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New Breakthroughs in Molecular Diagnostic Tools for Human Diseases

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A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Pathology, Diagnostics, and Therapeutics".

Deadline for manuscript submissions: 20 July 2025 | Viewed by 9135

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Special Issue Editors

Prof. Dr. Vittorio Fineschi

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Department of Anatomical, Histological, Forensic and Orthopaedic Sciences, Sapienza University of Rome, Viale Regina Elena 336, 00161 Rome, Italy
Interests: forensic pathology; sudden cardiac death; molecular mechanisms; traumatic brain injury & oxidative stress; immunohistochemistry; drugs of abuse; oxidative stress; maternal mortality; miRNA; forensic pathologies
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Dr. Aniello Maiese

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Department of Surgical Pathology, Medical, Molecular and Critical Area, Institute of Legal Medicine, University of Pisa, 56126 Pisa, Italy
Interests: forensic pathology; autopsy; histology; immunohistochemistry; miRNAs
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Major technological advances have made diagnostics much more accurate, particularly in light of genetic and proteomic developments. Despite laboratory development, the clinical autopsies and diagnoses of the cause of death remain a multidisciplinary approach requiring the use of technology and scientific knowledge with irreplaceable human input. Currently, the decisive genetic causality in many cardiovascular, neoplastic, and metabolic diseases is known. Naturally occurring cardiac deaths share complex features such as incomplete penetrance and molecularly variable expressivity; moreover, upon macro/microscopic evaluation, they are not easy to diagnose as they sometimes present nonspecific pictures. Multiple abnormalities in genes related to both potassium and cardiac sodium channels are involved in many cases of sudden cardiac death. Proteomic studies are therefore likely to provide new insights into the cellular mechanisms involved in organ dysfunction and may also provide new diagnostics for many diseases and for sudden cardiac death. The proteomic investigations of diseases have identified candidate proteins altered with pathologic states, therefore complementing the traditional biochemical observations of the past. Advanced laboratory techniques can be utilized to develop molecular diagnostic tools that calibrate technology selections against diagnostic hypotheses, and specific ancillary methods to support and confirm diagnosis can be utilized. With this in mind, this Special Issue aims to promote a debate on the contribution of genetic, epigenetic, and technological methods through the sharing of the current development status of molecular diagnostic tools. We encourage the establishment of Hospital Mortality Review Committees for a systematic proactive approach to the continuous improvement of the safety of care and the quality of care. The outlined survey includes methodologies extended to further centers with the aim of placing greater weight on the results obtained by ensuring their influence in health policy planning. In this Special Issue on “New Breakthroughs in Molecular Diagnostic Tools for Human Diseases”, we invite front-line researchers, pathologists, and investigators to submit original research and review articles regarding topics that include, but are not limited to, the following:

molecular pathology;
molecular diagnostics;
genetics;
epigenetics;
proteomics;
immunohistochemistry;
thrombo-embolic disease;
sudden cardiac death;
the molecular pathology of traumatic injuries;
hypoxic–ischemic brain damage;
the experimental models of injury and diseases;
the targeted therapy of injuries and diseases;
drug abuse;
histological diagnosis;
the epigenetics of organs injuries;
miRNA and the prognosis of diseases;
novel approaches and proofs of principle in therapy.

Prof. Dr. Vittorio Fineschi
Dr. Aniello Maiese
Guest Editors

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Keywords

molecular pathology
molecular diagnostics
genetics
epigenetics
proteomics
immunohistochemistry
thrombo-embolic disease
sudden cardiac death
molecular pathology of traumatic injuries
hypoxic–ischemic brain damage
experimental models of injury and diseases
targeted therapy of injuries and diseases
drug abuse
histological diagnosis
epigenetics of organs injuries
miRNA and prognosis of diseases
novel approaches and proofs of principle in therapy

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Published Papers (8 papers)

