Clinical and pathological characterization of FLNC-related myofibrillar myopathy caused by founder variant c.8129G>A in Hong Kong Chinese.

FLNC-related myofibrillar myopathy could manifest as autosomal dominant late-onset slowly progressive proximal muscle weakness; involvements of cardiac and/or respiratory functions are common. We describe 34 patients in nine families of FLNC-related myofibrillar myopathy in Hong Kong ethnic Chinese diagnosed over the last 12 years, in whom the same pathogenic variant c.8129G>A (p.Trp2710*) was detected. Twenty-six patients were symptomatic when diagnosed; four patients died of pneumonia and/or respiratory failure. Abnormal amorphous material or granulofilamentous masses were detected in half of the cases, with mitochondrial abnormalities noted in two-thirds. We also show by haplotype analysis the founder effect associated with this Hong Kong variant, which might have occurred 42 to 71 generations ago or around Tang and Song dynasties, and underlain a higher incidence of myofibrillar myopathy among Hong Kong Chinese. The late-onset nature and slowly progressive course of the highly penetrant condition could have significant impact on the family members, and an early diagnosis could benefit the whole family. Considering another neighboring founder variant in FLNC in German patients, we advocate development of specific therapies such as chaperone-based or antisense oligonucleotide strategies for this particular type of myopathy.

Dried Blood Spot Postmortem Metabolic Autopsy With Genotype Validation for Sudden Unexpected Deaths in Infancy and Childhood in Hong Kong.

Background Inborn errors of metabolism (IEM) are collectively rare but potentially preventable causes of sudden unexpected death (SUD) in infancy or childhood, and metabolic autopsy serves as the final tool for establishing the diagnosis. We conducted a retrospective review of the metabolic and molecular autopsy on SUD and characterized the biochemical and genetic findings. Methodology A retrospective review of postmortem metabolic investigations (dried blood spot acylcarnitines and amino acid analysis, urine metabolic profiling where available, and next-generation sequencing on a panel of 75 IEM genes) performed for infants and children who presented with SUD between October 2016 and December 2021 with inconclusive autopsy findings or autopsy features suspicious of underlying IEM in our locality was conducted. Clinical and autopsy findings were reviewed for each case. Results A total of 43 infants and children aged between zero days to 10 years at the time of death were referred to the authors' laboratories throughout the study period. One positive case of multiple acyl-CoA dehydrogenase deficiency was diagnosed. Postmortem reference intervals for dried blood spot amino acids and acylcarnitines profile were established based on the results from the remaining patients. Conclusions Our study confirmed the importance of metabolic autopsy and the advantages of incorporating biochemical and genetic testing in this setting.

Inborn errors of metabolism and expanded newborn screening: review and update.

Inborn errors of metabolism (IEM) are a phenotypically and genetically heterogeneous group of disorders caused by a defect in a metabolic pathway, leading to malfunctioning metabolism and/or the accumulation of toxic intermediate metabolites. To date, more than 1000 different IEM have been identified. While individually rare, the cumulative incidence has been shown to be upwards of 1 in 800. Clinical presentations are protean, complicating diagnostic pathways. IEM are present in all ethnic groups and across every age. Some IEM are amenable to treatment, with promising outcomes. However, high clinical suspicion alone is not sufficient to reduce morbidities and mortalities. In the last decade, due to the advent of tandem mass spectrometry, expanded newborn screening (NBS) has become a mandatory public health strategy in most developed and developing countries. The technology allows inexpensive simultaneous detection of more than 30 different metabolic disorders in one single blood spot specimen at a cost of about USD 10 per baby, with commendable analytical accuracy and precision. The sensitivity and specificity of this method can be up to 99% and 99.995%, respectively, for most amino acid disorders, organic acidemias, and fatty acid oxidation defects. Cost-effectiveness studies have confirmed that the savings achieved through the use of expanded NBS programs are significantly greater than the costs of implementation. The adverse effects of false positive results are negligible in view of the economic health benefits generated by expanded NBS and these could be minimized through increased education, better communication, and improved technologies. Local screening agencies should be given the autonomy to develop their screening programs in order to keep pace with international advancements. The development of biochemical genetics is closely linked with expanded NBS. With ongoing advancements in nanotechnology and molecular genomics, the field of biochemical genetics is still expanding rapidly. The potential of tandem mass spectrometry is extending to cover more disorders. Indeed, the use of genetic markers in T-cell receptor excision circles for severe combined immunodeficiency is one promising example. NBS represents the highest volume of genetic testing. It is more than a test and it warrants systematic healthcare service delivery across the pre-analytical, analytical, and post-analytical phases. There should be a comprehensive reporting system entailing genetic counselling as well as short-term and long-term follow-up. It is essential to integrate existing clinical IEM services with the expanded NBS program to enable close communication between the laboratory, clinicians, and allied health parties. In this review, we will discuss the history of IEM, its clinical presentations in children and adult patients, and its incidence among different ethnicities; the history and recent expansion of NBS, its cost-effectiveness, associated pros and cons, and the ethical issues that can arise; the analytical aspects of tandem mass spectrometry and post-analytical perspectives regarding result interpretation.

