Synchronized long-read genome, methylome, epigenome, and transcriptome for resolving a Mendelian condition.

Resolving the molecular basis of a Mendelian condition (MC) remains challenging owing to the diverse mechanisms by which genetic variants cause disease. To address this, we developed a synchronized long-read genome, methylome, epigenome, and transcriptome sequencing approach, which enables accurate single-nucleotide, insertion-deletion, and structural variant calling and diploid de novo genome assembly, and permits the simultaneous elucidation of haplotype-resolved CpG methylation, chromatin accessibility, and full-length transcript information in a single long-read sequencing run. Application of this approach to an Undiagnosed Diseases Network (UDN) participant with a chromosome X;13 balanced translocation of uncertain significance revealed that this translocation disrupted the functioning of four separate genes (NBEA, PDK3, MAB21L1, and RB1) previously associated with single-gene MCs. Notably, the function of each gene was disrupted via a distinct mechanism that required integration of the four 'omes' to resolve. These included nonsense-mediated decay, fusion transcript formation, enhancer adoption, transcriptional readthrough silencing, and inappropriate X chromosome inactivation of autosomal genes. Overall, this highlights the utility of synchronized long-read multi-omic profiling for mechanistically resolving complex phenotypes.

Long-Term Efficacy of T3 Analogue Triac in Children and Adults With MCT8 Deficiency: A Real-Life Retrospective Cohort Study.

CONTEXT: Patients with mutations in thyroid hormone transporter MCT8 have developmental delay and chronic thyrotoxicosis associated with being underweight and having cardiovascular dysfunction. OBJECTIVE: Our previous trial showed improvement of key clinical and biochemical features during 1-year treatment with the T3 analogue Triac, but long-term follow-up data are needed. METHODS: In this real-life retrospective cohort study, we investigated the efficacy of Triac in MCT8-deficient patients in 33 sites. The primary endpoint was change in serum T3 concentrations from baseline to last available measurement. Secondary endpoints were changes in other thyroid parameters, anthropometric parameters, heart rate, and biochemical markers of thyroid hormone action. RESULTS: From October 15, 2014 to January 1, 2021, 67 patients (median baseline age 4.6 years; range, 0.5-66) were treated up to 6 years (median 2.2 years; range, 0.2-6.2). Mean T3 concentrations decreased from 4.58 (SD 1.11) to 1.66 (0.69) nmol/L (mean decrease 2.92 nmol/L; 95% CI, 2.61-3.23; P < 0.0001; target 1.4-2.5 nmol/L). Body-weight-for-age exceeded that of untreated historical controls (mean difference 0.72 SD; 95% CI, 0.36-1.09; P = 0.0002). Heart-rate-for-age decreased (mean difference 0.64 SD; 95% CI, 0.29-0.98; P = 0.0005). SHBG concentrations decreased from 245 (99) to 209 (92) nmol/L (mean decrease 36 nmol/L; 95% CI, 16-57; P = 0.0008). Mean creatinine concentrations increased from 32 (11) to 39 (13) µmol/L (mean increase 7 µmol/L; 95% CI, 6-9; P < 0.0001). Mean creatine kinase concentrations did not significantly change. No drug-related severe adverse events were reported. CONCLUSIONS: Key features were sustainably alleviated in patients with MCT8 deficiency across all ages, highlighting the real-life potential of Triac for MCT8 deficiency.

Disease characteristics of MCT8 deficiency: an international, retrospective, multicentre cohort study.

