Padlock Assay in Transthyretin Amyloidosis: A Feasibility Study.

Two different single nucleotide substitutions in intron 2 give rise to novel HLA-DQB1*03:02:01 alleles.

Mutations of the hepatocyte nuclear factor 4 alpha (HNF-4alpha) gene have been demonstrated in maturity-onset diabetes of the young (MODY) 1 families. To investigate the possibility that the HNF-4alpha gene contributes to the onset of non-insulin-dependent diabetes mellitus (NIDDM) in Japanese patients, we screened all exons and flanking introns of this gene for mutations in 100 patients with NIDDM diagnosed after 25 years of age. We identified two missense mutations: M49V in exon 1c and T1301 in exon 4; and two nucleotide substitutions in introns: cytosine to thymidine at -5 nt in intron 1b and adenine to thymidine at -21 nt in intron 5. We screened an additional 220 diabetic subjects for the polymorphism in intron 1b. The c/t substitution in intron 1b was associated with NIDDM. This substitution in the polypyrimidine tract, an important cis-acting element directing intron removal, is likely to influence pre-mRNA splicing of this gene. T1301 in exon 4 was observed in only two diabetic subjects. This mutation could influence the conformation of this peptide, resulting in changes in ligand binding domain function. M49V in exon 1c was found in both diabetic and non-diabetic subjects; isoforms HNF-4alpha 4, 5, and 6 with this mutation may impair glucose metabolism in tissue. In contrast to the primary cause of nonsense and missense mutations of the HNF-4alpha gene in MODY1, the nucleotide substitution in intron 1b may partially contribute to development of NIDDM in combination with other genetic and environmental factors.

The newly discovered HLA-C*04:01:01:186 allele differs from HLA-C*04:01:01:01 by a single nucleotide substitution in intron 3.

The HLA-DQB1*03:01:01:76 allele differs from HLA-DQB1*03:01:01:03 by a single nucleotide substitution in intron 5.

The HLA-B*39:01:01:31 allele differs from HLA-B*39:01:01:03 by a single nucleotide substitution in intron 2.

BackgroundAlport syndrome is an inherited kidney disease caused by variants in the COL4A3, COL4A4, or COL4A5 genes, resulting in type IV collagen abnormalities. While autosomal dominant variants in COL4A3 and COL4A4 are increasingly being diagnosed, X-linked Alport syndrome caused by COL4A5 variants predominates. Single nucleotide substitutions in introns positioned at first and second from the last nucleotide (called a consensus sequence) of exons always cause aberrant splicing. However, whether intronic variants at the third to fifth positions from the last nucleotide of exons can cause aberrant splicing is unclear. MethodsWe identified 11 intronic variants positioned at the third, fourth, and fifth nucleotides from the exon 3′ end in COL4A5 from our Alport syndrome cohort (January 2006 to July 2022). We conducted in vitro splicing assays using minigenes and in silico splicing analysis using commercial splicing prediction software, and evaluated mRNA sequences obtained from patients’ samples when available. ResultsAll 11 patients showed aberrant splicing patterns in the minigene splicing assays. In vivo analysis of 6 patients corroborated these findings. The commercial splicing prediction software accurately predicted splicing changes in 10 variants. ConclusionsThis study shows that 11 intronic variants at the third to fifth positions in COL4A5 introns cause aberrant splicing. This finding highlights the importance of evaluating such variants for the diagnosis and prognosis of X-linked Alport syndrome. Further investigation is warranted to confirm the pathogenicity of these variants and their effect on the prognosis of the kidney in X-linked Alport syndrome.

The HLA-DQB1*05:01:01:37 allele differs from HLA-DQB1*05:01:01:02 by a single nucleotide substitution in intron 2.

The HLA-DQB1*06:02:01:40 allele differs from HLA-DQB1*06:02:01:01 by a single nucleotide substitution in intron 2.

The HLA-DPA1*02:02:02:22 allele differs from HLA-DPA1*02:02:02:01 by a single nucleotide substitution in intron 1.

The HLA-DQB1*03:01:01:75 allele differs from HLA-DQB1*03:01:01:03 by a single nucleotide substitution in intron 2.

Author SummaryThe molecular identification of genes and mutations affecting complex traits and disorders has proven to be very challenging in humans as well as in model organisms. These so-called quantitative traits arise from interactions between two or more genes and their environment, and can be mapped to their underlying genes via closely linked stretches of DNA called quantitative trait loci (QTL). Previously, we identified a single nucleotide substitution in a noncoding region of the insulin-like growth factor 2 gene (IGF2) in pigs that is underlying a major QTL affecting muscle growth, heart size, and fat deposition. The mutation disrupts interaction with an unknown nuclear protein acting as a repressor of IGF2 transcription. In the present study, we have isolated a zinc finger protein of unknown function and show that it regulates the expression of IGF2. The protein, which we named ZBED6, is encoded by a domesticated DNA transposon that was inserted into the genome prior to the radiation of placental mammals. ZBED6 is exclusive to placental mammals and highly conserved among species. Our functional characterization of ZBED6 shows that it has a broad tissue distribution and may affect the expression of thousands of other genes, besides IGF2, that control fundamental biological processes. We postulate that ZBED6 is an important transcription factor affecting development, cell proliferation, and growth in placental mammals.

The HLA-DPA1*02:01:01:37 allele differs from HLA-DPA1*02:01:01:19 by a single nucleotide substitution in intron 1.

The HLA-DQB1*05:02:01:17 allele differs from HLA-DQB1*05:02:01:01 by a single nucleotide substitution in intron 3.

The HLA-DQB1*03:01:01:69 allele differs from HLA-DQB1*03:01:01:03 by a single nucleotide substitution in intron 2.

The HLA-DQB1*06:02:01:41 allele differs from HLA-DQB1*06:02:01:01 by a single nucleotide substitution in intron 2.