Male infertility affects approximately 17% of all men and represents a complex disorder in which not only semen parameters such as sperm motility, morphology, and number of sperm are highly variable, but also testicular phenotypes range from normal spermatogenesis to complete absence of germ cells. Genetic factors significantly contribute to the disease but chromosomal aberrations, mostly Klinefelter syndrome, and microdeletions of the Y-chromosome have remained the only diagnostically and clinically considered genetic causes. Monogenic causes remain understudied and, thus, often unidentified, leaving the majority of the male factor couple infertility pathomechanistically unexplained. This has been changing mostly because of the introduction of exome sequencing that allows the analysis of multiple genes in large patient cohorts. As a result, pathogenic variants in single genes have been associated with non-syndromic forms of all aetiologic sub-categories in the last decade. This review highlights the contribution of exome sequencing to the identification of novel disease genes for isolated (non-syndromic) male infertility by presenting the results of a comprehensive literature search. Both, reduced sperm count in azoospermic and oligozoospermic patients, and impaired sperm motility and/or morphology, in asthenozoospermic and/or teratozoospermic patients are highly heterogeneous diseases with well over 100 different candidate genes described for each entity. Applying the standardized evaluation criteria of the ClinGen gene curation working group, 70 genes with at least moderate evidence to contribute to the disease are highlighted. The implementation of these valid disease genes in clinical exome sequencing is important to increase the diagnostic yield in male infertility and, thus, improve clinical decision-making and appropriate genetic counseling. Future advances in androgenetics will continue to depend on large-scale exome and genome sequencing studies of comprehensive international patient cohorts, which are the most promising approaches to identify additional disease genes and provide reliable data on the gene-disease relationship.

The CSF1R gene, located on chromosome 5, encodes a 108 kDa protein and plays a critical role in regulating myeloid cell function. Mutations in CSF1R have been identified as a cause of a rare white matter disease called adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP, also known as CSF1R-related leukoencephalopathy), characterized by progressive neurological dysfunction. This study aimed to broaden the genetic basis of ALSP by identifying novel CSF1R variants in patients with characteristic clinical and imaging features of ALSP. Genetic analysis was performed through whole-exome sequencing or panel analysis for leukodystrophy genes. Variant annotation and classification were conducted using computational tools, and the identified variants were categorized following the recommendations of the American College of Medical Genetics and Genomics (ACMG). To assess the evolutionary conservation of the novel variants within the CSF1R protein, amino acid sequences were compared across different species. The study identified six previously unreported CSF1R variants (c.2384G>T, c.2133_2919del, c.1837G>A, c.2304C>A, c.2517G>T, c.2642C>T) in seven patients with ALSP, contributing to the expanding knowledge of the genetic diversity underlying this rare disease. The analysis revealed considerable genetic and clinical heterogeneity among these patients. The findings emphasize the need for a comprehensive understanding of the genetic basis of rare diseases like ALSP and underscored the importance of genetic testing, even in cases with no family history of the disease. The study\'s contribution to the growing spectrum of ALSP genetics and phenotypes enhances our knowledge of this condition, which can be crucial for both diagnosis and potential future treatments.

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.

Molecular genetics enables more precise diagnoses of skeletal dysplasia and other skeletal disorders (SDs). We investigated the clinical utility of multigene panel testing for 5011 unrelated individuals with SD in the United States (December 2019-April 2022). Median (range) age was 8 (0-90) years, 70.5% had short stature and/or disproportionate growth, 27.4% had a positive molecular diagnosis (MDx), and 30 individuals received two MDx. Genes most commonly contributing to MDx were FGFR3 (16.9%), ALPL (13.0%), and COL1A1 (10.3%). Most of the 112 genes associated with ≥1 MDx were primarily involved in signal transduction (n = 35), metabolism (n = 23), or extracellular matrix organization (n = 17). There were implications associated with specific care/treatment options for 84.4% (1158/1372) of MDx-positive individuals; >50% were linked to conditions with targeted therapy approved or in clinical development, including osteogenesis imperfecta, achondroplasia, hypophosphatasia, and mucopolysaccharidosis. Forty individuals with initially inconclusive results became MDx-positive following family testing. Follow-up mucopolysaccharidosis enzyme activity testing was positive in 14 individuals (10 of these were not MDx-positive). Our findings showed that inclusion of metabolic genes associated with SD increased the clinical utility of a gene panel and confirmed that integrated use of comprehensive gene panel testing with orthogonal testing reduced the burden of inconclusive results.

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.

Limb girdle muscular dystrophies (LGMD) are a genetically heterogeneous group of muscular dystrophies. The study presents an overview of molecular characteristics of a large cohort of LGMD patients who are representative of the Czech LGMD population. We present 226 LGMD probands in which 433 mutant alleles carrying 157 different variants with a supposed pathogenic effect were identified. Fifty-four variants have been described only in the Czech LGMD population so far. LGMD R1 caplain3-related is the most frequent subtype of LGMD involving 53.1% of patients with genetically confirmed LGMD, followed by LGMD R9 FKRP-related (11.1%), and LGMD R12 anoctamin5-related (7.1%). If we consider identified variants, then all but five were small-scale variants. One large gene deletion was identified in the LAMA2 gene and two deletions in each of CAPN3 and SGCG. We performed comparison our result with other published studies. The results obtained in the Czech LGMD population clearly differ from the outcome of other LGMD populations in two aspects-we have a more significant proportion of patients with LGMD R1 calpain3-related and a smaller proportion of LGMD R2 dysferlin-related.

Hemophilia A (HA) is one of the most widespread, X-linked, inherited bleeding disorders, which results from defects in the F8 gene. Nowadays, more than 3500 different pathogenic variants leading to HA have been described. Mutation analysis in HA is essential for accurate genetic counseling of patients and their relatives. We analyzed patients from 273 unrelated families with different forms of HA. The analysis consisted of testing for intron inversion (inv22 and inv1), and then sequencing all functionally important F8 gene fragments. We identified 101 different pathogenic variants in 267 patients, among which 35 variants had never been previously reported in international databases. We found inv22 in 136 cases and inv1 in 12 patients. Large deletions (1-8 exons) were found in 5 patients, and we identified a large insertion in 1 patient. The remaining 113 patients carried point variants involving either single nucleotide or several consecutive nucleotides. We report herein the largest genetic analysis of HA patients issued in Russia.

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.