BACKGROUND:Virtual panel analysis (VPA) of exome data is a common approach for the molecular diagnosis of congenital heart disease (CHD). However, differences in gene panel composition and patient inclusion criteria limit the evaluation of its diagnostic utility. This study aims to assess the diagnostic yield of VPA in a cohort of patients with CHD across 3 academic centers. METHODS:We collected clinical data including phenotypic features and family history, from 853 probands with CHD who underwent VPA analysis at the Center for Medical Genetics Ghent (525 probands; 471 genes), the University Medical Center Groningen (195 probands; 345 genes), and the University Medical Center Utrecht (133 probands; 55 genes). We evaluated the diagnostic yield by comparing the 3 centers with respect to panel composition and clinical presentation. RESULTS:The Center for Medical Genetics Ghent reported a higher diagnostic yield (9.9%) compared with the University Medical Center Groningen (7.2%) and the University Medical Center Utrecht (5.3%). In all centers, the diagnostic yield was higher in patients presenting with a syndromic constellation and did not differ significantly between the sporadic and familial cases. In 1.7% of the 536 nonsyndromic probands, a molecular cause was identified that typically is associated with syndromic CHD. Twelve genes showed likely pathogenic or pathogenic variants in multiple patients and contributed to 56.2% of the identified causes. CONCLUSIONS:We report an overall diagnostic yield of VPA for CHD of 8.6%, to which only a few genes contribute significantly, highlighting the complex origin of CHD. Since panel size, gene panel content, and local practices largely affect the diagnostic yield, we propose a (minimum) core gene panel for suspected isolated CHD, as well as a coordinated testing strategy for CHD to improve diagnosis and counseling and to catalyze collaborative efforts.

RESEARCH QUESTION:What are the effects of integrating preimplantation genetic testing for aneuploidy into preimplantation genetic testing for monogenic/single gene disorders (PGT-M), termed 'comprehensive PGT' (coPGT-M)? DESIGN:A retrospective cohort of frozen embryo transfer cycles, derived from intracytoplasmic sperm injection, with single embryo transfers (SET) performed between 2019 and 2022 in patients undergoing their first round of assisted reproductive technology. CoPGT-M was implemented from July 2021. Only cycles with at least one biopsied blastocyst were included. Outcomes were compared between 701 embryos from 126 PGT-M cycles and 801 embryos from 131 coPGT-M cycles. RESULTS:The median female age at oocyte retrieval was similar between the PGT-M and coPGT-M cohorts [29.5 (IQR 27.0-33.0) versus 29.5 (IQR 28.0-33.0) years], as were male age, body mass index, and anti-Müllerian hormone concentration. The PGT-M cohort had a significantly lower median number of cumulus-oocyte complexes [15.0 (IQR 10.0-20.0) versus 15.5 (IQR 11.0-26.0); P = 0.039], while the number of zygotes, blastocysts biopsied, and blastocysts eligible for transfer after genetic analysis were comparable. The percentage of cycles resulting in at least one live birth (PGT-M 42.1% versus coPGT-M 51.9%) and the median number of embryo transfers required to reach the first live birth among patients who achieved a live birth [PGT-M 1 (IQR 1-2) versus coPGT-M 1 (1-2)] were comparable. Generalized estimating equation analysis, adjusted for embryo quality, showed higher odds of live birth per SET [adjusted OR (aOR) 1.86, 95% CI 1.21-2.87; P = 0.005] and lower odds of pregnancy loss (aOR 0.50, 95% CI 0.27-0.93; P = 0.028) in the coPGT-M cohort. CONCLUSIONS:While cumulative outcomes per cycle were similar, coPGT-M was associated with significantly higher odds of live birth per embryo transfer and lower risk of pregnancy loss, indicating clinical benefit, even in younger patients.

Genetic defects in the genes encoding fibrillar collagen types I, II, III, V, and XI result in a variety of heritable connective tissue disorders. Next-generation sequencing (NGS) applications have facilitated the screening of these disease genes, but the interpretation of the obtained variants can be challenging. We describe a protocol for both targeted gene panel and exome-wide library preparation, bioinformatic pipeline, and variant classification and interpretation of germline variants in types I, II, III, V, and XI collagen.

