Tatton-Brown-Rahman syndrome (TBRS) is a rare autosomal dominant overgrowth syndrome first reported in 2014 and associated with mutations in the DNA methyltransferase 3A gene (DNMT3A). Seventy‐eight individuals have been reported to date with a consistent phenotype of somatic overgrowth, dysmorphic features and intellectual disability. Peripheral neuropathy was not described in these cases.
We report a patient with a new pathogenic DNMT3A variant (c.2036G>A; p.R688H) causing TBRS associated with an axonal length-dependent sensory-motor polyneuropathy. Other causes of neuropathy were excluded by laboratory and genetic work-up.
We postulate that this patient’s neuropathy is caused by the DNMT3A variant he harbors and review the association of peripheral neuropathy with dysfunction of the DNA methyltransferase pathway and with other overgrowth syndromes.
CAH is a group of rare autosomal recessive disorders caused by the deficiency of any one of five key enzymes responsible for adrenal steroidogenesis. The main cause of CAH is the deficiency in CYP21A2 (95%) followed by CYP11B1 (5%) and very rarely HSD3B2, CYP17A1, and STAR. The most severe of the classical forms result in hyponatremia and hyperkalemia and can lead to death in early infancy, if not diagnosed and treated in time. Other complications include excessive or deficient production of sex steroids and alteration of primary or secondary sex characteristics in some affected individuals. Here, we report clinical, endocrine and molecular evaluations of a newborn with ambiguous genitalia.
Electrolytes and steroid precursors were measured as per standard procedures. Molecular investigations included rapid sex chromosome determination (QF-PCR), full chromosomal analysis by CGH array and molecular analysis of 62 genes associated with disorders of sex development (Blueprint Genetics, Abnormal Genitalia/Disorders of Sex Development panel).
A newborn, whose fetal gender had been reported as female, was evaluated at birth for ambiguous genitalia presenting as an incomplete fusion of labioscrotal folds and a 3cm genital tubercle. Pregnancy had been uneventful. Family history was notable for distant parental consanguinity, and a maternal cousin with “a need for salt during stress”. Rapid sex chromosome determination reported the presence of a Y chromosome.
While under investigation, the baby started presenting with vomiting and poor weight gain at 10 days of life. Clinical evaluations revealed mild hypotension, hyperkalemia hyponatremia. 17-hydroxyprogesterone (17OHP) levels were elevated (1449.0 nmol/L, Ref: 0.0-49.9 nmol/L). Abdominal ultrasound showed mild prominence of the adrenal glands and bilateral gonads in the low inguinal canal. Treatment was promptly initiated with corticosteroids.
CGH chromosomal analysis did not reveal any pathogenic copy number variation (CNV). Subsequent NGS sequencing and CNV analysis of 62 DSD genes identified a rare apparently homozygous inframe deletion variant in HSD3B2 (NM_000198.3(HSD3B2):c.687_713del (p.Trp230_Ala238del])). This variant is absent from the reference population database gnomAD, but had previously been described in individuals of 4 unrelated families of the same ethnic background as the patient’s family, in association with similar clinical features as the patient.
HSD3B2 encodes 3β-Hydroxysteroid dehydrogenase (3β-HSD) type II which is expressed almost exclusively in adrenals and gonads. The deficiency of this enzyme causes a very rare type of CAH characterized by severe impairment of steroid biosynthesis in both these tissues. Affected newborns exhibit signs of glucocorticoid and mineralocorticoid deficiencies with varying degrees of salt loss. Moreover, external genitalia exhibit incomplete masculinization in male newborns and normal sexual differentiation or partial virilization in affected females.
While the vast majority of patients with ambiguous genitalia and CAH present as virilized females, molecular investigations are invaluable to establish the underlying etiology of this condition in 46,XY infants, as CAH is rarely suspected in an undervirilized male.
Background: Minimal change nephrotic syndrome (MCNS) is the most common chronic glomerular disorder in children. It has been postulated that a humoral factor of immune cell origin injures podocytes leading to pathogenesis. However, the identity of both the pathogenic factor and its cellular source remains unknown. Rituximab (RTX), a B-cell-depleting anti-CD20 monoclonal antibody, was recently found to be effective in maintaining a long-term remission of proteinuria in patients, thereby implicating B-cells in MCNS pathogenesis. Thus, we hypothesized that a pathogenic B-cell population may drive aberrant immune responses that lead to podocyte injury.
Objectives: In this longitudinal observational study, we profiled the immune systems of children with MCD during relapses of proteinuria and remission achieved by glucocorticoids, RTX or other second line agents. We aimed to elucidate a RTX-sensitive B-cell profile that correlates with disease activity.
Methods: Peripheral blood mononuclear cells (PBMC) were collected from 22 children with GC- sensitive idiopathic nephrotic syndrome (8.5±3.3 y.o.). Clinical time points of sample collection were denoted as follows: in relapse before receiving any treatment (Relapse, N=6), in remission post-RTX after B-cell recovery (RTX-rem, N=21), in remission after treated with GC (GC-rem, N=10), and in remission while receiving MMF (MMF-rem, N=3). Cells were stained and analyzed by flow cytometry for multiple B-cell subsets.
Results: The proportion of B-cells (% of total lymphocytes) in periphery in Relapse was significantly higher as compared with RTX-rem (13.1% vs. 5.5%, p<0.01), but was not different from GC-rem (10.5%) or MMF-rem (11.8%). The B-cell profile in RTX-rem showed dominant expansion of transitional naïve B-cells (42.3% vs. 13.3% in Relapse, p<0.001) with a negligible resurgence of the isotype-switched memory B-cells (1.2% vs. 7.1% in Relapse, p<0.01) indicating an immature B-cell profile. In contrast, B-cell profiles in GC-rem and MMF-rem were similar to Relapse, with low levels of transitional naïve B-cells and higher levels of isotype-switched memory B-cells (a mature B-cell profile).
Conclusions: We demonstrate that RTX, but not GC or MMF, induces B-cell contraction and an immature B-cell profile in patients with MCD. The results suggest a possibility that an immature B-cell profile induced by RTX, but not by GC or MMF, may allow longer protection against relapses. Further studies are required to uncover the precise role of B-cells in MCD pathogenesis, including their crosstalk with T-cells, their clonality and secretory profiles.
Genetic leukoencephalopathies are considered rare in the adult population. However, several lines of evidence suggest that there is a larger than expected number of adult patients with genetic leukoencephalopathies who are currently not diagnosed.
The study of leukoencephalopathies - either genetically-proven or unsolved - relies greatly on MRI pattern-recognition, which allowed the characterization of the majority of genetic white matter disorders. The advent of next generation sequencing (NGS) revolutionized the field, by disclosing new phenotypes associated to known genetic white matter conditions and identifying novel genes responsible for unsolved leukoencephalopathies.
The goals of our research are
1. the application of advanced neuroimaging tools to define and characterize white matter abnormalities of known and novel genetic leukoencephalopathies
2. the clinical and demographic characterization of subjects with adult genetic leukoencephalopathies
3. the identification of genes responsible for new forms of hereditary white matter disorders, using NGS techniques.
We apply an integrated approach which combined clinical phenotyping with MRI pattern-recognition and NGS data.
