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An international group recommends that papers relating phenotypes to genotypes involving mutations in the X chromosome gene MECP2 should provide a minimum data set reporting the range of disturbances frequently encountered in Rett Syndrome. A simple scoring system is suggested which will facilitate comparison among the various clinical profiles. Features are described which should prompt screening for MECP2 mutations.

Imprinted genes within the Prader-Willi/Angelman syndrome region of human chromosome 15q11-q13 are regulated by a mechanism involving allele-specific DNA methylation. Since transcriptional regulation by DNA methylation involves histone deacetylation, we explored whether differences in histone acetylation exist between the two parental alleles of SNRPN and other paternally expressed genes in the region by using a chromatin immunoprecipitation assay with antibodies against acetylated histones H3 and H4. SNRPN exon 1, which is methylated on the silent maternal allele, was associated with acetylated histones on the expressed paternal allele only. SNRPN intron 7, which is methylated on the paternal allele, was not associated with acetylated histones on either allele. The paternally expressed genes NDN, IPW, PWCR1 and MAGEL2 were not associated with acetylated histones on either allele. Treatment of the lymphoblastoid cells with trichostatin A, a histone deacetylase inhibitor, did not result in any changes to SNRPN expression or association of acetylated histones with exon 1. Treatment with 5-aza-deoxycytidine (5-aza-dC), which inhibits DNA methylation, resulted in activation of SNRPN expression from the maternal allele, but was not accompanied by acetylation of histones. Our finding of allele-specific association of acetylated histones with the SNRPN exon 1 region, which encompasses the imprinting center, suggests that histone acetylation at this site may be important for regulation of SNRPN and of other paternally expressed genes in the region. On the silent allele, 5-aza-dC treatment altered SNRPN expression, but not association with acetylated histones, suggesting that histone acetylation is a secondary event in the process of gene reactivation by CpG demethylation.

We present the results of an international collaborative study aimed at estimating the ratio of male to female mutation rates in Duchenne muscular dystrophy based on the method of C. Müller and T. Grimm. With a sample size of 295, this ratio is found to be very close to 1, thus giving evidence for equal mutation rates in males and females in Duchenne muscular dystrophy.

Upon our request, the Society has been admitted to full membership in FASEB (the Federation of American Societies of Experimental Biology), the nation's largest association of biomedical scientists. Made up of 19 societies, with a combined total of 63,000 members, FASEB has a strong voice on matters of legislative and public interest and has campaigned successfully for increased biomedical research funding. We have appointed ASHG representatives to the FASEB Board of Directors and to seven different committees dealing with research funding, public affairs, and science policy.In national debates about guidelines and regulations, many issues have come to the forefront and will continue to be addressed by the Society, its committees, and its members. These include the overhaul of the NIH peer-review system, the establishment of PubMed Central, the derivation and use of human pluripotent stem cells in research, the use of archived human tissues for research, federal oversight of genetic testing, and patenting of human genes and of genetic diagnostic tests. The protection of human subjects from research risks—including the protection of the privacy of genetic information collected as part of family studies—and issues of informed consent procedures for mentally disabled persons have caught public attention. With the formation of the Office of Human Research Protection (OHRP) under the Secretary of HHS—replacing the Office of Protection from Research Risks (OPRR) under the NIH Director—there will be increased scrutiny of informed-consent procedures and of the role of Institutional Review Boards. These developments are likely to have considerable impact on researchers in human genetics.Under John Carey, an Ad Hoc Committee on Consumer Issues has been formed of researchers and leaders of advocacy groups. With the support of ASHG, a workshop was held to address consumer issues surrounding participation in clinical genetic research. The combined voice of researchers and consumer advocates will be needed to ensure that the progress of research will not be impeded by—and will be conducted within the framework of—existing federal regulations. The ASHG Social Issues Committee, under Mark Rothstein, has developed a statement on Genetic Testing in Adoption that will soon be released. As Social Issue Committees of other genetics organizations (ACMG, NSGC, and ISONG) are dealing with similar topics, the Board strongly encouraged joint sessions and activities of the ASHG committee with these committees.To improve communication between the ASHG officers, the membership, and the public, a Communications Committee has been established by the Board to redesign our Web site, making it more visible, informative, and interactive. This effort should enable ASHG members to be informed of critical issues in a timely fashion and to act in influencing public policy. Active participation of the ASHG membership will be essential for some of these complex issues.With increasing globalization, the communication between human genetics societies around the world is facilitated by the formation of the International Federation of Human Genetics Societies (IFHGS). The Federation currently has three full members—the regional societies ASHG, ESHG (European Society of Human Genetics), and HGSA (Human Genetics Society of Australasia)—and over 30 national societies as corresponding members. As I am beginning a three-year term as President of the IFHGS, I look forward to expanding the activities of this global organization in the information age. The new IFHGS web site is http://www.faseb.org/genetics/ifhgs/index.html. Elaine Strass, the ASHG Executive Director, deserves recognition for her expert management of the Society's office in Bethesda and special gratitude for serving as IFHGS Executive Secretary as well.

Rett syndrome (RTT, MIM 312750) is a progressive neurodevelopmental disorder and one of the most common causes of mental retardation in females, with an incidence of 1 in 10,000-15,000 (ref. 2). Patients with classic RTT appear to develop normally until 6-18 months of age, then gradually lose speech and purposeful hand use, and develop microcephaly, seizures, autism, ataxia, intermittent hyperventilation and stereotypic hand movements. After initial regression, the condition stabilizes and patients usually survive into adulthood. As RTT occurs almost exclusively in females, it has been proposed that RTT is caused by an X-linked dominant mutation with lethality in hemizygous males. Previous exclusion mapping studies using RTT families mapped the locus to Xq28 (refs 6,9,10,11). Using a systematic gene screening approach, we have identified mutations in the gene (MECP2 ) encoding X-linked methyl-CpG-binding protein 2 (MeCP2) as the cause of some cases of RTT. MeCP2 selectively binds CpG dinucleotides in the mammalian genome and mediates transcriptional repression through interaction with histone deacetylase and the corepressor SIN3A (refs 12,13). In 5 of 21 sporadic patients, we found 3 de novo missense mutations in the region encoding the highly conserved methyl-binding domain (MBD) as well as a de novo frameshift and a de novo nonsense mutation, both of which disrupt the transcription repression domain (TRD). In two affected half-sisters of a RTT family, we found segregation of an additional missense mutation not detected in their obligate carrier mother. This suggests that the mother is a germline mosaic for this mutation. Our study reports the first disease-causing mutations in RTT and points to abnormal epigenetic regulation as the mechanism underlying the pathogenesis of RTT.

