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Study Question: Is it possible to develop a comprehensive pipeline for all-in-one preimplantation genetic testing (PGT), also suitable for parents-only haplotyping and, for the first time, third-party reproduction?Summary Answer: Optimized reduced representation sequencing (RRS) by GENType, along with a novel analysis platform (Hopla), enables cheap, accurate and comprehensive PGT of blastocysts, even without the inclusion of additional family members or both biological parents for genome-wide embryo haplotyping.What Is Known Already: Several haplotyping strategies have proven to be effective for comprehensive PGT. However, these methods often rely on microarray technology, whole-genome sequencing (WGS) or a combination of strategies, hindering sample throughput and cost-efficiency. Moreover, existing tools (including other RRS-based strategies) require both prospective biological parents for embryo haplotyping, impeding application in a third-party reproduction setting.Study Design, Size, Duration: This study included a total of 257 samples. Preliminary technical validation was performed on 81 samples handpicked from commercially available cell lines. Subsequently, a clinical validation was performed on a total of 72 trophectoderm biopsies from 24 blastocysts, tested for a monogenic disorder (PGT-M) (n = 15) and/or (sub)chromosomal aneuploidy (PGT-SR/PGT-A) (n = 9). Once validated, our pipeline was implemented in a diagnostic setting on 104 blastocysts for comprehensive PGT.Participants/materials, Setting, Methods: Samples were whole-genome amplified (WGA) and processed by GENType. Quality metrics, genome-wide haplotypes, b-allele frequencies (BAFs) and copy number profiles were generated by Hopla. PGT-M results were deduced from relative haplotypes, while PGT-SR/PGT-A results were inferred from read-count analysis and BAF profiles. Parents-only haplotyping was assessed by excluding additional family members from analysis and using an independently diagnosed embryo as phasing reference. Suitability for third-party reproduction through single-parent haplotyping was evaluated by excluding one biological parent from analysis. Results were validated against reference PGT methods.Main Results and the Role Of Chance: Genome-wide haplotypes of single cells were highly accurate (mean > 99%) compared to bulk DNA. Unbalanced chromosomal abnormalities (>5 Mb) were detected by GENType. For both PGT-M as well as PGT-SR/PGT-A, our technology demonstrated 100% concordance with reference PGT methods for diverse WGA methods. Equally, for parents-only haplotyping and single-parent haplotyping (of autosomal dominant disorders and X-linked disorders), PGT-M results were fully concordant. Furthermore, the origin of trisomies in PGT-M embryos was correctly deciphered by Hopla.Limitations, Reasons For Caution: Intrinsic to linkage-analysis strategies, de novo single-nucleotide variants remain elusive. Moreover, parents-only haplotyping is not a stand-alone approach and requires prior diagnosis of at least one reference embryo by an independent technology (i.e. direct mutation analysis) for haplotype phasing. Using a haplotyping approach, the presence of a homologous recombination site across the chromosome is biologically required to distinguish meiotic II errors from mitotic errors during trisomy origin investigation.Wider Implications Of the Findings: We offer a generic, fully automatable and accurate pipeline for PGT-M, PGT-A and PGT-SR as well as trisomy origin investigation without the need for personalized assays, microarray technology or WGS. The unique ability to perform single-parent assisted haplotyping of embryos paves the way for cost-effective PGT in a third-party reproduction setting.Study Funding/competing Interest(s): L.D.W. is supported by the Research Foundation Flanders (FWO; 1S74619N). L.R. and B.M. are funded by Ghent University and M.B., S.S., K.T., F.V.M. and A.D. are supported by Ghent University Hospital. Research in the N.C. lab was funded by Ghent University, VIB and Kom op Tegen Kanker. A.D.K and N.C. are co-inventors of patent WO2017162754A1. The other authors have no conflicts of interest.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Abstract Background The high mental health care consumption rates of divorced singles may constitute a heavy burden on the public health care system. This raises the question of whether their higher health care use stems from a greater need, or whether there are other factors contributing to these high consumption rates. We examine both health care use and subjective unmet need (perceiving a need for care without seeking it) because of social or emotional problems of the divorced singles, the repartnered divorcees, and the married. Moreover, we investigate how health care use and subjective unmet need relate to each other. Methods We conduct several gender specific logistic regressions employing data from the Divorce in Flanders Survey (N men = 2884; N women = 3317). Results Results show that the divorced singles have more contact with professional health care providers (general practitioners, psychiatrists, and psychologists) because of social or emotional problems, and more often perceive unmet needs. The higher health care use rates and greater subjective unmet needs can largely be attributed to higher levels of depressive symptoms. Surprisingly, we find that non-frequent health care users more often perceive a subjective unmet need than frequent health care users and those who have not contacted any health care provider. Conclusion The single divorced consult health care providers more often because of social or emotional problems and they also perceive unmet needs more often.

