ESHG Concurrent Sessions

Increasingly, human dignity is relegated to the rank of a standard ethical principle or human right and then used as an unexplained source of blanket prohibitions (e.g. reproductive cloning, creation of embryos for research, germ line therapy ….). Once considered the source of other human rights and outside the range of any normative hierarchy, the commonplace political and legislative use of the concept of human dignity in the context of human genetics risks turning it into a banality. Drawing on the origins of the concept and tracing its legal and ethical interpretations and use since the 1948 Universal Declaration of Human Rights, we will criticize this trend and offer a different perspective that ensures its fundamental and overarching nature.

Prenatal diagnosis (PND) in adult-onset disorders: survey of cases and attitudes in Portugal.

J. Sequeiros¹^,2, J. Rocha¹^,3, J. Pinto-Basto¹, J. Leal Loureiro¹^,4, T. Coelho¹^,5, A. Lopes¹^,6;
¹UnIGENe-IBMC, Univ. Porto, PORTUGAL, ²ICBAS - Univ. Porto, Porto, PORTUGAL, ³C.Gen.Clín., Porto, PORTUGAL, ⁴Hosp.S.Teotónio, Viseu, PORTUGAL, ⁵H.G.S.António, Porto, PORTUGAL, ⁶H.M.Lemos, Porto, PORTUGAL.

Prenatal diagnosis in adult-onset diseases may be controversial due to several decades of a (physically) healthy life. We surveyed all 33 national centres offering PND, for number of requests and their attitudes regarding termination of pregnancy (TOP): 26 (79%) replied.
50 cases were ascertained (including 34 for FAP-TTRMet30, 3 HD, 3 MJD, 3 SCA2, 1 DRPLA, 3 DM). 29 amniocenteses were performed. Several were simultaneous requests for pre-symptomatic testing (PST), a very difficult situation, given time constraints and the potential for three distressful situations (PST, PND, TOP). Pregnancy was not terminated despite a 'carrier' result at least in 4 cases.
Concerning attitudes of the 'Commisions for TOP', 9 answers were "unanimous", 10 "by majority" and 5 were only the director's; only 1/3 had a medical geneticist; 7 found the current law satisfactory, but 15 did not; 13 interpreted it as permiting TOP, while 9 did not (although 5 of these would practice it!). Only 5 centres would not accept TOP for any adult-onset disease, 19 centres would perform TOP for all diseases, 1 for all but FAP (liver transplant is now a 'therapeutical' option); 14 centres would not accept for PND if couple not considering TOP, but 10 would.
The non-termination of a carrier foetus will result in a pre-symptomatic test for the unborn child (precluded by ethical and/or legal reasons). PND in adult-onset diseases needs tactful counselling, special sensibility and intensive psychosocial evaluation and support. We propose a specific protocol of consults and evaluations. Current law may need redefinition.

S. Patton¹, R. Elles¹, D. Barton², E. Dequeker³, C. Mueller⁴, M. Losekoot⁵, B. Bakker⁵, B. Rautenstrauss⁶, M. Simoni⁷, V. Biancalana⁸, P. Vogt⁹, M. Voelckel¹⁰, D. Lohmann¹¹;
¹European Molecular Genetics Quality Network, Manchester, UNITED KINGDOM, ²National Centre for Medical Genetics, Dublin, IRELAND, ³University of Leuven, Leuven, BELGIUM, ⁴University of Wuerzburg, Wuerzburg, GERMANY, ⁵Leiden University Medical Centre, Leiden, NETHERLANDS, ⁶Universität Erlangen-Nürnberg, Erlangen, GERMANY, ⁷Institute of Reproductive Medicine of the University, Münster, GERMANY, ⁸University Louis Pasteur, Strasbourg, FRANCE, ⁹University of Heidelberg, Heidelberg, GERMANY, ¹⁰Hôpital d'enfants de la Timone, Marseille, FRANCE, ¹¹University of Essen, Essen, GERMANY.

Genetic testing for inherited disorders is now a routine part of laboratory medicine. Studies of the reliability of such testing have indicated significant levels of inaccuracy in laboratory reports, arising from errors in sample identification, genotyping or interpretation. These errors can have significant consequences for prenatal diagnosis and carrier testing for example. External Quality Assessment (EQA), or Proficiency testing, is one approach to quantifying these errors and can be used to raise the standards of output from laboratories. The European Molecular Genetics Quality Network (EMQN) runs EQA schemes for ten different genetic disorders. Each scheme is designed to test the ability of laboratories to interpret data in the light of clinical information supplied with a referral, and to produce a clear and accurate report. Laboratories from all the European Union countries have participated in these exercises. In 2001, the schemes evaluated 315 returns from laboratories, a 33% increase on 2000. Error rates (no. of diagnostic errors/no. of cases analysed) varied between 0.7% and 7.6%. The causes of errors include incorrect genotyping, sample swaps and incorrect interpretation of technically correct results. Examples of results and errors will be presented. The errors identified indicate a clear need for EQA to measure current standards of proficiency and encourage laboratories to raise their technical performance.

K. R. Rasmussen¹, C. Lundsteen², H. Ropers³, N. Tommerup¹;
¹Wilhelm Johannsen Centre for Functional Genome Research, Panum Institute, University of Copenhagen, Copenhagen, DENMARK, ²Dept. of Clinical Genetics, Rigshospitalet, Copenhagen, DENMARK, ³Max-Planck-Institute for Molecular Genetics, Berlin, GERMANY.

Mendelian Cytogenetics Network (MCN) is a global collaboration involving >300 cytogenetic laboratories, aimed at the systematic collection of data and material from disease-associated balanced chromosomal rearrangements (DBCRs). A five year grant from the Danish National Research Foundation establishing the Wilhelm Johannsen Centre for Functional Genome Research at the University of Copenhagen has ensured continued and extended support of MCN for 2001-2006. This will include improved facilities for assisted help with retrieval of DBCRs from cytogenetic archives, improved mapping facilities in collaboration with the Max-Planck Institute for Molecular Genetics, Berlin (MPI) and systematic high resolution comparative genome hybridization (CGH) of DBCR-cases within MCN. The online version of the associated database MCNdb (http://mcndb.imbg.ku.dk) at the University of Copenhagen has been developed into a working tool for the participating laboratories: New DBCRs can be submitted online. We have improved the query interface, where clinical information and breakpoint data on more than 2100 published and unpublished DBCRs can be queried and displayed together with relevant information drawn from the OMIM morbid map, data on cytogenetic microdeletions/duplications and the murine phenotypic map, thus improving the chance to identify relevant DBCRs. YAC clones can be selected and ordered from MPI (http://www.molgen.mpg.de/~abt_rop/neurogenetics/chromosome_rearrangements.html) for FISH mapping of specific breakpoints, and the breakpoints of DBCRs are linked to the human genome map by a direct interface between MCNdb and the UCSC Human Genome Browser (http://genome.cse.ucsc.edu/goldenPath/hgTracks.html), enabling rapid identification of sequenced BAC clones for planning of FISH mapping and for direct visualization of candidate genes for specific phenotypes and diseases.

B. Zimmermann¹, W. Holzgreve¹, F. Wenzel², S. Hahn¹;
¹University Women's Hospital, Basel, SWITZERLAND, ²University of Basel, Basel, SWITZERLAND.

A significant proportion of clinical genetics is involved with the analysis of gross chromosomal anomalies. In prenatal diagnosis a major concern are aneuploidies, of which Down’s syndrome is the most important in live births. The detection of these gross changes is still time consuming despite modern technologies such as FISH or quantitative fluorescent PCR.
For this purpose we have developed a novel alternative using real time quantitative PCR using genetic loci in the Down's region of chromosome 21 and a control locus on chromosome 12. This locus was chosen in such a manner that it should also detect cases of Down's syndrome resulting from unbalanced Robertsonian translocations.
The assessment of the ratio of these two loci by multiplex real time PCR has shown that this technique can be used for the reliable and rapid distinction of trisomy 21 from karyotypically normal tissue (refer to Figure 1). We have now extended this test to detect trisomy 18, and it can be readily extended to examine the most common other fetal aneuploidies (13, 16, X and Y) or instances of chromosomal loss or gain. Furthermore, since it permits the rapid automatic analysis of numerous samples it is very well suited for high-throughput diagnostic settings.

Many different and increasingly sophisticated (and time consuming) technologies have been applied in order to separate foetal cells from maternal, but to date none of these have achieved the ultimate goal of obtaining a pure sample of all types of cells of foetal origin. We describe a novel approach for identification of foetal cells in maternal blood samples using an in vitro telomere depletion assay (TDA). We present the principle for this new assay together with preliminary experimentation substantiating its practicability and future potential.
Maternal blood samples (10-20 ml) are collected in EDTA tubes. Foetal cells are firstly enriched using a Triple Density Gradient and then fixed in 3:1 methanol:acetic acid. The cells are spread on clean glass slides and aged on a hot plate (40-50°C) for 2 h. Enzymatic digestion of telomeric DNA sequences is carried out in situ, by application of 2-3 units of BAL 31 enzyme (New England Bio Labs) in 50µl buffer at 37°C for 10 mins. The enzymatic reaction is stopped by washing in 2xSSC at room temperature; the slides are then dehydrated through an ethanol series and air dried. Telomeres are identified by FISH with a pantelomeric DNA probe. Foetal and adult cell nuclei are differentiated by their respective telomere fluorescence: foetal nuclei are expected to be brightly fluorescing while adult nuclei should contain little or no telomere fluorescence. Foetal sex, identified by subsequent FISH using the Y probe, was congruent in an initial series of 12 pregnancies.

Mutations of the RET / GDNF / HASH1 signalling pathway in congenital central hypoventilation syndrome (CCHS, Ondine's curse)

L. de Pontual¹, V. Nepote², T. Attié-Bitach¹, H. Trang², M. Simonneau², M. Vekemans¹, A. Munnich¹, C. Gaultier², S. Lyonnet¹, J. Amiel¹;
¹Necker-Enfants Malades Hospital, Paris, FRANCE, ²R. Debré Hospital, Paris, FRANCE.

CCHS is a hitherto unexplained congenital disorder of the metabolic control of breathing. Hirschsprung disease is associated with CCHS in 25% of the cases (Haddad syndrome, HS, MIM 209880), suggesting a common defect of neural crest derived cells. The RET, GDNF and HASH1 genes were regarded as candidate genes in CCHS due to: i) their role in early neuronal differentiation, ii) the phenotype of homozygous knock-out mice, and iii) their expression in the central and peripheral nervous systems of mouse embryo.
Thirty patients were screened for RET, GDNF and HASH1 genes mutations by SSCP and direct DNA sequencing (23 CCHS and 7 HS cases). We identified a heterozygous nucleotidic variation of one of the 3 tested genes in 6/30 patients: i) a P1039L mutation of the RET gene (HS case), ii) a recurrent GDNF gene mutation of the in 2 CCHS patients (R93W), and, iii) 2 heterozygous polyalanine tract contractions of 5/13 and 8/13 codons as well as a de novo P18T missense mutation of the HASH1 gene in 2 CCHS and 1 HS cases. These DNA variations were not found in 180 control chromosomes and concern amino acids conserved in mammalians. Although polyalanine expansions are well documented in human, contractions have not been hitherto reported. In vitro studies will investigate the putative role of these mutations on HASH1 function. Finally, these findings support the view of the involvement of genes participating to the HASH1 / RET / GDNF signalling pathway and an oligogenic inheritance of CCHS.

P. Griseri¹, B. Pesce¹, G. Patrone¹, F. Puppo¹, M. Sancandi¹, J. Osinga², R. Hofstra², M. Devoto³, R. Ravazzolo¹, I. Ceccherini¹;
¹Ist. G.Gaslini, Genova, ITALY, ²Dep. Medical genetics, Groningen, NETHERLANDS, ³Dip. Oncologia, Biologia e genetica, Genova, ITALY.

Hirschsprung disease, a common genetic disorder characterized by intestinal obstruction secondary to enteric aganglionosis, demonstrates a complex pattern of inheritance, with the RET proto-oncogene as a major gene and several different susceptibility loci, related to Ret-signaling pathway or other neural-crest cells developmental programs. To test whether HSCR phenotype could result by additive effect of multiple genetic defects, we investigated the role of a polymorphic RET variant, 2508C>T, in exon 14 of the gene, characterized by low frequency among HSCR patients and over-representation in individuals affected by sporadic medullary thyroid carcinoma. Typing several different loci across the RET gene, we were able to determine that not the single SNP variant, but a whole conserved haplotype displays anomalous distribution and non-random segregation in HSCR families. We provide genetic evidences about a low-penetrant protective role of this haplotype in HSCR pathogenesis and demonstrate a possible functional effect linked to RET mRNA expression.

