Molecular Vision 2007; 13:2183-2193 <http://www.molvis.org/molvis/v13/a248/> Received 10 July 2007 | Accepted 17 November 2007 | Published 27 November 2007

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Novel truncating mutations of the CHM gene in Chinese patients with choroideremia

Shea Ping Yip,^1,2 Tsz Shan Cheung,¹ Man Yu Chu,¹ Suk Chun Cheung,³ Kam Wah Leung,^2,3 Kin Ping Tsang,² Stephen T.S. Lam,^2,4 Chi Ho To^2,3
 
 

¹Department of Health Technology and Informatics, The Hong Kong Polytechnic University, Hung Hom; ²Scientific and Medical Advisory Committee, Retina Hong Kong, Lai Chi Kok; ³School of Optometry, The Hong Kong Polytechnic University, Hung Hom; and ⁴Clinical Genetic Service, Department of Health, Hong Kong SAR Government, Hong Kong SAR, China

Correspondence to: Shea Ping Yip, Department of Health Technology and Informatics, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong SAR, China; Phone: +852 3400 8571; FAX: +852 2362 4365; email: shea.ping.yip@polyu.edu.hk

Abstract

Purpose: Choroideremia (CHM) is an X-linked retinal degenerative disorder caused by mutations in the CHM gene. The mutations result in malfunction of the Rab escort protein 1 (REP-1). In this study, mutational analysis of the CHM gene was performed on five Chinese families clinically diagnosed with CHM.

Methods: Denaturing high performance liquid chromatography was used for mutation screening for all 15 exons and flanking intron regions of the CHM gene. Mutations were confirmed and characterized with DNA sequencing. Second samples were later collected for extraction of mRNA and proteins from leukocytes. A non-radioactive protein truncation test (PTT) was developed and used to characterize the truncating nature of the mutations. Immunoblot analysis of proteins extracted from leukocytes was also performed.

Results: Five mutations were identified in these five families, each with one distinct mutation: three frameshift, one nonsense, and one splicing. Two of these were novel mutations: c.627dupA in exon 5 and c.703-1G>C in intron 5. The truncating nature of the mutations was experimentally proved by PTT for four families with second samples collected. In particular, c.703-1G>C spliced exon 5 directly to exon 7 and deleted the entire exon 6 from the transcript. Direct immunoblot analysis failed to detect REP-1 in males affected by CHM, but demonstrated its presence in female carriers and homozygous normal females.

Conclusions: This is the first study reporting mutations in the CHM gene in Chinese families. Mutational analysis was performed at the DNA, mRNA and protein levels. Five truncating mutations were found, and two of these were novel.

Introduction

Choroideremia (CHM; MIM 303100) is an X-linked recessive inherited disease characterized by progressive degeneration of the choroid, retinal pigment epithelium, and retina [1]. Patients usually develop night blindness in childhood, followed by progressive loss of peripheral vision in the second and third decades of life, eventually culminating in blindness. CHM is rare with an estimated prevalence of 1 in 100,000 and accounts for about 4% of the blind population.

The CHM gene is located at chromosome Xq21.2 [2,3]. The gene has 15 exons spanning about 150 kb (kb) of genomic DNA, produces a messenger RNA of about 5.5 kb, and is expressed in many tissue types including retina, choroid, retinal pigment epithelium, and lymphocytes [2,4]. It encodes an intracellular protein of 653 amino acids, called Rab escort protein-1 (REP-1) [5].

REP-1 is one of the proteins involved in the post-translational lipid modification of many intracellular proteins, a process important in the trafficking of proteins between various vesicular compartments of the cell [6]. Rab proteins are small proteins that bind guanosine triphosphate and regulate trafficking of proteins between various membranous organelles in the eukaryotic cell. The activity of Rab proteins relies on their association with the cytoplasmic side of cellular membranes, and this association is dependent on the prenylation of Rab proteins. REP-1 binds to newly synthesized Rab proteins, presents them to the enzyme Rab geranylgeranyl transferase, and then escorts the prenylated Rab proteins to their target membranes [7,8]. Rab geranylgeranyl transferase is not able to interact directly with nascent Rab proteins without the mediation of REP-1. The importance of REP-1 was highlighted when the gene encoding REP-1 was found to be the gene mutated in CHM [2-4].

