Persistent left superior vena cava : an overlooked feature of CHARGE syndrome ?

CHARGE is a well-characterized syndrome (OMIM 2148400) associated with multiple congenital anomalies including cardiovascular malformations. Mutations in CHD7 are the most common cause of CHARGE syndrome. Persistent left superior vena cava (LSVC) has been described in patients with CHARGE syndrome in one study of LSVC associations. A retrospective chart review was conducted for all patients with CHARGE syndrome, diagnosed by Blake criterion features and/or the presence of a pathogenic CHD7 mutation. Echocardio grams were performed on a clinical basis for all patients and were systematically reviewed and classified. Persistent LSVC was present in 50% of patients with CHARGE syndrome (4/8) and was seen in 3 out of 33 patients seen by cardiovascular genetics with 22q11.2 deletion syndrome. Persistent LSVC is a common finding in patients with CHARGE syndrome and its presence may increase the index of suspicion in patients with other characteristic congenital anomalies.


Introduction
CHARGE syndrome (OMIM 2148400) is an autosomal dominant, multiple congenital anomaly syndrome.Typically, the occurrence is sporadic with rare familial cases.The term CHARGE itself is an acronym of common features in this syndrome, including ocular Coloboma, Heart defects, choanal Atresia, Retardation of growth and development, Genitourinary and Ear anomalies including deafness.The prevalence of CHARGE syndrome is estimated to be one in 8500-10,000 live births. 1 Cardiovascular malformations (CVMs) that are typically associated with this syndrome include conotruncal malformations [tetralogy of Fallot, double outlet right ventricle, D-transposition of the great arteries (TGA)], septal defects (atrial septal defects, ventriculoseptal defects), right ventricular outflow tract obstruction (RVOTO, e.g., pulmonary valve stenosis), and patent ductus arteriosus. 2 CHARGE syndrome was formerly a clinical diagnosis based on particular major and minor findings.The schema most generally used was proposed by Blake et al., 3 and there is an additional clinical criterion schema proposed by Verloes et al. (Table 1). 4 Both systems utilize similar criteria: endocrine abnormalities (delayed pubertal development, gonadotropin or growth hormone deficiency), developmental delay or intellectual disability, and cranial nerve dysfunction, in addition to CVM.
Since 2004, the clinical diagnosis of CHARGE syndrome has been supported by sequencing of CHD7, 5 Chromodomain Helicase DNA-binding protein active in the developing embryo.Among patients with a clinical diagnosis of CHARGE syndrome by Blake and/or Verloes criteria, over 90% have pathogenic mutations, deletions, or duplications of CHD7. 6atients with truncating mutations of CHD7 are more likely to both meet clinical criteria (Blake and/or Verloes), have CVMs, choanal atresia, and cleft lip/palate than patients with missense mutations of CHD7. 7here is difficulty in making a clinical diagnosis of CHARGE syndrome in the cardiac intensive care unit (CICU) because newborns may not have observable threshold criterion features.Unifying diagnoses impact management and therefore timely diagnoses impact care.Additional congenital anomalies or specific CVMs that increase suspicion for CHARGE syndrome would be helpful in the assessment of newborns in the CICU.
The presence of left superior vena cava (LSVC) is common in the general population (0.1-1.7%) 8 and may be an asymptomatic congenital anomaly discovered in the course of evaluation for other conditions.LSVC is more commonly seen in individuals with concurrent CVMs (10%). 8,9In particular, SVC abnormalities are common in individuals with heterotaxy syndromes, 10 who may have a LSVC with or without a persistent right superior vena cava (bilateral SVC with or without a communicating vein).There is a high prevalence of extracardiac findings in patients with LSVC (51%), and of these, 21% may have heterotaxy syndrome. 8reviously, Postema and colleagues reported a case series of 102 consecutive patients with LSVC in which five patients (5%) with CHARGE syndrome were identified. 11In addition, unspecified SVC anomalies were reported in 5 patients (8%) with CHARGE in an epidemiologic survey. 1 Larger studies examining cardiac features in CHARGE syndrome have not routinely documented SVC findings, 2,[12][13][14] thus the true frequency of LSVC in patients with CHARGE syndrome is unknown.
We reviewed patients with CHARGE syndrome seen by the cardiovascular genetics service at CCHMC to determine the frequency of venous anomalies.We hypothesized that there is an increased frequency of LSVC in patients with a diagnosis of CHARGE syndrome.