Mitochondrial diseases in Hong Kong: prevalence, clinical characteristics and genetic landscape.

OBJECTIVE: To determine the prevalence of mitochondrial diseases (MD) in Hong Kong (HK) and to evaluate the clinical characteristics and genetic landscape of MD patients in the region. METHODS: This study retrospectively reviewed the phenotypic and molecular characteristics of MD patients from participating public hospitals in HK between January 1985 to October 2020. Molecularly and/or enzymatically confirmed MD cases of any age were recruited via the Clinical Analysis and Reporting System (CDARS) using relevant keywords and/or International Classification of Disease (ICD) codes under the HK Hospital Authority or through the personal recollection of treating clinicians among the investigators. RESULTS: A total of 119 MD patients were recruited and analyzed in the study. The point prevalence of MD in HK was 1.02 in 100,000 people (95% confidence interval 0.81-1.28 in 100,000). 110 patients had molecularly proven MD and the other nine were diagnosed by OXPHOS enzymology analysis or mitochondrial DNA depletion analysis with unknown molecular basis. Pathogenic variants in the mitochondrial genome (72 patients) were more prevalent than those in the nuclear genome (38 patients) in our cohort. The most commonly involved organ system at disease onset was the neurological system, in which developmental delay, seizures or epilepsy, and stroke-like episodes were the most frequently reported presentations. The mortality rate in our cohort was 37%. CONCLUSION: This study is a territory-wide overview of the clinical and genetic characteristics of MD patients in a Chinese population, providing the first available prevalence rate of MD in Hong Kong. The findings of this study aim to facilitate future in-depth evaluation of MD and lay the foundation to establish a local MD registry.

Neurological manifestations in m.3243A>G-related disease triggered by metformin.

INTRODUCTION: m.3243A>G-related disease has multi-systemic manifestations including diabetes mellitus. It is uncertain whether metformin would trigger neurological manifestations of this disease. This study aims to review the diagnosis and management of m.3243A>G-related diabetes genetically confirmed by our laboratory and to evaluate the risk of metformin use triggering neurological manifestations. METHODS: Cases with m.3243A>G detected between 2009 and 2020 were reviewed. Cases with diabetes mellitus were included. Cases with mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) before diabetes onset were excluded. Odds ratio was calculated for association between metformin use and newly developed neurological manifestations. RESULTS: Sixteen patients were identified. Odds ratio for metformin use was 3.50 [0.37-33.0; p = 0.3287]. One illustrative case with clear causal relationship between metformin use and neurological manifestations was described in detail. CONCLUSION: m.3243A>G-related diabetes mellitus is underdiagnosed. Red flags including positive family history, short stature, low body weight and hearing loss are often overlooked. Early diagnosis allows regular systemic assessment. In the era of precision medicine and novel therapies, it is prudent to avoid metformin as it could trigger neurological manifestations in this condition. Coenzyme Q10, DPP-IV inhibitors, SGLT2 inhibitors and GLP-1 receptor agonists may be considered.

Fatal viral infection-associated encephalopathy in two Chinese boys: a genetically determined risk factor of thermolabile carnitine palmitoyltransferase II variants.

