Aicardi–Goutières syndrome is an inflammatory disease occurring due to mutations in any of TREX1, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, ADAR or IFIH1. We report on 374 patients from 299 families with mutations in these seven genes. Most patients conformed to one of two fairly stereotyped clinical profiles; either exhibiting an in utero disease-onset (74 patients; 22.8% of all patients where data were available), or a post-natal presentation, usually within the first year of life (223 patients; 68.6%), characterized by a sub-acute encephalopathy and a loss of previously acquired skills. Other clinically distinct phenotypes were also observed; particularly, bilateral striatal necrosis (13 patients; 3.6%) and non-syndromic spastic paraparesis (12 patients; 3.4%). We recorded 69 deaths (19.3% of patients with follow-up data). Of 285 patients for whom data were available, 210 (73.7%) were profoundly disabled, with no useful motor, speech and intellectual function. Chilblains, glaucoma, hypothyroidism, cardiomyopathy, intracerebral vasculitis, peripheral neuropathy, bowel inflammation and systemic lupus erythematosus were seen frequently enough to be confirmed as real associations with the Aicardi-Goutieres syndrome phenotype. We observed a robust relationship between mutations in all seven genes with increased type I interferon activity in cerebrospinal fluid and serum, and the increased expression of interferon-stimulated gene transcripts in peripheral blood. We recorded a positive correlation between the level of cerebrospinal fluid interferon activity assayed within one year of disease presentation and the degree of subsequent disability. Interferon-stimulated gene transcripts remained high in most patients, indicating an ongoing disease process. On the basis of substantial morbidity and mortality, our data highlight the urgent need to define coherent treatment strategies for the phenotypes associated with mutations in the Aicardi–Goutières syndrome-related genes. Our findings also make it clear that a window of therapeutic opportunity exists relevant to the majority of affected patients and indicate that the assessment of type I interferon activity might serve as a useful biomarker in future clinical trials.

Copy number changes and CpG methylation of various genes are hallmarks of tumor development but are not yet widely used in diagnostic settings. The recently developed multiplex ligation-dependent probe amplification (MLPA) method has increased the possibilities for multiplex detection of gene copy number aberrations in a routine laboratory. Here we describe a novel robust method: the methylation-specific MLPA (MS-MLPA) that can detect changes in both CpG methylation as well as copy number of up to 40 chromosomal sequences in a simple reaction. In MS-MLPA, the ligation of MLPA probe oligonucleotides is combined with digestion of the genomic DNA–probe hybrid complexes with methylation-sensitive endonucleases. Digestion of the genomic DNA–probe complex, rather than double-stranded genomic DNA, allowed the use of DNA derived from the formalin treated paraffin-embedded tissue samples, enabling retrospective studies. To validate this novel method, we used MS-MLPA to detect aberrant methylation in DNA samples of patients with Prader–Willy syndrome, Angelman syndrome or acute myeloid leukemia.

Context:P450 oxidoreductase deficiency (PORD) is a unique congenital adrenal hyperplasia variant that manifests with glucocorticoid deficiency, disordered sex development (DSD), and skeletal malformations. No comprehensive data on genotype-phenotype correlations in Caucasian patients are available.Objective:The objective of the study was to establish genotype-phenotype correlations in a large PORD cohort.Design:The design of the study was the clinical, biochemical, and genetic assessment including multiplex ligation-dependent probe amplification (MLPA) in 30 PORD patients from 11 countries.Results:We identified 23 P450 oxidoreductase (POR) mutations (14 novel) including an exonic deletion and a partial duplication detected by MLPA. Only 22% of unrelated patients carried homozygous POR mutations. p.A287P was the most common mutation (43% of unrelated alleles); no other hot spot was identified. Urinary steroid profiling showed characteristic PORD metabolomes with variable impairment of 17α-hydroxylase and 21-hydroxylase. Short cosyntropin testing revealed adrenal insufficiency in 89%. DSD was present in 15 of 18 46,XX and seven of 12 46,XY individuals. Homozygosity for p.A287P was invariably associated with 46,XX DSD but normal genitalia in 46,XY individuals. The majority of patients with mild to moderate skeletal malformations, assessed by a novel scoring system, were compound heterozygous for missense mutations, whereas nearly all patients with severe malformations carried a major loss-of-function defect on one of the affected alleles.Conclusions:We report clinical, biochemical, and genetic findings in a large PORD cohort and show that MLPA is a useful addition to POR mutation analysis. Homozygosity for the most frequent mutation in Caucasians, p.A287P, allows for prediction of genital phenotype and moderate malformations. Adrenal insufficiency is frequent, easily overlooked, but readily detected by cosyntropin testing.

