Genetic disturbances in patients with bodily isomerism from a single center : clinical implications of affected genes and potential impact of ciliary dyskinesia

So-called heterotaxy or isomerism is characterized by abnormal lateralization and malformations of the bodily organs. The mechanism is unclear, although there is growing evidence that ciliary dyskinesia is involved. We reviewed genetic findings from patients with isomerism to determine if affected genes were known to be associated with isomerism and ciliary dyskinesia and determine associations between genotype and clinical findings. We identified patients with isomerism cared for over a 16year period. Characteristics were compared between those with and without identified mutations. A total of 83 patients with isomerism were identified. Of those who had genotyping, 14/27 had mutations identified, most frequently involving the CFC1 and NODAL genes. Specific mutations were associated with clinical findings, with NODAL mutations often portending need for increased clinical support. Genes associated with isomerism and/or ciliary dyskinesia were identified in the cohort. Specific gene mutations may help predict clinical course.


Introduction
Isomerism is a unique clinical entity in which there is mirror imagery of the thoracic organs within the same individual, which has also been characterized by the more ambiguous term heterotaxy. 1,2As a result, both bronchuses are either long and hyparterial, making them morphologically left, or short and eparterial, meaning that they are morphologically right.4][5][6][7] In the heart, isomerism is unequivocally present with the atrial appendages.[10] Examination of large numbers of autopsied specimens shows that this is a more reliable method of segregating heterotaxy than use of splenic anatomy. 11,12Isomerism in the heart, furthermore, is also found in the conduction system, with sinus nodes known to be bilaterally present in the setting of right isomerism. 13win atrioventricular nodes may also be present in either subset, this potentiating in many cases to arrhythmias. 14,15The arrangement of the abdominal organs, however, although markedly different from the usual or mirrorimages lateralized arrangements, shows minimal evidence of isomerism. 8he mechanism underscoring an isomeric formation of the thoracic organs is unclear, although emerging data indicates that isomerism may be the result of ciliary dyskinesia, with dyskinesia being found in at least twofifths of afflicted patients. 16While the ciliary ultrastructure can be normal, the ciliary beat is abnormal.This may alter leftward differentiation of embryonic flow at the node during early development.Additionally, formation of nonmotile cilia may impact intracellular signaling, which could lead to functional abnormalities not only during embryogenesis, but also later in life. 17,18][24][25][26][27][28][29][30][31][32] Ciliary dyskinesia and isomerism are now known to be associated with specific genetic mutations, some being common to both.Genes such as Lefty1 and Pitx2 were initially demonstrated to be associated with isomerism, with additional genes subsequently identified. 33,34he primary aim of this study, therefore, was to determine if mutations were present in a cohort of patients from a single center known to have isomeric features, and to quantify what proportion of affected genes have been previously associated with either isomerism, ciliary dyskinesia, or both.Additionally, we sought to assess the association of specific affected genes with anatomic and clinical outcomes.

Selection of patients
We reviewed medical records of all patients cared for since 1998 with isomerism at Children's Hospital of Wisconsin.This was the year that electronic medical records were implemented at our institution.The dataset included patients born in or after 1998, as well as those born earlier who transitioned their care to our heart center after 1998.Patients were identified through multiple searches so as to ensure the highest yield of patients.Medical records, medical billing data, and the echocardiography database, cardiac catheterization database, cardiothoracic surgical database were queried for isomerism, heterotaxy, asplenia, multiple spleens, and polysplenia.The resulting lists of patients were then combined, and redundant entries removed.The remaining patients were reviewed, and included if they had both congenital heart disease and recognized features of isomerism.Congenital heart disease was defined as any intracardiac lesion and/or an abnormality of venous return to the heart.We considered isomerism to be present when there was a cardiovascular malformation in addition to evidence of abnormal arrangement of the abdominal organs, including the spleen, or overt evidence of pulmonary isomerism.The diagnosis of right or left isomerism was based on our subsequent review of the clinical and anatomic data.
Next, we identified which patients had undergone genetic testing other than a chromosomal karyotype.These included heterotaxy gene panels conducted by polymerase chain reaction, ciliopathy gene panel conducted by The details of cardiac anatomy were obtained primarily from the echocardiographic findings, although data from computed tomographic, magnetic resonance, and cardiac catheterization studies was also available for some patients.The splenic status was based on findings from abdominal ultrasonography.At our institution, genetic evaluation of patients with isomerism is done on a case by case basis.The proposed methodology was approved by the Institutional Review Board at our institution.

