del)] and exert a dominant negative effect on wild-type PDGFB. All three mutations were located in highly conserved regions among multiple species and predicted to be pathogenic, as evaluated by at least eight common genetic variation scoring systems. This study identified three novel mutations in SLC20A2 and PDGFB, which broadened and enriched the PFBC mutation spectrum.

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Biallelic variants in COPB1 cause a novel, severe intellectual disability syndrome with cataracts and variable microcephaly

Genome Medicine ◽

10.1186/s13073-021-00850-w ◽

2021 ◽

Vol 13 (1) ◽

Author(s):

William L. Macken ◽

Annie Godwin ◽

Gabrielle Wheway ◽

Karen Stals ◽

Liliya Nazlamova ◽

...

Keyword(s):

Genome Sequencing ◽

Donor Site ◽

Xenopus Tropicalis ◽

Splice Donor Site ◽

Homologous Region ◽

Disease Genes ◽

Coat Proteins ◽

Splice Donor ◽

Severe Intellectual Disability

Abstract Background Coat protein complex 1 (COPI) is integral in the sorting and retrograde trafficking of proteins and lipids from the Golgi apparatus to the endoplasmic reticulum (ER). In recent years, coat proteins have been implicated in human diseases known collectively as “coatopathies”. Methods Whole exome or genome sequencing of two families with a neuro-developmental syndrome, variable microcephaly and cataracts revealed biallelic variants in COPB1, which encodes the beta-subunit of COPI (β-COP). To investigate Family 1’s splice donor site variant, we undertook patient blood RNA studies and CRISPR/Cas9 modelling of this variant in a homologous region of the Xenopus tropicalis genome. To investigate Family 2’s missense variant, we studied cellular phenotypes of human retinal epithelium and embryonic kidney cell lines transfected with a COPB1 expression vector into which we had introduced Family 2’s mutation. Results We present a new recessive coatopathy typified by severe developmental delay and cataracts and variable microcephaly. A homozygous splice donor site variant in Family 1 results in two aberrant transcripts, one of which causes skipping of exon 8 in COPB1 pre-mRNA, and a 36 amino acid in-frame deletion, resulting in the loss of a motif at a small interaction interface between β-COP and β’-COP. Xenopus tropicalis animals with a homologous mutation, introduced by CRISPR/Cas9 genome editing, recapitulate features of the human syndrome including microcephaly and cataracts. In vitro modelling of the COPB1 c.1651T>G p.Phe551Val variant in Family 2 identifies defective Golgi to ER recycling of this mutant β-COP, with the mutant protein being retarded in the Golgi. Conclusions This adds to the growing body of evidence that COPI subunits are essential in brain development and human health and underlines the utility of exome and genome sequencing coupled with Xenopus tropicalis CRISPR/Cas modelling for the identification and characterisation of novel rare disease genes.

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A 5′ splice site mutation affecting the pre-mRNA splicing of two upstream exons in the collagen COL1A1 gene. Exon 8 skipping and altered definition of exon 7 generates truncated proα1(I) chains with a non-collagenous insertion destabilizing the triple helix

Biochemical Journal ◽

10.1042/bj3020729 ◽

1994 ◽

Vol 302 (3) ◽

pp. 729-735 ◽

Cited By ~ 21

Author(s):

J F Bateman ◽

D Chan ◽

I Moeller ◽

M Hannagan ◽

W G Cole

Keyword(s):

