RESEARCH ARTICLE
Blastogenetic Associations: General Considerations Mark Lubinsky* 6003 W. Washington Blvd, Wauwatosa, Wisconsin 53213 Manuscript Received: 15 May 2015; Manuscript Accepted: 3 June 2015
Associations of anomalies, with VACTERL as the prototype, have been the source of much debate, including questions about the validity and definition of this category. Evidence is presented for a teratologic basis for associations involving interactions between disruptive events and specific vulnerabilities. Because the embryo is organized in time and space, differences in the timing, location, and severity of exposures will create variable sequelae for any specific vulnerability, creating associations. The blastogenetic stage of development involves distinct properties that affect the nature of associations arising during this time, including relatively undifferentiated developmental fields and causally nonspecific malformations. With this, single anomalies can be part of the spectrum of findings that comprise a specific association. A specific defect defines a subset of disturbances, biasing frequencies of other defects. Processes are basic, integrated, and general, so disruptions are often lethal, and can have multiple effects, accounting for high incidences of multiple anomalies, and overlaps between associations. Blastogenetic disturbances also do not affect the late “fine tuning” of minor anomalies, although pathogenetic sequences can occur. This model suggests that certain combinations of congenital anomalies can arise from causally nonspecific teratogenetic fields determined by timing, location, and vulnerabilities, rather than polytopic developmental fields. Ó 2015 Wiley Periodicals, Inc.
Key words: associations; blastogenesis; developmental fields; lethality; malformations; multiple congenital anomalies; VACTERL
INTRODUCTION VATER, an acronym later expanded to VACTERL (Vertebral, Anorectal, Cardiac, Tracheo-Esophageal, Renal, and Limb), is the prototypic association, that is, a non-random tendency for anomalies to cluster together. However, with variable findings, uncertain boundaries, and no “gold standard” for diagnosis, the VACTERL association is an empirically defined, ongoing source of confusion [Solomon, 2011], and even the validity of associations as biological entities has been questioned [Spranger et al., 1982; Hennekam et al., 2013]. The initial interest in VATER reflected teratologic concerns in the early post-thalidomide era, with an emphasis on developmental relationships that differentiate associations from other categories, such as syndromes [Lubinsky, 1986]. Here, this is used to develop a
Ó 2015 Wiley Periodicals, Inc.
How to Cite this Article: Lubinsky M. 2015. Blastogenetic associations: General considerations. Am J Med Genet Part A 167A:2589–2593.
general framework, as well as a specific model of associations arising during the stage of blastogenesis. This formulation identifies associations as biologically valid classifications of congenital anomalies, and can be used to delineate specific groups including, but not limited to, VACTERL.
ASSOCIATIONS: GENERAL DEFINITIONS Associations represent teratogenic effects on normal developmental organization, making them “abnormal markers of normal embryologic relationships” [Lubinsky, 1986], and specific associations reflect particular vulnerabilities to disruptions. While disturbances may arise from specific agents, findings are typically causally non-specific. Since development involves systems organized in time and space, effects also depend on the timing and location of disturbances, and severity of exposure. Therefore, phenotypes cannot be explained by exposures alone, and reflect these parameters as well. With this, associations are aggregates of multiple teratogenic outcomes. This is seen in fetal thalidomide and alcohol exposures, which, although often labeled “syndromes,” represent associations because of their teratogenic origins. Thalidomide teratogenicity appears to be primarily based on anti-angiogenic factors [Holmes, 2002]. Effects occur during a critical period from 20 to 36 days post-fertilization, but exposures at different days have different effects, so that timing constrains findings, and whether or not they coincide [Miller et al., 2009]. Similarly, early and late fetal alcohol exposures have different consequences, and the amount ingested is also important. Vulnerabilities also contribute chronic alcoholism affects metabolic Conflict of interest: none Correspondence to: Mark Lubinsky, M.D., 6003 W. Washington Blvd., Wauwatosa, WI, 53213. E-mail: [email protected] Article first published online in Wiley Online Library (wileyonlinelibrary.com): 14 July 2015 DOI 10.1002/ajmg.a.37239
2589
2590 processing, increasing fetal effects as maternal liver damage progresses [May and Gossage, 2011]. With this definition, it also follows that, paradoxically, a single finding can be part of an association, for example, a child with an appropriate exposure still has a thalidomide embryopathy, even with only one manifestation. With a known cause, delineation is generally straightforward, but difficulties arise when this is unknown. However, it is still possible to describe specific entities as products of a defined pathogenesis [Botto et al., 1997] or as effects on a particular developmental process, such as segmentation or fusion [Martı´nez-Frı´as, 1995]. This definition differentiates associations from syndromes, which are causally defined [Opitz and Neri, 2013], even if phenotypically ascertained, for example, Down syndrome versus trisomy 21. As a result, anomalies that are part of a specific syndrome are related to a cause, and not to each other (Fig. 1 in Lubinsky, 1994), while associations also involve teratogenic parameters that bias frequencies ascertained through different component anomalies (Fig. 2 in Lubinsky, 1994), biases that were noted in the original description of VATER [Quan and Smith, 1972].
