Johan T. den Dunnen

High‐Resolution Melting Analysis (HRMA)—More than just sequence variant screening

Transition of the double‐stranded DNA molecule to its two single strands, DNA denaturation or melting, has been used for many years to study DNA structure and composition. Recent technological advances have improved the potential of this technology, especially to detect variants in the DNA sequence. Sensitivity and specificity were increased significantly by the development of so‐called saturating DNA dyes and by improvements in the instrumentation to measure the melting behavior (improved temperature precision combined with increased measurements per time unit and drop in temperature). Melt analysis using these new instruments has been designated high‐resolution melting curve analysis (HRM or HRMA). Based on its ease of use, simplicity, flexibility, low cost, nondestructive nature, superb sensitivity, and specificity, HRMA is quickly becoming the tool of choice to screen patients for pathogenic variants. Here we will briefly discuss the latest developments in HRMA and review in particular other applications that have thus far received less attention, including presequence screening, single nucleotide polymorphism (SNP) typing, methylation analysis, quantification (copy number variants and mosaicism), an alternative to gel‐electrophoresis and clone characterization. Together, these diverse applications make HRMA a multipurpose technology and a standard tool that should be present in any laboratory studying nucleic acids. Hum Mutat 30:1–7, 2009.

Theoretic applicability of antisense-mediated exon skipping for Duchenne muscular dystrophy mutations†‡

Antisense‐mediated exon skipping aiming for reading frame restoration is currently a promising therapeutic application for Duchenne muscular dystrophy (DMD). This approach is mutation specific, but as the majority of DMD patients have deletions that cluster in hotspot regions, the skipping of a small number of exons is applicable to relatively large numbers of patients. To assess the actual applicability of the exon skipping approach, we here determined for deletions, duplications and point mutations reported in the Leiden DMD mutation database, which exon(s) should be skipped to restore the open reading frame. In theory, single and double exon skipping would be applicable to 79% of deletions, 91% of small mutations, and 73% of duplications, amounting to 83% of all DMD mutations. Exon 51 skipping, which is being tested in clinical trials, would be applicable to the largest group (13%) of all DMD patients. Further research is needed to determine the functionality of different in‐frame dystrophins and a number of hurdles has to be overcome before this approach can be applied clinically. Hum Mutat 0, 1–7, 2009.

Complex SNP-related sequence variation in segmental genome duplications.

There is uncertainty about the true nature of predicted single-nucleotide polymorphisms (SNPs) in segmental duplications (duplicons) and whether these markers genuinely exist at increased density as indicated in public databases. We explored these issues by genotyping 157 predicted SNPs in duplicons and control regions in normal diploid genomes and fully homozygous complete hydatidiform moles. Our data identified many true SNPs in duplicon regions and few paralogous sequence variants. Twenty-eight percent of the polymorphic duplicon sequences we tested involved multisite variation, a new type of polymorphism representing the sum of the signals from many individual duplicon copies that vary in sequence content due to duplication, deletion or gene conversion. Multisite variations can masquerade as normal SNPs when genotyped. Given that duplicons comprise at least 5% of the genome and many are yet to be annotated in the genome draft, effective strategies to identify multisite variation must be established and deployed.

Deletion and duplication screening in the DMD gene using MLPA.

We have designed a multiplex ligation-dependent probe amplification (MLPA) assay to simultaneously screen all 79 DMD gene exons for deletions and duplications in Duchenne and Becker muscular dystrophy (DMD/BMD) patients. We validated the assay by screening 123 unrelated patients from Serbia and Montenegro already screened using multiplex PCR. MLPA screening confirmed the presence of all previously detected deletions. In addition, we detected seven new deletions, nine duplications, one point mutation, and we were able to precisely determine the extent of all rearrangements. To facilitate MLPA-based screening in laboratories lacking specific equipment, we designed the assay such that it can also be performed using agarose gel analysis and ethidium bromide staining. The MLPA assay as described provides a simple and cheap method for deletion and duplication screening in DMD/BMD patients. The assay outperforms the Beggs and Chamberlain multiplex-PCR test, and should be considered as the method of choice for an initial DNA analysis of DMD/BMD patients.

The protein truncation test: A review

Only changes in the DNA sequence manifesting deleterious effects at a functional level provide disease-causing mutations. Consequently, mutation-scanning techniques applied on a protein level would be most informative. However, because of a lack of functional knowledge and powerful methods, most currently applied techniques try to resolve mutations at the DNA level. The protein truncation test (PTT) provides a rare exception, targeting mutations that generate shortened proteins, mainly premature translation termination. PTT has several attractive characteristics, including pinpointing the site of a mutation, good sensitivity, a low false-positive rate, and, more importantly, the near-exclusive highlighting of disease-causing mutations. In addition, PTT facilitated the detection of a new mutation type, i.e., a sequence change generating a hypermutable region surfacing in the RNA. The main technical problems are related to the fact that PTT generally uses an RNA target, including the difficulties that arise from the potential differential expression and stability of the transcripts derived from the two alleles present. The PTT has hardly evolved from the method originally described, with multiplexing and N-terminal protein tagging forming the only innovating modifications. To implement high-throughput screens using PTT, major improvements of the basic procedure will be required.Only changes in the DNA sequence manifesting deleterious effects at a functional level provide “disease‐causing” mutations. Consequently, mutation‐scanning techniques applied on a protein level would be most informative. However, because of a lack of functional knowledge and powerful methods, most currently applied techniques try to resolve mutations at the DNA level. The protein truncation test (PTT) provides a rare exception, targeting mutations that generate shortened proteins, mainly premature translation termination. PTT has several attractive characteristics, including pinpointing the site of a mutation, good sensitivity, a low false‐positive rate, and, more importantly, the near‐exclusive highlighting of disease‐causing mutations. In addition, PTT facilitated the detection of a new mutation type, i.e., a sequence change generating a hypermutable region surfacing in the RNA. The main technical problems are related to the fact that PTT generally uses an RNA target, including the difficulties that arise from the potential differential expression and stability of the transcripts derived from the two alleles present. The PTT has hardly evolved from the method originally described, with multiplexing and N‐terminal protein tagging forming the only innovating modifications. To implement high‐throughput screens using PTT, major improvements of the basic procedure will be required. Hum Mutat 14:95–102, 1999.

