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Meet the Inventor of NEXTGENPCR [Interview Part 1 of 2]
Recently, Dana Pfister Sullivan, a product manager at Canon BioMedical, sat down with Gert de Vos, the inventor of the NEXTGENPCR instrument and Director at Molecular Biology Systems, B.V. (MBS). Gert has master’s degrees in biology and physics from Leiden University in the Netherlands. After teaching physics in Curaçao, Gert returned to the Netherlands and embarked on a career as an entrepreneur and inventor in the life sciences. 
 
DS: How did you come up with the idea for NEXTGENPCR?
 
Well, I was in a meeting focused on the detection of pathogens in milk. During the meeting, we were discussing gel electrophoresis and touched briefly on PCR. One person at the table mentioned PCR chips. I was aware of how PCR chips worked, specifically how the liquid or sample is pumped through different temperature zones, and also that they required nanoliter volumes rather than microliter volumes. I thought, wouldn’t it be great if we could do something similar — not only move the liquid around, but instead move the whole enclosure. That is when I got the idea — so then I went home, and we started on prototypes for what would become NEXTGENPCR. The first experiment we did was around ten minutes, and it worked brilliantly.  I said, “Wow, we have something here!” 
 
DS: How is NEXTGENPCR different than standard end-point PCR instruments?
 
In a typical PCR instrument there is an aluminum or silver block with cavities that holds the individual tubes of the PCR plate. These tubes are made of polypropylene, and they have a 200 to 300 micron wall to help hold their shape. The block is heated to the desired temperature, usually 95°C for denaturation, and then cooled down to the desired annealing temperature. What happens then is interesting; the cooling is done by Peltier elements. The moment you start to use a Peltier element for cooling, the other side gets hot; so, you have to cool a lot more than just the block. When the other side gets hot, the efficiency tumbles; so, while it is initially cooling at eight degrees per second, after a few seconds it slows down to two to three degrees  per second. Therefore, it takes some time for the instrument to achieve the correct annealing temperature. Also, the sample inside the tube relies on convection to reach the correct temperature. Rather than use the common Peltier technology, NEXTGENPCR uses temperature zones that eliminate the time it takes to heat and cool during a three-step PCR.  
 
DS:  How does NEXTGENPCR achieve such fast PCR?
 
The technology differs from typical thermocyclers in two ways that enable fast PCR. One, the instrument has three temperature zones, one each for denaturing, annealing, and extension. Two, we decrease the thickness of the well so that the liquid can reach the needed temperature quicker. Our plates have polypropylene enclosures where the PCR mix has been added inside. These enclosures are squeezed between two blocks at the correct temperature. So, if the sample has to go from 95°C to 60°C, the plate moves from the 95°C temperature zone to the 60°C temperature zone. The instrument then presses the blocks together, slightly deforming the enclosures, which allows the PCR reagents to not only mix instantly but also come to the desired temperature, essentially removing the ramp time.
 
DS: What was important to you when you were designing NEXTGENPCR? 
 
That is an excellent question. While developing the NEXTGENPCR instrument, I met an expert in the PCR market. I think he sold his first PCR instrument in 1984 or 1985. He told me this is potentially a market-disruptive technology, but not in its current shape. He noted the need to have a microplate format because then it will be compatible with all the current downstream applications. We started to work together because of what he said. We started making an instrument that accepts microplate formats. Our plate matches the rim of your typical microplate; in the middle there is a polypropylene sheet with either 96 or 384 wells. Our blocks are flat faced, so it does not matter what plate format you use in the instrument; there are no changes needed. 
 
In addition to the plate, it was important to achieve high-speed PCR while also considering those things important to a user such as how much space the instrument takes on the bench, how much energy is used, and, in the end, how much the device will cost. 
 
DS: How are the plates and consumables used with the NEXTGENPCR instrument different than standard 96- and 384-well plates?
 
The plates are the same size as a typical microplate because we use the same outside rim. They adhere to the standard defined by the Society for Laboratory Automation and Screening, so they fit in all robotics – both upstream and downstream. However, they differ in the center where we have inserted polypropylene film that has either 96 or 384 wells with a well thickness of 30 to 40 microns. The user’s current PCR mix is pipetted into the wells and then sealed with a heat sealer to close the wells. 
 
