Search

Orofacial cleft (OC) is a common congenital anomaly in humans, which has lifelong implications for affected individuals. This disorder can be classified as syndromic or non-syndromic depending on the presence or absence of additional physical or neurodevelopmental abnormalities, respectively. Non-syndromic cleft is often non-familial in nature and has a complex aetiology, whereas syndromic forms tend to be monogenic. Although individual OC-related syndromes have been frequently described in the medical literature, there has not been a comprehensive review across syndromes, thereby leaving a gap in our knowledge, which this paper aims to address. Six hundred and three patients with cleft-related human phenotype ontology terms were identified within the Deciphering Developmental Disorders study. Genes carrying pathogenic/likely pathogenic variants were identified and reviewed enabling a diagnostic yield of 36.5%. In total, 124 candidate genes for syndromic OC were identified, including 34 new genes that should be considered for inclusion in clinical clefting panels. Functional enrichment and gene expression analyses identified three key processes that were significantly overrepresented in syndromic OC gene lists: embryonic morphogenesis, protein stability and chromatin organization. Comparison with non-syndromic OC gene networks led us to propose that chromatin remodelling specifically contributes to the aetiology of syndromic OC. Disease-driven gene discovery is a valid approach to gene identification and curation of gene panels. Through this approach, we have started to unravel common molecular pathways contributing to syndromic orofacial clefting.

Introduction: Since 2005, disease-related human genetic diversity has been intensively characterized using genome-wide association studies (GWAS). Understanding how and by whom this work was performed may yield valuable insights into the generalizability of GWAS discoveries to global populations and how high-impact genetics research can be equitably sustained in the future. Materials and Methods: We mined the NHGRI-EBI GWAS Catalog (2005–2022) for the most burdensome non-communicable causes of death worldwide. We then compared (i) the geographic, ethnic and socioeconomic characteristics of study populations; (ii) the geographic and socioeconomic characteristics of the regions within which researchers were located and (iii) the extent to which male and female investigators undertook and led the research. Results: The research institutions leading the work are often US-based (37%), while the origin of samples is more diverse, with the Nordic countries having contributed as much data to GWAS as the United States (~17% of data). The majority of first (60%), senior (75%) and all (66%) authors are male; although proportions vary by disease and leadership level, male co-authors are the ubiquitous majority. The vast majority (91%) of complex trait GWAS has been performed in European ancestry populations, with cohorts and scientists predominantly located in medium-to-high socioeconomically ranked countries; apart from East Asians (~5%), other ethnicities rarely feature in published GWAS. See: https://hugofitipaldi.shinyapps.io/gwas_results/ to browse all results. Conclusion: Most GWAS cohorts are of European ancestry residing outside the United States, with a smaller yet meaningful proportion of East Asian ancestry. Papers describing GWAS research are predominantly authored by male scientists based in medium-to-high income countries.

Advances in molecular biology and genetics have allowed researchers to probe function and dysfunction at the level of the individual gene and/or protein. The integration of such information into an understanding of function has been a challenge facing all molecular geneticists and has contributed to the widening gap between molecular and systems scientists. In the neurosciences, this rift is especially dramatic. The ability to simultaneously monitor changes in the expression of thousands of genes using DNA microarrays has the potential to provide a global or ‘genomic’ view of brain function/ dysfunction. Two important barriers in the application of this technology to the study of neurological disease have been the availability of high-powered analytical approaches and appropriate genetic models. This issue of Human Molecular Genetics contains six contributions that approach these hurdles. These papers are centered on the application of DNA microarrays to survey alterations in gene expression associated with the expression of mutant polyglutamine proteins. A consortium headed by Dr Jim Olson of the Fred Hutchinson Cancer Research Center in Seattle and funded by the Hereditary Disease Foundation initiated this effort. Thus, this work is focused on the effects of expressing a mutant form of the Huntington disease (HD) gene product, huntingtin. Three papers, two by Luthi-Carter and colleagues (1,2) and one from Chan and co-workers (3), examine polyglutamineinduced changes in gene expression using several mouse models of HD. In the Luthi-Carter et al. contributions, mice expressing a truncated form of mutant huntingtin are examined. The first paper provides data that, in R6/2 mice (the often designated Bates mouse), a considerable number of genes have an altered pattern of expression and this pattern shows little sign of regional specificity (1). These are consistent with the previous pathological data indicating that this mouse model of HD shows little sign of the regional-specificity of disease as seen in patients. The second paper from the Luthi-Carter group compares gene expression changes between mouse models of dentatorubral–pallidoluysian atrophy and HD (2). At least in the cerebella of these mice, there was a considerable overlap in the genes showing altered gene expression. Thus, polyglutamine-induced changes in gene expression can be identified that are independent of the protein context in which the glutamine tract is located. Perhaps such context-independent genes reflect pathways that are common among the different polyglutamine

Polygenic risk scores (PRSs) have become the standard for quantifying genetic liability in the prediction of disease risks. PRSs are generally constructed as weighted sum scores of risk alleles using effect sizes from genome-wide association studies as their weights. The construction of PRSs is being improved with more appropriate selection of independent single-nucleotide polymorphisms (SNPs) and optimized estimation of their weights but is rarely reflected upon from a theoretical perspective, focusing on the validity of the risk score. Borrowing from psychometrics, this paper discusses the validity of PRSs and introduces the three main types of validity that are considered in the evaluation of tests and measurements: construct, content, and criterion validity. This introduction is followed by a discussion of three topics that challenge the validity of PRS, namely, their claimed independence of clinical risk factors, the consequences of relaxing SNP inclusion thresholds and the selection of SNP weights. This discussion of the validity of PRS reminds us that we need to keep questioning if weighted sums of risk alleles are measuring what we think they are in the various scenarios in which PRSs are used and that we need to keep exploring alternative modeling strategies that might better reflect the underlying biological pathways.

Transcriptome-wide association studies increase the yield of loci associated with disease phenotypes by focusing on expression quantitative trait loci (eQTL). The major source of eQTL data for is the Gene and Tissue Expression (GTEx) project, which is comprised entirely of adults, mainly those >50 years of age at death. Since gene expression levels differ by developmental stage, it is not clear whether eQTLs derived from adult data sources are best suited for use in young-onset diseases such as pediatric cancers. To fill in this knowledge gap, we performed a large-scale eQTL mapping analysis in the GenCord study with newborn samples and compared it with GTEx. Under matched conditions, we found around 80% of the eQTLs in one study can be replicated in the other. However, among all eQTLs identified in GenCord (GTEx), 584 (1045) showed statistically significant differences in effect sizes in GTEx (GenCord). We further investigated how using fetal eQTL data can facilitate the genetic association study of acute lymphoblastic leukemia. GenCord and GTEx identified the same genetic loci with statistical significance; however, the overall association pattern was only weakly correlated. Our paper demonstrates age-differential eQTLs and shows their potential influence on childhood leukemia research.

