A novel GC-rich human macrosatellite VNTR in Xq24 is differentially methylated on active and inactive X chromosomes.

A new X chromosome-specific repetitive sequence, a 3 kilobase HindIII clone with a base composition of 63% C+G, has been isolated. The sequence is organized as a hypervariable tandem repeat cluster ranging in size from 150-350 kilobases, with outlying single copies. This locus, designated DXZ4 and mapped to chromosome band Xq24, may consist of as many as 50 variable-length alleles. It represents a class of variable number of tandem repeat polymorphism which may be termed 'macrosatellite'. The cluster is highly methylated on the active X chromosome and hypomethylated on the inactive X.

Molecular evolution of the human interleukin-8 receptor gene cluster.

Interleukin-8 (IL-8) is the prototype for a family of at least eight neutrophil chemoattractants whose genes map to human chromosome 4q13-q21. Two human IL-8 receptors, IL8RA and IL8RB, are known from cDNA cloning; IL8RA is a promiscuous receptor for at least two other related ligands, GRO alpha and NAP-2. We now report cloning of the genes for IL8RA, IL8RB and a recently inactivated pseudogene of receptor A (IL8RAP). These form a cluster of only three genes in the superfamily of G protein-coupled receptors (GPCRs) and map to 2q34-q35. The coevolutionary diversity displayed by the IL-8 ligand-receptor complex--ligand promiscuity for IL-8, receptor promiscuity for IL8RA, gene duplication for both ligands and receptors and gene extinction in the case of IL8RAP--is unprecedented for the GPCR superfamily.

Small nuclear ribonucleoprotein polypeptide N (SNRPN), an expressed gene in the Prader-Willi syndrome critical region.

Prader-Willi syndrome (PWS) is associated with paternally derived chromosomal deletions in region 15q11-13 or with maternal disomy for chromosome 15. Therefore, loss of the expressed paternal alleles of maternally imprinted genes must be responsible for the PWS phenotype. We have mapped the gene encoding the small nuclear RNA associated polypeptide SmN (SNRPN) to human chromosome 15q12 and a processed pseudogene SNRPNP1 to chromosome region 6pter-p21. Furthermore, SNRPN was mapped to the minimal deletion interval that is critical for PWS. The fact that the mouse Snrpn gene is maternally imprinted in brain suggests that loss of the paternally derived SNRPN allele may be involved in the PWS phenotype.

Maternal imprinting of the mouse Snrpn gene and conserved linkage homology with the human Prader-Willi syndrome region.

Prader-Willi syndrome (PWS) is associated with paternal gene deficiencies in human chromosome 15q11-13, suggesting that PWS is caused by a deficiency in one or more maternally imprinted genes. We have now mapped a gene, Snrpn, encoding a brain-enriched small nuclear ribonucleoprotein (snRNP)-associated polypeptide SmN, to mouse chromosome 7 in a region of homology with human chromosome 15q11-13 and demonstrated that Snrpn is a maternally imprinted gene in mouse. These studies, in combination with the accompanying human mapping studies showing that SNRPN maps in the Prader-Willi critical region, identify SNRPN as a candidate gene involved in PWS and suggest that PWS may be caused, in part, by defects in mRNA processing.

African origin of an intragenic deletion of the human P gene in tyrosinase positive oculocutaneous albinism.

Oculocutaneous albinism (OCA) is a genetically heterogeneous hypopigmentation disorder. One of the two major autosomal recessive forms involves the tyrosinase gene (OCA1), while the other form (OCA2) has recently been associated with alterations of the P gene on chromosome 15. OCA2 is about twice as common as OCA1 in African and African-American populations. We now describe an interstitial deletion that removes a single exon of the P gene. In a large family from an inbred population of tri-racial origin, all individuals with OCA2 were found to be homozygous for this allele. Moreover, the same mutant P allele was detected in several unrelated African American individuals with OCA2, but not in Caucasians with OCA2. The detection of the same allele in two unrelated Africans with OCA2 indicates an African origin for this allele.

Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2.

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.

A mouse model for Prader-Willi syndrome imprinting-centre mutations.

