Facioscapulohumeral muscular dystrophy (FSHD) molecular diagnosis: from traditional technology to the NGS era.

Facioscapulohumeral muscular dystrophy (FSHD) is a genetic neuromuscular disorder which mainly affects the muscles of the face, shoulder, and upper arms. FSHD is generally associated with the contraction of D4Z4 macrosatellite repeats on 4q35 chromosome or mutations in SMCHD1, which are responsible of the toxic expression of DUX4 in muscle tissue. Despite the recent application of NGS techniques in the clinical practice, the molecular diagnosis of FSHD is still performed with dated techniques such as Southern blotting. The diagnosis of FSHD requires therefore specific skills on both modern and less modern analytical protocols. Considering that clinical and molecular diagnosis of FSHD is challenging, it is not surprising that only few laboratories offer a comprehensive characterization of FSHD, which requires the education of professionals on traditional techniques even in the era of NGS. In conclusion, the study of FSHD provides an excellent example of using classical and modern molecular technologies which are equally necessary for the analysis of DNA repetitive traits associated with specific disorders.

Identification of Duchenne/Becker muscular dystrophy mosaic carriers through a combined DNA/RNA analysis.

OBJECTIVE: The Duchenne/Becker muscular dystrophy (DMD) carrier screening includes the evaluation of mutations in DMD gene, and the most widely used analysis is the multiplex ligation-dependent probe amplification (MLPA) for the DMD deletions/duplications detection. The high frequency of de novo mutations permits to estimate a risk up to 20% of mosaicisms for mothers of sporadic DMD children. The purpose of this study is to evaluate alternative analytical strategy for the detection of mosaics carrier women, in order to improve the recurrence risk estimation. METHOD: Different DNA and RNA analyses were conducted on samples from a woman that conceived a DMD fetus without previous family history of dystrophynopathy. RESULTS: Standard MLPA analysis failed to identify mosaicism, even if MLPA doses suggested it. Electrophoresis and direct sequencing conducted on RNA permitted to detect two different amplicons of cDNAs, demonstrating the presence of somatic mosaicism. Subsequent detection of a second affected fetus confirmed the mosaic status on the mother. CONCLUSION: The implementation of RNA analysis in diagnostic algorithm can increase the sensitivity of carrier test for mothers of sporadic affected patients, permitting detection of mosaic status. A revision of analytical guidelines is needed in order to improve the recurrence risk estimation and support prenatal genetic counseling.

Common sequence variants in the LOXL1 gene in pigment dispersion syndrome and pigmentary glaucoma.

BACKGROUND: Single nucleotide polymorphisms (SNPs) within the LOXL1 gene are associated with pseudoesfoliation syndrome and pseudoesfoliation glaucoma. The aim of our study is to investigate a potential involvement of LOXL1 gene in the pathogenesis of pigment dispersion syndrome (PDS) and pigmentary glaucoma (PG). METHODS: A cohort of Caucasian origin of 84 unrelated and clinically well-characterised patients with PDS/PG and 200 control subjects were included in the study. Genomic DNA from whole blood was extracted and the coding and regulatory regions of LOXL1 gene were risequenced in both patients and controls to identify unknown sequence variations. Genotype and haplotype analysis were performed with UNPHASED software. The expression levels of LOXL1 were determined on c-DNA from peripheral blood lymphocytes by quantitative real-time RT-PCR. RESULTS: A significant allele association was detected for SNP rs2304722 within the fifth intron of LOXL1 (Odds ratio (OR = 2.43, p-value = 3,05e-2). Haplotype analysis revealed the existence of risk and protective haplotypes associated with PG-PDS (OR = 3.35; p-value = 1.00e-5 and OR = 3.35; p-value = 1.00e-4, respectively). Expression analysis suggests that associated haplotypes can regulate the expression level LOXL1. CONCLUSIONS: Haplotypes of LOXL1 are associated with PG-PDS independently from rs1048661, leading to a differential expression of the transcript.

Innovations in Medicine: Exploring ChatGPT's Impact on Rare Disorder Management.