This report describes two unrelated prenatal cases of Shwachman-Diamond syndrome (SDS) presenting primarily with severe skeletal anomalies. SDS is a rare autosomal recessive disorder characterized by a triad of bone marrow dysfunction, skeletal abnormalities, and exocrine pancreatic dysfunction. The most common postnatal features include faltering growth, short stature, and neutropenia resulting in recurrent infections. Prenatal presentations could be scarce as the most common features are typically not apparent before birth. Molecular diagnosis of SDS relies on the identification of biallelic loss-of-function pathogenic variants in the SBDS gene. However, molecular genetic analysis is hampered by the presence of a pseudogene (SBDSP1), which can lead to misalignment or gene conversion events. In both reported cases, initial genetic testing was inconclusive. Subsequently, through clinical phenotype reassessment and expanded molecular analysis, the diagnosis of SDS by germline pathogenic SBDS variants (c.258+2T>C p.(?) and c.184A>T p.Lys62Ter) was established. These cases underscore the diagnostic complexity of SDS in prenatal settings and the necessity of comprehensive molecular analysis when facing severe skeletal anomalies suggesting small thoracic skeletal dysplasia and are further supported by an added literature review that expands the prenatal phenotype.

Abstract Aims Genetic testing is routinely recommended in dilated cardiomyopathy (DCM), yet the prevalence and implications of pathogenic/likely pathogenic (P/LP) variants in patients with DCM and left bundle branch block (LBBB) remain unclear. We therefore investigated the electromechanical profile of genotyped patients with DCM and LBBB, and its relationship with cardiac resynchronization therapy (CRT) response and clinical outcomes. Methods Patients with LBBB were selected from a multicenter cohort of 1206 consecutive DCM patients undergoing genetic testing. All underwent sequencing of 20 clinically validated DCM-related genes (ClinGen) and comprehensive electro- and echocardiographic phenotyping, including speckle-tracking strain analysis, categorizing septal strain curves into five stages (LBBB-0 to LBBB-4). CRT response was assessed as end-systolic volume (ESV) reduction and left ventricular ejection fraction (LVEF) improvement. The clinical endpoints were a composite of all-cause mortality, heart transplantation/left ventricular assist device implantation, and heart failure hospitalization (HFH). Results Among 347 DCM patients with LBBB (median age 60[53-68], median LVEF 31%[23-39]), 22 (6%) exhibited P/LP variants. Genotype-positive patients less frequently fulfilled strict LBBB criteria (Strauss:P<0.001) and exhibited less mechanical dyssynchrony (predominantly LBBB-0/1;P<0.001). They showed attenuated reverse remodeling after CRT (ΔLVEF 1%[-6-6] vs. 14%[7-22];P<0.001) and worse clinical outcomes (both composite outcome and HFH;P<0.001). Conversely, advanced LBBB stages excluded an underlying rare genetic variant. P/LP variant status, LVEF, and LBBB stage independently predicted CRT response and composite outcome. Conclusions Genetic testing has a low diagnostic yield in patients with DCM and LBBB. Genotype-positive variants exhibit a distinct electromechanical profile, characterised by atypical electrocardiographic features and markedly reduced mechanical dyssynchrony, poor CRT response, and worse long-term outcomes. Integrating genetic and electromechanical phenotyping may improve individualized risk stratification and management.

