Conquering the complex world of human septins: implications for health and disease.

Septins are highly conserved filamentous proteins first characterized in budding yeast and subsequently identified in must eukaryotes. Septins can bind and hydrolyze GTP, which is intrinsically related to their formation of septin hexamers and functional protein interactions. The human septin family is composed of 14 loci, SEPT1-SEPT14, which encode dozens of different septin proteins. Their central GTPase and polybasic domain regions are highly conserved but they diverge in their N-terminus and/or C-terminus. The mechanism by which the different isoforms are generated is not yet well understood, but one can hypothesize that the use of different promoters and/or alternative splicing could give rise to these variants. Septins perform diverse cellular functions according to tissue expression and their interacting partners. Functions identified to date include cell division, chromosome segregation, protein scaffolding, cellular polarity, motility, membrane dynamics, vesicle trafficking, exocytosis, apoptosis, and DNA damage response. Their expression is tightly regulated to maintain proper filament assembly and normal cellular functions. Alterations of these proteins, by mutation or expression changes, have been associated with a variety of cancers and neurological diseases. The association of septins with cancer results from alterations of expression in solid tumors or translocations in leukemias [mixed lineage leukemia (MLL)]. Expression changes in septins have also been associated with neurological conditions such as Alzheimer's and Parkinson's disease, as well as retinopathies, hepatitis C, spermatogenesis and Listeria infection. Pathogenic mutations of SEPT9 were identified in the autosomal dominant neurological disorder hereditary neuralgic amyotrophy (HNA). Human septin research over the past decade has established their importance in cell biology and human disease. Further functional characterization of septins is crucial to our understanding of their possible diagnostic, prognostic, and therapeutic applications.

Understandings of basic genetics in the United States: results from a national survey of black and white men and women.

AIM: This study examined understandings of basic genetic concepts among Americans. METHOD: In a national telephone survey of 1,200 Americans with equal representation among Black and White men and women, subjects responded to 8 items developed by a multidisciplinary team of experts that assessed understanding of basic concepts in multiple domains, including inheritance, genetics and race, and genetics and behavior. RESULTS: Over 70% of subjects responded correctly on items about the genetic similarity of identical twins and siblings. Less than half of subjects responded correctly on all other items. Understanding of genetics was lowest in three areas: types/locations of genes in the body (29% correct), a genetic basis for race (25% correct), and the influence of single genes on behaviors (24% correct). Logistic regression models controlling for age and education showed some differences by race and gender on specific items but also showed that understandings are generally similar across these groups. CONCLUSION: Misunderstandings about genetics are common among Black and White American men and women. Responses appear to reflect personal experiences, group values and interests. These findings emphasize the need for initiatives to improve the public's genetic literacy as well as a need for further investigation in this domain.

MicroRNAs in development and disease.

Since the discovery of microRNAs (miRNAs) in Caenorhabditis elegans, mounting evidence illustrates the important regulatory roles for miRNAs in various developmental, differentiation, cell proliferation, and apoptosis pathways of diverse organisms. We are just beginning to elucidate novel aspects of RNA mediated gene regulation and to understand how heavily various molecular pathways rely on miRNAs for their normal function. miRNAs are small non-protein-coding transcripts that regulate gene expression post-transcriptionally by targeting messenger RNAs (mRNAs). While individual miRNAs have been specifically linked to critical developmental pathways, the deregulated expression of many miRNAs also has been shown to have functional significance for multiple human diseases, such as cancer. We continue to discover novel functional roles for miRNAs at a rapid pace. Here, we summarize some of the key recent findings on miRNAs, their mode of action, and their roles in both normal development and in human pathology. A better understanding of how miRNAs operate during the normal life of a cell as well as in the pathogenesis of disease when deregulated will provide new avenues for diagnostic, prognostic, and therapeutic applications.

Novel autosomal dominant mandibulofacial dysostosis with ptosis: clinical description and exclusion of TCOF1.

