Rare diseases are diverse in type and phenotype complex. Approximately 50% of rare disease patients can not receive a definite diagnosis through conventional genetic testing such as whole-exome sequencing（WES）. Long-read sequencing（LRS），as an emerging sequencing technology，offers new opportunities for the diagnosis of rare diseases. With continuous technological advancements，standardized analysis processes and the construction of reference databases，LRS is expected to break through bottlenecks and become a key technology for promoting precise diagnosis of rare diseases. This review focuses on the basic principles，advantages of LRS technology，its current application status and challenges in the diagnosis of rare diseases，and discusses the application prospects of LRS in the molecular diagnosis of rare diseases in combination with related papers in the Precision Detection and Diagnosis of Genetic Diseases special issue.

Objective To investigate the relationship between 22q11.2 microdeletions and microduplications and clinical phenotypes，and to provide a reference for clinical genetic counseling. Methods From January 2015 to May 2023，19 patients diagnosed with 22q11.2 microdeletions and microduplications by chromosomal microarray analysis（CMA） at Lianyungang Maternal and Child Health Care Hospital were enrolled. The clinical phenotypes，pregnancy follow-up outcomes and pathogenic genes of these patients were analyzed. The follow-up for live-born newborns of 19 pregnant women was conducted. Results Among the 19 pregnant women，8 cases had 22q11.2 microdeletions，and 11 cases had microduplications. The main pathogenic genes involved were TBX1 and CRKL. Among the 8 pregnant women with 22q11.2 microdeletions，5 fetuses had abnormal heart development. Among the 11 pregnant women with 22q11.2 microduplications，no heart-related abnormalities were found. Among the 8 pregnant women with 22q11.2 microdeletions，5 cases terminated the pregnancy，2 cases had spontaneous abortions，and 1 case had a birth（at the time of the follow-up，the newborn was 6 months old，and no obvious abnormalities were observed in the phenotype）. Among the 11 pregnant women with 22q11.2 microduplications，4 cases terminated the pregnancy，and 7 cases had births（a 4-year-old boy with delayed fontanel closure，the phenotypes of the other 6 cases were not significantly abnormal at the time of the follow-up）. Conclusions Compared with microduplications，microdeletions show a wider range of clinical phenotypes and a stronger association with congenital heart defects（CHD）.

Objective To investigate the role of genomic copy number variation sequencing（CNV-seq） combined with short tandem repeat（STR） typing in the genetic etiology of early spontaneous abortion. Methods A total of 529 patients with spontaneous abortion in Lianyungang Maternal and Child Health Hospital from January 2022 to December 2023 were enrolled. All the patients' products of abortion tissue samples were collected，and CNV-seq combined with STR typing was used to determine chromosomal copy number variation（CNV）. Chromosomal aneuploidy and CNV >100 kb were analyzed. Peripheral blood G-banding karyotype analysis was performed on the biological parents of the products of abortion tissue samples with abnormal D and G group chromosomes and large CNV. Results Among the 529 products of abortion tissue samples from patients with spontaneous abortion，322 cases of chromosomal abnormalities were determined，including 279 cases of chromosomal number abnormalities（240 cases of chromosomal aneuploidy and 39 cases of chromosomal triploidy） and 43 cases of CNV abnormalities（11 cases of submicroscopic CNV and 32 cases of large CNV）. Among the recurrent CNV，3 cases of 15q11.2 microdeletion syndrome and 2 cases of 16p11.2 microdeletion syndrome were inherited from fathers or mothers with no obvious clinical phenotypes. Peripheral blood karyotype analysis detected 6 cases of chromosomal balanced translocation carriers and 2 cases of chromosomal Robertsonian translocation carriers. Conclusions The combination of CNV-seq and STR typing can eliminate maternal contamination and detect triploidy abnormalities，which can better improve the overall detection rate of genetic etiology in spontaneous abortion. For the products of abortion tissue samples with D and G group aneuploidy and large CNV abnormalities，peripheral blood karyotype analysis of parents can timely detect chromosomal balanced translocations，providing timely guidance for precise clinical management and subsequent reproduction after spontaneous abortion and avoiding recurrent spontaneous abortion.

