Mutations in the very low-density lipoprotein receptor VLDLR cause cerebellar hypoplasia and quadrupedal locomotion in humans

Abstract

Quadrupedal gait in humans, also known as Unertan syndrome, is a rare phenotype associated with dysarthric speech, mental retardation, and varying degrees of cerebrocerebellar hypoplasia. Four large consanguineous kindreds from Turkey manifest this phenotype. In two families (A and D), shared homozygosity among affected relatives mapped the trait to a 1.3-Mb region of chromosome 9p24. This genomic region includes the VLDLR gene, which encodes the very low-density lipoprotein receptor, a component of the reelin signaling pathway involved in neuroblast migration in the cerebral cortex and cerebellum. Sequence analysis of VLDLR revealed nonsense mutation R257X in family A and single-nucleotide deletion c2339delT in family D. Both these mutations are predicted to lead to truncated proteins lacking transmembrane and signaling domains. In two other families (B and C), the phenotype is not linked to chromosome 9p. Our data indicate that mutations in VLDLR impair cerebrocerebellar function, conferring in these families a dramatic influence on gait, and that hereditary disorders associated with quadrupedal gait in humans are genetically heterogeneous.

Fig. 1.

Phenotypic (A) and cranial radiologic (B) presentation of quadrupedal gait in families A and D. (A) Affected brothers VI:20 and VI:18 and cousin VI:25 in family A (Upper) and the proband II:2 in family D (Lower) display palmigrate walking. This is different from quadrupedal knuckle-walking of the great apes (2). The hands make contact with the ground at the ulnar palm, and consequently this area is heavily callused as exemplified by VI:20. Strabismus was observed in all affected individuals. (B) Coronal and midsagittal MRI sections of VI:20, demonstrating vermial hypoplasia, with the inferior vermial portion being completely absent. Inferior cerebellar hypoplasia and a moderate simplification of the cerebral cortical gyri are noted. The brainstem and the pons are particularly small (Left and Center). Similar findings are observed for II:2 (Right).

Fig. 2.

Homozygosity mapping of cerebellar hypoplasia and quadrupedal locomotion to chromosome 9p24 (A) and identification of the VLDLR c769C → T mutation in family A (B) and of the VLDLR c2339delT mutation in family D (C). (A) Pedigree of family A; filled symbols represent the affected individuals. Squares indicate males, and circles indicate females. Black bars represent the haplotype coinherited with the quadrupedal phenotype in the family. Recombination events in individuals VI:16 (obligate carrier) and VII:4 (normal sibling) positioned the disease gene between markers rs7847373 and rs10968723. Physical positions and pairwise lod scores for each marker are shown on the upper left. Z_max represents the maximum lod score obtained at θ = 0.00 cM. (B and C) Sequences of critical regions of VLDLR for wild-type and homozygous mutant genotypes.

Fig. 3.

Functional domains of VLDLR with positions of the mutations relative to the exons (A), domains (B), and the analysis of VLDLR transcript (C and D). (A) The gene consists of 19 exons. Arrows indicate the locations of the mutations. (B) VLDLR consists of ligand-binding type repeat (LBTR), epidermal growth factor repeat (EGFR) I–III, YWTD β-propeller (YWTD), O-linked sugar domain (OLSD), transmembrane domain (TD), and cytoplasmic domain (CD) (34) (www.expasy.org/uniprot/P98155). (C) Restriction-based analysis with HphI revealed the presence of only the mutant (347 bp) and both the mutant and wild type (396 and 347 bp; please note that the 49-bp fragment is not visible) VLDLR transcripts in patient VI:20 and carrier V:18 (both from family A), respectively. M is a DNA size marker. (D) Quantitative RT-PCR analysis of VLDLR transcript from peripheral blood samples of all probands in families A and D and controls was performed. Relative expression ratios were normalized according to the housekeeping gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and the endothelial marker KDR (kinase insert domain receptor). ΔCt values were calculated from duplicate samples and were converted to linear scale (35). Control denotes “VLDLR expression in controls,” VLDLR-GAPDH denotes “VLDLR expression in patients normalized to GAPDH,” and finally VLDLR-KDR denotes “VLDLR expression in patients normalized to KDR.”