Abstract Background Little is known about differences in professional care seeking based on marital status. The few existing studies show more professional care seeking among the divorced or separated compared to the married or cohabiting. The aim of this study is to determine whether, in a sample of the European general population, the divorced or separated seek more professional mental health care than the married or cohabiting, regardless of self-reported mental health problems. Furthermore, we examine whether two country-level features--the supply of mental health professionals and the country-level divorce rates--contribute to marital status differences in professional care-seeking behavior. Methods We use data from the Eurobarometer 248 on mental well-being that was collected via telephone interviews. The unweighted sample includes 27,146 respondents (11,728 men and 15,418 women). Poisson hierarchical regression models were estimated to examine whether the divorced or separated have higher professional health care use for emotional or psychological problems, after controlling for mental and somatic health, sociodemographic characteristics, support from family and friends, and degree of urbanization. We also considered country-level divorce rates and indicators of the supply of mental health professionals, and applied design and population weights. Results We find that professional care seeking is strongly need based. Moreover, the divorced or separated consult health professionals for mental health problems more often than people who are married or who cohabit do. In addition, we find that the gap between the divorced or separated and the married or cohabiting is highest in countries with low divorce rates. Conclusions The higher rates of professional care seeking for mental health problems among the divorced or separated only partially correlates with their more severe mental health problems. In countries where marital dissolution is more common, the marital status gap in professional care seeking is narrower, partially because professional care seeking is more common among the married or cohabiting.

Competing Interests: Conflicts of interest Dr Callewaert is a senior clinical investigator of the Research Foundation Flanders. Drs Beyens, Weytens, Pottie, De Meulemeester, Aelbrecht, De Feyter, De Schepper, Van Holm, and Symoens have no conflicts of interest to declare.

Study Question: What is the frequency of PLCZ1 , ACTL7A , and ACTL9 variants in male patients showing fertilization failure after ICSI, and how effective is assisted oocyte activation (AOA) for them?, Summary Answer: Male patients with fertilization failure after ICSI manifest variants in PLCZ1 (29.09%), ACTL7A (14.81%), and ACTL9 (3.70%), which can be efficiently overcome by AOA treatment with ionomycin., What Is Known Already: Genetic variants in PLCZ1 , and more recently, in ACTL7A , and ACTL9 male genes, have been associated with total fertilization failure or low fertilization after ICSI. A larger patient cohort is required to understand the frequency at which these variants occur, and to assess their effect on the calcium ion (Ca 2+ ) release during oocyte activation. AOA, using ionomycin, can restore fertilization and pregnancy rates in patients with PLCZ1 variants, but it remains unknown how efficient this is for patients with ACTL7A and ACTL9 variants., Study Design Size Duration: This prospective study involved two patient cohorts. In the first setting, group 1 (N = 28, 2006-2020) underwent only PLCZ1 genetic screening, while group 2 (N = 27, 2020-2023) underwent PLCZ1, ACTL7A , and ACTL9 genetic screening. Patients were only recruited when they had a mean fertilization rate of ≤33.33% in at least one ICSI cycle with at least four MII oocytes. Patients underwent a mouse oocyte activation test (MOAT) and at least one ICSI-AOA cycle using calcium chloride (CaCl 2 ) injection and double ionomycin exposure at our centre. All patients donated a saliva sample for genetic screening and a sperm sample for further diagnostic tests, including Ca 2+ imaging., Participants/materials Setting Methods: Genetic screening was performed via targeted next-generation sequencing. Identified variants were classified by applying the revised ACMG guidelines into a Bayesian framework and were confirmed by bidirectional Sanger sequencing. If variants of uncertain significance or likely pathogenic or pathogenic variants were found, patients underwent additional determination of the sperm Ca 2+ -releasing pattern in mouse (MOCA) and in IVM human (HOCA) oocytes. Additionally, ACTL7A immunofluorescence and acrosome ultrastructure analyses by transmission electron microscopy (TEM) were performed for patients with ACTL7A and/or ACTL9 variants., Main Results and the Role of Chance: Overall, the frequency rate of PLCZ1 variants was 29.09%. Moreover, 14.81% of patients carried ACTL7A variants and 3.70% carried ACTL9 variants. Seven different PLCZ1 variants were identified (p.Ile74Thr, p.Gln94*, p.Arg141His, p.His233Leu, p.Lys322*, p.Ile379Thr, and p.Ser500Leu), five of which are novel. Interestingly, PLCZ1 variants p.Ser500Leu and p.His233Leu occurred in 14.55% and 9.09% of cases. Five different variants were found in ACTL7A (p.Tyr183His, p.Gly214Ser, p.Val340Met, p.Ser364Glnfs*9, p.Arg373Cys), four of them being identified for the first time. A novel variant in ACTL9 (p.Arg271Pro) was also described. Notably, both heterozygous and homozygous variants were identified.The MOCA and HOCA tests revealed abnormal or absent Ca 2+ release during fertilization in all except one patient, including patients with PLCZ1 heterozygous variants. TEM analysis revealed abnormal acrosome ultrastructure in three patients with ACTL7A variants, but only patients with homozygous ACTL7A variants showed reduced fluorescence intensity in comparison to the control.AOA treatment significantly increased the fertilization rate in the 19 patients with detected variants (from 11.24% after conventional ICSI to 61.80% after ICSI-AOA), as well as positive hCG rate (from 10.64% to 60.00%) and live birth rate (from 6.38% to 37.14%), resulting in 13 healthy newborns. In particular, four live births and two ongoing pregnancies were produced using sperm from patients with ACTL7A variants., Limitations Reasons for Caution: Genetic screening included exonic and outflanking intronic regions, which implies that deep intronic variants were missed. In addition, other male genes or possible female-related factors affecting the fertilization process remain to be investigated., Wider Implications of the Findings: Genetic screening of PLCZ1 , ACTL7A , and ACTL9 offers a fast, cost-efficient, and easily implementable diagnostic test for total fertilization failure or low fertilization after ICSI, eliminating the need for complex diagnostic tests like MOAT or Ca 2+ analysis. Nonetheless, HOCA remains the most sensitive functional test to reveal causality of uncertain significance variants. Interestingly, heterozygous PLCZ1 variants are sufficient to cause inadequate Ca 2+ release during ICSI. Most importantly, AOA treatment using CaCl 2 injection followed by double ionomycin exposure is highly effective for this patient group, including those with ACTL7A variants, who also display a Ca 2+ -release deficiency., Study Funding/competing Interests: This study was supported by the Flemish Fund for Scientific Research (FWO) (TBM-project grant T002223N awarded to B.H.) and by the Special Research Fund (BOF) (starting grant BOF.STG.2021.0042.01 awarded to B.H.). A.C.B., R.R.G., C.C., E.V.D.V., A.R., D.S., L.L., P.C., S.S., A.B., and F.V.M. have nothing to disclose. B.H. reports a research grant from FWO and BOF, and reports being a board member of the Belgian Ethical Committee on embryo research., Trial Registration Number: N/A., Competing Interests: A.C.B., R.R.G., C.C., E.V.D.V., A.R., D.S., L.L., P.C., S.S., A.B., and F.V.M. have nothing to disclose. B.H. reports a research grant from the FWO and BOF and reports being a board member of the Belgian Ethical Committee on embryo research., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology.)

