Unraveling novel mRNA transcripts of the human DNA N-glycosylase 1 (NTHL1) gene with the implementation of an innovative targeted DNA-seq assay.

The human NTHL1 gene encodes a DNA glycosylase that plays a key role in the base excision repair (BER) pathway, repairing oxidative DNA damage and maintaining genome integrity. The physiological activity of NTHL1 is crucial in preventing genetic alterations that can lead to cancer. In this study, we employed an innovative targeted DNA sequencing (DNA-seq) methodology to explore the transcriptional landscape of the NTHL1 gene, revealing previously uncharacterized alternative splicing events and novel exons. Our designed approach provided significantly improved sequencing depth and coverage, enabling the identification of novel NTHL1 mRNA transcripts. Bioinformatics analysis confirmed all annotated splice junctions of the main NTHL1 transcripts (v.1 - v.3) and revealed novel mRNA transcripts (NTHL1 v.4 - v.9) derived from splicing events between annotated exons as well as mRNAs containing previously uncharacterized exons (NTHL1 v.10 - v.14). Quantitative PCR analysis highlighted a diverse expression pattern of these novel transcripts across different human cell lines, suggesting cell-specific roles and regulatory mechanisms. Notably, NTHL1 v.5 was overexpressed in luminal A breast cancer cells (MCF-7), while v.13 was prominent in triple negative (BT-20), HER2 + breast cancer (SK-BR-3), prostate, colorectal cancer cells and HEK-293 cells. Our findings suggest that specific novel NTHL1 transcripts may encode protein isoforms with distinct structural features, as indicated by ribosome profiling datasets, while others containing premature termination codons could function as long non-coding RNAs. These insights enhance our understanding of NTHL1 regulatory role and its potential as a biomarker and therapeutic target in human malignancies. This study underscores the importance of exploring the transcriptional diversity of NTHL1 to fully elucidate its role in cancer pathobiology.

Preimplantation Genetic Testing Inhibits the Transmission of Pathogenic Variants Associated With Cerebral White Matter Disease.

Hereditary white matter disease is a series of progressive genetic diseases that mainly affect the white matter of the central nervous system. The development of molecular genetics enables the clinical diagnosis, carrier detection, and prenatal diagnosis of hereditary white matter disease. Here, we block the transmission of pathogenic variants in ABCD1 and NOTCH3 in a family with cerebral white matter disease via preimplantation genetic testing (PGT). Pathogenic genes were identified based on clinical manifestations, genetic background, and the results of targeted gene capture sequencing. A blastocyst biopsy was performed, and multiple annealing and looping-based amplification (MALBAC), next-generation sequencing (NGS), and single nucleotide polymorphism (SNP) arrays were used to analyze ploidy and the state of the gene mutations. The proband (III:1) had hemizygous mutations in ABCD1 (c.323C>A (p.Ser108 *) and c.775C>T (p.Arg259Trp)) and heterozygous mutations in NOTCH3 (c.1630C>T (p.Arg544Cys)), which were maternally inherited (II:2). After genetic analysis, a euploid blastocyst without ABCD1 and NOTCH3 variations was transferred. A healthy male baby was born at full term, and the results of prenatal diagnosis by amniocentesis in the second trimester verified the results of PGT. To our knowledge, this is the first report of simultaneously blocking the transmission of pathogenic variants in ABCD1 and NOTCH3 via PGT. This report highlights the feasibility and effectiveness of PGT in preventing cerebral adrenoleukodystrophy (cALD) and cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) and provides valuable insights for the diagnosis and treatment of similar cases.

The application status of sequencing technology in global respiratory infectious disease diagnosis.

Next-generation sequencing (NGS) has revolutionized clinical microbiology, particularly in diagnosing respiratory infectious diseases and conducting epidemiological investigations. This narrative review summarizes conventional methods for routine respiratory infection diagnosis, including culture, smear microscopy, immunological assays, image techniques as well as polymerase chain reaction(PCR). In contrast to conventional methods, there is a new detection technology, sequencing technology, and here we mainly focus on the next-generation sequencing NGS, especially metagenomic NGS(mNGS). NGS offers significant advantages over traditional methods. Firstly, mNGS eliminates assumptions about pathogens, leading to faster and more accurate results, thus reducing diagnostic time. Secondly, it allows unbiased identification of known and novel pathogens, offering broad-spectrum coverage. Thirdly, mNGS not only identifies pathogens but also characterizes microbiomes, analyzes human host responses, and detects resistance genes and virulence factors. It can complement targeted sequencing for bacterial and fungal classification. Unlike traditional methods affected by antibiotics, mNGS is less influenced due to the extended survival of pathogen DNA in plasma, broadening its applicability. However, barriers to full integration into clinical practice persist, primarily due to cost constraints and limitations in sensitivity and turnaround time. Despite these challenges, ongoing advancements aim to improve cost-effectiveness and efficiency, making NGS a cornerstone technology for global respiratory infection diagnosis.

