qPCR diagnostics in the BioVariance lab

The abbreviation „qPCR“ stands for “quantitative polymerase chain reaction”. SARS-CoV-2 is a so-called RNA virus, that means that its genome consists of RNA and not of DNA like in animals or humans. An organism can be clearly identified by analyzing its genome, which is the main point of the PCR analysis. A pharyngeal sample contains RNA in the form of potential viruses as well as DNA in the form of human mucosal cells. Therefore, the contained RNA is isolated and purified before the actual PCR. This RNA is then converted to DNA using the enzyme reverse transcriptase, because the PCR can only work with DNA. After this process the actual PCR starts: The process being called “amplification”, characteristic segments of the produced DNA are multiplied using the enzyme polymerase. BioVariance targets 3 specific DNA segments, while 2 of them belong to genes found in SARS-CoV-2: The RdRP gene, which exists only in SARS-CoV-2 specifically. And the E gene, which occurs generally in the subgenus Sarbecovirus, whereto also SARS-CoV-2 belongs. The evidence of the RdRP gene alone would be enough to identify a sample as positive, but BioVariance presupposes the detection of both virus genes for the generation of a positive report to avoid wrong-positive results. The third targeted segment, the RNAse P gene, is a human gene, which serves as an internal control factor for checking whether the sample contains enough biological material to avoid wrong-negative results.

Evaluation of the PCR analysis

  But how can we check whether the amplification is successfull? Using a special PCR cycler (see Fig. 1), fluorescence markers are embedded into the multiplied DNA sequences during the PCR. Every target sequence is associated with another fluorescence colour. The more DNA is produced by the polymerase, the stronger is the fluorescence of the sample in the real time measurement. For generating an unequivocal result – no matter if negative or positive – the fluorescence signal of the human RNAse P gene must be sufficient. If there is additional fluorescence of both virus genes, the sample will be marked positive. The Ct value serves as a scale unit for the number of existing genes. It describes the number of DNA multiplication steps being necessary to differentiate the fluorescence signal clearly from the so-called background noise and to generate a reliable test signal. The lower the Ct value is, the higher is the number of existing target genes and thus the number of virus particles. Conversely, a high Ct value shows a low concentration of virus particles. During the PCR, the fluorescence signal arises until it peaks after about 1 hour. After this peak it flattens out because the added components for DNA multiplication are used up. This process creates the typical sigmoidal curve of the fluorescence (see Fig. 2).  

  But even the best procedure is subjected to limitations. In about 1 % of cases the result of the PCR is not unequivocal, because the sample material is insufficient or the fluorescence signal is too low, for example. In these cases, the PCR is repeated to gain a clear result.

Identification of virus variants using qPCR

In the BioVariance lab all positive samples additionally undergo a special variant PCR analysis to identify the respective SARS-CoV-2 variant, targeting several SARS-CoV-2 specific gene segments containing the yet known mutations. These analyses revealed that in the last weeks the former virus, the so-called wildtype, was gradually replaced by the British variant B.1.1.7, today called “Alpha“, in the county of Tirschenreuth. Furthermore, positive samples are also sent to a partner lab of BioVariance for further variant analyses, so that new and yet unknown mutations can be identified.

Screening of schools using pooled gargle samples

In May 2021, BioVariance has introduced a new pooling procedure, with several schools in the county of Tirschenreuth participating in the context of a study. This procedure does not need the pupils to take a nose or pharyngeal sample, but only to gargle with some tap water in the morning at home. At school, these gargle samples are “pooled” per class, that means mixed in one sample cup (see Fig. 3). Thus, only one PCR analysis has to be done for each class. Only if a pool sample is positive, the gargle samples of the referring pupils will be analyzed individually. This process allows great savings of lab resources while maintaining a high quality of results.  

Quality management in the BioVariance lab

BioVariance regularly faces external evaluations to ensure the quality and safety of their PCR analyses. For these so-called ring trials, the lab receives several unknown samples from a central office. Only if all samples are identified correctly as negative or positive, the lab will pass the trial and gain a certificate. In May 2021, BioVariance has passed again the external test from the European Society for External Quality Assessment (ESfEQA). Further, all incoming samples are treated under sterile conditions and conforming with strict hygienic regulations in the BioVariance lab, avoiding the contamination of the workstations and a possible infection of employees (see Fig. 4).  

Speed of PCR diagnostics in the BioVariance lab

In Germany, it usually takes 24-48 hours until a tested person receives the PCR result. In the BioVariance lab, results can be produced in less than 3 hours after the samples arrived in the lab. So 99 % of the tested people receive their result at the same day. Two special components allow BioVariance to reach this speed: On the one hand, BioVariance uses an optimized procedure for the RNA extraction which guarantees high efficiency, precision and safety. On the other hand, the IT specialists of BioVariance developed a purpose-built software called TubeLab enabling the digital monitoring of the lab processes, which clearly accelerated and facilitated the handling of samples. Using TubeLab for a transparent tracing and documentation, the BioVariance lab meets the highest requirements. Besides, the partner lab doing the further sequencing of the positive samples also uses TubeLab for monitoring their PCR analyses since April 2020. BioVariance also increased its personnel and expanded the lab team by 2 technical lab employees and 2 student assistants from the scientific area.

