Subtle mutations in the SMN1 gene in Chinese patients with SMA: p.Arg288Met mutation causing SMN1 transcript exclusion of exon7.

BACKGROUND: Proximal spinal muscular atrophy (SMA) is a common neuromuscular disorder resulting in death during childhood. Around 81~95% of SMA cases are a result of homozygous deletions of survival motor neuron gene 1 (SMN1) gene or gene conversions from SMN1 to SMN2. Less than 5% of cases showed rare subtle mutations in SMN1. Our aim was to identify subtle mutations in Chinese SMA patients carrying a single SMN1 copy. METHODS: We examined 14 patients from 13 unrelated families. Multiplex ligation-dependent probe amplification analysis was carried out to determine the copy numbers of SMN1 and SMN2. Reverse transcription polymerase chain reaction (RT-PCR) and clone sequencing were used to detect subtle mutations in SMN1. SMN transcript levels were determined using quantitative RT-PCR. RESULTS: Six subtle mutations (p.Ser8LysfsX23, p.Glu134Lys, p.Leu228X, p.Ser230Leu, p.Tyr277Cys, and p.Arg288Met) were identified in 12 patients. The p.Tyr277Cys mutation has not been reported previously. The p.Ser8LysfsX23, p.Leu228X, and p.Tyr277Cys mutations have only been reported in Chinese SMA patients and the first two mutations seem to be the common ones. Levels of full length SMN1 (fl-SMN1) transcripts were very low in patients carrying p.Ser8LysfsX23, p.Leu228X or p.Arg288Met compared with healthy carriers. In patients carrying p.Glu134Lys or p.Ser230Leu, levels of fl-SMN1 transcripts were reduced but not significant. The SMN1 transcript almost skipped exon 7 entirely in patients with the p.Arg288Met mutation. CONCLUSIONS: Our study reveals a distinct spectrum of subtle mutations in SMN1 of Chinese SMA patients from that of other ethnicities. The p.Arg288Met missense mutation possibly influences the correct splicing of exon 7 in SMN1. Mutation analysis of the SMN1 gene in Chinese patients may contribute to the identification of potential ethnic differences and enrich the SMN1 subtle mutation database.

Application and evaluation of artificial intelligence TPS-assisted cytologic screening system in urine exfoliative cytology / 中华病理学杂志

Objective: To explore the application of manual screening collaborated with the Artificial Intelligence TPS-Assisted Cytologic Screening System in urinary exfoliative cytology and its clinical values. Methods: A total of 3 033 urine exfoliated cytology samples were collected at the Henan People's Hospital, Capital Medical University, Beijing, China. Liquid-based thin-layer cytology was prepared. The slides were manually read under the microscope and digitally presented using a scanner. The intelligent identification and analysis were carried out using an artificial intelligence TPS assisted screening system. The Paris Report Classification System of Urinary Exfoliated Cytology 2022 was used as the evaluation standard. Atypical urothelial cells and even higher grade lesions were considered as positive when evaluating the recognition sensitivity, specificity, and diagnostic accuracy of artificial intelligence-assisted screening systems and human-machine collaborative cytologic screening methods in urine exfoliative cytology. Among the collected cases, there were also 1 100 pathological tissue controls. Results: The accuracy, sensitivity and specificity of the AI-assisted cytologic screening system were 77.18%, 90.79% and 69.49%; those of human-machine coordination method were 92.89%, 99.63% and 89.09%, respectively. Compared with the histopathological results, the accuracy, sensitivity and specificity of manual reading were 79.82%, 74.20% and 95.80%, respectively, while those of AI-assisted cytologic screening system were 93.45%, 93.73% and 92.66%, respectively. The accuracy, sensitivity and specificity of human-machine coordination method were 95.36%, 95.21% and 95.80%, respectively. Both cytological and histological controls showed that human-machine coordination review method had higher diagnostic accuracy and sensitivity, and lower false negative rates. Conclusions: The artificial intelligence TPS assisted cytologic screening system has achieved acceptable accuracy in urine exfoliation cytologic screening. The combination of manual screening and artificial intelligence TPS assisted screening system can effectively improve the sensitivity and accuracy of cytologic screening and reduce the risk of misdiagnosis.

