Facioscapulohumeral muscular dystrophy-Reproductive counseling, pregnancy, and delivery in a complex multigenetic disease.

Reproductive counseling in facioscapulohumeral muscular dystrophy (FSHD) can be challenging due to the complexity of its underlying genetic mechanisms and due to incomplete penetrance of the disease. Full understanding of the genetic causes and potential inheritance patterns of both distinct FSHD types is essential: FSHD1 is an autosomal dominantly inherited repeat disorder, whereas FSHD2 is a digenic disorder. This has become even more relevant now that prenatal diagnosis and preimplantation genetic diagnosis options are available for FSHD1. Pregnancy and delivery outcomes in FSHD are usually favorable, but clinicians should be aware of the risks. We aim to provide clinicians with case-based strategies for reproductive counseling in FSHD, as well as recommendations for pregnancy and delivery.

Embryo tracking system for high-throughput sequencing-based preimplantation genetic testing.

STUDY QUESTION: Can the embryo tracking system (ETS) increase safety, efficacy and scalability of massively parallel sequencing-based preimplantation genetic testing (PGT)? SUMMARY ANSWER: Applying ETS-PGT, the chance of sample switching is decreased, while scalability and efficacy could easily be increased substantially. WHAT IS KNOWN ALREADY: Although state-of-the-art sequencing-based PGT methods made a paradigm shift in PGT, they still require labor intensive library preparation steps that makes PGT cost prohibitive and poses risks of human errors. To increase the quality assurance, efficiency, robustness and throughput of the sequencing-based assays, barcoded DNA fragments have been used in several aspects of next-generation sequencing (NGS) approach. STUDY DESIGN, SIZE, DURATION: We developed an ETS that substantially alleviates the complexity of the current sequencing-based PGT. With (n = 693) and without (n = 192) ETS, the downstream PGT procedure was performed on both bulk DNA samples (n = 563) and whole-genome amplified (WGAed) few-cell DNA samples (n = 322). Subsequently, we compared full genome haplotype landscapes of both WGAed and bulk DNA samples containing ETS or no ETS. PARTICIPANTS/MATERIALS, SETTING, METHODS: We have devised an ETS to track embryos right after whole-genome amplification (WGA) to full genome haplotype profiles. In this study, we recruited 322 WGAed DNA samples derived from IVF embryos as well as 563 bulk DNA isolated from peripheral blood of prospective parents. To determine possible interference of the ETS in the NGS-based PGT workflow, barcoded DNA fragments were added to DNA samples prior to library preparation and compared to samples without ETS. Coverages and variants were determined. MAIN RESULTS AND THE ROLE OF CHANCE: Current PGT protocols are quality sensitive and prone to sample switching. To avoid sample switching and increase throughput of PGT by sequencing-based haplotyping, six control steps should be carried out manually and checked by a second person in a clinical setting. Here, we developed an ETS approach in which one step only in the entire PGT procedure needs the four-eyes principal. We demonstrate that ETS not only precludes error-prone manual checks but also has no effect on the genomic landscape of preimplantation embryos. Importantly, our approach increases efficacy and throughput of the state-of-the-art PGT methods. LIMITATIONS, REASONS FOR CAUTION: Even though the ETS simplified sequencing-based PGT by avoiding potential errors in six steps in the protocol, if the initial assignment is not performed correctly, it could lead to cross-contamination. However, this can be detected in silico following downstream ETS analysis. Although we demonstrated an approach to evaluate purity of the ETS fragment, it is recommended to perform a pre-PGT quality control assay of the ETS amplicons with non-human DNA, such that the purity of each ETS molecule can be determined prior to ETS-PGT. WIDER IMPLICATIONS OF THE FINDINGS: The ETS-PGT approach notably increases efficacy and scalability of PGT. ETS-PGT has broad applicative value, as it can be tailored to any single- and few-cell sequencing approach where the starting specimen is scarce, as opposed to other methods that require a large number of cells as the input. Moreover, ETS-PGT could easily be adapted to any sequencing-based diagnostic method, including PGT for structural rearrangements and aneuploidies by low-pass sequencing as well as non-invasive prenatal testing. STUDY FUNDING/COMPETING INTEREST(S): M.Z.E. is supported by the EVA (Erfelijkheid Voortplanting & Aanleg) specialty program (grant no. KP111513) of Maastricht University Medical Centre (MUMC+), and the Horizon 2020 innovation (ERIN) (grant no. EU952516) of the European Commission. TRIAL REGISTRATION NUMBER: N/A.

