Delineation of molecular findings by whole-exome sequencing for suspected cases of paediatric-onset mitochondrial diseases in the Southern Chinese population.

BACKGROUND: Mitochondrial diseases (MDs) are a group of clinically and genetically heterogeneous disorders characterized by defects in oxidative phosphorylation. Since clinical phenotypes of MDs may be non-specific, genetic diagnosis is crucial for guiding disease management. In the current study, whole-exome sequencing (WES) was performed for our paediatric-onset MD cohort of a Southern Chinese origin, with the aim of identifying key disease-causing variants in the Chinese patients with MDs. METHODS: We recruited Chinese patients who had paediatric-onset MDs and a minimum mitochondrial disease criteria (MDC) score of 3. Patients with positive target gene or mitochondrial DNA sequencing results were excluded. WES was performed, variants with population frequency ≤ 1% were analysed for pathogenicity on the basis of the American College of Medical Genetics and Genomics guidelines. RESULTS: Sixty-six patients with pre-biopsy MDC scores of 3-8 were recruited. The overall diagnostic yield was 35% (23/66). Eleven patients (17%) were found to have mutations in MD-related genes, with COQ4 having the highest mutation rate owing to the Chinese-specific founder mutation (4/66, 6%). Twelve patients (12/66, 18%) had mutations in non-MD-related genes: ATP1A3 (n = 3, two were siblings), ALDH5A1, ARX, FA2H, KCNT1, LDHD, NEFL, NKX2-2, TBCK, and WAC. CONCLUSIONS: We confirmed that the COQ4:c.370G>A, p.(Gly124Ser) variant, was a founder mutation among the Southern Chinese population. Screening for this mutation should therefore be considered while diagnosing Chinese patients suspected to have MDs. Furthermore, WES has proven to be useful in detecting variants in patients suspected to have MDs because it helps to obtain an unbiased and precise genetic diagnosis for these diseases, which are genetically heterogeneous.

Reference intervals of spot urine copper excretion in preschool children and potential application in pre-symptomatic screening of Wilson disease.

The objectives were to determine the reference intervals of spot urine copper excretion indexes in pre-school children and to evaluate their utility in screening for Wilson disease (WD). With spot urine collected from a control sample of preschool children (aged 3-7 years, n=153), the reference intervals of spot urine copper excretion indexes and their biological variation were defined. In order to investigate their utility performance in screening for WD in this age group, multiple spot urine samples from six WD patients who were diagnosed at presymptomatic stage were also analysed and compared. Cut-off values useful for detection of WD were defined by receiver operator curve (ROC) analysis. Biological (inter-individual) variation of spot urine copper indexes expressed as coefficient of variation (CVg) were around 60% at this age group, which was moderate and similar to other clinically useful urine tests, such as urine albumin excretion ratio. Spot urine copper excretion strongly correlated with both urine creatinine and osmolality. Linear regression against both creatinine and osmolality showed that ∼94% of data points in healthy preschool children fell within the prediction interval, suggesting that both were useful normalisation factors. ROC showed that copper to osmolality ratio was the best index with an area under curve (AUC) greater than 0.98. Cut-off values of 0.5 µmol/L, 0.1 µmol/mmol and 0.00085 µmol/mOsmol (32 µg/L, 56 µg/g creatinine and 0.054 µg/mOsmol, respectively, in conventional units) for spot urine copper concentration, copper to creatinine ratio and copper to osmolality ratio, respectively, have potential application in the differentiation of WD patients. Based on the data, a new WD screening strategy targeting preschool children is proposed. Application of a bivariate screening strategy using spot urine copper concentration and urine osmolality may be useful in a population-wide screening program for WD among preschool children.

Paralog Studies Augment Gene Discovery: DDX and DHX Genes.

Members of a paralogous gene family in which variation in one gene is known to cause disease are eight times more likely to also be associated with human disease. Recent studies have elucidated DHX30 and DDX3X as genes for which pathogenic variant alleles are involved in neurodevelopmental disorders. We hypothesized that variants in paralogous genes encoding members of the DExD/H-box RNA helicase superfamily might also underlie developmental delay and/or intellectual disability (DD and/or ID) disease phenotypes. Here we describe 15 unrelated individuals who have DD and/or ID, central nervous system (CNS) dysfunction, vertebral anomalies, and dysmorphic features and were found to have probably damaging variants in DExD/H-box RNA helicase genes. In addition, these individuals exhibit a variety of other tissue and organ system involvement including ocular, outer ear, hearing, cardiac, and kidney tissues. Five individuals with homozygous (one), compound-heterozygous (two), or de novo (two) missense variants in DHX37 were identified by exome sequencing. We identified ten total individuals with missense variants in three other DDX/DHX paralogs: DHX16 (four individuals), DDX54 (three individuals), and DHX34 (three individuals). Most identified variants are rare, predicted to be damaging, and occur at conserved amino acid residues. Taken together, these 15 individuals implicate the DExD/H-box helicases in both dominantly and recessively inherited neurodevelopmental phenotypes and highlight the potential for more than one disease mechanism underlying these disorders.

