Berry Oncology (China)

Transcranial electrical stimulation (tES) has been widely used in neuroscience research, and the spatial focusing and penetration of the process are currently the main constraints on the effectiveness of treatment. A high-definition electrical stimulation (HD-tES) device with envelope waves was designed. The device utilized a 4×1 electrode structure and was designed with an impedance adjustment circuit to evenly distribute the current among the four return channels. The output performance and safety of the device were verified in invitro experiments. The spatial focusing of the 4×1 electrode structure and the high penetration advantage of envelope waves are explored through simulations. Finally, experiments were performed on 10 healthy adults. The 4×1 electrode structure has the best spatial focusing effect. Current frequencies above 1 kHz may have higher tissue penetration. In addition, the safety of envelope wave stimulation has been verified in human trials, and no adverse reactions occurred during stimulation. The low and medium frequency (<10 kHz) envelope wave HD-tES device is expected to have a positive impact in the field of medicine and neuroscience.

BackgroundThe molecular genetic diagnosis of congenital adrenal hyperplasia (CAH) is very challenging due to the high homology between the CYP21A2 gene and its pseudogene CYP21A1P.MethodologyThis study aims to assess the clinical efficacy of targeted long-read sequencing (T-LRS) by comparing it with a control method based on the combined assay (NGS, Multiplex ligation-dependent probe amplification and Sanger sequencing) and to introduce T-LRS as a first-tier diagnostic test for suspected CAH patients to improve the precise diagnosis of CAH.ResultsA large cohort of 562 participants including 322 probands and 240 family members was enrolled for the perspective (96 probands) and prospective study (226 probands). The comparison analysis of T-LRS and control method have been performed. In the perspective study, 96 probands were identified using both the control method and T-LRS. Concordant results were detected in 85.42% (82/96) of probands. T-LRS performed more precise diagnosis in 14.58% (14/96) of probands. Among these, a novel 4141 kb deletion involving CYP21A2 and TNXB was established. A new diagnosis was improved by T-LRS. The duplications were also precisely identified to clarify the misdiagnosis by MLPA. In the prospective study, Variants were identified not only in CYP21A2 but also in HSD3B2 and CYP11B1 in 226 probands. Expand to 322 probands, the actual frequency of duplication haplotype (1.55%) could be calculated due to the accurate genotyping. Moreover, 75.47% of alleles with SNVs/indels, 22.20% of alleles with deletion chimeras.ConclusionT-LRS has higher resolution and reduced cost than control method with accurate diagnosis. The clinical utility of L-LRS could help to provide precision therapy to CAH patients, advance the life-long management of this complex disease and promote our understanding of CAH.

Despite the well-documented mutation spectra of β-thalassemia, the genetic variants and haplotypes of globin gene clusters modulating its clinical heterogeneity remain incompletely illustrated. Here, a targeted long-read sequencing (T-LRS) is demonstrated to capture 20 genes/loci in 1,020 β-thalassemia patients. This panel permits not only identification of thalassemia mutations at 100% of sensitivity and specificity, but also detection of rare structural variants (SVs) and single nucleotide variants (SNVs) in modifier genes/loci. The highly homologous regions of α-/β-globin gene clusters are then phased and 3 novel haplotypes in HBG1/HBG2 region are reported in this population of β-thalassemia patients. Furthermore, one of the haplotypes is associated with ameliorated symptoms of β-thalassemia. Similarly, 5 major haplotypes are identified in HBA1/HBA2 homologous region while one of them is found highly linked with deletional α-thalassemia mutations. Finally, rare mutations in erythroid transcription factors in DNMT1 and KLF1 associated with increased expression of fetal hemoglobin and reduced transfusion dependencies are identified. This study presents the largest T-LRS study for β-thalassemia patients to date, facilitating precise clinical diagnosis and haplotype phasing of globin gene clusters.

