Vascular structure and integrity are determined by connective tissue elements, which include elastic fibers, collagen and several glycoproteins. Cardiovascular abnormalities resulting from inherited defects of many of these components are well known, e.g. Marfan syndrome (fibrillin-1) (MFS; MIM #154700), Williams-Beuren syndrome (elastin) (WBS; MIM#194050), vascular Ehlers-Danlos syndrome (type III collagen) (vEDS; MIM # 130050), and hereditary cutis laxa syndromes (elastin and fibulin-4 and 5) (ADCL1 and ARCL1A; MIM #123700, 219100). However, some of the most dramatic vascular manifestations observed occur in a group of rare disorders which includes Arterial Tortuosity Syndrome (ATS; MIM #208050), Loeys-Dietz Syndrome (LDS;MIM #609192) and Autosomal Recessive Cutis Laxa (ARCL) type I. ATS is an autosomal recessive disorder attributed to mutations in the SLC2A10 gene (chromosome 20q13)[GenBank:AF321240.1] [1]; LDS is an autosomal dominant disorder associated with heterozygous mutations in genes encoding the transforming growth factor beta receptors (TGFBR) 1 and 2 [GenBank:GU143401.1 and GU143402.1] [2]. It has been shown that perturbations in the TGF beta pathway contribute to the pathogenesis of some of the above-mentioned disorders. Despite the dramatic vascular phenotype and associated morbidities [3–6] ATS and LDS are reported to have a natural history permitting survival of several patients to adulthood [2, 5–7]. ARCL Type I is characterized by cutis laxa, pulmonary emphysema,umbilical and inguinal hernias and gastrointestinal and vesico-urinary tract diverticuli, described in association with mutations of fibulin-5 [8] [GenBank:BC022280.1] and fibulin-4 [9] [GenBank:AF109121.1]. Though each of these disorders has characteristic phenotypic features [2, 3, 5, 7–16], the dominating common features are dilatation, elongation and tortuosity of the large and medium sized arteries.

The large arteries (aorta and its branches, pulmonary arteries) are distinguished by their elasticity, which allows them to expand and recoil with cardiac contraction and relaxation, maintaining blood pressure and hemodynamics. This property is largely attributable to elastic fibers [17, 18] present as sheets (lamellae) in the tunica media as well as, in sparser quantities, in internal elastic lamina and along with collagen in the tunica adventitia. Loss of elasticity can result in deformation in response to the constant hemodynamic stresses. While elastic fibers constitute only 2-3% of skin tissue, they contribute to nearly 50% of dry weight of elastic arteries [19]. Mature elastic fibers are composed of central amorphous elastin (derived from its soluble precursor, tropoelastin), surrounded by a scaffold of microfibrils.Microfibrils contain numerous proteins such as microfibrillar-associated glycoprotein, fibrillin and fibulin [17, 18]. Recent studies have shown direct structural and regulatory roles of fibulin-4 and −5 in elastogenesis [20, 21]. In addition, it has been postulated that mutations in fibulin-4 and −5 may impair elastogenesis through modulation of the TGF β signaling pathways [22]. The steps in elastogenesis and role of different molecules in formation and stabilization of elastic fibers are complex and incompletely understood [17].

This study characterizes a cohort of twenty-two infants from an identical ethno-social background with a lethal syndromic vasculopathy associated with a novel mutation in FBLN4, confirming fibulin-4 as a critical determinant in human elastogenesis.

This study describes the spectrum of clinical features, natural history, and molecular genetics of a disorder resulting from a novel mutation in the gene encoding the fibulin-4 protein in a unique population subset.

Fibulins constitute a recently recognized family of seven extracellular glycoproteins that interact with many extracellular matrix proteins including components of basement membranes and elastic fibers [24]. They are widely distributed throughout the body, especially in elastic-fiber rich tissues (aorta, lungs etc.). Fibulins contain contiguous calcium-binding epidermal growth factor-like domains (cbEGF) and a characteristic C-terminal fibulin domain [25] (Figure 4B. Fibulins-4 and −5 are implicated in elastogenesis [21, 22, 26].