Protein truncation test (PTT) to rapidly screen the DMD gene for translation terminating mutations

We have developed a rapid and sensitive method to screen the Duchenne muscular dystrophy (DMD) mRNA for translation terminating mutations by a combination of RT-PCR (Reverse Transcription and Polymerase Chain Reaction) and in vitro transcription/translation applied to white blood cell mRNA. This technique was termed the protein truncation test (PTT). Here we demonstrate the detection of a point mutation in a DMD patient and his mother, a carrier. The PTT can also be used for carrier detection when no patient material is available, or in the case of spontaneous mutations. We developed a protocol to screen the total coding region of the DMD gene in 5-10 PTT reactions. Furthermore, PTT could be of diagnostic value in any disease where premature terminations form a substantial part of the total mutation spectrum.

Detecting copy number changes in genomic DNA - MAPH and MLPA

Publisher Summary Copy number changes, that is, deletions, duplications, and amplifications in genomic DNA are involved in many genetic diseases. However, detection of these rearrangements is rather difficult, mainly because the assay applied should be quantitative. This chapter describes multiplex amplifiable probe hybridization (MAPH) and multiplex ligation-dependent probe amplification (MLPA). These are the new methodologies developed specifically to detect copy number changes in many target sequences simultaneously. The MAPH and MLPA protocols are also outlined in the chapter. For both MAPH and MLPA, the methods of data analysis are effectively identical. The polymerase chain reaction (PCR) products are separated by electrophoresis and each probe is quantified with the relative amount of each product being proportional to the copy number of the locus being tested. The MAPH and MLPA have focused primarily on screening either single genes for exonic deletions and duplications or chromosomal regions for rearrangements. New MLPA/MAPH assays must be developed that will focus not only on specific genes but also on high-resolution analysis of chromosomal regions associated with a range of diseases.

Morphology of a human-derived YAC in yeast meiosis

In meiosis of human males DNA is packaged along pachytene chromosomes about 20 time more compactly than in meiosis of yeast. Nevertheless, a human-derived yeast artificial chromosome (YAC) shows the same degree of compaction of DNA as endogenous chromosomes in meiotic prophase nuclei of yeast. This suggests that in yeast meiosis, human and yeast DNA adopt a similar organization of chromatin along the pachytene chromosome cores. Therefore meiotic chromatin organization does not seem to be an inherent chromosomal property but is governed by the host-specific cellular environment. We suggest that there is a correlation between the less dense DNA packaging and the increased rate of recombination that has been reported for human-derived YACs as compared with human DNA in its natural environment.

Protein Truncation Test

The protein truncation test detects mutations at the protein level that lead to premature translation termination. The method has evolved considerably since is original publication in this manual. This thoroughly revised unit describes what is now the preferred method for performing the protein truncation test. Transcription and translation are performed in separate reactions; during translation, biotin‐labeled or N‐terminally tagged proteins are synthesized. The translation products are detected on immunoblots via chemiluminescence. An Alternate Protocol using coupled in vitro transcription/translation and radiolabeled proteins is also presented.

Using systematic nomenclature for CFTR variants: Improvement needed

The discovery of the gene involved in Cystic Fibrosis (CF) in 1989 was one of the first successes of the use of DNA markers and linkage analysis to determine the cause of a genetic disease. DNA-based assays were developed rapidly to screen the CFTR gene in thousands of patients, identifying the disease-causing variants and confirming diagnoses. At the time, no standards existed regarding how to report variants. As a consequence, the CF field developed their own legacy system. When world-wide standards were developed—what are now known as the “HGVS Recommendations for the Description of Sequence Variants” (Hum Mutat 8: 203-06, 1996; Hum Mutat 15: 1–12, 2000; www.hgvs.org/mutnomen)—the CF-field was reluctant to adopt them. The ultimate consequence was that newcomers in the field have had difficulty understanding the legacy descriptions used. Even worse, if one is not aware of this problem and takes the legacy descriptions at face value, one may end up at the wrong position in the CFTR gene. In this issue, Berwouts et al. (Hum Mutat 32: 1197–1203, 2011) evaluate laboratory reports from 217 participants in a CF study. They show that only 6% exclusively used the internationally accepted HGVS system,while 79% used traditional descriptions (only 15% used both). More disturbingly, 5% of the reports used nomenclature that was evaluated as being seriously incorrect and/or misleading, and only 24% gave the correct cDNA description and cited the nucleotide reference sequence used. The authors concluded that there is an urgent need for more consistent and correct description of the variants identified and recommend that all variants should be reported using both systems. A great help toward this end is that, since April 2010, the CFTR Gene Variant Database (http://www.genet.sickkids.on.ca/cftr/) started to list their variants using HGVS nomenclature. After reading the paper I wonder whether these issues have lead to diagnostic errors: it is hard to believe these have not occured. Although we hate them, we do need standards and we also should use them.