SummaryFibrillin-1 (FBN1) contains 47 epidermal growth factor (EGF)–like domains characterized by six conserved cysteine residues. Cysteine substitutions that disrupt one of the three disulfide bonds are frequent causes of Marfan syndrome (MFS). We identified 19 new substitutions involving cysteine residues in each of the six positions of EGF-like domains. Allele-specific mRNA assays revealed equal abundance of mutant and normal FBN1 transcripts in all 10 individuals studied. Quantitative pulse-chase analysis of fibrillin protein was performed on 25 mutant fibroblast strains with substitutions of 22 different cysteine residues in 18 different EGF-like domains spanning the entire gene. Normal synthesis and stability of mutant fibrillin molecules was seen in 20/25 individuals, 11 of whom showed delayed intracellular processing and/or secretion. In the remaining five cases, the mutant protein was apparently unstable. In four of these five cases, the second or third disulfide bond of EGF-like domains immediately preceding an 8-cysteine or hybrid domain was affected. All but two mutations caused severe reduction of matrix deposition, which was attributed to a dominant-negative effect of mutant molecules. For genotype/phenotype comparisons, clinical data on 25 probands and 19 mutation-positive family members were analyzed. Ocular manifestations were among the most consistent features (ectopia lentis in 86%, myopia in 80%). Nine mutations encoded by exons 26–32 resulted in early-onset classic MFS and, in one case, neonatal-lethal MFS. Mutations outside this region were associated with variable clinical phenotypes, including individuals with fibrillinopathies not meeting diagnostic criteria for MFS.

Williams-Beuren syndrome (WBS; OMIM 194050) is caused by heterozygous deletions of approximately 1.6 Mb of chromosomal sub-band 7q11.23. The deletions are rather uniform in size as they arise spontaneously by inter- or intrachromosomal crossover events within misaligned duplicated regions of high sequence identity that flank the typical deletion. This review will discuss the status of the molecular characterization of the deletion and flanking regions, the genes identified in the deletion region and their possible roles in generating the complex multi-system clinical phenotype.

Neuregulin-2 (NRG2) is a novel member of the neuregulin family of growth and differentiation factors. Through interaction with the ErbB family of receptors, neuregulin-2 induces the growth and differentiation of epithelial, neuronal, glial and other types of cells. In this study, we have cloned the human neuregulin-2 gene, and determined its genomic structure and alternative splicing patterns. By using radiation hybrid mapping panels, the human NRG2 gene was mapped to the D5S658–D5S402 region within 5q23–q33, close to an autosomal recessive form of demyelinating Charcot-Marie-Tooth (CMT) disease. The NRG2 gene was found to be on two yeast artificial chromosomes overlapping the candidate interval and was, thus, considered a good positional candidate for this form of CMT. When the entire neuregulin-2 coding sequence and splice junctions were explored, however, no mutation was identified in one CMT family linked to 5q23–q33. In addition, three intronic single nucleotide polymorphisms were identified in the NRG2 gene. Genotyping in two families localized the NRG2 gene outside of the revised candidate interval between D5S402–D5S210 and excluded NRG2 as the gene responsible for this form of CMT disease.

The frizzled gene family is conserved from insects to mammals and codes for putative Wnt receptors that share a cysteine-rich extracellular domain and seven transmembrane domains. We previously identified a novel frizzled gene, FZD3, now renamed FZD9, in the Williams-Beuren syndrome (WBS) deletion region at chromosomal band 7q11.23 and showed that its product can interact with the Drosophila wingless protein. Here, we report the characterization of the mouse homolog Fzd9. The Fzd9 gene produces a 2.4-kb transcript encoding a 592-amino-acid protein with 95% identity to the human FZD9. Fzd9 was mapped to the conserved syntenic region on distal mouse chromosome 5. By RNA in situ hybridization studies of whole-mount embryos and sections we delineated the temporal and spatial expression patterns in the neural tube, trunk skeletal muscle precursors (myotomes), limb skeletal anlagen, craniofacial regions, and nephric ducts. In adult mouse tissue, the Fzd9 transcript is abundantly present in heart, brain, testis, and skeletal muscle. In testis, Fzd9 is expressed in all spermatogenic cell types. Immunohistochemical studies of cells transfected with a Fzd9 expression construct confirm that Fzd9 is a membrane protein. These results suggest potential Wnt ligands of Fzd9, a role of Fzd9 in skeletal muscle specification, and contributions of FZD9 to the WBS phenotype.

The K homology-type splicing regulatory protein, KSRP, activates splicing through intronic splicing enhancer sequences. It is highly expressed in neural cells and is required for the neural-specific splicing of the c-src N1 exon. In this study, we mapped the gene (gene symbols KHSRP and Khsrp) to human chromosome 19 by using radiation hybrid panels and to mouse chromosome 17 by studying an interspecific backcross panel. Human KHSRP is a positional candidate gene for familial febrile convulsion and Cayman type cerebellar ataxia. Comparative analysis of the human and mouse genomes indicates that the KHSRP gene is located in regions of conserved synteny between the two species.

We have identified a novel gene (WBSCR9) within the common Williams-Beuren syndrome (WBS) deletion by interspecies sequence conservation. The WBSCR9 gene encodes a roughly 7-kb transcript with an open reading frame of 1483 amino acids and a predicted protein product size of 170.8 kDa. WBSCR9 is comprised of at least 20 exons extending over 60 kb. The transcript is expressed ubiquitously throughout development and is subject to alternative splicing. Functional motifs identified by sequence homology searches include a bromodomain; a PHD, or C4HC3, finger; several putative nuclear localization signals; four nuclear receptor binding motifs; a polyglutamate stretch and two PEST sequences. Bromodomains, PHD motifs and nuclear receptor binding motifs are cardinal features of proteins that are involved in chromatin remodeling and modulation of transcription. Haploinsufficiency for WBSCR9 gene products may contribute to the complex phenotype of WBS by interacting with tissue-specific regulatory factors during development.

The SWI/SNF-related, matrix-associated, actin-dependent regulators of chromatin (SMARC), also called BRG1-associated factors, are components of human SWI/SNF-like chromatin-remodeling protein complexes. We mapped five human SMARC genes toregions on four different human chromosomes, SMARCC1 to 3p23-p21, SMARCC2 to 12q13-q14, SMARCD1 to 12q13-q14, SMARCD2 to 17q23-q24, and SMARCD3 to 7q35-q36. SMARCC1, SMARCC2, and SMARCD1 are assigned to chromosomal regions that are frequently involved in somatic rearrangements in human cancers. SMARCD1 was mapped to the critical region of Allgrove syndrome; however, no mutation was identified in one Allgrove syndrome family studied.

Paralemmin is a newly identified protein that is associated with the plasma membrane and with intracellular membranes through a lipid anchor. It is abundant in brain, is expressed at intermediate levels in the kidney and in endocrine cells, and occurs at low levels in many other tissues. As it is a candidate for genetic disorders that affect membrane functions, we have determined the structure of the human paralemmin gene, PALM, showing that it is organized into nine exons. Moreover, we have performed chromosomal assignments of the human and mouse paralemmin genes, localizing them to regions of homology at human 19p13.3 and the central mouse chromosome 10. Finally, mutation analysis using RNA from mice homozygous for the mutant genes grizzled (gr), mocha (mh), mocha 2J (mh2J), jittery (ji) and hesitant (ji(hes)), which map to this area, excluded mutations in their Palm coding sequences.

