Compound-heterozygous GRIN2A null variants associated with severe developmental and epileptic encephalopathy.

We report on an 8-year-old girl with severe developmental and epileptic encephalopathy due to the compound heterozygous null variants p.(Gln661*) and p.(Leu830Profs*2) in GRIN2A resulting in a knockout of the human GluN2A subunit of the N-methyl-D-aspartate receptor. Both parents had less severe GRIN2A-related phenotypes and were heterozygous carriers of the respective null variant. Functional investigations of both variants suggested a loss-of-function effect. This is the first description of an autosomal recessive, biallelic type of GRIN2A-related disorder. Nonetheless, there are marked parallels to two previously published families with severe epileptic encephalopathy due to homozygous null variants in GRIN1 as well as various knockout animal models. Compared to heterozygous null variants, biallelic knockout of either GluN1 or GluN2A is associated with markedly more severe phenotypes in both humans and mice. Furthermore, recent findings enable a potential precision medicine approach targeting GRIN-related disorders due to null variants.

Characterization of two novel intronic OPA1 mutations resulting in aberrant pre-mRNA splicing.

BACKGROUND: We report two novel splice region mutations in OPA1 in two unrelated families presenting with autosomal-dominant optic atrophy type 1 (ADOA1) (ADOA or Kjer type optic atrophy). Mutations in OPA1 encoding a mitochondrial inner membrane protein are a major cause of ADOA. METHODS: We analyzed two unrelated families including four affected individuals clinically suspicious of ADOA. Standard ocular examinations were performed in affected individuals of both families. All coding exons, as well as exon-intron boundaries of the OPA1 gene were sequenced. In addition, multiplex ligation-dependent probe amplification (MLPA) was performed to uncover copy number variations in OPA1. mRNA processing was monitored using RT-PCR and subsequent cDNA analysis. RESULTS: We report two novel splice region mutations in OPA1 in two unrelated individuals and their affected relatives, which were previously not described in the literature. In one family the heterozygous insertion and deletion c.[611-37_611-38insACTGGAGAATGTAAAGGGCTTT;611-6_611-16delCATATTTATCT] was found in all investigated family members leading to the activation of an intronic cryptic splice site. In the second family sequencing of OPA1 disclosed a de novo heterozygous deletion c.2012+4_2012+7delAGTA resulting in exon 18 and 19 skipping, which was not detected in healthy family members. CONCLUSION: We identified two novel intronic mutations in OPA1 affecting the correct OPA1 pre-mRNA splicing, which was confirmed by OPA1 cDNA analysis. This study shows the importance of transcript analysis to determine the consequences of unclear intronic mutations in OPA1 in proximity to the intron-exon boundaries.

SDHA mutation with dominant transmission results in complex II deficiency with ocular, cardiac, and neurologic involvement.

Isolated defects of the mitochondrial respiratory complex II (succinate dehydrogenase, SDH) are rare, accounting for approximately 2% of all respiratory chain deficiency diagnoses. Here, we report clinical and molecular investigations of three family members with a heterozygous mutation in the large flavoprotein subunit SDHA previously described to cause complex II deficiency. The index patient presented with bilateral optic atrophy and ocular movement disorder, a progressive polyneuropathy, psychiatric involvement, and cardiomyopathy. Two of his children presented with cardiomyopathy and methylglutaconic aciduria in early childhood. The daughter deceased at the age of 7 months due to cardiac insufficiency. The 30-year old son presents with cardiomyopathy and developed bilateral optic atrophy in adulthood. Of the four nuclear encoded proteins composing complex II (SDHA, SDHB, SDHC, SDHD) and currently known assembly factors SDHAF1 and SDHAF2 mainly recessively inherited mutations have been described in SDHA, SDHB, SDHD, and SDHAF1 to be causative for mitochondrial disease phenotypes. This is the second report presenting autosomal dominant inheritance of a SDHA mutation.© 2016 Wiley Periodicals, Inc.

The Vacuolar H+-ATPase B1 Subunit Polymorphism p.E161K Associates with Impaired Urinary Acidification in Recurrent Stone Formers.

