Long-read sequencing and optical mapping generates near T2T assemblies that resolves a centromeric translocation.

Long-read genome sequencing (lrGS) is a promising method in genetic diagnostics. Here we investigate the potential of lrGS to detect a disease-associated chromosomal translocation between 17p13 and the 19 centromere. We constructed two sets of phased and non-phased de novo assemblies; (i) based on lrGS only and (ii) hybrid assemblies combining lrGS with optical mapping using lrGS reads with a median coverage of 34X. Variant calling detected both structural variants (SVs) and small variants and the accuracy of the small variant calling was compared with those called with short-read genome sequencing (srGS). The de novo and hybrid assemblies had high quality and contiguity with N50 of 62.85 Mb, enabling a near telomere to telomere assembly with less than a 100 contigs per haplotype. Notably, we successfully identified the centromeric breakpoint of the translocation. A concordance of 92% was observed when comparing small variant calling between srGS and lrGS. In summary, our findings underscore the remarkable potential of lrGS as a comprehensive and accurate solution for the analysis of SVs and small variants. Thus, lrGS could replace a large battery of genetic tests that were used for the diagnosis of a single symptomatic translocation carrier, highlighting the potential of lrGS in the realm of digital karyotyping.

Extending the phenotypes associated with TRIO gene variants in a cohort of 25 patients and review of the literature.

The TRIO gene encodes a rho guanine exchange factor, the function of which is to exchange GDP to GTP, and hence to activate Rho GTPases, and has been described to impact neurodevelopment. Specific genotype-to-phenotype correlations have been established previously describing striking differentiating features seen in variants located in specific domains of the TRIO gene that are associated with opposite effects on RAC1 activity. Currently, 32 cases with a TRIO gene alteration have been published in the medical literature. Here, we report an additional 25, previously unreported individuals who possess heterozygous TRIO variants and we review the literature. In addition, functional studies were performed on the c.4394A > G (N1465S) and c.6244-2A > G TRIO variants to provide evidence for their pathogenicity. Variants reported by the current study include missense variants, truncating nonsense variants, and an intragenic deletion. Clinical features were previously described and included developmental delay, learning difficulties, microcephaly, macrocephaly, seizures, behavioral issues (aggression, stereotypies), skeletal problems including short, tapering fingers and scoliosis, dental problems (overcrowding/delayed eruption), and variable facial features. Here, we report clinical features that have not been described previously, including specific structural brain malformations such as abnormalities of the corpus callosum and ventriculomegaly, additional psychological and dental issues along with a more recognizable facial gestalt linked to the specific domains of the TRIO gene and the effect of the variant upon the function of the encoded protein. This current study further strengthens the genotype-to-phenotype correlation that was previously established and extends the range of phenotypes to include structural brain abnormalities, additional skeletal, dental, and psychiatric issues.

Symptoms and ECG changes precede sudden cardiac death in hypertrophic cardiomyopathy-A nationwide study among the young in Sweden.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is a major cause of sudden cardiac death (SCD) in the young. We aimed to characterize detailed family history, symptoms, hospital utilization and ECG changes before SCD. METHODS: We extracted all cases suffering SCD with HCM from the SUDDY cohort, which includes all cases of SCD between 2000-2010 in Sweden among individuals aged 0-35 years along with their controls. We gathered data from mandatory national registries, autopsy reports, medical records, ECGs (including military conscripts), and detailed family history from an interview-based questionnaire (with relatives, post-mortem). RESULTS: Thirty-eight cases (7 female), mean age 22 years, with HCM were identified. Among these, 71% presented with possible cardiac symptoms (chest pain [26%], syncope [22%], palpitations [37%]), before death; 69% received medical care (vs 21% in controls) within 180 days before death. The majority (68%) died during recreational activity (n = 14) or exercise/competitive sports (n = 12). Fifteen (39%) had a known cardiac disorder prior to death, with HCM being diagnosed pre-mortem in nine cases. 58% presented with abnormal ECG recordings pre-mortem, and 50% had a positive family history (1st-3rd generation) for heart disease. CONCLUSION: In this comprehensive, nationwide study of SCD due to HCM, 87% (33/38) of cases had one or more abnormality prior to death, including cardiac symptoms, a positive family history, known cardiac disease or ECG abnormalities. They sought medical care prior death, to a larger extent than controls. These findings suggest that cardiac screening should be expanded beyond competitive athletes to aid SCD prevention in the young population with HCM.

