Sanger Sequencing Research Articles

Knockdown-resistant mutations in head lice (Pediculus humanus capitis) collected from schoolchildren in Riyadh, Saudi Arabia

Head lice infestation remains one of the most common child problems. This problem is not only attributed to the ability of head lice to spread rapidly but also because of the head lice resistance that develops from incomplete or improper treatment. Pyrethroids are a group of medications that have been widely used for the treatment of head lice. However, in recent years, several countries reported knockdown resistance (kdr). The aim of this study is to evaluate the frequency of pyrethroid-resistant mutations in Riyadh City, Saudi Arabia. To do that, Sanger sequencing was employed to find the frequency of mutated alleles in the voltage-sensitive sodium channel gene (VSSC). The result showed that the frequency of the resistant alleles in T917I was 0.83. In addition, three new mutations (L920F, V966F, and F967L) were detected in the examined samples. These findings highlight the significant prevalence of pyrethroid-resistant mutations, which may be attributed to the cultural and tourism openness that leads to the abundance of numerous job opportunities for different workforces from all around the world.

Familial Oculoauriculovertebral Spectrum: A Genomic Investigation of Autosomal Dominant Inheritance.

Oculoauriculovertebral spectrum (OAVS) encompasses abnormalities on derivatives from the first and second pharyngeal arches including macrostomia, hemifacial microsomia, micrognathia, preauricular tags, ocular, and vertebral anomalies. We present genetic findings on a 3-generation family affected with macrostomia, preauricular tags and ptosis following an autosomal dominant pattern. We generated whole-genome sequencing data for the proband, affected father, and unaffected paternal grandmother followed by Sanger sequencing on 23 family members for the top candidate gene mutations. We performed parent and sibling-based transmission disequilibrium tests (TDTs) and burden analysis via a penalized linear mixed model, for segregation and mutation burden, respectively. Next, via bioinformatic tools we predicted protein function, mutation pathogenicity, and pathway enrichment to investigate the biological relevance of mutations identified. Rare missense mutations in SIX1, KDR/VEGFR2, and PDGFRA showed the best segregation with the OAVS phenotypes in this family. When considering any of the 3 OAVS phenotypes as an outcome, SIX1 had the strongest associations in parent-TDTs and sib-TDTs (P = 0.025, P = 0.052) (unadjusted P-values). Burden analysis identified SIX1 (RC = 0.87) and PDGFRA (RC = 0.98) strongly associated with OAVS severity. Using phenotype-specific outcomes, sib-TDTs identified SIX1 with uni- or bilateral ptosis (P = 0.049) and ear tags (P = 0.01), and PDGFRA and KDR/VEGFR2 with ear tags (both P < 0.01). SIX1, PDGFRA, and KDR/VEGFR2 are strongly associated to OAVS phenotypes. SIX1 has been previously associated with OAVS ear malformations and is co-expressed with EYA1 during ear development. Efforts to strengthen the genotype-phenotype co-relation underlying the OAVS are key to discover etiology, family counseling, and prevention.

Updates on the molecular spectrum of MEFV variants in lebanese patients with Familial Mediterranean Fever

Familial Mediterranean Fever (FMF) is a hereditary autoinflammatory disorder, particularly present in the Mediterranean populations, and associated with pathogenic variants in the MEFV gene. This study aims to investigate the distribution of MEFV variants in a large cohort of Lebanese patients, and to explore the genotype-phenotype correlation among affected individuals. A retrospective analysis was conducted on 3,167 patients referred for MEFV sequencing at the Medical Genetics and Genomics Center(CGGM) at Saint-Joseph University of Beirut-Lebanon, from 2006 to 2023. Sanger sequencing was used to detect MEFV variants, focusing initially on hot-spot exons. Among the 3,167 patients, 46.73% (N = 1,480) carried at least one MEFV variant. The most common variants detected were M694V and V726A, both found in 28.98% of cases, followed by E148Q(27.83%) and M694I(13.98%). Moreover, Shiites and Sunni Muslims, and individuals from South and North Lebanon exhibited the highest frequency of variants. Interestingly, family history was found to be significantly higher in patients having two MEFV variants than those with one variant (p = 0.0026). The most commonly reported symptoms were fever(78%), abdominal pain(88%), joint pain(65%), and thoracic pain(46%). The genotype-phenotype correlation analysis revealed a more severe phenotype in patients carrying the M694V or V726A mutations compared to those with the homozygous E148Q genotype. This study, the largest in Lebanon, highlights the high prevalence of MEFV variants, particularly M694V and V726A, in FMF patients. Our data provide valuable insights into the genetic landscape of FMF in Lebanon and emphasize the importance of early genetic screening for a better disease management and genetic counselling.

