Long-range PCR amplification-based targeted enrichment & next generation sequencing: A cost-effective testing strategy for lysosomal storage disorders

Background & objectives: Lysosomal storage disorders (LSDs) are genetic metabolic disorders which result from deficiency of lysosomal enzymes or defects in other lysosomal components. Molecular genetic testing of LSDs is required for diagnostic confirmation when lysosomal enzyme assays are not available or not feasible to perform, and for the identification of the disease causing genetic variants. The aim of this study was to develop a cost-effective, readily customizable and scalable molecular genetic testing strategy for LSDs. Methods: A testing method was designed based on the in-house creation of selective amplicons through long range PCR amplification for targeted capture and enrichment of different LSD genes of interest, followed by next generation sequencing of pooled samples. Results: In the first phase of the study, standardization and validation of the study protocol were done using 28 samples of affected probands and/or carrier parents (group A) with previously identified variants in seven genes, and in the second phase of the study, 30 samples of enzymatically confirmed or biopsy-proven patients with LSDs and/or their carrier parents who had not undergone any prior mutation analysis (group B) were tested and the sequence variants identified in them through the study method were validated by targeted Sanger sequencing. Interpretation & conclusions: This testing approach was found to be reliable, easily customizable and cost-effective for the molecular genetic evaluation of LSDs. The same strategy may be applicable, especially in resource poor settings, for developing cost-effective multigene panel tests for other conditions with genetic heterogeneity.

Genetics and muscle pathology in the diagnosis of muscular dystrophies: An update

Muscular dystrophies are a clinically and genetically heterogeneous group of disorders involving the skeletal muscles. They have a progressive clinical course and are characterized by muscle fiber degeneration. Congenital muscular dystrophies (CMD) include dystroglycanopathies, merosin-deficient CMD, collagen VI-deficient CMD, SELENON-related rigid spine muscular dystrophy, and LMNA-related CMD. Childhood and adult-onset muscular dystrophies include dystrophinopathies, limb-girdle muscular dystrophies, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, and myotonic dystrophy. Traditionally, muscle biopsy and histopathology along with special pathology techniques such as immunohistochemistry or immunoblotting were used for the diagnosis of muscular dystrophies. However, recent advances in molecular genetic testing, especially the next-generation sequencing technology, have revolutionized the diagnosis of muscular dystrophies. Identification of the underlying genetic basis helps in appropriate management and prognostication of the affected individual and genetic counseling of the family. In addition, identification of the exact disease-causing mutations is necessary for accurate prenatal genetic testing and carrier testing, to prevent recurrence in the family. Mutation identification is also essential for initiating mutation-specific therapies (which have been developed recently, especially for Duchenne muscular dystrophy) and for enrolment of patients into ongoing therapeutic clinical trials. The 'genetic testing first' approach has now become the norm in most centers. Nonetheless, muscle biopsy-based testing still has an important role to play, especially for cases where genetic testing is negative or inconclusive for the etiology.

Advances in epigenetic modification genes in rare diseases with abnormal cardiac development / 国际儿科学杂志

Epigenetic modification genes are defined as genes whose products modify the epigenome directly through DNA methylation, histone modification or chromatin remodeling.More and more studies have shown that mutations in epigenetic modification genes are an important etiology of rare diseases with abnormal cardiac development.And these diseases usually affect multiple organs including heart due to the change of epigenetic components.Moreover, children′s lives and health are often threatened by a lack of effective drugs and complex cardiovascular malformations.This article reviews advances in molecule genetics of Tatton-Brown-Rahman syndrome, Kabuki syndrome, Rubinstein-Taybi syndrome, CHARGE syndrome and Sifrim-Hitz-Weiss syndrome, and mainly elaborates the mechanism of cardiovascular malformations caused by mutations in corresponding epigenetic modification genes, providing more comprehensive reference for clinical diagnosis and management.