The molecular cloning and developmental expression of mouse LTBP-2 are presented here. We established the identity of the cDNA by sequence comparison (80% identity with human LTBP-2) and by chromosome localization (mouse chromosome 12, band D, a region of conserved synteny with the human LTBP-2 gene). In contrast to LTBP-1 and LTBP-3, mouse LTBP-2 apparently is a more modular protein, with proline/glycine-rich sequences always alternating with clusters of cysteine-rich structural motifs. We found for the first time that LTBP-2 gene expression in mouse embryos was restricted to cartilage perichondrium and blood vessels, a somewhat surprising result since other LTBP genes are widely expressed in rodent tissues. Therefore, mouse LTBP-2 may play a critical role in the assembly of latent TGF-β complexes in developing elastic tissues such as cartilage and blood vessel.

The Marfan database is a software that contains routines for the analysis of mutations identified in the FBN1 gene that encodes fibrillin-1. Mutations in this gene are associated not only with Marfan syndrome but also with a spectrum of overlapping disorders. The third version of the Marfan database contains 137 entries. The software has been modified to accommodate four new routines and is now accessible on the World Wide Web at http://www.umd.necker.fr.

In individuals with the Marfan syndrome (MFS), mutations have been identified in the fibrillin-1 gene (FBN1) at 15q21.1. A proline-to-alanine change at position 1148 in exon 27 (Pro1148Ala) has been reported in probands with MFS, aortic aneurysm or Marfanoid-craniosynostosis. It was suggested that this mutation could be a risk factor for aortic dilatation, since it was rarely observed in control populations. To investigate further the pathogenicity of this substitution, we screened 416 unrelated control individuals by allele-specific oligonucleotide (ASO) hybridization. We found 16 individuals who carried the alanine allele (3.8%), 3 of whom were homozygous. Five were of Latin American and eight were of Asian extraction. We also screened 133 probands with MFS, aortic aneurysm or related connective tissue disorders and found 4 (3%) that were heterozygous for the 1148Ala allele. All positive results were confirmed by DNA sequencing. In 20 individuals with 1148Ala, we confirmed the association with the rarer A allele at the IVS27-5G-->A polymorphism. Our results suggest that the Pro1148Ala change is a polymorphism of ancient evolutionary origin that is more prevalent in Asian and Latin American than in Caucasian or African populations.

Williams syndrome (WS) is a developmental disorder with a characteristic personality and cognitive profile that is associated, in most cases, with a 2 Mb deletion of part of chromosome band 7q11.23. By applying CpG island cloning methods to cosmids from the deletion region, we have identified a new gene, called FZD3. Dosage blotting of DNA from 11 WS probands confirmed that it is located within the commonly deleted region. Sequence comparisons revealed that FZD3, encoding a 591 amino acid protein, is a novel member of a seven transmembrane domain receptor family that are mammalian homologs of the Drosophila tissue polarity gene frizzled. FZD3 is expressed predominantly in brain, testis, eye, skeletal muscle and kidney. Recently, frizzled has been identified as the receptor for the wingless (wg) protein in Drosophila. We show that Drosophila as well as human cells, when transfected with FZD3 expression constructs, bind Wg protein. In mouse, the wg homologous Wnt1 gene is involved in early development of a large domain of the central nervous system encompassing much of the midbrain and rostral metencephalon. The potential function of FZD3 in transmitting a Wnt protein signal in the human brain and other tissues suggests that heterozygous deletion of the FZD3 gene could contribute to the WS phenotype.

Homeobox genes play important roles in limb development. Backfoot is a recently identified mammalian homeobox gene whose temporal and spatial expression pattern during limb development suggests that it is a key component for specifying the identify and structure of the hindlimb. Here we report the chromosomal mapping of the Backfoot locus in human (BFT) and mouse (Bft). Using single-strand conformation analysis of PCR products amplified from a panel of somatic cell hybrid lines and two radiation hybrid (RH) panels, we have physically mapped BFT to human chromosome 5, closely linked to STS markers D5S2543, D5S458, D5S1947, and D5S1995 on the Stanford G3 RH map and to AFMA057VG5 and AFM350YB1 on the Gene-Bridge 4 RH map. Linkage analysis of a mouse inter-specific backcross panel (C57BL/6J x Mus musculus spretus) has localized Bft to the central part of mouse chromosome 13. The map position of Bft is near two mouse limb mutant loci defined as dumpy and mdac.

Here we describe two mammalian transcription factors selectively expressed in the central nervous system. Both proteins, neuronal PAS domain protein (NPAS) 1 and NPAS2, are members of the basic helix–loop–helix-PAS family of transcription factors. cDNAs encoding mouse and human forms of NPAS1 and NPAS2 have been isolated and sequenced. RNA blotting assays demonstrated the selective presence of NPAS1 and NPAS2 mRNAs in brain and spinal cord tissues of adult mice. NPAS1 mRNA was first detected at embryonic day 15 of mouse development, shortly after early organogenesis of the brain. NPAS2 mRNA was first detected during early postnatal development of the mouse brain. In situ hybridization assays using brain tissue of postnatal mice revealed an exclusively neuronal pattern of expression for NPAS1 and NPAS2 mRNAs. The human NPAS1 gene was mapped to chromosome 19q13.2–q13.3, and the mouse Npas1 gene to chromosome 7 at 2 centimorgans. Similarly, the human NPAS2 gene was assigned to chromosome 2p11.2–2q13, and the mouse Npas2 gene to chromosome 1 at 21–22 centimorgans. The chromosomal regions to which human NPAS1 and NPAS2 map are syntenic with those containing the mouse Npas1 and Npas2 genes, indicating that the mouse and human genes are true homologs.

A significant proportion of cases of GH deficiency (5-30%) may be due to genetic causes. At least four Mendelian types of isolated GH deficiency (IGHD) have been delineated based on the mode of inheritance and the degree of GH deficiency, with IGHD type IA being the most severe. A 2 year-old girl, the second child of consanguineous parents, with short stature was diagnosed with IGHD type IA. The analysis of the genomic DNA of this patient, performed by polymerase chain reaction (PCR) amplification of the flanking regions of the GH-1 gene, showed a homozygous deletion of 7.0 kb of sequence including the GH-1 gene. She was treated with biosynthetic GH resulting in long-lasting catch-up growth during at least three years, despite a clinically irrelevant appearance of low binding capacity GH antibodies. Growth hormone-binding protein (GHBP) levels were normal at the time of diagnosis. In addition, GHBP plasma levels did not show any significant change during the three years of therapy with GH. Diagnosis of carrier status in family relatives was done by genotyping GH gene alleles by PCR amplification from blood spots on filter paper.