Resource availability for CNS tumor diagnostics in the Asian Oceanian region: A survey by the Asian Oceanian Society of Neuropathology committee for Adapting Diagnostic Approaches for Practical Taxonomy in Resource-Restrained Regions (AOSNP-ADAPTR).

The shift toward a histo-molecular approach in World Health Organization classification of central nervous system tumors (WHO CNS5) emphasizes the critical role of molecular testing, such as next-generation sequencing (NGS) and DNA methylation profiling, for accurate diagnosis. However, implementing these advanced techniques is particularly challenging in resource-constrained countries. To address this, the Asian Oceanian Society of Neuropathology committee for Adapting Diagnostic Approaches for Practical Taxonomy in Resource-Restrained Regions (AOSNP-ADAPTR) was initiated to help pathologists in resource-limited regions to implement WHO CNS5 diagnoses using simpler diagnostic tools, mainly immunohistochemistry. An online survey by this group assessed the in-house/local availability of diagnostic resources and outsourcing capabilities across the Asian Oceanian region, covering 19 countries. Of 318 responding centers, the availability of molecular techniques in-house/locally was limited in lower middle-income countries (LMICs), with 29% having fluorescence in situ hybridization, 10.7% Sanger sequencing, and only 9.4% NGS. DNA methylation was largely unavailable in-house/locally in of LMICs, while in the whole region, its availability stood at a meager 4.4%. Though outsourcing for all diagnostic tests was an easily accessible alternative, outsourcing for NGS and DNA methylation was uncommon because of the financial burden on patients being a significant obstacle. The survey categorized centers into five resource levels (RLs) based on the in-house/local access to diagnostic techniques, which highlighted the variability and disparity in diagnostic capabilities across the region. High-income countries had 80% of centers with advanced RL IV, V resources, in contrast to LMICs, where 70.5% of centers fell into RL I-III. This comprehensive evaluation emphasizes the need for tailored resource level guidelines, with the aim of improving diagnostic accuracy and treatment as well as advocating better healthcare infrastructure in resource-constrained areas. However, the survey's reliance on responses from better-resourced centers may underestimate challenges in lower-resource settings, stressing the need for broader outreach and support.

Identification of Rickettsia species in cattle ticks in selected regions of urban Ghana.

Ticks continue to invade new regions spreading pathogens of zoonotic and veterinary importance. Diverse tick species have been reported in Ghana due to the continuous trade of livestock. In this study, ticks were collected from cattle in three sites within Southern Ghana. The 1489 ticks collected were morphologically identified and further confirmed using a molecular assay that amplifies the 660-bp segment of the mitochondrial cytochrome c oxidase subunit 1 (COI) gene. These ticks were pooled and screened for pathogens of zoonotic importance. From the 215 tick pools screened, Rickettsia DNA was detected in 34 (15.81%). Sanger sequencing revealed the occurrence of Rickettsia africae (14.42%), the causative agent of African tick-bite fever, and Rickettsia aeschlimannii (1.39%). Regular monitoring of tick populations is essential to generate data for creating effective control and preventive measures.

First report of a cat (Felis catus) infected by Leishmania (Viannia) guyanensis from Southeastern Brazil.

Feline leishmaniasis is increasingly recognized as a disease affecting cats worldwide, with notable prevalence in regions surrounding the Mediterranean Basin and in Brazil. An approximately one-year-old domestic shorthair cat was presented with small papules and nodules (2mm) on the nasal planum. Physical examination and routine blood analyses were normal. The cat tested negative for FIV and FeLV. Skin biopsies of the lesions were conducted, removing the nodules entirely due to their small size. The samples were submitted for histopathological and molecular analysis. Histopathology confirmed the presence of Leishmania amastigotes, while PCR-RFLP and Sanger sequencing of the hsp70 gene identified the infecting species as Leishmania guyanensis. The patient has since been under continuous clinical monitoring every six months, with no recurrence observed to date. This is the first documented case of L. guyanensis infection in a domestic cat, underscoring the need for improved diagnostic accuracy and a broader understanding of feline leishmaniasis.

