Daily Archives: January 3, 2022

When Keegan Joines was born as a low-birthweight baby, his parents saw the rough start as a mere bump in the road.

He plumped up quickly and everything seemed fine, said his mom, Susan Joines, an elementary school assistant and pediatric nurse by trade who lives in Castle Rock.

But by the time he was a year and a half, the Joineses were noticing developmental delays, including in walking and speech. A year later he would be diagnosed with Type 1 diabetes, an autoimmune disorder that destroys the bodys ability to create insulin, a vital hormone that allows the body to use glucose for energy.

We noticed more global abnormalities and we always just kept thinking, Something is related here. These are not all separate instances occurring.

Little did they know that, half a decade later, Keegan would be diagnosed with a rare genetic disorder KCNJ11, which affects the pancreas and the brain, resulting in developmental delay and juvenile-onset diabetes; the diagnosis was one of only 30 identified cases in the world at the time similar to his.

Even with the diagnosis, the search for answers continues, as the Joineses await therapies that could unlock his potential, allowing him to develop beyond the kindergarten-to-first grade level he functions at as a 10-year-old or could have no effect at all.

Unfortunately we dont know what his future is and you never can, even with a well-researched disorder, his mom said. Of course, the skys the limit for these kids. But, without having much research, we just dont know what to expect.

Theres just not enough kids like him to know.

Diagnostic odyssey

Alone, rare disease can be isolating. The U.S. National Institutes of Health defines a rare disorder as one that affects fewer than 200,000 people nationwide a definition created by Congress in the 1983 Orphan Drug Act, which established financial incentives for drug companies to develop medications for such conditions.

Collectively, however, rare is common, with approximately 7,000 known diseases affecting an estimated 25 million-30 million Americans nearly 10% of the population, according to the National Human Genome Research Institute. Worldwide,263 million to 446 million people are affected by rare disorders at any point in time between 3.5% and 6% of the global population, according to a 2019 article in the European Journal of Human Genetics.

With nearly 1 in 10 Americans and Coloradans affected by rare disorders, we likely encounter them daily at schools, at grocery shops, at places of worship, at workplaces. Some prefer to keep quiet, realizing the likelihood of being misunderstood is much greater than that of finding common ground. Others become vocal advocates, on a quest to raise awareness of a disease very few if any others have been diagnosed with. Others yet are oblivious to their disorders, on a quest for an answer to their health maladies that may never materialize.

The quest for a diagnosis the Joineses were on seemed foreign, rare, esoteric. But patients with rare disorders, on average, spent six to eight years and often untold thousands of dollars searching for an answer. While waiting, there's the uncertain no man's land of "undiagnosed," a label that can call into question one's symptoms and even one's sanity.

And when a diagnosis is finally received, it's not always the right one.

It's hard to diagnose people (with rare disorders) it takes a really long time, said Dr. Anne Pariser, director of the Office of Rare Diseases Research at the National Institutes of Healths National Center for Advancing Translational Sciences.

This happens so often in rare diseases. We call it the diagnostic odyssey.

Beyond frustration, accompanying the diagnostic odyssey are consequences that have the potential to take a toll on health and finances.

Being undiagnosed carries a substantial monetary and also human cost, Pariser said. People are treated for the wrong disease. They don't receive therapies that may be available, or there arent specific ways that we can intervene to lessen suffering.

Pariser cited a 2019 study by the EveryLife Foundation for Rare Diseases that estimated the economic cost of nearly 400 rare diseases in the U.S. that year at nearly $1 trillion, surpassing the estimated economic burdens for diabetes, heart disease and cancer among the costliest common chronic diseases.

Theres a mental cost, as well. Without a diagnosis, Susan Joines spent years attributing Keegans issues to pregnancy complications.

She blamed herself.

I had to go on beta blockers just for myself to survive because I wasnt profusing to him well, she said. He just wasnt thriving super well in my body. As we started seeing the gap widening between him and his peers, I was just like, I should have done better. That mom guilt just never goes away, because it always feels like you could do more. Even with your neurotypical kids, you always feel like youre not doing things well enough.