Artificial intelligence (AI) is rapidly transforming the field of medicine, announcing a new era of innovation and efficiency. Among AI programs designed for general use, ChatGPT holds a prominent position, using an innovative language model developed by OpenAI. Thanks to the use of deep learning techniques, ChatGPT stands out as an exceptionally viable tool, renowned for generating human-like responses to queries. Various medical specialties, including rheumatology, oncology, psychiatry, internal medicine, and ophthalmology, have been explored for ChatGPT integration, with pilot studies and trials revealing each field's potential benefits and challenges. However, the field of genetics and genetic counseling, as well as that of rare disorders, represents an area suitable for exploration, with its complex datasets and the need for personalized patient care. In this review, we synthesize the wide range of potential applications for ChatGPT in the medical field, highlighting its benefits and limitations. We pay special attention to rare and genetic disorders, aiming to shed light on the future roles of AI-driven chatbots in healthcare. Our goal is to pave the way for a healthcare system that is more knowledgeable, efficient, and centered around patient needs.

Distribution of the C9orf72 hexanucleotide repeat expansion in healthy subjects: a multicenter study promoted by the Italian IRCCS network of neuroscience and neurorehabilitation.

Introduction: High repeat expansion (HRE) alleles in C9orf72 have been linked to both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD); ranges for intermediate allelic expansions have not been defined yet, and clinical interpretation of molecular data lacks a defined genotype-phenotype association. In this study, we provide results from a large multicenter epidemiological study reporting the distribution of C9orf72 repeats in healthy elderly from the Italian population. Methods: A total of 967 samples were collected from neurologically evaluated healthy individuals over 70 years of age in the 13 institutes participating in the RIN (IRCCS Network of Neuroscience and Neurorehabilitation) based in Italy. All samples were genotyped using the AmplideXPCR/CE C9orf72 Kit (Asuragen, Inc.), using standardized protocols that have been validated through blind proficiency testing. Results: All samples carried hexanucleotide G4C2 expansion alleles in the normal range. All samples were characterized by alleles with less than 25 repeats. In particular, 93.7% of samples showed a number of repeats ≤10, 99.9% ≤20 repeats, and 100% ≤25 repeats. Conclusion: This study describes the distribution of hexanucleotide G4C2 expansion alleles in an Italian healthy population, providing a definition of alleles associated with the neurological healthy phenotype. Moreover, this study provides an effective model of federation between institutes, highlighting the importance of sharing genomic data and standardizing analysis techniques, promoting translational research. Data derived from the study may improve genetic counseling and future studies on ALS/FTD.

Co-Inheritance of Pathogenic Variants in PKD1 and PKD2 Genes Determined by Parental Segregation and De Novo Origin: A Case Report.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary renal disease, and it is typically caused by PKD1 and PKD2 heterozygous variants. Nonetheless, the extensive phenotypic variability observed among affected individuals, even within the same family, suggests a more complex pattern of inheritance. We describe an ADPKD family in which the proband presented with an earlier and more severe renal phenotype (clinical diagnosis at the age of 14 and end-stage renal disease aged 24), compared to the other affected family members. Next-generation sequencing (NGS)-based analysis of polycystic kidney disease (PKD)-associated genes in the proband revealed the presence of a pathogenic PKD2 variant and a likely pathogenic variant in PKD1, according to the American College of Medical Genetics and Genomics (ACMG) criteria. The PKD2 nonsense p.Arg872Ter variant was segregated from the proband's father, with a mild phenotype. A similar mild disease presentation was found in the proband's aunts and uncle (the father's siblings). The frameshift p.Asp3832ProfsTer128 novel variant within PKD1 carried by the proband in addition to the pathogenic PKD2 variant was not found in either parent. This report highlights that the co-inheritance of two or more PKD genes or alleles may explain the extensive phenotypic variability among affected family members, thus emphasizing the importance of NGS-based techniques in the definition of the prognostic course.

A Splicing Variant in RDH8 Is Associated with Autosomal Recessive Stargardt Macular Dystrophy.