CHM is caused by mutations in the CHM gene encoding REP-1 [5]. These mutations include large deletions, translocations, an L1 insertion, and a variety of small mutations (nonsense, frameshift, and splicing mutations). Virtually all subtle mutations in the CHM gene give rise to a premature stop codon, which results in the dysfunction or the absence of REP-1. CHM mutations have been reported in European, North American, and Japanese families, and are documented in the Human Gene Mutation Database. This study reports the mutational analysis of five Chinese CHM families at the DNA, RNA, and protein levels, and identifies two novel truncating mutations.

Methods

Subjects

Five probands and their family members (3.5-88 years old, 17 male, and 13 female, all in good general health except for ocular findings in affected and carrier individuals) were recruited on a voluntary basis from Retina Hong Kong (formerly called the Hong Kong Retinitis Pigmentosa Society) as part of the Hong Kong Patients' Register of Retinal Degeneration (formerly called Hong Kong RP Registry). All participating subjects underwent full eye examination at the Optometry Clinic of The Hong Kong Polytechnic University. The clinical tests included visual acuity, refraction, fundus examination, visual field testing (30-2 or 24-2 patterns of Humphrey II Visual Field Analyzer; Carl Zeiss, Dublin, CA), and fundus photodocumentation (Topcon TRC-50X; Topcon Optical, Tokyo, Japan). A panel composed of an ophthalmologist and several optometrists reviewed all ocular findings and confirmed the clinical diagnosis of CHM in affected individuals. The diagnosis of CHM was based on the fundus characteristics of diffuse retinochoroidal degeneration and the corresponding visual field defects. A clinical geneticist (STSL) obtained detailed family history from each family recruited and offered genetic counseling. The study adhered to the tenets of the Declaration of Helsinki, and written informed consent was obtained from every subject.

Anonymous DNA samples from 100 healthy Chinese blood donors (16-47 years old, 44 male, and 57 female) were also available from a previous study for determining the allele frequencies of the mutations found in this study.

Isolation of nucleic acids and protein from leukocytes

Venous blood was collected from participants and DNA extracted with a salting-out method [9] with two modifications. First, whole blood was mixed with erythrocyte lysis buffer (155 mM NH₄Cl, 10 mM KHCO₃, 1 mM EDTA, pH 7.4) before the harvested white cell pellet was mixed with nucleus lysis buffer as described [9]. Second, incubation with proteinase K was carried out overnight at 55 °C, instead of 37 °C. DNA samples were measured for concentration at 260 nm and then stored below -20 °C until use.

After the initial identification of mutations at the DNA level, a second fresh venous blood sample was obtained with additional written consent from ten participants (five affected, four carrier, and one normal) from four families; one family could not be contacted at this stage. Total RNA was extracted from fresh whole blood with a commercial kit (High Pure RNA Isolation Kit; Roche, Mannheim, Germany) according to the manufacturer's instructions, and then used immediately for the synthesis of complementary DNA (cDNA; process to follow). The newly synthesized cDNA was stored at -20 °C until use. In addition, leukocytes were harvested from fresh blood samples with a separating medium (Histopaque-1077; Sigma-Aldrich, St. Louis, MO). Leukocytes were washed twice with phosphate-buffered solution and lysed at 4 °C in a cocktail containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 1mM EDTA, 0.1% sodium dodecyl sulphate (SDS) and protease inhibitor cocktail (Sigma-Aldrich) [10]. After lysis of the leukocytes, the cell debris was spun down at 16,000 g at 4 °C, and the supernatant protein concentration was determined using the Bradford assay. Protein was extracted from the harvested leukocytes according to a published protocol [10] with minor modifications, and the protein concentration determined using the Bradford assay. The protein-containing supernatant was stored at -80 °C until use.