Its especially hard with a kiddo with extra needs because there's always so much you feel like you should or could be doing.

Complicating each patients search for answers is the reality that every rare-disease patient is unique. A patients symptoms can be caused by a single gene or chromosomal abnormality; multiple genetic errors; nongenetic factors; or a combination thereof. Even those considered to have the same disorder can have similar but distinct genetic errors that result in different presentations and health outcomes.

When we think about rare disease, each patient is essentially unique in their characteristics and that makes studying them, diagnosing them and understanding the public health impacts of rare disease patients very, very challenging, said Melissa Haendel, chief research informatics officer at the University of Colorado Anschutz Medical Campus and director of the National Center for Data to Health.

Case in point: Youll find differing estimates of the number of rare diseases, depending on the source and the country the data originates in more than 7,000, according to the National Institutes of Health; between 5,000 and 8,000, according to the World Health Organization; more than 6,000, according to Rare Diseases Europe.

Why do we care about how many rare diseases there are? Despite having 10% of the population potentially having a rare disease, the inability to count them really underlies an inability to identify them in the first place,Haendel said.

Its not the count that we care about. The fact that we cant count them is an indication of our inability to understand and define them, to diagnose them, to treat them.

Zebras, not horses

For Elliott Wellnitz, 3, of Colorado Springs, the diagnostic odyssey was blessedly short seven or eight months, as his mom, Christine, recalls.

A few things were off during the pregnancy Christine had only one artery in her umbilical cord instead of two, and Elliott was born prematurely but we didnt know at the time we were going to have a special-needs kid, she recalls.

But soon medical providers began to point out other anomalies a widely spaced big toe, port wine stains under his lips, an abnormally large head.

A basic genetic test showed no abnormalities, nor did a more sophisticated test.

The Wellnitzs were sent home with a tank of oxygen, a myriad of specialist appointments and no answers.

Several months later they learned he had Megalencephaly-capillary malformation syndrome an exceedingly rare genetic syndrome involving developmental delay, intellectual disability, poor muscle tone, parts of the body that are larger than usual and epilepsy. The disorder places patients at risk of fluid buildup in the brain and of cancerous tumors.

For Christine, the diagnosis has meant wearing more hats than parents already wear those of honorary therapist and educator, and that of an actual nurse.

When we got out of the hospital, no one pointed me in the right direction, said Christine, a hospice nurse whose husband stays home and cares for their son. I kind of had to figure it all out on my own. To me, thats the most frustrating aspect of this journey.

Susan Joines ran a group for special needs families where she lived last, working as a parent liaison to the early childhood education system. She found that many parents of children with rare disorders are desperate for answers and support and help.

I feel like very rarely do we get all three of those or even two out of three of those from providers.

Shes encountered a wide variety of personality types in doctors over countless visits, from the kind who say, Wait, give it time, theyll be fine, and then theyre 16 and end up with an autism diagnosis to the ones who say, Here you go, heres your childs list of problems, and well see you later, completing writing a kid off.

The former have served as roadblocks, the latter sources of immense hurt and devastation.

A psychologist once administered an IQ test on Keegan without Susan's permission or understanding, then delivered dismal news.

"Essentially they just said he was in the bottom 15th percentile, so probably under 40," she said of his score. "Just basically, 'Here is his IQ, and most likely he'll never live alone, he'll never be able to have a job or live independently,' and they just kind of left it there. We were like, 'Should we follow up with you?' They were like, 'No, this is it.'

"I feel like they gave him a life sentence."

With myriad rare disorders and such fuzzy definitions of many, its often a struggle to find a provider equipped to diagnose, no less treat, a rare disorder.

The problem at least partially originates in medical schools, where doctors in training are taught that when you hear hoofbeats, think horses, not zebras common things occur commonly. Dont give somebody a rare diagnosis if they probably have a common problem, Pariser said.

Providers need to be trained to look for zebra triggers, she said, invoking a symbol of the rare disease community: zebras, which each have a unique stripe pattern, as humans do fingerprints.