A Novel Frameshift Mutation of HBB Causing Dominant ß-Thalassemia in a Chinese Individual.

We reported a rare ß-thalassemia patient, a 41-year-old Chinese male with small cell hypopigmentation anemia, jaundice and splenomegaly as the main clinical symptoms. By using Next-Generation Sequencing (NGS), we identified a novel de novo HBB mutation(c.358_365dup, p.Phe123Alafs*39) which resulted in an abnormally prolonged ß-globin chain comprising 159 amino acid residues. The secondary and three-dimensional structures of the ß-globin predicted that the novel prolonged ß-globin chain has a considerable risk of instability in the hemoglobin, and leads to clinical phenotype. This study contributes to the enrichment of the genetic pathogenic mutation database for thalassemia and underscores the significance of NGS in the screening of mutations for thalassemia families.

Integrative Analysis of CUT&Tag and RNA-Seq Data Through Bioinformatics: A Unified Workflow for Enhanced Insights.

Cleavage Under Targets and Tagmentation (CUT&Tag) is a recent methodology used for robust epigenomic profiling that, unlike conventional chromatin immunoprecipitation (ChIP-Seq), requires only a limited amount of cells as starting material. RNA sequencing (RNA-Seq) reveals the presence and quantity of RNA in a biological sample, describing the continuously changing cellular transcriptome. The integrated analysis of transcriptional activity, histone modifications, and chromatin accessibility via CUT&Tag is still in its infancy compared to the well-established ChIP-Seq. This chapter describes a robust bioinformatics methodology and workflow to perform an integrative CUT&Tag/RNA-Seq analysis.

Pure Hereditary Spastic Paraplegia in a Patient With a Novel Heterozygous KIDINS220 Gene Mutation.

This case presents a somewhat unique and different phenotype of hereditary spastic paraplegia from previously reported kinase D-interacting substrate of 220 kDa (KIDINS220) gene mutation-related disease. We report a unique putative causative heterozygous mutation in KIDINS220 in a pure hereditary spastic paraplegia (HSP) patient expanding the HSP group further. We also deliberate on how our case was different from prior KIDINS220-related pathologies including spastic paraplegia, intellectual disability, nystagmus, and obesity (SINO) syndrome, and the observation of KIDINS220 and aquaporin-4 (AQP4) downregulation in the ventricular ependymal lining of idiopathic normal pressure hydrocephalus (iNPH) patients. These findings warrant further investigations of the biology of KIDINS220. With the advent of new gene editing technologies like Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), variants such as ours provide an opportunity for targeted precision medicine.

ESMO Recommendations on clinical reporting of genomic test results for solid cancers.

BACKGROUND: Genomic tumour profiling has a crucial role in the management of patients with solid cancers, as it helps selecting and prioritising therapeutic interventions based on prognostic and predictive biomarkers, as well as identifying markers of hereditary cancers. Harmonised approaches to interpret the results of genomic testing are needed to support physicians in their decision making, prevent inequalities in precision medicine and maximise patient benefit from available cancer management options. METHODS: The European Society for Medical Oncology (ESMO) Translational Research and Precision Medicine Working Group assembled a group of international experts to propose recommendations for preparing clinical genomic reports for solid cancers. These recommendations aim to foster best practices in integrating genomic testing within clinical settings. After review of available evidence, several rounds of surveys and focused discussions were conducted to reach consensus on the recommendation statements. Only consensus recommendations were reported. Recommendation statements were graded in two tiers based on their clinical importance: level A (required to maintain common standards in reporting) and level B (optional but necessary to achieve ideal practice). RESULTS: Genomics reports should present key information in a front page(s) followed by supplementary information in one or more appendices. Reports should be structured into sections: (i) patient and sample details; (ii) assay and data analysis characteristics; (iii) sample-specific assay performance and quality control; (iv) genomic alterations and their functional annotation; (v) clinical actionability assessment and matching to potential therapy indications; and (vi) summary of the main findings. Specific recommendations to prepare each of these sections are made. CONCLUSIONS: We present a set of recommendations aimed at structuring genomics reports to enhance physician comprehension of genomic profiling results for solid cancers. Communication between ordering physicians and professionals reporting genomic data is key to minimise uncertainties and to optimise the impact of genomic tests in patient care.