Polymerase chain reaction amplification, and screening and characterization of mutations

Seventeen primer pairs were designed to amplify all 15 exons and their immediate flanking intron sequences of the CHM gene (Table 1). The fragments ranged in size from 236 basepairs (bp) to 419 bp. Each fragment was amplified by polymerase chain reaction (PCR) in a 25 μl reaction mixture containing 50 ng genomic DNA, 0.3 mM of each primer (0.5 mM for fragment chm10), 0.2 mM of each dNTP, 1.5 or 2.5 mM MgCl₂ (see Table 1), 0.5 U AmpliTaq Gold Polymerase and 1X Gold buffer (both from Applied Biosystems, Foster City, CA). Amplification was performed in a thermal cycler (GeneAmp PCR System 9700, Applied Biosystems) with the following cycling conditions: one cycle of initial denaturation at 95 °C for 5 min, 35 cycles of 94 °C for 20 s, 58 °C or 60 °C (see Table 1) for 30 s and 72 °C for 30 s, and a final extension at 72 °C for 7 min.

For denaturing high performance liquid chromatography (DHPLC) analysis, a 30 μl mixture was prepared by mixing 10 μl each of the PCR products from two different samples with one part of the PCR product from a healthy normal control. This mixing protocol doubled the screening throughput without sacrificing the detection sensitivity (unpublished data). Each of these two samples were separately mixed with a normal sample and re-analyzed if the initial analysis indicated the presence of possible sequence variations. PCR products or their mixtures were analyzed with the Wave DNA Fragment Analysis System (Transgenomic, Omaha, NE) using a DNASep column (Transgenomic), as described in our previous report [11]. Briefly, PCR products were denatured for 5 min at 95 °C and then cooled to 25 °C at a rate of 1 °C/min to allow for heteroduplex formation. Next, aliquots of denatured products were automatically injected into the column and eluted with a linear acetonitrile (ACN) gradient in 0.1 M triethylammonium acetate (TEAA) buffer, pH 7.4, at a constant flow rate of 0.9 ml/min. The gradient was a range of proportions (percent) of buffer B increased at a rate of 2%/min and created by mixing buffers A and B, where buffer A was 0.1 M TEAA with 0.025% ACN and buffer B 25% ACN in 0.1 M TEAA. The acetonitrile gradient was calculated automatically by the software Wavemaker (version 4.1, Transgenomic) on the basis of the DNA sequence and hence was specific to each PCR product (Table 1). Each fragment was analyzed by DHPLC at 2-6 column temperatures (Table 1), which were determined by the number of melting domains in the fragment.

All PCR fragments that produced a chromatogram other than a typical one-peak profile were subjected to direct DNA sequencing using dRhodamine Terminator Cycle Sequencing Ready Reaction Kit and ABI Prism 310 Genetic Analyzer (both from Applied Biosystems) according to the manufacturer's instructions. Sequencing was performed in both directions.

To determine whether the mutations were present in the general Chinese population in Hong Kong, we genotyped the mutations in 100 DNA samples from healthy blood donors. Genotyping was performed either with DHPLC, using the conditions best for detecting the mutations, or by restriction analysis using AluI or Mva1269I (Fermentas UAB, Vilnius, Lithuania) according to the manufacturer's instructions.

Protein truncation test

CHM cDNA was first synthesized from total RNA, and then used to produce two separate, but overlapping PCR fragments with two rounds of PCR amplification (Figure 1 and Table 2). The nested PCR products were used to produce CHM polypeptide by in vitro coupled transcription-translation. The translated polypeptides were separated by polyacrylamide gel electrophoresis (PAGE) and detected by immunoblotting.

