Genetic testing for mitochondrial disease: the United Kingdom best practice guidelines.

Primary mitochondrial disease describes a diverse group of neuro-metabolic disorders characterised by impaired oxidative phosphorylation. Diagnosis is challenging; >350 genes, both nuclear and mitochondrial DNA (mtDNA) encoded, are known to cause mitochondrial disease, leading to all possible inheritance patterns and further complicated by heteroplasmy of the multicopy mitochondrial genome. Technological advances, particularly next-generation sequencing, have driven a shift in diagnostic practice from 'biopsy first' to genome-wide analyses of blood and/or urine DNA. This has led to the need for a reference framework for laboratories involved in mitochondrial genetic testing to facilitate a consistent high-quality service. In the United Kingdom, consensus guidelines have been prepared by a working group of Clinical Scientists from the NHS Highly Specialised Service followed by national laboratory consultation. These guidelines summarise current recommended technologies and methodologies for the analysis of mtDNA and nuclear-encoded genes in patients with suspected mitochondrial disease. Genetic testing strategies for diagnosis, family testing and reproductive options including prenatal diagnosis are outlined. Importantly, recommendations for the minimum levels of mtDNA testing for the most common referral reasons are included, as well as guidance on appropriate referrals and information on the minimal appropriate gene content of panels when analysing nuclear mitochondrial genes. Finally, variant interpretation and recommendations for reporting of results are discussed, focussing particularly on the challenges of interpreting and reporting mtDNA variants.

Mitochondrial Strokes: Diagnostic Challenges and Chameleons.

Mitochondrial stroke-like episodes (SLEs) are a hallmark of mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS). They should be suspected in anyone with an acute/subacute onset of focal neurological symptoms at any age and are usually driven by seizures. Suggestive features of an underlying mitochondrial pathology include evolving MRI lesions, often originating within the posterior brain regions, the presence of multisystemic involvement, including diabetes, deafness, or cardiomyopathy, and a positive family history. The diagnosis of MELAS has important implications for those affected and their relatives, given it enables early initiation of appropriate treatment and genetic counselling. However, the diagnosis is frequently challenging, particularly during the acute phase of an event. We describe four cases of mitochondrial strokes to highlight the considerable overlap that exists with other neurological disorders, including viral and autoimmune encephalitis, ischemic stroke, and central nervous system (CNS) vasculitis, and discuss the clinical, laboratory, and imaging features that can help distinguish MELAS from these differential diagnoses.

Diagnosing Mitochondrial Disorders Remains Challenging in the Omics Era.

OBJECTIVE: We hypothesized that novel investigative pathways are needed to decrease diagnostic odysseys in pediatric mitochondrial disease and sought to determine the utility of clinical exome sequencing in a large cohort with suspected mitochondrial disease and to explore whether any of the traditional indicators of mitochondrial disease predict a confirmed genetic diagnosis. METHODS: We investigated a cohort of 85 pediatric patients using clinical exome sequencing and compared the results with the outcome of traditional diagnostic tests, including biochemical testing of routine parameters (lactate, alanine, and proline), neuroimaging, and muscle biopsy with histology and respiratory chain enzyme activity studies. RESULTS: We established a genetic diagnosis in 36.5% of the cohort and report 20 novel disease-causing variants (1 mitochondrial DNA). Counterintuitively, routine biochemical markers were more predictive of mitochondrial disease than more invasive and elaborate muscle studies. CONCLUSIONS: We propose using biochemical markers to support the clinical suspicion of mitochondrial disease and then apply first-line clinical exome sequencing to identify a definite diagnosis. Muscle biopsy studies should only be used in clinically urgent situations or to confirm an inconclusive genetic result. CLASSIFICATION OF EVIDENCE: This is a Class II diagnostic accuracy study showing that the combination of CSF and plasma biochemical tests plus neuroimaging could predict the presence or absence of exome sequencing confirmed mitochondrial disorders.

A Quality Improvement Initiative to Reduce Surgical Site Infections in Patients Undergoing Delayed Sternal Closure After Pediatric Cardiac Surgery.