Strategic success in Tirschenreuth

With the help of the administrative district office and the health office, the administrative barriers for the establishment of the diagnostic lab as from 26.02.2021 in Waldsassen could be removed within a very short time. Short ways between the test station and the lab are indispensable for rapid testing. In the best case, both stations are located in the same building like in Waldsassen. And thanks to the BRK, 4 permanent and several mobile test stations could be established. More than 40.000 pharyngeal samples from people living in the county of Tirschenreuth have been analyzed since the commissioning of the lab. Using the highly efficient PCR diagnostics, infected people could be identified and quarantined within a few hours. This fact also had a strong positive influence on the local incidence. In February 2021 when the lab was commissioned, the 7-day-incidence was more than 350 infections per 100.000 inhabitants in the county of Tirschenreuth. Now, 15 weeks later, the region is the first county in Bavaria to show a 7-day-incidence of 0. The effective collaboration of the administrative district office, the health office, the BRK and the BioVariance lab as well as the consistent endurance of the population made it possible to signficantly reduce new infections and to finally conquer the high incidence in the county of Tirschenreuth.

Future orientation of the BioVariance lab

BioVariance aims to expand the application possibilities in the lab and to integrate the diagnostics of cancer diseases into their portfolio, additionally to the existing diagnostics of infectious diseases. The company has always focused on the personalization of cancer diseases by sequencing tumors and using the homemade software OncoVariant. This pioneering step allows the selection of the most suitable therapy for cancer patients individually to achieve the greatest possible effectiveness. Further, BioVariance plans to take advantage of the mass spectrometry, which allows the measurement of metabolic products for example.   Conntact person: Kerstin Hammer

Der Beitrag BioVariance Express-PCR-Lab conquers incidence in the county of Tirschenreuth erschien zuerst auf BioVariance - data-driven diagnostics.

Generally, drugs are only allowed to be prescribed when special agencies have tested them on safety, effectiveness and quality and have approved them on corresponding indications. The prescription of drugs without considering these facts is called “off-label use”. ^[1] The relevant aspects for a drug approval are not only the application area and route of administration, but also dosage, moment of intake and the duration of the treatment. Off-label use is not permitted officially, but requires special care. General risks like decreased effectiveness or side effects are harder to conceive due to a lack of study data on effectiveness and safety. ^[1,2] In new-born, off-label use concerns up to 90 % of all patients, while it’s about 50 % in children and about 25 % in adults. ^[3,4] Insurances may take over the accruing costs only in these particular cases: ^[1]

• Severe or life-threatening diseases
• Reasonable assumption that the prescribed drug will induce a therapy success
• Other therapy options are not available

Off-label use in children and adolescents

Off-label use is common especially in paediatrics, because clinical studies regarding drug intake in young patients are often lacking. Children and adolescents differ in body size and functions from each other and from adults. Thus, drugs which are originally approved for adults can cause overdosage or severe side effects in children. ^[2,5] But despite that, ill children have to be treated sufficiently and so the treating physicians are forced to act in this legal grey area. When children are included in clinical studies, there have to be considered special ethical and legal facts. For the participation in a clinical study the consent of the participant (children with an age of 8 years, dependent on development) and his legal representative has to be recorded. In contrast to adult studies, a remuneration is not allowed in studies with children. Further, single indications will probably never get a full approval in rare diseases, because the number of patients in relevant age groups is too low to conduct clinical studies. ^[5] To improve the current situation of healthcare for children and adolescents regarding approved drugs, the European Union implemented the regulation (EC) Nr. 1901/2006 in the year 2007. The aim was to maintain and extend the number of drugs approved for the treatment of young patients under 18 years. ^[6] A Pediatric Commission (PDCO) was established for the valuation of paediatric concepts of drug companies. Since the implementation of the regulation clinical studies were increasingly conducted, about 100 new indications were authorized and more than 270 new drugs were approved for the treatment of children (see Fig. 1). ^[2,7]  

  In the year 2016, most of the off-label prescriptions were recognized for patients between 1 and 13 years. The highest number was listed for patients in the age group 6-13 years. Off-label use in dosage, indication and duration of the treatment were highest among all patients (see Fig. 2). ^[8]  

Advancement through digitalization

With the paediatric off-label use being necessary way too often, several countries implemented special projects dealing with the development of web-based platforms. These platforms are meant to support physicians in treating their young patients. Well-known examples are „Kinderformularium“ from the Netherlands, the „British National Formulary for Children“ from Great Britain, „Kispiportal“ from Switzerland and „Drugbase“ from Germany. ^[9,10,11,12]

• „Kinderformularium“ is an open access paediatric database for physicians and pharmacists. It currently includes information about dosage, application form, side effects, contraindications and corresponding references for more than 730 drugs. ^[9]
• The „British National Formulary for Children“ was established for health professionals by the Royal College of Paediatrics and Child Health (RCPCH) and the Neonatal and Paediatric Pharmacists Group (NPPG). It deals with prescriptions and care for young patients and offers information about more than 900 drugs and 200 treatment options. ^[10]
• „Kispiportal“ is the official database of the Universitäts-Kinderspital in Zurich. After entering patient’s data, the correct dosage of a certain drug is calculated automatically. ^[11]
• „Drugbase“ offered by Wissenschaftliche Verlagsgesellschaft Stuttgart includes several reference works in the fields of medicine and pharmacy, for example the Red List, drug profiles and paediatric dosage charts. ^[12]

These comprehensive and easily accessible databases will improve therapies for young patients by avoiding side effects and unnecessary expenses and also by shortening the duration of the treatments.  

