Characterization of a distinct lethal arteriopathy syndrome in twenty-two infants associated with an identical, novel mutation in FBLN4 gene, confirms fibulin-4 as a critical determinant of human vascular elastogenesis

Twenty-two patients (11 males, 11 females) were studied. All patients belonged to unrelated families from the same ethno-religious group (Muslim) hailing from the same geographical area, northern coastal (“Malabar”) belt of the southern Indian state of Kerala. Muslims of Malabar constitute a distinct community, commonly called “Mappilas”.

Table  1 shows the clinical profiles, family history and phenotypic features (Figure 1A, 1B). All patients had normal birth weights (median 2.96 kg). Two infants presented initially with an incidentally detected murmur, all others presented with cardiorespiratory distress. Six patients presented in neonatal period, two of them with severe cardiorespiratory distress and refractory shock within hours of birth. Vasculopathy was diagnosed at a median age of 1.75 months (1 day to 15.5 months). Antenatal history, routine fetal scans, and birth history were unremarkable in all except for one patient (proband 20) who was detected to have an excessively dilated aorta and pulmonary artery in third trimester fetal ultrasonogram. None of the other patients had undergone third-trimester fetal ultrasonography.

Eight of the patients were born out of third degree consanguineous marriages (between first cousins). Four families (probands 1, 14, 18, 19) had a history of death of a previous infant (age at death 4, 11, 12 and 24 months,respectively), all of whom were noted to have respiratory distress in association with at least one of the following - dilated great arteries (n = 2), loose skin (n = 2), or inguinal/abdominal hernia (n = 2). The mother of proband 18 had history of third trimester fetal loss from unknown cause.

The phenotype included variable combination of features, as listed in Table  1. The facial appearance was characteristic,with long philtrum and thin vermillion observed in 90%; other features being hypertelorism (57%), micrognathia (43%), prominent eyes (43%), sagging cheeks (43%), high arched palate (38%), hooded eyelids (29%), broad forehead (29%) and dysplastic/low set pinnae (19%). Cutis laxa with redundant skin folds was seen in 52%, most prominent in the axillary and anterior abdominal skin. Skin was hyperextensible in two patients. Thin long digits (48%), generalized hypotonia (43%), and herniae (19%) were the other common physical features observed. 67%(n = 14) of the patients had 5 or more of the listed features, 90.5%(n = 19) had 3 or more, while only 9.5% (n = 2) had less than 3.

Pulsatile arterial vascular swellings (measuring 1.5 cm-3 cm) were a striking finding in 8 patients, most prominently visible in the axilla,suprasternal, supraclavicular or submandibular locations (Additional file 1). No major skeletal abnormalities were noted in any patient.

Delay in attainment of gross motor milestones was observed in all infants older than four months, mostly in association with generalized hypotonia and debility.

The chest roentgenogram showed mediastinal widening with aortic dilatation (Figure 1C). Lung volumes were compromised by the enlarged great arteries within the thorax. The findings on cardiovascular imaging are summarized in Table  2 and shown in Figures 1D, 2A-F, 3Ai, 3Aii and Additional file 2. Most striking in all patients were the impressive abnormalities of the aorta and pulmonary arteries, and their branches. While ascending and descending thoracic aorta, and the main pulmonary artery, were elongated and dilated in all, some sites like the aortic isthmus,the origins of the arch branches (branchiocephalic, carotid and subclavian arteries), abdominal aorta and its branches and the branch pulmonary arteries,were prone to stenosis. All patients who had peripheral pulmonary stenosis showed consequent right ventricular hypertension.

Parental cardiovascular evaluation by echo or MRI was normal in all the nine cases studied.

Cranial CT revealed elongation and tortuosity of cerebral arteries in nine patients, and signs of cerebral ischemia in three of these. The latter three had stenosis of the origin of common carotid arteries. Retinal vessels were found to be prominent but non-tortuous in both the patients subjected to retinal examination (probands 5 and 6).