Sternal wound infections (SWI) in delayed sternal closure (DSC) patients are a healthcare burden after congenital heart surgery. There are no guidelines specific for pediatric DSC patients to prevent this costly complication. The hypothesis was that the modifications to a bundled approach for DSC patients would decrease the SWI rate. For this prospective cohort study, DSC patients were postoperatively admitted to a pediatric cardiac care unit from February 2017 to January 2018. Using a modified protocol for prevention of SWI, the infection rates pre- and post-modified protocol were compared. The primary outcome measure was SWI. Secondary outcome measures were compliance with modifications. Retrospective review of cases in pre-protocol modification era from January 1, 2014 to December 31, 2016 showed 377 pediatric cardiopulmonary bypass cases and 39 (10.4%) underwent DSC. During the post-protocol modification era, there were 129 cardiopulmonary bypass cases and 17 (13%) DSC cases. The SWI rate in DSC were 7.7% and 0% for pre-intervention and post-intervention, respectively (p = 0.52). The Bayesian confidence interval with Jeffreys prior gives a 95% confidence interval of 1.5% to 18.3% for pre-intervention and 0 to 13.5% for post-intervention. Compliance with the protocol bundle during the post protocol era was 93-100%. Although preliminary results are not statistically significant due to cohort size, the economic burden and increased LOS for each SWI is clinically significant. The early results of reduced infections for DSC patients using a modified bundle approach appear promising. Continued study and a multicenter project would be beneficial.

Efforts to Reduce Infections in Delayed Sternal Closure Patients: A Survey of Pediatric Practice.

BACKGROUND: Pediatric patients with sternum left open after cardiac surgery experience a higher risk for sternal wound infection (SWI). These infections are costly for programs, payers, and patients and their families. Despite efforts by individual programs to reduce infections in patients undergoing delayed sternal closure (DSC), there are no established guidelines that address preventive procedures. The purpose of this study was to determine the practice of pediatric cardiac surgery programs to prevent infection in their DSC patients and if preventive measures were associated with less infections. METHODS: A 33 question survey on institutional practices was sent to chief surgeons at pediatric cardiac surgery programs in the United States. RESULTS: Twenty-eight (35%) surgical programs responded. The mean number of pediatric cardiac bypass operations performed by programs in 2016 was 227 (range: 69-872). Data represented 6,484 patients <18 years of age who underwent cardiac surgery with 807 (12%) of those undergoing DSC. One hundred fifty-eight (2.4%) of all patients and 51 (6.3%) of the DSC patients developed a SWI. Patients with DSC who received preoperative baths were less likely to become infected (5.9% vs 15.8%; P = .015). Patients in programs with feeding protocols had fewer infections (5.7% vs 14.8%; P = .008). CONCLUSIONS: The results of this survey of children's cardiac surgery programs describe their practices to reduce infection rates in DSC patients. A multicenter project on wound care and closure techniques that might impact this costly complication is needed.

Expanding the molecular and phenotypic spectrum of truncating MT-ATP6 mutations.

OBJECTIVE: To describe the clinical and functional consequences of 1 novel and 1 previously reported truncating MT-ATP6 mutation. METHODS: Three unrelated probands with mitochondrial encephalomyopathy harboring truncating MT-ATP6 mutations are reported. Transmitochondrial cybrid cell studies were used to confirm pathogenicity of 1 novel variant, and the effects of all 3 mutations on ATPase 6 and complex V structure and function were investigated. RESULTS: Patient 1 presented with adult-onset cerebellar ataxia, chronic kidney disease, and diabetes, whereas patient 2 had myoclonic epilepsy and cerebellar ataxia; both harbored the novel m.8782G>A; p.(Gly86*) mutation. Patient 3 exhibited cognitive decline, with posterior white matter abnormalities on brain MRI, and severely impaired renal function requiring transplantation. The m.8618dup; p.(Thr33Hisfs*32) mutation, previously associated with neurogenic muscle weakness, ataxia, and retinitis pigmentosa, was identified. All 3 probands demonstrated a broad range of heteroplasmy across different tissue types. Blue-native gel electrophoresis of cultured fibroblasts and skeletal muscle tissue confirmed multiple bands, suggestive of impaired complex V assembly. Microscale oxygraphy showed reduced basal respiration and adenosine triphosphate synthesis, while reactive oxygen species generation was increased. Transmitochondrial cybrid cell lines studies confirmed the deleterious effects of the novel m.8782 G>A; p.(Gly86*) mutation. CONCLUSIONS: We expand the clinical and molecular spectrum of MT-ATP6-related mitochondrial disorders to include leukodystrophy, renal disease, and myoclonic epilepsy with cerebellar ataxia. Truncating MT-ATP6 mutations may exhibit highly variable mutant levels across different tissue types, an important consideration during genetic counseling.

