上海人群面肩肱型肌营养不良症相关位点的D4Z4多态性

目的 对上海人群4p35位点D4Z4重复序列进行研究，分析D4Z4的多态性。方法 191名正常上海人的基因组DNA经EooR I/Bln I双酶水解后，应用脉冲场凝胶电泳及Southem印迹测定其染色体4p35位点的D4Z4片段长度，并对短的D4Z4片段Kpn I酶进行部分酶切以计数其D4Z4串联重复序列数。结果 在191名正常上海人群中，有17人（占8.9％）携带短的D4Z4片段，其长度在22－34kb之间；其中16人携带的短D4Z4片段位于4q35位点，1人携带的短D4Z4片段为4q35→10q26。结论 面肩肱型肌营养不良症的发病虽与4q35位点D4Z4片段的串联重复序列数减少有关，但上海人群中携带4q35位点短的D4Z4片段个体的比例明显高于西方人群，提示其他因素可能也参与面肩肱型肌营养不良症的发病。     

Analysis of the tandem repeat locus D4Z4 associated with facioscapulohumeral muscular dystropothhy

The sequence of the tandem repeat sequence (D4Z4) associatedwith facioscapulohumeral muscular dystrophy (FSHD) has beendetermined: each copy of the 3.3 kb repeat contains two homeoboxesand two previously described repetitive sequences, LSau anda GC-rich low copy repeat designated hhspm3. By Southern blotting,FISH and isolation of cDNA and genomic clones we show that thereare repeat sequences similar to D4Z4 at other locations in thehuman genome. Southern blot analysis of primate genomic DNAindicates that the copy number of D4Z4-like repeats has increasedmarkedly within the last 25 million years. Two cDNA clones wereisolated and found to contain stop codons and frameshifts withinthe homeodomains. An STS was produced to the cDNAs and analysisof a somatic cell hybrid panel suggests they map to chromosome14. No cDNA clones mapping to the chromosome 4q35 D4Z4 repeatshave been Identified, although the possiblilty that they encodea protein cannot be ruled out. Although D4Z4 may not encodea protein, there is an association between deletions withinthis locus and FSHD. The D4Z4 repeats contain LSau repeats andare adjacent to 68 bp Sau3A repeats. Both of these sequencesare associated with heterochromatic regions of DNA, regionsknown to be involved in the phenomenon of position effect variegation.We postulate that deletion of D4Z4 sequences could produce aposition effect.     

Common epigenetic changes of D4Z4 in contraction‐dependent and contraction‐independent FSHD

Facioscapulohumeral muscular dystrophy (FSHD), caused by partial deletion of the D4Z4 macrosatellite repeat on chromosome 4q, has a complex genetic and epigenetic etiology. To develop FSHD, D4Z4 contraction needs to occur on a specific genetic background. Only contractions associated with the 4qA161 haplotype cause FSHD. In addition, contraction of the D4Z4 repeat in FSHD patients is associated with significant D4Z4 hypomethylation. To date, however, the methylation status of contracted repeats on nonpathogenic haplotypes has not been studied. We have performed a detailed methylation study of the D4Z4 repeat on chromosome 4q and on a highly homologous repeat on chromosome 10q. We show that patients with a D4Z4 deletion (FSHD1) have D4Z4‐restricted hypomethylation. Importantly, controls with a D4Z4 contraction on a nonpathogenic chromosome 4q haplotype or on chromosome 10q also demonstrate hypomethylation. In 15 FSHD families without D4Z4 contractions but with at least one 4qA161 haplotype (FSHD2), we observed D4Z4‐restricted hypomethylation on chromosomes 4q and 10q. This finding implies that a genetic defect resulting in D4Z4 hypomethylation underlies FSHD2. In conclusion, we describe two ways to develop FSHD: (1) contraction‐dependent or (2) contraction‐independent D4Z4 hypomethylation on the 4qA161 subtelomere. Hum Mutat 30:1–11, 2009. © 2009 Wiley‐Liss, Inc.     

Methylation of the FSHD syndrome-linked subtelomeric repeat in normal and FSHD cell cultures and tissues.

