Mutations in HPSE2 cause urofacial syndrome

Abstract

Urinary voiding dysfunction in childhood, manifesting as incontinence, dysuria, and urinary frequency, is a common condition. Urofacial syndrome (UFS) is a rare autosomal recessive disease characterized by facial grimacing when attempting to smile and failure of the urinary bladder to void completely despite a lack of anatomical bladder outflow obstruction or overt neurological damage. UFS individuals often have reflux of infected urine from the bladder to the upper renal tract, with a risk of kidney damage and renal failure. Whole-genome SNP mapping in one affected individual defined an autozygous region of 16 Mb on chromosome 10q23-q24, within which a 10 kb deletion encompassing exons 8 and 9 of HPSE2 was identified. Homozygous exonic deletions, nonsense mutations, and frameshift mutations in five further unrelated families confirmed HPSE2 as the causative gene for UFS. Mutations were not identified in four additional UFS patients, indicating genetic heterogeneity. We show that HPSE2 is expressed in the fetal and adult central nervous system, where it might be implicated in controlling facial expression and urinary voiding, and also in bladder smooth muscle, consistent with a role in renal tract morphology and function. Our findings have broader implications for understanding the genetic basis of lower renal tract malformations and voiding dysfunction.

Figure 1

Identification of Intragenic Deletion in HPSE2 in an Affected UFS Patient (A) The pedigree of family 1 indicates the individuals who were screened by PCR for a deletion breakpoint, indicated by ^∗. Filled-in symbols represent affected individuals, and symbols containing a dot indicate heterozygote. SNP6.0 array performed on IV:4 is indicated by an arrow. (B) Photograph of affected siblings demonstrating characteristic grimace upon smiling. (C) SNP6.0 array copy number analysis illustrating 11 probe deletions in HPSE2. Log2ratio, copy number state (CNState), and red bar all indicate the deleted region, and an ideogram of chromosome 10 with the position of the deletion is indicated by a red triangle.

Figure 2

Identification of HPSE2 Mutations in UFS Families Pedigrees and genomic sequence chromatograms of families 2, 3, 4, and 6. In family 2, a homozygous nonsense mutation in exon 10 (p.R472X) was identified; in family 3, a frameshift mutation as a result of a homozygous single base pair insertion in exon 1 (p.A20RfsX45) was identified; in family 4, a homozygous nonsense mutation in exon 3 (p.R153X) was identified; and in family 6, a 2 bp deletion at the end of exon 10 resulting in a frameshift (p.N489PfsX126) was identified. Pedigree of family 5 and agarose gel reveal no amplification of exon 3 in the affected individual. M represents the 100 bp DNA ladder, + represents the positive control, and – represents the negative control. All individuals screened are indicated by ^∗, filled-in symbols represent affected individuals, and symbols containing a dot indicate heterozygote.

Figure 3

Protein Structure of Heparanase 2 Model of the predicted 50 kDa subunit of human heparanase 2 (residues 195–592) based on the crystal structure of the family 51 α-L-arabinofuranosidase (Protein Data Bank code 1PZ2). The part of the structure that is encoded by exons 8 and 9 is shown in marine blue. The N and C termini are indicated. This figure was prepared with the program PyMOL (DeLano Scientific LLC).

Figure 4

Expression of HPSE2 in Human and Murine Tissues (A) RT-PCR demonstrating expression of HPSE2 in embryonic brain tissue at Carnegie stages 16 and 21. (B) Direct in situ RT-PCR detection of HPSE2 mRNA expression human bladder tissue. Strong brown staining demonstrates expression in longitudinal smooth muscle tissue. (C) RT-PCR demonstrating expression of Hpse2 in E15 and adult mouse bladder, brain, kidney, and ureter. Hpse1 expression at E15 in the murine bladder, kidney, and ureter are positive, with no expression in the brain. In adult mouse tissue, Hpse1 is expressed in the bladder, but not in the brain. Hpse1 expression in the adult kidney is positive, but very weak, and the ureter is negative.