The NPHP1 gene deletion associated with juvenile nephronophthisis is present in a subset of individuals with Joubert syndrome

Abstract

Joubert syndrome (JS) is an autosomal recessive multisystem disease characterized by cerebellar vermis hypoplasia with prominent superior cerebellar peduncles (the "molar tooth sign" [MTS] on axial magnetic resonance imaging), mental retardation, hypotonia, irregular breathing pattern, and eye-movement abnormalities. Some individuals with JS have retinal dystrophy and/or progressive renal failure characterized by nephronophthisis (NPHP). Thus far, no mutations in the known NPHP genes, particularly the homozygous deletion of NPHP1 at 2q13, have been identified in subjects with JS. A cohort of 25 subjects with JS and either renal and/or retinal complications and 2 subjects with only juvenile NPHP were screened for mutations in the NPHP1 gene by standard methods. Two siblings affected with a mild form of JS were found to have a homozygous deletion of the NPHP1 gene identical, by mapping, to that in subjects with NPHP alone. A control subject with NPHP and with a homozygous NPHP1 deletion was also identified, retrospectively, as having a mild MTS and borderline intelligence. The NPHP1 deletion represents the first molecular defect associated with JS in a subset of mildly affected subjects. Cerebellar malformations consistent with the MTS may be relatively common in patients with juvenile NPHP without classic symptoms of JS.

Figure 1

Midline sagittal T1 images (A, C, and E) and associated axial T2 images through the level of the superior cerebellar peduncles (SCPs) (B, D, and F) in subjects indicated above each column. Note the superiorly positioned fourth ventricle with moderate inferior vermis hypoplasia (small white arrows) on the sagittal images of subjects K76-1 (A) and K84-1 (C), compared with the normal fourth ventricle with mild inferior vermis deficiency in subject K89-1 (E). Elongated SCPs are demonstrated on axial images (B and D, black arrows), with associated mild MTS, compared with normal SCPs (F).

Figure 2

Southern analysis of EcoRI-digested genomic DNA from members of family K76 and from control subjects, confirming the presence of a homozygous deletion of NPHP1 in the two affected daughters (blackened circles). The probe is a 405-bp PCR fragment, including exons 9–10 (forward primer: 5′-TGGAAAGCAAGTTCTTAGTAAGCAGCG-3′, reverse primer: 5′-GGCAGAATTTGGACTTGCTACCTTGA-3′). The number of wild-type alleles in control samples is indicated by the genotype symbols underneath the black line (below “NPHP1”). Dosage is apparent by intensity of the 3.94-kb band that hybridizes to the intragenic NPHP1 probe. A ß-actin control probe detected a band of almost equal intensity for all samples, confirming equal loading of DNA (data not shown).

Figure 3

Haplotype analysis in family K76, showing markers compatible with linkage to the 2q13 region surrounding NPHP1. The deleted STS marker 9657T is indicated in affected subjects K76-1 and K76-2. The 290-kb deletion is mediated by two inverted duplicated segments (invdup) of ∼330 kb each, via a complex mechanism.

Figure 4

Comparison of the NPHP1 deletion in subjects with JS (K76-1 and K76-2) and in a control subject with the classic deletion (K84-1). The results of STS deletion mapping are indicated in C and D as ticks (to indicate the marker was present in these three subjects) and crosses (to indicate the marker was absent in all three subjects). A, Scale bar indicating 850 kb of contiguous DNA sequence at 2q13, isolated from the UCSC genome browser. B, BAC clone contig of the interval. (Note that clone 385J6 flanks both ends, demonstrating the difficulty presented by genomic duplications in generating accurate contigs.) C, Map of STS markers in the region that was used for deletion mapping. The NPHP1 deletion interval is indicated by a gray bar, and arrow diagrams outline the repeat regions: large blue arrows indicate the 330-kb inverted repeat, and short black arrows indicate the three copies of the 45-kb repeat, of which the two direct repeats are known to mediate unequal crossover. D, Gene locations included within the region encompassing the 45-kb repeats, mapped with orientation indicated by arrowheads. The shaded gray box indicates the only unique DNA sequence of the deleted region, which includes LOC205251, NPHP1, and all but the 3′ portion of the BENE gene.