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Research

14 pages, 1374 KiB

Open AccessArticle

Combined First-Trimester PAPP-A and Free β-hCG Levels for the Early Diagnosis of Placenta Accreta Spectrum and Placenta Previa: A Case-Control Study

by Vera Belousova, Irina Ignatko, Irina Bogomazova, Evdokiya Zarova, Svetlana Pesegova, Anastasia Samusevich, Madina Kardanova, Oxana Skorobogatova, Tatiana Kuzmina, Natalia Kireeva and Anna Maltseva

Int. J. Mol. Sci. 2025, 26(13), 6187; https://doi.org/10.3390/ijms26136187 - 27 Jun 2025

Viewed by 178

Abstract

Placenta accreta spectrum (PAS) and placenta previa (PP) are severe obstetric disorders associated with high maternal and perinatal morbidity. Early diagnosis of both conditions remains challenging, particularly in cases with subtle imaging findings. This study was aimed to evaluate the diagnostic value of first-trimester maternal serum levels of pregnancy-associated plasma protein-A (PAPP-A) and free beta subunit of human chorionic gonadotropin (β-hCG) in predicting PAS and PP. In this retrospective case–control study, a total of 100 pregnant women were included: 36 with PAS, 32 with PP, and 32 healthy controls. Serum levels were measured at 11–13⁶ weeks of gestation. Both biomarkers were significantly altered in pathological groups compared to controls: PAPP-A was lower in PP (3.04 [1.42–4.52] IU/L) and PAS (3.63 [2.51–5.39] IU/L) vs. controls (5.34 [3.72–8.41] IU/L; p < 0.001), while β-hCG was higher in PP (45.4 [40.1–54.9] IU/L) and PAS (51.4 [32.3–74.8] IU/L) vs. controls (33.5 [22.7–54.1] IU/L; p = 0.044 and p < 0.001, respectively). ROC analysis demonstrated that combined biomarker modeling improved diagnostic accuracy over single-marker use, with AUCs reaching 0.85 (sensitivity 85.2%, specificity 72%) for PAS and 0.88 (sensitivity 100%, specificity 72%) for PP. These findings support the integration of biochemical screening into first-trimester risk assessment protocols. Incorporating maternal serum biomarkers may enhance early identification of high-risk pregnancies, allow timely referral to specialized care, and reduce adverse outcomes. Further prospective studies are warranted to validate the utility of this dual-marker approach across diverse populations and clinical settings. Full article

(This article belongs to the Special Issue New Breakthroughs in Molecular Diagnostic Tools for Human Diseases)

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16 pages, 1858 KiB

Open AccessArticle

Characterisation of the ABO Blood Group Phenotypes Using Third-Generation Sequencing

by Fredrick M. Mobegi, Samuel Bruce, Naser El-Lagta, Felipe Ayora, Benedict M. Matern, Mathijs Groeneweg, Lloyd J. D’Orsogna and Dianne De Santis

Int. J. Mol. Sci. 2025, 26(12), 5443; https://doi.org/10.3390/ijms26125443 - 6 Jun 2025

Viewed by 444

Abstract

Third-generation sequencing (TGS), also known as long-read sequencing, has become a promising tool in clinical and research laboratories because it delivers high-resolution results with unmatched throughput. Specialised immunohematology laboratories currently employ sequencing-based methods to characterise rare ABO blood group phenotypes that cannot be identified through serology and genotyping methods. However, routine clinical application of these methods remains elusive due to the absence of validated laboratory protocols and bioinformatics tools. In this study, we have developed and validated a TGS-based workflow for comprehensive determination of the clinically relevant ABO phenotypes from DNA isolated from buccal swabs or whole blood. The region spanning exons 2 to 7 of the ABO gene were amplified and sequenced on MinION 10.4.1 flow cells. Predicted ABO phenotypes were initially determined based on single-nucleotide variants at gDNA261 (rs8176719), gDNA796 (rs8176746), and gDNA803 (rs8176747). However, certain O subtypes lacked the distinguishing deletion (rs8176719) and instead exhibited variations in exon 7 at gDNA802 (rs41302905) and gDNA805, caused by gDNA804 (rs782782485), which differentiate them from A alleles sharing the same nucleotides at gDNA261, gDNA796, and gDNA803. These additional variants were added to the analysis pipeline to identify the additional subtypes. DNA sequence data were sufficient to distinguish between the four clinically relevant ABO blood group phenotypes based on five polymorphic positions. While high sequencing coverage allowed for higher resolution genetic analysis, as few as 20 reads are sufficient for determining the ABO genotype and predicted phenotype of an individual. Typing results generated by this pipeline showed remarkable concordance with both serological results and molecular typing results by an independent laboratory, indicating its accuracy and reliability. This study demonstrates a comprehensive characterisation of clinically relevant ABO blood genotypes and predicted phenotypes using TGS methods. The approach provided a scalable and precise method for routine ABO blood group screening and aided in the development of pioneering bioinformatics tools suitable for clinical and research application. Full article