Imprinting in the 15q11-q13 region involves an 'imprinting centre' (IC), mapping in part to the promoter and first exon of SNRPN. Deletion of this IC abolishes local paternally derived gene expression and results in Prader-Willi syndrome (PWS). We have created two deletion mutations in mice to understand PWS and the mechanism of this IC. Mice harbouring an intragenic deletion in Snrpn are phenotypically normal, suggesting that mutations of SNRPN are not sufficient to induce PWS. Mice with a larger deletion involving both Snrpn and the putative PWS-IC lack expression of the imprinted genes Zfp127 (mouse homologue of ZNF127), Ndn and Ipw, and manifest several phenotypes common to PWS infants. These data demonstrate that both the position of the IC and its role in the coordinate expression of genes is conserved between mouse and human, and indicate that the mouse is a suitable model system in which to investigate the molecular mechanisms of imprinting in this region of the genome.

The gene for the peripheral myelin protein PMP-22 is a candidate for Charcot-Marie-Tooth disease type 1A.

Charcot-Marie-Tooth disease type 1A (CMT1A) is an autosomal dominant peripheral neuropathy associated with a large DNA duplication on the short arm of human chromosome 17. The trembler (Tr) mouse serves as a model for CMT1A because of phenotypic similarities and because the Tr locus maps to mouse chromosome 11 in a region of conserved synteny with human chromosome 17. Recently, the peripheral myelin gene Pmp-22 was found to carry a point mutation in Tr mice. We have isolated cDNA and genomic clones for human PMP-22. The gene maps to human chromosome 17p11.2-17p12, is expressed at high levels in peripheral nervous tissue and is duplicated, but not disrupted, in CMT1A patients. Thus, we suggest that a gene dosage effect involving PMP-22 is at least partially responsible for the demyelinating neuropathy seen in CMT1A.

The new Ghent criteria for Marfan syndrome: what do they change?

The diagnosis of Marfan syndrome (MFS) is challenging and international criteria have been proposed. The 1996 Ghent criteria were adopted worldwide, but new diagnostic criteria for MFS were released in 2010, giving more weight to aortic root aneurysm and ectopia lentis. We aimed to compare the diagnosis reached by applying this new nosology vs the Ghent nosology in a well-known series of 1009 probands defined by the presence of an FBN1 mutation. A total of 842 patients could be classified as MFS according to the new nosology (83%) as compared to 894 (89%) according to the 1996 Ghent criteria. The remaining 17% would be classified as ectopia lentis syndrome (ELS), mitral valve prolapse syndrome or mitral valve, aorta, skeleton and skin (MASS) syndrome, or potential MFS in patients aged less than 20 years. Taking into account the median age at last follow-up (29 years), the possibility has to be considered that these patients would go on to develop classic MFS with time. Although the number of patients for a given diagnosis differed only slightly, the new nosology led to a different diagnosis in 15% of cases. Indeed, 10% of MFS patients were reclassified as ELS or MASS in the absence of aortic dilatation; conversely, 5% were reclassified as MFS in the presence of aortic dilatation. The nosology is easier to apply because the systemic score is helpful to reach the diagnosis of MFS only in a minority of patients. Diagnostic criteria should be a flexible and dynamic tool so that reclassification of patients with alternative diagnosis is possible, requiring regular clinical and aortic follow-up.

Structure and functional expression of the human macrophage inflammatory protein 1 alpha/RANTES receptor.

The chemokine beta family is comprised of at least six distinct cytokines that regulate trafficking of phagocytes and lymphocytes in mammalian species; at least one of these, macrophage inflammatory protein 1 alpha (MIP-1 alpha), also regulates the growth of hematopoietic stem cells. We now show that MIP-1 alpha and the related beta chemokine, RANTES, induce transient alterations in intracellular Ca2+ concentration in polymorphonuclear leukocytes that can be reciprocally and specifically desensitized, suggesting a common receptor. Moreover, we have now cloned both the cDNA and the gene for this receptor, functionally expressed the receptor in Xenopus oocytes, and mapped the gene to human chromosome 3p21. Transcripts for the receptor were found in mature and immature myeloid cells as well as B cells. The receptor is a member of the G protein-coupled receptor superfamily. It has approximately 33% amino acid identity with receptors for the alpha chemokine, interleukin 8, and may be the human homologue of the product of US28, an open reading frame of human cytomegalovirus.