DS: You mentioned that energy usage was an important factor in the design. How is the instrument able to save energy?
 
That is simple to explain — since the instrument has three temperature zones, we don’t need to change the temperature of the blocks. This means we isolate the blocks in the denaturing zone and the extension zone, and in the annealing zone we are able to cool at a certain rate so that the instrument is capable of touchdown and stepdown experiments. Because of this isolation, the instrument is running at 150 to 170 watts rather than 800 to 1200 watts that a standard cycler uses. Then, if you take into account that a PCR experiment lasts between five and 20 times shorter with NEXTGENPCR, you can imagine the amount of energy you use. It could be up to 100 times less compared to other PCR instruments.
 

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This is only the first part of our interview with Gert. Come back next week for the conclusion of our interview. In the meantime, download the application note that explains how a 100 bp fragment was amplified in less than 2 minutes using NEXTGENPCR.
 

 

DOWNLOAD APPLICATION NOTE

 

 
Products mentioned are for Research Use Only.  Not for use in diagnostic procedures.
The NEXTGENPCR products are manufactured by Molecular Biology Systems, B.V.
All referenced product names, and other marks, are trademarks of their respective owner.

 

 

 

 

CYP2D6 Genetic Variations and Drug Metabolism
The CYP2D6 gene encodes for the cytochrome P450 2D6 enzyme, which is responsible for metabolizing as many as 25% of all prescribed drugs.1 Variation in the CYP2D6 gene is notoriously high from person to person and between different ethnic groups. The high degree of genetic variability coupled with the number of medications the enzyme metabolizes makes the gene one of the most important pharmacogenetic targets. 
 
The therapeutic effect of drugs that are transformed into their active form by the cytochrome P450 2D6 enzyme, such as codeine, can be drastically altered due to variations of the CYP2D6 gene. As an example, an individual with the ultrarapid metabolizer phenotype that is prescribed codeine would likely create too much of the active metabolite, morphine, and suffer toxic side effects. Inversely, someone with a poor or intermediate metabolizer phenotype may not receive the complete pain relief expected due to less morphine being biologically available. 
 
Metabolic phenotypes as a result of CYP2D6 variation fall into four categories: poor, intermediate, normal, and ultrarapid metabolizers. To assign a metabolic phenotype, pharmacogenetic researchers must run a genotyping panel as well as a copy number analysis of the CYP2D6 gene in order to generate an activity score. The first step in assigning an activity score requires knowing which mutations, designated as star alleles or "*alleles", exist in the sample. In addition,  deletions or duplications of the gene must be considered. There are over 100 different star alleles for the CYP2D6 gene, and some of the more common star alleles and their respective activity levels are shown below.
 

 

 Activity level  Activity score per allele  Common *alleles
 Normal  1  *1, *2
 Reduced  0.5  *9, *10, *17, *29, *41
 Nonfunctional  0  *3, *4, *5, *6, *7, *8, *14, *36


Poor, intermediate, normal, and ultrarapid metabolizer phenotypes are defined by the activity score values of each of the alleles present in the sample added together, and, if duplication or deletion of alleles exist, they must be taken into consideration. Examples of genotypes and how the associated activity score is calculated and used for the assignment to a metabolizer phenotype can be seen in the scale below. Note in the ultrarapid metabolizer example that the copy number of 2 for one allele adds its score twice.

Activity Score Scale

 

 

While the CYP2D6 gene and its functionality in biology can seem complex, genotyping samples for the common mutations can be done in any lab with an HRM-enabled thermocycler. The Novallele genotyping assays for CYP2D6 targets currently available, as well as homozygous mutant and wild-type characterized controls are listed in the table below. If there are additional CYP2D6 targets that you would like to investigate in your pharmacogenetic research, let us know by emailing us.   