Multicenter Quality Improvement Project to Prevent Sternal Wound Infections in Pediatric Cardiac Surgery Patients.

BACKGROUND: Children undergoing cardiac surgery are at risk for sternal wound infections (SWIs) leading to increased morbidity and mortality. Single-center quality improvement (QI) initiatives have demonstrated decreased infection rates utilizing a bundled approach. This multicenter project was designed to assess the efficacy of a protocolized approach to decrease SWI. METHODS: Pediatric cardiac programs joined a collaborative effort to prevent SWI. Programs implemented the protocol, collected compliance data, and provided data points from local clinical registries using Society of Thoracic Surgery Congenital Heart Surgery Database harvest-compliant software or from other registries. RESULTS: Nine programs prospectively collected compliance data on 4,198 children. Days between infections were extended from 68.2 days (range: 25-82) to 130 days (range: 43-412). Protocol compliance increased from 76.7% (first quarter) to 91.3% (final quarter). Ninety (1.9%) children developed an SWI preprotocol and 64 (1.5%) postprotocol, P = .18. The 657 (15%) delayed sternal closure patients had a 5% infection rate with 18 (5.7%) in year 1 and 14 (4.3%) in year 2 P = .43. Delayed sternal closure patients demonstrated a trend toward increased risk for SWI of 1.046 for each day the sternum remained open, P = .067. Children who received appropriately timed preop antibiotics developed less infections than those who did not, 1.9% versus 4.1%, P = .007. CONCLUSION: A multicenter QI project to reduce pediatric SWIs demonstrated an extension of days between infections and a decrease in SWIs. Patients who received preop antibiotics on time had lower SWI rates than those who did not.

Cryptic Amyloidogenic Elements in the 3' UTRs of Neurofilament Genes Trigger Axonal Neuropathy.

Abnormal protein aggregation is observed in an expanding number of neurodegenerative diseases. Here, we describe a mechanism for intracellular toxic protein aggregation induced by an unusual mutation event in families affected by axonal neuropathy. These families carry distinct frameshift variants in NEFH (neurofilament heavy), leading to a loss of the terminating codon and translation of the 3' UTR into an extra 40 amino acids. In silico aggregation prediction suggested the terminal 20 residues of the altered NEFH to be amyloidogenic, which we confirmed experimentally by serial deletion analysis. The presence of this amyloidogenic motif fused to NEFH caused prominent and toxic protein aggregates in transfected cells and disrupted motor neurons in zebrafish. We identified a similar aggregation-inducing mechanism in NEFL (neurofilament light) and FUS (fused in sarcoma), in which mutations are known to cause aggregation in Charcot-Marie-Tooth disease and amyotrophic lateral sclerosis, respectively. In summary, we present a protein-aggregation-triggering mechanism that should be taken into consideration during the evaluation of stop-loss variants.

Thromboelastography--does it impact blood component transfusion in pediatric heart surgery?