M. C. Babron¹, F. Clerget-Darpoux², H. Ascher³, P. Ciclitira⁴, J. Partanen⁵, L. M. Sollid⁶, L. Greco⁷;
¹INSERM U535, Le Kremlin Bicetre, FRANCE, ²INSERM U535, Le Kremlin-Bicêtre, FRANCE, ³Göteborg University, Göteborg, SWEDEN, ⁴St Thomas' Hospital, London, UNITED KINGDOM, ⁵Finnish Red Cross Blood Transfusion Service, Helsinki, FINLAND, ⁶University of Oslo, Oslo, NORWAY, ⁷University Federico II, Napoli, ITALY.

Identification of genetic risk factors for multifactorial disease such as celiac disease (CD), is often carried out through systematic linkage analysis on the whole genome. Each study highlights regions of interest, which nevertheless rarely achieve the genome-wide level of significance. However, some studies tend to pinpoint the same broad area of the genome.
A meta-analysis method GSMA (Wise et al, 1999) was proposed to globally interpret full genome scan results. It has then been extended to take into account replication studies on more restricted genome regions (Wise, 2001). Briefly, this method is based on the sum of the ranks of the linkage statistics obtained for a set of chromosome bins.
Four genome scans has been carried out by the partners of the European Cluster on CD, using the same statistic. Each partner also carried out replication studies on additional sample of families.
GSMA was first applied to the genome scans only. Apart from the well-known risk factor in the HLA region on 6q, regions 2q, 5q, 11q and 14q were significant at the 5% level. Region 5q was pinpointed in 3 out of the 4 scans, 11q in 2 out of the 4. Region 2q contains the CTLA4/CD28 cluster whose role in CD has been suggested by other studies. Accounting for the different replication studies carried out by the European partners, strengthens the evidence for region 5q.
This study was funded by the Commission of the European Communities (QLRT-1999-00037).

Genetic Dissection of the HLA Region using Haplotypes of Tasmanians with Multiple Sclerosis

J. P. Rubio¹^,2, M. Bahlo¹^,2, H. Butzkueven¹, I. A. F. van der Mei³^,2, M. M. Sale³^,2, J. L. Dickinson³, P. Groom³^,2, L. J. Johnson¹^,2, R. D. Simmons⁴, B. Tait⁵, M. Varney⁵, B. Taylor⁶, T. Dwyer³, R. Williamson⁷, N. M. Gough⁸, T. J. Kilpatrick¹, T. P. Speed¹, S. J. Foote¹;
¹The Walter and Eliza Hall Institute of Medical Research, Melbourne, AUSTRALIA, ²The Cooperative Research Centre for the Discovery of Genes for Common Human Diseases, Melbourne, AUSTRALIA, ³Menzies Centre for Population Health Research, Hobart, AUSTRALIA, ⁴Australian National Register of MS families, Canberra, AUSTRALIA, ⁵The Victorian Transplantation and Immunogenetics service, Melbourne, AUSTRALIA, ⁶The Royal Hobart Hospital, Hobart, AUSTRALIA, ⁷Murdoch Children's Research Institute, Melbourne, AUSTRALIA, ⁸The Cooperative Research Centre for Discovery of Genes for Common Human Diseases, Melbourne, AUSTRALIA.

Association of the HLA class II haplotype, DRB1*1501-DQB1*0602, is the most consistently replicated finding of multiple sclerosis (MS) genetic studies. However, the high level of linkage disequilibrium (LD) in the HLA region has hindered the identification of other loci that single marker tests for association are unlikely to resolve. In order to address this issue we generated haplotypes spanning 11-12 megabases (~5cM) across the entire HLA region. The haplotypes, inferred by also genotyping relatives of 152 MS cases and 105 controls of Tasmanian ancestry, define a genomic segment from D6S276 to D6S291 including 13 microsatellite markers integrated with allele typing data for DRB1 and DQB1. Association to the DRB1*1501-DQB1*0602 haplotype was replicated. In addition, we found that the class I/extended class I region, defined by a genomic segment of ~350 kb between MOGCA and D6S265, harbours genes that independently increase risk and provide protection from MS. Log linear modelling analysis of constituent-haplotypes representing genomic regions containing class I (MOGCA-D6S265), class III (TNFa-TNFd-D6S273) and class II (DRB1-DQB1) genes indicated that having class I and class II susceptibility variants on the same haplotype provides an additive effect on risk. Moreover, we found no evidence for a disease locus in the class III region. We propose that the types of statistical approaches outlined here for the analysis of haplotypes will assist in defining the HLA’s contribution to MS. More broadly, these methods provide the basis for gene localisation through the dissection of haplotypes associated with other phenotypes.

Genetic epidemiology of carotid artery thickness in type 2 diabetes families of the Diabetes Heart Study

D. W. Bowden, C. D. Langefeld, L. A. Lange, L. E. Wagenknecht, J. J. Carr, B. I. Freedman, S. S. Rich;
Wake Forest University School of Medicine, Winston-Salem, NC.

Carotid artery intimal medial thickness (IMT) is a strong predictor of subsequent cardiovascular morbidity. The role of genetic factors in thickening of the carotid wall remains largely unknown. We hypothesize that in families with multiple members having diabetes, carotid IMT is influenced by both inherited and environmental factors. Familial aggregation of carotid IMT in the presence of type 2 diabetes was studied in 252 individuals with type 2 diabetes from 122 families enrolled in the Diabetes Heart Study. Common carotid artery IMT was measured by high-resolution B-mode ultrasonography. Other measured factors included lipid levels, body mass index, fasting glucose, hemoglobin A1c, albumin-creatinine ratio, and self-reported medical history. Heritability estimates were obtained using the variance component approach implemented in the software SOLAR. Tests of association between carotid IMT and these variables were performed using mixed model analysis that accounts for familial correlation. The sample was 89% Caucasian (11% African American), 59% female and had a mean±SD for age and duration of diabetes of 60.6±10.4 and 11.2±7.9, respectively. In a multivariate model, carotid IMT was positively associated with age (p=0.001), male gender (p=0.001), African American ethnicity (p=0.020), smoking (p=0.001), hypertension (p=0.040), and total cholesterol (p=0.019). Adjusting for age, gender and ethnicity, we estimated the heritability (h2±SE) for carotid IMT to be 0.32±0.17 (P=0.030). Further adjusting for total cholesterol, hypertension status and current smoking status yielded an estimate of 0.41±0.16 (P = 0.005). These data provide empirical evidence that sub-clinical cardiovascular disease has a significant genetic component.

A. Jannot¹^,2, L. Essioux², M. G. Reese², F. Clerget-Darpoux¹;
¹Unité INSERM 535, Le Kremlin-Bicêtre, FRANCE, ²ValiGen, La Défense, FRANCE.

To detect the effect of a candidate gene in a multifactorial trait, one acknowledged strategy is to genotype all its known intragenic SNPs and to test the association between each SNP separately and the trait. One difficulty is that the model underlying the association of the gene to the trait can be more complex and involve a combination of intragenic SNPs. The effect of a gene might not be detected if each SNP has only small marginal effect on the trait. A second possible strategy is to test the association of the trait with the combination formed by the whole set of SNPs. Because only some of the genotyped SNPs belongs to the functional combination, the information used for this test is diluted. To overcome this drawback, a third strategy is to test the association with all the possible combinations of variable numbers of SNPs. When the functional combination is tested, the information is not diluted. The gain of power by using this strategy is balanced by the correction for multiple testing. Herein, we performed simulation studies based on genotypic data taken from three genes to compare these three strategies. Different models of correspondence between a quantitative trait and the genotype were considered. We found that the strategy that gives the best power is very model-dependent.

Neuronal intranuclear inclusions in a new cerebellar tremor/ataxia syndrome among fragile X carriers

P. J. Hagerman¹, C. M. Greco¹, F. Tassone¹, A. Chudley², M. R. Del Bigio², S. Jacquemont¹, M. Leehey³, R. J. Hagerman¹;
¹University of California, Davis, School of Medicine, Davis, CA, ²University of Manitoba, Winnipeg, MB, CANADA, ³University of Colorado Health Sciences Center, Denver, CO.

Fragile X syndrome is generally regarded as a non-progressive neurodevelopmental disorder in which premutation carriers (~55 to 200 CGG repeats; FMR1 gene) are largely unaffected. However, neurological findings of progressive action tremor, ataxia, cognitive decline, and generalized brain atrophy have recently been described in some adult males with premutation alleles. Neurohistological studies on the brains of four adult male carriers (range: 70-135 CGG repeats) who had the neurological findings reveal ubiquitin-positive, intranuclear inclusions in both neuronal and astrocytic cells, with highest frequencies (~40% of neuronal nuclei) in the hippocampus. Intranuclear inclusions were absent from Purkinje cells, although inclusions were present in a small number of neurons in the dentate nucleus and diffusely in cerebellar astrocytes. The presence of intranuclear inclusions in all brains examined to date is strongly supportive of association with the premutation alleles. Results from additional brains, including a carrier female with no neurological findings, will be presented. The mechanistic basis for the inclusions is not known. FMRP (lymphocyte) levels are generally near normal in the mid-premutation range, although FMR1 mRNA levels are elevated by 2 to 5-fold in this range. The absence of an abnormal protein product in fragile X carriers suggests that the ubiquitin-positive inclusions may reflect a general cellular (neuronal) response in which the cell’s protein-degradative capacity is exceeded as a consequence of altered gene regulation. The intranuclear inclusions observed with the CAG (polyglutamine) repeat disorders (e.g., Huntington’s, the SCAs) may reflect a similar (general) mechanism. These models will be discussed.

Up to now, molecular studies have identified 7 genes that are specifically mutated in MRX families: FMR2, OPHN1, PAK3, IL1RAPL1, TM4SF2, and ARHGEF6. Furthermore, in 2 MRXS genes mutations have been found in families with MRX: RSK2 and MECP2. On average, each MRX gene is mutated in approximately 1% of patients tested. The high heterogeneity in MRX makes it necessary to screen more than 100 probands per candidate gene. In order to have access to such a large patient panel, the European XLMR consortium was established. We collected approximately 200 well-characterized XLMR families, in which the mental retardation occurs as a trait compatible with X-linked inheritance. We exchanged DNA and cell lines of families that have been localized to the X chromosome with significant lod scores of 2 or more, smaller families with a lod score less than 2, and pairs of 3 or 2 affected male family members. In addition, mentally retarded patients with X-chromosomal rearrangements have been collected and analyzed in order to identify novel candidate genes for MRX. An overview of the collected families, as well as linkage data and mutation analysis in 7 of the MRX genes (GDI1, OPHN1, PAK3, IL1RAPL1, TM4SF2, ARHGEF6, MECP2) will be reported. The patient collection of the European XLMR consortium will be a valuable resource for the identification of novel MRX genesin the postgenome era. Extended collaborative efforts will be required to handle the massive testing of candidate genes

Mutations of the human ortholog of Aristaless cause X-linked mental retardation and epilepsy.

P. Stromme¹, M. E. Mangelsdorf¹^,2, M. A. Shaw¹, K. M. Lower¹^,2, S. M. S. Lewis³, H. Bruyere³, V. Lutcherath⁴, A. K. Gedeon¹, R. H. Wallace¹, I. E. Scheffer⁵, G. Turner⁶, M. Partington⁶, S. Frints⁷, J. Fryns⁷, G. R. Sutherland¹^,2, J. C. Mulley¹^,2, J. Gecz¹^,2;
¹Women's and Children's Hospital, Adelaide, AUSTRALIA, ²University of Adelaide, Adelaide, AUSTRALIA, ³Children’s and Women’s Health Center of British Columbia and the University of British Columbia, Vancouver, BC, CANADA, ⁴Central Hospital of Rogaland, Stavanger, NORWAY, ⁵Monash Medical Centre and Royal Children’s Hospital, Melbourne, AUSTRALIA, ⁶Hunter Genetics and University of Newcastle, Waratah, AUSTRALIA, ⁷University Hospital Leuven, Leuven, BELGIUM.

Mental retardation and epilepsy are common, often debilitating conditions of the human brain. Frequently associated, epilepsy and mental retardation are heterogeneous conditions. Where causes are primarily genetic, major advances have been made in the unravelling of their molecular basis. The human X-chromosome alone is estimated to harbour more than 100 genes in which mutations cause mental retardation. At least eight autosomal genes for idiopathic epilepsy have been identified, and many more for conditions where epilepsy is a feature. We have identified mutations in a novel human X-chromosome linked, Aristaless related homeobox gene (ARX), in nine X-linked families with mental retardation and epilepsy. Among these were 4 families with X-linked infantile spasms syndrome (ISSX or West syndrome; MIM 308350), 2 families with Partington syndrome (PRTS; MIM 309510, and S. Frints, unpublished), one family with X-Linked Myoclonic Epilepsy with Spasticity and Intellectual Disability (XMESID; I. Scheffer, unpublished), one MRX family (M. Partington, unpublished), and one family with syndromic XLMR (M. Partington, unpublished). Two recurrent mutations found in seven families, result in expansion of polyalanine tracts of the ARX protein. Such mutations are likely to cause protein aggregation similar to other polyalanine and polyglutamine disorders. Additionally, a missense mutation within the ARX homeodomain and a truncation mutation were identified. The ARX gene has emerged as yet another major contributor to X-chromosome linked mental retardation, similar to genes like FMR1, FMR2, ATRX, or MECP2.

