PLACENTAL DEFECTS ARE HIGHLY PREVALENT IN EMBRYONIC LETHALS

It is widely known that a functional placenta is vital for normal embryonic development, but how much it may contribute to embryonic lethality has never before been systematically studied. Our research, published in Nature, demonstrates for the first time a remarkable co-association between embryonic lethality and placental defects.


A healthy placenta is vital to sustain normal pregnancy, ensuring proper supply of nutrients and oxygen to the baby. Abnormalities in the placenta can therefore have serious repercussions on fetal development, even causing miscarriage. Despite this, remarkably little is known about the identity of genes essential for a normal, functioning placenta and even less about the extent to which placental abnormalities contribute to defects that can arise as the fetus develops.

EXTENT AND IMPACT

We screened more than 100 mouse mutant lines in which affected embryos die before or immediately at birth. Almost 70% showed serious abnormalities in the placenta; in extreme cases this resulted in a placenta incapable of supporting embryo development beyond an early stage (Figure 1), in others, abnormalities in the developing embryo were accompanied by abnormalities in the placenta.

FIGURE 1 – mouse mid-gestation embryos and placentas shown at the same magnification


LEFT: a normal, wild-type (WT) genotype. RIGHT: Nubpl mutation (MUT) shows a growth-retarded and developmentally delayed embryo that will not survive until birth.

The placentas are stained for a marker of the exchange surface (MCT4, in green) across which nutrients are transported from the mother to the embryo. Note the complete absence of this cell type from the MUT placenta. Red staining is for a cell surface protein (CDH1) demarcating the cells underneath the MCT4-positive layer (arrows), which are greatly reduced in number in the MUT placenta.

EMBRYO AND PLACENTA DEFECTS ARE LINKED

Not only do these results identify a large number of genes essential for normal development of the placenta; in addition they show an intriguing link between placental defects and abnormalities affecting the brain , heart and vascular system of the embryo itself. The research, led by Dr Myriam Hemberger and her colleagues at the Babraham Institute demonstrates how common placental abnormalities are when embryos develop abnormally.

RESCUING EMBRYONIC LETHALITY

The team examined in detail three different genes that cause embryonic lethality, and showed that for two of them the loss of the gene affected proper differentiation of placental cell types. For one of these genes they were also able to show that embryo death was a direct result of gene loss in the placenta, by providing the mutant embryo with a genetically normal placenta, which prevented embryo death.

Although the DMDD study uses mice, the results are likely to be just as relevant for studying human pregnancy and the role the placenta may play in pregnancy complications and the origins of birth defects in newborn babies.


REFERENCES

The Advance Online Publication on Nature, ‘Placentation defects are highly prevalent in embryonic lethal mouse mutants is available now .


All image and phenotype data gathered by the DMDD programme is freely available to the scientific community at dmdd.org.uk. The research described in this blog post was funded by the Wellcome Trust with support from the Francis Crick Institute.

JOIN US AT THE BSCB/BSDB/GENETICS SOCIETY MEETING


The joint BSCB/BSDB/Genetics Society Spring Meeting is taking place at the University of Warwick from 2 – 5 April, and DMDD will be there to exhibit our data. Come and visit our stand to find out more about phenotype data for embryonic lethal knockout mice.

Emily, Dorota, Emma and Jenna from the DMDD team will be on hand to give you a demo of the data and answer any questions you might have. You can even take away a free mug if you sign up to our email newsletter.

 

DMDD promotional mugs
Sign up to our mailing list at the meeting to get a free DMDD mug.

 

On Wednesday 5 April, Myriam Hemberger from the Babraham Institute will speak about her team’s work to phenotype placentas from DMDD embryonic lethal lines. Plus if you’re interested in our recent publications, you can visit one of the three posters we’re presenting:

Poster 66: Staging mouse embryos harvested on embryonic day 14 (E14.5). (Original article in Journal of Anatomy).

Poster 77: Highly variable penetrance of abnormal phenotypes in embryonic lethal knockout mice. (Original article in Wellcome Open Research).

Poster 83: Interpreting neonatal lethal phenotypes in mouse mutants.

 

We’re looking forward to meeting you!

