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Human Molecular Genetics, 2003, Vol. 12, No. 15 1917-1925
DOI: 10.1093/hmg/ddg198
© 2003 Oxford University Press

Hereditary sensory neuropathy is caused by a mutation in the delta subunit of the cytosolic chaperonin-containing t-complex peptide-1 (Cct4 ) gene

Ming-Jen Lee1,{dagger}, Dennis A. Stephenson1,2, Michael J. Groves1, Mary G. Sweeney1, Mary B. Davis1, Shu-Fang An1, Henry Houlden1, Mustafa A. M. Salih3, Vincent Timmerman4, Peter de Jonghe4, Michaela Auer-Grumbach5, Emilio Di Maria6, Francesco Scaravilli1, Nicholas W. Wood1 and Mary M. Reilly1,*

1Division of Clinical Neurology and Department of Molecular Pathogenesis, Institute of Neurology, Queen Square, London, WC1N 3BG, UK, 2McLaughlin Research Institute, 1520 23rd Street South, Great Falls, Montana 59405, USA, 3Division of Paediatric Neurology, Department of Paediatrics, College of Medicine, PO Box 2925, King Saudi University, Saudi Arabia, 4Molecular Genetics Department, Flanders Interuniversity Institute for Biotechnology (VIB), University of Antwerp (UIA), Belgium, 5Institute of Medical Biology and Human Genetics, Karl-Franzens University, Graz, Austria and 6Department of Neurosciences, Ophthalmology and Genetics, Section of Medical Genetics, University of Genova, Via Benedetto XV, 6-16132 Genova, Italy

Received February 7, 2003; Accepted June 4, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
A spontaneous autosomal recessive mutation was identified in the Sprague–Dawley rat strain with an early onset sensory neuropathy. The main clinical features of the mutation (mutilated foot, mf ), detectable shortly after birth, include ataxia, insensitivity to pain and foot ulceration. The pathological features include a severe reduction in the number of sensory ganglia and fibres. This mutant is therefore an excellent model for human hereditary sensory neuropathies. Here, we demonstrate that the mf locus maps to the distal end of rat chromosome 14, a region syntenic to human 2p13–p16 and proximal mouse 11. Sequence analysis of four candidate genes in this interval revealed a 1349G>A mutation in the chaperonin (delta) subunit 4 (Cct4) gene associated with the mf mutant. This change resulted in the substitution of a highly conserved cysteine for tyrosine at amino acid 450. Although we did not identify a mutation in the human CCT4 gene in a set of HSN patients, this result clearly demonstrates the pathological consequences of a defect in Cct4, a subunit of CCT (cytosolic chaperonin-containing t-complex peptide-1), involved in folding tubulin, actin and other cytosolic proteins. This is the first report of a mutation in a molecular chaperonin causing a hereditary neuropathy and raises the possibility that mis-folding proteins may be a cause of this group of neuropathies.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The hereditary neuropathies are a large and heterogeneous group of diseases in which the neuropathy can either be the sole or primary part of the disease, or part of a more widespread neurological or multi-system disorder (1). The former group includes the more common hereditary neuropathies, Charcot–Marie–Tooth disease [CMT, also known as hereditary motor and sensory neuropathy (HMSN)] and the hereditary sensory neuropathies [HSN, also known as the hereditary sensory and autonomic neuropathies (HSAN)].

Major advances in the understanding of the genetics of both these disease groups have occurred in the last decade. CMT is the more common disease and is usually classified into type 1 (demyelinating) and type 2 (axonal). Although some genes have been identified in CMT type 2, most of the advances have occurred in the identification of the genes causing CMT type 1. These include the identification of a 1.5 Mb duplication of chromosome 17, containing the peripheral myelin protein 22 gene (PMP22) as the cause of the common type of autosomal dominant CMT type 1 (2).