ARX, a novel prd-class-homeobox gene highly expressed in the telencephalon, is mutated in X-linked mental deficiency

T. Bienvenu, K. Poirier, L. Ben Jeema, V. Desportes, C. Moraine, H. van Bokhoven, V. Kalscheuer, S. Frinyts, J. Gecz, H. Chaabouni, J. P. Fryns, C. Beldjord, J. Chelly;
Laboratoire de Génétique et Physiopathologie des Retards Mentaux. Institut Cochin. CHU Cochin, Paris, FRANCE.

Investigation of a critical region in Xp22.1 involved in a non-specific X-linked mental retardation family (MRX54) enabled us to demonstrate that the disease-related gene encodes a novel homeobox protein, Aristeless (ARX). Further screening detected de novo and inherited mutations in eleven families affected with non specific MR, Only missense mutations and in frame duplication/insertion leading to expansion of polyamine tracts of the ARX protein were identified. DNA binding assay showed that ARX binds to palindromic sites containing two core TAAT homeodomain sites. In contrast to the other genes involved in XMR, ARX expression is specific to the telencephalon and ventral thalamus, and completely lacking in cerebellum. The absence of detectable brain malformation in MR patients suggest that ARX has a specialized role in physiological processes underlying cognitive development.

I. Meloni¹, M. Muscettola¹, M. Raynaud², I. Longo¹, M. Bruttini¹, M. Moizard², M. Gomot², J. Chelly³, V. des Portes³, J. Fryns⁴, H. Ropers⁵, B. Magi⁶, C. Bellan⁷, N. Volpi⁸, H. G. Yntema⁹, S. E. Lewis¹⁰, J. E. Schaffer¹⁰, A. Renieri¹;
¹University of Siena, Dept. of Molecular Biology, Medical Genetics, Siena, ITALY, ²Service de Génétique, Inserm U316, Tours cédex, FRANCE, ³Inserm U129-ICGM, CHU Cochen, Paris, FRANCE, ⁴University of Leuven, Genetics Department, Leuven, BELGIUM, ⁵Max-Planck Institute for Molecular Genetics, Berlin, GERMANY, ⁶University of Siena, Department Molecular Biology, Biochemistry, Siena, ITALY, ⁷University of Siena, Pathology, Siena, ITALY, ⁸University of Siena, Department Biomedical Science, Siena, ITALY, ⁹Department of Human Genetics, Nijmegen, NETHERLANDS, ¹⁰Washington University, Departments of Internal Medicine, Molecular Biology & Pharmacology, St. Louis, MO.

X-linked mental retardation (XLMR) is an inherited condition in which the failure to develop cognitive abilities is due to mutations in one gene on the X chromosome. In the last XLMR update up to 136 conditions leading to “syndromic” or “specific” MR (MRXS) and 66 entries leading to “nonspecific” MR (MRX) are listed. For 9 of the 66 MRX entries the causative gene has been identified. The recent discovery of the contiguous gene deletion syndrome ATS-MR, characterized by Alport syndrome and mental retardation, pointed at Xq22.3 as a region containing one mental retardation gene. Comparison of the deletion extent between ATS-MR patients and patients with ATS alone allowed us to define a mental retardation critical region of about 380 kb containing four genes. We report here the identification of two point mutations, one missense and one splice site change, in the FACL4 gene in two families with nonspecific mental retardation. Analysis of enzymatic activity on lymphoblastoid cell lines from both patients demonstrated a marked reduction in activity compared to normal cells, demonstrating that both mutations are null mutations. All carrier females with either FACL4 point mutations or genomic deletions showed a completely skewed X-inactivation, suggesting a role of the gene in survival advantage. FACL4 is the tenth gene mutated in MRX and the first involving a fatty acid metabolic pathway.

Mutations in the Creatine Transporter Gene (SLC6A8) in Xq28 Cause X-Linked Mental Retardation: The Important Role of Creatine Metabolism in Brain Function

C. E. Schwartz¹, K. A. Hahn¹, G. S. Salomons², D. Tackels-Horne¹, T. C. Wood¹, H. A. Taylor¹, R. J. Schroer¹, H. A. Lubs³, C. Jakobs², R. L. Olson¹, K. R. Holden¹, R. E. Stevenson¹;
¹Greenwood Genetic Center, Greenwood, SC, ²VU University Medical Center, Amsterdam, NETHERLANDS, ³University of Miami School of Medicine, Miami, FL.

An X-linked mental retardation (XLMR) family with severe mental retardation, speech and behavioral abnormalities and seizures in affected males has been found to have a G1141C mutation in the creatine transporter gene SLC6A8 (GenBank NM_005629). This mutation results in a glycine being replaced by an arginine (G381R) and alternative splicing since the G→C transversion occurs at the -1 position of the 5’ splice junction of intron 7. Two female relatives who are heterozygous for the SLC6A8 mutation also exhibit mild mental retardation with behavior and learning problems. Males with the mutation have highly elevated creatine in their urine and creatine uptake by fibroblasts from affected males was impaired reflecting the deficiency in creatine transport.
Based on an observed 30% excess of males in the MR population, XLMR should result in a frequency of 15% of all cases of MR. However, many surveys report less than 5% of cases resulting from XLMR. Numerous factors may be responsible for this discrepancy, a major one being few XLMR entities have an associated metabolic abnormality, which might bring them to attention. The ability to measure elevated creatine in urine or serum makes it possible to screen for SLC6A8 deficiency in males with MR of unknown etiology.
The SLC6A8 finding, in conjunction with the association of MR with the creatine biosynthesis defects (AGAT and GAMT deficiencies), clearly indicates the importance of creatine metabolism in brain function. Furthermore, all three of these errors in creatine metabolism can be detected early and may be amenable to treatment.

Mutation In Neurotrypsin is Responsible For Autosomal Recessive Non-specific Mental Retardation

F. Molinari, M. Rio, A. Munnich, L. Colleaux;
INSERM U393, Hopital Necker-Enfants Malades, Paris, FRANCE.

Mental retardation (MR) is the most common developmental disability, affecting largely 2% of the general population. The causes of MR are diverse, but an autosomal recessive mode of inheritance may account for a significant proportion of mentally retarded individuals. The extreme genetic heterogeneity of idiopathic MR and the unavailability of large family pedigrees of nonsyndromic autosomal recessive MR has limited the use of genetic linkage to identify the disease causing genes. While a large number of X-linked mental retardation genes have been found, none of the numerous genes involved in autosomal recessive MR have been hitherto identified. Here we report the identification of the first gene involved in autosomal recessive isolated MR using homozygosity mapping in an inbred family. Genome-wide search provided evidence for linkage to a region of 13 Mb on chromosome 4q24 between markers D4S1564 and D4S402. This interval encompasses the gene PRSS12 (also known as BSSP-3) encoding a brain-specific serine protease named neurotrypsin. We identified a 4 base-pair deletion (del ACGT1391-1394) within the coding sequence that segregates with the disease. This mutation is likely a null allele as it is predicted to result in a shortened protein lacking the catalytic domain.
Our results provide the first evidence for an association between cognitive impairment and a defect in proteolytic activity of brain serine protease.

Prospective screening for cytogenetic anomalies, including subtelomeric rearrangements, in children with mental retardation of unknown etiology: The Amsterdam experience

C. D. M. van Karnebeek, C. Koevoets, R. C. M. Hennekam, J. M. N. Hoovers;
Department of Clinical Genetics, Academic Medical Center, Amsterdam, NETHERLANDS.

The frequency of subtelomeric rearrangements in individuals with unexplained mental retardation (MR) is uncertain, as most studies have been retrospective and case retrieval often biased towards cases more likely to have a chromosome anomaly. After a pilot study in a group of cases selected on the basis of suspicion for a chromosome anomaly, to study the applicability of the technique (subtelomeric rearrangement in 5/30[16.7%]), a prospective study was performed in a consecutive cohort of cases with unexplained MR in an academic tertiairy center. Inclusion criteria: age<18 yrs at referral; IQ<;80; no etiologic diagnosis after complete work-up. In 266 karyotyped children, anomalies were detected in 22(8.3%;7 numerical,15 structural); of 39 cases analyzed by FISH for specific interstitial microdeletions, anomalies were found in 8. FISH analyses for subtelomeric microdeletions were performed in 184 children (44% moderate-profound MR;51% familial MR), and 1(0.5%) rearrangement was identified in a non-familial MR female with mild MR (de novo deletion 12qter). The number of probable polymorphisms(n=11;6%) was considerable.A higher total number of malformations and minor anomalies were present in the cytogenetic anomaly group(n=29) versus the group without anomalies(n=183)(p<0.05).We conclude that the frequency of cytogenetic anomalies in this prospective tertiary center study was high(1 in 10). However, the frequency of subtelomeric rearrangements was low. Possible explanations are provided. Previously proposed selection criteria for efficient subtelomeric screening were not effective in our cohort. The low yield, heavy workload, and high costs of presently available screening techniques mandates development and application of new techniques such as micro-arrays.

Screening Cryptic Telomeric Rearrangements In Children With Idiopathic Mental Retardation Using An Automated Fluorescent Genotyping Strategy.

M. Rio¹, F. Molinari¹, S. Heuertz¹, C. Turleau², M. de Blois², O. Raoul², M. Prieur², S. Romana², M. Vekemans², A. Munnich¹^,2, L. Colleaux¹;
¹INSERM U393, Hopital Necker-Enfants Malades, Paris, FRANCE, ²Department of Genetics, Hopital Necker-Enfants Malades, Paris, FRANCE.

Mental retardation is a common condition that affects largely 2 % of the general population. However, its origin remains poorly understood. Recent studies have demonstrated that cryptic unbalanced subtelomeric rearrangements contribute to a significant proportion of idiopathic syndromic mental retardation cases. Because of the limited sensitivity of routine analyses, we developed a novel strategy based upon automated fluorescent genotyping to search for non-Mendelian segregation of telomeric markers. Here we report a 10 % rate of cryptic subtelomeric rearrangements in a large series of 150 probands with severe idiopathic syndromic mental retardation and normal RHG-GTG banded karyotype. Fourteen children were found to carry deletions or duplications of one or more chromosome telomeres and two children had uniparental disomy. This study clearly demonstrates that fluorescent genotyping is a sensitive and cost-effective method that not only detects telomere rearrangements but also provides the unique opportunity to detect uniparental disomies. Our results provide evidence for the prevalence of the paternal origin of the rearrangements and emphasize the phenotypic variability of these subtelomeric rearangements. Finally we suggest giving consideration to systematic examination of subtelomeric regions in the diagnostic work-up of patients with unexplained syndromic mental retardation.

Screening of dysmorphic and mentally retarded subjects with high resolution comparative genomic hybridization

M. Kirchhoff¹, H. Rose¹, M. Dunø², T. Gerdes¹, C. Lundsteen¹;
¹Department of Clinical Genetics, Rigshospitalet, Copenhagen, DENMARK, ²Fertility Clinic, Rigshospitalet, Copenhagen, DENMARK.

We have improved the sensitivity and specificity of comparative genomic hybridization (CGH) by using dynamic standard reference intervals instead of fixed thresholds.
In our clinical cytogenetics laboratory we use this high resolution CGH (HR-CGH) as a diagnostic tool for screening of dysmorphic and mentally retarded subjects with normal or apparently balanced G-banded karyotypes. So far we found that among 207 patients with a normal conventional karyotype 23 (11%) had small deletions or duplications, of which 18 were were interstitial and five were terminal. In addition one mosaic (47,XX+9/46,XX) was detected. Among 25 translocation carriers six deletions (24%) were detected in five patients. Four had deletions at translocation breakpoints and two had deletions elsewhere in the genome. Our data indicates the existence of a large number of interstitial abnormalities which at present can only be detected by screening the genome with HR-CGH.
By the use of an ABI 7000 we aim to characterize the abnormalities with regard to size and location. We have previously shown that HR-CGH is capable of detecting deletions as small as 3 Mb. We suspect that some of the abnormalities detected by HR-CGH in the dysmorphic and mentally retarded subjects may be this small or even smaller, thus they may be close to the theoretical detection limit of CGH which has been estimated to be about 2 Mb.
We will present results of the screening of dysmorphic and mentally retarded subjects with HR-CGH as well as results of the further characterization of certain abnormalities.

Screening for telomeric rearrangements in mental retardation patients using CGH-array.

S. P. du Manoir¹, R. Redon¹, T. Hussenet¹, S. Wicker¹, L. Colleaux², S. Struski¹;
¹INSERM U184, IGBMC, Illkirch, C.U. de Strasbourg, FRANCE, ²Inserm U393, Hopital NECKER-ENFANTS MALADES, Paris, FRANCE.

