- Victor Martinez-Glez added an answer:3Can any body tell me about how many genes are there for ectodermal dysplasia?
i didnot find any research paper regarding this.
You can find a good review in: (also attached)
Ectodermal dysplasias: a clinical and molecular review. García-Martín P1, Hernández-Martín A, Torrelo A. Actas Dermosifiliogr. 2013 Jul-Aug;104(6):451-70.
- Gregory Dressler added an answer:3Can someone advise on a query regarding autosomal dominant and recessive traits?
Why the recessive diseases do occur early in early childhood and dominant ones at about the age of 40?
The answers are really disease specific. Recessive phenotype just means that it takes two mutant alleles to initiate disease and that heterozygous individuals are generally asymptomatic. Dominantly inherited diseases occur through several mechanisms. 1- haploidinsufficiency is when a single normal allele is not enough and often can result in a congenital or progressive phenotype. Examples of this are Aniridia or Charge syndrome. 2- A dominant negative mutation occurs when a single mutant allele creates a protein that interferes with the normal protein made from the WT allele. Examples of this are Alport's syndrome, in which a mutant collagen chain disturbs the assembly of the collagen heterotrimer. 3- Loss of heterozygosity is when during development or in the lifespan of the individual, the remaining normal allele is lost in a fraction of cells. Examples of this are Autosomal Dominant PKD or Wilm's tumor. Does that make sense?Following
- Fawaz Naji Saleh Al-Shaheri added an answer:4How can I detect mutations of a recessive gene in both chromosomes by Sanger sequencing?
I am looking for germ-line mutations in patients with Colorectal Adenomcarcinoma, how can I identify that mutations are monoallelic ar biallelic using DNA Sanger Sequencing and what is the Bioinformatic tool to differentiate between pathogenic from non-pathogenic mutations.Thank you so much Paul Rutland . I really appreciate your efforts to answer my question. Your answers are very useful and I am really grateful for your help.Following
- Sanja Cirkovic added an answer:4What are the EMNQ recommendations about nomenclature of the large (whole exons) del/dup?
I have a question for those who has been recently involved in EMNQ schemes for the diseases that included large (whole exons) gene deletions/duplications.
Do you used official nomenclature of HGVS or some other nomenclature to describe it?
How can you check if the formula is right?
Can you give some example, perhaps?
Thank you in advance.
It's about MLPA results. There are at least three kinds of results that we are getting (for the kits we used):
1. deletion/duplication of chromosome region, designated as: rsa "chromosome region"("name of the MLPA kit")x"number of copies"
2. copy number of the exons in the specific gene (all exons are analyzed)
3. copy number of the exon/exons (not all exons of the gene are analyzed).
The first case is simple and is officially recommended by ISCN 2013.
The other two cases are problematic.
According to https://mutalyzer.nl/ and HGVS sites (A+1_B-1)_(C+1_D-1) designation for exon del/dup is still a proposal, not recommendation of HGVS.
I suppose that the traditional designation is still officially recommended and in the use.
I just wanted to hear more about it from the "clinical lab" people and their experience.
- Deeann Visk added an answer:6Can the monozygotic twins have different blood types?I really want to know the answer.
I think that something similar to what Marya G Zlatnik described took place during embryonic development of the monozygotic twins with different blood types. Please refer to my blog about this phenomena for more details--see link below. Personally, I find the likelihood of post-zygotic mutations to be very low. Chimerism is a more likely explanation. This would mean that the "monozygotic" twins are not actually from one fertilized egg, but two!
Nature is indeed complex and perplexing.
- Nicole Gillain-Martin added an answer:3What is the next test you would order for a patient with isolated ALP elevation?
One of my patients, brought me an ALP=669 in an exam order by a psychiatrist because of the use of valproic acid (ALT, AST, GGT and Bilirubin are normal). I have already ordered serum Calcium, serum total protein and albumin, TSH and PTH. I wander if it is essential to order a bone scan.