Contact Person: Kerstin Hammer Sources

Der Beitrag Off-Label Use in Paediatrics erschien zuerst auf BioVariance - data-driven diagnostics.

This blog post answers the questions of what rare diseases are in general, in which medical areas they’re involved, how many people are affected and the number of medications, which are available now.

Definition and characteristics

This collective term stands for many different diseases which don’t share a consistent pathology.^1,2 All these mulit-systemic diseases have in common that they’re chronic, progressive, degenerative and life-threatening.² The quality of life is usually limited and there’s no effective cure.²

In Europe, a disease is defined as an rare disease if there aren’t more than 5 in 10,000 patients suffering from them.¹ Due to the lack of information regarding the cause and course of the disease, 40% of all patients receive a misdiagnosis and thus no effective therapy.³ On average, it takes five years to get a correct diagnosis.³

Worldwide there’re about 8,000 rare diseases.³ However, due to the better understanding in human genetics, the number of known diseases is increasing annually.^4,5

Cause

8 out of 10 diseases are triggered by a genetic defect.^1,5,6 Due to the genetic anomalies a whole cascade of complaints are released, often leaving the source uncovered.³ However, meanwhile there’re clear assignments for 6,084 rare diseases, which are associated with around 4,000 genes (as of 2016).⁵ Rare diseases can also be caused by infections or environmental influences, often it’s also an interaction of several factors.⁶

Medical areas

Rare diseases are involved in many medical areas:

• Cancer: acute lymphocytic leukemia,⁴ renal cell cancer,⁷ hepatocellular carcinoma,⁷ carcinoma of the adrenal cortex⁷
• Cardiovascular diseases: pulmonary hypertension¹
• Metabolic disorders: cystic fibrosis, phenylketonuria, Fabry disease⁴
• Neurological disorders: Neuronal ceroid lipofuscinosis,¹ narcolepsy⁷
• Infectious diseases: tuberculosis,⁴
• Autoimmune diseases: lupus, Sjögren syndrome¹

Selected samples for rare diseases

An example for a rare disease is narcolepsy with cataplexy among adults. About 185,00 people are affected in Europe.⁷ It’s a lifelong neurological disorder in which the sleep wake rhythm is disturbed.⁸ This is why it’s also called sleeping sickness.⁸ One of the symptoms is catalepsy.⁹ Patients suffer from short episodes of muscle failure, such as relaxation of the facial muscles or a complete collapse of the entire body.⁹ In contrast to epilepsy, those affected are fully conscious.⁹ The reason for this disease is a combination of genetic predispositions and environmental influences.¹⁰ Recent studies have revealed that there’s a lack of the neurotransmitter hypcretin in patients suffering from narcolepsy.¹⁰ This substance transmits signals from nerve cell to nerve cell and is involved in the regulation of the sleep-wake cycle.¹⁰ Misregulation of the immune response is thought to destroy the cells producing hypcretin.¹⁰ Narcolepsy occurs in 4 out of 10,000 people.¹¹

Another example is acromegaly,⁷ also called gigantism,¹² which is responsible for changing the external appearance, metabolism and internal organs.¹³ The disease is caused by a tumor in the Hypophyse leading to an overproduction of growth hormones.^12,13 Stimulation of IGF-1 in the liver promotes the growth of cartilage, bones, muscles and connective tissue.¹³ Among other things, this leads to an enlargement of hands, feet and tongue as well as facial features.¹³

Epidemiology

Worldwide there’re 410 million people affected by rare diseases.⁶ 30 million are living in Europe,^5,6 corresponding to 6-7% of the European population.⁴ At least half of all affected people are children.^1,2,5 30% die before the age of five years.²

Comparision of rare and frequent diseases¹

Orphan drugs

For a long time there was no research in the field of rare diseases, that’s why they are also called orphan diseases. The drugs for the treatment are referred as orphan drugs.¹

Challenges in the development

In order to be able to develop effective drugs for certain diseases, it’s necessary to understand all related molecular processes leading to the disease.¹ However, one characteristic of rare diseases is the lack of knowledge regarding the progression of the disease.¹ Thus, basic research is needed in this field in order to be able to find and produce appropriate drugs.¹ Furthermore, drug testing in clinical trials is difficult because often there aren’t enough patients for the study due to the rarity of the disease.¹ Additionally this process is aggravated by the fact that many people don’t know about their disease because of the lack of knowledge of the physicians.¹

Orphan drug status

In order to acquire an orphan drug status, the related disease have to fullfill following characteristics:¹

• The disease have to be rare¹
• The disease have to be severe, this means life-threating or leading to chronic disability.¹
• The drug must have an additional benefit.¹

The European Medicines Agency (EMA) decides if a drug gets the orphan drug status or not.¹