CAP18 is an antimicrobial protein found in specific granules of PMNs. The human CAP18 (HCAP18) gene was cloned from a human genomic phage library. Sequence analysis revealed the HCAP18 gene to have 4 exons spanning 3 kb, including 700 bp of upstream DNA. Using 3′ RACE no homologs of human HCAP18 were found in human bone marrow or leukocyte populations. By PCR analysis of a somatic cell mapping panel and fluorescence in situ hybridization of a genomic clone to metaphase chromosomes the gene was mapped to chromosome band 3p21.3. Like several other genes expressed late in PMN development the CAP18 gene did not contain typical TATA box or CCAAT sequences. Expression in Cos 7 cells permitted limited mapping of the promoter function in upstream fragments of the HCAP18 gene. Western blot, Northern blot and RT-PCR analysis show HCAP18 to be produced specifically in granulocytes. This work forms the groundwork for future analysis of the genetic regulation of this antimicrobial protein during PMN differentiation.

Marfan syndrome (MFS), a heritable connective tissue disorder, is caused by mutations in the gene coding for fibrillin-1 (FBN1), an extracellular matrix protein. One of the three major categories of FBN1 mutations involves exon-skipping. To rapidly detect such mutations, we developed a long RT-PCR method. Either three segments covering the entire FBN1 coding sequence or a single 8.9 kb FBN1 coding segment were amplified from reverse-transcribed total fibroblast RNA. Restriction fragment patterns of these RT-PCR products were compared and abnormal fragments were directly sequenced. Six exon-skipping mutations were identified in a panel of 60 MFS probands. All skipped exons encode calcium binding epidermal growth factor (EGF)-like domains and maintain the reading frame. In five probands, exon-skipping was due to point mutations in splice site sequences, and one had a 6 bp deletion in a donor splice site. Pulse-chase analysis of labelled fibrillin protein revealed normal levels of synthesis but significantly reduced matrix deposition. This dominant-negative effect of the mutant monomers is considered in the light of current models of fibrillin assembly. Probands with this type of FBN1 mutation include the most severe forms of MFS, such as neonatally lethal presentations.

A high-resolution radiation hybrid map of human chromosome 18 has been developed by testing DNA samples of 92 radiation hybrids (RH) from a previously characterized chromosome 18-specific RH panel. Half of the 159 STS markers were tested on RH DNA amplified by primer extension preamplification. This map includes 20 genes and 95 polymorphic markers, most of which have heterozygosity frequencies greater than 65% and, therefore, allow integration with genetic maps. The framework map consists of 137 markers ordered with odds of 1000:1 or better and spaced on average 580 kb apart. A YAC contig map of 18p was generated independently by STS content mapping of YACs using the same primers as for RH mapping. The RH map and the contig map are concordant.

UDP-galactose:ceramide galactosyltransferase (CGT) (EC 2.4.1.62) catalyzes the final step in the synthesis of galactocerebroside, a glycosphingolipid characteristically abundant in myelin. In this report, we describe the isolation of genomic clones spanning the mouse CGT gene. The mouse CGT gene consists of six exons that span a minimum of 70 kb of DNA and that encode a 541 amino acid translation product with extensive sequence similarity to the rat CGT enzyme and to UDP-glucuronosyltransferases (UGT). The 5′-untranslated region of the mouse CGT gene is encoded by a separate exon located approximately 25 kb upstream of the first protein-encoding exon. Furthermore, the genomic organization of the five coding region exons of the mouse CGT gene resembles that of the human UGT1 and rat UGT2B1 genes. Finally, analysis of somatic cell hybrids by PCR and fluorescencein situhybridization to metaphase chromosomes has localized the mouse CGT gene to chromosome 3, bands E3–F1.

Comparative studies of genes in the pseudoautosomal region (PAR) of human and mouse sex chromosomes have thus far been very limited. The only comparisons that can presently be made indicate that the PARs of humans and mice are not identical in terms of gene content. Here we describe additional comparative studies of human pseudoautosomal genes and their mouse homologs. Using a somatic cell hybrid mapping panel, we have assigned the mouse homolog of the human pseudoautosomal inter-leukin 3 receptor alpha subunit (IL3RA) gene to mouse Chromosome (Chr) 14. Attempts to clone the mouse homolog of the human pseudoautosomal adenine nucleotide translocase-3 (ANT3) gene resulted in the isolation of the murine homologs of the human ANT1 and ANT2 genes. The mouse Ant1 and Ant2 genes are very similar in sequence to their human homologs, and we have mapped them to mouse Chromosomes (Chrs) (8 and X respectively) that exhibit conserved synteny with the chromosomes on which the human genes are located. In contrast, the homolog of ANT3 appears to be either very divergent or absent from the mouse genome. Southern blot analysis of DNA from a variety of mammalian species shows restricted conservation of human pseudoautosomal genes, a trend that also applies to the two cloned mouse homologs of these genes and to neighboring human genes in distal Xp22.3. Our observations combined with those of other workers lead us to propose a model for the evolution of the PAR that includes both rapid sequence evolution and the incremental reduction in size of the region during mammalian evolution.

Marfan syndrome (MFS), a multisystem autosomal-dominant disorder, is characterized by mutations of the fibrillin-1 (FBN1) gene and by abnormal patterns of synthesis, secretion, and matrix deposition of the fibrillin protein. To determine the sensitivity and specificity of fibrillin protein abnormalities in the diagnosis of MFS, we studied dermal fibroblasts from 57 patients with classical MFS, 15 with equivocal MFS, 8 with single-organ manifestations, and 16 with other connective tissue disorders including homocystinuria and Ehlers-Danlos syndrome. Abnormal fibrillin metabolism was identified in 70 samples that were classified into four different groups based on quantitation of fibrillin synthesis and matrix deposition. Significant correlations were found for phenotypic features including arachnodactyly, striae distensae, cardiovascular manifestations, and fibrillin groups II and IV, which included 70% of the MFS patients. In addition, these two groups were associated with shortened "event-free" survival and more severe cardiovascular complications than groups I and III. The latter included most of the equivocal MFS/single manifestation patients with fibrillin abnormalities. Our results indicate that fibrillin defects at the protein level per se are not specific for MFS, but that the drastically reduced fibrillin deposition, caused by a dominant-negative effect of abnormal fibrillin molecules in individuals defined as groups II and IV, is of prognostic and possibly diagnostic significance.

Signals transduced by the T cell antigen receptor (TCR) regulate developmental transitions in the thymus and also mediate the immunologic activation of mature, peripheral T cells. In both cases TCR stimulation leads to the assembly of the NFAT transcription complex as a result of the calcium-dependent nuclear translocation of cytosolic subunits, NFATc, and the Ras/protein kinase C-dependent induction of a nuclear subunit, NFATn. To further understand the diverse roles of antigen receptor signaling throughout T cell development, we have identified a new NFATc family member, NFATc3, that is expressed at highest levels in the thymus. NFATc3 is the product of a gene on murine chromosome 8 that is not linked to the other NFATc genes. NFATc3, like other NFATc family members, contains a conserved rel similarity domain, and also defines a region conserved among NFATc family members, the SP repeat region, characterized by the repeated motif SPxxSPxxSPrxsxx (D/E)(D/E)swl. NFATc3 activates NFAT site-dependent transcription when overexpressed, yet exhibits a pattern of DNA site specificity distinct from other NFATc proteins. Additionally, thymic NFATc3 undergoes modifications in response to agents that mimic T cell receptor signaling, including a decrease in apparent molecular mass upon elevation of intracellular calcium that is inhibited by the immunosuppressant FK506. Given the preferential expression of NFATc3 in the thymus, NFATc family members may regulate distinct subsets of genes during T cell development.