Full length genome sequencing and phylogenetic analysis of two strains of Getah virus / 中国热带医学

@#Abstract: Objective　The main aim of the study is to sequence the complete genome of two Getah virus strains (GS11-155 and HNDZ1712-1) isolated in Gansu Province and Hainan Province in 2011 and 2017 respectively and analyze the molecular and genetic evolution of the two strains compared with M1, which was first isolated in 1964 in Hainan Province, China. Methods　Genome of two newly isolated Getah viruses were sequenced by virus gene amplification technique, and the genomic database of Getah viruses was established. The molecular characteristics and genetic evolution of the viruses were analyzed by bioinformatics software. Results　The genome length of two new isolated Getah virus strains (GS11-155 and HNDZ1712-1) was 11 690 nt and 11 621 nt, respectively. Both strains had the structural characteristics of Alphavirus genome. Although the nucleotide sequence lengths of structural genes, non-structural genes and non-coding junction regions of the two strains were identical, the nucleotide sequence lengths of the 5' and 3' non-coding regions of the viral genomes were a few different. The 3'UTR repeats elements in the genomes of the two virus strains did not change. It was 97.7% and 98.1% different of nucleotide and amino acid homology between both strains of Getah virus, HNDZ1712-1 isolated in 2017 and M1 isolated in 1964 in Hainan Province. Interesting, Gansu 2011 cluster and Hainan 2017 cluster were emerged leading by both strains GS11-155 and HNDZ1712-1 respectively, those two clusters totally independent with M1 virus isolated from Hainan in 1964 in whole genome phylogenetic analysis first. Conclusions　Although the HNDZ1712-1 was also isolated from mosquito samples in Hainan Province, it was in a completely different evolutionary branch from the M1 isolated from Hainan Island in 1964, and was closely related to the strain isolated from Gansu Province (GS11-155) thousands of kilometers away. It is suggested that the two new strains of Getah virus are different from the Getah virus isolated in 1964.

Standardization of an organic DNA extraction method from dried blood spots and its downstream molecular applications in neonatal screening and diagnostic confirmation of lysosomal disorders

Introduction: Dried blood spot (DBS) samples have been used for diagnostic purposes since their introduction in the neonatal screening of phenylketonuria almost 50 years ago. The range of its application has been extended to modern approaches, such as next-generation sequencing (NGS) for molecular genetic testing. This study aimed to evaluate the use of a standardized organic method for DNA extraction from DBS samples in the diagnostic setting.Methods: The clinical applicability of the method was tested using 3 samples collected from a newborn screening project for lysosomal storage diseases, allowing the determination of the genotype of the individuals. DNA was extracted from 3 3-mm diameter DBS punches. Quality, purity, and concentration were determined, and method performance was assessed by standard polymerase chain reaction, restriction length polymorphism, Sanger sequencing, and targeted NGS.Results: Results were compared with the ones obtained from DNA samples extracted following the internally validated in-house extraction protocol that used 6 3-mm punches of DBS and samples extracted from whole blood.Conclusion: This organic method proved to be effective in obtaining high-quality DNA from DBS, being compatible with several downstream molecular applications, in addition to having a lower cost per sample

Molecular genetic analysis of primary renal epithelial tumours with granular oncocytic cytoplasms / Análisis genético molecular de tumores epiteliales renales primarios con citoplasma oncocítico granular

SUMMARY: The group of primary renal tumours with granular-oncocytic cytoplasm is a very heterogeneous group, in its histological origin and biological behavior resulting in many diagnostic problems. In this study 57 renal epithelial tumours with granular oncocytic cells were analyzed using fluorescence in situ hybridisation (FISH), array comparative genomic hybridisation (aCGH) and polymerase chain reaction (PCR). The results of analysis in renal oncocytoma (RO) did not indicate the presence of the gene mutations or chromosomal abnormalities. Sporadic renal hybrid oncocytic/chromophobe tumours (HOCT) had multiple numerical aberrations of chromosomes 1, 2, 6, 9, 10, 13, 17, 20, 21 and 22. This type of tumour had no mutations in the VHL, c-kit, PDGFRA, and FLCN genes. Oncocytic papillary renal cell carcinoma (O-PRCC) had numerical abnormalities of chromosomes 7 and 17 and the loss of the Y chromosome. Cytogenetic analysis of 20 pigmented microcystic chromophobe renal cell carcinomas (PMChRCC) showed monosomy as the most frequent aberration in all analyzed chromosomes 1, 2, 5, 10, 13, 17 and 21. One case of chromophobe renal cell carcinoma (ChRCC) with hyaline globules had a mutation in the distal part of exon 3 of the VHL gene. Absence of genetic disorders in usual RO is common result, but we have established absence of genetic disorders even in rare variants. Variety of genetic alterations detected in sporadic renal HOCT proves it to be a separate entity, not a variant of ChRCC, while PMChRCC is an uncommon variant of ChRCC. O-PRCC is a subtype of papillary renal cell carcinoma.