Materials and Methods
A retrospective case series of consecutive patients with a clinical and/or molecular diagnosis of CHARGE syndrome and 22q11.2deletion syndrome followed by the cardiovascular genetics service at CCHMC from 7/2010-12/2012 was completed with Institutional Review Board approval.Blake criteria were ascertained and typical CHARGE was used as the standard for clinical diagnosis.We also evaluated for Verloes criteria in all patients as these are weighted differently (Table 1).Pathogenic mutations of CHD7 were the standard for laboratory-confirmed diagnosis.All initial echocardiograms were reviewed by a cardiologist, and CVMs were classified using an established taxonomy that identifies both specific lesions and groups of related lesions. 15s a comparison cohort, patients with 22q11.2deletion syndrome, a condition with similar phenotype, 16 were evaluated similarly.

Results
Eight patients were ascertained with CHARGE syndrome (4 female, 7 Caucasian non-Hispanic, 1 African American).There was an equal distribution of gender and median age at diagnosis of approximately 3 months (0.26 years), however two subjects were ascertained much later at 1.73 years and 12.30 years in a cardiac neurodevelopmental clinic (Table 2).The 22q11.2 deletion syndrome comparison group had 33 patients with demographics similar to the CHARGE cohort (18 female, 30 Caucasian with one Hispanic, 2 African American, 1 Asian).All patients with 22q11 Deletion syndrome had typical deletions by fluorescent in situ hybridization (FISH) or microarray studies, were consecutively ascertained during the same time period, and were also followed by the cardiovascular genetics service.
Patient 1 did not meet Blake criteria, but did meet Verloes criteria as atypical CHARGE (Table 2).We did include this patient based on the pathogenic molecular findings of a splice site mutation that would truncate CHD7; interestingly, this patient did not have persistent LSVC.
The one patient without a pathogenic CHD7 mutation, Patient 4, also had normal microarray and 22q11 FISH testing.LSVC was present and this patient had typical features of CHARGE including choanal stenosis, growth delay (length 0.2% at 6 months of life), striking ear anomalies (right microtia, left anotia, absent left tympanic membrane, malrotated left middle ear ossicles fused to the inferior wall, dysmorphic stapes, hypoplastic oval window, right incus fused to lateral wall, superior semicircular canal partially absent, dysmorphic, and large undeveloped horizontal semicircular canal), severe to profound hearing loss bilaterally (conductive hearing loss ipsilateral to anotia), cranial nerve anomalies (absent right facial nerve and tortuous left facial nerve) with broad forehead and square facies.Cardiac findings are listed in Table 3.No colobomas were seen on ophthalmologic evaluation.This patient also had additional rare and severe craniofacial findings including absent left internal carotid artery and absent left parotid gland, some of which have been previously described in the case report literature in association with CHARGE syndrome. 17his patient met Blake and Verloes criteria for a clinical diagnosis of typical CHARGE syndrome, and may be one of the <10% of patients meeting this standard that do not have mutations of CHD7. 7ll patients had unique phenotypic features.Molecular findings of all patients showed a preponderance of splice site and premature stop codon mutations.

Discussion and Conclusions
Early diagnosis of genetic syndromes is important for optimal patient care in the CICU setting, as knowledge of a unifying diagnosis informs medical decision making, perioperative care, identification and management of comorbidities, improved attention to feeding difficulty in patients with increased risk of dysphagia, and postoperative rehabilitative therapy.
Establishing a diagnosis of CHARGE syndrome in a newborn may be delayed by failure to meet full clinical diagnostic schema in infancy, delay in molecular testing, and/or features that overlap with more common or wellknown genetic syndromes such as 22q11.2deletion syndrome.Usually in newborn care, unless suggested by genetics consult, teams will not routinely order an ophthalmology consult that may detect a retinal coloboma, facial bone imaging for unilateral choanal atresia, or audiology testing that might lead to the diagnosis of CHARGE syndrome in a newborn with conotruncal defects.Identification of additional features, which increase suspicion for CHARGE syndrome and assist in delineating it from other common syndromes, would assist in identifying patients who should have CHD7 sequencing, and in appropriate diagnosis of patients who may be too young to meet the clinical criteria of CHARGE syndrome.
This study identifies LSVC as a possible new phenotypic association for CHARGE syndrome.Echocardiograms are routinely performed to