BLASTOGENETIC PROPERTIES AND ASSOCIATIONS While associations can arise at different times in development, the embryonic stage can influence their properties. As a result, associations that arise during blastogenesis, including VACTERL, share certain distinct characteristics that make them worth examining as a group. Blastogenesis involves early development from fertilization through gastrulation [Opitz et al., 2002], but, since “disturbances may occur in processes that are active prior to the visible embryogenesis of an organ,” and later during morphogenesis sequential defects can also occur [Stevenson and Hunter, 2013], timing may not always be clear. This especially applies to the determination of cell pattern and fate, which affects subsequent morphogenetic events [Lubinsky, 2015]. With this, the assignment of a particular anomaly to blastogenesis may involve some uncertainty. Still, developmental properties reflect relatively undifferentiated developmental fields at this stage [Martı´nez-Frı´as, 1994, 1995], rather than later, more differentiated Anlagen that exist as semiindependent modular units [Galis and Metz, 2001]. These fields are “spatially coordinated, temporally synchronized, epimorphically hierarchical, phylogenetically constrained, self-organizing units of the embryo.” Since field defects are causally nonspecific [Opitz et al., 2002], and the same anomaly may have different causes in different associations, there are no distinct pathognomonic defects, for example, the characteristic CHARGE syndrome ear, differentiating it from an association [Lubinsky, 1994]. Blastogenetic processes are basic, integrated, and general, so disruptions can have multiple, and often lethal, effects [Opitz et al., 2002]. At least 70% of all conceptions are lost before term, six out of seven unrecognized before the first missed menstrual cycle, mostly with aneuploidy [Macklon et al., 2002], which affects about 50% of recognized pregnancy failures [van den Berg et al., 2012]. But there are also embryos with malformations and normal chromosomes,
AMERICAN JOURNAL OF MEDICAL GENETICS PART A for example, in 221 successfully karyotyped early missed abortions, out of 56 with normal chromosomes, 20 had growth disorganization and 20 had single or multiple anomalies, 3 with amniotic bands [Philipp et al., 2003]. It therefore appears that these disruptions are relatively common, but mostly as early embryonic lethality. This can have major consequences for analysis, for example, holoprosencephaly was found in 1 in 250 conceptuses, but in only about 1 in 16,000 live births, [Nishimura et al., 1968; Ming and Muenke, 2002], making liveborn cases ascertained through birth defect registries highly biased. Because blastogenetic processes are integrated and inter-dependent, disturbances of one can affect others secondarily, causing overlaps between associations. This vulnerability to multiple effects may help explain a higher rate of multiple blastogenetic anomalies than the frequencies of isolated findings would suggest [Khoury et al., 1990; Botto et al., 1997; Opitz et al., 2002]. Galis and Metz [2001] present evidence for a particular teratogenic vulnerability during the phylotypic stage (when vertebrate embryos most resemble each other roughly, neurulation through somite formation) towards the end of blastogenesis. Animal studies show a peak susceptibility during this stage, with considerably lower effects before and after. However, low levels with early exposures may represent the “all-or-none” phenomenon, where processes are so basic that any effects are lethal, leaving only untouched survivors [Brent, 1999]. Additional overlap involves the midline, which appears to be particularly vulnerable to disruption [Opitz and Gilbert, 1982; Lubinsky, 2015]. Khoury et al. [1990] noted a somewhat general tendency for anomalies arising at the midline to associate, and Martı´nez-Frı´as [1995] found clinical and epidemiological evidence for a variety of clusters representing different “essential events of blastogenesis, such as fusion, lateralization, decussation, segmentation, morphogenetic movements, asymmetry formation, etc,” that could be at least roughly separated [Martı´nez-Frı´as, 1994, 1995]. With early disturbances, late “fine tuning” should be unaffected, so that minor anomalies suggest other factors. This particularly applies to aneuploidy. As Quan and Smith [1972] noted, VATER defects “may occur as part of a broader pattern of malformation in which the prognosis may vary,” and Smith, who drew attention to the importance of minor anomalies in clinical genetics [Opitz and Neri, 2013], omitted them from his analysis of VATER. Empirically, 79% of clinical geneticists in a recent survey felt that “dysmorphic” facial features with VACTERL would alter the diagnostic impression [Solomon et al., 2012]. Similarly, if other conditions are excluded and the brain is structurally normal, intelligence should be unaffected [Lubinsky, 1994], which again seems to be the case [Opitz et al., 2002]. Finally, blastogenetic development may be particularly sensitive to intrinsic effects such as fluctuating asymmetry, “small random perturbations” affecting bilaterally active processes, causing local deviations that accumulate separately, leading to asymmetry [Livshits et al., 1998; van Dongen, 2006]. Opitz and Utkus [2001] and Opitz and Pysher [2008] noted a possible role in the genesis of asymmetric anomalies, and Fraser [1996] emphasized potential effects of dyssynchrony and increased variance on a
LUBINSKY destabilized developmental system, while Wilson [1988] suggested heterochronicity, issues involving the co-ordination of timing.