Objective To analyze the genetic analysis results and pregnancy outcome follow-up results of 11 fetuses with chromosome 12 copy number variation（CNV）. Methods From January 2017 to December 2023，6 002 pregnant women who underwent interventional prenatal diagnosis [chromosome karyotype analysis and single nucleotide polymorphism array（SNP-array）] at Quanzhou Women's and Children's Hospital were enrolled. Among them，11 pregnant women with fetal chromosome 12 abnormalities were determined. Among them，case 2 fetus was verified by fluorescence in situ hybridization（FISH）. Results The prenatal diagnosis karyotype result of case 1 fetus was 46，XN，der（1）t（1；12）（p36.3；q24.1），that of case 2 fetus was 47，XN，+dic（12）（q12）[3]/46，XN[97]，and that of case 3 fetus was 47，XN，+i（12）（p10）. Case 4 fetus had failed amniotic fluid culture. Case 5-case 11 fetuses had no obvious abnormalities in karyotype. Case 1 fetus had 19.6 Mb duplication and 1.7 Mb deletion in the chromosome 12 12q24.13q24.33 and chromosome 1 1p36.33p36.32，case 2 fetus had 40.2 Mb tetrasomic duplication in the chromosome 12 12p13.33q12，case 3 fetus had 34.6 Mb tetrasomic duplication in the chromosome 12 12p13.33p11.1，and the chromosome CNV of case 1-case 3 fetuses were all pathogenic CNV. Case 4 fetus had 2.2 Mb duplication in the chromosome 12 12p13.31，which was a possible pathogenic CNV. Case 5-case 11 fetuses had 327.7 kb-1.0 Mb CNV in the chromosome 12，all of which were of unknown clinical significance. Case 1，case 4，case 6-case 10 fetuses were inherited from phenotypically normal parents，case 2，case 3，case 5 fetuses had CNV as new mutations，and case 11 fetus was not verified. Case 2 fetus had approximately 82% of cells during division as tetrasomic cells at the 12pter probe site fragment. Case 1-case 4 fetuses' pregnant women terminated the pregnancy；case 5，case 7，case 10，case 11 fetuses' pregnant women continued the pregnancy，and the newborns were healthy after birth；case 6，case 8 fetuses' pregnant women continued the pregnancy，and the newborns showed abnormalities after birth，received medical intervention，and are currently healthy；case 9 fetus' pregnant woman continued the pregnancy，and the fetus was lost to follow-up after birth. Conclusions The use of multiple different determination techniques in prenatal diagnosis can increase the determination rate of chromosome abnormalities，clarify the specific situation of CNV，assist in pathogenicity interpretation，evaluate prognosis，and guide pregnancy.

Objective To investigate the genetic cause of a fetus diagnosed with congenital cataract（CC） by prenatal ultrasound，and to analyze the pathogenic gene variations and prenatal ultrasound manifestations of Nance-Horan syndrome. Methods A sample of amniotic fluid from a pregnant woman at 24⁺³ weeks of gestation who was suspected of having bilateral CC in the fetus，as well as peripheral blood samples from the pregnant woman and her husband，were collected for family Trio-whole-exome sequencing（Trio-WES）. The candidate variations were verified by Sanger sequencing. The variations were analyzed by bioinformatics，and the distribution frequency of the variations was evaluated by searching public variation databases（dbSNP database，1000 Genomes database，ExAC database，gnomAD database and ESP database）. The literature reports of the variations were retrieved from disease-related databases（HGMD database，ClinVar database and OMIM database）. The pathogenicity of the variations was evaluated by protein prediction tools. Results The Trio-WES results showed that a hemizygous frameshift variation [NM_001291867.2：c.3799dup（p.Cys1267Leufs*18）] was present in the X chromosome of the fetus，and the pregnant woman was a heterozygous carrier of this variation，while her husband had the wild type. The c.3799dup（p.Cys1267Leufs*18） variation was not included in the public variation databases and disease-related databases，and it was a new variation. No other pathogenic variations were determined. Combined with the ultrasound characteristics of bilateral CC in the fetus，Nance-Horan syndrome caused by NHS gene variation was diagnosed. After genetic counseling，the pregnant woman chose to terminate the pregnancy. Conclusions Trio-WES can achieve precise molecular diagnosis of Nance-Horan syndrome in the fetal period. The c.3799dup（p.Cys1267Leufs*18） is a new variation of NHS gene，enriching the variation spectrum of this disease，and providing a reference for prenatal diagnosis and reproductive strategies.