Mutations in the vacuolar-type H(+)-ATPase B1 subunit gene ATP6V1B1 cause autosomal-recessive distal renal tubular acidosis (dRTA). We previously identified a single-nucleotide polymorphism (SNP) in the human B1 subunit (c.481G>A; p.E161K) that causes greatly diminished pump function in vitro To investigate the effect of this SNP on urinary acidification, we conducted a genotype-phenotype analysis of recurrent stone formers in the Dallas and Bern kidney stone registries. Of 555 patients examined, 32 (5.8%) were heterozygous for the p.E161K SNP, and the remaining 523 (94.2%) carried two wild-type alleles. After adjustment for sex, age, body mass index, and dietary acid and alkali intake, p.E161K SNP carriers had a nonsignificant tendency to higher urinary pH on a random diet (6.31 versus 6.09; P=0.09). Under an instructed low-Ca and low-Na diet, urinary pH was higher in p.E161K SNP carriers (6.56 versus 6.01; P<0.01). Kidney stones of p.E161K carriers were more likely to contain calcium phosphate than stones of wild-type patients. In acute NH4Cl loading, p.E161K carriers displayed a higher trough urinary pH (5.34 versus 4.89; P=0.01) than wild-type patients. Overall, 14.6% of wild-type patients and 52.4% of p.E161K carriers were unable to acidify their urine below pH 5.3 and thus, can be considered to have incomplete dRTA. In summary, our data indicate that recurrent stone formers with the vacuolar H(+)-ATPase B1 subunit p.E161K SNP exhibit a urinary acidification deficit with an increased prevalence of calcium phosphate-containing kidney stones. The burden of E161K heterozygosity may be a forme fruste of dRTA.

Evaluation of genome-wide loci of iron metabolism in hereditary hemochromatosis identifies PCSK7 as a host risk factor of liver cirrhosis.

Genome-wide association studies (GWAS) have revealed genetic determinants of iron metabolism, but correlation of these with clinical phenotypes is pending. Homozygosity for HFE C282Y is the predominant genetic risk factor for hereditary hemochromatosis (HH) and may cause liver cirrhosis. However, this genotype has a low penetrance. Thus, detection of yet unknown genetic markers that identify patients at risk of developing severe liver disease is necessary for better prevention. Genetic loci associated with iron metabolism (TF, TMPRSS6, PCSK7, TFR2 and Chr2p14) in recent GWAS and liver fibrosis (PNPLA3) in recent meta-analysis were analyzed for association with either liver cirrhosis or advanced fibrosis in 148 German HFE C282Y homozygotes. Replication of associations was sought in additional 499 Austrian/Swiss and 112 HFE C282Y homozygotes from Sweden. Only variant rs236918 in the PCSK7 gene (proprotein convertase subtilisin/kexin type 7) was associated with cirrhosis or advanced fibrosis (P = 1.02 × 10(-5)) in the German cohort with genotypic odds ratios of 3.56 (95% CI 1.29-9.77) for CG heterozygotes and 5.38 (95% CI 2.39-12.10) for C allele carriers. Association between rs236918 and cirrhosis was confirmed in Austrian/Swiss HFE C282Y homozygotes (P = 0.014; ORallelic = 1.82 (95% CI 1.12-2.95) but not in Swedish patients. Post hoc combined analyses of German/Swiss/Austrian patients with available liver histology (N = 244, P = 0.00014, ORallelic = 2.84) and of males only (N = 431, P = 2.17 × 10(-5), ORallelic = 2.54) were consistent with the premier finding. Association between rs236918 and cirrhosis was not confirmed in alcoholic cirrhotics, suggesting specificity of this genetic risk factor for HH. PCSK7 variant rs236918 is a risk factor for cirrhosis in HH patients homozygous for the HFE C282Y mutation.

Mutations in SDHD lead to autosomal recessive encephalomyopathy and isolated mitochondrial complex II deficiency.

