A disulfidptosis-related lncRNA prognostic model to predict survival and response to immunotherapy in lung adenocarcinoma.

Background: Lung adenocarcinoma (LUAD) is the major subtype of lung cancer and has a poor prognosis. Disulfidptosis is a novel regulated cell death form characterized by aberrant disulfide stress and actin network collapse. This study aimed to identify disulfidptosis-related lncRNAs, and predict LUAD patients' prognosis and response to antitumor therapies by establishing a disulfidptosis-related lncRNA model. Methods: Transcriptome and clinical data of LUAD patients were obtained from the TCGA database. Pearson correlation and Cox regression analysis was used to identify disulfidptosis-related lncRNAs associated with overall survival. LASSO regression analysis was adopted to construct the prognostic model. GO, KEGG and GSEA analysis was used to identify cellular pathways related to this model. Immune cell infiltration was investigated by ESTIMATE and CIBERSORT algorithms. Tumor mutational burden (TMB) and its association with model-derived risk score were analyzed using simple nucleotide variation data. Patients' response to immunotherapy and other antineoplastic drugs was predicted by the TIDE algorithm and GDSC tool, respectively. Results: We identified 127 disulfidptosis-related lncRNAs, and a prognostic model that consists eight of them (KTN1-AS1, AL365181.3, MANCR, LINC01352, AC090559.1, AC093673.1, AP001094.3, and MHENCR) was established and verified. The prognostic model could stratify LUAD patients into two distinct risk-score groups. A high risk score was an independent prognosis factor indicating poor overall survival, and correlated with reduced immune cell infiltration, high TMB, and lower activity of tumor immune response. Immune checkpoint blockade might bring more survival benefits to the high-risk LUAD patients, whereas low-risk patients might be more responsive to targeted therapy and diverse kinase inhibitors. Conclusion: We established a disulfidptosis-related lncRNA model that can be exploited to predict the prognosis, tumor mutational burden, immune cell infiltration landscape, and response to immunotherapy and targeted therapy in LUAD patients.

Whole-exome sequencing identifies genetic variants of hearing loss in 113 Chinese families.

BACKGROUND: Hearing loss is a group of diseases with high genetic heterogeneity. About 160 genes have been reported to be associated with hereditary hearing loss. METHODS: 113 families with hearing loss were collected, and WES was used to detect SNV, InDel, CNV and mitochondrial gene variants. For some probands with negative WES test results, the copy number of STRC and OTOA were determined by using real-time fluorescence quantitative PCR. RESULTS: Pathogenic or likely pathogenic variants were found in 54 probands, of which 98% (53/54) were SNVs or InDels and 2% (1/54) were CNVs, a positive rate of 48%. 16 families (14%) were detected with candidate variants of uncertain significance. 19 novel pathogenic or likely pathogenic variants and 22 candidate variants of uncertain significance were identified in this study. The most common hearing loss gene in the families was GJB2, accounting for 28% (15/53), followed by SLC26A4 and MYO15A, accounting for 21% (11/53) and 11% (6/53), respectively. Heterozygous gene deletion was detected in 3 probands, including 2 with STRC and 1 with OTOA in 43 families with WES negative test. CONCLUSION: Genetic etiology was clarified in 54 families. All of these findings broadened the mutation spectrum of hearing loss genes, thus providing new variant information for the future diagnosis of patients with hearing loss.

Identification of four novel mutations in BTK from six Chinese families with X-linked agammaglobulinemia.

BACKGROUND: The defect of Bruton's tyrosine kinase (BTK) gene resulted in X-linked agammaglobulinemia (XLA), which is characterized by recurrent bacterial infections, immunodeficiency with low B-cell numbers and immunoglobulin. Diagnosis of XLA depends on clinical phenotype and genetic testing. METHODS: Six unrelated Chinese families with high suspicion of XLA were enrolled in this study. Potential pathogenic variants were detected and validated by Whole Exome Sequencing (WES) and Sanger Sequencing. Western blot, Quantitative PCR (qPCR) analysis and immunofluorescence analysis were used to evaluate the preliminary function of candidate BTK variants. RESULTS: A total of six variants were identified, four of which were not reported before. The novel missense mutation(c.1900 T > G) and deletion(c.897delG) were found that the mutant protein and mRNA expression levels have fallen by Western Blot and qPCR identification. We also constructed minigene expression vector to determine the deletion (c.1751-6_1755delttctagGGGTT) resulting a 35 bp skipping in exon 18. Meanwhile, the break point of gross deletion (Exon2-5) discovered based on WES was confirmed to be located at site ChX:101367539_101376531 through qPCR and Gap-PCR. CONCLUSION: This study makes definitive diagnosis for 6 families with suspected XLA and further expands the spectrum of BTK mutations, providing new information for the diagnosis of the disease.

Detection of Spinal Muscular Atrophy Using a Duplexed Real-Time PCR Approach With Locked Nucleic Acid-Modified Primers.