Study Question: To what extent can genotype analysis aid in the classification of (mosaic) aneuploid embryos diagnosed through copy number analysis of a trophectoderm (TE) biopsy?, Summary Answer: In a small portion of embryos, genotype analysis revealed signatures of meiotic or uniform aneuploidy in those diagnosed with intermediate copy number changes, and signatures of presumed mitotic or putative mosaic aneuploidy in those diagnosed with full copy number changes., What Is Known Already: Comprehensive chromosome screening (CCS) for preimplantation genetic testing has provided valuable insights into the prevalence of (mosaic) chromosomal aneuploidy at the blastocyst stage. However, diagnosis of (mosaic) aneuploidy often relies solely on (intermediate) copy number analysis of a single TE biopsy. Integrating genotype information allows for independent assessment of the origin and degree of aneuploidy. Yet, studies aligning both datasets to predict (putative mosaic) aneuploidy in embryos remain scarce., Study Design Size Duration: A single TE biopsy was collected from 1560 embryos derived from 221 couples tested for a monogenic disorder (n = 218) or microdeletion-/microduplication syndrome (n = 3). TE samples were subjected to both copy number and genotyping analysis., Participants/materials Setting Methods: Copy number and SNP genotyping analysis were conducted using GENType. Unbalanced chromosomal anomalies ≥10 Mb (or ≥20 Mb for copy number calls <50%) were classified by degree, based on low-range intermediate (LR, 30-50%), high-range intermediate (HR, 50-70%) or full (>70%) copy number changes. These categories were further subjected to genotyping analysis to ascertain the origin (and/or degree) of aneuploidy. For chromosomal gains, the meiotic division of origin (meiotic I/II versus non-meiotic or presumed mitotic) was established by studying the haplotypes. The level of monosomy (uniform versus putative mosaic) in the biopsy could be ascertained from the B-allele frequencies. For segmental aneuploidies, genotyping was restricted to deletions., Main Results and the Role of Chance: Of 1479 analysed embryos, 24% (n = 356) exhibited a whole-chromosome aneuploidy, with 19% (n = 280) showing full copy number changes suggestive of uniform aneuploidy. Among 258 embryos further investigated by genotyping, 95% of trisomies with full copy number changes were identified to be of meiotic origin. For monosomies, a complete loss of heterozygosity (LOH) in the biopsy was observed in 97% of cases, yielding a 96% concordance rate at the embryo level (n = 248/258). Interestingly, 4% of embryos (n = 10/258) showed SNP signatures of non-meiotic gain or putative mosaic loss instead. Meanwhile, 5% of embryos (n = 76/1479) solely displayed HR (2.5%; n = 37) or LR (2.6%; n = 39) intermediate copy number changes, with an additional 2% showing both intermediate and full copy number changes. Among embryos with HR intermediate copy number changes where genotyping was feasible (n = 25/37), 92% (n = 23/25) showed SNP signatures consistent with putative mosaic aneuploidy. However, 8% (n = 2/25) exhibited evidence of meiotic trisomy (9%) or complete LOH in the biopsy (7%). In the LR intermediate group, 1 of 33 (3%) genotyped embryos displayed complete LOH. Furthermore, segmental aneuploidy was detected in 7% of embryos (n = 108/1479) (or 9% (n = 139) with added whole-chromosome aneuploidy). These errors were often (52%) characterized by intermediate copy number values, which closely aligned with genotyping data when examined (94-100%)., Large Scale Data: N/A., Limitations Reasons for Caution: The findings were based on single TE biopsies and the true extent of mosaicism was not validated through embryo dissection. Moreover, evidence of absence of a meiotic origin for a trisomy should not be construed as definitive proof of a mitotic error. Additionally, a genotyping diagnosis was not always attainable due to the absence of a recombination event necessary to discern between meiotic II and non-meiotic trisomy, or the unavailability of DNA from both parents., Wider Implications of the Findings: Interpreting (intermediate) copy number changes of a single TE biopsy alone as evidence for (mosaic) aneuploidy in the embryo remains suboptimal. Integrating genotype information alongside the copy number status could provide a more comprehensive assessment of the embryo's genetic makeup, within and beyond the single TE biopsy. By identifying meiotic aberrations, especially in presumed mosaic embryos, we underscore the potential value of genotyping analysis as a deselection tool, ultimately striving to reduce adverse clinical outcomes., Study Funding/competing Interests: L.D.W. was supported by the Research Foundation Flanders (FWO; 1S74621N). M.B., K.T., F.V.M., S.J., A.V.T., V.S., D.S., A.D., and S.S. are supported by Ghent University Hospital. B.M. was funded by Ghent University. The authors have no conflicts of interest., Competing Interests: The authors have no conflicts of interest to declare., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology.)