First strand cDNA was synthesized from total RNA by reverse transcription with the primer NR3 (Table 2). A 12 μl reaction mixture containing 100 ng RNA (freshly extracted), 0.5 mM of each dNTP, and 0.5 mM primer NR3 was prepared, denatured at 65 °C for 5 min, and then cooled in ice. This cooled mixture was mixed with a 7 μl solution containing 10 mM dithiothreitol, first-strand buffer (1X, 50 mM Tris-HCl, pH8.3, 75 mM KCl, 3 mM MgCl₂; Invitrogen, Carlsbad, CA), 40 units RNAguard Ribonuclease Inhibitor (porcine; GE Healthcare, Piscataway, NJ), and then further incubated at 42 °C for 2 min. One μl of Superscript II Reverse Transcriptase (200 units; Invitrogen) was added, and the final 20 μl mixture incubated at 42 °C for 50 min for cDNA synthesis. The enzyme was then inactivated by heating at 70 °C for 15 min. Note that the concentrations shown here refer to those in a final volume of 20 μl.

In the first round PCR, a 25 μl reaction mixture was prepared: 1 μl cDNA, 0.2 mM of each dNTP, 0.3 mM of each of the forward and reverse primers (NF1 and NR2 for fragment CHM-1.1, and NF2 and NR3 for fragment CHM2.1; Table 2), 5 mM MgCl₂, 1 unit FastStart Taq DNA polymerase (Roche), and 1X PCR buffer (50 mM Tris-HCl, 10 mM KCl, 5 mM (NH4)₂SO₄, pH 8.3). Amplification was started with an initial denaturation of 95 °C for 5 min, followed by 30 cycles of 95 °C for 1 min, 60 °C for 1 min, and 72 °C for 3 min, plus a final extension at 72 °C for 7 min. The first PCR product CHM-1.1 (1,500 bp) spanned from exon 1 to the beginning of exon 12 while CHM-2.1 (1,382 bp) covered the last portion of exon 5 and extended beyond the stop codon in exon 15.

In the second round PCR, a 25 μl reaction mixture was also prepared as described in the previous section but with the following modifications: 2 μl of first round PCR product as template, 0.1 μM of each primer (CHM-1FT7myc and CHM-2R for CHM1.2, and CHM-2FT7myc and CHM-3R for CHM-2.2; Table 2) and 2 mM MgCl₂. The nested amplification was performed using the cycling conditions given in the previous paragraph. Nested PCR products were analyzed in 1% agarose gel to check size and specificity. In addition, the sequences of the nested PCR products were also confirmed by DNA sequence analysis using BigDye Terminator Cycle Sequencing Ready Reaction Kit (version 1.1, Applied Biosystems) according to the manufacturer's instructions. This served to confirm the presence of ATG initiation codon and other codons in the correct reading frame, and the presence of the respective mutations in the cDNA (i.e. mRNA) level. The forward primers of both primer pairs for the second round PCR included the sequences encoding a c-myc reporter peptide that enabled the in vitro translated protein to be detected easily by anti-c-myc antibody [12].

Translated polypeptides were produced from the nested PCR products by in vitro coupled transcription-translation using the TnT T7 Wheat Germ Extract System (Promega, Madison, WI) according to the manufacturer's protocol. The translated products were separated in a 15% polyacrylamide gel in the presence of sodium dodecyl sulphate (SDS-PAGE) at 200 V for 90 min with SE260 Mighty Small Mini-gel System (Hoefer, San Francisco, CA), and then transferred onto a polyvinylidene difluoride (PVDF) membrane (GE Healthcare) with a Mini Trans Blot Cell (Bio-Rad, Hercules, CA) at 100 V for 90 min.

The PVDF membrane was blocked overnight at 4 °C in a solution containing 0.5% Tween 20 and 5% non-fat milk. The c-myc-tagged translated products were detected by incubating the membrane as follows: 2 h in a solution containing 2 μg/ml anti-c-myc (9E10, mouse monoclonal; Santa Cruz Biotechnology, Santa Cruz, CA), then 1 h with the secondary antibody rabbit anti-mouse immunoglobulins (Z0259, polyclonal; Dako, Glostrup, Denmark) at a dilution of 1 in 1000, and finally 1 h with the tertiary antibody goat anti-rabbit IgG conjugated with horseradish peroxidase (sc-2004, polyclonal; Santa Cruz) at a dilution of 1 in 1000, with several washes with 0.5% Tween 20 in Tris-buffered saline between antibodies. The blot was developed with Enhanced Chemiluminescence (GE Healthcare) and exposed to Hyperfilm (GE Healthcare).