 

 

 
 Star allele  dbSNP number  HGVS (c.)  GenBank number   Activity  Assay  Control set
 *2  rs16947  c.886C>T  2850C>T  Normal  40054  40422
 *2  rs1135840  c.1457G>C  4180G>C  Normal  40390  40730
 *2A  rs1080985  c.-1584C>G  -1584C>G  Normal  40237  40595
 *4  rs3892097  c.506-1G>A  1846G>A  Nonfunctional  40403  40743
 *6  rs5030655  c.454delT  1707delT  Nonfunctional  40129  40491
 *7  rs5030867  c.971A>C  2935A>C  Nonfunctional  40061  40429
 *8  rs5030865  c.505G>T  1758G>T  Nonfunctional  40378  40719
 *9  rs5030656  c.841_843delAAG  2615-2617delAAG  Reduced  40365  40707
 *10  rs1065852  c.100C>T  100C>T  Reduced  40327  40678
 *11  rs5030863  c.883G>C  883G>C  Nonfunctional  40405  40745
 *12  rs5030862  c.124G>A  124G>A  Nonfunctional  40796  40811
 *17  rs28371706  c.320C>T  1023C>T  Reduced  40156  40517
 *29  rs59421388  c.1012G>A  3183G>A  Reduced  40188  40546
 *41  rs28371725  c.985+39G>A  2988G>A  Reduced  40315  40669
 *52  rs28371733  c.3877G>A  3877G>A  Reduced  40404  40744
 

To learn more, register to download our infographic about pharmacogenetics.  

References:
1. Owen, R. P., Sangkuhl, K., Klein, T. E., & Altman, R. B. (2009). Cytochrome P450 2D6. Pharmacogenetics and genomics, 19(7), 559.

For research use only.  Not for use in diagnostic procedures.
Tables, graphs, and diagrams are for illustration purposes only. 
Nothing herein constitutes medical advice.

 

The APOE gene — Mutations Lead to Increased Risk of Alzheimer’s Disease and Recurrent Stroke

By definition, the APOE gene is responsible for the synthesis of the protein apolipoprotein E, a member of the lipoprotein family. This family of proteins is responsible for metabolism, movement, and clearance of fat-soluble vitamins and cholesterols across multiple body systems. However, when speaking about the APOE gene and its well characterized mutations, the conversation usually focuses around the gene's role in neurological diseases, especially Alzheimer’s disease. In large cohort studies, mutations in the APOE gene, specifically those that relate to the E4 allele, have been shown to increase the risk of developing Alzheimer’s disease1

Table 1. The table shows the genotype designations with the associated mutation and the Alzheimer’s disease risk correlation and allele frequency as determined by the cohort study.1

 Genotype  c.388T>C
 (rs429358)
 c.526C>T
 (rs7412)
 Disease risk   Allele frequency
 E2/E2  T:T  T:T  40% less likely  8.40%
 E2/E3  T:T  T:C  40% less likely  
 E2/E4  C:T  C:T  2.6 times more likely  
 E3/E3  T:T  C:C  Average risk  77.90%
 E3/E4  T:C  C:C  2-3 times more likely  
 E4/E4  C:C  C:C  12 times more likely  13.7%, 40% in AD population

 

While the concordance of allele frequency to the risk of disease has been thoroughly researched and reproduced, there are exceptions to this rule. Specific populations, such as Nigerians, have one of the highest E4 allele frequencies yet Alzheimer’s disease remains rare in this group.

 
While the risk of developing Alzheimer’s disease based on one’s APOE allele type is well characterized, recent research has shown that these mutations play a role well beyond disease prediction. A 2017 publication from the Munoz lab shows that females with Alzheimer’s disease that also carry homozygous E4 alleles have a higher likelihood of developing a more severe disease phenotype2. Females with this high-risk genotype were more likely to experience delusions and/or hallucinations in addition to the more common characteristics of Alzheimer’s disease. To learn more about this phenomenon, visit the ClinicalOMICS webinar page and register to watch a webinar sponsored by Canon BioMedical featuring Dr. David Munoz from the University of Toronto. 
 
