Patient 1 (PKD-238) is an 8 year-old boy diagnosed with ARPKD at week 19 of gestation, when routine prenatal ultrasound showed diffusely hyperechoic and enlarged fetal kidneys with loss of corticomedullary differentiation. The amount of amniotic fluid was initially normal but progressively decreased. Fetal growth was normal, and no other anomalies were detected. The patient was delivered vaginally at 36 weeks without complications. Urine output was normal. Serum creatinine peaked at 1.6 mg/dL on the third day of life, decreased to 0.4 mg/dL at 1 month, and remained normal for the first 6 years of life. Severe hypertension, diagnosed on the first day of life, was difficult to manage with 2 antihypertensives, and left ventricular hypertrophy developed. This resolved after 2 years of age, when the hypertension became easier to control. Hyponatremia required oral sodium chloride supplements until 7 months of age. Marked polyuria and polydipsia were present. A normocytic normochromic anemia appeared at 1 month and persisted despite iron supplementation. Liver enzymes and liver ultrasound were unremarkable at birth. By age 5 years, liver enzymes were elevated; ALT and AST ranged between 100 and 250 U/L and AP between 450 and 500 U/L. Synthetic function of the liver remained intact. A liver biopsy at age 7 years showed CHF (). Central hypotonia and motor delays were noted after 6 months. At 12 months, a diagnosis of global developmental delay and speech apraxia prompted an extensive evaluation, which included a normal eye examination. An MRI of the brain performed at 14 months of age was reported as normal, resulting in persistence of the diagnosis of ARPKD, with probable cerebral palsy. Head size, weight, and height remained at the 50th percentile.
Figure 1 Summary of kidney, liver, and brain findings of patients 1 through 3. Ultrasound image shows normal kidney A, measuring 8 cm at 5 years of age, in comparison with the kidney of a typical patient with ARPKD B, measuring 14 cm at age 5 years. Patients’ (more ...)
On evaluation at the NIH Clinical Center at age 8 years, ultrasound revealed enlarged hyperechoic kidneys with small macrocysts in the cortex and medulla (). Other laboratory findings are presented in . The liver was diffusely hyperechoic and inhomogeneous on ultrasound, without cysts. The spleen was mildly enlarged at 11.5 cm. Ophthalmologic examination showed mild, slow torsional nystagmus when fixating clockwise. The retina, optic nerve, and anterior chamber were normal. Snellen visual acuity was 20/50 OD and 20/40 OS. On the Wechsler Intelligence Scale for Children-Fourth Edition (WISC-IV), the verbal comprehension index was 63 (1st percentile). His perceptual reasoning index was 61 (1st centile). A full-scale IQ could not be derived because he did not complete the test. Sequencing of the PKHD1 gene revealed no pathogenic variations. Given the milder CNS phenotype and frequent absence of polydactyly in reported patients with MKS3 mutations, we sequenced the MKS3 gene. The boy was compound heterozygous for a novel splice site mutation in exon 2, c.224-2 A>T, and a missense mutation in exon 18, c.1843 T>C, resulting in amino acid substitution p.Cys615Arg. Segregation analysis showed that the c.224-2 A>T splice site mutation was inherited maternally and the Cys615Arg missense mutation was inherited paternally. Retrospective evaluation of his brain MRI obtained at 14 months of age, initially interpreted as normal, demonstrated features of MTS ().
Laboratory findings at NIH evaluation
Patient 2 (PKD-271) is a 6.5 year-old girl with large hyperechoic kidneys with scattered cysts on prenatal ultrasound. There was no oligohydramnios and the pregnancy, delivery, and neonatal course were uncomplicated. Postnatal ultra-sound confirmed the prenatal findings. At age 1 week, she had hypertension that was difficult to control, resulting in transient left ventricular hypertrophy despite 2 antihypertensive medications. Renal function was normal. At 4 months of age, a neuro-ophthalmological evaluation, prompted by the history of oculomotor apraxia (OMA) in her older brother (patient 3), revealed OMA and mild generalized hypotonia. She has had chronic normochromic normocytic anemia since early infancy and remained on iron supplementation with minimal response. There was polyuria and polydipsia. Her growth was normal. She developed chronic elevation of liver enzymes (ALT and AST >200 U/L and AP >400 U/L) and splenomegaly. At the NIH evaluation, her glomerular function was impaired, normochromic normocytic anemia persisted, and liver enzymes were elevated (). Synthetic function of the liver was intact. Eye examination was normal except for slight slowing of saccades. Snellen visual acuity was 20/25 OD and 20/32 OS. ERG was normal. Cognitive evaluation showed slight articulation problems, but she was 100% comprehensible. On the WISC-IV, she obtained a full-scale IQ of 95 (37th percentile; average range) with variable index scores; verbal comprehension, working memory, perceptual reasoning, and processing speed were 99, 102, 98, and 83, respectively.