Diagnosis of Spinal Muscular Atrophy: A Simple Method for Quantifying the Relative Amount of Survival Motor Neuron Gene 1/2 Using Sanger DNA Sequencing / 中华医学杂志(英文版)

Background@#Spinal muscular atrophy (SMA) is caused by homozygous deletion or compound heterozygous mutation of survival motor neuron gene 1 (SMN1), which is the key to diagnose SMA. The study was to establish and evaluate a new diagnostic method for SMA.@*Methods@#A total of 1494 children suspected with SMA were enrolled in this study. Traditional strategy, including multiplexed ligation-dependent probe amplification (MLPA) and TA cloning, was used in 1364 suspected SMA children from 2003 to 2014, and the 130 suspected SMA children were tested by a new strategy from 2015 to 2016, who were also verified by MLPA combined with TA cloning. The SMN1 and SMN2 were simultaneously amplified by polymerase chain reaction using the same primers. Mutation Surveyor software was used to detect and quantify the SMN1 variants by calculating allelic proportions in Sanger sequencing. Finally, turnaround time and cost of these two strategies were compared.@*Results@#Among 1364 suspected SMA children, 576 children had SMN1 homozygous deletion and 27 children had SMN1 compound heterozygous mutation. Among the 130 cases, 59 had SMN1 homozygous deletion and 8 had heterozygous deletion: the SMN1-specific peak proportion on exon 7 was 34.6 ± 1.0% and 25.5 ± 0.5%, representing SMN1:SMN2 to be 1:2 and 1:3, respectively. Moreover, five variations, including p.Ser8Lysfs *23 (in two cases), p.Leu228*, p.Pro218Hisfs *26, p.Ser143Phefs*5, and p.Tyr276His, were detected in 6/8 cases with heterozygous deletion, the mutant allele proportion was 31.9%, 23.9%, 37.6%, 32.8%, 24.5%, and 23.6%, which was similar to that of the SMN1-specific site on exon 7, suggesting that those subtle mutations were located in SMN1. All these results were consistent with MLPA and TA cloning. The turnaround times of two strategies were 7.5 h and 266.5 h, respectively. Cost of a new strategy was only 28.5% of the traditional strategy.@*Conclusion@#Sanger sequencing combined with Mutation Surveyor analysis has potential application in SMA diagnosis.

Analysis of chromosomal karyotypes, age and height on diagnosis of 232 patients with Turner syndrome / 中华实用儿科临床杂志

Objective To study the distribution characteristics of chromosomal karyotypes,age and height on diagnosis of patients with Turner syndrome(TS).Methods Karyotypes analysis was performed in 1015 girls with short stature who visited the hospital in recent 27 years.And statistical analysis of the chromosomal karyotypes,age and height on the diagnosis of patients with TS were performed by using the SPSS 11.0 software.Results 1.Two hundred and thirty-two (22.9％) patients were diagnosed with TS.2.A total of 26 kinds of abnormal karyotypes were detected in 232 patients with TS and the 45,X (32.4％,75/232 cases) and 46,Xi (Xq) (16.4％,38/232 cases) were the more common karyotypes.In addition,there were 111 cases(47.8％) with 21 kinds of mosaicisms which were related to the number and/or structural anomalies of one X chromosome.3.The median(range) age on the diagnosis was (10.90 ±4.55) years old.TS was mainly discovered during adolescence.The diagnosis rates at different ages were as follows:＜2 years 15 cases(6.4％),2-＜7 years 35 cases(15.1％),7-＜ 12 years 93 cases(40.1％) and 12-＜ 18 years 89 cases(38.4％).4.The median height Z score was-3.60 ± 1.20.There was a significant negative linear correlation between age and Z score of height on the diagnosis of TS (r =-0.613,P ＜ 0.000).5.Eighty-six point five percent (64/74 cases)TS patients with age over 13 years presented pubertal delay at diagnosis,which occurred only in 27.2 ％(22/81 cases) of the control group,and there existed a significant difference(OR =16.297,P =0.000).Conclusions 1.Chromosomal karyotypes in TS are in diverse ness.2.In TS patients,as the age increases,delay of height and pubertal development could be more obvious to observe and more characteristic manifestation of pubertal development would appear.3.The diagnosis of TS was later in children in our cotmtry than that in the developed countries.The analysis of chromosomal karyotypes should be detected earlier in the girls with unexplained short stature,height below-2 Z score of the mean age or the height below the specific lower limit of the peers,so as to benefit the diagnose of TS earlier.

Detection of homozygous deletions in spinal muscular atrophy with genomic DNA sequencing / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To detect homozygous deletions of survival motor neuron (SMN) gene with genomic DNA sequencing, and to assess the value of genetic testing for the diagnosis of spinal muscular atrophy (SMA).</p><p><b>METHODS</b>Polymerase chain reaction (PCR) was used for amplifying SMN gene in 100 SMA patients and 110 controls. Four different bases (g.31957, g.32006, g.32154 and g.32269) between SMN1 and SMN2 within the amplified segments were identified with genomic DNA sequencing. Homozygous deletion of SMN1 or SMN2 was determined by the presence or absence of base peaks at such four sites. Multiplex ligation-dependent probe amplification (MLPA) was carried out to confirm the results of genomic DNA sequencing.</p><p><b>RESULTS</b>In the 100 SMA samples, only SMN2 specific base peaks were detected at the four sites, for which the copy numbers of SMN1 and SMN2 was 0:2 or 0:3, suggesting homozygous deletion of SMN1 gene. By contrast, only SMN1 specific base peaks were detected in 5 samples, for which the ratio of SMN1:SMN2 was 2:0, indicating homozygous deletion of SMN2. At four different sites, SMN1/SMN2 heterozygous peaks were detected in the remaining 105 samples, for which SMN1:SMN2was 2:2, suggesting non-deletion of SMN1 or SMN2. The results of sequencing were consistent with those of MLPA.</p><p><b>CONCLUSION</b>Genomic DNA sequencing is a rapid, accurate and economic method for the diagnosis of homozygous deletion of SMA.</p>