Myotonic dystrophy type 1 (DM1) is a multi-systemic disease resulting in severe muscle weakening and wasting. DM1 is caused by expansion of CTG repeats in the 3′ untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. We have developed an inducible, skeletal muscle-specific mouse model of DM1 (CUG(960)) that expresses 960 CUG repeat-expressing animals (CUG(960)) in the context of human DMPK exons 11–15. CUG(960) RNA-expressing mice induced at postnatal day 1, as well as adult-onset animals, show clear, measurable muscle wasting accompanied by severe histological defects including central myonuclei, reduced fiber cross-sectional area, increased percentage of oxidative myofibers, the presence of nuclear RNA foci that colocalize with Mbnl1 protein, and increased Celf1 protein in severely affected muscles. Importantly, muscle loss, histological abnormalities and RNA foci are reversible, demonstrating recovery upon removal of toxic RNA. RNA-seq and protein array analysis indicate that the balance between anabolic and catabolic pathways that normally regulate muscle mass may be disrupted by deregulation of platelet derived growth factor receptor β signaling and the PI3K/AKT pathways, along with prolonged activation of AMP-activated protein kinase α signaling. Similar changes were detected in DM1 skeletal muscle compared with unaffected controls. The mouse model presented in this paper shows progressive skeletal muscle wasting and has been used to identify potential molecular mechanisms underlying skeletal muscle loss. The reversibility of the phenotype establishes a baseline response for testing therapeutic approaches.

Missense mutations in the gene, MAP3K1, are a common cause of 46,XY gonadal dysgenesis, accounting for 15-20% of cases [Ostrer, 2014, Disorders of sex development (DSDs): an update. J. Clin. Endocrinol. Metab., 99, 1503-1509]. Functional studies demonstrated that all of these mutations cause a protein gain-of-function that alters co-factor binding and increases phosphorylation of the downstream MAP kinase pathway targets, MAPK11, MAP3K and MAPK1. This dysregulation of the MAP kinase pathway results in increased CTNNB1, increased expression of WNT4 and FOXL2 and decreased expression of SRY and SOX9. Unique and recurrent pathogenic mutations cluster in three semi-contiguous domains outside the kinase region of the protein, a newly identified N-terminal domain that shares homology with the Guanine Exchange Factor (residues Met164 to Glu231), a Plant HomeoDomain (residues Met442 to Trp495) and an ARMadillo repeat domain (residues Met566 to Glu862). Despite the presence of the mutation clusters and clinical data, there exists a dearth of mechanistic insights behind the development imbalance. In this paper, we use structural modeling and functional data of these mutations to understand alterations of the MAP3K1 protein and the effects on protein folding, binding and downstream target phosphorylation. We show that these mutations have differential effects on protein binding depending on the domains in which they occur. These mutations increase the binding of the RHOA, MAP3K4 and FRAT1 proteins and generally decrease the binding of RAC1. Thus, pathologies in MAP3K1 disrupt the balance between the pro-kinase activities of the RHOA and MAP3K4 binding partners and the inhibitory activity of RAC1.

Nanopore sequencing has been available to researchers for a little over 3 years. Recently, the milestone of sequencing and assembling a human genome on this platform was achieved for the first time. Significant improvements to the platform in yield and accuracy, coupled with higher throughput nanopore sequencers, mean that human genome sequencing at scale is now possible. Here, a brief recent history of the nanopore platform is provided, key papers and innovations are highlighted and some of the challenges for the future are discussed.

Very recently, two papers have presented intriguing data suggesting that prevention of transmission of human mitochondrial DNA (mtDNA) disease is possible. [Craven, L., Tuppen, H.A., Greggains, G.D., Harbottle, S.J., Murphy, J.L., Cree, L.M., Murdoch, A.P., Chinnery, P.F., Taylor, R.W., Lightowlers, R.N. et al. (2010) Pronuclear transfer in human embryos to prevent transmission of mitochondrial DNA disease. Nature, 465, 82–85. Tachibana, M., Sparman, M., Sritanaudomchai, H., Ma, H., Clepper, L., Woodward, J., Li, Y., Ramsey, C., Kolotushkina, O. and Mitalipov, S. (2009) Mitochondrial gene replacement in primate offspring and embryonic stem cells. Nature, 461, 367–372.] These recent advances raise hopes for families with mtDNA disease; however, the successful translational of these techniques to clinical practice will require further research to test for safety and to maximize efficacy. Furthermore, in the UK, amendment to the current legislation will be required. Here, we discuss the clinical and scientific background, studies we believe are important to establish safety and efficacy of the techniques and some of the potential concerns about the use of these approaches.

Nephronophthisis is a hereditary nephropathy characterized by interstitial fibrosis and cyst formation. It is caused by mutations in NPHP genes encoding the ciliary proteins, nephrocystins. In this paper, we investigate the function of nephrocystin-4, the product of the nphp4 gene, in vivo by morpholino-mediated knockdown in zebrafish and in vitro in mammalian kidney cells. Depletion of nephrocystin-4 results in convergence and extension defects, impaired laterality, retinal anomalies and pronephric cysts associated with alterations in early cloacal morphogenesis. These defects are accompanied by abnormal ciliogenesis in the cloaca and in the laterality organ. We show that nephrocystin-4 is required for the elongation of the caudal pronephric primordium and for the regulation of cell rearrangements during cloaca morphogenesis. Moreover, depletion of either inversin, the product of the nphp2 gene, or of the Wnt-planar cell polarity (PCP) pathway component prickle2 increases the proportion of cyst formation in nphp4-depleted embryos. Nephrocystin-4 represses the Wnt-β-catenin pathway in the zebrafish cloaca and in mammalian kidney cells in culture. In these cells, nephrocystin-4 interacts with inversin and dishevelled, and regulates dishevelled stability and subcellular localization. Our data point to a function of nephrocystin-4 in a tight regulation of the Wnt-β-catenin and Wnt-PCP pathways, in particular during morphogenesis of the zebrafish pronephros. Moreover, they highlight common signalling functions for inversin and nephrocystin-4, suggesting that these two nephrocystins are involved in common physiopathological mechanisms.

Contains fulltext : 89133.pdf (Publisher’s version ) (Closed access) Myotonic dystrophy (DM) is a complex, dominantly inherited, multisystem disorder and the archetypal example of an RNA gain-of-function disease. Unstable expansions of (CTG*CAG)n or (CCTG*CAGG)n repeat tracts in the DMPK and ZNF9 genes cause the two known subtypes of myotonic dystrophy, DM1 and DM2, for which no cure or effective molecular treatment exists. Focus in therapeutic development is currently on toxic, expanded (C/CUG)n RNAs. A series of recent papers provide proof of concept of promising strategies using antisense oligonucleotides or small organic compounds aimed at either complete elimination of expanded (CUG)n RNA transcripts or prevention of detrimental protein binding to thermodynamically stable (C/CUG)n hairpin structures. These developments offer new hope to patients with DM, even though several hurdles still have to be overcome before they can be introduced into clinical practice.