BACKGROUND: Treacher Collins syndrome (TCS), the most common type of mandibulofacial dysostosis (MFD), is genetically homogeneous. Other types of MFD are less common and, of these, only the Bauru type of MFD has an autosomal dominant (AD) mode of inheritance established. Here we report clinical features of a kindred with a unique AD MFD with the exclusion of linkage to the TCS locus (TCOF1) on chromosome 5q31-q32. METHODS: Six affected family members underwent a complete medical genetics physical examination and two affected subjects had skeletal survey. All available medical records were reviewed. Linkage analysis using the markers spanning the TCOF1 locus was performed. One typically affected family member had a high resolution karyotype. RESULTS: Affected subjects had significant craniofacial abnormalities without any significant acral changes and thus had a phenotype consistent with a MFD variant. Distinctive features included hypoplasia of the zygomatic complex, micrognathia with malocclusion, auricular abnormalities with conductive hearing loss, and ptosis. Significantly negative two point lod scores were obtained for markers spanning the TCOF1 locus, excluding the possibility that the disease in our kindred is allelic with TCS. High resolution karyotype was normal. CONCLUSIONS: We report a kindred with a novel type of MFD that is not linked to the TCOF1 locus and is also clinically distinct from other types of AD MFD. Identification of additional families will facilitate identification of the gene causing this type of AD MFD and further characterisation of the clinical phenotype.

Genomic characterization of human SEC14L1 splice variants within a 17q25 candidate tumor suppressor gene region and identification of an unrelated embedded expressed sequence tag.

Human SEC14L1 shows partial sequence homology to the budding yeast SEC14 protein and the Japanese flying squid retinal-binding protein and was previously generally localized to 17q25. We more precisely mapped SEC14L1 within a discrete region of 17q25 that likely harbors at least one putative breast and ovarian tumor suppressor gene. We determined that this gene consists of 18 exons ranging in size from 70 bp (exon 11) to 3088 bp (exon 17) and spanning at least 58 kb of DNA. Exon 17 contained a highly polymorphic variable number of tandem repeats (VNTR) and was present only in the larger ubiquitously expressed 5.5-kb transcript. The 3.0-kb ubiquitously expressed transcript included sequences at the beginning of exon 17 (designated exon 17a) and the end of exon 17 (designated exon 18), but lacked the internal 2439 bp of exon 17, including the VNTR. This alternative splicing resulted in a predicted protein of 719 residues from the smaller transcript with four more terminal amino acids than the 715 residue protein predicted from the larger transcript. EST H49244 spanned exon 11 of SEC14L1 and was specifically expressed in human peripheral blood leukocytes. One intragenic single nucleotide polymorphism (SNP) was confirmed. SEC14L1 contained the CRAL/TRIO domain also found in alpha-tocopherol transfer protein (TTPA) and cellular retinaldehyde-binding protein (CRALBP). As retinoids have been shown to inhibit the growth of breast cancer cells, loss of the proposed SEC14L1 retinal-binding function may contribute to breast tumorigenesis. As TTPA and CRALBP have been implicated in retinitis pigmentosa (RP), altered SEC14L1 expression may contribute to RP in previously unlinked families. Coding exon-specific PCR primers were designed to aid in future expression and mutational analyses.

Virtual genome scan: a tool for restriction landmark-based scanning of the human genome.

There is substantial interest in implementing technologies that allow comparisons of whole genomes of individuals and of tissues and cell populations. Restriction landmark genome scanning (RLGS) is a highly resolving gel-based technique in which several thousand fragments in genomic digests are visualized simultaneously and quantitatively analyzed. The widespread use of RLGS has been hampered by difficulty in deriving sequence information for displayed fragments and a lack of whole-genome sequence-based framework for interpreting RLGS patterns. We have developed informatics tools for comparisons of sample derived RLGS patterns with patterns predicted from the human genome sequence and displayed as Virtual Genome Scans (VGS). The tools developed allow sequence prediction of fragments in RLGS patterns obtained with different restriction enzyme combinations. The utility of VGS is demonstrated by the identification of restriction fragment length polymorphisms, and of amplifications, deletions, and methylation changes in tumor-derived CpG islands and the characterization of an amplified region in a breast tumor that spanned <230 kb on 17q23.

Systemic lupus erythematosus in a man with Noonan syndrome.