Multi-Omic Investigations of a 17-19 Translocation Links MINK1 Disruption to Autism, Epilepsy and Osteoporosis.

Balanced structural variants, such as reciprocal translocations, are sometimes hard to detect with sequencing, especially when the breakpoints are located in repetitive or insufficiently mapped regions of the genome. In such cases, long-range information is required to resolve the rearrangement, identify disrupted genes and, in symptomatic carriers, pinpoint the disease-causing mechanisms. Here, we report an individual with autism, epilepsy and osteoporosis and a de novo balanced reciprocal translocation: t(17;19) (p13;p11). The genomic DNA was analyzed by short-, linked- and long-read genome sequencing, as well as optical mapping. Transcriptional consequences were assessed by transcriptome sequencing of patient-specific neuroepithelial stem cells derived from induced pluripotent stem cells (iPSC). The translocation breakpoints were only detected by long-read sequencing, the first on 17p13, located between exon 1 and exon 2 of MINK1 (Misshapen-like kinase 1), and the second in the chromosome 19 centromere. Functional validation in induced neural cells showed that MINK1 expression was reduced by >50% in the patient's cells compared to healthy control cells. Furthermore, pathway analysis revealed an enrichment of changed neural pathways in the patient's cells. Altogether, our multi-omics experiments highlight MINK1 as a candidate monogenic disease gene and show the advantages of long-read genome sequencing in capturing centromeric translocations.

Spontaneous Coronary Artery Dissection and Papillary Muscle Rupture in Patient With Undiagnosed Vascular Ehler-Danlos Syndrome.

We present the case of a woman with acute coronary syndrome on the basis of spontaneous coronary artery dissection causing a papillary muscle rupture with severe mitral regurgitation and acute heart failure. The patient subsequently underwent successful emergent surgery of both the mitral and tricuspid valves. Postoperatively, the patient was diagnosed with vascular Ehlers-Danlos syndrome. (Level of Difficulty: Advanced.).

Family History and Warning Symptoms Precede Sudden Cardiac Death in Arrhythmogenic Right Ventricular Cardiomyopathy (from a Nationwide Study in Sweden).

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited cardiac disease explaining about 4% of sudden cardiac death (SCD) cases in the young in Sweden. This study aimed to describe the circumstances preceding SCD in all victims <35 years of age who received an autopsy-confirmed diagnosis of ARVC from January 1, 2000, to December 31, 2010, in Sweden (n = 22). Data on demographics, medical and family history, circumstances of death, and anatomopathological findings were collected from several compulsory national health registries, clinical records, family interviews, and autopsy reports. Registry-based data were compared with age-matched, gender-matched, and geographically-matched population controls. During the 6 months preceding SCD, 15 cases (68%) had experienced symptoms of cardiac origin, mainly syncope or presyncope (54%) and chest discomfort (27%). A total of 8 cases (36%) had sought medical care because of cardiac symptoms. The occurrence of hospital visits was significantly increased in cases compared with controls (odds ratio 4.62 [1.35 to 15.8]). A total of 10 cases (45%) had a family history of SCD. The most common activity at the time of death was exercise (41%). A complete cardiac investigation was seldom performed; only 1 case was diagnosed with ARVC before death. In conclusion, in this nationwide study, we observed a high prevalence of symptoms of cardiac origin, healthcare use, and family history of SCD preceding SCD in the young caused by ARVC. Increased awareness of these warning signals in younger patients is critical to improving risk stratification and early disease detection.

Cohort profile: the Swedish study of SUDden cardiac Death in the Young (SUDDY) 2000-2010: a complete nationwide cohort of SCDs.