The fine-scale structure of the majority of copy number variation (CNV) regions remains unknown. The killer immunoglobulin receptor (KIR) gene complex exhibits significant CNV. The evolutionary plasticity of the KIRs and their broad biomedical relevance makes it important to understand how these immune receptors evolve. In this paper, we describe haplotype re-arrangement creating novel loci at the KIR complex. We completely sequenced, after fosmid cloning, two rare contracted haplotypes. Evidence of frequent hybrid KIR genes in samples from many populations suggested that re-arrangements may be frequent and selectively advantageous. We propose mechanisms for formation of novel hybrid KIR genes, facilitated by protrusive non-B DNA structures at transposon recombination sites. The heightened propensity to generate novel hybrid KIR receptors may provide a proactive evolutionary measure, to militate against pathogen evasion or subversion. We propose that CNV in KIR is an evolutionary strategy, which KIR typing for disease association must take into account.

Common diseases such as type 2 diabetes and coronary heart disease result from a complex interplay of genetic and environmental factors. Recent developments in genomics research have boosted progress in the discovery of susceptibility genes and fueled expectations about opportunities of genetic profiling for personalizing medicine. Personalized medicine requires a test that fairly accurately predicts disease risk, particularly when interventions are invasive, expensive or have major side effects. Recent studies on the prediction of common diseases based on multiple genetic variants alone or in addition to traditional disease risk factors showed limited predictive value so far, but all have investigated only a limited number of susceptibility variants. New gene discoveries from genome-wide association studies will certainly further improve the prediction of common diseases, but the question is whether this improvement is sufficient to enable personalized medicine. In this paper, we argue that new gene discoveries may not evidently improve the prediction of common diseases to a degree that it will change the management of individuals at increased risk. Substantial improvements may only be expected if we manage to understand the complete causal mechanisms of common diseases to a similar extent as we understand those of monogenic disorders. Genomics research will contribute to this understanding, but it is likely that the complexity of complex diseases may ultimately limit the opportunities for accurate prediction of disease in asymptomatic individuals as unraveling their complete causal pathways may be impossible.

Neuronal nicotinic acetylcholine receptors have been implicated in various measures of nicotine dependence. In this paper, we present findings from an exploratory study of single nucleotide polymorphisms (SNPs) in the CHRNB3 and CHRNA6 genes with tobacco and alcohol phenotypes, including frequency of use and three subjective response factors occurring shortly after initiation of use. Subjects were 1056 ethnically diverse adolescents ascertained from clinical and community settings. The most significant associations were found between two CHRNB3 SNPs (rs4950 and rs13280604) and the three subjective response factors to initial tobacco use. These findings were replicated in a separate community sample of 1524 families participating in the National Longitudinal Study of Adolescent Health. Both CHRNB3 SNPs were found to be associated with similar measures of subjective response to tobacco. These results indicate that early subjective response to nicotine may be a valuable endophenotype for genetic studies aimed at uncovering genes contributing to nicotine use and addiction.

Neural tube defects (NTDs) are a heterogeneous group of common severe congenital anomalies which affect 1-2 infants per 1000 births. Most genetic and/or environmental factors that contribute to the pathogenesis of human NTDs are unknown. Recently, however, pathogenic mutations of VANGL1 and VANGL2 genes have been associated with some cases of human NTDs. Vangl genes encode proteins of the planar cell polarity (PCP) pathway that regulates cell behavior during early stages of neural tube formation. Homozygous disruption of PCP genes in mice results in a spectrum of NTDs, including defects that affect the entire neural axis (craniorachischisis), cranial NTDs (exencephaly) and spina bifida. In this paper, we report the dynamic expression of another PCP gene, Fuzzy, during neural tube formation in mice. We also identify non-synonymous Fuzzy amino acid substitutions in some patients with NTDs and demonstrate that several of these Fuzzy mutations affect formation of primary cilia and ciliary length or affect directional cell movement. Since Fuzzy knockout mice exhibit both NTDs and defective primary cilia and Fuzzy is expressed in the emerging neural tube, we propose that mutations in Fuzzy may account for a subset of NTDs in humans.

PKD2 is mutated in 15% of patients with autosomal dominant polycystic kidney disease (ADPKD). Polycystin-2 (PC2), the PKD2 protein, is a nonselective Ca 2+ -permeable cation channel which may function at the cell surface and ER. Nevertheless, the factors that regulate the dynamic translocation of PC2 between the ER and other compartments are not well understood. Constitutive phosphorylation of PC2 at a single C-terminal site (Ser 812 ) has been previously reported. Since we were unable to abolish phospholabelling of PC2 in HEK293 cells by site-directed mutagenesis of Ser 812 or all 5 predicted phosphorylation sites in the C-terminus, we hypothesised that PC2 could also be phosphorylated at the N-terminus. In this paper, we report the identification of a new phosphorylation site for PC2 within its N-terminal domain (Ser 76 ) and demonstrate that this residue is phosphorylated by glycogen synthase kinase 3 (GSK-3). The consensus recognition sequence for GSK-3 (Ser 76 /Ser 80 ) is evolutionarily conserved down to lower vertebrates. In the presence of specific GSK-3 inhibitors, the lateral plasma membrane pool of endogenous PC2 redistributes into an intracellular compartment in MDCK cells without a change in primary cilia localization. Finally, co-injection of wild-type but not a S76A/S80A mutant PKD2 capped mRNA could rescue the cystic phenotype induced by an antisense morpholino oligonucleotide to pkd2 in zebrafish pronephric kidney. We conclude that surface localization of PC2 is regulated by phosphorylation at a unique GSK-3 site in its N-terminal domain in vivo and in vitro. This site is functionally significant for the maintenance of normal glomerular and tubular morphology.

Mitochondrial DNA (mtDNA) polymerase gamma (Polg) is a heterodimeric enzyme containing a Pol I-like catalytic core (PolgA) and an accessory subunit. Mutations in POLGA, affecting the stability of mtDNA, have been identified in several human pathologies such as progressive external ophthalmoplegia and Alpers' syndrome. Extensive literature shows mitochondrial toxicity effects nucleoside analogue reverse transcriptase inhibitors used in the treatment of HIV and chronic hepatitis B as a consequence of an inhibitory effect on Polg. We have previously shown that mice with an error-prone version of PolgA accumulate higher levels of somatic mtDNA mutations resulting in a premature aging phenotype. In the present paper, we demonstrate PolgA deficiency in mouse embryos causes an early developmental arrest between embryonic days 7.5 and 8.5 associated with severe mtDNA depletion. Heterozygous knockout mice have half the wild-type levels of PolgA transcripts and a slight reduction in mtDNA levels but develop normally. Surprisingly, amounts of PolgA transcripts in heterozygous knockout mice are increased in response to artificially elevated mtDNA copy number, revealing a possible regulatory link between mtDNA maintenance and PolgA expression. Our results show that Polg indeed is the only DNA polymerase capable of maintaining mtDNA in mammalian mitochondria. In addition, presence of Polg is absolutely essential for the organogenesis during mammalian embryonic development.