I fully agree with Mrs Poggiali,: it is essential to achieve electrophoresis of iso-PAL will specify hepatic or bone origine. Macromolecular could be present, but should not be responsible for such an increase without being accompanied by an increase of GGT.
Still think a transient hyperphosphatasemia (very rare in adults) and check the persistence of high value
This patient is not suffering from kidney failure, I supposeFollowing
- Stefanie Huhn added an answer:4Can anyone help regarding incidental findings according to ACMG recommendation?
I have a question about how to report incidental findings in exome samples for the clinical diagnostic purposes.
I have read the ACMG recommendations for reporting incidental findings in patients and tried to filter out the pathogenic variants in the 56 genes suggested to be checked by ACMG.
When I filter out the variants using common polymorphisms in dbSNP142, the remaining pathogenic variants are mostly not so much. However, without this filtration there would be around 20 remaining variants on average that should be checked manually to investigate their probable pathogenicity.
Most of these variants are the common polymorphisms (reported through GWAS studies) which are associated with more common disorders and not the rare variants of monogenic disorders.
Since there is no accurate recommendation about the type of variants that should be reported to patients, I was wondering whether we should check these associated polymorphisms manually and investigate their effects in these disorders and then report them to patients, or we should just report the rare pathogenic variants (DM variants in HGMD) as incidental findings?
I am looking forward to hearing from you.Following
- Charlotte Brasch-Andersen added an answer:7What is the best way to check the results of my MLPA experiments?
Hi! I am currently dealing with the result of single exon 24 probe seems deleted (0.5:1), in my MLPA experiments in one one patient. The nearest probe in this gene is for exon 15 and it shows normal copy number.
Diagnosis is uncertain, and the parents and siblings have normal copy number of all analyzed exons in that region.
I have checked these results twice (another sample of DNA of the same person) and they are reproducible.
So, I have to screen this region for potential SNP and other point mutation, as well as for actually deleted exon/exons.
What do you suggest, what is the best strategy for this?
Do I have to do sequencing first or to use another MLPA kit with more probes in this region?
And what to sequence first: all unchecked exons 16-24 or the nearest 23-24 (24 is the last in this gene)?
In our Laboratory, we perform fragment analyses (STR, MLPA), but this could be our first time in sequencing...
As you may assume, we need the cheapest and the fastest way to do it.
I hope I was clear..
Thank you in advance!
I had the same problem with that link when I tested it, just before writting to you. Never had those problems before. Wrote the guy that designed it and asked if he cancelled the www. Waiting for a reply.
Yes, I have been designing synthetic probes for 5 years in my clinical work to verify arrayCGH data. It work very nice.
Where did you see that the gene (NSD1?) had 24 exons? Was that in a MLPA kit or the databases? UCSC shows two isoforms. One has 23 exons, the other has 24 exons. The DGV track in UCSC also shows that duplications from exon 4 (aprox) to exon 23/24 are not rare in the general population.
I'll be happy to help you, but this discussion is probably too specific for an open forum now. Why don't you email me and I'll see if I can help you if you wish to do custom designed MLPA probes for you patients.
- Joep De Ligt added an answer:4Any suggestions about some tools for CNV calling analysis on whole Exome sequencing data?
I am doing some CNV calling analysis on whole Exome sequencing data. Can anyone recommend some tools for CNV on WES data?
I have done some literature search, and VarScan2 is one of tools suitable for this type of data, but does VarScan2 have to take disease-normal paired data? Can I run VarScan2 on normal and disease, respectively, and merge the results at a later step? I know this would not make much difference in terms of the disease-specific CNVs identified, but I am trying to fit the CNV calling to a pipeline, and I need to stick on the ways the existing pipeline works.
Recommendation on other tools are welcome.
As is clear from the earlier answers there is no one tool that will do the job. Some tools are better at detecting small CNVs while others excel only in detecting rare events, also see this thread on biostar: https://www.biostars.org/p/13807/
About two years back we reviewed several Exome specific tools for their poer to detect known clinically relevant CNVs in comparison to a high resolution aCGH platform (see link 1). Since then many new tools have been developed and tested, each performing better in a particular area. Look up a recent review (such as link 2 and 3) to get the ins and outs.
