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Induced pluripotent stem cell (iPSC)-derived kidney organoids are a potential tool for the regeneration of kidney tissue. They represent an early stage of nephrogenesis and have been shown to successfsully vascularize and mature further in vivo. However, there are concerns regarding the long-term safety and stability of iPSC derivatives. Specifically, the potential for tumorigenesis may impede the road to clinical application. To study safety and stability of kidney organoids, we analyzed their potential for malignant transformation in a teratoma assay and following long-term subcutaneous implantation in an immune-deficient mouse model. We did not detect fully functional residual iPSCs in the kidney organoids as analyzed by gene expression analysis, single-cell sequencing and immunohistochemistry. Accordingly, kidney organoids failed to form teratoma. Upon long-term subcutaneous implantation of whole organoids in immunodeficient IL2Ry−/−RAG2−/− mice, we observed tumor formation in 5 out of 103 implanted kidney organoids. These tumors were composed of WT1+CD56+ immature blastemal cells and showed histological resemblance with Wilms tumor. No genetic changes were identified that contributed to the occurrence of tumorigenic cells within the kidney organoids. However, assessment of epigenetic changes revealed a unique cluster of differentially methylated genes that were also present in undifferentiated iPSCs. We discovered that kidney organoids have the capacity to form tumors upon long-term implantation. The presence of epigenetic modifications combined with the lack of environmental cues may have caused an arrest in terminal differentiation. Our results indicate that the safe implementation of kidney organoids should exclude the presence of pro-tumorigenic methylation in kidney organoids.

Approximately 25% of all uveal melanoma (UM) contain driver mutations in the gene encoding the spliceosome factor SF3B1, and whilst patients with such SF3B1 mutations generally have an intermediate risk on developing metastatic disease, a third of these patients develop early metastasis within 5 years after diagnosis. We therefore investigated whether clinical and/or genetic variables could be indicative of short progression-free survival (PFS < 60 months) or long PFS (PFS ≥ 60 months) for SF3B1-mutated (SF3B1mut) UM patients. We collected 146 SF3B1mut UM from our Rotterdam Ocular Melanoma Studygroup (ROMS) database and external published datasets. After stratification of all SF3B1mut UM using short PFS vs. long PFS, only largest tumor diameter (LTD) was significantly larger (mean: 17.7 mm (±2.8 SD) in the short PFS SF3B1mut group vs. the long PFS group (mean: 14.7 (±3.7 SD, p = 0.001). Combined ROMS and The Cancer Genome Atlas (TCGA) transcriptomic data were evaluated, and we identified SF3B1mut-specific canonical transcripts (e.g., a low expression of ABHD6 indicative for early-onset metastatic disease) or distinct expression of SF3B1mut UM aberrant transcripts, indicative of early- or late-onset or no metastatic SF3B1mut UM.

__Background:__ Patients born with esophageal atresia (EA) have a higher incidence of infantile hypertrophic pyloric stenosis (IHPS), suggestive of a relationship. A shared etiology makes sense from a developmental perspective as both affected structures are foregut derived. A genetic component has been described for both conditions as single entities and EA and IHPS are variable components in several monogenetic syndromes. We hypothesized that defects disturbing foregut morphogenesis are responsible for this combination of malformations. __Methods:__ We investigated the genetic variation of 15 patients with both EA and IHPS with unaffected parents using exome sequencing and SNP arraybased genotyping, and compared the results to mouse transcriptome data of the developing foregut. __Results:__ We did not identify putatively deleterious de novo mutations or recessive variants. However, we detected rare inherited variants in EA or IHPS disease genes or in genes important in foregut morphogenesis, expressed at the proper developmental time-points. Two pathways were significantly enriched (p < 1 × 10−5): proliferation and differentiation of smooth muscle cells and self-renewal of satellite cells. __Conclusions:__ None of our findings could fully explain the combination of abnormalities on its own, which makes complex inheritance the most plausible genetic explanation, most likely in combination with mechanical and/or environmental factors. As we did not find one defining monogenetic cause for the EA/IHPS phenotype, the impact of the corrective surgery could should be further investigated.