Huntington's disease (HD) is an incurable and fatal neurodegenerative disorder. Improvements in the objective measurement of HD will lead to more efficient clinical trials and earlier therapeutic intervention. We hypothesized that abnormalities seen in the R6/2 mouse, a greatly accelerated HD model, might highlight subtle phenotypes in other mouse models and human HD. In this paper, we identify common gene expression changes in skeletal muscle from R6/2 mice, Hdh(CAG(150)) homozygous knock-in mice and HD patients. This HD-triggered gene expression phenotype is consistent with the beginnings of a transition from fast-twitch to slow-twitch muscle fiber types. Metabolic adaptations similar to those induced by diabetes or fasting are also present but neither metabolic disorder can explain the full phenotype of HD muscle. The HD-induced gene expression changes reflect disease progression. This raises the possibility that muscle gene expression may be used as an objective biomarker to complement clinical HD-rating systems. Furthermore, an understanding of the molecular basis of muscle dysfunction in HD should provide insight into mechanisms involved in neuronal abnormalities and neurodegeneration.

Beside the well-known polyglutamine expansions involved in several neurodegenerative disorders, convergent recent findings pointed to the expansion of polyalanine stretches as a disease mechanism in congenital malformations, skeletal dysplasia and nervous system anomalies. Polyalanine stretches have been predicted in roughly 500 human proteins among which nine have been ascribed to disease phenotype by expansion of polyalanines. The function of polyalanine stretches is largely unknown. This paper aims to review the rapidly growing evidences for a disease-causing mechanism common to expansion of homopolymeric tracts whatever the amino acid involved is.

The muscular dystrophies and congenital myopathies are inherited diseases of the skeletal muscle, which lead to a loss of muscle function and are often fatal. While many of the loci involved are already known, these conditions remain incurable, and genetic models are being developed in an effort to understand the pathological mechanisms involved. Recently several papers have shown that the zebrafish, which is now widely used in developmental genetic studies, will provide a useful addition to our toolkit in this regard. Here we describe these studies, including a zebrafish model of what is potentially the novel pathological mechanism of muscle attachment failure in Duchenne and other muscular dystrophies.

The population of the province of Newfoundland and Labrador is genetically isolated. This isolation is evidenced by an overabundance of several monogenic disorders. The Newfoundland population, like that of other isolates, is now the focus of interest for identification of genes implicated in common diseases. However, the utility of such populations for this purpose remains unproven. In this paper, we review the current genetic architecture of the province, with respect to geographic isolation, homogeneity, founder effect, genetic drift and extended linkage disequilibrium. Based on these factors, we propose that the population of Newfoundland offers many advantages for genetic mapping of common diseases, compared with admixed populations, and even compared with other isolates.

Migration of post-mitotic neurons from the ventricular zone to the cortical plate during embryogenesiscomprises one of the most critical stages in brain development. Deficiency of this process often results inmajor brain malformations, including human lissencephaly (smooth brain). Since discovery of the firstgenetic cause of lissencephaly, deletions of chromosome 17p13.3 in Miller–Dieker syndrome, rapid progressin our understanding of neuronal migration has been made based on advances in both brain imagingtechnology and molecular genetics. This progress has resulted in a new system of classification that beganwith pathological descriptions and has evolved to include patterns on brain imaging, causative genes andmost recently the molecular pathways and proposed modes of migration involved. In this review, wesummarize current knowledge regarding five genes that cause or contribute to human lissencephaly,including LIS1, 14-3-3e, DCX, RELN and ARX. Each of these is associated with a characteristic pattern ofmalformation that involves the cerebral cortex and sometimes other brain structures. Based on detailedgenotype–phenotype analysis, we can now infer the most likely causative gene based on brain imaging andother clinical findings, and inversely are becoming able to predict clinical severity based on the specificmutations detected. We also hypothesize, for the first time, a relationship between the specific type oflissencephaly observed and deficiency of specific modes of neuronal migration.INTRODUCTIONMigration of post-mitotic neurons from the ventricular zone toform the cortical plate comprises one of the most critical stagesin brain development. Our understanding of this complexprocess has progressed based on studies of human malformationsand mouse mutants with deficient neuronal migration, particu-larly the malformation known as lissencephaly (LIS) or ‘smooth-brain’. LIS is characterized by a smooth or nearly smoothcerebral surface. It encompasses a spectrum of gyral malforma-tions from complete agyria (absent gyri) to regional pachygyria(broad gyri), and merges with subcortical band heterotopia(SBH). LIS is always associated with an abnormally thick cortex,reduced or abnormal lamination and diffuse neuronal heterotopia(1,2). SBH or ‘double cortex’ consists of circumferential bandsof heterotopic neurons located just beneath the cortex andseparated from it by a thin band of white matter (3,4).Several different types of LIS have been recognized. Themost common type, known as classical LIS (previously type I),has a very thick 10–20mm cortex and no other major brainmalformations. This is the only type associated with SBH. Lesscommon types are associated with agenesis of the corpuscallosum (ACC) or severe cerebellar hypoplasia (5,6).In this paper, we review recent discoveries regarding themolecular mechanisms that regulate or effect neuronalmigration to the cerebral cortex, emphasizing those derivedfrom studies in humans with LIS. Other genes and proteinsidentified in humans with periventricular nodular heterotopiaor cobblestone complex malformations (previously typeII LIS), or in mouse mutants have been reviewed elsewhere(7–11).HUMAN LISSENCEPHALY GENESTo date, five genes have been identified that cause or contributeto LIS in humans: LIS1, 14-3-3e ,DCX RELN and ARX. Theirrespective subtypes and syndromes are shown in Figures 1and 2, and can usually be distinguished based on detailedanalysis of the phenotype as shown in Figure 3.

Rapid developments in the elucidation of simple Mendelian traits in humans, the complexity of genotype-phenotype relationships, and the growing appreciation of complex genetic traits have conspired to focus interest on the role of modifier genes in humans. This paper reviews categories of genetic modifiers and their effects and then discusses non-Mendelian inheritance patterns involving modifier genes. Although genetic models from many disease classes of human and model systems will be considered, we focus this review on the implications for the understanding of pleiotropic malformation syndromes. Genetic modifiers have so far been molecularly defined in relatively few malformation syndromes, but the rapid acknowledgement of their critical role in human development is an exciting advance in contemporary attempts to understand the relationship of phenotype and genotype.