BACKGROUND: Defects of the mitochondrial respiratory chain complex II (succinate dehydrogenase (SDH) complex) are extremely rare. Of the four nuclear encoded proteins composing complex II, only mutations in the 70 kDa flavoprotein (SDHA) and the recently identified complex II assembly factor (SDHAF1) have been found to be causative for mitochondrial respiratory chain diseases. Mutations in the other three subunits (SDHB, SDHC, SDHD) and the second assembly factor (SDHAF2) have so far only been associated with hereditary paragangliomas and phaeochromocytomas. Recessive germline mutations in SDHB have recently been associated with complex II deficiency and leukodystrophy in one patient. METHODS AND RESULTS: We present the clinical and molecular investigations of the first patient with biochemical evidence of a severe isolated complex II deficiency due to compound heterozygous SDHD gene mutations. The patient presented with early progressive encephalomyopathy due to compound heterozygous p.E69 K and p.*164Lext*3 SDHD mutations. Native polyacrylamide gel electrophoresis and western blotting demonstrated an impaired complex II assembly. Complementation of a patient cell line additionally supported the pathogenicity of the novel identified mutations in SDHD. CONCLUSIONS: This report describes the first case of isolated complex II deficiency due to recessive SDHD germline mutations. We therefore recommend screening for all SDH genes in isolated complex II deficiencies. It further emphasises the importance of appropriate genetic counselling to the family with regard to SDHD mutations and their role in tumorigenesis.

Loss of insulin-induced activation of TRPM6 magnesium channels results in impaired glucose tolerance during pregnancy.

Hypomagnesemia affects insulin resistance and is a risk factor for diabetes mellitus type 2 (DM2) and gestational diabetes mellitus (GDM). Two single nucleotide polymorphisms (SNPs) in the epithelial magnesium channel TRPM6 (V(1393)I, K(1584)E) were predicted to confer susceptibility for DM2. Here, we show using patch clamp analysis and total internal reflection fluorescence microscopy, that insulin stimulates TRPM6 activity via a phosphoinositide 3-kinase and Rac1-mediated elevation of cell surface expression of TRPM6. Interestingly, insulin failed to activate the genetic variants TRPM6(V(1393)I) and TRPM6(K(1584)E), which is likely due to the inability of the insulin signaling pathway to phosphorylate TRPM6(T(1391)) and TRPM6(S(1583)). Moreover, by measuring total glycosylated hemoglobin (TGH) in 997 pregnant women as a measure of glucose control, we demonstrate that TRPM6(V(1393)I) and TRPM6(K(1584)E) are associated with higher TGH and confer a higher likelihood of developing GDM. The impaired response of TRPM6(V(1393)I) and TRPM6(K(1584)E) to insulin represents a unique molecular pathway leading to GDM where the defect is located in TRPM6.

Predicting parameters of airway dynamics generated from inspiratory and expiratory plethysmographic airway loops, differentiating subtypes of chronic obstructive diseases.

BACKGROUND: The plethysmographic shift volume-flow loop (sRaw-loop) measured during tidal breathing allows the determination of several lung function parameters such as the effective specific airway resistance (sReff), calculated from the ratio of the integral of the resistive aerodynamic specific work of breathing (sWOB) and the integral of the corresponding flow-volume loop. However, computing the inspiratory and expiratory areas of the sRaw-loop separately permits the determination of further parameters of airway dynamics. Therefore, we aimed to define the discriminating diagnostic power of the inspiratory and expiratory sWOB (sWOBin, sWOBex), as well as of the inspiratory and expiratory sReff (sReff IN and sReff EX), for discriminating different functional phenotypes of chronic obstructive lung diseases. METHODS: Reference equations were obtained from measurement of different databases, incorporating 194 healthy subjects (35 children and 159 adults), and applied to a collective of 294 patients with chronic lung diseases (16 children with asthma, aged 6-16 years, and 278 adults, aged 17-92 years). For all measurements, the same type of plethysmograph was used (Jaeger Würzburg, Germany). RESULTS: By multilinear modelling, reference equations of sWOBin, sWOBex, sReff IN and sReff EX were derived. Apart from anthropometric indices, additional parameters such as tidal volume (VT), the respiratory drive (P0.1), measured by means of a mouth occlusion pressure measurement 100 ms after inspiration and the mean inspiratory flow (VT/TI) were found to be informative. The statistical approach to define reference equations for parameters of airway dynamics reveals the interrelationship between covariants of the actual breathing pattern and the control of breathing. CONCLUSIONS: We discovered that sWOBin, sWOBex, sReff IN and sReff EX are new discriminating target parameters, that differentiate much better between chronic obstructive diseases and their subtypes, especially between chronic obstructive pulmonary disease (COPD) and asthma-COPD overlap (ACO), thus strengthening the concept of precision medicine.

