BACKGROUND: Distinguishing paracentric inversions (PAIs) from chromosomal insertions has traditionally relied on fluorescent in situ hybridization (FISH) techniques, but recent advancements in high-throughput sequencing have enabled the use of genome sequencing for such differentiation. In this study, we present a 38-year-old male carrier of a paracentric inversion on chromosome 2q, inv (2)(q31.2q34), whose partner experienced recurrent miscarriages.
METHODS: FISH analysis confirmed the inversion, and genome sequencing was employed for detailed characterization.
RESULTS: Preimplantation genetic testing (PGT) revealed that all assessed embryos were balanced, consistent with the low risk of unbalanced offspring associated with PAIs. While PAI carriers traditionally exhibit low risk of producing unbalanced offspring, exceptions exist due to crossover events within the inversion loop. Although the sample size was limited, the findings align with existing sperm study data, supporting the rare occurrence of unbalanced progeny in PAI carriers.
CONCLUSIONS: This study highlights the possibility of characterizing PAIs using genome sequencing to enable correct reproductive counseling and PGT decisions. Detailed characterization of a PAI is crucial for understanding potential outcomes and guiding PGT strategies, as accurate knowledge of the inversion size is essential for appropriate method selection in PGT. Given the very low risk of unbalanced offspring in PAI carriers, routine PGT may not be warranted but should be considered in specific cases with a history of unbalanced progeny or recurrent miscarriages. This study contributes to our understanding of PAI segregation and its implications for reproductive outcomes.

The potential of genome sequencing (GS), which allows detection of almost all types of genetic variation across nearly the entire genome of an individual, greatly expands the possibility for diagnosing genetic disorders. The opportunities provided with this single test are enticing to researchers and clinicians worldwide for human genetic research as well as clinical application. Multiple studies have highlighted the advantages of GS for genetic variant discovery, emphasizing its added value for routine clinical use. We have implemented GS as first-line genetic testing for patients with rare diseases. Here, we report on our experiences in establishing GS as a reliable diagnostic method for almost all types of genetic disorders, from validating diagnostic accuracy of sequencing pipelines to clinical implementation in routine practice.

For more than five decades, all newborns in Germany have been offered a screening examination for the early detection of congenital treatable diseases. Since its inception, about 35 million children have been screened in this way.Originally, screening exams only included early detection of phenylketonuria, which, without timely treatment, would lead to mental retardation that could no longer be corrected. The bacteriological Guthrie test allowed the detection of elevated concentrations of phenylalanine. The methods used today are the result of decades of development. They have been expanded to include tests to determine enzyme activities, immunoassays for the early detection of important hormonal disorders such as congenital hypothyroidism, and high-pressure liquid chromatography for the diagnosis of pathologic hemoglobins. The very sophisticated tandem mass spectrometry enables the simultaneous detection of amino acid and fatty acid compounds. Steroids can also be identified. The specificity can be further increased by combining tandem mass spectrometry with chromatographic pre-separation. In recent years, chemical-analytical analyses have been supplemented by genetic diagnostic methods such as quantitative or qualitative polymerase chain reaction (PCR).The current state of laboratory technology is by no means final. Both classical analytics and especially genetic methods are facing further rapid development. Although the expansion of screening is also a consequence of technical development, the inclusion of further congenital diseases is fundamentally dependent on the given therapy. But it is precisely here that many innovations are currently being investigated. Gene therapy is at the forefront of interest.
UNASSIGNED: Seit mehr als 5 Jahrzehnten wird allen Neugeborenen in Deutschland eine Vorsorgeuntersuchung zur Früherkennung angeborener behandelbarer Krankheiten angeboten. Seit Beginn sind so etwa 35 Mio. Kinder untersucht worden.Anfangs ging es nur um die Früherkennung der Phenylketonurie, die ohne frühzeitige Behandlung zu nicht mehr korrigierbarer geistiger Behinderung führt. Der bakteriologische Guthrie-Test erlaubte den Nachweis erhöhter Konzentrationen von Phenylalanin. Die heute eingesetzten Methoden sind das Ergebnis einer über Jahrzehnte verlaufenden Entwicklung. Hinzugekommen sind Tests zur Bestimmung von Enzymaktivitäten, Immunoassays zur Früherkennung wichtiger hormoneller Störungen wie der angeborenen Schilddrüsenunterfunktion sowie Hochdruck-Flüssigkeits-Chromatografie zur Identifizierung pathologischer Hämoglobine. Die sehr anspruchsvolle Tandem-Massenspektrometrie ermöglicht die gleichzeitige Erfassung von Aminosäuren und Derivaten organischer Säuren und Fettsäuren. Auch Steroide können damit identifiziert werden. Die Spezifität lässt sich durch Kombination mit chromatografischer Vortrennung noch erhöhen. In den letzten Jahren wurden die chemisch-analytischen Untersuchungen ergänzt durch gendiagnostische Verfahren, wie beispielsweise quantitative oder qualitative Polymerasekettenreaktion (PCR).Der Stand der Labortechnik ist keineswegs endgültig. Sowohl die klassische Analytik als auch besonders die genetischen Verfahren stehen vor einer weiteren rasanten Entwicklung. Während die Ausweitung des Screenings auch eine Folge der technischen Entwicklung ist, hängt die Einbeziehung weiterer angeborener Erkrankungen grundsätzlich von einer jeweiligen Therapie ab. Aber gerade hier werden gegenwärtig viele Neuerungen erprobt. Im Vordergrund des Interesses steht dabei die Gentherapie.