BACKGROUND: Disordered thyroid hormone transport, due to mutations in the SLC16A2 gene encoding monocarboxylate transporter 8 (MCT8), is characterised by intellectual and motor disability resulting from cerebral hypothyroidism and chronic peripheral thyrotoxicosis. We sought to systematically assess the phenotypic characteristics and natural history of patients with MCT8 deficiency. METHODS: We did an international, multicentre, cohort study, analysing retrospective data from Jan 1, 2003, to Dec 31, 2019, from patients with MCT8 deficiency followed up in 47 hospitals in 22 countries globally. The key inclusion criterion was genetically confirmed MCT8 deficiency. There were no exclusion criteria. Our primary objective was to analyse the overall survival of patients with MCT8 deficiency and document causes of death. We also compared survival between patients who did or did not attain full head control by age 1·5 years and between patients who were or were not underweight by age 1-3 years (defined as a bodyweight-for-age Z score <-2 SDs or <5th percentile according to WHO definition). Other objectives were to assess neurocognitive function and outcomes, and clinical parameters including anthropometric characteristics, biochemical markers, and neuroimaging findings. FINDINGS: Between Oct 14, 2014, and Jan 17, 2020, we enrolled 151 patients with 73 different MCT8 (SLC16A2) mutations. Median age at diagnosis was 24·0 months (IQR 12·0-60·0, range 0·0-744·0). 32 (21%) of 151 patients died; the main causes of mortality in these patients were pulmonary infection (six [19%]) and sudden death (six [19%]). Median overall survival was 35·0 years (95% CI 8·3-61·7). Individuals who did not attain head control by age 1·5 years had an increased risk of death compared with patients who did attain head control (hazard ratio [HR] 3·46, 95% CI 1·76-8·34; log-rank test p=0·0041). Patients who were underweight during age 1-3 years had an increased risk for death compared with patients who were of normal bodyweight at this age (HR 4·71, 95% CI 1·26-17·58, p=0·021). The few motor and cognitive abilities of patients did not improve with age, as evidenced by the absence of significant correlations between biological age and scores on the Gross Motor Function Measure-88 and Bayley Scales of Infant Development III. Tri-iodothyronine concentrations were above the age-specific upper limit in 96 (95%) of 101 patients and free thyroxine concentrations were below the age-specific lower limit in 94 (89%) of 106 patients. 59 (71%) of 83 patients were underweight. 25 (53%) of 47 patients had elevated systolic blood pressure above the 90th percentile, 34 (76%) of 45 patients had premature atrial contractions, and 20 (31%) of 64 had resting tachycardia. The most consistent MRI finding was a global delay in myelination, which occurred in 13 (100%) of 13 patients. INTERPRETATION: Our description of characteristics of MCT8 deficiency in a large patient cohort reveals poor survival with a high prevalence of treatable underlying risk factors, and provides knowledge that might inform clinical management and future evaluation of therapies. FUNDING: Netherlands Organisation for Health Research and Development, and the Sherman Foundation.

Homozygous 15q13.3 microdeletion in a child with hypotonia and impaired vision: A new report and review of the literature.

Although there are numerous reports of heterozygous 15q13.3 microdeletion, homozygous 15q13.3 microdeletion is rare. We report a new patient with homozygous microdeletion of 15q13.2q13.3 and review the previous literature reports. Common clinical features include encephalopathy, hypotonia, developmental delay, cortical vision impairment, optic nerve abnormality, epilepsy, and abnormal electroencephalogram (EEG) findings.

Longitudinal expression profiling of CD4+ and CD8+ cells in patients with active to quiescent giant cell arteritis.

BACKGROUND: Giant cell arteritis (GCA) is the most common form of vasculitis affecting elderly people. It is one of the few true ophthalmic emergencies but symptoms and signs are variable thereby making it a challenging disease to diagnose. A temporal artery biopsy is the gold standard to confirm GCA, but there are currently no specific biochemical markers to aid diagnosis. We aimed to identify a less invasive method to confirm the diagnosis of GCA, as well as to ascertain clinically relevant predictive biomarkers by studying the transcriptome of purified peripheral CD4+ and CD8+ T lymphocytes in patients with GCA. METHODS: We recruited 16 patients with histological evidence of GCA at the Royal Victorian Eye and Ear Hospital, Melbourne, Australia, and aimed to collect blood samples at six time points: acute phase, 2-3 weeks, 6-8 weeks, 3 months, 6 months and 12 months after clinical diagnosis. CD4+ and CD8+ T-cells were positively selected at each time point through magnetic-assisted cell sorting. RNA was extracted from all 195 collected samples for subsequent RNA sequencing. The expression profiles of patients were compared to those of 16 age-matched controls. RESULTS: Over the 12-month study period, polynomial modelling analyses identified 179 and 4 statistically significant transcripts with altered expression profiles (FDR < 0.05) between cases and controls in CD4+ and CD8+ populations, respectively. In CD8+ cells, two transcripts remained differentially expressed after 12 months; SGTB, associated with neuronal apoptosis, and FCGR3A, associatied with Takayasu arteritis. We detected genes that correlate with both symptoms and biochemical markers used for predicting long-term prognosis. 15 genes were shared across 3 phenotypes in CD4 and 16 across CD8 cells. In CD8, IL32 was common to 5 phenotypes including Polymyalgia Rheumatica, bilateral blindness and death within 12 months. CONCLUSIONS: This is the first longitudinal gene expression study undertaken to identify robust transcriptomic biomarkers of GCA. Our results show cell type-specific transcript expression profiles, novel gene-phenotype associations, and uncover important biological pathways for this disease. In the acute phase, the gene-phenotype relationships we have identified could provide insight to potential disease severity and as such guide in initiating appropriate patient management.

