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.
Mutations in the PKHD1 gene are responsible for autosomal recessive polycystic kidney disease (ARPKD). Using exon scanning by denaturing high-performance liquid chromatography (dHPLC) or bidirectional sequencing of all exons constituting the longest open reading frame, the mutation detection rate reaches approximately 82% and minor lesion mutations include truncating, splice, and missense mutations.The main aim of this study was to screen ARPKD patients in whom only one pathogenic PKHD1 mutation was identified after bidirectional sequencing of the longest open reading frame, for gene copy number alterations by employing multiplex ligation-dependent probe amplification complemented with quantitative real-time polymerase chain reaction.Sixteen ARPKD probands were studied in whom only one clearly pathogenic mutation was previously identified. One patient with a suspected homozygous deletion of the exons 1-37 was also included in this cohort. Three distinct PKHD1 germ-line deletions were identified. Two of these deletions encompassed multiple exons of PKHD1 extending far beyond the 5' and 3' untranslated regions of the gene, and spanning at least 170 and 470 kb, respectively. The third 3.7 kb intragenic deletion affected only exons 20-21 of the PKHD1 gene. Thus, this is the first report presenting analysis of the entire PKHD1 longest open reading frame for gene deletions/duplications in a select cohort of ARPKD patients, in whom previously only one mutation was identified after bidirectional sequencing of the entire longest open reading frame.The data indicate that multiplex ligation-dependent probe amplification is a sensitive and rapid method to identify PKHD1 deletions. Our study demonstrates that dosage analysis will increase the PKHD1 mutation detection rate and should be performed as a complementary assay in patients suspected to have ARPKD in the absence of two clear pathogenic mutations.
Spinal Muscular Atrophy (SMA) is a disorder characterized by the degeneration of motor neurons in the spinal cord, leading to muscular atrophy. In the majority of cases, SMA is caused by the homozygous absence of the SMN1 gene. The disease severity of SMA is strongly influenced by the copy number of the closely related SMN2 gene. In addition, an SMN variant lacking exons 7 and 8 has been reported in 8% and 23% of healthy Swedish and Spanish individuals respectively. We tested 1255 samples from the 1000 Genomes Project using a new version of the multiplex ligation-dependent probe amplification (MLPA) P021 probemix that covers each SMN exon. The SMN variant lacking exons 7 and 8 was present in up to 20% of individuals in several Caucasian populations, while being almost completely absent in various Asian and African populations. This SMN1/2Δ7–8 variant appears to be derived from an ancient deletion event as the deletion size is identical in 99% of samples tested. The average total copy number of SMN1, SMN2 and the SMN1/2Δ7–8 variant combined was remarkably comparable in all populations tested, ranging from 3.64 in Asian to 3.75 in African samples.
Legius syndrome, is a recently identified autosomal dominant disorder caused by loss of function mutations in the SPRED1 gene, with individuals mainly presenting with multiple café-au-lait macules (CALM), freckling and macrocephaly. So far, only SPRED1 point mutations have been identified as the cause of this syndrome. To determine if copy number changes (CNCs) are a cause of Legius syndrome, we have used a Multiplex Ligation-dependent Probe Amplification (MLPA) assay covering all SPRED1 exons in a cohort of 510 NF1-negative patients presenting with multiple CALMs with or without freckling, but no other NF1 diagnostic signs. Four different deletions were identified by MLPA and confirmed by quantitative PCR, reverse transcriptase PCR and/or array CGH: a deletion of exon 1 and the SPRED1 promoter region in a proband and two first-degree relatives; a deletion of the entire SPRED1 gene in a sporadic patient; a deletion of exon 2-6 in a proband and her father; and an ∼6.6 Mb deletion on chromosome 15 that spans SPRED1 in a sporadic patient. Deletions account for ∼10% of the 40 detected SPRED1 mutations in this cohort of 510 individuals. These results indicate the need for dosage analysis to complement sequencing-based SPRED1 mutation analyses.
Aim: To develop a SULT1A1 multiplex ligation-dependent probe amplification assay and to investigate multi-ethnic copy number variant frequencies. Methods: A novel multiplex ligation-dependent probe amplification assay was developed and tested on 472 African–American, Asian, Caucasian, Hispanic and Ashkenazi Jewish individuals. Results: The frequencies of atypical total copy number (i.e., greater or less than two) were 38.7% for Hispanics, 38.9% for Ashkenazi Jewish, 43.2% for Caucasians, 53.6% for Asians and 64.1% for African–Americans. Heterozygous SULT1A1 deletion carriers (slow sulfators) were most common among Caucasians (8.4%), whereas African–Americans had the highest frequencies of three or more copies (rapid sulfators; 60.9%). Conclusion: Different ethnic and racial populations have varying degrees of SULT1A1-mediated sulfation activity, which warrants further research and that may have utility for drug response prediction among SULT1A1-metabolized medications.
