In Vitro Splicing Assay Proves the Pathogenicity of Intronic Variants in MRAP

Introduction: Familial glucocorticoid deficiency (FGD) is characterised by isolated glucocorticoid deficiency in a patient who retains normal mineralocorticoid production. FGD causing mutations in the MC2R accessory protein, MRAP, are often splice-site or nonsense mutations resulting in a truncated protein. Many of these mutations occur at the canonical donor splice-site of intron 3, where it has been shown previously that c.106 + 2_3dupTA, for example, results in skipping of the first coding exon with unknown consequences at the protein level. Patients and methods: DNA was isolated from three consanguineous individuals diagnosed with early onset FGD (0 - 13 months) with high ACTH and/or low cortisol levels and underwent whole exome sequencing. The proband in family 1 (P1) presented at 13 months and had a hyperpigmented sibling who died in neonatal period due to adrenal failure. Patient 2 (P2), who also had a family history of adrenal insufficiency, was noted to be hyperpigmented at birth with markedly raised ACTH, patient 3 (P3) was noted to have diffuse hyperpigmentation in the early neonatal period and on formal testing at 16m was found to have low serum cortisol. Variants were confirmed using Sanger sequencing and predicted splice-site mutations were investigated using an in vitro splicing assay. Results: Homozygous mutations in MRAP were identified in all three cases which were heterozygous in their parents. Previously described mutations, c.106 + 1delG (chr21:33671388delG; rs1476574441; CD050155) in P1 and c.106 + 2dupT (Chr21: 33671390_91insT; rs761576317; CI118288) in P2 at the canonical donor splice-site of intron 3, were identified, with the former predicted to destroy the splice site and the latter to weaken it. These mutations in vitro resulted in the complete skipping of exon 3, which contains the translational start site, and presumably result in no protein product. A novel homozygous mutation in intron 4, c.206 + 5G>T; (chr21:33679055G>T rs1064796398) was identified in P3, but was not predicted to alter splicing. In vitro, this mutation negates the canonical donor splice site and creates two different alternative sites, both resulting in frameshifts and predicted early termination of the protein (p.Val44fs*50, p.Pro72fs*90). Conclusion: All mutations reported here are predicted to produce no protein, either because the start site is excluded (for c.106 + 1delG and c.106 + 2dupT) or because the transcripts are likely to undergo nonsense mediated decay (for c.206 + 5G>T), resulting in the early onset FGD seen in the patients. Splice prediction protocols, although effective for variants within 2bp of exon/intron boundaries may not predict the true outcome of a base change whereas the splice assay conclusively revealed the effect of all three variants allowing us to assign pathogenicity to them.

Complete CSN1S2 Characterization, Novel Allele Identification and Association With Milk Fatty Acid Composition in River Buffalo

The αs2-casein is one of the phosphoproteins secreted in all ruminants' milk, and it is the most hydrophilic of all caseins. However, this important gene (CSN1S2) has not been characterized in detail in buffaloes with only two alleles detected (reported as alleles A and B), and no association studies with milk traits have been carried out unlike what has been achieved for other species of ruminants. In this study, we sequenced the whole gene of two Mediterranean river buffalo homozygotes for the presence/absence of the nucleotide C (g.7539G>C) realized at the donor splice site of exon 7 and, therefore, responsible for the skipping of the same exon at mRNA level (allele B). A high genetic variability was found all over the two sequenced CSN1S2 alleles. In particular, 74 polymorphic sites were found in introns, six in the promoter, and three SNPs in the coding region (g.11072C>T, g.12803A>T, and g.14067A>G) with two of them responsible for amino acid replacements. Considering this genetic diversity, those found in the database and the SNP at the donor splice site of exon 7, it is possible to deduce at least eight different alleles (CSN1S2 A, B, B1, B2, C, D, E, and F) responsible for seven different possible translations of the buffalo αs2-casein. Haplotype data analysis suggests an evolutionary pathway of buffalo CSN1S2 gene consistent with our proposal that the published allele CSN1S2 A is the ancestral αs2-CN form, and the B2 probably arises from interallelic recombination (single crossing) between the alleles D and B (or B1). The allele CSN1S2 C is of new identification, while CSN1S2 B, B1, and B2 are deleted alleles because all are characterized by the mutation g.7539G>C. Two SNPs (g.7539G>C and g.14067A>G) were genotyped in 747 Italian buffaloes, and major alleles had a relative frequency of 0.83 and 0.51, respectively. An association study between these SNPs and milk traits including fatty acid composition was carried out. The SNP g.14067A>G showed a significant association (P < 0.05) on the content of palmitic acid in buffalo milk, thus suggesting its use in marker-assisted selection programs aiming for the improvement of buffalo milk fatty acid composition.

A de novo synonymous variant in EFTUD2 disrupts normal splicing and causes mandibulofacial dysostosis with microcephaly: case report

Background Mandibulofacial dysostosis with microcephaly (MFDM) is a rare autosomal dominant genetic disease characterized by intellectual and growth retardations, as well as major microcephaly, induced by missense and splice site variants or microdeletions in the EFTUD2 gene. Case presentation Here, we investigate the case of a young girl with symptoms of MFDM and a normal karyotype. Whole-exome sequencing of the family was performed to identify genetic alterations responsible for this phenotype. We identified a de novo synonymous variant in the EFTUD2 gene. We demonstrated that this synonymous variant disrupts the donor splice-site in intron 9 resulting in the skipping of exon 9 and a frameshift that leads to a premature stop codon. Conclusions We present the first case of MFDM caused by a synonymous variant disrupting the donor splice site, leading to exon skipping.