The aim of this study was exploration of the genetic background of conjunctival melanoma (CM) and correlation with recurrent and metastatic disease. Twenty-eight CM from the Rotterdam Ocular Melanoma Study group were collected and DNA was isolated from the formalin-fixed paraffin embedded tissue. Targeted next-generation sequencing was performed using a panel covering GNAQ, GNA11, EIF1AX, BAP1, BRAF, NRAS, c-KIT, PTEN, SF3B1, and TERT genes. Recurrences and metastasis were present in eight (29%) and nine (32%) CM cases, respectively. TERT promoter mutations were most common (54%), but BRAF (46%), NRAS (21%), BAP1 (18%), PTEN (14%), c-KIT (7%), and SF3B1 (4%) mutations were also observed. No mutations in GNAQ, GNA11, and EIF1AX were found. None of the mutations was significantly associated with recurrent disease. Presence of a TERT promoter mutation was associated with metastatic disease (p-value = 0.008). Based on our molecular findings, CM comprises a separate entity within melanoma, although there are overlapping molecular features with uveal melanoma, such as the presence of BAP1 and SF3B1 mutations. This warrants careful interpretation of molecular data, in the light of clinical findings. About three quarter of CM contain drug-targetable mutations, and TERT promoter mutations are correlated to metastatic disease in CM.

The aim of this study was exploration of the genetic background of conjunctival melanoma (CM) and correlation with recurrent and metastatic disease. Twenty-eight CM from the Rotterdam Ocular Melanoma Study group were collected and DNA was isolated from the formalin-fixed paraffin embedded tissue. Targeted next-generation sequencing was performed using a panel covering GNAQ, GNA11, EIF1AX, BAP1, BRAF, NRAS, c-KIT, PTEN, SF3B1, and TERT genes. Recurrences and metastasis were present in eight (29%) and nine (32%) CM cases, respectively. TERT promoter mutations were most common (54%), but BRAF (46%), NRAS (21%), BAP1 (18%), PTEN (14%), c-KIT (7%), and SF3B1 (4%) mutations were also observed. No mutations in GNAQ, GNA11, and EIF1AX were found. None of the mutations was significantly associated with recurrent disease. Presence of a TERT promoter mutation was associated with metastatic disease (p-value = 0.008). Based on our molecular findings, CM comprises a separate entity within melanoma, although there are overlapping molecular features with uveal melanoma, such as the presence of BAP1 and SF3B1 mutations. This warrants careful interpretation of molecular data, in the light of clinical findings. About three quarter of CM contain drug-targetable mutations, and TERT promoter mutations are correlated to metastatic disease in CM.

Autosomal‐dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease, leading to kidney failure in most patients. In approximately 85% of cases, the disease is caused by mutations in PKD1. How dysregulation of PKD1 leads to cyst formation on a molecular level is unknown. Induced pluripotent stem cells (iPSCs) are a powerful tool for in vitro modeling of genetic disorders. Here, we established ADPKD patient‐specific iPSCs to study the function of PKD1 in kidney development and cyst formation in vitro. Somatic mutations are proposed to be the initiating event of cyst formation, and therefore, iPSCs were derived from cystic renal epithelial cells rather than fibroblasts. Mutation analysis of the ADPKD iPSCs revealed germline mutations in PKD1 but no additional somatic mutations in PKD1/PKD2. Although several somatic mutations in other genes implicated in ADPKD were identified in cystic renal epithelial cells, only few of these mutations were present in iPSCs, indicating a heterogeneous mutational landscape, and possibly in vitro cell selection before and during the reprogramming process. Whole‐genome DNA methylation analysis indicated that iPSCs derived from renal epithelial cells maintain a kidney‐specific DNA methylation memory. In addition, comparison of PKD1+/− and control iPSCs revealed differences in DNA methylation associated with the disease history. In conclusion, we generated and characterized iPSCs derived from cystic and healthy control renal epithelial cells, which can be used for in vitro modeling of kidney development in general and cystogenesis in particular., Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease. Here, we generated induced pluripotent stem cells (iPSCs) of ADPKD patients to study kidney development and cyst formation in vitro. These iPSCs revealed germline and autosomal mutations implicated in ADPKD and displayed an epigenetic memory of kidney epithelial cells, providing powerful models to study ADPKD in vitro.

Huntington disease (HD) is an autosomal dominant, neurodegenerative disease caused by a CAG repeat expansion within the coding sequence of the HTT gene, resulting in a highly toxic protein with an expanded polyglutamine stretch that forms typical protein aggregates throughout the brain. We generated human induced pluripotent stem cells (hiPSCs) from two HD patients using non-integrating Sendai virus (SeV). The hiPSCs display a normal karyotype, express all pluripotency markers, have the same CAG repeat expansion as the original fibroblasts and are able to differentiate into the three germ layers in vitro.