If you're looking for tools that are modified for pipeline usage take a look at the tools/pipeline developed by the Sanger https://github.com/cancerit/. cgpBattenberg is propably the one you would be interested in for this question
The key to almost all tools is a high quality control sample or pool of samples that capture the enrichment/sequencing biases of your experimental setup. The question which tool will then best suit your needs depends on several factors like if you want to detect rare events (likely a Z-Score based approach) or shared events (more likely a HMM). And also on if you have a specific application in mind, e.g. tools optimised for cancer samples usually assume a single reference sample while more general tools usually require a pool.
Hope this helps in your decision making.
- Minghuan Jiang added an answer:2Is the Pharmacogenetics testing cost-effective?Some success has been achieved in recent years in establishing the clinical utility of the pharmacogenetic testing though it remains questionable whether it is cost- effective or not.
Jiang M, You J H S. Review of pharmacoeconomic evaluation of genotype-guided antiplatelet therapy[J]. Expert opinion on pharmacotherapy, 2015, 16(5): 771-779.
This is really an important question!
I think whether genotype-guided therapy is cost-effective depends the studied topic. The upper paper is about cost-effectiveness of genotype-guided antiplatelet therapy in ACS patients for your reference.
- Constantin Polychronakos added an answer:3Is microdeletion 2q11.1q1q11.2 pathogenic for CNV / VOUS, a susceptibility factor or just incidental finding?
We discovered the same microdeletion 2q11.1q11.2 for a fetus presenting with cleft lip/ cardiac malformation/ clinodactyly. It is inherited from a father no yet seen in consultation. Is this variant can be considered as a putative cause of a syndrome with malformations and ID.
I assume that you already looked at the databasaes (e.g. DESIPHER) and came up empty. Then, get the transmitting father's phenotype. If the father has the same or very similar features there is a reasonable chance that this is causal. In that case, look further in the family for any history suggesting dominant inheritantce. In dominant pedigrees, it does not take too many affected to get a LOD of 3, the definitive proof.
If no onr in the father'd family is unaffected, it could be irrelevant or a "susceptibility factor" requiring additional genetic or envirnomental input. In that case, you are dealing with a complex trait and you will need anywhere from dozens to thousands (depending on the strength of the effect) of similarly affected probands to prove it. Discouraging, I know, but such are the limitations of human knowledge.Following
- Go J Yoshida added an answer:2What brings about the genotype-phenotype or signal-phenotype decorrelation?
Neurofibromatosis type-I (NF-1), also referred to as von Recklinghausen's disease is a developmental disorder caused by germline mutations of NF1 genes encoding inneurofibromin, a gene that is involved in the RAS pathway (RASopathy). NF-1 exhibits several clinical features such as multiple café-au-lait spots, Lisch nodules, optic glioma etc. The definition of Rasopathy is that there are several resembling diseases in which Ras signal is constitutively activated. RASopathy is composed of Legius syndrome, Noonan syndrome, Costello syndrome etc. For instance, Legius syndrome in which SPRED1 gene has a loss-of-functional mutation, thereby the negative regulation of Ras signaling pathway is no longer works. However, Legius syndrome shows multiple café-au-lait spots much the same as NF1, but also exhibits the different clinical features as compared with NF1 such as macrocephaly and ADHD (mild mental disorder). I have always wondered what is responsible for the genotype-phenotype decorrelation. Considering the case of RASopathy, in which hyperactivated Ras signal in the germline level shows different phenotypes, the correlation of signal activation disorder and the clinical symptoms is not always correlated. I would be appreciated if you give me some opinion on what makes the signal-phenotype decorrelation.