Myhre syndrome (MS, MIM 139210) is a rare multisystemic disorder caused by recurrent pathogenic missense variants in SMAD4. The clinical features have been mainly documented in childhood and comprise variable neurocognitive development, recognizable craniofacial features, a short stature with a pseudo-muscular build, hearing loss, thickened skin, joint limitations, diverse cardiovascular and airway manifestations, and increased fibrosis often following trauma or surgery. In contrast, adults with MS are underreported obscuring potential clinical variability. Here, we describe 24 adults with MS, including 17 diagnosed after the age of 18 years old, and we review the literature on adults with MS. Overall, our cohort shows a milder phenotype as well as lower mortality rates compared to what has been published in literature. Individuals with a codon 500 variant in SMAD4 present with a more pronounced neurodevelopmental and systemic phenotype. However, in contrast to the literature, we observe cardiovascular abnormalities in individuals with the p.(Arg496Cys) variant. In addition, we describe scoliosis as a new manifestation and we report fertility in two additional males with the p.(Arg496Cys). In conclusion, our study contributes novel insights into the clinical variability of MS and underscores the importance of variant-specific considerations, and we provide recommendations for the management of MS in adulthood., (© 2024. The Author(s), under exclusive licence to European Society of Human Genetics.)

Introduction: Counseling osteogenesis imperfecta (OI) pregnancies is challenging due to the wide range of onsets and clinical severities, from perinatal lethality to milder forms detected later in life., Methods: Thirty-eight individuals from 36 families were diagnosed with OI through prenatal ultrasonography and/or postmortem clinical and radiographic findings. Genetic analysis was conducted on 26 genes associated with OI in these subjects that emerged over the past 20 years; while some genes were examined progressively, all 26 genes were examined in the group where no pathogenic variations were detected., Results: Prenatal and postnatal observations both consistently showed short limbs in 97%, followed by bowing of the long bones in 89%. Among 32 evaluated cases, all exhibited cranial hypomineralization. Fractures were found in 29 (76%) cases, with multiple bones involved in 18 of them. Genetic associations were disclosed in 27 families with 22 (81%) autosomal dominant and five (19%) autosomal recessive forms, revealing 25 variants in six genes (COL1A1, COL1A2, CREB3L1, P3H1, FKBP10, and IFITM5), including nine novels. Postmortem radiological examination showed variability in intrafamily expression of CREBL3- and P3H1-related OI., Conclusion: Prenatal diagnosis for distinguishing OI and its subtypes relies on factors such as family history, timing, ultrasound, genetics, and postmortem evaluation., (© 2024 S. Karger AG, Basel.)