In the past five years this work led to a) the description of a cohort of 68 adult subjects with leukoencephalopathy of probable genetic origin, b) the identification of the causal mutations (16 in total, 11 novel) in genes known to be associated with leukoencephalopathies (23.7% of 59 subjects), and c) the broadening of clinical and imaging phenotypes of known disorders: POLR3-related disorders, vanishing white matter disease, Krabbe disease, MTFMT-related disorders. We demonstrated that MRI family studies can be crucial in adult leukoencephalopathies to define the modality of transmission of unclear white matter disorders within families.
In conclusion, we documented that adult genetic leukoencephalopathies are an emerging problem in clinical neurosciences. MRI family studies and the recognition of disease-specific MRI features are critical to guide the diagnostic process. Despite the access to NGS techniques, more than 70% of our subjects remain without a diagnosis. The international sharing of MRI and NGS on adult leukoencephalopathies will allow the identification of subjects with shared phenotypes or mutated genes and ultimately lead to the description of new genetic entities.
Arthrogryposis multiplex congenita (AMC) affects 1 in 3000 live births and is characterized by congenital contractures affecting at least two different body areas including upper and lower limbs, spine and jaw. AMC is related to fetal akinesia, which can be caused by environmental factors (i.e. maternal illness, limited space), single gene changes (autosomal dominant, autosomal recessive, X-linked), chromosomal abnormalities and various syndromes. Treatment includes casting, splints, exercise, and/or surgery, and thus a multidisciplinary approach (e.g., orthopedics, rehabilitation, genetics, social work) is recommended. Considering the rarity of AMC, evidence is lacking regarding best practice. As such, the AMC research program at Shriners Hospitals for Children – Canada (SHC-Canada) aims to improve the clinical interventions for children with AMC using a collaborative approach.
To achieve our objective, four research projects have been undertaken.
Project 1: Since the inception of the monthly AMC clinic in May 2016, there are 124 active patients (mean age=6.9 years, range=0.2-18.7 years). Overall, families have expressed being satisfied with this multidisciplinary clinic.
Project 2: At SHC-Canada, 19 children have been enrolled in the registry, and three have undergone whole genome sequencing. SHC-Philadelphia has recruited one participant to date; SHC-Northern California and SHC-Portland are ready to begin recruiting.
Project 3: Clinicians, researchers, caregivers, youth and adults with AMC are collaborating to develop expert guidance recommendations for rehabilitation, using knowledge syntheses, expert opinion, consensus, and validation using AGREE II.
Project 4: Six youth shared their life experiences using PhotoVoice. Facilitators for living with AMC included people, objects and a service dog, while challenges included activity limitations, built structures, and obstacles in the natural environment. These findings can inform health care practitioners about the needs and perspectives of youth with AMC, and empower youth to have their voice heard.
Involving all stakeholders in the development of a rare disease research program increases relevance of the research to clinicians and youth/families, facilitates dissemination of findings, and generates collaborations for future research avenues.
Importance: Adenopathy and extensive skin patch overlying plasmacytoma (AESOP) syndrome is a paraneoplastic syndrome characterized by a cutaneous vascular patch overlying a plasmacytoma and systemic manifestations. It is thought to be an early stage of Polyneuropathy, Organomegaly, Endocrinopathy, Monoclonal gammopathy, and Skin changes (POEMS) syndrome, which is a rare, but potentially fatal multisystemic disease that is associated with plasma cell dyscrasia. Thus, a high index of suspicion is required to identify patients with AESOP as they may present with early POEMS, which is curable if detected early.
Objective: To report additional cases of AESOP syndrome, describe dermatoscopic and histologic findings of the cutaneous patch and review all up to date literature on AESOP syndrome.
Design: Case series from a single tertiary care center and literature review.
Participants: Here, we present the second case series of 3 patients with AESOP syndrome who all met the diagnostic criteria for POEMS. The diagnosis was suspected based on the presence of the violaceous cutaneous patch along with symptoms of systemic involvement (fatigue, weight loss, weakness, hypothyroidism). Dermoscopy revealing regular dilated parallel capillaries was suggestive of a benign/reactive vascular process. Histopathology in all 3 cases showed reactive vascular proliferation with a characteristic 90⁰ branching. To date only 20 cases of AESOP have been published including ours. All patients presented with cutaneous lesions (violaceous patch and others) and most, at least 15/20, met the diagnostic criteria for POEMS. When clinical follow up was reported, most patients had a favorable prognosis with partial or complete symptom resolution following treatment of the underlying plasmocytoma.
Homozygous familial hypercholesterolemia (HoFH) is an orphan disease characterized by extremely high levels of plasma LDL-C levels. Affected patients develop clinical atherosclerotic cardiovascular disease (ASCVD) in youth and survival > 30 years of age was unusual until the advent of medications and extracorporeal LDL filtration (apheresis) techniques. HoFH is an autosomal co-dominant condition defined as an LDL-C > 13 mmol/L in adults without treatment and homozygous or compound heterozygous mutations of the LDLR gene. HoFH has a genetic probability of 1/250,000 and the rare diseases inventory Orphanet estimates its worldwide prevalence at 0.1/100,000 individuals. Canada is known to have several regions with a founder effect for HoFH and we recently identified 79 cases across the country. Data from other countries show a median survival of HoFH patients at < 40 years of age.
Clinical outcomes in HoFH patients, especially ASCVD events (fatal and non-fatal myocardial infarctions, strokes and peripheral vascular disease) and severe calcific aortic stenosis, are difficult to capture, in part because of the rarity of the disorder and the lack of registry focusing on this disease.
As we previously demonstrated in a 25 HoFH patients cohort, all HoFH patients develop premature severe aortic calcifications and 24% of them underwent aortic valve replacement for severe aortic stenosis. These patients are frequently seen as surgical management challenges as they present with porcelain aorta.
Here, we present a systematic review of the literature on the risk of calcific aortic stenosis and aortic valve replacement in HoFH patients.
We plan to use this data at provincial and national levels, in help with the Canadian Organization for Rare Diseases (CORD) and the Réseau Québecois des maladies orphelines (RQMO), to provide HoFH patients access to care, including PCSK9 inhibitors, orphan drugs such as lomitapide and treatment techniques such as extracorporeal LDL filtration (apheresis). Early aggressive treatment, including the use of LDL apheresis, seams to prevent the rapid progression of the disease and prolongs the patient’s life. Strategies to identify and monitor aortic calcification in HoFH patients by non-invasive techniques are still to be establish.
Leukodystrophies (LD) are a group of rare genetic disorders that are characterized by abnormal cerebral white matter, (i.e. myelin). This research project focuses on describing one of the most common hypomyelinating leukodystrophies (HLD), RNA polymerase III-related HLD (POLR3-HLD or 4H: Hypomyelination, Hypodontia, Hypogonadotropic Hypogonadism). 4H is a relatively new disease with little information available about the disease course. The onset of symptoms is typically in childhood, and there is presently no known cure. Extensive efforts are underway to elucidate the pathophysiology and to develop therapies. However, the lack of natural history studies for 4H represents a major obstacle for future clinical trials.
By using major clinical and MRI outcomes to predict disease progression and identify surrogate markers, this proposed project will allow for the characterization of 4H leukodystrophy patient populations. The development of a rare disease database using the Longitudinal Online Research and Imaging System (LORIS) is underway to facilitate recording and organizing vast volumes of clinical, genomic, and imaging data in order to improve international collaboration.