RESUMEN: El grupo de tumores renales primarios con citoplasma granular-oncocítico es un grupo muy heterogéneo, en su origen histológico y comportamiento biológico, resultando en problemas de diagnóstico. En el estudio se analizaron 57 tumores epiteliales renales con citoplasma oncocítico granular mediante hibridación fluorescente in situ (FISH), hibridación genómica comparativa de matriz (aCGH) y reacción en cadena de la polimerasa (PCR). Los resultados del análisis en oncocitoma renal (RO) no indicaron la presencia de mutaciones genéticas ni anomalías cromosómicas. Los tumores oncocíticos / cromófobos híbridos renales esporádicos (HOCT) tenían múltiples aberraciones numéricas de los cromosomas 1, 2, 6, 9, 10, 13, 17, 20, 21 y 22. No se observaron mutaciones en este tipo de tumor en el VHL, c-kit, PDGFRA y genes FLCN. El carcinoma de células renales papilar oncocítico (O-PRCC) tenía anomalías numéricas de los cromosomas 7 y 17 y la pérdida del cromosoma Y. El análisis citogenético de 20 carcinomas de células renales cromófobos microquísticos pigmentados (PMChRCC) mostró que la monosomía era la aberración más frecuente en todos los cromosomas analizados 1, 2, 5, 10, 13, 17 y 21. Un caso de carcinoma de células renales cromófobo (CCRc) hialino tenía una mutación en la parte distal del exón 3 del gen VHL. La ausencia de trastornos genéticos en la OI habitual es un resultado común, pero hemos establecido la ausencia de trastornos genéticos incluso en variantes raras. Varias alteraciones genéticas detectadas en esporádica HOCT renal demuestran que es una entidad separada, no una variante de ChRCC, mientras que PMChRCC es una variante poco común de ChRCC. O-PRCC es un subtipo de carcinoma papilar de células renales.

Progresses on diagnostic criteria and genetic features of synchronous multiple primary lung cancer / 中国胸心血管外科临床杂志

@#With the broad application of high-resolution computed tomography (CT) and high rates of early lung cancer screening, the number of patients diagnosed with synchronous multiple primary lung cancer (sMPLC) has been increasing. It becomes of great prominence to distinct sMPLC from intrapulmonary metastases in clinical practice. An increasing number of studies have developed high-throughput sequencing based genetic approaches to specify the molecular characteristics of sMPLC, which contributes to a better understanding of its tumorigenesis. The genetic profile of sMPLC also benefits its diagnosis, which mainly relies on its clinicopathological criteria. Here, we summarize the progresses on the diagnostic criteria for sMPLC, and also molecular features of sMPLC from the perspective of clonality analysis.

Clinical and genetic study of a child with 15q11.2 microduplication / 中华医学遗传学杂志

Objective@#To explore the genetic basis of a child with developmental delay and intellectual disability.@*Methods@#Peripheral blood samples of the child and his parents were collected for routine G-band karyotyping analysis and single nucleotide polymorphism array (SNP array) assay. Amniotic fluid sample was collected during the next pregnancy for prenatal diagnosis.@*Results@#No karyotypic abnormality was found in the child and his parents. SNP array showed that the child has carried a 855.3 kb microduplication in 15q11.2. His mother carried the same duplication but had no phenotypic anomaly. No microdeletion/microduplication was found in his father. Upon prenatal diagnosis, no abnormalities was found with the chromosomal karyotype and SNP array result of the fetus.@*Conclusion@#15q11.2 microduplication may result in developmental delay and intellectual disability, for which CYFIP1 may be a candidate gene. However, the duplication may increase the risk but with a low penetrance. This should attract attention during clinical consultation.