HSN (HSAN) is less common than CMT but many of the causative genes for HSN have now been identified. HSN (HSAN) can be divided into five types although types 4 and 5 may be allelic and three causative genes have been identified. HSN I (HSAN I) is an autosomal dominant disorder, characterized predominantly by progressive degeneration of the dorsal root ganglia leading initially to distal sensory loss with motor involvement occurring later. Many patients with HSN I have been shown to have a mutation in the long chain base subunit-1 of serine palmitoyltransferase (SPTLC1) (3), but the genetic basis for many other patients with HSN I has yet to be identified. HSNII (HSAN II) is an autosomal recessive early-onset mainly sensory neuropathy for which no loci or genes have yet been described. HSN III (HSAN III, Riley–Day syndrome, familial dysautomonia) is another autosomal recessive sensory neuropathy with prominent autonomic involvement due to mutations in the gene encoding the I{kappa}B kinase complex-associated protein (IKAP) (4). HSN IV (HSAN IV) and HSN V (HSAN V) are both autosomal recessive sensory neuropathies presenting with congenital insensitivity to pain. Pathology suggests that type IV is mainly associated with loss of unmyelinated fibres whereas type V is due to the loss of small myelinated fibres. The two diseases may be allelic as a mutation in the causative gene for type IV (tyrosine kinase A receptor, TRKA) (5) has also been described in one patient with type V (6). There are many forms of HSN (especially some patients with type 1 and all patients with type II) for which the causative genes have not been identified.

Clinically some forms of axonal CMT (CMT type 2) are very similar to HSN and most of the genes for CMT type 2 remain unidentified. Therefore the underlying pathogenesis for many forms of the axonal inherited neuropathies (including many types of HSN and CMT 2) have yet to be clearly understood.

Animal models of the hereditary neuropathies offer a unique opportunity to increase our understanding of this complex group of disorders. We have previously described one such model, an autosomal recessive early-onset sensory neuropathy in the rat (7). This mutation, mutilated foot (mf ), has been clinically and pathologically well characterized (7,8). Clinical features of the disease include ataxia, insensitivity to pain and ulceration of the feet (7). Pathological features include a severe reduction in the number of sensory ganglia and fibres (8). These features suggest that this animal mutant is an excellent model for the human HSN, especially HSN types I and II. Reduction in the number of sensory ganglia in the mf rat is preceded by excess apoptosis during late fetal (15 days post-coitus) to early post-natal (2 days after birth) development (8). Despite extensive clinical and pathological characterization, little was known about the genetic defect or its underlying mechanism. To gain a better understanding of the disease process in the mutant rat and to see if this knowledge could further our understanding of the human HSNs/CMTs, we undertook to identify the molecular defect using a positional cloning strategy. The results of this study are reported here.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Genetic characterization of the mf-SD rat strain
An initial screen of the mf-SD rat strain was undertaken to determine the degree of polymorphic variation at a group of microsatellite repeat loci compared with established allele sizes for the other genetically defined strains of rat (www-genome.wi.mit.edu/rat/public/). A group of 51 microsatellite repeat loci (RatMap® pairs) (9–11) was used in this initial screen. Of the 51 examined, 48 displayed some polymorphic variation between the mf-SD and LE strains. These differences ranged from two to 68 nucleotides in some instances. Given this level of variation, an intercross was established between the two strains and the F1 offspring backcrossed to the mf-SD parental strain to produce the N2 offspring for genetic linkage analysis. A total of 536 N2 offspring were produced. These offspring were examined clinically (7) 7 days after birth to determine whether they were affected. Of the 536 N2 offspring, 258 were classified as mf homozygotes by these clinical criteria. This frequency is consistent with an autosomal recessive mode of inheritance ({chi}2(1)=0.746) in accordance with previous findings for this disease gene (7,8).