SNARE proteins comprise a conserved protein family responsible for catalyzing membrane fusion during vesicle traffic. Syntaxin18 (STX18) is a poorly characterized endoplasmic reticulum (ER)-resident t-SNARE. Recently, together with TANGO1 and SLY1, its involvement was shown in ER to Golgi transport of collagen II during chondrogenesis. We report a fetus with a severe osteochondrodysplasia in whom we identified a homozygous substitution of the highly conserved p.Arg10 to Pro of STX18. CRISPR/Cas9-mediated Stx18 deficiency in zebrafish reveals a crucial role for Stx18 in cartilage and bone development. Furthermore, increased expression of multiple components of the Stx18 SNARE complex and of COPI and COPII proteins suggests that Stx18 deficiency impairs antero- and retrograde vesicular transport in the crispant stx18 zebrafish. Taken together, our studies highlight a new candidate gene for a recessive form of osteochondrodysplasia, thereby possibly broadening the SNAREopathy phenotypic spectrum and opening new doors toward future research avenues. © 2023 American Society for Bone and Mineral Research (ASBMR)., (© 2023 American Society for Bone and Mineral Research (ASBMR).)

Neuronal TDP-43-positive inclusions are neuropathological hallmark lesions in frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Pathogenic missense variants in TARDBP, the gene encoding TDP-43, can cause ALS and cluster in the C-terminal prion-like domain (PrLD), where they modulate the liquid condensation and aggregation properties of the protein. TDP-43-positive inclusions are also found in rimmed vacuole myopathies, including sporadic inclusion body myositis, but myopathy-causing TDP-43 variants have not been reported. Using genome-wide linkage analysis and whole exome sequencing in an extended five-generation family with an autosomal dominant rimmed vacuole myopathy, we identified a conclusively linked frameshift mutation in TDP-43 producing a C-terminally altered PrLD (TDP-43 p.Trp385IlefsTer10 ) (maximum multipoint LOD-score 3.61). Patient-derived muscle biopsies showed TDP-43-positive sarcoplasmic inclusions, accumulation of autophagosomes and transcriptomes with abnormally spliced sarcomeric genes (including TTN and NEB) and increased expression of muscle regeneration genes. In vitro phase separation assays demonstrated that TDP-43 Trp385IlefsTer10 does not form liquid-like condensates and readily forms solid-like fibrils indicating increased aggregation propensity compared to wild-type TDP-43. In Drosophila TDP-43 p.Trp385IlefsTer10 behaved as a partial loss-of-function allele as it was able to rescue the TBPH (fly ortholog of TARDBP) neurodevelopmental lethal null phenotype while showing strongly reduced toxic gain-of-function properties upon overexpression. Accordingly, TDP-43 p.Trp385IlefsTer10 showed reduced toxicity in a primary rat neuron disease model. Together, these genetic, pathological, in vitro and in vivo results demonstrate that TDP-43 p.Trp385IlefsTer10 is an aggregation-prone partial loss-of-function variant that causes autosomal dominant vacuolar myopathy but not ALS/FTD. Our study genetically links TDP-43 proteinopathy to myodegeneration, and reveals a tissue-specific role of the PrLD in directing pathology., (© 2023. The Author(s).)