Objective To investigate the clinical phenotype and genetic characteristics of X-linked spondyloepiphyseal dysplasia tarda（SEDT） families caused by trafficking protein particle complex subunit 2（TRAPPC2） gene variations. Methods Totall，3 families with X-linked SEDT were enrolled from Shanghai Children's Medical Center of Shanghai Jiao Tong University School of Medicine from January 2019 to September 2024. The clinical data of family members were collected，and whole-exome sequencing（WES） was performed，and bioinformatics analysis was conducted. Suspected mutations were verified by Sanger sequencing. The variations were rated according to the relevant guidelines of American College of Medical Genetics and Genomics（ACMG）. Results The probands of all the 3 families were male，and they presented with growth retardation. X-ray of the spine showed flattened vertebrae，depression of the anterior upper and lower margins of the vertebrae，and hump-like protrusions in the middle and posterior parts. The probands of family 1 and family 2 had hemizygous variations c.271_275del（p.Gln91Argfs*9） in TRAPPC2 gene（NM_001011658.4），which were pathogenic variations. The mothers of the probands in these 2 families had heterozygous mutations at this site，while the fathers did not detect the mutation. The proband of family 3 had a hemizygous variation c.191_192delTG（p.Val64Glyfs*24） in the TRAPPC2 gene（NM_001011658.4），and no mutations were found at this site in the parents，and it was considered as a putative de novo variation. Recombinant human growth hormone（rhGH） treatment was used，but the efficacy was poor. Conclusions The SEDT caused by TRAPPC2 gene variations is late-onset and progressive，resulting in disproportionate short stature and premature degeneration of joints. For children with onset in adolescence，if there are abnormal upper and lower body measurements or mismatch in the interphalangeal distance，SEDT should be vigilant. WES is helpful for clarifying the cause.

Objective To investigate the distribution characteristics of common genetic variations of deafness in newborns in Tongzhou District of Jiangsu Province，and to evaluate the effectiveness of deafness gene screening. Methods A total of 11 256 newborns who underwent deafness gene screening in Tongzhou District of Jiangsu Province from June 2018 to December 2022 were enrolled. All the newborns' heel blood was collected and prepared into dried blood spots. The 15 variation sites of 4 deafness genes（GJB2，GJB3，SLC26A4，mtDNA12SrRNA） were determined using microarray chips. The samples with determined variation sites were verified by Sanger sequencing. The parents of newborns with determined genetic variations were followed up by phone. Results Among the 11 256 newborns，514 cases were determined as carriers of genetic variations（including 9 cases of double gene variations），with a total variation rate of 4.57%. Among them，253 cases had GJB2 gene variations（2.25%），27 cases had GJB3 gene variations（0.23%），209 cases had SLC26A4 gene variations（1.86%），and 34 cases had mtDNA 12SrRNA variations（0.30%）（P<0.001）. The top 3 variation sites determined were GJB2 gene c.235delC（1.67%），SLC26A4 gene c.IVS7-2A>G（1.45%） and GJB2 gene c.299delG（0.39%）. There were 261 cases of male newborns（261/5 711，4.57%） and 253 cases of female newborns（253/5 545，4.56%） as carriers of genetic variations. Among the follow-up patients of the genetic variation newborns，2 cases had hearing loss（GJB2 gene c.235delC homozygous variation and SLC26A4 gene c.IVS7-2A>G+c.1226G>A heterozygous variation）. Conclusions The common types of deafness genes in newborns in Tongzhou District of Jiangsu Province are SLC26A4 gene c.IVS7-2A>G，GJB2 gene c.235delC and GJB2 gene c.299delG. Conducting deafness susceptibility gene screening can help improve the determination rate of hearing impairment in newborns.

