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1、专家笔谈 Mapping and identification of disease responsible genes based on genetic resource reservation and bioinformatics approach Jia-hui XIA，Kun XIA，Qian PAN，Zhi-gao LONG，He-ping DAI，Ling-qian WU，De-sheng LIANG，Fang CAI （National Lab of Medical Genetics of China，Central South University， Changsha 4100
2、78， China） KEY WORDS Karyotyping；Chromosome mapping；Cloning， molecular；Genome Mapping and identification of disease associated genes will demonstrate the genetic basis for the human genetic disorders，and provide the fundamental data for elucidation of pathogenesis mechanism of the disorders. Genetic
3、 re- sources，including pedigree information，blood sample，and tissues，etc. ，are essential materials for finding of the linked locus and gene for certain genetic disease. Genome wide scanning，positional cloning and candidate ap- proach are most widely used methods or strategy，by which，thousands of dis
4、eases responsible genes have been i- dentified. National laboratory of medical genetics of China（NLMG）has initiated the study on genetic resources collection，mapping and identification of disease associated gene since 1970s，here we summarize the major find- ings in this area achieved by NLMG. 1 Coll
5、ection，reservation and database administration of human genetic resources NLMG has initiated the collection and reservation of family with genetic disorders since 1972，and established a network for family collection all over China. The families with monogenic disease and chromosomal anomalies are th
6、e most concentrated. To date，1 753 families with 116 disorders including 4 794 individuals（2 395 patients） have been collected and reserved. Besides those， 1 703 firstly reported abnormal karyotypes of Chinese population were assigned and partial suffered individuals were sampled. With these genetic
7、 resources，4 novel loci and 2 re- sponsible genes for genetic disease were identified. In order to promote the application and administration of these samples， 3 databases were set up recently. The first one is Human Abnormal Karyotype Database，which present all karyotypes and clinical data for coll
8、ected chro- mosomal diseases. The special chromosomal rearrangement usually can help us to find out the new loci or genes for mental retardation，developmental abnormality，or other diseases. The second is a Database for Chinese Family with Genetics Diseases，which contains the detailed information abo
9、ut the families we collected，and can provide clinical and genetic data for the study on mapping and cloning. The third is the Human Mendelian Hereditary Dis- eases Database. This database gathers all research progress on different Mendelian Hereditary Diseases， and we can easily find the progress fo
10、r certain disease and help us to decide whether a family is valuable for further study and propose a satisfied strategy. All these three database were all upgraded recently，and a new system based on Brow- ser/ Server（B/ S）was also set up，which is hopefully promote the domestic and international coll
11、aboration on ge- netic disease study. 2 Identification of a nasopharyngeal carcinoma （NPC）marker chromosome In 1972，XIA et al. set up the techniques for cytogenetics earliest in China and started to investigate the mechanism of the chromosomal diseases. In 1975，an novel enormous chromosome was ident
12、ified in two lymphocyte like cell lines of NPC，G-banding analysis revealed the abnormal chromosome was a translocated and rearranged chromosome formed by chromosome 1 and 3，t （1； 3）（q44； p11） 1. Interestingly，in 1976，XIA et al. found same marker chromosome in both pathological tissue of an outdoor N
13、PC patient and established lymphocyte like cell line of a Guangzhou patient 1. In 1987，same marker chromo- some was further identified in peripheral blood of a NPC patient 2. These findings suggested a novel marker chro- mosome for NPC and established the association between NPC with chr1q and chr3p
14、. In 2004，XIONG et al.3identified a susceptibility locus at chromosome 3p21 linked to NPC by genome scan- ning in 18 families with NPC，and which is identical to and evidences the findings obtained by XIA et al. 3 Mapping of human testis determine factor （TDF）gene After the cytogenetics platform was
15、established，Xia et al. carried series population study in order to find out the cytogentic mechanism of chromosomal syndrome. In 1981，two special cases with sex organ abnormity were di- agnosed clinically and the karyotype were further analyzed 4. In one patient，no testis was detected，and the chro-
16、mosomal analysis by high-resolution chromosome banding technique revealed this patient carried an abnormal Y chromosome，dic （Y） ，but the bright band（Yp11. 32）at the terminal of the short arm was lost. The other patient show similar recombinant Y chromosome，however，the bright band at the terminal of
17、the short arm did exist，and the testis was normal developed. These two cases and their chromosomal analysis results indicated a possibility that Corresponding author s e-mail， nlmglcy xysm. net 12 北京大学学报 （医学版） JOURNAL OF PEKING UNIVERSITY （HEALTH SCIENCES） Vol.38 No. 1 Feb. 2006 the terminal of the