Guanylate cyclase activating protein-1 (GCAP1) is required for activation of retinal guanylate cyclase-1 (RetGC1), which is essential for recovery of photoreceptor cells to the dark state. In this paper, experimentally derived observations are reported that help in explaining why a proline--leucine mutation at position 50 of human GCAP1 results in cone-rod dystrophy in a family carrying this mutation. The primary amino acid sequence of wild-type GCAP1 was mutated using site-directed mutagenesis to give a leucine at position 50. In addition, serine replaced a glutamic acid residue at position 6 to promote N-terminal myristoylation, yielding the construct GCAP1 E6S/P50L. The enzyme was over-expressed in Escherichia coli cells, isolated and purified before being used in assays with RetGC1, characterized by circular dichroism (CD) spectroscopy, and investigated for protease resistance and thermal stability. Assays of cyclic guanosine monophosphate (cGMP) synthesis from guanosine triphosphate by RetGC1 in the presence of E6S/P50L showed that E6S/P50L could activate RetGC1 and displayed similar calcium sensitivity to wild-type GCAP1. In addition, E6S/P50L and wild-type GCAP1 possess similar CD spectra. However, there was a marked increase in the susceptibility to protease degradation and also a reduction in the thermal stability of E6S/P50L as observed by both the cGMP assay and CD spectroscopy. It is therefore suggested that although GCAP1 E6S/P50L has a similar activity and calcium dependency profile to the wild-type GCAP1, its lower stability could reduce its cellular concentration, which would in turn alter [Ca2+] and result in death of cells.

Pompe's disease or glycogen storage disease type II (GSDII) belongs to the family of inherited lysosomal storage diseases. The underlying deficiency of acid alpha-glucosidase leads in different degrees of severity to glycogen storage in heart, skeletal and smooth muscle. There is currently no treatment for this fatal disease, but the applicability of enzyme replacement therapy is under investigation. For this purpose, recombinant human acid alpha-glucosidase has been produced on an industrial scale in the milk of transgenic rabbits. In this paper we demonstrate the therapeutic effect of this enzyme in our knockout mouse model of GSDII. Full correction of acid alpha-glucosidase deficiency was obtained in all tissues except brain after a single dose of i.v. enzyme administration. Weekly enzyme infusions over a period of 6 months resulted in degradation of lysosomal glycogen in heart, skeletal and smooth muscle. The tissue morphology improved substantially despite the advanced state of disease at the start of treatment. The results have led to the start of a Phase II clinical trial of enzyme replacement therapy in patients.

In this paper, we report a novel heterozygous mutation of A285V codon conversion on exon 4 of the desmin (DES), using whole exome sequencing (WES) in an isolated proband with documented dilated cardiomyopathy (DCM). This mutation is predicted to cause three-dimensional structure changes of DES. Immunohistological and electron microscopy studies demonstrated diffuse abnormal DES aggregations in DCM-induced-pluripotent stem cell (iPSC)-derived cardiomyocytes, and control-iPSC-derived cardiomyocytes transduced with A285V-DES. DCM-iPSC-derived cardiomyocytes also exhibited functional abnormalities in vitro. This is the first demonstration that patient-specific iPSC-derived cardiomyocytes can be used to provide histological and functional confirmation of a suspected genetic basis for DCM identified by WES.

Blepharophimosis syndrome (BPES) is an autosomal dominant disorder of craniofacial development, the features of which are small palpebral fissures (blepharophimosis), drooping eyelids (ptosis) and a skin fold arising from the lower eyelid (epicanthus inversus). The chromosomal localization and identity of the BPES locus is not known with certainty. In the current paper, DNA samples from three individuals with a clinical history of BPES, two with interstitial deletions (cases 1 and 2) and one with a balanced translocation (case 3) all involving chromosome 3q23, were analyzed. Allele loss studies using short tandem repeat markers in cases 1 and 2 suggested that the region between the markers D3S1292 and D3S1306 was deleted in both cases. Subsequently, the derived chromosomes resulting from the translocation in case 3 were segregated in interspecific somatic cell hybrids. Analysis of the resultant hybrids showed that D3S1615 was retained in the derived chromosome 3, whereas D3S1316 was retained in the derived chromosome 4. In neither case was the marker present in the reciprocal hybrid. These results indicate that the BPES critical region lies in the D3S1615-D3S1316 interval.

The CFTR gene, in which more than 300 mutations have been described, displays a spectrum of mutations which varies according to ethnic and geographic origin of patients. In this paper we report an exhaustive study of the 27 exons and exon/intron boundaries of a sample of 35 CF patients from Bulgaria which is situated in the south east of Europe. We have used denaturing gradient gel electrophoresis assay followed by DNA sequencing and we report the identification of six previously undescribed CFTR alleles.

The product of the tumor suppressor gene p53 binds to DNA and activates transcription from promoters containing its consensus binding site. This activity has been hypothesized to be responsible for its biological effects. However, the total number and nature of human genomic sites with which p53 can functionally interact is unknown. In this paper, we have used a Saccharomyces cerevisiae-based screen to identify human genomic sequences that activate transcription from an adjacent reporter gene in a p53-dependent manner (p53-tagged sites, PTS). Fifty-seven different PTS were identified, and the total number of such sites in the human genome was predicted to be between 200 and 300. Almost all contained two adjacent copies of the previously defined consensus 5'-PuPuPuC(A/T)(T/A)GPyPyPy-3'. Spacing between the copies was found to be critical for sequence-specific transcriptional activation in vivo. These results further refine the nature of the genomic sequences likely to be most important for p53-mediated tumor suppression.

In order to characterize the non-delta F508 mutations that account for 36% of cystic fibrosis (CF) chromosomes in Southern France in a sample of 137 patients, we have systematically screened the entire coding region and adjacent sequences of the cystic fibrosis transmembrane conductance regulator (CFTR) gene by the single strand conformation polymorphism (SSCP) technique followed by direct sequencing of the mutant DNAs. We identified 13 novel mutations (9 reported in this paper) and 4 novel rare nucleotide sequence variations. Forty different mutations including delta F508, located in 15 exons, account for only 91.2% of mutants in a population originating from Southern France, in contrast with a recent report on the Celtic population of Brittany demonstrating that 90% of mutations can be detected with only three mutations. We present a very large spectrum of different CF mutations identified in a small geographical area.

