Clinical and molecular characterization of Diastrophic Dysplasia in the Portuguese population.

SLC26A2-related dysplasias encompass a spectrum of diseases: from lethal achondrogenesis type 1B (ACG1B; MIM #600972) and atelosteogenesis type 2 (AO2; MIM #256050) to classical diastrophic dysplasia (cDTD; MIM #222600) and recessive multiple epiphyseal dysplasia (rMED; MIM #226900). This study aimed at characterizing clinically, radiologically and molecularly 14 patients affected by non-lethal SLC26A2-related dysplasias and at evaluating genotype-phenotype correlation. Phenotypically, eight patients were classified as cDTD, four patients as rMED and two patients had an intermediate phenotype (mild DTD - mDTD, previously 'DTD variant'). The Arg279Trp mutation was present in all patients, either in homozygosity (resulting in rMED) or in compound heterozygosity with the known severe alleles Arg178Ter or Asn425Asp (resulting in DTD) or with the mutation c.727-1G>C (causing mDTD). The 'Finnish mutation', c.-26+2T>C, and the p.Cys653Ser, both frequent mutations in non-Portuguese populations, were not identified in any of the patients of our cohort and are probably very rare in the Portuguese population. A targeted mutation analysis for p.Arg279Trp and p.Arg178Ter in the Portuguese population allows the identification of approximately 90% of the pathogenic alleles.

Evidence for catabolic pathway of propionate metabolism in CNS: expression pattern of methylmalonyl-CoA mutase and propionyl-CoA carboxylase alpha-subunit in developing and adult rat brain.

Methylmalonyl-CoA mutase (MCM) and propionyl-CoA carboxylase (PCC) are the key enzymes of the catabolic pathway of propionate metabolism and are mainly expressed in liver, kidney and heart. Deficiency of these enzymes leads to two classical organic acidurias: methylmalonic and propionic aciduria. Patients with these diseases suffer from a whole spectrum of neurological manifestations that are limiting their quality of life. Current treatment does not seem to effectively prevent neurological deterioration and pathophysiological mechanisms are poorly understood. In this article we show evidence for the expression of the catabolic pathway of propionate metabolism in the developing and adult rat CNS. Both, MCM and PCC enzymes are co-expressed in neurons and found in all regions of the CNS. Disease-specific metabolites such as methylmalonate, propionyl-CoA and 2-methylcitrate could thus be formed autonomously in the CNS and contribute to the pathophysiological mechanisms of neurotoxicity. In rat embryos (E15.5 and E18.5), MCM and PCC show a much higher expression level in the entire CNS than in the liver, suggesting a different, but important function of this pathway during brain development.

Autosomal dominant nemaline myopathy: a new phenotype unlinked to previously known genetic loci.

We report a large family with a mild form of autosomal dominant nemaline myopathy and a new phenotype. Onset of symptoms was in infancy with hypotonia and motor delay. Weakness involved neck flexors, abdominal and proximal limb muscles. There was no bulbar, respiratory or foot dorsiflexion weakness and no slowness in movement. Patients had remarkably good physical endurance and no limitation in daily activities, but were slow runners since childhood. Nemaline rods were seen in less than 5% of muscle fibres. No linkage to the five known nemaline myopathy genes (alpha-tropomyosin-3, nebulin, alpha-actin, troponin T1 and beta-tropomyosin), to the ryanodine receptor gene (associated with core-rod myopathy) or to the 15q21-23 locus was found.

Adamts-1 is essential for the development and function of the urogenital system.

Successful ovulation and implantation processes play a crucial role in female fertility. Adamts-1, a matrix metalloproteinase with disintegrin and thrombospondin motifs, has been suggested to be regulated by the progesterone receptor in the hormonal pathway leading to ovulation. With the primary aim of investigating the role of Adamts-1 in female fertility, we generated Adamts-1 null mice. Forty-five percent of the newborn Adamts-1 null mice die, with death most likely caused by a kidney malformation that becomes apparent at birth. Surviving female null mice were subfertile, whereas males reproduced normally. Ovulation in null females was impaired because of mature oocytes remaining trapped in ovarian follicles. No uterine phenotype was apparent in Adamts-1 null animals. Embryo implantation occurred normally, the uteri were capable of undergoing decidualization, and no morphological changes were observed. These results demonstrate that a functional Adamts-1 is required for normal ovulation to occur, and hence the Adamts-1 gene plays an important role in female fertility, primarily during the tissue remodeling process of ovulation.