Assessment of functional diversities in patients with Asthma, COPD, Asthma-COPD overlap, and Cystic Fibrosis (CF).

The objectives of the present study were to evaluate the discriminating power of spirometric and plethysmographic lung function parameters to differenciate the diagnosis of asthma, ACO, COPD, and to define functional characteristics for more precise classification of obstructive lung diseases. From the databases of 4 centers, a total of 756 lung function tests (194 healthy subjects, 175 with asthma, 71 with ACO, 78 with COPD and 238 with CF) were collected, and gradients among combinations of target parameters from spirometry (forced expiratory volume one second: FEV1; FEV1/forced vital capacity: FEV1/FVC; forced expiratory flow between 25-75% FVC: FEF25-75), and plethysmography (effective, resistive airway resistance: sReff; aerodynamic work of breathing at rest: sWOB), separately for in- and expiration (sReffIN, sReffEX, sWOBin, sWOBex) as well as static lung volumes (total lung capacity: TLC; functional residual capacity: FRCpleth; residual volume: RV), the control of breathing (mouth occlusion pressure: P0.1; mean inspiratory flow: VT/TI; the inspiratory to total time ratio: TI/Ttot) and the inspiratory impedance (Zinpleth = P0.1/VT/TI) were explored. Linear discriminant analyses (LDA) were applied to identify discriminant functions and classification rules using recursive partitioning decision trees. LDA showed a high classification accuracy (sensitivity and specificity > 90%) for healthy subjects, COPD and CF. The accuracy dropped for asthma (~70%) and even more for ACO (~60%). The decision tree revealed that P0.1, sRtot, and VT/TI differentiate most between healthy and asthma (68.9%), COPD (82.1%), and CF (60.6%). Moreover, using sWOBex and Zinpleth ACO can be discriminated from asthma and COPD (60%). Thus, the functional complexity of obstructive lung diseases can be understood, if specific spirometric and plethysmographic parameters are used. Moreover, the newly described parameters of airway dynamics and the central control of breathing including Zinpleth may well serve as promising functional marker in the field of precision medicine.

Immunoreactive trypsinogen in healthy newborns and infants with cystic fibrosis.

OBJECTIVE: Newborn screening (NBS) for cystic fibrosis (CF) was introduced in Switzerland in 2011 based on an immunoreactive trypsinogen (IRT)-DNA-IRT protocol. CF diagnosis was confirmed by sweat test and/or genetics but remained inconclusive for some newborns (cystic fibrosis transmembrane conductance regulator related metabolic syndrome (CRMS)/CF screen positive, inconclusive diagnosis (CFSPID)). We aimed to (1) Describe IRT levels in healthy newborns in the first year of life and by gestational age (GA), and (2) Compare IRT at two time points between healthy newborns and newborns with CF and CRMS/CFSPID. DESIGN: Retrospective study. SETTING: National NBS database. PATIENTS: All children with an IRT measurement by heel prick test from 2011 to 2019. INTERVENTIONS: None. MAIN OUTCOME MEASURES: IRT values were extracted from the National NBS Laboratory, and clinical characteristics of positively screened children from the CF-NBS database. Second IRT assessment in positively screened children was usually performed after 18-24 days. We calculated internal IRT Z-Scores and multiples of the median to compare our results across different laboratory tools. RESULTS: Among 815 899 children; 232 were diagnosed with CF, of whom 36 had meconium ileus (MI); 27 had CRMS/CFSPID. Among all samples analysed, mean IRT Z-Scores were higher for newborns with GA <33 weeks and ≥43 weeks (all Z-Scores >0.11) compared with term babies (all Z-Scores ≤0.06). Repeated IRT Z-Scores after a median (IQR) of 19 (17-22) days remained high for infants with CF with or without MI but decreased for infants with CRMS/CFSPID. CONCLUSIONS: Measurement of a second IRT value can help distinguish between children with CRMS/CFSPID and CF, early in life.

qPCR-based mitochondrial DNA quantification: influence of template DNA fragmentation on accuracy.