The gene responsible for facioscapulohumeral muscular dystrophy (FSHD), an autosomal dominant neuromuscular condition, has been mapped to chromosome 4. Until recently, the two closest available markers were D4S139 and D4S163 but a new marker (p13E-11) which recognizes de novo rearrangements in isolated cases of FSHD characterized by shorter EcoRI fragments has been now identified. Linkage analysis in FSHD families with p13E-11 shows that usually a smaller fragment segregates with the disease gene among the affected individuals from each genealogy. In the present paper, we report the results from linkage analysis with the marker loci D4S163 and D4S139 in 6 FSHD families and with p13E-11 in these and in 6 other additional Brazilian families (total of 12). The results from such analysis do not suggest genetic heterogeneity for FSHD in our population. In 11 out of the 12 families studied with p13E-11, a shorter specific EcoRI band was found to segregate in all affected patients from each genealogy. In one family, the normal individuals had a smaller EcoRI fragment than the affected ones. The size of the EcoRI fragments detected with p13E-11 varied from 13.5 to 29 kb but was constant within each genealogy. Our results suggest that the use of the marker p13E-11 for preclinical and prenatal diagnosis should be done only in families in which it is possible to identify the fragments segregating among the affected individuals.

Aniridia is a congenital malformation of the eye, chiefly characterised by iris hypoplasia, which can cause blindness. The PAX6 gene was isolated as a candidate aniridia gene by positional cloning from the smallest region of overlap of aniridia-associated deletions. Subsequently PAX6 intragenic mutations were demonstrated in Smalleye, a mouse mutant which is an animal model for aniridia, and six human aniridia patients. In this paper we describe four additional PAX6 point mutations in aniridia patients, both sporadic and familial. These mutations highlight regions of the gene which are essential for normal PAX6 function. In addition, the frequency at which we have found PAX6 mutations suggests that lesions in PAX6 will account for most cases of aniridia.

The gene responsible for Huntington disease (HD) has been localized to a 2.2 million base pair (Mbp) region between the loci D4S10 and D4S98 on the short arm of human chromosome 4. As part of a strategy originally designed to clone the gene based on its chromosomal location, we and others previously identified overlapping yeast artificial chromosome (YAC) clones covering most of this region. While these YAC clones were useful for initially obtaining long-range clone continuity, a number of features of the YACs indicated that smaller clones are generally more useful in the subsequent steps of the positional cloning strategy. In this paper, we use these YAC clones to generate sets of overlapping cosmid clones covering most of the HD region. We isolated a large number of cosmids by screening a chromosome 4-specific cosmid library with labeled DNA from a minimal overlapping set of YAC clones. These cosmid clones were further analyzed by restriction mapping and hybridization experiments, leading to the assembly of 185 cosmids into eleven contigs covering more than 1.65 Mbp and to a fine-structure restriction map of the region. Nine of these contigs cover 90 percent of the 1.7 Mbp subregion between loci D4S125 and D4S98 where the HD gene is now known to lie. The detailed restriction map and the cosmid clones should facilitate the identification and localization of cDNAs and polymorphic markers, and they provide reagents for large scale DNA sequencing of this region of the human genome. Our results suggest that this strategy should be generally useful for converting YAC clones into cosmid contigs and generating high-resolution restriction maps of genomic regions of interest.

The molecular defect in Duchenne muscular dystrophy is well established as being due to mutations at Xp21 which disrupt the normal synthesis of the 14kb dystrophin mRNA. More recently, several groups have identified a 4.8kb transcript from this locus which shares exons with the carboxy-terminal region of the dystrophin gene. In this paper we present evidence for an additional 2.2kb mRNA transcript. The 5' untranslated region and first 7 amino acids are identical to that published for the 4.8kb transcript. The position of the translational stop codon and 3' untranslated region is similar to that previously described as the truncated fetal dystrophin isoform. This 2.2kb mRNA has a similar tissue distribution to that described for the 4.8kb mRNA but unlike the other transcripts from the DMD locus, the 2.2kb mRNA is expressed in early development. The relevance of this transcript in the clinical expression of muscular dystrophy and developmental delay is discussed.

Glycogen storage disease type II (GSDII) or Pompe disease is an autosomal recessive disorder caused by acid alpha-glucosidase (GAA) deficiency, leading to lysosomal glycogen accumulation. Affected individuals store glycogen mainly in cardiac and skeletal muscle tissues resulting in fatal hypertrophic cardiomyopathy and respiratory failure in the most severe infantile form. Enzyme replacement therapy has already proved some efficacy, but results remain variable especially in skeletal muscle. Substrate reduction therapy was successfully used to improve the phenotype in several lysosomal storage disorders. We have recently demonstrated that shRNA-mediated reduction of glycogen synthesis led to a significant reduction of glycogen accumulation in skeletal muscle of GSDII mice. In this paper, we analyzed the effect of a complete genetic elimination of glycogen synthesis in the same GSDII model. GAA and glycogen synthase 1 (GYS1) KO mice were inter-crossed to generate a new double-KO model. GAA/GYS1-KO mice exhibited a profound reduction of the amount of glycogen in the heart and skeletal muscles, a significant decrease in lysosomal swelling and autophagic build-up as well as a complete correction of cardiomegaly. In addition, the abnormalities in glucose metabolism and insulin tolerance observed in the GSDII model were corrected in double-KO mice. Muscle atrophy observed in 11-month-old GSDII mice was less pronounced in GAA/GYS1-KO mice, resulting in improved exercise capacity. These data demonstrate that long-term elimination of muscle glycogen synthesis leads to a significant improvement of structural, metabolic and functional defects in GSDII mice and offers a new perspective for the treatment of Pompe disease.

Dyskeratosis congenita (DC) is characterized by multiple features including mucocutaneous abnormalities, bone marrow failure and an increased predisposition to cancer. It exhibits marked clinical and genetic heterogeneity. DKC1 encoding dyskerin, a component of H/ACA small nucleolar ribonucleoprotein (snoRNP) particles is mutated in X-linked recessive DC. Telomerase RNA component (TERC), the RNA component and TERT the enzymatic component of telomerase, are mutated in autosomal dominant DC, suggesting that DC is primarily a disease of defective telomere maintenance. The gene(s) involved in autosomal recessive DC remains elusive. This paper describes studies aimed at defining the genetic basis of AR-DC. Homozygosity mapping in 16 consanguineous families with 25 affected individuals demonstrates that there is no single genetic locus for AR-DC. However, we show that NOP10, a component of H/ACA snoRNP complexes including telomerase is mutated in a large consanguineous family with classical DC. Affected homozygous individuals have significant telomere shortening and reduced TERC levels. While a reduction of TERC levels is not a universal feature of DC, it can be brought about through a reduction of NOP10 transcripts, as demonstrated by siRNA interference studies. A similar reduction in TERC levels is also seen when the mutant NOP10 is expressed in HeLa cells. These findings identify the genetic basis of one subtype of AR-DC being due to the first documented mutations in NOP10. This further strengthens the model that defective telomere maintenance is the primary pathology in DC and substantiates the evidence in humans for the involvement of NOP10 in the telomerase complex and telomere maintenance.

