Genetic Testing Research Articles

Incidence of hip problems in developmental central hypotonia: A scoping review.

To describe what is known about hip problems in individuals with developmental central hypotonia. Searches were conducted in five databases to October 2023. Down syndrome was excluded from this analysis of less well-known genetic diagnoses. At least two reviewers independently screened titles, abstracts, read full-text articles, and extracted data. Of 89 full-text articles, 79 met inclusion criteria. Studies included 544 individuals aged 1 month to 63 years with Kabuki, 49, XXXXY, Prader-Willi, PURA, Koolen de Vries, Emanuel, TRPM3, Wolf-Hirschhorn, and other rare syndromes. Most diagnoses may be associated with a combination of differences in hip structure or stability that are evident at birth, or develop in early infancy, with increasing hip dysplasia and subluxation over time. Joint or ligamentous laxity was most reported along with hypotonia and hypermobility as risk factors. Limited data were identified about conservative or surgical intervention and outcomes in these populations. Children with significant hypotonia, with or without a confirmed genetic diagnosis, are at increased risk of hip problems that may be missed with standard neonatal screening. Ultrasound is recommended between 6 weeks and 6 months, and annual orthopaedic review with regular radiographs for older children and adults with significant and persistent hypotonia.

Association of HPA Antigens with Immune Thrombocytopenia: A Case-Control Study by PCR-SSP Method

Background: Human platelet antigens (HPAs) play a clinically significant role in alloimmunization and the development of immune-mediated disorders such as immune thrombocytopenia (ITP), fetal and neonatal alloimmune thrombocytopenia (FNAIT), and post-transfusion purpura (PTP). Understanding the genetic profiles of HPAs is critical for preventing and treating these conditions. Given the limitations of serological methods in determining HPA genotypes, this study aims to investigate the association between the genotypes of HPA1, HPA2, HPA3, HPA4, and HPA15 antigens and autoimmune thrombocytopenia in Lorestan Province, utilizing the PCR-SSP method. Materials and Methods: This case-control study involved 80 individuals diagnosed with ITP and 120 healthy controls. DNA samples were extracted using a commercial DNA extraction kit, with concentrations quantified via a Nanodrop spectrophotometer. Genotyping was performed using the PCR-SSP method with specific primers for each HPA gene. The genotype data were verified using previously established sample sets. The frequencies of each HPA genotype were recorded, and a comparative analysis was conducted between the patient and control groups to evaluate the study hypothesis. Results: The results revealed that individuals carrying the HPA2b allele had a 5.31-fold increased risk of developing ITP, a statistically significant finding (P < 0.05, OR = 5.31). Similarly, the presence of the HPA15b allele was associated with a 6.54-fold increased risk (P < 0.05, OR = 6.54). Conclusion: These findings, in conjunction with previous studies, suggest the need for larger-scale investigations across different populations. Such research could aid in the early diagnosis and prediction of thrombocytopenia severity, inform treatment strategies, and facilitate the removal of pathogenic antibodies from circulation.

Expanding the Role of Molecular Genetic Expertise in Crime Detection and Investigation

Molecular genetic testing using DNA plays a crucial role in the field of justice. Beyond the analysis of genomic DNA sequences, advanced methods such as mitochondrial DNA analysis, epigenetic marker identification, and RNA profiling are being developed. A particularly promising approach is forensic phenotyping, which involves predicting human characteristics from DNA samples, including appearance, biogeographic origin, and age.

Lynch syndrome diagnostic testing pathways in endometrial cancers: a nationwide English registry-based study

BackgroundFor female patients with Lynch syndrome (LS), endometrial cancer (EC) is often their first cancer diagnosis. A testing pathway of somatic tumour testing triage followed by germline mismatch repair (MMR)...

Methodological approaches in 16S sequencing of female reproductive tract in fertility patients: a review.

The female genital tract microbiome has become a particular area of interest in improving assisted reproductive technology (ART) outcomes with the emergence of next-generation sequencing (NGS) technology. However, NGS assessment of microbiomes currently lacks uniformity and poses significant challenges for accurate and precise bacterial population representation. As multiple NGS platforms and assays have been developed in recent years for microbiome investigation-including the advent of long-read sequencing technologies-this work aimed to identify current trends and practices undertaken in female genital tract microbiome investigations. Areas like sample collection and transport, DNA extraction, 16S amplification vs. metagenomics, NGS library preparation, and bioinformatic analysis demonstrated a detrimental lack of uniformity. The lack of uniformity present is a significant limitation characterised by gap discrepancies in generation and interpretation of results. Minimal consistency was observed in primer design, DNA extraction techniques, sample transport, and bioinformatic analyses. With third-generation sequencing technology highlighted as a promising tool in microbiota-based research via full-length 16S rRNA sequencing, there is a desperate need for future studies to investigate and optimise methodological approaches of the genital tract microbiome to ensure better uniformity of methods and results interpretation to improve clinical impact.