Orphan drug regulation

Developing medicines for rare diseases is an economical risk, because it involves time-consuming research, low chances of success and low turnover potential.¹ A regulation introduced in the year 2000 in Europe and 1983 in the USA should improve that.¹ The orphan drug regulation protects an approved drug for ten years against generic. Generic drugs which don’t differ in their composition and mode of action from the original drug are therefore not approved.¹

Progress

Significant progress has already been made in this area. An example is the metabolic disorder cystic fibrosis. Here, the predicted average survial of a newborn has increased from one year to 50 years by using so-called CFTR modulators.¹⁴

Current status

There’re 158 drugs for 131 diseases authorized in the European Union (as of 2019).^3,4 Cancer treatment accounts for the largest share, followed by drugs for metabolic disorders.⁴

Nearly half of all drugs are also authorized for children:¹

There’re 23 drugs approved for rare diseases affecting a maximum of 1,000 patients in Germany.³ A total of 93 orphan drugs were prescribed in 2017,⁴ which accounts for approximately 3.7% of total health insurance expenditures.^1,3,4

The share of orphan drugs is approximately 33% of all new drugs approved in Germany between 2014 and 2017.^1,4 This number is increasing dramatically in the past year due to the increasig focus on rare diseases.^1,4

So a lot has happened in this field in the past years. An important point was the decryption of the human genome in 2003 as part of the human genome project.⁴ Currently there’re 1,900 active substances against rare diseases in clinical developent.³

The fact that there’s still a great need for research on rare diseases will be shown by the next blog.

Contact Person:

Kristina Schraml (kristina.schraml@biovariance.com)

Sources

Der Beitrag Rare diseases erschien zuerst auf BioVariance - data-driven diagnostics.

Although the European epilepsy day has been over for a few days, it’s still important to understand this neurological disease. Therefore, this blog post discusses the defintion of epilepsy, the development, consequences and causes of various forms of epileptic seizures and the first advances in personalized treatment.

Definition

Epilepsy is a neurological disorder associated with a malfunction of the brain.¹ It’s not a single disease but rather the result of a multidue of diseases sharing a common tendency to epileptic seizures.^2,3 In such seizures the communication between the nerve cells is disturbed leading to a temporary, synchronous discharge of larger nerve cells.⁴ The diagnosis epilepsy is made when such seizures occur multiple times without apparent triggers.¹

Nerve cells

Nerve cells (neurons) are part of the nervous system. Depending on the localization in the body a distinction between the central nervous system including the neural pathways in the brain and spinal cord, and the peripheral nervous system is made.⁵ The human body contains several billions of neurons, only our brain contains 100 billion nerve cells.^5,6 These are interconnected by electrical and chemical signals.⁶ Nerve cells consists of a body and several appendages.⁵ The dendrites act as antennas, through which the body receives the signals.⁵ Via the longer extension, the axon, the signals are transmitted.⁵  

Formation and consequences

When an epileptic seizure is happen, this interaction is temporarily disturbed and the brain is excessively active.⁶ Neurons of the cerebal cortex suddenly send signals simultaneously and highly synchronously, forcing their own rhythm to other cells.² Thus, parts of the brain are overly active for a time, depending on the nature of the seizure.¹ This is followed by the post-phase, in which the cells are no longer pathological unloading themselves, but still show abnormalities.¹

The consequences are different, depending on

• Where the nerve cells are lying in the brain²
• How many nerve cells are involved²
• Which body functions they control²

A single seizure isn’t dangerous.² However, in some rare cases a cardiovascular failure called SUDEP (sudden death of epilepsy patients) can occure.²

Causes

The cause of a seizure is sometimes directly attributable to a specific trigger, for example genetic changes, a stroke or a brain tumor.² Often, however, the exact development of the disease is obscure.¹

• Cryptogenic epilepsy: no specific cause can be found, although there’s evidence in the brain.¹
• Idiopathic epilepsy: there’s no evidence of a cause or pathological change in the brain.¹

• Genetic epilepsy: genetic alterations for example at binding sites (receptors) for neurotransmitters¹
• Symptomatic/structural/metabolic epilepsy: structural changes in the brain, for example congenital malformations, brain damage acquired at birth, stroke, inflammation or metabolic disorders (Diabetes).¹

Classification

Epileptic seizures may vary in severity.² From a few seconds of mild muscle twitching to several minutes of spasms and loss of consciousness.² In general, seizures can be grouped into three categories:

Focal seizures always start at a specific point in the brain, for example at an injury.⁷ Most of them beginn with a so-called aura, in which unusual sensory impressions such as visual disturbances, concentration disorders or dizziness are recorded.⁷ The complex-focal form is also associated with a loss of consciousness.⁷

Generalized seizures spread from the beginning throughout the brain, leading to temporary disturbances of consciousness.⁷ Here, the cause can rarely be found.