Hypothesis: Disease progression of 4H can be measured using LORIS by recording (1) specific clinical characteristics and (2) the 4H MRI severity score, an MRI scoring system developed to quantify disease severity in 4H.
Using a prospective international study design, specific objectives include: (1) building a multi-modal (e.g. genetics, MRI imaging, and patient determined outcomes) rare disease database, (2) delineating the natural history of the disease in a cohort of 400 patients, and (3) developing an online predictive tool to estimate trajectories of important disease milestones for patients and families.
Development of this rare disease database using LORIS has commenced. With the help of international collaborators, we have gathered data from over 300 patients and aim to reach a cohort of 400 patients. With LORIS’ “Instrument Builder” module and its scoring functionality, 90 clinical instruments have been created and made ready for data entry.
Demographic data, patient/family determined outcomes, and data from past medical records will be used to complete data capture of major outcomes. Clinically acquired MRI will be scored using our previously validated 4H MRI severity score, which analyzes hypomyelination and cerebral, cerebellar, and brainstem atrophy. Our primary outcome will be time to loss of ambulation.
Functional scales such as the Gross Motor Function Classification System, the Eating and Drinking Classification System, the Manual Ability Classification System, and the Communication Function Classification System, have been implemented. Sixty pediatric quality of life measures, intelligence, stress, and pain reported outcomes are complete. Detailed phenotyping of family history, developmental history, clinical investigations, rehabilitation, clinical evolution, and clinical examinations were created with guidance from pediatric neurologists.
Along with behavioral data, the LORIS database stores neuroimaging data that is to be integrated with the functional scales data to allow querying and dissemination. The imaging data runs through a robust pre-processing pipeline that administers quality control before insertion.
A major obstacle in rare disease research is overcoming small cohorts. Developing an online database that international collaborators can access and contribute to from all over the world will be invaluable for increasing cohort sizes, discerning surrogate markers, and improving natural history data. Creating a predictive tool using this natural history data will life-changing for affected patients and their families. I would enable clinicians to inform them of expected disease course and to be able to evaluate response to emerging treatment strategies.
BACKGROUND: Focal cortical dysplasias (FCDs) are congenital structural abnormalities of the brain, and represent the most common cause of medication-resistant focal epilepsy in children and adults. When possible, surgical resection of affected tissue is performed as treatment. Recent studies have shown that somatic mutations (i.e. mutations arising in the embryo) in mTOR pathway genes underlie some FCD cases. Specific therapies targeting the mTOR pathway are available. However, testing for somatic mTOR pathway mutations in FCD tissue is not performed on a clinical basis, and the contribution of such mutations to the pathogenesis of FCD remains unknown.
AIM: To investigate the feasibility of screening for somatic mutations in FCD tissue and determine the proportion and spatial distribution of FCDs which are due to low-level somatic mTOR pathway mutations.
METHODS: We performed ultra-deep sequencing of 13 mTOR pathway genes using a custom HaloPlexHS target enrichment kit in 24 resected histologically-confirmed FCD specimens.
RESULTS: We identified causal variants in 58.3% (14/24) of patients at an alternate allele frequency of 0.62–33.7%. The spatial mutation frequency correlated with the FCD lesion’s size and severity.
CONCLUSIONS: Screening FCD tissue using a custom panel results in a high yield, and should be considered clinically given the important potential implications regarding surgical resection, medical management and genetic counselling.
Introduction. Peroxisome biogenesis disorders are primarily autosomal recessive disorders caused by mutations in any of 13 PEX genes involved in peroxisome assembly and function. Defects in PEX16, an integral peroxisome membrane protein essential for de novo peroxisome biogenesis, result in a spectrum of clinical features with varying levels of severity. Indeed, while 5 patients with PEX16 were reported to have the severe Zellweger Syndrome phenotype, many more cases of PEX16 patients with unique milder phenotypes and longer survival have been reported. We report, here, an additional 6 unrelated probands, including a sibling pair, with this milder PEX16 presentation, and provide further clinical and cellular characterization of this atypical phenotype.
Methods. Patients were recruited through our institutional research ethics board-approved study, Longitudinal Natural History Study of Patients with Peroxisome Biogenesis Disorders (ClinicalTrials.gov Identifier: NCT01668186). Patient medical records were reviewed, and clinical values were queried in a system-specific manner. Immunoblotting was done according to standard protocol to quantify PEX16 protein in patient primary fibroblasts with PEX16 defects. A green fluorescent protein tagged peroxisome targeting signal 1 (GFP-PTS1) reporter assay was performed to study peroxisome import function in one patient cell line.
Results. The patients in our series largely do not have the vision and hearing deficits, adrenal insufficiency, or renal stones that are commonly seen in PEX1,6,26 mutations. They are also distinguished from the mild phenotypes observed in PEX2,10,12, notably with their unique presentation of hypertonia. Indeed, hypertonia is uniformly present in our patient series, with 3 (and one sibling) out of 6 probands having dystonia. Four patients were tried on carbidopa/levodopa treatment, with 3 having partial but significant improvements in their dystonia, hypertonia, and tremors. One patient experienced side effects (bad dreams, excessive crying, and dizziness) with no significant neurological improvement, and discontinued the medication. Immunoblotting showed reduced PEX16 protein levels in patient primary fibroblasts with PEX16 mutations. A GFP-PTS1 reporter assay showed localization of the GFP-PTS1 reporter with PEX14 peroxisome membrane protein in one patient cell line (PEX16-p.H231r/H231R), suggesting intact peroxisome import function.
Conclusion. This case series highlights hypertonia as a common presentation in patients with PEX16 defects and contributes to the phenotype of this group by describing dystonia in a larger set of patients. We also further characterize the cellular phenotype of PEX16 cell lines by showing reduced PEX16 protein levels as well as intact peroxisome import function. Given the significant functional improvement in response to carbidopa/levodopa treatment in three of the patients reported here, we recommend a trial of carbidopa/levodopa in patients with PEX16 defects and hypertonia.
Although there has been significant progress in our understanding of Lysosomal Storage Diseases (LSDs), biomarkers for diagnosis and monitoring therapies remain limited. Sphingolipids, a family of lipids including ceramide, have been reported to be elevated in LSDs patients. In the case of Mucopolysaccharidoses (MPSs), glycosaminoglycans (GAGs) build-up or inflammation can lead to a restructuring of micro-domains in cellular membranes where ceramide is mainly found, eventually causing increased levels of ceramide and sphingosine-1-phosphate. This is consistent with previous studies that have established a relationship between elevated levels of these two sphingolipids in MPS rat models and enhanced apoptosis in cartilage as well as hyperplasia in synovial tissue. Considering that there is a founder effect of MPSs and acid ceramidase deficiency (Farber disease) in Quebec (Canada), we decided to focus on those two groups of disorders. In our laboratory at the MUHC, we have developed methods to screen and quantify various ceramide species, GAGs and pro-inflammatory cytokines from biological fluids. Using these methods and our unique access to a large cohort of LSDs patients, our objective is to identify specific species of ceramide, or panel combination of several ceramide molecules that could serve as biomarkers for the disease. We hereby present a comparative and correlation study of biochemical substances in serum of LSDs patients. Levels of various classes of ceramide (VLC and VVLC, ceramide, dihydroceramide, hexosylceramide lactosylceramide) and pro-inflammatory cytokines were measured using state-of-the-art liquid chromatography tandem mass spectrometry (LC-MS/MS) and ELISA based techniques (Quansys), respectively. By investigating levels of different ceramide species, we have thus far found significantly increased levels of some species and decreased levels of others compared to healthy controls. This finding suggests that certain ceramide species may be further investigated as biomarkers for LSDs and could potentially indicate clinical symptoms of disease severity.