Objective To investigate the clinical characteristics and the gene mutation site of a family with Wiedemann-Steiner syndrome（WDSTS） caused by a new mutation in KMT2A gene. Methods The clinical data and peripheral blood samples of the proband and family members were collected. Genomic DNA was extracted，and whole-exome sequencing，datum analysis and Sanger sequencing verification were performed. Results The clinical characteristics of the proband included intellectual disability，special facial features，growth and development retardation and multiple hair on the limbs. The proband，his mother and his younger brother all carried a heterozygous frameshift mutation in the KMT2A gene at 11q23.3（NM_005933.4）：c.3061_3062delAA/p.Lys1021fs*17，and it was a new mutation in the KMT2A gene. After Sanger sequencing verification，it was determined that KMT2A gene c.3061_3062delAA/p.Lys1021fs*17 was pathogenic. Conclusions A new mutation site in the KMT2A gene c.3061_3062delAA/p.Lys1021fs*17 has been found，expanding the pathogenic gene and phenotypic spectrum of WDSTS.

Objective To investigate the clinical significance of tripartite motif-containing protein 2（TRIM22） gene expression in patients with ulcerative colitis（UC）. Methods The UC dataset GSE119600 was downloaded from the GEO database for bioinformatics analysis. Totally，62 patients with UC（UC group） and 60 healthy subjects（healthy control group） at the First Affiliated Hospital of Nanjing Medical University from December 2023 to June 2024 were enrolled. The relative expression levels of serum TRIM22 gene were determined. UC patients were classified into non-active group（22 cases） and active group（40 cases） according to the modified Mayo score. All the patients were classified into mild，moderate and severe groups according to the ulcerative colitis endoscopic index of severity（UCEIS） score. The active UC patients were classified into good prognosis group（29 cases）and poor prognosis group（11 cases）based on the endoscopic reexamination results after 3 months of treatment. Spearman correlation analysis was used to evaluate the correlation between serum TRIM22 and laboratory routine indicators in UC patients. Logistic regression analysis was used to assess the influencing factors of UC. Receiver operating characteristic（ROC） curve was used to evaluate the efficacy of TRIM22 in judging the severity and poor prognosis of UC patients. Results TRIM22 was involved in anti-viral responses，interferon-mediated signaling pathways and inflammatory bowel diseases. The relative expression level of serum TRIM22 gene of UC group was higher than that of healthy control group（P<0.001）. The relative expression level of serum TRIM22 gene in active group was higher than that in non-active group（P=0.012）. The relative expression level of serum TRIM22 gene in severe group was higher than those in mild and moderate groups（P<0.001）. Compared with before treatment，the relative expression level of serum TRIM22 gene in good prognosis group after 3 months of treatment was decreased（P=0.025）. Elevated C-reactive protein（CRP） levels and TRIM22 expression were independent risk factors for the occurrence of UC [odds ratios（OR） were 1.10 and 1.69，95% confidence intervals（CI） were 1.85-9.40 and 1.15-2.48，P<0.05]. The areas under curves（AUC） of TRIM22 for judging the active stage of UC，severe disease condition and poor prognosis were 0.71，0.77 and 0.76，respectively. Conclusions TRIM22 is expressed in the serum of UC patients and is related to the severity of the disease. TRIM22 is expected to become a potential molecular marker for UC.

Objective To investigate the relationship between the expression of preoperative serum long non-coding RNA（lncRNA） CDKN2B-AS1 and miR-383-5p and the N stage and postoperative recurrence of thyroid cancer. Methods A total of 104 patients with thyroid cancer and 129 patients with benign thyroid diseases（control group） were enrolled from Nanshi Hospital of Nanyang from April 2021 to May 2023. The clinical data and laboratory determination results were collected，and the relative expression levels of preoperative serum lncRNA CDKN2B-AS1 and miR-383-5p were determined. According to the final pathological examination results，the patients were classified into N0 stage group（61 cases） and N1 stage group（43 cases）. The patients with thyroid cancer were followed up for 1 year after surgery，and the recurrence situation was classified into non-recurrence group（71 cases） and recurrence group（33 cases）. Receiver operating characteristic（ROC） curve was used to evaluate the efficacy of various indicators in determining the preoperative N stage and postoperative recurrence of thyroid cancer patients. Multivariate Logistic regression analysis was used to evaluate the influencing factors of postoperative recurrence in thyroid cancer patients. Results Compared with control group，the relative expression levels of preoperative serum lncRNA CDKN2B-AS1 in N0 stage group and N1 stage group were increased（P<0.05），and the relative expression levels of miR-383-5p were decreased（P<0.05）. Compared with N0 stage group，the relative expression levels of lncRNA CDKN2B-AS1 in N1 stage group were increased（P<0.05），and the relative expression levels of miR-383-5p were decreased（P<0.05）. The preoperative N1 stage proportion，thyroglobulin（Tg） and lncRNA CDKN2B-AS1 in recurrence group were higher than those in non-recurrence group（P<0.001），and miR-383-5p was lower than that in non-recurrence group（P<0.001）. There was no statistical significance in the other indicators between the 2 groups（P>0.05）. The areas under curves（AUC） of single and combined determinations of lncRNA CDKN2B-AS1 and miR-383-5p for determining the N1 stage of thyroid cancer and the AUC for determining postoperative recurrence were 0.809，0.814，0.919 and 0.856，0.833，0.962，respectively. The AUC of Tg for determining postoperative recurrence was 0.776. The lncRNA CDKN2B-AS1 increasing，miR-383-5p decreasing and preoperative N1 stage were independent risk factors for postoperative recurrence in thyroid cancer patients [odds ratios（OR） were 2.914，0.826 and 2.902，95% confidence intervals（CI） were 1.494-5.685，0.739-0.924 and 1.499-5.618，P<0.01]. Conclusions The expression of preoperative serum lncRNA CDKN2B-AS1 and miR-383-5p in thyroid cancer patients is related to the N stage and postoperative recurrence，and they may be used as biological indicators for the assessment of the condition and prognosis of thyroid cancer.

