Mutations in FLVCR2 are associated with proliferative vasculopathy and hydranencephaly-hydrocephaly syndrome (Fowler syndrome)

Abstract

Proliferative vasculopathy and hydranencephaly-hydrocephaly syndrome (PVHH), also known as Fowler syndrome, is an autosomal-recessively inherited prenatal lethal disorder characterized by hydranencephaly; brain stem, basal ganglia, and spinal cord diffuse clastic ischemic lesions with calcifications; glomeruloid vasculopathy of the central nervous system and retinal vessels; and a fetal akinesia deformation sequence (FADS) with muscular neurogenic atrophy. To identify the molecular basis for Fowler syndrome, we performed autozygosity mapping studies in three consanguineous families. The results of SNP microarrays and microsatellite marker genotyping demonstrated linkage to chromosome 14q24.3. Direct sequencing of candidate genes within the target interval revealed five different germline mutations in FLVCR2 in five families with Fowler syndrome. FLVCR2 encodes a transmembrane transporter of the major facilitator superfamily (MFS) hypothesized to be involved in regulation of growth, calcium exchange, and homeostasis. This is the first gene to be associated with Fowler syndrome, and this finding provides a basis for further studies to elucidate the pathogenetic mechanisms and phenotypic spectrum of associated disorders.

Figure 1

Pedigrees and Linkage Analysis Results for Three Consanguineous Families with Fowler Syndrome The results of the microsatellite marker analysis for chromosome 14 of the three Pakistani families 1, 2 and 3 are shown (localization of markers according to NCBI build 36.3). The markers of the assumed common homozygous haplotype are highlighted (red box).

Figure 2

Sequences of the FLVCR2 Mutations (A) Chromatograms of mutation c.1289C>G in Pakistani families 1, 2 and 3. Top row: The sequence chromatogram of a control sample with wild-type allele. Middle row: The sequence chromatogram of an affected fetus with the homozygous variant (c.1289G/G). Bottom row: The sequence chromatogram of a parent with the heterozygous mutation (c.1289C/G). (B) Chromatograms of mutations c.329_334 del (panel left) and c.1192C>G (panel right) in families 4. Top row: The sequence chromatogram of a control sample with wild-type alleles. Bottom row: The sequence chromatogram of an affected fetus with the heterozygous deletion and heterozygous C to G substitution (c.1192C/G). (C) Chromatograms of variants c.473C>A (panel left) and c.839C>G (panel right) in families 5. Top row: The sequence chromatogram of a control sample with wild-type alleles. Bottom row: The sequence chromatogram of an affected fetus with the heterozygous changes (c.473C/A and c.839C/G).

Figure 3

Conservation of Mutated FLVCR2 Amino Acid Residues Seven representative sequences of Bilateria for FLVCR2 are aligned (^∗predicted amino acid sequences). The affected amino acid residues are shown in red and bold. Residues matching the human FLVCR2 sequence are in gray and those not matching the sequence are in white. Residues which are conserved in all seven sequences are in dark gray. (A) Leu398 in exon 6 and Thr430 in exon 7. (B) Pro280 in exon 3. (C) Asn110_Phe112 in exon 1.

Figure 4

Localization of Mutations in a Schematic Diagram of the FLVCR2 Gene Product that Comprises Twelve Transmembrane Domains Asn110_Phe112delinsIle and Leu398Val are predicted to be in an extracellular loop whereas Pro280Arg and Thr430Arg are probably in an intracellular loop. The nonsense mutation Ser158X is probably located in transmembrane domain 3. The mutated amino acid residues are shown in red circles and the surrounding amino acids are in white circles.

Figure 5

Embryonal Expression of Mfsd7c the Mouse Ortholog of FLVCR2 (A) Endoglin expression at E12.5 by in situ hybridization showing expression in endothelial cells (blue) throughout the embryo. (B) Magnification of the embryo in panel A showing the form of endothelial cells at the hindbrain level. (C) Flvcr2 expression in an E14.5 embryo taken from Genepaint (ID: EH275 Gene: BC011209). Note the similarity in form to endoglin expression. (D) Sagittal brain image taken from the Allen Brain Atlas (Allen Brain Atlas [Internet]. Seattle (WA): Allen Institute for Brain Science. © 2004– [cited 1/2/10], (Section ID BC011209_80)). (E) Transverse image of the spinal cord taken from the Allen Brain Atlas (Allen Brain Atlas [Internet]. Seattle (WA): Allen Institute for Brain Science. © 2004– [cited 1/2/10], (Section ID 100009650-BC011209-4.0-116)). (F) Magnification of the image of D at the brain stem level, showing expression in endothelial-like cells. The analysis of endoglin expression was performed as described previously.