HUWE1 variants cause dominant X-linked intellectual disability: a clinical study of 21 patients.

Whole-gene duplications and missense variants in the HUWE1 gene (NM_031407.6) have been reported in association with intellectual disability (ID). Increased gene dosage has been observed in males with non-syndromic mild to moderate ID with speech delay. Missense variants reported previously appear to be associated with severe ID in males and mild or no ID in obligate carrier females. Here, we report the largest cohort of patients with HUWE1 variants, consisting of 14 females and 7 males, with 15 different missense variants and one splice site variant. Clinical assessment identified common clinical features consisting of moderate to profound ID, delayed or absent speech, short stature with small hands and feet and facial dysmorphism consisting of a broad nasal tip, deep set eyes, epicanthic folds, short palpebral fissures, and a short philtrum. We describe for the first time that females can be severely affected, despite preferential inactivation of the affected X chromosome. Three females with the c.329 G > A p.Arg110Gln variant, present with a phenotype of mild ID, specific facial features, scoliosis and craniosynostosis, as reported previously in a single patient. In these females, the X inactivation pattern appeared skewed in favour of the affected transcript. In summary, HUWE1 missense variants may cause syndromic ID in both males and females.

DNM1 encephalopathy: A new disease of vesicle fission.

OBJECTIVE: To evaluate the phenotypic spectrum caused by mutations in dynamin 1 (DNM1), encoding the presynaptic protein DNM1, and to investigate possible genotype-phenotype correlations and predicted functional consequences based on structural modeling. METHODS: We reviewed phenotypic data of 21 patients (7 previously published) with DNM1 mutations. We compared mutation data to known functional data and undertook biomolecular modeling to assess the effect of the mutations on protein function. RESULTS: We identified 19 patients with de novo mutations in DNM1 and a sibling pair who had an inherited mutation from a mosaic parent. Seven patients (33.3%) carried the recurrent p.Arg237Trp mutation. A common phenotype emerged that included severe to profound intellectual disability and muscular hypotonia in all patients and an epilepsy characterized by infantile spasms in 16 of 21 patients, frequently evolving into Lennox-Gastaut syndrome. Two patients had profound global developmental delay without seizures. In addition, we describe a single patient with normal development before the onset of a catastrophic epilepsy, consistent with febrile infection-related epilepsy syndrome at 4 years. All mutations cluster within the GTPase or middle domains, and structural modeling and existing functional data suggest a dominant-negative effect on DMN1 function. CONCLUSIONS: The phenotypic spectrum of DNM1-related encephalopathy is relatively homogeneous, in contrast to many other genetic epilepsies. Up to one-third of patients carry the recurrent p.Arg237Trp variant, which is now one of the most common recurrent variants in epileptic encephalopathies identified to date. Given the predicted dominant-negative mechanism of this mutation, this variant presents a prime target for therapeutic intervention.

Recurrent duplications of 17q12 associated with variable phenotypes.

The ability to identify the clinical nature of the recurrent duplication of chromosome 17q12 has been limited by its rarity and the diverse range of phenotypes associated with this genomic change. In order to further define the clinical features of affected patients, detailed clinical information was collected in the largest series to date (30 patients and 2 of their siblings) through a multi-institutional collaborative effort. The majority of patients presented with developmental delays varying from mild to severe. Though dysmorphic features were commonly reported, patients do not have consistent and recognizable features. Cardiac, ophthalmologic, growth, behavioral, and other abnormalities were each present in a subset of patients. The newly associated features potentially resulting from 17q12 duplication include height and weight above the 95th percentile, cataracts, microphthalmia, coloboma, astigmatism, tracheomalacia, cutaneous mosaicism, pectus excavatum, scoliosis, hypermobility, hypospadias, diverticulum of Kommerell, pyloric stenosis, and pseudohypoparathryoidism. The majority of duplications were inherited with some carrier parents reporting learning disabilities or microcephaly. We identified additional, potentially contributory copy number changes in a subset of patients, including one patient each with 16p11.2 deletion and 15q13.3 deletion. Our data further define and expand the clinical spectrum associated with duplications of 17q12 and provide support for the role of genomic modifiers contributing to phenotypic variability.