NEW PHENOTYPE DATA FOR EMBRYOS AND PLACENTAS

Today, DMDD has released many new images and phenotypes for embryos and placentas from embryonic lethal knockout mouse lines. We now hold data on 70 mutant lines that have been phenotyped in detail using the Mammalian Phenotype ontology. The resulting data is freely available to the scientific community and is a potential goldmine of information about the genetic basis of developmental disorders.

The new data is accompanied by several exciting updates to our website. These include the ability to search for phenotypes by anatomy terms and the release of additional data about gene knockouts that are lethal very early in embryonic development. Highlights of the release, including examples of interesting phenotypes, can be found below.

 

SEARCH FOR PHENOTYPES USING ANATOMY TERMS

Following a major update to our search tool, users of the DMDD database can now search for phenotypes by anatomy term. This new functionality is designed to help researchers of specific organ or tissue types to quickly identify all phenotypes that are relevant to their studies. Choose from embryo and adult anatomy terms for both humans and mice.

 

Image of the DMDD search box
New search functunality gives users the option to search for phenotypes by anatomy term.

 

FIND GENE KNOCKOUTS THAT ARE LETHAL BEFORE E9.5

Around a third of the knockout lines studied by the DMDD programme have been found to cause lethality before 9.5 days of gestation. Although it is not possible for us to image and phenotype embryos from these lines, we have added them to our database and they can be found using the ‘search’ tool.

For a full list of lines that are lethal before E9.5, visit our Early lethals page.

 

EXPLORE THE NEW EMBRYO PHENOTYPE DATA

In our latest release we’ve made phenotypes available for 7 new knockout lines. These include Cfap53, which is known to be involved in left-right asymmetric patterning in humans. In mouse embryos we identified the phenotype ‘abdominal situs ambiguus’, in which the abdominal organs have neither the usual nor the mirror-image arrangement.

We have also released data on Fut8. This gene is linked to Leukocyte Adhesion Deficiency, a syndrome with symptoms including microcephaly and abnormality of the tongue and palate. In the mouse we identified various phenotypes related to the hypoglossal nerve, which controls movements of the tongue.

Some further highlights from the phenotypes released today include spinal haemorrhage in a Fut8 knockout embryo, a perimembraneous ventricular septal defect in an Arid1b knockout embryo and abnormal lens epithelium morphology in an Actn4 knockout embryo.

 

Click to view larger image.
A Fut8 knockout embryo found to have a spinal haemorrhage.

 

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Abnormal lens epithelium morphology in an Actn4 embryo. Lens epithelial cells are the parental cells responsible for growth and development of the lens.

 

 

In total, 162 distinct phenotypes were identified across 91 new mutant and wild-type control embryos. Phenotype data for a total of 81 new placentas has also been released.

 

A FULL LIST OF THE NEW DATA

HREM embryo image stacks added for Dennd4c, Dnajc8 and Pigf.

Embryo phenotypes added for Actn4, Arid1b, Cfap53, Cyp11a1, Dmxl2, Fut8 and Mfsd7c.

Placenta images and phenotypes added for B9d2, Cbx6, Commd10, Coq4, Dcx, Dhx35, Fam160a1, Gpatch1, Mfsd7c, Oaz1 and Smg1.

 

All image and phenotype data from the DMDD programme can be accessed at dmdd.org.uk. For assistance, please email contact@dmdd.org.uk.

HOW MICE ARE KEY TO UNDERSTANDING GENETIC HEART CONDITIONS

Nearly 1 in every 100 babies is born with a heart defect. These are the most common type of birth defect and can range from simple, symptomless cases to life-threatening conditions that require treatment within hours of birth.

Environmental influences such as excessive alcohol consumption or exposure to other toxins are known to cause heart defects. But many are also the result of faulty genes that can be passed on from one generation to the next. Identifying the genes involved is key to understanding when a baby might be at risk, and could also help us develop new ways to treat or prevent these heart defects. But with more than 20,000 genes in the human genome there are a mind-boggling number of possibilities.

A screen of mouse embryos by the DMDD programme (Deciphering the Mechanisms of Developmental Disorders) has identified a huge number of genes related to heart defects and other developmental abnormalities, and is now a potential goldmine of information on the genetic basis of heart conditions. Part of an open data initiative by the Wellcome Trust, the project has made all of its data available online, with a goal to spark further research into heart defects and rare disease.