Influenza-associated encephalopathy (IAE) is a potentially fatal neurological complication of influenza infection usually in the presence of high and persistent fever. Thermolabile carnitine palmitoyltransferase II enzyme (CPT-II) predisposes IAE, so far only described in Japanese. As the genetic origins of Japanese and Chinese are alike, similar genetic risk factors in CPT-II are expected. We report the first two unrelated Chinese patients of thermolabile CPT-II variants that underlain the persistent high fever-triggered viral infection-associated encephalopathy, multi-organ failure and death. Elevated (C16:0+C18:1)/C2 acylcarnitines ratio and the CPT2 susceptibility variant allele [p.Phe352Cys; p.Val368Ile] were detected. The asymptomatic family members of one patient also had abnormal long-chain acylcarnitines. In our experience of biochemical genetics, the elevated (C16:0+C18:1)/C2 acylcarnitines ratio is unusual and specific for thermolabile CPT-II variants. Allele frequency of [p.Phe352Cys; p.Val368Ile] among Hong Kong Chinese was 0.104, similar to Japanese data, and [p.Phe352Cys] has not been reported in Caucasians. This may explain the Asian-specific phenomenon of thermolabile CPT-II-associated IAE. We successfully demonstrated the thermolabile CPT-II variants in patients with viral infection-associated encephalopathy in another Asian population outside Japanese. The condition is likely under-recognized. With our first cases, it is envisaged that more cases will be diagnosed in subsequent years. The exact pathogenic mechanism of how other factors interplay with thermolabile CPT-II variants and high fever leading to IAE, is yet to be elucidated. Fasting and decreased intake during illness may aggravate the disease. Further studies including high risk and neonatal screening are warranted to investigate its expressivity, penetrance and temperature-dependent behaviors in thermolabile CPT-II carriers. This may lead to discovery of the therapeutic golden window by aggressive antipyretics and L-carnitine administration in avoiding the high mortality and morbidity of IAE.

Urine organic acid as the first clue towards aromatic L-amino acid decarboxylase (AADC) deficiency in a high prevalence area.

BACKGROUND: Aromatic L-amino acid decarboxylase deficiency is a rare neurometabolic disease due to impaired decarboxylation of neurotransmitter precursors to its active form. CASE: We retrospectively reviewed 8 cases from 2008 to 2019 with cerebrospinal fluid neurotransmitter analysis performed at our centre. All cases had an elevated urine vanillactic acid and, in most cases, with N-acetylvanilalanine detected. Cerebrospinal fluid analysis showed low downstream metabolites vanillylmandelic acid, homovanillic acid but high 3-O-methyl-L-DOPA, 5-hydroxytryptophan. Cerebrospinal fluid pterins were normal. Genotyping in DDC confirms the diagnosis. Urine organic acid analysis provided the first clue to diagnosis in four of the cases, which then triggered cerebrospinal fluid neurotransmitter and genetic analysis. We also developed a diagnostic decision support system to assist the interpretation of the mass spectrometry data from urine organic acids. CONCLUSIONS: Urine organic acid could be essential in guiding subsequent investigations for the diagnosis of aromatic L-amino acid decarboxylase deficiency. We propose to screen suspected cases first with urine organic acids, specifically looking for vanillactic acid and N-acetylvanilalanine. Suggestive findings should be followed with target analysis for c.714 + 4A > T in ethnically Chinese patients. The assistive tool allowed expedite interpretation of profile data generated from urine organic acids analysis. It may also reduce interpreter's bias when peaks of interest are minor peaks in the spectrum.

Flexi-Myo Panel Strategy: Genomic Diagnoses of Myopathies and Muscular Dystrophies by Next-Generation Sequencing.