BackgroundThe clinical diagnosis of Fabry Disease (FD) can be challenging due to the clinical heterogeneity, especially in females. Patients with FD often experience a prolonged interval between the onset of symptoms and receiving a diagnosis. Genetic testing is the gold standard for precise diagnosis of FD, however conventional genetic testing could miss deep intronic variants and large deletions or duplications. Although next-generation sequencing, which analyzes numerous genes, has been successfully used for FD diagnosis and can detect complex variants, an effective and rapid tool for identifying a wide range of variants is imminent, contributing to decrease the diagnostic delay.MethodsThe comprehensive Analysis of FD (CAFD) assay was developed for FD genetic diagnosis, employing long-range PCR coupled with long-read sequencing to target the full-length GLA gene and its flanking regions. Its clinical performance was assessed through a comparative analysis with Sanger sequencing.ResultsGenetic testing was performed on 82 individuals, including 48 probands and 34 relatives. The CAFD assay additionally identified variants in two probands: one had a novel and de novo pathogenic variant with a 1715 bp insertion in intron 4, and the other carried two deep intronic VUS variants in cis-configuration also in intron 4. In total, CAFD identified 47 different variants among 48 probands. Of these, 42 (89.36%, 42/47) were pathogenic, while 5 (10.64%, 5/47) were VUS. Sixteen (34.04%, 16/47) of the variants were novel, including 15 SNV/Indels and one large intronic insertion. Pedigree analysis of 21 probands identified four de novo disease-causing variants. Hence, FD exhibits not only variable clinical presentations but also a wide spectrum of variants. Utilizing a comprehensive testing algorithm for diagnosing FD, which includes enzyme activity, clinical features, and genetic testing, the diagnostic yield of CAFD is 97.92% (47/48), which is higher than that of conventional Sanger sequencing, at 95.83% (46/48).ConclusionThe duration between initial clinical presentation and diagnosis remains long and winding. CAFD provides precise diagnosis for a wide spectrum of GLA variants, promoting timely diagnosis and appropriate treatment for FD patients.

In southern China, α-thalassemia is the most prevalent hereditary monogenic disorder, and deletion variants are the predominant form. Conventional thalassemia diagnosis techniques are numerous, however they are all limited in their ability to detect rare deletions. Here, we discuss a family who sought genetic counseling during their fourth pregnancy after experiencing Hb Bart’s hydrops fetalis in two of their previous pregnancies. To ascertain the thalassemia genotype, the family members underwent hematological testing, routine genetic analysis and multiplex ligation-dependent probe amplification (MLPA). The precise deletion locations could not be identified, while MLPA detected an unknown copy number variant. Lastly, a rare 11.1 kb deletion located in the HBA gene (Chr16: 170,832–182,004, GRch38/hg38) was directly identified by single-molecule real-time technology (SMRT) sequencing. Furthermore, we confirmed the compound heterozygosity of --11.1 allele and --SEA allele, which contributed to the explanation of the Hb Bart’s hydrops fetalis syndrome in the fetuses from the second and third pregnancies. We have first verified a compound heterozygosity for --11.1 allele and --SEA allele. This study may provide a reference strategy for the discovery of rare and potentially novel thalassemia variants using a comprehensive method combining SMRT sequencing and conventional diagnostic technology, improving the accuracy and efficacy of thalassemia diagnosis.

BackgroundTo explore reasons for the failure of noninvasive prenatal test (NIPT) for cell‐free fetal DNA (cffDNA) in maternal peripheral blood, and discuss appropriate treatment schemes after the failure of the test.MethodsAltogether 41,136 pregnant women participated in NIPT. Blood samples were taken again from pregnant women who failed the first blood collection upon their informed consent. Prenatal genetic counseling or prenatal diagnosis was recommended for pregnant women with final NIPT failure.ResultsThe first failure rate of NIPT was 0.737% (303/41136), and the reason for the failure was the low ratio of cffDNA in 135 (44.6%) of the 303 pregnant women. After the second or third blood sampling, the final failure rate was 0.182% (75/41136). The low ratio of cffDNA was the main reason for test failure in 42 (56.0%) of the 75 pregnant women who finally failed NIPT, among whom 44 (58.7%) had underlying diseases, including 21 (47.7%) with more than two coexisting underlying diseases. Only 27 (36.0%) of the 75 pregnant women with NIPT failure underwent interventional prenatal diagnosis.ConclusionsThe main reason for NIPT failure was the low ratio of cffDNA. Postponing the gestational weeks of blood collection may improve the success rate. Resampling and retesting upon informed consent in pregnant women who failed the first test could improve the success rate. For pregnant women who finally failed NIPT, it is suggested strengthening the genetic counseling, prenatal examination, and ultrasound evaluation, and carry out interventional prenatal diagnosis if necessary.

