Karyotyping? etc

[jem]

H!

So this is a slightly unusual post. But I was wondering if anyone could offer any advice?

To get to the point, I appear to have been born with an intersex condition, I've had a number of hidden surgeries, etc and I'm still unraveling a bunch of lies having been told to me by the medical profession (and this happens a lot to other's too).

Anywho, I'm looking to do karotyping on myself and perhaps some others... and I was wondering what is the procedure for doing this? I've had a lot of frustrations and difficulties going through the local health service to get these tests done, mainly because they simply don't want to do it as it costs money and is probably deemed by most doctors or endros to be a waste of time and indeed that can be the case, (they also generally don't have a clue about dsds/intersex conditions).  I actually work in a biomedical facility but I'm a physicist primarily, I dabble in tissue engineering growing cells on scaffolds, etc. So I've been asking around here and in the medical school about getting these tests done but I've been told its not so easy, contamination, etc etc 

So does anyone know of any papers / procedures for doing these tests? I have access to every type of equipment here. :-)

Ta

Jem

[Josiah Zayner]

Why karyotyping?

Have you thought about just attempting to sequence parts of your own DNA?

[Matt Harbowy]

Jem:

I'm interested in this as well. I think we need to come up with an open protocol for this. I assume the limiting step is a fluorescence microscope, which is $$$$, but this would make an interesting DIY build and application, even if just for simple curiosity. 

I think Eric Harness has been working on a fluorescent microscope at BioCurious, I have cc'ed them, correct me if I'm talking out of school.

Sorry for the list </aol> but mostly this is a "me too".

Matt Harbowy -hberg...@gmail.com

650 243 8467 - @hbergeronx on Twitter

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[Dakota Hamill]

Hey, I'd actually be really interested in that as well.  I'm actually low on certain pituitary peptide hormones and although thankfully it didn't affect the development of important organs, it has had a noticeable physical and mental impact.  

I was looking at some journal papers having to do with androgen insensitivity and 5-alpha-reductase mutations, though based on some blood-work, that might not be the issue.  

I spent an hour sifting through the NCBI database looking at the corresponding genes, and looked through the corresponding papers, but never found the primers they kept saying they were using.  Never found supplemental information either.

Karyotyping is expensive and from what my endocrinologist said, is only warranted in certain cases when things like Kallman's or Kleinfelter's syndrome is a potential diagnosis.  There are similar conditions in females, but the name escapes me.

Though my experience wasn't as horrendous as some other people's accounts I've read, finding an endo that will thoroughly investigate hormone problems relating to sex hormones is hard.

Steroids have such a bad rap that few doctors will treat males in the low-normal range, and even fewer will try to do Clomid HPTA restarts or use recombinant hCG for leydig cell stimulation.  I'm actually going to be investing some $ into RPRX which is in Phase 3 trials of the single isomer from clomiphene citrate (Androxal) to help boost T levels naturally without estrogen side effects from the normal Clomid which has both antagonistic and agonistic properties.

I've actually become pretty obsessed with the endocrine system because of everything I've been going through, and trying to think up better ways to do testing.  

Right now, immunoassays are generally used for a lot of hormone panels, and while LCMS-MS is more sensitive and accurate, it is more expensive.  There are variations in any analytical testing, and basing a potential life-long treatment off ONE panel is a terrible idea.  It's just a "snap shot" of your endocrine system.

Also, you can get private blood-work done in EVERY STATE IN THE US Except Massachusetts (where I live) and one other state which I can't remember.  

Fuck me for wanting to be pro-active about my health right?

After reaching out and finding many forums, I've come across a lot of people that feel quite dissatisfied with the way their treatment for certain conditions is handled.  Medicine is complex and there isn't always a smoking gun, but having access to cheaper blood testing and potential DNA tests could allow for the acquisition of a larger data set, versus 1 or 2 blood tests and then, here inject this for the rest of your life!

While I was diagnosed with http://en.wikipedia.org/wiki/Hypogonadotropic_hypogonadism another endo thinks I should be re-tested for thyroid conditions because basically 90% of the women on my mothers side have Hashimotos Antibodies and are hypothyroid.

I also took a shitload of corticosteroids when I had mono-nucleosis as a kid for a month and ever since my adrenals have always shown high-outside of range levels of cortisol and ATHC.