Real-time PCR (qPCR) is the method of choice for quantification of mitochondrial DNA (mtDNA) by relative comparison of a nuclear to a mitochondrial locus. Quantitative abnormal mtDNA content is indicative of mitochondrial disorders and mostly confines in a tissue-specific manner. Thus handling of degradation-prone bioptic material is inevitable. We established a serial qPCR assay based on increasing amplicon size to measure degradation status of any DNA sample. Using this approach we can exclude erroneous mtDNA quantification due to degraded samples (e.g. long post-exicision time, autolytic processus, freeze-thaw cycles) and ensure abnormal DNA content measurements (e.g. depletion) in non-degraded patient material. By preparation of degraded DNA under controlled conditions using sonification and DNaseI digestion we show that erroneous quantification is due to the different preservation qualities of the nuclear and the mitochondrial genome. This disparate degradation of the two genomes results in over- or underestimation of mtDNA copy number in degraded samples. Moreover, as analysis of defined archival tissue would allow to precise the molecular pathomechanism of mitochondrial disorders presenting with abnormal mtDNA content, we compared fresh frozen (FF) with formalin-fixed paraffin-embedded (FFPE) skeletal muscle tissue of the same sample. By extrapolation of measured decay constants for nuclear DNA (λnDNA) and mtDNA (λmtDNA) we present an approach to possibly correct measurements in degraded samples in the future. To our knowledge this is the first time different degradation impact of the two genomes is demonstrated and which evaluates systematically the impact of DNA degradation on quantification of mtDNA copy number.

Duplication of the sodium channel gene cluster on 2q24 in children with early onset epilepsy.

PURPOSE: Sodium channel gene aberrations are associated with a wide range of seizure disorders, particularly Dravet syndrome. They usually consist of missense or truncating gene mutations or deletions. Duplications involving multiple genes encoding for different sodium channels are not widely known. This article summarizes the clinical, radiologic, and genetic features of patients with 2q24 duplication involving the sodium channel gene cluster. METHODS: A systematic review of the literature and report of two cases. KEY FINDINGS: Nine individuals with 2q24 duplication involving the sodium channel gene cluster are described (seven female, two male). All presented with severe seizures refractory to anticonvulsant drugs. Seizure onset was in the neonatal period in eight patients with SCN1A-involvement, in infancy in one patient with SCN2A and SCN3A, but no SCN1A involvement. Seizure activity decreased and eventually stopped at 5-20 months of age. Seizures recurred at the age of 3 years in the patient with SCN2A and SCN3A, but no SCN1A involvement. Eight patients had a poor neurodevelopmental outcome despite seizure freedom. SIGNIFICANCE: This article describes a distinct seizure disorder associated with a duplication of the sodium gene cluster on 2q24 described in otherwise healthy neonates and infants with severe, anticonvulsant refractory seizures and poor developmental outcome despite seizure freedom occurring at the age of 5-20 months.

Targeted next generation sequencing as a diagnostic tool in epileptic disorders.

PURPOSE: Epilepsies have a highly heterogeneous background with a strong genetic contribution. The variety of unspecific and overlapping syndromic and nonsyndromic phenotypes often hampers a clear clinical diagnosis and prevents straightforward genetic testing. Knowing the genetic basis of a patient's epilepsy can be valuable not only for diagnosis but also for guiding treatment and estimating recurrence risks. METHODS: To overcome these diagnostic restrictions, we composed a panel of genes for Next Generation Sequencing containing the most relevant epilepsy genes and covering the most relevant epilepsy phenotypes known so far. With this method, 265 genes were analyzed per patient in a single step. We evaluated this panel on a pilot cohort of 33 index patients with concise epilepsy phenotypes or with a severe but unspecific seizure disorder covering both sporadic and familial cases. KEY FINDINGS: We identified presumed disease-causing mutations in 16 of 33 patients comprising sequence alterations in frequently as well as in less commonly affected genes. The detected aberrations encompassed known and unknown point mutations (SCN1A p.R222X, p. E289V, p.379R, p.R393H; SCN2A p.V208E; STXBP1 p.R122X; KCNJ10 p.L68P, p.I129V; KCTD7 p.L108M; KCNQ3 p.P574S; ARHGEF9 p.R290H; SMS p.F58L; TPP1 p.Q278R, p.Q422H; MFSD8 p.T294K), a putative splice site mutation (SCN1A c.693A> p.T/P231P) and small deletions (SCN1A p.F1330Lfs3X [1 bp]; MFSD8 p.A138Dfs10X [7 bp]). All mutations have been confirmed by conventional Sanger sequencing and, where possible, validated by parental testing and segregation analysis. In three patients with either Dravet syndrome or myoclonic epilepsy, we detected SCN1A mutations (p.R222X, p.P231P, p.R393H), even though other laboratories had previously excluded aberrations of this gene by Sanger sequencing or high-resolution melting analysis. SIGNIFICANCE: We have developed a fast and cost-efficient diagnostic screening method to analyze the genetic basis of epilepsies. We were able to detect mutations in patients with clear and with unspecific epilepsy phenotypes, to uncover the genetic basis of many so far unresolved cases with epilepsy including mutation detection in cases in which previous conventional methods yielded falsely negative results. Our approach thus proved to be a powerful diagnostic tool that may contribute to collecting information on both common and unknown epileptic disorders and in delineating associated phenotypes of less frequently mutated genes.