Purpose To investigate the prevalence and prognostic value of SF3B1 and EIF1AX mutations in uveal melanoma (UM) patients. Design Case series. Participants Cohort of 151 patients diagnosed with and treated for UM. Methods SF3B1 and EIF1AX mutations in primary tumors were investigated using whole-exome sequencing (n = 25) and Sanger sequencing (n = 151). For the detection of BAP1 mutations, a previously reported cohort of 90 patients was extended using BAP1 sequencing or immunohistochemistry. Main Outcome Measures The status of SF3B1 , EIF1AX , and BAP1 in tumors of patients were correlated to clinical, histopathologic, and genetic parameters. Survival analyses were performed for patients whose tumors had SF3B1 , EIF1AX , and BAP1 mutations. Results Patients with tumors harboring EIF1AX mutations rarely demonstrated metastases (2 of 28 patients) and overall had a longer disease-free survival (DFS; 190.1 vs. 100.2 months; P SF3B1 mutation had an increased metastatic risk compared with those without an SF3B1 mutation (DFS, 132.8 vs. 174.4 months; P = 0.008). Patients with such a mutation were more prone to demonstrate late metastases (median, 8.2 years; range, 23–145 months). Patients with UM and loss of BAP1 expression had a significantly decreased survival (DFS, 69.0 vs. 147.9 months; P Conclusions According to our data, patients with UM can be classified into 3 groups, of which EIF1AX -mutated tumors and tumors without BAP1 , SF3B1 , or EIF1AX mutations are associated with prolonged survival and low metastatic risk, SF3B1 -mutated tumors are associated with late metastasis, and tumors with an aberrant BAP1 are associated with an early metastatic risk and rapid decline in patient DFS.

Hereditary Cerebral Hemorrhage with Amyloidosis-Dutch type (HCHWA-D) is an autosomal dominant hereditary disease caused by a point mutation in exon 17 of the APP gene. We generated human induced pluripotent stem cells (hiPSCs) from a symptomatic HCHWA-D patient by using non-integrating Sendai virus (SeV). The newly generated hiPSCs express all pluripotency markers, have a normal karyotype, carry the Dutch mutation, can differentiate in the three germ layers in vitro and are SeV free.

Spinocerebellar ataxia type 1 (SCA1) is a hereditary neurodegenerative disease caused by a CAG repeat expansion in exon 8 of the ATXN1 gene. We generated induced pluripotent stem cells (hiPSCs) from a SCA1 patient and his non-affected sister by using non-integrating Sendai Viruses (SeV). The resulting hiPSCs are SeVfree, express pluripotency markers, display a normal karyotype, retain the mutation (length of the CAG repeat expansion in the ATXN1 gene) and are able to differentiate into the three germ layers in vitro.

Uveal melanoma (UM) is the most common primary intraocular malignancy in the Western world. Recurrent mutations in GNAQ, GNA11, CYSLTR2, PLCB4, BAP1, EIF1AX, and SF3B1 are described as well as non‐random chromosomal aberrations. Chromothripsis is a rare event in which chromosomes are shattered and rearranged and has been reported in a variety of cancers including UM. SNP arrays of 249 UM from patients who underwent enucleation, biopsy or endoresection were reviewed for the presence of chromothripsis. Chromothripsis was defined as ten or more breakpoints per chromosome involved. Genetic analysis of GNAQ, GNA11, BAP1, SF3B1, and EIF1AX was conducted using Sanger and next‐generation sequencing. In addition, immunohistochemistry for BAP1 was performed. Chromothripsis was detected in 7 out of 249 tumors and the affected chromosomes were chromosomes 3, 5, 6, 8, 12, and 13. The mean total of fragments per chromosome was 39.8 (range 12‐116). In 1 UM, chromothripsis was present in 2 different chromosomes. GNAQ, GNA11 or CYSLTR2 mutations were present in 6 of these tumors and 5 tumors harbored a BAP1 mutation and/or lacked BAP1 protein expression by immunohistochemistry. Four of these tumors metastasized and for the fifth only short follow‐up data are available. One of these metastatic tumors harbored an SF3B1 mutation. No EIF1AX mutations were detected in any of the tumors. To conclude, chromothripsis is a rare event in UM, occurring in 2.8% of samples and without significant association with mutations in any of the common UM driver genes.