Critical importance of fibulin-4 in elastogenesis has been shown in animal experimental models. Fibulin-4 knock-out mice showed severe vascular and lung defects and perinatal lethality due to abolition of elastogenesis [27]. Suppression of elastogenesis in fibulin-4 knock-down mice also resulted in vascular tortuosity and aneurysms resembling human arterial tortuosity syndromes [22, 28]. The role of fibulin-4 in human elastogenesis has been recently demonstrated experimentally in human foreskin fibroblast model [20]. However, reports of mutations in FBLN4 resulting in clinical disease in humans are rare. Hucthagowder et al. [9] identified a missense mutation (c.169 G > A; p.E57K)in FBLN4 in a child with multiple fractures, cutis laxa, vascular tortuosity and aneurysms, emphysema, inguinal and diaphragmatic hernia and joint laxity. Compound heterozygous mutations ((c.835C > T (p.R279C) /c.1070_1073dupCCGC) in FBLN4 causing a severe phenotype including cutis laxa and aortic aneurysm and tortuosity resulting in death at 27 days age was reported by Dasouki et al. [29]. Hoyer et al. reported a homozygous missense mutation (p. Cys267Tyr) in a female neonate with fetal overgrowth and oligohydramnios, arachnodactly,contractures, cutis laxa, microcephaly, spina bifida and extreme perinatal bradycardia leading to death [30]. This group of FBLN4 mutation-positive patients was doubled by the identification of 2 homozygous (c.376 G > A; p. Glu126Lys,and c.1189 G > A; p. Ala397Thr) and 1 compound heterozygous mutations (c.377A > T: p. Glu126Val, and c.577delC; p. Gln193Ser fs X12) in three patients by Renard et al. [31]. These patients were clinically characterized by major cardiovascular defects (aortic aneurysms, arterial tortuosity, and stenosis) and a marked absence of severe skin involvement. All mutations in FBLN4 discussed here are depicted in Figure 4B.

Mechanisms whereby fibulin-4 absence or deficiency results in aneurysm formation are not established. While Chen Q et al. [20] and Choudhary et al. [21] postulated direct structural role for fibulin-4 in elastic fiber assembly, Hanada et al. [22] postulated TGF beta upregulation as possible mechanism in the murine model. Renard et al. provided the first evidence of altered TGF beta signaling in humans with FBLN4 mutations [31]. Huang et al. observed that while Fbln4 null mice develop lethal aortic aneurysms, deficiency of other elastic elements like fibulin-5 and elastin were not associated with as severe aneurysm formation or lethality. They postulated a dual role for fibulin-4, in elastic fiber formation, as well as terminal differentiation and maturation of SMC (smooth muscle cells) in aortic wall [32].

We describe 22 new patients with an identical novel mutation in FBLN4, with severe vascular phenotype and lethal outcomes, providing the largest clinical evidence so far, for vital role of fibulin-4 in human elastogenesis.

The first mutation identified was homozygous single base substitution (c.608A > C) in 21/22 probands, resulting in p. Asp203Ala substitution which affects the highly conserved DVNE consensus sequence of the fourth cbEGF domain (Figure 4A). The consensus sequence is critical in binding calcium ions, which are essential for the structural and binding properties of the fibulin-4 protein. The second mutation, an argine 227 to cysteine substitution, of the compound heterozygote patient (proband 8) results in substitution of a positively charged arginine to a disulfide containing cysteine which may foster incorrect disulfide bond formation or interfere with other charge interactions important for protein structure stability, or functional interactions potentially leading to altered secretion into the extracellular matrix. The causal nature of the mutations is further supported by the following facts: (1) the mutations were not present in 200 control alleles, (2) they affected highly conserved amino acids and nucleotides (Figure 4A), (3) by its de novo occurrence (p. Arg227Cys), (4) the mutations are predicted to have a pathogenic effect based on in silico prediction software (SIFT:deleterious (score 0.00); Polyphen2: probably damaging (score 1.000); Align GVGD:class65 (GV 0.00 – GD 125.75)), and (5) the mutations lead to an amino acid change which results in a moderate (p. Asp203Ala, Grantham score 126, acidic to hydrophobic amino acid) to large (p. Arg227Cys, Grantham score 180, basic to hydrophobic amino acid) physicochemical difference.

