Somatic, germ-cell, and whole sequence DNA in cell lineage and disease profiling

Curator: Larry H Bernstein, MD, FCAP

In humans, mitochondrial DNA spans about 16,500 DNA building blocks (base pairs), representing a small fraction of the total DNA in cells. Mitochondrial DNA contains 37 genes, essential for normal mitochondrial function and thirteen of them provide instructions for making enzymes involved in inner membrane function. The remaining 24 genes are transcribed into transfer RNA (tRNA) and ribosomal RNA (rRNA), which are needed to transfer amino acids into proteins.

Somatic mutations occur in the DNA of certain cells during a person’s lifetime and typically are not passed to future generations.  They differ from germ-line mutations that have a lineal descent from the maternal parent, and they occur later in life.  Mutations in the sperm DNA are not carried on to future generations, as the sperm mitochondria are destroyed after the egg is fertilized.

There is limited evidence linking somatic mutations in mitochondrial DNA with certain cancers, including breast, colon, stomach, liver, and kidney tumors. These mutations might also be associated with cancer of blood-forming tissue (leukemia) and cancer of immune system cells (lymphoma).  There are many heritable diseases that are related to germ-line mutations, and germ-line mutations have a role in many common diseases.  Mitochondrial DNA is particularly vulnerable to the effects of reactive oxygen species (ROS), and with a limited ability of the mitochondrion to repair itself, ROS easily damage mitochondrial DNA.  The repair mechanism is tied to ubiquitinylation system.  A  list of disorders associated with mitochondrial genes  is provided from Wikipedia.

Inherited changes in mitochondrial DNA may be associated with pathologies in growth and development, and multiorgan system disorders, as mutations disrupt the mitochondria’s ability to generate the cell’s energy. The effects of these conditions are most pronounced in organs and tissues with high energy requirements (such as the heart, brain, and muscles). Although the health consequences of inherited mitochondrial DNA mutations vary widely, some frequently observed features include muscle weakness and wasting, problems with movement, diabetes, kidney failure, heart disease, loss of intellectual functions (dementia), hearing loss, and abnormalities involving the eyes and vision.

A buildup of somatic mutations in mitochondrial DNA has been considered to have a role in or associated with increased risk of certain age-related disorders such as heart disease, Alzheimer disease, and Parkinson disease, and the severity of many mitochondrial disorders is thought to be associated with the percentage of mitochondria affected by a particular genetic change. Consequently, the progressive accumulation of these mutations over a person’s lifetime may play a role in aging.

Mitochondrial DNA is typically diagrammed as a circular structure with genes and regulatory regions labeled.

Mitochondrial DNA

http://ghr.nlm.nih.gov/html/images/chromosomeIdeograms/mitochondria/wholeMitochondria.jpg

Additional Resources:

• Additional NIH Resources – National Institutes of Health

NHGRI Talking Glossary: Mitochondrial DNA

• Gene Reviews – Clinical summary (6 links)
• Research Resources – Tools for researchers (5 links)
• OMIM – Genetic disorder catalog (2 links)
• Genetics Education
• Human Genome Project
• Resources for Genetics Researchers

mtDNA : The Eve Gene –  by Stephen Oppenheimer

Mutations are a cumulative dossier of our own maternal prehistory. The main task of DNA is to copy itself to each new generation. We can use these mutations to reconstruct a genetic tree of mtDNA, because each new mtDNA mutation in a prospective mother’s ovum will be transferred in perpetuity to all her descendants down the female line. Each new female line is thus defined by the old mutations as well as the new ones.

By looking at the DNA code in a sample of people alive today, and piecing together the changes in the code that have arisen down the generations, biologists can trace the line of descent back in time to a distant shared ancestor. Because we inherit mtDNA only from our mother, this line of descent is a picture of the female genealogy of the human species.

formation of gene trees

The diagram above shows the drawing of gene trees using single mutations

http://www.bradshawfoundation.com/journey/images/gene-diagram3.gif

Not only can we retrace the tree, but by taking into account here the sampled people came from, we can see where certain mutations occurred – for example, whether in Europe, or Asia, or Africa. What’s more, because the changes happen at a statistically consistent (though random) rate, we can approximate the time when they happened.  This has made it possible, during the late 1990s and in the new century, for us to do something that anthropologists of the past could only have dreamt of: we can now trace the migrations of modern humans around our planet.