RNU4-2-Related Neurodevelopmental Disorder Is Associated With a Recognisable Facial Gestalt.

De novo heterozygous variants in RNU4-2, a component of the major spliceosome, were recently found to cause a novel neurodevelopmental disorder. Preliminary evidence suggests that this newly discovered syndrome is one of the most common monogenic causes of neurodevelopmental disorders. It is characterised by developmental delay and intellectual disability, microcephaly, short stature and hypotonia. However, much remains to be elucidated regarding the phenotype of the affected individuals. We report on four novel individuals affected by the condition, two of which were identified following targeted sequencing based solely on the facial features that were similar to those of the first patient we identified. This strongly suggests that this syndrome entails a recognisable morphological phenotype, which is particularly relevant for resource-limited regions where whole genome sequencing is not readily available, and in view of retro-active selection/prioritisation of individuals with hitherto negative genetic testing.

A method for determining potential parental contamination: linkage disequilibrium-based log-likelihood ratio analysis for IVF-PGT

BackgroundAt present, embryologists are attempting to use conventional in vitro fertilization (cIVF) as an alternative to intracytoplasmic sperm injection (ICSI) for preimplantation genetic testing (PGT). However, the potential parental contamination origin of sperm cells and cumulus cells is considered the main limiting factor in the inability of cIVF embryos to undergo PGT.MethodsIn this study, we established an IVF-PGTA assay for parental contamination tests with a contamination prediction model based on allele frequencies and linkage disequilibrium (LD) to compute the log-likelihood ratio (LLR) under competing ploidy hypotheses, and then verified its sensitivity and accuracy. Finally, comparisons of the effectiveness of SNP-based analysis and LLR-based IVF-PGTA among 40 cIVF embryos was performed, based on both statistical analysis of the parental contamination rate and chromosomal ploidy concordance rate between TE biopsy and ICM isolations.ResultsWith IVF-PGTA assay, biopsies with 10% maternal contamination could be detected accurately, and contamination caused by sperm cells could be eliminated completely. Utilizing LLR-based or single Nucleotide Polymorphism (SNP) -based analyses, our comprehensive examination of 40 clinically discarded fresh cIVF embryos revealed an absence of paternal contamination. Strikingly, the LLR-based analysis uniquely revealed a mere instance of 24% maternal contamination within the trophectoderm cell (TE) biopsy of 5* embryo. Furthermore, it was solely through this analysis that embryo (9-F) was identified as a triploid of paternal origin.ConclusionsIn this study, we developed a new bioinformatics analysis method for identifying parental contamination during IVF-PGT, especially for couples with nonmale factor infertility.

Genetic Panel Reveals Coexisting Neuromuscular Disorders in Patients With Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy is a genetically based neuromuscular disorder characterized by progressive physical impairment and cardiomyopathy in children, leading to fatal cardiac or respiratory failure. Duchenne muscular dystrophy shares some overlapping clinical features with other disorders, complicating clinical differentiation. We hypothesized that some Duchenne muscular dystrophy patients may have a secondary neuromuscular disorders that could negatively skew data during pharmaceutical clinical trials and lead to incomplete treatment plans. Consecutive genetic panels on 353 patients were reviewed. Thirty-two (32; 9.1%) patients with Duchenne muscular dystrophy were identified. Three (3; 9.4%) were found to have at least 1 genetically confirmed secondary neuromuscular disorder. Overlooking these coexisting disorders could lead to unexpected treatment failures, potentially affecting medication efficacy in trials or commercial use. Secondary neuromuscular disorders should be considered in Duchenne muscular dystrophy patients before clinical trial enrollment or treatment planning, with expanded genetic testing, such as whole exome sequencing or whole genome sequencing, likely to reveal even more secondary disorders.