Noonan syndrome is a multiple congenital anomaly condition characterized by craniofacial anomalies, short stature, cardiac malformations, and normal peripheral blood karyotype analysis. Prior reports of individuals with Noonan syndrome have revealed an association with several autoimmune diseases, including vasculitis and anterior uveitis, but no reports of systemic lupus erythematosus (SLE). Here we present the first case report of a 21-year-old man with a clinical diagnosis of Noonan syndrome and a recent history of mitral valve dysfunction and systemic lupus erythematosus. We discuss his findings in the context of known features of Noonan syndrome and propose that individuals with Noonan syndrome be regularly monitored for associated autoimmune phenomena.

Fusion of a novel gene, RCC17, to the TFE3 gene in t(X;17)(p11.2;q25.3)-bearing papillary renal cell carcinomas.

A subset of childhood and young adult renal cell carcinomas displays a recurrent translocation t(X;17)(p11;q25) as the sole cytogenetic abnormality. In two young girls, we demonstrate that this translocation results in the fusion of a novel gene, designated RCC17, at chromosome 17q25, to the transcription factor TFE3 located on the Xp11 chromosomal region. In both cases, the t(X;17) fuses the NH(2)-terminal region of RCC17 to the COOH-terminal part of TFE3 including the basic helix-loop-helix DNA-binding domain and the leucine zipper dimerization domain. The reciprocal fusion transcript TFE3/RCC17 is also expressed. RCC17 encodes a putative protein of 553 amino acids. It is ubiquitously expressed in normal adult tissues. No significant similarity was found with other fusion partners of TFE3 or with any relevant functional protein domains, precluding informed speculation about the normal function of this gene.

Overexpressed genes/ESTs and characterization of distinct amplicons on 17q23 in breast cancer cells.

17q23 is a frequent site of gene amplification in breast cancer. Several lines of evidence suggest the presence of multiple amplicons on 17q23. To characterize distinct amplicons on 17q23 and localize putative oncogenes, we screened genes and expressed sequence tags (ESTs) in existing physical and radiation hybrid maps for amplification and overexpression in breast cancer cell lines by semiquantitative duplex PCR, semiquantitative duplex RT-PCR, Southern blot, and Northern blot analyses. We identified two distinct amplicons on 17q23, one including TBX2 and another proximal region including RPS6KB1 (PS6K) and MUL. In addition to these previously reported overexpressed genes, we also identified amplification and overexpression of additional uncharacterized genes and ESTs, some of which suggest potential oncogenic activity. In conclusion, we have further defined two distinct regions of gene amplification and overexpression on 17q23 with identification of new potential oncogene candidates. Based on the amplification and overexpression patterns of known and as of yet unrecognized genes on 17q23, it is likely that some of these genes mapping to the discrete amplicons function as oncogenes and contribute to tumor progression in breast cancer cells.

Direct molecular diagnosis of multiple endocrine neoplasia type 1.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant syndrome characterized by the predisposition to develop both peptic ulcer disease and a wide variety of endocrine tumors usually in adolescence and adulthood. Specifically, hyperplasia and/or tumors (most often adenomas) of the parathyroid, pancreatic islet cells, anterior pituitary, and adrenal cortical glands are classically described in affected individuals who have MEN1 (1,2). MEN1 is a highly penetrant disorder whose onset is generally during adult life with the occurrence of at least one, but most often more than one, of the aforementioned tumors. The age-related penetrance of this disorder based on analysis in 63 unrelated kindreds is 7, 52, 87, 98, 99, and 100% by 10, 20, 30, 40, 50, and 60 yr, respectively (3). The disorder is estimated to occur in approx 1 in 30,000 to 1 in 50,000 individuals. Most cases are associated with a positive family history of the disorder, but new germline mutations have been identified in a small percentage of individuals having a negative family history of the disorder but classic features of MEN1 (3-7).

Successful bone marrow transplantation in a patient with Schimke immuno-osseous dysplasia.

Early death in Schimke immuno-osseous dysplasia often results from renal failure and/or cell-mediated immunodeficiency. Kidney transplants have improved renal function, but effective therapy for the immunodeficiency has not yet been reported. We describe markedly improved marrow function 2 years after bone marrow transplantation in a boy with Schimke immunoosseous dysplasia.

Characterisation and genetic mapping of a new X linked deafness syndrome.

