Germ-line mutational analysis of the TSC2 gene in 90 tuberous-sclerosis patients.

Ninety patients with tuberous-sclerosis complex (TSC) were tested for subtle mutations in the TSC2 gene, by means of single-strand conformational analysis (SSCA) of genomic DNA. Patients included 56 sporadic cases and 34 familial probands. For all patients, SSCA was performed for each of the 41 exons of the TSC2 gene. We identified 32 SSCA changes, 22 disease-causing mutations, and 10 polymorphic variants. Interestingly, we detected mutations at a much higher frequency in the sporadic cases (32%) than in the multiplex families (9%). Among the eight families for which linkage to the TSC2 region had been determined, only one mutation was found. Mutations were distributed equally across the gene; they included 5 deletions, 3 insertions, 10 missense mutations, 2 nonsense mutations, and 2 tandem duplications. We did not detect an increase in mutations either in the GTPase-activating protein (GAP)-related domains of TSC2 or in the activating domains that have been identified in rat tuberin. We did not detect any mutations in the exons (25 and 31) that are spliced out in the isoforms. There was no evidence for correspondence between variability of phenotype and type of mutation (missense versus early termination). Diagnostic testing will be difficult because of the genetic heterogeneity of TSC (which has at least two causative genes: TSC1 and TSC2), the large size of the TSC2 gene, and the variety of mutations. More than half of the mutations that we identified (missense, small in-frame deletion, and tandem duplication) are not amenable to the mutation-detection methods, such as protein-truncation testing, that are commonly employed for genes that encode proteins with tumor-suppressor function.     

Angiomyolipoma have common mutations in TSC2 but no other common genetic events

Renal angiomyolipoma are part of the PEComa family of neoplasms, and occur both in association with Tuberous Sclerosis Complex (TSC) and independent of that disorder. Previous studies on the molecular genetic alterations that occur in angiomyolipoma are very limited. We evaluated 9 angiomyolipoma for which frozen tissue was available from a consecutive surgical series. Seven of 8 samples subjected to RT-PCR-cDNA sequencing showed mutations in TSC2; none showed mutations in TSC1 or RHEB. Six of the seven mutations were deletions. We searched for 983 activating and inactivating mutations in 115 genes, and found none in these tumors. Similarly analysis for genomic regions of loss or gain, assessed by Affymetrix SNP6.0 analysis, showed no abnormalities. Loss of heterozygosity in the TSC2 region was commonly seen, except in patients with low frequency TSC2 mutations. We conclude that sporadic renal angiomyolipoma usually have mutations in TSC2, but not TSC1 or RHEB, and have no other common genomic events, among those we searched for. However, chromosomal translocations and gene fusion events were not assessed here. TSC2 inactivation by mutation is a consistent and likely necessary genetic event in the pathogenesis of most angiomyolipoma.     

Identification of 54 large deletions/duplications in TSC1 and TSC2 using MLPA, and genotype-phenotype correlations

Tuberous sclerosis (TSC) is an autosomal dominant disorder caused by mutations in either of two genes, TSC1 and TSC2. Point mutations and small indels account for most TSC1 and TSC2 mutations. We examined 261 TSC DNA samples (209 small-mutation-negative and 52 unscreened) for large deletion/duplication mutations using multiplex ligation-dependent probe amplification (MLPA) probe sets designed to permit interrogation of all TSC1/2 exons, as well as 15–50 kb of flanking sequence. Large deletion/duplication mutations in TSC1 and TSC2 were identified in 54 patients, of which 50 were in TSC2, and 4 were in TSC1. All but two mutations were deletions. Only 13 deletions were intragenic in TSC2, and one in TSC1, so that 39 (73%) deletions extended beyond the 5′, 3′ or both ends of TSC1 or TSC2. Mutations were identified in 24% of small-mutation-negative and 8% of unscreened samples. Eight of 54 (15%) mutations were mosaic, affecting 34–62% of cells. All intragenic mutations were confirmed by LR-PCR. Genotype/phenotype analysis showed that all (21 of 21) patients with TSC2 deletions extending 3′ into the PKD1 gene had kidney cysts. Breakpoints of intragenic deletions were randomly distributed along the TSC2 sequence, and did not preferentially involve repeat sequence elements. Our own 20-plex probe sets gave more robust performance than the 40-plex probe sets from MRC-Holland. We conclude that large deletions in TSC1 and TSC2 account for about 0.5 and 6% of mutations seen in TSC patients, respectively, and MLPA is a highly sensitive and accurate detection method, including for mosaicism. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Germ-line mosaicism in tuberous sclerosis: how common?