18、short arm of Y chromosome，might play an important role in the testis formation and develop- ment. Though the human TDF gene had been mapped to the cetromere region at that time，our findings provided strong evidence that the TDF should be located at Yp11. 32 4. In 1990，Berta et al.5identified the TDF
19、 gene just at Yp11. 32， which finally confirmed the mapping of the TDF， and revealed it was a effective method to investi- gate some genetic disorders using cytogentics strategy. 4 Identification of the first gene for genetic disease in China： GJB3 for neurological deafness Hearing impairment is the
20、 most commonly occurring condition that affects the ability of humans to communi- cate. More than 50% of the cases of profound early-onset deafness are caused by genetic factors. Previous reports revealed mutations in the gene encoding gap junction protein played an important role in the pathogenesi
21、s of hearing loss. To study the possible involvement of other members of the connexin family in hereditary hearing impairment， we screened human dbEST，and identified two overlapping ESTs，which were assembled into a contig with 83% i- dentity to rat and mouse Gjb3. By nested PCR the full-length cDNA
22、encoding a 270aa peptide was finally cloned. Similarity analysis showed the protein encoded by this novel gene was a new member of gap junction protein，which was termed GJB3. By genome library screening and FISH GJB3 was mapped to chromosome 1p33-p35. The data- base searching against OMIM and Human
23、Mendelian Hereditary Diseases Database established in our lab identified at least four diseases：sensorineural deafness，erythrokeratodermia，Charcot-Marie-Tooth disease and ptosis linked to GJB3 locus. To detect possible disease-causing mutations of GJB3，the entire coding region of GJB3 was then scree
24、ned in 42 families with above hereditary diseases. Two mutations，including one missense mutation and one nonsense mutation were detected in two sensorineural deafness families characterized by bilateral high-frequency hearing impairment. In a family from Zhejiang province，a GA substitution at positi
25、on 547 of GJB3，resulting in a glutamine-to-lysine change at position 183，occurred in 4 of 7 genetically related family members from 3 genera- tions. In another family from the Hunan province，four genetically related individuals carried a CT mutation at nt 538 of GJB3， resulting in a stop codon at aa
26、 180. Sequence analysis of the entire coding region of GJB3 in 208 ran- domly selected，unrelated，normal individuals showed no missense or nonsense mutations. In adult rat，expression of Gjb3 was detected in cortex，spinal cord and inner ear，suggesting a role of Gjb3 in normal hearing. Our results sugg
27、est that mutations in GJB3 may be involved in pathogenesis of sensorineural deafness. Residues R180 and E183 are well conserved among all cloned connexins. The change of acidic glutamine to a basic lysine at the bound- ary between the second extracellular domain and the fourth transmembrane domain o
28、f GJB3 may affect the anchorage of this protein in the membrane. The R180X mutation in NDF005 results in a truncated protein lacking the fourth transmembrane domain and the third intracellular domain at the carboxy terminus of GJB3. This study provides evi- dence that mutations in GJB3 may cause an
29、autosomal dominant form of non-syndromic hearing impairment at high frequencies 6. 5 Identification of the gene PITX2 for ring dermoid of cornea（RDC） RDC（MIM180550）is an autosomal dominantly inherited syndrome characterized by bilateral，annular limbal dermoids with corneal and conjunctival extension
30、. The genetic basis of RDC is unknown. In our study，a large Chi- nese family with 17 individuals affected by the RDC was collected. Linkage analysis and further fine mapping iden- tified a maximum two point lod score of 6. 72（ =0）for D4S2989. Haplotype analysis was constructed，and the RDC locus was
31、mapped to a 15-cM interval between D4S1572 and D4S1522. Three potential candidate genes for RDC were chosen for further mutation examination based on both their tissue specific expression and their roles in regulating cell proliferation，differentiation and migration that may play important roles in
32、RDC pathogenesis. These include IDAX（NM 025212） ， TM4SF9（NM005723） ， and PITX2（NM 153427） . Mutation screening finally iden- tified a heterozygous mutation of guanine to adenine（185GA）in PITX2，which showed a perfect segregation with the disease in all 17 affected individuals in the family. PITX2， a
33、downstream target of wnt/ -catenin pathway， encodes a homeodomain transcription factor required for normal development of multiple organs，including eye， heart and pituitary. The PITX2 G185A mutation found in RDC patients results in a substitution of arginine by histi- dine at amino acid 62（R62H）loca