Cloning, mapping, and characterization of activated leukocyte-cell adhesion molecule (ALCAM), a CD6 ligand.

Antibody-blocking studies have demonstrated the role of CD6 in thymocyte-thymic epithelial (TE) cell adhesion. Here we report that CD6 expressed by COS cells mediates adhesion to TE cells and that this interaction is specifically blocked with an anti-CD6 monoclonal antibody (mAb) or with a mAb (J4-81) that recognized a TE cell antigen. We isolated and expressed a cDNA clone encoding this antigen and show that COS cells transfected with this cDNA bind a CD6 immunoglobulin fusion protein (CD6-Rg). This antigen, which we named ALCAM (activated leukocyte-cell adhesion molecule) because of its expression on activated leukocytes, appears to be the human homologue of the chicken neural adhesion molecule BEN/SC-1/DM-GRASP. The gene was mapped to human chromosome 3q13.1-q13.2 by fluorescence in situ hybridization of cDNA probes to metaphase chromosomes. We prepared an ALCAM-Rg fusion protein and showed that it binds to COS cell transfectants expressing CD6, demonstrating that ALCAM is a CD6 ligand. The observations that ALCAM is also expressed by activated leukocytes and that both ALCAM and CD6 are expressed in the brain suggest that ALCAM-CD6 interactions may play a role in the binding of T and B cells to activated leukocytes, as well as in interactions between cells of the nervous system.

Blast-1 possesses a glycosyl-phosphatidylinositol (GPI) membrane anchor, is related to LFA-3 and OX-45, and maps to chromosome 1q21-23.

Blast-1 is a human activation-associated glycoprotein expressed on the surface of leukocytes. Analysis of a translated sequence from a Blast-1 cDNA reveals a single hydrophobic sequence which could traverse the plasma membrane, but is devoid of charged residues that might represent a cytoplasmic tail. Consistent with this characteristic, Blast-1 is demonstrated here to be anchored to the cell surface through a glycosyl-phosphatidylinositol (GPI)-containing lipid. Comparison of Blast-1 to other GPI-anchored membrane proteins revealed a striking primary and secondary structure similarity with MRC OX45 and the lymphocyte function antigen LFA-3. The degree of overall amino acid sequence homology reveals that OX45 is a rat homologue of Blast-1. The greatest homology to LFA-3 occurs between their NH2-terminal Ig-like domains. Evidence is presented that demonstrates that Blast-1 and LFA-3 possess a disulfide-bonded second domain. These common characteristics demonstrate a structural and evolutionary relationship between Blast-1, OX45, LFA-3, and CD2, which in turn suggests a functional role for Blast-1 in cell-cell interactions in the immune response. The gene for Blast-1 has been localized to chromosome 1 q21-q23, indistinguishable from the CD1 cluster of Ig superfamily genes, raising the possibility that they may be linked.

Human peroxisomal targeting signal-1 receptor restores peroxisomal protein import in cells from patients with fatal peroxisomal disorders.

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.

Pathogenic FBN1 mutations in 146 adults not meeting clinical diagnostic criteria for Marfan syndrome: further delineation of type 1 fibrillinopathies and focus on patients with an isolated major criterion.

Mutations in the FBN1 gene cause Marfan syndrome (MFS) and have been associated with a wide range of milder overlapping phenotypes. A proportion of patients carrying a FBN1 mutation does not meet diagnostic criteria for MFS, and are diagnosed with "other type I fibrillinopathy." In order to better describe this entity, we analyzed a subgroup of 146 out of 689 adult propositi with incomplete "clinical" international criteria (Ghent nosology) from a large collaborative international study including 1,009 propositi with a pathogenic FBN1 mutation. We focused on patients with only one major clinical criterion, [including isolated ectopia lentis (EL; 12 patients), isolated ascending aortic dilatation (17 patients), and isolated major skeletal manifestations (1 patient)] or with no major criterion but only minor criteria in 1 or more organ systems (16 patients). At least one component of the Ghent nosology, insufficient alone to make a minor criterion, was found in the majority of patients with isolated ascending aortic dilatation and isolated EL. In patients with isolated EL, missense mutations involving a cysteine were predominant, mutations in exons 24-32 were underrepresented, and no mutations leading to a premature truncation were found. Studies of recurrent mutations and affected family members of propositi with only one major clinical criterion argue for a clinical continuum between such phenotypes and classical MFS. Using strict definitions, we conclude that patients with FBN1 mutation and only one major clinical criterion or with only minor clinical criteria of one or more organ system do exist but represent only 5% of the adult cohort.