New insights into minor splicing-a transcriptomic analysis of cells derived from TALS patients.

Minor intron splicing plays a central role in human embryonic development and survival. Indeed, biallelic mutations in RNU4ATAC, transcribed into the minor spliceosomal U4atac snRNA, are responsible for three rare autosomal recessive multimalformation disorders named Taybi-Linder (TALS/MOPD1), Roifman (RFMN), and Lowry-Wood (LWS) syndromes, which associate numerous overlapping signs of varying severity. Although RNA-seq experiments have been conducted on a few RFMN patient cells, none have been performed in TALS, and more generally no in-depth transcriptomic analysis of the ∼700 human genes containing a minor (U12-type) intron had been published as yet. We thus sequenced RNA from cells derived from five skin, three amniotic fluid, and one blood biosamples obtained from seven unrelated TALS cases and from age- and sex-matched controls. This allowed us to describe for the first time the mRNA expression and splicing profile of genes containing U12-type introns, in the context of a functional minor spliceosome. Concerning RNU4ATAC-mutated patients, we show that as expected, they display distinct U12-type intron splicing profiles compared to controls, but that rather unexpectedly mRNA expression levels are mostly unchanged. Furthermore, although U12-type intron missplicing concerns most of the expressed U12 genes, the level of U12-type intron retention is surprisingly low in fibroblasts and amniocytes, and much more pronounced in blood cells. Interestingly, we found several occurrences of introns that can be spliced using either U2, U12, or a combination of both types of splice site consensus sequences, with a shift towards splicing using preferentially U2 sites in TALS patients' cells compared to controls.

Diagnostic exome-based preconception carrier testing in consanguineous couples: results from the first 100 couples in clinical practice.

PURPOSE: Consanguineous couples are at increased risk of being heterozygous for the same autosomal recessive (AR) disorder(s), with a 25% risk of affected offspring as a consequence. Until recently, comprehensive preconception carrier testing (PCT) for AR disorders was unavailable in routine diagnostics. Here we developed and implemented such a test in routine clinical care. METHODS: We performed exome sequencing (ES) for 100 consanguineous couples. For each couple, rare variants that could give rise to biallelic variants in offspring were selected. These variants were subsequently filtered against a gene panel consisting of ~2,000 genes associated with known AR disorders (OMIM-based). Remaining variants were classified according to American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines, after which only likely pathogenic and pathogenic (class IV/V) variants, present in both partners, were reported. RESULTS: In 28 of 100 tested consanguineous couples (28%), likely pathogenic and pathogenic variants not previously known in the couple or their family were reported conferring 25% risk of affected offspring. CONCLUSION: ES-based PCT provides a powerful diagnostic tool to identify AR disease carrier status in consanguineous couples. Outcomes provided significant reproductive choices for a higher proportion of these couples than previous tests.

Liquid biopsy: state of reproductive medicine and beyond.

Liquid biopsy is the process of sampling and analyzing body fluids, which enables non-invasive monitoring of complex biological systems in vivo. Liquid biopsy has myriad applications in health and disease as a wide variety of components, ranging from circulating cells to cell-free nucleic acid molecules, can be analyzed. Here, we review different components of liquid biopsy, survey state-of-the-art, non-invasive methods for detecting those components, demonstrate their clinical applications and discuss ethical considerations. Furthermore, we emphasize the importance of artificial intelligence in analyzing liquid biopsy data with the aim of developing ethically-responsible non-invasive technologies that can enhance individualized healthcare. While previous reviews have mainly focused on cancer, this review primarily highlights applications of liquid biopsy in reproductive medicine.

Metabolic events in HIV-infected patients using abacavir are associated with erythrocyte inosine triphosphatase activity.