PURPOSE: The rationale behind the SUDden cardiac Death in the Young (SUDDY) cohort was to provide a complete nationwide, high-quality platform with integrated multisource data, for clinical and genetic research on sudden cardiac death (SCD) in the young, with the ultimate goal to predict and prevent SCD. PARTICIPANTS: The cohort contains all SCD victims <36 years, in Sweden during the period 2000-2010. We assigned five population-based controls per case, together with parents of cases and controls, in total 15 633 individuals. Data of all individuals were extracted from multiple mandatory registries; the National Patient Registry, the Medical Birth Registry, the Prescribed Drug registry, the Cause of Death registry, the Multigeneration Registry, combined with socioeconomic data from Statistics Sweden. From SCD victims, the autopsy report, medical records, ECGs, parental information and biological samples were gathered. FINDINGS TO DATE: We identified 903 individuals diagnosed with SCD (67% men, 33% women). The cases comprised 236 infants <1 year of age (26%), 90 individuals aged 1-15 years (10%), 186 individuals aged 15-25 years (21%) and 391 aged 25-35 years (43%). Hospitalisations and outpatient clinic visits due to syncope were significantly more common among cases than controls. DNA obtained from dried blood spots tests (DBS) stored from birth was equally suitable as venous blood samples for high-throughput genetic analysis of SCD cases. FUTURE PLANS: We will explore the SUDDY cohort for symptoms and healthcare consumption, socioeconomic variables and family history of SCD. Furthermore, we will perform whole exome sequencing analysis on DNA of cases obtained from DBS or postmortem samples together with parental blood samples in search for gene variants associated with cardiac disease. The genetic analysis together with data compiled in the nationwide cohort is expected to improve current knowledge on the incidence, aetiology, clinical characteristics and family history of SCD.

Novel missense ACAN gene variants linked to familial osteochondritis dissecans cluster in the C-terminal globular domain of aggrecan.

The cartilage aggrecan proteoglycan is crucial for both skeletal growth and articular cartilage function. A number of aggrecan (ACAN) gene variants have been linked to skeletal disorders, ranging from short stature to severe chondrodyplasias. Osteochondritis dissecans is a disorder where articular cartilage and subchondral bone fragments come loose from the articular surface. We previously reported a missense ACAN variant linked to familial osteochondritis dissecans, with short stature and early onset osteoarthritis, and now describe three novel ACAN gene variants from additional families with this disorder. Like the previously described variant, these are autosomal dominant missense variants, resulting in single amino acid residue substitutions in the C-type lectin repeat of the aggrecan G3 domain. Functional studies showed that neither recombinant variant proteins, nor full-length variant aggrecan proteoglycan from heterozygous patient cartilage, were secreted to the same level as wild-type aggrecan. The variant proteins also showed decreased binding to known cartilage extracellular matrix ligands. Mapping these and other ACAN variants linked to hereditary skeletal disorders showed a clustering of osteochondritis dissecans-linked variants to the G3 domain. Taken together, this supports a link between missense ACAN variants affecting the aggrecan G3 domain and hereditary osteochondritis dissecans.

Case Report: Bilateral Epiphysiodesis Due to Extreme Tall Stature in a Girl With a De Novo DNMT3A Variant Associated With Tatton-Brown-Rahman Syndrome.

Objective: To present a rare clinical case of a patient with Tatton-Brown-Rahman syndrome and the outcome of tall stature management with bilateral epiphysiodesis surgery at the distal femur and proximal ends of tibia and fibula. Study Design: Clinical case report. Results: This is a 20-year-old female with a history of proportional tall stature, developmental psychomotor and language delay with autism spectrum behavior and distinctive facial features. At 12 years and 2 months of age she was in early puberty and 172.5 cm tall (+ 2.8 SDS) and growing approximately 2 SDS above midparental target height of 173 cm (+ 0.9 SDS). A bone age assessment predicted an adult height of 187.1 cm (+3.4 SDS). To prevent extreme tall stature, bilateral epiphysiodesis surgery was performed at the distal femur and proximal ends of tibia and fibula at the age of 12 years and 9 months. After the surgery her height increased by 12.6 cm to 187.4 cm of which approximately 10.9 cm occurred in the spine whereas leg length increased by only 1.7 cm resulting in a modest increase of sitting height index from 50% (-1 SDS) to 53% (+ 0.5 SDS). Genetic evaluation for tall stature and intellectual disability identified a de novo nonsense variant in the DNMT3A gene previously associated with Tatton-Brown-Rahman syndrome. Conclusion: Tatton-Brown-Rahman syndrome should be considered in children with extreme tall stature and intellectual disability. Percutaneous epiphysiodesis surgery to mitigate extreme tall stature may be considered.