TERATOGENETIC FIELDS Specific vulnerabilities can be thought of as teratogenetic, as opposed to developmental, fields. Here, developmental fields can be disrupted by a single event because of timing, location, or susceptibility, either individually or in combination. For example, hypoperfusion may cause amyoplasia, bowel atresias, and gastroschisis [Hall et al., 2014], even though they have little in common developmentally. Galis and Metz [2001] noted that “induced anomalies appear to be mainly influenced by the timing rather than by the nature of the teratological treatment” so, like developmental fields, teratogenetic fields seem to be causally nonspecific. teratogenetic commonalities may be a more accurate explanation for certain multiple anomalies, such as acrorenal findings, than a polytopic developmental field [Kroes et al., 2004].
PATTERNS AND RELATIONSHIPS Evans found clusters within VACTERL suggesting “continuous multidimensional spectra with different patterns of anomalies or even single defects at different end points” [1982; see also, Evans et al., 1985]. In keeping with a role for spatial factors, there appear to be cranial and caudal distinctions [Botto et al., 1997]. Timing is also demonstrable. Cardiovascular anomalies in VACTERL can be roughly ordered temporally, with dextrocardia the earliest and aortic arch anomalies the latest findings. Lubinsky and Moeschler [1987] found different VACTERL clusters with different cardiac defects, consistent with a role for timing. Similarly, there are different patterns of association of specific morphological types of aberrant bronchi with different cardiovascular anomalies [Evans, 1990]. Patterns support blastogenetic associations such as VACTERL as aggregates of multiple teratogenic events [Lubinsky, 1986]. With this, a specific defect defines a subset of disturbances with a greater or lesser risk for other findings, so ascertainment through different anomalies should result in different frequencies of other defects. In the original VATER cohort, for example, an imperforate anus occurred in 3% of patients ascertained with radial dysplasias, versus 10% with tracheo-esophageal fistulas [Quan and Smith, 1972]. A series of multiple events affecting changing developmental relationships “explains why associations are continuous and overlapping, why similar patterns may be seen after a variety of teratogenic insults, (and) why isolated malformations may be part of the same spectrum as a specific association” [Evans et al., 1993]. Patterns differentiate associations from syndromes. Since anomalies in a specific syndrome arise from a shared cause, the presence of one should not affect the presence of another expected and observed concurrences should be equal, unless some additional factor is operating (e.g., sequences or severity factors). As a result, anomalies that are part of a specific syndrome should be transitive- if A associates with B, and B with C, then A should associate proportionately with C. However, in associations,
2591 multiple events each capable of producing only a subset of components limit transitivity (Fig. 3 in Lubinsky, 1994); for example, with thalidomide exposures, critical periods for strabismus and triphalangeal thumb both overlap with inner ear anomalies, but not with each other [Miller et al., 2009]: A associates with B, and B with C, but A does not associate with C. Overall, the closer two primordia are in time and space, the greater the degree of transitivity, making it possible to map the relationships of anomalies within associations such as VACTERL. The simplest pattern involves derivatives of a specific area at a defined time, and the MURCS association [Duncan et al., 1979] is probably representative. For more complicated systems, using numerical taxonomy, Evans and co-workers found that cardiovascular and costovertebral anomalies had a strong negative association, even though both were often found with aberrant bronchi [Evans, 1990], while renal and limb defects “rarely occur together unless other malformations are present” [see also Evans et al., 1992]. Similarly, 48% of 244 patients with imperforate anus had genitourinary anomalies; incidence and severity correlated with the level of fistulae between tracts, increasing from low to high; the same relationship held for the lumbosacral vertebral abnormalities found in 29% of patients. If both anal and spinal findings occurred together, the incidence of genitourinary abnormalities increased [Rich et al., 1988]. Given their frequencies as isolated findings, most blastogenetic anomalies show unexpectedly high associations with each other [Khoury et al., 1990], consistent with disturbances of integrated systems. However, this is not non-specific; within the general class of multiple malformations, there are both positive and negative associations, indicating separate clusters within this group [Khoury et al., 1990; Botto et al., 1997]. These different clusters can represent vulnerabilities to different processes [Martı´nez-Frı´as, 1994, 1995]. These tendencies towards overlap are commonly ignored as new, infrequent, anomalies are suggested as possible VACTERL components, often on the basis of a single case. Also, for overlaps, a “hit” on an early, relatively undifferentiated field potentially affects more than one subsequent field [Martı´nezFrı´as et al., 1998]. This may especially apply to renal, genital, and ano-rectal malformations, which form a cluster that may be part of a link between VACTERL and sirenomelia [Sch€ uler and Salzano, 1994].