Gene dose effect: regional mapping of human nuceloside phosphorylase on chromosome 14.

Quantitative analyses of erythrocyte nucleoside phosphorylase in four unrelated cases of partial trisomy 14 indicate that the structural gene for this enzyme maps in the chromosome region 14q11 leads to 14q21.

The human genes for S-adenosylhomocysteine hydrolase and adenosine deaminase are syntenic on chromosome 20.

Human-Chinese hamster cell hybrids and a monoclonal antibody to human S-adenosylhomocysteine hydrolase were used to identify chromosome 20 as the location of the human gene for this enzyme. The gene for adenosine deaminase had previously been mapped to this chromosome. The activity of S-adenosylhomocysteine hydrolase is dependent in vivo on that of adenosine deaminase, since the substrates for the deaminase, adenosine and deoxyadenosine, respectively, inhibit and inactivate S-adenosylhomocysteine hydrolase in genetic or drug-induced adenosine deaminase deficiency. This functional dependence and the likelihood that S-adenosylhomocysteine hydrolase, a eukaryotic enzyme, arose later than adenosine deaminase, which occurs in prokaryotes as well as eukaryotes, suggest that the occurrence of their genes on the same chromosome may have evolutionary significance. In addition, the unusual capacity of S-adenosylhomocysteine hydrolase to form stable complexes with adenosine and its cofactor, nicotinamide adenine dinucleotide, suggest that evolution of its gene may have involved recombination of a portion of the adenosine deaminase gene with an adenine nucleotide domain-coding sequence of another preexisting gene.

Linkage groups II and XII of the mouse: cytological localization by fluorochrome staining.

The T(2;12) 163H translocation involves two chromosomes which carry the two groups of genes known as linkage groups II and XII. Staining with quinacrine mustard reveals that these chromosomes are numbers 10 and 19 of the fluorescent karyotype of the normal mouse. Linkage group II is assigned to chromosome 10, linkage group XII to chromosome 19.

Gene for human insulin receptor: localization to site on chromosome 19 involved in pre-B-cell leukemia.

Consistent chromosomal translocations in neoplastic cells may alter the expression of proto-oncogenes that are located near the breakpoints. The complementary DNA sequence of the human insulin receptor is similar to those of the EGF receptor (erbB oncogene) and products of the src family of oncogenes. With in situ hybridization and Southern blot analysis of somatic cell hybrid DNA, the human insulin receptor gene was mapped to the distal short arm of chromosome 19 (bands p13.2----p13.3), a site involved in a nonrandom translocation in pre-B-cell acute leukemia.

The human gene for the beta subunit of nerve growth factor is located on the proximal short arm of chromosome 1.

Fragments of the recently cloned human gene for the beta subunit of nerve growth factor (beta-NGF) were used as hybridization probes in analyzing two sets of rodent-human somatic cell hybrids for the presence of human beta-NGF sequences. Results from the first set of hybrids assigned the human beta-NGF gene to chromosome 1 and ruled out the presence of sequences of comparable homology on any other chromosome. With the second set of hybrids, which contained seven different, but overlapping, regions of chromosome 1, the NGF locus was mapped to band 1p22.

Oncogene from human EJ bladder carcinoma is located on the short arm of chromosome 11.

The human cellular homolog of the transforming DNA sequence isolated from the bladder carcinoma cell line EJ was localized on the short arm of human chromosome 11 by Southern blot analysis of human-rodent hybrid cell DNA. This locus contains human sequences homologous to the Harvey murine sarcoma virus v-Ha-ras oncogene.