BACKGROUND: Hereditary forms of hearing loss are classified as syndromic, when deafness is associated with other clinical features, or non-syndromic, when deafness occurs without other clinical features. Many types of syndromic deafness have been described, some of which have been mapped to specific chromosomal regions. METHODS: Here we describe a family with progressive sensorineural hearing loss, cognitive impairment, facial dysmorphism, and variable other features, transmitted by apparent X linked recessive inheritance. Haplotype analysis of PCR products spanning the X chromosome and direct sequencing of candidate genes were used to begin characterising the molecular basis of features transmitted in this family. Comparison to known syndromes involving deafness, mental retardation, facial dysmorphism, and other clinical features was performed by review of published reports and personal discussions. RESULTS: Genetic mapping places the candidate locus for this syndrome within a 48 cM region on Xq1-21. Candidate genes including COL4A5, DIAPH, and POU3F4 were excluded by clinical and molecular analyses. CONCLUSIONS: The constellation of clinical findings in this family (deafness, cognitive impairment, facial dysmorphism, variable renal and genitourinary abnormalities, and late onset pancytopenia), along with a shared haplotype on Xq1-21, suggests that this represents a new form of syndromic deafness. We discuss our findings in comparison to several other syndromic and non-syndromic deafness loci that have been mapped to the X chromosome.

Expression and mutational analyses of the human MAD2L1 gene in breast cancer cells.

Breast cancer is a heterogeneous disorder in which most tumors display some degree of aneuploidy, especially those at later stages of the disease. Aneuploidy and associated chromosome instability may be important in the progression of mammary tumorigenesis. Aneuploidy is prevented during normal cell division in part through regulation of a mitotic spindle checkpoint where mitotic arrest prevents segregation of misaligned chromosomes into daughter cells at anaphase. Mitotic arrest genes, including the MAD family, which was originally characterized in yeast, help regulate normal function of the mitotic spindle checkpoint. Decreased expression of the human gene MAD2L1 was previously reported in a breast cancer cell line exhibiting chromosome instability and aneuploidy. To explore further the potential role of MAD2L1 in breast cancer, we analyzed MAD2L1 gene expression in 13 minimally to grossly aneuploid human breast cancer cell lines and found significant differences of expression in three lines. Sequence analysis of MAD2L1 cDNA in these as well as nine additional aneuploid breast cancer and five immortalized normal human mammary epithelial cell lines revealed one heterozygous frameshift (572 del A) mutation in a cancer cell line that demonstrated a high level of transcript expression. In addition, two 3'UTR sequence variants were noted in breast cancer cell lines. The 572 del A mutation creates a truncated MAD2 protein product. Further functional studies in primary breast tumors are therefore warranted to determine the potential role MAD2L1 may play in breast cancer.

Isolation and mapping of a human septin gene to a region on chromosome 17q, commonly deleted in sporadic epithelial ovarian tumors.

Allele losses from chromosome 17 are common in sporadic ovarian tumors. Previously, we reported high rates of LOH (up to 70%) from 17q25 at the marker THH59 in a bank of malignant ovarian tumors. We have extended this study to 70 tumors with 17 markers from the long arm of chromosome 17. In most cases, the data are consistent with whole chromosome loss, but we have identified a minimal region of deletion that is centered around 4 microsatellites with zero recombination at map position 106.9 cM. A P1/BAC contig across the region (approximately 200 kb) was constructed and used to determine the precise position and order of the microsatellites. The contig was shown to hybridize to 17q25 by fluorescence in situ hybridization analysis. The DNA sequence of the entire contig was determined and analyzed by BLAST searches. A 4-kb cDNA was subsequently identified with homology to the yeast, Drosophila and mammalian septin family of genes. We have designated this gene Ovarian/Breast (Ov/Br) septin. Two splice variants were demonstrated within the 200-kb contig, which differ only at exon 1. Within the contig, approximately 45% of the septin alpha transcript was identified and 38% of the septin beta transcript. The septins are a family of genes involved in cytokinesis and cell cycle control. Their known functions are consistent with the hypothesis that the human 17q25 septin gene is a candidate for the ovarian tumor suppressor gene.

Mutation and expression analysis of human BUB1 and BUB1B in aneuploid breast cancer cell lines.