Going beyond Alzheimer’s disease, the APOE  E4 genotype has also been shown to potentially affect the likelihood of recurrence in individuals who have suffered a stroke. A research study at the University of Edinburgh reported in The Lancet Neurology shows that using the APOE genotype in addition to more common prognostic tools, such as CT scans, could give physicians more confidence in segmenting out which individuals have a higher risk of having another stroke3. This study was quite limited in the number of participants, and the authors mention their desire to expand these investigations across multiple external sites in the future.   
 
If, after reading up on the APOE gene and its role in neurogenetics, your lab wants to conduct research on the APOE gene, we have products to help. Genotyping samples for the common APOE mutations can be done in any lab with an HRM-enabled thermocycler. The Novallele™ genotyping assays for APOE targets currently offered, as well as characterized controls, are listed in the table below.
 
 Assay name  Protein change  Assay catalog number  Control set catalog number
 APOE c.388T>C Novallele Genotyping Assay  Cys130Arg  40394  40734
 APOE c.526C>T Novallele Genotyping Assay  Arg176Cys  40395  40735
 
Novallele genotyping assays and reagents are For Research Use Only. Not for use in diagnostic procedures. 
 
References:
 
1. Liu, C. C., Kanekiyo, T., Xu, H., & Bu, G. (2013). Apolipoprotein E and Alzheimer disease: risk, mechanisms and therapy. Nature Reviews Neurology, 9(2), 106. 
2. Kim, J., E Fischer, C., A Schweizer, T., & G Munoz, D. (2017). Gender and Pathology-Specific Effect of Apolipoprotein E Genotype on Psychosis in Alzheimer’s Disease. Current Alzheimer Research, 14(8), 834-840. 
3. Rodrigues, M. A., Samarasekera, N., Lerpiniere, C., Humphreys, C., McCarron, M. O., White, P. M., & Smith, C. (2018). The Edinburgh CT and genetic diagnostic criteria for lobar intracerebral haemorrhage associated with cerebral amyloid angiopathy: model development and diagnostic test accuracy study. The Lancet Neurology.

 

Nothing herein constitutes medical advice.

 

 

 

A Guide to the Novallele™ HRM Analyzer
A helpful high-resolution (HRM) analysis software package should be easy to use, make the output simple to understand, and provide publication-quality results. The Novallele HRM Analyzer software offers an intuitive and efficient alternative to the often cryptic data analysis software that comes standard with many thermocyclers. 
 
The Novallele HRM Analyzer is free, cloud-based software that offers genotyping and copy number analysis through an intuitive user interface. Step one is to upload data as a text file by either dragging and dropping a file or using standard file uploading procedures. The software currently accepts text files that are unedited and exported from the Bio-Rad CFX, QuantStudio™, LightCycler® 480 System, or even the ring-based Rotor-Gene® Q. Sample names transfer with the data so hovering over the well positions with your mouse allows easy selection of no-template control (NTC) and allele control wells for review. Samples are selected or deselected by clicking, and entire rows can be added or removed as required for analysis. The commonly used normalization bars, T1,T2,T3, and T4, are shown on the raw melt window. The four-panel view lets you monitor the normalization on both the normalized melt curve window and the derivative melt curve window (Figure 1). If you struggle properly placing the normalization bars, using the derivative curves is an immense help. 
 
Novallele HRM Software screen shot

Figure 1. Sample analysis of QuantStudio 7 genotyping data of one row of samples minus NTCs as shown in the "Plate Selection" panel. The upper-left graph shows raw melt data with normalization bars. The upper-right graph shows normalized melt data. The lower-left graph shows derivative curves, and the lower-right graph shows the difference curves without the cluster analysis. 

The truly novel feature of the Novallele HRM Analyzer is the unique functionality of its clustering feature (Figure 2). Although other thermocycler software packages offer this feature, clustering is usually cumbersome to apply and rarely allows you to modify the cluster number or  label the unknown samples. In contrast, the Novallele HRM Analyzer clustering tool provides up and down arrows to change the cluster numbers, a simple checkbox to set the baseline value, and a dropdown list of labels for control samples as well as the unknowns that only require a click to assign a genotype or copy number. The graphs are easily saved with a right click and an especially nice feature is that the statistics behind the data can be downloaded to an excel sheet. Both the numerical values for the derivative curves and clusters are available for download; thus, all analyzed data can be saved in two different formats: graphical and numeric. 