Two-thirds of cases of tuberous sclerosis complex (TSC) are sporadic and usually are attributed to new mutations, but unaffected parents sometimes have more than one affected child. We sought to determine how many of these cases represent germ-line mosaicism, as has been reported for other genetic diseases. In our sample of 120 families with TSC, 7 families had two affected children and clinically unaffected parents. These families were tested for mutations in the TSC1 and TSC2 genes, by Southern blotting and by single-strand conformational analysis. Unique variants were detected in six families. Each variant was present and identical in both affected children of a family but was absent in both parents and the unaffected siblings. Sequencing of the variants yielded two frameshift mutations, one missense mutation, and two nonsense mutations in TSC2 and one nonsense mutation in TSC1. To determine which parent contributed the affected gametes, the families were analyzed for linkage to TSC1 and TSC2, by construction of haplotypes with markers flanking the two genes. Linkage analysis and loss-of-heterozygosity studies indicated maternal origin in three families, paternal origin in one family, and either being possible in two families. To evaluate the possibility of low-level somatic mosaicism for TSC, DNA from lymphocytes of members of the six families were tested by allele-specific PCR. In all the families, the mutant allele was detected only in the known affected individuals. We conclude that germ-line mosaicism was present in five families with mutations in the TSC2 gene and in one family with the causative mutation in the TSC1 gene. The results have implications for genetic counseling of families with seemingly sporadic TSC.     

Evidence that lymphangiomyomatosis is caused by TSC2 mutations: chromosome 16p13 loss of heterozygosity in angiomyolipomas and lymph nodes from women with lymphangiomyomatosis.

Lymphangiomyomatosis (LAM) is a rare disease, of unknown etiology, affecting women almost exclusively. Lung transplantation is the only consistently effective therapy for LAM. Microscopically, LAM consists of a diffuse proliferation of smooth muscle cells. LAM can occur without evidence of other disease (referred to as "sporadic LAM") or in association with tuberous sclerosis complex (TSC). TSC is an autosomal dominant tumor suppressor gene syndrome characterized by seizures, mental retardation, and tumors in the brain, heart, skin, and kidney. Renal angiomyolipomas occur in approximately 50% of sporadic LAM patients and in 70% of TSC patients. Loss of heterozygosity (LOH) in the chromosomal region for the TSC2 gene occurs in 60% of TSC-associated angiomyolipomas. Because of the similar pulmonary and renal manifestations of TSC and sporadic LAM, we hypothesized that LAM and TSC have a common genetic basis. We analyzed renal angiomyolipomas, from 13 women with sporadic LAM, for LOH in the regions of the TSC1 (chromosome 9q34) and TSC2 (chromosome 16p13) genes. TSC2 LOH was detected in seven (54%) of the angiomyolipomas. We also found TSC2 LOH in four lymph nodes from a woman with retroperitoneal LAM. No TSC1 LOH was found. Our findings indicate that the TSC2 gene may be involved in the pathogenesis of sporadic LAM. However, genetic transmission of LAM has not been reported. Women with LAM may have low-penetrance germ-line TSC2 mutations, or they may be mosaic, with TSC2 mutations in the lung and the kidney but not in other organs.     

Spectrum of small mutations in the dystrophin coding region.