REALITY AND DELINEATION A Morphology Consortium [Hennekam et al., 2013] concluded that “There are relatively few associations; VACTERL/VATER and MURCS are the two that this group currently recognizes. Historically, a number of associations have been found to be syndromes, the best example being the former CHARGE association. Once the causative gene was identified, [this designation] was changed to CHARGE syndrome. We anticipate that this will happen for most, if not all, associations.” Unfortunately, there are a number of difficulties with this statement, and even in the same issue of the journal, two consortium members still felt that associations are “real biological entities” [Opitz and Neri, 2013].
2592 Here, Botto et al. [1997] presented “evidence for . . . a common pathway for patterns of VATER and other types of defects in at least a subset of infants with multiple congenital anomalies,” bringing “the concept of association at least to a pathogenetic, if not a causal, level” [Opitz and Neri, 2013]. This gives us biologically defined associations. Also, I am unaware of associations redefined as syndromes with the identification of a specific cause. CHARGE, the example cited, was recognized as “a variable syndrome with a more specific etiology” [Lubinsky, 1994] before the gene was found, based on specific findings incompatible with associations, including: 1. Physiological abnormalities in addition to structural defects, with a deceleration of initially normal length and head circumference soon after birth. 2. Atypical isolated defects, especially a characteristic ear seen in few if any other conditions. 3. Minor anomalies, such as a “square-shaped” face, and an interdigital palmar crease. 4. Familial cases, with similar findings within kindreds. 5. A “tight” clustering of findings.
AMERICAN JOURNAL OF MEDICAL GENETICS PART A Because blastogenetic processes are intertwined in development, there can be considerable overlap between different associations. Also, malformations are causally heterogeneous, so that a specific finding can occur in different situations, and represent more than one association. Despite these factors, blastogenetic associations can be at least roughly delineated and differentiated from each other, and it may even be possible to map the embryological relationships between certain components. There are also clinical criteria, for example, because they arise from early events, blastogenetic associations lack the minor anomalies that arise from later “fine tuning” of development, and, if there are no structural abnormalities of the brain, intelligence is normal. Evidence suggests a role for causally nonspecific teratogenetic fields defined by timing, location, and vulnerabilities. A single teratogenetic field can affect multiple developmental fields that are otherwise unrelated. Genetic susceptibilities may also potentiate non-genetic stochastic factors, but Mendelian forms of standard associations such as VACTERL are exceptional.
ACKNOWLEDGMENTS Another concern is that “delimitation of individual entities such as VATER, MURCS, etc. is an arbitrary exercise since all overlap” [Opitz et al., 2002]. However, while overlaps are characteristic of blastogenetic associations, different types reflecting different developmental processes can still be observed and documented [Martı´nez-Frı´as, 1995]. And with this, different patterns are important clues to pathogenetic processes and possible causes. Finally, although blastogenetic associations have teratogenic causes, genes may exist that can predispose to non-genetic stochastic factors. Such genes appear to be infrequent, with low penetrance and variable expressivity, and a limited number of possible derivative anomalies, for example, orofacial clefts and neural tube defects [Czeizel, 1981]. With this, suggested genetic causes for VACTERL often represent midline defects that lack the relationships expected with associations [Lubinsky, 2015]. While Mendelian mutations may result in syndromes with many of the same components as a specific association, relationships between those components should differ. Therefore, a substantial causal role for developmental genes in associations such as VACTERL [Reutter and Ludwig, 2013], is unlikely, although involvement in pathogenesis is likely [Stevenson and Hunter, 2013].
CONCLUSIONS Associations are biologically valid entities that reflect developmental relationships, and that differ from syndromes in the relationships between anomalies. While associations can be causally defined by teratogenic agents acting on specific vulnerabilities, other parameters, such as timing, are necessary to understand the effects in individual patients. With delineation based on vulnerabilities, both isolated and multiple anomalies can be part of the same association. Early embryonic properties, such as developmental fields, characterize a group of blastogenetic associations, such as VACTERL.
I thank Dr. JM Opitz for his suggestions, comments, and support for this and subsequent articles on associations.