Prognostic values of molecular subtypes and SWI/SNF protein expression in de-differentiated/undifferentiated endometrial carcinoma.

Classification and risk stratification of endometrial carcinoma (EC) has transitioned from histopathological features to molecular classification, e.g. the ProMisE classifier, identifying four prognostic subtypes: POLE mutant (POLEmut) with almost no recurrence or disease-specific death events, mismatch repair deficient (MMRd) and no specific molecular profile (NSMP), with intermediate outcome and p53 abnormal (p53abn) with poor outcomes. However, the applicability of molecular classification is unclear in rare but aggressive histotypes of EC, e.g. de-differentiated and undifferentiated endometrial cancers (DD/UDEC). Here, we aim to assembled a cohort of DD/UDEC from a single institution and analysed the prognostic significance of ProMisE molecular subtypes and the expression of SWItch/sucrose non-fermentable (SWI/SNF) chromatin remodelling complex members, previously implicated in the pathogenesis of DD/UDEC. We accrued 88 DD/UDEC cases, assessed POLE status by Sanger sequencing and performed immunohistochemistry for p53, mismatch repair and SWI/SNF proteins on the tissue microarrays assembled. Assignment of molecular subtypes was possible in 80 tumours; POLE sequencing failed in the remaining eight cases. There were 12 (15%) POLEmut, 44 (55%) MMRd, 14 (17.5%) p53abn and 10 (12.5%) NSMP DD/UDEC. POLEmut DD/UDECs had excellent outcomes, but the other three molecular subtypes all had poor outcomes, with no significant differences among them. The loss of one or more SWI/SNF proteins [AT-rich interactive domain-containing protein 1A (ARID1A), ARID1B, SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member 4 (SMARCA4), SMARCA2], observed in 66% (55 of 83) cases, was not of prognostic significance. These results indicate that all molecular subtypes of DD/UDEC except POLEmut behave in an aggressive fashion. Further study is needed to determine whether these molecular alterations can be targeted with adjuvant therapy, in order to improve outcomes of patients with DD/UDEC.

Occurrence of Pestalotiopsis sp. on Ornamental Plants Camellia japonica L. in Romanian Public Gardens

Camellias are evergreen shrubs native to the eastern and southern regions of Asia. In Romania, camellias are popular ornamental plants grown in pots. Pestalotiopsis species have a worldwide distribution and are pathogenic to a wide range of hosts. They also represent an important group of endophytes capable of producing a variety of bioactive secondary metabolites. This paper reports the occurrence of grey blight and twig or branch dieback caused by Pestalotiopsis sp. on Camellia japonica L. grown in pots and used in the landscaping of a public garden located in Iasi city, Romania (GPS coordinates: 47°09′24.0″ N 27°35′15.6″ E). Pestalotiopsis species were identified based on morphological characteristics and Sanger sequencing. Genomic analysis of the fungal isolate coded P_CJ_24, obtained from living plants, was based on ribosomal internal transcribed spacer region amplification using the LSU primers. This first report of Pestalotiopsis species infection of ornamental plants in Romania suggests that new plant pathogens can be introduced in new environmental conditions through plant pods and their incidence increases with the widespread of plant material among gardens.

Congenital muscular dystrophies and myopathies: the leading cause of genetic muscular disorders in eleven Chinese families

BackgroundCongenital muscular dystrophies (CMDs) and myopathies (CMYOs) are a clinically and genetically heterogeneous group of neuromuscular disorders that share common features, such as muscle weakness, hypotonia, characteristic changes on muscle biopsy and motor retardation. In this study, we recruited eleven families with early-onset neuromuscular disorders in China, aimed to clarify the underlying genetic etiology.MethodsEssential clinical tests, such as biomedical examination, electromyography and muscle biopsy, were applied to evaluate patient phenotypes. Whole exome sequencing was used to screen for prospective variants responsible for muscular diseases, followed by co-segregation analysis in the families and prenatal diagnosis via Sanger sequencing, if appropriate. Nanopore sequencing and optical genome mapping were applied for detecting complicated genome rearrangements.ResultsFourteen variants in nine genes (ATL1, LMNA, KLHL40, FKRP, DMD, ACTA1, MSTO1, RYR1 and LAMA2) associated with congenital muscular conditions were identified in the ten families mentioned above. Among them, five novel variants, c.1153_1155del in LMNA, c.577 A > G in ACTA1, c.694T > G in MSTO1, c.5938del in LAMA2, and a rare genome fusion ogm[GRCh38] fus(X; X)(p22.31;p21.1) involving DMD, have not been recorded in public databases to the best of our knowledge.ConclusionsCMDs and CMYOs were probably responsible for most of the cases (9/11) of genetic muscular defects in this study. Molecular analysis is highly beneficial for the precise diagnosis, genetic counseling and prenatal diagnosis of families suspected of having genetic muscular disorders.Clinical trial numberNot applicable.

