MKS3-related ciliopathy with features of autosomal recessive polycystic kidney disease, nephronophthisis, and Joubert Syndrome

Abstract

Objectives: To describe 3 children with mutations in a Meckel syndrome gene (MKS3), with features of autosomal recessive polycystic kidney disease (ARPKD), nephronophthisis, and Joubert syndrome (JS).

Study design: Biochemical evaluations, magnetic resonance and ultrasound imaging, electroretinograms, IQ testing, and sequence analysis of the PKHD1 and MKS3 genes were performed. Functional consequences of the MKS3 mutations were evaluated by cDNA sequencing and transfection studies with constructs of meckelin, the protein product of MKS3.

Results: These 3 children with MKS3 mutations had features typical of ARPKD, that is, enlarged, diffusely microcystic kidneys and early-onset severe hypertension. They also exhibited early-onset chronic anemia, a feature of nephronophthisis, and speech and oculomotor apraxia, suggestive of JS. Magnetic resonance imaging of the brain, originally interpreted as normal, revealed midbrain and cerebellar abnormalities in the spectrum of the "molar tooth sign" that characterizes JS.

Conclusions: These findings expand the phenotypes associated with MKS3 mutations. MKS3-related ciliopathies should be considered in patients with an ARPKD-like phenotype, especially in the presence of speech and oculomotor apraxia. In such patients, careful expert evaluation of the brain images can be beneficial because the brain malformations can be subtle.

Trial registration: ClinicalTrials.gov NCT00068224.

Conflict of interest statement

The authors declare no conflicts of interest. Registered with www.clinicaltrials.gov (NCT00068224).

Figures

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’ kidney ultrasound findings (dots outline the kidneys) (left panel of C through E) and kidney growth over the years are shown (average of right and left), measured on ultrasound and plotted against age (right panel of C through E). Bold solid red line indicates the patients’ kidney length for age, with the normal mean indicated by a bold gray line, +2 SD by a solid gray line and −2 SD by a dotted gray line. Kidneys of patient 1 C, remained large (10.3 cm) at 8 years of age. Kidneys of patient 2 measured 9.2 cm at age 2 years 10 months D, continued above 2 SD until approximately 4 years of age, and then declined toward normal size as she progressed into end-stage renal failure. Kidneys of patient 3 measured 11.1 cm at age 4 years, 2 months E, remained large at approximately 4 SD until 5 years of age, and declined sharply in size as he progressed into end-stage renal disease. All 3 patients (C through E) exhibited enlarged, diffusely hyperechoic kidneys with loss of corticomedullary differentiation and a few scattered macrocysts (arrow) indistinguishable from ARPKD kidneys (B). The liver biopsies of patient 1 F and patient 2 G show congenital hepatic fibrosis characterized by expanded portal areas with persistence of embryonic bile duct structures (arrows). The extracted kidney of patient 3 at low H and high I magnification shows cysts scattered throughout the cortex and medulla and accumulating at the corticomedullary junction (arrow, H). Lymphoid aggregates with chronic interstitial nephritis, glomerulosclerosis, and tubular atrophy (H and I) are shown. Normal brain MRI on axial cut through the superior cerebellar peduncles J is shown. The “molar tooth sign” seen in JSRD K caused by the combination of a hypoplastic cerebellar vermis (white arrow), elongated and thickened superior cerebellar peduncles (black arrow), and abnormally deep interpeduncular fossa is shown. The brain MRI of patient 1 L, initially reported as normal, in fact shows a subtle molar tooth sign (white arrow). The brain MRI of patient 3, M initially reported as normal, shows slightly elongated and pointed superior cerebellar peduncles (white arrow) with hypoplastic and dysplastic vermis on retrospective reevaluation.

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 (red), transfected wild-type HA-tagged meckelin (green), endogenous GRP94 (a marker of the endoplasmic reticulum; blue), and DAPI (grey). z-Stacks were taken every 0.5 μm and cells were assessed for immunostaining in the apical region (within the first 2.5 μm of the cell, above the nucleus) or in the mid-cell regions (below the apical region, but above the last 1 μm of the cell). The igures show individual z-stacks from the apical and mid-cell regions, as indicated. Endogenous meckelin colocalized with wild-type HA-tagged meckelin at both cilia (arrowheads) and at the apical cell surface (arrow). Wild-type HA-tagged meckelin is absent from mid-cell subcellular locations. Note that endogenous meckelin colocalizes with some components of the endoplasmic reticulum. Bottom panels: Immunostaining as above, but transfected with HA-tagged meckelin containing the p.C615R mutation. Mutant meckelin does not appear to extensively colocalize with endogenous meckelin at the apical cell surface and is aggregated in the mid-cell region (arrows). Scale bars = 5 μm.

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 to the transcript lacking exon 2, A. Direct sequencing of the RT-PCR product from patient 1 shows skipping from exon 1 to exon 3, B. Localization of the p.Cys615Arg mutation (red sphere) to the intracellular loop of the meckelin protein, C, in comparison with the location of the mutations reported in MKS (blue diamonds) and JSRD patients (green diamonds) is shown. Modified from Khaddour et al.