Novallele HRM software screen shot

Figure 2. SMN1 copy number data from the RotorGene Q that was analyzed using the clustering feature. An example of downloadable statistics is shown in the upper-left table, hiding the raw melt data. The normalized melt curves and derivative curves are shown with the cluster analysis turned on as compared to Figure 1. The ring layout of the samples is in the upper-right table with the cluster identification in the table below.

In summary, the Novallele HRM Analyzer offers an easy-to-use, complete analysis method for both genotyping and copy number variation. This simple software includes all the common features found in thermocycler programs plus the extra benefits of numerical data tracking and adjustable clustering baselines. The Novallele HRM Analyzer doesn’t require any heroic data formatting, as a text file is sufficient, and the import works for four qPCR instruments. More detailed instructions and examples can be found in the online guide. Check out the Novallele HRM Analyzer for free today!

All referenced product names and other marks, are trademarks of their respective owners.
Nothing herein constitutes medical advice.

Genotyping Your Way to Better Medicine [Free Infographic]
With a shift toward precision medicine, physicians are no longer using a "one size fits all model" to treat patients. People do not respond to drugs in a uniform and predictable manner, and single nucleotide polymorphisms (SNPs) or other genomic insertions or deletions can alter a person’s metabolic pathways that function in the breakdown of a drug. 
 
Pharmacogenetics is the study of drug response variability in relation to specific genes. By combining pharmacology and genetics, pharmacogenetic researchers work to develop and understand safe, effective medications and dosages that are tailored according to an individual’s DNA makeup.
 
Register to download our infographic about pharmacogenetics, drug metabolism, and examples of gene influences on particular drugs.


Tables, graphs, and diagrams are for illustration purposes only. 
Nothing herein constitutes medical advice.

Updates in Newborn Screening of SMA across the U.S.A.
Across the U.S.A., state-sponsored pilot studies are underway to lay the groundwork for adding spinal muscular atrophy (SMA) to the nationwide Recommended Uniform Screening Panel (RUSP). In preparation for the possibility that SMA is added to the RUSP, states like New York, Utah, Massachusetts, and North Carolina have conducted or will begin studies to see how labs would integrate SMA testing into their newborn screening workflows.    
 
Taking screening efforts into their own hands, Missouri passed Senate Bill 50 in July 2017 that provides all newborns in the state will get screened for SMA along with more common, routinely tested diseases such as beta thalassemia, cystic fibrosis, and medium-chain acyl-CoA dehydrogenase (MCAD) deficiency. In late December 2017, Minnesota followed suit and became the second state to offer statewide newborn screening of SMA. Soon thereafter in late January 2018, Utah announced that every newborn in the state would be tested for SMA.
 
In addition to state-sponsored studies, industry organizations and advocacy groups representing various facets of SMA healthcare have also begun to ratchet up pressure to begin nationwide SMA screenings. In February 2017, the American College of Obstetricians and Gynecologists (ACOG) released their guidance recommending carrier screening for all women expand to include SMA-related deletions. In February 2017, Cure SMA launched the SMA Newborn Screening Coalition that will advocate at both the state and federal level in an effort to put SMA screening on the RUSP.
 
In late-breaking news, the Advisory Committee on Heritable Disorders in Newborns and Children testified on February 9, 2018 to the Department of Health and Human Services that they recommended adding SMA to the RUSP. 
 
One reason that things are moving so quickly to get states and the nation at large on board for newborn screening of SMA is the recently approved drug Spinraza®. When used on children diagnosed with SMA at a point when they are still pre-symptomatic, the drug has demonstrated, in some cases, that these children can achieve age-appropriate motor function milestones. Newborn screening for SMA will be integral in getting pre-symptomatic SMA-affected children on this drug within days of birth instead of waiting until symptoms arise months or even years later.
 
To learn more about therapy advances in treating SMA-affected individuals, check out our recent blog titled Developments in Spinal Muscular Atrophy (SMA) Therapeutics.