Immunoblot analysis of fresh protein isolated from leukocytes

Protein (20 μg) extracted from leukocytes was loaded onto a 10% polyacrylamide gel and separated by SDS-PAGE at 200 V for 90 min with SE260 Mighty Small Mini-gel System (Hoefer). Transfer to a PVDF membrane was carried out in a Mini Trans Blot Cell (Bio-Rad) at 200 V for 120 min. The membrane was blocked as described in the previous paragraph. Immunological detection of REP-1 was performed as described in the previous paragraph but with the following exceptions. The primary antibody was anti-REP-1 (2F1, mouse monoclonal; Santa Cruz) at 2 μg/ml; the secondary antibody was bovine anti-mouse IgG conjugated with horseradish peroxidase (polyclonal; Santa Cruz) at a 1 in 1000 dilution; and the blot was developed after the secondary antibody.

Results

Thirty individuals from five Chinese families participated in the study (Figure 2). Of these, 26 underwent eye examination and gave blood for molecular genetic study: seven with CHM, seven female carriers, and 12 with normal ocular findings. Four others (three carrier and one normal participant) had eye examinations but did not donate blood for molecular studies.

All of the CHM patients had minimal residual visual field (less than central 5 °) and severe chorioretinal degeneration in their fundi. The CHM female carriers showed different extents of pigment changes in their fundi, but apparently with good visual function both in terms of visual acuity and visual field.

Screening and identification of mutations

DHPLC analysis of the CHM exons and their flanking intronic regions PCR-amplified from genomic DNA revealed aberrant chromatograms for fragments chm2, chm5.2, chm6, chm8, and chm13 (Table 1 and Figure 3). Subsequent sequence analysis identified one polymorphism and five mutations; the mutations were each found in one family. For the sake of consistency, mutations are denoted at the DNA and protein levels according to recommended nomenclature system [13] and its subsequent online revisions (genomic, nomenclature for the description of sequence variations).

The five CHM mutations included one duplication, two deletions, and two base substitutions (Figure 3 and Table 3). The mutations each segregated with the disease status within the respective families under study (Figure 2). All seven subjects affected with CHM were males hemizygous for the mutations concerned. Females with pigment changes in the fundus were heterozygous for the mutation present in the respective families-a finding consistent with their carrier status. Subjects with normal ocular findings did not have any mutations. In addition, none of the five mutations was found in a group of 100 healthy subjects. Two mutations were genotyped by restriction analysis (c.703-1G>C by AluI and c.1019C>A by Mva1269I) while the other three were typed by DHPLC.

One single nucleotide polymorphism (C/T; rs1015148) was identified in fragment chm2 at position +80 within intron 2, and had already been documented in public databases like dbSNP. This was not followed up.

Effects of the CHM mutations

Protein truncation test (PTT) was used to determine if the mutations identified were truncating in nature or not. Only four families provided a subsequent sample for additional testing, and family C7812 could not be contacted.

As expected, the duplication (c.627dupA) identified in family C7809 produced a truncated protein (about 26 kDa) in vitro translated from CHM-1.2, but yielded a wildtype protein (about 49 kDa) from CHM-2.2 because this mutation did not fall within the region covered by CHM-2.2 (Figure 1, Figure 4 and Table 3). On the other hand, the four-base deletion (c.1584_1587delTGTT) identified in family 9051 produced a truncated protein (about 36 kDa) from CHM-2.2, yet generated a wildtype protein (about 56 kDa) from CHM-1.2 because this mutation was outside the region covered by CHM-1.2. The nonsense mutation (c.1019G>A) identified in family C8161 was covered by both CHM-1.2 and CHM-2.2, and thus produced truncated proteins of 40 kDa and 14 kDa, respectively, translated from them. Cycle sequencing of cDNA from these samples confirmed the nature of mutations involved in the PTT and the initial findings based on the sequencing of genomic DNA. One interesting observation about the PTT results was that the in vitro translated truncated protein was detected in a much smaller amount than the corresponding wildtype protein in female heterozygous carriers (CHM-1.2 and CHM-2.2 for II:1 in family 8161, and CHM-1.2 for II:4 in family 7809; Figure 4).

