NIPA1 gene mutations cause autosomal dominant hereditary spastic paraplegia (SPG6)

Abstract

The hereditary spastic paraplegias (HSPs) are genetically heterogeneous disorders characterized by progressive lower-extremity weakness and spasticity. The molecular pathogenesis is poorly understood. We report discovery of a dominant negative mutation in the NIPA1 gene in a kindred with autosomal dominant HSP (ADHSP), linked to chromosome 15q11-q13 (SPG6 locus); and precisely the same mutation in an unrelated kindred with ADHSP that was too small for meaningful linkage analysis. NIPA1 is highly expressed in neuronal tissues and encodes a putative membrane transporter or receptor. Identification of the NIPA1 function and ligand will aid an understanding of axonal neurodegeneration in HSP and may have important therapeutic implications.

Figure 1

a, Representative SPG6 sequence of normal and affected subjects from the ADHSP-ARK and ADHSP-IRQ kindreds. PCR products (from 50 ng of DNA) were purified through Sephadex G-50 columns, were analyzed by agarose gel electrophoresis, and were sequenced either by use of the ABI PRISM dRhodamine Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems) on an ABI PRISM 3100 Genetic Analyzer, according to the manufacturer’s instructions, or through the University of Pennsylvania DNA sequencing facility. The arrows mark the position of the NIPA1 mutation (cDNA nucleotide 159). b, Pedigrees of ADHSP-ARK1 and ADHSP-IRQ1. Letters refer to sequence at nucleotide 159 of the NIPA1 cDNA sequence. Clinical features (Fink et al. 1995a), muscle biopsy (Hedera et al. 2000 ), and genetic features (Fink et al. 1995b) of ADHSP-ARK1 have been reported elsewhere. The ADHSP-IRQ1 kindred was ascertained through the University of Michigan Neurology Outpatient Clinic.

Figure 2

a, SPG6 occurs in regions deleted in PWS and AS. SPG6 is mapped to chromosome 15q11, proximal to the imprinted PWS and AS domains, within a region containing four nonimprinted genes. These genes, including NIPA1, map within class I deletions in patients with PWS and AS but are intact in class II deletions (Chai et al. [in this issue]). BP = breakpoint hotspot; cen = centromere; tel = telomere. b, NIPA1 secondary structure analysis. Nine predicted transmembrane (TM) domains (Chai et al. [in this issue]) are shown. The NIPA1 T45R mutation occurs at the interface of TM1 and the first putative outside loop. c, Expression of NIPA1 by northern blot analysis. NIPA1 transcripts of 2.2 kb and 7.5 kb are found in all tissues but are greatly enriched in neuronal tissues. Probes were prepared from TA-cloned RT-PCR products of human NIPA1 (167 bp) and exons 2–3 of the control SNURF-SNRPN gene, were labeled with α-³²P-dCTP, and were hybridized in ExpressHyb (Clontech) at 65°C to northern blots (Clontech), according to the manufacturer’s protocol.