Unravelling undiagnosed rare disease cases by HiFi long-read genome sequencing.

Solve-RD is a pan-European rare disease (RD) research program that aims to identify disease-causing genetic variants in previously undiagnosed RD families. We utilised 10-fold coverage HiFi long-read sequencing (LRS) for detecting causative structural variants (SVs), single nucleotide variants (SNVs), insertion-deletions (InDels), and short tandem repeat (STR) expansions in extensively studied RD families without clear molecular diagnoses. Our cohort includes 293 individuals from 114 genetically undiagnosed RD families selected by European Rare Disease Network (ERN) experts. Of these, 21 families were affected by so-called 'unsolvable' syndromes for which genetic causes remain unknown, and 93 families with at least one individual affected by a rare neurological, neuromuscular, or epilepsy disorder without genetic diagnosis despite extensive prior testing. Clinical interpretation and orthogonal validation of variants in known disease genes yielded thirteen novel genetic diagnoses due to de novo and rare inherited SNVs, InDels, SVs, and STR expansions. In an additional four families, we identified a candidate disease-causing SV affecting several genes including an MCF2 / FGF13 fusion and PSMA3 deletion. However, no common genetic cause was identified in any of the 'unsolvable' syndromes. Taken together, we found (likely) disease-causing genetic variants in 13.0% of previously unsolved families and additional candidate disease-causing SVs in another 4.3% of these families. In conclusion, our results demonstrate the added value of HiFi long-read genome sequencing in undiagnosed rare diseases.

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.

Refining the 9q34.3 microduplication syndrome reveals mild neurodevelopmental features associated with a distinct global DNA methylation profile.

Precise regulation of gene expression is important for correct neurodevelopment. 9q34.3 deletions affecting the EHMT1 gene result in a syndromic neurodevelopmental disorder named Kleefstra syndrome. In contrast, duplications of the 9q34.3 locus encompassing EHMT1 have been suggested to cause developmental disorders, but only limited information has been available. We have identified 15 individuals from 10 unrelated families, with 9q34.3 duplications <1.5 Mb in size, encompassing EHMT1 entirely. Clinical features included mild developmental delay, mild intellectual disability or learning problems, autism spectrum disorder, and behavior problems. The individuals did not consistently display dysmorphic features, congenital anomalies, or growth abnormalities. DNA methylation analysis revealed a weak DNAm profile for the cases with 9q34.3 duplication encompassing EHMT1, which could segregate the majority of the affected cases from controls. This study shows that individuals with 9q34.3 duplications including EHMT1 gene present with mild non-syndromic neurodevelopmental disorders and DNA methylation changes different from Kleefstra syndrome.

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.

Exploring uncertainties regarding unsolicited findings in genetic testing.

OBJECTIVES: Non-normative uncertainty (uncertainty about empirical facts) and normative uncertainty (uncertainty about moral values or beliefs) regarding unsolicited findings (UFs) might play an important role in clinical genetics. Identifying normative uncertainty is of special interest since it might guide towards novel directions for counseling practice. This study aims to gain insight into the role of non-normative and normative uncertainty regarding UFs, as expressed by counselees and counselors. METHODS: We performed a secondary qualitative analysis of interviews with counselees (n = 20) and counselors (n = 20) who had been confronted with UFs. Following a deductive approach, we used Han et al.'s existing theoretical framework of uncertainty, in which we additionally incorporated normative uncertainty. RESULTS: Major issues of non-normative uncertainty were practical and personal for counselees, whilst counselors' uncertainty pertained mainly to scientific issues. Normative uncertainty was a major theme throughout the interviews. We encountered the moral conflicts of autonomy vs. beneficence and non-maleficence and of autonomy vs. truthfulness. CONCLUSION: Non-normative uncertainty regarding UFs highlights the need to gain more insight in their penetrance and clinical utility. This study suggests moral conflicts are a major source of feelings of uncertainty in clinical genetics. PRACTICE IMPLICATIONS: Exploring counselees' non-normative uncertainties and normative conflicts seems a prerequisite to optimize genetic counseling.