 

Nothing herein constitutes medical or legal advice.

 

 

6 Key Benefits of High-Resolution Melting

Since sequencing the human genome, scientists have identified specific mutations of particular genes that affect human health. Through in-depth investigation of genetic variants, scientists continue to identify and understand disease states and develop new drugs, such as targeted or personalized therapeutics. There are a variety of genetic variant detection methods such as complicated full-genome technologies, like next-generation sequencing, and tailored approaches, like hydrolysis probe PCR and high-resolution melting (HRM).

HRM is a simplified detection method that can provide a fast answer for known genetic mutations, such as single-nucleotide polymorphisms (SNPs) and indels. HRM detects fluorescence changes based on target melting - the exact fluorescence change varies depending on the DNA sequence and assay used. When compared to the reference DNA, a shift in melting temperature or a change in the melt curve shape indicates a genetic variation.

 

 

Here are six key benefits of HRM:

 

 

Accurate

HRM is highly accurate. In one study that looked at 35 publications, HRM was found to be a highly sensitive technology able to detect disease-associated mutations in humans. Overall, the summary sensitivity was 97.5%. HRM specificity showed considerable heterogeneity between studies.

 

Icon-Dollar-Sign-Blue

Cost effective

HRM is cheaper per sample when compared to other genotyping methods, such as Sanger sequencing, which makes HRM ideal for large-scale genotyping projects.

 

 

Fast

Requiring just an hour from sample to answer, HRM is quick because there is no need to process or separate PCR products. Once PCR is finished, an HRM step is performed, and analysis is completed using the free, web-based Novallele HRM Analyzer.

 

 

Minimal reagents

HRM generates little waste and only requires the reagents needed for a PCR reaction thus eliminating the need for HPLC solvents or denaturing gradient gel electrophoresis.

 

 

Icon-PCR-Machine-Blue

Simple

No special instrumentation is required for HRM. HRM analysis can be done on any thermocycler with real-time and fluorescent capture capabilities. A high-resolution melt step can be added to the end of a PCR run and analyzed immediately after completion.

 

 

Versatile

HRM can be used to detect SNPs and indels as well as methylation analysis and mutation scanning for copy number analysis.

 

Reference: Li B-S, Wang X-Y, Ma F-L, Jiang B, Song X-X, et al. (2011) Is High Resolution Melting Analysis (HRMA) Accurate for Detection of Human Disease Associated Mutations? A Meta Analysis. PLoS ONE 6(12): e28078.

 

 

Female-Lab-Tech-Viewing-Computer-Monitor

Want to learn more? Register to watch a
free HRM analysis webinar.

 

 

Developments in Spinal Muscular Atrophy (SMA) Therapeutics

 

In a our last blog, Genetic Basis of Spinal Muscular Atrophy (SMA), we described the lack of functional survival motor neuron (SMN) protein as the cause of SMA. Full-length transcripts that yield functional SMN protein are primarily synthesized by the SMN1 gene. Affected individuals have zero copies of the SMN1 gene and, therefore, lack enough functional SMN protein to protect their motor neurons from undergoing atrophy. The SMN2 gene acts as a backup but only produces a small amount of functional protein due to a splicing variation that limits production of the full-length transcript. Those affected by SMA but with high copy numbers of SMN2 can produce enough functional SMN protein to delay the onset of the disease or lessen disease severity. 

Numerous therapeutics in various states of development can be grouped into two main categories:

 

  • Therapies that address the genetic basis of the disease  These therapies attempt to either correct a mutated or replace an absent SMN1 gene or increase the amount of functional protein derived from the SMN2 gene.       
  • Therapies that protect the physical systems affected by the lack of SMN protein  These therapies work to either shield motor neurons normally affected by the lack of functional SMN proteins or prevent or restore muscle loss in SMA-affected systems.

The diagram below from the Cure SMA organization outlines current SMA drugs in development.