Facioscapulohumeral muscular dystrophy (FSHD) has an unusual molecular etiology. In a putatively heterochromatic subtelomeric region of each chromosome 4 homologue (4q35), unaffected individuals have 11 to about 95 tandem copies of a complex 3.3-kb repeat (D4Z4). Most FSHD patients have less than 10 copies at one allelic 4q35. This has been proposed to lead to the loss of heterochromatinization and, thereby, inappropriate gene expression by position effects, explaining the dominant nature of FSHD and the role of a decreased number of copies of D4Z4 at 4q35 but not at 10q26. Consistent with the proposed heterochromatinization of this repeat, by Southern blot analysis, we found that SmaI, MluI, SacII, and EagI sites in D4Z4 are highly methylated in normal and FSHD cell lines and somatic tissues, including skeletal muscle. Like repeated DNA sequences in the juxtacentromeric heterochromatin of chromosomes 1, 9, and 16, D4Z4 was hypomethylated at numerous CpGs in sperm and in cell lines from patients with an unrelated DNA methyltransferase deficiency syndrome (ICF; immunodeficiency, centromeric region instability, facial anomalies) in contrast to its hypermethylation in non-ICF postnatal somatic tissues. Our data on FSHD samples suggest that the disease-associated 4q35 D4Z4 repeats, which constitute a small percentage of the total D4Z4 repeats, are not generally hypomethylated relative to the other repeats of this sequence. However, in individuals not affected with FSHD, the hypermethylation of tandem, high-copy-number D4Z4 repeats might help stabilize heterochromatinization at allelic 4q35 regions just as hypermethylation elsewhere in the genome has been linked to chromatin compaction.     

A large patient study confirming that facioscapulohumeral muscular dystrophy (FSHD) disease expression is almost exclusively associated with an FSHD locus located on a 4qA-defined 4qter subtelomere

Facioscapulohumeral muscular dystrophy (FSHD), an autosomal dominant disorder, represents the third most common human muscular dystrophy. The FSHD disease locus, at chromosome 4q35, is associated with large contractions of the polymorphic repeat sequence array D4Z4. In addition to FSHD disease association with large D4Z4 deletions, a biased interaction with a specific 4qter subtelomeric sequence has been described in patients. Two distinct 4qter subtelomeres, defined as types 4qA and 4qB, have been identified and shown to be equally prevalent in the Caucasian population. In almost all 4q35-linked patients with FSHD, however, disease expression only occurs when large D4Z4 deletions are located on 4qA-defined 4qter subtelomeres. Conversely, large D4Z4 repeat contractions situated on 4qB-defined subtelomeres either are not disease-causing or exhibit a greatly reduced disease penetrance. This study was initiated to confirm this direct FSHD disease association data by measuring the frequency of type 4qA-defined and 4qB-defined subtelomeric sequences in a large cohort of 164 unrelated patients with FSHD from Turkey and the UK, all known to have large D4Z4 deletions. An almost complete association was found between large D4Z4 repeat array deletions located on 4qA-defined 4qter subtelomeres and disease expression in our large FSHD patient cohort. The observed failure of probes 4qA and 4qB to hybridise to two patient-derived DNA samples confirms the presence of an additional rare type of 4qter subtelomeric sequence in humans.     

Exclusion mapping of chromosomal regions which cross hybridise to FSHD1A associated markers in FSHD1B

Facioscapulohumeral muscular dystrophy (FHSD) is a genetically heterogeneous, autosomal dominant primary disease of muscle. The predominant form of FSHD, which has been designated FSHD1A, has been localised to the 4q34 region of human chromosome 4. The disease locus (loci) for the remaining FSHD families, which are not linked to chromosome 4 and have been designated FSHD1B, has not yet been identified.

The D4F104S1 marker which detects copies of a 3·2 kb tandem repeat (D4Z4) which contains several types of repetitive sequences, including Hox gene-like elements, has been shown to be closely linked to the chromosome 4 FHSD disease locus. The loss of an integral number of the 3·2 kb tandem repeats has been associated with FSHD1A. When hybridised to chromosomal spreads these sequences cross hybridise with heterochromatin on acrocentric chromosomes and specific areas of human chromosomes 1, 3, and 10. Potentially these specific regions of cross hybridisation may be linked to FSHD1B. To examine this possiblity we have carried out linkage studies in our largest FSHD1B family. In this paper we exclude these areas of specific cross hybridisation as disease loci for FSHD1B.

     

A region on human chromosome 4 (q35.1-->qter) induces senescence in cell hybrids and is involved in cervical carcinogenesis