• People suffering from absences lose consciousness for a few seconds. These occur mainly in children and adolescents.⁷
• A myoclonic seizure is always associated with twitching of certain muscle groups, for example the hand, arm or the trunk. The consciousness is maintained.⁷
• Clonic seizures last up to several minutes, in which the muscle groups on the trunk contract rhythmically.⁷
• In tonic seizures, the muscles cramp together without twitching, the affected often lose consciousness. The duration is only a few seconds.⁷
• The tonic-clonic seizure also called grand mal starts with an aura, followed by a loss of consciousness. Camps take place all over the body, as well as rhythmic twitches on arms, legs and head. Foamy saliva can come out of the mouth. The skin turns blue as a sign of hypoxia.⁷
• In atonic seizures, muscle tension in parts of the muscles decreases.⁷

Epidemiology

There are between 500,000 and 600,000 people confronted with epilepsy in Germany. This corresponds to 0.6% of the population.^3,8 Wordwide there are almost 50 million epileptics.⁹ Industrialized countries, such as Germany, register between 40 and 70 new cases per 100,000 inhabitants annually.^1,3

The neurological disease can occur at any age,² but the risk is particularly high in the first five years of life, accounting for two-thirds of all epilepsy cases.³ For children, it’s the most common disease of the central nervous system.¹ From the age of 20 the risk decreases while it rises again at the age of 60.^3,10 Age epilepsy is the third morst common brain disease among older people.¹⁰

Liftetime risk is understood as the probability of come down with certain disorders in the course of life. In the case of epilepsy this is 3%, in the case of an epileptic seizure 10% up to the age of 80.² Experts estimate that 5-10% of all people experience an epileptic seizure at least once in their lives.^3,6

This risk is also increased among people with certain previous illnesses. 15% of all patients suffering a stroke have epileptic seizures, 10% of them develop epilepsy.³

Treatment

Patients whose epilepsy is genetically determined have the same life expectancy as healthy people.⁶ The seizures don’t damage the brain, but other illnesses, such as depressions can be favored.⁶ The so-called status epilepticus isn’t common, but life-threatening.² This causes seizures of long duration in which the brain can be damaged.²

There is the option of a surgery for patients suffering from focal seizures.¹ In the so-called epilepsy surgery parts of the brain are removed, especially if the origin lies in the temporal lobe.¹

Anticonvulsants inhibit the excessive activity of nerve cells and reduce the risk of suffering from a seizure.¹ However, they have only a symptomatic effect and can’t cure the disease.¹ There’re currently almost 20 antiepileptic drugs on the market⁹, with another 36 drugs under development.¹¹

Another problem with anticonvulsants is that only two-thirds of all patients feel a positive effect of the prescribed medication and they can live without further seizures.⁸ 3 out of 10 patients still suffer from epileptic seizures or experience side effects.^6,8 In such cases, a genetic analysis can help to predict the efficacy of drugs.⁸

Progress

Personalized medicine

It has been shown that patients with particular hereditary disposition of the immune system have an increased risk of severe and rare skin reactions after taking oxacarbazepine or carbamazepine.¹² This is a matter of a gene of the HLA system.¹³ Carrier of this gene variant are mainly Han Chinese, Thai, Japanese and other asian populations, but also 5% of the europeans.¹² Since 2014 or rather 2013 a genetic test for prescribing the antiepileptic drug is recommended to exclude these side effects.¹³

New found epilepsy genes

The International League against Epilepsy (ILAE) perfomed an analysis of genetic data from more than 15,000 epileptics and nearly 30,000 unaffected people.¹⁴ This allowed them to discover 21 risk genes with different functions involved in the development of the neurological disease.¹⁴ One of the functions is the association with ion channels, which transmit the signals in the nervous system.¹⁴ Mutations limit the functionality of proteins, which is the basis of epilepsy.¹⁴

These findings can be used to personalize the medicine to get therapies more targeted.

Contact person:
Kristina Schraml (kristina.schraml@biovariance.com)

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Der Beitrag Epilepsy – chaos inside the head erschien zuerst auf BioVariance - data-driven diagnostics.

The blog post this week is about CRISPR/Cas and because of the World-AIDS-Day last Saturday it is also about first successes in HIV treatment with this genome editing tool and about a message that has shocked many people last month.

DNA structure

The blog on Black Friday already concerned about chromosomes and DNA. Just as a quick reminder: DNA is a long, chain-like polymer composed of individual buiding blocks called nucleotides. A nucleotide consists of a phophate, a sugar and one of four bases (adenine (A), cytosine (C), guanine (G) and thymine (T)). The structure was decrypted in 1953 by Watson and Crick.¹ They found that the DNA forms a double helix. Two such chain-like polymers, also called single strands, assemble in opposite directions called complementary. The backbone is formed by the phosphate and the sugar, while the bases pair with each other via hydrogen bonds, always adenine with thymine and cytosine with guanine.

CRISPR/Cas – Definition

To come to CRISPR: CRISPR stands for clustered regularly interspaced short palindromic repeats. In this case, a palindrome is a sequence in which the bases on one DNA strand are in the same order as those on the complementary strand – only in the opposite direction.

Although such sequences are preceded by a promotor responsible for transcribing DNA into RNA and then translating into proteins, they don’t yield a funtional protein. The point of it wasn’t known for a long time.

Already in 1987 Yoshizumi Ishino discovered the existence of repeating DNA segments in the bacterial strain Escherichia coli K12. He identified similar sequences of each 29 nucleotides interrupted by a sequence of 32 nucleotides, which was variable.² In 1993 a similar motif was discovered in the DNA of Mycobacterium tuberculosis, one of the vectors of tuberculosis.³ In 2002 similar structures were observed in the genome of many bacteria and archaea belonging to the prokaryotes and the term CRISPR was coined. In addition, a group of genes close to the CRISPR sequence was found in all studied organsims and therefore designated as CRISPR-associated genes (Cas-genes).⁴ In 2005 it was found out that the sequences located between the palindromic sequences, termed spacers, are identical to the DNA of viruses.^5,6,7

According to estimates, there are approximately 4-6×10³⁰ prokaryotes on earth and about ten time more viruses exist. As these organisms depend on host cells for survival, as mentioned in the previous blog they kill about half of the prokaryontes every two days.