Gene fusions, mainly between TMPRSS2 and ERG, are frequent early genomic rearrangements in prostate cancer (PCa). In order to discover novel genomic fusion events, we applied whole-genome paired-end sequencing to identify structural alterations present in a primary PCa patient (G089) and in a PCa cell line (PC346C). Overall, we identified over 3800 genomic rearrangements in each of the two samples as compared with the reference genome. Correcting these structural variations for polymorphisms using whole-genome sequences of 46 normal samples, the numbers of cancer-related rearrangements were 674 and 387 for G089 and PC346C, respectively. From these, 192 in G089 and 106 in PC346C affected gene structures. Exclusion of small intronic deletions left 33 intergenic breaks in G089 and 14 in PC346C. Out of these, 12 and 9 reassembled genes with the same orientation, capable of generating a feasible fusion transcript. Using PCR we validated all the reliable predicted gene fusions. Two gene fusions were in-frame: MPP5-FAM71D in PC346C and ARHGEF3-C8ORF38 in G089. Downregulation of FAM71D and MPP5-FAM71D transcripts in PC346C cells decreased proliferation; however, no effect was observed in the RWPE-1-immortalized normal prostate epithelial cells. Together, our data showed that gene rearrangements frequently occur in PCa genomes but result in a limited number of fusion transcripts. Most of these fusion transcripts do not encode in-frame fusion proteins. The unique in-frame MPP5-FAM71D fusion product is important for proliferation of PC346C cells.

X chromosome inactivation (XCI) is a mechanism specifically initiated in female cells to silence one X chromosome, thereby equalizing the dose of X-linked gene products between male and female cells. XCI is regulated by a locus on the X chromosome termed the X-inactivation centre (XIC). Located within the XIC is XIST , which acts as a master regulator of XCI. During XCI, XIST is upregulated on the inactive X chromosome and chromosome-wide cis spreading of XIST leads to inactivation. In mouse, the Xic comprises Xist and all cis -regulatory elements and genes involved in Xist regulation. The activity of the XIC is regulated by trans -acting factors located elsewhere in the genome: X-encoded XCI activators positively regulating XCI, and autosomally encoded XCI inhibitors providing the threshold for XCI initiation. Whether human XCI is regulated through a similar mechanism, involving trans -regulatory factors acting on the XIC has remained elusive so far. Here, we describe a female individual with ovarian dysgenesis and a small X chromosomal deletion of the XIC. SNP-array and targeted locus amplification (TLA) analysis defined the deletion to a 1.28 megabase region, including XIST and all elements and genes that perform cis -regulatory functions in mouse XCI. Cells carrying this deletion still initiate XCI on the unaffected X chromosome, indicating that XCI can be initiated in the presence of only one XIC. Our results indicate that the trans -acting factors required for XCI initiation are located outside the deletion, providing evidence that the regulatory mechanisms of XCI are conserved between mouse and human. This article is part of the themed issue ‘X-chromosome inactivation: a tribute to Mary Lyon’.

In a screening project of patients with (complex) craniosynostosis using genomic arrays, we identified two patients with craniosynostosis and microcephaly with a deletion in the 2p15p16.1 chromosomal region. This region has been associated with a new microdeletion syndrome, for which patients have various features in common, including microcephaly and intellectual disability. Deletions were identified using Affymetrix 250K SNP array and further characterized by fluorescence in situ hybridization (FISH) analysis and qPCR. The deletions in our two patients overlapped within the 2p15p16.1 microdeletion syndrome area and were 6.8 and 6.9 Mb in size, respectively. FISH and qPCR confirmed the presence of only one copy in this region. Finemapping of the breakpoints indicated precise borders in our patients and were further finemapped in two other previously reported patients. Clinical features of patients with deletions in the 2p15p16.1 region vary. Including data from our patients, now eight out of nine reported patients have microcephaly, one of the major features, and all had intellectual disability. The current reported two patients add different forms of craniosynostosis to the clinical spectrum of this recently recognized microdeletion syndrome. © 2013 Wiley Periodicals, Inc.

textabstractAutosomal dominant polycystic kidney disease (ADPKD) is a common genetic disorder that frequently results in renal fallure due to progressive cyst development. The major locus, PKD1, maps to 16p13.3. We identified a chromosome translocation associated with ADPKD that disrupts a gene (PBP) encoding a 14 kb transcript in the PKD1 candidate region. Further mutations of the PBP gene were found in PKD1 patients, two deletions (one a de novo event) and a splicing defect, confirming that PBP is the PKD1 gene. This gene is located adjacent to the TSC2 locus in a genomic region that is reiterated more proximally on 16p. The duplicate area encodes three transcripts substantially homologous to the PKD1 transcript. Partial sequence analysis of the PKD1 transcript shows that it encodes a novel protein whose function is at present unknown.

