The proband is an 11-year-old girl who is the second of three children of parents that are 1st degree cousins. The father and mother had no history of repeated urinary tract infections. A 13 year old brother and 8 year old sister have had no urological abnormalities or urinary tract infections, and each had normal ultrasound, urinalysis and urine culture investigations (data not shown). The siblings also do not demonstrate an inverted facial expression when smiling.
The proband was born at term following a normal vaginal delivery after an uneventful pregnancy. Birth weight was 3200 g and length 49 cm. Development and health were normal until 11 months of age when she developed a febrile E. coli
urinary tract infection and was treated with oral antibiotics. Renal ultrasonography showed mildly enlarged kidneys, with ureteral, pelvic and calyceal dilatation, and a large trabeculated bladder. Voiding cystourethrography showed a trabeculated bladder and massive grade V bilateral vesicoureteral reflux into markedly dilated ureters without any sign of anatomical obstruction (). The patient had a normal spinal MRI. At that time the diagnosis was idiopathic neurogenic bladder with high grade stage V reflux. This child had repeated pyelonephritis requiring IV antibiotic therapy during the next 6 months. Ureteral reimplantation was performed at age 18 months and she was maintained on prophylactic trimethoprim – sulfamethoxazole and oxybutynin for the next 2 years. Intermittent self catheterization three times per day using clean technique was performed by the parents. The child was stable for the next 2 years and she was free of urinary tract infections but started to exhibit severe constipation and Type II procidentia (rectal prolapse). Complete rectal prolapse signifies the intussusception of the rectal wall in its entirety through the anal sphincter. Type II refers to a complete full-thickness prolapse, and is further divided by severity. First degree is a high or concealed intussusception. Second degree is externally visible with straining, and third degree is externally visible at all times [1
]. The patient had Type II second degree rectal prolapse with need for manual reduction after each defecation. With continuous use of lactulose as a stool softener the symptoms declined and rectal prolapse became intermittent with spontaneous reduction. The frequency of intermittent catheterization was increased to 5–6 times per day to reduce the risk of urinary tract infection after urodynamic studies showed a sharp rise in bladder pressure to more than 100mmHg with volumes greater than 85 ml.
Figure 1 Radiographic documentation of vesicoureteral reflux. Voiding cystourethogram showing neurogenic bladder with a trabeculated, thickened bladder wall and increased bladder capacity. During filling a bilateral grade five reflux into the palvicalceal system (more ...)
At age 5 ½ years the diagnosis of Ochoa was recognized when she showed the characteristic, inverted, facial expression at a routine examination (, age 9). At the age of 7 she was also noted to have slow growth, with height and weight falling below the 25th centile. Psychomotor development has been normal. The child presently performs intermittent self catheterization 7–8 times per day and has an active life. She takes oxybutynin, nitrofurantoin and lactulose daily, remains free of upper urinary tract infections and is able to hold her urine for a reasonable period of time. Routine biochemistry, including urea and creatinine as well as urine microalbumin remain within the normal ranges. She has had four episodes of orthostatic hypotension and presyncope but no syncopal episodes. Workup included a normal EKG and a normal Holter monitor except for sinus tachycardia during a presyncopal attack that was documented during the Holter monitor. She had an exaggerated heart rate increase in response to postural change which is diagnostic of postural tachycardia syndrome POTS [2
]. Autonomic reflexes have been otherwise preserved.
Photograph of proband at 9 years old. Note characteristic facial grimace, which occurs when laughing or smiling.
Family sample collection and molecular studies were approved by the University of Michigan Institutional Review Board and the family described provided informed consent. To exclude a gene dosage abnormality within the 10q24 genomic region, we performed a comparative genomic hybridization experiment using a 244K Agilent chromosomal microarray (data not shown) according to previously described methods in the Michigan Medical Genetics Laboratories [3
]; results for the proband were normal. Homozygosity mapping was performed based on genotype data collected using the Affymetrix Human SNP Array 6.0, followed by calculation of nonparametric likelihood ratio-based z-scores as described previously [4
]. We next performed variant discovery in the exomes by using the Agilent Sure Select target enrichment system, followed by paired-end sequencing on the Illumina Genome Analyzer II to generate 36-nt sequence reads across ~160,000 coding exons in the human genome. We processed the sequence data in two pipelines, one involving read alignment with BWA [5
] and variant calling with the Genome Analysis Toolkit (GATK) from the Broad institute, the other by using the CLC Genomics Workbench™ software (CLC-bio, Aarhus, Denmark). We focused our analysis on reads mapping to the 10q24 region.
Molecular genetic evaluation disclosed long runs of homozygosity for a number of chromosomal regions including a ~18 Mb region in 10q23–q24 (data not shown) mapped previously by Wang et al. [6
] to harbor an Ochoa syndrome gene. We obtained 58,829,100 36-nt Illumina GA-II reads (in 29,414,550 read pairs); 28.8 million reads were correctly mapped pairs and there were 4% duplicate read pairs. Examination of the reads within the 10q23q24 region revealed 7 of 9 reads in exon 10 of HPSE2 demonstrating a 5 base pair deletion (). PCR amplification and Sanger sequencing confirmed a homozygous 5 bp deletion in the proband, and both parents are heterozygous mutation carriers (). The 5-bp deletion is located in exon 10 of the HPSE gene at c.1374_1378delTGTGC, which causes a frameshift beginning at codon 458 and predicts the replacement of the C-terminal 132 amino acids with a novel 153 amino acid peptide. The mutated sequence has been submitted to GenBank, Accession number: BankIt1402480 Ochoa HQ438075.
Paired-end sequencing identifies HPSE2 deletion. Genome Analysis Toolkit screen shot of a portion of HPSE2 exon 10 showing 36 base pair reads with a 5 base pair deletion (gap between the grey bars) encountered in 7/9 reads.
Figure 4 Confirmation of paired-end sequencing result using Sanger sequencing. PCR amplification and sequencing of genomic DNA from the affected proband and her parents revealed that she is homozygous for the 5 base pair deletion and confirms the high-throughput (more ...)