Left Atrial Function in Patients with Titin Cardiomyopathy.

BACKGROUND: Truncating variants in titin (TTNtv) are the most prevalent genetic etiology of dilated cardiomyopathy (DCM). Although TTNtv has been associated with atrial fibrillation, it remains unknown whether and how left atrial (LA) function differs between patients with DCM with and without TTNtv. We aimed to determine and compare LA function in patients with DCM with and without TTNtv and to evaluate whether and how left ventricular (LV) function affects the LA using computational modeling. METHODS AND RESULTS: Patients with DCM from the Maastricht DCM registry that underwent genetic testing and cardiovascular magnetic resonance (CMR) were included in the current study. Subsequent computational modeling (CircAdapt model) was performed to identify potential LV and LA myocardial hemodynamic substrates. In total, 377 patients with DCM (n = 42 with TTNtv, n = 335 without a genetic variant) were included (median age 55 years, interquartile range [IQR] 46-62 years, 62% men). Patients with TTNtv had a larger LA volume and decreased LA strain compared with patients without a genetic variant (LA volume index 60 mLm-2 [IQR 49-83] vs 51 mLm-2 [IQR 42-64]; LA reservoir strain 24% [IQR 10-29] vs 28% [IQR 20-34]; LA booster strain 9% [IQR 4-14] vs 14% [IQR 10-17], respectively; all P < .01). Computational modeling suggests that while the observed LV dysfunction partially explains the observed LA dysfunction in the patients with TTNtv, both intrinsic LV and LA dysfunction are present in patients with and without a TTNtv. CONCLUSIONS: Patients with DCM with TTNtv have more severe LA dysfunction compared with patients without a genetic variant. Insights from computational modeling suggest that both intrinsic LV and LA dysfunction are present in patients with DCM with and without TTNtv.

Further delineation of the rare GDACCF (global developmental delay, absent or hypoplastic corpus callosum, dysmorphic facies syndrome): genotype and phenotype of 22 patients with ZNF148 mutations.

BACKGROUND: Pathogenic variants in the zinc finger protein coding genes are rare causes of intellectual disability and congenital malformations. Mutations in the ZNF148 gene causing GDACCF syndrome (global developmental delay, absent or hypoplastic corpus callosum, dysmorphic facies; MIM #617260) have been reported in five individuals so far. METHODS: As a result of an international collaboration using GeneMatcher Phenome Central Repository and personal communications, here we describe the clinical and molecular genetic characteristics of 22 previously unreported individuals. RESULTS: The core clinical phenotype is characterised by developmental delay particularly in the domain of speech development, postnatal growth retardation, microcephaly and facial dysmorphism. Corpus callosum abnormalities appear less frequently than suggested by previous observations. The identified mutations concerned nonsense or frameshift variants that were mainly located in the last exon of the ZNF148 gene. Heterozygous deletion including the entire ZNF148 gene was found in only one case. Most mutations occurred de novo, but were inherited from an affected parent in two families. CONCLUSION: The GDACCF syndrome is clinically diverse, and a genotype-first approach, that is, exome sequencing is recommended for establishing a genetic diagnosis rather than a phenotype-first approach. However, the syndrome may be suspected based on some recurrent, recognisable features. Corpus callosum anomalies were not as constant as previously suggested, we therefore recommend to replace the term 'GDACCF syndrome' with 'ZNF148-related neurodevelopmental disorder'.

A recurrent de novo MAX p.Arg60Gln variant causes a syndromic overgrowth disorder through differential expression of c-Myc target genes.