I am greatly appreciated for your kind comment. As you have explained, the concept of Waddington's epigenetic landscape is critical to understand the reversible differentiation with higher plasticity. Temporal and tissue-dependent characteristics of gene expression pattern seems to make the decorrelation between genotype/ signal abnormality and phenotype/ clinical symptoms.Following
- Cingeetham Vinod added an answer:3Can someone suggest a protocol for primary breast cancer cell culture for Karyotyping?Seeking an established protocol to culture short term primary breast cancer cells for karyotyping. I request you to suggest a best mitogen to enhance the cell division and its conc. to be used.
@ Betty G Dunn: Madam i have succeeded in culturing the primary breast cancer cell but now im stuck at hypotonic solution, I have treated cells with various concs of KCl and also used detergents along with KCl as suggested by various papers but all affords gone into vain. a very few no. of plates are obtained like 5-6 metaphase plates per slide, so i need your suggestion to increase the plate number.Following
- Thorsten Gerstner added an answer:12Correlating epilepsy with chromosomal aberration?We are conducting a review study on a relationship between chromosomal aberrations and childhood epilepsy. Has anyone had registered cases with chromosomal abnormalities and epilepsy?
and more and more of the early encephalopathic epilepsies are known for a genetic background. I do a genetic panel of encephalopathic epilepsies in all my patients who are suspect for that kind of epilepsy. That includes ARX, CDKL5, FOXG1, KCNQ2, MECP2, PCDH19, PNKP, POLG, SCN1A, SLC9A6, STXBP1, SYNGAP1, UBE3A. And i have found more and more of these genetic epilepsies the last to yearsFollowing
- Kirsi Vaali added an answer:1I am looking for anyone that is either conducting active research or has completed research on the AUTS2 gene 7q11.22 interuptions.I am in the process of compiling as much information as possible on AUTS2. If you are actively conducting research and need participants please contact me. If you have completed any research published or unpublished I would like to compare data and possibly see if any new findings have been made that I have not yet come across. I am particularly interested in the syndromic qualities that are not autism related. That information is well documented I am looking for things like: Microcephly, Short Stature, facial dysmorphisms, systemic findings, immunological, otolaryngological (specifically hearing losses), visual abnormalities, brain malformation, and neurocognitive deficits, Gastro findings, and any other connections to this gene's interuption that are considered syndromic in nature.Hi Cheri,
We are trying to start a study of autism in which the role of diet will be studied. We will take cases that are permanently living 24/7 in health care units, many of these are aggressive in behavior and the intention is to affect the behavior with gluten-free and milk-free diet. We will do electro encelographical measurements, behavioral evaluations, assay f-calprotectin, fecal microbiota (still not confirmed), serum anti-MAG, anti-gangliosides, S100, Vit D and B12, T3, Fe and ferritin, amylase-trypsin inhibitor IgG Abs, milk protein-specific Abs, and eventually metabolomics with 100 different metabolites. At the moment we are applying ethical permission (the first meeting will be at the end of July), and we do not yet have funding for these assays. We can run the study and collect the samples (when we have received the ethical permission) but will need to leave most of the assays for later if we will not get funding for this.
University of HelsinkiFollowing
- Bhairavi Srinageshwar asked a question:OpenIncorporation of ddGTP?Why does ddGTP gets incorporated faster than other three ddNTPs during sequencing PCR?Following
- Jafar Mohseni added an answer:9Why is mtDNA particularly susceptible to mutation?Can anybody please explain to me why mitochondrial DNA is particularly susceptible to mutation? I know that in the most organisms mtDNA lacks intron and repetitive DNA. Can anybody explain further? Thanks in advance.The POLG low fidelity and higher exposure rate to Oxidative agent made mtDNA pron to mutations. Despite of them, mtDNA is the most informative in phylogentic studies and sequencing based mutation rate for mtDNA in control region is .0043 per generation.Following
- Corrado Testolin added an answer:2Does anybody have information about the genes involved in Aicardi syndrome?We have a clinical case and now we're doing a review article in molecular topics in Aicardi syndrome.I suggest this linK:
WWW.ORPHA.NET is a very good syndrome database where you can find information about syndromes and links to genetic Lab almost all over the worldFollowing
- Mert Burak Öztürk added an answer:5Is there any software or website for matching sequences and diseases?For example, you got some DNA sequences from patient and you saw that there are some mutations. Thus, you wanted to check that sequences with possible diseases. Wıth this software/website you enter that sequences and it gives you possible diseases. Do you know like that program?Thanks to everyone for your interest. I decied to use firstly, UCSC to understand that sequences belong to which gene and secondly, HGMD to see the possible diseases with using that gene symbol.Following
- Bhairavi Srinageshwar added an answer:5What is the difference between Mosaicism and Chimaerism?--Thank You!Following
- Hadi Bayat added an answer:4Can somebody suggest a protocol for DNA extraction from amniotic fluid ?.Dear Bhairavi
Here is a good protocol:
1. Divide the sample of amniotic fluid into labelled 2 mL eppendorf tubes (the number of tubes will depend on the volume of sample received). Spin in microcentrifuge at 13,000 rpm for 10 min. alternatively, centrifuge the cells in a larger tube, remove supernatant and transfer entire pellet to single eppendorf using PBS and then centrifuge eppendorf as above.