34、ted in the conserved DNA binding homeodomain，and likely changes its tran- scriptional activity. Mutations in PITX2 are associated with multiple dominantly inherited diseases related to mal- function of the eyes，including Riger syndrome，iridogoniodysgenesis，iris hypoplasia，and Peter s anomaly. Our st
35、udy suggests that mutation in PITX2 is linked to another eye disease 7. 6 Mapping of other 4 novel loci for genetic diseases The families with different monogenic genetic disorders collected and reserved in our lab provide us important genetic resources to conduct mapping of disease linked loci. The
36、 major strategy in our lab for mapping is genome wide scanning and linkage analysis. During past years，we carried gene scan in a few big families with 5 different diseases，including disseminate superficial actinic porokeratosis type & （DSAP1 & DSAP2） ，autosomal dominant nonsyndromic hearing loss（DFN
37、A52） ，and autosomal dominant Charcot-Marie-Tooth disease type 2 （CMT2L） . Here we report the brief findings for these studies. 6. 1 Identification and fine mapping of a locus for disseminated superficial actinic porokeratosis type （DSAP1） 22 北京大学学报 （医学版） JOURNAL OF PEKING UNIVERSITY （HEALTH SCIENCES
38、） Vol. 38 No. 1 Feb. 2006 at chromosome 12q23. 2-24. 1 Disseminated superficial actinic porokeratosis（DSAP）is an autosomal dominant cutaneous disorder character- ized by many uniformly small， minimal， annular， anhidrotic and keratotic lesions. The genetic basis for this disease is unknown. Using a g
39、enomewide search in a 7-generation Chinese kindred，we identified a locus at chromosome 12q23. 2-24. 1 responsible for DSAP（DSAP1） . The fine mapping study indicates that the DSAP1 is located within a 9. 6-cM region between markers D12S1727 and D12S1605，with a maximum two-point LOD score of 20. 53（ =
40、 0. 00）at D12S78 8. Furthermore，another five-generation family with DSAP was collected from Anhui province， genome-wide scan and linkage analysis firstly established the linkage of this family to chromosome 12q. Fine map- ping and hapolotype construction finally refine the DSAP1 locus to a 4. 4-cM i
41、nterval at chromosome 12q23. 2- 24. 1 9. By candidate approach，we screened more than 40 genes in this region for disease associated mutation， however，no any mutation was detected except some a group of SNPs. Recently，Zhang et al.10identified a mis- sense mutation，p. Ser63Asn in SSH1 in one family， a
42、 frameshift mutation， p. Ser19CysfsX24 in an alternative vari- ant（isoform f）of SSH1 in another family，and a frameshift mutation，p. Pro27ProfsX54 in the same alternative va- riant in one non-familial case with DSAP1. We then screened SSH1 gene for mutation in two families we studied， unfortunately，n
43、o mutation was found yet， suggesting anther gene localized to this locus may contribute to the patho- genesis of DSAP1，too. 6. 2 A novel locus（DSAP2）for disseminated superficial actinic porokeratosis maps to chromosome 15q25. 1-26. 1 After the first locus for DSAP was localized to chromosome 12q23.
44、2-24. 1，we collected another three-genera- tion Chinese family with DSAP. Linkage analysis was carried out in this family using 15 microsatellite markers be- tween D12S1671 to D12S369 on chromosome 12q，and followed by a genomewide scan with 382 microsatellite markers from autosomes. Our results reve
45、aled the locus in this family is not linked to chromosome12q，suggesting DSAP is a genetic heterogeneous disorder. The further genomewide scan and fine mapping localized a novel locus， DSAP2，to a 6. 4-cM region between markers D15S1023 and D15S1030 at chromosome 15q25. 1-26. 1 11. 6. 3 A novel locus
46、for autosomal dominant nonsyndromic hearing loss identified at 5q31. 1-32 in a Chinese pedi- gree Hearing impairment is an extremely heterogeneous disorder. More than 100 loci and 40 related genes for non- syndromic hearing loss have been identified. In this study，we performed a genomewide scan in a
47、 five-generation Chinese pedigree characterized by autosomal dominant inheritance with bilateral，postlingual，progressive and sen- sorineural non-syndromic hearing impairment. A linkage of the disorder in this family to chromosome 5q was firstly established. Fine mapping indicated that the disease ge
48、ne was located within an 8. 8-cM region between makers D5S2056 and D5S638，with a maximum two-point LOD score of 6. 89 at D5S2017. By candicate gene approach， mutation screening of DIAPH1 and POU4F3 genes at 5q31 was performed，no mutation was found，suggesting this is a novel deafness locus，which has
49、been named DFNA52 12. 6. 4 A new locus for autosomal dominant Charcot-Marie-Tooth disease type 2（CMT2L）maps to chromosome 12q24 Charcot-Marie-Tooth disease（CMT）is one of the most common inherited neurological disorders with a preva- lence estimated at 1/2 500. The axonal form of this disorder is referred to as Charcot-Marie-Tooth type 2 disease （CMT2） . Recently， a large Chinese family with CMT2 was found in the Hunan and Hubei provinces of China. The known loci for CMT1A，CMT2D，CMT1B（the same locus is also responsible for CMT2I and CMT2J） ，CMT2A