Limitation of PCR-RFLP method for the detection of genetic mutations in spinal muscular atrophy / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To explore the applicability and limitation of PCR-restriction fragment length polymorphism (PCR-RFLP) method for genetic diagnosis of spinal muscular atrophy (SMA).</p><p><b>METHODS</b>PCR-RFLP was applied to detect potential deletion in exons 7 and 8 of SMN1 gene in 935 suspected cases with SMA. Multiplex ligation-dependent probe amplification(MLPA) was carried out to analyze dosage alteration of SMN1 gene in 339 of such cases. To confirm the accuracy of PCR-RFLP method for homozygous and heterozygous deletions detection, the consistency of PCR-RFLP and MLPA results were assessed with a Pearson Chi-square test.</p><p><b>RESULTS</b>Homozygous deletion of exon 7 of SMN1 was detected in 590 suspected cases. The rate of diagnosis was therefore 63.1% (590/935). For the 339 suspected cases, PCR-RFLP and MLPA respectively identified 194 and 196 homozygous deletions in the exon 7 of SMN1 gene, suggesting a good consistency (98.9%)(Chi-square = 0.2, P = 0.88). However, only 4 of 339 cases was found to carry a heterozygous deletion of SMN1 exon 7 by PCR-RFLP, in contrast with 17 detected by MLPA. The consistency only reached 23.5%, for which statistical significance was detected (Chi-square = 8.29, P< 0.01).</p><p><b>CONCLUSION</b>Although PCR-RFLP is a simple, specific and efficient method for SMA diagnosis, it has obvious limitation for the diagnosis of 5%-10% SMA patients who have carried a compound heterozygous mutation.</p>

Compound heterozygous mutation in two unrelated cases of Chinese spinal muscular atrophy patients / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Infantile proximal spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder. Approximately 90% - 95% cases of SMA result from homozygous deletion of survival motor neuron gene 1 (SMN1) and 5% cases are caused by compound heterozygous mutation (a SMN1 deletion on one allele and a subtle mutation on the other allele).</p><p><b>METHODS</b>In this research, two unrelated patients were clinically diagnosed according to the criteria of proximal SMA. Genetic diagnosis was performed to detect the homozygous deletion of exon 7 of SMN1 by PCR-restriction fragment length polymorphism (RFLP) and genomic sequencing. Multiplex ligation-dependent probe amplification (MLPA) analysis was carried out to measure copy numbers of SMN1, SMN2 and neuronal apoptosis inhibitor protein (NAIP) in the patients. Further sequencing of SMN1 allele-specific PCR (AS-PCR) and SMN1 clones were also performed to analyze the point mutation of SMN1 gene. Additionally, the pedigree analysis of these two families was carried out to identify the transmission of the mutation.</p><p><b>RESULTS</b>The inconsistent results using PCR-RFLP and genomic sequencing showed homozygous deletion of exon 7 of SMN1 and heterozygous deletion accompanied with a suspicious mutation in SMN1 gene, respectively. MLPA analysis of these two cases exhibited one SMN1 copy deletion. One identical c.863G > T (p.Arg288Met) mutation was found in two cases by sequencing the SMN1 clones, which confirmed that both cases were SMA compound heterozygotes. One case showed partial conversion to form hybrid SMN (SMN2 I7/SMN1 E8) identified by clones sequencing and another case carrying 3 SMN2 implied complete conversion from SMN1 to SMN2.</p><p><b>CONCLUSION</b>p.Arg288Met is more a disease-causing mutation than a polymorphism variation, and children with this mutation may have more severe phenotypes.</p>

