LC-MS/MS in clinical chemistry: Did it live up to its promise?: Consideration from the Dutch EQAS organisation.

BACKGROUND: Over the past decade the use of LC-MS/MS has increased significantly in the hospital laboratories. Clinical laboratories have switched from immunoassays to LC-MS/MS methods due to the promise of improvements in sensitivity and specificity, better standardization with often non-commutable international standards, and better between-laboratory comparison. However, it remains unclear whether routine performance of the LC-MS/MS methods have met these expectations. METHOD: This study examined the EQAS results, from the Dutch SKML, of serum cortisol, testosterone, 25OH-vitaminD and cortisol in urine and saliva over 9 surveys (2020 to first half of 2021). RESULTS: The study found a significant increase in the number of compounds and in the number of results measured in the different matrices, with LC-MS/MS over a period of eleven years. In 2021, approximately 4000 LC-MS/MS results were submitted (serum: urine: saliva = 58:31:11%) compared to only 34 in 2010. When compared to the individual immunoassays, the LC-MS/MS based methods for serum cortisol, testosterone and 25OH-vitaminD showed comparable but also higher between-laboratory CVs in different samples of the surveys. For cortisol, testosterone and 25OH-vitaminD the median CV was 6.8%, 6.1% and 4.7% respectively for the LC-MS/MS compared to 3.9-8.0%,4.5-6.7%, and 7.5-18.3% for immunoassays. However, the bias and imprecision of the LC-MS/MS was better than that of the immunoassays. CONCLUSION: Despite the expectation that LC-MS/MS methods would result in smaller between-laboratory differences, as they are relatively matrix independent and better to standardize, the results of the SKML round robins do not reflect this for some analytes and may be in part explained by the fact that in most cases laboratory developed tests were used.

Prevalence of detectable biotin in The Netherlands in relation to risk on immunoassay interference.

Despite the increasing awareness about biotin interference with immunoassays, so far, only two studies have quantified the prevalence of elevated biotin in patient populations. In a US study, over 7% had biotin concentrations exceeding 10 ng/mL, whereas in an Australian study only 0.8% of ED samples contained biotin exceeding 10 ng/mL. At present, representative data for the European population are lacking. In this study, we investigated biotin prevalence in The Netherlands in a representative cohort of routine laboratory requests in our laboratory using an LC-MS/MS assay for quantification of biotin in human plasma. In our study, we found 0.2% of samples exceeding 10 ng/mL of biotin, a finding more or less in line with the Australian data. Even though the biotin prevalence appears to be low, with concomitant low to moderate biotin concentrations, it is by no means a rare phenomenon. Laboratories like ours are likely to experience biotin positive samples on a daily basis with variable impact on patient care depending on the analytical bias from the immunoassay platform used. Our simple and robust LC-MS/MS assay for quantification of biotin in human samples may contribute to better understanding of the systemic concentrations seen after moderate- and high-dose biotin supplementation and the extent of immunoassay interference.

Diabetes-associated mitochondrial DNA mutation A3243G impairs cellular metabolic pathways necessary for beta cell function.

AIMS/HYPOTHESIS: Mitochondrial DNA (mtDNA) mutations cause several diseases, including mitochondrial inherited diabetes and deafness (MIDD), typically associated with the mtDNA A3243G point mutation on tRNALeu gene. The common hypothesis to explain the link between the genotype and the phenotype is that the mutation might impair mitochondrial metabolism expressly required for beta cell functions. However, this assumption has not yet been tested. METHODS: We used clonal osteosarcoma cytosolic hybrid cells (namely cybrids) harbouring mitochondria derived from MIDD patients and containing either exclusively wild-type or mutated (A3243G) mtDNA. According to the importance of mitochondrial metabolism in beta cells, we studied the impact of the mutation on key parameters by comparing stimulation of these cybrids by the main insulin secretagogue glucose and the mitochondrial substrate pyruvate. RESULTS: Compared with control mtDNA from the same patient, the A3243G mutation markedly modified metabolic pathways leading to a high glycolytic rate (2.8-fold increase), increased lactate production (2.5-fold), and reduced glucose oxidation (-83%). We also observed impaired NADH responses (-56%), negligible mitochondrial membrane potential, and reduced, only transient ATP generation. Moreover, cybrid cells carrying patient-derived mutant mtDNA exhibited deranged cell calcium handling with increased cytosolic loads (1.4-fold higher), and elevated reactive oxygen species (2.6-fold increase) under glucose deprivation. CONCLUSIONS/INTERPRETATION: The present study demonstrates that the mtDNA A3243G mutation impairs crucial metabolic events required for proper cell functions, such as coupling of glucose recognition to insulin secretion.