It turns out that the oldest changes in our mtDNA took place in Africa 150,000 – 190,000 years ago. Then new mutations start to appear in Asia, about 60,000 – 80,000 years ago. This tells us that modern humans evolved in Africa, and that some of us migrated out of Africa into Asia after 80,000 years ago.  A method established in 1996, which dates each branch of the gene tree by averaging the number of new mutations in daughter types of that branch, has stood the test of time.

A final point on the methods of genetic tracking of migrations: it is important to distinguish this new approach to tracing the history of molecules on a DNA tree, known as phylogeography (literally ‘tree-geography’), from the mathematical study of the history of whole human populations, which has been used for decades and is known as classical population genetics.

The two disciplines are based on the same Mendelian biological principles, but have quite different aims and assumptions, and the difference is the source of much misunderstanding and controversy. The simplest way of explaining it is that phylogeography studies the prehistory of individual DNA molecules, while population genetics studies the prehistory of populations. Put another way, each human population contains multiple versions of any particular DNA molecule, each with its own history and different origin.

gene-diagram

The diagram above shows the tracing of gene spread geographically.
Green disks represent migrant new growth on the tree
http://www.bradshawfoundation.com/journey/images/gene-diagram4.gif

http://www.bradshawfoundation.com/journey/eve.html

David Moskowitz, MD, PhD
Founder and President, GenoMed

Germline genes make the best drug targets

• They operate earliest in the disease pathway
• Unlike tissue-expressed genes, which operate years after the disease began
• But which everybody else is using as drug targets

Variation in germline DNA is where all disease starts

• Cancer patients overexpress oncogenes and underexpress tumor suppressors

beginning in their germline DNA

• Mutations in tumor DNA are “private”
• Each tumor is a “snowflake”

Tumor-expressed genes can be compensatory, not causative

• “Passengers, not drivers”
• We have the drivers

Tumorigenesis SNPs

Using a SNPnet™ covering only 1/3 of the genome, we found about

2,500 genes associated with each of 6 different cancers in whites

• Nobody else has found any yet
• This will change in 2-3 years

We estimate 10,000 genes per cancer

What cellular program takes up 1/3-1/2 of the genome?

What program takes up >1/3 of the genome?

• Differentiation…

Does sporadic cancer arise when a tissue stem cell fails to differentiate?

• In the embryo, the surrounding tissue expresses “fields”

Lent C. Johnson published a “field” based hypothesis of bone tumors that coincides with differentiation at the

1. METAPHYSIS
2. HYPOPHYSIS

and the type CELL – chondroblast, osteoblast, giant cell (osteoclast), fibroblast

Orthopedic surgeons use magnetic fields for healing

• of powerful transcription factors.
• Not so in adult life: a proliferating tissue stem cell is literally on its own.

Germlines hold the key to effective “differentiation therapy”

• Ideal for patients with stage 3-4 cancer
• Examples of differentiation therapy:

1. 1,25-vitamin D and
2. retinoic acid

Non-toxic but more effective treatment for late stage disease,

GenoMed’s 2,500 cancer-causing genes:

• ½ are oncogenes,
• ½ are tumor suppressors

Design inhibitors to oncogenes

• Screen 1st for toxicity;
• genomic epidemiology guarantees clinical efficacy

Jewish Heritage Written in DNA

By Kate Yandell | Sept 9, 2014

Fully sequenced genomes of more than 100 Ashkenazi people clarify the group’s history and provide a reference for researchers and physicians trying to pinpoint disease-associated genes.

A whole-genome sequence study from 128 healthy Jewish people is aimed at identifying disease-associated variants in the jewish population of Ashkenazi ancestry, according to a study published Sept 9 in Nature Communications. The library of sequences confirms earlier conclusions about Ashkenazi history hinted at by more limited DNA sequencing studies. The sequences point to an approximate 350-person bottleneck in the Ashkenazi population as recently as 700 years ago (1400 A.D.), and suggest that the population has a mixture of European and Middle Eastern ancestry.

The study “provides a very nice reference panel for the very unique population of Ashkenazi Jews,” said Alon Keinan, who studies human population genomics at Cornell University in New York. Keinan
is acknowledged in the study but was not involved in the research.