Cyclin D2 (CCND2) stabilization underpins a range of macrocephaly-associated disorders through mutation of CCND2 or activating mutations in upstream genes encoding PI3K-AKT pathway components. Here, we describe three individuals with overlapping macrocephaly-associated phenotypes who carry the same recurrent de novo c.179G>A (p.Arg60Gln) variant in Myc-associated factor X (MAX). The mutation, located in the b-HLH-LZ domain, causes increased intracellular CCND2 through increased transcription but it does not cause stabilization of CCND2. We show that the purified b-HLH-LZ domain of MAXArg60Gln (Max∗Arg60Gln) binds its target E-box sequence with a lower apparent affinity. This leads to a more efficient heterodimerization with c-Myc resulting in an increase in transcriptional activity of c-Myc in individuals carrying this mutation. The recent development of Omomyc-CPP, a cell-penetrating b-HLH-LZ-domain c-Myc inhibitor, provides a possible therapeutic option for MAXArg60Gln individuals, and others carrying similar germline mutations resulting in dysregulated transcriptional c-Myc activity.

Prevalence of chromosomal alterations in first-trimester spontaneous pregnancy loss.

Pregnancy loss is often caused by chromosomal abnormalities of the conceptus. The prevalence of these abnormalities and the allocation of (ab)normal cells in embryonic and placental lineages during intrauterine development remain elusive. In this study, we analyzed 1,745 spontaneous pregnancy losses and found that roughly half (50.4%) of the products of conception (POCs) were karyotypically abnormal, with maternal and paternal age independently contributing to the increased genomic aberration rate. We applied genome haplarithmisis to a subset of 94 pregnancy losses with normal parental and POC karyotypes. Genotyping of parental DNA as well as POC extra-embryonic mesoderm and chorionic villi DNA, representing embryonic and trophoblastic tissues, enabled characterization of the genomic landscape of both lineages. Of these pregnancy losses, 35.1% had chromosomal aberrations not previously detected by karyotyping, increasing the rate of aberrations of pregnancy losses to 67.8% by extrapolation. In contrast to viable pregnancies where mosaic chromosomal abnormalities are often restricted to chorionic villi, such as confined placental mosaicism, we found a higher degree of mosaic chromosomal imbalances in extra-embryonic mesoderm rather than chorionic villi. Our results stress the importance of scrutinizing the full allelic architecture of genomic abnormalities in pregnancy loss to improve clinical management and basic research of this devastating condition.

Clonal Hematopoiesis Has Prognostic Value in Dilated Cardiomyopathy Independent of Age and Clone Size.

BACKGROUND: Clonal hematopoiesis (CH) gives rise to mutated leukocyte clones that induce cardiovascular inflammation and thereby impact the disease course in atherosclerosis and ischemic heart failure. CH of indeterminate potential refers to a variant allele frequency (VAF; a marker for clone size) in blood of ≥2%. The impact of CH clones-including small clone sizes (VAF <0.5%)-in nonischemic dilated cardiomyopathy (DCM) remains largely undetermined. OBJECTIVES: The authors sought to establish the prognostic impact of CH in DCM including small clones. METHODS: CH is determined using an ultrasensitive single-molecule molecular inversion probe technique that allows detection of clones down to a VAF of 0.01%. Cardiac death and all-cause mortality were analyzed using receiver-operating characteristic curve-optimized VAF cutoff values. RESULTS: A total of 520 DCM patients have been included. One hundred and nine patients (21%) had CH driver mutations, of which 45 had a VAF of ≥2% and 31 <0.5%. The median follow-up duration was 6.5 years [IQR: 4.7-9.7 years]. DCM patients with CH have a higher risk of cardiac death (HR: 2.33 using a VAF cutoff of 0.36%, 95% CI: 1.24-4.40) and all-cause mortality (HR: 1.72 using a VAF cutoff of 0.06%, 95% CI: 1.10-2.69), independent of age, sex, left ventricular ejection fraction, and New York Heart Association classification. CONCLUSIONS: CH predicts cardiac death and all-cause mortality in DCM patients with optimal thresholds for clone size of 0.36% and 0.06%, respectively. Therefore, CH is prognostically relevant, independent of clone size in patients with DCM.

An E280K Missense Variant in KCND3/Kv4.3-Case Report and Functional Characterization.