Unraveling the genetic mysteries of spinal muscular atrophy in Chinese families

ObjectiveSpinal muscular atrophy (SMA) is a motor neuron disorder encompassing 5q and non-5q forms, causing muscle weakness and atrophy due to spinal cord cell degeneration. Understanding its genetic basis is crucial for genetic counseling and personalized treatment options.MethodsThis study retrospectively analyzed families of patients suspected of SMA at our institution from February 2006 to March 2024. Various molecular techniques, including multiplex ligation-dependent probe amplification analysis, long-range polymerase chain reaction (PCR) combined with nested PCR, Sanger sequencing, and whole-exome sequencing were employed to establish a thorough genetic variant profile in 680 Chinese pedigrees with clinically suspected SMA.ResultsOut of 680 families suspected of having SMA, 675 exhibited mutations in the SMN1 gene, while three families were linked to mutations in the IGHMBP2 gene. One family exhibited a genetic variation in the NEB gene, and another family exhibited a variation in the SCO2 gene. Among the families with mutations in the SMN1 gene, 645 families exhibited either E7‒E8 or E7 homozygous deletion. Some families displayed E7‒8 heterozygous deletions along with other mutations, such as E1 or E1‒6 heterozygote deletion and point mutations. Furthermore, one family demonstrated a compound-heterozygous double mutation, while another carried a type “2 + 0” mutation alongside a point mutation.ConclusionsThis study comprehensively analyzed the genetics of suspected familial SMA cases in the Chinese population, providing insights into the molecular genetic mechanisms of SMA and the utility of various detection techniques. The findings revealed important implications for genetic counseling, prenatal diagnosis, and targeted therapies in clinical practice.

Whole-exome sequencing identifies novel mutation in intrahepatic cholestasis of pregnancy: A case report and literature review.

Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy-specific liver disease characterized by pruritus and elevated total bile acid (TBA) levels. The most serious impact of ICP is sudden unexplained intrauterine fetal death, especially when an associated TBA ≥ 100 µmol/L is confirmed.We report a case of a 27-year-old female patient with early-onset severe refractory ICP. Whole-exome sequencing and mutation analyses were performed to obtain genetic data on the patient and her mother. Sanger sequencing was performed to screen the mutation site. Computer-based algorithms were applied to predict the pathogenesis of the identified mutation. Subsequently, we conducted a literature review to characterize the pathological features and perinatal management of severe refractory ICP, especially ICP with genetic susceptibility.A heterozygous mutation in the ABCD3 gene: c.130C > T/p.Pro44Ser was detected in this patient. Through the analysis of pathogenicity prediction software, the mutations were disease-causing. This is the first report to identify the novel p.Pro44Ser mutations of ABCD3 gene in ICP patients.Our report provides new insights into the genetic architecture of ICP involving ABCD3 variants. Early-onset severe refractory ICP is rare and mutations in bile acid metabolism genes might accentuate the phenotype. Emphasized perinatal management and screening for potential pathogenicity sites of variants that drive specific recognition of ICP is necessary.

Discovery of novel 'sugarcane totivirus 1' from Saccharum officinarum by high-throughput sequencing.