Genetic instability is a hallmark feature of breast, colorectal and other types of cancers. One type characterized by chromosomal instability is thought to be important in the pathogenesis of many solid tumors displaying aneuploidy. Two related protein kinases and homologues of the yeast checkpoint genes, hBUB1 and hBUB1B, have been implicated in the pathogenesis of colorectal cancers. Mutations in hBUB1 have demonstrated a dominant negative effect by disrupting the mitotic checkpoint when transfected into euploid colon cancer cell lines. In Brca2 deficient murine cells, Bub1 mutants potentiate growth and cellular transformation. This would suggest that aneuploidy in solid tumors including breast, could be the result of defects in mitotic checkpoint genes and may be responsible for a chromosomal instability phenotype contributing to tumor progression. We conducted mutational analysis of 19 aneuploid breast cancer cell lines. No mutations were found but we identified nine sequence variations including five previously unreported sequence variants in hBUB1B, two of which affect restrictions sites. None of these nucleotide changes predict significant changes in the predicted protein structure. Expression analysis by Northern blot of breast cell lines showed variable expression of hBUB1 and hBUB1B genes. This suggest that while regulation of expression of these genes may be important in cancer, the lack of putative deleterious mutations in the coding sequence does not support a frequent role for mutant hBUB1 and hBUB1B alleles in the pathogenesis of breast cancer.

Genomic and expression analyses of alternatively spliced transcripts of the MLL septin-like fusion gene (MSF) that map to a 17q25 region of loss in breast and ovarian tumors.

We previously defined a common region of 17q25 loss in breast and ovarian tumors, suggesting localization of at least one putative tumor suppressor gene. Genomic clones from the interval were used to isolate candidate transcripts. One novel transcript had strong homology to a septin family of GTPase genes involved in cytokinesis. This gene was recently identified as a myeloid/lymphoid leukemia (MLL) fusion protein partner in acute myeloid leukemia and was named MSF (MLL septin-like fusion). As this gene may play roles in both leukemogenesis and tumorigenesis, it is essential to understand its structure and normal expression. We cloned two human alternative transcripts and identified a third database variant of MSF. RNA expression studies with a probe common to the three novel sequences showed differential expression of 4.0- and 3.0-kb transcripts in all adult and fetal tissues tested. A probe spanning sequence unique to one MSF variant detected specific expression of the 4.0-kb transcript in all tissues. Another probe unique to a different MSF variant detected a 4.0-kb transcript only in skeletal muscle. Proteins of 422 and 586 amino acids were predicted from the novel alternate transcripts and included both a xylose isomerase 1 domain and a GTPase domain. Nine common exons, three alternatively spliced exons, and six polymorphisms were identified.

Support for the availability of prenatal testing for neurological and psychiatric conditions in the psychiatric community.

Faculty and residents of the University of Michigan Department of Psychiatry, members of the Alliance for the Mentally Ill (AMI), and university students were surveyed to elicit attitudes toward the availability of prenatal testing and genetic therapy or enhancement for early- and late-onset psychiatric diseases compared to neurological disorders and human traits. They were asked to complete a written questionnaire designed to assess their opinions as to whether prenatal testing and genetic therapy or enhancement should be applied to 16 selected "disease" phenotypes or human traits: eight early- and late-onset psychiatric conditions, four neurological disorders, and four human traits. Twenty-two percent returned the written survey. The majority of all respondents supported the availability of prenatal testing for well-defined, serious psychiatric or neurological phenotypes and found testing for human traits less desirable. The percentages of respondents supporting availability of testing increased if in utero curative gene therapy was available. Response to the survey differed on the basis of gender and age, as well as personal versus professional familiarity with the condition. The results of this pilot study suggest that a majority of the population, including psychiatrists, will support the public availability of prenatal diagnosis for serious psychiatric or neurological phenotypes, even if no in utero curative therapy is available. Support for testing for human traits was not strongly endorsed.

Absence of PTEN germ-line mutations in men with a potential inherited predisposition to prostate cancer.