Aims: Muscle disorders are clinically and genetically heterogeneous. Investigations, including plasma creatine kinase, electromyography, and nerve conduction velocity studies are often nonspecific, whereas muscle biopsy might be limited by sampling bias and variable histopathology. Next-generation sequencing is now generally considered an important diagnostic tool for muscle disorders, with decreased costs and improved diagnostic yield. Inclusion of a large number of genes in the analysis might, however, generate a large number of ambiguous results and create unnecessary confusion for clinicians and patients. Methods: An ethnic Chinese patient presented at age 10 with tip-toe walking. Upon examination the patient had a waddling gait, a tight Achilles tendon with pes cavus. A muscle biopsy showed the presence of minicores with disruption of the myofibrillary network and Z-bands. Sequencing was performed using the Flexi-Myo panel, which provides coverage for 85 myopathic genes. Reporting of sequencing results was decided by the responsible chemical pathologists based on the available clinical and genetic information. Results: A previously identified heterozygous in-frame deletion was detected in MYH7, which confirmed the diagnosis of Laing myopathy. No variants of uncertain significance required reporting. Conclusion: We describe the effectiveness of our Flexi-Myo panel approach for the diagnosis of muscle disorders, which confirmed diagnosis of Laing myopathy in what had been a clinically ambiguous presentation. This approach enables efficient genomic testing for muscle diseases in adults and children with satisfactory diagnostic yield and sufficient sensitivity, whereas avoiding the reporting of ambiguous results. Similar strategies might also be implemented for other groups of disorders.

Two related Chinese Fabry disease patients with a p.N215S pathological variant who presented with nephropathy.

Fabry disease is an X-linked lysosomal storage disease resulting from a mutation in the GLA gene that encodes α-galactosidase A. The p.N215S (c.644A > G [p.Asn215Ser]) genotype is the most common later-onset variant reported in individuals of European or North American descent. It is usually referred to as a cardiac variant, although manifestations in other organ systems have been observed. In this report, we describe a nephropathy presentation in two related Chinese Fabry disease patients with p.N215S.

The co-occurrence of serologically proven myasthenia gravis and Miller Fisher/Guillain Barré overlap syndrome: a case report.

The co-occurrence of myasthenia gravis (MG) and Guillain Barré syndrome (GBS) is uncommon with a few reported cases in the literature. There is only one reported case of MG and Miller Fisher variant of GBS. We described an 84 year old Chinese woman with underlying seropositive myasthenia gravis (MG) who presented with ophthalmoplegia, areflexia and acute neuromuscular weakness. She was proved to have co-occurrence of MG and GBS/Miller Fisher overlap syndrome with positive anti-GQ1b antibody. The unusual finding in this patient raises an interesting question on their pathogenesis with the possibility that prior activation of the immune system may predispose the development of autoantibodies against other antigens within the same set of muscles.

Practical Aspects in Genetic Testing for Cardiomyopathies and Channelopathies.

Genetic testing has an increasingly important role in the diagnosis and management of cardiac disorders, where it confirms the diagnosis, aids prognostication and risk stratification and guides treatment. A genetic diagnosis in the proband also enables clarification of the risk for family members by cascade testing. Genetics in cardiac disorders is complex where epigenetic and environmental factors might come into interplay. Incomplete penetrance and variable expressivity is also common. Genetic results in cardiac conditions are mostly probabilistic and should be interpreted with all available clinical information. With this complexity in cardiac genetics, testing is only indicated in patients with a strong suspicion of an inheritable cardiac disorder after a full clinical evaluation. In this review we discuss the genetics underlying the major cardiomyopathies and channelopathies, and the practical aspects of diagnosing these conditions in the laboratory.

Founder Mutation c.1516A>C in KLHL40 Is a Frequent Cause of Nemaline Myopathy With Hyponatremia in Ethnic Chinese.

KLHL40-related nemaline myopathy is a severe autosomal recessive muscle disorder. The current study describes 4 cases of KLHL40-related nemaline myopathy in Hong Kong ethnic Chinese presenting within 3 years, which are confirmed with clinicopathologic features and genetic studies. The incidence is estimated to be at least 1 in 45 226 livebirths (at least 1 in 41 608 among ethnic Chinese livebirths) in Hong Kong. Hyponatremia appears to be another common feature in these patients. Salient histological features include nemaline bodies ranging from 200 to 500 nm in diameters on ultrastructural examination as well as negative KLHL40 immunohistochemistry; type II fiber predominance is obvious in 2 cases. We demonstrate the founder effect associated with genetic variant c.1516A>C (p.Thr506Pro) by polymorphic marker analysis, which revealed a 0.56-0.75-Mb or 0.41-0.78-cM shared haplotype encompassing the disease allele. The mutation is believed to have occurred around 412 generations ago or 6220 BCE, as estimated using DMLE+ and a formula described by Boehnke. We believe the founder variant might possibly underlie a sizable portion of nemaline myopathy in ethnic Chinese. Analysis of the KLHL40 gene may be considered as the first-tier testing of congenital myopathy in this ethnic group.