“One might have thought that, after many years of genetic studies relating to Ashkenazi Jews . . . there would be little room for additional insights,” Karl Skorecki of the Rambam Healthcare Campus
in Israel who also was not involved in the study wrote in an e-mail to The Scientist. The study, he added, provides “a powerful further validation and further resolution of the demographic history of
the Ashkenazi Jews in relation to non-Jewish Europeans that is reassuringly consistent with inferences drawn from two decades of studies using uniparental regions . . . and from array-based data.”

Itsik Pe’er, coauthor of the new study and an associate professor of computer science at Columbia University in New York City, recalled that several years ago, he and his colleagues kept running into the same problem as they tried to understand the genetics of disease in Ashkenazi populations. They were comparing their Ashkenazi samples to the only control genomes that were available, which were of largely non-Jewish European origin. The Ashkenazi genomes had variation that was absent in these general European genomes, making it hard to distinguish rare variants in Ashkenazi people.

“Technology is there to tell us everything in that [Ashkenazi] patient’s genome, but the genome was not there to distinguish the variants that are there and to tell us whether they are normal or whether we should get worried,” said Pe’er. Pe’er’s group teamed up with researchers from additional universities and hospitals in the U.S., Belgium, and Israel to sequence a collection of healthy Ashkenazi people’s genomes. The panel of reference sequences performs better than a group of European genomes at filtering out harmless variants from Ashkenazi Jewish genomes, thereby making it easier to identify potentially harmful ones. According to Pe’er, researchers will also be able to use the panel to infer
more complete sequences from partially sequenced genomes by looking for familiar sequences from the reference genomes.

The team also used its data to better understand the history of the Ashkenazi Jewish people through analyzing both level of similarity within Ashkenazi genomes and between Ashkenazi and non-Jewish
European genomes. By analyzing the length of identical DNA sequences that Ashkenazi individuals share, the researchers were able to estimate that 25 to 32 generations ago, the Ashkenazi Jewish population shrunk to just several hundred people, before expanding rapidly to eventually include the millions of Ashkenazi Jews alive today. Further, the researchers concluded that modern Ashkenazi Jews likely have an approximately even mixture of European and Middle Eastern ancestry. This suggests that after the Jewish people migrated from the Middle East to Europe, they recruited people from local European populations.

These results are compatible with those of prior work on mitochondrial DNA (mtDNA), which is passed on maternally. This prior work suggested that Ashkenazi men from the Middle East intermarried with local European women. The Ashkenazi population “hasn’t been likely as isolated as at least some researchers considered,” said Keinan.

Finally, the newly sequenced genomes shed light on the deeper history of Europe, showing that the European and Middle Eastern portions of Ashkenazi ancestry diverged just around 20,000 years ago.

“This is, I think, the first evidence from whole human genomes that the most important wave of settlement from the Near East was most likely shortly after the Last Glacial Maximum  . . . and, notably, before the Neolithic transition—which is what researchers working on mitochondrial DNA have been arguing for some years,” Martin Richards, an archeogeneticist at the University of Huddersfield in the U.K., told The Scientist in an e-mail.

Skorecki noted that the new study “demonstrates the utility of sequencing whole genomes in a diverse population… with sufficient numbers of samples, parent population information, and
computational analytic power, we can expect important and surprising utilities for personal genomic and insights in terms of human demographic history from whole genomes.”

1. Carmi et al., “Sequencing an Ashkenazi reference panel supports population-targeted personal genomics and illuminates Jewish and European origins,” Nature
   Communications,
   http://dx.doi.org:/10.1038/ncomms5835, 2014.

Added Layers of Proteome Complexity

By Anna Azvolinsky | July 17, 2014

Scientists discover a broad spectrum of alternatively spliced human protein variants within a well-studied family of genes.

There may be more to the human proteome than previously thought. Some genes are known to have several different alternatively spliced protein variants, but the Scripps Research Institute’s Paul Schimmel and his colleagues have now uncovered almost 250 protein splice variants of an essential, evolutionarily conserved family of human genes. The results were published today (July 17) in Science.