Background and Objectives: Owing to their extensive clinical and molecular heterogeneity, hereditary neurologic diseases in adults are difficult to diagnose. The current knowledge about the diagnostic yield and clinical utility of exome sequencing (ES) for neurologic diseases in adults is limited. This observational study assesses the diagnostic value of ES and multigene panel analysis in adult-onset neurologic disorders., Methods: From January 2019 through April 2022, ES-based multigene panel testing was conducted in 1,411 patients with molecularly unexplained neurologic phenotypes at the Ghent University Hospital. Gene panels were developed for ataxia and spasticity, leukoencephalopathy, movement disorders, paroxysmal episodic disorders, neurodegeneration with brain iron accumulation, progressive myoclonic epilepsy, and amyotrophic lateral sclerosis. Single nucleotide variants, small indels, and copy number variants were analyzed. Across all panels, our analysis covered a total of 725 genes associated with Mendelian inheritance., Results: A molecular diagnosis was established in 10% of the cases (144 of 1,411) representing 71 different monogenic disorders. The diagnostic yield depended significantly on the presenting phenotype with the highest yield seen in patients with ataxia or spastic paraparesis (19%). Most of the established diagnoses comprised disorders with an autosomal dominant inheritance (62%), and the most frequently mutated genes were NOTCH3 (13 patients), SPG7 (11 patients), and RFC1 (8 patients). 34% of the disease-causing variants were novel, including a unique likely pathogenic variant in APP (Ghent mutation, p.[Asn698Asp]) in a family presenting with stroke and severe cerebral white matter disease. 7% of the pathogenic variants comprised copy number variants detected in the ES data and confirmed by an independent technique., Discussion: ES and multigene panel testing is a powerful and efficient tool to diagnose patients with unexplained, adult-onset neurologic disorders., Competing Interests: The authors report no relevant disclosures. Full disclosure form information provided by the authors is available with the full text of this article at Neurology.org/NG., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

Osteogenesis imperfecta (OI) is a genetically and clinically heterogeneous disorder characterized by bone fragility and reduced bone mass generally caused by defects in type I collagen structure or defects in proteins interacting with collagen processing. We identified a homozygous missense mutation in SEC16B in a child with vertebral fractures, leg bowing, short stature, muscular hypotonia, and bone densitometric and histomorphometric features in keeping with OI with distinct ultrastructural features. In line with the putative function of SEC16B as a regulator of trafficking between the ER and the Golgi complex, we showed that patient fibroblasts accumulated type I procollagen in the ER and exhibited a general trafficking defect at the level of the ER. Consequently, patient fibroblasts exhibited ER stress, enhanced autophagosome formation, and higher levels of apoptosis. Transfection of wild-type SEC16B into patient cells rescued the collagen trafficking. Mechanistically, we show that the defect is a consequence of reduced SEC16B expression, rather than due to alterations in protein function. These data suggest SEC16B as a recessive candidate gene for OI., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)

Bi-allelic mutations in the gene coding for human trans-membrane anterior-posterior transformation protein 1 (TAPT1) result in a broad phenotypic spectrum, ranging from syndromic disease with severe skeletal and congenital abnormalities to isolated early-onset cataract. We present here the first patient with a frameshift mutation in the TAPT1 gene, resulting in both bilateral early-onset cataract and skeletal abnormalities, in addition to several dysmorphic features, in this way further expanding the phenotypic spectrum associated with TAPT1 mutations. A tapt1a/tapt1b double knock-out (KO) zebrafish model generated by CRISPR/Cas9 gene editing revealed an early larval phenotype with eye malformations, loss of vision, increased photokinetics and hyperpigmentation, without visible skeletal involvement. Ultrastructural analysis of the eyes showed a smaller condensed lens, loss of integrity of the lens capsule with formation of a secondary lens and hyperplasia of the cells in the ganglion and inner plexiform layers of the retina. Transcriptomic analysis pointed to an impaired lens development with aberrant expression of many of the crystallin and other lens-specific genes. Furthermore, the phototransduction and visual perception pathways were found to be significantly disturbed. Differences in light perception are likely the cause of the increased dark photokinetics and generalized hyperpigmentation observed in this zebrafish model. In conclusion, this study validates TAPT1 as a new gene for early-onset cataract and sheds light on its ultrastructural and molecular characteristics., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Infertility in couples is a common problem, with both female and male factors contributing to similar extents. Severe, congenital disorders affecting fertility are, however, rare. While folliculogenesis and spermatogenesis are generally orchestrated via different mechanisms, some genetic anomalies can impair both female and male gametogenesis. Minichromosome maintenance complex component 9 (MCM9) is involved in DNA repair and mutations of the MCM9 gene have been previously reported in females with premature ovarian insufficiency (POI). MCM9 is also an emerging cancer risk gene. We performed next-generation and Sanger sequencing of fertility and related genes and hormonal and imaging studies in a kindred whose members had POI and disordered spermatogenesis. We identified a homozygous pathogenic MCM9 variant, c.394C>T (p.Arg132*) in three sisters affected by POI due to ovarian dysgenesis and their brother who had normal pubertal development but suffered from non-obstructive azoospermia. Testicular biopsy revealed Sertoli cell-only testicular histopathology. No evidence of early onset cancer was found in the homozygotic family members, but they were all young (<30 years) at the time of the study. In the male patient the homozygous MCM9 variant led to normal pubertal development and hormonal levels but caused a Sertoli-cell-only syndrome with non-obstructive azoospermia. In the homozygous females studied, the clinical, hormonal, and gonadal phenotypes revealed ovarian dysgenesis consistent with previous reports. Active screening for potential colorectal and other cancer risks in the homozygotic MCM9 subjects has been instigated.