Analysis of survival motor neuron gene conversion in patients with spinal muscular atrophy / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the type and frequency of gene conversion from SMN1 to SMN2 in Chinese patients affected with spinal muscular atrophy (SMA), and to explore the relationship between gene conversion and clinical phenotype.</p><p><b>METHODS</b>Non-homozygous deletion of SMN1 gene exon 8 was screened among 417 patients with SMN1 exon 7 homozygous deletions. To analyze and verify the types of gene conversion, genomic DNA sequencing, multiplex ligation-dependent probe amplification (MLPA), and gene subcloning and sequencing were carried out.</p><p><b>RESULTS</b>Thirty-one patients (7.4% of all) with non-homozygous deletions of SMN1 exon 8 were detected. Through series of experiments, the fusion genes SMN1/SMN2 in all cases were delineated. Five types of gene conversions were identified, which included SMN2-I7b/SMN1 E8, SMN2-I7a/SMN1 I7b, SMN2-E7/SMN1 I7a, SMN1 I6/SMN2 E7/SMN1 I7a and SMN2-E7/SMN1 I7a/SMN2 I7b. Such conversions were found in the type I-III patients. For 10 patients with type I-III SMA and 3 copies of SMN2 gene produced by conversion, the average survival age was 5 year and 4 months.</p><p><b>CONCLUSION</b>Partial conversions of SMN1 gene have been found among Chinese SMA patients. The type of conversion and frequency seem to be different from those of other races. Gene conversion to some extent may impact on survival time and rate of SMA patients, especially type I SMA.</p>

Mutation analysis of SMN1 gene in patients with spinal muscular atrophy / 中华儿科杂志

<p><b>OBJECTIVE</b>Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder. About 80% - 90% of SMA patients are missing both copies of SMN1, and 5% - 10% of patients are compound heterozygotes. In the present study, we aimed to analyze survival motor neuron 1 (SMN1) gene mutation in three patients with spinal muscular atrophy and their families to explore the effect of mutation on SMN protein function and the relationship between mutation and clinical phenotype.</p><p><b>METHOD</b>According to the international criterion, all patients were diagnosed by a neurologist. Patient 1 is a 5 years old boy with SMA type II. Patient 2, female, 2.5 years old, was SMA type II. Patient 3, female, 9 years old, was SMA type III. The brother of patient 3 was SMA type II, too. The age at last examination was 14 years. Genomic DNA was extracted from peripheral blood leukocytes by using standard phenol/chloroform method and total RNA was extracted from whole blood with QIAamp RNA Blood Mini Kit. PCR/RFLP was used to detect the homozygosis deletion of the SMN1 exon 7, and multiplex ligation-dependent probe amplification (MLPA) were performed to analyze the gene dosage of SMN1 and SMN2 for each patient and his/her family members; reverse transcriptase (RT)-PCR and clone sequencing were conducted for identifying the point mutation of SMN1 in three patients. The sequencing of genomic DNA and MLPA were carried out in the 3 families members to confirm the transition of mutation.</p><p><b>RESULT</b>No homozygous deletion of the SMN1 exon 7 was observed in any member of the 3 families. Case 1 and case 2 had one SMN1 copy compound with c.400G > A (p.Glu134Lys) mutation on it and SMN2 was two copies, respectively. Case 3 and her brother also had one copy of SMN1 and two copies of SMN2, and a mutation c.689C > T (p.Ser230Leu) occurred on the retained SMN1. All point mutations were from their fathers and deletion come from their mothers for SMN1 gene.</p><p><b>CONCLUSION</b>In this work, p.Glu134Lys and p.Ser230Leu mutations were identified in three unrelated families and p.Glu134Lys from two patients was first discovered in Chinese SMA. The p.Glu134Lys mutation within the SMN Tudor domain prevents the binding of SMN and Sm. The fact that p.Ser230Leu results in a polar amino acid substituted for non-polar amino acid possibly affects the structure of SMN and then damages its function. SMN1 point mutation analysis is not only advantageous to the diagnosis of those patients with heterozygous deletion of SMN1, but will be beneficial to the prenatal diagnosis and genetic counseling for their families.</p>

Point mutation analysis of SMN1 gene in patients with spinal muscular atrophy / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To identify the point mutations in survival motor neuron gene 1 SMN1 gene and confirm the existence of compound heterozygous mutations in Chinese patients with spinal muscular atrophy (SMA).</p><p><b>METHODS</b>Three unrelated patients were diagnosed and clinically typed according to the criteria of proximal SMA established by the International SMA Consortium. Multiplex ligation-dependent probe amplification (MLPA) analysis was carried out to measure the copy numbers of SMN1, SMN2 and neuronal apoptosis inhibitory protein gene (NAIP)in the patients. The point mutation analysis of SMN1 gene was performed by reversed transcript-polymerase chain reaction (RT-PCR) and cloning sequencing. The MLPA assay and point mutation analysis were also performed in the family members to confirm the transmission of the mutations.</p><p><b>RESULTS</b>Two point mutations were identified in the present study, i.e., the p.Leu228X in one patient and p.Arg288Met in two patients. The mutation p.Arg288Met was first reported in Chinese and p.Leu228X was first reported in Mainland Chinese. The case carrying p.Leu228X mutation was diagnosed as SMA I with 2 copies of SMN2, and the cases with p.Arg288Met were diagnosed as SMA I and SMA II , respectively, with 3 copies of SMN2 gene.</p><p><b>CONCLUSION</b>The mutations p.Leu228X and p.Arg288Met caused severe clinical phenotypes, SMA I or SMA II. This study suggested that the compound heterozygous mutations of SMN1 existed in Chinese SMA patients, which was rarely reported previously in Chinese. It was necessary to detect the point mutation in SMN1 for genetic diagnosis of those patients with heterozygous deletion of SMN1, which would be beneficial to prenatal diagnosis and genetic counseling in these families.</p>