Objectives: Abacavir use has been associated with an increased risk of cardiovascular disease (CVD) and metabolic events in HIV-infected patients, although this finding was not consistently found. It is unclear whether abacavir only increases this risk in subpopulations of HIV-infected patients. It may be hypothesized that inosine 5'-triphosphate pyrophosphohydrolase (ITPase), an enzyme involved in the metabolism of purine analogues used in HIV treatment, plays a role in the risk of CVD and metabolic events in HIV-infected patients. Methods: ITPase activity and ITPA genotype were determined in 393 HIV-infected patients. ITPase activity <4 mmol IMP/mmol Hb/h was considered decreased. ITPA polymorphisms tested were: c.94C>A (rs1127354) and c.124 + 21A>C (rs7270101). ORs were determined using generalized estimating equation models for developing CVD in patients who had ever been exposed to abacavir, tenofovir or didanosine and for developing metabolic events in patients currently using these drugs. Results: In patients using abacavir, metabolic events were associated with ITPase activity. No association was demonstrated for tenofovir or didanosine. The OR for metabolic events was 3.11 in patients using abacavir with normal ITPase activity (95% CI 1.34-7.21; P = 0.008) compared with patients with decreased ITPase activity [adjusted for age, BMI, cumulative duration of combination ART (cART) use and the use of PI and NNRTI]. CVD was not associated with ITPase activity or ITPA genotype. Conclusions: This study shows, for the first time, that ITPase activity is associated with the occurrence of metabolic events in patients using abacavir. Further studies are needed to confirm this association and to elucidate the possible mechanism.

Evolution of Dihydropyrimidine Dehydrogenase Diagnostic Testing in a Single Center during an 8-Year Period of Time.

OBJECTIVE: Fluoropyrimidine treatment can be optimized based on dihydropyrimidine dehydrogenase (DPD) activity. DPD dysfunction leads to increased exposure to active metabolites, which can result in severe or even fatal toxicity. METHODS: We provide an overview of 8 years of DPD diagnostic testing (n = 1194). RESULTS: Within the study period, our diagnostic test evolved from a single-enzyme measurement using first a radiochemical and then a nonradiochemical assay by ultra HPLC-MS in peripheral blood mononuclear cells with uracil, to a combined enzymatic and genetic test (ie, polymerase chain reaction) followed by Sanger sequence analysis of 4 variants of the DPYD gene (ie, DPYD*2A, DPYD*13, c.2846A>T, and 1129-5923C>G; allele frequencies 0.58%, 0.03%, 0.29%, and 1.35%, respectively). Patients who have 1 of the 4 variants tested (n = 814) have lower enzyme activity than the overall patient group. The majority of patients with the DPYD*2A variant (83%) consistently showed decreased enzyme activity. Only 24 (25.3%) of 95 patients (tested for 4 variants) with low enzyme activity carried a variant. Complete DPYD sequencing in a subgroup with low enzyme activity and without DPYD*2A variant (n = 47) revealed 10 genetic variants, of which 4 have not been described previously. We did not observe a strong link between DPYD genotype and enzyme activity. CONCLUSIONS: Previous studies have shown that DPD status should be determined before treatment with fluoropyrimidine agents to prevent unnecessary side effects with possible fatal consequences. Our study in combination with literature shows that there is a discrepancy between the DPD enzyme activity and the presence of clinically relevant single nucleotide polymorphisms. At this moment, a combination of a genetic and enzyme test is preferable for diagnostic testing. (Curr Ther Res Clin Exp. 2018; 79:XXX-XXX).

PTCH1 isoform 1b is the major transcript in the development of basal cell nevus syndrome.

Basal cell nevus syndrome (BCNS) is an autosomal dominant disorder most commonly caused by a germline mutation in the PTCH1 gene. PTCH1 is known to have different isoforms with different functional properties and expression patterns among tissues. We detected a novel, pathogenic de novo mutation in PTCH1 isoform 1b (c.114delG) in a BCNS patient. Furthermore, we elucidated the specific expression pattern of PTCH1 isoforms in normal skin, BCC and peripheral blood by studying expression of different PTCH1 isoforms. Human skin showed expression of isoforms 1b and 1d, while peripheral blood additionally showed 1a and 1e expression. BCCs showed expression of all isoforms. Here we report a patient with a novel, isoform 1b specific mutation in PTCH1 and thereby distinguish PTCH1 isoform 1b as the major transcript in the development of BCNS.

Mutation-specific effects in germline transmission of pathogenic mtDNA variants.