 

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Photographs of isolated mouse embryo hearts. On the right is a ‘normal’ heart — this mouse was not missing any genes. On the left, due to a missing gene, the heart has a defect called ‘Double Outlet Right Ventricle’. Here, the heart’s two major arteries (the pulmonary artery and the aorta) both connect to the right ventricle. In a normal heart the aorta connects to the left ventricle.

INACTIVATING GENES TO UNDERSTAND RARE DISEASE

The DMDD team studies mouse embryos that have been bred to have a single one of their 20,000 genes inactivated – a process that’s known as knocking out a gene. As each embryo grows, any abnormalities in the way it develops are likely to be due to the missing gene and this provides powerful information about the sort of birth defects that the gene could be linked to. Although we might look very different, mice and humans are thought to share around 98% of our genes, so the effects of a missing gene on a developing mouse can tell us a lot about what we might expect if the same faulty gene is found in humans.

We concentrate specifically on genes that when knocked out cause a mouse embryo to die before birth.  On the face of it, studying these genes might not seem so important to people living with rare genetic diseases – these people have already survived past birth. But genes like these are a rich source of information about human genetic diseases.

Many rare disease patients have mutations that act as genetic dimmer switches, increasing or decreasing a gene’s activity rather than completely turning it off. A particular gene may only be partially turned on (called a hypomorph mutation) or it may be turned on more than normal (known as a hypermorph mutation). If we are able to understand the effect of fully turning off a gene, we can then begin to infer what might happen to patients who have a hypomorph or hypermorph mutation.

 

Like genetic dimmer switches, gene mutations can increase or decrease the activity of a gene, or knock it out completely.

 

By the end of the project, DMDD will have studied the effects of 250 different gene knockouts – it’s a huge opportunity to learn more about genetic causes of rare diseases in humans. And the biggest surprise in the results so far is the overwhelming prevalence of defects in the developing embryo hearts.


IDENTIFYING HEART DEFECTS

So far the team have analysed more than 200 embryos, each with one of 42 different genes knocked out. But unexpectedly, more than 80% of the gene deletions resulted in heart defects. Using an imaging technique called High Resolution Episcopic Microscopy the embryos were reconstructed in incredible 3D detail and studied down to the level of individual nerves and blood vessels. In the image below, the developing embryo heart is less than 2 mm across – smaller than the thickness of a matchstick – yet even the tiniest abnormality can be picked up.

“Even though we know that heart defects are common, we were really surprised that they were caused by more than 80% of our gene deletions. The data is a potential goldmine of information about the genetic basis of many different types of heart condition.” Dr Tim Mohun, DMDD.

 

Click to view larger image.
A 3D model of a mouse embryo heart using data from High Resolution Episcopic Microscopy.

 

The most common defects were problems with the walls that separate the right and left chambers of the heart. But there were also many defects in the heart valves, the outflow vessels (which carry blood out of the heart to the body or the lungs) and in the structure of the heart itself.

Several of the gene knockouts result in developmental defects that mimic known human genetic disorders. For example knocking out the genes Psph or Psat1 causes a range of developmental defects that appear similar to Neu-Laxova syndrome, a serious condition that leads to miscarriage or neonatal death. Tim Mohun commented “we know that many rare genetic diseases cause problems with the heart as it develops. Having so much new data about heart defects is exciting, because there is the potential for us to learn more about rare disease.”


THE PLACENTA: A NEW WAY TO UNDERSTAND THE HEART?

In the first study of its kind the placentas from a large collection of knockout embryos have also been analysed, and the results show an unexpected link between the placenta and the heart. Around a third of gene knockouts that cause placental abnormalities also cause a heart defect in the developing embryo. A more detailed statistical study of the data (publication in progress) has shown that this is a genuine link. Myriam Hemberger of the Babraham Institute, who performed the work as part of the DMDD programme, said “it could be that the restricted nutrient supply or blood circulation defects caused by an abnormal placenta adversely affect heart development. It does suggest there is more we could learn about some rare heart conditions by studying the placenta.

 [The results] suggest there is more we could learn about some rare heart conditions by studying the placenta. Myriam Hemberger, DMDD.


Initial analysis of the DMDD embryo and placenta data has shown it to be a rich resource for those studying rare disease and developmental disorders. But, unexpectedly, it may shed particular light onto the genetic basis of heart disease.