Not available.

Objective: Variants of the polycystic kidney and hepatic disease 1 (PKHD1) gene are associated with autosomal recessive polycystic kidney disease (ARPKD). This study aimed to identify the genetic causes in a Chinese pedigree with ARPKD and design a minigene construct of the PKHD1 gene to investigate the impact of its variants on splicing. Methods: Umbilical cord samples from the proband and peripheral blood samples from his parents were collected, and genomic DNA was extracted for whole-exome sequencing (WES). Bioinformatic analysis was used to identify potential genetic causes, and Sanger sequencing confirmed the existence of variants within the pedigree. A minigene assay was performed to validate the effects of an intronic variant on mRNA splicing. Results: Two variants, c.9455del (p.N3152Tfs*10) and c.2408-13C>G, were identified in the PKHD1 gene (NM_138694.4) by WES; the latter has not been previously reported. In silico analysis predicted that this intronic variant is potentially pathogenic. Bioinformatic splice prediction tools revealed that the variant is likely to strongly impact splice site function. An in vitro minigene assay revealed that c.2408-13C>G can cause aberrant splicing, resulting in the retention of 12 bp of intron 23. Conclusion: A novel pathogenic variant of PKHD1, c.2408-13C>G, was found in a fetus with ARPKD, which enriches the variant spectrum of the PKHD1 gene and provides a basis for genetic counseling and the diagnosis of ARPKD. Minigenes are optimal to determine whether intron variants can cause aberrant splicing.