STUDY QUESTION: Does germline selection (besides random genetic drift) play a role during the transmission of heteroplasmic pathogenic mitochondrial DNA (mtDNA) mutations in humans? SUMMARY ANSWER: We conclude that inheritance of mtDNA is mutation-specific and governed by a combination of random genetic drift and negative and/or positive selection. WHAT IS KNOWN ALREADY: mtDNA inherits maternally through a genetic bottleneck, but the underlying mechanisms are largely unknown. Although random genetic drift is recognized as an important mechanism, selection mechanisms are thought to play a role as well. STUDY DESIGN, SIZE, DURATION: We determined the mtDNA mutation loads in 160 available oocytes, zygotes, and blastomeres of five carriers of the m.3243A>G mutation, one carrier of the m.8993T>G mutation, and one carrier of the m.14487T>C mutation. PARTICIPANTS/MATERIALS, SETTING, METHODS: Mutation loads were determined in PGD samples using PCR assays and analysed mathematically to test for random sampling effects. In addition, a meta-analysis has been performed on mutation load transmission data in the literature to confirm the results of the PGD samples. MAIN RESULTS AND THE ROLE OF CHANCE: By applying the Kimura distribution, which assumes random mechanisms, we found that mtDNA segregations patterns could be explained by variable bottleneck sizes among all our carriers (moment estimates ranging from 10 to 145). Marked differences in the bottleneck size would determine the probability that a carrier produces offspring with mutations markedly different than her own. We investigated whether bottleneck sizes might also be influenced by non-random mechanisms. We noted a consistent absence of high mutation loads in all our m.3243A>G carriers, indicating non-random events. To test this, we fitted a standard and a truncated Kimura distribution to the m.3243A>G segregation data. A Kimura distribution truncated at 76.5% heteroplasmy has a significantly better fit (P-value = 0.005) than the standard Kimura distribution. For the m.8993T>G mutation, we suspect a skewed mutation load distribution in the offspring. To test this hypothesis, we performed a meta-analysis on published blood mutation levels of offspring-mother (O-M) transmission for the m.3243A>G and m.8993T>G mutations. This analysis revealed some evidence that the O-M ratios for the m.8993T>G mutation are different from zero (P-value <0.001), while for the m.3243A>G mutation there was little evidence that the O-M ratios are non-zero. Lastly, for the m.14487T>G mutation, where the whole range of mutation loads was represented, we found no indications for selective events during its transmission. LARGE SCALE DATA: All data are included in the Results section of this article. LIMITATIONS, REASON FOR CAUTION: The availability of human material for the mutations is scarce, requiring additional samples to confirm our findings. WIDER IMPLICATIONS OF THE FINDINGS: Our data show that non-random mechanisms are involved during mtDNA segregation. We aimed to provide the mechanisms underlying these selection events. One explanation for selection against high m.3243A>G mutation loads could be, as previously reported, a pronounced oxidative phosphorylation (OXPHOS) deficiency at high mutation loads, which prohibits oogenesis (e.g. progression through meiosis). No maximum mutation loads of the m.8993T>G mutation seem to exist, as the OXPHOS deficiency is less severe, even at levels close to 100%. In contrast, high mutation loads seem to be favoured, probably because they lead to an increased mitochondrial membrane potential (MMP), a hallmark on which healthy mitochondria are being selected. This hypothesis could provide a possible explanation for the skewed segregation pattern observed. Our findings are corroborated by the segregation pattern of the m.14487T>C mutation, which does not affect OXPHOS and MMP significantly, and its transmission is therefore predominantly determined by random genetic drift. Our conclusion is that mutation-specific selection mechanisms occur during mtDNA inheritance, which has implications for PGD and mitochondrial replacement therapy. STUDY FUNDING/COMPETING INTEREST(S): This work has been funded by GROW-School of Oncology and Developmental Biology. The authors declare no competing interests.

Preimplantation genetic diagnosis for mitochondrial DNA mutations: analysis of one blastomere suffices.