Genomic localization of the mf locus
A subset of 34 affected N2 offspring were used in a genome-wide screen to ascribe linkage of the mf locus to a specific autosome. This was performed using rat microsatellite repeat loci spaced ~20 cM across the genome. Despite the level of variation suggested by our initial characterization, not all the chosen loci gave informative polymorphisms between the two parental strains (LE and mf-SD). The genetic data for those that did were analyzed using MapManager® (12) software. These data are presented in Table 1 and Figure 1. Two or more microsatellite repeat loci defined 14 of the 20 autosomes. Most loci displayed a recombination frequency of 30% or more with the mf locus (LOD scores ranging from 0 to 1.3). Significantly, two loci on chromosome 14, D14Rat39 and D14Rat22, gave recombination frequencies of 17.6 and 0.0, respectively. LOD scores for these two loci with the mf locus were 3.4 and 9.3. These data indicate that the mf locus is located on chromosome 14 in a region associated with D14Rat39 and D14Rat22.


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  		Table 1. Screened markers, frequency of recombination and LOD scores for the affected 34 N2 rats
 


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  		Figure 1. Genetic linkage map identifying the relative position of the mf locus. Left-hand map displays the relative position of the microsatellite loci used to assign the mutant to chromosome 14 based upon the consensus map for these loci (Table 1). In this study, the mutant locus was non-recombinant with D14Rat22 in a sample of 34 affected backcross offspring. The right-hand map provides a higher resolution map in the region of D14Rat22 employing additional loci and more back-cross animals. The numbers in parentheses provide the recombinant fraction observed between the microsatellite repeat locus and the mutant locus (e.g. seven recombinants were detected between D14Rat21 and the mutant locus from a total 174 back-cross offspring typed for both loci). In this more detailed map, the mf locus was recombinant with D14Rat22 (2/220) but non-recombinant with D14Got72 (0/536). As D14Got72 is flanked by D14Got73 (proximally) and D14Got76 (distally), the position of the mf locus is defined to lie within a critical region identified by these two loci. The genetic distance, in centiMorgans, was calculated by dividing the number of recombinant individuals by the total number of back-cross offspring characterized for the two neighbouring loci multiplied by 100.

 
Refined mapping of the mf locus on chromosome 14
Given the assignment of the mf locus to chromosome 14, the linkage analysis was expanded to include all 536 N2 progeny and additional microsatellite repeat loci associated with the chromosome in the region of D14Rat22. Eight loci were added to the linkage map in this analysis: D14Rat21, D14Rat49, D14Got73, D14Got72, D14Got76, D14Rat32, D14Rat131 and D14Rat132 (Fig. 1). The order of loci on the genetic map was determined by minimizing the number of multiple crossover events.

Although D14Rat22 was non-recombinant with the mf locus in the initial linkage analysis, both loci were shown to recombine (2/220) with one another in this larger set of N2 progeny. Nonetheless, four (D14Got73, D14Got72, D14Got76, D14Rat32) of the nine new loci served to redefine the relative location of the mf locus. D14Got72 was shown to be non-recombinant with mf (0/536) whilst 2/536 recombinants separated D14Got73, D14Got76, D14Rat32 and the mf locus. Further, four out of 536 recombinants separated D14Got73 from D14Got76 and D14Rat32, placing them on either side of the mf locus. Given these data, we concluded that the mf locus lies within a genetic distance of 0.7±0.4 cM defined by D14Got73 at the proximal margin and D14Got76/D14Rat32 at the distal margin. Furthermore, the mf locus lies within 0.6 cM of D14Got72 at the 95% confidence interval.

Comparative mapping studies using sequence database searches
Given the genetic map that defines the critical region containing the mf locus, the unique sequences associated with closely linked microsatellite repeat loci were use in a BLAST search of both the mouse and human genome sequence databases. Although no sequence similarities were observed in the human sequence database associated with the microsatellite repeat loci, searches of the Celera® mouse genome database did reveal sequence similarities (Table 2). The probability of a random match (P-values in the range of 3x10-50 to 0.26) for these sequences suggests that such events are, in the main, extremely unlikely. This would therefore imply that they are true matches.