Chinese Siblings with Prader-Willi Syndrome Inherited from Their Paternal Grandmother

Background: Prader-Willi syndrome (PWS) is a complex neurobehavioral disorder causedby failure of expression of paternally inherited genes in the PWS region of chromosome 15.Case characteristics: Two siblings who both met the inclusion criteria for clinical diagnosisof PWS during neonatal period. Outcome: Molecular genetic analysis demonstrated a 417-kb microdeletion within the 15q11.2 region inherited from siblings’ paternal grandmother,involving key genes of PWS, except for UBE3A, which may explain why their father andpaternal grandmother had a normal phenotype. Conclusion: The findings may be helpfulfor better understanding of the underlying mechanism of this rare imprinting defect

Clinicopathologic characteristics and molecular genetic analysis of 17 cases with multilocular cystic clear cell neoplasm of low malignant potential / 临床与实验病理学杂志

Purpose To investigate the clinicopathological features, immunophenotype, molecular genetic alteration in multilocular cystic clear cell neoplasm of low malignant potential. Methods 17 cases of multilocular cystic clear cell neoplasm of low malignant potential with complete clinical data, systematic measurement and follow-up were retrospectively studied. Histopathological evaluation and immunophenotyping were examed by HE staining and EnVision two steps methods, chromosome 3p deletion was analyzed by interphase fluorescence in situ hybridization. Results In 17 cases, there were 12 males and 5 females, and the ratio of male and female was 2.4: 1. The prevalence age was at a range of 28-73 years, and the average age is54 years. Mostly of them were found by incidental or physical exmanination. Microscopically, most cysts were lined by a single layer of tumor cells with clear cytoplasm, small nuclear, and no obvious nucleoli. According to WHO/ISUP nuclear grade, they were level I. Clear cell groups similar to cells lined cysts were seen within the fibrous septa. Immunohistochemically, tumor cells were positive for CK(AE1/AE3), CK7, EMA, vimentin, CD10, CAIX, PAX-2, and PAX-8, but negative for CD68. Ki-67 index were less than 10％. The loss of heterozygosity of 3p chromosome was detected in 11 cases and the rate of the loss of heterozygosity was 64.7％. Conclusion Multilocular cystic clear cell neoplasm of low malignant potential is a relatively rare type of renal cell carcinoma with low malignant potential and a good prognosis. It is suggested that tumor cells may be derived from tumor stem cells with pluripotent potential in renal tubules based on the immunophenotypes. Multilocular cystic clear cell renal cell carcinoma and renal clear cell carcinoma is similar in immunophenotype and molecular genetics, which suggesting that it may be a special histologic subtype of renal clear cell carcinoma.

Application of mutated inbred animal models in the experimental teaching of medical genetics / 中国比较医学杂志

Objective Mutated inbred animal model is introduced to the practical course of genetic diagnosis in the hope that medical students are able to apply what they have learned to clinical cases, based on a deep understanding of principle and technology on gene mutation detection. Methods We integrated DNA extraction, polymerase chain reaction, agarose gel electrophoresis, and gel imaging analysis into a comprehensive experiment and arranged 4-year-programme undergraduates majoring in preclinical medical sciences to conduct it with the purpose of investigating the internal relations between phenotype and genotype in a hairless Uncv mouse model. Subsequently, the questionnaire aimed at evaluating learning effect on the part of students was handed out and their feedbacks were analyzed. Results More than 90% of respondents are satisfied with the general learning effect. Especially, 98. 7% of students support the enhancing effect of the new teaching mode on their research skills and 96% consider the practical course helpful to their problem-solving ability. Conclusions The introduction of mutated inbred animal model to the practical system of molecular diagnostics proves beneficial to boost students' learning effect and scientific research quality. Our practice also provokes thoughts on the further utilization of animal models in teaching system of medical sciences.

Application of mutated inbred animal models in the experimental teaching of medical genetics / 中国比较医学杂志

Objective Mutated inbred animal model is introduced to the practical course of genetic diagnosis in the hope that medical students are able to apply what they have learned to clinical cases, based on a deep understanding of principle and technology on gene mutation detection. Methods We integrated DNA extraction, polymerase chain reaction, agarose gel electrophoresis, and gel imaging analysis into a comprehensive experiment and arranged 4-year-programme undergraduates majoring in preclinical medical sciences to conduct it with the purpose of investigating the internal relations between phenotype and genotype in a hairless Uncv mouse model. Subsequently, the questionnaire aimed at evaluating learning effect on the part of students was handed out and their feedbacks were analyzed. Results More than 90% of respondents are satisfied with the general learning effect. Especially, 98. 7% of students support the enhancing effect of the new teaching mode on their research skills and 96% consider the practical course helpful to their problem-solving ability. Conclusions The introduction of mutated inbred animal model to the practical system of molecular diagnostics proves beneficial to boost students' learning effect and scientific research quality. Our practice also provokes thoughts on the further utilization of animal models in teaching system of medical sciences.