Is additional ploidy screening (PGT-A) on blastocysts screened for monogenic conditions (PGT-M) of any added value? Additional ploidy screening improved live birth rates per single embryo transfer and significantly reduced pregnancy loss odds, with no impact on cumulative cycle outcomes. Numerical chromosomal abnormalities in embryos are key contributors to implantation failure, miscarriage, and delayed pregnancy. Preimplantation genetic testing for aneuploidy (PGT-A) aims to improve outcomes by excluding aneuploid embryos, though its utility remains debated. Since July 2021, our center has implemented comprehensive PGT-M (coPGT-M) to simultaneously screen for monogenic conditions and aneuploidies, allowing only unaffected, euploid embryos for transfer. This approach may shorten time to pregnancy and reduce miscarriage risk. This single-center retrospective cohort study analyzed first-rank cycles conducted from December 2019 to October 2022. Outcomes - including biochemical pregnancy, clinical pregnancy, live birth rates, and pregnancy loss - were compared between 126 PGT-M and 131 coPGT-M cycles (226 PGT-M and 177 coPGT-M single embryo transfers [SETs]). Clinical follow-up extended through December 31, 2024. Exclusively first-rank cycles with ≥1 biopsied blastocyst were included. Exclusion criteria encompassed cycles with no oocytes retrieved, no embryos suitable for biopsy, use of non-autologous or cryopreserved oocytes, and translocation carriers. Statistical analyses included Student’s t-tests, Chi-square tests, Fisher’s exact tests, and generalized estimating equations (GEE) models for transfer-specific comparisons. A total of 126 PGT-M and 131 coPGT-M cycles were included, with both groups demonstrating comparable baseline characteristics, including female age (30.4±4.2 vs. 30.3±3.9 years, p = 0.810), AMH (3.00±2.58 vs. 2.93±2.22 mcg/L, p = 0.805), BMI (23.6±4.2 vs. 24.6±4.4 kg/m², p = 0.095), and mode of inheritance of the monogenic condition. Laboratory outcomes were comparable, with the mean number of biopsied blastocysts being 5.6±3.8 in the PGT-M group and 6.1±4.7 in the coPGT-M group (p = 0.308), while the mean number of embryos eligible for transfer were also similar (resp. 2.6±2.2 vs. 2.4±2.0, p = 0.424). At least one ongoing pregnancy or live birth was achieved by 42.1% of patients after PGT-M and 51.9% after coPGT-M (p = 0.114), with a comparable mean number of transfers required (1.55±0.75 vs. 1.47±0.80, p = 0.592). The odds of having an ongoing pregnancy or live birth per SET was 86% higher in the coPGT-M group compared to the PGT-M group (odds ratio 1.86 [CI 1.21;2.87], p = 0.005). Additionally, the odds of experiencing a pregnancy loss were 50% lower in the coPGT-M group compared to the PGT-M group (odds ratio 0.5, [CI 0.27;0.93], p = 0.028). These findings highlight the enhanced outcomes associated with coPGT-M in terms of higher live birth rates and reduced miscarriage odds per single embryo transfer. Not all eligible embryos had been transferred at the time of analysis, and ongoing follow-up will provide more comprehensive cumulative data, likely further strengthening the conclusions. Cumulative outcomes per cycle were comparable; however, coPGT-M showed higher per-SET pregnancy success and lower miscarriage risk. These findings support the potential clinical benefits of incorporating PGT-A into PGT-M protocols, even in younger patients. No