[Missed hyperinsulinaemia in a patient with an insulinoma]. / Gemiste hyperinsulinemie bij een patiënt met insulinoom.

In a 57-year-old man with symptomatic hypoglycaemias which gave cause to suspect an insulinoma, normal insulin levels were initially found. A repeated fasting assay at another hospital did, however, reveal the expected hyperinsulinaemia. Scans revealed an abnormality in the pancreas. After surgical removal of the insulin-producing tumour the patient made a quick recovery. The diagnosis of organic hyperinsulinaemia is established by demonstrating inappropriately high serum-insulin concentrations during fasting hypoglycaemia. The diagnostic normative values are based on the classic polyclonal method of determination. This new highly-specific insulin assay has no cross-reactivity with pro-insulin, which is often produced disproportionately more by an insulinoma. As a result of this false-normal insulin values are found. Therefore new normative values are needed for the newer insulin assays when diagnosing an insulin-producing islet cell tumour. Pro-insulin and C-peptide assays may play a useful role in this.

[From gene to disease; mutations in the WFS1-gene as the cause of juvenile type I diabetes mellitus with optic atrophy (Wolfram syndrome)]. / Van gen naar ziekte; mutaties in het WFS1-gen als oorzaak van juveniele type-1-diabetes mellitus met opticusatrofie (Wolfram-syndroom).

Wolfram syndrome patients are mainly characterised by juvenile onset diabetes mellitus and optic atrophy. A synonym is the acronym DIDMOAD: diabetes insipidus, diabetes mellitus, optic atrophy, deafness. Diabetes insipidus and sensorineural high-frequency hearing impairment are important additional features. This rare autosomal recessively inherited neurodegenerative syndrome is caused by mainly inactivating mutations in the WFS1 gene. It is located at chromosome 4p16 and encodes wolframin, a transmembrane protein. No function has yet been ascribed to this protein.

[From gene to disease; mutation in mitochondrial DNA and maternally inherited diabetes mellitus with deafness (MIDD)]. / Van gen naar ziekte; mutatie in mitochondrieel DNA en maternaal overervende diabetes mellitus met doofheid (MIDD).

MIDD is a maternally inherited disorder with diabetes and impaired hearing due to a reduced perception of high tones. The disorder is caused by an A to G mutation at position 3243 in mitochondrial DNA. Approximately 1.3% of insulin-dependent diabetic patients in the Netherlands has this mutation. The main defect in these patients seems to be a reduced secretion of insulin by the pancreas in response to glucose stimulation.

Molecular and clinical aspects of mitochondrial diabetes mellitus.

This review provides a compact overview on the contribution of mutations in mtDNA to the pathogenesis of diabetes mellitus, with emphasis on the A3243G mutation in the tRNA(Leu, UUR) gene. This mutation associates in most individuals with maternally inherited diabetes and deafness (MIDD) whereas in some other carriers the MELAS syndrome or a progressive kidney failure is seen. Possible pathogenic mechanisms are discussed especially the question why particular mutations in mtDNA associate with distinct clinical entities. Mutations in mtDNA can affect the ATP production, thereby leading to particular clinical phenotypes such as muscle weakness. On the other hand mtDNA mutations may also alter the intracellular concentration of mitochondrial metabolites which can act as signalling molecules, such as Ca or glutamate. This situation may contribute to the development of particular phenotypes that are associated with distinct mtDNA mutations.