BACKGROUND: The administration of blood products during pediatric cardiac surgery is common. We sought to determine if thromboelastography (TEG) is a cost-effective tool to reduce blood product transfusion in open pediatric cardiac surgery. MATERIALS AND METHODS: A retrospective case-control study was undertaken for 150 pediatric cardiac patients requiring cardiopulmonary bypass from January 2010-May 2012, in a University-affiliated pediatric hospital. Fifty sequential patients operated on when TEG was used were compared with 100 control patients before TEG availability. Groups were matched 2:1 for age and risk adjustment for congenital heart surgery score. Blood product utilization was compared between groups, as were outcomes metrics such as postoperative complications, length of stay, and hospital costs of transfusions. RESULTS: Demographic variables, risk adjustment for congenital heart surgery score classifications, and cardiopulmonary bypass times were similar between groups. Red cell and plasma transfusion were comparable between groups. TEG patients saw a substantial reduction in the administration of platelet (1 versus 2.2 U; P < 0.0001) and cryoprecipitate (0.7 versus 1.7 U; P < 0.0001) transfusions. A greater than 50% reductions in hospital costs of platelet ($595 versus $1309) and cryoprecipitate ($39 versus $94) transfusions were observed in the TEG group. Mortality, length of stay, ventilator requirements, postoperative bleeding, and thrombotic events were equivalent. CONCLUSIONS: Intraoperative TEG use reduced platelet and cryoprecipitate transfusions without an increase in postoperative complications. TEG is a cost-effective method to direct blood product replacement.

Paediatric single mitochondrial DNA deletion disorders: an overlapping spectrum of disease.

BACKGROUND: Single large-scale mitochondrial DNA (mtDNA) deletions (SLSMDs) are amongst the most frequently diagnosed mtDNA disorders in childhood, yet their natural history remains poorly understood. We report the natural history of a large multicentre cohort of such children. METHODS: We reviewed case notes from three different UK centres to determine the clinical course of 34 patients (16 female, 18 male) with childhood-onset mitochondrial disease caused by SLSMDs. Kaplan-Meier analysis was used to compare survival of patients presenting with haematological features (Pearson syndrome) and those with nonhaematological presentations. RESULTS: The most frequent initial presentation was with isolated ptosis (16/34, 47%). Eleven (32%) patients presented with transfusion-dependent anaemia soon after birth and were diagnosed with Pearson syndrome, whilst ten were classified as having Kearns-Sayre syndrome, three as having progressive external ophthalmoplegia (PEO) and seven as having PEO-plus. Three patients did not conform to any specific mitochondrial syndrome. The most frequently affected organ during the disease course was the kidney, with documented tubular or glomerular dysfunction in 17 of 20 (85%) cases who had detailed investigations. SLSMDs were present in blood and/or urine cells in all cases tested, indicating that muscle biopsy is not necessary for diagnosis in the paediatric age range. Kaplan-Meier survival analysis revealed significantly worse mortality in patients with Pearson syndrome compared with the rest of the cohort. CONCLUSIONS: Mitochondrial disease caused by SLSMDs is clinically heterogeneous, and not all cases conform to a classical mitochondrial syndrome. Multisystem disease is the norm, with anaemia, renal impairment and endocrine disturbance being the most frequent extraneurological features. SLSMDs should be considered in the differential diagnosis of all children presenting with ptosis.

Distal myopathy with cachexia: an unrecognised phenotype caused by dominantly-inherited mitochondrial polymerase γ mutations.

BACKGROUND: The myopathy associated with mutations in the nuclear-encoded mitochondrial DNA maintenance gene POLG, coding for the catalytic subunit of DNA polymerase, is typically proximal with early ophthalmoplegia. RESULTS: We report two unrelated patients in whom a distal, mainly upper limb, myopathy was the predominant and early clinical feature. One patient also suffered with marked cachexia. DNA genomic sequence analysis identified novel dominant heterozygous missense POLG mutations (Leu896Arg and Tyr951His) located within the conserved catalytic polymerase domain of the protein in both cases. CONCLUSIONS: Distal upper limb myopathy/cachexia is not previously described with dominant POLG mutations and our observations further highlight the diverse clinical spectrum of POLG-related mitochondrial disorders. These data indicate that dominant POLG mutations should be considered in the differential diagnosis of distal upper limb predominant myopathy.

The routine use of chest radiographs after chest tube removal in children who have had cardiac surgery.