Epidemiological studies have demonstrated that men with a family history of prostate cancer are at an increased risk for this disease. This important observation has led a number of research teams, including our own, to collect DNA samples and clinical data from prostate cancer families, with the goal of localizing and characterizing prostate cancer susceptibility genes. The candidate tumor suppressor gene PTEN (also called MMAC1) has recently been shown to be somatically altered in several common malignancies, including cancers of the brain, kidney, skin, thyroid, endometrium, breast, and prostate. Germ-line mutations in this gene, which maps to chromosome 10q23, have been associated with Cowden disease, an autosomal dominant cancer predisposition syndrome that is characterized by multiple hamartomas. Although prostate cancer is not typically associated with Cowden disease, previous studies of sporadic prostate cancers demonstrate loss of heterozygosity at 10q23 loci in approximately 25% of cases. We, therefore, hypothesized that germ-line mutations in the PTEN gene may predispose to prostate cancer in a subset of families, particularly those in which cancers of the breast, kidney, and/or thyroid also segregate. To test this hypothesis, DNA was isolated from whole blood of 11 prostate cancer patients from 10 unrelated families. Four of the 10 families met the previously established clinical criteria for hereditary prostate cancer. Eight of the II men had at least one second primary malignancy, including cases of neuroendocrine cancer, glioblastoma multiforme, melanoma, kidney, and thyroid cancer. Although we identified some common as well as some unique polymorphisms, no nonsense or missense mutations were identified in any of the 11 samples. To further examine the possibility that PTEN mutations contribute to prostate cancer predisposition, we also studied the probands from each of 10 families with early-onset and/or multiple individuals with prostate cancer. Sequence analysis of the PTEN gene in these 10 men also revealed no mutations or novel polymorphisms. We conclude that germ-line mutations in the PTEN are unlikely to contribute in a significant way to the inherited predisposition to prostate cancer.

An integrated physical and gene map of human distal chromosome 17q24-proximal 17q25 encompassing multiple disease loci.

Genetic mapping studies suggest that a small interval on human chromosome distal 17q24-proximal 17q25 harbors genes involved in sporadic breast and ovarian tumorigenesis and in the autosomal dominant disorders hereditary neuralgic amyotrophy and tylosis with esophageal cancer. Prior to this study, isolated genomic clones and markers were assigned to this interval but integrated physical maps were not available. We improved resolution by isolating 52 additional clones and developing 24 additional markers. Genomic clones spanning distal 17q24-proximal 17q25 were organized into a contig with two gaps that encompassed 14 existing genetic markers, 8 known genes (GALR2, AANAT, ENVL, SFRS2, SEC14L, DNAH17, API4, and TK1), and 11 previously identified expressed sequence tags. This integrated map provides a foundation for identifying additional candidate genes for the disorders mapped to this interval.

Distal chromosome 17q loss in Barrett's esophageal and gastric cardia adenocarcinomas: implications for tumorigenesis.

The molecular genetic mechanisms underlying esophageal cancer are poorly understood. However, a novel gene that may be involved in esophageal carcinogenesis was recently localized by others to distal 17q by linkage analysis of kindreds with palmoplantar keratoderma and squamous cell carcinoma of the esophagus. To help determine whether a distal 17q gene may also be involved in the pathogenesis of primary Barrett's esophageal and gastric cardia adenocarcinomas, we performed loss of heterozygosity (LOH) analysis of 21 Barrett's and 18 gastric cardia adenocarcinomas at loci spanning 17q: cen-BRCA1-SSTR2-D17S2058-D17S929-D17S722-+ ++D17S937-D17S802-tel. Over 50% of the Barrett's and cardia adenocarcinomas demonstrated loss of an allele at one or more informative distal 17q markers. One common overlapping region of loss involved loci mapped to distal 17q24-proximal 17q25, which tentatively defines a potential chromosomal region distal to BRCA1 involved in the pathogenesis or progression of both types of adenocarcinomas. LOH analysis of DNA from matched microdissected sections of Barrett's metaplasia suggested that loss of D17S2058 in this region may be an early event in the malignant transformation of Barrett's metaplasia. No statistically significant correlations between 17q LOH and tumor stage or patient survival were noted. In summary, LOH mapping of 17q in Barrett's and cardia adenocarcinomas suggests the existence of at least one putative distal 17q tumor suppressor gene involved in the pathogenesis of these tumors.