Focusing on the 20-gene family of aminoacyl tRNA synthetases (AARSs), the team captured AARS transcripts from human tissues—some fetal, some adult—and showed that many of these messenger RNAs (mRNAs) were translated into proteins. Previous studies have identified
several splice variants of these enzymes that have novel functions, but uncovering so many more variants was unexpected, Schimmel said. Most of these new protein products lack the catalytic domain but retain other AARS non-catalytic functional domains. “The main point is that a vast new area of biology, previously missed, has been uncovered,”
said Schimmel.

“This is an incredible study that fundamentally changes how we look at the protein-synthesis machinery,” Michael Ibba, a protein translation researcher at Ohio State University who was not involved in the work, told The Scientist in an e-mail. “The unexpected and potentially vast
expanded functional networks that emerge from this study have the potential to influence virtually any aspect of cell growth.”

The team—including researchers at the Hong Kong University of Science and Technology, Stanford University, and aTyr Pharma, a San Diego-based biotech company that Schimmel co-founded—comprehensively captured and sequenced the AARS mRNAs from six human tissue types using high-throughput deep sequencing. While many of the transcripts were expressed in each of the tissues, there was also some tissue specificity.

Next, the team showed that a proportion of these transcripts, including those missing the catalytic domain, indeed resulted in stable protein products: 48 of these splice variants associated with polysomes. In vitro translation assays and the expression of more than 100 of these variants in cells confirmed that many of these variants could be made into
stable protein products.

The AARS enzymes—of which there’s one for each of the 20 amino acids—bring together an amino acid with its appropriate transfer RNA (tRNA) molecule. This reaction allows a ribosome to add the amino acid to a growing peptide chain during protein translation. AARS
enzymes can be found in all living organisms and are thought to be among the first proteins to have originated on Earth.

To understand whether these non-catalytic proteins had unique biological activities, the researchers expressed and purified recombinant AARS fragments, testing them in cell-based assays for proliferation, cell differentiation, and transcriptional regulation, among other
phenotypes. “We screened through dozens of biological assays and found that these variants operate in many signaling pathways,” said Schimmel.

“This is an interesting finding and fits into the existing paradigm that, in many cases, a single gene is processed in various ways [in the cell] to have alternative functions,” said Steven Brenner, a computational genomics researcher at the University of California, Berkeley.

The team is now investigating the potentially unique roles of these protein splice variants in greater detail—in both human tissue as well as in model organisms. For example, it is not yet clear whether any of these variants directly bind tRNAs.

“I do think [these proteins] will play some biological roles,” said Tao Pan, who studies the functional roles of tRNAs at the University of Chicago. “I am very optimistic that interesting biological functions will come out of future studies on these variants.”

Brenner agreed. “There could be very different biological roles [for some of these proteins]. Biology is very creative that way, [it’s] able to generate highly diverse new functions using combinations of existing protein domains.” However, the low abundance of these variants
is likely to constrain their potential cellular functions, he noted.

Because AARSs are among the oldest proteins, these ancient enzymes were likely subject to plenty of change over time, said Karin Musier-Forsyth, who studies protein translational
at the Ohio State University. According to Musier-Forsyth, synthetases are already known to have non-translational functions and differential localizations. “Like the addition of post-translational modifications, splicing variation has evolved as another way to repurpose protein function,” she said.

One of the protein variants was able to stimulate skeletal muscle fiber formation ex vivo and upregulate genes involved in muscle cell differentiation and metabolism in primary human skeletal myoblasts. “This was really striking,” said Musier-Forsyth. “This suggests
that, perhaps, peptides derived from these splice variants could be used as protein-based therapeutics for a variety of diseases.”

W.S. Lo et al., “Human tRNA synthetase catalytic nulls with diverse functions,” Science, http://dx.doi.org:/10.1126/science.1252943, 2014.

It’s Not Only in DNA’s Hands

By Ilene Schneider  LabRoots   Aug 22, 2014

Blood stem cells have the potential to turn into any type of blood cell, whether it is the oxygen-carrying red blood cells or the immune system’s many types of white blood cells that help fight infection. How exactly is the fate of these stem cells regulated? Preliminary findings from research conducted by scientists from the Weizmann Institute of Science and the Hebrew University are starting to reshape the conventional understanding of the way blood stem cell fate decisions are controlled, thanks to a new technique for epigenetic analysis developed at these institutions. Understanding epigenetic mechanisms (environmental influences other than genetics) of cell fate could lead to the deciphering of the molecular mechanisms of many diseases,
including immunological disorders, anemia, leukemia, and many more. The study of epigenetics also lends strong support to findings that environmental factors and lifestyle play a more prominent
role in shaping our destiny than previously realized.