Genetic and functional insights into CDA-I prevalence and pathogenesis.

BACKGROUND: Congenital dyserythropoietic anaemia type I (CDA-I) is a hereditary anaemia caused by biallelic mutations in the widely expressed genes CDAN1 and C15orf41. Little is understood about either protein and it is unclear in which cellular pathways they participate. METHODS: Genetic analysis of a cohort of patients with CDA-I identifies novel pathogenic variants in both known causative genes. We analyse the mutation distribution and the predicted structural positioning of amino acids affected in Codanin-1, the protein encoded by CDAN1. Using western blotting, immunoprecipitation and immunofluorescence, we determine the effect of particular mutations on both proteins and interrogate protein interaction, stability and subcellular localisation. RESULTS: We identify six novel CDAN1 mutations and one novel mutation in C15orf41 and uncover evidence of further genetic heterogeneity in CDA-I. Additionally, population genetics suggests that CDA-I is more common than currently predicted. Mutations are enriched in six clusters in Codanin-1 and tend to affect buried residues. Many missense and in-frame mutations do not destabilise the entire protein. Rather C15orf41 relies on Codanin-1 for stability and both proteins, which are enriched in the nucleolus, interact to form an obligate complex in cells. CONCLUSION: Stability and interaction data suggest that C15orf41 may be the key determinant of CDA-I and offer insight into the mechanism underlying this disease. Both proteins share a common pathway likely to be present in a wide variety of cell types; however, nucleolar enrichment may provide a clue as to the erythroid specific nature of CDA-I. The surprisingly high predicted incidence of CDA-I suggests that better ascertainment would lead to improved patient care.

A novel heterozygous variant in FGF9 associated with previously unreported features of multiple synostosis syndrome 3.

Human multiple synostoses syndrome 3 is an autosomal dominant disorder caused by pathogenic variants in FGF9. Only two variants have been described in FGF9 in humans so far, and one in mice. Here we report a novel missense variant c.566C > G, p.(Pro189Arg) in FGF9. Functional studies showed this variant impairs FGF9 homodimerization, but not FGFR3c binding. We also review the findings of cases reported previously and report on additional features not described previously.

Celiprolol Treatment in Patients with Vascular Ehlers-Danlos Syndrome.

OBJECTIVE: Vascular Ehlers-Danlos syndrome (vEDS) is a rare monogenetic disease caused by pathogenic variants in procollagen 3A1. Arterial rupture is the most serious clinical manifestation. A randomised controlled trial, the Beta-Blockers in Ehlers-Danlos Syndrome Treatment (BBEST) trial, reported a significant protective effect of the beta blocker celiprolol. The aim was to study the outcome of celiprolol treatment in a cohort of Swedish patients with vEDS. METHODS: Uppsala is a national referral centre for patients with vEDS. They are assessed by vascular surgeons, angiologists, and clinical geneticists. Family history, previous and future clinical events, medication, and side effects are registered. Celiprolol was administered twice daily and titrated up to a maximum dose of 400 mg daily. Logistic regression was used to analyse predictors of vascular events. RESULTS: Forty patients with pathogenic sequence variants in COL3A1 were offered treatment with celiprolol in the period 2011-2019. The median follow up was 22 months (range 1-98 months); total follow up was 106 patient years. In two patients, uptitration of the dose is ongoing. Of the remaining 38, 26 (65%) patients reached the target dose of 400 mg daily. Dose uptitration was unsuccessful in six patients because of side effects; one died before reaching the maximum dose, and five terminated the treatment. Five major vascular events occurred; four were fatal (ruptured ascending aorta; aortic rupture after type B dissection; ruptured cerebral aneurysm; and ruptured pulmonary artery). One bled from a branch of the internal iliac artery, which was successfully coiled endovascularly. The annual risk of a major vascular event was 4.7% (n = 5/106), similar to the treatment arm of the BBEST trial (5%) and lower than in the control arm of the same trial (12%). No significant predictor of vascular events was identified. CONCLUSION: Treatment with celiprolol is tolerated in most patients with vEDS. Despite fatal vascular events, these observations suggest that celiprolol may have a protective effect in vEDS.

High frequency of intermediary alleles in the HTT gene in Northern Sweden - The Swedish Huntingtin Alleles and Phenotype (SHAPE) study.