Prospects for liquid biopsy approaches in lymphomas.

Analytes within liquid biopsies have emerged as promising alternatives to traditional tissue biopsies for various malignancies, including lymphomas. This review explores the clinical applications of one such liquid biopsy analyte, circulating tumor DNA (ctDNA) in different types of lymphoma, focusing on its role in diagnosis, disease monitoring, and relapse detection. Advancements in next-generation sequencing (NGS) and machine learning have enhanced ctDNA analysis, offering a multi-omic approach to understanding tumor genetics. In lymphoma, ctDNA provides insights into tumor heterogeneity, aids in genetic profiling, and predicts treatment response. Recent studies demonstrate the prognostic value of ctDNA and its potential to improve patient outcomes by facilitating early disease detection and personalized treatment strategies Despite these advancements, challenges remain in optimizing sample collection, processing, assay sensitivity, and overall consensus workflows in order to facilitate integration into routine clinical practice.

Correlation analysis between driver gene mutation and clinicopathological features in lung adenocarcinoma based on real-world cumulative clinical data.

Background: Driver genes are essential predictors of targeted therapeutic efficacy. Detecting driver gene mutations in lung adenocarcinoma (LUAD) patients can help to screen for targeted drugs and improve patient survival benefits. This study aims to investigate the mutation characterization of driver genes and their correlation with clinicopathological features in LUAD. Methods: A total of 440 LUAD patients were selected from Sir Run Run Shaw Hospital between July 2019 and September 2022. Postoperative tissue specimens were analyzed for gene mutations using next-generation sequencing technology, focusing, including epidermal growth factor receptor EGFR, ALK, ROS1, RET, KRAS, MET, BRAF, HER2, PIK3CA and NRAS. At the same time, clinicopathological data were collected and organized for multidimensional correlation analysis. Results: Of 440 LUAD patients, driver gene mutations were not detected in 48 patients. The proportion of patients with driver gene mutations was as high as 89.09%. The top three driver genetic mutations were EGFR, KRAS, and MET. Sixty-nine types of EGFR mutations were detected and distributed in the protein tyrosine kinase catalytic domain (56, 81.16%), Furin-like cysteine-rich region (9, 13.04%), receptor binding domain (3, 4.35%), and EGFR transmembrane domain (1, 1.45%). Single gene locus mutation occurred in 343 LUAD patients, but the mutation gene types covered all tested genes. Our findings showed that EGFR mutations were more commonly observed in non-smoking and female patients (P<0.01), KRAS mutations were more prevalent in male patients and smokers (P<0.01), ROS1 mutations had larger tumor diameters (P<0.01) and RET mutations were more prevalent in smokers (P<0.05). Conclusions: LUAD patients exhibit diverse genetic mutations, which may co-occur simultaneously. Integrated analysis of multiple mutations is essential for accurate diagnosis and effective treatment of the disease. The use of NGS can significantly expand our understanding of gene mutations and facilitate integrated analysis of multiple gene mutations, providing critical evidence for targeted treatment methods.

Molecular alterations and prognosis of breast cancer with cutaneous metastasis.