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  		Table 2. Relative position of chromosome 14 MapRat® pairs sequence on mouse chromosome 11
 
Of the dozen or so loci mapping to the same region on rat chromosome 14, nine (D14Rat130, D14Rat21, D14Rat95, D14Rat139, D14Rat138, D14Rat22, D14Got73, D14Rat131, D14Rat132) identified sequences in the mouse genome that mapped to the proximal region of chromosome 11 (Table 2), encompassing a physical distance of about 10 Mb. Furthermore, five of these loci (D14Rat21, D14Rat22, D14Got73, D14Rat131, D14Rat132) were used to define the genetic position of the mutant locus (Fig. 1). The physical distance for these five loci on mouse chromosome 11 is ~4.5 Mb. Of these five, two (D14Got73, D14Rat131) flank the mf locus and define a physical distance of 2.9 Mb in the mouse.

Although the mf locus was non-recombinant with D14Got72, the BLAST search failed to identify similar sequences in the mouse genome. However, the primer pairs defining this locus were used to screen a rat BAC library for BAC clones containing this sequence. Three clones were identified in this screen (RPCIB657O1162, RPCIB657O13359 and RPCIB657B21324). These clones were sequenced using primers for the T7 and Sp6 promoter sequences flanking the genomic insert. A BLAST search with these BAC end sequences against the mouse genome database revealed unique sequence similarities to four of the six sequences (Table 2). These four sequences defining the ends of two of the BAC clones (RPCIB657O13359 and RPCIB657B21324) also identified sequences in the proximal region of mouse chromosome 11 (Table 2). The relative location of the two end sequences from both clones effectively defined the sizes of the genomic inserts (i.e. 139 and 154 kb). Furthermore, these sequences fell within the region flanked by those associated with D14Got73 and D14Rat131 thus, indirectly, placing D14Got72 in the position we would anticipate from the genetic data.

As a significant number of rat microsatellite repeat loci (including D14Got73, D14Got72 and D14Rat131 that defines the position of the mf locus) found similarities with mouse sequences in the same region of the mouse genome that preserves the same order, it would suggest that this region is conserved in the two species. At least six mouse genes are known to map in the interval containing the rat loci D14Rat22, D14Got73, D14Got72 and D14Rat131 (Fig. 2): soluble malate dehydrogenase (Mor2), Drosophila orthodenticle homologue 1 (Otx1), beta-1,3-N-acetylglucosaminyltransferase 1 (B3gnt), chaperonin containing TCP1, delta subunit 4 (Cct4), avian reticuloendotheliosis viral oncogene homologue (Rel ) and B-cell CLL/lymphoma 11A zinc finger protein (Bcl11a). Five (Mor2, B3gnt, Cct4, Rel and Bcl11a) have recognized human homologues (MDH1, B3GNT, CCT4, REL and BCL11A). These human homologues all map to chromosome 2p15. Furthermore, the physical distance between the genes is approximately equivalent in the two species (Fig. 2). Given this conservation between mouse chromosome 11 and human chromosome 2p15, it is reasonable to assume that the same genes will also be present in the conserved interval of rat chromosome 14.



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  		Figure 2. Comparative genetic map of mouse chromosome 11 displaying the position of the unique sequences associated with the rat microsatellite loci and human chromosome 2p15. The physical map of proximal mouse chromosome 11 is shown on the right and the equivalent region of human chromosome 2p15 is shown on the left. The relative positions of the rat microsatellite repeat loci with similar sequences on mouse chromosome 11 are identified to the right-hand side of the physical map of mouse chromosome 11. Similarly, the BAC end sequences containing the rat microsatellite repeat locus D14Got72 are shown on the right-hand side of mouse map (B21324-T7+B21324-Sp6 and O13359-Sp6+O13359-T7 defining the BAC clones RPCIB657B21324 and RPCIB657O13359, respectively). Mouse genes (Mor2, B3gnt, Cct4, Rel and Bcl11a) with recognized homologues in the human genome are placed on the left-hand side of the mouse map and the equivalent human genes (MDH1, B3GNT, CCT4, REL and BCL11A, respectively) are placed on the right-hand side of the human map with lines linking the genes. Genes with, as yet, no known homologues in the other species are placed on the outside of the two linkage maps (i.e. RPL21, LAMRI, XPO1, PEX13 and PAPOLG in human and Otx1 in the mouse). The approximate physical between various loci are given in kilobases. In some instances, the physical distance between loci is reasonably well conserved between the two species (e.g. MDH1 to B3GNT1 is 1365.4 kb in human and 1248 kb in the mouse for Mor2 and B3gnt).