Biological material from the oral cavity is an excellent source of samples for genetic diagnostics. This is because collection is quick, easy-to-access, and non-invasive. We have set-up clinical whole genome sequence testing for patients with suspected hereditary disease. Beside the excellent quality of human DNA that can be isolated from such samples, we observed the presence of non-human DNA sequences at varying percentages. We investigated the proportion of non-human mapped reads (NHMR) sequenced from buccal swabs and saliva, the type of microbial genomes from which they were derived, and impact on molecular classification. Read sequences that did not map to the human reference genome were aligned to complete reference microbial reference sequences from the National Center for Biotechnology Information\'s (NCBI) RefSeq database using Kraken2. Out of 765 analyzed samples over 80% demonstrated more than 5% NHMRs. The majority of NHMRs were from bacterial genomes (average 69%, buccal swabs and 54% saliva), while the proportion of viruses was low, averaging 0.32% (buccal swabs) and 0.07% (saliva). We identified more than 30 different bacterial families of which Streptococcus mitis and Rothia mucilaginosa were the most common species. Importantly, the level of contamination did not impact the diagnostic yield.

Monogenic autoinflammatory diseases (AID) encompass a growing group of inborn errors of the innate immune system causing unprovoked or exaggerated systemic inflammation. Diagnosis of monogenic AID requires an accurate description of the patients\' phenotype, and the identification of highly penetrant genetic variants in single genes is pivotal. We performed whole exome sequencing (WES) of 125 pediatric patients with suspected monogenic AID in a routine genetic diagnostic setting. Datasets were analyzed in a step-wise approach to identify the most feasible diagnostic strategy. First, we analyzed a virtual gene panel including 13 genes associated with known AID and, if no genetic diagnosis was established, we then analyzed a virtual panel including 542 genes published by the International Union of Immunological Societies associated including all known inborn error of immunity (IEI). Subsequently, WES data was analyzed without pre-filtering for known AID/IEI genes. Analyzing 13 genes yielded a definite diagnosis in 16.0% (n = 20). The diagnostic yield was increased by analyzing 542 genes to 20.8% (n = 26). Importantly, expanding the analysis to WES data did not increase the diagnostic yield in our cohort, neither in single WES analysis, nor in trio-WES analysis. The study highlights that the cost- and time-saving analysis of virtual gene panels is sufficient to rapidly confirm the differential diagnosis in pediatric patients with AID. WES data or trio-WES data analysis as a first-tier diagnostic analysis in patients with suspected monogenic AID is of limited benefit.

