Sali M. K. Farhan

Exome sequencing identifies NFS1 deficiency in a novel Fe-S cluster disease, infantile mitochondrial complex II/III deficiency

Iron‐sulfur (Fe‐S) clusters are a class of highly conserved and ubiquitous prosthetic groups with unique chemical properties that allow the proteins that contain them, Fe‐S proteins, to assist in various key biochemical pathways. Mutations in Fe‐S proteins often disrupt Fe‐S cluster assembly leading to a spectrum of severe disorders such as Friedreichs ataxia or iron‐sulfur cluster assembly enzyme (ISCU) myopathy. Herein, we describe infantile mitochondrial complex II/III deficiency, a novel autosomal recessive mitochondrial disease characterized by lactic acidemia, hypotonia, respiratory chain complex II and III deficiency, multisystem organ failure and abnormal mitochondria. Through autozygosity mapping, exome sequencing, in silico analyses, population studies and functional tests, we identified c.215G>A, p.Arg72Gln in NFS1 as the likely causative mutation. We describe the first disease in man likely caused by deficiency in NFS1, a cysteine desulfurase that is implicated in respiratory chain function and iron maintenance by initiating Fe‐S cluster biosynthesis. Our results further demonstrate the importance of sufficient NFS1 expression in human physiology.

Old gene, new phenotype: mutations in heparan sulfate synthesis enzyme, EXT2 leads to seizure and developmental disorder, no exostoses

Background Heparan sulfate proteoglycans are vital components of the extracellular matrix and are essential for cellular homeostasis. Many genes are involved in modulating heparan sulfate synthesis, and when these genes are mutated, they can give rise to early-onset developmental disorders affecting multiple body systems. Herein, we describe a consanguineous family of four sibs with a novel disorder, which we designate as seizures-scoliosis-macrocephaly syndrome, characterised by seizures, intellectual disability, hypotonia, scoliosis, macrocephaly, hypertelorism and renal dysfunction. Methods Our application of autozygosity mapping and whole-exome sequencing allowed us to identify mutations in the patients. To confirm the autosomal-recessive mode of inheritance, all available family members were genotyped. We also studied the effect of these mutations on protein expression and function in patient cells and using an in vitro system. Results We identified two homozygous mutations p.Met87Arg and p.Arg95 Cys in exostosin 2, EXT2, a ubiquitously expressed gene that encodes a glycosyltransferase required for heparan sulfate synthesis. In patient cells, we observed diminished EXT2 expression and function. We also performed an in vitro assay to determine which mutation has a larger effect on protein expression and observed reduced EXT2 expression in constructs expressing either one of the mutations but a greater reduction when both residues were mutated. Conclusions In short, we have unravelled the genetic basis of a new recessive disorder, seizures-scoliosis-macrocephaly syndrome. Our results have implicated a well-characterised gene in a new developmental disorder and have further illustrated the spectrum of phenotypes that can arise due to errors in glycosylation.

Exome Sequencing: New Insights into Lipoprotein Disorders

Several next generation sequencing platforms allow for a DNA-to-diagnosis protocol to identify the molecular basis of monogenic dyslipidemias. However, recent reports of the application of whole genome or whole exome sequencing in families with severe dyslipidemias have largely identified genetic variants in known lipid genes. To date, high-throughput DNA sequencing in families with previously uncharacterized monogenic dyslipidemias, have failed to reveal new genes for regulation of plasma lipids. This suggests that rather than sequencing whole genomes or exomes, most patients with monogenic dyslipidemias could be diagnosed using a more dedicated approach that focuses primarily on genes already known to act within lipoprotein metabolic pathways.

Genetics 101 for Cardiologists: Rare Genetic Variants and Monogenic Cardiovascular Disease

Monogenic diseases have a distinctive familial inheritance that follows Mendels laws, showing patterns like dominant, recessive, or X-linked. There are > 7000 monogenic diseases curated in databases, and together they account for up to 10% of all illnesses encountered in the emergency room or clinic. Despite the rarity of individual monogenic conditions, mapping their causative genes and mutations is important for several reasons. First, knowing the causative gene and mutation could provide actionable information for genetic counselling. Sometimes, knowing the gene and mutation allows for early diagnosis in affected families, which is important if there is an evidence-based intervention. Second, the implication of a mutant gene as being causative for a clinical phenotype provides strong evidence of the importance of the gene product in a cellular or biochemical pathway. Discovery of new molecular pathways in families with rare diseases can serve as the first step toward developing rational therapies to help not only affected families, but also patients with less extreme, nongenetic forms of the same condition. For instance, the study of rare patients with familial hypercholesterolemia helped in developing statin drugs, initially as a treatment for familial hypercholesterolemia but now a widely used therapy to reduce low-density lipoprotein cholesterol and cardiovascular disease risk.