Diabetic nephropathy (DN) is one of the most serious complications of diabetes, accounting for the majority of patients with end-stage renal disease. The molecular pathogenesis of DN involves multiple pathways in a complex, partially resolved manner. The paper presents an exploratory epistatic study for DN. Association analysis were performed on 231 SNP loci in a cohort of 264 type 2 diabetes patients, followed by the epistasis analysis using the multifactor dimensionality reduction and the genetic algorithm with Boolean algebra. A two-locus epistatic effect of EGFR and RXRG was identified, with a cross-validation consistency of 91.7%.

With the genome sequences of numerous species at hand, we have the opportunity to discover how evolution has acted at each and every nucleotide in our genome. To this end, we must identify sets of nucleotides that have descended from a common ancestral nucleotide. The problem of identifying evolutionary-related nucleotides is that of sequence alignment. When the sequences under consideration are entire genomes, we have the problem of multiple whole-genome alignment. In this paper, we first state a series of definitions for homology and its subrelations between single nucleotides. Within this framework, we review the current methods available for the alignment of multiple large genomes. We then describe a subset of tools that make biological inferences from multiple whole-genome alignments.

The discovery in 1999 that Rett syndrome (RTT) is caused by mutations in a gene encoding the methyl-CpG-binding repressor protein MECP2 provided a significant breakthrough in the understanding of this devastating disease. The subsequent production of Mecp2 knockout mice 2 years later provided an experimental resource to better understand how mutations in the MECP2 gene result in RTT. This paper reviews the recent progress in understanding when and where MeCP2 function becomes important in the developing brain, why MeCP2 protein levels are crucial, which genes are normally silenced by MeCP2, and how misexpression of these targets might lead to the clinical manifestations of RTT.

Alzheimer's disease (AD) phenotype complexity raises the question whether genetic features remain unknown. Although a few percentage of patients are familial cases linked to mutations in amyloid precursor protein, presenilin 1 or presenilin 2 genes, the remainder are considered mainly sporadic late-onset cases with a complex etiology. However, changes in gene expression or other genetic features of the individual can clearly contribute to develop the illness. Consequently, in this paper we have focused on the identification of new genes, the expression of which is altered in AD. We used the technique of differential display reverse transcriptase-polymerase chain reaction (DDRT-PCR) in order to study the gene expression differences in brain tissue from patients in an advanced stage of AD. After studying medial septum and hippocampus brain areas, we found an inhibition of the KIAA0471 gene expression in three out of six AD patients, including one with a presenilin 1 gene mutation. This gene encodes for a large protein that presents, in its predicted form, 95% homology with IDN4-GGTR sequences. These results may provide significant clues for understanding the molecular mechanisms underlying septohippocampal neurodegeneration. In addition, they may open a new area of research for diagnostic and therapeutic tools, the relevance of which is also considered.

Contains fulltext : 57812.pdf (Publisher’s version ) (Closed access) NADH-ubiquinone oxidoreductase (complex I) deficiency is amongst the most encountered defects of the mitochondrial oxidative phosphorylation (OXPHOS) system and is associated with a wide variety of clinical signs and symptoms. Mutations in complex I nuclear structural genes are the most common cause of isolated complex I enzyme deficiencies. The cell biological consequences of such mutations are poorly understood. In this paper we have used blue native electrophoresis in order to study how different nuclear mutations affect the integrity of mitochondrial OXPHOS complexes in fibroblasts from 15 complex I-deficient patients. Our results show an important decrease in the levels of intact complex I in patients harboring mutations in nuclear-encoded complex I subunits, indicating that complex I assembly and/or stability is compromised. Different patterns of low molecular weight subcomplexes are present in these patients, suggesting that the formation of the peripheral arm is affected at an early assembly stage. Mutations in complex I genes can also affect the stability of other mitochondrial complexes, with a specific decrease of fully-assembled complex III in patients with mutations in NDUFS2 and NDUFS4. We have extended this analysis to patients with an isolated complex I deficiency in which no mutations in structural subunits have been found. In this group, we can discriminate between complex I assembly and catalytic defects attending to the fact whether there is a correlation between assembly/activity levels or not. This will help us to point more selectively to candidate genes for pathogenic mutations that could lead to an isolated complex I defect.

The possibility that immune responses to autoantigens may contribute to the development of atopic disease has been largely ignored. In this paper, we describe the chromosomal localization of the gene for squamous cell carcinoma-associated reactive antigen for cytotoxic T cells (SART-1). The SART-1 gene localized to a region of 11q12-13 showing strong linkage to atopy in previous studies. Further analysis of this gene revealed the presence of at least 20 exons of varying lengths and four novel single-nucleotide polymorphisms, one of which resulted in an amino acid substitution. Association analysis in families recruited on the basis of affected sib pairs for asthma reveal significant association for both coding region polymorphisms with atopy. We therefore hypothesize that polymorphic variation within the SART-1 gene may account for individuals developing atopy.

The upper respiratory tract, including the nasal and oral cavities, is the major route of entry of pathogens into the body, and early recognition of bacterial products in this region is critical for host defence. A well-established family of four proteins involved in this process are bactericidal/permeability-increasing protein (BPI) and lipopolysaccharide-binding protein (LBP), which are central to the host defence against bacteria, and cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP), which have also been implicated in this response. In this paper, we demonstrate the existence of a related family of seven human proteins, which we designate PLUNC proteins. The PLUNC proteins are encoded by adjacent genes found within a 300 kb region of chromosome 20, suggesting that they may be under transcriptional control of shared genomic elements, and expression data shows that these proteins are found in overlapping regions of the pulmonary, nasopharyngeal and oral epithelium, sites where the previously described BPI family members are not expressed. Whereas the BPI family are predicted to share very closely similar three-dimensional structures, the PLUNC family is predicted to have much greater variability in the N-terminal domain, corresponding to the active domain of BPI, thus creating the notion of a BPI/PLUNC structural superfamily. We suggest that members of the PLUNC family may function in the innate immune response in regions of the mouth, nose and lungs, which are sites of significant bacterial exposure.

Microsatellite polymorphisms provide highly informative readily detectable markers for human linkage studies. This paper reports apparent non-Mendelian inheritance of a dinucleotide repeat polymorphism due to allele non-amplification. The previously hidden allele was revealed and Mendelian inheritance restored when a new CA strand primer was used for amplification. Sequencing of the hidden allele identified a single base substitution at the 3'-most position of the binding site for the original CA strand primer. Allele non-amplification is a potential source of confusion in linkage studies employing polymorphisms detected by the polymerase chain reaction. It should be considered whenever apparent non-Mendelian inheritance or non-paternity are encountered.