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Blastogenetic Associations: General Considerations Mark Lubinsky* 6003 W. Washington Blvd, Wauwatosa, Wisconsin 53213 Manuscript Received: 15 May 2015; Manuscript Accepted: 3 June 2015
Associations of anomalies, with VACTERL as the prototype, have been the source of much debate, including questions about the validity and definition of this category. Evidence is presented for a teratologic basis for associations involving interactions between disruptive events and specific vulnerabilities. Because the embryo is organized in time and space, differences in the timing, location, and severity of exposures will create variable sequelae for any specific vulnerability, creating associations. The blastogenetic stage of development involves distinct properties that affect the nature of associations arising during this time, including relatively undifferentiated developmental fields and causally nonspecific malformations. With this, single anomalies can be part of the spectrum of findings that comprise a specific association. A specific defect defines a subset of disturbances, biasing frequencies of other defects. Processes are basic, integrated, and general, so disruptions are often lethal, and can have multiple effects, accounting for high incidences of multiple anomalies, and overlaps between associations. Blastogenetic disturbances also do not affect the late “fine tuning” of minor anomalies, although pathogenetic sequences can occur. This model suggests that certain combinations of congenital anomalies can arise from causally nonspecific teratogenetic fields determined by timing, location, and vulnerabilities, rather than polytopic developmental fields. Ó 2015 Wiley Periodicals, Inc.
Key words: associations; blastogenesis; developmental fields; lethality; malformations; multiple congenital anomalies; VACTERL
INTRODUCTION VATER, an acronym later expanded to VACTERL (Vertebral, Anorectal, Cardiac, Tracheo-Esophageal, Renal, and Limb), is the prototypic association, that is, a non-random tendency for anomalies to cluster together. However, with variable findings, uncertain boundaries, and no “gold standard” for diagnosis, the VACTERL association is an empirically defined, ongoing source of confusion [Solomon, 2011], and even the validity of associations as biological entities has been questioned [Spranger et al., 1982; Hennekam et al., 2013]. The initial interest in VATER reflected teratologic concerns in the early post-thalidomide era, with an emphasis on developmental relationships that differentiate associations from other categories, such as syndromes [Lubinsky, 1986]. Here, this is used to develop a
Ó 2015 Wiley Periodicals, Inc.
How to Cite this Article: Lubinsky M. 2015. Blastogenetic associations: General considerations. Am J Med Genet Part A 167A:2589–2593.
general framework, as well as a specific model of associations arising during the stage of blastogenesis. This formulation identifies associations as biologically valid classifications of congenital anomalies, and can be used to delineate specific groups including, but not limited to, VACTERL.
ASSOCIATIONS: GENERAL DEFINITIONS Associations represent teratogenic effects on normal developmental organization, making them “abnormal markers of normal embryologic relationships” [Lubinsky, 1986], and specific associations reflect particular vulnerabilities to disruptions. While disturbances may arise from specific agents, findings are typically causally non-specific. Since development involves systems organized in time and space, effects also depend on the timing and location of disturbances, and severity of exposure. Therefore, phenotypes cannot be explained by exposures alone, and reflect these parameters as well. With this, associations are aggregates of multiple teratogenic outcomes. This is seen in fetal thalidomide and alcohol exposures, which, although often labeled “syndromes,” represent associations because of their teratogenic origins. Thalidomide teratogenicity appears to be primarily based on anti-angiogenic factors [Holmes, 2002]. Effects occur during a critical period from 20 to 36 days post-fertilization, but exposures at different days have different effects, so that timing constrains findings, and whether or not they coincide [Miller et al., 2009]. Similarly, early and late fetal alcohol exposures have different consequences, and the amount ingested is also important. Vulnerabilities also contribute chronic alcoholism affects metabolic Conflict of interest: none Correspondence to: Mark Lubinsky, M.D., 6003 W. Washington Blvd., Wauwatosa, WI, 53213. E-mail: [email protected] Article first published online in Wiley Online Library (wileyonlinelibrary.com): 14 July 2015 DOI 10.1002/ajmg.a.37239
2589
2590 processing, increasing fetal effects as maternal liver damage progresses [May and Gossage, 2011]. With this definition, it also follows that, paradoxically, a single finding can be part of an association, for example, a child with an appropriate exposure still has a thalidomide embryopathy, even with only one manifestation. With a known cause, delineation is generally straightforward, but difficulties arise when this is unknown. However, it is still possible to describe specific entities as products of a defined pathogenesis [Botto et al., 1997] or as effects on a particular developmental process, such as segmentation or fusion [Martı´nez-Frı´as, 1995]. This definition differentiates associations from syndromes, which are causally defined [Opitz and Neri, 2013], even if phenotypically ascertained, for example, Down syndrome versus trisomy 21. As a result, anomalies that are part of a specific syndrome are related to a cause, and not to each other (Fig. 1 in Lubinsky, 1994), while associations also involve teratogenic parameters that bias frequencies ascertained through different component anomalies (Fig. 2 in Lubinsky, 1994), biases that were noted in the original description of VATER [Quan and Smith, 1972].