Can mito-TALENs selectively eliminate mitochondrial DNA (mtDNA) carryover following pronuclear transfer (PNT) in a mouse model carrying a heteroplasmic mtDNA mutation? Mito-TALENs effectively reduced mitochondrial DNA mutation load in embryos following pronuclear transfer, without compromising developmental outcomes. Maternally inherited mitochondrial diseases can arise from mutations in mtDNA. PNT can reduce the transmission risk by replacing the cytoplasm of an affected zygote with that of a healthy donor. However, a small amount of mutant mtDNA, known as carryover, remains in the reconstructed embryo, potentially leading to disease reversion if it re-expands overtime. To address this, ongoing efforts focus on eliminating residual pathogenic mtDNA. Mitochondrial-targeted TALENs (mito-TALENs) have been shown to selectively reduce mutant mtDNA in various models. Combining PNT with mito-TALENs may further reduce heteroplasmy and mitigate mtDNA genetic drift, offering a promising strategy to prevent disease recurrence. A murine model with a heteroplasmic mutation in the mitochondrially encoded tRNA alanine gene (mt-Ta, m.5024C>T) was used. Oocytes and zygotes from wild-type (WT, n = 143) and mutant (n = 161) female mice were retrieved, and divided into experimental groups: PNT, PNT with mRNA injection (mito-TALEN or GFP), mRNA injection alone, and unmanipulated controls. Mito-TALEN monomers were designed to target the mutant allele. GFP mRNA injection was used as control for embryo development. mRNAs were produced in vitro. Oocytes and zygotes were collected from superovulated WT and mutant female mice. Oocytes were fertilised using piezo-ICSI. PNT was performed between mutant and WT zygotes. mRNA was injected into WT, mutant, and PNT zygotes. Resulting embryos were cultured to the blastocyst stage in vitro. mtDNA heteroplasmy was analysed across different groups using digital PCR (dPCR). Assay validation was performed with synthetic templates of known mutation loads (0.01–50%). Results were compared with next-generation sequencing (NGS). dPCR reliably detected mutation loads as low as 0.1%. Unmanipulated mutant embryos had mean heteroplasmy levels of 68.59% (SD = 7.76%; n = 31). Mito-TALEN mRNA injection significantly reduced mean mutant load to 40.23% (SD = 9.90%, n = 32, p < 0.0001), whereas GFP mRNA injection had no effect (70.09%, SD = 9.25%, n = 14) showing similar heteroplasmy levels to unmanipulated mutant embryos. Blastocyst rates were comparable across groups, including unmanipulated and mRNA-injected (mito-TALEN and GFP) WT embryos, indicating no adverse effects of mRNA injections. Colocalisation of mito-TALENs with mitochondria was confirmed using MitoTracker staining. PNT was carried out using mutant zygotes as karyoplasts (nuclear donors) and WT zygotes as cytoplasts (mitochondrial donors). Control PNT blastocysts exhibited mtDNA carryover levels of 2.42% (SD = 0.82%, n = 25), while PNT blastocysts injected with mito-TALEN mRNA showed significantly reduced carryover levels to 0.86% (SD = 0.42%, n = 24), with some as low as 0.33% (p < 0.0001). No differences in development rates were observed between groups. At high mutation levels (20–80%), dPCR and NGS results were strongly concordant. However, in PNT embryos, dPCR accurately quantified minor alleles below 1%, whereas NGS produced more false positives and failed to detect mutations below this threshold. In WT samples, dPCR detected minimal mutant alleles (∼0.07%), while NGS reported ∼0.6% mutation loads. This study describes preliminary results and is limited by its small sample size. While dPCR accurately quantified minor alleles, NGS failed to detect mutation levels below 1%, highlighting the need for caution when using less sensitive methodologies for mtDNA mutation load quantification, particularly for nuclear transfer techniques. Our results demonstrate that the sequential application of PNT and mito-TALENs effectively reduces mtDNA carryover, offering a promising strategy for safer application of nuclear transfer. dPCR provides precise mtDNA quantification, outperforming NGS at low mutation levels with superior accuracy and fewer false positives. No

Focal facial dermal dysplasia (FFDD) type IV is a rare inherited facial defect caused by biallelic variants in CYP26C1. This study reports two novel Belgian FFDD type IV cases, both homozygous for a recurrent CYP26C1 frameshift variant, with a common 700 kb haplotype, indicating a founder effect.

The etiology of congenital heart disease (CHD) is complex, comprising both genetic and environmental factors. Despite documented familial occurrences, the genetic etiology remains largely elusive. Trio exome sequencing identified a heterozygous FLT4 splice site variant in two families with respectively tetralogy of Fallot (TOF), and variable CHD comprising both the TOF spectrum and aortic coarctation. In the first family, Sanger sequencing on cDNA confirmed aberrant splicing for the c.985+1G > A variant. In the second family, transcriptome sequencing uncovered altered splicing for the c.1657+6T > C variant, despite normal targeted Sanger sequencing. In conclusion, our study establishes FLT4 splice site variants as a molecular cause of both left and right-sided isolated CHD, with incomplete penetrance. RNA-sequencing emerges as a valuable technique in unraveling the missing inheritability of CHD.

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 (Ca2+) 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 (CaCl2) 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 Ca2+ 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 Ca2+-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 Ca2+ 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.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 Ca2+ 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.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 Ca2+ 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 Ca2+ 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 Ca2+ release during ICSI. Most importantly, AOA treatment using CaCl2 injection followed by double ionomycin exposure is highly effective for this patient group, including those with ACTL7A variants, who also display a Ca2+-release deficiency.STUDY FUNDING/COMPETING INTEREST(S) 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.