To investigate the prevalence and prognostic value of SF3B1 and EIF1AX mutations in uveal melanoma (UM) patients.Case series.Cohort of 151 patients diagnosed with and treated for UM.SF3B1 and EIF1AX mutations in primary tumors were investigated using whole-exome sequencing (n = 25) and Sanger sequencing (n = 151). For the detection of BAP1 mutations, a previously reported cohort of 90 patients was extended using BAP1 sequencing or immunohistochemistry.The status of SF3B1, EIF1AX, and BAP1 in tumors of patients were correlated to clinical, histopathologic, and genetic parameters. Survival analyses were performed for patients whose tumors had SF3B1, EIF1AX, and BAP1 mutations.Patients with tumors harboring EIF1AX mutations rarely demonstrated metastases (2 of 28 patients) and overall had a longer disease-free survival (DFS; 190.1 vs. 100.2 months; P0.001). Within the patient group with disomy 3, UM patients with an SF3B1 mutation had an increased metastatic risk compared with those without an SF3B1 mutation (DFS, 132.8 vs. 174.4 months; P = 0.008). Patients with such a mutation were more prone to demonstrate late metastases (median, 8.2 years; range, 23-145 months). Patients with UM and loss of BAP1 expression had a significantly decreased survival (DFS, 69.0 vs. 147.9 months; P0.001).According to our data, patients with UM can be classified into 3 groups, of which EIF1AX-mutated tumors and tumors without BAP1, SF3B1, or EIF1AX mutations are associated with prolonged survival and low metastatic risk, SF3B1-mutated tumors are associated with late metastasis, and tumors with an aberrant BAP1 are associated with an early metastatic risk and rapid decline in patient DFS.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by seizures, mental retardation and the development of hamartomas in a variety of organs and tissues. The disease is caused by mutations in either the TSC1 gene on chromosome 9q34, or the TSC2 gene on chromosome 16p13.3. Here we describe a deletion encompassing the TSC1 gene and two neighboring transcripts on chromosome 9q34 in six affected individuals from a family with TSC. To our knowledge, this is the first report of such a large deletion at the TSC1 locus and indicates that screening for similar mutations at the TSC1 locus is warranted in individuals with TSC.

Summary In placental mammals, balanced expression of X-linked genes is accomplished by X chromosome inactivation (XCI) in female cells. In humans, random XCI is initiated early during embryonic development. To investigate whether reprogramming of female human fibroblasts into induced pluripotent stem cells (iPSCs) leads to reactivation of the inactive X chromosome (Xi), we have generated iPSC lines from fibroblasts heterozygous for large X-chromosomal deletions. These fibroblasts show completely skewed XCI of the mutated X chromosome, enabling monitoring of X chromosome reactivation (XCR) and XCI using allele-specific single-cell expression analysis. This approach revealed that XCR is robust under standard culture conditions, but does not prevent reinitiation of XCI, resulting in a mixed population of cells with either two active X chromosomes (Xas) or one Xa and one Xi. This mixed population of XaXa and XaXi cells is stabilized in naive human stem cell medium, allowing expansion of clones with two Xas., Graphical Abstract, Highlights • Robust X chromosome reactivation in human iPSCs with large X-chromosomal deletions • Female human iPSCs with two active X chromosomes • Expansion of human iPSCs with two active X chromosomes in naive human stem cell medium, In this article, Gribnau and colleagues used fibroblasts, obtained from female carriers heterozygous for large X-chromosomal deletions, to generate human induced pluripotent stem cells (hiPSCs) and show that reprogramming leads to robust reactivation of the inactive X chromosome.