BLASTOGENETIC PROPERTIES AND ASSOCIATIONS While associations can arise at different times in development, the embryonic stage can influence their properties. As a result, associations that arise during blastogenesis, including VACTERL, share certain distinct characteristics that make them worth examining as a group. Blastogenesis involves early development from fertilization through gastrulation [Opitz et al., 2002], but, since “disturbances may occur in processes that are active prior to the visible embryogenesis of an organ,” and later during morphogenesis sequential defects can also occur [Stevenson and Hunter, 2013], timing may not always be clear. This especially applies to the determination of cell pattern and fate, which affects subsequent morphogenetic events [Lubinsky, 2015]. With this, the assignment of a particular anomaly to blastogenesis may involve some uncertainty. Still, developmental properties reflect relatively undifferentiated developmental fields at this stage [Martı´nez-Frı´as, 1994, 1995], rather than later, more differentiated Anlagen that exist as semiindependent modular units [Galis and Metz, 2001]. These fields are “spatially coordinated, temporally synchronized, epimorphically hierarchical, phylogenetically constrained, self-organizing units of the embryo.” Since field defects are causally nonspecific [Opitz et al., 2002], and the same anomaly may have different causes in different associations, there are no distinct pathognomonic defects, for example, the characteristic CHARGE syndrome ear, differentiating it from an association [Lubinsky, 1994]. Blastogenetic processes are basic, integrated, and general, so disruptions can have multiple, and often lethal, effects [Opitz et al., 2002]. At least 70% of all conceptions are lost before term, six out of seven unrecognized before the first missed menstrual cycle, mostly with aneuploidy [Macklon et al., 2002], which affects about 50% of recognized pregnancy failures [van den Berg et al., 2012]. But there are also embryos with malformations and normal chromosomes,
AMERICAN JOURNAL OF MEDICAL GENETICS PART A for example, in 221 successfully karyotyped early missed abortions, out of 56 with normal chromosomes, 20 had growth disorganization and 20 had single or multiple anomalies, 3 with amniotic bands [Philipp et al., 2003]. It therefore appears that these disruptions are relatively common, but mostly as early embryonic lethality. This can have major consequences for analysis, for example, holoprosencephaly was found in 1 in 250 conceptuses, but in only about 1 in 16,000 live births, [Nishimura et al., 1968; Ming and Muenke, 2002], making liveborn cases ascertained through birth defect registries highly biased. Because blastogenetic processes are integrated and inter-dependent, disturbances of one can affect others secondarily, causing overlaps between associations. This vulnerability to multiple effects may help explain a higher rate of multiple blastogenetic anomalies than the frequencies of isolated findings would suggest [Khoury et al., 1990; Botto et al., 1997; Opitz et al., 2002]. Galis and Metz [2001] present evidence for a particular teratogenic vulnerability during the phylotypic stage (when vertebrate embryos most resemble each other roughly, neurulation through somite formation) towards the end of blastogenesis. Animal studies show a peak susceptibility during this stage, with considerably lower effects before and after. However, low levels with early exposures may represent the “all-or-none” phenomenon, where processes are so basic that any effects are lethal, leaving only untouched survivors [Brent, 1999]. Additional overlap involves the midline, which appears to be particularly vulnerable to disruption [Opitz and Gilbert, 1982; Lubinsky, 2015]. Khoury et al. [1990] noted a somewhat general tendency for anomalies arising at the midline to associate, and Martı´nez-Frı´as [1995] found clinical and epidemiological evidence for a variety of clusters representing different “essential events of blastogenesis, such as fusion, lateralization, decussation, segmentation, morphogenetic movements, asymmetry formation, etc,” that could be at least roughly separated [Martı´nez-Frı´as, 1994, 1995]. With early disturbances, late “fine tuning” should be unaffected, so that minor anomalies suggest other factors. This particularly applies to aneuploidy. As Quan and Smith [1972] noted, VATER defects “may occur as part of a broader pattern of malformation in which the prognosis may vary,” and Smith, who drew attention to the importance of minor anomalies in clinical genetics [Opitz and Neri, 2013], omitted them from his analysis of VATER. Empirically, 79% of clinical geneticists in a recent survey felt that “dysmorphic” facial features with VACTERL would alter the diagnostic impression [Solomon et al., 2012]. Similarly, if other conditions are excluded and the brain is structurally normal, intelligence should be unaffected [Lubinsky, 1994], which again seems to be the case [Opitz et al., 2002]. Finally, blastogenetic development may be particularly sensitive to intrinsic effects such as fluctuating asymmetry, “small random perturbations” affecting bilaterally active processes, causing local deviations that accumulate separately, leading to asymmetry [Livshits et al., 1998; van Dongen, 2006]. Opitz and Utkus [2001] and Opitz and Pysher [2008] noted a possible role in the genesis of asymmetric anomalies, and Fraser [1996] emphasized potential effects of dyssynchrony and increased variance on a
LUBINSKY destabilized developmental system, while Wilson [1988] suggested heterochronicity, issues involving the co-ordination of timing.