Direct medical cost of newly diagnosed stable coronary artery disease in Hong Kong.

BACKGROUND: Stable coronary artery disease (CAD) affects approximately 7% of the population of Hong Kong and is associated with substantial healthcare costs. OBJECTIVE: We aimed to evaluate the first-year direct medical cost for a patient with newly diagnosed stable CAD at a tertiary care public hospital in Hong Kong and to identify CAD-related resource consumption pattern among different patient subgroups. METHODS: 89 consecutive patients with newly diagnosed stable CAD at our institution from January 2007 to December 2009 were retrospectively analysed. Direct medical costs including hospitalisation, clinic visits, diagnostic tests, laboratory tests, invasive procedures and medications were calculated for 1 year after diagnosis. Mann-Whitney tests were performed to compare median costs in patients with and without hypertension, diabetes mellitus and hyperlipidaemia, and in patients undergoing coronary intervention and those who were not. RESULTS: The mean first-year total direct medical cost of newly diagnosed stable CAD per patient was US$11 477. Hospitalisation was the dominant cost item accounting for 29.2% of the total cost. The total cost for patients who underwent invasive coronary procedure was higher than those treated medically alone (US$14 787 vs US$6121, p<0.001). Hyperlipidaemia was associated with higher incremental costs than hypertension and diabetes mellitus (p<0.001). (1US$=7.8HK$). CONCLUSIONS: Huge healthcare expenses are incurred in the first year of stable CAD diagnosis from the perspective of the local public healthcare system. Healthcare costs are highest among patients with hyperlipidaemia and those undergoing invasive coronary procedures (even discounting costs for procedural consumables). Strategies for cost saving and preventive measures should be implemented to lower healthcare expenditure associated with CAD.

Methylglyoxal-modified collagen promotes myofibroblast differentiation.

Fibrosis is a frequent complication of diabetes mellitus in many organs and tissues but the mechanism of how diabetes-induced glycation of extracellular matrix proteins impacts the formation of fibrotic lesions is not defined. As fibrosis is mediated by myofibroblasts, we investigated the effect of collagen glycation on the conversion of human cardiac fibroblasts to myofibroblasts. Collagen glycation was modeled by the glucose metabolite, methylglyoxal (MGO). Cells cultured on MGO-treated collagen exhibited increased activity of the α-smooth muscle actin promoter and enhanced expression of α-smooth muscle actin, ED-A fibronectin and cadherin, which are markers for myofibroblasts. In cells remodeling floating or stress-relaxed collagen gels, MGO treatment promoted more contraction (p<0.025) than vehicle controls, which was MGO dose-dependent. Transwell assays showed that cell migration was increased by MGO-treated collagen (p<0.025). In shear-force detachment assays, cells on MGO-treated collagen were less adherent than untreated collagen, and the formation of high affinity, ß1 integrin-dependent adhesions was inhibited. MGO-collagen-induced expression of SMA was dependent on TGF-ß but not on Rho kinase. We conclude that collagen glycation augments the formation and migration of myofibroblasts, critical processes in the development of fibrosis in diabetes.

Familial deletion within NLGN4 associated with autism and Tourette syndrome.

Neuroligin 4 (NLGN4) is a member of a cell adhesion protein family that appears to play a role in the maturation and function of neuronal synapses. Mutations in the X-linked NLGN4 gene are a potential cause of autistic spectrum disorders, and mutations have been reported in several patients with autism, Asperger syndrome, and mental retardation. We describe here a family with a wide variation in neuropsychiatric illness associated with a deletion of exons 4, 5, and 6 of NLGN4. The proband is an autistic boy with a motor tic. His brother has Tourette syndrome and attention deficit hyperactivity disorder. Their mother, a carrier, has a learning disorder, anxiety, and depression. This family demonstrates that NLGN4 mutations can be associated with a wide spectrum of neuropsychiatric conditions and that carriers may be affected with milder symptoms.

Ube3a mRNA and protein expression are not decreased in Mecp2R168X mutant mice.

