American Journal of Medical Genetics Part C (Seminars in Medical Genetics) 169C:209–215 (2015) INTRODUCTION

Making Abnormalities Treatable Conditions

JANNINE DEMARS CODY* AND DANIEL ESTEN HALE

Individuals affected by the classic chromosome deletion syndromes which were first identified at the beginning of the genetic age, are now positioned to benefit from genomic advances. This issue highlights five of these conditions (4p-, 5p-, 11q-, 18p-, and 18q-). It focuses on the increased in understanding of the molecular underpinnings and envisions how these can be transformed into effective treatments. While it is scientifically exciting to see the phenotypic manifestations of hemizygosity being increasingly understood at the molecular and cellular level, it is even more amazing to consider that we are now on the road to making chromosome abnormalities treatable conditions. © 2015 Wiley Periodicals, Inc.

KEY WORDS: chromosome abnormalities; dosage; haploinsufficiency; hemizygosity How to cite this article: Cody JD,Hale DE. 2015. Making chromosome abnormalities treatable conditions. Am J Med Genet Part C Semin Med Genet 169C:209–215.

WHAT IS THE GOAL OF empiric, for example, hearing aids for changes in many other medical disci- THIS ISSUE? those that are hearing impaired, rather plines. The problematic and pervasive than scientific, for example, therapeutic attitude, recently voiced in the Septem- Emerging science on chromosomal gene up-regulation. This journal issue ber 2014 issue of Discover Magazine,inan abnormalities suggests that we now will provide the most contemporary article about a child with 22q11.2 have the opportunity not only to expand information on genotype/phenotype deletion is, “But there is no treatment our understanding of these multigene correlation, thus setting the stage for for contiguous gene syndromes like disorders, but also, for the first time, to new and more integrative approaches 22q11.2 deletion; too many and develop specific targeted interventions. for both chromosome abnormality complex biological systems are af- The goal of this series of articles on five research and treatment. fected.” The authors in this Seminars in of the earliest described chromosome Medical Genetics issue obviously disagree deletions (4p-, 5p-, 11q-, 18p-, and with the widely-held sentiment and will WHAT IS THE MAJOR 18q-) is to fundamentally shift the suggest possible pathways to effective CHALLENGE? medical discussions and research direc- treatments. tions about chromosomal abnormalities One of the major challenges confronting We also wished to address the away from phenotypic description and the study of chromosome abnormalities perception problem in a more literal toward genetically informed treatment. is that they suffer from a perception sense. The images shown in this issue are Although these conditions have been problem. This perception, set when of people with the classic chromosome well described for almost 50 years, they were first described decades ago, abnormalities who were all photo- current treatment remains largely has not evolved, despite startling graphed by Rick Guidotti of Positive

Jannine DeMars Cody, PhD is a Professor in the Department of Pediatrics and Director of the Clinical Research Center. Additionally, she is the Founder and President of the Chromosome 18 Registry and Research Society, a lay advocacy group for the families and friends of people with chromosome 18 conditions. She earned her PhD from the UT Health Science Center and has focused her career on making the chromosome 18 conditions treatable. Daniel E. Hale, MD earned his medical degree from the University of Texas at Houston and is a Professor and Chief of the Division of Endocrinology and Diabetes in the Department of Pediatrics at the UT Health Science Center. He is the Medical Director of the Chromosome 18 Clinical Research Center and the Chromosome 18 Registry & Research Society. He has been a part of the Chromosome 18 team for 21 years specializing in the endocrine and growth issues of children with chromosome 18 abnormalities. Drs. Cody and Hale have authored over 50 publications on the chromosome 18 conditions. In an effort to create a scientific community and ignite research interest on all chromosome abnormalities, they along with Dr. John Carey, hosted the World Congress on Chromosome Abnormalities in 2004 with over 500 families and scientists in attendance. *Correspondence to: Jannine D. Cody, Ph.D., Department of Pediatrics, UT Health Science Center, 7703 Floyd Curl Drive, San Antonio 78229, TX. E-mail: [email protected] DOI 10.1002/ajmg.c.31447 Article first published online 3 August 2015 in Wiley Online Library (wileyonlinelibrary.com).