Splice Site ◽

De Novo ◽

Donor Site ◽

Mrna Splicing ◽

Splice Donor Site ◽

Sequence Motif ◽

Type I ◽

Splice Donor ◽

Site Mutation ◽

Definition Of

A heterozygous de novo G to A point mutation in intron 8 at the +5 position of the splice donor site of the gene for the pro alpha 1(I) chain of type I procollagen, COL1A1, was defined in a patient with type IV osteogenesis imperfecta. The splice donor site mutation resulted not only in the skipping of the upstream exon 8 but also unexpectedly had the secondary effect of activating a cryptic splice site in the next upstream intron, intron 7, leading to re-definition of the 3′ limit of exon 7. These pre-mRNA splicing aberrations cause the deletion of exon 8 sequences from the mature mRNA and the inclusion of 96 bp of intron 7 sequence. Since the mis-splicing of the mutant allele product resulted in the maintenance of the correct codon reading frame, the resultant pro alpha 1(I) chain contained a short non-collagenous 32-amino-acid sequence insertion within the repetitive Gly-Xaa-Yaa collagen sequence motif. At the protein level, the mutant alpha 1(I) chain was revealed by digestion with pepsin, which cleaved the mutant procollagen within the protease-sensitive non-collagenous insertion, producing a truncated alpha 1(I). This protease sensitivity demonstrated the structural distortion to the helical structure caused by the insertion. In long-term culture with ascorbic acid, which stimulates the formation of a mature crosslinked collagen matrix, and in tissues, there was no evidence of the mutant chain, suggesting that during matrix formation the mutant chain was unable to stably incorporated into the matrix and was degraded proteolytically.

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A Novel Splice Donor Site in thegag-polGene Is Required for HIV-1 RNA Stability

Journal of Biological Chemistry ◽

10.1074/jbc.m513698200 ◽

2006 ◽

Vol 281 (27) ◽

pp. 18644-18651 ◽

Cited By ~ 20

Author(s):

Martin Lützelberger ◽

Line S. Reinert ◽

Atze T. Das ◽

Ben Berkhout ◽

Jørgen Kjems

Keyword(s):

Donor Site ◽

Rna Stability ◽

Splice Donor Site ◽

Splice Donor ◽

Hiv 1

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A splice donor site mutation in HOXD13 underlies synpolydactyly with cortical bone thinning

Gene ◽

10.1016/j.gene.2013.09.040 ◽

2013 ◽

Vol 532 (2) ◽

pp. 297-301 ◽

Cited By ~ 2

Author(s):

Xiuyan Shi ◽

Chunyan Ji ◽

Lihua Cao ◽

Yuhong Wu ◽

Yuyang Shang ◽

...

Keyword(s):

Cortical Bone ◽

Donor Site ◽

Splice Donor Site ◽

Splice Donor ◽

Site Mutation ◽

Splice Donor Site Mutation

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Human immunodeficiency virus type 1 splicing at the major splice donor site is controlled by highly conserved RNA sequence and structural elements

Journal of General Virology ◽

10.1099/jgv.0.000288 ◽

2015 ◽

Vol 96 (11) ◽

pp. 3389-3395 ◽

Cited By ~ 4

Author(s):

Nancy Mueller ◽

Bep Klaver ◽

Ben Berkhout ◽

Atze T. Das

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Donor Site ◽

Splice Donor Site ◽

Structural Elements ◽

Splice Donor ◽

Rna Sequence ◽

Immunodeficiency Virus

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A Deep Exon Cryptic Splice Site Promotes Aberrant Intron Retention in a Von Willebrand Disease Patient

International Journal of Molecular Sciences ◽

10.3390/ijms222413248 ◽

2021 ◽

Vol 22 (24) ◽

pp. 13248

Author(s):

John G. Conboy

Keyword(s):

Secondary Structure ◽

Splice Site ◽

Intron Retention ◽

Donor Site ◽

Von Willebrand Disease ◽

Splice Donor Site ◽

Cryptic Splice Site ◽

Splice Donor ◽

Nucleotide Mutation ◽

Von Willebrand

A translationally silent single nucleotide mutation in exon 44 (E44) of the von Willebrand factor (VWF) gene is associated with inefficient removal of intron 44 in a von Willebrand disease (VWD) patient. This intron retention (IR) event was previously attributed to reordered E44 secondary structure that sequesters the normal splice donor site. We propose an alternative mechanism: the mutation introduces a cryptic splice donor site that interferes with the function of the annotated site to favor IR. We evaluated both models using minigene splicing reporters engineered to vary in secondary structure and/or cryptic splice site content. Analysis of splicing efficiency in transfected K562 cells suggested that the mutation-generated cryptic splice site in E44 was sufficient to induce substantial IR. Mutations predicted to vary secondary structure at the annotated site also had modest effects on IR and shifted the balance of residual splicing between the cryptic site and annotated site, supporting competition among the sites. Further studies demonstrated that introduction of cryptic splice donor motifs at other positions in E44 did not promote IR, indicating that interference with the annotated site is context dependent. We conclude that mutant deep exon splice sites can interfere with proper splicing by inducing IR.

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