Trinucleotide (CAG) repeat expansions longer than 39 in the huntingtin (HTT) gene cause Huntington's disease (HD). The frequency of intermediate alleles (IA) with a length of 27-35 in the general population is not fully known, but studied in specific materials connected to the incidence of HD. The Swedish Huntingtin Alleles and Phenotype (SHAPE) study aims to assess the frequency of trinucleotide repeat expansions in the HTT gene in north Sweden. 8260 individuals unselected for HD from the counties of Norr- and Västerbotten in the north of Sweden were included. DNA samples were obtained and analysis of the HTT gene was performed, yielding data on HTT gene expansion length in 7379 individuals. A high frequency of intermediate alleles, 6.8%, was seen. Also, individuals with repeat numbers lower than ever previously reported (<5) were found. These results suggest a high frequency of HD in the norther parts of Sweden. Subsequent analyses may elucidate the influence of IA:s on traits other than HD.

A progressive and complex clinical course in two family members with ERF-related craniosynostosis: a case report.

BACKGROUND: ERF-related craniosynostosis are a rare, complex, premature trisutural fusion associated with a broad spectrum of clinical features and heterogeneous aetiology. Here we describe two cases with the same pathogenic variant and a detailed description of their clinical course. CASE PRESENTATION: Two subjects; a boy with a BLSS requiring repeated skull expansions and his mother who had been operated once for sagittal synostosis. Both developed intracranial hypertension at some point during the course, which was for both verified by formal invasive intracranial pressure monitoring. Exome sequencing revealed a pathogenic truncating frame shift variant in the ERF gene. CONCLUSIONS: Here we describe a boy and his mother with different craniosynostosis patterns, but both with verified intracranial hypertension and heterozygosity for a truncating variant of ERF c.1201_1202delAA (p.Lys401Glufs*10). Our work provides supplementary evidence in support of previous phenotypic descriptions of ERF-related craniosynostosis, particularly late presentation, an evolving synostotic pattern and variable expressivity even among affected family members.

Exercise related sudden cardiac death (SCD) in the young - Pre-mortal characterization of a Swedish nationwide cohort, showing a decline in SCD among athletes.

AIMS: To study the frequency, etiology, and premortal abnormalities in exercise-related sudden cardiac death (SCD) in the young in Sweden. METHODS: All subjects with SCD in 10-35-year olds in Sweden during 2000-10, were included (n = 514). Information about each case was retrieved from death certifications, autopsy- and medical records. The number of SCD in athletes was compared to national figures from 1992-99. RESULTS: Exercise-related SCD occurred in 12% (62/514) of the SCD-population, a majority being men (56/62; 90%). Cardiopulmonary resuscitation (CPR) was started in 87% (54/62). In total, 48% (30/62), had a cardiac diagnosis, symptoms, family history and/or ECG-changes, before the fatal event. The most prevalent autopsy diagnosis was sudden arrhythmic death syndrome (15/62; 24%). The frequency of hypertrophic cardiomyopathy (HCM) and arrhythmogenic right ventricular cardiomyopathy (ARVC) was significantly higher in exercise-related SCD compared to non-exertional SCD. Exercise-related SCD was more common in athletes (21/29) than in non-athletes (41/485) (P < 0.0001). The total number of SCDs/year in athletes 15-35 years old, are approximately halved in 2000-10 compared to the years 1992-99. CONCLUSION: The increased risk of exercise-related SCD in HCM and ARVC underlines the importance of early detection and eligibility recommendations. There is a major reduction in deaths among athletes in the 2000s, compared to the previous decade. These results may partly be explained by improved acute preparedness for sudden cardiac arrest (CPR, defibrillation), but as a substantial percentage have preceding risk factors, such as symptoms and ECG-abnormalities, increased cardiac screening and increased general awareness, may also play a role.

Expanding the phenotypic spectrum of osteogenesis imperfecta type V including heterotopic ossification of muscle origins and attachments.

