Molecular analysis of the SMN gene mutations in spinal muscular atrophy patients in China.

Spinal muscular atrophy (SMA) is one of the most common autosomal recessive diseases. Survival motor neuron1 (SMN1) is the SMA disease-determining gene. We examined the molecular basis of SMA in 113 Chinese SMA patients. Homozygous exon 7 and 8 deletions in SMN1 were detected by PCR-RFLP. Heterozygous deletion of SMN1 was analyzed based on variation of the sequencing peak height of the two different base pairs of exons 7 and 8 between SMN1 and SMN2. Subtle mutation was detected by genomic sequencing in the patients with heterozygous deletion of SMN1. In our study, the rate of deletion of SMN1 exon 7 and/or 8 was 91.2%; the rate of subtle mutations was 1.8%. We detected the same subtle mutation (p.Leu228X) of SMN exon 5 in two patients (one type I, one type III). The p.Ser8LysfsX23 and p.Leu228X mutations accounted for 13 of the 23 families with subtle mutations reported in the SMN1 gene of Chinese SMA. This is the first report where the phenotype of SMA-type III is associated with p.Leu228X. We found two subtle mutation hotspots (p.Ser8LysfsX23 and p.Leu228X) of SMN1 exons 1 and 5 in Chinese SMA patients. These two mutations have not been reported from America or Europe. It is proposed that the distribution of subtle mutations of SMN1 of SMA is associated with ethnicity or geographic origin.

The polymorphisms of HLA-DR and TNF B loci in northern Chinese Han nationality and susceptibility to systemic lupus erythematosus.

With the aid of methods of polymerase chain reaction/sequence specific primers (PCR/SSP) and polymerase chain reaction-restriction fragment length polymorphism (PCR/RFLP), the allelic polymorphism of HLA-DR and TNFB loci and susceptibility to systemic lupus erythematosus (SLE) in northern Chinese Han nationality were studied. The genetic analysis of 51 patients with SLE and 106 healthy controls indicated that frequencies of DR2 and DR3 alleles were significantly increased in SLE patients (P < 0.05 and < 0.005, relative risks of 1.77 and 4.01 respectively), which represent candidate susceptible genes or useful marker for SLE. The frequency of DR5 was found to decrease in SLE patients compared with control population (P < 0.025, relative risk = 0.38). It might be an antagonistic or protective allele or a marker for such allele. Analysis of 51 patients with SLE and 80 healthy control also revealed that the frequency of TNFB*2 allele was significantly increased (P < 0.05, RR = 1.70). Therefore TNFB*2 gene may also be a susceptibility gene or a marker gene for SLE in northern Chinese Han nationality. It was also investigated the association between HLA-DR, TNF B alleles and Patient plasmic SC5b-9 levels, auto-antibodies (anti-SSA, SSB, Sm, RNP, ds DNA and ANA) and SLE complications (SLE nephritis, SLE pneumonitis and SLE encephalopathy), no relationship was found.

[Study on some susceptible genes of systemic lupus erythematosus in Han nationality of China].

In order to explore the allelic polymorphism of HLA-DR and TNF B loci and susceptibility to systemic lupus erythematosus (SLE) in the Han nationality of northern China with the aid of methods of polymerase chain reaction/sequence specific primers (PCR/SSP) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) respectively. The findings from a case-control study on 151 blood samples (45 from the cases and 106 from the controls) indicated that there were significantly higher frequency of DR2 (P < 0.05, RR = 1.56) and DR3 (P < 0.01, RR = 2.69), which represent candidate susceptible genes or useful markers for SLE. The DR5 allele in the samples (P < 0.05, RR = 0.43) might be an antagonistic or protective allele, or a marker for such allele. The frequency of TNF B * 1 and TNF B * 2 alleles was investigated in 45 SLE patients and 80 healthy controls and it was found that the frequency of TNF B * 2 allele was significantly higher in the patient group (P < 0.05, RR = 1.84). It might also be a suspicious susceptible allele or a marker for such allele. The frequency of HLA polymorphisms in various clinical/immunological subsets of our patient population was also determined. Clinical findings used include plasma SC5b-9 level, SSA, SSB, Sm, RNP, ANA antibodies, and SLE complications (SLE nephritis, pneumonia & encephalopathy). It turned out that there was a positive association between HLA-DR2 allele and SLE nephritis (P < 0.05, RR = 1.32).

[SC6 antigen for the diagnosis of pancreatic cancer before operation].

SC6 antigen, defined by monoclonal antibody SC6 purified by our laboratory, was measured with a sandwich immunoradiometric assay. Its normal upper limit was considered 41 U/ml. Levels of serum SC6 antigen, a new tumor marker for pancreatic cancer, were assayed in 42 patients who were diagnosed as having pancreatic cancer before operation, acute pancreatitis in 16, and chronic pancreatitis in 7, involving 40 healthy controls. The concentration of SC6 antigen of 19 from the 42 patients was higher than normal upper limit. 15 of the 19 patients was last diagnosed as having pancreatic cancer by operation and pathology. The sensitivity, specificity and predictive value of this assay for pancreatic cancer was 76.2% (15/21), 94% and 75%. The levels of SC6 antigen in 4 patients with pancreatic cancer were observed in different times. The false positive rate of serum SC6 antigen was lower than that of CA19-9. The results show that the antigen is significant for diagnosis, prognosis and observation of the change of serum SC6 antigen for pancreatic cancer. It may be of help to find early pancreatic cancer.