BRCA1 and BRCA2 tumor suppressors protect against endogenous acetaldehyde toxicity.

Maintenance of genome integrity requires the functional interplay between Fanconi anemia (FA) and homologous recombination (HR) repair pathways. Endogenous acetaldehyde, a product of cellular metabolism, is a potent source of DNA damage, particularly toxic to cells and mice lacking the FA protein FANCD2. Here, we investigate whether HR-compromised cells are sensitive to acetaldehyde, similarly to FANCD2-deficient cells. We demonstrate that inactivation of HR factors BRCA1, BRCA2, or RAD51 hypersensitizes cells to acetaldehyde treatment, in spite of the FA pathway being functional. Aldehyde dehydrogenases (ALDHs) play key roles in endogenous acetaldehyde detoxification, and their chemical inhibition leads to cellular acetaldehyde accumulation. We find that disulfiram (Antabuse), an ALDH2 inhibitor in widespread clinical use for the treatment of alcoholism, selectively eliminates BRCA1/2-deficient cells. Consistently, Aldh2 gene inactivation suppresses proliferation of HR-deficient mouse embryonic fibroblasts (MEFs) and human fibroblasts. Hypersensitivity of cells lacking BRCA2 to acetaldehyde stems from accumulation of toxic replication-associated DNA damage, leading to checkpoint activation, G2/M arrest, and cell death. Acetaldehyde-arrested replication forks require BRCA2 and FANCD2 for protection against MRE11-dependent degradation. Importantly, acetaldehyde specifically inhibits in vivo the growth of BRCA1/2-deficient tumors and ex vivo in patient-derived tumor xenograft cells (PDTCs), including those that are resistant to poly (ADP-ribose) polymerase (PARP) inhibitors. The work presented here therefore identifies acetaldehyde metabolism as a potential therapeutic target for the selective elimination of BRCA1/2-deficient cells and tumors.

Isolated persistent elevation of alanine transaminase for early diagnosis of pre-symptomatic Wilson's disease in Chinese children.

BACKGROUND: Recent studies presented a contradictory approach for the investigation of pediatric patients with an isolated increase in alanine transaminase. While classical teaching advised for a thorough investigation, recent studies suggested the yield on further investigation was low and thus not necessary. Yet the approach to the same clinical problem may need to be different due to variable disease prevalence rates among different ethnic populations. For the population with a higher prevalence rate of genetic liver diseases like Wilson's disease, an abnormal liver function may be the first presenting feature for some patients. METHODS: We reviewed 10 Chinese children with Wilson's disease who were diagnosed at a presymptomatic stage because of an isolated persistent elevation of alanine transaminase. RESULTS: All 10 patients did not have overt symptoms of liver impairment or neurological deficit. They were picked up incidentally with an abnormal liver function test. All patients were started on treatment shortly after diagnosis, and they remained well and symptom-free on the latest follow-up. CONCLUSIONS: This case series illustrated that an isolated persistent elevation of alanine transaminase is an important clue to the early diagnosis of pre-symptomatic Wilson's disease. It is particularly relevant in the Asian population where the disease is more prevalent.

Lupus nephritis in Chinese children--a territory-wide cohort study in Hong Kong.

We report a multicenter study of Chinese children in Hong Kong with systemic lupus erythematosus (SLE) nephritis. Children were included if: they fulfilled the ACR criteria, had significant proteinuria or casturia, were Chinese and younger than 19 years and had been diagnosed with SLE between January 1990 and December 2003. Investigators in each center retrieved data on clinical features, biopsy reports, treatment and outcome of these patients. There were 128 patients (eight boys, 120 girls; mean age: 11.9+/-2.8 years). About 50% presented with multisystem illness and 40% with nephritic/nephrotic symptoms. Negative anti-dsDNA antibodies were found in 6% of the patients. Renal biopsy revealed WHO Class II, III, IV and V nephritis in 13 (10%), 22 (17%), 69 (54%) and 13 (10%) patients, respectively. The clinical severity of the nephritis did not accurately predict renal biopsy findings. The follow-up period ranged from 1 to 16.5 years (mean+/-SD: 5.76+/-3.61 years). During the study five patients died (two from lupus flare, one from cardiomyopathy, two from infections). Four patients had endstage renal failure (ESRF) (one died during a lupus flare). All deaths and end-stage renal failure occurred in the Class IV nephritis group. Chronic organ damage was infrequent in the survivors. The actuarial patient survival rates at 5, 10 and 15 years of age were 95.3, 91.8, and 91.8%, respectively. For Class IV nephritis patients, the survival rates without ESRF at 5, 10, and 15 years were 91.5, 82.3 and 76%, respectively. The survival and chronic morbidity rates of the Chinese SLE children in the present study are comparable to those of other published studies.