So....yeah...things can get very complicated very quickly.

I do continue to read about potential genes to be sequenced from my own genome, as I feel like that might be more "definitive" than one or two blood tests, but so far I've found it hard to find the corresponding genes, and data representing "normal" and "mutant".

I'll admit i didn't look very hard though as I've been busy with other things.

I guess it's wierd saying that all over the internet and knock on wood it never comes back to bite me in the ass!  But hey, we all have something off with us, and we're just a big ol sack of chemicals and feedback loops, so if we have the knowledge, I say go for it when it comes to finding better ways to test or diagnose certain conditions.

Obviously, still consult a doctor, but knowledge is power.  And the more data and knowledge we can gather before putting someone on pharmaceuticals for long periods of time, the better.

[jem]

Hei,

thanks for your replies!

Well the reason for karyotyping is that the closest I really have to a diagnosis is "gonadal dysgenesis" which could mean anything... eg pais (seems like a probable scenario), etc , so this test would be the way to go so I can narrow down possibilities. I want to know the truth and I want to know why I was sterile and what health problems I could end up facing later.... I think it would be great if there was an easier cheaper way to test for stuff like this, that is if you find out you will have problems with fertility etc, you can plan accordingly.

So in answer to sequencing parts of my dna, allow me to digress and tell a little story, recently one of the masters students came into my lab and we were talking about the bioprinters I work on, he talked about his thesis and how a nanosensor he was developing hadn't worked so I was talking to him about how to get it to work. He looked at me like I was speaking ancient greek until I realized he wasn't a physicist,  he was a chemist!  So going back to the question asked, Josiah, I'm not a biologist, I wouldn't know where to start! Most of my knowledge is in sensors and robotics. I'm merely a dabbler in chemistry and biology but I would like to know more.

Matt : I have access to flo microscopes, I used a confocal microscope for my thesis, however I have to pay to use them here... or rather its taken from the budget.. or I could always bribe someone with chocolate... everyone loves chocolate. :-)

Dakota : thanks for the answer,  my experience with medical professionals is that they always assume they know more than you do and condescendingly dismiss your data. I don't trust medical professionals in general, especially after lying to me and covering up surgeries. The doctors motto I believe is something like " above all else, do no harm" which is a total joke. That's why I'd like to ideally do my own tests and share with others what I have done.

So can anyone point to any papers that illustrate the process? As I'm based in a uni, I can download and share.

Thanx

Jem

[Dakota Hamill]

Yes it is unfortunately sometimes difficult to find a doctor who will listen to what you have to say with an open mind, and not just an open ear.  I've had the best luck at teaching hospitals, although that can differ I am sure.  I'd say showing up with knowledge in hand ready to propose a hypothesis and subsequent specific test might be your best bet in some cases.   I've had the best luck showing up with knowledge in hand or head and approaching the subject with delivery in medical or chemical terms, and in general have gotten a good response once the doctor realizes you aren't another Joe Smoe reading WebMD and convincing yourself you have cancer and 100 other diseases.

Or, maybe they are silently still thinking they know better but will appease the patient to give the illusion of them being cooperative and believing anything you just said.  

I don't know medicine so I can't comment fully, but I would say that scientists would probably make pretty damn good doctors.  The lack of analysis and data sets gathered prior to a diagnosis and treatment regiment I've experienced is pretty scary.  At least at the teaching hospital, the specialist wanted 3 sets of tests spaced 1 week apart to get an average of levels, whereas another one took one set of tests and said here, take this forever.

There is also the problem of waiting months in between seeing specialists, and if one sucks, you still have to pay!

But anyway, for your specific case, just keep googling and finding tons of papers relating to what it is you want to do.  I think a lot of the legwork is going to have to be done by you, if you want something bad enough, you'll figure out a way to do it.  Obviously having a personal relationship to the cause is an even bigger motivator.  

I guess I'm still not entirely sure what exactly you want to come up with a test for, just gonadal dysgensis or other intersex conditions in general?  

When you say hide surgeries, do you mean you had procedures done when you were to young to remember and your doctors/parents didn't want you to know about them for fear they might impact your perception of yourself?

Sorry if that's a personal question but I'm just trying to understand where you're coming from.