We want (doctors) to think of zebras, and we want them to think of zebras when they start seeing certain clusters of things: young age, high (medical system) utilization, multiple consults, having to travel great distances. Also, some of these what we call basket (medical) codes, like developmental delay or motor delay.

Rare diseases we have many, many diseases that affect small numbers of patients each and very few treatments. But when you consider this collectively, it really is a large public health problem.

How long is my child going to live?

Even with a diagnosis, the future is uncertain for Keegan.

Now 10, he performs in school three to four years behind grade level, even with extensive developmental services, Susan said.

Weve exhausted just about everything we think we can think to do for him. He struggles greatly with academics. He still cant write his name. He still isnt reading well," she said, adding that, regardless, Keegan is a joyful, humorous child who loves life, his family, school and friends.

To get him to recognize the word the thats a (school) goal for him, to have an 85% success rate on just recognizing that word. Hes in a severe special-needs program. We arent seeing progress like we hope. Just from the little bit we do know, early to late elementary school is typically, developmentally, where they see him maxing out."

For Christine Wellnitz, receiving a diagnosis was comfortingbut it didnt come with a road map of what to expect.

It was definitely a huge relief when there was actually a name for what he had, she said. I cried. But there wasnt a whole lot of information, even on life expectancy.

How long is my child going to live?

A Facebook group Christine joined, for those affected and their families, has a couple of older patients in their 30s and 40s, and that makes me happy.

But beyond that, we dont really know, and thats pretty scary. I always tell my husband, 'Right now were doing pretty good, and things are going pretty well, but I never hold my breath,' because every time I think that, something else pops up, or we need to see another specialist.

Elliotts pediatrician is supportive but doesnt have much to offer in the way of specialized knowledge.

Our pediatric doctor basically just goes with whatever I want, Christine said. If I call and say, I think I need this, she usually does it. I think part of it is because Im a nurse. I love our pediatric doctor, but I feel like there should be more specialists in town.

I really have no idea exactly where my child is when it comes to development. "I just know that hes somewhat delayed.

When it comes to enrolling her child in school and ensuring he receives proper education and support, I dont even know what the next steps are. I hear the school districts arent great when it comes to working with special needs children, and that just puts fear in my heart.

"I havent heard of one good district in this town, unfortunately."

For Susan Joines, looking to the future is equally tough.

My husband and I frequently think we might be forever-nesters, she said. Theres such a grieving process we will never put a cap on him, of course, but it is a process of this potential grief of maybe not achieving the life we hope for him. He may never drive. He may never graduate. He may never have a job. He may never get married. We may never see grandkids from him.

"Maybe we will," she added hopefully.

Susan leans on her personal faith in the fact that God made her son for a purpose and has plans for him.

"But its definitely not without grief," she said. "Its the heaviest and hardest thing we deal with on a regular basis."

Continued here:
Diagnostic odyssey: The lonely road walked by thousands of Coloradans with rare disorders - Colorado Springs Gazette

Abstract

Nuclear noncoding RNAs (ncRNAs) are key regulators of gene expression and chromatin organization. The progress in studying nuclear ncRNAs depends on the ability to identify the genome-wide spectrum of contacts of ncRNAs with chromatin. To address this question, a panel of RNADNA proximity ligation techniques has been developed. However, neither of these techniques examines proteins involved in RNAchromatin interactions. Here, we introduce RedChIP, a technique combining RNADNA proximity ligation and chromatin immunoprecipitation for identifying RNAchromatin interactions mediated by a particular protein. Using antibodies against architectural protein CTCF and the EZH2 subunit of the Polycomb repressive complex 2, we identify a spectrum of cis- and trans-acting ncRNAs enriched at Polycomb- and CTCF-binding sites in human cells, which may be involved in Polycomb-mediated gene repression and CTCF-dependent chromatin looping. By providing a protein-centric view of RNADNA interactions, RedChIP represents an important tool for studies of nuclear ncRNAs.