Characteristics of the cohort
For our baseline analysis, we categorized the cardiac diagnoses into primary and secondary lists.The primary diagnosis was the lesion considered to be the most hemodynamically significant, while the secondary diagnosis included other associated lesions.For instance, if a patient had an atrioventricular septal defect with common atrioventricular valvar orifice, double outlet right ventricle, and an interrupted inferior caval vein, then the atrioventricular septal defect was coded as the primary diagnosis, with double outlet right ventricle and interrupted inferior caval vein listed as secondary diagnoses.If a patient simply had interruption of the inferior caval vein, then this was coded as the primary diagnosis.It was implicit that those with bilateral superior caval veins of necessity had a left-sided superior caval vein.

Inferences regarding presence of isomeric right or left atrial appendages
The intracardiac features considered to indicate presence of isomeric right as opposed to left atrial appendages were assessed independently by two authors (RL and RHA), with cognizance taken also of the findings in the other systems of organs.These included, but were not limited to, overall cardiac anatomy, venoatrial connections, and pulmonary and splenic anatomy.The results of the independent assessments were then compared, and any differences discussed, with a consensus being reached by both authors regarding the likely presence of isomeric right as opposed to left atrial appendages.In some instances, the assessors agreed that there was probably usual arrangement of the atrial appendages, despite the presence of known bronchial isomerism.
An aggregate of several features was used to help determine whether isomerism of the right or left atrial appendages was present.Interruption of the inferior caval vein, for example, is known to be associated more with isomerism of the left atrial appendage.In contrast, absence of the coronary sinus and totally anom-alous pulmonary venous connection are uniformly present in the setting of right isomerism.The presence of multiple spleens, or absence of a spleen, was also used to help segregate the subsets of isomerism.The bronchial branching pattern was also used when available.

Genetic testing
General microarray testing was done utilizing an array done within the institution itself.Whole genome array-based analysis was done to identify losses or gains of DNA variants (copy number variants) within the human genome using the Affymetrix Cytoscan HD array.Array data was then analyzed using Affymetrix Chromosome Analysis Suite.The array contains 2,696,550 genetic markers, including 743,305 single nucleotide polymorphisms and approximately 1,953,246 non-polymorphic markers for the detection of copy number variants.When the array identified a duplication less than or equal to 500kb or a deletion than or equal to 200kb, quantitative polymerase chain reaction was used to confirm losses or gains.For each segment confirmed with quantitative polymerase chain reaction, an Applied Biosystems TaqMan Gene Copy Number Assay was used.Assay data was analyzed using the Applied Biosystems CopyCaller Software.Each assay is run with positive controls and a non-template control.

Statistical analysis
To identify patients who differed from each other in regard to the arrangement of their organs, we compared the findings between patients as being likely to have isomerism of the right or left atrial appendages as opposed to usual or mirror-imaged atrial arrangement, using chi-squared analysis for categorical data and independent T-tests or Mann-Whitney-U tests where appropriate.Characteristics were also compared between those shown to have isomerism of the right or left bronchuses.
Characteristics, including intracardiac and extracardiac anomalies, were compared between those with and without an identified genetic mutation using chi square analysis for descriptive variables.Continuous variables were compared using a Mann-Whitney-U test.On this basis, patients were categorized into groups of NODAL mutation only, CFC1 mutation only, CFC1 combined with NODAL muta-tion, mutation in another gene, or no mutation.These groups were then compared by chi square analysis for descriptive variables, and analysis of variance for continuous variables.

Results
A total of 83 subjects met criteria for inclusion with the diagnosis of isomerism.All of these patients had karyotypes done.Of these, 27 (32.5%)had undergone genetic analysis other than a karyotype as well (Figure 1).This additional analysis consisted of a general microarray in all 27 of these patients.8 of these patients also had a heterotaxy panel sent in addition to the karyotype and general microarray.Review of genetic testing identified known mutations in 14 (51.9%) of the 27 patients (Table 1).