ß 2015 Wiley Periodicals, Inc. 210 AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) INTRODUCTION

Exposure. This program is dedicated to the discovery that principle contributors be upregulated is only a small percentage portraying the beauty of individuals to normal genetic variation (i.e., with- of the total. regardless of their differences. And we, out overt adverse phenotypic effect) are in like fashion, dedicate this issue to gene copy number variants (CNVs). HAPLOINSUFFICIENCY these individuals, many of whom we These CNVs, identified in large control know as truly remarkable people from populations, show that 91.24% of the Three basic mechanisms have been amazing families, who embrace their genes in the OMIM Morbid Map and suggested for haploinsufficiency; sub- challenges with a smile and welcome us 91.18% of transcripts are overlapped by unit imbalance, metabolic rate limiting into their lives with open arms and CNVs [MacDonald et al., 2013]. If 91% steps, and developmental regulation hearts. of genes and transcripts are found in [Wilkie, 1994; Veitia and Birchler, The critical breakthrough to trans- CNVs in an apparently normal popula- 2010]. As we learn more about the form simple phenotypic description tion, then these data imply that, at a molecular mechanism of specific exam- into effective interventions was the minimum, 91% of gene products are not ples these mechanisms are likely to be mapping of the . Hence, sufficiently sensitive to hemizygosity to both expanded and refined. we now know exactly which genes are be haplosufficient and produce an One example of haploinsufficiency present in an abnormal number of copies abnormal phenotype. resulting from subunit imbalance is in an individual. The next step is to Second, the 1000 Genomes project illustrated by components of determine the consequences, or lack of demonstrates that each individual has cell adhesion complexes such as consequences, when a particular gene is roughly 80 heterozygous and 20 homo- d-catenin (cadherin-associated protein), present in an abnormal copy number, zygous loss of function mutations delta 2 (CTNND2) on 5p15.2 at 11 Mb. thus defining the dosage sensitive genes. [MacArthur et al., 2012]. A heterozygous d-catenin is involved in neural cell This is our current challenge. Once loss-of-function mutation would be adhesion and dendritic spine formation those critical genes are identified, the functionally equivalent to hemizygosity. through its role with cadherin to create a potential pathways to treatment will Additionally, 26% of genes were deter- cell adhesion complex [Yuan et al., become clear. mined to have heterozygous loss-of- 2013]. Reduced levels of d-catenin function mutations in an Icelandic could disrupt the integrity of the population [Sulem et al., 2015]. Together adhesion complex resulting in failed HOW DO CHROMOSOME these suggest that many cellular processes cell adhesion and signal transduction. ABNORMALITIES CAUSE are unaffected when there is a single Hemizygosity of this gene (in 5p-) leads DISEASE? functional copy of a gene. to intellectual disability and impaired The molecular mechanisms leading to Third, the converse of the dosage angiogenesis [DeBusk et al., 2010; adverse outcomes in hemizygosity (i.e., insensitive hypotheses is data on the Turner et al., 2015]. haploinsufficiency) are fundamentally percent of genes that are dosage sensi- Haploinsufficiency caused by a different from those of most single tive. These data show that 5% of all metabolic rate limiting step is illustrated gene diseases. In recessive conditions, genes have very low heterozygosity by CYB5A on 18q22.3 at 70 Mb. there are no functional copies of a gene [Samocha et al., 2014]. This means CYB5A enhances the activity of Cyto- and in most dominant conditions, that these genes are evolutionarily con- chrome P450 17A1 (CYP17,20 lyase) mutations confer an adverse gain of strained with little functional coding which is in the biosynthetic pathway to function. In contrast, in hemizygous variation demonstrating the evolution- dihydrotestosterone. Males who are conditions, there is almost always one ary pressure for their being present in hemizygous for this enzyme are at risk perfectly normal gene. This raises the two functional copies. for cryptorchidism, hypospadias, and/or question; “How hard can it be to Given these estimates, Table I micropenis [Cody et al., 2014]. upregulate a gene?” shows the number of hemizygous genes An example of developmental reg- The major challenge is the sheer for each of the conditions described in ulation haploinsufficiency can be found number of genes involved in the this issue and the estimated number of in individuals with hemizygosity for the relatively large chromosome abnormal- dosage sensitive genes supporting proc- FLI-1 gene on 11q24.3 at 127 Mb. This ities described in this issue. As many as esses that are haploinsufficient. These winged helix-turn-helix transcription 300 contiguous genes may be hemi- numbers are shown in comparison to factor is involved in the regulation of zygous in any affect individuals. How- the actual numbers of dosage sensitive megakaryopoiesis. Hemizygosity of the ever, this is not as big a challenge as it and conditionally dosage sensitive genes FLI-1 gene can result in Paris–Trousseau may appear on first consideration. As we based on current data. thrombocytopenia [Hart et al., 2000]. proposed several years ago, [Cody and These data support the concept that Hale, 2011] emerging data from three the task of devising treatments for the OTHER MECHANISMS different lines of research continue to classic chromosome abnormalities may support our hypothesis that only 5–10% not be as big a challenge as originally In addition to haploinsufficiency, there of genes are dosage sensitive. First, was assumed because the number of genes to are other potential mechanisms by INTRODUCTION AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) 211