 
Gene sequence analysis
The rat homologues to three of the five mouse genes (Cct4, Rel and Bcl11a) were sequenced from RT–PCR product generated from RNA isolated from both wild-type and mf/mf liver tissue. No variation in sequence was detected in either the Rel or Bcl11a gene between the wild-type and mutant homozygotes. However, the substitution of a guanine for an adenine at nucleotide 1349 was observed in the rat Cct4 between the wild-type and mutant samples (Fig. 3A) using the primers described in Table 3. Sequencing both forward and reverse strands as well as demonstrating that both nucleotides were present in sequence from known heterozygotes confirmed this change. Similarly, sequence analysis of this gene in animals from the mapping study demonstrated that the guanine nucleotide always co-segregated with the mutant phenotype even in the recombinant individuals involving D14Got73 and D14Got76/D14Rat32. To exclude the possibility that this change was a neutral polymorphism associated with the SD-mf strain, the same gene was sequenced in several different strains of rat (i.e. LE, SHR, WKY, BN, DNA, F334, Hooded, PVG). In each case, adenine was the observed at nucleotide 1349 rather than the guanine associated with the Sprague–Dawley (SD)-mf strain.





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  		Figure 3. Nature of the modification in Cct4 associated with the mf mutant. (A) Electropherograms for the region of the rat Cct4 coding sequence that displays a detectable change between wild-type (upper pherogram) and mutant (lower pherogram). The middle pherogram is the same sequence from a heterozygote. At nucleotide 1349 the wild-type sequence is reported as G, the homozygous mutant has an A (arrow) whereas the known heterozygote has both nucleotides (arrow). The entire coding sequence of the rat Cct4 is available through GenBank (Accession no. AY223861). (B) A translation of the same region of the open reading frame into an amino acid sequence. The wild-type peptide sequence with the relevant codons is presented in the upper sequence whereas the mutant translation is provided in the lower sequence. At the third codon, the wild-type sequence defines a cysteine (TGT) whereas the equivalent codon (arrow) in the mutant defines a tyrosine (TAT). Full translation of the coding sequence is provided in the GenBank submission. (C) Provides a ClustalW analysis of the same region for the same gene from a diverse group of species ranging from yeast to human. In every species depicted, the relevant cysteine is highly conserved (amino acid 450 in mouse, rat and human). A tyrosine at amino acid 450 (arrow) in the mutant rat is inconsistent with the evolutionary conserved cysteine.

 

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  		Table 3. Cct4 gene specific primers
 
The 1832 nucleotide sequence obtained for the rat Cct4 gene (GenBank accession no. AY223861) displays 93% identity with the mouse Cct4 gene (NCBI accession no. NM_009837.1) and 90% identity with the corresponding human gene (NCBI accession no. AF026291). This level of conservation confirms that the rat sequence is derived from the rat Cct4 gene and not from a related gene. Translation of the sequence indicates that the change associated with the mutant causes the substitution of a cysteine (CYS: TGT) for a tyrosine (TYR: TAT) at amino acid 450 (Fig. 3B). Comparison of the protein sequence with those in the peptide sequence database (Fig. 3C) indicates that this cysteine is highly conserved across species, genera, families and kingdoms.

Cct4 gene expression
To gain insight into the probable reason for the specific effect on the sensory nervous system, we undertook to ascertain whether expression of the rat Cct4 was affected differentially in tissues from both mutant and wild-type individuals. Northern slot blots and RT–PCR (Fig. 4) established that Cct4 is ubiquitously expressed in both mutant and wild-type tissues, including the dorsal root ganglia. This would imply that the defect is not due to abnormal Cct4 gene expression in the mutant animal.