[jem]

Hi dakota,

I've had a lot of weird medical problems, the details of which I shan't go into. Yes I had surgeries when I was too young to remember and surgery later when I did remember but discovered later that they were doing something else.. The surgeries probably explain the development issues I had and medical problems encountered later on. I've had multiple scans, nothing too strange encountered, just minor stuff related to development issues, its been suggested I have an mri but I expect nothing significant will be found and again its another cost I can't really afford and another test I don't really want (I'm pretty sick of hospitals, pun intended)

For me this is more out of curiosity and eliminating a possibility.

One endro suggested a probable scenario and he could be right but I kind of disagree, he was speculating, and I would like to see proof, so I gave bloods for analysis, a karyotype test was supposed to have been carried out but it was either 1. not done or 2. they lost the paperwork (which is quite normal here).

[Nathan McCorkle]

Check out colchicine and giemsa stain, the first sets up cultured
cells for karyotyping and the latter stains the chromosomes.

www.pitt.edu/~super7/31011-32001/31081.ppt

If you don't have the time:
https://www.scienceexchange.com/services/karyotyping

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[jem]

coolio

Thanks for the info, it looks like one very convoluted process and perhaps time critical? I'd have to steal er borrow stuff from the other labs/groups  plus I would need to book time on the microscopes.. It would be good to see this process being done in minute detail like a video or something. I'll have a gander and see if I can find some more info later...

[Alexey Zaytsev]

Hey.

Not to say that you should not do karyotyping if you'd really like to,
but it might be easier (and a lot more reliable) to rather look at the
DNA sequence. There are companies that do genotyping for just 100
dollars, and that gives you a lot of additional fun and medical
information. Or if you like DIY, and have access to a PCR machine,
doing a few PCRs with primers specific to Y chromosome should be very
easy, cheap and reliable.

> https://groups.google.com/d/msgid/diybio/1717e021-0fa0-41b6-857a-ec4ed01e6174%40googlegroups.com.

[Cathal Garvey]

Genotyping by SNP (a la 23andpatentme) will not identify chromosomal
translocations, and might even miss some kinds of chromosomal number
abnormality (i.e. two X chromosomes, if they are the same parental
chromosome? Lemme think about that..).

So, given that many intersex or sex-developmental conditions are caused
by chromosome-level oddities, if you have for example a
clean-translocation you may only observe it by sequencing if you
sequence the point at which translocation occurred, which probably
varies a lot.

Now, quantitative or real-time PCR might pick up copy number variation,
so you could use that; there might even be someone on campus who'll do
it as a favour if you identify the areas you'd like to check for copy
number (start with marker genes on both sex chromosomes, for example)
and buy the primers in?

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[Josiah Zayner]

PCR can for sure work. I don't know if it is better than Karyotyping. I guess that will just depend on the disorder.

Because there are alot of disorders that are not just sex chromosome duplication.translocation related I would go here: http://en.wikipedia.org/wiki/Disorders_of_sex_development

or somewhere similar. Find which closely matches your symptoms.

then goto scholar.google.com and search for the disorder and PCR. Should give you primers and protocols, &c.

for example: http://scholar.google.com/scholar?q=PCR+turner+syndrome&btnG=&hl=en&as_sdt=0%2C5&as_ylo=1990&as_yhi=2001

I searched between the years 1990 and 2001. The earlier the better. PCR came about in the late 80s so your results won't be bogged down with Q-PCR, QF-PCR, real-time PCR, &c.

[jemma redmond]

That's really useful, thanks!

Btw the pseudo acronym dsd is considered controversial, most people with "Dsds" consider themselves intersex or hermaphrodites, not disordered, although some will use the term but refer to it as "differences". Medical professional's coined the term DSD's so they could fix stuff that didn't really need to be fixed.

It's a sore subject for a lotta people. Personally I'm kinda meh about it. Tomato, tomato whatever.

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[SC]

Hi Jem,

If your goal is to simply obtain your karyotype, it would save a lengthy learning curve if you contracted out for it.  Forget the whole medical doctor thing, those folks have a whole different vibe going on.   Approach it as obtaining research data.  Google "karyotype service", and you'll find a whole bunch of places that will do it for less than you'd imagine. 

Best of luck to you!