TABLE I. Hemizygous Versus Dosage Sensitive Genes

Known number of Number of genes in Estimated number of Known number of conditional dosage Initial Condition hemizygous region dosage sensitive genes dosage sensitive genes sensitive genes Description 4p- 28 7 0 0 Cooper and Hirschhorn [1961] 5p- 314 16–28 5 6 Lejeune et al. [1963] 11q- 150 15 6 0 Jacobsen et al. [1973] 18p- 66 3–6 7 5 de Grouchy et al. [1966] 18q-(largest 101 5–9 13 2 de Grouchy terminal et al. [1964] deletion) 18q-reference 37 2–35 2 group

which hemizygosity can lead to an mechanism of the common phenotypes inactive. There are two ways in which abnormal phenotype. These include a associated with a hemizygous deletion. this could happen. First, the existence of revealed recessive mutation, a condi- Secondly, abnormal gene dosage regulatory elements in a hemizygous tional or compound effect and a may be a risk factor or conditional factor region for genes that are nearby and not position effect. A mechanism for the contributing to, but not solely causing, themselves hemizygous may render the infrequent phenotypes associated with a an abnormal phenotype. For example, gene functionally hemizygous. Second, hemizygous region could be through hemizygosity of the TWSG1 gene on the formation of a neo-telomere at a revealed recessive mutations when the 18p is associated with holoprosence- breakpoint could mask a gene within remaining single copy of a gene has an phaly, but only in the presence of a heterochromatin. This position effect inactivating mutation. This is not second mutation in the SHH gene on mechanism would not necessarily apply expected to be a common mechanism chromosome 7 [Billington et al., 2014]. to all copy number changes but would because human genomes each typically Less direct connections to disease may be dependent on the breakpoint and the have approximately 100 loss-of-func- include gene deletions identified more morphology of individual genes and tion mutations [MacArthur et al., commonly in people with conditions their regulatory elements near the 2012]. If there are approximately such as autism than in controls [Krumm breakpoint [Ibn-Salem et al., 2014]. 20,000 genes in the human genome et al., 2013]. Hemizygosity could be one then 100/20,000 genes or 0.5% of of many factors that change the risk for WHAT IS THE ROAD TO genes would have a loss-of-function of a condition without being a direct MAKING THESE mutation. Therefore, phenotypes cause. This means that copy number TREATABLE CONDITIONS? occurring in 0.5% of the cohort with variations identified in self-declared GENOTYPE/PHENOTYPE a particular deletion might be due to a normal individuals, may still be risk MAPPING revealed recessive mutation. The study factors for disease or variants that are cited above also determined that each conditional and requiring an additional The well-established approach for genome harbors approximately 20 ho- genetic variant to be present in order to identifying dosage sensitive genes in mozygous loss-of-function mutations cause disease. This is distinct from cohorts of patients with hemizygosity indicating that not all revealed recessive genotypes with low penetrance because is genotype/phenotype mapping or mutations will have a deleterious effect. in conditional genotypes a secondary more Phenotypic characteristics, or more variant is always required in order to appropriately termed; phenotype extreme versions of common pheno- produce an abnormal phenotype. mapping. The goal is to link a specific types occurring with a low frequency, Thirdly, an abnormal phenotype a specific phenotype with a specific may be the result of a revealed recessive could result from functional hemizygos- gene within a hemizygous region. mutation. Conversely, revealed reces- ity when the position effect of a gene(s) This is an iterative process that hones sive mutations are unlikely to be the near the deletion breakpoint renders it in on smaller and smaller-shared 212 AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) INTRODUCTION hemizygous regions to identify critical are the prototype. This is illustrated by mapping and ultimately gene dosage regions within which the causative congenital aural atresia in the absence annotation. gene lies, until a single gene is of microtia. This phenotype is not identified. There are three factors found in conjunction with numerous ANNOTATING THE that are essential for this approach to syndromes, but is a common pheno- GENOME—GENE DOSAGE be successful. The first is high reso- type in people with terminal deletions MAP lution genotyping to determine ex- of 18q. In addition, the tools for actly which genes are hemizygous. assessing this condition are well stand- One goal of the work that is described in This is now standard practice in ardized. Thus this is an excellent and the five articles in this series is to today’s molecular genetic laborato- relatively precise phenotype for phe- establish a paradigm for determining ries. The second is a cohort of a large notype mapping and consequently the the consequence of having only a single number of affected individuals with a critical region and then the gene were copy of a gene. These consequences may particular chromosome abnormalities successfully identified on 18q- range from having no detectable dele- with a variety of deletion sizes and [Feenstra et al., 