Human papillomavirus (HPV) types 16 and 18 are known to play a major role in cervical carcinogenesis. Additional genetic alterations are required for the development and progression of cervical cancer. Previously, we showed that the introduction of an entire human chromosome 4 into HPV-immortalized cells by microcell-mediated chromosome transfer (MMCT) can induce senescence in cell hybrids. In the present study, we established eight new murine donor cell lines harboring different fragments of the human chromosome 4. These were tested for their ability to induce senescence by MMCT into HPV16-immortalized keratinocytes (HPK II) and cervical carcinoma cells (HeLa). By exclusion, we could identify a region for a putative senescence gene or genes at 4q35.1-->qter. Further evidence that this locus may be involved in cervical carcinogenesis was obtained by studying sections of high-grade cervical intraepithelial neoplasias (CIN2/3) and cervical cancers from 87 women using a combination of interphase fluorescence in situ hybridization (I-FISH) and microsatellite PCR. I-FISH indicated copy number loss at 4q34-->qter. Microsatellite analysis showed that loss of one or more alleles at chromosome 4 was more frequent in the cervical carcinomas than in the CINs. Loss of heterozygosity (LOH) affected four areas, D4S412 (4p16.3), D4S2394 (4q28.2), D4S3041 (4q32.3), and D4S408 (4q35.1), and was highest at D4S408. LOH at terminal 4q has been reported previously for cervical carcinomas and other human malignancies. This is the first report associating allelic loss at 4q34-->qter with high-grade intraepithelial neoplasia and cervical carcinoma, and the first experimental evidence that this locus or these loci can induce senescence in cervical carcinoma cells and HPV16-immortalized cells.     

Association analysis of chromosome 1 migraine candidate genes

Background  

Migraine with aura (MA) is a subtype of typical migraine. Migraine with aura (MA) also encompasses a rare severe subtype Familial Hemiplegic Migraine (FHM) with several known genetic loci. The type 2 FHM (FHM-2) susceptibility locus maps to chromosome 1q23 and mutations in the ATP1A2 gene at this site have recently been implicated. We have previously provided evidence of linkage of typical migraine (predominantly MA) to microsatellite markers on chromosome 1, in the 1q31 and 1q23 regions. In this study, we have undertaken a large genomic investigation involving candidate genes that lie within the chromosome 1q23 and 1q31 regions using an association analysis approach.     

Inter- and intrachromosomal sub-telomeric rearrangements on 4q35: implications for facioscapulohumeral muscular dystrophy (FSHD) aetiology and diagnosis

The autosomal dominant myopathy facioscapulohumeral muscular dystrophy (FSHD) is causally related to a short Eco RI fragment detected by probe p13E-11. This remnant fragment is the result of a deletion of an integral number of tandemly arrayed 3.3 kb repeat units (D4Z4) on 4q35. Despite intensive efforts, no transcribed sequences have been identified within this array. Previously, we have shown that these repeats on 4q35 have been exchanged for a similar highly homologous repeat locus on 10q26 in 20% of the population and that a short chromosome 10-like array on 4q35 also results in FSHD. Here, we describe the hybrid structure of some of these repeat arrays, reflecting additional sub-telomeric instability. In three healthy individuals carrying a 4-like repeat on chromosome 10 or vice versa, one repeat array was shown to consist of hybrid clusters of 4-derived and 10-derived repeat units. Moreover, employing pulsed field gel electrophoresis analysis, we identified two unrelated individuals carrying deletions of a chromosomal segment (p13E-11) proximal to the repeat locus. These deletions were not associated with FSHD. In one of these cases, however, an expansion of the deletion into the repeat array was observed in one of his children suffering from FSHD. These data provide additional evidence for instability of this sub-telomeric region and suggests that the length of the repeat, and not its intrinsic properties, is crucial to FSHD. Moreover, they are in agreement with the hypothesis that FSHD is caused by a position effect in which the repeat structure influences the expression of genes nearby. Therefore, the region deleted proximal to the repeat locus in healthy individuals can be instrumental to refine the critical region for FSHD1.      

面肩肱型肌营养不良症发病机制的研究进展

面肩肱型肌营养不良症,是一种常染色体显性遗传疾病,至今尚未找到其致病基因。大部分面肩肱型肌营养不良症患者和4q35区域3.3-kb的串联重复序列Z4D4的整倍缺失紧密连锁,几乎所有面肩肱型肌营养不良症患者,Southern杂交片段小于35 kb(少于11个D4Z4重复序列),而正常人群该片段为350 kb(11-150个D4Z4重复序列)。通过分子生物学研究与生物信息学分析,在4q35区域内,排除了FRG1、FRG2、ALP、ANT1、DUX4、YY I、HMGB2及Nu-c lolin等几个可能的候选基因;有关肩肱型肌营养不良症发病机制的位置变异效应假说,需要更多的证据支持;另一假说认为,面肩肱型肌营养不良症患者,D4Z4区域内类似沉默子的序列与转录抑制复合物相结合,由于D4Z4的缺失,该复合物不能形成并导致D4Z4上游基因的过表达,有关基因的过表达通过某种不明机制导致FSHD疾病的发生;D4Z4的缺失使4qter在细胞核内的定位异常,使许多基因的表达不正常,从而引起一系列的病理变化,并最终导致FSHD疾病的发生,也是FSHD发病的可能性机制之一。FSHD的发病相关基因和发病机制的研究有待深入。     