BACKGROUND: Preimplantation genetic diagnosis (PGD) is a reproductive strategy for mitochondrial DNA (mtDNA) mutation carriers, strongly reducing their risk of affected offspring. Embryos either without the mutation or with mutation load below the phenotypic threshold are transferred to the uterus. Because of incidental heteroplasmy deviations in single blastomere and the relatively limited data available, we so far preferred relying on two blastomeres rather than one. Considering the negative effect of a two-blastomere biopsy protocol compared with a single-blastomere biopsy protocol on live birth delivery rate, we re-evaluated the error rate in our current dataset. METHODS: For the m.3243A>G mutation, sufficient embryos/blastomeres were available for a powerful analysis. The diagnostic error rate, defined as a potential false-negative result, based on a threshold of 15%, was determined in 294 single blastomeres analysed in 73 embryos of 9 female m.3243A>G mutation carriers. RESULTS: Only one out of 294 single blastomeres (0.34%) would have resulted in a false-negative diagnosis. False-positive diagnoses were not detected. CONCLUSION: Our findings support a single-blastomere biopsy PGD protocol for the m.3243A>G mutation as the diagnostic error rate is very low. As in the early preimplantation embryo no mtDNA replication seems to occur and the mtDNA is divided randomly among the daughter cells, we conclude this result to be independent of the specific mutation and therefore applicable to all mtDNA mutations.

A comprehensive strategy for exome-based preconception carrier screening.

PURPOSE: Whole-exome sequencing (WES) provides the possibility of genome-wide preconception carrier screening (PCS). Here, we propose a filter strategy to rapidly identify the majority of relevant pathogenic mutations. METHODS: Our strategy was developed using WES data from eight consanguineous and five fictive nonconsanguineous couples and was subsequently applied to 20 other fictive nonconsanguineous couples. Presumably pathogenic variants based on frequency and database annotations or generic characteristics and mutation type were selected in genes shared by the couple and in the female's X-chromosome. Unclassified variants were not included. RESULTS: This yielded an average of 29 (19-51) variants in genes shared by the consanguineous couples and 15 (6-30) shared by the nonconsanguineous couples. For X-linked variants, the numbers per female were 3 (1-5) and 1 (0-3), respectively. Remaining variants were verified manually. The majority were able to be quickly discarded, effectively leaving true pathogenic variants. CONCLUSION: We conclude that WES is applicable for PCS, both for consanguineous and nonconsanguineous couples, with the remaining number of variants being manageable in a clinical setting. The addition of gene panels for filtering was not favorable because it resulted in missing pathogenic variants. It is important to develop and continuously curate databases with pathogenic mutations to further increase the sensitivity of WES-based PCS.Genet Med advance online publication 27 October 2016.

PGD for the m.14487 T>C mitochondrial DNA mutation resulted in the birth of a healthy boy.

We report on the first PGD performed for the m.14487 T>C mitochondrial DNA (mtDNA) mutation in the MT-ND6 gene, associated with Leigh syndrome. The female carrier gave birth to a healthy baby boy at age 42. This case adds to the successes of PGD for mtDNA mutations.

Hypertrophic remodelling in cardiac regulatory myosin light chain (MYL2) founder mutation carriers.

AIMS: Phenotypic heterogeneity and incomplete penetrance are common in patients with hypertrophic cardiomyopathy (HCM). We aim to improve the understanding in genotype-phenotype correlations in HCM, particularly the contribution of an MYL2 founder mutation and risk factors to left ventricular hypertrophic remodelling. METHODS AND RESULTS: We analysed 14 HCM families of whom 38 family members share the MYL2 c.64G > A [p.(Glu22Lys)] mutation and a common founder haplotype. In this unique cohort, we investigated factors influencing phenotypic outcome in addition to the primary mutation. The mutation alone showed benign disease manifestation with low penetrance. The co-presence of additional risk factors for hypertrophy such as hypertension, obesity, or other sarcomeric gene mutation increased disease penetrance substantially and caused HCM in 89% of MYL2 mutation carriers (P = 0.0005). The most prominent risk factor was hypertension, observed in 71% of mutation carriers with HCM and an additional risk factor. CONCLUSION: The MYL2 mutation c.64G > A on its own is incapable of triggering clinical HCM in most carriers. However, the presence of an additional risk factor for hypertrophy, particularly hypertension, adds to the development of HCM. Early diagnosis of risk factors is important for early treatment of MYL2 mutation carriers and close monitoring should be guaranteed in this case. Our findings also suggest that the presence of hypertension or another risk factor for hypertrophy should not be an exclusion criterion for genetic studies.

Next-Generation Sequencing in Oncology: Genetic Diagnosis, Risk Prediction and Cancer Classification.