A five-year-old girl presented with headache attacks, clumsiness, and a history of transient gait disturbances. She and her father, mother, twin sister, and brother underwent neurological evaluation, neuroimaging, and exome sequencing covering 357 genes associated with movement disorders. Sequencing revealed the new variant KCND3 c.838G>A, p.E280K in the father and sisters, but not in the mother and brother. KCND3 encodes voltage-gated potassium channel D3 (Kv4.3) and mutations have been associated with spinocerebellar ataxia type 19/22 (SCA19/22) and cardiac arrhythmias. SCA19/22 is characterized by ataxia, Parkinsonism, peripheral neuropathy, and sometimes, intellectual disability. Neuroimaging, EEG, and ECG were unremarkable. Mild developmental delay with impaired fluid reasoning was observed in both sisters, but not in the brother. None of the family members demonstrated ataxia or parkinsonism. In Xenopus oocyte electrophysiology experiments, E280K was associated with a rightward shift in the Kv4.3 voltage-activation relationship of 11 mV for WT/E280K and +17 mV for E280K/E280K relative to WT/WT. Steady-state inactivation was similarly right-shifted. Maximal peak current amplitudes were similar for WT/WT, WT/E280K, and E280K/E280K. Our data indicate that Kv4.3 E280K affects channel activation and inactivation and is associated with developmental delay. However, E280K appears to be relatively benign considering it does not result in overt ataxia.

Systemic Inflammation and Normocytic Anemia in DOCK11 Deficiency.

BACKGROUND: Increasing evidence links genetic defects affecting actin-regulatory proteins to diseases with severe autoimmunity and autoinflammation, yet the underlying molecular mechanisms are poorly understood. Dedicator of cytokinesis 11 (DOCK11) activates the small Rho guanosine triphosphatase (GTPase) cell division cycle 42 (CDC42), a central regulator of actin cytoskeleton dynamics. The role of DOCK11 in human immune-cell function and disease remains unknown. METHODS: We conducted genetic, immunologic, and molecular assays in four patients from four unrelated families who presented with infections, early-onset severe immune dysregulation, normocytic anemia of variable severity associated with anisopoikilocytosis, and developmental delay. Functional assays were performed in patient-derived cells, as well as in mouse and zebrafish models. RESULTS: We identified rare, X-linked germline mutations in DOCK11 in the patients, leading to a loss of protein expression in two patients and impaired CDC42 activation in all four patients. Patient-derived T cells did not form filopodia and showed abnormal migration. In addition, the patient-derived T cells, as well as the T cells from Dock11-knockout mice, showed overt activation and production of proinflammatory cytokines that were associated with an increased degree of nuclear translocation of nuclear factor of activated T cell 1 (NFATc1). Anemia and aberrant erythrocyte morphologic features were recapitulated in a newly generated dock11-knockout zebrafish model, and anemia was amenable to rescue on ectopic expression of constitutively active CDC42. CONCLUSIONS: Germline hemizygous loss-of-function mutations affecting the actin regulator DOCK11 were shown to cause a previously unknown inborn error of hematopoiesis and immunity characterized by severe immune dysregulation and systemic inflammation, recurrent infections, and anemia. (Funded by the European Research Council and others.).

Comprehensive de novo mutation discovery with HiFi long-read sequencing.