BACKGROUND: It is routine to obtain a chest radiograph (CXR) after removal of a chest tube (CT) to assess for pneumothorax. Retrospective studies have shown that clinical signs were present in most children with pneumothorax and were an indication for a CXR. OBJECTIVE: Our objective was to determine if clinical indicators of pneumothorax are sufficient predictors of the need for CT reinsertion in children who have had a CT removed after cardiac surgery. METHODS: The prospective study included a physical assessment before CT removal, using a two-person technique, which was repeated 2 hours after CT removal. Based on assessment findings, a decision was made regarding whether a CXR was indicated. The routine CXR was then obtained and read by a pediatric intensivist who was blinded to the decision of the investigator. RESULTS: Sixty CTs were removed in 53 children. No false-positive predictions were made, because none of the children was predicted to have a pneumothorax requiring chest tube reinsertion, and none developed a significant pneumothorax (95% confidence interval: 0, 5%). CONCLUSIONS: The low rate of pneumothoraces in this study may be been related to how the CT was placed in surgery, the type of CT used, or the method of removal. In this study the risk of developing a pneumothorax requiring CT reinsertion after CT removal was at most 5% and therefore low enough to consider obtaining a CXR for symptomatic children only.

Prevention of sternal wound infection in pediatric cardiac surgery: a protocolized approach.

BACKGROUND: Sternal wound infections (SWIs) are a costly complication for children after cardiac surgery, increasing morbidity, mortality, and financial cost. There are no pediatric guidelines to reduce the incidence of SWI in this vulnerable population. METHODS: A quality improvement, multidisciplinary team was formed, and a protocol to prevent SWI was developed. A prospective review of patients who underwent pediatric cardiac surgery was conducted over a two-year period to follow adherence to the protocol and incidence of SWI. The Centers for Disease Control definitions for surgical site infections were used to determine the depth and presence of infection. RESULTS: Three hundred and eight children <18 years of age had sternotomies during the study period. There was a reduction in all SWI between the first and second years of the study (odds ratio [OR] = 0.35; confidence interval [CI] 95% 0.12-1.01; P = .059). Delayed sternal closure (DSC) was associated with increased risk of SWI (OR = 5.4; CI 95% 2.13-14.9; P ≤ .001). Institution of a protocol in patients with DSC was associated with decreased infections during the second year (first year: n = 7 (14%), second year: n = 2 (4%), P = .14). CONCLUSIONS: Institution of a protocol was associated with a decreased number of infections in children. A multicenter study of a bundled protocol approach to SWI prevention is needed. Children with DSC had a significantly higher risk of developing a wound infection. Initiating strategies to reduce SWI with a focus on children with DSC may result in improved overall infection rates.

Sternal wound infections in pediatric congenital cardiac surgery: a survey of incidence and preventative practice.

BACKGROUND: Guidelines exist for prevention of sternal wound infections (SWI) in adults. There are no guidelines for pediatric patients and limited reports on SWI incidence. The purpose of this study was to determine the incidence of, and preventative practice regarding pediatric SWIs with a long-term aim to develop best practice guidelines. METHODS: Eighty-nine congenital heart programs were sent a 31 question on-line survey regarding pediatric SWI. RESULTS: Thirty eight (43%) of the 89 programs responded. They reported 8,774 pediatric congenital procedures with a mean SWI rate of 1.53% (range, 0 to 9.09). Mean yearly volume was 237 operations (range, 50 to 720). Neither program size nor delayed sternal closure was associated with increased incidence of SWI. Variations in preoperative measures, antibiotic regimens, and wound care did not statistically impact incidence of SWI. Programs with protocols to monitor and control blood glucose levels postoperatively had statistically lower infection rates (1.04 vs 2.35, p = 0.03), and those that sent mediastinal cultures at time of delayed sternal closure reported lower infection rates (1.34 vs 1.74, p = 0.051). CONCLUSIONS: This report provides a multiinstitutional SWI incidence from pediatric programs of 1.53%. Despite variations in clinical practice between programs, this survey revealed two strategies resulting in reduced SWIs; protocol-based management of glucose levels and mediastinal wound cultures sent at time of closure. Pediatric programs do not consistently follow adult preventative guidelines. Multicenter randomized research is needed to formulate preventative guidelines to reduce the incidence of pediatric SWI.