(This article belongs to the Special Issue New Breakthroughs in Molecular Diagnostic Tools for Human Diseases)

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13 pages, 2603 KiB

Open AccessArticle

Metabolic Imaging of Hyperpolarized [1-¹³C]Pyruvate in a Ferret Model of Traumatic Brain Injury

by Dirk Mayer, Abubakr Eldirdiri, Amanda L. Hrdlick, Boris Piskoun, Joshua C. Rogers, Aditya Jhajharia, Minjie Zhu, Julie L. Proctor, Ulrich H. Leiste, William L. Fourney, Jody C. Cantu, Gary Fiskum and Molly J. Goodfellow

Int. J. Mol. Sci. 2025, 26(11), 5327; https://doi.org/10.3390/ijms26115327 - 1 Jun 2025

Viewed by 449

Abstract

It is increasingly recognized that early perturbation of energy metabolism might have important implications in management and ultimately the neurological outcome in patients with traumatic brain injury (TBI). At the same time, treatments and screening tools successfully developed in preclinical TBI models have failed to translate to the clinic. As ferrets possess primate-like gyrencephalic brains that may better replicate the human response to neurologic injury, the goal of this study was to noninvasively measure brain energy metabolism after injury in a ferret model of TBI. To this end, metabolic imaging of hyperpolarized (HP) [1-¹³C]pyruvate (Pyr) and its conversion to lactate (Lac) and bicarbonate (Bic) was performed in ferrets before and after combined under-vehicle blast and controlled cortical impact injury. Reduced Bic/Pyr, reflecting reduced pyruvate dehydrogenase activity, was detected 8–10 days post-injury whereas no difference in Lac/Pyr was observed. These results demonstrate the feasibility of using metabolic imaging of HP [1-¹³C]Pyr to measure perturbations in brain energy metabolism in a novel highly translatable animal model of TBI. The method may contribute to both improved understanding of injury mechanisms and more effective drug development. Full article

(This article belongs to the Special Issue New Breakthroughs in Molecular Diagnostic Tools for Human Diseases)

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11 pages, 2086 KiB

Open AccessCommunication

Evaluation of Apoptotic Caspase-3 Immunopositivity in Human Model of Asphyxial Death

by Fabio Del Duca, Michele Treglia, Raffaele La Russa, Stefania De Simone, Luigi Cipolloni, Aniello Maiese and Paola Frati

Int. J. Mol. Sci. 2025, 26(7), 3317; https://doi.org/10.3390/ijms26073317 - 2 Apr 2025

Viewed by 442

Abstract

The pathological mechanisms underlying the ligature mark in hanging involve the skin layers and an ischemic mechanism. The apoptotic process develops whenever ischemic mechanisms affect the dermal and epidermal layers. Effector caspase-3 appears to play a crucial role in both acute and chronic pressure-induced skin ischemia. The aim of this study is to identify the role of caspase-3 as a marker of supravitality in the diagnosis of premortem hanging. Skin samples from ligature marks in hanging cases were collected to investigate this apoptotic process. The caspase-3 levels in compressed skin were significantly higher compared to those found in healthy skin (p < 0.005). The apoptotic process in ischemic epidermal cells begins with stable mechanical stress, as seen in the hanging model. Caspase-3 expression seems to vary from minutes after the initial stress input. Caspase-3 activation is an ATP-dependent process and can only occur if the victim was alive before the pressure was applied. Caspase-3 is a reliable marker of supravitality in ligature marks in premortem hanging cases. Full article