Common CFTR haplotypes and susceptibility to chronic pancreatitis and congenital bilateral absence of the vas deferens.

CFTR mutations enhance susceptibility for idiopathic chronic pancreatitis (ICP) and congenital bilateral absence of the vas deferens (CBAVD); however, it is unknown why CFTR heterozygotes are at increased disease risk. We recently showed that common CFTR variants are associated with aberrantly spliced transcripts. Here, we genotyped for common CFTR variants and tested for associations in two ICP (ICP-A: 126 patients, 319 controls; ICP-B: 666 patients, 1,181 controls) and a CBAVD population (305 patients, 319 controls). Haplotype H10 (TG11-T7-470V) conferred protection (ICP-A: OR 0.19, P<0.0001; ICP-B: OR 0.78, P = 0.06; CBAVD OR 0.08, P<0.001), whereas haplotype H3 (TG10-T7-470M) increased disease risk (ICP-A: OR 8.34, P = 0.003; ICP-B: OR 1.88, P = 0.007; CBAVD: OR 5.67, P = 0.01). The risk of heterozygous CFTR mutations carriers for ICP (OR 2.44, P<0.001) and CBAVD (OR 14.73, P<0.001) was fully abrogated by the H10/H10 genotype. Similarly, ICP risk of heterozygous p.Asn34Ser SPINK1 mutation carriers (OR 10.34, P<0.001) was compensated by H10/H10. Thus, common CFTR haplotypes modulate ICP and CBAVD susceptibility alone and in heterozygous CFTR and p.Asn34Ser mutation carriers. Determination of these haplotypes helps to stratify carriers into high- and low-risk subjects, providing helpful information for genetic counseling.

Recurrent rearrangements of chromosome 1q21.1 and variable pediatric phenotypes.

BACKGROUND: Duplications and deletions in the human genome can cause disease or predispose persons to disease. Advances in technologies to detect these changes allow for the routine identification of submicroscopic imbalances in large numbers of patients. METHODS: We tested for the presence of microdeletions and microduplications at a specific region of chromosome 1q21.1 in two groups of patients with unexplained mental retardation, autism, or congenital anomalies and in unaffected persons. RESULTS: We identified 25 persons with a recurrent 1.35-Mb deletion within 1q21.1 from screening 5218 patients. The microdeletions had arisen de novo in eight patients, were inherited from a mildly affected parent in three patients, were inherited from an apparently unaffected parent in six patients, and were of unknown inheritance in eight patients. The deletion was absent in a series of 4737 control persons (P=1.1x10(-7)). We found considerable variability in the level of phenotypic expression of the microdeletion; phenotypes included mild-to-moderate mental retardation, microcephaly, cardiac abnormalities, and cataracts. The reciprocal duplication was enriched in nine children with mental retardation or autism spectrum disorder and other variable features (P=0.02). We identified three deletions and three duplications of the 1q21.1 region in an independent sample of 788 patients with mental retardation and congenital anomalies. CONCLUSIONS: We have identified recurrent molecular lesions that elude syndromic classification and whose disease manifestations must be considered in a broader context of development as opposed to being assigned to a specific disease. Clinical diagnosis in patients with these lesions may be most readily achieved on the basis of genotype rather than phenotype.