Simultaneous A8344G heteroplasmy and mitochondrial DNA copy number quantification in myoclonus epilepsy and ragged-red fibers (MERRF) syndrome by a multiplex molecular beacon based real-time fluorescence PCR.

The association of a particular mitochondrial DNA (mtDNA) mutation with different clinical phenotypes is a well-known feature of mitochondrial diseases. A simple genotype-phenotype correlation has not been found between mutation load and disease expression. Tissue and intercellular mosaicism as well as mtDNA copy number are thought to be responsible for the different clinical phenotypes. As disease expression of mitochondrial tRNA mutations is mostly in postmitotic tissues, studies to elucidate disease mechanisms need to be performed on patient material. Heteroplasmy quantitation and copy number estimation using small patient biopsy samples has not been reported before, mainly due to technical restrictions. In order to resolve this problem, we have developed a robust assay that utilizes Molecular Beacons to accurately quantify heteroplasmy levels and determine mtDNA copy number in small samples carrying the A8344G tRNA(Lys) mutation. It provides the methodological basis to investigate the role of heteroplasmy and mtDNA copy number in determining the clinical phenotypes.

Characterization of a novel mitochondrial DNA deletion in a patient with a variant of the Pearson marrow-pancreas syndrome.

We have recently diagnosed a patient with anaemia, severe tubulopathy, and diabetes mellitus. As the clinical characteristics resembled Pearson marrow-pancreas syndrome, despite the absence of malfunctioning of the exocrine pancreas in this patient, we have performed DNA analysis to seek for deletions in mtDNA. DNA analysis showed a novel heteroplasmic deletion in mtDNA of 8034bp in length, with high proportions of deleted mtDNA in leukocytes, liver, kidney, and muscle. No deletion could be detected in mtDNA of leukocytes from her mother and young brother, indicating the sporadic occurrence of this deletion. During culture, skin fibroblasts exhibited a rapid decrease of heteroplasmy indicating a selection against the deletion in proliferating cells. We estimate that per cell division heteroplasmy levels decrease by 0.8%. By techniques of fluorescent in situ hybridisation (FISH) and mitochondria-mediated transformation of rho(o) cells we could show inter- as well as intracellular variation in the distribution of deleted mtDNA in a cell population of cultured skin fibroblasts. Furthermore, we studied the mitochondrial translation capacity in cybrid cells containing various proportions of deleted mtDNA. This result revealed a sharp threshold, around 80%, in the proportion of deleted mtDNA, above which there was strong depression of overall mitochondrial translation, and below which there was complementation of the deleted mtDNA by the wild-type DNA. Moreover, catastrophic loss of mtDNA occurred in cybrid cells containing 80% deleted mtDNA.

HLA-DQ polymorphism and degree of heteroplasmy of the A3243G mitochondrial DNA mutation in maternally inherited diabetes and deafness.

AIM: Maternally inherited diabetes and deafness (MIDD) associates with a mutation at position 3243 in mitochondrial DNA. Phenotypic expression of MIDD includes Type 1-like and Type 2-like diabetes. This study examined whether HLA-DQ phenotype and the degree of heteroplasmy in leucocyte and oral mucosa DNA influence clinical expression of the 3242 mutation. METHODS: In a group of 20 unrelated probands with MIDD, eight with Type 1- like diabetes, 12 with Type 2-like diabetes, HLA-DQ type and degree of heteroplasmy for the 3243 mutation were determined. HLA-DQA1/DQB1 phenotypes were categorized as predisposing, neutral or protective for autoimmune-mediated Type 1 diabetes. RESULTS: No differences were observed between Type 1 and Type 2-like MIDD groups with respect to the cumulative frequency of protective and predisposing HLA-DQ types. Predisposing HLA-DQ types are more prevalent in MIDD patients than in the control population (P < 0.05). Degrees of heteroplasmy for the 3243 mutation showed large variations in patients, ranging from 1 to 52% in leucocyte DNA. A strong correlation was seen between heteroplasmy in leucocyte DNA and DNA from oral mucosa cells (r = 0.89, P < 0.001). No correlation was observed between the degree of heteroplasmy and diabetic phenotype, even when group size was extended with diabetic relatives of patients with MIDD. The age of diagnosis of diabetes was not correlated with heteroplasmy, but the degree of heteroplasmy tended to decrease with age. CONCLUSIONS: The phenotype of diabetes in MIDD appears to be independent of HLA-DQ phenotype and degree of heteroplasmy in leucocyte and oral mucosa DNA indicating that other, as yet unknown, factors modulate clinical expression of the 3243 mutation.