The substitution at the junction between intron 5 and exon 6 (c.703-1G>C) turned out a truncated protein (about 52 kDa) slightly shorter than the corresponding wildtype protein (about 56 kDa) in vitro translated from CHM-1.2 (Figure 4). This substitution did not generate any protein from CHM-2.2. Sequencing of cDNA indicated the absence in the transcript of the whole exon 6 (Figure 3C), which has 117 bases and encodes 39 complete amino acids (amounting to about 4 kDa). In other words, c.703-1G>C was a splicing mutation that resulted in exon skipping and completely deleted exon 6 from the transcript. Deletion of exon 6 removed the annealing sites of the forward primers of CHM-2.1 and CHM-2.2 (Figure 1) and hence explained the absence of protein translated from CHM-2.2.

Immunoblot analysis of proteins extracted from leukocytes

Immunoblot analysis revealed that no REP-1 could be detected in hemizygous males affected by CHM (Figure 5). Yet, REP-1 was detected in heterozygous female carriers and a homozygous normal female.

Discussion

This study screened, identified, and characterized the mutations in the CHM gene in clinically diagnosed choroideremia patients and their family members at three levels: DNA, mRNA (or cDNA), and protein. Initial screening by DHPLC of PCR-amplified fragments and subsequent DNA sequencing identified five mutations: one duplication (a type of insertion), two deletions, and two base substitutions (Figure 3 and Table 3). This is the first study to report mutations in the CHM gene in Chinese families. In addition, two of these mutations are novel and have not been reported before.

The two novel mutations are the duplication c.627dupA in exon 5 and the substitution c.703-1G>C at the acceptor splice site of intron 5. The duplication is predicted to produce a shift in the reading frame of the protein coding sequence and consequently a premature termination codon at a position 12 codons downstream (p.Pro210ThrfsX12; Table 3). Indeed, it was experimentally proved to be truncating in nature (Figure 4). On the other hand, c.703-1G>C was predicted to be a splicing mutation producing aberrant splicing because it mutates one of the invariant dinucleotides at the acceptor site of splicing. It is, however, difficult to predict the exact outcome of the aberrant splicing without additional investigation. Yet this mutation spliced exon 5 directly to exon 7 and skipped the whole exon 6 completely, as evident from the results of cDNA sequencing and PTT (Figure 3 and Figure 4). In other words, this resulted in an in-frame deletion of 117 nucleotides from the mature transcript or 39 amino acids from the mutant polypeptide (p.Leu235_Gln273del; Table 3), if any. The substitution c.703-1G>C was also the first splicing mutation identified in the intron 5 of the CHM gene. As documented in the Human Gene Mutation Database and this study to date, splicing mutations have been found in all CHM introns except intron 8.

It is interesting to note that the same in-frame deletion of the entire exon 6 from the mature transcript was also reported in a Caucasian CHM patient who, however, harbored a completely different type of mutation [14]. In this case, the coding region of exon 6 in genomic DNA was disrupted by the insertion of an L1 element (about 6 kb long) in reverse orientation between nucleotides 737 and 738, and was thus skipped as a result of aberrant splicing. L1 elements are retrotransposons that can move around the human genome in a process involving transcription, reverse transcription and re-integration into a new chromosomal site [15]. It is also of interest to note that, in both cases, the missing peptide of 39 amino acids (residues 235 to 273) falls within the sequence conserved region 2 (SCR2) of REP-1; SCR2 is one of the several regions conserved between Rab escort proteins and Rab GDP dissociation inhibitors [16]. The amino acids of SCR2 were found to form parts of a hydrophobic pocket that bound one of the prenyl groups attached to the carboxyl terminus of Rab proteins-this is part of the process involved in post-translational prenylation of intracellular proteins [17].