Genetic and clinical study on 17 cases of Angelman syndrome with deletion of 15q11-13 / 中华儿科杂志

<p><b>OBJECTIVE</b>Angelman syndrome (AS) is a neurodevelopmental genetic disorder that maps to 15q11-13. The primary phenotypes are attributable to loss of expression of imprinted UBE3A gene within this region which can arise by means of a number of mechanisms. The purpose of this study was to make a genetic diagnosis and to analyze the clinical features in suspected patients with AS.</p><p><b>METHOD</b>A total of 17 cases were diagnosed clinically as AS including 7 males and 10 females. The age at the time of diagnosis ranged from 8 months to 5 years. Genetic diagnosis was made by methylation-specific PCR (MS-PCR), linkage analysis by short tandem repeat (STR) and chromosome karyotype analysis. According to the international diagnostic criteria of AS, the related characteristic clinical features of the AS patients with deletion of 15q11-13 were analyzed and summarized.</p><p><b>RESULT</b>Deletion of 15q11-13 was confirmed by genetic diagnosis in 17 AS patients. No abnormal findings were observed when they were born. Developmental delay in movement, speech impairments and happy disposition were observed in 100% (17/17) AS patients. And the severe speech deficit was much easier and more obvious to observe than movement. About 80% (14/17) - 90% (15/17) AS patients presented frequent clinical characteristics, such as seizures and abnormal EEG. However, microcephaly could only be observed in 35% (6/17) AS patients. Regarding the associated findings of AS, 41% (7/17) - 77% (13/17) AS patients could be observed with flat occiput/occipital groove, prognathia, wide mouth, wide-spaced teeth, frequent drooling, excessive mouth behaviors, hypopigmented skin, light hair compared to parents, flexed arm position during ambulation and sleep disorder etc. These features occurred at a higher frequency in those patients of > 2 years old group than that of < 2 years old group.</p><p><b>CONCLUSION</b>The testing strategies of MS-PCR and STR linkage analysis combined with chromosome karyotype analysis were appropriate to the molecular genetic diagnosis of AS. In our analysis of clinical features, there was a lower rate of small head circumference (HC) in 35% patients compared with 80% patients in Caucasian with microcephaly, which might be attributable to the phenotypic heterogeneity in different races. And the birth history, movement and speech development and main clinical features of the Chinese AS patients were consistent with those of other studies. Clinical analysis in patients of different age groups showed that findings associated with AS would be more easily observed with the age increasing. Genetic diagnosis should be performed in clinically suspected AS patients.</p>

A study on genetic diagnosis for Angelman syndrome / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the genetic diagnosis of Angelman syndrome(AS), and provide information for clinic diagnosis and counseling to AS families.</p><p><b>METHODS</b>Methylation specific-PCR (MS-PCR) was used for primary diagnosis of 16 clinically suspected AS cases, and linkage analysis by short tandem repeat (STR) was applied to detect the molecular genetic defect in the nuclear families.</p><p><b>RESULTS</b>In this study, 10 AS patients were identified by MS-PCR, and 9 of them with maternal deletion in chromosome 15q11-q13, 1 with imprinting defect in chromosome 15q11-q13 were confirmed by STR linkage analysis.</p><p><b>CONCLUSION</b>Most of the AS patients could be confirmed by MS-PCR. And STR linkage analysis can detect the molecular defect of AS. It is very important for disease diagnosis, genetic counseling and prenatal diagnosis to perform the related genetic diagnosis.</p>

Characteristics of the PAH gene mutation in Chinese patients with phenylketonuria in Xinjiang / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the characteristics of the PAH gene mutation in patients with phenylketonuria (PKU) in Xinjiang area.</p><p><b>METHODS</b>The mutations in exons 3, 5, 6, 7, 11 and 12 and the flanking intronic sequence of the PAH gene were detected by PCR/SSCP analysis and direct DNA sequencing in 46 PKU patients.</p><p><b>RESULTS</b>Twenty different mutations were found in 68/92 alleles (73.9%). The prevalent mutations of R243Q, EX6 96A>G, R111X, Y356X and V399V were similar to that of Northern China populations. The mutations F161S, L255S, P281L, and R413P were significantly different from that in other Chinese populations. It was the second time that E280G and A434D mutations were reported in the world, that L255S, P281L, R261Q, and I65T mutations were found in China. Thirteen different mutations were first found in Chinese Uygur, which showed a distinct ethnic characteristics.</p><p><b>CONCLUSION</b>The study showed not only a distinct and conservative, but also a crossed and syncretic genetic characteristics in Xinjiang Uygur population. The results suggest that Xinjiang could be an ideal genetic resource repertoire for studying diversity of gene mutations, heterogeneity of PAH gene, human origins and migration.</p>