Clinical features of KCNB1 gene variation related developmental and epileptic encephalopathy

Objective: To summarize the clinical features of epilepsy and (or) developmental delay associated with KCNB1 gene variants in children. Methods: A case series study was conducted on 24 children with KCNB1 gene variants associated with epilepsy and (or) developmental delay who were treated at the Children's Medical Center of Peking University First Hospital and the Department of Neurology of Shenzhen Children's Hospital from July 2015 to June 2024. The manifestations of seizures, electroencephalogram (EEG) and genetic test results of those children were analyzed. Results: All the KCNB1 gene variants were de novo, involving 20 different variation, including 15 missense variations, 3 frameshift variations and 2 nonsense variations. There were 7 novel variations. Among the 24 developmental and epileptic encephalopathy children, there were 14 boys and 10 girls. The last follow-up age ranged from 9 months to 13 years and 9 months. Seizures were present in 21 children (88%), with onset ranging from 1 month to 7 years, and 76% (16/21) began before 2 years of age. The seizure types included focal seizures in 15 children (71%), epileptic spasms, myoclonic seizures, and generalized tonic-clonic seizures in 6 children respectively, atypical absence seizures in 4 children, and myoclonic atonic seizures in 1 child. Seventeen children (81%) had a cluster of seizures and 5 had a history of focal status epilepticus with impaired consciousness. All 24 children had varying degrees of developmental delay, with 3 presenting solely developmental delay. EEG abnormalities were present in all the 21 children with seizures, including focal or multifocal discharges in 20 children, generalized discharges in 10 children, hypsarrhythmia in 2 children, and electrical status epilepticus during sleep in 3 children. Magnetic resonance imaging abnormalities were found in 5 of the 24 children. Among the 21 children with seizures, 57% (12/21) achieved seizure control. Conclusions: KCNB1 gene variants are predominantly de novo missense variation. Most affected children present with epilepsy, though some may exhibit only developmental delay. Epilepsy often begins before 2 years of age, with focal seizures being the most common type. About 80% of patients experience clustered seizures. Although most patients achieve seizure control, they still exhibit varying degrees of developmental delay, consistent with developmental epileptic encephalopathy.

Comparative Analysis of Fluorescence In Situ Hybridization and Next-Generation Sequencing in Sperm Evaluation: Implications for Preimplantation Genetic Testing and Male Infertility

Pre-implantation genetic testing (PGT) is a crucial process for selecting embryos created through assisted reproductive technology (ART). Couples with chromosomal rearrangements, infertility, recurrent miscarriages, advanced maternal age, known single-gene disorders, a family history of genetic conditions, previously affected pregnancies, poor embryo quality, or congenital anomalies may be candidates for PGT. Preimplantation genetic testing for aneuploidies (PGT-A) enables the selection and transfer of euploid embryos, significantly enhancing implantation rates in assisted reproduction. Fluorescence in situ hybridization (FISH) is the preferred method for analyzing biopsied cells to identify these abnormalities. While FISH is a well-established method for identifying sperm aneuploidy, NGS offers a more comprehensive assessment of genetic material, potentially enhancing our understanding of male infertility. Chromosomal abnormalities, arising during meiosis, can lead to aneuploid sperm, which may hinder embryo implantation and increase miscarriage rates. This review provides a comparative analysis of fluorescence in situ hybridization (FISH) and next-generation sequencing (NGS) in sperm evaluations, focusing on their implications for preimplantation genetic testing. This analysis explores the strengths and limitations of FISH and NGS, aiming to elucidate their roles in improving ART outcomes and reducing the risk of genetic disorders in offspring. Ultimately, the findings will inform best practices in sperm evaluations and preimplantation genetic testing strategies.

A Frameshift Variant in ANKRD24 Implicates Its Role in Human Non-Syndromic Hearing Loss.

Hearing loss (HL) is the most prevalent sensorineural disorders, affecting about one in 1000 newborns. Over half of the cases are attributed to genetic factors; however, due to the extensive clinical and genetic heterogeneity, many cases remain without a conclusive genetic diagnosis. The advent of next-generation sequencing methodologies in recent years has greatly helped unravel the genetic etiology of HL by identifying numerous genes and causative variants. Despite this, much remains to be uncovered about the genetic basis of sensorineural hearing loss (SNHL). Here, we report an Iranian consanguineous family with postlingual, moderate-to-severe autosomal recessive SNHL. After first excluding plausible variants in known deafness-causing genes using whole exome sequencing, we reanalyzed the data, using a gene/variant prioritization pipeline established for novel gene discovery for HL. This approach identified a novel homozygous frameshift variant c.1934_1937del; (p.Thr645Lysfs*52) in ANKRD24, which segregated with the HL phenotype in the family. Recently, ANKRD24 has been shown to be a pivotal constituent of the stereocilia rootlet in cochlea hair cells and interacts with TRIOBP, a protein already implicated in human deafness. Our data implicate for the first time, ANKRD24 in human nonsyndromic HL (NSHL) and expands the genetic spectrum of HL.