Mammalian genomes contain numerous regulatory DNA sites with unknown target genes. We used mice with an extra beta-globin locus control region (LCR) to investigate how a regulator searches the genome for target genes. We find that the LCR samples a restricted nuclear subvolume, wherein it preferentially contacts genes controlled by shared transcription factors. No contacted gene is detectably upregulated except for endogenous beta-globin genes located on another chromosome. This demonstrates genetically that mammalian trans activation is possible, but suggests that it will be rare. Trans activation occurs not pan-cellularly, but in 'jackpot' cells enriched for the interchromosomal interaction. Therefore, cell-specific long-range DNA contacts can cause variegated expression. [KEYWORDS: Animals, DNA/ genetics/ metabolism, GATA1 Transcription Factor/metabolism, Gene Expression, In Situ Hybridization, Fluorescence, Kruppel-Like Transcription Factors/metabolism, Locus Control Region, Mice, Mice, Transgenic, RNA, Messenger/genetics/metabolism, Transcriptional Activation, beta-Globins/genetics]

Tuberous sclerosis (TSC) is an autosomal dominant multisystem disorder with loci assigned to chromosomes 9 and 16. Using pulsed-field gel electrophoresis (PFGE), we identified five TSC-associated deletions at 16p13.3. These were mapped to a 120 kb region that was cloned in cosmids and from which four genes were isolated. One gene, designated TSC2, was interrupted by all five PFGE deletions, and closer examination revealed several intragenic mutations, including one de novo deletion. In this case, Northern blot analysis identified a shortened transcript, while reduced expression was observed in another TSC family, confirming TSC2 as the chromosome 16 TSC gene. The 5.5 kb TSC2 transcript is widely expressed, and its protein product, tuberin, has a region of homology to the GTPase-activating protein GAP3.

In this paper we present the detailed clinical and cytogenetic analysis of a prenatally detected complex Congenital Diaphragmatic Hernia (CDH) patient with a mosaic unbalanced translocation (5;12). High-resolution whole genome SNP array confirmed a low-level mosaicism (20%) in uncultured cells, underlining the value of array technology for identification studies. Subsequently, targeted Fluorescence In-Situ Hybridization in postmortem collected tissues demonstrated a similar low-level mosaicism, independently of the affected status of the tissue. Thus, a higher incidence of the genetic aberration in affected organs as lung and diaphragm cannot explain the severe phenotype of this complex CDH patient. Comparison with other described chromosome 5p and 12p anomalies indicated that half of the features presented in our patient (including the diaphragm defect) could be attributed to both chromosomal areas. In contrast, a few features such as the palpebral downslant, the broad nasal bridge, the micrognathia, microcephaly, abnormal dermatoglyphics and IUGR better fitted the 5p associated syndromes only. This study underlines the fact that low-level mosaicism can be associated with severe birth defects including CDH. The contribution of mosaicism to human diseases and specifically to congenital anomalies and spontaneous abortions becomes more and more accepted, although its phenotypic consequences are poorly described phenomena leading to counseling issues. Therefore, thorough follow-up of mosaic aberrations such as presented here is indicated in order to provide genetic counselors a more evidence based prediction of fetal prognosis in the future.

Tuberous sclerosis (TSC) is a heterogeneous multisystem disorder with loci on 9q34 (TSC1) and 16p13.3 (TSC2). The TSC2 gene has recently been isolated, while the TSC1 gene has been mapped to a 5-cM region between the markers D9S149 and D9S114. In our effort to localise and clone TSC1, we have obtained three adjacent cosmid contigs that cover the core of the candidate region. The three contigs comprise approximately 600 kb and include 80 cosmids, 2 P1 clones, 1 YAC, 5 anonymous markers and 4 sequence-tagged sites. The ABO blood group locus, the Surfeit gene cluster, the dopamine beta-hydroxylase gene (DBH) and VAV2, a homologue of the vav oncogene, have all been mapped within the contigs. Exon trapping and mutation screening experiments, aimed at identifying the TSC1 gene, are currently in progress.

Fragile X syndrome is the most frequent form of inherited mental retardation and is associated with a fragile site at Xq27.3. We identified human YAC clones that span fragile X site-induced translocation breakpoints coincident with the fragile X site. A gene (FMR-1) was identified within a four cosmid contig of YAC DNA that expresses a 4.8 kb message in human brain. Within a 7.4 kb EcoRI genomic fragment, containing FMR-1 exonic sequences distal to a CpG island previously shown to be hypermethylated in fragile X patients, is a fragile X site-induced breakpoint cluster region that exhibits length variation in fragile X chromosomes. This fragment contains a lengthy CGG repeat that is 250 bp distal of the CpG island and maps within a FMR-1 exon. Localization of the brain-expressed FMR-1 gene to this EcoRI fragment suggests the involvement of this gene in the phenotypic expression of the fragile X syndrome.