Johannes Zschocke

A survey of tools for variant analysis of next-generation genome sequencing data

Recent advances in genome sequencing technologies provide unprecedented opportunities to characterize individual genomic landscapes and identify mutations relevant for diagnosis and therapy. Specifically, whole-exome sequencing using next-generation sequencing (NGS) technologies is gaining popularity in the human genetics community due to the moderate costs, manageable data amounts and straightforward interpretation of analysis results. While whole-exome and, in the near future, whole-genome sequencing are becoming commodities, data analysis still poses significant challenges and led to the development of a plethora of tools supporting specific parts of the analysis workflow or providing a complete solution. Here, we surveyed 205 tools for whole-genome/whole-exome sequencing data analysis supporting five distinct analytical steps: quality assessment, alignment, variant identification, variant annotation and visualization. We report an overview of the functionality, features and specific requirements of the individual tools. We then selected 32 programs for variant identification, variant annotation and visualization, which were subjected to hands-on evaluation using four data sets: one set of exome data from two patients with a rare disease for testing identification of germline mutations, two cancer data sets for testing variant callers for somatic mutations, copy number variations and structural variations, and one semi-synthetic data set for testing identification of copy number variations. Our comprehensive survey and evaluation of NGS tools provides a valuable guideline for human geneticists working on Mendelian disorders, complex diseases and cancers.

A European multicenter study of phenylalanine hydroxylase deficiency: Classification of 105 mutations and a general system for genotype-based prediction of metabolic phenotype

Phenylketonuria (PKU) and mild hyperphenylalaninemia (MHP) are allelic disorders caused by mutations in the gene encoding phenylalanine hydroxylase (PAH). Previous studies have suggested that the highly variable metabolic phenotypes of PAH deficiency correlate with PAH genotypes. We identified both causative mutations in 686 patients from seven European centers. On the basis of the phenotypic characteristics of 297 functionally hemizygous patients, 105 of the mutations were assigned to one of four arbitrary phenotype categories. We proposed and tested a simple model for correlation between genotype and phenotypic outcome. The observed phenotype matched the predicted phenotype in 79% of the cases, and in only 5 of 184 patients was the observed phenotype more than one category away from that expected. Among the seven contributing centers, the proportion of patients for whom the observed phenotype did not match the predicted phenotype was 4%-23% (P<.0001), suggesting that differences in methods used for mutation detection or phenotype classification may account for a considerable proportion of genotype-phenotype inconsistencies. Our data indicate that the PAH-mutation genotype is the main determinant of metabolic phenotype in most patients with PAH deficiency. In the present study, the classification of 105 PAH mutations may allow the prediction of the biochemical phenotype in >10,000 genotypes, which may be useful for the management of hyperphenylalaninemia in newborns.

Natural history, outcome, and treatment efficacy in children and adults with glutaryl-CoA dehydrogenase deficiency.

Glutaryl-CoA dehydrogenase (GCDH) deficiency is a rare inborn disorder of l-lysine, l-hydroxylysine, and l-tryptophan metabolism complicated by striatal damage during acute encephalopathic crises. Three decades after its description, the natural history and how to treat this disorder are still incompletely understood. To study which variables influenced the outcome, we conducted an international cross-sectional study in 35 metabolic centers. Our main outcome measures were onset and neurologic sequelae of acute encephalopathic crises. A total of 279 patients (160 male, 119 female) were included who were diagnosed clinically after clinical presentation (n = 218) or presymptomatically by neonatal screening (n = 23), high-risk screening (n = 24), or macrocephaly (n = 14). Most symptomatic patients (n = 185) had encephalopathic crises, characteristically resulting in bilateral striatal damage and dystonia, secondary complications, and reduced life expectancy. First crises usually occurred during infancy (95% by age 2 y); the oldest age at which a repeat crisis was reported was 70 mo. In a few patients, neurologic disease developed without a reported crisis. Differences in the diagnostic criteria and therapeutic protocols for patients with GCDH deficiency resulted in a huge variability in the outcome worldwide. Recursive partitioning demonstrated that timely diagnosis in neurologically asymptomatic patients followed by treatment with l-carnitine and a lysine-restricted diet was the best predictor of good outcome, whereas treatment efficacy was low in patients diagnosed after the onset of neurologic disease. Notably, the biochemical phenotype did not predict the clinical phenotype. Our study proves GCDH deficiency to be a treatable disorder and a good candidate for neonatal screening.

MLPA analysis for the detection of deletions, duplications and complex rearrangements in the dystrophin gene: potential and pitfalls

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are common X-chromosomal recessive disorders caused by mutations in the dystrophin gene. Using the novel multiplex ligation-dependent probe amplification (MLPA) method we performed retrospective and prospective analyses in a total of 193 individuals. Deletions or duplications were identified in 14 out of 90 families previously tested negative by multiplex PCR or FISH analysis. Partially incorrect results were subsequently identified in two families: the loss of exon 38 signal in one case was due to a p.Q1802X nonsense mutation, whilst in another patient an apparent deletion of exon 37 (coinciding with a duplication of exons 46–53) was caused by a p.R1735C polymorphism. In one case we found a complex rearrangement involving a duplication of two regions: dupEX45–48 and dupEX54–55. We conclude that MLPA is a highly sensitive and rapid alternative to multiplex PCR. It can be used on blood samples, chorionic villi and paraffin-embedded tissue. The ease of detection of duplications and the application for female carrier analysis are clearly the main advantages of the method. However, apparent single exon deletions detected by MLPA should be checked by an independent method. Complex rearrangements such as double mutations on the same allele are rare.