Higher oxygen content and transport characterize high-altitude ethnic Tibetan women with the highest lifetime reproductive success

We chose the "natural laboratory" provided by high-altitude native ethnic Tibetan women who had completed childbearing to examine the hypothesis that multiple oxygen delivery traits were associated with lifetime reproductive success and had genomic associations. Four hundred seventeen (417) women aged 46 to 86 y residing at ≥3,500 m in Upper Mustang, Nepal, provided information on reproductive histories, sociocultural factors, physiological measurements, and DNA samples for this observational cohort study. Simultaneously assessing multiple traits identified combinations associated with lifetime reproductive success measured as the number of livebirths. Women with the most livebirths had distinctive hematological and cardiovascular traits. A hemoglobin concentration near the sample mode and a high percent of oxygen saturation of hemoglobin raised arterial oxygen concentration without risking elevated blood viscosity. We propose ongoing stabilizing selection on hemoglobin concentration because extreme values predicted fewer livebirths and directional selection favoring higher oxygen saturation because higher values had more predicted livebirths. EPAS1, an oxygen homeostasis locus with strong signals of positive natural selection and a high frequency of variants occurring only among populations indigenous to the Tibetan Plateau, associated with hemoglobin concentration. High blood flow into the lungs, wide left ventricles, and low hypoxic heart rate responses aided effective convective oxygen transport to tissues. Women with physiologies closer to unstressed, low altitude values had the highest lifetime reproductive success. This example of ethnic Tibetan women residing at high altitudes in Nepal links reproductive fitness with trait combinations increasing oxygen delivery under severe hypoxic stress and demonstrates ongoing natural selection.

Association of selected gene variants with nonsyndromic orofacial clefts in Kuwait

Introduction and objectivesNon-syndromic orofacial clefts (NSOFCs) are complex congenital abnormalities involving both environmental and genetic factors involved in orofacial development. This study aimed to investigate the genetic association of specific genetic variants at different CYRIA gene loci with the development of NSOFCs in Kuwait. MethodsFour genetic variants (rs7552, rs3758249, rs3821949, and rs3917201) at four selected gene loci (CYRIA, FOXE1, MSX1, and TGFB3) were genotyped in a total of 240 DNA samples (patients (n = 114) and random controls (n = 126)) employing TaqMan® allele discrimination assay. For each variant and its genotype, the frequencies were determined and tested for Hardy-Weinberg Equilibrium. Genotype frequencies was compared between patients and controls using Pearson’s test. Logistic regression analyses were employed to test for the associations of the four selected variants with the occurrence of NSOFCSs. ResultsSignificant differences in the distribution of genotypes between cases and controls, rs7552, rs3821949, and rs3917201 were found to have a positive association with NSOFCs. After adjusting for gender, the GG genotype of the rs7552 variant, the AG genotype of the rs3821949 variant, and the CC genotype of the rs3917201 variant showed nearly a two-fold increased risk of NSOFC (p < 0.05). ConclusionThis study reports significant findings on the contribution and modest effect of CYRIA rs7552, MSX1 rs3821949, and TGFB3 rs3917201 in the development of NSOFCs. Our findings provide further evidence on the molecular mechanism and the role of the selected genes in NSOFCs.

Applying nanopore sequencing technology in Paracoccidioides sp.: a high-quality DNA isolation method for next-generation genomic studies.

Paracoccidioidomycosis is a severe systemic endemic mycosis caused by Paracoccidioides spp. which mainly affects individuals in Latin America. Progress in Paracoccidioides genomics has been slow, as evidenced by the incomplete reference databases available. Next-generation sequencing is a valuable tool for epidemiological surveillance and genomic characterization. With the ability to sequence long reads without the need for prior amplification, Oxford Nanopore Technology (ONT) offers several advantages, but high-quality and high-quantity DNA samples are required to achieve satisfactory results. Due to the low concentration of Paracoccidioides DNA in clinical samples and inefficient culture isolation methods, DNA extraction can be a significant barrier to genomic studies of this genus. This study proposes a method to obtain a high-coverage de novo genome assembly for Paracoccidioides using an improved DNA extraction method suitable for sequencing with ONT. The assembly obtained was comparable in size to those constructed from available data from Illumina technology. To our knowledge, this is the first genome assembly of Paracoccidioides sp. of such a large size constructed using ONT.