In about 7% of idiopathic severe or moderate children mental retardation, cryptic subtelomeric rearrangements are found (Knight J.L. et al, Lancet, 1999).
Several strategies to screen for subtle telomeric rearrangements have been proposed as FISH or CGH on metaphases chromosomes, automated fluorescent genotyping and MAPH telomeric assay. To date, it remains to be demonstrated that any of these approaches is sufficiently reliable and efficient to detect low level gains such as trisomy in large scale studies settings.
To evaluate the efficiency of CGH-array to detect telomeric anomalies, we generated a chip containing the set of telomere-specific PAC clones isolated by Flint 's group. Preliminary experiments with DNA from cell lines containing constitutional known chromosomal abnormalities showed that monosomic and trisomic segments are reliably detected.
To further validate the CGH-array approach, we perform a blind test on a series of 18 DNA from patients with idiopathic mental retardation. These patients have been previously tested for telomere integrity using fluorescent genotyping at the Hopital Necker, Paris (L. Colleaux).
Among the 10 first samples analysed, abnormalities found include gains of 13 (13q telomere), gain of 18 (18p and 18q telomere), loss of 2q and gain of 3p plus several cases without aberrations. Sensibility and specificity of the CGH array will be evaluated and compared with the results previously obtained using fluorescent genotyping.

Heterozygous submicroscopic inversions involving olfactory receptor-gene clusters mediate the recurrent t(4;8)(p16;p23) translocation

S. Giglio¹, V. Calvari¹, G. Gregato¹, G. Gimelli², S. Camanini¹, R. Giorda³, A. Ragusa⁴, S. Guerneri⁵, A. Selicorni⁶, M. Stumm⁷, H. Tönnies⁸, M. Ventura⁹, M. Zollino¹⁰, G. Neri¹⁰, J. Barber¹¹, D. Wieczorek¹², M. Rocchi⁹, O. Zuffardi¹^,13;
¹Biologia Generale e Genetica Medica, Università di Pavia, Pavia, ITALY, ²Laboratorio di Citogenetica, Istituto Gaslini, Genova, ITALY, ³IRCCS E. Medea, Bosisio Parini, Lecco, ITALY, ⁴IRCCS Oasi Maria SS, Troina, Enna, ITALY, ⁵Laboratorio di Genetica, Istituti Clinici di Perfezionamento, Milano, ITALY, ⁶Clinica Pediatrica Università di Milano, Milano, ITALY, ⁷Institut fur Humangenetik, Otto-von-Guericke-Universität, Magdeburg, GERMANY, ⁸Department of Human Genetics, Charité, Campus Virchow, Humboldt-Universität, Berlin, GERMANY, ⁹Institute of Genetics, Bari, ITALY, ¹⁰Istituto di Genetica Medica, Universita Cattolica, Roma, ITALY, ¹¹Wessex Regional Genetics Laboratory, Salisbury Health Care Trust, Salisbury District Hospital, Salisbury, UNITED KINGDOM, ¹²Institut für Humangenetik, Universitaetsklinikum Essen, Essen, GERMANY, ¹³IRCCS Policlinico San Matteo, Pavia, ITALY.

The t(4;8)(p16;p23) translocation, either in the balanced or unbalanced form, has been reported in more than 14 cases. The detection of this rearrangement in routine cytogenetics is difficult. Its incidence, therefore, may be underestimated, and it could represent the second most frequent translocation in man, after the t(11q;22q). Der(4) patients have the Wolf-Hirschhorn syndrome whereas the der(8) subjects show a different spectrum of dysmorphic features. We had recently reported an inversion polymorphism of two olfactory receptors (OR)-gene clusters at 8p23 triggering de novo chromosomal rearrangements. Two OR-gene clusters are also present at 4p16. We thus investigated whether OR polymorphisms at 4p16 and 8p23 were involved in the t(4;8)(p16;p23). In one balanced and six unbalanced cases, we demonstrated that the translocation breakpoints fall at the OR-gene clusters. Heterozygous submicroscopic inversions at both the 4p-OR- and 8p-OR-gene clusters were found in all the five mothers at whose meiosis the translocation occurred. The heterozygous 4p16 inversion was found in 12% control subjects. We have previously found 8p inversion heterozygosity in 26% control subjects. In agreement with statistical expectation, 2.5% of the population was found double heterozygous. Our results suggest that rearrangements involving non-homologous chromosomes can occur as a consequence of specific genomic polymorphisms.

Complex chromosome rearrangement with neocentromere formation in a fetus with IUGR.

P. C. Warburton, J. Barwell, M. Splitt, D. Maxwell, C. Mackie Ogilvie;
Guy's Hospital, London, UNITED KINGDOM.

Neocentromeres are rare functional centromeres formed within non-centromeric chromosomal regions. We present a case of neocentromere formation detected prenatally. The karyotype was: 47,XY,del(4)(p15.3q21.1),+r(4)(p15.3q21.1).ish del(4)(D4S3360+,WHS+,D4Z1-,4qsubtel+),r(4)(D4S3360-,WHS-,D4Z1+,4qsubtel-)de novo. The fetus was missing a normal chromosome 4 but had a ring chromosome, consisting of the pericentromeric region of chromosome 4, and a deleted chromosome 4, the reciprocal product of the ring formation. In situ hybridization established that the chromosome 4 pericentromeric heterochromatin sequences were located on the ring chromosome whilst the Wolf-Hirschhorn critical region and chromosome 4 subtelomeric regions were present on the deleted chromosome. A constriction was observed in band 4q21 of the deleted chromosome 4, indicating that a neocentromere had been formed in this band, allowing stable segregation during cell division.
This chromosome abnormality was detected in cultured amniocytes from a 20 week pregnancy presenting with intrauterine growth retardation and echogenic bowel. The pregnancy resulted in intrauterine death at 33-34 weeks. On delivery, the baby weighed 640g (below the 0.4^th centile) and was macerated. Anal atresia and neck webbing were evident on clinical examination. The umbilical cord had two vessels. The placenta had a small infarct. Post mortem was not performed and no further tissue was available for karyotyping.
The case will be presented and discussed in the context of current understanding of centromere structure and function and of previously reported cases of neocentromere formation.

A. Shaw¹, I. van der Burgt², H. G. Brunner², K. Noordam², K. Kalidas¹, A. H. Crosby¹, A. Ion¹, S. Jeffery¹, M. A. Patton¹, M. Tartaglia³, B. D. Gelb³;
¹St George's Hospital Medical School, London, UNITED KINGDOM, ²University Hospital, Nijmegen, NETHERLANDS, ³Mount Sinai School of Medicine, New York, NY.

Background.
Noonan Syndrome (NS) is a multiple congenital abnormality syndrome characterised by short stature, typical facial appearance, pulmonary stenosis (PS), hypertrophic cardiomyopathy (HCM), pectus deformities and cryptorchidism. It has previously been mapped to 12q24 but NS is known to be genetically heterogeneous.
Mutations in the non-receptor protein tyrosine phosphatase gene PTPN11 have recently been shown to cause Noonan Syndrome in some patients. Molecular analysis may be useful to identify affected individuals, allowing more accurate genetic counselling and awareness of potential complications. Presence of the PTPN11 mutation may also predict the likelihood of specific features of NS.
Methods.
We reviewed the clinical and molecular findings of 127 patients who fulfilled the diagnostic criteria of NS. The incidence of the mutation was then compared with the presence of the major clinical features.
Results.
Mutations were identified in 52 (41%) of the group and were mostly concentrated in 2 exons. 70% of the mutation group had PS and 4% had HCM compared to 47% and 23% of the non mutation group (p=0.01 and 0.03) respectively. Frequency of short stature and pectus deformities was similar in the two groups. 76% of those with the mutation attended a mainstream school as opposed to 67% of those without and cryptorchidism was found in 84% and 68% respectively (not significant).
Conclusions.
Mutations in PTPN11 have improved the phenotypic recognition of NS and are associated with a high incidence of PS. HCM appears to be more common in other genetic forms of the syndrome.

A chromosomal translocation family and mutation detection identifies MAF as a new human disease gene in ocular anterior segment development

R. V. Jamieson¹^,2, R. Perveen¹, B. Kerr¹, M. Carette¹, N. Farrar¹, D. Donnai¹, F. Munier³, G. C. M. Black¹^,4;
¹St Mary's Hospital, Manchester, UNITED KINGDOM, ²Children's Hospital at Westmead, Sydney, AUSTRALIA, ³Hopital Ophtalmique Jules Gonin, Lausanne, SWITZERLAND, ⁴Manchester Royal Eye Hospital, Manchester, UNITED KINGDOM.

Chromosomal rearrangements, particularly those that are balanced, can provide a vital clue to the localisation of a gene of functional significance in the causation of the associated phenotype. There are many genes involved in the development of the anterior segment of the eye and this is reflected in the marked heterogeneity in the genetic causation of hereditary congenital cataract and anterior segment dysgenesis. We identified a three-generation family with cataract and Peters anomaly, where there was a chromosomal rearrangement in balanced and unbalanced forms with breakpoints at 16q23.2 and 5p15.3. Cloning of the 16q23.2 breakpoint identified a break through the genomic control domain of MAF, a basic region leucine zipper (bZIP) transcription factor, expressed in mammalian lens development. This breakpoint transected the common fragile site, FRA16D. Screening of other families and individuals with similar ocular phenotypes identified an R288P mutation in the highly evolutionarily conserved DNA-binding domain of MAF, in a three-generation family with cataract, microcornea and iris coloboma. The mutation co-segregated with disease in the family and was not present in 496 normal control chromosomes. The results in these two families indicate that the lens development gene, MAF, is a human disease gene in congenital cataract and anterior segment abnormality. These findings further implicate MAF in lens development and emphasise the importance of the lens in formation of the ocular anterior segment. The presence of iris coloboma in one of the families broadens the possible role of MAF in development of the anterior segment.

Analysis of the phenotypic abnormalities in Lymphoedema Distichiasis Syndrome in 74 patients with FOXC2 mutations or linkage to 16q24

S. Mansour, G. Brice, V. Murday, S. Jeffery, P. Mortimer;
St George's Hospital Medical School, London, UNITED KINGDOM.

Lymphoedema-Distichiasis Syndrome (LD)(OMIM 153400) is a rare, primary lymphoedema of pubertal onset, associated with distichiasis. Causative mutations have now been described in FOXC2, a forkhead transcription factor gene. Numerous clinical associations have been reported with this condition including congenital heart disease, ptosis, varicose veins, cleft palate and spinal extradural cysts. In this paper we now report clinical findings in 74 affected individuals, from 18 families and 6 isolated cases. All of these individuals were shown to have mutations in FOXC2 with the exception of one family who had two affected individuals with lymphoedema and distichiasis and linkage consistent with the16q24 locus.
The presence of lymphoedema was highly penetrant. Males had an earlier onset of lymphoedema and a significantly increased risk of complications. Lymphatic imaging confirmed the earlier suggestion that LD is associated with hyperplasia of the lymphatics rather than the hypo or aplasia seen in other forms of primary lymphoedema. Distichiasis was 94.2% penetrant, but not always symptomatic. Associated findings included ptosis (31%), congenital heart disease (6.8%) and cleft palate (4%). Other than distichiasis, the most commonly occurring anomaly was varicose veins of early onset (49%). This has not been previously reported and suggests a possible developmental role for FOXC2 in both venous and lymphatic systems. This is the first gene that has been implicated in the aetiology of varicose veins.

Mutations in the SIP1 gene cause a distinctive dysmorphic syndrome with or without HSCR

D. Mowat¹^,2, M. Wilson³, S. Worthington⁴, H. Kaarianen⁵, C. Curry⁶, S. Aftimos⁷, J. Clayton-Smith⁸, D. Donnai⁸, S. Braddock⁹, C. Barrey¹⁰, F. Dastot-Le Moal¹¹, V. Cacheux¹¹, M. Goossens¹¹;
¹Sydney Children's Hospital, Sydney, AUSTRALIA, ²University of New South Wales, Sydney, AUSTRALIA, ³The Children's Hospital at Westmead, Sydney, AUSTRALIA, ⁴Genetic Services of Western Australia, Subiaco, AUSTRALIA, ⁵The Family Federation of Finland, Helsinki, FINLAND, ⁶Valley Children's Hospital/UCSF, Madera, CA, ⁷Northern Regional Genetic Service, Auckland, NEW ZEALAND, ⁸St Mary's Hospital, Manchester, UNITED KINGDOM, ⁹University of Missouri-Columbia, Missouri, MO, ¹⁰Hopital Saint-Camille, Bry-sur-Marne, FRANCE, ¹¹INSERM u468 et service de Biochimie et Genetique, Hopital Henri Mondor, Creteil, FRANCE.