The process of differentiation – in which a stem cell becomes a specialized mature cell – is controlled by a cascade of events in which specific genes are turned “on” and “off” in a highly regulated and accurate order. The instructions for this process are contained within the DNA itself in short regulatory sequences.

• These regulatory regions are normally in a “closed” state, masked by special proteins called histones to ensure against unwarranted activation. Therefore, to access and “activate”
  the instructions,
• this DNA mask needs to be “opened” by epigenetic modifications of the histones so it can be read by the necessary machinery.

In a paper published in Science, Dr. Ido Amit and David Lara-Astiaso of the Weizmann Institute’s Department of Immunology, along with Prof. Nir Friedman and Assaf Weiner of the Hebrew University of Jerusalem, charted – for the first time – histone dynamics during blood development. Thanks to the new technique for epigenetic profiling they developed, in which just a handful of cells – as few as 500 – can be sampled and analyzed accurately, they have identified the exact
DNA sequences, as well as the various regulatory proteins, that are involved in regulating the process of blood stem cell fate.

This research has also yielded unexpected results: As many as

• 50% of these regulatory sequences are established and opened during intermediate stages of cell development.

The meaning of the research is that epigenetics can be active at stages in which it had been thought that cell destiny was already set. “This changes our whole understanding of the process of blood stem cell fate decisions,” says Lara-Astiaso, “suggesting that the process is more
dynamic and flexible than previously thought.”

Although this research was conducted on mouse blood stem cells, the scientists believe that the mechanism may hold true for other types of cells. “This research creates a lot of excitement in the field, as it sets the groundwork to study these regulatory elements in humans,” says Weiner.

Largest Cancer Genetic Analysis Reveals New Way of Classifying Cancer

http://www.biosciencetechnology.com/news/2014/08/largest-cancer-genetic-analysis-reveals-new-way-classifying-cancer

Thu, 08/07/2014 – 2:24pm

Researchers with The Cancer Genome Atlas (TCGA) Research Network have completed the largest, most diverse tumor genetic analysis ever conducted, revealing a new approach to classifying cancers. The work, led by researchers at the UNC Lineberger Comprehensive
Cancer Center at the University of North Carolina at Chapel Hill and other TCGA sites, not only

• revamps traditional ideas of how cancers are diagnosed and treated, but could also have
• a profound impact on the future landscape of drug development.

“We found that one in 10 cancers analyzed in this study would be classified differently using this new approach,” said Chuck Perou, PhD, professor of genetics and pathology, UNC Lineberger member and senior author of the paper, which appears online Aug. 7 in Cell.
“That means that

• 10 percent of the patients might be better off getting a different therapy—that’s huge.”

Since 2006, much of the research has identified cancer as not a single disease, but many types and subtypes and has defined these disease types based on the tissue—breast, lung, colon, etc.—in which it originated. In this scenario, treatments were tailored to which
tissue was affected, but questions have always existed because some treatments work, and fail for others, even when a single tissue type is tested.

In their work, TCGA researchers analyzed more than 3,500 tumors across 12 different tissue types to see how they compared to one another — the largest data set of tumor genomics ever assembled, explained Katherine Hoadley, PhD, research assistant professor
in genetics and lead author. They found that

• cancers are more likely to be genetically similar based on the type of cell in which the cancer originated, compared to the type of tissue in which it originated. 

This is fundamental premise of pathology! (Larry Bernstein)  It goes back to Rudolph Virchow. 

“In some cases, the cells in the tissue from which the tumor originates are the same,” said Hoadley. “But in other cases, the tissue in which the cancer originates is made up of multiple types of cells that can each give rise to tumors. Understanding the cell in which the cancer originates appears to be very important in determining the subtype of a tumor
and, in turn, how that tumor behaves and how it should be treated.”

Perou and Hoadley explain that the new approach may also shift how cancer drugs are developed, focusing more on the development of drugs targeting larger groups of cancers with genomic similarities, as opposed to a single tumor type as they are currently developed.