Indigenous Australians, genomics, and the law

This article utilises three key characteristics of human genetic information—namely, its ubiquitous nature, its familial relationships and its predictive capacity—to explore how such research continues to exacerbate existing health inequalities for Indigenous Australians. By utilising substantive equality as its theoretical framework, this article explores key regulatory issues as follows: first, and regarding genetic information’s ubiquity, the article discusses how property laws in Australia fail to adequately protect Indigenous Australians from unauthorised usage of historical DNA samples; second, the familial nature of genetic information can leave Indigenous Australian’s liable to hyper-precise genetic definitions of Aboriginality, leading to a potentially adverse impact on claims of Native Title, due to the application of a biological definition to something which has always been more contextual and flexible; finally, the predictive element of genetic information is explored, and it is suggested that without proper regulation, could lead to increased genetic discrimination.

A novel variant BCL11B mutation in a pediatric patient with difficult‐to‐treat eosinophilic esophagitis

AbstractEosinophilic esophagitis (EoE) is an immunoinflammatory disease of the esophagus attributable to a complex interaction between genetic and environmental factors. While several genetic risk variants have been linked with EoE, we report a novel association between B‐cell lymphoma/leukemia 11B genetic mutation in a child with dysmorphic facies, developmental delays, atopic comorbidities, and difficult‐to‐treat EoE. After a prolonged course of EoE and multiple esophagogastroduodenoscopies with biopsies, this patient achieved clinical and histologic remission on a combination of swallowed topical steroids and high‐dose proton pump inhibitor (PPI) therapy. However, her EoE relapsed when we attempted to wean her off PPI, and it was finally controlled after adding PPI back to her regimen. This report underscores the importance of genetic testing in patients with unusual clinical features and difficult‐to‐treat EoE. Relevant to real‐world clinical practice, this case also raises the question of the treatment goals in children with EoE and underlying genetic mutation(s).

Delayed-onset cord1 progressive retinal atrophy in English Springer Spaniels genetically affected with the RPGRIP1 variant.

Cone-rod dystrophy (cord1) is a form of progressive retinal atrophy. It is linked to an RPGRIP1 genetic variant which is the third most common canine disease variant thus far. While the variant affects various breeds, it is highly prevalent in English Springer Spaniels (ESSs). Yet its clinical and pathological implications remain equivocal. Herein, we study the retinal phenotype in ESSs genetically affected with the RPGRIP1 variant. Over 4 years, 494 ESSs (123 affected) were enrolled. Owner-perceived vision was collected via a questionnaire. Ophthalmic examination included fundus photography. In selected ESSs, retinal function and structure were assessed using electroretinography (ERG, 148 dogs) and optical coherence tomography (OCT, 4 dogs). Ophthalmoscopic changes included peripheral hypo-reflective lesions often with distinct borders progressing centripetally culminating in generalized retinal atrophy. Cross-sectional study revealed declining photopic ERG amplitudes with age in the affected group but not in controls. OCT indicated progressive photoreceptor loss. Despite ophthalmoscopic, ERG, or OCT abnormalities, most affected dogs were not visually impaired per their owners. In a fraction of afflicted ESSs, vision/globe-threatening complications were documented including cataracts, lens luxation, and glaucoma. In ESSs, the RPGRIP1 variant is associated with insidious pathology with delayed-onset visual defects. The subtle phenotype without apparent visual deficit until the final years of life, if at all, may have caused underdiagnosis of cord1. Still, DNA testing remains informative, and ERG and OCT indicate progressive pathology. Peripheral fundus examination and photopic ERG are particularly useful for early detection and monitoring of cord1.

Association of polymorphism of NLRP3, ICAM-1, PTPN22, INS genes in childhood onset type 1 diabetes in a Pakistani population.