Malignant hyperthermia is a life-threatening disorder, which can be prevented by avoiding certain anesthetic agents. Pathogenic variants in the skeletal muscle ryanodine receptor 1-gene are linked to malignant hyperthermia. We retrospectively studied 15 patients who presented to our clinic with symptoms of muscle dysfunction (weakness, myalgia or cramps) and were later found to have a variant in the skeletal muscle ryanodine receptor 1-gene. Symptoms, creatine kinase levels, electromyography, muscle biopsy and in vitro contracture test results were reviewed. Six out of the eleven patients, with a variant of unknown significance in the skeletal muscle ryanodine receptor 1-gene, had a positive in vitro contracture test, indicating malignant hyperthermia susceptibility. In one patient, with two variants of unknown significance, both variants were required to express the malignant hyperthermia-susceptibility trait. Neurologists should consider screening the skeletal muscle ryanodine receptor 1-gene in patients with myalgia or cramps, even when few to no abnormalities on ancillary testing., Competing Interests: Declaration of Competing Interest J.D.B. and S.H. are a member of the European Reference Network for Neuromuscular Diseases. There is no conflict of interest. No targeted funding was received for this study., (Copyright © 2022. Published by Elsevier B.V.)

EMILIN1 (elastin-microfibril-interface-located-protein-1) is a structural component of the elastic fiber network and localizes to the interface between the fibrillin microfibril scaffold and the elastin core. How EMILIN1 contributes to connective tissue integrity is not fully understood. Here, we report bi-allelic EMILIN1 loss-of-function variants causative for an entity combining cutis laxa, arterial tortuosity, aneurysm formation, and bone fragility, resembling autosomal-recessive cutis laxa type 1B, due to EFEMP2 (FBLN4) deficiency. In both humans and mice, absence of EMILIN1 impairs EFEMP2 extracellular matrix deposition and LOX activity resulting in impaired elastogenesis, reduced collagen crosslinking, and aberrant growth factor signaling. Collagen fiber ultrastructure and histopathology in EMILIN1- or EFEMP2-deficient skin and aorta corroborate these findings and murine Emilin1 -/- femora show abnormal trabecular bone formation and strength. Altogether, EMILIN1 connects elastic fiber network with collagen fibril formation, relevant for both bone and vascular tissue homeostasis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

The Ehlers-Danlos syndromes (EDS) are a heterogeneous group of heritable connective tissue diseases. The autosomal recessive kyphoscoliotic EDS results from deficiency of either lysyl hydroxylase 1 (encoded by PLOD1), crucial for collagen cross-linking; or the peptidyl-prolyl cis-trans isomerase family FK506-binding protein 22 kDa (FKBP22 encoded by FKBP14), a molecular chaperone of types III, IV, VI, and X collagen. This study reports the clinical manifestations of three probands with homozygous pathogenic FKBP14 variants, including the previously reported c.362dupC; p.(Glu122Argfs*7) variant, a novel missense variant (c.587A>G; p.(Asp196Gly)) and a start codon variant (c.2T>G; p.?). Consistent clinical features in the hitherto reported individuals (n = 40) are kyphoscoliosis, generalized joint hypermobility and congenital muscle hypotonia. Severe vascular complications have been observed in 12.5%. A previously unreported feature is microcornea observed in two probands reported here. Both the c.587A>G and the c.362dupC variant cause complete loss of FKBP22. With immunocytochemistry on dermal fibroblasts, we provide the first evidence for intracellular retention of types III and VI collagen in EDS-FKBP14. Scratch wound assays were largely normal. Western blot of proteins involved in the unfolded protein response and autophagy did not reveal significant upregulation in dermal fibroblasts., (© 2022 Wiley Periodicals LLC.)

Congenital heart defects (CHD) are the most common congenital anomalies in liveborn children. In contrast to syndromic CHD (SCHD), the genetic basis of isolated CHD (ICHD) is complex, and the underlying pathogenic mechanisms appear intricate and are incompletely understood. Next to rare Mendelian conditions, somatic mosaicism or a complex multifactorial genetic architecture are assumed for most ICHD. We performed exome sequencing (ES) in 73 parent-offspring ICHD trios using proband DNA extracted from cardiac tissue. We identified six germline de novo variants and 625 germline rare inherited variants with 'damaging' in silico predictions in cardiac-relevant genes expressed in the developing human heart. There were no CHD-relevant somatic variants. Transmission disequilibrium testing (TDT) and association testing (AT) yielded no statistically significant results, except for the AT of missense variants in cilia genes. Somatic mutations are not a common cause of ICHD. Rare de novo and inherited protein-damaging variants may contribute to ICHD, possibly as part of an oligogenic or polygenic disease model. TDT and AT failed to provide informative results, likely due to the lack of power, but provided a framework for future studies in larger cohorts. Overall, the diagnostic value of ES on cardiac tissue is limited in individual ICHD cases.