The unique social background of our patients is intriguing. The northern coast of Kerala (Malabar) has a population of about 17.5 million, and a large proportion of the Muslims in Kerala (about 24%) [33] (Figure 5). The ports of Malabar were historically important as hubs of sea trade in ancient and medieval India. Mappilas of Malabar are historically regarded as the first Muslims in India, many of them originally descended from Arab settlers who brought the religion to this region as early as 7^th century AD [34]. Consanguineous marriages are common among this distinct sub-population.Most marriages are arranged within the Mappila community even in the current era.The presence of a shared haplotype including the FBLN4 gene among the families in our cohort strongly suggests a common ancestry for all probands. Limited size of the shared haplotype block (max 5.85 Mb), favors the existence of a relatively old founder. The significance of this unique historical/ethnic background needs further evaluation. No major studies exist on the genetic profile or diseases specific to this community. The finding that all parents tested (except father of proband 8) in this cohort were heterozygous for the same mutation, bearing normal phenotype,suggests that the mutation is not lethal in its heterozygous state and therefore allows transmission.

All homozygous mutant patients showed early presentation and high lethality. Only 4/22 homozygous mutants currently survive. Correlation of aortic isthmic hypoplasia with early clinical presentation and death implicates severity of coarctation as an important predictor of outcomes. Neonatal presentation and death in four, as well as the 3^rd trimester antenatal detection of vascular dilatation in one patient implies presence of advanced vascular lesions at birth in at least some of these patients. Although the terminal event in most appeared to be worsening cardiorespiratory failure, inability to subject these patients to autopsy limits our understanding. Notably, proband 8 bearing a compound heterozygous mutation is surviving with no major symptoms despite having vascular abnormalities similar to the homozygotes (Z scores: aortic root, +8.14; ascending aorta, +12.14; isthmus,-1.55; MPA, +3.41). Even though survival may be linked to absence of severe coarctation, the overall asymptomatic status and longevity of this patient as compared to the homozygous mutant patients with similar aortic isthmic Z-scores sets this patient clinically apart. There may be a role for factors other than just physical deformation of arteries and coarctation, in determining mortality and morbidity.

While the phenotypic features overlapped with those of ATS, LDS and ARCL Type I,severe vascular abnormalities were the dominating feature in all. Aortic dilatation was more impressive than pulmonary artery dilatation, and more rapidly progressive,reflecting greater hemodynamic stresses within the aorta. Aortic isthmus was relatively narrow in all patients (median Z score −2) and in sharp contrast to the ascending aorta (median Z scores: +8.6) and descending aorta. Obstructive lesions were also noted in the branch pulmonary arteries, origins of arch branches and abdominal aorta. Reasons for this pattern of differential vascular growth is unclear and could point to differences in elastic fiber content at different sites.Ventricular hypertrophy seen in most patients was probably secondary to the obstructive lesions (coarctation, peripheral pulmonary stenosis).

Interestingly, in the only case where a fetal molecular diagnosis was established through amniocentesis, no arterial deformation was observed on prenatal ultrasonogram (17 weeks) or, fetal autopsy (18 weeks). Family opted for termination of pregnancy in view of two previous infant deaths (both at 4 months of age), one of whom was a proven homozygous mutant. Although in the absence of a large study of controls in this specific population, it is difficult to prove that all homozygotes for the mutation would have lethal vasculopathy, the normal vascular phenotype in this fetus could indicate that early fetal diagnosis is dependent on mutation analysis. It could also be an indicator of progressive nature of the vasculopathy through fetal life, possibly in response to hemodynamic stresses and other molecular factors. It has previously been demonstrated in fetal calf model that while elastic fibers at term are large and predominantly made up of central amorphous elastin, those in very early fetal life consist of only microfibrils;progressive accumulation of elastin occurs with advancing gestation [35]. It is therefore likely that vascular deformation occurring due to abnormal elastic fiber formation would become manifest only in later pregnancy.

There exists a hitherto undescribed syndrome of arterial dilatation and tortuosity with lethal outcomes in the Malabar Mappila community, related to a novel mutation in the FBLN4 gene. The actual disease burden in this community is likely to be much higher and needs extensive studies. Additionally, it has been once again shown that fibulin-4 is critical to human elastogenesis, vascular integrity, and survival.