Distinguishing the 4qA and 4qB variants is essential for the diagnosis of facioscapulohumeral muscular dystrophy in the Chinese population

Facioscapulohumeral muscular dystrophy (FSHD) is the third most common inherited muscular dystrophy with markedly clinical variability and complex genetic cause. Several reports pertaining to the Caucasian population have confirmed that there are 4qA and 4qB variants of the 4qter subtelomere, and FSHD is uniquely associated with the 4qA variant. However, few data relevant to the Chinese population have been published. In present paper, detailed clinical and genetic re-evaluations were performed in members of four special families who had been initially diagnosed as atypical or asymptomatic FSHD based only on the D4Z4 repeat length analysis. The FSHD-sized D4Z4 repeats in the probands from families 1, 2 and 3 were identified as 4qB variants. These patients were further confirmed as limb-girdle muscular dystrophy (LGMD2) or myotonic dystrophy (DM1) by molecular analyses. Specifically, we identified a 4qB variant on chromosome 10 in the healthy members of the fourth FSHD family with complex D4Z4 rearrangements of two exchanged repeat arrays. For the first time, we demonstrated in the Chinese population that D4Z4 contractions on the 4qB variant do not cause FSHD and 4qB variant on chromosome 10 might also represent intermediate structures in the transition from 4q to 10q. Furthermore, our results emphasize that D4Z4 repeat length analysis alone is not sufficient for the diagnosis of FSHD, especially when used as an exclusion criterion. This analysis should be accompanied by 4qA/4qB variant determination and integrated chromosome assignments, especially in patients with obscure and unclassified myopathies similar to atypical forms of FSHD.     

A new locus for autosomal dominant retinitis pigmentosa on the short arm of chromosome 17

Retinitis pigmentosa (RP) is a group of genetically and clinicallyheterogeneous retinopathies, some of which have been shown toresult from mutations in two different known retinal genes,rhodopsin (3q) and peripherin-rds (6p). Three additional anonymousloci at 7p, 7q and pericentric 8 have been implicated by linkagestudies. There are still, however, a few families in which allknown loci have been excluded. In this report we present dataindicating a location, on the short arm of chromosome 17, forthe autosomal dominant RP (ADRP) locus in a large South African(SA) family of British ancestry. Positive two-point lod scoreshave been obtained for nine markers (D17S938, Z = 5.43; D17S796,Z = 4.82; D17S849, Z = 3.6; D17S786, Z = 3.55; TP53, Z = 3.55;D17S578, Z = 3.29; D17S960, Z = 3.16; D17S926, Z = 1.51; D17S804,Z = 0.47 all at      

Highly significant linkage to chromosome 3q13.31 for rhinitis and related allergic diseases

Background

Allergic diseases such as asthma and rhinitis have closely related phenotypes and often occur with atopy. They show strong familial and intra‐individual clustering, suggesting overlapping disease aetiology. Various loci and candidate genes have been suggested to underlie allergy. Many or all are still inconclusive. Following genome‐wide scans on multiple phenotypes, we previously suggested that chromosome 3q13.12–q21.2 harbours an allergy locus.

Objective

To identify candidate loci in the Danish population, two additional independent sets of sib‐pair families were fine‐scale mapped in candidate regions showing maximum likelihood scores (MLS) ⩾1.5 in the genome‐wide scans.

Results

Twenty eight microsatellite markers in a denser map on chromosome 3q were analysed in 236 allergy sib‐pair families including 125 sib pairs with rhinitis. We report significant evidence for linkage to chromosome 3q13.31 for rhinitis (MLS 5.55, identity by descent (IBD) 63.9%) and atopy (increased specific immunoglobulin E) (MLS 3.71, IBD 61.7%). We obtained an MLS of 5.1 (IBD 67.3%) at 3q13.31 when sib pairs with both rhinitis and atopy were analysed.

Conclusion

This study reports the first statistically significant evidence for a genetic susceptibility locus for rhinitis and to our knowledge shows the most significant evidence to date of linkage for any allergy phenotype.     

A new locus for autosomal dominant posterior polar cataract in Moroccan Jews maps to chromosome 14q22-23

Background

Posterior polar cataract is a clinically distinctive opacity located at the back of the lens. It is commonly acquired in age related cataract, and may infrequently occur in pedigrees with congenital cataract. To date, five loci for autosomal dominant congenital posterior polar cataract have been identified. These include two genes, CRYAB and PITX3, on chromosomes 11q and 10q respectively, and three loci with as yet unknown genes on chromosomes 1p, 16q and 20p.