Clinical characteristics
When we compared characteristics between those with and without an identified mutation, those with an identified mutation were more likely to have bilaterally right bronchuses (Table 1).There was no statistically significant difference, however, in the inferred morphology of the atrial appendages, or cardiac anatomic features.
The need for mechanical ventilatory support before initial cardiac surgical palliation and the number of days of postoperative mechanical ventilatory support after initial cardiac palliation was higher in those with an identified mutation (Table 2).Mechanical ventilatory support prior to initial cardiac palliation was required in 6 (50%) of those with an identified mutation, but in only 1 (9%) of those without (P=0.033).Median duration of postoperative mechanical ventilatory support after initial cardiac palliation was 7.5 days in those with an identified mutation, and 3 days in those without (P=0.045).Clinical outcomes of bacteremia, recurrent otitis media, recurrent pneumonia, chronic lung disease, asthma, need for supplemental oxygen at home, need for extracorporeal membrane oxygenation at home, and mortality did not differ between the groups (Table 2).

Genetic characteristics
By combining the positive karyotypes and the microarrays, a total of 13 genes were noted to have mutations.Of the 13 genes, 10 (76.9%) have been demonstrated to have associations with either ciliary abnormalities or isomerism in mice, 8 (61.5%) with ciliary abnormalities, and 8 (61.5%) with isomerism.Of these 13 genes, 5 (38.5%) have been demonstrated to be associated with ciliary abnormalities or isomerism in humans, 2 (15.4%) with ciliary abnormalities, and 3 (23.1%)with isomerism (Table 3).A CFC1 mutation was present in 5 (35.7%) of all patients with identified mutations, while a NODAL mutation was present in 3 (21.4%),making these the two most commonly affected genes in our cohort.In 2 (14.3%) patients, both CFC1 and NODAL were mutated (Table 4).When the cohort was segregated into subgroups of CFC1 mutation only, NODAL mutation only, both CFC1 and NODAL mutation, other affected gene, or no mutation, there were statistically significant differences noted between the groups.Those with a NODAL mutation only, CFC1 mutation only, other mutation, or no mutation tended to be associated with right isomerism, while those with Frequencies presented as absolute number (percentage) while numeric values are presented as median (minimum to maximum).The denominator for some variables is lower than the total due to missing data.Those with a NODAL mutation only, or CFC1 mutation only, tended to have a right-sided stomach with left-sided liver, while those with a mutation in another gene tended to have right or left sided stomachs with midline livers (P=0.038).Arrhythmias also differed between the various mutations.Supraventricular tachycardia was more commonly noted in those with a NODAL mutation only, CFC1 mutation only, and those with mutations in other genes, but was not present in those with both a NODAL and CFC1 mutation (P=0.024).Sinus nodal dysfunction was most frequently noted in those with only a CFC1 mutation, while ectopic atrial tachycardia was most frequently noted in those with mutations in other genes (P=0.024).A mutation in NODAL only was associated with increased length of hospitalization for initial cardiac palliation, with a median of 134 days.Those with a mutation in both NODAL and CFC1 had an even greater length of hospitalization for initial cardiac palliation, with a median of 177 days (P=0.031).Mutation in NODAL only was also associated with increased length of hospitalization for second cardiac intervention, with a median of 134 days.Those with a mutation in both NODAL and CFC1 had an even greater length of hospitalization for second cardiac palliation, with a median of 235 days (P=0.023).Those with a mutation in NODAL, CFC1, or both NODAL and CFC1, also required increased length of mechanical ventilatory support after second cardiac palliation (P=0.030).

Article
Table 3. Genes identified in cohort and their association with isomerism and ciliary dyskinesia.