BACKGROUND: Osteogenesis imperfecta (OI) is a clinical and genetic heterogeneous group of connective tissue disorders, characterized by bone fragility and a propensity to fracture. METHODS: In this report we describe the clinical phenotype of two patients, a 28-year-old woman and her mother (54 years old), both with a history of short stature and multiple fractures. RESULTS: Exome sequencing revealed the recurring IFITM5:c.-14 C>T variant causing OI type V. Both patients had several fractures during childhood. CT-scan and scintigraphy showed ossification of the origin and attachment of muscles and hypertrophic callus formation. CONCLUSION: Ossification of the origin and attachment of muscles seems to be part of the phenotype in patients with OI type V.

Early activating somatic PIK3CA mutations promote ectopic muscle development and upper limb overgrowth.

PIK3CA-related overgrowth spectrum is a group of rare genetic disorders with asymmetric overgrowth caused by somatic mosaic PIK3CA mutations. Here, we report clinical data and molecular findings from two patients with congenital muscular upper limb overgrowth and aberrant anatomy. During debulking surgery, numerous ectopic muscles were found in the upper limbs of the patients. DNA sequencing, followed by digital polymerase chain reaction, was performed on DNA extracted from biopsies from hypertrophic ectopic muscles and identified the somatic mosaic PIK3CA hotspot mutations c.3140A > G, p.(His1047Arg) and c.1624G > A, p.(Glu542Lys) in a male (patient 1) and a female (patient 2) patient, respectively. Patient 1 had four ectopic muscles and unilateral isolated muscular overgrowth while patient 2 had 13 ectopic muscles and bilateral isolated muscular overgrowth of both upper limbs, indicating that her mutation occurred at early pre-somitic mesoderm state. The finding of PIK3CA mutations in ectopic muscles highlights the importance of PIK3CA in cell fate in early human embryonic development. Moreover, our findings provide evidence that the disease phenotype depends on the timing of PIK3CA mutagenesis during embryogenesis and confirm the diagnostic entity PIK3CA-related muscular overgrowth with ectopic accessory muscles.

Exome sequencing in Crisponi/cold-induced sweating syndrome-like individuals reveals unpredicted alternative diagnoses.

Crisponi/cold-induced sweating syndrome (CS/CISS) is a rare autosomal recessive disorder characterized by a complex phenotype (hyperthermia and feeding difficulties in the neonatal period, followed by scoliosis and paradoxical sweating induced by cold since early childhood) and a high neonatal lethality. CS/CISS is a genetically heterogeneous disorder caused by mutations in CRLF1 (CS/CISS1), CLCF1 (CS/CISS2) and KLHL7 (CS/CISS-like). Here, a whole exome sequencing approach in individuals with CS/CISS-like phenotype with unknown molecular defect revealed unpredicted alternative diagnoses. This approach identified putative pathogenic variations in NALCN, MAGEL2 and SCN2A. They were already found implicated in the pathogenesis of other syndromes, respectively the congenital contractures of the limbs and face, hypotonia, and developmental delay syndrome, the Schaaf-Yang syndrome, and the early infantile epileptic encephalopathy-11 syndrome. These results suggest a high neonatal phenotypic overlap among these disorders and will be very helpful for clinicians. Genetic analysis of these genes should be considered for those cases with a suspected CS/CISS during neonatal period who were tested as mutation negative in the known CS/CISS genes, because an expedited and corrected diagnosis can improve patient management and can provide a specific clinical follow-up.

Detailed analysis of HTT repeat elements in human blood using targeted amplification-free long-read sequencing.

Amplification of DNA is required as a mandatory step during library preparation in most targeted sequencing protocols. This can be a critical limitation when targeting regions that are highly repetitive or with extreme guanine-cytosine (GC) content, including repeat expansions associated with human disease. Here, we used an amplification-free protocol for targeted enrichment utilizing the CRISPR/Cas9 system (No-Amp Targeted sequencing) in combination with single molecule, real-time (SMRT) sequencing for studying repeat elements in the huntingtin (HTT) gene, where an expanded CAG repeat is causative for Huntington disease. We also developed a robust data analysis pipeline for repeat element analysis that is independent of alignment of reads to a reference genome. The method was applied to 11 diagnostic blood samples, and for all 22 alleles the resulting CAG repeat count agreed with previous results based on fragment analysis. The amplification-free protocol also allowed for studying somatic variability of repeat elements in our samples, without the interference of PCR stutter. In summary, with No-Amp Targeted sequencing in combination with our analysis pipeline, we could accurately study repeat elements that are difficult to investigate using PCR-based methods.