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Polynucleotide kinase phosphatase (PNKP), encoded by the PNKP gene, is a DNA processing enzyme involved in double-strand break and single-strand break repair pathways, which are essential for genome stability and for the correct development and maintenance of human nervous system. PNKP biallelic loss-of-function variants have been associated with a broad spectrum of neurological anomalies, ranging from congenital microcephaly with intellectual disability and seizures (MCSZ), to later onset forms of ataxia-oculomotor apraxia (AOA4) or peripheral neuropathy (CMT2B2). We report the atypical clinical manifestations of a patient with severe microcephaly, short stature, developmental delay, conductive hearing loss, and tracheoesophageal malformation, in the absence of seizures. Whole exome sequencing analysis identified two novel, compound heterozygous splice-site variants in the PNKP gene (NM_007254.4): c.1448+1G > A and c.199-8_199-5del. To demonstrate the effect of both variants on the splicing process and prove their pathogenicity, we performed a hybrid minigene assay, which successfully highlighted a deleterious impact on the transcript, particularly regarding the c.199-8_199-5del variant. The uncommon clinical features of the proband and the identification of two newly associated pathogenic variants add further evidence to the allelic and phenotypic heterogeneity of the PNKP locus., (© 2024 The Author(s). American Journal of Medical Genetics Part B: Neuropsychiatric Genetics published by Wiley Periodicals LLC.)

Purpose: Pathogenic LZTR1 variants cause schwannomatosis and dominant/recessive Noonan syndrome (NS). We aim to establish an association between heterozygous loss-of-function LZTR1 alleles and isolated multiple café-au-lait macules (CaLMs)., Methods: A total of 849 unrelated participants with multiple CaLMs, lacking pathogenic/likely pathogenic NF1 and SPRED1 variants, underwent RASopathy gene panel sequencing. Data on 125 individuals with heterozygous LZTR1 variants were collected for characterizing their clinical features and the associated molecular spectrum. In vitro functional assessment was performed on a representative panel of missense variants and small in-frame deletions., Results: Analysis revealed heterozygous LZTR1 variants in 6.0% (51/849) of participants, exceeding the general population prevalence. LZTR1-related CaLMs varied in number, displayed sharp or irregular borders, and were generally isolated but occasionally associated with features recurring in RASopathies. In 2 families, CaLMs and schwannomas co-occurred. The molecular spectrum mainly consisted of truncating variants, indicating loss-of-function. These variants substantially overlapped with those occurring in schwannomatosis and recessive NS. Functional characterization showed accelerated protein degradation or mislocalization, and failure to downregulate mitogen-activated protein kinase signaling., Conclusion: Our findings expand the phenotypic variability associated with LZTR1 variants, which, in addition to conferring susceptibility to schwannomatosis and causing dominant and recessive NS, occur in individuals with isolated multiple CaLMs., Competing Interests: Conflict of Interest The authors declare no conflicts of interest., (Copyright © 2024 American College of Medical Genetics and Genomics. Published by Elsevier Inc. All rights reserved.)

Sequence variants in Eyes Shut Homolog (EYS) gene are one of the most frequent causes of autosomal recessive retinitis pigmentosa (RP). Herein, we describe an Italian RP family characterized by EYS-related pseudodominant inheritance. The female proband, her brother, and both her sons showed typical RP, with diminished or non-recordable full-field electroretinogram, narrowing of visual field, and variable losses of central vision. To investigate this apparently autosomal dominant pedigree, next generation sequencing (NGS) of a custom panel of RP-related genes was performed, further enhanced by bioinformatic detection of copy-number variations (CNVs). Unexpectedly, all patients had a compound heterozygosity involving two known pathogenic EYS variants i.e., the exon 33 frameshift mutation c.6714delT and the exon 29 deletion c.(5927þ1_5928-1)_(6078þ1_6079-1)del, with the exception of the youngest son who was homozygous for the above-detailed frameshift mutation. No pathologic eye conditions were instead observed in the proband's husband, who was a heterozygous healthy carrier of the same c.6714delT variant in exon 33 of EYS gene. These findings provide evidence that pseudodominant pattern of inheritance can hide an autosomal recessive RP partially or totally due to CNVs, recommending CNVs study in those pedigrees which remain genetically unsolved after the completion of NGS or whole exome sequencing analysis., (© 2024. The Author(s).)

Cystic fibrosis is a highly prevalent genetic disorder caused by biallelic pathogenic variants in the CFTR gene, causing an altered function of the exocrine glands and a subsequent spectrum of hypofunctional and degenerative manifestations. The increasing availability of carrier screening programmes, the enhanced life expectancy of patients due to improved treatment and care strategies and the development of more precise and affordable molecular diagnostic tools have prompted a rise in demand of prenatal diagnosis procedures for at-risk couples, including Preimplantation Genetic Testing (PGT). However, challenges remain: heterogeneity among screening programmes, nuances of variant interpretation and availability of novel treatments demand a considerate and knowledgeable approach to genetic counselling. In this work, we retrospectively evaluated the molecular data of 92 unselected couples who received a diagnosis of CFTR-related status and were referred to the genetics clinic at the University Hospital of Padua for genetic counselling on eligibility for PGT. A total of 50 couples were considered eligible for the procedure based on risk of transmitting biallelic pathogenic variants. We report and discuss our experience with this case series in the context of the Italian medical care system and present an overview of the most relevant issues regarding genetic counselling for PGT in CFTR-related disorders.