All data from the DMDD programme is freely available at dmdd.org.uk. For further information please email contact@dmdd.org.uk.

 

MEET THE DMDD TEAM – MYRIAM HEMBERGER

In a series of interviews we’re hearing from members of the DMDD programme. Who are they? What inspires them? And what do they hope that DMDD will achieve? This month we speak to Myriam Hemberger, who leads the team’s analysis of placentas from embryonic lethal knockout mouse lines.


What has been your main area of research in your career so far?

Ever since my PhD I have been interested in studying the mechanisms that underpin placental development. Over the years we have worked on identifying chromosome “hotspots” harbouring genes that are important for placentation, studying the physiological processes that ensure the normal function of the placenta, and exploring mechanisms for gene regulation known as “epigenetic mechanisms” that make placental cells different to any other cell type found in an embryo .

 

What inspired you to devote your career to understanding placental development?

The placenta is absolutely essential for reproduction. The earliest cells of the placental “trophoblast” lineage allow the embryo to implant, while later on in pregnancy the placenta is the organ solely responsible for providing nutrient and gas supply to the embryo as it grows. The importance of the placenta is reflected by the fact that defects in its function can cause some of the most common and serious pregnancy complications, such as preeclampsia, fetal growth restriction, or even miscarriage. Even increased risk of childhood and adult diseases, such as cardiovascular disease or diabetes, may originate in a malfunctional placenta. The placenta has a long–lasting impact on our health and wellbeing, but it has often been under-appreciated in research.

I have always been fascinated by the types of cell that make up the placenta. Some of them have truly extraordinary capacities: they can become gigantic in size by amplifying their genome dramatically and invade into foreign tissue, completely remodelling the structure of blood vessels (an ability only shared by metastatic tumour cells). In the placenta, invasive behaviour is needed to attach the embryo to the wall of the uterus and to access maternal blood supply. These (and many other) intriguing features captured my imagination, sparking my research in this area.

 

What do you think is the most exciting recent development in your field?

A major milestone was Professor Janet Rossant’s work to successfully isolate and maintain stem cells from the early mouse embryo that are specific for the placenta (so-called trophoblast stem cells). The field of placental research has really been propelled forwards by the possibility to grow and manipulate these cells in culture. Recent advances mean that we now understand trophoblast stem cells and their differentiation capacity in much more detail, but there is still much to learn about what makes these cells so special and distinct from any other cell type in the embryo itself.

 

What is the biggest outstanding problem in your field that you wish could be solved?

Perhaps the biggest question in the field is whether such a stem cell population exists in the early human placenta and, if so, how to propagate it. This would open up the possibility to derive patient-specific stem cells and identify, in unprecedented detail, precisely which aspects of placental development are failing in specific cases of complicated pregnancy.

 

Why did you decide to become involved with the DMDD programme?

DMDD was proposed as a systematic screen of embryonic lethal mouse knockouts – one of the most detailed screens of its kind. But instead of screening only the embryos, a key part of the proposal was to consider the placenta as an essential (and often overlooked) organ system that must form during early development in order for an embryo to reach full gestation. It meant that we would also screen for placental phenotypes in addition to embryo phenotypes. It was a hugely exciting opportunity to make an impact on the field, as it allowed us to determine just how many genes contribute to the formation of the placenta and are therefore important to ensure normal development of the embryo.

 

What do you hope the DMDD programme will achieve?

Our placental analysis has already highlighted that a far greater number of genes than previously known are necessary for placental development . We find that an extraordinarily high proportion of embryonic lethal knockouts show a placental phenotype and, at least in some cases, this will mean that the placenta was either the cause or a contributing factor of embryonic lethality. One of the most important and personally rewarding achievements of the programme would be to raise awareness of this result, and to encourage others to include the placenta in studies aimed at finding the causes of developmental disorders.

 

What are you most proud of achieving outside of your research?

I am proud to think that some of my outreach work might have inspired young people to be fascinated by biology in the broadest sense. Ultimately, the precise field that sparks their interest is secondary – what’s important is that young people discover and develop an admiration of biological processes, for example the development of a fertilised egg to become a complete embryo and placenta. Engaging a new generation of scientists is personally rewarding, but it’s also really important to ensure the advancement of science in the future.

 

Myriam Hemberger is a group leader in epigenetics research at the Babraham Institute.