BACKGROUND: Long-read sequencing (LRS) techniques have been very successful in identifying structural variants (SVs). However, the high error rate of LRS made the detection of small variants (substitutions and short indels < 20 bp) more challenging. The introduction of PacBio HiFi sequencing makes LRS also suited for detecting small variation. Here we evaluate the ability of HiFi reads to detect de novo mutations (DNMs) of all types, which are technically challenging variant types and a major cause of sporadic, severe, early-onset disease. METHODS: We sequenced the genomes of eight parent-child trios using high coverage PacBio HiFi LRS (~ 30-fold coverage) and Illumina short-read sequencing (SRS) (~ 50-fold coverage). De novo substitutions, small indels, short tandem repeats (STRs) and SVs were called in both datasets and compared to each other to assess the accuracy of HiFi LRS. In addition, we determined the parent-of-origin of the small DNMs using phasing. RESULTS: We identified a total of 672 and 859 de novo substitutions/indels, 28 and 126 de novo STRs, and 24 and 1 de novo SVs in LRS and SRS respectively. For the small variants, there was a 92 and 85% concordance between the platforms. For the STRs and SVs, the concordance was 3.6 and 0.8%, and 4 and 100% respectively. We successfully validated 27/54 LRS-unique small variants, of which 11 (41%) were confirmed as true de novo events. For the SRS-unique small variants, we validated 42/133 DNMs and 8 (19%) were confirmed as true de novo event. Validation of 18 LRS-unique de novo STR calls confirmed none of the repeat expansions as true DNM. Confirmation of the 23 LRS-unique SVs was possible for 19 candidate SVs of which 10 (52.6%) were true de novo events. Furthermore, we were able to assign 96% of DNMs to their parental allele with LRS data, as opposed to just 20% with SRS data. CONCLUSIONS: HiFi LRS can now produce the most comprehensive variant dataset obtainable by a single technology in a single laboratory, allowing accurate calling of substitutions, indels, STRs and SVs. The accuracy even allows sensitive calling of DNMs on all variant levels, and also allows for phasing, which helps to distinguish true positive from false positive DNMs.

ANK2 loss-of-function variants are associated with epilepsy, and lead to impaired axon initial segment plasticity and hyperactive network activity in hiPSC-derived neuronal networks.

PURPOSE: To characterize a novel neurodevelopmental syndrome due to loss-of-function (LoF) variants in Ankyrin 2 (ANK2), and to explore the effects on neuronal network dynamics and homeostatic plasticity in human-induced pluripotent stem cell-derived neurons. METHODS: We collected clinical and molecular data of 12 individuals with heterozygous de novo LoF variants in ANK2. We generated a heterozygous LoF allele of ANK2 using CRISPR/Cas9 in human-induced pluripotent stem cells (hiPSCs). HiPSCs were differentiated into excitatory neurons, and we measured their spontaneous electrophysiological responses using micro-electrode arrays (MEAs). We also characterized their somatodendritic morphology and axon initial segment (AIS) structure and plasticity. RESULTS: We found a broad neurodevelopmental disorder (NDD), comprising intellectual disability, autism spectrum disorders and early onset epilepsy. Using MEAs, we found that hiPSC-derived neurons with heterozygous LoF of ANK2 show a hyperactive and desynchronized neuronal network. ANK2-deficient neurons also showed increased somatodendritic structures and altered AIS structure of which its plasticity is impaired upon activity-dependent modulation. CONCLUSIONS: Phenotypic characterization of patients with de novo ANK2 LoF variants defines a novel NDD with early onset epilepsy. Our functional in vitro data of ANK2-deficient human neurons show a specific neuronal phenotype in which reduced ANKB expression leads to hyperactive and desynchronized neuronal network activity, increased somatodendritic complexity and AIS structure and impaired activity-dependent plasticity of the AIS.

Diagnostic and prognostic relevance of using large gene panels in the genetic testing of patients with dilated cardiomyopathy.

It was previously suggested that increasing the number of genes on diagnostic gene panels could increase the genetic yield in patient with dilated cardiomyopathy (DCM). We explored the diagnostic and prognostic relevance of testing DCM patients with an expanded gene panel. The current study included 225 consecutive DCM patients who had no genetic diagnosis after a 48-gene cardiomyopathy-panel. These were then evaluated using an expanded gene panel of 299 cardiac-associated genes. A likely pathogenic/pathogenic (P/LP) variant was detected in 13 patients. Five variants were reclassifications of variants found in genes which were already detected using the 48 gene panel. Only one of the other eight variants could explain the phenotype of the patient (KCNJ2). The panel detected 186 VUSs in 127 patients (of which 6 also had a P/LP variant). The presence of a VUS was significantly associated with the combined end-point of mortality, heart failure hospitalization, heart transplantation or life-threatening arrhythmias(HR, 2.04 [95% CI, 1.15 to 3.65]; p = 0.02). The association of a VUS with prognosis remained when we only included VUSs in robust DCM-associated genes (high suspicious VUSs), but disappeared when we only included VUSs in non-robust DCM-associated genes (low suspicious VUSs), highlighting the importance of weighing of VUSs. Overall, the use of large gene panels for genetic testing in DCM does not increase the diagnostic yield, although a VUS in a robust DCM-associated gene is associated with an adverse prognosis. Altogether, current diagnostic gene panels should be limited to the robust DCM-associated genes.