Patient 3 (PKD-272) is the 10 year-old brother of patient 2. He was born at term after a normal pregnancy. The delivery and neonatal period were uneventful. At 12 months he was diagnosed with oculomotor apraxia associated with central hypotonia and mild developmental delay. He made progress with speech and physical therapy. At age 3, a screening ultrasound, prompted by the diagnosis of polycystic kidney disease in his younger sister (patient 2), showed enlarged hyperechoic kidneys; at the same visit, hypertension was diagnosed. Two antihypertensive medications achieved borderline blood pressure control. Renal function was normal. A brain MRI performed at age 22 months was interpreted as normal. He began to have decline in glomerular function at age 5.5 and underwent kidney transplantation at age 8 years. The extracted native kidneys exhibited moderate chronic interstitial nephritis, glomerulosclerosis, tubular atrophy, and nephrocalcinosis as well as cysts, mostly accumulated at the corticomedullary junction but also scattered throughout the cortex and medulla (). He had polyuria and polydipsia and chronic normochromic normocytic anemia since early infancy. His growth was normal. He developed chronic elevation of liver enzymes and splenomegaly and had an episode of cholangitis at age 5 years. A liver biopsy performed at 5.5 years showed CHF (). Eye evaluation at NIH revealed slight slowing of the saccades, without significant evidence of OMA. His vision was 20/20 OU. The optic discs and retina were normal. On ERG, mixed responses were slightly subnormal but cone responses were normal. The full-scale IQ of patient 3 was 76, with index scores of 81, 71, 94, and 75, respectively.
Sequencing of PKHD1 in patients 2 and 3 was negative. Similarly, testing for the common NPHP1 gene deletion and for NPHP2 mutations was negative. Sequencing of the MKS3 gene showed that both siblings were homozygous and their parents were heterozygous for the missense mutation c.1843 T>C, p.Cys615Arg in exon 18 (). On retrospective review, subtle findings within the spectrum of the MTS were noted on patient 3’s brain MRI performed at 22 months ().
Figure 2 Immunofluorescent confocal microscopy of the polarized, ciliated IMCD3 cell-line, after transfection with an HA-epitope tagged construct of meckelin with the p.C615R mutation, compared with wild-type. Top panels: Immunostaining of endogenous meckelin (more ...)
To document the consequences of the splice mutation, we amplified and sequenced the RT PCR product of RNA extracted from the transformed peripheral blood lymphocytes of patient 1 and demonstrated that the c.224-2 A>T mutation resulted in skipping of exon 2 (; available at www.jpeds.com
) with a subsequent frameshift in exon 3, resulting in premature truncation after 79 amino acids. To assess subcellular localization of wild-type and mutant (p.Cys615Arg) meckelin, we used immunofluorescent confocal microscopy of the polarized, ciliated IMCD3 cell-line. Both endogenous and transfected wild-type HA epitope-tagged meckelin are folded in the ER and transported efficiently to the apical cell surface and primary cilia (, top panel). Very little of the wild-type protein is found at mid-cell z-slices in the confocal images. However, the HA-tagged meckelin containing the C615R mutation is partially retained at the mid-cell regions (, bottom panel), possibly in lysosomes, although it can also be transported to the apical cell surface. There was no colocalization of this mutant protein with primary cilia (data not shown).
Figure 3 MKS3 mutations found in patients 1 through 3. Analysis of the c.224-2 A>T splice site mutation at the RNA level (A and B) is shown. Expected wild-type size (483 bp) is seen in the control, whereas in patient 1 the smaller band (394 bp) corresponds (more ...)