Type 1 diabetes (T1D) is an organ-specific autoimmune disorder characterized by the destruction of pancreatic β cells, leading to absolute insulin deficiency. The genes NLRP3, ICAM-1, PTPN22, and INS are reportedly associated with T1D in other populations. However, the genetic pattern of T1D in the Pakistani population is not clear. This study aimed to find the association of polymorphisms in the PTPN22, INS, NLRP3, and ICAM-1 genes with T1D susceptibility in the Pakistani population. This case-control study includes 100 T1D patients (3-14 years), recruited randomly from the pediatric endocrinology department of Fatima Memorial Hospital, Lahore, Pakistan and 100 age-matched healthy controls were selected from different localities of the same population. The polymorphisms in PTPN22 (rs601, rs33996649, rs2488457), INS (rs80356664), NLRP3 (rs10754558, rs35829419), and ICAM-1 (rs1799969, rs5498) genes were genotyped by Sanger sequencing. The genotypic and allelic frequencies, haplotypes, and linkage disequilibrium were computed using the genetic toolset PLINK to investigate their relationship to T1D. The results indicate that the occurrence of the GT genotype of the rs33996649 variant is significantly higher in children with T1D compared to a control group of healthy individuals (P = 0.001, OR: 2.0, 95% CI = 0.15-0.45). Furthermore, the CT genotype of rs2488457 was notably associated with T1D patients (P = 0.007, OR: 2.8, 95% CI = 0.56-0.67). The CG genotype of rs80356664 showed a slight association with T1D (P = 0.03, OR: 1.9, 95% CI = 0.35-0.59). The prevalence of the AT genotype of rs10754558 showed a strong association with T1D (P = 0.005, OR: 3.4, 95% CI = 0.45-0.69). The TG genotype of rs5498 was also strongly associated with T1D (P = 0.009, OR: 2.8, 95% CI = 0.75-0.89). The present study provides evidence that SNPs in the PTPN22, INS, NLRP3, and ICAM-1 genes are associated with the development of T1D. Further research is needed to explore their potential use in genetic screening and personalized medication.

Diagnosis of lactose intolerance: concordance between 13910-C/T genotype and lactose tolerance test in a Danish population

The association between the MCM6-13910-C/T polymorphism and lactose intolerance in individuals of European descent is well known. However, the notion that having a single versus a double allelic mutation might influence one’s phenotype has been hypothesized. This study investigated whether patients with the three genotypes C/C, C/T, T/T differed in response to a lactose tolerance test (LTT) in a Danish setting. Anonymized data on 603 individuals with results for both genetic test and LTT were investigated. Mean delta glucose values were plotted for the time points of the LTT (0, 15, 30, 45 and 60 min) for the C/C, C/T and T/T genotype, respectively. Further, the agreement between the three genotypes and the diagnostic interpretation of the LTT were examined using a cut-off of > 1.4 mmol/L rise in glucose. In subjects with the C/C genotype, mean glucose delta levels were markedly lower compared to both the C/T and T/T genotypes at all time points. Overall, a difference between mean glucose delta values among the C/T and T/T genotype could not be shown. Using a LTT cut-off of > 1.4 mmol/L, the proportions of lactose intolerant LTT results for each genotype were as follows: 58% among C/C, 5% among C/T, and 7% among T/T. In a Danish healthcare setting, the C/C genotype was on average associated with a smaller glucose response during a LTT when compared to the C/T and T/T genotypes. A marked difference in the LTT response among the C/T and T/T genotype was not observed.

Diagnostic Approach to Children with Unexplained Global Developmental Delay in Pediatric Neurology Outpatient Clinic.

Global developmental delay (GDD) is a common pediatric disorder that affects up to 3% of children. Due to the heterogeneous etiology of GDD, diagnostic procedures and algorithms are complex and diverse. The aim of our study was to investigate the diagnostic yield of genetic, metabolic, and imaging studies in establishing the etiology of unexplained GDD (UGDD). In this retrospectively observational study, we examined the medical records of all children diagnosed with UGDD at the Department of Pediatric Neurology, University Medical Centre Ljubljana, Slovenia, between January and December 2019. We evaluated the effectiveness of various genetic, metabolic, and magnetic resonance imaging (MRI) tests in identifying the underlying cause of GDD. Additionally, we assessed subgroups of patients to determine whether any of the studied tests were particularly beneficial based on their clinical symptoms. A total of 123 patients met the inclusion criteria, with a median age of 4.3 years (range, 0-16 years), of which 71 (57.7%) were males. Genetic diagnosis was established in 47.1% (58/123) of patients. Metabolic laboratory testing did not identify a metabolic disease in any of the tested participants (114/123) and MRI was critical for diagnosis in only 1/81 (1.2%) patient. Our findings strongly suggest that genetic testing surpasses MRI and metabolic testing in establishing the etiology of UGDD in a pediatric neurology outpatient setting. This information will help guide the diagnostic evaluation of these children.