One striking example of the genetic differences within a single tissue type is breast cancer.
The breast, a highly complex organ with multiple types of cells, gives rise to multiple types of breast cancer; luminal A, luminal B, HER2-enriched and basal-like, which was previously known. In this analysis, the basal-like breast cancers looked more like ovarian cancer
and cancers of a squamous-cell type origin, a type of cell that composes the lower-layer of a tissue, rather than other cancers that arise in the breast.

“This latest research further solidifies that basal-like breast cancer is an entirely unique disease and is completely distinct from other types of breast cancer,” said Perou. In addition, bladder cancers were also quite diverse and might represent at least three different disease types that also showed differences in patient survival.

As part of the Alliance for Clinical Trials in Oncology, a national network of researchers conducting clinical trials, UNC researchers are already testing the effectiveness of carboplatin—a common treatment for ovarian cancer—on top of standard of care chemotherapy for triple-negative breast cancer (TNBC) patients, of which 80 percent are the basal-like subtype. The results of this study (called CALGB40603)
were just published on Aug. 6 in the Journal of Clinical Oncology and showed a benefit of carboplatin in TNBC patients. This new clinical trial result suggests that there may be great value in comparing clinical results across tumor types for which this study highlights as having common genomic similarities.

As participants in TCGA, UNC Lineberger scientists have been involved in multiple individual tissue type studies including most recently an analysis of a comprehensive genomic profile of lung adenocarcinoma. Perou’s seminal work in 2000 led to the first discovery of breast
cancer as not one, but in fact, four distinct subtypes of disease.  These most recent findings should continue to lay the groundwork for what could be the next generation of cancer diagnostics.

Source: University of North Carolina at Chapel Hill School of Medicine

New Gene Tied to Breast Cancer Risk

Wed, 08/06/2014

Marilynn Marchione – AP Chief Medical Writer – Associated Press

It’s long been known that faulty BRCA genes greatly raise the risk for breast cancer. Now, scientists say a more recently identified, less common gene can do the same.

Mutations in the gene can make breast cancer up to nine times more likely to develop, an international team of researchers reports in this week’s New England Journal of Medicine.

About 5 to 10 percent of breast cancers are thought to be due to bad BRCA1 or BRCA2 genes. Beyond those, many other genes are thought to play a role but how much each one raises risk has not been known, said Dr. Jeffrey Weitzel, a genetics expert at City of Hope Cancer Center
in Duarte, Calif.

The new study on the gene- called PALB2 – shows “this one is serious,” and probably is the most dangerous in terms of breast cancer after the BRCA genes, said Weitzel, one of leaders of the study.

It involved 362 members of 154 families with PALB2 mutations – the largest study of its kind. The faulty gene seems to give a woman a 14 percent chance of breast cancer by age 50 and 35 percent by age 70 and an even greater risk if she has two or more close relatives with the disease.

That’s nearly as high as the risk from a faulty BRCA2 gene, Dr. Michele Evans of the National Institute on Aging and Dr. Dan Longo of the medical journal staff write in a commentary in the journal.

The PALB2 gene works with BRCA2 as a tumor suppressor, so when it is mutated, cancer can flourish.

How common the mutations are isn’t well known, but it’s “probably more than we thought because people just weren’t testing for it,” Weitzel said. He found three cases among his own breast cancer
patients in the last month alone.

Among breast cancer patients, BRCA mutations are carried by 5 percent of whites and 12 percent of Eastern European (Ashkenazi) Jews. PALB2 mutations have been seen in up to 4 percent of families with a history of breast cancer.

 Men with a faulty PALB2 gene also have a risk for breast cancer that is eight times greater than men in the general population.

Testing for PALB2 often is included in more comprehensive genetic testing, and the new study should give people with the mutation better information on their risk, Weitzel said. Doctors say that people with faulty cancer genes should be offered genetic counseling and may want to consider more frequent screening and prevention options, which can range from hormone-blocking pills to breast removal.

The actress Angelina Jolie had her healthy breasts removed last year after learning she had a defective BRCA1 gene.

The study was funded by many government and cancer groups around the world and was led by Dr. Marc Tischkowitz of the University of Cambridge in England. The authors include Mary-Clare King, the University of Washington scientist who discovered the first breast
cancer predisposition gene, BRCA1.