Duchenne and Becker muscular dystrophies (DMD and BMD) are caused by defects in the dystrophin gene. About two-thirds of the affected patients have large deletions or duplications, which occur in the 5' and central portion of the gene. The nondeletion/duplication cases are most likely the result of smaller mutations that cannot be identified by current diagnostic screening strategies. We screened approximately 80% of the dystrophin coding sequence for small mutations in 158 patients without deletions or duplications and identified 29 mutations. The study indicates that many of the DMD and the majority of the BMD small mutations lie in noncoding regions of the gene. All of the mutations identified were unique to single patients, and most of the mutations resulted in protein truncation. We did not find a clustering of small mutations similar to the deletion distribution but found > 40% of the small mutations 3' of exon 55. The extent of protein truncation caused by the 3' mutations did not determine the phenotype, since even the exon 76 nonsense mutation resulted in the severe DMD phenotype. Our study confirms that the dystrophin gene is subject to a high rate of mutation in CpG sequences. As a consequence of not finding any hotspots or prevalent small mutations, we conclude that it is presently not possible to perform direct carrier and prenatal diagnostics for many families without deletions or duplications.     

Mutational analysis in a cohort of 224 tuberous sclerosis patients indicates increased severity of TSC2, compared with TSC1, disease in multiple organs

Tuberous sclerosis (TSC) is a relatively common hamartoma syndrome caused by mutations in either of two genes, TSC1 and TSC2. Here we report comprehensive mutation analysis in 224 index patients with TSC and correlate mutation findings with clinical features. Denaturing high-performance liquid chromatography, long-range polymerase chain reaction (PCR), and quantitative PCR were used for mutation detection. Mutations were identified in 186 (83%) of 224 of cases, comprising 138 small TSC2 mutations, 20 large TSC2 mutations, and 28 small TSC1 mutations. A standardized clinical assessment instrument covering 16 TSC manifestations was used. Sporadic patients with TSC1 mutations had, on average, milder disease in comparison with patients with TSC2 mutations, despite being of similar age. They had a lower frequency of seizures and moderate-to-severe mental retardation, fewer subependymal nodules and cortical tubers, less-severe kidney involvement, no retinal hamartomas, and less-severe facial angiofibroma. Patients in whom no mutation was found also had disease that was milder, on average, than that in patients with TSC2 mutations and was somewhat distinct from patients with TSC1 mutations. Although there was overlap in the spectrum of many clinical features of patients with TSC1 versus TSC2 mutations, some features (grade 2-4 kidney cysts or angiomyolipomas, forehead plaques, retinal hamartomas, and liver angiomyolipomas) were very rare or not seen at all in TSC1 patients. Thus both germline and somatic mutations appear to be less common in TSC1 than in TSC2. The reduced severity of disease in patients without defined mutations suggests that many of these patients are mosaic for a TSC2 mutation and/or have TSC because of mutations in an as-yet-unidentified locus with a relatively mild clinical phenotype.     

Visualization of mosaicism in tissues of normal and mismatch-repair-deficient mice carrying a microsatellite-containing transgene

We tested for azoospermia factor (AZF) deletions 17 loci corresponding to AZF subintervals a-d in 17 cases of testicular tumors occurring in Finns. While DNA samples from 48 CEPH and 32 Finnish males showed no deletions, patients with testicular cancer displayed AZF deletion mosaicisms in various non-tumor tissues (13 cases) and specific deletion haplotypes in tumor tissues (10 cases). Two of the cases with AZF deletions were testicular non-Hodgkin lymphomas indicating that Y-microdeletions appear also in malignancies other than seminoma and non-seminoma tumors. In good agreement with this assumption, we detected one AZF deletion in normal cells from 1 of 5 HNPCC cases, heterozygous for an MLH1 mutation. We propose that AZF deletions occur in early embryogenesis due to mutations of TSPY, mismatch repair (MMR), or X-specific genes. Since fathers of testicular, tumor cases did not exhibit AZF deletions, we assumed they were not carriers of the mutation inducing AZF deletion-mosaicisms. Therefore, tumor cases should have received the MMR gene or X mutations via the maternal lineage, or for the case of TSPY and MMR genes via a sperm carrying a mutation occurred in the paternal germ-cell line. We consider AZF microdeletions in non-tumor cells to be part of a broader pattern of chromosome instability producing susceptibility to testicular tumors. Clonal transformation and expansion of one of these tumor-susceptible cell lineages give rise to testicular tumors showing genome anomalies characteristic of testicular cancers (i12p, LOH and genetic imbalance for various autosomal regions, Y- and autosomal MSI, specific AZF deletion haplotypes).     

