Molecular Characterization Of SMN1 Copy Number In The Chinese Population And Rapid Genotyping Of Spinal Muscular Atrophy Determinants

Background and ObjectiveSpinal muscular atrophy(SMA) is one of the most common recessive neuromuscular disorders in the world.In all populations studied worldwide,carrier frequency is about 1/40-1/50.Its clinical features are symmetric proximal muscle weakness and atrophy.On the basis of age of onset and clinical severity,SMA is divided into four groups:TypeⅠis a infantile acute form with the greatest severity; TypeⅡis a infantile chronic form;TypeⅢis a mild childhood and adolescent form; TypeⅣis an adult form.SMA has great relationship with survival motor neuron (SMN) gene located in 5q13 which include SMN1 and SMN2.Mutations in SMN1,the telemetric copy,are associated with SMA.However,SMN2,the centromeric copy,is a disease-modifying gene which can ameliorate the clinical severity of SMA in a copy-dependent manner.94%of SMA patients are homozygous absence of the SMN1 exon7 which is caused by deletion or SMN1-to-SMN2 conversion.The remaining 6% may be homozygote for small intragenic mutations,or heterozygote for absence of the SMN1 exon7 on one allele and a small intragenic mutation on the other,or not caused by SMN1 gene.Detection of SMN1/SMN2 copy numbers is an important method for carrier screening and diagnosis.These years,new technologies on SMN1/SMN2 quantitative detection have improved clinical genetics,genetic epidemiology, population screening,and prenatal diagnosis of SMA.Molecular genetic of SMA in Western people has been studied widely and deeply.However,the data on Chinese SMA is not enough,including genetic epidemiology survey based on a large population in mainland China.On the basis of modified DHPLC quantitative detection methodology focused on SMN copy number,we identified SMN copy number quantitatively in 1712 southern Chinese newborn cord blood samples and genotyped 25 SMA core families.Our research elucidated Chinese SMA incidence, genetic load,and SMN gene variant and would be the basis for the plan of carrier screening and prenatal diagnosis.Because present SMA gene diagnosis methodology can not fulfill the demand of clinical diagnosis,we developed a reverse dot blot technology for rapid genotyping deletion type SMA,combined with DHPLC,which could serve SMA clinical diagnosis better.Materials and methods1712 newborn cord blood samples were used for molecular epidemiology survey. Among them,1220 samples were collected in Shaoguan from October 8,2006 to December 28,2007,and 492 samples were collected in Guangzhou from March 15, 2002 to July 20,2002 by a simple random sampling way.95%of 1712 newborns belonged to southern Chinese.SMN copy numbers were detected by semi-quantitative DHPLC method.On the basis of SMN copy numbers,we could determine sample genotype,carrier frequency and genetic load.Genotypes and allele frequencies of different mutation site and variant site were calculated.The population data was analyzed by Hardy Weinberg equilibrium.In order to evaluate distributional characteristic of SMA mutation and genotyping methodology,we collected 25 SMA core families which were diagnosed by clinical features.All of them were detected by semi-quantitative DHPLC method. Based on the differences of exon7/8 in SMN1/2,we developed a reverse dot blot technology which could identify deletional type SMA.Using blind analysis,we evaluated the reverse dot blot technology by 181 normal and SMA carrier samples which were diagnosed by DNA sequencing and DHPLC.Some samples with abnormal DHPLC profile were analyzed by DNA direct sequencing.The functional effect of this mutation was accessed at the mRNA level by real-time quantitative reverse-transcript PCR(RT-PCR) technology.Results and discussion 1712 samples could be divided into 13 groups according to the different SMN1: SMN2 ratio and SMN1/2 copy number.There were 12,17,8 and 1 samples in SMN1: SMN2=1:2,1:3,1:1 and 1:4 group,respectively.All the 4 groups belonged to SMA carrier and the population frequency is 2.22%.There were 993 and 461 samples in 2:2 and 2:1 group,respectively.The 2 groups were normal individual and detection rate was 84.64%.Furthermore,in the 1712 samples,we identified 7 gene variants and small deletion,including IVS6-50A＞G 18,IVS6-24dupT 6,IVS7+287dupG 3, c.875C＞G 1,IVS7+288intC 1,IVS7+137G＞T 1,and EX8+204de12 1.Among the 25 SMA patients of the 25 core families,23 were SMN1 homozygous deletion and 2 were heterozygous deletion.One heterozygous deletion patient was proved to be a carrier of mutation c.835-1G＞A which was a splicing mutation located in the acceptor site of intron 6.Among the 44 parents of the 25 core families,19,12,8,3,land 1 were with SMN1:SMN2 ratio 1:3,1:2,1:1,2:1,1+1~D:2 and 1+1~D:1,respectively.No parent had 1:4.The SMA baby with c.835-1G＞A fell ill in 6 month old and died in 10 month old.The point mutation was inherited from his father and deletional mutation from his mother.RT-PCR could prove the transcript of mutation c.835-1G＞A was SMN△7.After retrieving PubMed,it was confirmed to be a novel SMA mutation,which had never been reported.This was the first time to identify a point mutation in Chinese SMA patient and the new knowledge could be important for clinical diagnosis and genetic counseling.Our research was the first SMA carrier screening in southern Chinese based on a large population.According to our investigation,we could calculate the SMA incidence of different genotype.SMN1 homozygous deletion was the main cause of SMA and the incidence was 12/100,000(95%CI,10.46/100,000～13.54/100,000). With the current annual one million newborns,the estimated number of SMA newborn each year in Guangdong was 120(95%CI,104.6～135.4).The predominant 3 genotypes in core families were coincident with the data from large population.In core families,we identified 3 carders with a gene ratio of SMN1:SMN2=2:1,which accounted for 6.82%(3/44).After DHPLC profile analysis and linkage analysis,the genotype of the 3 carriers was proved to be "2+0".That meant one chromosome carried two-copies of SMN1 and the other chromosome carried no one.We should think highly of the gene ratio of SMN1:SMN2=2:1 in genetic counseling and population screening,because it was the second-ranking gene frequency in Chinese which accounted for 27%and some of them were SMA carrier.We identified a point mutation in 25 SMA patients,thus,the non-deletional mutation maybe play an important role in the molecular basis of Chinese SMA.Because the complexity of SMN copy number and genotype distribution,it was very difficult to fulfill population screening and clinical diagnosis correctly.In order to improve the molecular diagnosis method,we developed a reverse dot blot technology which based on PCR amplification and amplicon' SNP sites of SMN gene. We evaluated 181 samples with 3 kinds of SMN genotypes compared with DNA sequencing and DHPLC.The sensitivity and specificity of the test was 92%and 100%,respectively.Reverse dot blot technology could discriminate homozygous deletion of SMN1 or SMN2 and would be promising in SMA prenatal diagnosis combined with DHPLC.Detecting SMA carrier with a genotype of "2+0" in individual with a gene ratio of SMN1:SMN2=2:1 and distinguishing gene ratio of SMN1:SMN2=2:2 from 1:1 are still great challenge for genetic testing.Our research tried some new method in overcoming the challenge and would enlighten the future study.The data of distribution and frequency of SMN copy number in southern Chinese will be valuable for evaluating SMA genetic load in Chinese.The development and application of related SMA genotyping technology would be the scientific and technologic basis for birth control plan of SMA patient in our country.