Study: http://www.nejm.org/doi/full/10.1056/NEJMoa1400382

Gene info: http://ghr.nlm.nih.gov/gene/PALB2

Structure of the DDB1–CRBN E3 ubiquitin ligase in complex with thalidomide

Eric S. Fischer, Kerstin Böhm, John R. Lydeard, Haidi Yang, …, J. Wade Harper, Jeremy L. Jenkins & Nicolas H. Thomä

Nature (07 Aug 2014); 512, 49–53  http://dx.doi.org:/10.1038/nature13527

Published online 16 July 2014

In the 1950s, the drug thalidomide, administered as a sedative to pregnant women, led to the birth of thousands of children with multiple defects. Despite the teratogenicity of thalidomide and its derivatives lenalidomide and pomalidomide,

• these immunomodulatory drugs (IMiDs) recently emerged as effective treatments for
  multiple myeloma and 5q-deletion-associated dysplasia.

• IMiDs target the E3 ubiquitin ligase CUL4–RBX1–DDB1–CRBN (known as CRL4CRBN) and
• promote the ubiquitination of the IKAROS family transcription factors IKZF1 and IKZF3 by CRL4CRBN.

Here we present crystal structures of the DDB1–CRBN complex bound to thalidomide,
lenalidomide and pomalidomide. The structure establishes that

• CRBN is a substrate receptor within CRL4CRBN and enantioselectively binds IMiDs.

Using an unbiased screen, we identified the

• homeobox transcription factor MEIS2 as an endogenous substrate of CRL4CRBN.

Our studies suggest that IMiDs block endogenous substrates (MEIS2) from binding to CRL4CRBN while the ligase complex is recruiting IKZF1 or IKZF3 for degradation.

This dual activity implies that

• small molecules can modulate an E3 ubiquitin ligase and thereby upregulate or downregulate the ubiquitination of proteins.

Curator’s Viewpoint:

The short pieces may not appear to be so closely connected, except for the last subject on the pharmaceutical targeting of an E3 ubiquitin ligase ubiquitination of proteins, but even in that case, we have to keep in mind that protein formation by amino acid transcription, remodeling, and recapture of amino acids are in equilibrium through ubiquitylation. So I put it there.  The DNA in populations ties some mutations to disease that is tied specifically to populations, not only the sephardic population, but in Asia as well.

The next article for consideration is methodological considerations.  The BRCA2 in the sephardic population is one of a number of mutations we can identify, extending to Tay Sachs disease, for instance.  How this might have occurred in the history of the jewish people is not so obvious, except perhaps in the segregation of the jewish population for centuries.  The mutation would be confined within the population with limited marriage outside of the jewish community.  It has been known for some time that there is a Cohen gene that traces back to the priests (Kohanim) of the Holy Temple, the descendents of Aaron (Aharon), the brother of Moses.  The priests would stand at the Ark and bless the congregation in the most holy convocation of Yom Kippur, according to tradition.  Marriages were arranged between the bride and the groom.  Of course, arranged marriages were also the case in other ethnic communities, and between the privileged.

That was dramatically the case during the reign of Queen Victoria of England, with Royal arrangements across Europe.
That would be a factor in the transmission of hemophilia, and in mental disorders in the Royal families. Haemophilia figured prominently in the history of European royalty in the 19th and 20th centuries. Britain’s Queen Victoria, through two of her five daughters (Princess Alice and Princess Beatrice), passed the mutation to various royal houses across the continent, including the royal families of Spain, Germany and Russia. Victoria’s son Prince Leopold, Duke of Albany suffered from the disease.  The Prince Leopold, Duke of Albany KG KT GCSI GCMG GCStJ (Leopold George Duncan Albert; 7 April 1853 – 28 March 1884) was the eighth child and fourth son of Queen Victoria and Prince Albert of Saxe-Coburg and Gotha. Leopold was later created Duke of Albany, Earl of Clarence, and Baron Arklow. He had haemophilia, which led to his death at the age of 30.  The sex-linked X chromosome disorder manifests almost entirely in males, although the gene for the disorder is located on the X chromosome and may be inherited from either mother or father. Expression of the disorder is much more common in males than in females. This is because, although the trait is recessive, males only inherit one X chromosome, from their mothers. Of course, this is classical Mendelian genetics. Victoria appears to have been a spontaneous or de novo mutation and is usually considered the source of the disease in modern cases of haemophilia among royalty. The mutation would probably be assumed today to have occurred at the conception of Princess Alice, as she was the only known carrier among Victoria and Albert’s first seven children. Leopold was a sufferer of haemophilia and her daughters Alice and Beatrice were confirmed carriers of the gene.