The diabetes-associated 3243 mutation in the mitochondrial tRNA(Leu(UUR)) gene causes severe mitochondrial dysfunction without a strong decrease in protein synthesis rate.

Cells harboring patient-derived mitochondria with an A-to-G transition at nucleotide position 3243 of their mitochondrial DNA display severe loss of respiration when compared with cells containing the wild-type adenine but otherwise identical mitochondrial DNA sequence. The amount and degree of leucylation of tRNA(Leu(UUR)) were both found to be highly reduced in mutant cells. Despite the low level of leucyl-tRNA(Leu(UUR)), the rate of mitochondrial translation was not seriously affected by this mutation. Therefore, decrease of mitochondrial protein synthesis as such does not appear to be a necessary prerequisite for loss of respiration. Rather, the mitochondrially encoded proteins seem subject to elevated degradation, leading to a severe reduction in their steady state levels. Our results favor a scheme in which the 3243 mutation causes loss of respiration through accelerated protein degradation, leading to a disequilibrium between the levels of mitochondrial and nuclear encoded respiratory chain subunits and thereby a reduction of functional respiratory chain complexes. The possible mechanisms underlying the pathogenesis of mitochondrial diabetes is discussed.

Functional and morphological abnormalities of mitochondria harbouring the tRNA(Leu)(UUR) mutation in mitochondrial DNA derived from patients with maternally inherited diabetes and deafness (MIDD) and progressive kidney disease.

AIMS/HYPOTHESIS: An A to G transition at nucleotide position 3243 in the mitochondrial tRNA Leu(UUR) gene has been identified in patients with maternally inherited diabetes and deafness, as well as in patients with mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes, chronic progressive external ophpthalmoplegia, cardiomyopathy and progressive kidney disease. Variations in the mitochondrial DNA haplotype as well as differences in the degree and distribution of heteroplasmy in a certain tissue are factors that may contribute to the variety in phenotypical expression of the 3243 tRNA(Leu)(UUR) mutation. We have done morphological and functional experiments on mitochondria carrying the 3243 mutation derived from patients with either maternally inherited diabetes and deafness or progressive kidney disease to prove the pathogenicity of the 3243 mutation and to examine whether the mtDNA haplotype modulates the pathobiochemistry of this mutation. METHODS: We constructed clonal cell lines that contain predominantly mutated or exclusively wild-type mtDNA with a distinct mtDNA haplotype by the methodology of mitochondria-mediated transformation. Cells lacking mitochondrial DNA (rho(o)) were used as recipients and donor mitochondria were derived from fibroblasts of a patient with either maternally inherited diabetes and deafness or progressive kidney disease. The fibroblasts from these clinically distinct patients carry different mitochondrial DNA haplotypes with the 3243 mutation in heteroplasmic form. RESULTS: Heteroplasmy in the clonal cybrid cells ranged from 0 to 100%, reflecting the heterogeneity of the mitochondrial donor cell. Cybrid cells containing predominantly mutant mitochondrial DNA showed lactic acidosis, poor respiration and marked defects in mitochondrial morphology and respiratory chain complex I and IV activities. No differences were observed in the extent of the mitochondrial dysfunction between the mutant cells derived from the two donors. CONCLUSION/INTERPRETATION: These results provide evidence for a pathogenic effect of the tRNA(Leu)(UUR) mutation in maternally inherited diabetes and deafness and progressive kidney disease, and show no evidence of a contribution of the mitochondrial DNA haplotype as a modulating the biochemical expression of the mutation.