2011]. terious effect to lethality. Great progress locations; the most informative being There will undoubtedly be indi- has been made in identifying specific those participants with interstitial viduals with hemizygosity for a critical genes over the past five decades; how- deletions. The more variety in the region who do not have a specific ever, there are still many new genes and size and position of hemizygous phenotype and who are therefore non- novel gene functions and mechanisms to regions, the more potential there is penetrant. Such calculations have clin- be explored. This is especially true for to define small regions. The third, and ical relevance with regard to the genetic genes that are present in atypical most important factor today, and counseling of future patients with hemi- numbers. As a means of tracking the assuredly the most complex, is precise zygosity for the region. The potential emerging science of each gene on a phenotyping. for non-penetrance is also the reason particular chromosome, we have devel- By precise phenotyping, we mean that the absence of a phenotype in oped “custom tracks” on the University several things. Firstly, the phenotype someone with hemizygosity cannot be of California Santa Cruz Genome should reflect as closely as possible its used to narrow a critical region. Browser. These custom tracks allow physiological basis. Second, the phe- It is also important to point out for a visual representation of the location notype is objectively observed and that not all phenotypes are evident in of critical regions and genes. These described. Third, the phenotype is the early years of life and even those tracks can also be labeled or color coded unique in that it is not associated that are evident may evolve over time. to indicate different meaning. In addi- with numerous known genetic causes. Therefore, the approach with the tion, they provide a mechanism to create The widely used phenotypes of “in- highest likelihood of yielding the linked pages containing details about tellectual disability,”“hypotonia,” and optimal information on any phenotype data from other studies such as knock- “failure to thrive” are so generic that is to to maintain ongoing relationships out mice or molecular genetics that they can be applied to almost all with a large cohort of individuals with specifically pertain to the effects of chromosome abnormality. The use of a shared region of hemizygosity. When abnormal gene dosage. As an example, such imprecise phenotypes led to the a new phenotype, or evolution of an we have created a Gene Dosage Map for concept that chromosome abnormal- established phenotype, is identified in chromosome 18 [Cody et al., 2009]. ities affected virtually all biological one individual, the entire cohort can Genes are categorized as haplosufficient, systems and therefore, the individual be assessed for that phenotype using a haploinsufficient, conditional haploin- hemizygous genes involved were not standardized protocol. Likewise, as the sufficient, or haplolethal and are revised relevant [Shapiro, 1989]. However, physiological or biological nature of a continually as new information becomes there are numerous causes of failure clinical phenotype is appreciated, addi- known. In this way, the coordinates of a to thrive, ranging from complex con- tional testing of the cohort may be region of hemizygosity from a diagnos- genital heart disease, to inherited necessary. Additionally, as new data tic molecular genetics laboratory report metabolic conditions, to hormonal emerges on the function of genes can be entered into the UCSC Genome deficits. Each of these involves different within the hemizygous region those Browser within the Gene Dosage Map biological systems and unique genes. genes with data supporting a haploin- custom tracks. The genes in the region New tools, such as MRI, and mass sufficiency mechanism could prompt in question are labeled by dosage spectrometry, and new understanding, assessment or reassessment of partic- category. At this point in time, 80% of such as executive function versus ipants. An example of this involves the the genes on chromosome 18 can be simple IQ, provide new insight and SMCHD1 gene on 18p described in given a dosage designation. allow increasing precision in this issue. Therefore, long-term rela- The data used to create the Gene phenotyping. tionships between scientist, partici- Dosage Map for chromosome 18 are As an example of a relatively pants, and lay advocacy groups are primarily informed by data on precise phenotype, rare malformations essential to the success of phenotype the heterozygous knockout mice, the INTRODUCTION AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) 213 existence of CNVs that encompass the increasing expression to 100% normal genetics, and mouse models of disease. gene of interest and data about the gene activity may not be necessary in order to Conceptually, there are three types of with regard to molecular mechanisms of effect a treatment. Thus, it is reasonable treatments. Standard treatments include disease. Additionally, we have extensive to assume that were modest changes in the use of medications that are already phenotype/genotype correlation data gene expression feasible, these changes efficacious in typical children, for exam- on our cohort of over 400 people with might modify