(This article belongs to the Special Issue New Breakthroughs in Molecular Diagnostic Tools for Human Diseases)

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10 pages, 819 KiB

Open AccessArticle

Quantitative Analysis of Pseudogene-Associated Errors During Germline Variant Calling

by Artem Podvalnyi, Arina Kopernik, Mariia Sayganova, Mary Woroncow, Gauhar Zobkova, Anna Smirnova, Anton Esibov, Andrey Deviatkin, Pavel Volchkov and Eugene Albert

Int. J. Mol. Sci. 2025, 26(1), 363; https://doi.org/10.3390/ijms26010363 - 3 Jan 2025

Cited by 1 | Viewed by 1315

Abstract

A pseudogene is a non-functional copy of a protein-coding gene. Processed pseudogenes, which are created by the reverse transcription of mRNA and subsequent integration of the resulting cDNA into the genome, being a major pseudogene class, represent a significant challenge in genome analysis due to their high sequence similarity to the parent genes and their frequent absence in the reference genome. This homology can lead to errors in variant identification, as sequences derived from processed pseudogenes can be incorrectly assigned to parental genes, complicating correct variant calling. In this study, we quantified the occurrence of variant calling errors associated with pseudogenes, generated by the most popular germline variant callers, namely GATK-HC, DRAGEN, and DeepVariant, when analysing 30x human whole-genome sequencing data (n = 13,307). The results show that the presence of pseudogenes can interfere with variant calling, leading to false positive identifications of potentially clinically relevant variants. Compared to other approaches, DeepVariant was the most effective in correcting these errors. Full article

(This article belongs to the Special Issue New Breakthroughs in Molecular Diagnostic Tools for Human Diseases)

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17 pages, 982 KiB

Open AccessArticle

Proteomic Analysis of Follicular Fluid in Polycystic Ovary Syndrome: Insights into Protein Composition and Metabolic Pathway Alterations

by Janusz Przewocki, Adam Łukaszuk, Grzegorz Jakiel, Izabela Wocławek-Potocka, Karolina Kłosińska, Jolanta Olszewska and Krzysztof Łukaszuk

Int. J. Mol. Sci. 2024, 25(21), 11749; https://doi.org/10.3390/ijms252111749 - 1 Nov 2024

Cited by 2 | Viewed by 1909

Abstract

This study explores the proteomic composition of follicular fluid (FF) from women undergoing oocyte retrieval for in vitro fertilisation (IVF), with a focus on the effects of polycystic ovary syndrome (PCOS). FF samples were collected from 74 patients, including 34 with PCOS and 40 oocyte donors. Proteomic profiling using machine learning identified significant differences in protein abundance between the PCOS and control groups. Of the 484 quantified proteins, 20 showed significantly altered levels in the PCOS group. Functional annotation and pathway enrichment analysis pointed to the involvement of protease inhibitors and immune-related proteins in the pathophysiology of PCOS, suggesting that inflammation and immune dysregulation may play a key role. Additionally, HDL assembly was identified as a significant pathway, with apolipoprotein-AI (APOA1) and alpha-2-macroglobulin (A2M) as the major proteins involved. Notably, myosin light polypeptide 6 was the most downregulated protein, showing the highest absolute fold change, and may serve as a novel independent biomarker for PCOS. Full article

(This article belongs to the Special Issue New Breakthroughs in Molecular Diagnostic Tools for Human Diseases)

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22 pages, 2270 KiB

Open AccessArticle

Analysis of miRNA Expression Profiles in Traumatic Brain Injury (TBI) and Their Correlation with Survival and Severity of Injury

by Francesca Consalvo, Martina Padovano, Matteo Scopetti, Donato Morena, Luigi Cipolloni, Vittorio Fineschi and Alessandro Santurro

Int. J. Mol. Sci. 2024, 25(17), 9539; https://doi.org/10.3390/ijms25179539 - 2 Sep 2024