Detecting and resolving position-dependent temperature effects in real-time quantitative polymerase chain reaction.

Real-time quantitative polymerase chain reaction (qPCR) depends on precise temperature control of the sample during cycling. In the current study, we investigated how temperature variation in plate-based qPCR instruments influences qPCR results. Temperature variation was measured by amplicon melting analysis as a convenient means to assess well-to-well differences. Multiple technical replicates of several SYBR Green I-based qPCR assays allowed correlation of relative well temperature to quantification cycle. We found that inadequate template denaturation results in an inverse correlation and requires increasing the denaturation temperature, adding a DNA destabilizing agent, or pretreating with a restriction enzyme. In contrast, inadequate primer annealing results in a direct correlation and requires lowering the annealing temperature. Significant correlations were found in 18 of 25 assays. The critical nature of temperature-dependent effects was shown in a blinded study of 29 patients for the diagnosis of Prader-Willy and Angelman syndromes, where eight diagnoses were incorrect unless temperature-dependent effects were controlled. A method to detect temperature-dependent effects by pairwise comparisons of replicates in routine experiments is presented and applied. Systematic temperature errors in qPCR instruments can be recognized and their effects eliminated when high precision is required in quantitative genetic diagnostics and critical complementary DNA analyses.

Molecular and biochemical characterisation of a novel mutation in POLG associated with Alpers syndrome.

BACKGROUND: DNA polymerase γ (POLG) is the only known mitochondrial DNA (mtDNA) polymerase. It mediates mtDNA replication and base excision repair. Mutations in the POLG gene lead to reduction of functional mtDNA (mtDNA depletion and/or deletions) and are therefore predicted to result in defective oxidative phosphorylation (OXPHOS). Many mutations map to the polymerase and exonuclease domains of the enzyme and produce a broad clinical spectrum. The most frequent mutation p.A467T is localised in the linker region between these domains. In compound heterozygote patients the p.A467T mutation has been described to be associated amongst others with fatal childhood encephalopathy. These patients have a poorer survival rate compared to homozygotes. METHODS: mtDNA content in various tissues (fibroblasts, muscle and liver) was quantified using quantitative PCR (qPCR). OXPHOS activities in the same tissues were assessed using spectrophotometric methods and catalytic stain of BN-PAGE. RESULTS: We characterise a novel splice site mutation in POLG found in trans with the p.A467T mutation in a 3.5 years old boy with valproic acid induced acute liver failure (Alpers-Huttenlocher syndrome). These mutations result in a tissue specific depletion of the mtDNA which correlates with the OXPHOS-activities. CONCLUSIONS: mtDNA depletion can be expressed in a high tissue-specific manner and confirms the need to analyse primary tissue. Furthermore, POLG analysis optimises clinical management in the early stages of disease and reinforces the need for its evaluation before starting valproic acid treatment.

Intestinal current measurement for diagnostic classification of patients with questionable cystic fibrosis: validation and reference data.

BACKGROUND: In questionable cystic fibrosis (CF), mild or monosymptomatic phenotypes frequently cause diagnostic difficulties despite detailed algorithms. CF transmembrane conductance regulator (CFTR)-mediated ion transport can be studied ex vivo in rectal biopsies by intestinal current measurement (ICM). OBJECTIVES: To describe reference values and validate ICM for the diagnostic classification of questionable CF at all patient ages. METHODS: ICM was performed in 309 rectal biopsies from 130 infants, children and adults including patients with known pancreatic-insufficient (PI)-CF (n=34), pancreatic-sufficient (PS)-CF (n=7), patients with an unclear diagnosis with mild CF symptoms, intermediate sweat test and/or CFTR mutation screening (n=61) and healthy controls (n=28). ICM was correlated to sweat chloride, extensive CFTR genotype and transcript analysis in the diagnostic group. The results were compared with previous ICM data in subjects with CF, congenital bilateral absence of the vas deferens, heterozygotes and controls. RESULTS: The cumulative chloride secretory response of DeltaI(sc,carbachol), DeltaI(sc,cAMP/forskolin) and DeltaI(sc,histamine) was the best diagnostic ICM parameter (cut-off 34 muA/cm(2) between patients with known PS-CF and controls), differentiating patients with questionable CF into PS-CF (n=6) and 'CF unlikely' (n=55) groups. Extensive genotype analysis detected two mutations (40% disease-causing) in 100% of individuals classified as PS-CF compared with 1.8% in those classified as 'CF unlikely'. CONCLUSIONS: This comprehensive investigation of CFTR function and genotype underlines the diagnostic value of ICM, especially for confirmation of CF in the absence of two disease-causing CFTR mutations, exclusion of CF despite intermediate sweat test and age groups unsuitable for nasal potential difference measurements. ICM is an important tool for functional assessment in CFTR mutations of unknown clinical relevance.

