Cystinuria in a Girl Presenting with a Hyperechogenic Colon Detected by Prenatal Ultrasonography and a New SLC3A1 Gene Mutation (p.Phe278Ser)

Cystinuria (OMIM 220 100) is an inborn metabolic disorder characterized by inadequate reabsorption of cystine and the dibasic amino acids ornithine, lysine and arginine caused by a defect in the subunits rBAT or b^0,+ AT of the amino acid transporter b^0,+, located in the renal proximal tubule and epithelial cells of the small intestine (T. Eggermann et al. Orphanet J Rare Dis 2012; 7:19). The overall estimated prevalence is 1/7000 neonates with significant ethnic variation. Even though the four above-named amino acids reach high urinary concentrations, only the poor solubility of cystine at low pH leads to urolithiasis, the clinical manifestation of the disease. In patients with cystinuria type A, caused by homozygous mutations in the SLC3A1 gene (2p16.3-p21), urolithiasis occurs at an average age of 12.2 (± 8.9) years (L. Dello Strologo et al. J Am Soc Nephrol 2002; 13 (10): 2547 – 2553).

In utero, an isolated hyper-echogenic colon before 36 weeks of gestation has been described as an ultrasonographic finding in fetuses with cystinuria by M. Brasseur-Daudruy et al. (Prenat Diagn 2006; 26: 1254 – 1255) first. The authors explained this phenomenon by cystine crystals that are formed in the prenatal kidneys, passed in high concentrations into the amniotic fluid and, are then swallowed again. The defective absorption mechanism of cystine in the small intestine leads to a high concentration of cystine in the colon which could account for the hyper-echogenic colon pattern. According to Amat et al. (Ultrasound Obstet Gynecol 2011; 38 (5): 543 – 547), not all fetuses with cystinuria develop a hyper-echogenic colon. As no genetic testing was performed in the named studies, a correlation between the prenatal finding of a hyper-echogenic colon and the prenatal diagnosis of cystinuria at the molecular level has not been possible to date.

In the current case we present a new pathogenic homozygous mutation, c.833T>C in exon 4 of SLC3A1 gene, in a girl with cystinuria, whose single symptom was an increasingly hyper-echogenic colon in the prenatal sonography as of 30 weeks of gestation ([Fig. 1]). The ultrasound examinations were performed with a GE Voluson E 8 Expert ultrasonic device by a DEGUM III qualified examiner.

Fig. 1 Ultrasonography image at 34 + 4 weeks of gestation showing the hyperechogenic colon (arrows) with a caliber of 13 mm. The adjacent small intestine has a normal echogenicity and caliber.

Abb. 1 Sonografie mit tomografischer Darstellung des hyperechogenen Colonrahmens (Pfeile) im Abdomenquerschnitt mit 34 + 4 SSW. Der Durchmesser des hyperechogenen Dickdarms beträgt 13 mm. Der Dünndarm zeigt hingegen keine auffällige Erweiterung und eine normale Echogenität.

In 2004, the second-grade consanguineous parents of Turkish origin lost a newborn due to an abdominal wall defect and in 2007, a second child due to extreme prematurity. Furthermore, a miscarriage had occurred. A surviving boy of the family was born in 2008 via cesarean section and is clinically healthy with normal urinary amino acid excretion.

In 2012, the 32-year-old woman gave birth to a female at 39 weeks of gestation via primary re-cesarean section. The newborn had an uneventful postnatal adaptation (APGAR 9/10/10) with a normal physical examination including growth charts: birth weight 3200 g (50^th percentile), length 51 cm (50^th percentile) and head circumference 35 cm (55^th percentile). The following diseases, known to be associated with hyper-echogenic bowel, were excluded in our patient: congenital cytomegalovirus infection by negative CMV-PCR in urine, cystic fibrosis by normal pilocarpin iontophoresis and normal immunoreactive trypsin in the newborn screening test, ileal atresia, anorectal and urinary tract malformations by uneventful enteral feeding, dejection and repeated normal ultrasonography of the abdomen in the meanwhile 1.5-year-old girl. Dysmorphisms indicating trisomy 13, 18 and 21 were not obvious at the various clinical examinations.

Cystinuria was diagnosed primarily by repeated analysis of free urinary physiological amino acids with a fully automated amino acid analyzer and ninhydrin detection (Biochrom 30, Biochrom Ltd., Cambridge UK). A highly elevated excretion of cystine: 246 – 531 µmol/mmol creatinine (min.-max; age-related reference range 12 – 39 µmol/mmol creatinine), ornithine: 532 – 1626 µmol/mmol creatinine (min.-max.; age-related reference range 0 – 19 µmol/mmol creatinine), lysine: 926 – 3266 µmol/mmol creatinine (min.-max.; age-related reference range 0 – 19 µmol/mmol creatinine) and arginine: 483 – 2507 µmol/mmol creatinine (min.-max.; age-related reference range 0 – 14 µmol/mmol creatinine) was found. In blood, these amino acids showed normal levels. On light microscopy, hexagonal cystine crystals were also seen in urine samples.

The diagnosis was confirmed on the molecular level by the finding of a homozygous mutation c.833T>C on exon 4 in the SLC3A1 gene by direct sequencing of the 10 coding exons using standard protocols. Both clinically asymptomatic parents were heterozygous for this mutation. This mutation has not yet been described in the literature or in databases. On the protein level this variant results in an amino acid substitution (p.Phe278Ser). Pathogenicity was confirmed by bioinformatic analyses using the Polymorphism Phenotyping v2 PolyPhen2 (http://genetics.bwh.harvard.edu/pph2/) and MutationTaster (URL http://www.mutationtaster.org) programs. We therefore postulate that the persistently high urinary excretion of cystine and the finding of cystine crystals are a consequence of the SLC3A1 mutation c.833T>C. As both SLC3A1 alleles showed the mutation, a transient neonatal cystinuria could be excluded according to Boutros et al. (Kidney Int 2005; 67(2):443 – 448). To prevent urolithiasis in our patient, we advised the parents to maintain a high fluid intake, even at night. Additionally we started an oral therapy with citrate (1.5 mmol/kg body weight/d). Until now (at 1.5 years of age), the urinary pH has been constantly > 7, electrolytes have been normal and the patient has not yet developed urolithiasis, while still excreting high concentrations of cystine, namely 247 µmol/mmol creatinine (min.-max; age-related reference range 5 – 13 µmol/mmol creatinine).

In summary Cystinuria is an inborn metabolic disorder leading to cystine urolithiasis in childhood or adolescence. In a few patients, a hyper-echogenic colon was detected prenatally by ultrasound before 36 weeks of gestation. However, the pathophysiological link between this prenatal symptom and the molecular level of cystinuria remains unclear. In the described case the homozygous mutation c.833T>C in exon 4 of the SLC3A1 gene is pathogenic for cystinuria and led to a hyper-echogenic colon prenatally.