Progression of the vascular lesions was documented in CT scans repeated after 11 and 6 months respectively for probands 4 and 5 as shown in Figures 3-Ai, Aii, Bi, Bii. Tracheal and esophageal compression observed on the CT correlated with their symptoms of positional (supine) dyspnea and dysphagia.

Hematoxylin-eosin and Orcein staining of a skin biopsy of proband 10 showed deficient and fragmented elastic fibers in the subcutaneous layers, as compared to an age-matched control (Figure 1E,F).

In the absence of a precise molecular diagnosis in the initial period of the study, patients were assigned a provisional clinical diagnosis of Arterial Tortuosity Syndrome [23] on the basis of overlapping physical and vascular phenotype.Therefore, initially molecular analysis of the SLC2A10 gene was performed, but no disease-causing mutation was identified. Patients did not present with Loeys-Dietz syndrome strictly speaking, nevertheless mutations in the TGFBR1/2 genes were excluded by direct sequencing of both genes. Subsequently, direct sequencing of the FBLN4 gene revealed an identical homozygous c.608A > C (p. Asp203Ala) mutation in exon 7 in all patients except for the 8^th proband who showed compound heterozygosity for this mutation with a c.679C > T (p. Arg227Cys) mutation in the same calcium binding epidermal growth factor (cbEGF) domain. Parental DNA of 19 probands were tested (both parents of 12 probands , and only maternal DNA of 7 probands). All parents tested were heterozygous for the same c.608A > C (p. Asp203Ala) mutation, except for the father of the compound heterozygote who did not bear any FBLN4 mutation, indicating that the p. Arg227Cys occurred de novo (with proven paternity). The presence of the mutations were excluded in 200 control alleles. In order to determine a common ancestry, we haplotyped several polymorphic markers surrounding the FBLN4 gene (D11S4205 – 0.2 Mb – D11S1883 – 2 Mb – FBLN4 – 1.9 Mb – D11S1889 – 0.1 Mb – D11S4155 – 1.35 Mb – D11S4113 – 0.5 Mb – D11S4095). These analyses showed a shared haplotype for the following three markers: D11S1889, D11S4155, and D11S4113 in all probands.

All patients were managed with symptomatic and supportive measures, using digoxin and diuretics where indicated, respiratory care, nutritional supplements and physiotherapy. Two patients (probands 1 and 2) received propranolol (2 mg/kg/day q 12 hours) and all the remaining homozygous mutant patients received losartan (0.5-1.5 mg/kg/day q 24 hours). Among the homozygous mutant patients (n = 21), 76.2% (n = 17)died, median age of death being 4 months (36 hours-30 months,average 6.9 months) (Figure 3C-D), including 4 neonatal deaths. Probands 9 and 16 had the shortest survival, dying at 36 hours and 48 hours of life respectively, following intractable cardiorespiratory failure and shock soon after birth. The other two neonatal deaths (probands 7 and 14) occurred at 15 days and 28 days,respectively, due to rapidly worsening cardiorespiratory failure. The remaining homozygous mutant patients also showed progressive worsening of symptoms;tachypnea and chest indrawing especially in the supine position, deterioration during respiratory infections, feeding difficulties, failure to thrive,increasing skin laxity (in patients who had cutis laxa initially), generalized hypotonia, and delayed motor development. Four patients (probands 12, 13, 21,22) developed generalized seizures on follow-up (in early infancy) and required anti-convulsants; all four had evidence of stenotic lesions in common carotid artery origins and/or in the smaller cerebral vessels (CT angiography). Only 6 of the 21 homozygous mutants survived beyond first year of life (probands 4,5,6,11,18,19), three of whom subsequently died between 14–17 months of age (probands 4,5,6). Proband 11 was the sole survivor beyond 2 years, dying eventually at 30 months. All patients who survived beyond first year of life showed preference for a lateral position while sleeping and leaning forward position while feeding, suggesting airway and esophageal compression (also demonstrated in CT/MRI). Arterial pulsations noted in axilla, neck and submandibular regions became more prominent on follow-up;newer pulsatile masses appeared in three patients on follow-up. The four surviving homozygous mutants, probands 18, 19, 21, 22 are currently aged 19, 22,2 and 2 months respectively. All four are receiving Losartan (1.5 mg/kg/day), digoxin and frusemide. Proband 21 developed hemopericardium and cardiac tamponade with intracranial bleed at 55 days of age requiring emergency pericardiocentesis and blood transfusion. CT Angiogram failed to reveal the site or cause of the pericardial and intra-cranial hemorrhage; coagulation parameters were normal. All the other three surviving patients have history of recurrent hospitalization for intermittent worsening of respiratory symptoms especially during respiratory infections, improving with medications. Proband 8, bearing the compound heterozygous mutations has remained asymptomatic and is currently 7 years old.