The majority of the eukaryotic genome is transcribed into coding and noncoding RNAs (ncRNAs). ncRNAs fulfill various functions both in the cytoplasm and the cell nucleus. Nuclear ncRNAs are attracted to different genomic regions and mediate the activation or repression of genes located in these regions and are also implicated in the genome three-dimensional organization (1, 2). In particular, recent studies indicate that RNA is essential for the chromatin targeting of Polycomb repressive complexes (3) and the organization of CTCF-dependent chromatin loops (4). However, these studies do not show which particular RNAs are involved.

Helpful in studying nuclear functions of ncRNAs are methods that map the sites of ncRNA associations with the genome. Initially developed for probing genomic interactions of one particular RNA, these methods are now available in an all-vs.-all version allowing simultaneous detection of the sites of chromosomal locations for all RNA molecules present in the nucleus (reviewed in ref. 1). An important drawback of these techniques, however, is that they do not disclose the proteins involved in RNADNA interactions.

To identify RNAs that could be involved in the functioning of DNA-bound proteins, we developed a hybrid approachRedChIPcombining an RNADNA proximity ligation technique [Red-C (5)] with chromatin immunoprecipitation (ChIP). Using antibodies against CTCF and EZH2, we identified various ncRNAs interacting with DNA at the sites of deposition of the above-mentioned proteins.

The RedChIP experimental procedure is analogous to HiChIP used for protein-centric mapping of DNADNA interactions (6), with the difference being that RNADNA interactions are analyzed instead of DNADNA interactions. Briefly, RNAproteinDNA complexes are cross-linked in living cells, and RNA and DNA fragments are ligated in situ using a bridge adapter. Ligated complexes are solubilized by sonication and subjected to IP using antibodies against a protein of interest. RNADNA chimeric molecules are then purified and sequenced, thus reporting RNADNA interactions that may be mediated by a particular protein and proteinDNA interactions that may be mediated by various RNAs (Fig. 1A). An aliquot of the material (input fraction) is processed without an IP step to record the total set of RNADNA interactions mediated by any proteins, as in a regular Red-C experiment.

RedChIP technique. (A) Outline of the experimental procedure. (B) A region of Chr17 encompassing Hoxb genes showing distribution of DNA and RNA portions in IP (RedChIP) and input (RedC) fractions from experiments with EZH2 and CTCF antibodies. Shown alongside are ChIP-seq peaks of EZH2 in H1-hESCs (human embryonic stem cells) and ChIP-seq peaks of CTCF in K562 cells as well as total RNA-seq profiles for H1-hESCs and K562 cells (from ENCODE). (C) Distribution of DNA portions around EZH2 peaks in hESCs and CTCF peaks in K562. (D) Ratio of the number of RNA contacts detected in different chromatin types in IP fraction to the number of RNA contacts detected in the same chromatin types in input fraction.

We used antibodies against EZH2, a catalytic subunit of the PRC2 complex, to study the Polycomb-dependent RNADNA interactome in human embryonic stem cells. We also used antibodies against CTCF to study the CTCF-dependent RNADNA interactome in human K562 cells. DNA portions of the chimeric molecules showed a clear preference for the binding sites of corresponding proteins in the IP fraction (Fig.1 B and C), indicating successful IP. Accordingly, we observed an enrichment of DNA portions in chromatin types typical for poised promoters and Polycomb-repressed regions in the IP fraction from the EZH2 experiment and an enrichment of DNA portions in chromatin types typical for insulators and promoters in the IP fraction from the CTCF experiment (Fig. 1D). Meanwhile, RNA portions of the chimeric molecules showed correlation with RNA-sequencing (RNA-seq) profiles both in IP and input fractions (Fig. 1B), reflecting the origination of RNA portions from various transcripts. We combined the contacts of RNA portions originating from a single gene, thus obtaining a whole-genome contact profile for each annotated RNA.

