Genomic full-length sequence of the novel HLA-C*06:364 allele.

HLA-C*06:364 differs from HLA-C*06:02:01:01 by a non-synonymous nucleotide substitution in exon 3.

The novel KIR2DL3*037 allele, identified by Sanger dideoxy nucleotide sequencing in a Chinese individual.

The novel KIR2DL3*037 allele differs from the closest allele KIR2DL3*00101 by a single missense mutation.

A single nucleotide substitution in exon 5 generated the novel KIR2DL3*00112 allele.

The novel KIR2DL3*00112 allele differs from the closest allele KIR2DL3*00101 by a single same sense mutation.

Recognition of the HLA-DQB1*03:96 allele in a Taiwanese individual.

One nucleotide substitution in codon 130 of HLA-DQB1*03:03:02:01 results in a novel allele HLA-DQB1*03:96.

The novel HLA-A*02:1152 and HLA-B*40:566 alleles identified in Russian bone marrow donors.

Identification of two novel HLA alleles in Russian bone marrow donors.

Multinational proficiency tests for EGFR exon 20 insertions reveal that the assay design matters.

Insertion mutations in exon 20 of the epidermal growth factor receptor gene (EGFR exon20ins) are rare, heterogeneous alterations observed in non-small cell lung cancer (NSCLC). With a few exceptions, they are associated with primary resistance to established EGFR tyrosine kinase inhibitors (TKIs). As patients carrying EGFR exon20ins may be eligible for treatment with novel therapeutics-the bispecific antibody amivantamab, the TKI mobocertinib, or potential future innovations-they need to be identified reliably in clinical practice for which quality-based routine genetic testing is crucial. Spearheaded by the German Quality Assurance Initiative Pathology two international proficiency tests were run, assessing the performance of 104 participating institutes detecting EGFR exon20ins in tissue and/or plasma samples. EGFR exon20ins were most reliably identified using next-generation sequencing (NGS). Interestingly, success rates of institutes using commercially available mutation-/allele-specific quantitative (q)PCR were below 30% for tissue samples and 0% for plasma samples. Most of these mutation-/allele-specific (q)PCR assays are not designed to detect the whole spectrum of EGFR exon20ins mutations leading to false negative results. These data suggest that NGS is a suitable method to detect EGFR exon20ins in various types of patient samples and is superior to the detection spectrum of commercially available assays.

Novel homozygous frameshift insertion variant in the last exon of the EDARADD causing hypohidrotic ectodermal dysplasia in two siblings: case report and review of the literature.

BACKGROUND: Hypohidrotic ectodermal dysplasia (HED) is a genetic disorder that results in the abnormal development of structures derived from ectodermal tissue. This rare condition predominantly affects the hair, nails, eccrine glands, and teeth. While HED can be caused by various genes, the EDA, EDAR, EDARADD, and WNT10A genes account for approximately 90% of cases. Notably, HED forms associated with variants in the EDA, EDAR, or EDARADD genes may exhibit similar phenotypes due to defects in a common signaling pathway. Proper interaction among the products of these genes is crucial for the activation of the nuclear factor (NF-κB) signaling pathway, which subsequently regulates the transcription of targeted genes. The EDARADD gene, in particular, harbors one of the rarest reported variants associated with HED. CASE PRESENTATION: Five-and two-years-old brothers born into consanguineous parents were examined at our outpatient medical genetics clinic at Sanliurfa Training and Research Hospital, Turkey. Both displayed the same classical phenotypic features of HED. The elder had a very sparse dark and brittle hair, sparse eyebrows and eyelashes, conical upper and lower premolar teeth with hypodontia, widely spaced teeth, very dry skin, mildly prominent forehead, and periorbital wrinkles. The younger one showed the same, but less severe, clinical features. After thorough examination and patient history evaluation, targeted next-generation sequencing analysis yielded the novel homozygous insertion variant c.322_323insCGGGC p.(Arg108ProfsTer7) in EDARADD. The mutation has not been reported to date in the literature. CONCLUSIONS: In this report, we present two siblings exhibiting classical HED symptoms and a novel insertion variant of the EDARADD gene, which leads to a frameshift introducing a stop codon. Both brothers inherited such mutation from their parents, who were heterozygous carriers of the same variant. The present study may shed light about the pathogenic mechanisms underlying HED, and expand the spectrum of EDARADD gene variants associated with this condition.

Age-dependent regulation of ELP1 exon 20 splicing in Familial Dysautonomia by RNA Polymerase II kinetics and chromatin structure.

