[Analysis of COL1A1 and COL1A2 gene variants in two fetuses with osteogenesis imperfecta].

OBJECTIVE: To explore the genetic basis of two fetuses with an osteogenesis imperfecta (OI) phenotype. METHODS: Two fetuses diagnosed at the Affiliated Hospital of Weifang Medical College respectively on June 11, 2021 and October 16, 2021 were selected as the study subjects. Clinical data of the fetuses were collected. Amniotic fluid samples of the fetuses and peripheral blood samples of their pedigree members were collected for the extraction of genomic DNA. Whole exome sequencing (WES) and Sanger sequencing were carried out to identify the candidate variants. Minigene splicing reporter analysis was used to validate the variant which may affect the pre-mRNA splicing. RESULTS: For fetus 1, ultrasonography at 17+6 weeks of gestation had revealed shortening of bilateral humerus and femurs by more than two weeks, in addition with multiple fractures and angular deformities of long bones. WES revealed that fetus 1 had harbored a heterozygous c.3949_3950insGGCATGT (p.N1317Rfs*114) variant in exon 49 of the COL1A1 gene (NM_000088.4). Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), it was classified as a pathogenic variant (PVS1+PS2+PM2_Supporting) for disrupting the downstream open reading frame resulting in premature translational termination, being de novo in origin, and lacking records in the population and disease databases.For fetus 2, ultrasonography at 23 weeks of gestation also revealed shortening of bilateral humerus and femurs by one and four weeks, respectively, in addition with bending of bilateral femurs, tibias and fibulas. Fetus 2 had harbored a heterozygous c.1557+3A>G variant in intron 26 of the COL1A2 gene (NM_000089.4). Minigene experiment showed that it has induced skipping of exon 26 from the COL1A2 mRNA transcript, resulting in an in-frame deletion (c.1504_1557del) of the COL1A2 mRNA transcript. The variant was inherited from its father and had been previously reported in a family with OI type 4. It was therefore classified as a pathogenic variant (PS3+PM1+PM2_Supporting+PP3+PP5). CONCLUSION: The c.3949_3950insGGCATGT (p.N1317Rfs*114) variant in the COL1A1 gene and c.1557+3A>G variant in the COL1A2 gene probably underlay the disease in the two fetuses. Above findings not only have enriched the mutational spectrum of OI, but also shed light on the correlation between its genotype and phenotype and provided a basis for genetic counseling and prenatal diagnosis for the affected pedigrees.

Increase of a group of PTC(+) transcripts by curcumin through inhibition of the NMD pathway.

Nonsense-mediated mRNA decay (NMD), an mRNA surveillance mechanism, eliminates premature termination codon-containing (PTC⁺) transcripts. For instance, it maintains the homeostasis of splicing factors and degrades aberrant transcripts of human genetic disease genes. Here we examine the inhibitory effect on the NMD pathway and consequent increase of PTC+ transcripts by the dietary compound curcumin. We have found that several PTC⁺ transcripts including that of serine/arginine-rich splicing factor 1 (SRSF1) were specifically increased in cells by curcumin. We also observed a similar curcumin effect on the PTC⁺ mutant transcript from a Tay-Sachs-causing HEXA allele or from a beta-globin reporter gene. The curcumin effect was accompanied by significantly reduced expression of the NMD factors UPF1, 2, 3A and 3B. Consistently, in chromatin immunoprecipitation assays, curcumin specifically reduced the occupancy of acetyl-histone H3 and RNA polymerase II at the promoter region (-376 to -247nt) of human UPF1, in a time- and dosage-dependent way. Importantly, knocking down UPF1 abolished or substantially reduced the difference of PTC(+) transcript levels between control and curcumin-treated cells. The disrupted curcumin effect was efficiently rescued by expression of exogenous Myc-UPF1 in the knockdown cells. Together, our data demonstrate that a group of PTC⁺ transcripts are stabilized by a dietary compound curcumin through the inhibition of UPF factor expression and the NMD pathway.

Control of alternative splicing by forskolin through hnRNP K during neuronal differentiation.