Next-generation sequencing (NGS) technology has expanded in the last decades with significant improvements in the reliability, sequencing chemistry, pipeline analyses, data interpretation and costs. Such advances make the use of NGS feasible in clinical practice today. This review describes the recent technological developments in NGS applied to the field of oncology. A number of clinical applications are reviewed, i.e., mutation detection in inherited cancer syndromes based on DNA-sequencing, detection of spliceogenic variants based on RNA-sequencing, DNA-sequencing to identify risk modifiers and application for pre-implantation genetic diagnosis, cancer somatic mutation analysis, pharmacogenetics and liquid biopsy. Conclusive remarks, clinical limitations, implications and ethical considerations that relate to the different applications are provided.

Cell-Free RNA Is a Reliable Fetoplacental Marker in Noninvasive Fetal Sex Determination.

BACKGROUND: Noninvasive genetic tests that use cell-free fetal DNA (cffDNA) are used increasingly in prenatal care. A low amount of cffDNA can have detrimental effects on the reliability of these tests. A marker to confirm the presence of fetal nucleic acids is therefore required that is universally applicable and easy to incorporate. METHODS: We developed a novel multiplex, single-tube, noninvasive fetal sex determination assay by combining amplification of AMELY cffDNA with one-step reverse transcription (RT)-PCR of trophoblast-derived cell-free RNA (cfRNA), which functions as a sex-independent fetoplacental marker. We tested plasma samples from 75 pregnant women in duplicate in a blinded fashion. The fetus was considered to be male in the case of a positive result for AMELY and cfRNA amplification in both RT-PCRs. The fetus was considered to be female in the case of negative AMELY and positive cfRNA result in both RT-PCRs. In other cases, the test was repeated. We compared the results with invasive prenatal testing and pregnancy outcomes. RESULTS: The AMELY cffDNA amplification and cfRNA result was unambiguous and identical in duplicate in 71 of 75 plasma samples (95%). Four samples (5%) required an extra replicate because of an absent fetoplacental marker. Thereafter, fetal sex was correctly determined in all 75 plasma samples. CONCLUSIONS: Amplification of trophoblast-derived cfRNA is a reliable marker for the confirmation of the presence of fetoplacentally derived nucleic acids in noninvasive fetal sex determination.

Genome-wide karyomapping accurately identifies the inheritance of single-gene defects in human preimplantation embryos in vitro.

PURPOSE: Our aim was to compare the accuracy of family- or disease-specific targeted haplotyping and direct mutation-detection strategies with the accuracy of genome-wide mapping of the parental origin of each chromosome, or karyomapping, by single-nucleotide polymorphism genotyping of the parents, a close relative of known disease status, and the embryo cell(s) used for preimplantation genetic diagnosis of single-gene defects in a single cell or small numbers of cells biopsied from human embryos following in vitro fertilization. METHODS: Genomic DNA and whole-genome amplification products from embryo samples, which were previously diagnosed by targeted haplotyping, were genotyped for single-nucleotide polymorphisms genome-wide detection and retrospectively analyzed blind by karyomapping. RESULTS: Single-nucleotide polymorphism genotyping and karyomapping were successful in 213/218 (97.7%) samples from 44 preimplantation genetic diagnosis cycles for 25 single-gene defects with various modes of inheritance distributed widely across the genome. Karyomapping was concordant with targeted haplotyping in 208 (97.7%) samples, and the five nonconcordant samples were all in consanguineous regions with limited or inconsistent haplotyping results. CONCLUSION: Genome-wide karyomapping is highly accurate and facilitates analysis of the inheritance of almost any single-gene defect, or any combination of loci, at the single-cell level, greatly expanding the range of conditions for which preimplantation genetic diagnosis can be offered clinically without the need for customized test development.

Detection of PTCH1 Copy-Number Variants in Mosaic Basal Cell Nevus Syndrome.

