Bi-allelic LETM1 variants perturb mitochondrial ion homeostasis leading to a clinical spectrum with predominant nervous system involvement.

Leucine zipper-EF-hand containing transmembrane protein 1 (LETM1) encodes an inner mitochondrial membrane protein with an osmoregulatory function controlling mitochondrial volume and ion homeostasis. The putative association of LETM1 with a human disease was initially suggested in Wolf-Hirschhorn syndrome, a disorder that results from de novo monoallelic deletion of chromosome 4p16.3, a region encompassing LETM1. Utilizing exome sequencing and international gene-matching efforts, we have identified 18 affected individuals from 11 unrelated families harboring ultra-rare bi-allelic missense and loss-of-function LETM1 variants and clinical presentations highly suggestive of mitochondrial disease. These manifested as a spectrum of predominantly infantile-onset (14/18, 78%) and variably progressive neurological, metabolic, and dysmorphic symptoms, plus multiple organ dysfunction associated with neurodegeneration. The common features included respiratory chain complex deficiencies (100%), global developmental delay (94%), optic atrophy (83%), sensorineural hearing loss (78%), and cerebellar ataxia (78%) followed by epilepsy (67%), spasticity (53%), and myopathy (50%). Other features included bilateral cataracts (42%), cardiomyopathy (36%), and diabetes (27%). To better understand the pathogenic mechanism of the identified LETM1 variants, we performed biochemical and morphological studies on mitochondrial K+/H+ exchange activity, proteins, and shape in proband-derived fibroblasts and muscles and in Saccharomyces cerevisiae, which is an important model organism for mitochondrial osmotic regulation. Our results demonstrate that bi-allelic LETM1 variants are associated with defective mitochondrial K+ efflux, swollen mitochondrial matrix structures, and loss of important mitochondrial oxidative phosphorylation protein components, thus highlighting the implication of perturbed mitochondrial osmoregulation caused by LETM1 variants in neurological and mitochondrial pathologies.

SMPD4 regulates mitotic nuclear envelope dynamics and its loss causes microcephaly and diabetes.

Biallelic loss-of-function variants in SMPD4 cause a rare and severe neurodevelopmental disorder with progressive congenital microcephaly and early death. SMPD4 encodes a sphingomyelinase that hydrolyses sphingomyelin into ceramide at neutral pH and can thereby affect membrane lipid homeostasis. SMPD4 localizes to the membranes of the endoplasmic reticulum and nuclear envelope and interacts with nuclear pore complexes (NPC). We refine the clinical phenotype of loss-of-function SMPD4 variants by describing five individuals from three unrelated families with longitudinal data due to prolonged survival. All individuals surviving beyond infancy developed insulin-dependent diabetes, besides presenting with a severe neurodevelopmental disorder and microcephaly, making diabetes one of the most frequent age-dependent non-cerebral abnormalities. We studied the function of SMPD4 at the cellular and organ levels. Knock-down of SMPD4 in human neural stem cells causes reduced proliferation rates and prolonged mitosis. Moreover, SMPD4 depletion results in abnormal nuclear envelope breakdown and reassembly during mitosis and decreased post-mitotic NPC insertion. Fibroblasts from affected individuals show deficient SMPD4-specific neutral sphingomyelinase activity, without changing (sub)cellular lipidome fractions, which suggests a local function of SMPD4 on the nuclear envelope. In embryonic mouse brain, knockdown of Smpd4 impairs cortical progenitor proliferation and induces premature differentiation by altering the balance between neurogenic and proliferative progenitor cell divisions. We hypothesize that, in individuals with SMPD4-related disease, nuclear envelope bending, which is needed to insert NPCs in the nuclear envelope, is impaired in the absence of SMPD4 and interferes with cerebral corticogenesis and survival of pancreatic beta cells.

Trans-2,3-enoyl-CoA reductase-like-related catecholaminergic polymorphic ventricular tachycardia with regular ventricular tachycardia and response to flecainide.

INTRODUCTION: We describe a unique case of TECRL-CPVT presented with cardiac arrest. METHODS: Post resuscitation, the patient developed regular ventricular tachycardia featuring a left purkinje system morphology. RESULTS: There was clear suppression of arrhythmia with the addition of flecainide and isolated ventricular ectopy causing secondary T-wave changes. CONCLUSION: A high index of suspicion was required to eventually make the diagnosis through whole exome sequencing.