Primary GH insensitivity (Laron syndrome) due to GH receptor deficiency (GHRD) is an autosomal recessive condition characterized by severe growth failure. Diverse alterations in the GHR gene have been reported in affected individuals. We report here the first family with GHRD from Russia, with two affected siblings and consanguineous parents. Analysis of blood spot DNA by polymerase chain reaction (PCR), denaturing gradiant gel electrophoresis, and nucleotide sequencing indicated that these siblings are homozygous for a nonsense mutation, R43X, in the GHR gene. The R43X mutation, which changes an arginine codon to a translational stop codon, occurs at a CpG dinucleotide mutational hotspot and has previously been identified in affected individuals of Mediterranean and Ecuadorian origin.

Two peroxisomal targeting signals, PTS1 and PTS2, are involved in the import of proteins into the peroxisome matrix. Human patients with fatal generalized peroxisomal deficiency disorders fall into at least nine genetic complementation groups. Cells from many of these patients are deficient in the import of PTS1-containing proteins, but the causes of the protein-import defect in these patients are unknown. We have cloned and sequenced the human cDNA homologue (PTS1R) of the Pichia pastoris PAS8 gene, the PTS1 receptor (McCollum, D., E. Monosov, and S. Subramani. 1993. J. Cell Biol. 121:761-774). The PTS1R mRNA is expressed in all human tissues examined. Antibodies to the human PTS1R recognize this protein in human, monkey, rat, and hamster cells. The protein is localized mainly in the cytosol but is also found to be associated with peroxisomes. Part of the peroxisomal PTS1R protein is tightly bound to the peroxisomal membrane. Antibodies to PTS1R inhibit peroxisomal protein-import of PTS1-containing proteins in a permeabilized CHO cell system. In vitro-translated PTS1R protein specifically binds a serine-lysine-leucine-peptide. A PAS8-PTS1R fusion protein complements the P. pastoris pas8 mutant. The PTS1R cDNA also complements the PTS1 protein-import defect in skin fibroblasts from patients--belonging to complementation group two--diagnosed as having neonatal adrenoleukodystrophy or Zellweger syndrome. The PTS1R gene has been localized to a chromosomal location where no other peroxisomal disorder genes are known to map. Our findings represent the only case in which the molecular basis of the protein-import deficiency in human peroxisomal disorders is understood.

Marfan syndrome (MFS) is an autosomal dominant connective-tissue disorder. It involves predominantly three systems (skeletal, ocular, and cardiovascular systems) and is characterized by highly variable expressivity. The diagnosis relies solely on clinical criteria requiring anomalies in at least two systems. By excluding the disease locus fibrillin 1 (FBN1) in a large French family with typical cardiovascular and skeletal anomalies, we raised the issue of genetic heterogeneity in MFS and the implication of a second locus (MFS2). Linkage analyses, performed in this family with dispersed anonymous DNA markers, have localized MFS2 to a region of less than 9 cM between D3S1293 and D3S1283 which map at 3p24.2-p25. In this region the highest lod score was obtained for marker D3S2336 at 4.89 (θ=0.05). LINKMAP analyses indicated that the most probable position for MFS2 was at D3S2335 (multipoint lod score in log base 10 = 5.65).

Microfibril-associated glycoprotein, MAGP (gene symbol MFAP2), is a component of connective tissue microfibrils and a candidate for involvement in the etiology of inherited connective tissue diseases. We have cloned a human MAGP cDNA that is highly homologous to the previously characterized bovine and murine genes. Like the bovine and murine loci, the human gene has eight coding exons, but it contains two alternatively used 5' untranslated exons, whereas only one untranslated exon was described in the bovine and murine Magp genes. By using rodent x human somatic cell hybrid panels and fluorescence chromosomal in situ hybridization, we have assigned the locus to human chromosome 1p36.1-p35. An insertion/deletion polymorphism has been identified within intron 7. Linkage analysis between this polymorphism and markers on distal chromosome 1 revealed that MAGP is tightly linked to the anonymous marker D1S170. Physical mapping revealed a distance of100 kb between the two markers. This information can be used to screen for linkage in families with microfibrillar abnormalities that are not linked to the fibrillin genes on chromosomes 15 or 5.

The human calcitonin receptor (CTR) is a transmembrane peptide with dual action as a receptor for the hormone calcitonin and as an extracellular calcium sensor. Therefore, CTR dysfunction could lead to disorders of calcium metabolism associated with hypercalcemia, such as the Williams syndrome (WS). WS is a developmental disorder caused by a deletion at chromosome 7q11.23 that includes the elastin locus (ELN). We have mapped the CTR gene (CALCR) to chromosome band 7q21.3 by polymerase chain reaction and singlestrand conformation analysis of somatic cell hybrids as well as fluorescence in situ hybridization (FISH) to metaphase chromosome spreads. Two-color FISH cohybridizing CTR and ELN probes confirmed that CALCR maps telomeric to ELN. Subsequent analysis of chromosome spreads from four WS patients revealed deletion of the ELN locus in all of them and normal hybridization of CTR probes to both chromosome 7 homologues, indicating that CALCR lies outside the deleted region.

A novel gene, isolated because of structural similarities to fibrillin, was called LTBP2 when its 4.6-kb transcript was found to encode a protein sequence related to the latent TGF-beta binding protein (LTBP1), which is encoded on human chromosome (Chr) 2, region p12-q22. We have assigned the human and murine LTBP2 loci to regions of conserved synteny on human Chr 11 and mouse Chr 19. By PCR analysis of somatic cell hybrid DNA and fluorescence in situ hybridization, LTBP2 was mapped to human Chr band 11q12 and Ltbp2 to mouse Chr band 19B. Differences between inbred strains were discovered by single-strand conformation analysis of PCR products from the 3' untranslated region. Analysis of BXD and AKXL recombinant inbred strains have placed Ltbp2 between D19Rp19 and Ly10 on proximal mouse Chr 19.

Cytogenetic and molecular studies in neuroblastoma suggest the presence of a tumour suppressor gene at the distal chromosome band 1p36. Previously, we hypothesised that a constitutional translocation involving the region 1p36 [t(1;17)(p36;q12-q21)] in a patient with neuroblastoma predisposed him to tumour development. Here we report the molecular delineation of the translocation breakpoints. Somatic cell hybrids containing the derivative chromosomes were used to determine the position of chromosome 1p and 17q DNA probes respective to the breakpoints using fluorescence in situ hybridisation. The 1p breakpoint was localised between the PND and D1S56 loci. The chromosome 17q breakpoint is flanked by NF1 and SCYA7, as proximal and distal marker, respectively. We redefined the translocation as t(1;17)(p36.31-13;q11.2-q12). The identification of flanking markers of the breakpoints is a prerequisite for breakpoint cloning and identification of a putative neuroblastoma suppressor gene.