Objective To investigate the roles of p53，p300 and miR-944 expressions in cancer tissues in the prognosis assessment of non-small cell lung cancer（NSCLC）patients. Methods Totally，40 NSCLC patients from Qingdao Cancer Hospital from June 2016 to June 2018 were enrolled. Cancer tissues and adjacent tissues（>5 cm away from tumor margin）were collected to determine the expressions of p53，p300 and the relative expression level of miR-944. The clinical data and laboratory determination results were collected. All the patients were followed up for 5 years and were classified into death group（10 cases）and survival group（30 cases）according to their survival status. Logistic regression analysis was used to evaluate the influencing factors of death in NSCLC patients. Receiver operating characteristic（ROC）curve was used to evaluate the efficacy of p53，p300 and miR-944 in predicting death in NSCLC patients. Kaplan-Meier survival curve was used to analyze the survival status of NSCLC patients. Results The positive rates of p53 and p300 and the relative expression level of miR-944 in cancer tissues were higher than those in adjacent tissues（P<0.05）. There was statistical significance in the positive rates of p53 and p300 and the relative expression level of miR-944 among NSCLC patients with different TNM stages，differentiation degrees and lymphatic metastasis（P<0.05）. There was statistical significance in TNM stage，smoking history，lymphatic metastasis，differentiation degree，albumin（Alb），the positive rates of p53 and p300 and the relative expression level of miR-944 between death group and survival group（P<0.05）. TNM stage Ⅲ+Ⅳ，smoking history，lymphatic metastasis，Alb≤35 g·L-1，low differentiation degree，positive p53，positive p300 and elevated miR-944 were all risk factors for death in NSCLC patients（P<0.05）. The areas under curves（AUC）of single and combined determinations of p53，p300 and miR-944 in predicting death in NSCLC patients were 0.641，0.686，0.677 and 0.878，respectively. The total survival rates of p53 negative group，p300 negative group and miR-944 low expression group were all higher than those of p53 positive group，p300 positive group and miR-944 high expression group（Log-rank χ2 values were 6.372，7.127，5.429，respectively，P<0.05）. Conclusions The p53，p300 and miR-944 are highly expressed in cancer tissues of NSCLC patients and are related to the clinical pathological characteristics and prognosis. They may serve as indicators for prognosis assessment in NSCLC patients.