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  		Figure 4. RT–PCR expression studies of the rat Cct4 gene in a spectrum of tissues from wild-type and mutant rats. The Cct4 amplified product (upper band of 362 bp) is shown with the product of the Gapdh gene (lower band of 321 bp). Gaphd is ubiquitously expressed and therefore provides an internal control for the quality and success of RT–PCR. Expression of the rat Cct4 is present in every tissue sample from both wild-type (upper panel) and mutant (lower panel) individuals. This would therefore indicate that Cct4 is ubiquitously expressed in the rat and that the mutation does not affect expression of the gene. These data were supported by northern slot blot analysis (data not show).

 
Analysis of human patients with hereditary sensory neuropathy
As the mf rat is an excellent animal model for HSN, we designed 11 pairs of primers to sequence CCT4 in 39 patients with HSN and CMT 2, including five patients with early-onset disease. Although this analysis failed to detect a mutation in the human CCT4 gene in these 39 individuals, it does not preclude the possibility that it may be the basis for some of the other HSN and CMT 2 patients.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Using conventional genetic techniques combined with sequence database searches, we efficiently identified the molecular defect associated with an animal model of human HSN. Although the genetic approach used 536 N2 animals, the resolution of the genetic map did not change significantly beyond that obtained by analysing half this number of offspring. What expedited the process was access to the genome databases of at least two species (human and mouse) and the ability to extrapolate from conserved linkage groups (syntenic regions) between those species to identify likely genes in another species where sequence data is limited.

The gene involved in the mutant phenotype was not an overtly obvious candidate. Given the expression profile and their involvement in apoptosis (13,14), both Rel and Bcl11a were more likely candidates than the Cct4 gene but neither gene displayed sequence variation between mutant and wild-type individuals (unpublished data). Evidence that the change in the Cct4 gene sequence is the causative mutation in the mf mutant is derived from several sources: (i) known heterozygotes have an A/G at nucleotide 1349; (ii) the change co-segregates with the mutant phenotype in the recombinant individuals involving D14Got73 and D14Got76/D14Rat32; (iii) the same change is not present in any of the other wild-type rat strains surveyed; and (iv) the translation of the mRNA sequence indicates that the modification leads to the substitution of a highly conserved cysteine for a tyrosine at amino acid 450. For this cysteine to be evolutionarily conserved, it would suggest that it plays an important role in protein structure, perhaps by forming a disulfide bridge with an internal cysteine or a cysteine from a molecule with which it interacts.

The chaperonin-containing tailless complex polypeptide (CCT) is a cytosolic molecular chaperone involved in folding tubulin, actin and other cytosolic proteins (15). Mutations in putative chaperone proteins, rather than chaperonins themselves, have been shown to be associated with neurological diseases such as hereditary spastic paraparesis, HSP (16). Thus, it is not surprising to find that mutations in other components of the protein-folding machinery also have pathological consequences.

CCT complex is composed of eight subunits, encoded by independent genes (17,18). The Cct4 (CCT{delta}) subunit is involved in binding the actin small domain (19). The folding process involves the binding and hydrolysis of ATP (19,20). It is unclear why the Cys450Tyr mutation should cause a sensory neuropathy. However, the mutation probably disrupts a disulfide bridge between internal cysteine residues leading to structural instability of the protein. This might therefore interfere with the subunits ability to bind and fold actin. Loss of correctly folded actin could lead to a disruption in the integrity of the cytoskeleton causing defects in axonal transport (21) and pathway finding (22). Failure to make suitable contact will lead to neuronal loss through apoptosis (22). This potential mechanism fits with the known observation that the mf mutation acts primarily on dorsal root ganglia causing excessive apoptosis (8).

Dopamine-induced apoptosis in chick embryonic sympathetic neurons (23) leads to up regulation of the CCT4 suggesting that it may have an independent function associated with apoptosis. Apoptosis is also a predicted secondary function of a mutation in serine palmitoyltransferase, long chain base subunit-1 (SPTLC1) gene that also causes hereditary sensory neuropathy 1 (HSN1) in humans (3).