The ring chromosome 15 syndrome is characterized by mild-to-severe growth failure. We evaluated the status of the insulin-like growth factor I receptor (IGF1R) gene, which had previously been assigned to band 15q26 in several patients with de novo ring 15 chromosomes, to investigate a possible correlation between disruption or loss of the IGF1R gene with the severe growth failure phenotype. The presence or absence of the IGF1R gene on the ring 15 chromosomes of five patients was ascertained by in situ hybridization and gene-dosage (Southern) blotting. The location of the breakpoints was determined by typing polymorphic markers from the distal end of the long arm of chromosome 15 in both the probands and their parents. Deletion mapping determined that all breakpoints were distal to D15S100 and that the IGF1R gene is located between D15S107 and D15S87. Three patients who had suffered severe growth failure in early childhood were hemizygous at the IGF1R locus, while one patient with borderline short stature had two copies of the IGF1R gene. The correlation between IGF1R gene dosage and growth retardation demonstrated here in our ring chromosome 15 patients suggests a possible role for heterozygous IGF1R gene mutations or deletions in other cases of unexplained growth failure.

Abstract 1737 Background: The somatic JAK2 V617F (V617F) mutation is found in approximately 95% of polycythemia vera (PV) and 50–60% of essential thrombocythemia (ET) and primary myelofibrosis (PMF) patients. Detection of this mutation occurs primarily through genomic analysis of peripheral blood samples of myeloproliferative neoplasm (MPN) patients. This study demonstrates a novel broad screening mechanism for detection of V617F from myeloid cells in saliva samples from an online cohort of MPN patients and from a broader database of genotyped individuals. Methods: MPN patients were recruited primarily through social media and patient-mediated outreach efforts. Patients gave IRB-approved consent and completed surveys through an online 23andMe account. Participation in the MPN research initiative was free. In collaboration with an uncompensated panel of academic experts, online surveys were developed to collect patient-reported data on diagnosis and results of genetic testing for the V617F mutation. Participants were genotyped on SNP arrays based on the Illumina Omni Express, with additional custom content, including four probes for the V617F mutation. We estimated V617F mutation burden as the median across the four probes of B/(A+B) where A and B were normalized intensities for the reference and mutated alleles, respectively. We estimated sensitivity and specificity of our estimated V617F allele ratio, using the self-reported V617F-positive individuals to approximate the distribution of true positives, and 23andMe members from the broad database (outside of the MPN research cohort) and younger than 40 as likely true negatives. Results: Web-based recruitment efforts led to the accrual of over 900 MPN patients of different subtypes over 12 months. Of these participants, 745 patients have been genotyped and provided MPN-related health history and outcomes through online surveys. The cohort is disproportionately female (71%), with an average age of 53.1 ± 14.9 years. More than 57% of patients indicated a classic MPN diagnosis (PV, ET, PMF, or post PV/ET MF), 19.2% reported an atypical MPN diagnosis (systemic mastocytosis or hypereosinophilic syndrome), 14.5% indicated chronic myelogenous leukemia, and 9.3% of the cohort reported other diagnoses. From this MPN cohort, a total of 345 participants reported mutation test results. Consistent with expectations, the V617F variant was reported by patients in nearly all PV cases (105 of 110 patients with a self-reported diagnosis of PV), and at a lower rate in ET and MF cases (59 of 95 patients and 48 of 61 patients, respectively). In addition to self-reported data, we were specifically able to detect the V617F mutation using SNP array data. We determined a detection threshold for the V617F variant at which 86% of self-reported V617F carriers were scored as positive (i.e. test sensitivity), and 99.975% of young 23andMe customers were scored as negative (i.e. test specificity). Participants with a classic diagnosis in the MPN cohort who carry the V617F variant tended to be older (mean age=59.5 versus 54.6 years) and were less likely to be female (62.5% versus 72.0%) than non-carriers. At this threshold, we detected 133 individuals who likely carry the V617F mutation among a broader set of 23andMe participants (n= 99,700) who were not specifically recruited for the MPN study, corresponding to a prevalence of 0.14%. We estimated the positive predictive value of the test in this cohort, or the expected proportion of positive test results that are true, as 81%. Within the general 23andMe cohort, the frequency of the V617F variant increases with participant age, from a rate of 0.017% for age 20 to 30, to 1.1% for age 80 to 90. We also observed that patients with a high allele burden of V617F frequently had acquired uniparental disomy of chromosome 9p, as determined from calculated heterozygosity scores and no-call rates for this chromosomal arm. Conclusion: We can detect V617F with high specificity and sensitivity from myeloid cells in saliva. This non-invasive source for DNA extraction offers new possibilities for detecting genomic alterations and for finding novel genetic associations in MPN patients as well as the general population. This study demonstrates the feasibility of online technology to significantly accelerate recruitment and research progress in MPNs and other rare diseases. Disclosures: Barnholt: 23andMe, Inc.: Employment. Hinds:23andMe: Employment, Equity Ownership, Patents & Royalties. Kiefer:23andMe, Inc.: Employment. Do:23andMe, Inc.: Employment, Equity Ownership, Patents & Royalties. Eriksson:23andMe, Inc.: Employment, Equity Ownership, Patents & Royalties. Mountain:23andMe, Inc.: Consultancy, Employment, Honoraria, Patents & Royalties. Francke:23andMe, Inc.: Employment, Honoraria, Research Funding; Locus Development: Consultancy, Membership on an entity's Board of Directors or advisory committees. Tung:23andMe, Inc.: Employment. Mesa:23andMe, Inc.: Unpaid advisor and collaborator Other. Gotlib:23andMe, Inc.: Unpaid advisor and collaborator Other. Zehnder:23andMe, Inc.: Unpaid advisor and collaborator Other.