In 1998 we delineated a new syndrome of characteristic facial features- multiple congenital anomaly- mental retardation- Hirschsprung disease (HSCR) subsequently found to be caused by "new" dominant mutations in the ZFHX1B (or SIP1) gene. All the mutation positive cases from the literature (where photographs are presented) and our series show the same distinctive facial phenotype. The characteristic facial features may be used as an indicator for mutational analysis of the ZFHX1B gene in children. The initial cases were ascertained in the presence of HSCR as well as mental retardation but it is now apparent that this is not an invariable component of the syndrome. We illustrate this with the description of two further mutation positive cases where HSCR is not present. We also demonstrate the evolving facial features with age in our series of fifteen mutation positive cases (ten previously unpublished) to aid other clinicians in recognition of this syndrome even in the absence of HSCR. A low recurrence risk can be given to the parents when mutation analysis is positive.
We review the genotype-phenotype correlations in the 32 published cases and our 10 new cases with ZFHX1B mutations or deletions. All intragenic mutations so far reported have led to a premature stop codon suggesting that the mechanism for the phenotype is haplo-insufficiency with an altered gene dosage effect. It is important to test patients with Goldberg-Shprintzen syndrome (HSCR-mental retardation-microcephaly), especially those reported with sibling recurrence, to establish whether this is a separate disorder.

P. D. Turnpenny¹^,2, J. Duncan², S. Ellard²;
¹Royal Devon & Exeter Hospital, Exeter, UNITED KINGDOM, ²University of Exeter, Exeter, UNITED KINGDOM.

Abnormal vertebral segmentation (AVS) is an important diagnostic handle and occurs in a wide variety of syndromes, e.g. Goldenhar/facio-auriculo-vertebral spectrum, VATER/VACTERL association, trisomy 8 mosaicism and maternal diabetes. However, the use of terminology in clinical practice is not consistent and the literature is correspondingly confusing for a wide variety of radiological phenotypes. The identification of a number of murine somitogenesis genes with important roles in normal development of the axial skeleton makes it possible to approach this complex field systematically. In man, mutations in genes of the Notch signalling pathway are the first to enable a classification based on genotype-phenotype correlation. Jagged1 is implicated in Alagille syndrome, which includes butterfly vertebrae, and DLL3 in autosomal recessive spondylocostal dysostosis (SCD), in which a consistent pattern of AVS throughout the spine occurs in association with rib fusions. We propose the designation SCD type 1 for cases due to mutated DLL3 and SCD type 2 for similar phenotypes not DLL3-linked. There is no definite evidence as yet that DLL3 is implicated in autosomal dominant forms of AVS. Mutations in ROR2 cause autosomal recessive Robinow syndrome, whilst Jarcho-Levin syndrome (spondylothoracic dysostosis/dysplasia) has been mapped to 2q32.1. These are specific recognisable entities which are usually distinguishable from the many sporadic cases of AVS. We propose a system of classification based on syndromic AVS, ‘pure‘ SCD due to disrupted somitogenesis, neural tube associated AVS, and a large group of unknowns which will become the focus of new research from murine derived candidate genes.

K. W. Kjaer¹, J. Hedeboe², M. Bugge¹, C. Hansen¹, K. Friis-Henriksen¹, J. M. Opitz³, N. Tommerup¹;
¹Wilhelm Johannsen Centre for Functional Genome Research, Panum Institute, University of Copenhagen, Copenhagen, DENMARK, ²Department of Orthopedic Surgery, Næstved Hospital, Næstved, DENMARK, ³Department of Pediatrics (Medical Genetics), Human Genetics, Obstetrics and Gynecology, University of Utah, Salt Lake City, UT.

We are establishing a national DNA/cell bank for congenital limb defects together with the major hospitals in Denmark, including ~100 families referred to genetic counselling at the Department of Medical Genetics, University of Copenhagen. Among these are the families reported by Thomsen (1927), Kemp and Ravn (1932) and Tage-Hansen (1938), and the 625 probands with reduction defects reported by Birch-Jensen (1949) in his doctoral thesis "Congenital Deformities of the Upper Extremities". Thomsen and Kemp/Ravn in two classical papers described two large families with autosomal dominant axial synpolydactyly (SPD) with remarkable phenotypic differences. Presently, we have extended these families to 10 and 8 generations (169 and 366 individuals), and identified the mutations as 9- and 7-residue polyalanine tract expansions of HOXD13, respectively. The developmental field affected by the shorter expansion involves digits 1-5 (including syndactyly 1-2 and duplication of the index finger) whereas the 9-residue polyalanine expansion affects digits 3-4 (synpolydactyly). The large sizes of these two families permits a detailed genotype-phenotype study, which so far confirms an enormous variability of expressivity. In one carrier of the 9-residue expansion, abnormal flexion creases were the only visible trait affected, suggesting that inclusion of this trait may increase the penetrance in other SPD families. The access to a large number of probands and families with limb defects, which often can be traced and reinvestigated over many generations, and the nation-wide public health-care system with centralized registration of all individuals in Denmark provides a unique back-bone for identifying novel limb defect genes and phenotypes.

Triallelic inheritance in Bardet-Biedl syndrome, a Mendelian recessive disorder.

N. Katsanis¹, S. J. Ansley¹, J. L. Badano¹, E. R. Eichers¹, R. A. Lewis¹^,2^,3^,4^,5, B. Hoskins⁶, P. J. Scambler⁶, W. S. Davidson⁷, P. L. Beales⁶, J. R. Lupski¹^,3^,5;
¹Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, ²Department of Ophthalmology, Baylor College of Medicine, Houston, TX, ³Department of Pediatrics, Baylor College of Medicine, Houston, TX, ⁴Department of Medicine, Baylor College of Medicine, Houston, TX, ⁵The Texas Children’s Hospital, Baylor College of Medicine, Houston, TX, ⁶Molecular Medicine Unit, Institute of Child Health, University College, London, UNITED KINGDOM, ⁷Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, CANADA.

Bardet-Biedl syndrome (BBS) is a genetically heterogeneous disorder characterized by multiple clinical features that include retinal dystrophy, polydactyly, obesity, developmental delay, and renal defects. Analysis of pedigree structures led historically to the hypothesis that this disorder is inherited in an autosomal recessive fashion; subsequent positional cloning efforts identified the first three of at least seven BBS genes (BBS2, BBS4 and BBS6). We have screened our cohort of 163 BBS families for mutations in these genes and, when possible by family size, have constructed haplotypes across all known BBS regions. We report the presence of three mutant alleles in affected individuals in several BBS pedigrees. Patients in three pedigrees have two mutations in BBS2 and one mutation in BBS6, and the converse occurred in a fourth pedigree. In a fifth pedigree, the patient inherited two BBS2 and two BBS4 mutations. Finally, four pedigrees carried a single BBS2 mutation but have been excluded genetically from BBS2, whereas another three pedigrees carry a single BBS4 mutation but have likewise been excluded genetically from BBS4. We propose that BBS may not be a single-gene recessive disorder but a complex trait, possibly requiring the participation of multiple loci to manifest the phenotype. Consistent with this hypothesis, in two pedigrees segregating three BBS mutant alleles, we have identified unaffected individuals who carry two BBS2 mutations but not a BBS6 mutation. This model of disease transmission may be important in the study of genetic heterogeneity in recessive disorders and for modeling gene interactions in complex traits.

A Mutation in ARH Gene and a Chromosome 13q Locus Influence Cholesterol Levels in a New Form of Digenic Recessive Familial Hypercholesterolemia

H. A. Al-Kateb¹^,2^,3, S. Baehring⁴, K. Hoffmann⁴, K. Strauch⁵, A. Busjahn⁴, G. Nurenberg⁴, M. Jouma², E. Bautz³, H. Dresel³, F. C. Luft⁴;
¹Max-Delbruck-Center for Molecular Medicine, Berlin, GERMANY, ²Damascus University, Damascus, SYRIAN ARAB REPUBLIC, ³Heidelberg University, Heidelberg, GERMANY, ⁴Max-Delbruck-Center, Berlin, GERMANY, ⁵Institute for Medical Biochemistry, Informatics and Epidemiology, Bonn, GERMANY.

The heterogeneity of familial hypercholesterolemia (FH) is being unravelled by the discovery of novel loci contributing to this disease. Recently; mutations in a new gene, termed LDL receptor adaptor protein have been discovered in families with recessive FH. We encountered a Syrian family in which the parents and 3 of their 6 children are apparently normal and have normal total and LDL cholesterol levels. In contrast, the other three offspring have high total and LDL cholesterol levels and large xanthomas. The extended pedigree allowed us to examine another 64 members of the family. We performed a wide-genome scan in the core family and found a significant linkage to chromosome 1 p36-p35. Surprisingly, we also found a linkage to chromosome 13q32-q22 with the same power as on the chromosome 1 locus. We, therefore, performed an interaction analysis and found supportive evidence that mutations at both loci are indispensable to display the phenotype. We also genotyped our twin panel for the informative markers at both loci and found that both loci contribute quantitatively to both total and LDL cholesterol level. We also identified the exon-intron positions of the LDL receptor adaptor protein and sequenced the gene in our family. The mutation in our family is a transversion mutation that affects the splice-accpetor site of intron 1 converting it from AG to AC. The gene on chromosome 1 locus has been already identified. Identifying chromosome 13 locus and elucidating the mechanisms by which these two genes act will be of a major importance.

Large deletion of GJB6 gene in deaf patients heterozygous for GJB2 gene : genotype and phenotype analysis

S. Marlin¹, F. Denoyelle², D. Feldmann³, I. Del Castillo⁴, S. Odent⁵, A. Joannard⁶, F. Moreno⁴, N. Garabedian², C. Petit⁷;
¹Unité de Génétique, Hôpital d'Enfants Armand Trousseau, AP-HP, Paris, FRANCE, ²Service d’ORL, Hôpital d'Enfants Armand Trousseau, AP-HP, Paris, FRANCE, ³Service de Biochimie, Hôpital d'Enfants Armand Trousseau, AP-HP, Paris, FRANCE, ⁴Unidad de Genetica Molecular, Hospital Jamon y Cajal, Madrid, SPAIN, ⁵Unité de Génétique, Hôpital de Pontchaillou, Rennes, FRANCE, ⁶Service de Pédiatrie, CHU, Grenoble, FRANCE, ⁷Unité de Génétique des Déficits Sensoriels, Institut Pasteur, Paris, FRANCE.

Hearing loss is the most frequent sensorial defect. At birth, 1/1000 child presents with a severe or profound hearing loss. 60% of the prelingual hearing loss are presumed to have a genetic origin. GJB2 gene represents the major genetic form of prelingual deafness as it accounts for 40% of the congenital hearing loss. GJB2 gene has been analysed in 206 independant patients with non syndromic prelingual hearing loss. 60 of them have a biallelic mutation in GJB2 (31 homozygous 35delG). Of the 36 deaf patients heterozygous for a mutation in GJB2 (12 patients 35delG/+, one V37I/+), 13 carry a deletion in trans implicating GJB6. In all families, the molecular anomalies in GJB2 and GJB6 segregate with the hearing impairement. We have determined the phenotype of the 13 composite GJB2/GJB6 heterozygous patients and have compared it to the clinical signs presented by the patient homozygous for GJB2 mutations.

J. E. A. Common, W. Di, I. M. Leigh, D. P. Kelsell;
Barts and The London Queen Mary's School of Medicine and Dentistry, London, UNITED KINGDOM.

Distinct mutations in four connexins, Cx26, Cx30.3, Cx30 and Cx31, have been found to underlie sensorineural non-syndromic hearing loss (NSHL) and/or three types of hyperproliferative epidermal disease: Vohwinkel's syndrome, Hidrotic Ectodermal Dysplasia (HED) and Erythrokeratoderma variabilis (EKV).
Using PCR based analysis with dHPLC technology, we have identified a number of new mutations (both dominant and recessive) and coding polymorphisms in these four connexin genes. These and previously identified mutations have been further characterised. GFP-tagged connexin fusion proteins have been used to study mutant connexins particularly with respect to junction assembly and channel function in keratinocytes and HeLa cells. After transfection, the wildtype GFP-Cx30 fusion protein was localised at the plasma membrane in a characteristic punctate pattern showing functional gap junctions between adjoining cells. A similar localisation was observed for the NSHL mutation Cx30Thr5Met. In contrast to the wildtype and Thr5Met, the HED associated Cx30Gly11Arg and Cx30Ala88Val fusion proteins were localised to the cytoplasm. Similar genotype-phenotype differences were observed when analysing Cx26 and Cx31 mutations.
In summary, localisation data indicate that skin disease associated mutations impair protein trafficking to the plasma membrane. In contrast, NSHL mutations are capable of forming gap junction like structures at the plasma membrane but preliminary dye transfer studies suggest defective channel activity. These data show that mutations in connexin molecules can result in distinct junctional assembly and channel properties that may account for the different effects of particular mutants on epidermal function and auditory transduction.