Gelsemium poisoning mediated by the non-toxic plant Cassytha filiformis parasitizing Gelsemium elegans.

INTRODUCTION: Gelsemium poisoning is caused by consumption of the deadly Gelsemium species such as Gelsemium elegans, leading to significant gastrointestinal, neurological and cardio-respiratory toxicities. In 2011 (Cluster 1) and 2012 (Cluster 2), the authors encountered two clusters of gelsemium poisoning after consumption of the non-toxic parasitic plant Cassytha filiformis. The current study aims to examine the mechanism of gelsemium poisoning mediated by a benign parasitic plant. METHODS: Qualitative analysis of toxic gelsemium alkaloids using liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed on the herbal and urine samples from both clusters to confirm exposure. Morphological examination, qualitative analysis of aporphine alkaloids using liquid chromatography-ion trap-time of flight mass spectrometry (LC-IT-TOF/MS) and Sanger sequencing were performed on the plant sample from Cluster 2 to confirm its identity. A field study was conducted in local countryside and C. filiformis was collected for histological, LC-MS/MS and LC-IT-TOF/MS analyses to study its interaction with G. elegans. RESULTS: Gelsemium alkaloids that are not naturally present in C. filiformis were detected in the patients' herbal and urine samples. Misidentification and contamination with G. elegans during the preparation process were excluded by morphological examination of the plant sample from Cluster 2. Its identity as C. filiformis was verified with LC-IT-TOF/MS and molecular analyses. Histological, LC-MS/MS and LC-IT-TOF/MS analyses of C. filiformis collected during the field study confirmed that its haustoria penetrated the vascular bundles of G. elegans and absorbed its gelsemium toxins. CONCLUSIONS: The non-toxic plant C. filiformis absorbed toxic gelsemium alkaloids from its host, G. elegans, and led to gelsemium poisoning in our patients. Our study provides new insights into the toxicology of such plants. Benign parasitic plants may lead to potentially life-threatening poisoning if it parasitizes toxic hosts and absorbs their phytotoxins. The public awareness of risks associated with the use of these medicinal parasitic plants should be raised.

2,4-Dinitrophenol: a threat to Chinese body-conscious groups.

2,4-Dinitrophenol (2,4-DNP), a yellowish compound, has historically been used in the manufacture of dyes, explosives, and fungicides. As it uncouples mitochondrial oxidative phosphorylation, the compound was also used as an antiobesity agent early in the past century. The compound was subsequently banned by the United States Food and Drug Administration in 1938 due to its potentially fatal adverse effects, including hyperthermia, cataract, agranulocytosis, hepatoxicity, nephrotoxicity, and cardiotoxicity. However, the popularity of 2,4-DNP as a slimming aid has appeared to increase again in recent years. The Hong Kong Hospital Authority Toxicology Reference Laboratory recently confirmed two cases of self-administered 2,4-DNP with different clinical presentations to hospitals in the area. Here we describe those two cases, in an attempt to underscore the potential of misuse of this substance by body-conscious groups among the Chinese population.

Non-invasive urinary screening for aromatic L-amino acid decarboxylase deficiency in high-prevalence areas: a pilot study.

BACKGROUND: The diagnosis of aromatic L-amino acid decarboxylase (AADC) deficiency, one of the pediatric neurotransmitter disorders, is classically made with plasma enzyme level or cerebrospinal fluid (CSF) neurotransmitter profile, while both are technically demanding and the latter requires the invasive lumbar puncture. So far less than 100 cases have been reported worldwide with 20% from Taiwan. It was postulated that the condition might have been under-diagnosed among Chinese populations and a non-invasive screening tool should be developed in areas with high prevalence. METHODS: Urine metabolic profiles performed by gas chromatography-mass spectrometry (GC-MS) in a 31-month period were retrospectively reviewed: those with vanilmandelic acid concentration lower than one percentile plus the presence of 3-o-methyldopa were defined as positive and the patients were further evaluated. RESULTS: Among 1046 metabolic profiles (from 845 patients) reviewed, 3 profiles from 2 patients were screened positive: both cases had compatible CSF neurotransmitter profiles and the diagnosis was further confirmed by genetic analysis of DDC gene. 13 negative urinary metabolic profiles from 7 patients who had CSF neurotransmitters analyzed were identified as controls: all 7 CSF neurotransmitter profiles were not compatible for AADC deficiency. CONCLUSIONS: The GC-MS-based urine metabolic profiling was shown to be a satisfactory screening tool for AADC deficiency. Further confirmation can be performed by mutation analysis in the DDC gene, thus avoiding risks of lumbar puncture. We advocate all ethnic Chinese patients presenting with dystonia have their urine organic acids analyzed before proceeding to CSF neurotransmitters analysis.