2. Remove supernatant.
3. Add 375 uL salting out buffer, 6.25 uL 20% SDS and 125 uL proteinase K (5 mg/mL)
4. Incubate Over night in 37C water bath or 30 - 60 min at 65C (flicking to resuspend constantly) to digest protein.
5. Label 3 X 1.5 mL microfuge tubes
6. To the digested sample add 400 uL phenol, invert and spin at 10,000 rpm in microcentrifuge for 5 min.
7. Transfer aqueous layer to another tube and add 400 uL 1:1 phenol:chloroform. Mix gently by inversion, then spin 10,000 rpm in microcentrifuge for 5 min.
8. Transfer aqueous layer to a new tube and add 400 uL chloroform. Mix gently by inversion, then spin 10,000 rpm in microcentrifuge for 5 min.
9. Transfer aqueous layer to a new microfuge tube and add 1/10 volume 3 M NaAc, pH 5.2 (40 uL) and 2.5 volumes of 100% ethanol (1 mL). Invert tube gently to precipitate DNA.
10. Spin at 10,000 rpm for 5 min and pour off ethanol.
11. Rinse the pellet once with 70% ethanol, centrifuge 10,000 rpm for 5 min. (Do not include this step if the pellet is very small)
12. Dry DNA pellet in laminar flow cabinet and resuspend in 20 - 50 uL of sterile water depending on size of pellet.
Salting out Lysis Buffer
1M Tris pH.7.5 10 mL
5M NaCl 80 mL
0.5 M Na2 EDTA 4 mL
Add dH2O to 1lt. and mix reagents with magnetic stirrer for short while. Label and date. Autoclave. Store at RT.
- Francesco Saverio Dioguardi asked a question:OpenArginase 1, is it only a liver's matter?Very nice paper, and thanks to cite one of my first papers!
But, I am sorry to notice that in this paper, again, arginase 1 activity is defined as limited mainly to liver. Arginase 1 expression is controlled by exogenous arginine and arginase 1 is present also in red blood cells (RBC), not only in liver! Therefore, Arginase 1 concentrations in RBC and availability of arginine as substrate for eNos and/or iNOs in may be of pivotal importance when evaluating peripheral vasomotor efficiency. Moreover, recycling of citrulline to arginine in peripheral endothelium is dependent both on availability of aspartate exported from citric acid cycle, which is blunted if beta-oxydation becomes prevalent on glucose full oxydation, and maintained expression of the enzymatic path.Following
- Felix L Nunez-Santana added an answer:5Can somebody share their GC rich kit protocol for PCR amplfication of difficult templates?see aboveFor GC-rich fragments and difficult templates, try using Taq DNA Polymerase with Q-solution.
The Q-Solution facilitates amplification of GC-rich templates or templates with a high degree of secondary structure by modifying the melting behavior of DNA. Use of this reagent often enables or improves suboptimal PCR. Unlike DMSO and other PCR additives, Q-Solution is used at a defined working concentration with any primer–template system and is not toxic.