Background: Arginase 1 deficiency (ARG1-D) is a serious, progressive disease with early mortality and high unmet medical need due to an autosomal recessive disorder of arginine metabolis. Clinical presentation is typically in childhood, with spasticity, intellectual disability, and/or seizures. Progressive spasticity is a key disease manifestation that is not commonly present in other urea cycle disorders. High levels of arginine and arginine-related metabolites are thought to cause the neurologic manifestations of ARG1-D. Episodic hyperammonemia occurs in some patients. Current disease management approaches utilizing severe protein restriction, essential amino acid supplementation, and ammonia scavengers have a limited impact on both plasma arginine levels and disease manifestations, with continued disease progression.
Objective: A literature review was performed to collate available published data to: 1) better define the clinical presentation of patients with ARG1-D; 2) review disease management approaches; 3) provide insights on the age and causes of early mortality.
Methods: A PubMed search was undertaken of unique case reports of patients with ARG1-D published in the English language between 1965 and 2018, followed by review of relevant data. Cases were defined as unique based on assessment of country of origin, sex, age at diagnosis, age at presentation, and age at symptom onset.
Results: The initial search identified 140 case reports of patients with ARG1-D. Patients had markedly elevated plasma arginine and arginine-derived metabolites when measured.
Conclusions: ARG1-D is a severe, progressive disease that presents in early childhood with prominent neurological manifestations that significantly impact daily functioning. Delays in diagnosis of 5 years or more were common. Increased disease awareness and plasma arginine measurement in patients presenting with spasticity, cognitive impairment, or seizures may lead to earlier diagnosis with better outcomes. Current disease management fails to substantially impact the marked elevations in plasma arginine levels, leading to continued disease progression, worsening impairment in daily functioning, diminished quality of life, severe disabilities, and early death. Emerging data from clinical trials demonstrate the potential of a human enzyme-based approach to improve plasma arginine control with accompanying evidence of improvements in important disease-related manifestations in patients with ARG1-D.
We have used forward genetics approach to identify novel genes that modulate inflammatory responses in neuroinflammation. We uncovered that the mutation L166P that affects the highly conserved residue L166 within the Bisphosphoglyceromutase (BPGM) gene, dramatically decreases protein stability. At steady state, BpgmL166P mutant animals show an erythrocytosis phenotype with high hematocrit, elevated hemoglobin, and mildly increased erythropoiesis. In addition, the 2,3-bisphosphoglycerate, a key allosteric regulator of hemoglobin/oxyhemoglobin in erythrocytes, produced by BPGM, is intensely reduced in Red blood cells (RBC). This induces an increased affinity of hemoglobin to oxygen, that mimics features of human primary polycythemia. Moreover, we found that BPGM mutated mice are protected against both cerebral malaria (induced by Plasmodium berghei ANKA; PbA) and blood-stage malaria (induced by Plasmodium chabaudi AS; PcA). Malaria protection is associated with reduced blood parasitemia, milder clinical symptoms and increased survival. However, upon infection with Plasmodium, BpgmL166P mutants display a much stronger erythroid response than controls, with increase in CD71+/Ter119+ erythroid progenitors in spleen and bone marrow. Enhanced erythroid response may help mutant mice compensate for Plasmodium-driven erythrocyte loss. In addition, studies of PbA and PcA-infected erythrocytes in vivo, point to a significant decrease in intracellular parasite maturation from ring stage to mature forms in BpgmL166P mutant cells. To address whether BPGM deficiency alters the erythrocyte intracellular milieu, rendering them less permissive to Plasmodium replication, we performed metabolome analysis of RBC from mutant and control mice. We fund that the level of both purine and pyrimidine nucleotides are dramatically decreased in mutated erythrocytes. This indicates that during intraerythrocytic stages, the parasite has very limited access to the host purine nucleotides which affects it growth and multiplication. Our study shows that BPGM deficient mice are a good model for studying primary polycythemia and that BPGM deficiency drastically affects RBC metabolism that reduces RBC permissiveness to parasite growth that ultimately confers significant protection against malarial infections.
Background: Ewing sarcoma (ES) is a rare bone and soft tissue tumour that mainly affects children, adolescents and young adults at a global rate of ~1.5 cases per million. ES tumours are characterized by a quiet and stable genome with rare damaging somatic nucleotide variants (SNV) and non-recurrent genomic rearrangements. The primary oncogenic driver event in ES is the fusion between EWSR1 and members of the E26 Transformation-Specific (ETS) family of transcription factors, most commonly FLI1. The chimeric protein produced, EWS-FLI1, cooperate with other genetic events to mediate oncogenesis in human mesenchymal stromal cells (hMSC), the putative cell of origin of ES. In the absence of cooperating events, EWS-FLI1 mediates cell senescence and/or apoptosis in human somatic cells. After 20 years since the discovery of EWS-FLI1, ES remains a clinical challenge with unacceptably low survival rates, which is primarily due to metastasis. The critical interplay between EWS-FLI1 and other somatic mutations that orchestrate disease initiation, progression and metastatic relapse is yet to be charted.
Methods: To unravel cooperating events in ES initiation and metastatic progression, the LH system was used to perform a genome-wide mutagenesis screen in hMSCs and non-metastatic chemonaïve ES lines of different genetic backgrounds. To decipher events cooperating in ES initiation, EWS-FLI1 was overexpressed in hMSC-LH. To further study events acquired during tumour progression in a clinical setting, orthotopic mouse models were generated using LH-ES lines and mouse-adapted neoadjuvant chemotherapy was performed.
Results: Upon activating the LH system, transposon-mediated mutagenesis activated oncogenes and inactivated tumour suppressor genes in the engineered models. In contrast to wildtype hMSC, hMSC-LH were able to tolerate EWS-FLI1 expression while mimicking a ES phenotype. Implanted ES-LH cells were capable of forming aggressive tumours in recipient mice with enhanced metastatic capacity compared to wildtype ES lines. Matched primary, relapse and metastatic tumours are collected and sequenced to perform pathway-oriented bioinformatic analysis.
Conclusion: The EWS-LH system was successful in generating stable cell lines that model Ewing sarcoma. Cooperating events delivered by LH provided tolerance to EWS-FLI1 in hMSC and enhanced aggresiveness and metastatic capacity of ES lines in-vivo.
Homozygous familial hypercholesterolemia (HoFH) is an orphan disease characterized by extremely high levels of plasma levels of LDL-C. Affected patients develop clinical atherosclerotic cardiovascular disease (ASCVD) in youth and survival > 30 years of age was unusual until the advent of medications and extracorporeal LDL filtration (apheresis) techniques. HoFH is an autosomal co-dominant condition defined as an LDL-C > 13 mmol/L in adults without treatment and homozygous or compound heterozygous mutations of the LDLR gene. HoFH has a genetic probability of ~1/250,000 and the rare diseases inventory Orphanet estimates its worldwide prevalence at 0.1/100,000 individuals. Canada is known to have several regions with a founder effect for HoFH and we recently identified 79 cases across the country. Data from other countries show a median survival of HoFH patients at < 40 years of age.