Large Heterologies Impose Their Gene Conversion Pattern onto Closely Linked Point Mutations

We have studied the meiotic non-Mendelian segregation (NMS) pattern of seven large heterologous combinations located in the b2 ascospore gene of Ascobolus. The NMS patterns of these aberration heterozygotes widely differ from each other and from those of point mutations located in the same genetic region. They give lower gene conversion frequencies than point mutations, no postmeiotic segregations (PMS), and either parity or disparity that favors the wild type allele. Two related deletions, G234 and G40, were studied for their effects on the conversion behavior of closely linked point mutations. We found that, when heterozygous, the deletions impose their own NMS pattern onto close mutations. These effects occur on both sides of the heterologies. The effects upon PMS and disparity of linked point mutations gradually disappear as point mutations become more distant. The effects on NMS frequencies and on aberrant 4:4 are polar. They persist for all mutations located downstream from the high conversion end of the gene. This last effect can reflect a blockage of symmetric hDNA formation by large heterologies, whereas the epistasis of the NMS pattern of large heterologies over that of closely linked point mutations suggests that large heterologies and point mutations undergo conversion by means of distinct pathways.     

A comprehensive,sensitive and economical approach for the detection of mutations in the <Emphasis Type="Italic">RB1</Emphasis> gene in retinoblastoma

Retinoblastoma (Rb) is the most common primary intraocular malignancy in children. It is brought about by the mutational inactivation of both alleles of RB1 gene in the developing retina. To identify the RB1 mutations, we analysed 74 retinoblastoma patients by screening the exons and the promoter region of RB1. The strategy used was to detect large deletions/duplications by fluorescent quantitative multiplex PCR; small deletions/insertions by fluorescent genotyping of RB1 alleles, and point mutations by PCR-RFLP and sequencing. Genomic DNA from the peripheral blood leucocytes of 74 Rb patients (53 with bilateral Rb, 21 with unilateral Rb; 4 familial cases) was screened for mutations. Recurrent mutations were identified in five patients with bilateral Rb, large deletions in 11 patients (nine with bilateral Rb and two with unilateral Rb), small deletions/insertions were found in 12 patients all with bilateral Rb, and point mutations in 26 patients (14 nonsense, six splice site, five substitution and one silent change). Three mutations were associated with variable expressivity of the disease in different family members. Using this method, the detection rates achieved in patients with bilateral Rb were 44/53 (83%) and with unilateral Rb, 5/21 (23.8%). This approach may be feasible for clinical genetic testing and counselling of patients.     

Restriction analysis of deletions and deletion mapping of point mutations in the ADE2 gene of Saccharomyces cerevisiae yeasts

The method of restriction analysis has been used to study the length of 10 deletion mutations in ADE2 locus of Saccharomyces cerevisiae. We showed that 7 deletions overlapped the whole transcribed region of the gene ADE2, while 3 deletions have one of the ends situated in this region. Four controlled sites were fixed on the genetic map of ADE2 locus, based on these results. Deletion mapping of great number of point mutations demonstrated non-random distribution of mutations of different types on the map of ADE2 locus.     