Mild trimethylaminuria caused by common variants in FM03 gene

Mild to transient trimethylaminuria is caused by common variants in the FMO3 gene leading to greatly reduced enzyme activity in vivo. FMO3 deficiency may have clinical relevance well beyond unpleasant body odour.

Diagnosis and management of glutaric aciduria type I

Glutaric aciduria type I (GA1) is a preventable cause of acute brain damage in early childhood, leading to a severe dystonic-dyskinetic disorder that is similar to cerebral palsy and ranges from extreme hypotonia to choreoathetosis to rigidity with spasticity. Degeneration of the putamen and caudate typically occurs between 6 and 18 months of age and is probably linked to changes in metabolic demand caused by normal maturational changes and superimposed catabolic stress. Recognition of this biochemical disorder before the brain has been injured is essential to outcome. Diagnosis depends upon the recognition of relatively nonspecific physical findings such as hypotonia, irritability and macrocephaly, and on performance of urine organic acid quantification by gas chromatography–mass spectrometry or selective searches of urine or blood specimens by tandem mass spectrometry for glutarylcarnitine. The diagnosis may also be suggested by characteristic findings on neuroimaging. In selected patients diagnosis can only be reached by enzyme assay. Specific current management by the authors of this paper includes pharmacological doses of L-carnitine, as well as dietary protein restriction. Metabolic decompensation must be treated aggressively to avoid permanent brain damage. Multicentre studies are needed to establish best methods of diagnosis and optimal therapy of this disorder.

Use of guidelines improves the neurological outcome in glutaric aciduria type I

To evaluate the effect of treatment according to current evidence‐based recommendations on the neurological outcome of patients with glutaric aciduria type I (GA‐I).

Mutation analysis in glutaric aciduria type I

Glutaric aciduria type 1 (GA1), resulting from the genetic deficiency of glutaryl-CoA dehydrogenase (GDH), is a relatively common cause of acute metabolic brain damage in infants. Encephalopathic crises may be prevented by carnitine supplementation and diet, but diagnosis can be difficult as some patients do not show the typical excretion of large amounts of glutaric and 3-hydroxyglutaric acids in the urine. We present a rapid and efficient denaturing gradient gel electrophoresis (DGGE) method for the identification of mutations in the glutaryl-CoA dehydrogenase (GCDH) gene that may be used for the molecular diagnosis of GA1 in a routine setting. Using this technique, we identified mutations on both alleles in 48 patients with confirmed GDH deficiency, while no mutations were detected in other patients with clinical suspicion of GA1 but normal enzyme studies. There was a total of 38 different mutations; 27 mutations were found in single patients only, and 21 mutations have not been previously reported. Fourteen mutations involved hypermutable CpG sites. The commonest GA1 mutation in Europeans is R402W, which accounts for almost 40% of alleles in patients of German origin.GCDH gene haplotypes were determined through the analysis of polymorphic markers in all families, and three CpG mutations were associated with different haplotypes, possibly reflecting independent recurrence. The high sensitivity of the DGGE method allows the rapid and cost efficient diagnosis of GA1 in instances where enzyme analyses are not available or feasible, despite the marked heterogeneity of the disease.

Pyridoxal 5 '-phosphate may be curative in early-onset epileptic encephalopathy

SummaryNeonatal epileptic encephalopathy can be caused by inborn errors of metabolism. These conditions are often unresponsive to treatment with conventional antiepileptic drugs. Six children with pyridox(am)ine-5′-phosphate oxidase (PNPO) deficiency presented with neonatal epileptic encephalopathy. Two were treated with pyridoxal 5′-phosphate (PLP) within the first month of life and showed normal development or moderate psychomotor retardation thereafter. Four children with late or no treatment died or showed severe mental handicap. All of the children showed atypical biochemical findings. Prompt treatment with PLP in all neonates and infants with epileptic encephalopathy should become mandatory, permitting normal development in at least some of those affected with PNPO deficiency.

Progressive Infantile Neurodegeneration Caused by 2-Methyl-3-Hydroxybutyryl-CoA Dehydrogenase Deficiency: A Novel Inborn Error of Branched-Chain Fatty Acid and Isoleucine Metabolism

We report a novel inborn error of metabolism identified in a child with an unusual neurodegenerative disease. The male patient was born at term and recovered well from a postnatal episode of metabolic decompensation and lactic acidosis. Psychomotor development in the first year of life was only moderately delayed. After 14 mo of age, there was progressive loss of mental and motor skills; at 2 years of age, he was severely retarded with marked restlessness, choreoathetoid movements, absence of directed hand movements, marked hypotonia and little reaction to external stimuli. Notable laboratory findings included marked elevations of urinary 2-methyl-3-hydroxybutyrate and tiglylglycine without elevation of 2-methylacetoacetate, mild elevations of lactate in CSF and blood, and a slightly abnormal acylcarnitine profile. These abnormalities became more apparent after isoleucine challenge. Enzyme studies showed absent activity of 2-methyl-3-hydroxybutyryl-CoA dehydrogenase (MHBD) in the mitochondrial oxidation of 2-methyl branched-chain fatty acids and isoleucine. Under dietary isoleucine restriction, neurologic symptoms stabilized over the next 7 months.