Clustering of Cardiac Transcriptome Profiles Reveals Unique: Subgroups of Dilated Cardiomyopathy Patients.

Dilated cardiomyopathy is a heterogeneous disease characterized by multiple genetic and environmental etiologies. The majority of patients are treated the same despite these differences. The cardiac transcriptome provides information on the patient's pathophysiology, which allows targeted therapy. Using clustering techniques on data from the genotype, phenotype, and cardiac transcriptome of patients with early- and end-stage dilated cardiomyopathy, more homogeneous patient subgroups are identified based on shared underlying pathophysiology. Distinct patient subgroups are identified based on differences in protein quality control, cardiac metabolism, cardiomyocyte function, and inflammatory pathways. The identified pathways have the potential to guide future treatment and individualize patient care.

Evolution of age-related mutation-driven clonal haematopoiesis over 20 years is associated with metabolic dysfunction in obesity.

BACKGROUND: Haematopoietic clones caused by somatic mutations with ≥2% variant allele frequency (VAF) increase with age and are linked to risk of haematological malignancies and cardiovascular disease. Recent observations suggest that smaller clones (VAF<2%) are also associated with adverse outcomes. Our aims were to determine the prevalence of clonal haematopoiesis driven by clones of variable sizes in individuals with obesity treated by usual care or bariatric surgery (a treatment that improves metabolic status), and to examine the expansion of clones in relation to age and metabolic dysregulation over up to 20 years. METHODS: Clonal haematopoiesis-driver mutations (CHDMs) were identified in blood samples from participants of the Swedish Obese Subjects intervention study. Using an ultrasensitive assay, we analysed single-timepoint samples from 1050 individuals treated by usual care and 841 individuals who had undergone bariatric surgery, and multiple-timepoint samples taken over 20 years from a subset (n = 40) of the individuals treated by usual care. FINDINGS: In this explorative study, prevalence of CHDMs was similar in the single-timepoint usual care and bariatric surgery groups (20.6% and 22.5%, respectively, P = 0.330), with VAF ranging from 0.01% to 31.15%. Clone sizes increased with age in individuals with obesity, but not in those who underwent bariatric surgery. In the multiple-timepoint analysis, VAF increased by on average 7% (range -4% to 24%) per year and rate of clone growth was negatively associated with HDL-cholesterol (R = -0.68, 1.74 E-04). INTERPRETATION: Low HDL-C was associated with growth of haematopoietic clones in individuals with obesity treated by usual care. FUNDING: The Swedish Research Council, The Swedish state under an agreement between the Swedish government and the county councils, the ALF (Avtal om Läkarutbildning och Forskning) agreement, The Swedish Heart-Lung Foundation, The Novo Nordisk Foundation, The European Research Council, The Netherlands Organisation for Scientific Research.

Prevalence and Clinical Consequences of Multiple Pathogenic Variants in Dilated Cardiomyopathy.