Structure of the human Lanosterol synthase gene and its analysis as a candidate for holoprosencephaly (HPE1).

Holoprosencephaly (HPE) is the most common birth defect of the brain in humans. It involves various degrees of incomplete separation of the cerebrum into distinct left and right halves, and it is frequently accompanied by craniofacial anomalies. The HPE1 locus in human chromosome 21q22.3 is one of a dozen putative genetic loci implicated in causing HPE. Here, we report the complete gene structure of the human lanosterol synthase (LS) gene, which is located in this interval, and present its mutational analysis in HPE patients. We considered LS an excellent candidate HPE gene because of the requirement for cholesterol modification of the Sonic Hedgehog protein for the correct patterning activity of this HPE-associated protein. Despite extensive pedigree analysis of numerous polymorphisms, as well as complementation studies in yeast on one of the missense mutations, we find no evidence that the LS gene is in fact HPE1, implicating another gene located in this chromosomal region in HPE pathogenesis.

Isolation and characterization of the mouse Aire gene.

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a rare autosomal recessive disorder characterized by Addison's disease and/or hypoparathyroidism and/or chronic mucocutaneous candidiasis. Patients may also have other clinical symptoms both within and outside the endocrine system, mainly as a result of autoimmunity against organ-specific autoantigens. The gene for APECED has recently been identified and termed AIRE (for AutoImmune REgulator). APECED is a model of organ-specific autoimmunity and isolation and characterization of the homologous mouse gene, Aire, will provide tools for dissection of the mechanisms underlying this human disorder and defining molecular pathways involved in organ-specific autoimmunity. We have isolated and completely sequenced the mouse Aire gene which is split into 14 exons over 13 kb and encodes a predicted protein of 552 amino acids. The predicted mouse and human AIRE proteins are 71% identical and contain motifs suggestive of a transcriptional regulator. Additional conserved motifs are emerging in the AIRE/Aire proteins including a nuclear localization signal, an "ASS" domain, and a "SAND" domain. The human and mouse AIRE promoters have conserved sites for several thymus-specific transcription factors and others important in hematopoesis, consistent with its expression in rare cells of the thymus medulla, lymph nodes, and fetal liver. We have mapped mouse Aire to mouse chromosome 10 by FISH, to the same region as Pwp2 and Pfkl, confirming synteny to the corresponding region of human chromosome 21.

Mutation analyses of North American APS-1 patients.

Autoimmune polyendocrinopathy syndrome type 1 (APS-1; MIM# 240300) is a rare autosomal recessively inherited disease characterised by destructive autoimmune diseases of endocrine glands. The gene responsible for APS-1, known as AIRE (for autoimmune regulator), was recently identified and contains motifs suggestive of a transcription regulator. To date, nine APS-1-associated mutations have been identified in the AIRE gene, including two common mutations R257X and 1094-1106del. In addition to these two mutations, we report seven novel mutations in 16 APS-1 patients from North America. We found that 1094-1106del and R257X were the most common mutations in this population of mixed geoethnic origin, accounting for 17/32 and 4/32 alleles, respectively. Haplotype analyses suggest that both are recurrent mutations, occurring on several different haplotypes with closely linked markers. All the novel mutations appear to be rare, occurring in only single APS-1 families. After examining all coding sequences and exon/intron boundaries of the AIRE gene, the other APS-1 allele remained unidentified in three patients. Genotype-phenotype correlations for APS-1 remain difficult, suggesting that other genetic or environmental factors, or both, influence the clinical presentation and disease progression in individual APS-1 patients.

Common mutations in autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy patients of different origins.