Discussion and Conclusions
We have identified mutations in approximately half of genotyped patients known to have thoracic isomerism.All but two of these mutations were detected by genetic testing other than chromosome karyotype.Nearly four-fifths of the affected genes are known to be associated with either ciliary dyskinesia or isomerism in mice, with two-fifths known to be associated with these features in humans.
The most frequently affected gene was CFC1, which has been noted to be associated with ciliary abnormalities and isomerism in mice, as well as isomerism in humans.There have been 43 cases of CFC1 mutations in humans reported, with 80% consistent with a diagnosis of left isomerism and the remainder with right isomerism.6][37][38] Thus far, very few descriptions have provided details of bronchial, pulmonary, or atrial appendage morphology.Our segregation of isomerism, therefore, was based on the aggregate of the other clinical features described.In this regard, previously reported cases have been associated mostly with presumed left isomerism, while the patients making up our current cohort were more likely to have right isomerism.
CFC1 mutations impair normal left-right patterning, as proteins encoded by CFC1 are cofactors for NODAL-related signals. 39NODAL mutations were the second most frequently affected gene in our cohort.Such mutations are associated with ciliary dyskinesia and isomerism in mice, but only with isomerism in humans based on published reports.In one analysis, NODAL mutations were associated with a higher occurrence of pulmonary atresia, and 90% of the findings were most consistent with right isomerism. 40In the mouse, Nodal is known to play a role in formation of both endoderm and mesoderm, impacting left-right patterning.The gene encodes a protein that is part of the transforming growth factor beta family, and induces its coreceptor Cripto.][43] Likely due to the lack of mesoderm, there is an increase in endodermal formulation.Mice null for Nodal arrest their development during gastrulation, whereas hypomorphic mice have abnormalities in left-right patterning. 44n our cohort, we identified several patients with mutations of both CFC1 and NODAL mutation.Those with NODAL mutations alone suffered increased morbidity, and those with a combined CFC1 and NODAL mutation had the longest hospitalization of any of our subgroups.To the best of our knowledge, this finding has not been previously described.It is, perhaps, related to underlying ciliary dyskinesia, particularly since need for mechanical ventilatory was increased in the setting of these mutations.We also identified mutations of CCDC39, DNAH11, NKX2.5, TBX1, ZIC3, DNAAF, SOX7, and GATA4 genes.6][47][48][49][50][51][52][53][54][55][56][57][58][59][60][61][62][63] We find it of significance, therefore, that a large proportion of affected genes are associated with not only isomerism, but also with ciliary dyskinesia.Previous studies have shown that over 40% of patients with isomerism also have ciliary dyskinesia. 16Nodal cilia in humans have a 9 + 0 arrangement, lacking a central pair of microtubules. 64These cilia rotate in a rotary pattern, rather than the whiplike pattern of the cilia more traditionally referred to as the motile cilia, which have a 9 + 2 arrangement of microtubules.Studies have shown that mutations in genes affecting the central pair of microtubules can cause laterality abnormalities while mutations in genes affecting portions of the cilia other than the central pair do not affect laterality. 65,66It is not only flow of embryonic fluid created by this rotary movement of cilia that affects laterality, however, but also impairment in ciliary sensory function.While the motile nodal cilia create the leftward flow of embryonic fluid, there is the requirement of downstream cilia to sense the flow, thus producing the co-called two-cilia hypothesis. 67iliary dyskinesia has effects outside of embryogenesis.Impairment in motile cilia can affect mucociliary clearance, which can increase sinupulmonary symptoms, and may mediate the need for increased mechanical ventilatory support. 19Impairment of motile cilia has also been implicated in the development of hydrocephalus in patients with isomerism.Impairment in sensory cilia may potentiate hepatic, renal, and splenic dysfunction, although this is yet to be delineated.
We recognize the limitations of our study, which was simply descriptive in demonstrating its inferred associations.We are not able directly to establish causality.While our data has demonstrated trends, future studies, with larger number of patients and additional genetic analysis, will be necessary to establish causality.Our study shows, nonetheless, that future studies focused on isomerism should contain detailed information about the patients, their clinical course, and the genetic data available.This will allow for all associations to be analyzed, and for trends to be noted.
We conclude that at least half of all patients with bodily isomerism are likely to have a mutation if analysed by microarray.Most genes identified in our patients are known to be associated with ciliary dyskinesia or isomerism.Identification of genes may, in the future, be predictive of clinical course.

Figure 1 .
Figure 1.Flowchart illustrating testing done in the cohort.
Nodal and CFC1 mutation tended to be associated with left isomerism (P=0.006).