Objective To analyze the serotypes，drug resistance and virulence gene carrying status of invasive group B Streptococcus（GBS） in newborns in Wenzhou，and to provide references for the diagnosis and treatment of neonatal GBS infection. Methods Totally，54 GBS isolates isolated from sterile sites of newborns in Class 3 Grade A hospitals in Wenzhou from 2019 to 2023 were collected. The isolates were identified，and in vitro drug susceptibility test was performed. The drug sensitivity tests were conducted，and high-throughput whole-genome sequencing（WGS） and multi-locus sequence typing（MLST） were performed. Results All the 54 GBS isolates were sensitive to penicillin，ampicillin，vancomycin and linezolid. The drug resistance rate to tetracycline was 87.3%，and the drug resistance rates to clindamycin and levofloxacin were 55.6% and 13.0%，respectively. A total of 7 serotypes were determined，with type Ⅲ being the predominant one（46.2%，25/54）. A total of 14 ST types were determined，with ST17 being the predominant one（24.1%，13/54）. The carrying rates of adhesion-related genes pavA，fbsA，lmb and srr2 were 100.0%，76.0%，87.0% and 27.8%，respectively. The carrying rates of invasion-related genes hylB，cylK，cylJ，cylI，cylE，cylA，cylZ，cylG，cylD，cylF，acpC and cylB were all 100.0%，while the carrying rates of cylX and CAMP were 90.7% and 96.3%，respectively. The carrying rate of immune evasion-related gene rib was 48.1%，and the carrying rates of neuA，neuB and neuC were all 100.0%. Conclusions The GBS isolates causing neonatal infections have a high resistance rate to tetracycline and clindamycin，and the carrying rates of virulence genes are also high.

Objective To analyze the human papillomavirus（HPV） genotypes of different cervical lesion patients in Shanghai and the results of cervical cytology tests（TCT），and to provide a reference for cervical cancer screening. Methods Totally，4 121 female HPV-infected individuals from Ruijin Hospital of Shanghai Jiao Tong University School of Medicine from July 2019 to December 2023 were enrolled. HPV genotypes were determined，and thinprep cytology test（TCT） was performed. Results Among the 4 121 female HPV-infected individuals，3 280 cases had normal TCT results，538 cases had grade Ⅰ cervical intraepithelial neoplasia （CIN），154 cases had grade Ⅱ CIN，92 cases had grade Ⅲ CIN，and 57 cases had cervical squamous cell carcinoma （SCC）. The top 3 HPV genotypes determined were HPV52 （15.83%），HPV58 （9.30%） and HPV16 （9.29%）. Among those with normal TCT results，CIN Ⅰ，CIN Ⅱ，CIN Ⅲ and SCC，HPV52 positive individuals accounted for 15.82%，15.84%，18.01%，15.11% and 12.16%，HPV58 positive individuals accounted for 8.87%，9.60%，14.22%，12.23% and 13.51%，and HPV16 positive individuals accounted for 7.00%，9.94%，30.81%，31.65% and 40.54%，respectively. The multiple HPV infection rate was 26.06%，and multiple HPV infections were found in those with normal TCT results，CIN Ⅰ，CIN Ⅱ，CIN Ⅲ and SCC，accounting for 23.48%，40.52%，25.97%，35.87% and 22.81%，respectively. Among HPV-infected individuals，high-risk HPV infection accounted for 80.40%，and high-risk HPV infection in those with normal TCT results，CIN Ⅰ，CIN Ⅱ，CIN Ⅲ and SCC accounted for 78.42%，84.51%，94.31%，94.24% and 95.95%，respectively. Conclusions The infection rate of HPV16 in CIN Ⅱ，CIN Ⅲ and SCC among female patients in Shanghai is relatively high. HPV52 is more common in those with normal TCT results and CIN Ⅰ. The HPV multiple infection rate is higher in CIN Ⅰ patients. HPV typing has certain clinical significance in the screening of cervical cancer and its precancerous lesions.

Streptococcus pneumoniae（Sp） can cause community-acquired infection，it is the first opportunistic pathogen of out-of-hospital infection，and it is an important pathogen causing community-acquired pneumonia（CAP）. Sp infection can cause pneumonia，otitis media，meningitis，bloodstream infection and other diseases. The pathogenic mechanism of Sp is mainly related to capsular polysaccharide（CPS），hemolysin，protease，biofilm，hydrogen peroxide and so on. At present，with the widespread application of broad-spectrum antimicrobials and immune agents and the increase of penicillin-resistant and multi-drug resistant isolates，the drug resistance is becoming increasingly serious. The main reason for Sp drug resistance is the mutation of penicillin-binding protein（PBP），which causes great obstacles to clinical diagnosis and treatment. For the antimicrobial treatment of Sp infection，penicillin is recommended as the first choice. The review focuses on the research progress of Streptococcus pneumoniae.