Purpose

To find the chromosomal location of a gene causing autosomal dominant congenital posterior polar cataract in three Moroccan Jewish families.

Methods

A whole genome scan was performed using microsatellite markers spaced at approximately 10 cM intervals. For fine mapping, five additional microsatellite markers were genotyped. Two‐point lod scores were calculated using MLINK software, from the LINKAGE program package. After linkage was established, several positional candidate genes were assessed by PCR based DNA sequencing.

Results

The new cataract locus was mapped to an 11.3 cM interval between D14S980 and D14S1069 on chromosome 14q22‐23. A maximum two point lod score of 5.19 at θ = 0 was obtained with the markersD14S274. The positional and functional candidate genes SIX1, SIX4, SIX6, OTX2, and ARHJ were excluded as the cause of cataract in these families.

Conclusion

An as yet unidentified gene associated with posterior polar cataract maps to the long arm of chromosome 14q22‐23.     

Evaluating the association of common APOA2 variants with type 2 diabetes

Background  

APOA2 is a positional and biological candidate gene for type 2 diabetes at the chromosome 1q21-q24 susceptibility locus. The aim of this study was to examine if HapMap phase II tag SNPs in APOA2 are associated with type 2 diabetes and quantitative traits in French Caucasian subjects.     

Monosomy of distal 4q does not cause facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is a hereditary neuromuscular disorder transmitted in an autosomal dominant fashion. FSHD has been located by linkage analysis in the most distal part of chromosome 4q. The disease is associated with deletions within a 3.2 kb tandem repeat sequence, D4Z4. We have studied a family in which an abnormal chromosome 4 segregates through three generations in phenotypically normal subjects. This chromosome is the derivative of a (4;D or G) (q35;p12) translocation. Molecular analysis of the region 4q35 showed the absence of the segment ranging from the telomere to locus D4F104S1. Probe p13E-11 (D4F104S1), which detects polymorphic EcoRI fragments containing D4Z4, in Southern blot analysis showed only one allele in the carriers of the abnormal chromosome 4. Probe p13E-11 EcoRI fragments are contained in the subtelomeric region of 4q and their rearrangements associated with FSHD suggested that the gene responsible for the muscular dystrophy could be subject to a position effect variegation (PEV) because of its proximity to subtelomeric heterochromatin. The absence of the 4q telomeric region in our phenotypically normal cases indicates that haploinsufficiency of the region containing D4Z4 does not cause FSHD.     

A 45,X sterile male with Yp disguised as 21p

An azoospermic male was found to have, by means of banding techniques, a 45,X karyotype including a monocentric chromosome 21 with an euchromatic short arm that looked similar to Yp. This rearranged chromosome was further characterized by FISH with a whole Y chromosome paint and the alphoid repeats DYZ3 and D13Z1/D21Z1; the former probe gave a positive signal onto such a peculiar arm without spreading into the long arm, whereas the alphoid repeats revealed an apparent compound centromere with Y- and 21-sequences. Therefore, an unbalanced Y;21 whole arm translocation was concluded and the karyotype written as 45,X.ish der(Y;21)(p10;q10)(wcpY+,DYZ3+,D13Z1/D21Z1+). This patient represents the first case of a Y;21 translocation in an apparent 45,X male, constitutes the fifth instance of a 45,X sterile male, and conforms to previously established karyotype-phenotype correlations.     

A susceptibility locus for human systemic lupus erythematosus (hSLE1) on chromosome 2q

To identify chromosomal regions containing susceptibility loci for systemic lupus erythematosus (SLE), we performed genome scans in families with multiple SLE patients from Iceland, a geographical and genetic isolate, and from Sweden. A number of chromosomal regions showed maximum lod scores (Z) indicating possible linkage to SLE in both the Icelandic and Swedish families. In the Icelandic families, five regions showed lod scores greater than 2.0, three of which (4p15-13, Z=3.20; 9p22, Z=2.27; 19q13, Z=2.06) are homologous to the murine regions containing the lmb2, sle2 and sle3 loci, respectively. The fourth region is located on 19p13 (D19S247, Z=2.58) and the fifth on 2q37 (D2S125, Z=2.06). Only two regions showed lod scores above 2.0 in the Swedish families: on chromosome 2q11 (D2S436, Z=2. 13) and 2q37 (D2S125, Z=2.18). The combination of both family sets gave a highly significant lod score at D2S125 of Z=4.24 in favor of linkage for 2q37. This region represents a new locus for SLE. Our results underscore the importance of studying well-defined populations for genetic analysis of complex diseases such as SLE.