The other three truncating mutations (two deletions and one nonsense) have been reported previously in different Caucasian populations in Europe and North America: c.652_655delTCAC [18], c.1584_1587delTGTT [18-22], and c.1019C>A [18,23]. These three mutations are expected to be truncating because of the nature of the mutations (deletion and nonsense). However, the present study provides the first experimental evidence (PPT and direct immunoblot analysis; Figure 4 and Figure 5) demonstrating the truncating nature of c.1584_1587delTGTT and c.1019C>A. It remains to be determined whether each of these mutations was recurrent mutations that occurred either independently in different populations (Caucasian and Chinese) in human history, or descended from a common ancestor and spread to different parts of the world. This can be investigated by studying the haplotypes carrying the respective mutations and polymorphic markers both within and flanking the CHM locus. For a few nonsense mutations identified in different families or populations, preliminary studies indicated that some were independent recurrent mutations while some might be descended from a common ancestor [18].

The CHM mutations identified in this study were predicted to be truncating and then experimentally proved to be genuinely truncating in an in vitro system by PTT where second samples were available for study (Table 3 and Figure 4). Although putative in vivo translated proteins were also predicted to be shortened, no REP-1 was immunologically detectable in the protein extracts prepared from leukocytes (Figure 5). There are several possible explanations for this finding. First, the truncated proteins might be present, but was not detected by the monoclonal 2F1 antibody which recognized the C-terminus of REP-1. This C-terminus region was missing in the putative truncated protein, if any, in the CHM patients reported here except the one with the c.703-1G>C mutation that causes skipping of exon 6 encoding amino acid residues 235-273 (Table 3). The 2F1 antibody was produced by immunizing mice with a recombinant fusion protein carrying the C-terminal 415 amino acids of human REP-1 [7]. In addition, some of our earlier PTT experiments showed that the 2F1 antibody could recognize the C-terminal one-third portion of REP-1, but not the N-terminal two-third portion of REP-1 (data not shown). Second, it is possible that the mRNA species carrying premature termination codon was destroyed rapidly by the mechanism of nonsense-mediated mRNA decay so that little, if any, truncated protein was translated in vivo. Nonsense-mediated mRNA decay is an mRNA quality-control mechanism that degrades abnormal mRNAs carrying premature termination codon and hence prevents the production of shortened protein [24]. This means that the mutant mRNA would be expected to be less in amount in vivo than the wildtype mRNA in female heterozygous carriers. This could then be reflected in the smaller amount of in vitro translated mutant protein than the corresponding wildtype protein in the PTT experiments (Figure 4). Third, it is also possible that the shortened protein might have been produced in vivo, but was somehow destroyed rapidly because of incomplete folding or other reasons so that no REP-1 was detected by in vitro testing. It is also possible that both mechanisms were operative in vivo. Further studies are required to address these issues.

In conclusion, five Chinese families with CHM were investigated to identify and characterize the disease-causing mutations in the CHM locus at three levels (DNA, mRNA, and protein). A non-radioactive PTT was successfully developed and used in this study. Five truncating mutations were found in these families, each with one distinct mutation. Two were novel mutations not previously reported: c.627dupA and c.703-1G>C.

Acknowledgements

This study was supported by a grant (G-YD74) from The Hong Kong Polytechnic University (PolyU), and the Hong Kong Patients' Register of Retinal Degeneration was established with financial support (HCPF number HF-Z40) from the Health Care and Promotion Fund of the Hong Kong SAR Government. SCC was also partly supported by a Dean Reserve Fund (Faculty of Health and Social Sciences, PolyU). The Wave DNA Fragment Analysis System was purchased with a PolyU Large Equipment Grant (G.53.27.9027). The authors thank Dr. Maria Wong for helping to collect the second set of blood samples for use in studying the mutations at the mRNA and protein levels. The mutation screening part of the study was presented as a poster in Human Genome Meeting 2005 in April 2005 in Kyoto, Japan.

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