Familial Dysautonomia (FD) is a rare disease caused by ELP1 exon 20 skipping. Here we clarify the role of RNA Polymerase II (RNAPII) and chromatin on this splicing event. A slow RNAPII mutant and chromatin-modifying chemicals that reduce the rate of RNAPII elongation induce exon skipping whereas chemicals that create a more relaxed chromatin exon inclusion. In the brain of a mouse transgenic for the human FD-ELP1 we observed on this gene an age-dependent decrease in the RNAPII density profile that was most pronounced on the alternative exon, a robust increase in the repressive marks H3K27me3 and H3K9me3 and a decrease of H3K27Ac, together with a progressive reduction in ELP1 exon 20 inclusion level. In HEK 293T cells, selective drug-induced demethylation of H3K27 increased RNAPII elongation on ELP1 and SMN2, promoted the inclusion of the corresponding alternative exons, and, by RNA-sequencing analysis, induced changes in several alternative splicing events. These data suggest a co-transcriptional model of splicing regulation in which age-dependent changes in H3K27me3/Ac modify the rate of RNAPII elongation and affect processing of ELP1 alternative exon 20.

[Correlation between the mutation spectrum of the UGT1A1 gene and clinical phenotype in patients with inherited hyperunconjugated bilirubinemia].

Objective: To analyze the distribution characteristics of UGT1A1 mutant genes (including enhancers, promoters, and exons 1-5) and further explore the correlation between UGT1A1 genotype and clinical phenotypes in patients with inherited hyperunconjugated bilirubinemia. Methods: Patients diagnosed with hereditary hyperunconjugated bilirubinemia at Nanjing Second Hospital from June 2015 to December 2022 were retrospectively analyzed. The UGT1A1 gene was examined using Sanger sequencing in all patients. Complete blood count, liver function, and abdominal imaging examinations were performed. Comparison of categorical variable data using χ(2) testor Fisher percision tests. Comparison of continaous veriable data with normal distribution using t-test. Results: 112 cases (male:female ratio 81:31, aged 9-70 years) had inherited hyperunconjugated bilirubinemia, with a total of 14 mutation sites identified, of which seven were confirmed mutations, and the frequency ranged from high to low: (TA)n accounted for 50%, c.211G>A (p.G71R) accounted for 49.10%, 1456T>G (p.Y486D) accounted for 16.96%, c.686C>A (p.R229W) accounted for 12.5%, 1091C>T (p.P364L) accounted for 8.04%, and c- 3279T>G accounted for 0.982%. Simultaneously, all patients had one to four mutations, of which only one mutation was the most common (55.36%), followed by two mutations (37.5%), and rare three and four mutations (5.36% and 1.78%). There was no statistical significance in total bilirubin (TBil) levels among the four groups (F=0.652, P=0.583). One mutation was most common in (TA)n and c.211G>A (p.G71R), among which TA6/TA7 (n=10) and TA7/TA7 (n=14) mutations were statistically significant in TBil (t=2.143, P=0.043). The c.211G>A (p.G71R) heterozygous (n=9) and isolated (n=15) mutation had no statistical significance in TBil (t=0.382, P=0.706). The GS group accounted for 75%, the intermediate group accounted for 16.9%, and the CNS-Ⅱ group accounted for 8%. TBil was statistically significant among the three groups (F=270.992, P<0.001). There was no statistically significant difference (χ(2)=3.317, P=0.19) between mutation 1 (44 cases, 14 cases, and 4 cases, respectively) and mutations ≥ 2 (40 cases, 5 cases, and 5 cases, respectively) in the GS group, intermediate group, and CNS-II group. Conclusion: The number of UGT1A1 gene mutation sites may have no synergistic effect on TBil levels in patients with inherited hyperunconjugated bilirubinemia. TA7/TA7 mutations are not uncommon, and TBil levels are relatively high.

Identification of the novel HLA-DQA1*02:32 allele by next-generation sequencing.

HLA-DQA1*02:32 differs from DQA1*02:01:01 by one nucleotide substitution in codon 210 in exon 4.

Characterisation of the novel HLA-C*15:274 allele using short and long-read sequencing technologies.

The novel HLA-C*15:274 allele, first described in a potential bone marrow donor from Brazil.

An interpretable model of pre-mRNA splicing for animal and plant genes.