Clinical outcomes in HoFH patients, especially ASCVD events (fatal and non-fatal myocardial infarctions, strokes and peripheral vascular disease) and severe calcific aortic stenosis, are difficult to capture, in part because of the rarity of the disorder and the lack of registry focusing on this disease. The objective of our project is to obtain a comprehensive registry of HoFH in Canada, estimate the cost to society caused by HoFH burden of disease in Canada, and implement changes to advocate access to specialized care for these patients.
Here, we present preliminary data on our HoFH registry, including patients’ medical history, levels of LDL-C, treatments and outcomes.
We plan to use this data at provincial and national levels, in help with the Canadian Organization for Rare Diseases (CORD) and the Réseau Québecois des maladies orphelines (RQMO), to provide HoFH patients access to care, including PCSK9 inhibitors, orphan drugs such as lomitapide, and treatment techniques such as extracorporeal LDL filtration (apheresis). This work will provide important new health-related knowledge about the determinants of ASCVD risk and phenotypic manifestations of HoFH in Canada and examine the quality of life and burden to the healthcare system.
Spontaneous Coronary Artery Dissection (SCAD) is a rare type of Acute Coronary Syndrome (ACS) that primarily affects younger individuals who do not exhibit “traditional” cardiovascular risk factors. Previous genetic and epidemiologic studies indicate that connective tissue disorders (CTDs) such as Marfan syndrome, Loeys-Dietz syndrome, and Ehlers-Danlos syndrome with known genetic causes are more prevalent among SCAD than other ACS patients, however, these conditions combined are thought to explain less than 0.5% of SCAD cases. The arteriopathy fibromuscular dysplasia (FMD) is also enriched in SCAD patients, and common variants at the PHACTR1/EDN1 locus previously associated to FMD are also associated with SCAD. While many patients report extreme emotional episodes or physical exertion at SCAD onset, these environmental factors may serve as a trigger in patients with an underlying genetic vascular pathology.
Previous genetic studies of SCAD have used CTD and familial thoracic aortic aneurysm and dissection (TAAD) gene panels to screen for causative mutations, however this approach achieved only an 8% rate of genetic diagnosis. In addition, due to the potential phenotypic heterogeneity of vascular pathology and low penetrance (only 1.2% of cases show familial inheritance) the use of trios, a common practice for Mendelian diseases, may not be useful. New approaches are needed to facilitate improved screening, diagnosis, treatment, and genetic counselling for SCAD patients.
We have Whole Genome Sequenced 45 SCAD patients in the GENESIS-PRAXY cohort, which consists of individuals who had an ACS event at or under age 55. SCAD individuals were significantly more likely to be female (p<.001) and have an earlier age of onset (p=.006) and less likely to smoke (p=0.15), be obese (p=.02), or have hypertension (p=.15), dyslipidemia (p=.06), or diabetes (p=.35). We constructed a pipeline to identify likely causal variants using previous reports of pathogenicity in ClinVar, computational predictions of variant deleteriousness, and allele frequencies in gnomAD. The pipeline identified Variants of Unknown Significance (VUS) in previously known candidate genes and other closely related genes in 20/45 patients.
SCAD may represent the first obvious clinical presentation of a mild CTD, resulting from a mutation that does not reach the ACMG pathogenic designation. As such, VUS in CTD genes should be given extra scrutiny in SCAD patients.
Primary ciliary dyskinesia (PCD) is a disease of impaired mucociliary clearance with symptoms from early infancy. Inhaled hypertonic saline (HS) promotes cough and secretion clearance and is a mainstay of treatment in cystic fibrosis (CF), improving lung function and rates of pulmonary exacerbation. Studies in CF infants have shown safety and potential benefit to early HS initiation; however, no recommendations exist on initiation of HS in infants with PCD.
A retrospective review of patients with a neonatal diagnosis of PCD was performed on all patients in the PCD clinic of the Montreal Children’s Hospital between 2011-2019. Detailed data was collected on therapies in the neonatal period, including HS, and clinical data was gathered for the first 2 years of life.
Of 34 pediatric PCD patients, 9 (26%) were diagnosed ≤1 month of age. Five of 9 (56%) patients began HS during their initial postnatal hospitalization, with HS initiated in all 9 by 3 years of age. HS was administered via facemask, using concentrations of 5% or 7%, with pre-treatment of salbutamol via metered dose inhaler and spacer, with normal chest auscultation before and after the initial dose. All patients had neonatal respiratory distress within 48 hours of birth requiring hospitalization and supplemental oxygen (median length supplemental oxygen: 17 days; median length neonatal hospitalization: 21 days). All patients had year-round daily cough and nasal congestion from early infancy. Quarterly outpatient pharyngeal swabs showed only one patient acquired pseudomonas aeruginosa at 2 years of age, which was eradicated with 1 month of inhaled tobramycin. Of the 9 patients on HS, 2 were admitted in the first 6 months of life for bronchitis or bronchiolitis, while the other 7 were not admitted in the first 2 years of life.
Inhaled HS is well tolerated when initiated in infants with PCD, even during periods of acute neonatal respiratory distress. No adverse events occurred when HS was administered with salbutamol pre-treatment, and rates of P. aeruginosa acquisition do not seem increased with regular nebulizer sterilization. Early initiation of HS may have beneficial clinical effects in PCD infants, including decreased hospitalization for respiratory illnesses, improved nasal hygiene, and other outcomes. Given the small sample size and lack of a control group in this review, studies are needed to further investigate the clinical benefits of HS in the early life of PCD patients.
Introduction: Peroxisome biogenesis disorders (PBD) are a heterogenous group of hereditary disorders caused by a defect in the biogenesis or functions of peroxisomes. Individuals affected with PBD manifest a complex spectrum of clinical phenotypes ranging from profound neurologic symptoms in newborns to progressive degenerative disease in children and adults. In spite of advancements in peroxisome biology, the spectrum of phenotypes remains poorly characterized and the natural history not yet systematically reported.
Objectives: Our goals are to better understand the natural progression of the disease symptoms in PBD in order to generate clinical care guidelines and to clearly identify endpoints for future interventional trials.
Methods: We collect medical records from PBD patients internationally; we input clinical data in a custom database allowing us to do large-scale analyses to define the progression of the disease symptoms.
Results: We currently have medical records on 134 patients from 0 to 45 years old with mutations in 8 different PEX genes. Our neurological review revealed that developmental outcome is correlated with genotype severity and with specific brain abnormalities observed on brain MRIs. We found that polymicrogyria were visible on brain MRIs in mildly affected patients, which challenges the current classification criteria for the disease severity. Our review on vision loss revealed that maculopathy and abnormal pigmentation in the peripheral retina are present in almost all PBD patients, regardless of their severity subgroup, while the rate of visual acuity regression correlates with genotype severity. We generated disease specific growth curves and found that PBD patients generally track at the lower end of the normal population growth curves. Liver disease profiles show highest elevations of liver function tests in infancy that decline with age, but some patients experience liver failure due to fibrosis and portal hypertension. We are currently reviewing the unique medical needs of adult patients with PBD. These retrospective analyses allow the identification of critical questions that will be addressed in prospective studies in preparation for future interventional trials.