Linkage analysis and exome sequencing identify a novel mutation in KCTD7 in patients with progressive myoclonus epilepsy with ataxia

Epilepsy affects approximately 1% of the worlds population. Genetic factors and acquired etiologies, as well as a range of environmental triggers, together contribute to epileptogenesis. We have identified a family with three daughters affected with progressive myoclonus epilepsy with ataxia. Clinical details of the onset and progression of the neurologic presentation, epileptic seizures, and the natural history of progression over a 10‐year period are described. Using autozygosity genetic mapping, we identified a high likelihood homozygous region on chromosome 7p12.1‐7q11.22. We subsequently applied whole‐exome sequencing and employed a rare variant prioritization analysis within the homozygous region. We identified p.Tyr276Cys in the potassium channel tetramerization domain–containing seven gene, KCTD7, which is expressed predominantly in the brain. Mutations in this gene have been implicated previously in epileptic phenotypes due to disturbances in potassium channel conductance. Pathogenicity of the mutation was supported by bioinformatic predictive analyses and variant cosegregation within the family. Further biologic validation is necessary to fully characterize the pathogenic mechanisms that explain the phenotypic causes of epilepsy with ataxia in these patients.

The Ontario Neurodegenerative Disease Research Initiative (ONDRI)

Because individuals develop dementia as a manifestation of neurodegenerative or neurovascular disorder, there is a need to develop reliable approaches to their identification. We are undertaking an observational study (Ontario Neurodegenerative Disease Research Initiative [ONDRI]) that includes genomics, neuroimaging, and assessments of cognition as well as language, speech, gait, retinal imaging, and eye tracking. Disorders studied include Alzheimers disease, amyotrophic lateral sclerosis, frontotemporal dementia, Parkinsons disease, and vascular cognitive impairment. Data from ONDRI will be collected into the Brain-CODE database to facilitate correlative analysis. ONDRI will provide a repertoire of endophenotyped individuals that will be a unique, publicly available resource.

Identification of a novel synaptic protein, TMTC3, involved in periventricular nodular heterotopia with intellectual disability and epilepsy

Abstract Defects in neuronal migration cause brain malformations, which are associated with intellectual disability (ID) and epilepsy. Using exome sequencing, we identified compound heterozygous variants (p.Arg71His and p. Leu729ThrfsTer6) in TMTC3, encoding transmembrane and tetratricopeptide repeat containing 3, in four siblings with nocturnal seizures and ID. Three of the four siblings have periventricular nodular heterotopia (PVNH), a common brain malformation caused by failure of neurons to migrate from the ventricular zone to the cortex. Expression analysis using patient-derived cells confirmed reduced TMTC3 transcript levels and loss of the TMTC3 protein compared to parental and control cells. As TMTC3 function is currently unexplored in the brain, we gathered support for a neurobiological role for TMTC3 by generating flies with post-mitotic neuron-specific knockdown of the highly conserved Drosophila melanogaster TMTC3 ortholog, CG4050/tmtc3. Neuron-specific knockdown of tmtc3 in flies resulted in increased susceptibility to induced seizures. Importantly, this phenotype was rescued by neuron-specific expression of human TMTC3, suggesting a role for TMTC3 in seizure biology. In addition, we observed co-localization of TMTC3 in the rat brain with vesicular GABA transporter (VGAT), a presynaptic marker for inhibitory synapses. TMTC3 is localized at VGAT positive pre-synaptic terminals and boutons in the rat hypothalamus and piriform cortex, suggesting a role for TMTC3 in the regulation of GABAergic inhibitory synapses. TMTC3 did not co-localize with Vglut2, a presynaptic marker for excitatory neurons. Our data identified TMTC3 as a synaptic protein that is involved in PVNH with ID and epilepsy, in addition to its previously described association with cobblestone lissencephaly.

Sequencing: The Next Generation—What Is the Role of Whole-Exome Sequencing in the Diagnosis of Familial Cardiovascular Diseases?

Since 2010, we have been living in the era of massively parallel genome sequencing, which generates genetic information from a single individual on a heretofore unimaginable scale. The effect of this technological advance on cardiovascular disease (CVD) is already being felt in the research laboratory, and might be on the cusp of translation into the clinic. In the best case scenario, understanding the genetic architecture underlying CVD might help predict clinical outcomes presymptomatically, and perhaps guide and tailor specific treatments that target pathways, which have become disrupted as the result of variation or mutation in the genome. The decreasing cost of next-generation sequencing (NGS) technologies has allowed human genome sequencing to proceed at unprecedented rates and volumes. The current cost of sequencing a human genome is approximately

KMT2D p.Gln3575His segregating in a family with autosomal dominant choanal atresia strengthens the Kabuki/CHARGE connection.

7500 and the technical work can be completed within a week. This very cost-effective approach can justify certain research and clinical applications, especially when compared with previous requirements of resources and time to accomplish the same task:

The ONDRISeq panel: custom-designed next-generation sequencing of genes related to neurodegeneration

2.7 billion and 13 years of effort resulted in completion of the first human genomic map in 2001, and by 2008 these metrics were down to