The presence of chromosome-specific low-copy repeats (LCRs) predisposes chromosome 22 to deletions and duplications. The current diagnostic procedure for detecting aberrations at 22q11.2 is chromosomal analysis coupled with fluorescence in situ hybridization (FISH) or PCR-based multiplex ligation dependent probe amplification (MLPA). However, there are copy number variations (CNVs) in 22q11.2 that are only detected by high-resolution platforms such as array comparative genomic hybridization (aCGH). We report on development of a high-definition MLPA (MLPA-HD) 22q11 kit that detects copy number changes at 37 loci on the long arm of chromosome 22. These include the 3-Mb region commonly deleted in DiGeorge/velocardiofacial syndrome (DGS/ VCFS), the cat eye syndrome (CES) region, and more distal regions in 22q11 that have recently been shown to be deleted. We have used this MLPA-HD probe set to analyze 363 previously wellcharacterized samples with a variety of different rearrangements at 22q11 and demonstrate that it can detect copy number alterations with high sensitivity and specificity. In addition to detection of the common recurrent deletions associated with DGS/VCFS, variant and novel chromosome 22 aberrations have been detected. These include duplications within as well as deletions distal to this region. Further, the MLPA-HD detects deletion endpoint differences between patients with the common 3-Mb deletion. The MLPA-HD kit is proposed as a cost effective alternative to the currently available detection methods for individuals with features of the 22q11 aberrations. In patients with the relevant phenotypic characteristics, this MLPA-HD probe set could replace FISH for the clinical diagnosis of 22q11.2 deletions and duplications.

Dihydropyrimidine dehydrogenase (DPD) is the initial enzyme acting in the catabolism of the widely used antineoplastic agent 5-fluorouracil (5FU). DPD deficiency is known to cause a potentially lethal toxicity following administration of 5FU. Here, we report novel genetic mechanisms underlying DPD deficiency in patients presenting with grade III/IV 5FU-associated toxicity. In one patient a genomic DPYD deletion of exons 21–23 was observed. In five patients a deep intronic mutation c.1129–5923C>G was identified creating a cryptic splice donor site. As a consequence, a 44 bp fragment corresponding to nucleotides c.1129–5967 to c.1129–5924 of intron 10 was inserted in the mature DPD mRNA. The deleterious c.1129–5923C>G mutation proved to be in cis with three intronic polymorphisms (c.483 + 18G>A, c.959–51T>G, c.680 + 139G>A) and the synonymous mutation c.1236G>A of a previously identified haplotype. Retrospective analysis of 203 cancer patients showed that the c.1129–5923C>G mutation was significantly enriched in patients with severe 5FU-associated toxicity (9.1%) compared to patients without toxicity (2.2%). In addition, a high prevalence was observed for the c.1129–5923C>G mutation in the normal Dutch (2.6%) and German (3.3%) population. Our study demonstrates that a genomic deletion affecting DPYD and a deep intronic mutation affecting pre-mRNA splicing can cause severe 5FU-associated toxicity. We conclude that screening for DPD deficiency should include a search for genomic rearrangements and aberrant splicing.Electronic supplementary materialThe online version of this article (doi:10.1007/s00439-010-0879-3) contains supplementary material, which is available to authorized users.