Quantitative 1-step DNA methylation analysis with native genomic DNA as template.

BACKGROUND: DNA methylation analysis currently requires complex multistep procedures based on bisulfite conversion of unmethylated cytosines or on methylation-sensitive endonucleases. To facilitate DNA methylation analysis, we have developed a quantitative 1-step assay for DNA methylation analysis. METHODS: The assay is based on combining methylation-sensitive FastDigest(R) endonuclease digestion and quantitative real-time PCR (qPCR) in a single reaction. The first step consists of DNA digestion, followed by endonuclease inactivation and qPCR. The degree of DNA methylation is evaluated by comparing the quantification cycles of a reaction containing a methylation-sensitive endonuclease with the reaction of a sham mixture containing no endonuclease. Control reactions interrogating an unmethylated locus allow the detection and correction of artifacts caused by endonuclease inhibitors, while simultaneously permitting copy number assessment of the locus of interest. RESULTS: With our novel approach, we correctly diagnosed the imprinting disorders Prader-Willi syndrome and Angelman syndrome in 35 individuals by measuring methylation levels and copy numbers for the SNRPN (small nuclear ribonucleoprotein polypeptide N) promoter. We also demonstrated that the proposed correction model significantly (P < 0.05) increases the assay's accuracy with low-quality DNA, allowing analysis of DNA samples with decreased digestibility, as is often the case in retrospective studies. CONCLUSIONS: Our novel DNA methylation assay reduces both the hands-on time and errors caused by handling and pipetting and allows methylation analyses to be completed within 90 min after DNA extraction. Combined with its precision and reliability, these features make the assay well suited for diagnostic procedures as well as high-throughput analyses.

Long-term gas exchange characteristics as markers of deterioration in patients with cystic fibrosis.

BACKGROUND AND AIM: In patients with cystic fibrosis (CF) the architecture of the developing lungs and the ventilation of lung units are progressively affected, influencing intrapulmonary gas mixing and gas exchange. We examined the long-term course of blood gas measurements in relation to characteristics of lung function and the influence of different CFTR genotype upon this process. METHODS: Serial annual measurements of PaO2 and PaCO2 assessed in relation to lung function, providing functional residual capacity (FRCpleth), lung clearance index (LCI), trapped gas (VTG), airway resistance (sReff), and forced expiratory indices (FEV1, FEF50), were collected in 178 children (88 males; 90 females) with CF, over an age range of 5 to 18 years. Linear mixed model analysis and binary logistic regression analysis were used to define predominant lung function parameters influencing oxygenation and carbon dioxide elimination. RESULTS: PaO2 decreased linearly from age 5 to 18 years, and was mainly associated with FRCpleth, (p < 0.0001), FEV1 (p < 0.001), FEF50 (p < 0.002), and LCI (p < 0.002), indicating that oxygenation was associated with the degree of pulmonary hyperinflation, ventilation inhomogeneities and impeded airway function. PaCO2 showed a transitory phase of low PaCO2 values, mainly during the age range of 5 to 12 years. Both PaO2 and PaCO2 presented with different progression slopes within specific CFTR genotypes. CONCLUSION: In the long-term evaluation of gas exchange characteristics, an association with different lung function patterns was found and was closely related to specific genotypes. Early examination of blood gases may reveal hypocarbia, presumably reflecting compensatory mechanisms to improve oxygenation.