Sugarcane is cultivated globally and affected by more than 125 pathogens, which lead to various plant diseases. In recent years, high-throughput sequencing (HTS)-based genome analyses have been broadly adopted for the discovery of both characterized and un-characterized viruses from plant samples. In this study, the HTS data of sugarcane pooled sample retrieved from sequence read archive (SRA) were de novo re-assembled using CLC Genomic Workbench. The genomic sequence of a novel dsRNA totivirus, 5,384 nucleotides (nt) long, excluding the 5' untranslated region (UTR) and 3' UTR, was discovered and named sugarcane totivirus 1 (STV 1). The genome contains two open reading frames (ORFs): a putative coat protein (CP) encoding 866 amino acids (aa) and RNA-dependent RNA polymerase (RdRp) encoding 824 aa. Phylogenetic studies based on the genomic sequences (nt), and the aa sequences of CP as well as RdRp regions revealed that STV 1 is closely related to other members of the genus Totivirus. Pairwise sequence identity of CP and RdRp aa sequences showed 30.0-51.5% and 26.3-47.2% similarity, respectively with other members of the family Totiviridae. The HTS results were further validated and confirmed through OneStep RT-PCR assay and Sanger sequencing. A novel totivirus (STV 1) in the genus Totivirus, family Totiviridae has been identified. This is the first report of dsRNA totivirus STV 1 associated with sugarcane from India.

Accelerating the diagnosis of Chinese cblC type MMA patients by multiplex PCR sequencing method.

CblC type methylmalonic aciduria (cblC disease) is the most common inborn error of vitamin B12 metabolism and due to mutations in the MMACHC gene. The earlier the diagnosis, the better the prognosis. Therefore, convenient and inexpensive detection method is needed. This study selected mutational hot-spot regions in the MMACHC gene which harbors more than 90% of mutant alleles responsible for cblC disease in China. Subsequently, a hot-spot regions multi-PCR Sanger sequencing method (HsRMSS) was designed. The accuracy and efficiency of HsRMSS was validated using samples from 20 cblC families with known MMACHC gene mutations and 50 healthy volunteers. In addition, patients' clinical phenotypes and molecular genetic features were analyzed. A total of 16 different mutations were identified in 20 cblC families. Among them, the most common mutations were c.609 G>A (26/80, 32.5%), c.567dupT (10/80, 12.5%), c.80A>G (8/80, 10.0%), c.658_660delAAG (8/80, 10.0%) and c.394C>T (6/80, 7.5%), which accounted for over 70% of disease alleles. The HsRMSS results were the same as the results using the whole exon sequencing, with a coincidence rate of 100%. The HsRMSS targeting the mutational hot-spots of MMACHC gene could be a promising tool to accurately and rapidly diagnose cblC disease in China. This study reported the development and validation of a hot-spot regions multi-PCR Sanger sequencing method for targeting hotspots which harbor most of the common MMACHC gene mutations reported in Chinese patients with cblC disease. The approach could have a potential clinical application as a rapid diagnosis and screening tool for suspected children with cblC type MMA and population carrier, owing to its high throughput, low cost, and high sensitivity and specificity.

Case Report: Prenatal diagnosis of novel compound heterozygous variants in WDR35 gene causing short-rib thoracic dysplasia 7 with or without polydactyly

BackgroundWhole exome sequencing (WES) technology has been increasingly used for the etiological diagnosis of fetuses with ultrasound anomalies. In this article, we report a novel deletion compound combined with a causative variant in WDR35 gene leading to short-rib thoracic dysplasia 7 (SRTD7) with or without polydactyly using WES.MethodsThis study involved a Chinese fetus with clinical features of skeletal dysplasia on ultrasound imaging, in whom chromosome abnormalities and copy number variants (CNVs) were detected by chromosomal microarray analysis (CMA), and sequence variants were detected by WES. The obtained results were further verified by Sanger sequencing or real time quantitative PCR (qPCR).ResultsNo chromosomal abnormality or CNVs were identified in the fetus by CMA. However, WES result revealed a 14.38-kb large novel deletion compound covering exon 7 to exon 12 combined with a missense variant NM_001006657.2:c.932G&amp;gt;T(p.W311l) in WDR35. Both variants were thought of as pathogenic, which was further confirmed by Sanger sequencing and qPCR. In addition, two compound heterozygous variants NM_015102.5:c.[1196A&amp;gt;G(p.E399G)];[1972C&amp;gt;T(p.R658*)] in NPHP4 gene were also identified in the fetus, which may be partially responsible for fetal kidney hyperechogenicity and oligohydramnios.ConclusionThis is the first study reporting a novel deletion compound combined with a causative missense variant in WDR35 leading to SRTD7. This finding may broaden the spectrum of variants of WDR35 gene and provide a valuable reference for clinical counseling of related abnormalities in pregnancies.