Pre-mRNA splicing is a fundamental step in gene expression, conserved across eukaryotes, in which the spliceosome recognizes motifs at the 3' and 5' splice sites (SSs), excises introns, and ligates exons. SS recognition and pairing is often influenced by protein splicing factors (SFs) that bind to splicing regulatory elements (SREs). Here, we describe SMsplice, a fully interpretable model of pre-mRNA splicing that combines models of core SS motifs, SREs, and exonic and intronic length preferences. We learn models that predict SS locations with 83 to 86% accuracy in fish, insects, and plants and about 70% in mammals. Learned SRE motifs include both known SF binding motifs and unfamiliar motifs, and both motif classes are supported by genetic analyses. Our comparisons across species highlight similarities between non-mammals, increased reliance on intronic SREs in plant splicing, and a greater reliance on SREs in mammalian splicing.

Identification of the novel HLA-DQB1*06:466 allele by next-generation sequencing in a Chinese cord blood donor.

HLA-DQB1*06:466 differs from HLA-DQB1*06:01:01:01 by a single nucleotide substitution at position 571G→A.

A novel HLA-C*03 variant, HLA-C*03:620, identified in a Chinese individual.

HLA-C*03:620 differs from the HLA-C*03:04:01:02 allele by one nucleotide substitution in the exon 3.

The novel HLA-DPB1*05:01:20 allele, identified by Sanger dideoxy nucleotide sequencing in a Chinese individual.

HLA-DPB1*05:01:20 differs from HLA-DPB1*05:01:01:01 by one nucleotide in exon 3.

The novel HLA-B*15:659 allele, identified by Sanger dideoxy nucleotide sequencing in a Chinese individual.

HLA-B*15:659 differs from HLA-B*15:02:01:01 by one nucleotide in exon 2.

Genomic full-length sequence of the HLA-B*37:46 allele was identified by full length group-specific sequencing.

Genomic full-length sequence of HLA-B*37:46 was identified by a group-specific sequencing approach in a Chinese individual.

Disentangling genetic effects on transcriptional and post-transcriptional gene regulation through integrating exon and intron expression QTLs.

Expression quantitative trait loci (eQTL) studies typically consider exon expression of genes and discard intronic RNA sequencing reads despite their information on RNA metabolism. Here, we quantify genetic effects on exon and intron levels of genes and their ratio in lymphoblastoid cell lines, revealing thousands of cis-QTLs of each type. While genetic effects are often shared between cis-QTL types, 7814 (47%) are not detected as top cis-QTLs at exon levels. We show that exon levels preferentially capture genetic effects on transcriptional regulation, while exon-intron-ratios better detect those on co- and post-transcriptional processes. Considering all cis-QTL types substantially increases (by 71%) the number of colocalizing variants identified by genome-wide association studies (GWAS). It further allows dissecting the potential gene regulatory processes underlying GWAS associations, suggesting comparable contributions by transcriptional (50%) and co- and post-transcriptional regulation (46%) to complex traits. Overall, integrating intronic RNA sequencing reads in eQTL studies expands our understanding of genetic effects on gene regulatory processes.

In vivo genome editing via CRISPR/Cas9-mediated homology-independent targeted integration for Bietti crystalline corneoretinal dystrophy treatment.

Bietti crystalline corneoretinal dystrophy (BCD) is an autosomal recessive chorioretinal degenerative disease without approved therapeutic drugs. It is caused by mutations in CYP4V2 gene, and about 80% of BCD patients carry mutations in exon 7 to 11. Here, we apply CRISPR/Cas9 mediated homology-independent targeted integration (HITI)-based gene editing therapy in HEK293T cells, BCD patient derived iPSCs, and humanized Cyp4v3 mouse model (h-Cyp4v3mut/mut) using two rAAV2/8 vectors via sub-retinal administration. We find that sgRNA-guided Cas9 generates double-strand cleavage on intron 6 of the CYP4V2 gene, and the HITI donor inserts the carried sequence, part of intron 6, exon 7-11, and a stop codon into the DNA break, achieving precise integration, effective transcription and translation both in vitro and in vivo. HITI-based editing restores the viability of iPSC-RPE cells from BCD patient, improves the morphology, number and metabolism of RPE and photoreceptors in h-Cyp4v3mut/mut mice. These results suggest that HITI-based editing could be a promising therapeutic strategy for those BCD patients carrying mutations in exon 7 to 11, and one injection will achieve lifelong effectiveness.

Identification of five new HLA-C alleles by next-generation sequencing.

Five novel HLA-C alleles detected by next-generation sequencing: HLA-C*02:02:73, -C*03:04:106, -C*06:382, -C*07:1114Q and -C*12:408.