Thus it was hypothesized that CRISPR defends bacteria and archaea against foreign DNA of viruses and thus functions as an immune system.^5,6,7 In 2007 this claim was confirmed by Barrangov and colleagues, who showed that bacteria infected with phages (groups of viruses) integrated parts of the virus DNA as spacers between the CRISPR regions and thus built up immunity to these pathogens: artificially inserted spacers containing the sequence of viruses made the bacteria resistant to the foreign DNA. The experiment also worked the other way around – the bacteria lost this adaptive restistance when they deleted the spacer regions again.⁸

So, it has been shown that prokaryotes have an adaptive immune system allowing them to remember DNA sequences from pathogens, similar to humans through antibodies and when they get re-infected, the DNA of the virus can be cut. In detail, the mechanism probably works like this: when it comes to an infection, the DNA of the virus is split into small fragments and inserted into the CRISPR sections. If it comes to a renewed attack, these segments are transcribed into RNA – therefore the upstream promotor – and the viral DNA is checked.If this matches the „stored one“, it is intersected by DNA-cutting proteins, the Cas proteins and thus destroyed.⁹

Genome Editing

This recognition and cutting of a sequence has been used in so-called Genome Editing for quite some time. Genome Editing is a precise tool that can be used to selectively alter DNA. Genes can thus be inserted into an existing sequence, be removed or be completely replaced. Even individual nucleotides can thus be changed.

A new era in molecular biology was the discovery of restriction enzymes in the 1970s: these enzymes recognize and cleave characteristic sequences.¹⁰ The problem here is that the target site is difficult to predict because the recognition sequence is usually only a few base pairs long and thus may occure more frequently in the genome.

CRISPR/Cas largely prevents such unwanted effects. In addition, it is relaible, fast and in cheap. The principle, like other Genome Editing methods, is based on the three following steps: first the site which should be changed in a targeted way has to be found in the genome. A probe or a vector is constructed containing the desired target sequence, also referred to as gRNA (guideRNA) and the sequence of the molecular scissors – the protein Cas9. Once arrived at the target sequence, Cas9 can cut the DNA double strand exactly at the desired site. Finally, the cell’s own repair system comes into play and rejoins the severed DNA strands.¹¹ Here, the cell has two possibilites: non-homologous end-joining (NHEJ) or homologous recombination (homology directed repair, HDR).

With NHEJ, random insertions or deletions of single nucleotides result in gene inactivation because the reading frame (ORF; the region encoding a potential protein) is thereby shifted. With HDR, a template is added to the cell to give the repair system the information what new sequences are to incorporate.

Gene therapy

So far, patients with defective genes have been treated by a classical gene therapy. They were treated with a virus carrying the sequence of the new functional gene and incorporates it into the affected cells. But also here the problem is that you can’t predict exactly where the gene is integrate in the genome.¹² With CRISPR this circumstance is avoided and the target site is exactly predetermined.

CRISPR/Cas in HIV therapy

There have been initial successes in the treatment with CRISPR/Cas, for example in HIV therapy. Although the infection with the HI virus is largely under control by retroviral therapy (ART), a complete cure doesn’t exist yet. Experiments have shown that CRISPR/Cas can effectively fight against HI viruses. Using the molecular scissors, the team led by Kamel Khalili and Won-Bin Young has removed viral DNA from more than 95% of infected cells in mice as well as in human immune cells transferred to humanized mice.¹³

However, there is still no prospect of an application on humans. Because even this method isn’t without risks. On the one hand, only 95% of the infected cells are removed, the rest is enough that the disease is breaking out again. On the other hand, a virus is used to transport the CRISPR DNA triggering an immune response in the human body and thus can only be used once. In addtion, the transport viruses are suspected to trigger tumors.¹⁴

This year, another working group has destroyed the regulatory genes of HIV by CRISPR/Cas so the virus can’t replicate in infected cells. They designed a vector expressing Cas9 and one of six different gRNAs. These vectors were transfected into cells expressing the regulatory proteins of the virus (Tat and Rev). As a result, they were able to record a significant reduction in the expression and funtion of the proteins without affecting the survival of the cells.¹⁵ These two publications show that CRISPR/Cas actually would be able to cure HIV-infected people.

Last month a message from China shocked many people. A scientist claims to have helped make the world’s first genome-edited babies. He Jiankui fertilized a woman with embroys, in which CRISPR/Cas was used to destroy the CCR5 gene. This gene encodes a receptor protein, HIV is able to attach in the case of infection and thus is esential for the virus. The aim of the work was not to prevent the transmission of the virus by the parents, but rather that the children don’t have to suffer the same fate as their parent and are thus resistant to the virus.¹⁶

Criticism came from all sides. An important argument is that there are also some HIV strains using another receptor protein (CXCR4) for infection.¹⁵ In addition, there is the ethical aspect. In Germany, USA and many other countries such manipulations are prohibited. The changes brought into the genome due to Genome Editing will also be passed on to subsequent generations, so the associated risks are scarcely to be estimate.¹⁷ Therefore, the use of this silver bullet remains to be enjoyed with great care.