Handbook: Table 5. Reaction Composition Using Taq DNA Polymerase, Q-Solution, and 10x PCR Buffer.
We also achieve far better results for this kinds of templates by replacing dGTP with 7-Deaza-2'-dGTP
You can obtain 7-Deaza-2'-dGTP from several vendor. i.e.
- Joep De Ligt added an answer:1Is possible to perform a batch search of SNPs in the 1000 genomes browser in order to get individual genotypes for the variants?I need to perform some ancestry analysis and I'd like to use the individuals from 1000 genomes as proxies for the test.Hi, the complete dataset underlying the browser can be downloaded in different formats, or accessed through their data slicer tool.
The 1000 genomes DataSlicer tool is available here; http://www.1000genomes.org/data-slicer.
And raw dat file locations are listed here; http://www.1000genomes.org/data
From your question I think vcf files with genotypes are what you are looking for, which are available here;
The files can be downloaded per chromosome and using tabix (http://samtools.sourceforge.net/tabix.shtml) you can extract the desired genomic positions.
Answers to these types of questions are often also listed on the projects FAQ page;
- Jose A Lopez-Escamez asked a question:OpenWould you recommend BIOgranat software for pathway-based analysis of WES data?We are trying to use this software to find novel variants in genes in proteins in the same pathway. Do you think that this strategy could be useful to find molecular targets in the same pathway?Following
- Ashraf Al ghzaly added an answer:1How to calculate genotype relative risk for AA and Aa using Genetic Power Calculator?When using Genetic Power calculator, besides inputting the high risk allele frequency A (I used the hapmap data), the value of genotype relative risk for AA and Aa were required. How could I get the value? Should I use the data of previous literature with positive association to calculate?you can use CaTS Power Calculator program.
-Downloaded from Michigan University Center for Statistical Genetics web site: http://www.sph.umich.edu/csg/abecasis/CaTSFollowing
- Zarrin Basharat added an answer:4How to predict nucleotide changes causing amino acid changes cause disease or not?I have already checked by using SIFT and Poly-Phen softwares. Is there any other software?Following
- Jaimin S Patel added an answer:10How to check amino acid change from an identified SNP?I have found an SNP in a gene under my research. In mutationtaster, it appears to be disease causing. How can I check for amino acid change caused by single bp change? Any online tool?Following
- Alka Ekbote added an answer:1Why does polymerase slippage occur when there are homopolymeric repeats during sequencing?-Slippage occurs when the growing strand temporarily dissociates from the template,In case of homopolymers it can reassociate at a different spot - say, one nucleotide forward or back from where it started. This will make every individual band becomes a family of closely-spaced peaks giving a 'roller coaster' look to the chromatogram. Which is often regarded as slippage.Following
- Charles E. De Bock added an answer:2Is there any possibility that retrovirally induced animals can shed the virus, which can in turn infect other animals in the lab?We are planning to induce chronic myeloid leukemia in mouse. Our plan is to induce CML by infecting bone marrow cells from donor mouse with retroviral vector, and then implanting into recipient mice. My question is, do these recipient mice pose any threat to other mice (non-transplanted) by shedding retroviral particles by any means? What I understood is that induced animals are not a threat to other animal, but I just wanted to confirm it from some literature before any final conclusions for lab ethical approval.In our laboratory we routinely carry out such experiments but for genes involved in ALL.
When you generate the retrovirus you will transfect two vectors into a packaging cell line. For example you will have pMIG or MSCV encoding your oncogene and you will transfect together with a packaging vector such sa Ecopac. This ecopac vector encodes the envelope proteins to generate the complete viral particle allowing infection of mouse/rat cells. However, the virus itself is replication defective - as the components of the virus were split between the two vectors you used.
You will then harvest the virus from the packaging cell line, transduce you lineage negative cells from donor mice and then inject these into your irradiated recipient mice. These donor cells are transduced but will not be able make virus.
So rest assured that your transduced mice will not be able to infect other mice at all.
All the best with your work.Following