certain consequences of ple, treating growth hormone defi- chromosome 18 copy number changes. the hemizygous state. While there are ciency with growth hormone For example, genes involved in domi- not yet examples of this in the five papers replacement therapy. Another potential nant loss-of-function conditions are in this series, there are a variety of source of treatment might be “repur- categorized as haploinsufficient. Con- medications already in use that increase posed drugs.” These are drugs that have versely, genes involved in recessive gene expression and ameliorate clinical been approved for human use, but were conditions are by definition haplosuffi- conditions. ineffective for the intended therapy. The cient as well as genes that are hemi- Therefore on a theoretical basis, timeline for the use of such therapies zygous and in CNVs in multiple people treatment should be straight forward. It conceivable could be much shorter in control populations. Data from all may not even be necessary to double the because preliminary human studies these sources are considered in order to expression of a hemizygous gene. It may have already been completed. Lastly, determine a gene dosage category. only be necessary to increase the there is the development of novel Genes can be reassigned to a different expression by a much lesser extent in compounds specifically developed for category as new information is order to attain normal functionality. treatment of a specific genetic defect. discovered. Even for those conditions that are These will obviously take the longest to One category which we anticipate not recognized until after birth in most develop. will grow is the “conditional dosage children, there is still a rationale for The rate limiting steps in this plan sensitive” category. Genes in this cat- understanding the causative genes, since are the ability to understand the path- egory do not cause an abnormal the same gene that plays a role in ophysiology of specific human pheno- phenotype by simply being present in differentiation early in embryonic life, types and the capacity to develop an abnormal copy number. Instead they may play a different role in the child. biomarkers for treatment endpoints required a second genetic or environ- Additionally, knowledge of a gene’s role that are relevant and meaningful for mental event before their effect is in development can impact the treat- cognitive and social impairments. These unmasked. As we study, more individ- ment plan. For example, the TSHZ1 gaps in knowledge, while expensive and uals with higher resolution phenotyping gene is responsible for causing congen- time consuming to address, are critical in and genotyping involving the entire ital aural atresia in 18q-. The treatment moving treatment forward. genome instead of just the hemizygous for this malformation is canaloplastic region, we will likely find that the surgery. However, in our natural history CONCLUSION majority of genes have an impact database, we have observed that a large somewhere along a continuum from number of these surgeries are unsuc- When these five conditions were first slightly shifting the risk of an abnormal cessful in the long-term because the ear described, it was thought these were phenotype to being a necessary but not canals collapse and additional surgery is the rare variants with catastrophic sufficient conditional component in required. If we better understood the effects. We now know that genome causing an abnormal phenotype. These growth factors involved in the failure of copy number changes are not necessa- more subtle effects will be more difficult the ear canal to form properly in the first rily rare and don’t necessarily have to define. place, we might be able to effect those catastrophic consequences. Indeed, 1 factors and improve the long-term of every 60 babies born has a de novo surgical outcome. genomic copy number change large MAKING ONE GENE DO The ultimate goal of the investi- enough to include at least 1 gene [Itsara THE WORK OF 2; HOW gators and families is to make chromo- et al., 2010] and that these deletions HARD CAN IT BE? some abnormalities treatable in order can involve any chromosome. This For single gene disorders, there are that those affected should have healthy suggests that gene copy number several examples in which modest and automatous lives. Reaching this goal changes are potentially the single increases in gene expression can alleviate will require investigation on several most common cause of congenital a medical condition One example is, simultaneous fronts as shown in Figure 1. disability. Except that we also know erythropoetic protoporphyria, caused The figure is organized to indicate the that genomic copy number variation is by loss of function mutations in both earliest steps at the top moving more a common genetic variant without copies of the FECH gene can be rescued toward treatment knowledge at the phenotypic effect. Resolving which if only 1 allele is able to sustain the bottom. This work will involve inves- changes have phenotypic consequences expression necessary for 30% enzyme tigators with expertise in clinical trials, and which do not is of considerable activity [Gouya et al., 2006]. Therefore, clinical evaluation, cell biology, importance. 214 AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) INTRODUCTION