Identification of a novel de novo STK11 mutation in a Chinese child with Peutz-Jeghers syndrome.

Peutz-Jeghers syndrome (PJS) is a rare autosomal dominant inherited disorder characterized by gastrointestinal polyposis and mucocutaneous pigmentation. PJS patients have an increased risk of cancer in multiple locations. Germ-line mutations in the STK11 gene have been found to be responsible for most PJS cases. DNA samples were obtained from a Chinese child with PJS, his clinically unaffected parents and 50 unrelated normal individuals, and the exons and flanking intronic sequences of the STK11 gene were analysed by polymerase chain reaction and direct sequencing. A novel de novo mutation (c.698_699insG; F234LfsX3) was identified in exon 5 of STK11, that resulted in a translational frameshift leading to termination at codon 236. This mutation was not found in the parents or unrelated individuals. These results enlarge the genotypic spectrum of STK11, particularly with regard to early onset, as observed in the present sporadic PJS case. This study may have important future implications for precise genotype-phenotype correlation research.

Active sites in complement component C3 mapped by mutations at indels.

Engineered mutants of human complement component C3 were used to test the idea that sites of length polymorphisms in protein families (indels) can guide a search for protein:protein interaction sites. Sequence changes were introduced at each of the 27 indels in the C3/4/5 protein family, and mutants at 26 indels were expressed by transiently transfected COS cells. Expressed proteins were assayed 1) for concentration, by ELISA and by autoradiography of radiolabeled protein; 2) for classical pathway hemolytic activity; 3) for susceptibility to proteolytic activation by the alternative pathway and cobra venom factor C3 convertases; and 4) for susceptibility to complement factor I in the presence of factor H. Most of the mutations did not appreciably alter expression or activity relative to wild-type C3, consistent with the idea that most indels occur at the protein surface. Mutations at four indels severely damaged C3 functional activity, but did not affect the stability or structure of the protein, as assessed by their effects on expression by COS cells and on susceptibility to cleavage by C3 convertases and factor I. These indels are therefore near functionally important amino acid residues; they represent good candidates for sites of protein:protein interactions. Mutation of the sequence at a fifth indel altered the equilibrium between the latent and reacted C3 conformations, and mutations at 4 other indels substantially decreased both protein activity and expression. The mutants provided an overview of the structural and functional roles played by different parts of C3.

Active sites in complement components C5 and C3 identified by proximity to indels in the C3/4/5 protein family.

We recently suggested that sites of length polymorphisms in protein families (indels) might serve as useful guides for locating protein:protein interaction sites. This report describes additional site-specific mutagenesis and synthetic peptide inhibition studies aimed at testing this idea for the paralogous complement C3, C4, and C5 proteins. A series of C5 mutants was constructed by altering the C5 sequence at each of the 27 indels in this protein family. Mutants were expressed in COS cells and were assayed for hemolytic activity and protease sensitivity. Mutants at five indels showed relatively normal expression but substantially reduced sp. act., indicating that the mutations damaged sites important for C5 function. Twenty-three synthetic peptides with C5 sequences and 10 with C3 sequences were also tested for the ability to inhibit C hemolytic activity. Three of the C5 peptides and one of the C3 peptides showed 50% inhibition of both C hemolytic and bactericidal activities at a concentration of 100 microM. In several cases both the mutational and peptide methods implicated the same indel site. Overall, the results suggest that regions important for function of both C3 and C5 lie proximal to residues 150-200 and 1600-1620 in the precursor sequences. Additional sites potentially important for C5 function are near residue 500 in the beta-chain and at two or three sites between the N-terminus of the alpha'-chain and the C5d fragment. One of the latter sites, near residue 865, appears to be important for proteolytic activation of C5.

Distal recognition site for classical pathway convertase located in the C345C/netrin module of complement component C5.

Previous studies focused on indels in the complement C345 protein family identified a number of potential protein-protein interaction sites in components C3 and C5. Here, one of these sites in C5, near the alpha-chain C terminus, was examined by alanine-scanning mutagenesis at 16 of the 18 non-alanine residues in the sequence KEALQIKYNFSF RYIYPLD. Alanine substitutions affected activities in the highly variable manner characteristic of binding sites. Substitutions at the lysine or either phenylalanine residue in the central KYNFSF sequence had the greatest effects, yielding mutants with <20% of the normal activity. These three mutants were also resistant to the classical pathway (CP) C5 convertase, with sensitivities roughly proportional to their hemolytic activities, but had normal susceptibilities to the cobra venom factor (CVF)-dependent convertase. Synthetic peptide MGKEALQIKYNFS-NH2 was found similarly to inhibit CP but not CVF convertase activation, and the effects of alanine substitutions in this peptide largely reflected those of the equivalent mutations in C5. These results indicate that residues KYNFSF form a novel, distal binding site for the CP, but not CVF convertase. This site lies approximately 880 residues downstream of the convertase cleavage site within a module that has been independently named C345C and NTR; this module is found in diverse proteins including netrins and tissue inhibitors of metalloproteinases.