Basal cell nevus syndrome (BCNS) is an inherited disorder characterized mainly by the development of basal cell carcinomas (BCCs) at an early age. BCNS is caused by heterozygous small-nucleotide variants (SNVs) and copy-number variants (CNVs) in the Patched1 (PTCH1) gene. Genetic diagnosis may be complicated in mosaic BCNS patients, as accurate SNV and CNV analysis requires high-sensitivity methods due to possible low variant allele frequencies. We compared test outcomes for PTCH1 CNV detection using multiplex ligation-probe amplification (MLPA) and digital droplet PCR (ddPCR) with samples from a BCNS patient heterozygous for a PTCH1 CNV duplication and the patient's father, suspected to have a mosaic form of BCNS. ddPCR detected a significantly increased PTCH1 copy-number ratio in the index patient's blood, and the father's blood and tissues, indicating that the father was postzygotic mosaic and the index patient inherited the CNV from him. MLPA only detected the PTCH1 duplication in the index patient's blood and in hair and saliva from the mosaic father. Our data indicate that ddPCR more accurately detects CNVs, even in low-grade mosaic BCNS patients, which may be missed by MLPA. In general, quantitative ddPCR can be of added value in the genetic diagnosis of mosaic BCNS patients and in estimating the recurrence risk for offspring.

Clinical Guideline for Preimplantation Genetic Testing in Inherited Cardiac Diseases.

BACKGROUND: Preimplantation genetic testing (PGT) is a reproductive technology that selects embryos without (familial) genetic variants. PGT has been applied in inherited cardiac disease and is included in the latest American Heart Association/American College of Cardiology guidelines. However, guidelines selecting eligible couples who will have the strongest risk reduction most from PGT are lacking. We developed an objective decision model to select eligibility for PGT and compared its results with those from a multidisciplinary team. METHODS: All couples with an inherited cardiac disease referred to the national PGT center were included. A multidisciplinary team approved or rejected the indication based on clinical and genetic information. We developed a decision model based on published risk prediction models and literature, to evaluate the severity of the cardiac phenotype and the penetrance of the familial variant in referred patients. The outcomes of the model and the multidisciplinary team were compared in a blinded fashion. RESULTS: Eighty-three couples were referred for PGT (1997-2022), comprising 19 different genes for 8 different inherited cardiac diseases (cardiomyopathies and arrhythmias). Using our model and proposed cutoff values, a definitive decision was reached for 76 (92%) couples, aligning with 95% of the multidisciplinary team decisions. In a prospective cohort of 11 couples, we showed the clinical applicability of the model to select couples most eligible for PGT. CONCLUSIONS: The number of PGT requests for inherited cardiac diseases increases rapidly, without the availability of specific guidelines. We propose a 2-step decision model that helps select couples with the highest risk reduction for cardiac disease in their offspring after PGT.

Genome sequencing as a generic diagnostic strategy for rare disease.

BACKGROUND: To diagnose the full spectrum of hereditary and congenital diseases, genetic laboratories use many different workflows, ranging from karyotyping to exome sequencing. A single generic high-throughput workflow would greatly increase efficiency. We assessed whether genome sequencing (GS) can replace these existing workflows aimed at germline genetic diagnosis for rare disease. METHODS: We performed short-read GS (NovaSeq™6000; 150 bp paired-end reads, 37 × mean coverage) on 1000 cases with 1271 known clinically relevant variants, identified across different workflows, representative of our tertiary diagnostic centers. Variants were categorized into small variants (single nucleotide variants and indels < 50 bp), large variants (copy number variants and short tandem repeats) and other variants (structural variants and aneuploidies). Variant calling format files were queried per variant, from which workflow-specific true positive rates (TPRs) for detection were determined. A TPR of ≥ 98% was considered the threshold for transition to GS. A GS-first scenario was generated for our laboratory, using diagnostic efficacy and predicted false negative as primary outcome measures. As input, we modeled the diagnostic path for all 24,570 individuals referred in 2022, combining the clinical referral, the transition of the underlying workflow(s) to GS, and the variant type(s) to be detected. RESULTS: Overall, 95% (1206/1271) of variants were detected. Detection rates differed per variant category: small variants in 96% (826/860), large variants in 93% (341/366), and other variants in 87% (39/45). TPRs varied between workflows (79-100%), with 7/10 being replaceable by GS. Models for our laboratory indicate that a GS-first strategy would be feasible for 84.9% of clinical referrals (750/883), translating to 71% of all individuals (17,444/24,570) receiving GS as their primary test. An estimated false negative rate of 0.3% could be expected. CONCLUSIONS: GS can capture clinically relevant germline variants in a 'GS-first strategy' for the majority of clinical indications in a genetics diagnostic lab.