Preimplantation genetic testing for neurofibromatosis type 1: molecular genetic aspects and impact on reproductive counseling.

The use of fetal exome sequencing in prenatal diagnosis: a points to consider document of the American College of Medical Genetics and Genomics (ACMG)

DNA-based screening and population health: a points to consider statement for programs and sponsoring organizations from the American College of Medical Genetics and Genomics (ACMG)

Study question What are the technical challenges and pitfalls of PGT for NF1 (n = 296 families) as experienced by three large European PGT centers? Summary answer The high (de novo) mutation rate and presence of germline mosaicism, especially in case of deletions, is challenging in PGT test development for NF1. What is known already NF1 is a neurocutaneous disorder with complete penetrance and extreme clinical variability. It is one of the most frequently requested autosomal dominant indications for PGT. The NF1 gene is large and has a high mutation rate. A high number of variants occur de novo, are mostly unique and more frequently associated with germline mosaicism in the index. These characteristics can pose difficulties in counseling, test development and PGT treatment. Large NF1 deletions are associated with a more severe phenotype and relative frequent mosaicism, but this has not been described in literature for intragenic deletions. Study design, size, duration Retrospective analysis of data on all test development procedures with the indication NF1 completed by August 2022 (n = 296). The data on test development were collected from medical files by the laboratories of the three participating PGT centers. The study was approved by the Medical and Ethical Review Committee of all three centers. The Medical Research Involving Human Subjects Act was not applicable. Participants/materials, setting, methods The three participating PGT centers were Maastricht University Medical Center (the Netherlands), University Hospital Brussels (Belgium) and Strasbourg University Hospital (France). Techniques applied were locus specific PCR-STR with or without the specific causative variant or genome wide haplotyping methods (NGS based OnePGT or karyomapping) or a combination of the above. Main results and the role of chance Test development was completed for 296 cases involving 228 different variants in the NF1 gene (125 truncating, 31 missense, 47 splice and 7 in-frame or synonymous variants, 8 larger deletions, 1 translocation, 9 awaiting further classification). We observed 36 recurring variants for 104 couples, of which 55 were sporadic cases, The majority of the 296 cases were sporadic (60%), signs of mosaicism were observed in 14 of these cases (8%). In our cohort, 6 sporadic cases involved a complete gene deletion (n = 1) or a deletion of ≥ 1 exons in the NF1 gene (n = 5). Mosaicism was detected in 4 of these cases (67%). In one case, the mutation was not detected in sperm cells during test set-up. PGT treatment was started for 10 couples with signs of mosaicism, in 6 no affected embryos were detected in 1-2 cycles and 1 resulted in a misdiagnosis because of phasing based on unaffected offspring. Two cases had NF1 caused by a different variant in the NF1 gene than their affected family members. A successful PGT test was developed in 289 cases (98%), 5 couples opted out during test development and in only 2 cases, development of a test was not possible for technical reasons. Limitations, reasons for caution The observed challenges and pitfalls depend on the techniques used and local procedures, so results may not be applicable to all PGT laboratories. However, due to the large sample size with exhaustive data and the fact that ESHRE guidelines were followed, there is probably little reason for caution. Wider implications of the findings Our findings could improve broader reproductive counseling of NF1 patients. Also, this large cohort contains previously unpublished variants in the NF1 gene, which is relevant for genetic diagnosis in NF1 patients in general. Our findings could possibly aid in test development for other autosomal dominant PGT indications with similar characteristics. Trial registration number Not applicable.

Neurofibromatosis type 1 (NF1) is an autosomal dominant disease with complete penetrance but high variable expressivity. NF1 is caused by loss-of-function mutations in the NF1 gene, a negative regulator of the RAS-MAPK pathway. The NF1 gene has one of the highest mutation rates in human disorders, which may explain the outbreak of independent de novo variants in the same family. Here, we report the co-occurrence of pathogenic variants in the NF1 and SPRED1 genes in six families with NF1 and Legius syndrome, using next-generation sequencing. In five of these families, we observed the co-occurrence of two independent NF1 variants. All NF1 variants were classified as pathogenic, according to the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG-AMP) guidelines. In the sixth family, one sibling inherited a complete deletion of the NF1 gene from her mother and carried a variant of unknown significance in the SPRED1 gene. This variant was also present in her brother, who was diagnosed with Legius syndrome, a differential diagnosis of NF1. This work illustrates the complexity of molecular diagnosis in a not-so-rare genetic disease.