PURPOSE: Cutaneous metastasis (CM) accounts for 5-30% of patients with breast cancer (BC) and presents unfavorable response to treatment and poor prognosis. A better understanding of the molecular alterations involved in metastasis is essential, which would help identify diagnostic and efficacy biomarkers for CM. MATERIALS: We retrospectively reviewed a total of 13 patients with histological or cytological diagnosis of breast cancer and CM. Clinical information was extracted from the medical records. The mutational landscape of matched primary tumors with their lymph nodes or CM tissues were analyzed using next-generation sequencing (NGS) of 425 cancer-relevant genes. All tissues were also analyzed by immunohistochemistry (IHC). The association of prognosis with various clinical and molecular factors was also evaluated. RESULTS: More than half of the patients were Ki67 low (< 50%, 53.7%). Most patients (12, 92.3%) had other metastasis sites other than skin. The median time from diagnosis to the presentation of CM (T1) was 15 months (range: 0-94 months) and the median time from CM to death (T2) was 13 months (range 1-78). The most frequently altered genes across the three types of tissues were TP53 (69.6%, 16/23), PIK3CA (34.8%, 8/23), and MYC (26.1%). The number of alterations in CM tends to be higher than in primary tumors (median 8 vs. 6, P = 0.077). Copy number loss in STK11, copy number gain in FGFR4, TERT, AR, FLT4 and VEGFA and mutations in ATRX, SRC, AMER1 and RAD51C were significantly enriched in CM (all P < 0.05). Ki67 high group (> 50%) showed significantly shorter T1 than the Ki67 low group (≤ 50%) (median 12.5 vs. 50.0 months, P = 0.036). TP53, PIK3CA mutations, and TERT amplification group were associated with inferior T2 (median 11 vs. 36 months, P = 0.065; 8 vs. 36 months, P = 0.013, 7 vs. 36 months, P = 0.003, respectively). All p values were not adjusted. CONCLUSION: We compared the genomic features of primary breast cancer tissues with their corresponding CM tissues and discussed potential genes and pathways that may contribute to the skin metastasis of advanced breast cancers patients. TP53, PIK3CA mutant, and TERT amplification may serve as biomarkers for poor prognosis for CM patients.

Corrigendum: Current phenotypic and genetic spectrum of syndromic deafness in Tunisia: paving the way for precision auditory health.

[This corrects the article DOI: 10.3389/fgene.2024.1384094.].

Tumor-informed deep sequencing of ctDNA detects minimal residual disease and predicts relapse in osteosarcoma.

Background: Current surveillance modalities of osteosarcoma relapse exhibit limited sensitivity and specificity. Although circulating tumor DNA (ctDNA) has been established as a biomarker of minimal residual disease (MRD) in many solid tumors, a sensitive ctDNA detection technique has not been thoroughly explored for longitudinal MRD detection in osteosarcoma. Methods: From August 2019 to June 2023, 59 patients diagnosed with osteosarcoma at the First Affiliated Hospital of Sun Yat-sen University were evaluated in this study. Tumor-informed MRD panels were developed through whole exome sequencing (WES) of tumor tissues. Longitudinal blood samples were collected during treatment and subjected to multiplex PCR-based next-generation sequencing (NGS). Kaplan-Meier curves and Log-rank tests were used to compare outcomes, and Cox regression analysis was performed to identify prognostic factors. Findings: WES analysis of 83 patients revealed substantial mutational heterogeneity, with non-recurrent mutated genes accounting for 58.1%. Tumor-informed MRD panels were successfully obtained for 85.5% of patients (71/83). Among 59 patients with successful MRD panel customization and available blood samples, 13 patients exhibited positive ctDNA detection after surgery. Patients with negative post-operative ctDNA had better event-free survival (EFS) compared to those with positive ctDNA, at 1-6 months after surgery, after adjuvant chemotherapy, and more than 6 months after surgery (p < 0.05). In both univariate and multivariate Cox regression analysis, ctDNA results emerged as a significant predictor of EFS (p < 0.05). ctDNA detection preceded positive imaging in 5 patients, with an average lead time of 92.6 days. Thirty-nine patients remained disease-free, with ctDNA results consistently negative or turning negative during follow-up. Interpretation: Our study underscores the applicability of tumor-informed deep sequencing of ctDNA in osteosarcoma MRD surveillance and, to our knowledge, represents the largest cohort to date. ctDNA detection is a significant prognostic factor, enabling the early identification of tumor relapse and progression compared to standard imaging, thus offering valuable insights in guiding osteosarcoma patient management. Funding: The Grants of National Natural Science Foundation of China (No. 82072964, 82072965, 82203798, 82203026), the Natural Science Foundation of Guangdong (No. 2023A1515012659, 2023A1515010302), and the Regional Combination Project of Basic and Applied Basic Research Foundation of Guangdong (No. 2020A1515110010).

Temporal changes in the morphological and microbial diversity of biofilms on the surface of a submerged stone in the Danube River.