Inherited glomuvenous malformations are caused by the combination of a germline and a somatic “second hit” mutation in the glomulin gene

P. Brouillard¹, M. Ghassibe¹, L. Boon², O. Enjolras³, J. Mulliken⁴, M. Vikkula¹;
¹Christian de Duve Institute of Cellular Pathology, Université catholique de Louvain, Brussels, BELGIUM, ²Center for Vascular Anomalies, Division of Plastic Surgery, Université catholique de Louvain, Brussels, BELGIUM, ³Consultation des Angiomes, Hôpital Lariboisière, Paris, FRANCE, ⁴Vascular Anomlies Center, Children's Hospital, Harvard Medical SChool, Boston, MA.

Glomuvenous malformations (GVMs), localized defects of vascular morphogenesis, are single or multiple bluish-purple lesions that occur mainly in skin. Histologically, the distended veins present smooth muscle-like "glomus cells" in the media. GVMs are usually dominantly inherited and, with the more common mucocutaneous venous malformations, they are among the most frequent lesions in centers that specialize in treatment of vascular anomalies (Vikkula et al., 1998). In another abstract submitted to this meeting, we describe the criteria for clinical differential diagnosis between common VM and GVM (Boon et al.).
Using positional cloning, we recently identified the causative gene that we named glomulin (Brouillard et al., AJHG in press). As 13 of the 14 mutations identified in 20 families cause premature stop codons, GVMs are likely to be caused by loss-of-function of glomulin. As these inherited vascular lesions are localized, we hypothesized that haploinsufficiency is not enough for the development of lesions, but a somatic second hit, leading to complete localized lack of glomulin, is needed (Knudson's double-hit hypothesis for retinoblastoma). We have now screened for somatic mutations in GVM lesions, and report on the identification of a truncating mutation that was different from the patient’s inherited genetic alteration and that was not seen in genomic DNA extracted from blood. Thus, it is a de novo somatic mutation in DNA of the GVM lesion. Other tissue samples are being tested to confirm this promising finding. These data support our hypothesis that GVMs are due to complete localized loss of glomulin function. (vikkula@bchm.ucl.ac.be)

Coding region mutations in three acyl-CoA dehydrogenase genes may have unforeseeable consequences due to disruption of potential splice enhancer sequences

B. S. Andresen¹, K. B. Nielsen², T. J. Corydon², L. D. Schroeder², J. Kjems², N. Gregersen²;
¹Aarhus University, Aarhus N., DENMARK, ²Aarhus University, Aarhus, DENMARK.

It is becoming clear that coding region mutations do not always exert their effect simply by changing the amino acid sequence of the encoded protein. In order to examine the frequency and mechanisms underlying missplicing caused by simple coding region mutations, we have analyzed cDNA from a large number of alleles with disease-causing mutations in either of three acyl-CoA dehydrogenase (MCAD, VLCAD and SBCAD) genes. In all three genes we have identified missense mutations that despite the fact that they are located far from the exon-intron junctions lead to exon skipping as the main molecular defect. These mutations were always located in exons with suboptimal splice sites indicating a requirement for splice enhancers. Using transfection studies in CHANG- and COS-cells with wild-type and mutant MCAD minigene constructs, we have so far reproduced the exon skipping caused by one of the missense mutations (362C>T). Computer analysis indicated that this mutation disrupts an exonic splice enhancer (ESE) consensus sequence recognized by SF2/ASF, and this was examined by mutagenesis of neighboring positions. The missplicing could be corrected by cotransfection of the splicefactor SF2/ASF, and other SR-proteins also had a correcting effect. Similarly, we are underway with minigene based analysis of SBCAD and VLCAD missense mutations that are indicated to cause exon skipping as a result of splice enhancer inactivation.
We conclude that it is not uncommon that simple coding region mutations lead to disruption of ESE sequences, which are necessary for correct splicing of exons with suboptimal splice consensus sequences.

C. Godfraind¹, E. Rousseau¹^,2, M. Ruchoux³, F. Scaravilli⁴, M. Vikkula²;
¹Division of Neuropathology, Cliniques universitaires St-Luc, Université catholique de Louvain, Brussels, BELGIUM, ²Laboratory of Human Molecular Genetics, Christian de Duve Institute of Cellular Pathology, Université catholique de Louvain, Brussels, BELGIUM, ³Department of Neuropathology, Hôpital R. Salingro, Lille, FRANCE, ⁴Institute of Neuropathology, London, UNITED KINGDOM.

Oligodendroglioma is a tumour originating from oligodendrocytes, the myelin forming cells in the central nervous system. This glioma preferentially occurs in adults. It is mostly located in cerebral hemispheres with a predilection to the frontal lobe. This lesion accounts for 5-33% of all gliomas. The wide range reported for tumour occurrence reflects inter-observer discordance in histological diagnosis. Genetic analysis of oligodendroglioma has associated 1p/19q-deletions to chemosensitivity. Recently, we and others have linked a specific histological definition to this subgroup of tumors, allowing the diagnosis to be made on histological criteria prior to any genetic analysis.
Now, we have studied 9p21 deletions, p14 and p16 methylation as well as p14 and p16 mutations in a series of 21 1p/19q-deleted oligodendrogliomas. On 8 1p/19q-deleted oligodendrogliomas, presenting angiogenesis and/or necrosis, 6 had a heterozygous and 2 a homozygous deletion of 9p21. Three of them also had methylation of p16, which in one case was associated with a p16 mutation and in another to p14 methylation. These results illustrate the implication of 9p21-deletion in angiogenesis and tumor necrosis of 1p/19q-deleted oligodendrogliomas and the putative role of p16. (vikkula@bchm.ucl.ac.be)

Cyclin L/Ania-6a, a Gene located at 3q25, is amplified and overexpressed in a Head and Neck Cancer Cell Line.

R. Redon¹, T. Hussenet¹, K. Caulee¹, D. Muller², J. Abecassis², S. du Manoir¹;
¹IGBMC, Illkirch, FRANCE, ²Centre Paul Strauss, Strasbourg, FRANCE.

DNA gains or amplifications on the long arm of chromosome 3 are recurrent in solid tumors from various origins, i.e. head and neck, lung, uterine cervix and ovary. To systematically map 3q amplicons in these tumors, we designed a chromosome 3 BAC/PAC array for high resolution Comparative Genomic Hybridization (or CGH array). We demonstrated, with cell lines containing constitutional chromosome 3 abnormalities, that CGH array allows the detection of low-level DNA copy number changes. By this method, we found a narrow (less than 6 Mb) high-level amplification at 3q25.3 in a head and neck cell line, Cal 27. Further mapping of the amplification by semi-quantitative PCR showed a core amplicon of 3Mb.
We performed the transcriptional comparison of Cal 27 and Hs 677.Tg, a normal head and neck cell line, with cDNA microarrays. Among 438 genes mapped on chromosome 3, the one showing the greatest overexpression (10-fold increase) is located in the core amplicon, and encodes a protein from the cyclin family, named cyclin L/ania-6a. According to these results, we propose cyclin L/ania-6a as a new oncogene at 3q25.3. Its involvement in head and neck cancer is currently evaluated by cyclin L expression measurement in a series of primary tumors.

Functional analysis of MMR gene mutations linked to hereditary non-polyposis colorectal cancer

M. Nyström-Lahti, R. Kariola, T. Raevaara, K. Lönnqvist;
University of Helsinki, Helsinki, FINLAND.

To date, five mismatch repair (MMR) genes, MLH1, MSH2, MSH6, MSH3, and PMS2 are known to be involved in human MMR function. Two of those, MLH1 and MSH2 are further the most common susceptibility genes in hereditary non-polyposis colorectal cancer (HNPCC), whereas MSH3 and PMS2 is not or only in few cases, respectively shown to be involved in HNPCC. Although, the ever-increasing number of mutations is reported in the MSH6 gene, the mutations are mainly linked to putative HNPCC families. Especially, the early age at onset and high microsatellite instability (MSI) in tumors, the main hallmarks in HNPCC, are not typical for MSH6 mutation carriers. Based on the less typical clinical and molecular features linked to especially MSH6 but also to some MLH1 and MSH2 mutation carriers and their cancers, there is a cause to ask what really is HNPCC syndrome. High MSI is a consequence of MMR defect in the cell and consistently, the pathogenicity of germline mutations in HNPCC is linked to malfunction of MMR. To address the question, we studied the functionality of mutated MMR proteins in an in vitro MMR assay. Our results are clinically relevant since they demonstrate that in the stable in vitro circumstances, when the amounts of the proteins are adequate for repair, many mutations found from putative HNPCC families do not affect the repair function, whereas all the tested mutations found from typical HNPCC families impart a MMR deficient phenotype on the altered polypeptide.

CHK2 1100delC is a low penetrance breast cancer susceptibility allele in non-carriers of BRCA1 or BRCA2 mutations

N. Rahman¹, H. Meijers-Heijboer², M. Schutte², N. Sodha¹, D. F. Easton³, M. R. Stratton and the Breast Cancer Linkage Consortium¹^,4;
¹Institute of Cancer Research, Surrey, UNITED KINGDOM, ²Erasmus Medical Center, Rotterdam, NETHERLANDS, ³CRC Genetic Epidemiology Unit, Cambridge, UNITED KINGDOM, ⁴Wellcome Trust Sanger Institute, Cambridge, UNITED KINGDOM.

Mutations in the two major breast cancer predisposition genes, BRCA1 and BRCA2, confer a high risk of breast and ovarian cancer but only account for a small fraction of breast cancer susceptibility. As part of a search for additional susceptibility genes, we analysed CHK2, a cell cycle checkpoint kinase that is implicated in DNA repair processes involving BRCA1 and p53. We show that CHK2 1100delC, a truncating variant that abrogates the kinase activity, has a frequency of 1.1% in healthy controls. This indicates that this variant cannot be acting as a high penetrance Li-Fraumeni predisposition allele, as previously postulated, because this syndrome is very rare. However, CHK2 1100delC is present in 5.1% of breast cancer cases from 718 BRCA1/2 negative breast cancer families (p=.00000003), including 13.5% of cases from families with male breast cancer cases (p=.00015). In contrast, the variant confers no increased cancer risk in carriers of BRCA1 or BRCA2 mutations. This suggests that the biological mechanisms underlying the elevated risk of breast cancer in CHK2 mutation carriers are already subverted in BRCA1/2 mutation carriers, consistent with the participation of the encoded proteins in the same pathway. We estimate that CHK2 1100delC confers an approximately two-fold increased breast cancer risk in women and 10-fold risk in men, and that approximately 1% of female breast cancer incidence, 9% of male breast cancer incidence and 0.5% of the familial aggregation of the disease is attributable to CHK2 1100delC.

E. Seemanová¹, P. Jarolím², J. Janda¹, J. Koutecký¹, J. Starý³, P. Seeman¹, R. Varon⁴, K. Sperling⁴;
¹Charles University, Prague, CZECH REPUBLIC, ²Institute of Haematology and Blood Transfusion, Prague, CZECH REPUBLIC, ³University Hospital Motol, Prague, CZECH REPUBLIC, ⁴Humboldt University, Berlin, GERMANY.

The chromosomal instability disorder Nijmegen Breakage Syndrome (NBS) is associated with extreme susceptibility to lymphoid malignancies due to a defect in DNA double strand break repair. Based on the „index-test-method“ we accumulated evidence that not only the homozygous patients, but also heterozygote individuals have an increased cancer risk. In order to verify this observation in an independent study, we initiated an epidemiological investigation to estimate the frequency of the major NBS1 mutation, 657del5, in new-borns; adult blood donors; and elderly over 70 years of age in comparison to its frequency among oncological patients, both children and adults, all of Czech origin.
The incidence of NBS heterozygotes was as follows: new-borns 1:158 (4/630), adult blood donors 1:226 (4/908), elderly 1:400, children with benign tumours 1:178 and with malignant tumours 1:174 (2/348), and adult patients with malignancies 1:293. In addition to the 13 heterozygotes for the 657del5 mutation, we found one NBS homozygote (5,5 years old) with ALL. The malignant tumours of the two heterozygous children were medulloblastoma and osteosarcoma. The adult patient suffered from a non-Hodgkin lymphoma. Clearly, the differences in the frequency of heterozygotes among the various groups are not significant. The number of individuals studied is sufficient to rule out that the general cancer risk in NBS heterozygotes is highly increased, however this does not exclude that the risk for a subset of tumours might be increased.
Supported by IGA NH/6439-3, by GA UK 45/2000 and by VZ 11100003

H. C. Duba¹, T. Kühr², A. Mehringer¹, M. Erdel¹, G. Utermann¹, J. Thaler²;
¹Inst.f.Med.Biologie und Humangenetik, Innsbruck, AUSTRIA, ²IV. Interne Abteilung, A.ö.Krankenhaus der Kreuzschwestern, Wels, AUSTRIA.