Except for proband 17, none of the deaths occurred at our center; events surrounding death were noted by interviewing the care givers. Six deaths were precipitated by respiratory infections, and one was preceded by multiple generalized seizures. Terminal events in all were worsening cardiorespiratory symptoms and general debility. Vascular rupture as a terminal event could not be established in any of these patients, however the possibility cannot be excluded. None of the families consented for autopsy.

Aortic isthmic hypoplasia (Z score ≤ −2, range −2.1 to −6.49, n = 9) correlated with early clinical presentation (≤ 2 months) and early death (≤ 4 months) (p value <0.0001).

This was a collaborative study conducted at Amrita Institute of Medical Sciences,Kochi, Kerala, India, and Center for Medical Genetics, Ghent, Belgium, between August 2004 and June 2011. Informed consent was obtained from families of all patients involved in the study.

The study group comprised 22 patients identified to have dilatation and tortuosity of the aorta and its branches on echocardiography and/or radiological studies, during the study period. All patients underwent detailed clinical,phenotypic and socio-demographic characterization, specialized cardiovascular imaging, molecular genetic studies, histopathological studies (wherever feasible), and clinical follow-up for outcomes. Phenotypic features were described by a clinical geneticist. Of three neonates, where sickness and death precluded clinical examination by the geneticist, phenotype was recorded from clinical photographs in two; no phenotypic details were available for the third neonate (proband 16). Retinal examination was done for two patients (probands 3 and 4).

Cardiovascular imaging was done using echocardiography and either 64-slice computed tomography (CT) (n = 20) or magnetic resonance imaging (MRI) (n = 2). Dimensions of the aorta and the pulmonary arteries were measured on CT or MRI at standard pre-defined locations. Z-scores were derived for selected measurements (aortic root at the sinuses of Valsalva,ascending aorta at the level of right pulmonary artery, isthmus just beyond the subclavian artery and main pulmonary artery) using published data sources [36‐38]. Cerebral arteries were screened by CT scan in the eleven patients most recently clinically evaluated. Parental cardiovascular imaging was done by echocardiography in two, and cardiac MRI in seven cases.

Molecular genetic studies were carried out using DNA isolated from the peripheral blood leucocytes from all patients, and one or both parents of 19 patients.Studies included exclusion of mutations of SLC2A10 (associated with ATS) and TGFBR1 and TGFBR2 (associated with LDS) in the first 3 patients (proband 1–3) recruited. Direct sequencing of FBLN4was done for all patients. DNA from 100 individuals was used as control.Haplotyping around the FLBN4 gene was performed by PCR amplification of the following markers: D11S1205, D11S1883, D11S1889, D11S4155, D11S4113, D11S4095.Fragment analysis was done on ABI 3730XL.

A skin biopsy was obtained from one patient (proband 10) from the anterolateral aspect of the right thigh and studied after hematoxylin-eosin, and Orcein staining using standard histopathological techniques.

Natural history was studied through clinical follow-up. CT scan was repeated for two (probands 4 and 5, 11 and 6 months after their respective initial CT evaluations) to determine rate of progression of arterial dilatation. Symptoms and circumstances surrounding deaths during follow-up were recorded.