尼罗罗非鱼β2m基因SNP位点和单倍型与无乳链球菌抗性的关联分析

β₂-微球蛋白(β₂-microglobulin, β₂m)作为MHCⅠ类分子的亚基, 在鱼类的免疫系统中发挥重要作用。实验采用直接测序法从P₀代尼罗罗非鱼(Oreochromis niloticus)的β₂m基因组序列中筛选到30个SNPs, 其中1个SNP位于5?UTR, 16个SNPs位于外显子区域(15个非同义突变位点, 1个同义突变位点), 9个SNPs位于内含子区域, 4个SNPs位于3?UTR。利用snapshot分型法对F₁代的102尾易感群体和102尾抗病群体进行基因分型, 并通过Popgen32和PIC-CALC软件统计分析尼罗罗非鱼β₂m基因序列的SNPs的H_e、H_o、N_e和PIC等遗传参数, 表明易感群体中7个SNPs属于中度多态水平(0.251代2个群体中的基因型频率和等位基因频率, 分析其与链球菌抗性或易感性状之间的相关性。结果表明: 24个SNPs的基因型和等位基因频率与无乳链球菌(Streptococcus agalactiae)抗性/易感性状显著相关(P<0.05)。通过连锁不平衡分析发现30个SNPs构成4个单倍块和14种单倍型。其中, 4个单倍型与无乳链球菌抗性性状显著相关(P<0.05), 4个单倍型与易感性状显著相关(P<0.05)。标签SNP分析发现, 单倍块2中的4个SNPs和单倍块3中的13个SNPs彼此之间高度连锁(r2>0.9), 这意味着我们在β₂m基因中发现2个htSNPs。研究筛选到的与链球菌抗性/易感性状相关的SNP位点及单倍型具有辅助尼罗罗非鱼抗链球菌病品种选育的潜力。     

The Gene Structures of Spontaneous Mutations Affecting a CAENORHABDITIS ELEGANS Myosin Heavy Chain Gene

We have isolated spontaneous mutations affecting the unc-54 major myosin heavy chain gene of Caenorhabditis elegans (variety Bristol). Spontaneous unc-54 mutants occur in C. elegans populations at a frequency of approximately 3 X 10(-7). We have studied the gene structure of 65 independent unc-54 mutations using filter-transfer hybridization techniques. Most unc-54 mutations (50 of 65) exhibit no abnormalities detected with these techniques; these mutations are small lesions affecting less than 100 base pairs. Approximately 17% of the mutations (11 of 65) are simple deletions, ranging in size from less than 100 base pairs to greater than 17 kilobases. One isolate contains two separate deletions, each of which affects unc-54. Two mutants contain tandem genetic duplications that include a portion of unc-54 and extend 8-10 kilobases beyond the 5' terminus of the mRNA. Conspicuously absent from our collection of spontaneous unc-54 mutations are any resulting from insertion of transposable genetic elements. Such mutants, if they occur, must arise at a frequency of less than 5 X 10(-9).     

Comprehensive mutation analysis of TSC1 and TSC2-and phenotypic correlations in 150 families with tuberous sclerosis

Tuberous sclerosis (TSC [MIM 191090 and MIM 191100]) is an autosomal dominant disorder characterized by hamartomas in many organs. Two thirds of cases are sporadic and are thought to represent new mutations. TSC is caused by mutations affecting either of the presumed tumor-suppressor genes, TSC1 and TSC2. Both appear to function as tumor suppressors, because somatic loss or intragenic mutation of the corresponding wild-type allele is seen in the associated hamartomas. Here we report the first comprehensive mutation analysis of TSC1 and TSC2 in a cohort of 150 unrelated TSC patients and their families, using heteroduplex and SSCP analysis of all coding exons and using pulsed-field gel electrophoresis and conventional Southern blot analysis and long PCR to screen for large rearrangements. Mutations were characterized in 120 (80%) of the 150 cases, affecting TSC1 in 22 cases and TSC2 in 98 cases. TSC1 mutations were significantly underrepresented in sporadic cases (P=. 000185). Twenty-two patients had TSC2 missense mutations that were found predominantly in the GAP-related domain (eight cases) and in a small region encoded in exons 16 and 17, between nucleotides 1849 and 1859 (eight cases), consistent with the presence of residues performing key functions at these sites. In contrast, all TSC1 mutations were predicted to be truncating, consistent with a structural or adapter role for the encoded protein. Intellectual disability was significantly more frequent in TSC2 sporadic cases than in TSC1 sporadic cases (P=.0145). These data provide the first representative picture of the distribution and spectrum of mutations across the TSC1 and TSC2 loci in clinically ascertained TSC and support a difference in severity of TSC1- and TSC2-associated disease.     