Mutations in the transcriptional repressor methyl CpG binding protein 2 (MeCP2) are responsible for most cases of Rett Syndrome (RS), a severe neurodevelopmental disorder characterized by developmental regression, minimal speech, seizures, postnatal microcephaly and hand stereotypies. Absence of the maternal copy of ubiquitin protein ligase 3A (UBE3A) results in Angelman syndrome, also a severe developmental disorder that shares some clinical features with RS. As MeCP2 regulates gene expression, this has led to the hypothesis that MeCP2 may regulate UBE3A expression; however, there are conflicting reports regarding the expression of Ube3a in MeCP2 null mutant mice. We have generated a novel MeCP2 mutant knock-in mouse with the mutation R168X, one of the most common mutations in patients with RS. These mice show features similar to RS, including hypoactivity, forelimb stereotypies, breathing irregularities, weight changes, hind limb atrophy, and scoliosis. The male mice experience early death. Analysis of Ube3a mRNA and protein levels in the Mecp2(R168X) male mice showed no significant difference in expression compared to their wild type littermates.

Atypical cases of Angelman syndrome.

Angelman syndrome (AS) is a profound disorder notable for mental retardation and severe language deficits that results from lack of function of the maternally inherited copy of the UBE3A gene. Chromosome deletions of 15q11q13, paternal uniparental disomy (UPD), UBE3A gene mutations, and imprinting center defects are all commonly recognized mechanisms that disrupt the function of the maternal copy of the UBE3A gene. We report here two patients with different atypical etiologies of AS. The first patient is a 3-year-old boy with global developmental delay, severe speech deficits, seizures, and very happy disposition. Southern blot analysis for the maternal and paternal chromosome 15 methylation products showed a mosaic methylation pattern, suggesting an imprinting center defect. The second patient is a 4(1/2)-year-old boy with global developmental delay, no expressive language, microcephaly, seizures, and ataxic gait. Array-based comparative genomic hybridization (CGH) demonstrated a loss in copy number for two overlapping clones encompassing the UBE3A gene, indicating a partial deletion within UBE3A. His mother, who was adopted, had an identical pattern, suggesting that her deletion was probably on her paternally imprinted allele. These patients illustrate the expanding spectrum of molecular findings in AS, reinforce the need to maintain suspicion when clinical features suggest AS but initial testing is normal, and show the power of CGH as a tool to uncover partial UBE3A deletions.

Patient with novel interstitial deletion of chromosome 3q13.1q13.3 and agenesis of the corpus callosum.

Interstitial deletions of the proximal long arm of chromosome 3 are rare. Only eight previously reported patients have deletions involving the proximal segment of 3q. Of these patients, three had agenesis of the corpus callosum and one had holoprosencephaly. We report here a patient with a small unique interstitial deletion of the long arm of chromosome 3 spanning 3q13.1q13.3. This patient has agenesis of the corpus callosum, global developmental delay, and distinctive facial features of a small nose, anteverted nares, and broad nasal root. Our patient provides further evidence that a gene involved in corpus callosum development or neuronal migration may reside in this region.

The use of betaine in the treatment of elevated homocysteine.

Elevation of homocysteine is implicated in multiple medical conditions, including classical homocystinuria, a variety of remethylation disorders, and most recently in coronary artery disease. Betaine is a methyl donor agent that is beneficial in lowering homocysteine through the remethylation of methionine. Betaine therapy alone has been shown to prevent vascular events in homocystinuria and may have clinical benefits in other hyperhomocysteinemic disorders when used as adjunctive therapy. Betaine does raise the methionine level and cerebral edema has occurred when plasma methionine exceeds 1000 micromol/L. Thus the plasma methionine as well as homocysteine must be monitored in patients receiving betaine.

A new helper phage and phagemid vector system improves viral display of antibody Fab fragments and avoids propagation of insert-less virions.

Phage display technology (PDT) is a powerful method for isolating functional gene products such as antigen-specific monoclonal antibodies (mAbs). To improve the effectiveness of PDT, we sought to optimize display of Fab-g3p (antibody fragment fused with viral gene 3 protein) on phagemid virions and to optimize the yield of such phage. To do so, we constructed a novel helper phage, Phaberge, having a conditional deficiency in g3p production. Unlike most other published g3p-deficient helper phage, Phaberge is produced at high levels, 10(11) PFU/ml. As compared to g3p-sufficient helper phage, Phaberge caused a 5-20-fold increase in display level. Another novel feature is that Phaberge only packages insert-containing, not insert-less, phagemid into infectious virions. This should prove useful in preserving quality of phagemid libraries during propagation. In addition, other parameters were also found to affect production of phagemid virions. In particular, the choice of bacterial host cell, phagemid construct and growth temperature had a substantial impact on display levels, but generally no effect on number of phagemid virions produced. In short, we have established a set of parameters that improve production and quality of phagemid virions which we expect to facilitate the isolation of mAbs or other gene products by PDT.