The molecular basis of cell signal-regulated alternative splicing at the 3' splice site remains largely unknown. We isolated a protein kinase A-responsive ribonucleic acid (RNA) element from a 3' splice site of the synaptosomal-associated protein 25 (Snap25) gene for forskolin-inhibited splicing during neuronal differentiation of rat pheochromocytoma PC12 cells. The element binds specifically to heterogeneous nuclear ribonucleo protein (hnRNP) K in a phosphatase-sensitive way, which directly competes with the U2 auxiliary factor U2AF65, an essential component of early spliceosomes. Transcripts with similarly localized hnRNP K target motifs upstream of alternative exons are enriched in genes often associated with neurological diseases. We show that such motifs upstream of the Runx1 exon 6 also bind hnRNP K, and importantly, hnRNP K is required for forskolin-induced repression of the exon. Interestingly, this exon encodes the peptide domain that determines the switch of the transcriptional repressor/activator activity of Runx1, a change known to be critical in specifying neuron lineages. Consistent with an important role of the target genes in neurons, knocking down hnRNP K severely disrupts forskolin-induced neurite growth. Thus, through hnRNP K, the neuronal differentiation stimulus forskolin targets a critical 3' splice site component of the splicing machinery to control alternative splicing of crucial genes. This also provides a regulated direct competitor of U2AF65 for cell signal control of 3' splice site usage.

Whole-exome sequencing identified a novel pathogenic mutation of the CYP4F22 gene in a Chinese patient with autosomal recessive congenital ichthyosis and in vitro study of the mutant CYP4F22 protein.

Autosomal recessive congenital ichthyosis (ARCI) is a heterogeneous group of rare cornification disorders. Of the 14 genes already known to cause ARCI, CYP4F22 is a relatively new genetic etiology, the mutation spectrum of which has yet to be profiled. Using whole-exome sequencing in family trios, we identified the compound heterozygous mutations, c.844C>T (p.R282W) and c.1189C>T (p.R397C), of the CYP4F22 gene (NM_173483.4) in a Chinese neonatal boy with a congenital ichthyosis phenotype. In combination with multiple in silico analyses and the following in vitro functional studies, we provided evidence to classify these two variations as pathogenic mutations and demonstrated that both variants significantly reduced the CYP4F22 protein amount. Interestingly, the reduction of both mutant CYP4F22 protein could be recovered by trichostatin A (TSA) treatment, suggesting some deacetylation factors involved in regulating the mutant CYP4F22 protein and implying TSA might be a potential candidate compound for congenital ichthyosis caused by CYP4F22 variations.

Multiple effects of curcumin on promoting expression of the exon 7-containing SMN2 transcript.

Survival of motor neuron 2 (SMN2) is a modifier gene for spinal muscular atrophy (SMA), a neurodegenerative disease caused by insufficient SMN protein mostly due to SMN1 defect. SMN2 is nearly identical to SMN1 but unfortunately only able to produce a small amount of SMN protein due to exon 7 skipping. The exon 7-containing SMN2 transcript (SMN2_E7+) can be increased by a dietary compound, curcumin, but the involved molecular changes are not clear. Here we have found that in fibroblast cells of a SMA type II patient, curcumin enhanced the inclusion of SMN2 exon 7. Examination of the potential splicing factors showed that curcumin specifically increased the protein and transcript levels of SRSF1. The increased SRSF1 protein was mainly nuclear and hyperphosphorylated. Interestingly, the curcumin effects on the SMN2 and SRSF1 transcripts were inhibited by a protein deacetylase inhibitor, trichostatin A. Moreover, in support of its role in the SMN2 splicing, knocking down SRSF1 reduced the inclusion of SMN2 exon 7. Thus, curcumin appears to have multiple effects on the SMN2 transcript and its splicing regulators, including the change of alternative splicing and transcript/protein level as well as phosphorylation. Protein deacetylases and phosphatases are likely involved in these effects. Interestingly, the effects all seem to favor production of the SMN2_E7+ transcript in SMA patient cells.

Aberrant splicing in neurological diseases.

Splicing of precursor messenger RNA (pre-mRNA) removes the intervening sequences (introns) and joins the expressed regions (exons) in the nucleus, before an intron-containing eukaryotic mRNA transcript can be exported and translated into proteins in the cytoplasm. While some sequences are always included or excluded (constitutive splicing), others can be selectively used (alternative splicing) in this process. Particularly by alternative splicing, up to tens of thousands of variant transcripts can be produced from a single gene, which contributes greatly to the proteomic diversity for such complex cellular functions as 'wiring' neurons in the nervous system. Disruption of this process leads to aberrant splicing, which accounts for the defects of up to 50% of mutations that cause certain human genetic diseases. In this review, we describe the different mechanisms of aberrant splicing that cause or have been associated with neurological diseases.

[Two novel EIF2AK3 mutations in a Chinese boy with Wolcott-Rallison syndrome].