The BRCA1 Ashkenazi founder mutations occur on common haplotypes and are not highly correlated with anonymous single nucleotide polymorphisms likely to be used in genome-wide case-control association studies

Background

We studied linkage disequilibrium (LD) patterns at the BRCA1 locus, a susceptibility gene for breast and ovarian cancer, using a dense set of 114 single nucleotide polymorphisms in 5 population groups. We focused on Ashkenazi Jews in whom there are known founder mutations, to address the question of whether we would have been able to identify the 185delAG mutation in a case-control association study (should one have been done) using anonymous genetic markers. This mutation is present in approximately 1% of the general Ashkenazi population and 4% of Ashkenazi breast cancer cases. We evaluated LD using pairwise and haplotype-based methods, and assessed correlation of SNPs with the founder mutations using Pearson's correlation coefficient.

Results

BRCA1 is characterized by very high linkage disequilibrium in all populations spanning several hundred kilobases. Overall, haplotype blocks and pair-wise LD bins were highly correlated, with lower LD in African versus non-African populations. The 185delAG and 5382insC founder mutations occur on the two most common haplotypes among Ashkenazim. Because these mutations are rare, even though they are in strong LD with many other SNPs in the region as measured by D-prime, there were no strong associations when assessed by Pearson's correlation coefficient, r (maximum of 0.04 for the 185delAG).

Conclusion

Since the required sample size is related to the inverse of r, this suggests that it would have been difficult to map BRCA1 in an Ashkenazi case-unrelated control association study using anonymous markers that were linked to the founder mutations.     

Rare BRCA1 haplotypes including 3’UTR SNPs associated with breast cancer risk

Genetic markers identifying women at an increased risk of developing breast cancer exist, yet the majority of inherited risk remains elusive. While numerous BRCA1 coding sequence mutations are associated with breast cancer risk, BRCA1 mutations account for less then 5% of breast cancer risk. Since 3' untranslated region (3'UTR) polymorphisms disrupting microRNA (miRNA) binding can be functional and can act as genetic markers of cancer risk, we tested the hypothesis that such polymorphisms in the 3'UTR of BRCA1 and haplotypes containing these functional polymorphisms may be associated with breast cancer risk. We sequenced the BRCA1 3'UTR from breast cancer patients to identify miRNA disrupting polymorphisms. We further evaluated haplotypes of this region including the identified 3'UTR variants in a large population of controls and breast cancer patients (n=221) with known breast cancer subtypes and ethnicities. We identified three 3'UTR variants in BRCA1 that are polymorphic in breast cancer populations, and haplotype analysis including these variants revealed that breast cancer patients harbor five rare haplotypes not generally found among controls (9.50% for breast cancer chromosomes, 0.11% for control chromosomes, p=0.0001). Three of these rare haplotypes contain the rs8176318 BRCA1 3'UTR functional variant. These haplotypes are not biomarkers for BRCA1 coding mutations, as they are found rarely in BRCA1 mutant breast cancer patients (1/129 patients= 0.78%). These rare BRCA1 haplotypes and 3'UTR SNPs may represent new genetic markers of breast cancer risk.     

Denaturing high-performance liquid chromatography (DHPLC) is a highly sensitive, semi-automated method for identifying mutations in the TSC1 gene

Sensitive and automated methods for the detection of DNA sequence variation are required for a wide variety of genetic studies. Diagnostic testing in human genetic disorders is one application of such methods. Tuberous sclerosis complex (TSC) is an autosomal dominant familial tumor syndrome characterized by the development of benign tumors (hamartomas) in multiple organs (OMIM # 19110, #191092). There is a high frequency of sporadic cases and significant demand from patients and families for genetic testing information. Two TSC genes have been identified (TSC1 and TSC2) and together account for all cases [1,2]. Here we report our methods for DHPLC analysis of the TSC1 gene and demonstrate the high sensitivity of this method in a blinded analysis of 21 TSC patients with known TSC1 mutations. In this series, DHPLC detected 27/28 (96%) known TSC1 sequence variations. The only sequence variation not identified by DHPLC in this study is a mosaic case.