Targeted inactivation of mouse RAD52 reduces homologous recombination but not resistance to ionizing radiation.

The RAD52 epistasis group is required for recombinational repair of double-strand breaks (DSBs) and shows strong evolutionary conservation. In Saccharomyces cerevisiae, RAD52 is one of the key members in this pathway. Strains with mutations in this gene show strong hypersensitivity to DNA-damaging agents and defects in recombination. Inactivation of the mouse homologue of RAD52 in embryonic stem (ES) cells resulted in a reduced frequency of homologous recombination. Unlike the yeast Scrad52 mutant, MmRAD52(-/-) ES cells were not hypersensitive to agents that induce DSBs. MmRAD52 null mutant mice showed no abnormalities in viability, fertility, and the immune system. These results show that, as in S. cerevisiae, MmRAD52 is involved in recombination, although the repair of DNA damage is not affected upon inactivation, indicating that MmRAD52 may be involved in certain types of DSB repair processes and not in others. The effect of inactivating MmRAD52 suggests the presence of genes functionally related to MmRAD52, which can partly compensate for the absence of MmRad52 protein.

[A new subtype of diabetes mellitus: maternally inherited diabetes and deafness (MIDD)]. / Een nieuw subtype van diabetes mellitus: via de moeder overgeërfde diabetes en doofheid (MIDD).

Diabetes mellitus comprises many subtypes, the pathogenesis of each of which involves a combination of inherited and environmental factors. Recently a new subtype of diabetes mellitus was recognized in a Dutch pedigree, designated as 'maternally inherited diabetes and deafness' (MIDD). Impaired hearing is an associated phenomenon of the disease. Approximately 1.3% of all diabetic cases in the Netherlands exhibit the MIDD subtype. MIDD shows a strictly maternal heredity. In MIDD there is a guanine-for-adenine substitution at position 3243 in mitochondrial DNA. Mitochondria carrying this mutation exhibit a decreased functionality. In carriers of the MIDD mutation the insulin secretion by the pancreas in response to stimulation by glucose is impaired.

Mutation in mitochondrial tRNA(Leu(UUR)) gene associated with progressive kidney disease.

Several studies show an association of a guanine for adenine substitution (A-->G) at position 3243 in mitochondrial DNA (mtDNA) with a recently recognized diabetic subtype designated maternally inherited diabetes and deafness (MIDD). This mutation shows heterogeneity in its phenotypic expression as is apparent from its association with several other syndromes. Screening for the 3243A-->G mutation in mtDNA was performed in those diabetic patients attending the Leiden University Hospital diabetics clinic who had a history of maternally inherited diabetes, sensorineural hearing loss, or both. Four individuals from three unrelated families were identified who suffered from progressive nondiabetic kidney disease in association with diabetes mellitus and hearing loss. The mode of inheritance suggested maternal transmission. The combination of renal failure and hearing loss had been misdiagnosed as Alport syndrome in three of the four individuals. Therefore, in addition to these three families, another 63 unrelated patients with possible Alport syndrome were selected at random. DNA from peripheral blood and other tissues from members of the three families and from the 63 additional Alport syndrome patients was examined for the presence of the 3243A-->G mutation in mtDNA. The mutation was detected in heteroplasmic form in the four patients and their maternal relatives. Also, one of the 63 suspected Alport syndrome patients showed heteroplasmy for the 3243 mutation. These data show the existence of a kidney disease that is characterized by the presence of the A-->G mutation at position 3243 in the mtDNA.

Genomic characterization of the mouse homolog of the Saccharomyces cerevisiae recombination and double-strand break repair gene RAD52.