We then focused on the analysis of contacts of individual RNAs in the experiment with EZH2 antibodies. We first aimed to identify cis-acting RNAs that fulfill their functions in the vicinity of an encoding gene. For each RNA, we selected a fraction of cis contacts established with DNA regions surrounding the gene (1 Mb of gene boundaries including the gene) and compared the number of cis contacts between EZH2-precipitated and input fractions. We found that the degree of enrichment in the IP fraction correlated with the percentage of contacts detected in Polycomb-specific and poised promoter-specific chromatin types but not any other chromatin types in the area under study (Fig. 2A). We identified 10 long intergenic ncRNAs (lincRNAs) with a fold enrichment of >1.3 in both replicates (Fig. 2C). Among the identified RNAs is Kcnq1ot1 (fold change = 1.5), a well-known example of antisense lincRNA involved in the Polycomb-mediated silencing of several genes in the same locus (7). High fold enrichment was also observed for AC078778, antisense lincRNAs from the HOXC locus. Notably, antisense RNAs, on average, demonstrate higher fold enrichment than other lincRNAs and mRNAs of protein-coding genes (Fig. 2G), indicating the enrichment of the group of antisense RNAs with RNAs that may mediate Polycomb targeting.

Identification of ncRNAs associated with genomic regions occupied by EZH2 in hESCs and by CTCF in K562 cells. (A) Ratio of the number of cis contacts of individual RNAs between EZH2-precipitated and input fractions (x axis) vs. the percentage of cis contacts detected in different chromatin types in EZH2-precipitated fraction (y axis). (B) The same as A for CTCF-precipitated fraction. (C and D) Ratio of the number of cis (C) or trans (D) contacts of individual RNAs between EZH2-precipitated and input fractions for rep1 and rep2. (E and F) Ratio of the number of cis (E) or trans (F) contacts of individual RNAs between CTCF-precipitated and input fractions for rep1 and rep2. (G) Distribution of fold changes from C for different RNA biotypes (antisense, n = 44; linc, n = 162; protein coding, n = 4,184). *P < 0.05, ***P < 0.001. (H) Intersection of RNAs enriched in RedChIP and fRIP-seq.

At the final step of the analysis, we searched for trans-acting RNAs that could participate in Polycomb functioning genome-wide. We compared the number of trans contacts (contacts with nonparental chromosomes) for each RNA between EZH2-precipitated and input fractions and looked for RNAs showing an elevated number of contacts in the IP fraction. The highest enrichment was observed for antisense RNA KIF5C-AS1, snRNA RNU5B-1, and SNORD3a RNA (Fig. 2D). These RNAs are good candidates to act as global mediators of Polycomb activity.

The above types of analysis were then performed for the data from the experiment with CTCF antibodies. In the analysis of cis-acting RNAs, we observed a correlation of RNA enrichment in the CTCF-precipitated fraction with the percentage of contacts detected in promoter-, enhancer-, and insulator-specific chromatin type (Fig. 2B). We identified seven lincRNAs with a fold enrichment of >1.3 in both replicates (Fig. 2E). These lincRNAs might participate in loading CTCF to its DNA sites and organization of promoter-enhancer specific and other chromatin loops within genomic loci from where lincRNAs are produced. In the analysis of trans-acting RNAs, the highest fold enrichment was observed for snRNA RNU12 (Fig. 2F). Notably, U12 RNA is the second top by the total number of contacts among all RNAs in K562 cells (1.1% of all contacts). The potential involvement of RNU12 RNA in the functions of CTCF requires further experimental evidence.

Remarkably, the set of RNAs enriched in RedChIP significantly intersects the set of RNAs enriched in RNA IP (formaldehyde RNA IP-sequencing, fRIP-seq) experiments (Fig. 2H). Importantly, 18 of 22 ncRNAs overrepresented in CTCF- and EZH2-RedChIP samples are fRIP-positive, indicating these ncRNAs indeed interact with the studied proteins.

Collectively, the present study results demonstrate the utility of the RedChIP protocol for identifying RNAs that may target nonhistone proteins to various locations on chromosomes or mediate interactions of these proteins with DNA. The identification of RNAs that are known to target Polycomb complexes to repressed genomic domains strongly supports the validity of the experimental approach, whereas identifying a set of RNAs possessing similar characteristics will stimulate studies of their possible role in Polycomb and CTCF functioning. The RedChIP technique can be used for identifying RNAs associated with genomic regions occupied by any protein of interest.