Analysis of inborn errors of metabolism: disease spectrum for expanded newborn screening in Hong Kong.

BACKGROUND: Data of classical inborn errors of metabolism (IEM) of amino acids, organic acids and fatty acid oxidation are largely lacking in Hong Kong, where mass spectrometry-based expanded newborn screening for IEM has not been initiated. The current study aimed to evaluate the approximate incidence, spectrum and other characteristics of classical IEM in Hong Kong, which would be important in developing an expanded newborn screening program for the local area. METHODS: The laboratory records of plasma amino acids, plasma acylcarnitines and urine organic acids analyses from year 2005 to 2009 inclusive in three regional chemical pathology laboratories providing biochemical and genetic diagnostic services for IEM were retrospectively reviewed. RESULTS: Among the cohort, 43 patients were diagnosed of IEM, including 30 cases (69%) of amino acidemias (predominantly citrin deficiency, hyperphenylalaninemia due to 6-pyruvoyl-tetrahydropterin synthase deficiency and tyrosinemia type I), 5 cases (12%) of organic acidemias (predominantly holocarboxylase synthetase deficiency) and 8 cases (19%) of fatty acid oxidation defects (predominantly carnitine-acylcarnitine translocase deficiency). The incidence of classical IEM in Hong Kong was roughly estimated to be at least 1 case per 4122 lives births, or 0.243 cases per 1000 live births. This incidence is similar to those reported worldwide, including the mainland of China. The estimated incidence of hyperphenylalaninemia was 1 in 29 542 live births. CONCLUSIONS: Our data indicate that it is indisputable for the introduction of expanded newborn screening program in Hong Kong. Since Hong Kong is a metropolitan city, a comprehensive expanded newborn screening program and referral system should be available to serve the neonates born in the area.

Role of postmortem genetic testing demonstrated in a case of glutaric aciduria type II.

Glutaric aciduria type II, or multiple acyl-CoA dehydrogenase deficiency, is a rare metabolic disorder inherited in an autosomal recessive manner. The condition can be caused by mutations in at least 3 genes, including ETFA, ETFB, and ETFDH. When this potentially lethal disorder is known for its clinical and biochemical heterogeneity, mutation analysis will be an invaluable part of diagnosis. We here described a Chinese adolescent boy who enjoyed good health earlier and presented at the age of 14 years with severe vomiting. His condition deteriorated rapidly and he succumbed shortly after. With a travel history before presentation and the late age of onset, diagnosis was particularly difficult. Findings in perimortem biochemical investigations and postmortem autopsy were guiding but not diagnostic. The diagnosis of glutaric aciduria type II was finally confirmed by mutation analysis performed by direct sequencing on genomic DNA from peripheral blood, which identified 2 different unreported missense mutations, c.502G>T (p.V168F) and c.786A>G (p.Q262R), in ETFA. The father and the mother were found to be heterozygous for the 2 mutations in ETFA respectively. Subsequent molecular family screening also ruled out the disease in his elder sister, who had a history of convulsion and a suspicious plasma acylcarnitine profile, and freed her from life-long supplementation. The case showed that molecular autopsies should be part of routine postmortem examination of unexplained sudden death in all age groups and DNA-friendly samples should be routinely collected and archived. In the era of personalized medicine with the power of modern genetics, molecular diagnosis should be obtained for heterogeneous diseases with different genetic defects but sharing similar clinical and/or biochemical phenotypes.