Abstract 1732 Background: In addition to chronic myelogenous leukemia (CML), the classic myeloproliferative neoplasms (MPNs) include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The JAK2 V617F mutation (V617F) can be detected through genomic analysis of peripheral blood in 95% of PV and 50–60% of ET and MF patients (pts). These MPNs are often associated with debilitating symptoms and decreased quality of life (QOL). Systemic mastocytosis (SM) is an uncommon MPN in which proliferation of mast cells can result in mediator symptoms, as well as organ damage in advanced disease. We sought to compare the symptom burden of individuals with CML and SM to pts with classic MPNs in a more quantitative manner using a web-based recruitment design. Methods: Individuals with MPN were recruited through a web-based outreach effort. Participants gave IRB-approved consent, and completed on-line surveys of demographic information and questions based on the validated MPN-symptom assessment form (MPN-SAF). In addition, pts submitted saliva samples which were genotyped with SNP arrays based on the Illumina OmniExpress BeadChip, including probes for V617F. Regression analysis was used to evaluate the correlation between symptom responses and genotype data for 495 pts (118 PV, 121 ET, 97 MF, 85 SM, and 74 CML). For each MPN diagnosis, mean scores were calculated based on six fatigue-focused questions (Fatigue-6) and for six other symptoms (Symptom-6): early satiety, abdominal pain/discomfort, inactivity, night sweats, pruritis, and bone pain (ranked from 0–10; 10 = worst). ET pts' scores were used as the reference in the regression analysis based on prior data showing that ET pts have the lowest symptom burden among the classic MPNs. Individual symptoms, Fatigue-6, Symptom-6, and QOL (ranked 0–10; 10=worst) were analyzed using regression analysis against age, gender, diagnosis and V617F status. Results: Median age was 56 years (7–87), with 70% women. V617F was detected in 63% of classic MPN pts (87% PV, 46% ET, 56% MF). Analysis of Fatigue-6 scores revealed that age in years was negatively correlated with reported fatigue (β=-0.007 per year, P=0.032), while female gender and SM diagnosis were positively correlated with fatigue (female: β=0.33, P=2.4×10−4; SM: β=0.46, P=7.6×10−4). This trend was also seen for the Symptom-6: age (β=-0.025, P=5.8×10−4), female gender (β=0.73, P=1.4×10−4), and SM (β=1.8, P=5.0×10−10). Both Fatigue-6 and Symptom-6 were positively correlated with QOL, but Fatigue-6 had a much stronger association with QOL than did Symptom-6 (β=1.3, P=1.5×10−28 vs. β=0.36, P=1.2×10−11). When analyzed individually, abdominal pain/discomfort, pruritis, and bone pain were significantly correlated with V617F (β=0.676, P=0.0371; β=0.768, P=0.0241; β=0.791, P=0.0341, respectively), though these associations were not significant when all six symptoms were evaluated together (Symptom-6: β=0.315, P=0.171). There was no statistically significant correlation between V617F and Symptom-6. In addition, there was no correlation between any symptom and a germline variant in telomerase reverse transcriptase (TERT) rs2853677, identified as a novel predisposition allele for MPNs using a genome-wide association study of this cohort. Conclusion: Using a novel, web-based recruitment design combining MPN symptom assessment and genotyping of saliva samples, we identified significant correlations between 3 specific symptoms (pruritis, abdominal pain/discomfort, and bone pain) with V617F. Fatigue was also more strongly associated than other symptoms with QOL. There was no association between symptoms and TERT. This design is a powerful tool for future studies seeking to correlate symptoms with genotyping analysis. Disclosures: Hinds: 23andMe: Employment, Equity Ownership, Patents & Royalties. Barnholt:23andMe, Inc.: Employment. Kiefer:23andMe, Inc.: Employment. Do:23andMe, Inc.: Employment, Equity Ownership, Patents & Royalties. Eriksson:23andMe, Inc.: Employment, Equity Ownership, Patents & Royalties. Mountain:23andMe, Inc.: Consultancy, Employment, Honoraria, Patents & Royalties, Research Funding. Francke:23andMe, Inc.: Employment, Honoraria, Research Funding; Locus Development: Consultancy, Membership on an entity's Board of Directors or advisory committees. Tung:23andMe, Inc.: Employment. Zehnder:23andMe, Inc.: Unpaid advisor and collaborator Other. Gotlib:23andMe, Inc.: Unpaid advisor and collaborator Other. Mesa:23andMe, Inc: Unpaid advisor and collaborator Other.

Abstract 707 Background: Myeloproliferative neoplasms (MPNs) are disorders that result in unregulated overproduction of one or more myeloid blood cell types by the bone marrow. Polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) comprise the three classic MPNs. The somatic JAK2 V617F mutation is present in 95% of PV patients, and 50–60% of ET and PMF patients. Past work has identified a germline haplotype in JAK2 associated with risk of developing a V617F-positive MPN (Kilpivaara et al; Olcaydu et al; Jones et al, Nat. Genet., 2009). Methods: We recruited a web-based participatory cohort of individuals with MPNs to better understand the genetic basis of these conditions. Participation in this MPN research initiative included free genotyping, and participants provided informed consent and responses to surveys online, under a protocol approved by the external AAHRPP-accredited IRB, Ethical & Independent Review Services. We enrolled and collected saliva samples from more than 700 participants with the following self-reported diagnoses: systemic mastocytosis (n=142), ET (n=145), PV (n=137), PMF (n=50), chronic myelogenous leukemia (n=108), and 97 with overlapping diagnoses (e.g. ET or PV with PMF). Participants were genotyped using a derivative of the Illumina HumanOmniExpress BeadChip with additional custom content, including probes for the JAK2 V617F variant. We performed a genome-wide association study (GWAS) of classic MPN diagnoses (at least one of ET, PV, or MF), using 407 cases from this MPN research initiative and 94,037 controls drawn from the broader 23andMe research participant community. Association was assessed by logistic regression adjusted for age, gender, and 5 principal components to model ancestry. Results: We replicated the known association between germline variation in JAK2 and classic MPNs (rs12340895: odds ratio=2.5, P=7.4 × 10−35). We also identified a novel genome-wide-significant association in TERT, or telomerase reverse transcriptase (rs2853677: odds ratio=1.6 per G allele, P=6.3 × 10−11). The association is consistent with an additive model on the logistic scale (odds ratio=2.6 for 2 versus 0 G alleles). Another variant in TERT, rs2736100, has previously been associated with several non-hematological cancers and with red blood cell count, and our lead SNP is in linkage disequilibrium with TERT variant rs2736100 (r2=0.54). While no other loci reached the threshold of genome-wide significance (P The rs2853677 TERT variant was associated with each of ET, PV, and PMF, with similar effect sizes. It was not associated with JAK2 V617F mutation status within the MPN cohort (odds ratio=1.0, P=1.0), while the germline JAK2 variant was preferentially associated with V617F-positive MPNs (odds ratio=2.1, P=3.0 × 10−6). The high-risk allele of rs2853677 was also predictive of V617F status among 23andMe participants who had not been recruited into the MPN community (odds ratio=1.6, P=0.0012). These results suggest that while variation in TERT facilitates either acquisition or proliferation of V617F-positive myeloid cells, it also predisposes to MPNs through a V617F-independent mechanism. Conclusion: We have identified a germline variant in TERT which is a new predisposition allele for classic MPNs. These results demonstrate the potential for web-based recruitment methods to contribute to genetic research for uncommon diseases. Disclosures: Hinds: 23andMe: Employment, Equity Ownership, Patents & Royalties. Barnholt:23andMe, Inc.: Employment. Zehnder:23andMe, Inc.: Unpaid advisor and collaborator Other. Kiefer:23andMe, Inc.: Employment. Do:23andMe, Inc.: Employment, Equity Ownership, Patents & Royalties. Eriksson:23andMe, Inc.: Employment, Equity Ownership, Patents & Royalties. Mountain:23andMe, Inc.: Consultancy, Employment, Honoraria, Patents & Royalties, Research Funding. Francke:23andMe, Inc.: Employment, Honoraria, Research Funding; Locus Development: Consultancy, Membership on an entity's Board of Directors or advisory committees. Tung:23andMe, Inc.: Employment. Mesa:23andMe, Inc: Unpaid advisor and collaborator Other. Gotlib:23andMe, Inc.: Unpaid advisor and collaborator Other.