Introduction: RASopathies are among the most prevalent genetic syndromes caused by variants in the Ras/MAPK signaling pathway, affecting various systems such as the heart, craniofacial features, skin, musculoskeletal system, hearing, and vision. They can also increase the risk of secondary malignancies. Despite clinical overlaps, distinguishing features are crucial for diagnosis, as different variants lead to distinct clinical implications. This study reviews the molecular and clinical characteristics of RASopathies, focusing on neurofibromatosis type 1 (NF1) and non-NF1 RASopathies. Methods: The study analyzed 76 patients referred to our outpatient clinic over a 6-year period, all of whom were clinically diagnosed with RASopathy and confirmed in most cases by molecular testing. Patient files, clinical photographs, and laboratory results were reviewed and analyzed. A targeted multigene next-generation sequencing panel test was performed, followed by Sanger sequencing for both confirmation and segregation analysis. Multiplex ligation-dependent probe amplification was conducted in a patient with normal sequence results but strong clinical suspicion, to identify potential deletions. Results: We identified 44 pathogenic, 25 likely pathogenic variants, and 6 variants of uncertain significance based on American College of Medical Genetics and Genomics (ACMG) criteria. Among these, 14 novel variants were found – 13 in the NF1 gene and one in SOS1. NF1 variants were present in 51 cases. Additional variants, likely to represent clinically significant findings, were identified in PTPN11 (n = 11), RAF1 (n = 4), SOS1 (n = 3), RIT1 (n = 3), KRAS (n = 1), NRAS (n = 1), SOS2 (n = 1), and BRAF (n = 1). Diagnoses included 49 patients with NF1, 21 with Noonan syndrome, 2 with neurofibromatosis-Noonan syndrome, 2 with Noonan syndrome with multiple lentigines, and 1 with cardiofaciocutaneous syndrome. Here, 12% of NF1 variants were located in exon 21, 36% of PTPN11 variants in exon 3, and 75% of RAF1 variants in exon 7. Conclusion: RASopathies have a broad molecular and clinical spectrum, complicating diagnosis and management. Accurate clinical correlation and molecular analysis are essential, as different RASopathy syndromes can result from variants in the same genes, while the same syndrome may arise from different genetic alterations. This study identifies novel variants and emphasizes the need for precise diagnostic approaches in these complex disorders.

The aim of this study is to investigate if Preimplantation Genetic Testing (PGT) can effectively identify unreported variants according to American College of Medical Genetics and Genomics (ACMG)to prevent citrullinemia type 1 affection. This study involves a detailed case analysis of a family with history of citrullinemia type 1, focusing on the use of PGT for monogenic diseases (PGT-M). The genetic variants were identified using ACMG guidelines, and PGT was employed to prevent the inheritance of these variants. The study included haplotype analysis and Sanger sequencing to confirm the results. The study identified previously unreported variations in the ASS1 gene causing citrullinemia type 1. PGT successfully prevented the transmission of these variants, resulting in the birth of a healthy fetus. However, challenges such as allele dropout (ADO) and gene recombination were encountered during haplotype analysis, which could potentially defeat the diagnosis. The study demonstrated that combining haplotype analysis with Sanger sequencing can enhance the accuracy of PGT. Preimplantation Genetic Testing (PGT) targeting likely pathogenic and pathogenic variants in the ASS1 gene, as rated by ACMG, allows the birth of healthy infants free from citrullinemia type 1. Additionally, the establishment of single haplotypes and Sanger sequencing can reduce the misdiagnosis rate caused by allele dropout (ADO) and genetic recombination.

The American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) have introduced an internationally shared framework for variant classification in genetic disorders. FVII deficiency is a rare inherited autosomal recessive bleeding disorder with sparse data concerning ACMG classification. To develop an approach which may improve the utility of molecular genetic test results, 129 patients with FVII deficiency were retrospectively assigned to six subgroups for exploratory analysis: F7 gene wildtype (group 1), ACMG 1 (benign variant) or ACMG 2 (likely benign variant), only (group 2), ACMG 3 (variant of uncertain significance) ± ACMG 1-2 heterozygous or not classified variant (group 3), ACMG 4 (likely pathogenic variant), or ACMG 5 (pathogenic variant) single heterozygous ± ACMG 1-3 single heterozygous (group 4), ACMG 4-5 homozygous or ≥2 ACMG 4-5 heterozygous or ≥1 ACMG 4-5 heterozygous plus either ACMG 1 c.1238G>A modifying variant homozygous or ≥2 ACMG 1-3 (group 5), FVII deficiency and another bleeding disorder (group 6). Eleven of 31 patients (35.5%) in group 5 had abnormal ISTH-BS (n = 7) and/or history of substitution with recombinant factor VIIa (n = 5) versus 4 of 80 patients (5.0%, n = 1 abnormal ISTH-BS, n = 3 substitution) in groups 1 (n = 2/22), 2 (n = 1/29), 3 (n = 0/9), and 4 (n = 1/20). Four of 18 patients (22.2%) with FVII deficiency and another bleeding disorder (group 6) had an abnormal ISTH-BS (n = 2) and/or history of substitution with recombinant factor VIIa (n = 3). Patients with a homozygous ACMG 4-5 variant or with specific combinations of heterozygous ACMG 4-5 ± ACMG 1-3 variants exhibited a high-risk bleeding phenotype in contrast to the remaining patients without another bleeding disorder. This result may serve as a basis to develop a genotype/phenotype prediction model in future studies.