The ACTA2 gene codes for alpha-smooth muscle actin, a critical component of the contractile apparatus of the vascular smooth muscle cells. Autosomal dominant variants in the ACTA2 gene have been associated to familial non-syndromic thoracic aortic aneurysm/dissection (TAAD). They are thought to act through a dominant-negative mechanism. These variants display incomplete penetrance and variable expressivity, complicating the validation of ACTA2 variants pathogenicity by family segregation studies. In this study, we developed a yeast based assay to test putative TAAD-associated ACTA2 variants. We identified five new heterozygous ACTA2 missense variants in TAAD patients through next generation sequencing. We decided to test their pathogenicity in Saccharomyces cerevisiae, since yeast actin is very similar to human alpha-smooth muscle actin, and the residues at which the TAAD-associated variants occur in ACTA2 are well conserved. A wild type yeast strain was transformed with a vector expressing the different mutant alleles, to model the heterozygous condition of patients. Then, we evaluated yeast growth by spot test and cytoskeletal and mitochondrial morphology by fluorescence microscopy. We found that mutant yeast strains displayed only mild growth defects but a significant increase in the percentage of cells with abnormal mitochondrial distribution and abnormal organization of the actin cytoskeleton compared to controls. All variants appeared to interfere with the activity of wild type actin in yeast, suggesting a dominant-negative pathogenic mechanism. Our results demonstrate the utility of using the yeast actin model system to validate the pathogenicity of TAAD-associated ACTA2 variants., (© 2024. The Author(s), under exclusive licence to European Society of Human Genetics.)

Purpose: To evaluate whether a second biopsy, following a first diagnostic failure on blastocysts tested for preimplantation genetic testing for monogenic diseases (PGT-M), allows to obtain genetic diagnosis and to what extent this procedure can influence clinical pregnancy and live birth rates compared to the PGT-M process with a successful genetic diagnosis from the first biopsy., Methods: Embryos from women who underwent PGT-M in an infertility centre and who had been transferred after two biopsies for genetic analysis (n = 27) were matched in a 1:1 ratio accordingly to women's age (± 1 year) and fertility status (fertile vs infertile), as well as with the study period, with embryos who were transferred after receiving a conclusive PGT result straight after the first biopsy (n = 27). The main evaluated outcome was clinical pregnancy rate following embryo transfers in which healthy embryos were transferred after only one biopsy and those in which an embryo was transferred after being re-biopsied. Live birth rate was the secondary outcome., Results: Clinical pregnancy rate was 52% (95% CI: 34-69) following the transfer of a single-biopsy blastocyst and 30% (95% CI: 16-48) following the transfer of a re-biopsied blastocyst. The likelihood to have a healthy baby was 33% (95% CI: 19-52) following the transfer of a blastocyst biopsied once and 22% (95% CI: 11-41) following the transfer of a re-biopsied blastocyst., Conclusions: The re-biopsy intervention seems to considerably reduce the pregnancy potential of a blastocyst. However, a greater sample size is necessary to clarify this issue definitively., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Heterozygous loss-of-function variants in CHD8 have been associated with a syndromic neurodevelopmental-disease spectrum, collectively referred to as CHD8-related neurodevelopmental disorders. Several different clinical manifestations, affecting neurodevelopmental and systemic domains, have been described, presenting with highly variable expressivity. Some expressions are well established and comprise autism spectrum disorders, psychomotor delay with cognitive impairment, postnatal overgrowth with macrocephaly, structural brain abnormalities, gastrointestinal disturbances, and behavioral and sleep-pattern problems. However, the complete phenotypic spectrum of CHD8-related disorders is still undefined. In 2021, our group described two singular female patients with CHD8-related neurodevelopmental disorder and striking dystonic manifestations, prompting the suggestion that dystonia should be considered a possible component of this condition., Case Series Presentation: We describe three additional unrelated female individuals, each carrying a different CHD8 frameshift variant and whose clinical presentations were primarily characterized by young-onset dystonia. Their dystonic manifestations were remarkably heterogeneous and ranged from focal, exercise-dependent, apparently isolated forms to generalized permanent phenotypes accompanied by spasticity and tremor. Neurocognitive impairment and autistic behaviors, typical of CHD8-related disorders, were virtually absent or at the mild end of the spectrum., Conclusions: This work validates our previous observation that dystonia is part of the phenotypic spectrum of CHD8-related neurodevelopmental disorders with potential female preponderance, raising new challenges and opportunities in the diagnosis and management of this condition. It also highlights the importance of in-depth neurologic phenotyping of patients carrying variants associated with neurodevelopmental disorders, as the connection between neurodevelopmental and movement disorders is proving closer than previously appreciated., (© 2024. The Author(s).)