Interferon-alpha alone or in combination with cytostatic drugs can induce major and durable cytogenetic responses in about 25% of chronic myelogenous leukaemia (CML) patients. Since these patients have a significant survival benefit, frequent follow up investigations have become clinically important. It has recently been shown by Mühlmann et al. [Gene Chromosome Canc 21; 90-100; 1998] that fluorescence in situ hybridisation (FISH) on peripheral blood samples reveals results comparable with conventional cytogenetics and reduces the number of bone marrow aspirations. We therefore have included a FISH examination in 3-monthly intervals in a prospective CML study where patients are treated with Interferon-alpha and YNK1 (oral cytarabine). For FISH we have used a highly sensitive BCR/ABL D-FISH probe. It has recently been shown by Huntly et al. [Blood, 98, 1732-1738, 2001] that with this probe deletions of the derivative chromosome 9 can be detected. These deletions provide a powerful and independent prognostic indicator in CML. From 138 of the 150 patients included in our study a total of 420 cytogenetic examinations have been performed. The majority were FISH analyses on peripheral blood samples. Additionally GTG/QFQ-banding and FISH on bone marrow samples have been performed in a subset of patients. We have detected deletions in 12,6% (16/138) of patients. A BCR/ABL sensitive D-FISH for detection of the Philadelphia chromosome and deletions of the derivative chromosome 9 should therefore be incorporated into future diagnostic strategies as well as management decisions of CML.

D. P. Germain¹, L. Caplan², C. M. Eng³, N. Guffon⁴, P. Lee⁵, G. Linthorst⁶, S. Waldeck⁷, W. R. Wilcox⁸, R. J. Desnick⁹;
¹Hopital Européen Georges Pompidou, Paris, FRANCE, ²Beath Israel Deaconess Medical Center, Boston, MA, ³Baylor College of Medicine, Houston, TX, ⁴Hopital Edouard Herriot, Lyon, FRANCE, ⁵University College Hospital, London, UNITED KINGDOM, ⁶Academisch Medisch Centrum, Amsterdam, NETHERLANDS, ⁷Hope Hospital Salford, Manchester, UNITED KINGDOM, ⁸Cedars-Sinai Burns and Allen Research Institute, Los Angeles, CA, ⁹Mount Sinai School of Medicine, New York, NY.

Background: Fabry disease, lysosomal alpha-galactosidase A (alpha-Gal A) deficiency, results from progressive accumulation of globotriaosylceramide (GL-3), particularly in the microvasculature, leading to failure of target organs, and to ischemic complications involving kidneys, heart and brain. Recombinant human alpha-GalA (r-h-alpha-GalA) enzyme replacement therapy was previously evaluated in a randomized placebo-controlled, double-blind study of 58 Fabry patients who received r-h-alpha-GalA or placebo every 2 weeks for 20 weeks. The accumulated GL-3 was cleared in renal (P<0.001), cardiac (P<0.001) and skin (P<0.001) capillary endothelial cells of the treated group (N Engl J Med 2001; 345: 9-16).
Methods: Of these patients, 55/58 (95%) have continued treatment with r-h-alpha-GalA (1 mg/kg q 2wk) for an additional 12 months in an open-label extension study.
Results: The drug was well tolerated except for minor, conservatively managed infusion reactions, the incidence of which decreased over time. The infusion reactions were associated with IgG antibody seroconversion that did not affect therapeutic effect. The IgG titers in 50% of patients decreased by at least four-fold with time. Mean infusion time decreased to 2.25 hr. Renal function remained stable throughout the study, and subset analysis of high-risk
populations (age>35 and creatinine clearance<80ml) also revealed stability
at 18 months. The median percent reduction in plasma GL-3 was 100% after 12 months. GL-3 was also cleared/reduced in various renal and skin cell types. Pain scores measured by the Short Form McGill Pain questionnaire improved with therapy.
Conclusions: Long term therapy with r-h-alpha-GalA is safe, well-tolerated, reverses the disease pathology, and is clinically beneficial.

L. Van Maldergem¹, J. Magré², T. Gedde-Dahl Jr³, E. Khallouf⁴, M. Lathrop⁵, J. Capeau², S. O'Rahilly⁶, .. and the Berardinelli-Seip Group¹;
¹Institut de Pathologie et de Génétique, Loverval, BELGIUM, ²Inserm U 402, Faculté de Médecine Saint Antoine, Paris, FRANCE, ³Institute of Forensic Medicine, Rikshospitalet, Oslo, NORWAY, ⁴Hotel-Dieu de France, Beirut, LEBANON, ⁵Centre de Génotypage, Evry, FRANCE, ⁶Department of Medicine and Clinical Biochemistry, Addenbrooke's Hospital, Cambridge, UNITED KINGDOM.

Generalised lipodystrophy of the Berardinelli-Seip type (BSCL) is a rare autosomal recessive human disorder with severe adverse metabolic consequences. A locus on chromosome 9 (BSCL1) has recently been identified, predominantly in African-American families. More recently, mutations in a previously undescribed gene of unknown function (BSCL2) on chromosome 11, termed seipin, have been found to be responsible for this disorder in a number of European and Middle Eastern families. We have studied the genotype/phenotype relationships in 71 affected subjects from 45 apparently unrelated pedigrees. In all subjects, hepatic dysfunction, hyperlipidaemia, diabetes mellitus and hypertrophic cardiomyopathy were significant contributors to morbidity with no clear differences in their prevalence between subjects with BSCL1, BSCL2 and those with evidence against linkage to either chromosomes 9 or 11 (designated BSCLX). BSCL1 appears to be a somewhat less severe disorder than BSCL2 with a lower incidence of premature death and a higher frequency of subjects with partial and/or delayed onset of lipodystrophy. Notably, subjects with BSCL2 had a significantly higher prevalence of intellectual impairment (36/45) than those with BSCL1 (2/22) (p<0.001) or BSCLX (0/3). In summary, generalised lipodystrophy is heterogenous in nature encompassing at least three autosomal recessive conditions. While the consequences for metabolic derangement, hepatic dysfunction and cardiac enlargement appear similar between these three groups, subjects with seipin mutations (BSCL2) appear to have a markedly higher prevalence of intellectual impairment and a higher incidence of premature death, findings which have major implications for genetic counselling

Lamin A/C mutations in Charcot-Marie-Tooth disorder identify a novel laminopathy in human and mouse

N. Levy¹, A. De Sandre-Giovannoli¹, M. Chaouch², S. Kozlov³, J. Vallat⁴, M. Tazir⁵, N. Kassouri², P. Szepetowski¹, T. Hammadouche⁶, I. Boccaccio¹, D. Grid⁷, C. L. Stewart³;
¹Inserm U491 : Génétique Médicale et Développement, Faculté de médecine de la Timone, Marseille, FRANCE, ²Service de Neurologie, CHU Ben Aknoun, Alger, ALGERIA, ³Cancer and Developmental Biology laboratory, NCI-Frederick, Frederick, MD, ⁴Service de neuropathologie, CHU Dupuytren, Limoges, FRANCE, ⁵Service de neurologie, CHU Mustapha, Alger, ALGERIA, ⁶Institut Pasteur, Alger, ALGERIA, ⁷Genethon III, Evry, FRANCE.

Charcot-Marie-Tooth disease (CMT) is one of the most common inherited neurological disorders, affecting 1/2500 individuals. On the basis of electrophysiologic criteria these motor and sensory peripheral neuropathies have been divided into two main groups: the demyelinating (CMT1), and the axonal types (CMT2). Homozygosity mapping, performed on 23 consanguineous algerian families including patients affected with axonal autosomal recessive Charcot-Marie-Tooth neuropathy, evidenced linkage to the 1q21.2-q21.3 region in three families. The maximal pairwise lod score, was 4.14 (q=0) at D1S2721 with the linkage interval extending from D1S305 to D1S2635. By using a candidate gene approach, homozygous LMNA (lamin A/C) founder mutation (C892T) was identified in all affected patients, causing an Arg>Cys subsitution at the highly conserved residue 298 (R298C). C892T is a founder mutation arisen on an ancestral haplotype that we identified as covering a 1.7 cM genetic distance. This change, predicted to impair protein-protein interactions, affects all 4 isoforms derived from the gene. The ultrastructural analysis of sciatic nerves from Lmna null mice evidenced a peripheral axonopathy highly resembling to AR-CMT2, while the heterozygous knock out mice nerves were unaffected or harboured only minor changes such as slight neurofilaments accumulation. We will present genetic, histopathologic, and functional data regarding lamins involvment in peripheral nerve structure and function. The responsability of LMNA, encoding lamin A/C nuclear envelope proteins, in the pathogenesis of myopathic phenotypes (Emery-Dreifuss myopathy, Limb Girdle Muscular Distrophy and Dilated Cardiomyopathy) and, now, of autosomal recessive axonal CMT, gives new important clues to the comprehension of nerve-muscle interactions and relationships.

Mutational Spectrum in the PEX7 Gene and Functional Analysis of Mutant Alleles in 78 Patients with Rhizomelic Chondrodysplasia Punctata Type 1

H. R. Waterham, A. Motley, P. Brites, L. Gerez, E. Hogenhout, J. Haasjes, R. Benne, H. F. Tabak, R. J. A. Wanders;
Academic Medical Center, University of Amsterdam, Amsterdam, NETHERLANDS.

Rhizomelic chondrodysplasia punctata (RCDP) is a genetic heterogeneous, autosomal recessive disorder of peroxisomal metabolism, clinically characterized by symmetrical shortening of the proximal long bones, cataracts, periarticular calcifications, multiple joint contractures, and psychomotor retardation. Most patients with RCDP have mutations in the PEX7 gene encoding peroxin 7, the cytosolic PTS2 receptor protein required for targeting a subset of enzymes to peroxisomes. These enzymes are deficient in cells of RCDP patients due to their mislocalisation to the cytoplasm.
Here, we report the mutational spectrum in the PEX7 gene of 78 patients (including 5 pairs of sibs) clinically and biochemically diagnosed with RCDP type I. We found 22 different mutations including 18 novel ones. Furthermore, we show by functional analysis that disease severity correlates with PEX7 allele activity: expression of 8 different alleles from severe RCDP patients failed to restore the targeting defect in RCDP fibroblasts, while two alleles found only in mild patients complemented the targeting defect upon overexpression. Surprisingly, one of the mild alleles comprises a duplication of nucleotides 45-52 predicted to lead to a frameshift at codon 17 and no functional peroxin 7. The ability of this allele to complement the targeting defect in RCDP cells suggests that frame restoration occurs resulting in full-length functional peroxin 7, which leads to amelioration of the predicted severe phenotype. This was confirmed in vitro by expression of the 8nt-duplication-containing sequence fused in different reading frames to the coding sequence of firefly luciferase in COS cells.

Mutations in DNAH5 cause primary ciliary dyskinesia and randomization of left-right asymmetry

H. Olbrich¹, K. Häffner¹, K. Andreas², A. Völkel¹, G. Sasmaz¹, R. Reinhardt³, S. Hennig³, H. Lehrach³, N. Konietzko⁴, M. Zariwala⁵, P. G. Noone⁵, M. Knowles⁵, H. M. Mitchison⁶, M. Meeks⁶, E. M. K. Chung⁶, F. Hildebrandt¹, R. Sudbrak³, H. Omran¹;
¹University Children's Hospital, Freiburg, GERMANY, ²Medizinische Hochschule, Hannover, GERMANY, ³Max-Planck Institute for Molecular Genetics, Berlin, GERMANY, ⁴Ruhrland-Klinik, Essen, GERMANY, ⁵University of North Carolina, Chapel Hill, NC, ⁶Royal Free and University College, London, UNITED KINGDOM.

Primary ciliary dyskinesia (PCD, MIM 242650) is characterized by recurrent infections of the lower and upper respiratory tract due to reduced mucociliary clearance. Other manifestations of the disesae are reduced fertility. Half of the affected off-spring exhibit a situs inversus because of randomization of left-right asymmetry. We previously localized a PCD locus to chromosome 5p, containing DNAH5 encoding a protein highly similar to the Chlamydomonas gamma- dynein heavy chain (DHC). We characterized the full-length 14-kb transcript of DNAH5. DNAH5 encodes a DHC containing a motor domain with six tandemly linked AAA (ATPases associated with diverse cellular activities) modules. Computational analysis identified three domains with strong prediction for coiled-coil domains in the c-terminal portion of the protein. Using in situ hybridization we show that the mouse ortholog Dnahc5 is expressed in respiratory epithelia, brain and the node. Sequence analysis in affected individuals of eight PCD-families with randomization of LR asymmetry identified mutations resulting in non-functional DNAH5 proteins.