Candida albicans (C.alb) is an opportunistic fungal pathogen with a propensity for systemic disease, as well as a high mortality rate and remains a major concern in hospital settings. Mutations in the gene for Caspase Recruitment Domain-containing protein 9 (CARD9) cause invasive candidiasis (IC) in the central nervous system (CNS). The CNS relies on bone marrow derived immune cells to fight IC, yet the hematopoietic response in the context of CARD9-deficiency has not been investigated. We evaluated quantitative and qualitative characteristics of emergency hematopoiesis during the course of infection in CARD9null mice. Bone marrow hematopoietic stem cell responses diverge in CARD9null and wild-type mice early in disease progression and correspond with impaired resistance to fungal growth and immunopathology in the brain of CARD9null mice. We show that CARD9-deficient mice are able to mount a robust stem cell response, but that bone marrow-derived macrophages demonstrate dysfunctional anti-fungal responses. Our work deepens our understanding of the immune response to C. alb in the context of CARD9-deficiency.
Cystinuria is a rare hereditary renal stone disease, characterized by defective reabsorption of cystine and dibasic amino acids at the brush border membrane of proximal renal tubules. In humans, cystinuria is caused by mutations in SLC7A9 (an incompletely dominant gene encoding a cystine selective transporter) or in SLC3A1 (a recessive gene encoding a subunit required for apical localization). Of the reported pathogenic mutations in these genes, 18% in SLC3A1 and 4% in SLC7A9 are nonsense mutations (NSM).
Interestingly, some aminoglycosides bind to the mammalian ribosome and relax translational fidelity at premature STOP codons. This permits insertion of a “near-cognate” tRNA that continues translation despite the NSM. Unfortunately, aminoglycoside antibiotics have inherent renal and cochlear toxicity, precluding their long-term use. However, Baasov et al have developed a new class of designer aminoglycosides with decreased mammalian toxicity and efficient readthrough capacity. We recently reported that a 5thgeneration compound, ELX-02, induces translational readthrough of the CTNSW138X NSM that causes the lysosomal disease, cystinosis. Furthermore, we found that ELX-02 permits readthrough of a tyrosinase NSM and restores melanin production in mutant albino mice.
Here, we hypothesize that ELX-02 might allow translational readthrough of NSM in SLC3A1 or SLC7A9. In collaboration with Dr. David Goldfarb at the New York Langone Health, we have identified a cohort of cystinuria NSM patients and have established a protocol to isolate proximal tubule cell lines from patient urine. Urinary cells are pelleted from 100ml of fresh urine, allowed to attach in monolayer culture and are immortalized with an SV40/lentivirus vector. Immortalized proximal tubular cells (PTC) are isolated by FACS, using CD10/CD13 fluorescent-tagged markers, and re-cultured. The proximal tubular identity of the sorted cells is confirmed by immunofluorescent staining for megalin, and RT-PCR confirmation of SLC3A1 and SLC7A9 expression.
We next established an in vitro method to assess uptake of cystine urinary PTC derived from healthy controls. Wildtype PTC were washed and cultured in cystine–free medium for an hour. The cells were then incubated with FITC-tagged cystine at varying concentrations (1-50uM) and times (15min-24hr). After washing, we analyzed cells for uptake of cystine-FITC using flow cytometry. WT cells exhibit saturable uptake of cystine-FITC (Km ~7-8uM) after a 1-hour incubation; this Km is in agreement with published Km values for apical cystine transport.
Using these methods, we will next assess the effect of ELX-02 on restoration of in vitro cystine transport in PTC isolated from our NSM cystinuria patients.
Introduction. Zellweger spectrum disorders (ZSDs) are a group of autosomal recessive disorders caused by mutations in any one of 13 PEX genes whose protein products are required for peroxisome assembly and function. More mildly affected individuals harboring the common PEX1-G843D mutation consistently develop a potentially blinding retinopathy. To test whether we could improve vision in these patients, we performed a proof-of-concept trial for PEX1 retinal gene augmentation therapy using our mouse model homozygous for the equivalent PEX1-G844D mutation. This model exhibits a gradual decline in scotopic full field flash electroretinogram (ffERG) response, a residual photopic ffERG response, diminished visual acuity (optokinetic reflex, OKR), and photoreceptor cell anomalies.
Methods/Results. AAV8.CMV.HsPEX1.HA vector was administered by subretinal injection to 2 mouse cohorts of 5 or 9 weeks of age; AAV8.CMV.GFP vector was used as a control in the contralateral eye. Successful expression of HsPEX1.HA protein in the photoreceptor layer with no gross histologic side effect was observed by immunohistochemistry. In vitro studies using PEX1-deficient mouse and human cell lines confirmed vector expression and successful recovery of peroxisome import after HsPEX1 delivery. Preliminary ffERG and OKR analyses on a subset of animals at 8 weeks post injection showed two-fold increase in photopic ffERG and a slight (p=0.054) improvement in visual acuity, respectively, in the PEX1-injected eyes. ffERGs were improved by 1.6 to 2.5-fold at 25-weeks of age in both cohorts (16 or 20 weeks post gene delivery). At 32 weeks of age (23 or 27 weeks post gene delivery), the average ffERG response in the PEX1-injected eyes was double that of GFP-injected eyes in both cohorts. LC-MS/MS was used to measure peroxisomal metabolites in whole retinas, revealing that in the younger cohort PEX1 gene augmentation reduced the average C26:0 lysophosphatidylcholine (lyso-PC) in PEX1-G844D mice, which is elevated due to peroxisome dysfunction.
Conclusions. Robust PEX1 protein expression with no gross histologic side effect was observed in homozygous PEX1-G844D mice injected subretinally at 5 or 9-weeks-of age with AAV8.CMV.hPEX1.HA. Neither the injection, nor exposure to the AAV8 capsid or the transgenic protein negatively altered the ffERG or OKR response. At 5-6 months after gene delivery, therapeutic vector-treated eyes showed improved ffERG compared to control eyes, on average, in both the younger and older cohorts, and peroxisome metabolite recovery indicated a broader recovery of retinal peroxisome functions. Overall, our results support the clinical potential of retinal gene augmentation to improve vision in patients with ZSD at both earlier and later stages of disease.
Background: Peroxisome biogenesis disorders (PBD) is an inherited genetic condition affecting children and adult. It is caused by global dysfunction of peroxisome metabolisms due to mutation in any one of 14 Pex genes, whose protein products are required for peroxisome assembly. This leads to a multisystem disorder, including sensorineural deficit and chronic hepatic dysfunction. A common mutation encodes PEX1-p.Gly843Asp (G843D), a misfolded protein with residual function associated with a milder disease course. In absence of curative therapy for PBD, it is necessary to identify an available and affordable intervention for patients. In vitro study has shown that accumulated C27 bile acid intermediates (due to peroxisome metabolic dysfunction) is hepatotoxic. Other evidences also suggested toxic C27 bile acid intermediates causing liver injury and dysfunction. Taken together, we propose that cholic acid supplementation will suppress biosynthesis of C27 bile acid precursors by feedback inhibition and halt liver disease progression.
Over the past year, I have characterized the liver disease in our PEX1-G844D knock-in mouse model (murine equivalent of the human G843D), which recapitulates many of the human disease liver features, including hepatomegaly, cholestasis, hepatitis, fibrosis and cancer. In addition, there were elevated serum liver transaminases indicating persistent liver injury. These results support translatability of our mouse model in testing potential treatments for PBD patients.