BACKGROUND: Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder mainly caused by homozygous deletions that include exon 7 of the survival motor neuron 1 (SMN1) gene. A nearby paralog gene, SMN2, obstructs the specific detection of SMN1. We optimized a duplexed real-time PCR approach using locked nucleic acid (LNA)-modified primers to specifically detect SMN1. METHODS: An LNA-modified primer pair with 3' ends targeting SMN1 specific sites c.835-44g and c.840C was designed, and its specificity was examined by real-time PCR and Sanger Sequencing. A duplexed real-time PCR approach for amplifying SMN1 and control gene albumin (ALB) was developed. A randomized double-blind trial with 97 fresh peripheral blood samples and 25 dried blood spots (DBS) was conducted to evaluate the clinical efficacy of the duplexed approach. This new approach was then used to screen 753 newborn DBS. RESULTS: The LNA-modified primers exhibited enhanced specificity and 6.8% increased efficiency for SMN1 amplification, compared with conventional primers. After stabilizing the SMN1 test by optimizing the duplexed real-time PCR approach, a clinical trial validated that the sensitivity and specificity of our new approach for detecting SMA patients and carriers was 100%. Using this new approach, 15 of the screened 753 newborns were identified as carriers via DBS, while the rest were identified as normal individuals. These data reveal a carrier rate of 1.99% in Hunan province, South Central China. CONCLUSIONS: We have developed a novel, specific SMN1 detection approach utilizing real-time PCR with LNA-modified primers, which could be applied to both prenatal carrier and newborn screening.

[Clinical practice guidelines for spinal muscular atrophy].

Spinal muscular atrophy (SMA) is one of the most common fatal autosomal recessive genetic disorders among infants. It is caused by mutations of motor neuron survival gene 1 (SMN1). The incidence of SMA among newborns is approximately 1/10 000 - 1/6000, and the carrier rate is 1/72 - 1/47 with an ethnic variance. Based on the time of onset and clinical phenotype, SMA can be divided into types I - IV. Approximately 95% of SMA patients have carried homozygous deletions of exon 7 of the (SMN1)] gene. For its significant phenotypic difference, abundant changes of (SMN1)] gene copy number, presence of pseudogene interference and high carrier rate, early diagnosis, genetic consultation, treatment and prevention of SMA can be difficult. This guideline summarizes the relevant research, guideline and consensus issued at home and abroad, clinical manifestations and pathogenesis of SMA patients, and experience in its diagnosis and genetic counseling, with an aim to promote a standardized diagnosis and treatment and reduce the births of children affected with the disease.

Identification of Five Novel Mutations Causing Rare Lysosomal Storage Diseases.

BACKGROUND Lysosomal storage diseases (LSDs), a group of rare inherited metabolic disorders, result from specific lysosomal proteins deficiencies in the degradation of biomacromolecule, including over 70 different diseases, most of which are autosomal recessive. LSDs are multisystem disorders, and the clinical manifestations are usually broad and severe, involving the skeletal system, central nervous system (CNS), cardiovascular system, etc. Besides, patients with some subtypes of LSD have distinctive facial features. MATERIAL AND METHODS We performed next generation sequencing on 4 suspected mucopolysaccharidosis (MPS) cases to determine the genetic causes of the disease. By in vitro molecular cell assay, such as real-time polymerase chain reaction (RT-PCR) and western blot, we tested the pathogenicity of candidate variants. RESULTS We detected 5 novel mutations in 4 patients. The mutations were: c.211_214del and c.1270C>T in GUSB; c.1284+1C>A and c.2404C>T in GNPTAB; and c.717C>A in FUCA1). We identified a rare mucopolysaccharidosis VII patient, a rare fucosidosis patient, and 2 rare mucolipidosis II patients, one of which was an atypical patient. We also present a new pathogenic conjecture about a small deletion in GUSB. CONCLUSIONS Our study described rare diseases in Chinese patients and our results enrich the phenotype spectrum of related diseases, as well as mutation spectrum of related genes, which might be significant for clinical disease diagnosis and prenatal diagnosis.

Diagnosis of Joubert Syndrome 10 in a Fetus with Suspected Dandy-Walker Variant by WES: A Novel Splicing Mutation in OFD1.

Joubert syndrome (JBTS) is a clinically and genetically heterogeneous group of ciliary diseases. To date, 34 subtypes of JBTS have been classified due to different causative genes or extra clinical features. Most of them are autosomal recessive, while only the subtype 10 (JBTS10) is a quite rare X-linked recessive disorder caused by OFD1 mutations with few reports. In this study, by using whole exome sequencing (WES), a novel OFD1 splicing mutation (c.2488+2T>C) was identified in a male fetus with suspected Dandy-Walker variant (DWV) and syndactyly, for whom abnormal karyotype and pathogenic CNV have been excluded. This mutation was inherited from the mother who has experienced two similar pregnancies before. An abnormal skipping of exon 18 in OFD1 mRNA was confirmed by RT-PCR and sequencing. Result from quantitative RT-PCR also showed that total OFD1 mRNA in the index fetus was significantly lower than the control. After a combined analysis of genetic testing results and genotype-phenotype correlations, the novel mutation c.2488+2T>C in OFD1 was considered to be the genetic cause for the affected fetus. Thus the diagnosis should be JBTS10 rather than the primary clinical diagnosis of DWV. We report the first prenatal case of JBTS10 in Chinese population, which not only helps the family to predict recurrence risks for future pregnancies but also provides more information for understanding such a rare disease. The results also present evidence that WES is an effective method in prenatal diagnosis for those fetuses with Joubert syndrome.