Epilithic biofilms are ubiquitous in large river environments and are crucial for biogeochemical processes, but their community structures and functions remain poorly understood. In this paper, the seasonal succession in the morphological structure and the taxonomic composition of an epilithic bacterial biofilm community at a polluted site of the Danube River were followed using electron microscopy, high-throughput 16S rRNA gene amplicon sequencing and multiplex/taxon-specific PCRs. The biofilm samples were collected from the same submerged stone and carried out bimonthly in the littoral zone of the Danube River, downstream of a large urban area. Scanning electron microscopy showed that the biofilm was composed of diatoms and a variety of bacteria with different morphologies. Based on amplicon sequencing, the bacterial communities were dominated by the phyla Pseudomonadota and Bacteroidota, while the most abundant archaea belonged to the phyla Nitrososphaerota and Nanoarchaeota. The changing environmental factors had an effect on the composition of the epilithic microbial community. Critical levels of faecal pollution in the water were associated with increased relative abundance of Sphaerotilus, a typical indicator of "sewage fungus", but the composition and diversity of the epilithic biofilms were also influenced by several other environmental factors such as temperature, water discharge and total suspended solids (TSS). The specific PCRs showed opportunistic pathogenic bacteria (e.g. Pseudomonas spp., Legionella spp., P. aeruginosa, L. pneumophila, Stenotrophomonas maltophilia) in some biofilm samples, but extended spectrum ß-lactamase (ESBL) genes and macrolide resistance genes could not be detected.

Targeted next-generation sequencing reveals the genetic mechanism of Chinese Marfan syndrome cohort with ocular manifestation.

BACKGROUND: Marfan syndrome (MFS) is a hereditary connective tissue disorder involving multiple systems, including ophthalmologic abnormalities. Most cases are due to heterozygous mutations in the fibrillin-1 gene (FBN1). Other associated genes include LTBP2, MYH11, MYLK, and SLC2A10. There is significant clinical overlap between MFS and other Marfan-like disorders. PURPOSE: To expand the mutation spectrum of FBN1 gene and validate the pathogenicity of Marfan-related genes in patients with MFS and ocular manifestations. METHODS: We recruited 318 participants (195 cases, 123 controls), including 59 sporadic cases and 88 families. All patients had comprehensive ophthalmic examinations showing ocular features of MFS and met Ghent criteria. Additionally, 754 cases with other eye diseases were recruited. Panel-based next-generation sequencing (NGS) screened mutations in 792 genes related to inherited eye diseases. RESULTS: We detected 181 mutations with an 84.7% detection rate in sporadic cases and 87.5% in familial cases. The overall detection rate was 86.4%, with FBN1 accounting for 74.8%. In cases without FBN1 mutations, 23 mutations from seven Marfan-related genes were identified, including four pathogenic or likely pathogenic mutations in LTBP2. The 181 mutations included 165 missenses, 10 splicings, three frameshifts, and three nonsenses. FBN1 accounted for 53.0% of mutations. The most prevalent pathogenic mutation was FBN1 c.4096G>A. Additionally, 94 novel mutations were detected, with 13 de novo mutations in 14 families. CONCLUSION: We expanded the mutation spectrum of the FBN1 gene and provided evidence for the pathogenicity of other Marfan-related genes. Variants in LTBP2 may contribute to the ocular manifestations in MFS, underscoring its role in phenotypic diversity.

Mutations in Genes Producing Nitric Oxide and Hydrogen Sulfide and Their Connection With Apoptotic Genes in Chronic Myeloid Leukemia.