Mutation analysis and one novel mutation detection of 6-pyruvoyl tetrahydropterin synthase gene in children with tetrahydrobiopterin deficiency / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the distribution character of the mutations of 6-pyruvoyl tetrahydropterin synthase (PTPS) gene and to provide effective basis for gene diagnosis of tetrahydrobiopterin deficiency (BH4D) in children with hyperphenylalaninemia.</p><p><b>METHODS</b>Direct sequencing was performed for screening the PTPS gene mutations in 5 patients with clinically suspected BH4D and their parents. The nature of the novel mutations were deduced by the sequences alignment and the structural analysis of mutant protein. Artificial construct restriction site was used to detect the novel mutation in the control samples. The dates of urinary pterin analysis and BH4 loading test were retrospectively analyzed after gene analysis.</p><p><b>RESULTS</b>Four PTPS gene mutations (N52S, P87S, D96N, and L127F) were detected in our study. The genotypes of four PTPS deficiency patients were identified as N52S/L127F, P87S/D96N, N52S/D96N, and D96N/ -. As a novel mutation that has not been reported previously, the mutation L127F was not detected in 50 normal controls. This novel mutation L127F was inherited from the patient's mother, and this mutant site was highly conserved by sequences alignment and the protein structural analysis. Four of the five cases with hyperphenylalaninemia and suspicious BH4D, whose urinary biopterin percentage was lower than 2% , were diagnosed as PTPS deficiency during 5-20 months old. The remaining one case was excluded from BH4D.</p><p><b>CONCLUSIONS</b>The mutant characterization of PTPS gene was coincident with other early studies in Chinese. The novel mutation L127F was considered as a pathogenetic mutation and associated with severe clinical phenotype.</p>

Spectrum of phenylalanine hydroxylase gene mutations and genotype-phenotype correlation in the patients with phenylketonuria in Beijing area of China / 中华儿科杂志

<p><b>OBJECTIVE</b>To identify the mutation spectrum and the distribution of minihaplotypes (STR/VNTR) of phenylalanine hydroxylase (PAH) gene and explore the correlations between genotype and phenotype of patients with phenylketonuria (PKU) in Beijing area of China.</p><p><b>METHOD</b>(1) Fifty cases with PKU were involved in this study. PKU was identified by the Neonatal Screening Center of Beijing. All 13 exons and their flanking intronic sequences of PAH gene of these patients were amplified and then subjected to SSCP analysis and direct sequencing. (2) The distribution of polymorphic locus of short tandem repeat (STR) and variable number tandem repeat (VNTR) was analyzed by PCR and denaturing gel electrophoresis. (3) The correlations between genotype and phenotype were studied by analysis of the matching rate between the expected and observed phenotypes. The predicted phenotype was determined on the basis of the sum of the assigned values of the two mutant alleles.</p><p><b>RESULTS</b>(1) A total of 34 different mutations were detected with the relative frequency of 95% among 50 PKU patients. The prevalent mutations in this study were: R243Q (20%), EX6-96A > G (11%), Y356X (9%), and V399V (7%). The next common mutations were R111X (5%), R413P (5%), R252Q (3%) and A434D (3%). Thirty-four detected mutations were distributed throughout the whole PAH gene, except exon 1, 8 and 13. Exon 7 and 11, with the mutant rate 34% and 19% respectively, seemed to be the hot mutant areas/regions of PAH gene. (2) The minihaplotypes (STR/VNTR) of 34 mutations were identified in this research. The STR and VNTR showed 8 and 3 alleles, respectively. Among them, 244 bp (44%) and 240 bp (34%) were the prevalent STR alleles. Meanwhile, the VNTR3 (83%) was the most common VNTR allele in PKU patients. (3) A better consistency (81.5%) between expected and observed phenotypes was revealed by analysis of correlation between genotype and phenotype. Especially in classic PKU, the consistency rate was up to 87.5%.</p><p><b>CONCLUSION</b>(1) The frequency distribution of common PAH gene mutations in Beijing region was close to that of Tianjin and Yunnan regions, while it was different from that of Southern regions of China, such as Guangzhou, especially Taiwan. The PAH mutation with a highly heterogeneous trait was also demonstrated in this study. (2) STR and VNTR minihaplotype will prove helpful to trace the origins of PAH mutations and to analyze the genetic drift. However, the most minihaplotypes of the STR/VNTR are similar, so it is necessary to associate some other polymorphic loci with the STR/VNTR minihaplotype to analyze the different mutations. (3) The fact that a better consistency existed between phenotypes and genotype with most PKU patients suggested that the study of the genotype of PKU patients would be helpful to the individualized treatment and to genetic counseling for their families.</p>