Contact Person:
Kristina Schraml (kristina.schraml@biovariance.com)

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Der Beitrag CRISPR/Cas in HIV therapy erschien zuerst auf BioVariance - data-driven diagnostics.

Because of the World-AIDS-Day today, there is a new post about HIV and AIDS in general, the major breakthrough in HIV therapy and a first success in personalized medicine.

HIV infection and AIDS

AIDS (acquired immunodeficiency syndrom) is triggered by the human immodeficiency virus (HIV), which belongs to the family of retroviruses. Viruses can only replicate in foreign cells, so-called host cells and are thus dependent on them.

Our immune system is responsible for fighting against pathogens such as fungi, bacteria or viruses and destroying dead or sick cells. If a human is infected with the HI virus, the immune system is damaged because the virus primarily attacks immune cells, so-called helper cells: Their own genetic material is incorporated into the genome of the host cell, the virus can thereby merge with it and kill them.

The helper cells, which belongs to the white blood cells, leucocytes, actually control other cells in the immune response, but can now due to the infection no longer perform this function. Thus, the immune system is weakend more and more.

The virus is transmitted via fresh wounds and mucous membranes through contact with body fluids such as blood, sperm, vaginal secretions and breast milk. Shortly after infection, the virus proliferaties rapidly and at the same time, the number of helper cells decreases as they are attacked and destroyed.¹ After this acute phase, the cronic phase follows, in which the equilibrium is relatively constant. In the case of an untreated infection, this eventually leads to an acquired immune deficiency syndrom after a long, usually symptom-free phase.¹ However, not every infection with the HI virus automatically leads to AIDS.

Due to the permanently weakend immune system, there are other risks for HIV-infected people. For example, they have a 50-fold increased risk of a infection with tuberculosis.² At the same time, tuberculosis infection can also accelerate the onset of AIDS.² Both infections are mutually reinforcing.² Tuberculosis is therfore the leading cause of death among people living with HIV.³

Epidemiology

Since the early 1980s, a veritable pandemic has developed. Worldwide, approximately 36.9 million people are infected with HIV (as of 2017), including more than 3.3 million children under the age of 15.⁴ So far, the virus has claimed a total of 39 million people (as of 2015). There are about 84,700 infected people in Germany.⁵

However, worldwide both the number of new infections as well as the number of AIDS deaths has steadily declined in recent years.

On the one hand, this is due to the ever-better education, on the other hand because of the successful antiretroviral therapy (ART): this suppresses the increase of the virus in the body and prevents developing AIDS. More than half of all HIV-positive people worldwide receive these life-saving drugs, with the result that the total number of AIDS-deaths was almost halved between 2005 and 2016.⁶ 

HAART

Although medications have been available since 1987, the death rate increased enormously at that time. The major breakthrough in HIV therapy was the introduction of highly active antiretroviral therapy (HAART) in 1993 combining at least three drugs. Since then, the mortality rate has fallen rapidly in some countries – even 94% in France.⁸ This effective combination treatment puts the virus below the detection limit – preventing the onset of AIDS. In addition, further transmission of the virus is prevented. However, according to a recent study, only 53% of respondents know about this fact.⁷

UNAIDS

UNAIDS, the United Nations HIV/AIDS program has set the goal of 90-90-90 in the fight against AIDS. This means that by the year 2020, 90% of all infected people worldwide should know about their disease, of which 90% should be in treatment, of which 90% should be below the detection limit and therefore no longer be able to pass on the virus.⁹ While many countries has reached this target, Germany has reached 86-86-93 in 2018.⁹ The major problem with the fulfillment of this campaign is no longer the treatment or prevention of the outbreak of AIDS, but the diagnoses:¹⁰ in Germany there are about 13,000 people infected, but don’t know this. So, much has to happen in the next few years.¹¹

Personalized Medicine

One of the active ingredients used in HAART is Abacavir, which has now been approved for personalized treatment.¹² Drugs containing this substance should only be prescribed after a genetic test has been perfomed, provided that it is negative.¹² This test is for the presence of the gene marker HLA-B*5701, which triggers a hypersensitivity reaction to the active substance. The HLA system (human leucocyte antigen) describes a group of genes that are central to the immune system. HLA-B belongs to the class I antigens, which, like all other HLA genes, lie on a 4 million base pair stretch on chromosome 6. Of this gene, at least 31 different alleles exist. In HIV patients with the allele HLA-B*5701 occure severe side effects, which can be lethal, after ingestion of Abacavir.¹² This is in the case of 5% of all patients.¹² The prescribed genetic test may exclude this hypersensitivity reaction and patients should prescribed other medicines which are harmless and effective for them.

Thus, also in this area a success in the direction of personalized medicine was recorded.

There will be more on this topic and another breakthrough in HIV therapy in the next blog.

Contact Person:
Kristina Schraml (kristina.schraml@biovariance.com)

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Der Beitrag World-AIDS-Day erschien zuerst auf BioVariance - data-driven diagnostics.