SMA drugs in development

 

You’ll notice that one therapeutic, Spinraza® (nusinersen), developed by Biogen/Ionis gained FDA approval in December of 2016. Spinraza works by correcting the splicing error associated with the SMN2 gene that reduces the amount of full-length SMN transcript. The drug accomplishes this by using an antisense oligonucleotide that corrects the alternate splicing present in SMN2 RNA. By correcting the splicing, the drug effectively converts the altered SMN2 mRNA to SMN1 mRNA, thus increasing the amount of full-length transcript available to create the functional SMN protein.

Next in the SMA blog series, we’ll look at the changing landscape of newborn screening for SMA. 

Nothing herein constitutes medical advice.

Image used with permission from Cure SMA (curesma.org).

Genetic Basis of Spinal Muscular Atrophy (SMA)

 

Individuals with spinal muscular atrophy (SMA) suffer from a nerve-wasting disorder that ultimately makes their skeletal muscles atrophy, leading to impaired movement and possibly death. The nerves affected in this disease are neuronal cells in proximity to the spinal cord. These nerves undergo degradation due to a lack of the survival motor neuron (SMN) protein.

The disease typically only affects individuals that have a homozygous deletion of the SMN1 gene (approximately 95% of cases); however, other cases of SMA can arise from having mutations within the SMN1 gene that effectively make the gene nonfunctional. SMA is an autosomal recessive inherited disease, meaning both of an individual’s parents must be carriers or affected by the disease to pass the disease on to their offspring. Generally, carriers of the disease have only one functional copy of the SMN1 gene. It is important to note that, in some cases, carriers of the disease have been found to have two copies of the SMN1 gene. These individuals have a duplication of SMN1 on one of their chromosomes and a deletion on the other chromosome. These silent carriers, or (2+0) carriers, have two functional copies of SMN1 but also the potential to give their offspring a chromosome with no SMN1 gene.

With an incidence rate of one in 10,000 live births, approximately 400 new cases of SMA occur every year in the U.S.A. There are multiple subtypes of SMA, and they are defined by the time of disease onset and phenotype of the disease. Details on the different SMA types can be seen in the table below. 

 

SMA type

 

Disease name

Time of onset

Phenotype

SMA Type 0

Not applicable

Birth

Born with a need for mechanical respiratory assistance. Life expectancy is a few weeks.

 

SMAType 1

Werdnig–Hoffmann disease

0–6 months

Disease presents early in life as babies are unable to sit without support. Breathing is also labored and may require mechanical ventilation. Life expectancy varies based on the disease phenotype and can range from a couple years to adolescence, potentially into adulthood.

 

SMA Type 2

Dubowitz syndrome

6-18 months

Affected children are able to sit on their own but are never able to stand or walk. Life expectancy is reduced, but most live into adulthood. 

 

SMA Type 3

Kugelberg–Welander disease

>12 months

Children affected can usually begin to walk without supports but may lose this ability at some stage. Life expectancy is close to normal.

 

SMA Type 4

Not Applicable

Adulthood

Usually sets in after 20 years of age and can affect the extremities of the body, usually requiring use of a wheelchair. Life expectancy is unaffected.

 

 

 

Disease severity can range tremendously as seen in the table above. One of the things that researchers and physicians are looking at from a prognostic value is the number of copies of the highly similar SMN2 gene. The SMN2 gene can make a limited amount of functional SMN protein, but those with only the SMN2 gene cannot produce enough functional SMN protein to fully ward off the disease. The reason the SMN2 gene produces less SMN protein is that the SMN2 gene varies by one nucleotide from the SMN1 gene, which yields a slightly different splicing pattern in the messenger RNA. Instead of all transcripts yielding normal, full-length, and functional SMN protein, SMN2 transcripts usually create degraded SMN proteins with only a small percentage being functional. 

 

  SMN1 vs SMN2 alternate splicing

 

SMN1 SMN2 Splicing Translation

 

Need to determine SMN1 or SMN2 copy numbers as part of your research? Download our Novallele copy number flyer to learn more about our simple and accurate method. 

Stay tuned for the next blog in the series. Next is an update on therapeutic successes and drugs in development for SMA-affected individuals.

 

Products mentioned are for Research Use Only. Not for use in diagnostic procedures.

Nothing herein constitutes medical advice.