Cells are fixed with formaldehyde, DNA is fragmented with NlaIII restriction enzyme, and the ends are blunted and A-tailed. RNA 3 ends are ligated to a biotinylated bridge adapter followed by ligation of the opposite ends of the bridges with DNA ends in spatial proximity. Ligated complexes are solubilized by sonication, immunoprecipitated, and washed. RNADNA chimeras are purified, and DNA is digested with MmeI restriction enzyme. After biotin pull-down, reverse transcription is initiated from the bridge with template switching at the RNA 5 end, allowing for the incorporation of an Illumina adapter. Another Illumina adapter is ligated to DNA ends, and the chimeras are amplified and paired-end sequenced. The detailed protocol and sequencing data processing are described in SI Appendix. For sample processing statistics, refer to Dataset S1. For read processing statistics, refer to Dataset S2.

Raw fastq reads and processed TSV files with contacts are available at Gene Expression Omnibus (accession no. GSE174474). The code for read processing is available as RedClib on GitHub: https://github.com/agalitsyna/RedClib.

This work was supported by the Russian Science Foundation (21-64-00001) and by the Russian Ministry of Science and Higher Education (075-15-2021-1062).

Author contributions: A.A. Gavrilov, E.L.A., and S.V.R. designed research; A.A. Gavrilov and E.B.D. performed research; A.A. Gavrilov, R.I.S., M.D.M., and A.A. Galitsyna analyzed data; and A.A. Gavrilov and S.V.R. wrote the paper.

The authors declare no competing interest.

This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.2116222119/-/DCSupplemental.

Read more:
RedChIP identifies noncoding RNAs associated with genomic sites occupied by Polycomb and CTCF proteins - pnas.org

GENE & CELL MEDICINE LTD located in Israel and Singapore started a new project:LifeBank Chain (LBC). The project LBC plans to build a genetic and cell data collaboration platform.

Genetic research seeks to understand the process of trait inheritance from parents to offspring.Human genetic research is revealing the nature of human bioinformatics and giving scientists a powerful approach to study various health issues of human life.

Cell research focuses on stem cell and immune cell therapies, which are an extremely promising approach for the treatment of many diseases with an immune component including cancer, autoimmune disease, and chronic inflammation.

The wide applications of these new biological technologies in the medical field greatly reshaped the traditional pharmaceutical industry, whose focus was not only put on the treatment of disease as before but also on gene diagnosis and prevention, which opened the door to the world of a personalized and precise medicine.

Blockchain is an emerging technology that has attracted increasing attention from both researchers and practitioners. The functionalities of blockchain technology and smart contracts provide an opportunity over the large gene and cell data to support genetic and cell data integrity and security while giving patients control over their own data.

LBC plans to build a genetic and cell data collaboration platform incorporating an extensible cross-chain service system based on individual and institutional nodes. The platform product service layer abstracts all typical kinds of gene and cell blockchain applications and provides the full functions and implementation framework of typical applications.

The goal ofLifeBank Chain (LBC) is to establish a global-level service platform for sharing and utilizing human genetic and cell data through secured blockchain technologies.The LBC blockchain is designed to provide genetic and cell research industry partners with enterprise-level blockchain infrastructure, industry solutions, and secure, reliable, and flexible blockchain services. LBC will work together with medical practitioners to provide full-solution ancillary reagent services and provide flexible and pioneering tools to simplify therapy workflow at every step of the medical process.

LBC will form a professional and shared social organization LBC Life Alliance inviting life technology companies, scientific research institutes, medical institutions, etc. to jointly solve medical, health, disease, and public health problems, and jointly build the application standards of gene and stem cell medical technology on the blockchain, and contribute to the cause of human health.

LifeBank Chain enables healthcare professionals to manage the medical data and do research in an auditable, transparent, and secure way on LBCs distributed network. LBC continues to closely monitor the evolution of genetics and cell therapy in different medical subspecialties around the world.

View post:
LifeBank Chain (LBC) Focuses on Research and Development in the Field of Genetics and Cell Science - Markets Herald