TERATOGENETIC FIELDS Specific vulnerabilities can be thought of as teratogenetic, as opposed to developmental, fields. Here, developmental fields can be disrupted by a single event because of timing, location, or susceptibility, either individually or in combination. For example, hypoperfusion may cause amyoplasia, bowel atresias, and gastroschisis [Hall et al., 2014], even though they have little in common developmentally. Galis and Metz [2001] noted that “induced anomalies appear to be mainly influenced by the timing rather than by the nature of the teratological treatment” so, like developmental fields, teratogenetic fields seem to be causally nonspecific. teratogenetic commonalities may be a more accurate explanation for certain multiple anomalies, such as acrorenal findings, than a polytopic developmental field [Kroes et al., 2004].
PATTERNS AND RELATIONSHIPS Evans found clusters within VACTERL suggesting “continuous multidimensional spectra with different patterns of anomalies or even single defects at different end points” [1982; see also, Evans et al., 1985]. In keeping with a role for spatial factors, there appear to be cranial and caudal distinctions [Botto et al., 1997]. Timing is also demonstrable. Cardiovascular anomalies in VACTERL can be roughly ordered temporally, with dextrocardia the earliest and aortic arch anomalies the latest findings. Lubinsky and Moeschler [1987] found different VACTERL clusters with different cardiac defects, consistent with a role for timing. Similarly, there are different patterns of association of specific morphological types of aberrant bronchi with different cardiovascular anomalies [Evans, 1990]. Patterns support blastogenetic associations such as VACTERL as aggregates of multiple teratogenic events [Lubinsky, 1986]. With this, a specific defect defines a subset of disturbances with a greater or lesser risk for other findings, so ascertainment through different anomalies should result in different frequencies of other defects. In the original VATER cohort, for example, an imperforate anus occurred in 3% of patients ascertained with radial dysplasias, versus 10% with tracheo-esophageal fistulas [Quan and Smith, 1972]. A series of multiple events affecting changing developmental relationships “explains why associations are continuous and overlapping, why similar patterns may be seen after a variety of teratogenic insults, (and) why isolated malformations may be part of the same spectrum as a specific association” [Evans et al., 1993]. Patterns differentiate associations from syndromes. Since anomalies in a specific syndrome arise from a shared cause, the presence of one should not affect the presence of another expected and observed concurrences should be equal, unless some additional factor is operating (e.g., sequences or severity factors). As a result, anomalies that are part of a specific syndrome should be transitive- if A associates with B, and B with C, then A should associate proportionately with C. However, in associations,
2591 multiple events each capable of producing only a subset of components limit transitivity (Fig. 3 in Lubinsky, 1994); for example, with thalidomide exposures, critical periods for strabismus and triphalangeal thumb both overlap with inner ear anomalies, but not with each other [Miller et al., 2009]: A associates with B, and B with C, but A does not associate with C. Overall, the closer two primordia are in time and space, the greater the degree of transitivity, making it possible to map the relationships of anomalies within associations such as VACTERL. The simplest pattern involves derivatives of a specific area at a defined time, and the MURCS association [Duncan et al., 1979] is probably representative. For more complicated systems, using numerical taxonomy, Evans and co-workers found that cardiovascular and costovertebral anomalies had a strong negative association, even though both were often found with aberrant bronchi [Evans, 1990], while renal and limb defects “rarely occur together unless other malformations are present” [see also Evans et al., 1992]. Similarly, 48% of 244 patients with imperforate anus had genitourinary anomalies; incidence and severity correlated with the level of fistulae between tracts, increasing from low to high; the same relationship held for the lumbosacral vertebral abnormalities found in 29% of patients. If both anal and spinal findings occurred together, the incidence of genitourinary abnormalities increased [Rich et al., 1988]. Given their frequencies as isolated findings, most blastogenetic anomalies show unexpectedly high associations with each other [Khoury et al., 1990], consistent with disturbances of integrated systems. However, this is not non-specific; within the general class of multiple malformations, there are both positive and negative associations, indicating separate clusters within this group [Khoury et al., 1990; Botto et al., 1997]. These different clusters can represent vulnerabilities to different processes [Martı´nez-Frı´as, 1994, 1995]. These tendencies towards overlap are commonly ignored as new, infrequent, anomalies are suggested as possible VACTERL components, often on the basis of a single case. Also, for overlaps, a “hit” on an early, relatively undifferentiated field potentially affects more than one subsequent field [Martı´nezFrı´as et al., 1998]. This may especially apply to renal, genital, and ano-rectal malformations, which form a cluster that may be part of a link between VACTERL and sirenomelia [Sch€ uler and Salzano, 1994].