Cousin marriage is marriage between people with a common grandparent or other more distant ancestor. In various cultures and legal jurisdictions,  Marriages between first and second cousins account for over 10% of marriages worldwide, and they are common in the Middle East, where in some nations they account for over half of all marriages. Proportions of first-cousin marriage in the United States, Europe and other Western countries like Brazil have declined since the 19th century, though even during that period they were not more than 3.63 percent of all unions in Europe. Cousin marriage is allowed throughout the Middle East for all recorded history, and is used mostly in Syria. It has often been chosen to keep cultural values intact through many generations and preserve familial wealth. In Iraq the right of the cousin has also traditionally been followed and a girl breaking the rule without the consent of the ibn ‘amm could have ended up murdered by him. The Syrian city of Aleppo during the 19th century featured a rate of cousin marriage among the elite of 24% according to one estimate, a figure that masked widespread variation: some leading families had none or only one cousin marriage, while others had rates approaching 70%. Cousin marriage rates were highest among women, merchant families, and older well-established families.  The percentage of Iranian cousin marriages increased from 34 to 44% between the 1940s and 1970s. Cousin marriage among native Middle Eastern Jews is generally far higher than among the European Ashkenazim, who assimilated European marital practices after the diaspora.

The essential elements of the marriage contract were now an offer by the man, an acceptance by the woman, and the performance of such conditions as the payment of dowry. According to anthropologist Ladislav Holý, cousin marriage is not an independent phenomenon but rather one expression of a wider Middle Eastern preference for agnatic solidarity, or solidarity with one’s father’s lineage.

A 2009 study found that many Arab countries display some of the highest rates of consanguineous marriages in the world, and that first cousin marriages which may reach 25-30% of all marriages. Research among Arabs and worldwide has indicated that consanguinity could have an effect on some reproductive health parameters such as postnatal mortality and rates of congenital malformations.

In the terraced streets of Bradford, Yorkshire, a child’s death is anything but rare. At the boy’s inquest, coroner Mark Hinchliffe said Hamza Rehman had died because his Pakistan-born parents (shopkeeper Abdul and housewife Rozina) are first cousins. Muslims have practiced marriages between first cousins in non-prohibited countries since the time of the Quran.

Four years before, Hamza’s older sister, three-month-old Khadeja, had died of the same brain disorder which causes fits, sickness and chest infections. The couple had another baby born with equally devastating neurological problems.

A heartbroken Mr Rehman told the inquest that he and his wife were unsure whether to have any more children. The coroner expressed deep sympathy before saying that Hamza’s death should serve as a warning to others.

I have diverged somewhat onto the genetic risks of consanguinous marriages, which George Darwin, son of Charles Darwin, argues were had a small effect in then English society.  But most importantly, we see the larger factor here of social and familial inheritance, and also the concept of cultural identity.

Insofar as the somatic and mitochondrial mutations are concerned, I call attention to the finding in the GWAS study above discussed that the results were supportive of the conclusions from mtDNA.  This gives some reason to consider whether sufficient information is obtained from the mtDNA, without the more robust GWAS.  One cannot fully consider this without some knowledge of the methodology of specimen preparation.

It is not difficult to prepare mitochondria from cells and obtain a very good preparation before further analysis, whether of the membrane structures, the enzymatic activity, or of the DNA and RNA polynucleotides.  The separation is easily achieved with differential centrifugation.  On the other hand, the finding of the basal layer of epithelium having a different signature than the superficial layer, established by the genomic studies, but known histologically for non-neoplastic tissue, is a matter for cell separation methods that are not easy.  It is from the lower layer of cells that we derive carcinoma in-situ.  These cells were identified in breast, are expected to be found in uterus, and were like the cells in ovarian-cancer, which suggested the use of a common treatment regimen as adjunct in triple negative breast cancer and ovarian cancer.  The importance of a suuficiently prepared cellular specimen as opposed to tissue specimen can’t be taken for granted.

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