Molecular analysis of survival motor neuron gene in 338 suspicious children patients with spinal muscular atrophy / 中华儿科杂志

<p><b>OBJECTIVE</b>Spinal muscular atrophy (SMA) is an autosomal recessive disorder that results in symmetrical muscle weakness and wasting due to degeneration of the anterior horns of the spinal cord. The clinical picture of SMA is variable and childhood SMA has been classified into 3 types on the basis of the age of onset and clinical course. The survival motor neuron (SMN) gene was mapped to chromosome 5q13. The SMN1 gene has been recognized to be responsible for SMA because of homozygous deletions or intragenic mutations in SMN1 results in childhood onset of SMA. The main objective of this study was to determine the deletion frequency of SMN1 gene and to apply gene analysis in children patients with SMA.</p><p><b>METHODS</b>The SMA patients were diagnosed and clinically typed according to the international diagnostic criteria, following up cases, and gene analysis. The PCR enzyme assay was used to detect the homozygous deletion of SMN1 gene in SMA patients. A dosage assay that combined multiplexed allele-specific PCR and DHPLC was used to determine the copy numbers of the SMN1 and SMN2 and detect SMN1 heterozygous deletion.</p><p><b>RESULTS</b>(1) A total of 267 patients with SMA were diagnosed from 338 suspicious cases and 143, 82, and 42 cases were typed as types I, II, and III, with the percentages of 53.6% (143/267), 30.7% (82/267) and 15.7% (42/267), respectively. (2) Results of the present study showed that 68.5% (183/267) of SMA patients had homozygous deletions of exons 7 and 8 of SMN1 gene and 12.7% (34/267) had homozygous deletions of only exon 7 of SMN1 gene. The SMN1 heterozygous deletion was confirmed in 12.4% (33/267) of SMA patients. Non-deletion SMA patients accounted for 6.4%(17/267). The homozygous deletions of only exon 8 of SMN1 gene could not be detected. (3) The rates of homozygous or heterozygous deletion in types I and II were very similar. The rate of homozygous deletion was lower in type III than that in type I or II and rate of heterozygous deletion of type III was higher than that in types I or II.</p><p><b>CONCLUSION</b>(1) The frequency and pattern of deletions in the Chinese children patients with SMA are significantly different from that observed in Caucasians populations. Further gene characterization and subtle mutations within the SMN1 gene need to be studied in order to define the molecular basis of SMA in the Chinese population. (2) The gene diagnosis is a special and non invasive method as compared with other methods. A total of 80% patients can be diagnosed through the analysis of the homozygous deletion of SMN1 gene. (3) The clinical diagnosis and gene detection need to be studied in future for the SMA patients with type III.</p>

The mutant spectrum of phenylalanine hydroxylase gene in Northern Chinese / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To understand the mutant spectrum of phenylalanine hydroxylase (PAH) gene in Northern Chinese.</p><p><b>METHODS</b>All the exons and flaking introns of PAH gene were detected by PCR-single strand conformation polymorphism (PCR/SSCP) and sequencing in 230 patients with phenylketonuria (PKU).</p><p><b>RESULTS</b>(1) A total of 75 different mutations were detected in 435 out of 460 mutant alleles (94.6%). Among them 3 mutations (S251-R252>SfsX89, Y387D and A389G) have not been reported previously. The mutations, R243Q, EX6-96A>G, R111X, R413P and Y356X, were the prevalent mutations with relative frequencies of 21.7%, 10.2%, 8.3%, 6.5%, and 6.1% respectively, followed by V399V(4.1%), IVS4-1G>A (3.5%), IVS7+2T>A (2.2%) and R241C(2.2%). Most mutations were detected in exons 3, 5, 6, 7, 11 and 12 and flaking introns of PAH gene. (2) Ten polymorphism sites were detected in the study. Four sites, IVS3-22C>T, IVS10+97G>A, Q232Q and V245V, had high relative frequencies of 56.7%, 75.9%, 89.0% and 81.9% respectively. It would suggest that the race diversity exists in PAH cDNA sequence.</p><p><b>CONCLUSION</b>The mutation spectrum of PAH gene in Northern Chinese is similar to other Asian populations but significantly different from European populations.</p>