Cited by 3 | Viewed by 1682

Abstract

Traumatic brain injury (TBI) is the leading cause of traumatic death worldwide and is a public health problem associated with high mortality and morbidity rates, with a significant socioeconomic burden. The diagnosis of brain injury may be difficult in some cases or may leave diagnostic doubts, especially in mild trauma with insignificant pathological brain changes or in cases where instrumental tests are negative. Therefore, in recent years, an important area of research has been directed towards the study of new biomarkers, such as micro-RNAs (miRNAs), which can assist clinicians in the diagnosis, staging, and prognostic evaluation of TBI, as well as forensic pathologists in the assessment of TBI and in the estimation of additional relevant data, such as survival time. The aim of this study is to investigate the expression profiles (down- and upregulation) of a panel of miRNAs in subjects deceased with TBI in order to assess, verify, and define the role played by non-coding RNA molecules in the different pathophysiological mechanisms of brain damage. This study also aims to correlate the detected expression profiles with survival time, defined as the time elapsed between the traumatic event and death, and with the severity of the trauma. This study was conducted on 40 cases of subjects deceased with TBI (study group) and 10 cases of subjects deceased suddenly from non-traumatic causes (control group). The study group was stratified according to the survival time and the severity of the trauma. The selection of miRNAs to be examined was based on a thorough literature review. Analyses were performed on formalin-fixed, paraffin-embedded (FFPE) brain tissue samples, with a first step of total RNA extraction and a second step of quantification of the selected miRNAs of interest. This study showed higher expression levels in cases compared to controls for miR-16, miR-21, miR-130a, and miR-155. In contrast, lower expression levels were found in cases compared to controls for miR-23a-3p. There were no statistically significant differences in the expression levels between cases and controls for miR-19a. In cases with short survival, the expression levels of miR-16-5p and miR-21-5p were significantly higher. In cases with long survival, miR-21-5p was significantly lower. The expression levels of miR-130a were significantly higher in TBI cases with short and middle survival. In relation to TBI severity, miR-16-5p and miR-21-5p expression levels were significantly higher in the critical–fatal TBI subgroup. Conclusions: This study provides evidence for the potential of the investigated miRNAs as predictive biomarkers to discriminate between TBI cases and controls. These miRNAs could improve the postmortem diagnosis of TBI and also offer the possibility to define the survival time and the severity of the trauma. The analysis of miRNAs could become a key tool in forensic investigations, providing more precise and detailed information on the nature and extent of TBI and helping to define the circumstances of death. Full article

(This article belongs to the Special Issue New Breakthroughs in Molecular Diagnostic Tools for Human Diseases)

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16 pages, 4642 KiB

Open AccessArticle

Usefulness and Limitations of PFGE Diagnosis and Nucleotide Sequencing Method in the Analysis of Food Poisoning Pathogens Found in Cooking Employees

by Mi-Na Park, Sang-Gu Yeo, Junhyuk Park, Yoomi Jung and Se-Min Hwang

Int. J. Mol. Sci. 2024, 25(7), 4123; https://doi.org/10.3390/ijms25074123 - 8 Apr 2024

Cited by 1 | Viewed by 1959

Abstract

In the case of a food poisoning outbreak, it is essential to understand the relationship between cooking workers and food poisoning. Many biological diagnostic methods have recently been developed to detect food poisoning pathogens. Among these diagnostic tools, this study presents PCR-based pulsed-field gel electrophoresis and nucleotide sequencing diagnostic analysis results for diagnosing food poisoning outbreaks associated with cooking employees in Chungcheongnam-do, Republic of Korea. Pulsed-field gel electrophoresis was useful in identifying the food poisoning outbreaks caused by Staphylococcus aureus and Enteropathogenic Escherichia coli. In the case of Norovirus, nucleotide sequencing was used to identify the relationship between cooking workers and the food poisoning outbreak. However, it is difficult to determine whether cooking employees directly caused the food poisoning outbreaks based on these molecular biological diagnostic results alone. A system is needed to integrate epidemiological and diagnostic information to identify a direct correlation between the food poisoning outbreak and cooking employees. Full article

(This article belongs to the Special Issue New Breakthroughs in Molecular Diagnostic Tools for Human Diseases)

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