Central nervous system (CNS) tumours with BCOR (BCL-6 transcriptional corepressor) internal tandem duplication (ITD) have emerged as a new molecular entity in recent years [1]. This was first reported by Sturm et al. in a study of 323 patients with CNS tumours, where this type of tumour was named 'high-grade neuroepithelial tumour with BCOR ITD' (HGNET BCOR ITD) [2]. In the fifth edition of the WHO classification of CNS tumours, it is now defined as 'a malignant CNS tumour characterized by a predominantly solid growth pattern, uniform oval or spindle-shaped cells with a dense capillary network and focal pseudorosette formation, and an ITD in exon 15 of the BCOR gene'. However, the complete clinicopathological and molecular features, as well as the ideal treatment and prognosis, have not been fully elucidated. Furthermore, in addition to BCOR-ITD, other BCOR gene variants, such as EP300::BCOR [3, 4] and BCOR::CREBBP [5] fusion, have recently been reported, broadening the genetic alteration spectrum for this entity. To date, this tumour has been mainly reported in children and infants, cases in adults are rarely reported and less understood. However, we have recently encountered two cases of adult HGNET harbouring the fusion of EP300::BCOR. Clinical, histopathological and immunohistochemical (IHC) features of both cases were investigated. Comprehensive molecular analyses were conducted, including methylation profiling, Sanger sequencing, targeted next-generation sequencing (NGS) and reverse transcription-polymerase chain reaction (RT-PCR). Both cases exhibited similar imaging and pathological characteristics, and in addition to BCOR gene fusions, deep sequencing revealed other genetic variations that may have significant clinical implications for HGNETs with BCOR fusions. The first patient (Case 1), a previously healthy, 54-year-old male, was asymptomatic and discovered to have a mass in the right frontal lobe during a physical examination 5 days prior. Magnetic resonance imaging (MRI) revealed a large mass with clear boundaries in the right frontal lobe, showing nodular enhancement signals within the tumour and a shift towards the left side of the midline(Figure 1A). Case 1 underwent a total resection, followed by radiation therapy (6000 cGy) and adjuvant temozolomide therapy. At the 16-month follow-up, there were no signs of tumour recurrence. The pathological examination of Case 1 revealed a predominantly ependymoma-like pattern, characterized by abundant perivascular pseudorosettes, where the tumour cells formed a nuclear-free zone around the blood vessels (Figure 1B). The tumour had a well-circumscribed border with sharp demarcation from the adjacent brain tissue. In some areas, short-fascicular or ribbon patterns were observed, and extensive calcified foci were also present (Figure 1D,E). The tumour exhibited high cellularity, nuclear atypia and increased mitosis in some regions (Figure 1C). Immunohistochemical staining showed that the tumour cells were mostly negative for GFAP and S-100, but focally positive for Olig2. Epithelial membrane antigen (EMA) staining revealed faint granular cytoplasmic stain, and Ki-67 was focally up to 30%. Other immunohistochemical markers showed that the tumour cells were diffusely positive for vimentin, CD56 and EGFR; partially positive for SATB2; sporadically positive for NeuN; and negative for BCOR, IDH1R132H and BRAF V600E (VE1) (Figure 1F,G). It was difficult to get an exact diagnosis based on the above results, so a methylation profiling was performed using the EPIC 850k BeadChip array (Illumina, San Diego, CA). By comparing the methylation profile with the selected reference cases from the published cohort (GSE109381) of 2801 previously published CNS tumours [6], this sample was invariably clustered together with the reference methylation class 'CNS tumour with BCOR internal tandem duplication' by unsupervised hierarchical clustering analysis (Figure 1H) and t-SNE analysis (Figure 1I). Methylation-based copy number profiling revealed gains in chromosomes 1q, 5 and 7; more importantly, we observed the chromosome 22q12.31 deletion, where EP300 is located, as previously described by Aldape et al. [7] (Figure 1J). In this case, however, Sanger sequencing did not identify any BCOR-ITD at exon 15. Several months later, we encountered the second patient (Case 2), a 43-year-old female who had been experiencing headaches for 10 days. Her headaches were associated with changes in posture, with pain occurring when lying flat but not when standing. MRI revealed a large, nodular demarcated and heterogeneous mass in the right frontotemporal lobe with an obvious mass effect (Figure 2A). Near-total resection of the lesion was achieved, and the patient received radiation therapy (5200 cGy) supplemented with adjuvant chemotherapy postoperatively. The patient had no signs of tumour progression at the 8-month follow-up. Histologically, Case 2 exhibited diverse appearances with a higher cellularity than Case 1. In some areas, the tumour cells were spindle or ovoid and densely arranged in sheets with branching capillary network (Figure 2B). The local area showed a high-grade ependymoma-like structure