REALITY AND DELINEATION A Morphology Consortium [Hennekam et al., 2013] concluded that “There are relatively few associations; VACTERL/VATER and MURCS are the two that this group currently recognizes. Historically, a number of associations have been found to be syndromes, the best example being the former CHARGE association. Once the causative gene was identified, [this designation] was changed to CHARGE syndrome. We anticipate that this will happen for most, if not all, associations.” Unfortunately, there are a number of difficulties with this statement, and even in the same issue of the journal, two consortium members still felt that associations are “real biological entities” [Opitz and Neri, 2013].
2592 Here, Botto et al. [1997] presented “evidence for . . . a common pathway for patterns of VATER and other types of defects in at least a subset of infants with multiple congenital anomalies,” bringing “the concept of association at least to a pathogenetic, if not a causal, level” [Opitz and Neri, 2013]. This gives us biologically defined associations. Also, I am unaware of associations redefined as syndromes with the identification of a specific cause. CHARGE, the example cited, was recognized as “a variable syndrome with a more specific etiology” [Lubinsky, 1994] before the gene was found, based on specific findings incompatible with associations, including: 1. Physiological abnormalities in addition to structural defects, with a deceleration of initially normal length and head circumference soon after birth. 2. Atypical isolated defects, especially a characteristic ear seen in few if any other conditions. 3. Minor anomalies, such as a “square-shaped” face, and an interdigital palmar crease. 4. Familial cases, with similar findings within kindreds. 5. A “tight” clustering of findings.
AMERICAN JOURNAL OF MEDICAL GENETICS PART A Because blastogenetic processes are intertwined in development, there can be considerable overlap between different associations. Also, malformations are causally heterogeneous, so that a specific finding can occur in different situations, and represent more than one association. Despite these factors, blastogenetic associations can be at least roughly delineated and differentiated from each other, and it may even be possible to map the embryological relationships between certain components. There are also clinical criteria, for example, because they arise from early events, blastogenetic associations lack the minor anomalies that arise from later “fine tuning” of development, and, if there are no structural abnormalities of the brain, intelligence is normal. Evidence suggests a role for causally nonspecific teratogenetic fields defined by timing, location, and vulnerabilities. A single teratogenetic field can affect multiple developmental fields that are otherwise unrelated. Genetic susceptibilities may also potentiate non-genetic stochastic factors, but Mendelian forms of standard associations such as VACTERL are exceptional.
ACKNOWLEDGMENTS Another concern is that “delimitation of individual entities such as VATER, MURCS, etc. is an arbitrary exercise since all overlap” [Opitz et al., 2002]. However, while overlaps are characteristic of blastogenetic associations, different types reflecting different developmental processes can still be observed and documented [Martı´nez-Frı´as, 1995]. And with this, different patterns are important clues to pathogenetic processes and possible causes. Finally, although blastogenetic associations have teratogenic causes, genes may exist that can predispose to non-genetic stochastic factors. Such genes appear to be infrequent, with low penetrance and variable expressivity, and a limited number of possible derivative anomalies, for example, orofacial clefts and neural tube defects [Czeizel, 1981]. With this, suggested genetic causes for VACTERL often represent midline defects that lack the relationships expected with associations [Lubinsky, 2015]. While Mendelian mutations may result in syndromes with many of the same components as a specific association, relationships between those components should differ. Therefore, a substantial causal role for developmental genes in associations such as VACTERL [Reutter and Ludwig, 2013], is unlikely, although involvement in pathogenesis is likely [Stevenson and Hunter, 2013].
CONCLUSIONS Associations are biologically valid entities that reflect developmental relationships, and that differ from syndromes in the relationships between anomalies. While associations can be causally defined by teratogenic agents acting on specific vulnerabilities, other parameters, such as timing, are necessary to understand the effects in individual patients. With delineation based on vulnerabilities, both isolated and multiple anomalies can be part of the same association. Early embryonic properties, such as developmental fields, characterize a group of blastogenetic associations, such as VACTERL.
I thank Dr. JM Opitz for his suggestions, comments, and support for this and subsequent articles on associations.
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