Diagnóstico molecular de enfermedades genéticas: del diagnóstico genético al diagnóstico genómico con la secuenciación masiva / Molecular diagnosis of genetic diseases: from genetic to genomic diagnosis using next generation sequencing

En la actualidad se conocen 8.000 enfermedades genéticas monogénicas. La mayoría de ellas son heterogéneas, por lo que el diagnóstico molecular por técnicas convencionales de secuenciación suele ser largo y costoso debido al gran número de genes implicados. El tiempo estimado para el diagnóstico molecular se encuentra entre 1 y 10 años, y este retraso impide que los pacientes reciban medidas terapéuticas y de rehabilitación específicas, que sus familiares entren en programas preventivos y que reciban asesoramiento genético. La secuenciación masiva está cambiando el modelo de diagnóstico molecular de los afectos, sin embargo, los médicos y profesionales de la salud se enfrentan al dilema de la selección del método más eficiente, con el menor coste sanitario y con la mayor precisión de sus resultados. El objetivo de este trabajo es revisar la tecnología de secuenciación masiva y definir las ventajas y los problemas en su utilización.

Currently 8000 monogenic genetic diseases are known. Most of them are heterogeneous, so their molecular diagnosis by conventional sequencing techniques is labour intensive and time consuming due to the large number of genes involved. The estimated time is between 1 and 10 years for molecular diagnosis and this delay prevents patients from receiving therapy and rehabilitation measures, and their families from entering prevention programs and being given genetic counselling. Next generation sequencing (NGS) is changing the model of molecular diagnosis of patients; however, doctors and health professionals are faced with the dilemma of choosing the most efficient method, with lower health care costs and the most accurate results. The aim of this paper is to review the NGS technology and define the advantages and problems in the use of this technology.

Next generation sequencing based molecular genetic analysis of a Chinese Han family with autosomal retinitis pigmentosa / 中华实验眼科杂志

Background Retinitis pigmeutosa (RP) is a progressive inheritance disease.It is characterized by highly genetical and phenotypical heterogeneity.With the rapid development of genomics,new methods are applied to the genetic screening of RP.Objective This study was to characterize the clinical features of a Chinese family with autosomal RP and to screen the candidate genes.Methods Twelve members from this family were included in the study.All participants underwent complete ophthalmologic examinations.Targeted-capture next generation sequencing (NGS) based molecular genetic analysis was performed on two patients of this RP family(Ⅱ5,Ⅱ 7).The DNA sample from the two patients was separately sequenced using custom capture gene chip,which includes 59 retinal disease genes.The sequencing results were analyzed by bioinformatics technology.Identified variations were verified in the rest family members by PCR and Sanger sequencing.This study was approved by Ethic Committee of West China Hospital,and informed consent was obtained from the subjects.Results Four members of this family were diagnosed as RP,and the rest were asymptomatic.Missense mutation (c.3065T＞C,p.Phe1022Ser) in USH2A and missense mutation (c.1699G＞A,p.Ala1319Gly) in PDE6A were found in two patients (Ⅱ 5 and Ⅱ7).The variants were not co-segregated with the phenotype of this family.The causative mutation was not found by the targeted-capture NGS based eye disease chip,but it ruled out a large number of candidate genes for RP.Conclusions Our study suggests that targeted-capture NGS based eye disease chip can quickly detect mutations in known RP genes.It can be a new applicable and efficient method for molecular genetic analysis of ocular disease.

Research progress of molecular genetic analysis in Schistosoma variation / 中国血吸虫病防治杂志

The development of molecular biology techniques makes important contributions to the researches of heritable varia-tion of Schistosoma. In recent years,the molecular genetic analysis in the Schistosoma variation researches mainly includes the re-striction fragment length polymorphism(RFLP),random amplified polymorphism technology(RAPD),microsatellite anchored PCR(SSR-PCR),and polymerase reaction single-strand conformation polymorphism(PCR-SSCP). This article reviews the re-search progress of molecular genetic analysis in Schistosoma variation in recent years.