Arrhythmogenic right ventricular dysplasia type 2 (ARVD2, OMIM 600996) is an autosomal dominant cardiomyopathy, characterized by partial degeneration of the myocardium of the right ventricle, electrical instability and sudden death. The disease locus was mapped to chromosome 1q42--q43. We report here on the physical mapping of the critical ARVD2 region, exclusion of two candidate genes (actinin 2 and nidogen), elucidation of the genomic structure of the cardiac ryanodine receptor gene (RYR2) and identification of RYR2 mutations in four independent families. In myocardial cells, the RyR2 protein, activated by Ca(2+), induces the release of calcium from the sarcoplasmic reticulum into the cytosol. RyR2 is the cardiac counterpart of RyR1, the skeletal muscle ryanodine receptor, involved in malignant hyperthermia (MH) susceptibility and in central core disease (CCD). The RyR2 mutations detected in the present study occurred in two highly conserved regions, strictly corresponding to those where mutations causing MH or CCD are clustered in the RYR1 gene. The detection of RyR2 mutations causing ARVD2, reported in this paper, opens the way to pre-symptomatic detection of carriers of the disease in childhood, thus enabling early monitoring and treatment.

We have demonstrated that the phenotype of the mouse mutant sex-linked fidget ( slf ) is caused by developmental malformations of the inner ear that result in hearing loss and vestibular dysfunction. Recently, pilot mapping experiments suggested that the mouse Brn4 / Pou3f4 gene co-segregated with the slf locus on the mouse X chromosome. These mapping data, in conjunction with the observation that the vertical head-shaking phenotype of slf mutants is identical to that observed in mice with a targeted deletion of the Brn4 gene, suggested that slf is a mutant allele of the Brn4 gene. In this paper, we have identified the nature of the slf mutation, and demonstrated that it is an X chromosomal inversion with one breakpoint close to Brn4. This inversion selectively eliminates the expression of the Brn4 gene in the developing inner ear, but not the neural tube. Finally, these results demonstrate that the slf mutation is a good mouse model for the most prevalent form of X-linked congenital deafness in man, which is associated with mutations in the human Brn4 ortholog, POU3F4.

Tricho-dento-osseous syndrome (TDO) is an autosomal dominant disorder characterized by abnormal hair, teeth and bone. The main clinical manifestations of TDO include taurodontism, enamel hypoplasia, kinky, curly hair at birth and increased thickness and density of the cranial bones. These pleiotropic clinical features suggest the role of a developmental gene modulating epithelial-mesenchymal interactions. We recently mapped the TDO locus to chromosome 17q21, a region that includes two members of the distal-less homeobox gene family, DLX3 and DLX7. In this paper we describe genomic cloning and sequencing of both human DLX3 and DLX7 and identification of a 4 bp deletion in human DLX3 which correlates with the TDO phenotype in six families. The observed mutation is predicted to cause a frameshift and premature termination codon, resulting in a functionally altered DLX3. This first report of a human mutation in the DLX genes is consistent with murine studies indicating their important role in the development of hair, teeth and bone.

Wilson disease (WD) is an autosomal recessive defect of copper transport characterized by massive accumulation of copper in the liver, which can lead to liver failure. Mutations in a copper transporting ATPase (WND or ATP7B) have been shown to cause the disease. The toxic milk mouse mutant (tx) accumulates copper in the liver in a manner similar to that observed in patients with WD. However, some physiological differences between tx mice and human WD patients have cast doubts on whether this mutant mouse is a valid model for WD. In this paper we report the isolation of cDNA clones encoding the murine homologue of WND. The predicted amino acid sequence is 1462 amino acids and contains the same functional domains identified in human and rat WND. As in the rat, the fourth metal binding domain is apparently non-functional. Similar levels of a 7.5 kb WND mRNA were detected in liver and kidney from normal and tx mice, indicating that transcription of this gene was unaffected in the mutant mice. The coding sequence of WND cDNA from the tx mouse liver identified a single nucleotide difference between the normal DL mouse and the tx which is predicted to change methionine 1356 in the eighth transmembrane domain to valine. This methionine is conserved in all copper ATPases including those from bacteria and yeast. The conclusion that this is the causative mutation is supported by the recent mapping of tx and WND to the same region of mouse chromosome 8. Thus the tx mouse is presented as a valid model for studies of the role of WND in copper transport and for investigation of different treatment strategies for WD.

Fanconi anemia (FA) is a genetically heterogeneous, autosomal recessive disorder characterized by a variety of congenital and skeletal malformations, progressive pancytopaenia and predisposition to malignancies. While the basic defect in this disease is not known, the cloning of the gene defective in FA group C patients (FAC) allows analysis of its expression pattern, which may provide clues about the functional properties of the protein. This paper describes the distribution of Fac transcripts during murine development (8-19.5 days p.c.), using RNA in situ hybridization. Fac is initially expressed (8-10 days p.c.) in the mesenchyme and its derivatives with osteogenic potential. The transcript is also apparent at later stages of bone development (13-19.5 days p.c.), localized to cells of the inner perichondrium, periosteum and zone of endochondral ossification. In the latter, Fac transcripts are seen in cells from both osteogenic and hematopoietic lineages. Fac mRNA is also seen in intramembranous cranial and facial bones. In addition, Fac signal is detected in non-skeletal tissues : brain, whisker follicles, lung, kidney, gut and stomach. Fac expression is high in progenitor cell populations but is downregulated in differentiating cells that give rise to connective tissue. The pattern of Fac expression is consistent with the skeletal and non-skeletal congenital abnormalities in FA patients. As well, expression in rapidly dividing progenitors is consistent with hypotheses regarding the nature of the basic defect in FA : a role of the protein in DNA repair or protection from oxygen toxicity.

Globoid cell leukodystrophy (GLD) of Krabbe disease results from mutations in the galactocerebrosidase (GALC) gene. Previously, we had identified a large deletion in the GALC gene together with a C to T polymorphism at cDNA position 502 in a significant number of cases of infantile Krabbe disease; however, the deletion breakpoint had not been found. In this paper we show that the deletion is approximately 30 kb starting near the middle of the 12 kb intron 10, and includes all of the coding region through exon 17 plus an additional 9 kb. The deletion junction contains a 4 bp direct repeat and is preceded by sequence identified as belonging to the Alu family of interspersed repetitive elements. Using genomic DNA and a PCR-based test to detect normal and deleted sequences at that location, a large number of patients with all clinical types of GLD were analyzed. Of 21 infantile patients found to be heterozygous for the 502T polymorphism reported previously, 15 had the deletion, one could not tested and five, including a Hmong child, did not have the deletion. Sixteen other infantile patients previously tested were found to be either homozygous (10) or heterozygous (6) for the deletion. In addition, five patients with juvenile and adult GLD were found to be heterozygous for the deletion. In every case tested, the deletion was always found on the same allele as the 502T polymorphism. However, other disease-causing mutations have been found on the 502T allele. With careful genotype analysis these families can receive improved genetic information including patient and carrier identification and preimplantation diagnosis.