The yeast Saccharomyces cerevisiae RAD52 gene is involved in recombination and DNA double-strand break repair. Recently, mouse and human homologs of the yeast RAD52 gene have been identified. Here we present the genomic organization of the mouse RAD52 gene. It consists of 12 exons ranging in size from 67 to 374 bp spread over a region of approximately 18 kb. The first ATG is located in exon 2. Analysis of the promoter region revealed no classical promoter elements such as CCAAT or TATA boxes. Transcriptional mapping analysis revealed one major transcription start point. Analogous to the situation in yeast, transcription of the RAD52 gene in human skin fibroblasts and mouse Ltk- cells was not induced by methyl methanesulfonate treatment. Furthermore, no specific alteration in human RAD52 expression levels throughout the cell cycle was observed.

Maternally inherited diabetes and deafness: a diabetic subtype associated with a mutation in mitochondrial DNA.

Diabetes mellitus is a common disease with variations in its clinical expression and different modes of pathogenesis. The purpose of this review is to discuss a recently identified diabetic subtype. Based on the triad diabetes, maternal inheritance and impaired hearing in this subtype we have proposed the name Maternally Inherited Diabetes and Deafness (MIDD). This diabetic subtype associates in the vast majority of cases with a single mutation in mitochondrial DNA, at position 3243. The clinical presentation of MIDD which can be IDDM-like or NIDDM-like, its frequency of occurrence, possible pathogenic mechanisms and the contribution of other mitochondrial DNA mutations to the development of diabetes will be discussed.

Detection of mitochondrial DNA deletions in human skin fibroblasts of patients with Pearson's syndrome by two-color fluorescence in situ hybridization.

Pearson's marrow/pancreas syndrome is a disease associated with a large mitochondrial DNA (mtDNA) deletion. The various tissues of a patient contain heteroplasmic populations of wild-type (WT) and deleted mtDNA molecules. The clinical phenotype of Pearson's syndrome is variable and is not correlated with the size and position of the deletion. The histo- and cytological distribution of WT and deleted mtDNA molecules may be factors that correlate with the phenotypical expression of the disease. Here we introduce a new application of two-color FISH to visualize WT and deleted mtDNA simultaneously in a cell population of in vitro cultured skin fibroblasts of two patients with Pearson's syndrome. At the third passage of culturing, fibroblasts showed a remarkable heterogeneity of WT and deleted mtDNA: about 90% of the cells contained almost 100% WT mtDNA, and 10% of the cells contained predominantly deleted mtDNA. At the tenth passage of culturing, fibroblasts showed a reduction of intercellular heteroplasmy from 10% to 1%, while intracellular heteroplasmy was maintained. This new approach enables detailed analysis of distribution patterns of WT and deleted mtDNA molecules at the inter- and intracellular levels in clinical samples, and may contribute to a better understanding of genotype-phenotype relationships in patients with mitochondrial diseases.

The molecular basis and clinical characteristics of Maternally Inherited Diabetes and Deafness (MIDD), a recently recognized diabetic subtype.

Diabetes mellitus comprises a number of diseases with hyperglycemia as hallmark. Currently, multiple genetic factors are being recognized which contribute to the development of diabetes or which may modulate its clinical expression. This review presents an overview of our current knowledge on a diabetic subtype which associates with a single mutation in mitochondrial DNA. Based on the triad of Maternal Inheritance, Diabetes and Deafness we propose the name Maternally Inherited Diabetes and Deafness (MIDD) for this syndrome. In Northwestern Europe MIDD affects approximately 1.3% of all diabetic individuals.

Mitochondrial diabetes mellitus: a review.

We review the relationship between various types of mitochondrial DNA mutations and the prevalence as well as the pathobiochemical and clinical features of mitochondrial diabetes mellitus. An A to G transversion mutation in the tRNA(Leu(UUR)) gene is associated with diabetes in about 1.5% of the diabetic population in different countries and races. Phenotypically this type of mitochondrial diabetes is combined with deafness in more than 60% and is clinically distinguishable with respect to several characteristics from the two idiopathic forms of diabetes. The underlying pathomechanism is probably a delayed insulin secretion due to an impaired mitochondrial ATP production in consequence of the mtDNA defect.