The core phenotype of Kleefstra syndrome (KS) is characterized by intellectual disability, childhood hypotonia, and a characteristic facial appearance. This can be caused by either submicroscopic 9q34 deletions or loss of function mutations of the EHMT1 gene. Remarkably, in three patients with a clinical suspicion of KS, molecular cytogenetic analysis revealed an interstitial 9q34 microdeletion proximal to the coding region of the EHMT1 gene based on the NM_ 024757.3 transcript. Because we found a mono-allelic EHMT1 transcript suggestive for haploinsufficiency of EHMT1 in two of these patients tested, we hypothesized that a deletion of regulatory elements or so far unknown coding sequences in the 5' region of the EHMT1 gene, might result in a phenotype compatible with KS. We further characterized the molecular content of deletions proximal to the transcript NM_ 024757.3 and confirmed presence of a novel predicted open reading frame comprising 27 coding exons (NM_ 024757.4). Further analysis showed that all three deletions included the presumed novel first exon of the EHMT1 gene. Subsequent testing of 75 individuals without previously detectable EHMT1 aberrations showed one additional case with a deletion comprising only this 5' part of the gene. These results have important implications for the genetic screening of KS and for studies of the functional significance of EHMT1.
Genotyping of HLA-DQ2.2, HLA-DQ2.5, and HLA-DQ8 is important in celiac disease (CD). The absence of these three genotypes has a strong negative predictive value.We designed multiplex ligation-dependent probe amplification (MLPA) for the combined detection of HLA-DQ2.2, HLA-DQ2.5, and HLA-DQ8. The MLPA probe mix was validated against a set of 59 samples characterized by conventional techniques.The MLPA assay genotyped all 59 samples correctly when compared to the results obtained by PCR-SSCP/HD or PCR-SSO and PCR-SSP.The MLPA assay provides a reliable single-reaction analysis of the CD risk genotypes HLA-DQ2.2, HLA-DQ2.5, and HLA-DQ8 allowing for stratification or exclusion of disease risk.
The 17q21.31 deletion syndrome phenotype can be caused by either chromosome deletions or point mutations in the KANSL1 gene. To date, about 60 subjects with chromosome deletion and 4 subjects with point mutation in KANSL1 have been reported. Prevalence of chromosome deletions compared with point mutations, genotype–phenotype correlations and phenotypic variability have yet to be fully clarified.
Methods
We report genotype–phenotype correlations in 27 novel subjects with 17q21.31 deletion and in 5 subjects with KANSL1 point mutation, 3 of whom were not previously reported.
Results
The prevalence of chromosome deletion and KANSL1 mutation was 83% and 17%, respectively. All patients had similar clinical features, with the exception of macrocephaly, which was detected in 24% of patients with the deletion and 60% of those with the point mutation, and congenital heart disease, which was limited to 35% of patients with the deletion. A remarkable phenotypic variability was observed in both categories, mainly with respect to the severity of ID. Cognitive function was within normal parameters in one patient in each group. Craniosynostosis, subependymal heterotopia and optic nerve hypoplasia represent new component manifestations.
Conclusions
In KANSL1 haploinsufficiency syndrome, chromosome deletions are greatly prevalent compared with KANSL1 mutations. The latter are sufficient in causing the full clinical phenotype. The degree of intellectual disability (ID) appears to be milder than expected in a considerable number of subjects with either chromosome deletion or KANSL1 mutation. Striking clinical criteria for enrolling patients into KANSL1 analysis include speech delay, distinctive facial dysmorphism, macrocephaly and friendly behaviour.
Abstract Background: Celiac disease (CD) is an inflammatory disorder of the small intestine induced by gluten ingestion. CD has a strong genetic association with human leukocyte antigen (HLA)-DQ2.5 and HLA-DQ8. The absence of HLA-DQ2.5 and HLA-DQ8 has a strong negative predictive value for CD. Genetic screening of HLA-DQ2.5 and HLA-DQ8 in patients at risk is of great value. Methods: We designed, developed, and validated a multiplex assay based on multiplex ligation-dependent probe amplification (MLPA) technology, allowing the simultaneous detection of DQA1*05-DQB1*02 , encoding HLA-DQ2.5, and DQA1*03-DQB1*03:02 , encoding HLA-DQ8. The amplified products were separated and identified using capillary electrophoresis. Results: When compared with a polymerase chain reaction followed by single-strand conformation polymorphism/ heteroduplex analysis, one discrepancy was found. Sequencing analysis showed that the developed MLPA assay result was correct. Furthermore, we demonstrated that the MLPA method is able to distinguish between the heterozygote and homozygote expression of HLA-DQ2.5 or HLA-DQ8. Conclusions: This study shows that it is possible to rapidly and accurately screen for the absence of HLA-DQ2.5 and HLA-DQ8 using MLPA, excluding patients at risk for CD for further serological or histological follow-up. In addition, MLPA might be an accurate tool to screen for other specific HLA types in the context of disease association in a diagnostic laboratory setting.
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