OBJECTIVE: Wolcott-Rallison syndrome (WRS) is a rare autosomal recessive disorder characterized by the association of permanent neonatal or early-infancy insulin-dependent diabetes, multiple epiphyseal dysplasia and growth retardation, and other variable multisystem clinical manifestations. Here we describe a Chinese boy affected by WRS. Genetic testing of his EIF2AK3 gene was performed in order to elucidate molecular variations and subsequently to provide credible genetic counseling for prenatal diagnosis in his family. METHOD: Based on analysis of a nine-year-old boy's clinical symptoms associated with biochemical examination and imaging, the diagnosis of WRS was therefore made. Genomic DNAs were extracted from peripheral blood leukocytes from the boy and his parents with their informed consent for genetic studies. All EIF2AK3 exons and intron-exon boundaries were amplified by Touch-down polymerase chain reaction (Touch-down PCR) and sequenced. RESULT: Direct sequencing of PCR products revealed the presence of a heterozygous T insertion (c.1408_1409insT) in exon 8 of the EIF2AK3 gene leading to frameshifting and termination, and another heterozygous T to A exchange (c.1596T > A) in exon 9 of the EIF2AK3 gene resulting in nonsense C532X mutation. CONCLUSION: Combining mutation screening of EIF2AK3 gene with clinical manifestations and effective examination may provide a reliable diagnostic method for patients. In this research, two novel mutations identified in the Chinese boy locate in the catalytic domain of the EIF2AK3 gene, disrupting the ability of autophosphorylation, leading to the truncated proteins that are unable to phosphorylate the natural substrate, which are responsible for the phenotype of Wolcott-Rallison syndrome.

[Analysis of ITD characteristics in acute myeloid leukemia patients with FLT3-ITD positive].

The aim of this study was to analyze the FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) allelic ratios (AR), number of ITD, ITD length and positions of ITD insertions in de novo acute myeloid leukemia (AML) patients with FLT3-ITD positive, and the relationship between mutant level and therapeutic efficacy. Genomic DNA was amplified by PCR, capillary electrophoresis was used to detect the ITD characteristics in 31 de novo AML patients, and DNA sequences analysis of FLT3-ITD(+) were performed in 13 patients. The results showed that the ratios of mutant to wild type FLT3 allele ranged from 0.01 to 2.8; 28 patients (90.32%) had a single ITD, the remaining 3 patients had more than one ITD; the ITD length ranged from 3 to 144 bp in all FLT3-ITD(+) patients. 13 sequence-analyzed patients, 4 patients were of pure duplications, and 2 patients had foreign bases inserted, and the other 7 patients were partial duplications. The ITD occurred in the regions from p.E573 to p.P606 of the FLT3 protein, with the majority clustered in a stretch between p.F590 and p.R595. The complete remission (CR) rate in AR < 0.5 patients (43.75%) were more prevalent as compared with AR ≥ 0.5 patients (16.67%) (p > 0.05). It is concluded that the ITD length and AR are vary widely. Some of the insertions are foreign bases, and all of the 13 sequences-analyzed ITD were concentrated on the juxtamembrane domain. The CR rate in patients of AR < 0.5 had no statistical significance compared with patients of AR ≥ 0.5.

[FMS-like tyrosine kinase 3 gene mutation in acute myeloid leukemia detected by denaturing PAGE and its clinical significance].

The aim of this study was to analyze the frequency of flt3 length mutation (flt3-LM) in de novo acute myeloid leukemia patients and the relationship between flt3-LM and chromosome alterations, FAB subgroups, as well as efficiency of therapy. Genomic DNA was amplified by PCR; 2% agarose gel or 8% denaturing PAGE were used to detect the length mutation of flt3 gene in 99 de novo acute myeloid leukemia patients; karyotyping in 72 AML patients was performed by G banding technique. The results showed that the flt3-LM was detected in 20.2% (20/99) patients by agarose gel electrophoresis, and in 29.9% (29/99) by denaturing PAGE. The flt3-LM was not detected in M(0) (only one patient was available), but flt3-LM occurrence in AML subtypes was as follow: in M(2) (9/30), M(3) (6/27), M(4) (4/14), M(5) (7/19), M(6) (3/8) respectively. flt3-LM in patients with normal karyotypes (39.13%) was more prevalent as compared with patients of abnormal karyotype (24.49%), but there was no statistical difference (p > 0.05). The complete remission (CR) rate in flt3-LM positive patients (36.36%) was lower than that in flt3-LM negative patients (62.75%) in the 73 patients (p < 0.05) whose karyotypic detection was performed. The distributions of flt3-LM were observed in 8 out of 40 CR patients, 8 out of 21 PR patients, and 6 out of 12 NR patients. It is concluded that the denaturing PAGE is more sensitive and reliable to detect the flt3-LM. The flt3 mutation represents a common genetic abnormality in AML patients, and the flt3-LM is associated with lower CR rate.