M. Witsch-Baumgartner¹, M. Gruber¹, P. Clayton², N. Clusellas³, D. Haas⁴, R. Kelley⁵, M. Giros³, H. Kraft¹, M. Krajewska-Walasek⁶, G. Utermann¹;
¹Institute of Medical Biology and Human Genetics, Innsbruck, AUSTRIA, ²Great Ormond Street Hospital for Children NHS Trust, London, UNITED KINGDOM, ³Institut Bioquimica Clinica, Barcelona, SPAIN, ⁴Department of Neuropediatrics and Metabolic Disease, University of Marburg, Marburg, GERMANY, ⁵Kennnedy Krieger Institute, Baltimore, MD, ⁶Department of Medical Genetics, The Children's Memorial Health Institute, Warszawa, POLAND.

The Smith-Lemli-Opitz Syndrome (SLOS; MIM 270400) is an autosomal recessive malformation/mental retardation (MR) syndrome which ranges in clinical severity from mild dysmorphism and moderate MR to severe congenital anomalies and intrauterine death. SLOS is caused by mutations in the delta 7 sterol-reductase gene (DHCR7; E.C. 1.3.1.21) which impair endogeneous cholesterol biosynthesis making the growing embryo dependent from exogeneous (maternal) sources of cholesterol. We here have investigated whether the apo E gene which is a major component of the cholesterol transport system in humans is a modifier of the SLOS. Common apo E genotypes and DHCR7 genotypes were determined in 103 biochemically characterized SLOS patients and in 47 of their mothers. The SLOS patients clinical severity score correlated significantly (p = 0.009) with the maternal but not the patients apo E genotype. In line with their effects on cholesterol levels the apo e4 allele was associated with a mild and the e2 allele with a severe SLOS phenotype (p = 0.023). The correlation of apo E genotype with disease severity persisted after stratification for DHCR7 genotype but disappeared when cholesterol concentrations were considered. The data suggest that apo E is involved in the transport of cholesterol from the mother to the embryo and expand the role of apo E and it´s disease associations to embryonic development and malformation.

Knockout mice carrying a deletion of the Mental Retardation gene Gdi1 show impaired associative memory and altered social behavior

D. Toniolo¹, P. D'Adamo², H. Welzl³, H. Lipp⁴, P. Chapman⁵, C. Tiveron⁶, D. Bottai¹, F. Valtorta⁷, F. Valtorta⁷, F. Valtorta⁷, F. Valtorta⁷;
¹IGBE-CNR, Pavia, ITALY, ²Dept. of Anatomy, University of Zurich, Zurich, SWITZERLAND, ³Dept. of Anatomy, U.of Zurich, Zurich, SWITZERLAND, ⁴Dept. of Anatomy, U. of Zurich, Zurich, SWITZERLAND, ⁵University of Cardiff, Cardiff, UNITED KINGDOM, ⁶Regina Elena Institute, Rome, ITALY, ⁷DIBIT-HSR, Milano, ITALY.

Genes for non-specific mental retardation (NSMR) are thought to be responsible for development of cognitive functions. One of the recently identified genes, GD1, encodes aGdi, one of the proteins controlling the activity of the small GTPases of the Rab family in vesicle fusion and intracellular trafficking. It was suggested that, in brain, the main role of aGDI was to interact with Rab3A, the Rab protein participating in synaptic vesicle fusion and neurotransmitter release.
To establish how lack of aGDI could cause mental retardation, we generated mice carrying a deletion of Gdi1 resulting in complete loss of aGDI. The mice were viable and fertile. Histological analysis of brains revealed trilamination of the infrapyramidal mossy fibers and disorganized CA3 pyramidal cells in the hippocampus of mutants, as the only visible defect.
The Gdi1 deficient mice were normal in many tasks to assess learning capacity and emotional behavior. They were impaired in tasks requiring formation of temporal associations suggesting defects in short term memory. They also show lowered aggression and altered social behavior. Our results show that in mice, as in humans, lack of Gdi1 spares most CNS functions and preferentially impairs only a few, involved in the coordination and interaction between associative forebrain structures. Biochemical and electrophysiological analysis showed that altered behavior of the mutant mice is not dependent on Rab3A whose level and intracellular distribution are not changed in the KO mice. It more likely depends on steps of exo/endocytosis involved in synaptic vesicle recycling.

The role of different mutations found in Opitz BBB/G syndrome patients on MID1 protein function

S. Schweiger¹, A. Trockenbacher², T. Lehmann¹, J. Winter¹, V. Suckow¹, S. Krauß¹, F. Majewski³, J. L. Whittaker⁴, H. H. Ropers¹, R. Schneider²;
¹Max-Planck Institute for Molecular Genetics, Berlin, GERMANY, ²Institute of Biochemistry, Innsbruck, AUSTRIA, ³University of Düsseldorf, Düsseldorf, GERMANY, ⁴Addenbrooke's NHS Trust, Cambridge, UNITED KINGDOM.

Opitz BBB/G syndrome (OS) is a malformation syndrome of the ventral midline. The protein encoded by the MID1 gene, which is responsible for X-linked OS, comprises five separate domains common to the RING finger protein family, i.e. the RING finger itself, two B-Boxes, a coiled-coil domain and a fibronectin III domain. The C-terminal B30.2 domain is found in a subset of these proteins. Most of the mutations identified to date cluster in this part of the MID1 protein. We have previously shown that MID1 associates with microtubules. MID1 proteins carrying a mutation in the C-terminal domain do not associate with microtubules but form cytoplasmic clots instead. Recently we found that MID1 is involved in targeting the ubiquitination machinery towards microtubule-associated PP2A by binding to its regulatory subunit a4. Binding of the a4 protein to MID1 is clearly restricted to the B-Box 1 of the protein.
Now we have identified a missense mutation in this protein domain in a patient with Opitz BBB/G syndrome. Immunoprecipitation experiments show that the binding affinity of a4 to the MID1 protein with this particular mutation is significantly reduced. Interestingly this contradicts the crystral structure-based model of Freemont et al. which proposes that the loop carrying the respective B-Box mutation is not directly involved in protein-protein interaction. In addition we found a missense mutation in the coiled-coil domain which is essential for MID1 homodimer formation and protein function. Preliminary observations indicate that a MID1 protein that carries the respective mutation in fact can no longer homodimerize.

ATRX and CBP gene silencing in human neuronal precursor cells by RNA interference

F. Megiorni, P. Indovina, B. Mora, M. Mazzilli;
Dept. of Experimental Medicine and Pathology - University 'La Sapienza', Rome, ITALY.

The ATR-X (a-thalassaemia/mental retardation-X linked) and Rubinstein-Taybi syndromes are two examples of mental retardation and multiple congenital malformation disorders caused by mutations in transcription regulators (ATRX and CBP, respectively). The presence of a determinate spectrum of affected tissues indicates that both the ATRX and CBP mutations modify the expression of a restricted class of genes, but these genes are as yet unidentified. A general tool to recognize the target genes of a transcriptional modulator could be the silencing of the regulator and the subsequent research of the genes that present altered expression. Since the main clinical feature of both syndromes is the mental retardation, we performed the gene silencing experiments in NT2 cells that provide a model of the human neuronal differentiation. To block ATRX and CBP gene function, we have chosen the new strategy of RNA interference. The NT2 cells were separately transfected with two small interfering RNAs, one for ATRX knock out and one for CBP blocking. The ATRX expression was specifically reduced of about 90% by the cognate siRNA, but not by the siRNA directed against CBP. Similarly, a CBP protein reduction of more than 90% was observed only when the CBP siRNA was used. The ATRX or CBP depleted NT2 cells represent an in vitro model system to study the pathogenetic mechanisms of the two syndromes by the identification of the ATRX and CBP target genes that could be involved in neuronal differentiation.

Transcriptome and Transcriptosomes: Gene Expression Analysis in Human Autosomal Aneuploidy

D. R. FitzPatrick¹, N. McGill¹, M. Shade², A. Carothers¹, N. Hastie¹;
¹MRC Human Genetics Unit, Edinburgh, UNITED KINGDOM, ²Lothian Regional Cytogenetics Laboratory, Edinburgh, UNITED KINGDOM.

Autosomal trisomies are common causes of early pregnancy loss, neonatal death and multiple congenital anomalies. In postnatal life only trisomies of chromosome 21, 13 or 18 are consistently detected. Each produces a distinct clinical syndrome with considerable variability in the severity and pattern of associated malformations, which cannot be accurately predicted by the karyotype. We perform transcriptome analyses using mRNA extracted from human trisomy 21 and 13 primary amniocytes. These cells are of fetal origin and easily available following routine diagnostic testing. A commercial microarray system with ~9000 different human cDNAs was used. A subset of the microarray-derived gene expression ratios were also confirmed using the LightCycler system. Compared to normal amniocytes relatively few (0.7-3.2%) genes show substantial misregulation and these differ between the trisomies. Remarkably, when all the ratios were averaged by chromosome ("transcriptosome") only the relevant trisomic transcriptosome showed significant differences. The levels of these up-regulations were 1.14 for trisomy 21 and 1.06 for trisomy 13. The majority of altered expression is, however, secondary and scattered around the genome. We then used the bioinformatics tool PubGene to arrange expression data into potentially interacting gene networks. For example, several different matrix metalloproteinases (MMP10, MMP7, MMP1, TIMP3) are >1.7-fold upregulated in trisomy 21 whereas several insulin-like growth factor binding protein-associated genes (IGFBP4, IGFBP5, SERPINE1, EDN1, CTGF & THBS1) were >1.7 fold down-regulated. We believe that transcriptome analysis holds great potential for unravelling the molecular basis of phenotypic variation and embryopathology in chromosomal disorders.

A. Reymond¹, V. Marigo², M. Yaylaoglu³, A. Leoni², C. Ucla¹, N. Scamuffa¹, S. Banfi², R. Lyle¹, C. Caccioppoli², G. Eichele³, S. E. Antonarakis¹, A. Ballabio²;
¹Division of Medical Genetics, University of Geneva Medical School, Geneva, SWITZERLAND, ²TIGEM, Naples, ITALY, ³Max Planck Institute, Hannover, GERMANY.

Down syndrome (DS), due to an extra copy of human chromosome 21 (HC21), is the most common genetic cause of mental retardation. To define where HC21 genes exert their function and identify their possible role in the DS phenotypes we performed a systematic analysis of the expression profile of 170 murine genes which represent (almost all) the orthologues of the HC21 genes. To obtain an high resolution expression pattern several complementary methods were combined: RT-PCR on a mouse cDNA panel of 12 adult tissues and 4 developmental stages; wholemount in situ of E9.5 and E10.5 embryos and section in situ of E14.5 embryos. These stages correspond to mid and late embryonic and fetal human periods, when the major organs and body regions are organized. Genes showing interesting and/or restricted expression pattern were analyzed further on appropriate sections at more developmental timepoints. 91% of the tested genes showed a clear expression pattern during murine development. In 37% of the cases expression was ubiquitous while 50% of the cDNAs showed a differential expression pattern in the embryonal tissues.
The topographical catalogue of expression of the murine orthologues of human chromosome 21 genes will be instrumental to the understanding of the pathogenesis of trisomy 21 as well as of other chromosome 21 linked disorders. Some of analyzed genes display a pattern of expression relevant to the congenital heart disease and/or to the mental retardation observed in DS. The entire data set will be made available to the scientific community via a web site.

Human mitochondrial DNA diversity in the Near and Middle East and in northeastern Africa: a phylogeographic approach

E. Metspalu¹, M. Reidla², J. Parik², T. Kivisild³, K. Tambets³, E. Usanga⁴, R. Villems³;
¹Tartu University and Estonian Biocentre, Tartu, ESTONIA, ²Tartu University, Tartu, ESTONIA, ³Estonian Biocentre, Tartu, ESTONIA, ⁴University of Kuwait, Kuwait, KUWAIT.

It is now well established that virtually all mtDNA variants outside Africa derive from a single sub-Saharan African mtDNA lineage L3. Descending from L3 internal nodes of the phylogenetic tree, primarily M and N, and a derivative of the latter, R, seem to give rise to all extant branches of human maternal lineages outside Africa, except the effects caused by more recent migrations. Yet it is still unclear where and when this initial diversification arose, including a question whether largely African-specific haplogroups like M1 and U6 have arisen initially in Africa, or do they reflect selective human migrations back to Africa. We have analysed 1519 mtDNAs from Egypt, Ethiopia, Oman, Yemen, Saudi-Arabia, Jordan, Lebanon, Syria and Kuwait, as well as 1263 mtDNAs from Anatolia, South Caucasus and Iran. Detailed phylogeographic mapping of mtDNA haplogroups was carried out together with coalescence age calculations, using an additional knowledge about the European, Central Asian, Siberian and Indian populations. It became clear that individual lineage clusters within universally present basic western Eurasian mtDNA haplogroups may have very different spread in this geographically contiguous area, likely explained by deep Palaeolithic isolation, including yet poorly understood effects of the LGM to the human presence in this region. Several western Eurasian haplogroups (subgroups thereof) that are present universally, display systematically their deepest, pre-LGM coalescence ages in Egypt, Ethiopia and in Anatolia and in the South Caucasus, not in the Middle East or peninsular Arabia. Diversification in internal nodes N, R and HV is discussed.