Hypothesis: Cholic acid supplementation will halt liver disease by inhibiting the accumulation of C27 bile acid intermediates.
Methods: We fed G844D homozygotes and controls at 4 weeks of age using 0.5% and 0.2% cholic acid-containing diet for one month. Blood was collected pretreatment and every week during the treatment to monitor serum levels of bile acids and other peroxisome metabolites using LC-MS. After one month of treatment, mice were sacrificed, and body weight was measured. Liver tissue and blood were collected. Liver function tests were performed to assess liver injury, Hepatic levels of bile acids and other peroxisome metabolites were also quantified. Liver pathology was assessed using various histological staining and immunofluorescence. Biochemical profiles, body weight and liver pathologies in treated mice were compared to the concurrent untreated cohort to examine the effects of cholic acid therapy.
Results: By comparing treated mice to concurrent untreated cohort, I observed reduced serum levels of C27 bile acid precursors and increased serum C24 mature cholic acid in PEX1-G844D homozygotes after cholic acid diet. I observed decreased signal of SOD2 in treated PEX1-G844D homozygotes, representing alleviated mitochondrial oxidative stress. However, increased amount of neutral lipids in livers of cholic acid fed mutant mice was observed, suggestive of aggregated steatosis. In addition, a higher occurrence of bile duct proliferation was found in 0.5% cholic acid fed mutant mice. Both cholic acid treated and concurrent untreated mutant mice developed intercellular fibrosis. No change was observed in number nor import function of peroxisomes after cholic acid diet.
Conclusion: Our experimental results showed that 0.2% and 0.5% cholic acid diet exhibited both beneficial and adverse effects in PEX1-G844D homozygotes. Cholic acid alleviated mitochondrial oxidative stress presumably through reducing the accumulation of C27 bile acid precursors. However, it also aggravated steatosis and bile duct proliferation in treated mutant mice, suggestive of hepatotoxicity of oral cholic acid at high dose. Cholic acid diet did not prevent development of fibrosis. We will reduce the concentration of cholic acid in diet for future experiments.
Subependymal giant cell astrocytoma (SEGA) is a rare low-grade brain tumor that is frequently associated with tuberous sclerosis complex (TSC). The tumor typically presents during the first two decades of life, originating from the wall of the lateral ventricle of the brain, which can interfere with CSF flow and cause hydrocephalus. TSC is an autosomal dominant condition characterized by the formation of mostly benign tumors in several organs including brain, skin, kidneys, heart, lungs and eyes. Phenotypic features are extremely variable, even among members of the same family. Pathogenic variations in the genes TSC1 or TSC2 are identified in 95% of patients who meet clinical diagnostic criteria. Most cases are sporadic (60 to 70%), and a significant proportion of cases without an identifiable genetic variation are felt to likely represent somatic mosaicism.
A previously well young man presented with a two week history of headaches and four days of diplopia. Papilledema was noted on ophthalmology evaluation. Cerebral CT scan showed a left frontal lesion of the lateral ventricle causing obstructive hydrocephalus. He underwent urgent tumor resection and derivation. On physical examination, two small clear papules on the nasal tip, one larger colored papule on right cheek as well as a single traumatic peri-ungual fibroma of the right big toe were noted. No other skin or dental findings were identified. Abdominal ultrasound was normal. Molecular analysis of TSC1 and TSC2 in blood leucocytes was reported as negative (read depth 20X).
RNA-Sequencing on the resected tumor uncovered a pathogenic stop-gain variant in TSC2, named NM_000548.4:c.3412C>T (p.Arg1138Ter), with a variant allele frequency of 33%. A second TSC2 variant was not identified in the tumor sample. Targeted deep sequencing (Illumina MiSeq platform) of TSC2 exon 30 on DNA extracted from blood, buccal swab and tumor detected the TSC2 variant at an allele frequency of 7.6%, 6.3% and 46.1%, respectively, confirming postzygotic mosaicism. Genome-wide DNA methylation array data of the tumor sample further confirmed the diagnosis of SEGA with a match score of 99%.
Conclusion: while isolated cases of SEGA have been reported, new molecular diagnostic technologies will likely uncovered TSC mosaicism in many patients who do not fulfill current clinical diagnosis criteria.
Lymphangioleiomyomatosis (LAM) is a rare disease caused by the abnormal proliferation of smooth muscle-like cells (LAM cells), which form microscopic tumors that cause cystic destruction of the lung. LAM cells exhibit loss of function of the tumor suppressor protein tuberin (TSC2), which leads to hyperactivation of ‘mammalian target of rapamycin’ (mTOR). Administration of rapamycin to patients can slow progression of disease, but this is not universally effective or curative. Other pathogenetic mechanisms, and potential therapeutic targets, include elevated activity of selected oncogenic G-protein coupled receptors (GPCRs), and high levels of neural crest-related proteins. In LAM nodules and model cell lines, we identified high levels of the urotensin receptor (UT), a neural crest-associated oncogenic GPCR, and its agonist urotensin II (UII). Model TSC2-deficient cell lines exhibited increased migration, invasion, and growth in vitro and in vivo. We hypothesized that loss of TSC2 biases urotensin receptor activity towards the activation of oncogenic G-proteins (i.e., Gq, G13, Gi) that promote the invasive behaviour of LAM cells. We evaluated urotensin receptor activity in response to increasing concentrations of UII in TSC2-deficient HEK 293T cells and parental controls. By bioluminescence resonance energy transfer (BRET) assay, UII preferentially activated BRET biosensors associated with the neoplastic phenotype (i.e., Gq/G13 signaling). The other known urotensin agonist ‘urotensin related peptide’ (URP) had no effect; biosensor activation was inhibited by UT antagonists, but not by the mTOR inhibitors rapamycin or torin-1. In TSC2-deficient cells UT activation by UII led to Gq, but not G13 or Gi, activation. In summary, loss of TSC2 leads to biased UT activity that favors pro-neoplastic G-protein signaling in mTOR-independent fashion. Biased G-protein signaling may represent a mechanism by which loss of TSC2 promotes mTORC1-independent cell migration, invasion, and metastasis in LAM.
Little data is known about the genetic bases of recurrent reproductive loss and infertility mainly due to their genetic heterogeneity. In fact, the genetics of reproductive loss and infertility has a lot of delays relative to other medical disciplines such as neurology, biochemistry, ophthalmology, and pediatric. The advent of next generation sequencing in the last decade has opened an unprecedented opportunity to tackle genetically heterogeneous conditions and allowed the recent identification of several genes responsible for infertility and reproductive loss in humans. Known genes explain the genetic etiology of a small fraction of these conditions and many other genes remain to be identified. The research work of my laboratory is focused on the identification of novel genes responsible for reproductive loss and the elucidation of their functional roles. The goal of our work is to better understand these conditions and be able to provide the patients with DNA testing to identify their causative genes and provide them with accurate gene counselling and assisted reproductive technologies tailored to their exact defects.
EML3 is one of 6 Echinoderm Microtubule-Associated Proteins (EMAPs) in mammals. Only two papers have been published on EML3 (Tegha-Dunghu et al., JCS, 2008; Luo et al., JBC, 2019). They showed, in cultured cells, that EML3 regulates the assembly of the mitotic spindle, and that its loss of function slows the cell cycle. Our interest in EML3 arose because of its predominant expression in adult retina. To begin to explore its function, we generated Eml3-null mice.