BACKGROUND: The developmental and epileptic encephalopathies (DEEs) are the most severe group of epilepsies which co-present with developmental delay and intellectual disability (ID). DEEs usually occur in people without a family history of epilepsy and have emerged as primarily monogenic, with damaging rare mutations found in 50% of patients. Little is known about the genetic architecture of patients with DEEs in whom no pathogenic variant is identified. Polygenic risk scoring (PRS) is a method that measures a person\'s common genetic burden for a trait or condition. Here, we used PRS to test whether genetic burden for epilepsy is relevant in individuals with DEEs, and other forms of epilepsy with ID.
METHODS: Genetic data on 2,759 cases with DEEs, or epilepsy with ID presumed to have a monogenic basis, and 447,760 population-matched controls were analysed. We compared PRS for \'all epilepsy\', \'focal epilepsy\', and \'genetic generalised epilepsy\' (GGE) between cases and controls. We performed pairwise comparisons between cases stratified for identifiable rare deleterious genetic variants and controls.
RESULTS: Cases of presumed monogenic severe epilepsy had an increased PRS for \'all epilepsy\' (p<0.0001), \'focal epilepsy\' (p<0.0001), and \'GGE\' (p=0.0002) relative to controls, which explain between 0.08% and 3.3% of phenotypic variance. PRS was increased in cases both with and without an identified deleterious variant of major effect, and there was no significant difference in PRS between the two groups.
CONCLUSIONS: We provide evidence that common genetic variation contributes to the aetiology of DEEs and other forms of epilepsy with ID, even when there is a known pathogenic variant of major effect. These results provide insight into the genetic underpinnings of the severe epilepsies and warrant a shift in our understanding of the aetiology of the DEEs as complex, rather than monogenic, disorders.
BACKGROUND: Science foundation Ireland, Human Genome Research Institute; National Heart, Lung, and Blood Institute; German Research Foundation.

UNASSIGNED: The X and Y chromosomes are responsible for the determination and differentiation of the gonads, and their numerical and structural abnormalities may cause the abnormal development of secondary sex characteristics. The presence of abnormalities concerning X chromosome can also contribute to many genetically heterogeneous diseases associated with cognitive impairment and intellectual disability.
UNASSIGNED: This study shows the effect of aberrations of the maternal X chromosome on the abnormal development of the child.
UNASSIGNED: Ten women aged 26 to 40 years were consulted in genetic counselling clinic and subsequently subjected to cytogenetic and molecular tests due to abnormal psychomotor development of their children, in whom structural aberrations of the X chromosome had been detected.
UNASSIGNED: Two women were diagnosed with changes in karyotype: 46,X,der(X)t(X;Y)(p22.3;q11.2) in one and 46,X,inv(X)(p21.2q13). Five women were diagnosed with microduplications in the short arm of the X chromosome; dupXp22.31 in one, and in four women dupXp22.33. The remaining three women were diagnosed with duplication in the long arm of the X chromosome; dupXq25 in one and dupXq26.3 in two women.
UNASSIGNED: Genetic analysis of the X chromosome, based on cytogenetic and molecular methods of the highest available resolution, is extremely important in women with reproductive failure. These methods allow establishing accurately the breakpoints and rearrangements in chromosomes, and assessment of the copy number variation (CNV) can explain phenotypic variability with apparently similar aberrations. A more precise characterization of the alterations is necessary for the correct genetic diagnosis, as well as determination of the carrier status and genetic risk in family members.

Spinocerebellar ataxia (SCA) is a heterogeneous group of neurodegenerative disorders with autosomal dominant inheritance. Genetic testing for SCA leads to diagnosis, prognosis and risk assessment for patients and their family members. While advances in sequencing and computing technologies have provided researchers with a rapid expansion in the genetic test content that can be used to unravel the genetic causes that underlie diseases, the large number of variants with unknown significance (VUSes) detected represent challenges. To minimize the proportion of VUSes, follow-up studies are needed to aid in their reclassification as either (likely) pathogenic or (likely) benign variants. In this study, we addressed the challenge of prioritizing VUSes for follow-up using (a combination of) variant segregation studies, 3D protein modeling, in vitro splicing assays and functional assays. Of the 39 VUSes prioritized for further analysis, 13 were eligible for follow up. We were able to reclassify 4 of these VUSes to LP, increasing the molecular diagnostic yield by 1.1%. Reclassification of VUSes remains difficult due to limited possibilities for performing variant segregation studies in the classification process and the limited availability of routine functional tests.