Background Despite advances in chronic myeloid leukemia (CML) genetics, the role of nitric oxide (NO) and hydrogen sulfide (H2S) gene mutations and their relationship to apoptotic genes is unclear. Therefore, this study investigated NO- and H2S-producing genes' mutations and their interactions with apoptotic genes using Sanger sequencing and next-generation sequencing (NGS). Methodology A complete blood count (CBC) was carried out to measure the total number of white blood cells, while IL-6 levels were assessed in both control and CML patients using an ELISA technique. Sanger sequencing was used to analyze mutations in the CTH and NOS3 genes, whereas NGS was applied to examine mutations on all chromosomes. Results White blood cell (WBC) and granulocyte counts were significantly higher in CML patients compared to controls (p<0.0001), and monocyte counts were similarly higher (p<0.05). Interleukin-6 (IL-6) levels were significantly elevated in CML patients than controls (p<0.0001), indicating a possible link to CML etiology or progression. Multiple mutations have been identified in both genes, notably in CTH exon 12 and the NOS3 genes VNTR, T786C, and G894T. This study also measured IL-6 concentrations using IL-6 assays, identifying its potential as a CML prognostic diagnostic. WBC counts, granulocyte counts, and mid-range absolute counts, or MID counts, were significantly higher in CML patients than in normal control individuals. NGS identified 1643 somatic and sex chromosomal abnormalities and 439 actively expressed genes in CML patients. The findings imply a genomic landscape beyond the BCR-ABL1 mutation in CML development compared to other databases. Conclusion In conclusion, this study advances the understanding of the genetic characteristics of CML by identifying mutations in the NO- and H2S-producing genes and their complex connections with genes involved in apoptosis. The comprehensive genetic profile obtained by Sanger sequencing and NGS provides possibilities for identifying novel targets for therapy and personalized treatments for CML, therefore contributing to developments in hematological diseases.

The novel HLA-A allele, HLA-A*03:478, identified in a Korean individual.

One nucleotide substitution in codon 320 of A*03:01:01:01 results in the novel allele, HLA-A*03:478.

Personalized Medicine in Pancreatic Cancer: The Promise of Biomarkers and Molecular Targeting with Dr. Michael J. Pishvaian.

Pancreatic cancer, with its alarming rising incidence, is predicted to become the second deadliest type of solid tumor by 2040, highlighting the urgent need for improved diagnostic and treatment strategies. Despite medical advancements, the five-year survival rate for pancreatic cancer remains about 14%, dropping further when metastasized. This review explores the promise of biomarkers for early detection, personalized treatment, and disease monitoring. Molecular classification of pancreatic cancer into subtypes based on genetic mutations, gene expression, and protein markers guides treatment decisions, potentially improving outcomes. A plethora of clinical trials investigating different strategies are currently ongoing. Targeted therapies, among which those against CLAUDIN 18.2 and inhibitors of Claudin 18.1, have shown promise. Next-generation sequencing (NGS) has emerged as a powerful tool for the comprehensive genomic analysis of pancreatic tumors, revealing unique genetic alterations that drive cancer progression. This allows oncologists to tailor therapies to target specific molecular abnormalities. However, challenges remain, including limited awareness and uptake of biomarker-guided therapies. Continued research into the molecular mechanisms of pancreatic cancer is essential for developing more effective treatments and improving patient survival rates.

Variety of genetic defects in GnRH and hypothalamic-pituitary signaling and development in normosmic patients with IHH.

Introduction: Normosmic isolated hypogonadotropic hypogonadism (nIHH) is a clinically and genetically heterogeneous disorder. Deleterious variants in over 50 genes have been implicated in the etiology of IHH, which also indicates a possible role of digenicity and oligogenicity. Both classes of genes controlling GnRH neuron migration/development and hypothalamic/pituitary signaling and development are strongly implicated in nIHH pathogenesis. The study aimed to investigate the genetic background of nIHH and further expand the genotype-phenotype correlation. Methods: A total of 67 patients with nIHH were enrolled in the study. NGS technology and a 38-gene panel were applied. Results: Causative defects regarded as at least one pathogenic/likely pathogenic (P/LP) variant were found in 23 patients (34%). For another 30 individuals, variants of unknown significance (VUS) or benign (B) were evidenced (45%). The most frequently mutated genes presenting P/LP alterations were GNRHR (n = 5), TACR3 (n = 3), and CHD7, FGFR1, NSMF, BMP4, and NROB1 (n = 2 each). Monogenic variants with solid clinical significance (P/LP) were observed in 15% of subjects, whereas oligogenic defects were detected in 19% of patients. Regarding recurrence, 17 novel pathogenic variants affecting 10 genes were identified for 17 patients. The most recurrent pathogenic change was GNRHR:p.Arg139His, detected in four unrelated subjects. Another interesting observation is that P/LP defects were found more often in genes related to hypothalamic-pituitary pathways than those related to GnRH. Conclusions: The growing importance of the neuroendocrine pathway and related genes is drawing increasing attention to nIHH. However, the underestimated potential of VUS variants in IHH etiology, particularly those presenting recurrence, should be further elucidated.