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED; OMIM *240300, also called APS 1,) is a rare autosomal recessive disorder that is more frequent in certain isolated populations. It is generally characterized by two of the three major clinical symptoms that may be present, Addison's disease and/or hypoparathyroidism and/or chronic mucocutaneous candidiasis. Patients may also have a number of other clinical symptoms including chronic gastritis, gonadal failure, and rarely, autoimmune thyroid disease and insulin-dependent diabetes mellitus. We and others have recently identified the gene for APECED, which we termed AIRE (for autoimmune regulator). AIRE is expressed in thymus, lymph nodes, and fetal liver and encodes a protein containing motifs suggestive of a transcriptional regulator, including two zinc finger motifs (PHD finger), a proline-rich region, and three LXXLL motifs. Six mutations, in cluding R257X, the predominant Finnish APECED allele, have been defined. R257X was also observed in non-Finnish APECED patients occurring on different chromosomal haplotypes suggesting different mutational origins. Here we present mutation analyses in an extended series of patients, mainly of Northern Italian origin. We have detected 12 polymorphisms, including one amino acid substitution, and two additional mutations, R203X and X546C, in addition to the previously described mutations, R257X, 1096-1097insCCTG, and a 13-bp deletion (1094-1106del). R257X was also the common mutation in the Northern Italian patients (10 of 18 alleles), and 1094-1106del accounted for 5 of 18 Northern Italian alleles. Both R257X and 1094-1106del were both observed in patients of four different geo-ethnic origins, and both were associated with multiple different haplotypes using closely flanking polymorphic markers showing likely multiple mutation events (six and four, respectively). The identification of common AIRE mutations in different APECED patient groups will facilitate its genetic diagnosis. In addition, the polymorphisms presented provide the tools for investigation of the involvement of AIRE in other autoimmune diseases, particularly those affecting the endocrine system.

Localization of a novel human RNA-editing deaminase (hRED2 or ADARB2) to chromosome 10p15.

RNA-editing deaminase 2 (RED2; ADARB2) is a newly identified potential double-stranded RNA adenosine deaminase. It is the third member of this family, which includes DRADA and RED1. Genes of this family are candidates for involvement in neurological diseases such as epilepsy, because of their expression patterns and described functions. All three described genes are well expressed in brain, and DRADA and RED1 have been shown to play a role in the editing of mRNAs coding for glutamate receptor subunits in vitro, thereby changing the properties of these channels, which are the main excitatory neurotransmitter receptors in brain. Here we report the mapping of the human RED2 (hRED2; ADARB2) gene. Using the sequence of rat RED2, we identified a homologous human expressed sequence tag, and subsequently designed primers in the 3' untranslated region of the hRED2 transcript to perform polymerase chain reaction amplification on two somatic cell hybrid mapping panels. This allowed us to localize hRED2 on chromosome 10p15; until now, no genetic diseases have been mapped in this region or in the syntenic mouse chromosomal region that may involve RED2.

Cloning of a human RNA editing deaminase (ADARB1) of glutamate receptors that maps to chromosome 21q22.3.

RED1 is a double-stranded RNA-specific editase characterized in the rat and is implicated in the editing of glutamate receptor subunit pre-mRNAs, particularly in the brain. Starting from human ESTs homologous to the rat RED1 sequence, we have characterized two forms of human RED1 cDNAs, one form coding for a putative peptide of 701 amino acids (similar to the shorter of two rat mRNAs) and a long form coding for a putative protein of 741 amino acids, the extra 120 bp of which are homologous to an AluJ sequence. Both forms were observed at approximately equal levels in cDNA clones and in seven different human tissues tested by RT-PCR. The human and rat short isoforms have 95 and 85% sequence identity at the amino acid and nucleotide levels, respectively. The human sequence (designated ADARB1 by the HGMW Nomenclature Committee) contains two double-stranded RNA-binding domains and a deaminase domain implicated in its editing action. Northern blot analysis detected two transcripts of 8.8 and 4.2 kb strongly expressed in brain and in many human adult and fetal tissues. ADARB1 maps to human chromosome 21q22.3, a region to which several genetic disorders map, including one form of bipolar affective disorder. Recently it was shown that heterozygous mice harboring an editing-incompetent glutamate receptor B allele have early onset fatal epilepsy. Since glutamate receptor channels are essential elements in synaptic function and plasticity and mediate pathology in many neurological disorders, and since RED1 is central in glutamate receptor channel control, ADARB1 is a candidate gene for diseases with neurological symptoms, such as bipolar affective disorder and epilepsy.