Research progress of genetic monitoring methods in guinea pig / 中国比较医学杂志

Guinea pig as a commonly used laboratory animal is widely used in various fields of biomedical research.The stability of genetic quality directly affects its development and application .Genetic testing is designed to confirm the genetic characteristics of each strain , to verify whether there are genetic mutations and other genetic contamination, to ensure that the test object meets the requirements of this strain .Along with the emerge of biochemical and molecular marker technology , a more convenient and reliable means is provided for research of genetic homozygosity , genetic type detection and genetic quality monitoring of guinea pigs .In this paper, the application and research progress of biochemical, cytological and molecular markers in studies of guinea pig diversity will be summarized , and provide some help for genetic testing guinea pig.

Biological characteristics of Echinococcus f elidis / 中国人兽共患病学报

This review focuses on biological characteristics of Echinococcus f elidis including molecular genetic markers , species status ,host coverage ,geographical distribution ,epidemiological implications ,phylogeny ,and evolution .The molecular genetic markers are involved in mitochondrial cox1 (cytochrome C oxidase subunit 1) and nad1 (nicotinamide adenine dinucle-otide dehydrogenase subunit 1) genes ,nuclear protein-coding gene sequences such as elp (ezrin-radixin-moesin-like protein) , e f1a (elongation factor 1 alpha) ,pepck (phosphoenolpyruvate carboxykinase ) ,pold (DNA polymerase delta ) ,and ribosome RNA gene sequences such as ITS1 and 18S rRNA .The establishment of species status is based on distinctly discriminated mor-phological characteristics such as hooks on the rostrum with apparent rugae ,the special definitive host (lions) ,and divergence of DNA sequences ,etc .between E . f elidis and other Echinococcus species .In brief ,the review has provided researchers and ex-perts in the field of echinococcosis with fundamental background knowledge and guidelines for future research directions ,clinical and epidemiological investigations ,and prevention and control of echinococcosis .

Strategy for the Customized Mass Screening of Genetic Sensorineural Hearing Loss in Koreans

Hearing loss is one of the most common sensorineural disorder. More than half of congenital bilateral profound deafness cases have been estimated to be attributed to genetic cause. Identification of genetic cause can provide valuable information. We developed new diagnostic strategy combining phenotype-driven candidate gene approach and targeted exome sequencing to find out the causative mutation of hearing loss. The causative mutation detection rates of this strategy were 78.1% and 54.8% in Korean multiplex families and sporadic severe to profound hearing loss families, respectively. The most frequent causative genes of Korean multiplex families were SLC26A4 and POU3F4. The other causative genes were MRNR1, WFS1, COCH, TECTA, MYO6, COL11A2, EYA4, GJB3, OTOF, STRC, MYO3A, and GJB2. The most frequent causative gene of Korean sporadic severe to profound hearing loss families was SLC26A4 followed by GJB2, CHD7, and CDH23. Based upon the results, the value of this strategy as a diagnostic tool seems to be promising. Although whole genome and exome sequencing have advanced as the development of next-generation sequencing, this new strategy could be a good screening and diagnostic tool to find the causative mutations.

Molecular Genetic Analysis of Partial 9p Trisomy in Two Chinese Families with Mental Retardation and Facial Anomaly / 华中科技大学学报（医学）（英德文版）

Mental retardation is defined by significant limitations in intellectual function and adaptive behavior that occur before 18 years of age.Many chromosomal diseases come with mental retardation.We reported two Chinese families with partial trisomy 9p and other chromosome partial monosomy,clinical features of mental retardation and mild facial and pinkie anomalies.In the family 1,we showed that the proband carried a trisomy 9p21.3→pter and monosomy 21q22.3→qter by using fluorescence in situ hybridization analysis.Molecular genetic analysis defined the precise breakpoint on chromosome 9p between markers D9S1846 and D9S171,an interval of about 2.9 Mb on 9p21.3,and the breakpoint on chromosome 21q between markers D21S1897 and D21S1446,a region of about 1.5 Mb on 21q22.3.In the family 2,a patient with trisomy 9p21.3→pter and monosomy 5p15.33→pter,and a de novo maternal balanced translocation between chromosomes 5 and 9 was identified in his mother.Cytogenetic and molecular genetic analysis defined the precise breakpoints on chromosome 9p21.3 and chromosome 5p15.33.Further clinical investigation found that any individual had no refractoriness eczema disease except the proband in this family.These results further implicate that trisomy 9p is associated with mental retardation,and that there may be key gene duplication on chromosome 9p21.3→9pter responsible for mental retardation and mild facial anomaly.This result has been applied successfully in prenatal diagnosis of the second family.