The vast majority of reported (likely) pathogenic missense variants in the genes coding for the fibrillar collagens leads to the substitution of one of the obligatory glycine residues in the Gly-Xaa-Yaa repeat sequence of the triple helical domain. Their phenotypic consequences and deleterious effects have been well-documented. However, with increasing access to molecular diagnostic testing based on next-generation sequencing techniques, such as sequencing of multi-gene panels and whole-exome sequencing, non-glycine substitutions are more frequently identified in individuals suspected to have a heritable collagen disorder, but their pathogenic effect is often difficult to predict.Some specific non-glycine substitutions in the proα1(I)- (p.(Arg312Cys)) and proα1(III)- (glutamic acid to lysine at different positions) collagen chain have been identified in a number of individuals presenting a phenotype showing features of both classical and vascular Ehlers-Danlos syndrome. The number of reported individuals with these defects is currently very low, and several of these non-glycine substitutions had initially been categorised as variants of unknown significance (VUS), complicating early diagnosis, accurate counselling, management guidelines, and correct classification. This collaborative study reports on the phenotype of 22 and 7 individuals harbouring these rare variants in COL1A1 and COL3A1, respectively, expanding our knowledge on clinical presentation, phenotypic variability, and natural history, and informing on the risk for potentially life-threatening events, such as vascular, gastro-intestinal, and pregnancy-related complications.

Purpose: Providing additional insights on the efficacy of human nuclear transfer (NT). Here, and earlier, NT has been applied to minimize transmission risk of mitochondrial DNA (mtDNA) diseases. NT has also been proposed for treating infertility, but it is still unclear which infertility indications would benefit. In this work, we therefore additionally assess the applicability of NT to overcome failed fertilization., Methods: Patient 1 carries a homoplasmic mtDNA mutation (m.11778G > A). Seventeen metaphase II (MII) oocytes underwent pre-implantation genetic testing (PGT), while five MII oocytes were used for spindle transfer (ST), and one in vitro matured (IVM) metaphase I oocyte underwent early pronuclear transfer (ePNT). Patients 2-3 experienced multiple failed intracytoplasmic sperm injection (ICSI) and ICSI-assisted oocyte activation (AOA) cycles. For these patients, the obtained MII oocytes underwent an additional ICSI-AOA cycle, while the IVM oocytes were subjected to ST., Results: For patient 1, PGT-M confirmed mutation loads close to 100%. All ST-reconstructed oocytes fertilized and cleaved, of which one progressed to the blastocyst stage. The reconstructed ePNT-zygote reached the morula stage. These samples showed an average mtDNA carry-over rate of 2.9% ± 0.8%, confirming the feasibility of NT to reduce mtDNA transmission. For patient 2-3 displaying fertilization failure, ST resulted in, respectively, 4/5 and 6/6 fertilized oocytes, providing evidence, for the first time, that NT can enable successful fertilization in this patient population., Conclusion: Our study showcases the repertoire of disorders for which NT can be beneficial, to overcome either mitochondrial disease transmission or failed fertilization after ICSI-AOA., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: Infantile Caffey disease is a rare disorder characterized by acute inflammation with subperiosteal new bone formation, associated with fever, pain, and swelling of the overlying soft tissue. Symptoms arise within the first weeks after birth and spontaneously resolve before the age of two years. Many, but not all, affected individuals carry the heterozygous pathogenic COL1A1 variant (c.3040C>T, p.(Arg1014Cys))., Methods: We sequenced COL1A1 in 28 families with a suspicion of Caffey disease and performed ultrastructural, immunocytochemical, and biochemical collagen studies on patient skin biopsies., Results: We identified the p.(Arg1014Cys) variant in 23 families and discovered a novel heterozygous pathogenic COL1A1 variant (c.2752C>T, p.(Arg918Cys)) in five. Both arginine to cysteine substitutions are located in the triple helical domain of the proα1(I) procollagen chain. Dermal fibroblasts (one patient with p.(Arg1014Cys) and one with p.(Arg918Cys)) produced molecules with disulfide-linked proα1(I) chains, which were secreted only with p.(Arg1014Cys). No intracellular accumulation of type I procollagen was detected. The dermis revealed mild ultrastructural abnormalities in collagen fibril diameter and packing., Conclusion: The discovery of this novel pathogenic variant expands the limited spectrum of arginine to cysteine substitutions in type I procollagen. Furthermore, it confirms allelic heterogeneity in Caffey disease and impacts its molecular confirmation., (© 2021. The Author(s), under exclusive licence to the American College of Medical Genetics and Genomics.)