Because of the World Diabetes Day on 14.11 the first blog post will be about diabetes in general, the consequences of the disease and the first progress towards precision medicine.

Diabetes

In a healthy person carbohydrates are degraded by the digestive system to glucose and get through the intestinal wall into the blood.¹ The hormones insulin and glucagon are responsible for the control of the blood sugar level so the concentration of glucose in the blood.¹ Insulin is formed in the so-called beta cells of the pancreas and transports glucose from the bloodstream into the cells.1 Through a process called glycolysis, energy (ATP) is extracted through degradation of glucose.¹ The sugar is therefore an important energy source for the brain or the red blood cells for example.¹

In the metabolic disease diabetes mellitus this natural process is disturbed resulting in a permanently increased concentration of blood sugar, also called hyperglycemia.² Due to the different causes the disease was divided into two main groups. Type 1 diabetes is based on an insulin deficiency which is through a destruction of the beta cells by the own immune system.² As a result of this autoimmune reaction insulin can no longer be formed.² This type is relatively rare with a proportion of 5%.^2,3 Type 2 diabetes is based on an insulin resistance.² That means insulin can be produced but the secretion oft he hormone is disturbed.² In addition to genetic predisposition lifestyle plays a central role.² As a result this form occurs mainly in older age.²

Epidemiology

Worldwide there are 425 million people living with diabetes, accounting for 8.8% of all 20-79 year olds.⁴

In Germany, the number of diabetics is about 7.6 million (as of 2017).⁵ Daily, 1,000 new cases are added⁶ leading to an increase up to 20 million in the next few years.⁷ Worldwide experts predict 629 million victims until 2045.³

Secondary diseases

The main problem is that diabetes is associated with a variety of secondary diseases. In the long term the metabolic disorder affects kidneys, nerves, retina and blood vessels.⁸ People with diabetes run a higher risk of strokes and heart attacks.⁸ Thus, 50% of patients with diabetes die from cardiovascular diseases.⁶ In addition it has been shown that life expectancy is significantly reduced in patients with diabetes and cardiovascular diseases.⁹ Studies also reveal a link to neurological disorders. The risk of a diabetic suffering from dementia is about 2.5 times higher than of a healthy person.¹⁰

These secondary diseases are also reflected in the costs of health care. They went up to 48 billion euros in 2009, 24% more than in 2000.¹¹ This also becomes clear from an example calculation of a patient: If the diabetes therapy costs about 700 euros per quarter, the resulting complications for example for an amputation costs 14,000 euros per quarter.⁸

Every day 50,000 amputations, 2,000 cases of blinding and 2,300 cases of renal insufficiency are caused by diabetes.⁸

The metabolic disease is also responible for more deaths than previously thought. A first study concerning with such a calculation in 2017 revealed 174,627 deaths in 2010 due to diabetes.¹² This accounts for 21% of all deaths in Germany.¹² Worldwide, 5,1 million people died in 2013, making it to one of the top 10 causes of mortality.¹²

Recent advances

Recent advances in medicine and an even better understanding of the molecular properties of diseases can also been observed in this area. So it is now known that in type 1 diabetes the predisposition plays a certain role.¹³ Meanwhile a variety of genes have been discovered showing a connection to the development of the disease.¹³ In addition, researchers have reworked the classification of the disorder.¹⁴ After evaluation of 15,000 data in consideration of for example age, BMI or long-term blood glucose level there are five different types of the disease.¹⁴ This creates new possibilities of therapy going in the direction of precision medicine. cked0 List T

Personalized medicine

Succes has already been achieved in the field of personalized medicine this year. The drug glibenclamid, which causes huge side effects in the therapy of type 2 diabetes, was first recommended for the treatment of neonatal diabetes.¹⁵ This genetic disease is relatively rare with an incidence of 1:100,000.¹⁶

In the system of blood sugar ATP-sensitive potassium channels play an important role. These transmembrane proteins consist of two rings, each with four subunits.¹⁷ Each subunit is encoded by one gene – KCNJ11 and ABCC8. If the blood sugar level drops meaning the concentration of glucose in the blood, less energy can be produced in the cells.¹⁸ The channel is open.¹⁸ On the other hand, if the concentration increases, glucose enters the cells, energy (ATP) can be formed and the channel is closed.¹⁸ This causes a depolarization and calcium channels gets open. Due to thhe influx of calcium ions into the cell, insulin is secreted, so released from the cells into the bloodstream.¹⁸

In the case of neonatal diabetes, there is a mutation in one of the coding genes of the potassium channels. The beta cells are able to produce insulin, but it can’t be secreted because the channel is always open due to the mutation. The depolarization and thus the openig of the calcium channels can’t take place. Glibenclamid can now block the potassium channel, if the mutation is in the gene KCNJ11, so further steps can pass off until the release of insulin.¹¹

Since May 2018 a genetic test is recommended with the diagnosis of neonatal diabetes to determine the present mutation.¹⁵ Thus, glibenclamid is one of 62 active ingredients already approved for personalized medicine.¹⁵

Contact Person:

Kristina Schraml (kristina.schraml@biovariance.com)

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Der Beitrag Diabetes – an underestimate danger erschien zuerst auf BioVariance - data-driven diagnostics.