dominated by pseudo-rosettes, as well as Homer Wright rosettes with brisk mitosis (Figure 2C). Palisading necrosis was easily observed (Figure 2D), and microcyst structures and calcifications were frequently present (Figure 2E). Immunohistochemical analysis revealed that the tumour cells were diffusely and strongly positive for vimentin, CD56 and SATB2, weakly positive for BCOR, and focally positive for NeuN and Olig2 (Figure 2F–I). In contrast, staining for GFAP and S-100 was completely negative in Case 2. However, the amount of tumour tissue from Case 2 was insufficient for methylation testing after NGS detection. Targeted NGS results: the DNA-based and RNA-based NGS analyses were used to profile the two samples. In Case 1, the EP300::BCOR fusion was identified at both DNA and RNA levels with breakpoints located in exon 31 of EP300 and exon 6 of BCOR. Additionally, Case 1 showed a BCOR::L3MBTL2 fusion only at the RNA level with breakpoints in exon 4 of BCOR and exon 2 of L3MBTL2. No other genetic alterations were identified in this case. In Case 2, the EP300::BCOR fusion was also identified at both DNA and RNA levels with breakpoints in exon 31 of EP300 and exon 4 of BCOR. Furthermore, additional molecular alterations in Case 2 included a TP53 p.R273C missense mutation, a NOTCH3 p.G37E point mutation and PDGFRA amplification at the DNA level, as well as a PDGFRA (exon 9) and FIP1L1 (exon 4) fusion at the RNA level. The graphical depiction of the BCOR fusion products for both cases is shown in Figures 1K,I and 2J. RT-PCR was performed to further validate all BCOR fusion transcripts mentioned above. Agarose electrophoresis and Sanger sequencing of the amplified products confirmed the presence of the breakpoints detected by RNA-seq (Figures 1M and 2K). No BCOR-ITD was detected in either sample. The two adult cases exhibited comparable radiological and pathologic characteristics that are consistent with those of CNS HGNET-BCOR tumours documented in the literature. Many cases reported in the literatures were located supratentorialy and showed diverse histologic features including ependymoma-like, oligodendroglioma-like or glioblastoma-like [3, 7]. They often demonstrated a low-grade morphology with high-grade transformation [4]. The IHC characteristics of BCOR-fusion tumours were variable. Tumour cells were reported to be immunopositive for Olig2, GFAP, NeuN, EMA, EGFR or beta-catenin, possibly suggesting a potential for multidiretional differentiation [3-5]. Although BCOR IHC staining can be helpful, it often does not correspond with genetic tests. Therefore, methylation profiling combined with NGS detection could significantly enhance the diagnostic accuracy. Recently, Aldape et al. published a retrospective study of gliomas with EP300 and BCOR gene fusions [7]. Their study profiled 21 patients with CNS tumours harbouring BCOR fusions (11 with the EP300 gene, 10 with other or undetermined genes). The median age was 30 years, and 71% of the tumours were well-defined (10 were supratentorial and 5 were in the posterior fossa). Recurring histologic appearances included perivascular pseudorosettes, myxoid changes, calcifications or branching capillary networks. The authors noted a partially overlapping methylation profile between the BCOR-fusion group and the ITD group. They also reported that the median PFS for BCOR-fusion tumours was 45 months, which is favourable compared to the BCOR-ITD tumours [8]. Our two reported cases provide additional evidence for this type of tumour, which may have been underestimated in adults previously. According to our NGS detecting results, besides of fusions of EP300::BCOR, Case 1 also showed a fusion of BCOR::L3MBTL2, which has been previously reported as a confirmed somatic mutation in a 24-year-old female patient diagnosed with high-grade IDH-wild-type glioma [5]. Simultaneous detection of BCOR::L3MBTL2 and EP300::BCOR fusion was also reported in a female patient with uterine sarcoma, which showed moderate nuclear atypia, active mitosis and abundant vascular components [9]. The fusion of FIP1L1::PDGFRA in Case 2 may hold significance for targeted therapy. Previous studies have shown that the FIP1L1-PDGFRA fusion gene is a crucial oncogenic driver of chronic eosinophilic leukemia, which can be effectively targeted by tyrosine kinase inhibitors like imatinib, sunitinib or sorafenib [10]. Treatment with these drugs has resulted in rapid complete remission for patients. Therefore, if the tumour in Case 2 recurs in the future, drugs such as imatinib could be considered as a potential treatment option. In conclusion, BCOR fusion tumours may be distinguished from BCOR-ITD tumours of the CNS by older age, greater glial differentiation, an ependymoma-like appearance and a higher likelihood of a better prognosis. Further studies of additional cases are strongly needed to precisely characterize the classification of CNS tumours with BCOR alterations. With the help of effective molecular assays such as methylation profiling and NGS analysis, an increasing number of adult BCOR fusion tumours may be detected. Data sharing is not applicable to this article as no new data were created or analyzed in this study.