Detection of the prevalent mutations of 6-pyruvoyl-tetrahydropterin synthase gene by PCR-RFLP analysis in Chinese patients / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To establish a single and rapid method for detecting the prevalent mutations of 6-pyruvoyl-tetrahydropterin synthase (PTPS) gene in Chinese patients with 6-pyruvoyl-tetrahydropterin synthase deficiency (PTPSD).</p><p><b>METHODS</b>PCR-restriction fragment length polymorphism (PCR-RFLP) was used to detect three prevalent PTPS gene mutations in 4 cases of tetrahydrobiopterin deficiency (BH4D) and their parents, which was performed by the artificial construct restriction site (ACRS). PCR-RFLP included Hpa I digestion for the detection of mutation N52S (155A to G), Hae III for P87S (259C to T) and Eco R I for D96N(286G to A). The mutations of PTPS gene were confirmed by direct sequencing.</p><p><b>RESULTS</b>The genotypes of 4 PTPS deficient patients were identified: N52S/-, P87S/D96N, N52S/D96N and D96N/-. The result of direct sequencing was coincident with that of PCR-RFLP analysis.</p><p><b>CONCLUSION</b>(1) The PCR-RFLP analysis formed by ACRS would be a good way for detecting the three prevalent PTPS gene mutations in Chinese patients with PTPSD. (2)The PTPS gene analysis is very important for all patients with hyperphenylalaninemia, which would be useful for early clinical diagnosis and correct treatment of BH4D patients.</p>

Mutation of growth hormone receptor gene in patients with short stature / 中华儿科杂志

<p><b>OBJECTIVES</b>The mutations of growth hormone receptor (GHR) gene results in growth hormone insensitivity (Laron syndrome) or partial growth hormone insensitivity. This study aimed to understand the relation between mutations of GHR gene and short stature with non-growth-hormone deficiency, and the clinical feature of the patients with the GHR gene mutations.</p><p><b>METHODS</b>(1) Forty-seven patients with non-growth-hormone deficiency and short stature were enrolled in this study, 33 were male and 14 female. The age of the patients were at a range of 2 - 16 years. (2) The mutations of GHR gene were identified by PCR-SSCP and DNA sequencing. (3) The characteristics of the GHR mutation was assumed by screening for the same mutations in patients' family members and the control samples.</p><p><b>RESULTS</b>(1) Four GHR mutations were identified in 5 patients with non-growth-hormone deficiency: H56R, G148E, IVS6-30, -31CA > TG and IVS8 + 10G > C. These mutations were located within the extracellular domain of GHR and not reported before. Five patients were the heterozygous of H56R, G148E, IVS6-30, -31CA > TG and IVS8 + 10G > C. The detection rate of mutant heterozygous individual accounted for 10.6% (5/47). The mutations were considered non-polymorphism by the GHR gene analysis in patients' family members and control samples. (2) Comparison of the amino acid sequence of different species and the position of the mutations H56R and G148E in the GHR protein structure suggested impact of the mutations on the protein function. (3) A polymorphism site was identified in exon 6 of GHR gene: G168G (GGA > GGG). The allelic frequency of G168G had no difference between the patients with non-growth-hormone deficiency and control samples but had significant difference between Chinese and Caucasian. It seems that the G168G was a polymorphism and has no relationship with the height stature. However, there was the allele diversity in different races.</p><p><b>CONCLUSION</b>The mutations of GHR gene were detected in the patients with non-growth-hormone deficiency. Special attention should be paid clinically to its potential pathogenesis for short stature.</p>

Ten novel mutations in the phenylalanine hydroxylase gene identified in Chinese patients with phenylketonuria / 中国医学科学院学报

<p><b>OBJECTIVE</b>To study the molecular basis of the phenylalanine hydroxylase (PAH) gene mutation in Chinese patients with phenylketonuria (PKU).</p><p><b>METHODS</b>Using PCR/SSCP and DNA sequencing, we studied the mutations in exons 3, 5, 7, 10, 11, 12 of PAH gene. Totally 120 unrelated children with PKU and their parents from the northern region of China were included in the analysis.</p><p><b>RESULT</b>Ten novel mutations were first time identified in Chinese PKU population as I65T, S70del, G239D, R241fsdelG, L255S, P281L, G346R, L367fsinsC, R400S and Ivsllnt2t-->c. The mutations G239D, R241fsdelG, R400S and Ivsllnt2t-->c have not been yet described in International PAH. In the present study we firstly identified the deletion, insertion and frameshift mutations of PAH gene in China PKU population. So far the mutant type of PAH gene in Chinese included: missense, nonsense, splice, silence, deletion, insertion and frameshift. Novel mutations mainly existed in exon 7: four in exon 7, two in exon 3, two in exon 11, one in exon 10 and one in intron 11. Each proportion of the ten novel mutations was very low (0.42%-1.3%).</p><p><b>CONCLUSION</b>This study demonstrated the high heterogeneity of the PAH gene and the variety of the mutant type of Chinese PKU population and confirmed the exon 7 was the hot spot of PAH gene mutation.</p>