Mutations at the mouse pink-eyed dilution locus, p, cause hypopigmentation. We have cloned the mouse p gene cDNA and the cDNA of its human counterpart, P. The region of mouse chromosome 7 containing the p locus is syntenic with human chromosome 15q11-q13, a region associated with Prader-Willi syndrome (PWS) and Angelman syndrome (AS), both of which involve profound imprinting effects. PWS patients lack sequences of paternal origin from 15q, whereas AS patients lack a maternal copy of an essential region from 15q. However, the critical regions for these syndromes are much smaller than the chromosomal region commonly deleted that often includes the P gene. Hypopigmentation in PWS and AS patients is correlated with deletions of one copy of the human P gene that is highly homologous with its mouse counterpart. A subset of PWS and AS patients also have OCA2. These patients lack one copy of the P gene in the context of a PWS or AS deletion, with a mutation in the remaining chromosomal homologue of the P gene. Mutations in both homologues of the P gene of OCA2 patients who do not have PWS or AS have also been detected.

Pulse-chase studies of [35S]cysteine-labeled fibrillin were performed on fibroblast strains from 55 patients with Marfan syndrome (MFS), including 13 with identified mutations in the fibrillin-1 gene and 10 controls. Quantitation of the soluble intracellular and insoluble extracellular fibrillin allowed discrimination of five groups. Groups I (n = 8) and II (n = 19) synthesize reduced amounts of normal-sized fibrillin, while synthesis is normal in groups III (n = 6), IV (n = 18), and V (n = 4). When extracellular fibrillin deposition is measured, groups I and III deposit between 35 and 70% of control values, groups II and IV < 35%, and group V > 70%. A deletion mutant with a low transcript level from the mutant allele and seven additional patients have the group I protein phenotype. Disease in these patients is caused by a reduction in microfibrils associated with either a null allele, an unstable transcript, or an altered fibrillin product synthesized in low amounts. In 68% of the MFS individuals (groups II and IV), a dominant negative effect is invoked as the main pathogenetic mechanism. Products made by the mutant allele in these fibroblasts are proposed to interfere with microfibril formation. Insertion, deletion, and exon skipping mutations, resulting in smaller fibrillin products, exhibit the group II phenotype. A truncated form of fibrillin of 60 kD was identified with specific fibrillin antibodies in one of the group II cell culture media. Seven of the nine known missense mutations, giving rise to abnormal, but normal-sized fibrillin molecules, are in group IV.

Marfan's syndrome is classically defined as an autosomal dominant disorder of connective tissue with variable manifestations involving primarily the cardiovascular, ocular, and musculoskeletal syst...

Eight different mutations were detected in the growth hormone (GH) receptor gene of patients with inherited GH receptor deficiency (GHRD; Laron syndrome) from five continents. All the mutations are located in the extracellular domain of the receptor and are predicted to cause gross structural abnormalities and non-functional receptor molecules. They include three nucleotide changes in the coding region causing translational stop signals, including the newly identified E183X mutation; two nucleotide changes in introns that affect splice junctions; two dinucleotide deletions that result in stop codons downstream; and one single nucleotide change that activates a donor splice site within an exon and results in a transcript missing 24 nucleotides. This latter mutation (E180splice) was first identified in a cohort of patients with GHRD from southern Ecuador. Based on the fact that the E180splice mutation generates a new cleavage site for the restriction enzyme MnlI, a simple diagnostic test has been developed that can be carried out on dried blood spots collected on filter paper. A total of 55 affected individuals from Ecuador has been found to be homozygous for this mutation. Asymptomatic carriers can also be detected, and 104 of 150 individuals screened were found to be carriers. Using this test, the E180splice mutation has recently been detected in one of two oriental Jewish patients from Israel.

Murine B lymphocytes, adipocytes, and olfactory neurons contain a DNA-binding protein that participates in the regulation of genes encoding tissue-specific components of signal transduction. Purification and cloning of this protein, termed early B-cell factor (EBF), from murine B lymphocytes and independent cloning of a protein, termed Olf-1, from olfactory neuronal cells revealed virtual complete amino acid sequence identity between these proteins. As a first step towards identifying a human genetic disorder or mouse mutation for which EBF could be a candidate gene, we have chromosomally mapped the corresponding locus in both species. By Southern hybridization analyses of somatic cell hybrid panels with murine cDNA probe, fluorescence chromosomal in situ hybridization (FISH) of human genomic clones, and analysis of recombinant inbred mouse strains, we have found single sites for EBF homologous sequences on human Chromosome (Chr) 5, band q34, and on proximal mouse Chr 11, in an evolutionarily conserved region.

The molecular basis and the locus responsible for most familial cases of isolated GH deficiency (IGHD) are still unknown. The GH-releasing hormone (GHRH) gene has been evaluated as a possible candidate in 23 unrelated families with IGHD, 14 of whom were classified as having autosomal recessive IGHD type IB and 9 of whom had autosomal dominant IGHD type II. Three highly polymorphic microsatellites (dinucleotide repeats), mapped close to GHRH on chromosome 20 by previous linkage studies, were analyzed as markers for the GHRH locus. All available family members were genotyped for D20S44 [no recombination with GHRH at a LOD (logarithm of the odds) score of 3.6]. Noninformative families were also genotyped for D20S45 and/or D20S54 (located at approximately 1 and 3 centiMorgan of genetic distance from GHRH, respectively). Twenty families were informative for linkage analysis with 1 or more of these markers. We found at least 1 obligate recombinant with discordance between phenotype and genotype in 19 of the 23 families (83%). There is only a very small chance (1-3% or less) that the discordances observed are due to recombination between the GHRH locus and the marker tested. Concordant segregation was seen in only 1 type IB family (4%). When probands from this and the 3 noninformative families were screened for sequence variants in the 5 exons of the GHRH gene by single strand conformation analysis, no abnormal patterns were observed. We conclude that mutations responsible for IGHD are not within or near the structural gene for GHRH on chromosome 20 in the 23 families studied. As linkage to the GH-1 gene has also been previously excluded in 65% of these families, mutations in a locus or loci unlinked to GH-1 and GHRH must be responsible for the majority of these IGHD families.

Moesin is a member of a recently discovered family of closely related proteins that includes ezrin, radixin, and merlin. It is widely expressed in different tissues and cells and has been localized to filopodia and other membranous protrusions that are important for cell-cell recognition and signaling and cell movement. Here, we have localized the coding gene (MSN) to Xq11.2-q12 by Southern and Western blot analyses of Chinese hamster × human somatic cell hybrids and by fluorescence chromosomal in situ hybridization. Moesin-like sequences were identified on chromosomes 5 and 6. The murine Msn locus was mapped to the X chromosome as well by studying a rodent × mouse hybrid panel. The structure of the human moesin gene has been determined. The 12 exons are distributed over >30 kb, and the exon/intron junctions demarcate individual highly conserved domains. Primer extension analysis revealed two major start transcription sites, 184 and 133 bp upstream of the initiation codon. The 5′-flanking region is GC-rich, lacks a TATA box, and contains four SP1 and one AP1 binding sites.