Despite the identification of the molecular defect associated with the mf mutant, more work needs to be done before the underlying mechanism associated with the disease is known. Nonetheless, it will be of significant interest to see if mutations of Cct4 or other chaperones are associated with human disease such as HSN and ulcero-mutilating neuropathies.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Animals
The mf rat (Sprague–Dawley) strain was kept in the Animal House at the Institute of Neurology, Queen Square, London, under specific pathogen-free conditions. Food and water were available ad libitum. Lewis (LE) rats were purchased from B&K Universal Ltd, East Yorkshire, UK. LE males were crossed to SD-mf females, the F1 (SDxLE) were backcrossed to SD-mf homozygotes (N2 progeny). The N2 from these crosses were phenotyped according to the criteria established by Chimelli and Scaravilli (7,8) and killed for DNA and RNA extraction 7 days after birth.

Human subjects
Thirty-nine patients were selected who had a previous clinical diagnosis compatible with hereditary sensory neuropathy or axonal CMT. These patients were selected from diverse ethnic backgrounds (including English, Arab, Belgian, Austrian and Italian) and included five patients with a very-early-onset sensory neuropathy thought to be autosomal recessive.

Genotype analysis
DNA from the back-crossed offspring was purified by proteinase K digestion and phenol/chloroform extraction (24). Genotypes were determined by PCR amplification of polymorphic markers containing simple sequence repeats (Rat MapPairs®, Research Genetics, Huntsville, AL, USA). The reaction conditions and amplification protocols were those recommended by the manufacturer. Genetic data was analysed using MapManager QTXb06 (12).

Screening of the rat BAC library
The rat BAC library was purchased from RZPD, Berlin, Germany and screened by PCR analysis to identify clones containing the microsatellite repeat loci closely linked to the mf locus. Clones containing the specific microsatellite repeat loci were grown overnight under chloramphenicol selection and DNA prepared by SDS/alkali extraction procedure and purified on the Qiagen Maxi-prep column (Qiagen, Hilden, Germany).

RT–PCR
Total RNA was extracted using trizol–chloroform extraction protocol (Invitrogen Ltd, Paisley, UK). The first-strand cDNA synthesis was performed using the SuperScript® first-strand synthesis system (Invitrogen Ltd, Paisley, UK) from an oligo-dT primer. Gene-specific oligonucleotides were used to PCR amplify specific segments of the gene for sequencing and for a probe for northern blot analysis. The specific primers for sequencing the gene are given in Table 3.

Sequence analysis
The sequencing of both BAC ends and RT-PCR products were performed using the Bigdye® Terminator Cycle Sequencing Ready reaction kit version 2 (Applied Biosystems, Foster City, CA, USA) using the 377 automatic sequencer. For sequencing the human CCT4, 11 pairs of primers (Table 3) were used to amplify the coding and flanking regions.

Northern slot blot hybridization
Total RNA was denatured by alkali treatment and slot blotted onto nylon membranes using BioRad slot blot apparatus. The blot was dried and hybridized with tailing probes in a solution containing 0.5 M phosphate buffer (pH 7.5), 5% SDS at 65°C overnight. It was then washed three times at room temperature in a solution containing 0.2xSSC, 0.1% SDS. Hybridization signal was detected by anti-digoxigenin-POD (Roche, Mannheim, Germany) peroxidase reaction using the Vector Nova RED® substrate kit (Burlingame, CA, USA).


   FOOTNOTES
 
* To whom correspondence should be addressed. Tel: +44 2078373611, ext. 3457; Fax: +44 2078132126; Email: m.reilly{at}ion.ucl.ac.uk Back

{dagger} Present address: Department of Medical Genetics, National Taiwan University Hospital, No. 7, Chung-Shan South Road, Taipei 100, Taiwan. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Reilly, M.M. and Hanna, M.G. (2002) Genetic neuromuscular disease. Neurol. Pract., 73 (suppl.), ii12–ii21.

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