BACKGROUND: Dilated cardiomyopathy (DCM) was considered a monogenetic disease that can be caused by over 60 genes. Evidence suggests that the combination of multiple pathogenic variants leads to greater disease severity and earlier onset. So far, not much is known about the prevalence and disease course of multiple pathogenic variants in patients with DCM. To gain insight into these knowledge gaps, we (1) systematically collected clinical information from a well-characterized DCM cohort and (2) created a mouse model. METHODS: Complete cardiac phenotyping and genotyping was performed in 685 patients with consecutive DCM. Compound heterozygous digenic (LMNA [lamin]/titin deletion A-band) with monogenic (LMNA/wild-type) and wild-type/wild-type mice were created and phenotypically followed over time. RESULTS: One hundred thirty-one likely pathogenic/pathogenic (LP/P) variants in robust DCM-associated genes were found in 685 patients with DCM (19.1%) genotyped for the robust genes. Three of the 131 patients had a second LP/P variant (2.3%). These 3 patients had a comparable disease onset, disease severity, and clinical course to patients with DCM with one LP/P. The LMNA/Titin deletion A-band mice had no functional differences compared with the LMNA/wild-type mice after 40 weeks of follow-up, although RNA-sequencing suggests increased cardiac stress and sarcomere insufficiency in the LMNA/Titin deletion A-band mice. CONCLUSIONS: In this study population, 2.3% of patients with DCM with one LP/P also have a second LP/P in a different gene. Although the second LP/P does not seem to influence the disease course of DCM in patients and mice, the finding of a second LP/P can be of importance to their relatives.

Autism spectrum disorder and brain volume link through a set of mTOR-related genes.

BACKGROUND: Larger than average head and brain sizes are often observed in individuals with autism spectrum disorders (ASDs). ASD and brain volume are both highly heritable, with multiple genetic variants contributing. However, it is unclear whether ASD and brain volume share any genetic mechanisms. Genes from the mammalian target of rapamycin (mTOR) pathway influence brain volume, and variants are found in rare genetic syndromes that include ASD features. Here we investigated whether variants in mTOR-related genes are also associated with ASD and if they constitute a genetic link between large brains and ASD. METHODS: We extended our analyses between large heads (macrocephaly) and rare de novo mTOR-related variants in an intellectual disability cohort (N = 2,258). Subsequently using Fisher's exact tests we investigated the co-occurrence of mTOR-related de novo variants and ASD in the de-novo-db database (N = 23,098). We next selected common genetic variants within a set of 96 mTOR-related genes in genome-wide genetic association data of ASD (N = 46,350) to test gene-set association using MAGMA. Lastly, we tested genetic correlation between genome-wide genetic association data of ASD (N = 46,350) and intracranial volume (N = 25,974) globally using linkage disequilibrium score regression as well as mTOR specific by restricting the genetic correlation to the mTOR-related genes using GNOVA. RESULTS: Our results show that both macrocephaly and ASD occur above chance level in individuals carrying rare de novo variants in mTOR-related genes. We found a significant mTOR gene-set association with ASD (p = .0029) and an mTOR-stratified positive genetic correlation between ASD and intracranial volume (p = .027), despite the absence of a significant genome-wide correlation (p = .81). CONCLUSIONS: This work indicates that both rare and common variants in mTOR-related genes are associated with brain volume and ASD and genetically correlate them in the expected direction. We demonstrate that genes involved in mTOR signalling are potential mediators of the relationship between having a large brain and having ASD.

Clinical geneticists' views on and experiences with unsolicited findings in next-generation sequencing: "A great technology creating new dilemmas".

Unsolicited findings (UFs) from diagnostic genetic testing are a subject of debate. The emerging consensus is that some UFs from genetic testing should be disclosed, but recommendations on UF disclosure generally leave room for variation in practice. This study aimed to explore clinical geneticists' views on and experiences with UFs during pretest counseling and UF disclosure. We interviewed 20 certified clinical genetics medical specialists and clinical genetics residents, working in 7 Dutch genetic centers. Participants indicated that discussing the probability of detecting UFs is an integral part of pretest counseling and informed consent. However, they expressed doubts about the degree to which this discussion should occur and about what information they should share with patients. They argued that the contents of their counseling should depend on the individual patient's capacity to understand information. These results endorse the importance of tailored pretest counseling alongside informed consent for optimal genetic consultations. While "medical actionability" is broadly accepted as an important criterion for the disclosure of UFs, participants experienced substantial uncertainty regarding this concept. This study underscores the need for further demarcation of what exactly constitutes medical actionability. Installation of an expert panel to help healthcare professionals decide what variants to disclose will support them when facing the dilemmas presented by UFs.