NEW EMBRYO PHENOTYPE DATA AVAILABLE

New image and phenotype data for embryos and placentas from embryonic lethal knockout mouse lines has been made available on the DMDD website today. The knockout data includes the ciliary gene Rpgrip1l as well as Atg16l1, a gene encoding a protein that forms part of a larger complex needed for autophagy. In total we have added HREM image data for 10 new lines, embryo phenotypes for 11 lines and placenta image and phenotype data for 6 lines.

The new data was released at the same time as enhancements to our website, which have been described in a separate blog post. Keep reading to see some highlights from the phenotype data.


DETAILED EMBRYO PHENOTYPES REVEALED

The comprehensive and detailed nature of DMDD embryo phenotyping means that we are able to identify a wide range of abnormalities. In the data released today, a total of 423 phenotypes were scored across 78 embryos. These included gross morphological defects such as exencephaly and edema, but also abormalities on a much smaller scale such as an unusually small dorsal root ganglion, absent hypoglossal nerve and narrowing of the semicircular ear canal.

In the image below, a Trim45 embryo at E14.5, was found to have abnormal optic cup morphology and aphakia (a missing lens).

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HREM imaging of a Trim45 knockout embryo reveals abnormal optic cup morphology and aphakia on the left side.

3D modelling of the exterior of an Rpgrip1l knockout embryo at E14.5 revealed a cleft upper lip, as well as polydactyly.

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A 3D HREM model of an Rpgrip1l embryo shows a cleft upper lip.

All phenotypes are searchable on the DMDD website, highlighted on relevant images, and the full-resolution image data is available to explore online.


SYSTEMATIC PLACENTAL ANALYSIS

DMDD also carries out systematic phenotyping of the placentas from knockout lines. The image below shows a Cfap53 knockout placenta at E14.5, which was found to have an aberrant fibrotic lesion. The density of fetal blood vessels was also considerably reduced, the overall effect being to reduce the nutrient flow from mother to embryo.

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Placental histology for the line Cfap53 shows a fibrotic lesion (large arrow) and several regions of reduced blood vessel density (small arrows).

 


GENE EXPRESSION PROFILES

Work is underway to measure the gene expression profiles for embryos from embryonic lethal knockout lines, a study that complements the morphological phenotype data we are gathering. One of our ultimate goals is to allow data users to explore correlations between gene, morphological phenotype and gene expression profile. The first part of this dataset was released recently – a temporal baseline gene expression profile for wild type embryos.

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Example expression profiles of Nacad and Pdzk1 with increasing somite number. The data shows that, at this depth of sequencing, Nacad is switched on during somitogenesis and Pdzk1 is switched off.

 

The expression data is now accessible via a dedicated wild type gene expression profiling page on the DMDD website, which also gives background information about the analysis. Mutant expression data will follow in the new year.


LINKS BETWEEN DMDD GENES AND HUMAN DISEASE

Many of the genes studied by the DMDD programme are known to have links to human disease, including several new lines that have been made available in this release.

Rpgripl1: in humans, mutations in RPGRIPL1 are known to cause Joubert Syndrome (type 7) and Meckel Syndrome (type 5), a rare disorder affecting the cerebellum.

Cfap53: the human ortholog of this gene is known to be associated with visceral heterotaxy-6, in which organs have an abnormal placement and/or orientation.

Arhgef7: in humans the ortholog is associated with Borjeson-Forssman-Lehmann Syndrome.

Arid1b: in humans, mutations in ARID1B are associated with Coffin-Siris Syndrome.

Embryonic lethal lines with no known links to human disease may also be novel candidate genes for undiagnosed genetic disorders. Visit the DMDD website to explore the phenotype data.


A FULL LIST OF NEW DATA

HREM embryo image data has been added for Actn4, Arid1b, Cfap53, Crim1, Cyp11a1, Dmxl2, Fut8, Gas2l2, Mfsd7c, Rala.

Embryo phenotype data has been added for Atg16l1, Capza2, Coro1c, Crim1, Cyfip2Gas2l2, Gm5544Rala, Rpgrip1l, Syt1, Trim45.

Placenta image and phenotype data has been added for Arhgef7, Arid1b, Fam21, Fut8, Med23, Timmdc1.