CircCCT2/miR-146a-5p/IRAK1 axis promotes the development of head and neck squamous cell carcinoma

BackgroundHead and neck squamous cell carcinoma (HNSCC), a highly invasive malignancy with a poor prognosis, is one of the most common cancers globally. Circular RNAs (circRNAs) have become key regulators of human malignancies, but further studies are necessary to fully understand their functions and possible causes in HNSCC.MethodsCircCCT2 expression levels in HNSCC tissues and cells were measured via qPCR. CircCCT2 was characterized by Sanger sequencing, qRT-PCR, RNase R & Actinomycin D treatment, nucleoplasmic separation and FISH experiments. CCK-8 and colony formation assays were performed to determine cell proliferation, and Transwell assays were used to determine migration and invasion. A xenograft tumor model was used to study the influence of circCCT2 on HNSCC in vivo. Dual-luciferase gene reporter, RIP, western blotting, and rescue experiments, were used to explore target-binding relationships and regulatory mechanisms.ResultsCircCCT2 was significantly upregulated in HNSCC tissues and cells. High circCCT2 levels were associated with advanced T stage, N stage, clinical stage and poor prognosis. Functionally, we verified that circCCT2 promotes HNSCC development in vitro and in vivo. Mechanistically, functioning as a competitive endogenous RNA (ceRNA) or miRNA sponge, circCCT2 binds directly to miR-146a-5p and increases interleukin-1 receptor-associated kinase 1 (IRAK1) levels, which enhances the malignant development of HNSCC by driving epithelial-mesenchymal transition (EMT).ConclusionCircCCT2 promotes HNSCC development through the miR-146a-5p/IRAK1 axis, revealing that circCCT2 is a potential biomarker and target for HNSCC.

Study of the Prevalence of Human Papillomavirus Genotypes in Jeddah, Saudi Arabia

Human papillomavirus (HPV), a common sexually transmitted infection, includes over 200 types, some linked to genital warts and various cancers, including cervical, anal, penile, and oropharyngeal cancers. In Saudi Arabia, an estimated 10.7 million women aged 15 years and older are at risk of HPV-related cervical cancer. This study assessed HPV prevalence, genotype distribution, awareness, and health behaviors among females in Jeddah, Saudi Arabia. Pap smear samples were collected from 106 women attending gynecology clinics at King Abdulaziz University Hospital and were analyzed using nested PCR and Sanger sequencing. Results showed a 15.1% HPV positivity rate, with genotypes of HPV 16, 6, and 58 identified. HPV 16 was the most prevalent (43.75%), particularly among women aged 35–44. In addition, over 60% of the participants were unaware of the link between HPV and cervical cancer, or the availability of vaccines, indicating significant knowledge gaps in the population. These findings underscore the urgent need for comprehensive HPV surveillance and targeted public health interventions in Saudi Arabia. This study emphasizes the importance of HPV vaccination programs, increased screening, and educational campaigns to address the high prevalence of high-risk genotypes, particularly HPV 16. Tailored strategies are crucial to reducing HPV-related health risks, especially for high-risk age groups and under-informed populations. The data provide a foundation for improving HPV prevention and reducing the burden of related diseases in the Saudi population.

High clinical utility of long-read sequencing for precise diagnosis of congenital adrenal hyperplasia in 322 probands

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.

Helminths infection of Schizothorax niger in Kashmir, India: morphological and molecular characterization.

The identification of helminth parasites in Schizothorax spp. from Kashmir, including Schyzocotyle acheilognathi, Pomphorhynchus kashmirensis, and Adenoscolex oreini, is hindered by morphological limitations and high intraspecific variation. While previous studies have relied on morphological diagnosis, a comprehensive molecular characterization is lacking. To address these limitations and achieve more accurate species identification, this study employs molecular techniques such as DNA sequencing and phylogenetic analysis. Genomic DNA was extracted using standardized protocols. Partial amplification of 18S, ITS, and 28S rRNA was carried out using specific primer sets, followed by Sanger sequencing. Genetic characteristics were analyzed using bioinformatic approaches, focusing on Nucleotide divergence and Phylogenetic relationships. Phylogenetic analysis confirmed the monophyly of S. acheilognathi, revealing close relationships with S. nayarensis. Interspecific variation ranged from 2.0 - 4.1% for 18S and 0.8 - 2.2% for 28S rDNA, while P. kashmirensis showed high genetic similarity to P. tereticollis and P. laevis with variations ranging from 0.1 - 1.5% for 18S and 2.3-6.0% for ITS rDNA. A. oreini forms a monophyletic lineage separate from Caryophyllaeides and Caryophyllaeus species. Our study resolved persistent taxonomic ambiguities within the order Bothriocephalidea, resulting in the reassignment of Schyzocotyle acheilognathi (previously Bothriocephalus acheilognathi) to the reinstated genus Schyzocotyle. This study underscores the utility of ribosomal gene sequencing in characterizing fish parasites and highlights the need for taxonomic re-evaluation of A. oreini within the Capingentidae family.