We found that most Eml3-KOs die shortly after birth, with two major phenotypes at E18.5: 1) All Eml3-null embryos have global developmental delay as early as E9.5. The delay is global since the embryos are smaller, have fewer somite pairs, and show delayed onset of neurogenesis and lung saccule formation.
We hypothesize that the reduced proliferation of mesenchymal cells causes the developmental delay. Indeed, we found that the proliferation of mouse embryonic fibroblasts from Eml3-null mice is slow. To define the effect of EML3 LOF on the cell cycle of mesenchymal cells, we are EdU-labelling E9.5-E11.5 embryos. Using EdU and a marker of mitotic cells will allow us to establish the phase(s) of the cell cycle that are impaired; initial results suggest that all stages are prolonged. 2) The forebrains of 70% of Eml3-null mice have scattered focal neuronal ectopias (FNEs), a defect in which newborn neurons migrate beyond the pial basement membrane (PBM; which separates the forebrain and meninges). In FNEs, the newborn neurons either pass through an already breached abnormal PBM, or punch through an intact PBM. To distinguish between these alternatives, we are examining the PBM of developing Eml3-null embryos looking whether the breaches are present before or after the onset of neuronal migration. To define the role of EML3 in PBM development, we asked where EML3 is expressed in the developing head at E9.5, when the PBM is forming. We showed that EML3 is expressed in the two cell types that contribute to PBM, the meningeal mesenchyme and the neuroepithelium. We are now deleting Eml3 specifically from each cell type, using cell-specific promoters driving Cre recombinase, to determine whether the FNEs are due to the absence of Eml3 function in the developing meningeal mesenchyme, neuroepithelium, or both. A prolonged cell cycle in either the developing forebrain neuroepithelium and/or meningeal mesenchyme may reduce the number of either cell type, both of which are essential for PBM formation.
In humans, the FNE phenotype is called cobblestone brain malformation (COB). COB is present in disorders such as Walker-Warburg syndrome and Fukuyama congenital muscular dystrophy. We noted that in a recent publication (Hu et al., MolPsych, 2019), three sisters with autosomal recessive severe developmental delay, profound intellectual disability, and infantile-onset seizures (but no COB and normal brain MRI) each carried homozygous Leucine732Phenylalanine alleles. We have found, using in vitro expression studies in 293 cells, that the L732F mutation impairs EML3 folding. We are currently introducing the L732F mutation into mice using CRISPR, to determine whether the mutation is disease-causing. No convincing putative EML3 LOF alleles were found in a cohort of 61 patients with unexplained COB or polymicrogyria, a comparable phenotype, nor in the exomes of 1000s of patients with putative or demonstrated Mendelian disease phenotypes who have had exome sequencing.
To elucidate the molecular function of EML3, we turned to the tissue where it is predominantly expressed in adults, the retina, specifically rod photoreceptors (rPRs). We created mice in which the Eml3 gene is specifically deleted from rPRs (Eml3-cKO) and observed defects in rPR physiology. Dark/light adaptation kinetics in Eml3-cKO mutant retinas are abnormal: the translocation kinetics of proteins that normally move between rPR compartments in different light conditions are altered, as determined by immunofluorescence microscopy studies.
We conclude that EML3 is required for post-natal survival, global embryonic development, brain development, and dark/light adaptation of the retina. A complete loss of EML3 function in humans may be embryonic lethal.
Wiskott-Aldrich syndrome (WAS) is a rare X-linked primary immunodeficiency caused by mutation in the WAS gene expressed exclusively in hematopoietic stem cells. It encodes the WAS protein (Wasp), which is important in relaying signals from the cell surface to the actin cytoskeleton. Classic WAS, in which the protein is absent, is characterized by microthrombocytopenia, eczema, recurrent infections, increased autoimmunity and malignancies. X-linked thrombocytopenia (XLT) occurs when the protein is decreased but not absent, and is characterized by thrombocytopenia, milder eczema and immunodeficiency, whereas gain-of-function mutations of Wasp results in X-linked neutropenia, characterized by neutropenia and variable myelodysplasia. Prognosis of classic WAS is poor, with 36% of patients dying from disease-related complications at a mean age of 8. However, with the use of hematopoietic stem cell transplantation (HSCT), long-term survival has increased to 90%. In a long-term case series of WAS, among patients who did not undergo HSCT, the projected median survival was 25 years for those with splenectomy, in contrast to 4 years in unsplenectomized patients. We herein describe a unique case of WAS spanning four decades, illustrating the natural history of the disease while highlighting the challenges in management throughout the years.
Our patient is a 43-year-old gentleman who has experienced many complications of WAS since childhood, including infections, autoimmunity and malignancy. At 5 years old, the patient had severe thrombocytopenia, which was treated with prednisone and splenectomy. At age 7, the patient had a stage II Wilms tumor and underwent radical nephrectomy and chemotherapy. The patient had meningitis at age 13 with residual hearing loss, followed by two subsequent episodes of pneumosepsis at ages 16 and 21, requiring intensive care unit admission. The diagnosis of Wiskott-Aldrich syndrome was eventually made at age 21. He was started on intravenous immunoglobulin (IVIG) and long-term penicillin antibiotic prophylaxis. He was well on follow-up until age 32, when he was diagnosed with nodal marginal zone B cell lymphoma and was treated with Rituximab. His disease relapsed with progressive cytopenias 7 years later and was treated with RCVP with very good partial response and was on maintenance Rituximab. Most recently, he had relapsed immune thrombocytopenia treated with IVIG and prednisone. While on prolonged steroids however, he developed pneumocystis carinii pneumonia and was put on septra. Following an exanthematous maculopapular rash from septra, it was switched to atovaquone. The patient is now maintained on IVIG, prophylactic antibiotics and PCP prophylaxis.
With more than 300 mutations identified in the WAS gene, the clinical presentation of the disease is heterogenous. Patients have quantitative and qualitative defects in cell-mediated and humoral immune deficiencies, putting them at risk of viral, bacterial and fungal infections. They can also have an abnormal innate immune response with impaired formation of immunological synapse and compromised cytolytic activity of NK cells. Thrombocytopenia can be explained by abnormal platelet formation as well as destruction by antiplatelet antibodies. Autoimmunity is estimated to develop in 40% of patients, possibly due to a defect in regulatory T cells. Malignancy may occur in 13% of patients, lymphoma being the most common. This could be due to decreased cytotoxic T cell killing of cancer cells.
When caring for these patients and their families, a multidisciplinary approach is important and should include genetic counseling, psychosocial support as well as subspecialist involvement, such as immunology, hematology/oncology, infectious disease and critical care. Patients should be immunized with conjugated and unconjugated vaccines while avoiding live and attenuated viral vaccines. Splenectomised patients should be placed on lifelong prophylactic penicillin. If transfusion of blood product is required, irradiated and cytomegalovirus-negative products should be used. Since patients with WAS have abnormal antibody responses, immunoglobulin replacement therapy is important in preventing severe infections. Given the many complications that can affect a patient with WAS as illustrated in our case, it is important to have close follow-ups and a proactive approach to maintain them as healthy as possible. With the appropriate care and timely interventions as mentioned, patients with WAS may have an excellent prognosis.