If you have questions about the DMDD programme or our data, please email contact@dmdd.org.uk.

DATA RELEASE – AUGUST 2016

New embryonic-lethal knockout mouse lines are now available on the DMDD database.

If you haven’t previously taken a look at our data (or even if you have) now would be a good time to explore our website.  We’ve added new embryo phenotype data and HREM images for many knockout lines, taking our total dataset to more than 4 million images of 550 embryos. We also have placental histology images and phenotypes available for over 100 mutant lines.

This post explores some of the phenotypes observed in the new data, and highlights new lines that could be relevant for clinicians researching rare diseases and developmental disorders. But there isn’t enough space here to include every interesting feature of the data – the best thing to do is to explore it yourself.


EMBRYO PHENOTYPES

Our phenotypers at the Medical University of Vienna have observed many interesting phenotypes in the new data.

Embryos from the line Adamts3 display both subcutaneous edema and bifid ureter. A bifid ureter is the most common malformation of the urinary system, [1] in which there is a duplex kidney drain into separate ureters. This observation highlights the incredible resolution of HREM images, which allow detailed phenotypes to be scored for each embryo.

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Bifid ureter (left side) observed in an Adamts3 mutant embryo. The red arrows highlight a single ureter on the right side, but two branches on the left side.

 

Embryos from the line H13 suffered from severe abnormalities in heart morphology, and had an abnormal heart position within the body. The stomach situs was also inverted, as shown in the image below. Note that severely malformed embryos often have different tissue characteristics, which can result in reduced image clarity.

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Comparison between a H13 mutant embryo (left) and its wild-type litter-mate (right). The yellow arrows indicate situs invertus of the stomach.

 

Embryos from the line Brd2 exhibited a profound ventricular septal defect, as shown in the video below.

 


PLACENTAL PHENOTYPES

Our placental image and phenotype dataset is growing rapidly and now contains more than 100 lines.

H13 knockout placentas were smaller than their wild-type counterparts and showed reduced vascularisation in the placental labyrinth, the region of the placenta that allows nutrient and gas exchange between the mother and the developing embryo.

Click to view larger image.
A comparison of the placenta from a H13 mutant embryo and that of its wild-type litter-mate.

Vascularisation of the labyrinth is crucial to allow the embryo to receive the oxygen and nutrients needed for normal development. This is just one example, but many more placental phenotypes are available on our website.


LINKS TO CLINICAL STUDIES

Systematic knockout mouse screens can offer a wealth of information about the genetic basis of rare diseases. Many DMDD lines have human orthologues known to be associated with developmental disorders, and the nature of our study means that it would not be possible to derive equivalent systematic data from human patients.

New knockout lines of potential clinical interest include:

Brd2: the human ortholog of this gene is associated with epilepsy, generalised, with febrile seizures plus, type 5.

Cog6: in humans, COG6 is linked to Shaheen Syndrome and congenital disorder of glycosylation, type IiI.

Npat: the human ortholog is associated with Ataxia-telangiectasia, a rare inherited disorder affecting the nervous and immune systems.

Nsun2: in humans, NSUN2 is linked to mental retardation, autosomal recessive 5 and Dubowitz syndrome.


A FULL LIST OF NEW DATA

Embryo phenotype data added for: Adamts3, Brd2, Cog6, Cpt2, Dhx35, H13, Mir96, Npat, Nsun2, Pdzk1.

HREM embryo images added for: Atg16l1, Capza2, Cog6, Coro1c, Cyfip2, Dhx35, Gm5544, Nadk2, Nrbp1, Rab21, Rpgrip1l, Syt1.

Placenta image and phenotype data added for: 1110037F02Rik, Actn4, Atg16l1, Camsap3, Capza2, Cfap53, Coro1c, Crim1, Crls1, Cyfip2, Dmxl2, Gm5544, Gtpbp3, H13, Nsun2, Rab21, Rala, Rpgrip1l, Syt1, Trim45.


REFERENCES

[1] Obstructed bifid ureteric system causing unilateral hydronephrosis, A. Bhamani1 and M. Srivastava2, Rev Urol. 2013, 15(3) p.131–134, PMC3821993.

1 Department of General Medicine, The Ipswich Hospital, Ipswich, UK.
2 Department of Radiology, Barking, Havering and Redbridge NHS Trust, Romford, Essex, UK.