Figure 1. There are five broad categories of research that will be involved in the development of treatments for chromosome abnormalities. These are labeled across the top of each column in red. The remainder of the figure is arranged as a general timeline from top to bottom indicating the types of studies that need to take place. The relationship of these studies with regard to gaining information from other areas of research and contributing information to other areas is indicated by the arrows. One example is shown in parentheses. GH, growth hormone.

Second, few of these chromosome consequences of a copy number change syndromes; each of which was identi- deletions involve recurrent copy num- is a more useful approach. In this way, fied at the beginning of the genetic age. ber changes; meaning that the majority any hemizygous region can be under- While these were some of the earliest of people with chromosome deletions stood by identifying the genes involved identified, they are by no means the have unique copy number changes. because the consequences of abnormal only, or archetypal, chromosome dele- This means that the tactic of gene dosage for those genes is known. tion conditions. However, they do binning by common genotype to define The manuscripts in this issue of have the longest history and conse- a “syndrome” is not a feasible approach Seminars in Medical Genetics review the quently a larger and older cohort of to clarity. Rather, a gene by gene current state of the science for five of individuals than many other such annotation to understanding the the classic chromosome deletion conditions. As such, these five INTRODUCTION AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) 215 conditions can serve a point of refer- DeBusk LM, Boelte K, Min Y, Lin PC. 2010. Habegger L, Pickrell JK, Montgomery SB, Heterozygous deficiency of delta-catenin Albers CA, Zhang ZD, Conrad DF, Lunter ence for other conditions involving impairs pathological angiogenesis. J Exp G, Zheng H, Ayub Q, DePristo MA, hemizygosity. Med 207:77–84. BanksE,HuM,HandsakerRE,Rosenfeld Lastly, but most importantly, the de Grouchy J, Royer P,Salmon C, Lamy M. 1964. JA, Fromer M, Jin M, Mu XJ, Khurana E, Partial deletion of the long arms of the Ye K, Kay M, Saunders GI, Suner MM, conditions highlighted in this issue chromosome 18. Pathol Biol (Paris) Hunt T, Barnes IH, Amid C, Carvalho- illustrate the challenges and opportuni- 12:579–582. Silva DR, Bignell AH, Snow C, Yngva- ties of the field. The challenges are de Grouchy J, Bonnette J, Salmon C. 1966. dottir B, Bumpstead S, Cooper DN, Xue Deletion of the short arm of chromosome Y, Romero IG, 1000 Genomes Project numerous but for the first time the steps 18. Ann Genet 9:19–26. Consortium, Wang J, Li Y, Gibbs RA, necessary to make chromosome abnor- Feenstra I, Vissers LELM, Pennings RJE, Nillessen McCarroll SA, Dermitzakis ET, Pritchard malities treatable can be defined, W, Pfundt R, Kunst HP, Admiraal RJ, JK, Barrett JC, Harrow J, Hurles ME, Veltman JA, van Ravenswaaij-Arts, Brunner Gerstein MB, Tyler-Smith C. 2012. planned, and executed. These defined HG, Cremers CWRJ. 2011. 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