Figure used with permission from Butchbach, M. E., & Burghes, A. H. (2004). Perspectives on models of spinal muscular atrophy for drug discovery. Drug Discovery Today: Disease Models1(2), 151-156.

Beginner’s Guide to Mitochondrial DNA Mutations [Free Infographic]

The mitochondria in our cells are responsible for creating 90% of the energy needed to support our day to day activities. When mutations occur, mitochondria can fail to produce enough energy, which can cause mitochondrial disorders or diseases.

Mitochondrial DNA (mtDNA) is more than just a circle of 37 genes. While there are two copies of nuclear DNA per cell, there are greater than 1000 copies of mtDNA. Mutation rates of mtDNA are 10–17 fold higher compared to those in nuclear DNA, and more than 250 pathogenic mtDNA mutations have been identified.

So how do mitochondrial mutations occur, and what happens when they do? Register to download our free infographic to learn more and explore  detection methods for mutations in the mitochondrial genome.  

The GBA Gene — Causing Gaucher Disease and Now Linked to Parkinson's Disease

Gaucher disease, which is caused by mutations in the GBA gene, is more common in the Ashkenazi Jewish population. An interesting link between Gaucher disease and a higher incidence of Parkinson’s disease has been tracked in the past with many hypotheses as to why such a link would exist.

 

One hypothesis is that since mutations in the GBA gene reduce the function of the glucocerebrosidase (GCase) protein, these GBA mutations can lead to a buildup of alpha-synuclein. When alpha-synuclein is present at a high enough concentration, it will clump and turn into Lewy bodies. Lewy bodies are seen in the brain cells of those affected by Parkinson's disease.

Another interesting finding reported by Creese et al. (1) is that GBA mutations can increase the risk of developing more severe disease pathology when it comes to Parkinson’s disease. A meta-analysis in the report uncovered that certain GBA mutations yielded a 2.4-fold increase in dementia, 1.8-fold increase in psychosis, and 2.2-fold increase in depression among patients with Parkinson’s disease. 

Knowing the possible connections of the GBA gene to Parkinson’s disease, pharmaceutical companies have targeted this gene and its effect on the GCase protein. One of these companies is Sanofi with the drug GZ/SAR402671. The drug aims to aid in the breakdown of lipids, including glucosylceramide, which would help prevent buildup of alpha-synuclein, even without a fully-functional GCase protein. 

Want to learn more about disease pathology and the risks associated with common mutations in the APOE and GBA genes? A webinar titled Genetics of Neurodegenerative Disorders - How Mutations Affect Disease Pathology is available on demand. Use the link below to visit the ClinicalOMICS webinar page and register to watch.

 

 

Reference:

1. Creese, B., Bell, E., Johar, I., Francis, P., Ballard, C., & Aarsland, D. (2017). Glucocerebrosidase mutations and neuropsychiatric phenotypes in Parkinson's disease and Lewy body dementias: Review and meta-analyses. American Journal of Medical Genetics Part B: Neuropsychiatric Genetics 177(2), 232–241.

Nothing herein constitutes medical advice.

5 Tips for Successful CRISPR Screening [Free Infographic]

CRISPR technology has revolutionized research, and gene modifications now make it much easier to create cell models. Genotyping using high-resolution melting (HRM) can simplify your workflow by rapidly confirming the creation of single-nucleotide polymorphisms (SNPs) and small indels. Use these five tips to help successfully screen your CRISPR clones:

 

  1. Optimize transfection efficiency so that the maximum number of cells receive Cas9 + gRNA.
    Pro Tip: Use GFP Cas9 to monitor transfection.
  2. Select a fast, easy genotyping technology, such as HRM analysis, suitable or detecting your edit with high specificity.
  3. Confirm successful edits at the first split and dilute.
    Pro Tip: Don’t stop at the first screen if only heterozygous mutants are detected.
  4. Split and dilute wells after subsequent clonal enrichment. Analyze each well using HRM.
  5. Discard all unedited pools.
     

If present, homozygous mutants are detected using HRM. Register to download the full infographic and learn more about screening CRISPR clones.

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