Table 1. Comparison of clinical criteria for CHARGE syndrome.
screen for cardiac defects in patients suspected of having CHARGE syndrome, so this information would be available and accessible to geneticists and cardiologists.In our small retrospective review we have found a high proportion of patients with CHARGE syndrome who had persistent LSVC, suggesting that this anomaly in the presence of another cardinal feature of CHARGE syndrome might trigger further clinical suspicion for this syndrome.Defining the frequency of LSVC in CHARGE is therefore important to this end.Other syndromes on the differential diagnosis for LSVC include heterotaxy, VACTERL association, aneuploidy, and 22q11.2deletion syndrome. 11onsideration of a potential unifying mechanism of the occurrence of LSVC in CHARGE patients suggests there is some degree of phenotypic overlap between CHARGE syndrome and laterality defects.Specific types of CVMs, including complex CVM are present in both situations, but CVMs that are common and largely present in patients without a genetic syndrome (such as conotruncal defects) do not necessarily alter the index of suspicion like specific CVMs.Our patient population had a higher than expected prevalence of TGA and right aortic arch.Most, but not all, patients with either of these CVMs also had LSVC.Interestingly, mal-arrangement in addition to structural defects is present in both conditions, suggesting consideration of arrangement may also facilitate identification of CHARGE patients.Patient 4 had additional cardiac features consistent with heterotaxy, including dextrocardia and total anomalous pulmonary venous connection.Laterality defects have been seen in CHD7 knockdown zebrafish, 18 as well as more typical cardiac anomalies similar to those generally seen in patients with CHARGE syndrome. 19It may be that CHD7 plays a role in left-right discrimination in human cardiac development.
Limitations of this study include the fact that all patients were ascertained based on their CVMs, and we are therefore unable to assess whether patients with CHARGE syndrome may have isolated LSVC.All of the patients in this study had CVMs, whereas 75% of patients with CHARGE syndrome have CVMs, 2 reflecting the ascertainment of patients from a cardiovascular genetics service.Additionally, the small sample size of individuals with CHARGE syndrome in this study may overestimate this association and not be generalizable.Arguing against sampling bias, however, is the high prevalence of CHARGE syndrome in an LSVC population study 11 and increased SVC anomalies in an epidemiologic study of patients with CHARGE syndrome. 1A point may be made that the frequency of this finding is related to the 10% prevalence of LSVC in patients with CVMs, however in reviewing our patient group with 22q11 dele- In summary, we have identified LSVC as a likely common cardiovascular malformation in CHARGE, suggesting CHD7 plays a role in venous development.Other findings suggestive of abnormal development of laterality were also found in our population (right aortic arch, dextrocardia, D-TGA).Taken together with the abnormal laterality development in zebrafish with CHD7 knockdown, an additional role for CHD7 in early embryonic left-right patterning may be considered.CHARGE syndrome should be considered when LSVC and laterality abnormalities are identified.
Goldenberg, Medical Genetics, Massachusetts General Hospital for Children, 175 Cambridge St., 5th Floor, Boston, MA 02114, USA.Tel.: 617.726.1742-Fax: 617.724.1911.E-mail: pgoldenberg2@partners.orgN o n -c o m m e r c i a l u s e o n l y

Table 2 .
Age at diagnosis, criterion features, and CHD7 sequencing results.Patient Age Coloboma Choanal atresia Ear Cranial nerve presence of Blake criterion finding; 0, denotes absence; ?, unable to assess due to patient's age or death; Coloboma, iris, retina, choroid, disc, microphthalmia; Choanal atresia, unilateral/bilateral, membranous/bony, stenosis/atresia; Characteristic ear, external ear (lop or cup shaped), middle ear (ossicular malformations, chronic serous otitis), mixed deafness, cochlear defects; Cranial nerve, I: anosmia, VII: facial palsy (unilateral/bilateral), VIII: sensorineural deafness, vestibular problems; IX/X: swallowing problems; GU (genitourinary), males (micropenis, cryptorchidism), females (hypoplastic labia), either (delayed, incomplete pubertal development); DD (developmental delay), Delayed motor milestones, hypotonia, intellectual disability; CV (vardiovascular) malformations, all types, especially conotruncal defects; AV canal defects, and aortic arch anomalies; Growth, short stature; Cleft, cleft lip and/or palate; TEF (tracheo-esophageal fistula), TE defects of all types; Face (facial features), broad forehead, facial asymmetry, full nasal tip; Blake and Verloes criteria, M: major criteria, m minor criteria; SC anomaly, presence of malformation of the semicircular canals; CHD7 sequencing, all mutations are pathogenic and result in a truncated protein except Patient 4; VUS, variant of unknown significance and benign polymorphism.N o n -c o m m e r c i a l u s e o n l y tion with CVMs (n=33) we found three patients with LSVC (11%), similar to the general presence of LSVC in individuals with CVMs.Larger studies of SVC anomalies in CHARGE syndrome patients are required to clarify these limitations.