Stargardt macular dystrophy is a genetic disorder, but in many cases, the causative gene remains unrevealed. Through a combined approach (whole-exome sequencing and phenotype/family-driven filtering algorithm) and a multilevel validation (international database searching, prediction scores calculation, splicing analysis assay, segregation analyses), a biallelic mutation in the RDH8 gene was identified to be responsible for Stargardt macular dystrophy in a consanguineous Italian family. This paper is a report on the first family in which a biallelic deleterious mutation in RDH8 is detected. The disease phenotype is consistent with the expected phenotype hypothesized in previous studies on murine models. The application of the combined approach to genetic data and the multilevel validation allowed the identification of a splicing mutation in a gene that has never been reported before in human disorders.

C9orf72-Related Neurodegenerative Diseases: From Clinical Diagnosis to Therapeutic Strategies.

Hexanucleotide expansion in C9orf72 has been related to several phenotypes to date, complicating the clinical recognition of these neurodegenerative disorders. An early diagnosis can improve the management of patients, promoting early administration of therapeutic supportive strategies. Here, we report known clinical presentations of C9orf72-related neurodegenerative disorders, pointing out suggestive phenotypes that can benefit the genetic characterization of patients. Considering the high variability of C9orf72-related disorder, frequent and rare manifestations are described, with detailed clinical, instrumental evaluation, and supportive therapeutical approaches. Furthermore, to improve the understanding of molecular pathways of the disease and potential therapeutical targets, a detailed description of the cellular mechanisms related to the pathological effect of C9orf72 is reported. New promising therapeutical strategies and ongoing studies are reported highlighting their molecular role in cellular pathological pathways of C9orf72. These therapeutic approaches are particularly promising because they seem to stop the disease before neuronal damage. The knowledge of clinical and molecular features of C9orf72-related neurodegenerative disorders improves the therapeutical application of known strategies and will lay the basis for the development of new potential therapies.

Tracking the Initial Diffusion of SARS-CoV-2 Omicron Variant in Italy by RT-PCR and Comparison with Alpha and Delta Variants Spreading.

The emergence of the Omicron SARS-CoV-2 variant caused public health concerns worldwide, raising the need for the improvement of rapid monitoring strategies. The present manuscript aimed at providing evidence of the utility of a diagnostic kit for the routine testing of SARS-CoV-2 infection as a cost-effective method for tracking the Omicron variant in Italy. The study was conducted on patients' naso-oropharyngeal-swab-derived RNA samples. These samples were subjected to RT-PCR using the TaqPath COVID-19 RT PCR CE IVD kit. Nonparametric testing and polynomial models fitting were used to compare the spreading of Alpha, Delta and Omicron variants. The samples of interest were correctly amplified and displayed the presence of S gene-target failure, suggesting that these patients carry the Omicron variant. The trend of diffusion was found to be significantly different and more rapid compared with that of the Alpha and Delta variants in our cohorts. Overall, these results highlight that the S gene target failure was a very useful tool for the immediate and inexpensive tracking of Omicron variant in the three weeks from the first detection. Thus, our approach could be used as a first-line screening to reduce the time and costs of monitoring strategies, facilitating the management of preventive and counteracting measures against COVID-19.

Analysis of Genetic Variants Associated with COVID-19 Outcome Highlights Different Distributions among Populations.

The clinical spectrum of SARS-CoV-2 infection ranges from asymptomatic status to mild infections, to severe disease and death. In this context, the identification of specific susceptibility factors is crucial to detect people at the higher risk of severe disease and improve the outcome of COVID-19 treatment. Several studies identified genetic variants conferring higher risk of SARS-CoV-2 infection and COVID-19 severity. The present study explored their genetic distribution among different populations (AFR, EAS, EUR and SAS). As a result, the obtained data support the existence of a genetic basis for the observed variability among populations, in terms of SARS-CoV-2 infection and disease outcomes. The comparison of ORs distribution for genetic risk of infection as well as for disease outcome shows that each population presents its own characteristics. These data suggest that each country could benefit from a population-wide risk assessment, aimed to personalize the national vaccine programs and the preventative measures as well as the allocation of resources and the access to proper therapeutic interventions. Moreover, the host genetics should be further investigated in order to realize personalized medicine protocols tailored to improve the management of patients suffering from COVID-19.