ACMG SF v3.0 list for reporting of secondary findings in clinical exome and genome sequencing: a policy statement of the American College of Medical Genetics and Genomics (ACMG)

Updated recommendations for CFTR carrier screening: A position statement of the American College of Medical Genetics and Genomics (ACMG)

The American College of Medical Genetics and Genomics (ACMG) previously developed guidance for the interpretation of sequence variants.1 In the past decade, sequencing technology has evolved rapidly with the advent of high-throughput next generation sequencing. By adopting and leveraging next generation sequencing, clinical laboratories are now performing an ever increasing catalogue of genetic testing spanning genotyping, single genes, gene panels, exomes, genomes, transcriptomes and epigenetic assays for genetic disorders. By virtue of increased complexity, this paradigm shift in genetic testing has been accompanied by new challenges in sequence interpretation. In this context, the ACMG convened a workgroup in 2013 comprised of representatives from the ACMG, the Association for Molecular Pathology (AMP) and the College of American Pathologists (CAP) to revisit and revise the standards and guidelines for the interpretation of sequence variants. The group consisted of clinical laboratory directors and clinicians. This report represents expert opinion of the workgroup with input from ACMG, AMP and CAP stakeholders. These recommendations primarily apply to the breadth of genetic tests used in clinical laboratories including genotyping, single genes, panels, exomes and genomes. This report recommends the use of specific standard terminology: ‘pathogenic’, ‘likely pathogenic’, ‘uncertain significance’, ‘likely benign’, and ‘benign’ to describe variants identified in Mendelian disorders. Moreover, this recommendation describes a process for classification of variants into these five categories based on criteria using typical types of variant evidence (e.g. population data, computational data, functional data, segregation data, etc.). Because of the increased complexity of analysis and interpretation of clinical genetic testing described in this report, the ACMG strongly recommends that clinical molecular genetic testing should be performed in a CLIA-approved laboratory with results interpreted by a board-certified clinical molecular geneticist or molecular genetic pathologist or equivalent.

The use of ACMG secondary findings recommendations for general population screening: a policy statement of the American College of Medical Genetics and Genomics (ACMG)

Does the law require reinterpretation and return of revised genomic results?

Highlights from Recent Cancer Literature

A Call for Discovery and Therapeutic Development for Cutaneous Neurofibromas

To characterize the adaptive behavior profile of children with neurofibromatosis type 1 (NF1) and determine its relationship to neuropsychological functioning and non-neoplastic T2-weighted hyperintense brain lesions on brain magnetic resonance imaging (MRI). In this cross-sectional study, we retrospectively reviewed neuropsychological reports from 104 children with NF1 (56 males, 48 females; mean age 10y 4mo; standard deviation [SD] 3y 4mo; range 3y 5mo-17y 6mo), and extracted data from a range of cognitive and behavioral measures, including the Adaptive Behavior Assessment System (ABAS). Brain MRI was retrospectively reviewed in 42 individuals. Adaptive Behavior Assessment System scores were continuously distributed and pathologically shifted by 0.79 to 1.26SD across Conceptual, Social, and Practical domains, and 46.5% of individuals had a composite score in the borderline or impaired range. Impairment in adaptive functioning was correlated with deficits in executive function (r=-9.543, p<0.001), externalizing problems (r=-0.366, p<0.001), and attention (r=-9.467, p=0.001). Cluster analysis revealed three distinct phenotypic subgroups, one of which exhibited normal cognitive ability, but impaired adaptive functioning, with persistent deficits in executive function, behavioral problems, and attention-deficit/hyperactivity disorder symptomatology. There was no relationship between ABAS scores and the number or location of unidentified bright objects. Adaptive functioning deficits are common among children with NF1 and are associated with impairment in other cognitive/behavioral domains, independent of general cognitive ability. Deficits in adaptive behavior are common in children with neurofibromatosis type 1 (NF1). Poor adaptive functioning is associated with impairments in executive function, externalizing behaviors, and attention, regardless of cognitive ability. The presence or location of unidentified bright objects do not predict adaptive behavior skills in children with NF1.