Replication Study and Meta-Analysis of the Contribution of Seven Genetic Polymorphisms in Immune-Related Genes to the Risk of Gastric and Colorectal Cancers.

Recently, it has been realized that immune processes participate in the pathogenesis of human cancers. A large number of genetic polymorphisms in immune-related genes have been extensively examined for their roles in the susceptibility of gastric cancer (GC) and colorectal cancer (CRC), including IL4 gene rs2070874, IL4RA gene rs1801275, IL18 gene rs187238, IL18RAP gene rs917997, IL17A gene rs8193036, IL23R gene rs1884444 and IL23R gene rs10889677. However, there is no consistent conclusion, which calls for further research. In this case-control study, these 7 genetic polymorphisms were genotyped by Sanger sequencing in a total of 1247 patients with cancer (GC/CRC: 460/787) and 800 healthy individuals. A total of 31 previous studies and our present study were included in this meta-analysis. The case-control study revealed that in Hubei Chinese population, rs2070874, rs187238 and rs10889677 were significantly associated with CRC risk, whereas only rs917997 was significantly associated with GC risk. The meta-analysis showed that rs2070874, rs917997, rs8193036 and rs1884444 were significantly associated with GC risk in Chinese population, whereas rs2070874 in total population, rs1801275 in Asian population and rs187238 in Chinese population were significantly associated with CRC risk. IL4 gene rs2070874, IL18RAP gene rs917997, IL17A gene rs8193036 and IL23R gene rs1884444 may serve as the susceptible factors for GC carcinogenesis in Chinese population. IL4 gene rs2070874 in total population, IL4RA gene rs1801275 in Asian population and IL18 gene rs187238 in Chinese population may be genetic biomarkers for CRC susceptibility.

Enhancing newborn screening sensitivity and specificity for missed NICCD using selected amino acids and acylcarnitines

PurposeTo enhance the detection rate of Neonatal Intrahepatic Cholestasis caused by Citrin Deficiency (NICCD) through newborn screening (NBS), we analyzed the metabolic profiles of missed patients and proposed a more reliable method for early diagnosis.MethodsIn this retrospective study, NICCD patients were classified into “Newborn Screening” (64 individuals) and “Missed Screening” (52 individuals) groups. Metabolic profiles were analyzed using the non-derivatized MS/MS Kit, and genetic mutations were identified via next-generation sequencing and confirmed by Sanger sequencing. Receiver Operating Characteristic (ROC) analysis evaluated the predictive value of amino acids and acylcarnitines in dried blood spots (DBS) for identifying missed patients including 40 missed patients and 17,269 healthy individuals, with additional validation using 12 missed patients and 454 healthy controls.ResultsThe age of diagnosis was significantly higher in the “Missed Screening” group compared to the “Newborn Screening” group (74.50 vs. 18.00 days, P < 0.001). ROC analysis revealed that citrulline had excellent diagnostic accuracy for missed patients, with an AUC of 0.970 and a cut-off value of 17.57 µmol/L. Additionally, glycine, phenylalanine, ornithine, and C8 were significant markers, each with an AUC greater than 0.70. A combination of these markers achieved an AUC of 0.996 with a cut-off value of 0.00195. Validation demonstrated a true positive rate of 91.67% and a true negative rate of 96.48%. Common SLC25A13 mutations in both groups were c.852_855del, IVS16ins3kb, and c.615 + 5G > A.ConclusionsCombining multiple metabolic markers during NBS significantly improves sensitivity and specificity for detecting missed NICCD cases. However, the relationship between genetic mutations and missed cases remains unclear.