LYRM7-associated mitochondrial complex III deficiency with non-cavitating leukoencephalopathy and stroke-like episodes.

Defects of respiratory chain complex III (CIII) result in characteristic but rare mitochondrial disorders associated with distinct neuroradiological findings. The underlying molecular defects affecting mitochondrial CIII assembly factors are few and yet to be identified. LYRM7 assembly factor is required for proper CIII assembly where it acts as a chaperone for the Rieske iron-sulfur (UQCRFS1) protein in the mitochondrial matrix and stabilizing it. We present here the seventeenth individual with LYRM7-associated mitochondrial leukoencephalopathy harboring a previously reported rare pathogenic homozygous LYRM 7 variant, c.2T>C, (p.Met1?). Like previously reported individuals, our 5-year-old male proband presented with recurrent metabolic and lactic acidosis, encephalopathy, and fatigue. Further, he has additional, previously unreported features, including an acute stroke like episode with bilateral central blindness and optic neuropathy, recurrent hyperglycemia and hypertension associated with metabolic crisis. However, he has no signs of psychomotor regression. He has been stable clinically with residual left-sided reduced visual acuity and amblyopia, and no more metabolic crises for 2-year-period while on the mitochondrial cocktail. Although the reported brain MRI findings in other affected individuals are homogenous, it is slightly different in our index, revealing evidence of bilateral almost symmetric multifocal periventricular T2 hyperintensities with hyperintensities of the optic nerves, optic chiasm, and corona radiata but with no cavitation or cystic changes. This report describes new clinical and radiological findings of LYRM7-associated disease. The report also summarizes the clinical and molecular data of previously reported individuals describing the full phenotypic spectrum.

Identification and characterization of novel MPC1 gene variants causing mitochondrial pyruvate carrier deficiency.

Pyruvate, the end product of glycolysis, is a key metabolic molecule enabling mitochondrial adenosine triphosphate synthesis and takes part in multiple biosynthetic pathways within mitochondria. The mitochondrial pyruvate carrier (MPC) plays a vital role in transporting pyruvate from the cytosol into the organelle. In humans, MPC is a hetero-oligomeric complex formed by the MPC1 and MPC2 paralogs that are both necessary to stabilize each other and form a functional MPC. MPC deficiency (OMIM#614741) due to pathogenic MPC1 variants is a rare autosomal recessive disease involving developmental delay, microcephaly, growth failure, and increased serum lactate and pyruvate. To date, two MPC1 variants in four cases have been reported, though only one with a detailed clinical description. Herein, we report three novel pathogenic MPC1 variants in six patients from three unrelated families, identified within European, Kuwaiti, and Chinese mitochondrial disease patient cohorts, one of whom presented as a Leigh-like syndrome. Functional analysis in primary fibroblasts from the patients revealed decreased expression of MPC1 and MPC2. We rescued pyruvate-driven oxygen consumption rate in patient's fibroblasts by reconstituting with wild-type MPC1. Complementing homozygous MPC1 mutant cDNA with CRISPR-deleted MPC1 C2C12 cells verified the mechanism of variants: unstable MPC complex or ablated pyruvate uptake activity. Furthermore, we showed that glutamine and beta-hydroxybutyrate were alternative substrates to maintain mitochondrial respiration when cells lack pyruvate. In conclusion, we expand the clinical phenotypes and genotypes associated with MPC deficiency, with our studies revealing glutamine as a potential therapy for MPC deficiency.

Mutations in ELAC2 associated with hypertrophic cardiomyopathy impair mitochondrial tRNA 3'-end processing.