Lack of functional evidence hampers variant interpretation, leaving a large proportion of individuals with a suspected Mendelian disorder without genetic diagnosis after whole genome or whole exome sequencing (WES). Research studies advocate to further sequence transcriptomes to directly and systematically probe gene expression defects. However, collection of additional biopsies and establishment of lab workflows, analytical pipelines, and defined concepts in clinical interpretation of aberrant gene expression are still needed for adopting RNA sequencing (RNA-seq) in routine diagnostics.
We implemented an automated RNA-seq protocol and a computational workflow with which we analyzed skin fibroblasts of 303 individuals with a suspected mitochondrial disease that previously underwent WES. We also assessed through simulations how aberrant expression and mono-allelic expression tests depend on RNA-seq coverage.
We detected on average 12,500 genes per sample including around 60% of all disease genes-a coverage substantially higher than with whole blood, supporting the use of skin biopsies. We prioritized genes demonstrating aberrant expression, aberrant splicing, or mono-allelic expression. The pipeline required less than 1 week from sample preparation to result reporting and provided a median of eight disease-associated genes per patient for inspection. A genetic diagnosis was established for 16% of the 205 WES-inconclusive cases. Detection of aberrant expression was a major contributor to diagnosis including instances of 50% reduction, which, together with mono-allelic expression, allowed for the diagnosis of dominant disorders caused by haploinsufficiency. Moreover, calling aberrant splicing and variants from RNA-seq data enabled detecting and validating splice-disrupting variants, of which the majority fell outside WES-covered regions.
Together, these results show that streamlined experimental and computational processes can accelerate the implementation of RNA-seq in routine diagnostics.

Skeletal dysplasia is typically diagnosed using a combination of radiographic imaging, clinical examinations, and molecular testing. Identifying a molecular diagnosis for an individual with a skeletal dysplasia can lead to improved clinical care, guide future medical management and treatment, and inform assessment of risk for familial recurrence. The molecular diagnostic utility of multi-gene panel testing using next-generation sequencing (NGS) has not yet been characterized for an unselected population of individuals with suspected skeletal dysplasia. In this study, we retrospectively reviewed patient reports to assess the diagnostic yield, reported variant characteristics, impact of copy number variation, and performance in prenatal diagnostics of panel tests for variants in genes associated with skeletal dysplasia and growth disorders.
Clinical reports of consecutive patients with a clinical indication of suspected skeletal dysplasia who underwent panel testing were examined. The 543 patients included in the study submitted samples for diagnostic genetic testing with an indication of suspected skeletal dysplasia or growth disorder and received one of three nested panel tests. A molecular diagnosis was established in 42.0% of patients (n = 228/543). Diagnostic variants were identified in 71 genes, nearly half of which (n = 35, 49.3%) contributed uniquely to a molecular diagnosis for a single patient in this cohort. Diagnostic yield was significantly higher among fetal samples (58.0%, n = 51/88) than postnatal samples (38.9%, n = 177/455; z = 3.32, p < 0.0009). Diagnostic variants in fetal cases were identified across 18 genes. Thirteen diagnostic CNVs were reported, representing 5.7% of diagnostic findings and ranging in size from 241-bp to whole chromosome aneuploidy. Additionally, 11.4% (36/315) of non-diagnostic patient reports had suspicious variants of unknown significance (VUS), in which additional family studies that provide segregation data and/or functional characterization may result in reclassification to likely pathogenic.
These findings demonstrate the utility of panel testing for individuals with a suspected skeletal dysplasia or growth disorder, with a particularly high diagnostic yield seen in prenatal cases. Pursuing comprehensive panel testing with high-resolution CNV analysis can provide a diagnostic benefit, given the considerable phenotype overlap amongst skeletal dysplasia conditions.