Mutations in either the mitochondrial or nuclear genomes are associated with a diverse group of human disorders characterized by impaired mitochondrial respiration. Within this group, an increasing number of mutations have been identified in nuclear genes involved in mitochondrial RNA metabolism, including ELAC2. The ELAC2 gene codes for the mitochondrial RNase Z, responsible for endonucleolytic cleavage of the 3' ends of mitochondrial pre-tRNAs. Here, we report the identification of 16 novel ELAC2 variants in individuals presenting with mitochondrial respiratory chain deficiency, hypertrophic cardiomyopathy (HCM), and lactic acidosis. We provide evidence for the pathogenicity of the novel missense variants by studying the RNase Z activity in an in vitro system. We also modeled the residues affected by a missense mutation in solved RNase Z structures, providing insight into enzyme structure and function. Finally, we show that primary fibroblasts from the affected individuals have elevated levels of unprocessed mitochondrial RNA precursors. Our study thus broadly confirms the correlation of ELAC2 variants with severe infantile-onset forms of HCM and mitochondrial respiratory chain dysfunction. One rare missense variant associated with the occurrence of prostate cancer (p.Arg781His) impairs the mitochondrial RNase Z activity of ELAC2, suggesting a functional link between tumorigenesis and mitochondrial RNA metabolism.

Tribal Founder EMC1 Variant in 5 Kuwaiti Families Expands Phenotypic Spectrum of EMC1-Related Disorder.

Background and Objectives: The endoplasmic reticulum (ER) membrane protein complex is a conserved multisubunit transmembrane complex that enables energy-independent insertion of newly synthesized membrane proteins into ER membranes, mediating protein folding, phospholipid transfer from ER to mitochondria, and elimination of misfolded proteins. The first subunit of EMC (EMC1) is encoded by EMC1. Both monoallelic de novo and biallelic EMC1 variants have been identified to cause cerebellar atrophy, visual impairment, and psychomotor retardation (CAVIPMR) [OMIM #616875]. Eight families with biallelic EMC1 variants and CAVIPMR have been reported. Here, we describe 8 individuals from 5 Kuwaiti families from the same tribe, with the previously reported homozygous pathogenic missense EMC1 variant [c.245C>T:p.(Thr82Met)] and CAVIPMR. Methods: Proband exome sequencing was performed in 3 families, while targeted molecular testing for EMC1 [c.245C>T:p.(Thr82Met)] variant was performed in the other 2 families based on strong clinical suspicion and tribal origin. Sanger sequencing confirmed variant segregation with disease in all families. Results: We identified 8 individuals from 5 Kuwaiti families with the homozygous pathogenic EMC1 variant [c.245C>T:p.(Thr82Met)] previously reported in a Turkish family with CAVIPMR. The variant was absent from Kuwait Medical Genetic Center database, thus unlikely to represent a population founder allelic variant. The average age at symptom onset was 11 weeks, with all families reporting either visual abnormalities, hypotonia, and/or global developmental delay (GDD) as the presenting features. Shared clinical features included GDD (8/8), microcephaly (8/8), truncal hypotonia (8/8), visual impairment (7/7), and failure to thrive (7/7). Other common features included hyperreflexia (5/6; 83%), peripheral hypertonia (3/5; 60%), dysmorphism (3/6; 50%), epilepsy (4/8; 50%), and chorea (3/8; 36%). Brain imaging showed cerebellar atrophy in 4/7 (57%) and cerebral atrophy in 3/6 (50%) individuals. Discussion: The presence of exact biallelic homozygous EMC1 variant in 5 Kuwaiti families from the same tribe suggests a tribal founder allelic variant. The clinical features in this study are consistent with the phenotypic spectrum of EMC1-associated CAVIPMR in previous reports. The presence of chorea, first noted in this study, further expands the phenotypic spectrum. Our findings emphasize the importance of targeted EMC1 variant [c.245C>T:p.(Thr82Met)] testing for infants from affected tribe who present with visual impairment, GDD, and hypotonia.

Biotin-thiamine responsive basal ganglia disease: a retrospective review of the clinical, radiological and molecular findings of cases in Kuwait with novel variants.

BACKGROUND: Biotin-thiamine-responsive basal ganglia disease (BTBGD) is a rare autosomal recessive neurometabolic disorder that is caused by biallelic pathogenic SLC19A3 variants and is characterized by subacute encephalopathy associated with confusion, convulsions, dysphagia, dysarthria, or other neurological manifestations. METHODS: A retrospective review of the data registry in Kuwait Medical Genetics Center for all cases diagnosed clinically and radiographically and confirmed genetically with BTBGD. RESULTS: Twenty one cases from 13 different families were diagnosed with BTBGD in Kuwait. Most cases (86%) presented with confusion, dystonia, convulsions, or dysarthria, while three individuals were diagnosed pre-symptomatically during familial targeted genetic screening. Symptoms resolved completely within 2-week of treatment in two-thirds of the symptomatic cases but progressed in six of them to a variety of severe symptoms including severe cogwheel rigidity, dystonia and quadriparesis due to delayed presentation and management. Neuroradiological findings of the symptomatic cases revealed bilateral central changes in the basal ganglia. Two novel homozygous missense SLC19A3 variants were detected in a Kuwaiti and a Jordanian individuals, in addition to the previously reported Saudi founder homozygous variant, c.1264A > G; p.(Thr422Ala) in the remaining cases. Age of diagnosis ranged from newborn to 32 years, with a median age of 2-3 years. All cases are still alive receiving high doses of biotin and thiamine. CONCLUSION: This is the first study reporting the phenotypic and genotypic spectrum of 21 individuals with BTBGD in Kuwait and describing two novel SLC19A3 variants. BTBGD is a treatable neurometabolic disease that requires early recognition and treatment initiation. This study highlights the importance of performing targeted molecular testing of the founder variant in patients presenting with acute encephalopathy in the region.

Early Diagnosis of Classic Homocystinuria in Kuwait through Newborn Screening: A 6-Year Experience.

Kuwait is a small Arabian Gulf country with a high rate of consanguinity and where a national newborn screening program was expanded in October 2014 to include a wide range of endocrine and metabolic disorders. A retrospective study conducted between January 2015 and December 2020 revealed a total of 304,086 newborns have been screened in Kuwait. Six newborns were diagnosed with classic homocystinuria with an incidence of 1:50,000, which is not as high as in Qatar but higher than the global incidence. Molecular testing for five of them has revealed three previously reported pathogenic variants in the CBS gene, c.969G>A, p.(Trp323Ter); c.982G>A, p.(Asp328Asn); and the Qatari founder variant c.1006C>T, p.(Arg336Cys). This is the first study to review the screening of newborns in Kuwait for classic homocystinuria, starting with the detection of elevated blood methionine and providing a follow-up strategy for positive results, including plasma total homocysteine and amino acid analyses. Further, we have demonstrated an increase in the specificity of the current newborn screening test for classic homocystinuria by including the methionine to phenylalanine ratio along with the elevated methionine blood levels in first-tier testing. Here, we provide evidence that the newborn screening in Kuwait has led to the early detection of classic homocystinuria cases and enabled the affected individuals to lead active and productive lives.

Categorized Genetic Analysis in Childhood-Onset Cardiomyopathy.

BACKGROUND: Childhood-onset cardiomyopathy is a heterogeneous group of conditions the cause of which is largely unknown. The influence of consanguinity on the genetics of cardiomyopathy has not been addressed at a large scale. METHODS: To unravel the genetic cause of childhood-onset cardiomyopathy in a consanguineous population, a categorized approach was adopted. Cases with childhood-onset cardiomyopathy were consecutively recruited. Based on the likelihood of founder mutation and on the clinical diagnosis, genetic test was categorized to either (1) targeted genetic test with targeted mutation test, single-gene test, or multigene panel for Noonan syndrome, or (2) untargeted genetic test with whole-exome sequencing or whole-genome sequencing. Several bioinformatics tools were used to filter the variants. RESULTS: Two-hundred five unrelated probands with various forms of cardiomyopathy were evaluated. The median age of presentation was 10 months. In 30.2% (n=62), targeted genetic test had a yield of 82.7% compared with 33.6% for whole-exome sequencing/whole-genome sequencing (n=143) giving an overall yield of 53.7%. Strikingly, 96.4% of the variants were homozygous, 9% of which were found in 4 dominant genes. Homozygous variants were also detected in 7 novel candidates (ACACB, AASDH, CASZ1, FLII, RHBDF1, RPL3L, ULK1). CONCLUSIONS: Our work demonstrates the impact of consanguinity on the genetics of childhood-onset cardiomyopathy, the value of adopting a categorized population-sensitive genetic approach, and the opportunity of uncovering novel genes. Our data suggest that if a founder mutation is not suspected, adopting whole-exome sequencing/whole-genome sequencing as a first-line test should be considered.